Any screen.

Any time.
Anywhere.
Activate the eBook version
of this title at no additional charge.

Expert Consult eBooks give you the power to browse and find content,
view enhanced images, share notes and highlights—both online and offline.

Unlock your eBook today.

1 Visit expertconsult.inkling.com/redeem Scan this QR code to redeem your
eBook through your mobile device:
2 Scratch off your code
3 Type code into “Enter Code” box

4 Click “Redeem”
5 Log in or Sign up
6 Go to “My Library”
Place Peel Off
It’s that easy! Sticker Here

For technical assistance:

email expertconsult.help@elsevier.com
call 1-800-401-9962 (inside the US)
call +1-314-447-8200 (outside the US)
Use of the current edition of the electronic version of this book (eBook) is subject to the terms of the nontransferable, limited license granted on
expertconsult.inkling.com. Access to the eBook is limited to the first individual who redeems the PIN, located on the inside cover of this book,
at expertconsult.inkling.com and may not be transferred to another party by resale, lending, or other means.
2015v1.0
Abeloff’s
CLINICAL
ONCOLOGY
This page intentionally left blank
 Abeloff’s
CLINICAL
ONCOLOGY
SIXTH EDITION
JOHN E. NIEDERHUBER, MD
Executive Vice President, Inova Health System
President and CEO, Genomics and Bioinformatics Research Institute
Fairfax, Virginia;
Professor, Department of Public Health Sciences
Member, Center for Public Health Genomics
University of Virginia School of Medicine
Charlottesville, Virginia;
Adjunct Professor, Oncology and Surgery
The Johns Hopkins University School of Medicine
Deputy Director
Johns Hopkins Clinical Research Network
Baltimore, Maryland

JAMES O. ARMITAGE, MD JAMES H. DOROSHOW, MD

Joe Shapiro Professor of Medicine Bethesda, Maryland
University of Nebraska Medical Center
Omaha, Nebraska

MICHAEL B. KASTAN, MD, PhD JOEL E.TEPPER, MD

Executive Director, Duke Cancer Institute
William and Jane Shingleton Professor,
Pharmacology and Cancer Biology
Professor of Pediatrics
Duke University School of Medicine
Durham, North Carolina
Hector MacLean Distinguished Professor of
Cancer Research
Department of Radiation Oncology
UNC Lineberger Comprehensive Cancer Center
University of North Carolina School of Medicine
Chapel Hill, North Carolina
ABELOFF’S CLINICAL ONCOLOGY, SIXTH EDITION ISBN: 978-0-323-47674-4

Copyright © 2020 by Elsevier, Inc. All rights reserved.

No part of this publication may be reproduced or transmitted in any form or by any means, electronic or
mechanical, including photocopying, recording, or any information storage and retrieval system, without
permission in writing from the publisher. Details on how to seek permission, further information about the
Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance
Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions.

This book and the individual contributions contained in it are protected under copyright by the Publisher
(other than as may be noted herein).

Notices

Knowledge and best practice in this field are constantly changing. As new research and experience
broaden our understanding, changes in research methods, professional practices, or medical treatment
may become necessary.
Practitioners and researchers must always rely on their own experience and knowledge in evaluating
and using any information, methods, compounds, or experiments described herein. In using such
information or methods they should be mindful of their own safety and the safety of others, including
parties for whom they have a professional responsibility.
With respect to any drug or pharmaceutical products identified, readers are advised to check the
most current information provided (i) on procedures featured or (ii) by the manufacturer of each product
to be administered, to verify the recommended dose or formula, the method and duration of
administration, and contraindications. It is the responsibility of practitioners, relying on their own
experience and knowledge of their patients, to make diagnoses, to determine dosages and the best
treatment for each individual patient, and to take all appropriate safety precautions.
To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors,
assume any liability for any injury and/or damage to persons or property as a matter of products liability,
negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas
contained in the material herein.

Previous editions copyrighted 2014, 2008, 2004, 2000, and 1995.

Library of Congress Control Number: 2018953655

Executive Content Strategist: Robin Carter

Content Development Manager: Laura Schmidt
Publishing Services Manager: Catherine Jackson
Senior Project Manager: Amanda Mincher
Design Direction: Bridget Hoette

Printed in China

Last digit is the print number: 9 8 7 6 5 4 3 2 1

1600 John F. Kennedy Blvd.

Ste 1600
Philadelphia, PA 19103-2899
 To my son, Matthew, and my wife, Kathy, who have and continue to make sacrifices so that I might pursue my passions in
medicine and research. To my colleagues at the National Cancer Institute, University of Virginia, Johns Hopkins, and across the
country, whose selfless dedication to patient care and cancer research is truly an inspiration to all. To the many students who have
trained with me over the years, to my patients, and to my colleagues at the Inova Translational Medicine Institute, who have given
me the opportunity to have this tremendously rewarding career. Lastly, to Tracey, and to Marty, who, in memory, inspire all who
knew them to work a little harder each day toward the elimination of the pain and suffering from this disease.
JOHN E. NIEDERHUBER, MD

To my wife, Nancy, for her love and support over 49 ½ years.

JAMES O. ARMITAGE, MD

To my wife, Robin Winkler Doroshow, MD, my classmate and greatest supporter, for her love, dedication, and commitment and
for the remarkable joy and caring she brings to her patients and to all around her. To my remarkable daughter, Deborah Doroshow,
MD, PhD, who is completing her training for a career in academic oncology; my fondest hope is that you will enjoy sharing with
and learning from those you help as much as I have. To my patients and colleagues at the City of Hope and the National Cancer
Institute who have all contributed so much of themselves to my continuing education as a physician and investigator, please accept
my appreciation and utmost gratitude.
JAMES H. DOROSHOW, MD

To my wife, Kathy, and my sons, Benjamin, Nathaniel, and Jonathan. You are the lights of my life. I also acknowledge all of my
mentors, colleagues, and patients, who have taught me so much. A special note of gratitude goes to Marty Abeloff, a mentor and an
inspiring role model for career and for life.
MICHAEL B. KASTAN, MD, PhD

To my wife, Laurie, who has been my soul mate for many years and has constantly reminded me of life’s priorities. To my family
including my daughters, Miriam and Abigail, and my grandchildren, Zekariah, Zohar, Samuel, Marcelo, Jonah, and Aurelio. They
have been an inspiration. To my many teachers through the years who have helped define and foster my professional career, but
especially Herman Suit and Eli Glatstein.
JOEL E. TEPPER, MD
This page intentionally left blank

Martin D. Abeloff, MD (1942-2007)
Martin D. Abeloff, a founding editor of Clinical Oncology, dedicated
his life to caring for patients with cancer and to teaching his art to
fellows, residents, and students. He was a brilliant and caring clinician,
an extremely effective leader, and a beloved mentor to many trainees
and young faculty.
Marty was born on April 4, 1942, in Shenandoah, Pennsylvania. He
received his BA from The Johns Hopkins University in 1963 and his
MD from The Johns Hopkins University School of Medicine in 1966.
He spent the next year as an intern at the University of Chicago Hospitals
and Clinics. His legacy in medicine was established on his return to
Baltimore in 1971 as a fellow in clinical oncology. He would spend the
rest of his career at The Johns Hopkins Hospital, achieving the rank of
Professor of Medicine in 1990. At various times, he served as the fel-
lowship training program director, chief of medical oncology, clinical
director of the cancer center, oncologist in chief at The Johns Hopkins
Hospital, and in 1992, was appointed the second director of The Johns
Hopkins Oncology Center, later renamed, thanks to Marty’s efforts, the
Sidney Kimmel Comprehensive Cancer Center.
It was during his time as cancer center director that Marty brought
to life the idea of a comprehensive, user-friendly textbook of oncology
Memoriam
that would be as valuable to the practicing oncologist as to the primary
care physician and physicians-in-training. The first edition of Clinical
Oncology was published in 1995 to a gratifying response. It is now
established as a cornerstone reference for those caring for patients
with cancer.
In the sixth edition, we continue Marty’s vision for an ever better,
unique, and accessible text so that future generations of oncologists
will remember his inspiration and leadership.
The editors again dedicate this text, which is already a recognized
tangible aspect of his legacy in medicine, as a living memorial to him.
Abeloff ’s Clinical Oncology will continue to serve as a reminder to all
its users of this extraordinary person and exemplary physician who
went before them.
John E. Niederhuber, MD
James O. Armitage, MD
James H. Doroshow, MD
Michael B. Kastan, MD, PhD
Joel E. Tepper, MD
vii
This page intentionally left blank
 Contributors

James L. Abbruzzese, MD, FACP, FASCO, DSc (hon) Dara L. Aisner, MD, PhD
Duke Cancer Institute Distinguished Professor of Medical Oncology Associate Professor of Pathology
Chief, Division of Medical Oncology, Department of Medicine CU Anschutz Medical Campus
Associate Director for Clinical Research, Duke Cancer Institute University of Colorado
Duke University Medical Center Aurora, Colorado
Durham, North Carolina
Michelle Alonso-Basanta, MD, PhD
Omar Abdel-Wahab, MD Helene Blum Assistant Professor
Associate Attending Department of Radiation Oncology
Department of Medicine University of Pennsylvania
Leukemia Service Philadelphia, Pennsylvania
Memorial Sloan Kettering Cancer Center
New York, New York Jesus Anampa, MD, MS
Assistant Professor
Ghassan K. Abou-Alfa, MD Department of Oncology
Attending Physician Montefiore Medical Center
Memorial Sloan Kettering Cancer Center Albert Einstein College of Medicine
Professor of Medicine Bronx, New York
Weill Cornell Medicine
New York, New York Megan E. Anderson, MD
Assistant Professor
Janet L. Abrahm, MD Department of Orthopaedic Surgery
Professor of Medicine Harvard Medical School
Harvard Medical School Attending Orthopedic Surgeon
Member, Division of Palliative Care Department of Orthopedic Surgery
Psychosocial Oncology and Palliative Care Boston Children’s Hospital
Dana-Farber Cancer Institute Attending Orthopedic Surgeon
Boston, Massachusetts Department of Orthopedic Surgery
Beth Israel Deaconess Medical Center
Jeffrey S. Abrams, MD Boston, Massachusetts
Associate Director, Cancer Therapy Evaluation Program
Division of Cancer Treatment and Diagnosis Emmanuel S. Antonarakis, MD
National Cancer Institute Associate Professor of Oncology
Rockville, Maryland Sidney Kimmel Comprehensive Cancer Center
Johns Hopkins University School of Medicine
Jeremy S. Abramson, MD, MMSc Baltimore, Maryland
Director, Center for Lymphoma
Hematology/Oncology Richard Aplenc, MD, PhD
Massachusetts General Hospital Department of Pediatrics
Assistant Professor Section Chief, Hematologic Malignancies
Department of Medicine Chief Clinical Research Officer
Harvard Medical School Children’s Hospital of Philadelphia
Boston, Massachusetts Philadelphia, Pennsylvania

ix
x Contributors

Frederick R. Appelbaum, MD Karen Basen-Engquist, PhD, MPH

Executive Vice President and Deputy Director Professor of Behavioral Science
Fred Hutchinson Cancer Research Center University of Texas MD Anderson Cancer Center
Professor Houston, Texas
Division of Medical Oncology
University of Washington Lynda Kwon Beaupin, MD
Seattle, Washington Director, Adolescent and Young Adult Program
Roswell Park Cancer Institute
Luiz H. Araujo, MD, PhD Buffalo, New York
Scientific Director
COI Institute for Research and Education Ross S. Berkowitz, MD
Brazilian National Cancer Institute William H. Baker Professor of Gynecology
Rio de Janeiro, Brazil Department of Obstetrics and Gynecology
Harvard Medical School
Ammar Asban, MD Director of Gynecologic Oncology
Surgical Resident Department of Obstetrics and Gynecology
Department of Surgery Brigham and Women’s Hospital
University of Alabama at Birmingham Boston, Massachusetts
Birmingham, Alabama
Donald A. Berry, PhD
Edward Ashwood, MD Professor of Biostatistics
President and CEO Department of Biostatistics
ARUP Laboratories The University of Texas MD Anderson Cancer Center
Professor of Pathology Houston, Texas
University of Utah
Salt Lake City, Utah Therese Bevers, MD
Professor of Clinical Cancer Prevention
Farrukh T. Awan, MD, MS Medical Director, Cancer Prevention Center
Associate Professor of Medicine The University of Texas MD Anderson Cancer Center
Hematology Houston, Texas
The Ohio State University
Columbus, Ohio John F. Boggess, MD
Professor of Obstetrics and Gynecology
Juliet L. Aylward, MD University of North Carolina
Associate Professor of Dermatology Chapel Hill, North Carolina
University of Wisconsin School of Medicine and Public Health
Madison, Wisconsin Julie R. Brahmer, MD, MSc
Professor of Oncology
Arjun V. Balar, MD Department of Oncology
Associate Professor of Medicine Johns Hopkins Kimmel Cancer Center
Division of Hematology/Oncology Baltimore, Maryland
Director, Genitourinary Cancers Program
New York University Perlmutter Cancer Center Janet Brown, MD, FRCP, MSc, MBBS, BSc
New York University Langone Medical Center Professor
New York, New York Academic Unit of Clinical Oncology, Oncology, and Metabolism
Weston Park Hospital
Courtney J. Balentine, MD University of Sheffield
Assistant Professor of Surgery Sheffield, United Kingdom
Dallas VA Hospital
University of Texas Southwestern Karen Brown, MD
Dallas, Texas Attending Physician
Memorial Sloan Kettering Cancer Center
Stefan K. Barta, MD, MS, MRCP(UK) Professor of Clinical Radiology
Associate Professor Weill Medical College at Cornell University
Hematology and Oncology New York, New York
Fox Chase Cancer Center
Philadelphia, Pennsylvania Powel Brown, MD, PhD
Professor and Chairman
Nancy Bartlett, MD Clinical Cancer Prevention
Professor of Medical Oncology The University of Texas MD Anderson Cancer Center
Washington University School of Medicine Houston, Texas
St. Louis, Missouri
 Contributors xi

Ilene Browner, MD Stephen J. Chanock, MD

Assistant Professor Director
Department of Oncology and Division of Geriatric Medicine Division of Cancer Epidemiology and Genetics
The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins National Cancer Institute
and Johns Hopkins Bayview Bethesda, Maryland
The Johns Hopkins University
Baltimore, Maryland Claudia I. Chapuy, MD
St. Elizabeth’s Medical Center
Paul A. Bunn, MD Dana-Farber Cancer Institute
Distinguished Professor of Medical Oncology Boston, Massachusetts
CU Anschutz Medical Campus
University of Colorado Vikash P. Chauhan, PhD
Aurora, Colorado Massachusetts Institute of Technology
Boston, Massachusetts
William R. Burns, MD
Assistant Professor of Surgery Herbert Chen, MD, FACS
University of Michigan Health System Chairman and Fay Fletcher Kerner Endowed Chair
Ann Arbor, Michigan Department of Surgery
University of Alabama at Birmingham
John C. Byrd, MD Surgeon-in-Chief
Professor of Internal Medicine–Hematology University of Alabama at Birmingham Health System
The Ohio State University Birmingham, Alabama
Columbus, Ohio
Ronald C. Chen, MD, MPH
Karen Cadoo, MD Associate Professor
Attending Medical Oncologist Department of Radiation Oncology
Gynecologic Medical Oncology and Clinical Genetic Services University of North Carolina at Chapel Hill
Memorial Sloan Kettering Cancer Center Chapel Hill, North Carolina
Weill Cornell Medical College
New York, New York Nai-Kong V. Cheung, MD, PhD
Enid A. Haupt Endowed Chair in Pediatric Oncology
David P. Carbone, MD, PhD Department of Pediatrics
Professor of Medicine Memorial Sloan-Kettering Cancer Center
Director, James Thoracic Center New York, New York
James Cancer Center
The Ohio State University Medical Center Jennifer H. Choe, MD, PhD
Columbus, Ohio Medical Instructor
Division of Medical Oncology
H. Ballentine Carter, MD Duke Cancer Institute
Professor of Urology Durham, North Carolina
Johns Hopkins University School of Medicine
Baltimore, Maryland Michaele C. Christian, MD
Cancer Therapy Evaluation Program (Retired)
Jorge J. Castillo, MD National Cancer Institute
Physician Rockville, Maryland
Hematologic Malignancies
Dana-Farber Cancer Institute Paul M. Cinciripini, PhD
Assistant Professor Professor and Chair of Behavioral Science
Harvard Medical School The University of Texas MD Anderson Cancer Center
Boston, Massachusetts Houston, Texas

Alfred E. Chang, MD Michael F. Clarke, MD

Hugh Cabot Professor of Surgery Professor of Medicine
University of Michigan Health System Division of Oncology
Ann Arbor, Michigan Stanford School of Medicine
Palo Alto, California
Eric Chang, MD, FASTRO
Professor and Chair of Radiation Oncology Robert E. Coleman, MBBS, MD
Keck School of Medicine of USC Academic Unit of Clinical Oncology
Los Angeles, California Weston Park Hospital
University of Sheffield
Sheffield, United Kingdom
xii Contributors

Robert L. Coleman, MD Jeffrey Crawford, MD

Professor and Executive Director, Cancer Network Research Professor of Medicine
Department of Gynecologic Oncology and Reproductive Medicine Division of Medical Oncology
The University of Texas MD Anderson Cancer Center Duke Cancer Institute
Houston, Texas Durham, North Carolina

Adriana M. Coletta, PhD, RD Kristy Crooks, PhD

Department of Behavioral Science Assistant Professor of Pathology
Center for Energy Balance in Cancer Prevention and Survivorship CU Anschutz Medical Campus
The University of Texas MD Anderson Cancer Center University of Colorado
Houston, Texas Aurora, Colorado

Jerry M. Collins, PhD Daniel J. Culkin, MD

Associate Director Professor
Division of Cancer Treatment and Diagnosis Department of Urology
National Cancer Institute University of Oklahoma Health Sciences Center
Bethesda, Maryland Oklahoma City, Oklahoma

Jean M. Connors, MD Brian G. Czito, MD

Hematology Division Professor of Radiation Oncology
Brigham and Women’s Hospital Duke University Medical Center
Dana-Farber Cancer Institute Durham, North Carolina
Harvard Medical School
Boston, Massachusetts Piero Dalerba, MD
Assistant Professor of Pathology and Cell Biology
Michael Cools, MD Assistant Professor of Medicine
Department of Neurosurgery Division of Digestive and Liver Diseases
University of North Carolina Columbia University College of Physicians and Surgeons
Chapel Hill, North Carolina New York, New York

Kevin R. Coombes, PhD Josep Dalmau, MD, PhD

Professor of Biomedical Informatics ICREA Research Professor
The Ohio State University Hospital Clínic/Institut d’Investigació Biomèdica August Pi i Sunyer
Columbus, Ohio (IDIBAPS)
Barcelona, Spain
Jorge Cortes, MD Adjunct Professor Neurology
The University of Texas MD Anderson Cancer Center University of Pennsylvania
Houston, Texas Philadelphia, Pennsylvania

Mauro W. Costa, MSc, PhD Mai Dang, MD, PhD

Research Scientist Instructor in Neurology
The Jackson Laboratory Children’s Hospital of Philadelphia
Bar Harbor, Maine Philadelphia, Pennsylvania

Anne Covey, MD Michael D’Angelica, MD

Attending Physician Attending Physician
Memorial Sloan Kettering Cancer Center Memorial Sloan Kettering Cancer Center
Professor of Radiology Professor of Surgery
Weill Medical College at Cornell University Weill Medical College at Cornell University
New York, New York New York, New York

Kenneth H. Cowan, MD, PhD Kurtis D. Davies, PhD

Director, Fred and Pamela Buffett Cancer Center Assistant Professor of Pathology
University of Nebraska Medical Center CU Anschutz Medical Campus
Omaha, Nebraska University of Colorado
Aurora, Colorado
Christopher H. Crane, MD
Vice Chairman
Attending Physician
Memorial Sloan Kettering Cancer Center
New York, New York
 Contributors xiii

Myrtle Davis, DVM, PhD James H. Doroshow, MD

Chief, Toxicology and Pharmacology Branch Bethesda, Maryland
Division of Drug Treatment and Diagnosis
National Cancer Institute Jay F. Dorsey, MD, PhD
National Institutes of Health Associate Professor of Radiation Oncology
Bethesda, Maryland University of Pennsylvania
Philadelphia, Pennsylvania
Nicolas Dea, MD, MSc, FRCSC
Spinal Neurosurgeon Marianne Dubard-Gault, MD, MS
Clinical Associate Professor Medical Genetics Fellow
Department of Surgery Department of Medicine
Vancouver General Hospital Memorial Sloan Kettering Cancer Center
University of British Columbia New York, New York
Vancouver, British-Columbia, Canada
Steven G. DuBois, MD, MS
Ana De Jesus-Acosta, MD Associate Professor
Assistant Professor of Oncology Department of Pediatrics
Sidney Kimmel Comprehensive Cancer Center Harvard Medical School
The Johns Hopkins University School of Medicine Attending Physician
Baltimore, Maryland Department of Pediatrics
Boston Children’s Hospital
Angelo M. DeMarzo, MD, PhD Dana Farber Cancer Institute
Professor of Pathology Boston, Massachusetts
Johns Hopkins University School of Medicine
Baltimore, Maryland Dan G. Duda, PhD, DMD
Associate Professor
Theodore L. DeWeese, MD Harvard Medical School
Professor and Director of Radiation Oncology and Molecular Boston, Massachusetts
Radiation Sciences
Johns Hopkins University School of Medicine Malcolm Dunlop, MD
Baltimore, Maryland MRC Institute of Genetics and Molecular Medicine
The University of Edinburgh
Maximilian Diehn, MD, PhD Western General Hospital
Associate Professor of Radiation Oncology Edinburgh, United Kingdom
Stanford University
Palo Alto, California Linda R. Duska, MD
University of Virginia Health System
Subba R. Digumarthy, MD Emily Couric Clinical Cancer Center
Massachusetts General Hospital Charlottesville, Virginia
Boston, Massachusetts
Madeleine Duvic, MD
Angela Dispenzieri, MD Professor and Deputy Chairman
Professor of Medicine and Laboratory Medicine Department of Dermatology
Mayo Clinic The University of Texas MD Anderson Cancer Center
Rochester, Minnesota Houston, Texas

Khanh T. Do, MD Imane El Dika, MD

Assistant Professor of Medicine Assistant Attending Physician
Harvard Medical School Memorial Sloan Kettering Cancer Center
Medical Oncology Instructor of Medicine
Dana-Farber Cancer Institute Weill Medical College at Cornell University
Boston, Massachusetts New York, New York

Konstantin Dobrenkov, MD Hashem El-Serag, MD, MPH

Clinical Director, Oncology Margaret M. and Albert B. Alkek Chair of the Department
Merck & Company, Inc. of Medicine
Kenilworth, New Jersey Professor of Gastroenterology and Hepatology
Baylor College of Medicine
Jeffrey S. Dome, MD, PhD Houston, Texas
Vice President, Center for Cancer and Blood Disorders
Children’s National Medical Center
Washington, D.C.
xiv Contributors

Jeffrey M. Engelmann, PhD Debra L. Friedman, MD, MS

Assistant Professor of Psychiatry and Behavioral Medicine Vanderbilt-Ingram Cancer Center
Medical College of Wisconsin Nashville, Tennessee
Milwaukee, Wisconsin
Arian F. Fuller, Jr., MD
David S. Ettinger, MD, FACP, FCCP Winchester Hospital
Alex Grass Professor of Oncology North Reading Medical
The Sidney Kimmel Comprehensive Cancer Center at Johns North Reading, Massachusetts
Hopkins Hospital
The Johns Hopkins University Lorenzo Galluzzi, PhD
Baltimore, Maryland Assistant Professor of Cell Biology in Radiation Oncology
Weill Cornell Medical College
Lola A. Fashoyin-Aje, MD, MPH New York, New York
Medical Officer
Office of Hematology and Oncology Products Mark C. Gebhardt, MD
Center for Drug Evaluation and Research Frederick W. and Jane M. Ilfeld Professor of Orthopaedic Surgery
U.S. Food and Drug Administration Harvard Medical School
Silver Spring, Maryland Surgeon-in-Chief
Department of Orthopedic Surgery
Eric R. Fearon, MD, PhD Beth Israel Deaconess Medical Center
Maisel Professor of Oncology Orthopedic Surgeon
Professor of Internal Medicine Department of Orthopedics
University of Michigan Medical School Children’s Hospital
Ann Arbor, Michigan Boston, Massachusetts

James M. Ford, MD Daniel J. George, MD

Professor of Medicine, Pediatrics, and Genetics Professor of Medicine
Division of Oncology and Medical Genetics Duke University Medical Center
Stanford University School of Medicine Durham, North Carolina
Stanford, California
Mark B. Geyer, MD
Wilbur A. Franklin, MD Assistant Attending
Professor Emeritus of Pathology Department of Medicine
CU Anschutz Medical Campus Leukemia Service and Cellular Therapeutics Center
University of Colorado Memorial Sloan Kettering Cancer Center
Aurora, Colorado Instructor in Medicine
Joan and Sanford I. Weill Department of Medicine
Phoebe E. Freer, MD Weill Cornell Medical College
Associate Professor New York, New York
Radiology and Imaging Sciences
University of Utah Hospitals/Huntsman Cancer Institute Amato J. Giaccia, PhD
Salt Lake City, Utah Jack, Lulu, and Sam Willson Professor of Cancer Biology
Department of Radiation Oncology
Boris Freidlin, PhD Stanford University School of Medicine
Division of Cancer Treatment and Diagnosis Stanford, California
National Cancer Institute
Bethesda, Maryland Mark R. Gilbert, MD
Senior Investigator and Chief
Alison G. Freifeld, MD Neuro-Oncology Branch
Professor of Internal Medicine National Cancer Institute
Infectious Diseases Division Bethesda, Maryland
University of Nebraska Medical Center
Omaha, Nebraska Whitney Goldner, MD
Associate Professor of Internal Medicine
Terence W. Friedlander, MD Division of Diabetes, Endocrinology, and Metabolism
Associate Clinical Professor University of Nebraska Medical Center
Medicine Omaha, Nebraska
UCSF Medical Center
San Francisco, California
 Contributors xv

Donald P. Goldstein, MD Missak Haigentz, MD

Professor of Obstetrics, Gynecology, and Reproductive Biology Montefiore Medical Center
Harvard Medical School Bronx, New York
Senior Scientist
Department of Obstetrics and Gynecology John D. Hainsworth, MD
Brigham and Women’s Hospital Chief Scientific Officer
Boston, Massachusetts Sarah Cannon Research Institute
Nashville, Tennessee
Annekathryn Goodman, MD
Massachusetts General Hospital Benjamin E. Haithcock, MD
Boston, Massachusetts Associate Professor of Surgery
University of North Carolina at Chapel Hill
Karyn A. Goodman, MD, MS Chapel Hill, North Carolina
Professor of Radiation Oncology
Grohne Chair in Clinical Cancer Research Christopher L. Hallemeier, MD
University of Colorado School of Medicine Assistant Professor of Radiation Oncology
Aurora, Colorado Mayo Clinic
Rochester, Minnesota
Kathleen Gordon, MD
Medical Director of Ophthalmology Samir Hanash, MD, PhD
IQVIA Evelyn & Sol Rubenstein Distinguished Chair for Cancer Prevention
Co-Chair Professor of Clinical Cancer Prevention
IQIVA Ophthalmology Center of Excellence The University of Texas MD Anderson Cancer Center
Clinical Associate Professor of Ophthalmology Houston, Texas
University of North Carolina at Chapel Hill
Chapel Hill, North Carolina Aphrothiti J. Hanrahan, PhD
Assistant Lab Member
Laura Graeff-Armas, MD, MS Human Oncology and Pathogenesis Program
Associate Professor of Internal Medicine Memorial Sloan Kettering Cancer Center
Division of Diabetes, Endocrine and Metabolism New York, New York
University of Nebraska Medical Center
Omaha, Nebraska James Harding, MD
Assistant Attending Physician
Alexander J. Greenstein, MD, MPH Memorial Sloan Kettering Cancer Center
Associate Professor of Surgery Assistant Professor of Medicine
Icahn School of Medicine at Mount Sinai Weill Medical College at Cornell University
New York, New York New York, New York

Stuart A. Grossman, MD Michael R. Harrison, MD

Professor of Oncology, Medicine, and Neurosurgery Assistant Professor of Medicine
The Sidney Kimmel Comprehensive Cancer Center at Johns Division of Medical Oncology
Hopkins Medicine Duke Cancer Institute
The Johns Hopkins University Durham, North Carolina
Baltimore, Maryland
Muneer G. Hasham, PhD
Stephan Grupp, MD, PhD Research Scientist
Section Chief, Cellular Therapy and Transplant The Jackson Laboratory
Director, Cancer Immunotherapy Frontier Program Bar Harbor, Maine
CCCR Director of Translational Research
Children’s Hospital of Philadelphia Ernest Hawk, MD, MPH
Philadelphia, Pennsylvania Boone Pickens Distinguished Chair for Early Prevention of Cancer
Vice President and Division Head
Arjun Gupta, MD Division of Cancer Prevention and Population Sciences
Assistant Instructor The University of Texas MD Anderson Cancer Center
Department of Internal Medicine Houston, Texas
University of Texas Southwestern Medical Center
Dallas, Texas Jonathan Hayman, MD
Department of Internal Medicine
Irfanullah Haider, MD, MBA Johns Hopkins Bayview Medical Center
Breast Imaging Baltimore, Maryland
Brigham and Women’s Hospital
Boston, Massachussetts
xvi Contributors

Jonathan E. Heinlen, MD Clifford A. Hudis, MD

Assistant Professor Chief Executive Officer
Department of Urology American Society of Clinical Oncology
University of Oklahoma Health Sciences Center Alexandria, Virginia
Oklahoma City, Oklahoma
Stephen P. Hunger, MD
N. Lynn Henry, MD, PhD Jeffrey E. Perelman Distinguished Chair
Associate Professor Department of Pediatrics
Internal Medicine Chief, Division of Oncology
University of Utah Pediatrics
Salt Lake City, Utah Children’s Hospital of Philadelphia
Philadelphia, Pennsylvania
Joseph Herman, MD †
Professor and Division Head ad-interim Arti Hurria, MD
Department of Radiation Oncology Professor
The University of Texas MD Anderson Cancer Center Department of Medical Oncology and Therapeutics Research
Houston, Texas City of Hope Comprehensive Cancer Center
Duarte, California
Brian P. Hobbs, PhD
Associate Staff David H. Ilson, MD, PhD
Quantitative Health Sciences and The Taussig Cancer Institute Attending Physician
Cleveland Clinic Gastrointestinal Oncology Service
Cleveland, Ohio Department of Medicine
Memorial Sloan-Kettering Cancer Center
Ingunn Holen, BSc, MSc, PhD New York, New York
Oncology
University of Sheffield Annie Im, MD
Sheffield, United Kingdom Assistant Professor of Medicine
Department of Hematology and Oncology
Leora Horn, MD, MSc UPMC Hillman Cancer Center
Associate Professor of Medicine Pittsburgh, Pennsylvania
Medicine–Hematology Oncology
Vanderbilt University
Gopa Iyer, MD
Assistant Attending Physician, Genitourinary Oncology Service
Nashville, Tennessee
Department of Medicine
Memorial Sloan Kettering Cancer Center
Neil S. Horowitz, MD
New York, New York
Department of Obstetrics and Gynecology
Division of Gynecologic Oncology
Elizabeth M. Jaffee, MD
Brigham and Women’s Hospital
The Dana and Albert “Cubby” Broccoli Professor of Oncology
Dana Farber Cancer Institute
Deputy Director, Sidney Kimmel Comprehensive Cancer Center at
Boston, Massachusetts
Johns Hopkins
Johns Hopkins University School of Medicine
Steven M. Horwitz, MD Baltimore, Maryland
Associate Attending
Department of Medicine, Lymphoma Service Reshma Jagsi, MD, DPhil
Memorial Sloan Kettering Cancer Center Professor and Deputy Chair
Assistant Professor of Clinical Medicine Radiation Oncology
Weill-Cornell Medical College University of Michigan
New York, New York Ann Arbor, Michigan

Odette Houghton, MD Rakesh K. Jain, PhD

Associate Professor A.W. Cook Professor of Tumor Biology
Department of Ophthalmology Department of Radiation Oncology
Mayo Clinic Harvard Medical School
Scottsdale, Arizona Director
E.L. Steele Laboratory for Tumor Biology
Scott C. Howard, MD, MSc Department of Radiation Oncology
Professor of Acute and Tertiary Care Massachusetts General Hospital
University of Tennessee Health Sciences Center Boston, Massachusetts
Memphis, Tennessee
†
Deceased.
 Contributors xvii

William Jarnagin, MD, FACS Hagop Kantarjian, MD

Winchester Hospital The University of Texas MD Anderson Cancer Center
North Reading Medical Houston, Texas
North Reading, Massachusetts
Giorgos Karakousis, MD
Aminah Jatoi, MD Assistant Professor of Surgery
Professor of Oncology University of Pennsylvania
Mayo Clinic Philadelphia, Pennsylvania
Rochester, Minnesota
Maher Karam-Hage, MD
Anuja Jhingran, MD Professor of Behavioral Science
The University of Texas MD Anderson Cancer Center The University of Texas MD Anderson Cancer Center
Houston, Texas Houston, Texas

David H. Johnson, MD Nadine M. Kaskas, MD

Chairman, Department of Internal Medicine Resident Physician
University of Texas Southwestern Medical School Department of Dermatology
Dallas, Texas The Warren Alpert Medical School of Brown University
Providence, Rhode Island
Brian Johnston, MD
Royal Victoria Hospital Michael B. Kastan, MD, PhD
Belfast, United Kingdom Executive Director, Duke Cancer Institute
Director, Cancer Immunotherapy Frontier Program
†
Patrick G. Johnston, MD William and Jane Shingleton Professor, Pharmacology and
Center for Cancer Research and Cell Biology Cancer Biology
School of Medicine, Dentistry, and Biomedical Sciences Professor of Pediatrics
Queen’s University Belfast Duke University School of Medicine
Belfast, United Kingdom Durham, North Carolina

Kevin D. Judy, MD Nora Katabi, MD

Professor of Neurosurgery Department of Pathology
Thomas Jefferson University Memorial Sloan-Kettering Cancer Center
Jefferson Medical College New York, New York
Philadelphia, Pennsylvania
Daniel R. Kaul, MD
Lisa A. Kachnic, MD Associate Professor of Infectious Disease
Professor and Chair of Radiation Oncology University of Michigan
Vanderbilt University Medical Center Ann Arbor, Michigan
Nashville, Tennessee
Scott R. Kelley, MD, FACS, FASCRS
Orit Kaidar-Person, MD Assistant Professor of Surgery
Ramban Medical Center Division of Colon and Rectal Surgery
Haifa, Israel Mayo Clinic
Rochester, Minnesota
Sanjeeva Kalva, MD, RPVI, FSIR
Chief, Interventional Radiology Nancy Kemeny, MD
Associate Professor of Radiology Attending Physician
University of Texas Southwestern Medical Center Memorial Sloan Kettering Cancer Center
Dallas, Texas Professor of Medicine
Weill Medical College at Cornell University
Deborah Y. Kamin, RN, MS, PhD New York, New York
Vice President
Policy and Advocacy Erin E. Kent, PhD, MS
American Society of Clinical Oncology Scientific Advisor
Alexandria, Virginia Outcomes Research Branch
Healthcare Delivery Research Program
Division of Cancer Control and Population Sciences
National Cancer Institute
Rockville, Maryland
ICF, Inc.
Fairfax, Virginia
†
Deceased.
xviii Contributors

Oliver Kepp, PhD Daniel A. Laheru, MD

Metabolomics and Cell Biology Platforms Ian T. MacMillan Professorship in Clinical Pancreatic Research
Gustave Roussy Cancer Campus Department of Medical Oncology
Villejuif, France The Johns Hopkins University School of Medicine
Baltimore, Maryland
Simon Khagi, MD
Assistant Professor Paul F. Lambert, PhD
University of North Carolina School of Medicine Professor of Oncology
Lineberger Comprehensive Cancer Center University of Wisconsin
Chapel Hill, North Carolina Madison, Wisconsin

Joshua E. Kilgore, MD Mark Lawler, PhD

Division of Gynecologic Oncology Chair in Translational Cancer Genomics
University of North Carolina School of Medicine Centre for Cancer Research and Cell Biology
Chapel Hill, North Carolina School of Medicine, Dentistry and Biomedical Sciences
Queen’s University Belfast
D. Nathan Kim, MD, PhD Belfast, United Kingdom
Associate Professor
Department of Radiation Oncology Jennifer G. Le-Rademacher, PhD
University of Texas Southwestern Medical Center Associate Professor of Biostatistics
Dallas, Texas Health Sciences Research
Associate Professor of Oncology
Bette K. Kleinschmidt-DeMasters, MD Mayo Clinic
Professor of Neurology, Neurosurgery, and Pathology Rochester, Minnesota
CU Anschutz Medical Campus
University of Colorado John Y.K. Lee, MD
Aurora, Colorado Associate Professor of Neurosurgery
University of Pennsylvania
Edward L. Korn, PhD Philadelphia, Pennsylvania
Biometric Research Program
National Cancer Institute Nancy Y. Lee, MD
Bethesda, Maryland Department of Radiation Oncology
Memorial Sloan-Kettering Cancer Center
Guido Kroemer, MD, PhD New York, New York
Team 11, Centre de Recherche des Cordeliers
Paris, France Susanna L. Lee, MD, PhD
Massachusetts General Hospital
Geoffrey Y. Ku, MD Boston, Massachusetts
Assistant Attending Physician
Gastrointestinal Oncology Service Jonathan E. Leeman, MD
Department of Medicine Department of Radiation Oncology
Memorial Sloan Kettering Cancer Center Memorial Sloan-Kettering Cancer Center
New York, New York New York, New York

Shivaani Kummar, MD Andreas Linkermann, MD

Professor of Medicine Department of Internal Medicine III
Director, Phase 1 Clinical Research Program Division of Nephrology
Stanford University University Hospital Carl Gustav Carus at the Technische
Palo Alto, California Universität Dresden
Dresden, Germany
Bonnie Ky, MD, MSCE
Assistant Professor of Medicine and Epidemiology Jinsong Liu, MD, PhD
Division of Cardiovascular Medicine Professor of Pathology
Senior Scholar The University of Texas MD Anderson Cancer Center
Center for Clinical Epidemiology and Biostatistics Houston, Texas
University of Pennsylvania School of Medicine
Philadelphia, Pennsylvania Simon Lo, MD, FACR
Professor and Vice Chair for Strategic Planning
Department of Radiation Oncology
Professor of Neurological Surgery
University of Washington School of Medicine
Seattle, Washington
 Contributors xix

Jason W. Locasale, PhD Amit Maity, MD

Associate Professor University of Pennsylvania
Department of Pharmacology and Cancer Biology Philadelphia, Pennsylvania
Duke University School of Medicine
Durham, North Carolina Neil Majithia, MD
Mayo Clinic
Charles L. Loprinzi, MD Rochester, Minnesota
Regis Professor of Breast Cancer Research
Department of Oncology Marcos Malumbres, PhD
Mayo Clinic Group Leader
Rochester, Minnesota Cell Division and Cancer
Spanish National Cancer Research Center (CNIO)
Maeve Lowery, MD Madrid, Spain
Professor of Translational Cancer Medicine
Trinity College Karen Colbert Maresso, MPH
Dublin, Ireland Program Director
Division of Cancer Prevention and Population Sciences
Emmy Ludwig, MD The University of Texas MD Anderson Cancer Center
Associate Attending Physician Houston, Texas
Memorial Sloan Kettering Cancer Center
Associate Professor of Medicine John D. Martin, PhD
Weill Medical College at Cornell University The University of Tokyo
New York, New York Tokyo, Japan

Matthew A. Lunning, DO Koji Matsuo, MD, PhD

Associate Professor of Internal Medicine Assistant Professor of Obstetrics and Gynecology
University of Nebraska Medical Center University of Southern California
Omaha, Nebraska Los Angeles, California

Robert A. Lustig, MD Natalie H. Matthews, MD

Professor of Clinical Radiation Oncology Department of Dermatology
University of Pennsylvania The Warren Alpert Medical School of Brown University
Philadelphia, Pennsylvania Providence, Rhode Island

Mitchell Machtay, MD Lauren Mauro, MD

University Hospitals Cleveland Medical Center Assistant Professor of Medicine
Case-Western Reserve University School of Medicine George Washington University School of Medicine
Cleveland, Ohio Washington, D.C.

Crystal Mackall, MD R. Samuel Mayer, MD, MEHP

Endowed Professor of Pediatrics and Medicine Associate Professor and Vice Chair for Education
Stanford University Physical Medicine and Rehabilitation
Director The Johns Hopkins University School of Medicine
Stanford Center for Cancer Cell Therapy Medical Director, Cancer Rehabilitation Program
Director Physical Medicine and Rehabilitation
Parker Institute for Cancer Immunotherapy at Stanford The Johns Hopkins Hospital
Associate Director Baltimore, Maryland
Stanford Cancer Institute
Stanford, California Worta McCaskill-Stevens, MD
Chief, Community Oncology and Prevention Trials Research Group
David A. Mahvi, MD Division of Cancer Prevention
General Surgery Resident National Cancer Institute
Brigham and Women’s Hospital Rockville, Maryland
Boston, Massachusetts
Megan A. McNamara, MD
David M. Mahvi, MD Assistant Professor of Medicine
Professor of Surgery Department of Medical Oncology
Chief, Surgical Oncology Duke University Medical Center
Medical University of South Carolina Durham, North Carolina
Charleston, South Carolina
xx Contributors

Neha Mehta-Shah, MD Jarushka Naidoo, MB, BCH

Assistant Professor Assistant Professor of Oncology
Washington University The Sidney Kimmel Comprehensive Cancer Center at Johns
St. Louis, Missouri Hopkins Hospital
Baltimore, Maryland
Robert E. Merritt, MD
Director, Thoracic Surgery Amol Narang, MD
James Cancer Center Assistant Professor of Radiation Oncology
The Ohio State University Medical Johns Hopkins University School of Medicine
Columbus, Ohio Baltimore, Maryland

Matthew I. Milowsky, MD Heidi Nelson, MD, FACS, FASCRS

Professor of Medicine Professor of Surgery
Division of Hematology/Oncology Division of Colon and Rectal Surgery
UNC Lineberger Comprehensive Cancer Center Mayo Clinic
Chapel Hill, North Carolina Rochester, Minnesota

Lori M. Minasian, MD William G. Nelson, MD, PhD

Deputy Director Professor and Director
Division of Cancer Prevention Sidney Kimmel Comprehensive Cancer Center
National Cancer Institute Johns Hopkins University School of Medicine
National Institutes of Health Baltimore, Maryland
Bethesda, Maryland
Suzanne Nesbit, PharmD, BCPS, CPE
Tara C. Mitchell, MD Clinical Pharmacy Specialist, Pain Management
Assistant Professor of Medicine Research Associate, Department of Oncology
University of Pennsylvania The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins
Philadelphia, Pennsylvania The Johns Hopkins Hospital
Baltimore, Maryland
Demytra Mitsis, MD
Medical Oncology and Hematology Fellow Mark Niglas, MD, FRCPC
Department of Medicine Clinical Fellow
Roswell Park Cancer Institute Department of Radiation Oncology
Buffalo, New York Sunnybrook Health Sciences Centre
Toronto, Ontario, Canada
Michelle Mollica, PhD, MPH, RN
Program Director Tracey O’Connor, MD
Division of Cancer Control and Population Sciences Associate Professor of Oncology
Healthcare Delivery Research Program Department of Medicine
National Cancer Institute Roswell Park Cancer Institute
Bethesda, Maryland Buffalo, New York

Margaret Mooney, MD Kenneth Offit, MD, MPH

Branch Chief, Clinical Investigations Branch Chief, Clinical Genetics Service
Cancer Therapy Evaluation Program Robert and Kate Niehaus Chair in Inherited Cancer Genomics
Division of Cancer Treatment and Diagnosis Memorial Sloan Kettering Cancer Center
National Cancer Institute New York, New York
Rockville, Maryland
Mihaela Onciu, MD
Farah Moustafa, MD Medical Director
Department of Dermatology OncoMetrix Laboratories
The Warren Alpert Medical School of Brown University Poplar Healthcare
Providence, Rhode Island Memphis, Tennessee

Lida Nabati, MD
Instructor in Medicine
Harvard Medical School
Senior Physician
Dana-Farber Cancer Institute
Boston, Massachusetts
 Contributors xxi

Eileen M. O’Reilly, MD Steven Z. Pavletic, MD, MS

Winthrop Rockefeller Chair in Medical Oncology Head, Graft-Versus-Host Disease and Autoimmunity Section
Section Head Hepatopancreaticobiliary & Neuroendocrine Cancers Experimental Transplantation and Immunology Branch
Gastrointestinal Oncology Service National Cancer Institute
Associate Director Bethesda, Maryland
David M. Rubenstein Center for Pancreatic Cancer Research
Attending Physician, Member Peter C. Phillips, MD
Memorial Sloan Kettering Cancer Center Professor of Neurology and Oncology
Professor of Medicine The Children’s Hospital of Philadelphia
Weill Medical College at Cornell University Philadelphia, Pennsylvania
New York, New York
Miriam D. Post, MD
Elaine A. Ostrander, PhD Associate Professor of Pathology
Chief and Distinguished Investigator CU Anschutz Medical Campus
Cancer Genetics and Comparative Genomics Branch University of Colorado
National Human Genome Research Institute Aurora, Colorado
Bethesda, Maryland
Amy A. Pruitt, MD
Lisa Pappas-Taffer, MD University of Pennsylvania
Assistant Professor of Clinical Dermatology Philadelphia, Pennsylvania
University of Pennsylvania
Philadelphia, Pennsylvania Christiane Querfeld, MD, PhD
Chief, Division of Dermatology
Drew Pardoll, MD, PhD Director, Cutaneous Lymphoma Program
Director, Bloomberg~Kimmel Institute for Cancer Immunotherapy Assistant Professor of Dermatology
Sidney Kimmel Comprehensive Cancer Center City of Hope
Johns Hopkins School of Medicine Duarte, California
Baltimore, Maryland
Vance A. Rabius, PhD
Jae H. Park, MD Research Director, Tobacco Treatment Program
Assistant Attending The University of Texas MD Anderson Cancer Center
Department of Medicine Houston, Texas
Leukemia Service and Cellular Therapeutics Center
Memorial Sloan Kettering Cancer Center S. Vincent Rajkumar, MD
Assistant Professor of Medicine Edward W. and Betty Knight Scripps Professor of Medicine
Joan and Sanford I. Weill Department of Medicine Division of Hematology
Weill Cornell Medical College Mayo Clinic
New York, New York Rochester, Minnesota

Anery Patel, MD Mohammad O. Ramadan, MD

Clinical Instructor Assistant Professor
Department of Internal Medicine Department of Urology
Division of Diabetes, Endocrine, and Metabolism University of Oklahoma Health Sciences Center
University of Nebraska Medical Center Oklahoma City, Oklahoma
Omaha, Nebraska
Erinn B. Rankin, PhD
Anish J. Patel, MD Assistant Professor of Radiation Oncology, Obstetrics,
Assistant Professor of Endocrinology and Gynecology
Department of Endocrinology Stanford University School of Medicine
University of Alabama at Birmingham Stanford, California
Birmingham, Alabama
Sushanth Reddy, MD
Steven R. Patierno, PhD Assistant Professor of Surgery
Deputy Director Department of Surgery
Duke Cancer Institute University of Alabama at Birmingham
Professor of Medicine Birmingham, Alabama
Professor of Pharmacology and Cancer Biology
Professor of Community and Family Medicine
Duke University Medical Center
Durham, North Carolina
xxii Contributors

Michael A. Reid, PhD Nadia Rosenthal, PhD

Postdoctoral Fellow Scientific Director
Department of Pharmacology and Cancer Biology The Jackson Laboratory
Duke University School of Medicine Bar Harbor, Maine
Durham, North Carolina Chair, Cardiovascular Science
National Heart and Lung Institute
Scott Reznik, MD Imperial College London
Associate Professor London, United Kingdom
Department of Cardiothoracic Surgery
University of Texas Southwestern Medical Center Meredith Ross, MD
Dallas, Texas Fellow
Department of Internal Medicine
Tina Rizack, MD, MPH Division of Diabetes, Endocrinology, and Metabolism
Hematologist/Oncologist University of Nebraska Medical Center
South County Health Omaha, Nebraska
Clinical Assistant Professor of Internal Medicine and Obstetrics
& Gynecology Julia H. Rowland, PhD
Alpert Medical School of Brown University Director, Office of Cancer Survivorship
Providence, Rhode Island Division of Cancer Control and Population Sciences
National Cancer Institute
Jason D. Robinson, PhD Rockville, Maryland
Associate Professor of Behavioral Science
The University of Texas MD Anderson Cancer Center Anthony H. Russell, MD
Houston, Texas Massachusetts General Hospital
Boston, Massachusetts
Leslie Robinson-Bostom, MD
Senior Attending Michael S. Sabel, MD, FACS
Department of Dermatology Associate Professor
The Warren Alpert Medical School of Brown University Surgery
Providence, Rhode Island University of Michigan
Ann Arbor, Michigan
Carlos Rodriguez-Galindo, MD
Departments of Global Pediatric Medicine and Oncology Arjun Sahgal, MD, FRCPC
St. Jude Children’s Research Hospital Professor of Radiation Oncology and Surgery
Memphis, Tennessee Deputy Chief, Department of Radiation Oncology
Sunnybrook Health Sciences Center
Paul B. Romesser, MD University of Toronto Faculty of Medicine
Department of Radiation Oncology Toronto, Ontario
Memorial Sloan-Kettering Cancer Center
New York, New York Ryan D. Salinas, MD
Resident Physician
Steven T. Rosen, MD Department of Neurosurgery
Provost & Chief Scientific Officer University of Pennsylvania
Director, Comprehensive Cancer Center and Beckman Philadelphia, Pennsylvania
Research Institute
Irell & Manella Cancer Center Director’s Distinguished Chair Erin E. Salo-Mullen, MS, MPH, CGC
Helen & Morgan Chu Director’s Chair, Beckman Research Institute Senior Genetic Counselor
City of Hope Clinical Genetics Service
Duarte, California Department of Medicine
Memorial Sloan Kettering Cancer Center
Myrna R. Rosenfeld, MD, PhD New York, New York
Senior Investigator, Neuroimmunology
Institut d’Investigació Biomèdica August Pi i Sunyer (IDIBAPS) Manuel Salto-Tellez, MD
Barcelona, Spain Center for Cancer Research and Cell Biology
Adjunct Professor Neurology School of Medicine, Dentistry, and Biomedical Sciences
University of Pennsylvania Queen’s University Belfast
Philadelphia, Pennsylvania Belfast, United Kingdom
 Contributors xxiii

Sydney M. Sanderson, BS Konstantin Shilo, MD

PhD Candidate Department of Pathology
Department of Pharmacology and Cancer Biology James Cancer Center
Duke University School of Medicine The Ohio State University Medical
Durham, North Carolina Columbus, Ohio

John T. Sandlund, MD Eric Small, MD

Member Professor of Medicine
Department of Oncology University of California, San Francisco
St. Jude Children’s Research Hospital San Francisco, California
Professor of Pediatrics
University of Tennessee College of Medicine Angela B. Smith, MD, MS, FACS
Memphis, Tennessee Associate Professor of Urology
Department of Urology
Victor M. Santana, MD University of North Carolina
Department of Oncology Chapel Hill, North Carolina
St. Jude Children’s Research Hospital
Memphis, Tennessee Stephen N. Snow, MD
Professor of Dermatology
Michelle Savage, MD Northwest Permanente
Department of Clinical Cancer Prevention Portland, Oregon
The University of Texas MD Anderson Cancer Center
Houston, Texas David B. Solit, MD
Geoffrey Beene Chair
Eric C. Schreiber, PhD Director, Center for Molecular Oncology
Associate Professor Member, Human Oncology and Pathogenesis Program
Department of Radiation Oncology Attending Physician, Genitourinary Oncology Service, Department
University of North Carolina School of Medicine of Medicine
Chapel Hill, North Carolina Memorial Sloan Kettering Cancer Center
New York, New York
Lynn Schuchter, MD
C. Willard Robinson Professor of Hematology and Oncology Anil K. Sood, MD
Professor of Medicine Professor and Vice Chair
University of Pennsylvania Department of Gynecologic Oncology and Reproductive Medicine
Philadelphia, Pennsylvania The University of Texas MD Anderson Cancer Center
Houston, Texas
Liora Schultz, MD
Department of Pediatric Oncology, Division of Oncology Enrique Soto-Perez-de-Celis, MD
Stanford University International Fellow
Stanford, California Department of Medical Oncology and Therapeutics Research
City of Hope
Michael V. Seiden, MD, PhD Duarte, California
Texas Oncology Researcher in Medical Science
The Woodlands, Texas Cancer Care in the Elderly Clinic
Department of Geriatrics
Morgan M. Sellers, MD, MS Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran
Icahn School of Medicine at Mount Sinai Mexico City, Mexico
New York, New York
Joseph A. Sparano, MD
Payal D. Shah, MD Associate Chairman
Assistant Professor Department of Oncology
Medicine Montefiore Medical Center
University of Pennsylvania Professor of Medicine and Women’s Health
Philadelphia, Pennsylvania Department of Medicine and Oncology
Albert Einstein College of Medicine
Jinru Shia, MD Bronx, New York
Member and Attending Pathologist
Memorial Sloan Kettering Cancer Center Vladimir S. Spiegelman, MD, PhD
Professor of Pathology and Laboratory Medicine Professor of Pediatrics and Pharmacology
Weill Medical College at Cornell University Department of Pediatrics
New York, New York Pennsylvania State University College of Medicine
Hershey, Pennsylvania
xxiv Contributors

Sheri L. Spunt, MD, MBA James E. Talmadge, PhD

Endowed Professor of Pediatric Cancer Professor of Pathology and Microbiology
Department of Pediatrics University of Nebraska Medical Center
Division of Hematology/Oncology Omaha, Nebraska
Stanford University School of Medicine
Stanford, California David T. Teachey, MD
Department of Pediatrics
Zsofia K. Stadler, MD Divisions of Hematology and Oncology
Assistant Attending Physician Children’s Hospital of Philadelphia
Department of Medicine Philadelphia, Pennsylvania
Memorial Sloan Kettering Cancer Center
Assistant Professor of Medicine Catalina V. Teba, MD
Weill Cornell Medical College University Hospitals Cleveland Medical Center
New York, New York Case-Western Reserve University School of Medicine
Cleveland, Ohio
David P. Steensma, MD
Institute Physician Ayalew Tefferi, MD
Department of Medical Oncology Department of Hematology
Dana-Farber Cancer Institute Mayo Clinic
Associate Professor of Medicine Rochester, Minnesota
Harvard Medical School
Boston, Massachusetts Bin Tean Teh, MD, PhD
Professor
Richard M. Stone, MD Division of Medical Sciences
Professor of Medicine National Cancer Centre Singapore
Harvard Medical School Professor, Cancer and Stem Cell Biology Program
Chief of Staff Duke–NUS Medical School
Department of Medical Oncology Singapore
Dana-Farber Cancer Institute
Boston, Massachusetts Joyce M.C. Teng, MD, PhD
Associate Professor of Dermatology and Pediatrics
Steven Kent Stranne, MD, JD Stanford of Medicine
Polsinelli PC Stanford University
Washington, D.C. Palo Alto, California

Kelly Stratton, MD Joel E. Tepper, MD

Assistant Professor Hector MacLean Distinguished Professor of Cancer Research
Department of Urology Department of Radiation Oncology
University of Oklahoma Health Sciences Center University of North Carolina School of Medicine
Oklahoma City, Oklahoma University of North Carolina Lineberger Comprehensive
Cancer Center
Bill Sugden, PhD Chapel Hill, North Carolina
Professor of Oncology
University of Wisconsin Premal H. Thaker, MD
Madison, Wisconsin Professor in Gynecologic Oncology
Division of Obstetrics and Gynecology
Andrew M. Swanson, MD Washington University School of Medicine
Assistant Professor of Dermatology St. Louis, Missouri
University of Wisconsin School of Medicine and Public Health
Madison, Wisconsin Aaron P. Thrift, PhD
Assistant Professor
Martin S. Tallman, MD Dan L. Duncan Comprehensive Cancer Center
Chief, Leukemia Service Department of Medicine, Gastroenterology Section
Memorial Sloan-Kettering Cancer Center Baylor College of Medicine
Professor of Medicine Houston, Texas
Joan and Sanford I. Weill Department of Medicine
Weill Cornell Medical College Arthur-Quan Tran, MD
New York, New York Division of Gynecologic Oncology
University of North Carolina School of Medicine
Chapel Hill, North Carolina
 Contributors xxv

Grace Triska, MS Richard L. Wahl, MD

Washington University School of Medicine Elizabeth Mallinckrodt Professor and Director
St. Louis, Missouri Mallinckrodt Institute of Radiology
Washington University School of Medicine
Donald Trump, MD, FACP St. Louis, Missouri
Chief Executive Officer and Executive Director
Inova Schar Cancer Institute Michael F. Walsh, MD, FAAP, FACMG, DABMG
Falls Church, Virginia Assistant Member
Department of Pediatrics and Medicine
Kenneth Tsai, MD, PhD Divisions of Solid Tumor and Clinical Genetics
Associate Member Memorial Sloan Kettering Cancer Center
Anatomic Pathology and Tumor Biology New York City, New York
H. Lee Moffitt Cancer Center and Research Institute
Tampa, Florida Thomas Wang, MD
Professor of Surgery
Chia-Lin Tseng, MD, FRCPC Department of Surgery
Assistant Professor University of Alabama at Birmingham
Radiation Oncologist Birmingham, Alabama
Sunnybrook Health Sciences Centre
Toronto, Ontario, Canada Jared Weiss, MD
Associate Professor of Medicine
Diane Tseng, MD, PhD Section Chief of Thoracic and Head/Neck Oncology
Department of Medicine Division of Hematology and Oncology
Division of Oncology University of North Carolina at Chapel Hill
Stanford University Chapel Hill, North Carolina
Stanford, California
Irving L. Weissman, MD
Sandra Van Schaeybroeck, MD Director, Institute for Stem Cell Biology and Regenerative Medicine
Center for Cancer Research and Cell Biology Director, Stanford Ludwig Center for Cancer Stem Cell Research
School of Medicine, Dentistry, and Biomedical Sciences and Medicine
Queen’s University Belfast Stanford University
Belfast, United Kingdom Palo Alto, California

Brian A. Van Tine, MD, PhD Shannon N. Westin, MD, MPH

Associate Professor Associate Professor of Gynecologic Oncology and
Internal Medicine Reproductive Medicine
Washington University in Saint Louis The University of Texas MD Anderson Cancer Center
St. Louis, Missouri Houston, Texas

Erin R. Vanness, MD Jeffrey D. White, MD

Associate Professor of Dermatology Associate Director, Office of Cancer Complementary and
University of Wisconsin School of Medicine and Public Health Alternative Medicine
Madison, Wisconsin Division of Cancer Treatment and Diagnosis
National Cancer Institute
Gauri Varadhachary, MD Bethesda, Maryland
Professor
Medical Director, Gastrointestinal Center Richard Wilson, MD
Executive Medical Director, Ambulatory Operations Center for Cancer Research and Cell Biology
Department of Gastrointestinal Medical Oncology School of Medicine, Dentistry, and Biomedical Sciences
The University of Texas MD Anderson Cancer Center Queen’s University Belfast
Houston, Texas Belfast, United Kingdom

Marileila Varella-Garcia, PhD Richard J. Wong, MD

Professor of Medicine and Medical Oncology Department of Surgery
CU Anschutz Medical Campus Memorial Sloan-Kettering Cancer Center
University of Colorado New York, New York
Aurora, Colorado
xxvi Contributors

Gary S. Wood, MD Timothy Zagar, MD

Professor and Chair of Dermatology Northeastern Radiation Oncology
University of Wisconsin School of Medicine and Public Health Glens Falls, New York
Middleton VA Medical Center
Madison, Wisconsin Elaine M. Zeman, PhD
Associate Professor
Yaohui G. Xu, MD, PhD Department of Radiation Oncology
Associate Professor of Dermatology University of North Carolina School of Medicine
University of Wisconsin School of Medicine and Public Health Chapel Hill, North Carolina
Madison, Wisconsin
Tian Zhang, MD
Meng Xu-Welliver, MD, PhD Assistant Professor of Medicine
Associate Professor of Radiation Oncology Duke University Medical Center
James Cancer Center Durham, North Carolina
The Ohio State University Medical Center
Columbus, Ohio James A. Zwiebel, MD
Cancer Therapy Evaluation Program (Retired)
Shlomit Yust-Katz, MD National Cancer Institute
Professor Rockville, Maryland
Davidoff Cancer Center
Rabin Medical Center
Petah Tikva, Israel

New insights into whole genome sequence variations and the genomic
structural alterations associated with cancer, including their downstream
effects on protein structure and function, are helping us to define specific
communication pathway changes that drive cancer initiation, progression,
metastasis, and resistance. We have learned that each individual and
each tumor may be unique. Individual physiognomies in terms of path
of progression and unique cellular communication pathway alterations
are continuing to define the nature of specific cancers and offer greater
opportunities for the development of highly prescriptive intervention(s).
In addition, we have a much greater understanding of the relationship
of the host’s tissues, the patient’s immune system, and the broad tumor
microenvironment, to the process of tumor development and progression
and their impact on tumor control. This new body of knowledge on
how the body’s immune system and the tumor’s microenvironment
are altered to support disease growth, invasion, and distant spread is
providing opportunities for the development of novel therapeutic
interventions. There is exciting new evidence to support the presence
of a special subclass of cells within the tumor that has properties of
“stemness,” which places them in the key role of maintaining tumor
growth and tumor spread. The cumulative effect of these advances—
where certain cancers can be prevented and where others will be detected
earlier and controlled—promises to be transformative in our effort to
conquer cancer.
The sixth edition of Abeloff ’s Clinical Oncology incorporates the
exciting advances in basic, translational, clinical, and epidemiologic
oncology. Each chapter begins with a summary highlighting the key
points and comprises a critical analysis of the literature and updated
clinical studies—authors present their own opinions in specially identified
boxes and algorithms.
Despite significant progress, the diagnosis of cancer remains devastat-
ing to patients and their families. Our goal is to provide a reference
textbook that is the most useful, understandable, attractive, and thorough
in presenting the principles of clinical oncology. It is meant to be equally
Preface
useful to students and trainees, experts in the various disciplines of
oncology, and as a reference text for physicians from other medical
disciplines and the various staff who regularly care for patients with
cancer. It is our hope that readers will find this scholarly textbook
properly balanced between the disciplines of science, clinical medicine,
and humanism and that it will serve them well in their efforts to prevent,
diagnose, and effectively treat their patients suffering from cancer.
The multidisciplinary nature of cancer care is, and will continue to
be, reflected in our editors. Experts in cancer biology, surgical oncology,
pediatric oncology, radiation oncology, medical oncology, and hema-
tologic malignancies directed the development of the content. Reflecting
the multispecialty approach necessary for optimal care of patients, the
majority of chapters are the joint product of several of these disciplines.
Engaging the very best subject matter authorities was a guiding principle
for the editors and we are deeply indebted to our outstanding authors
who, in a most diligent and thoughtful way, have brought their knowledge
and skills to the sixth edition of Abeloff ’s Clinical Oncology.
ACKNOWLEDGMENTS
This sixth edition represents a highly collaborative and dynamic effort
between the editors and Elsevier. We are greatly indebted to Laura
Schmidt, Kathleen Schlom, and Kristi Batchelor for their creative input
and guidance and for turning the principles behind this text into a
reality. Finally, we want to express our gratitude to our many contributing
authors for their dedication to this project, their generosity of time,
and, of course, their very valuable friendship.
John E. Niederhuber, MD
James O. Armitage, MD
James H. Doroshow, MD
Michael B. Kastan, MD, PhD
Joel E. Tepper, MD
xxvii
This page intentionally left blank

Part I Science and Clinical Oncology
Section A Biology and Cancer
1 Molecular Tools in Cancer Research,  2
Mauro W. Costa, Muneer G. Hasham,
and Nadia Rosenthal
2 Intracellular Signaling, 24
Aphrothiti J. Hanrahan, Gopa Iyer,
and David B. Solit
3 Cellular Microenvironment and Metastases,  47
Erinn B. Rankin and Amato J. Giaccia
4 Control of the Cell Cycle,  56
Marcos Malumbres
5 Pathophysiology of Cancer Cell Death,  74
Lorenzo Galluzzi, Andreas Linkermann, Oliver Kepp,
and Guido Kroemer
6 Cancer Immunology, 84
Diane Tseng, Liora Schultz, Drew Pardoll,
and Crystal Mackall
7 Stem Cells, Cell Differentiation, and Cancer,  97
Piero Dalerba, Maximilian Diehn, Irving L. Weissman,
and Michael F. Clarke
8 Tumor Microenvironment: Vascular and
Extravascular Compartment,  108
Rakesh K. Jain, John D. Martin, Vikash P. Chauhan,
and Dan G. Duda
9 Cancer Metabolism, 127
Michael A. Reid, Sydney M. Sanderson,
and Jason W. Locasale
Section B Genesis of Cancer
10 Environmental Factors,  139
Steven R. Patierno
11 DNA Damage Response Pathways and
Cancer, 154
James M. Ford and Michael B. Kastan
Contents
12 Viruses and Human Cancer,  165
Paul F. Lambert and Bill Sugden
13 Genetic Factors: Hereditary Cancer Predisposition
Syndromes, 180
Michael F. Walsh, Karen Cadoo,
Erin E. Salo-Mullen, Marianne Dubard-Gault,
Zsofia K. Stadler, and Kenneth Offit
14 Genetic and Epigenetic Alterations in
Cancer, 209
Bin Tean Teh and Eric R. Fearon
Section C Diagnosis of Cancer
15 Pathology, Biomarkers, and Molecular
Diagnostics, 225
Wilbur A. Franklin, Dara L. Aisner, Kurtis D. Davies,
Kristy Crooks, Miriam D. Post, Bette K.
Kleinschmidt-DeMasters, Edward Ashwood,
Paul A. Bunn, and Marileila Varella-Garcia
16 Imaging, 254
Richard L. Wahl
Section D Clinical Trials
17 Biostatistics and Bioinformatics in Clinical
Trials, 284
Brian P. Hobbs, Donald A. Berry,
and Kevin R. Coombes
18 Clinical Trial Designs in Oncology,  296
Edward L. Korn and Boris Freidlin
19 Structures Supporting Cancer Clinical Trials,  308
Jeffrey S. Abrams, Margaret Mooney,
James A. Zwiebel, Worta McCaskill-Stevens,
Michaele C. Christian, and James H. Doroshow
20 Oncology and Health Care Policy,  317
Steven Kent Stranne, Clifford A. Hudis,
and Deborah Y. Kamin
Section E Prevention and Early Detection
21 Discovery and Characterization of Cancer Genetic
Susceptibility Alleles,  323
Stephen J. Chanock and Elaine A. Ostrander
xxix
xxx Contents
22 Lifestyle and Cancer Prevention,  337
Karen Basen-Engquist, Powel Brown,
Adriana M. Coletta, Michelle Savage,
Karen Colbert Maresso, and Ernest Hawk
23 Screening and Early Detection,  375
Therese Bevers, Hashem El-Serag, Samir Hanash,
Aaron P. Thrift, Kenneth Tsai,
Karen Colbert Maresso, and Ernest Hawk
24 Nicotine Dependence: Current Treatments and
Future Directions,  399
Jeffrey M. Engelmann, Maher Karam-Hage,
Vance A. Rabius, Jason D. Robinson,
and Paul M. Cinciripini
Section F Treatment
25 Cancer Pharmacology,  411
Jerry M. Collins
26 Therapeutic Targeting of Cancer Cells: Era of
Molecularly Targeted Agents,  420
Khanh T. Do and Shivaani Kummar
27 Basics of Radiation Therapy,  431
Elaine M. Zeman, Eric C. Schreiber,
and Joel E. Tepper
28 Hematopoietic Stem Cell Transplantation,  461
Annie Im and Steven Z. Pavletic
29 Gene Therapy in Oncology,  470
James E. Talmadge and Kenneth H. Cowan
30 Therapeutic Antibodies and Immunologic
Conjugates, 486
Konstantin Dobrenkov and Nai-Kong V. Cheung
31 Complementary and Alternative Medicine,  500
Jeffrey D. White
Part II Problems Common to Cancer
and Therapy
Section A Hematologic Problems and Infections
32 Disorders of Blood Cell Production in Clinical
Oncology, 514
Jennifer H. Choe and Jeffrey Crawford
33 Diagnosis, Treatment, and Prevention of
Cancer-Associated Thrombosis,  523
Claudia I. Chapuy and Jean M. Connors
34 Infection in the Patient With Cancer,  544
Alison G. Freifeld and Daniel R. Kaul
Section B Symptom Management
35 Hypercalcemia, 565
Anery Patel, Laura Graeff-Armas, Meredith Ross,
and Whitney Goldner
36 Tumor Lysis Syndrome,  572
Scott C. Howard
37 Cancer-Related Pain,  581
Suzanne Nesbit, Ilene Browner,
and Stuart A. Grossman
38 Cancer Cachexia,  593
Jennifer G. Le-Rademacher and Aminah Jatoi
39 Nausea and Vomiting,  598
John D. Hainsworth
Section C Treatment Complications
40 Oral Complications,  607
Neil Majithia, Christopher L. Hallemeier,
and Charles L. Loprinzi
41 Dermatologic Toxicities of Anticancer
Therapy, 621
Natalie H. Matthews, Farah Moustafa,
Nadine M. Kaskas, Leslie Robinson-Bostom,
and Lisa Pappas-Taffer
42 Cardiovascular Effects of Cancer Therapy,  649
Lori M. Minasian, Myrtle Davis, and Bonnie Ky
43 Reproductive Complications,  665
Demytra Mitsis, Lynda Kwon Beaupin,
and Tracey O’Connor
44 Paraneoplastic Neurologic Syndromes,  676
Josep Dalmau and Myrna R. Rosenfeld
45 Neurologic Complications,  688
Shlomit Yust-Katz, Simon Khagi,
and Mark R. Gilbert
46 Endocrine Complications,  707
Donald Trump
47 Pulmonary Complications of Anticancer
Treatment, 715
Mitchell Machtay and Catalina V. Teba
Section D Posttreatment Considerations
48 Rehabilitation of Individuals With Cancer,  725
R. Samuel Mayer
49 Survivorship, 732
Julia H. Rowland, Michelle Mollica, and Erin E. Kent
50 Second Malignant Neoplasms,  741
Debra L. Friedman

51 Caring for Patients at the End of Life,  751
Lida Nabati and Janet L. Abrahm
Section E Local Effects of Cancer and Its Metastasis
52 Acute Abdomen, Bowel Obstruction,
and Fistula,  764
William R. Burns and Alfred E. Chang
53 Superior Vena Cava Syndrome,  775
Arjun Gupta, D. Nathan Kim, Sanjeeva Kalva,
Scott Reznik, and David H. Johnson
54 Spinal Cord Compression,  786
Mark Niglas, Chia-Lin Tseng, Nicolas Dea,
Eric Chang, Simon Lo, and Arjun Sahgal
55 Brain Metastases and Neoplastic Meningitis,  794
Orit Kaidar-Person, Michael Cools,
and Timothy Zagar
56 Bone Metastases,  809
Robert E. Coleman, Janet Brown, and Ingunn Holen
57 Lung Metastases,  831
Jonathan Hayman, Jarushka Naidoo,
and David S. Ettinger
58 Liver Metastases,  846
David A. Mahvi and David M. Mahvi
59 Malignancy-Related Effusions,  863
Lola A. Fashoyin-Aje and Julie R. Brahmer
Section F Special Populations
60 Cancer in the Elderly: Biology, Prevention,
and Treatment,  874
Enrique Soto-Perez-de-Celis and Arti Hurria†
61 Special Issues in Pregnancy,  882
Tina Rizack and Jorge J. Castillo
62 Human Immunodeficiency Virus (HIV) Infection
and Cancer,  894
Jesus Anampa, Stefan K. Barta, Missak Haigentz,
and Joseph A. Sparano
Part III Specific Malignancies
Section A Central Nervous System
63 Cancer of the Central Nervous System,  906
Jay F. Dorsey, Ryan D. Salinas, Mai Dang,
Michelle Alonso-Basanta, Kevin D. Judy,
Amit Maity, Robert A. Lustig, John Y.K. Lee,
Peter C. Phillips, and Amy A. Pruitt
†
Deceased.
Contents xxxi
Section B Head, Neck, and Eye
64 Ocular Tumors,  968
Odette Houghton and Kathleen Gordon
65 Cancer of the Head and Neck,  999
Jonathan E. Leeman, Nora Katabi,
Richard J. Wong, Nancy Y. Lee,
and Paul B. Romesser
Section C Skin
66 Melanoma, 1034
Tara C. Mitchell, Giorgos Karakousis,
and Lynn Schuchter
67 Nonmelanoma Skin Cancers: Basal Cell and
Squamous Cell Carcinomas,  1052
Yaohui G. Xu, Juliet L. Aylward,
Andrew M. Swanson, Vladimir S. Spiegelman,
Erin R. Vanness, Joyce M.C. Teng,
Stephen N. Snow, and Gary S. Wood
Section D Endocrine
68 Cancer of the Endocrine System,  1074
Ammar Asban, Anish J. Patel, Sushanth Reddy,
Thomas Wang, Courtney J. Balentine,
and Herbert Chen
Section E Thoracic
69 Cancer of the Lung: Non–Small Cell Lung Cancer
and Small Cell Lung Cancer,  1108
Luiz H. Araujo, Leora Horn, Robert E. Merritt,
Konstantin Shilo, Meng Xu-Welliver,
and David P. Carbone
70 Diseases of the Pleura and Mediastinum,  1159
Orit Kaidar-Person, Timothy Zagar,
Benjamin E. Haithcock, and Jared Weiss
71 Cancer of the Esophagus,  1174
Geoffrey Y. Ku and David H. Ilson
Section F Gastrointestinal
72 Cancer of the Stomach,  1197
Geoffrey Y. Ku and David H. Ilson
73 Cancer of the Small Bowel,  1211
Morgan M. Sellers and Alexander J. Greenstein
74 Colorectal Cancer,  1219
Mark Lawler, Brian Johnston,
Sandra Van Schaeybroeck, Manuel Salto-Tellez,
Richard Wilson, Malcolm Dunlop,
and †Patrick G. Johnston
xxxii Contents
75 Cancer of the Rectum,  1281
Scott R. Kelley and Heidi Nelson
76 Cancer of the Anal Canal,  1300
Karyn A. Goodman, Lisa A. Kachnic,
and Brian G. Czito
77 Liver and Bile Duct Cancer,  1314
Ghassan K. Abou-Alfa, William Jarnagin,
Imane El Dika, Michael D’Angelica, Maeve Lowery,
Karen Brown, Emmy Ludwig, Nancy Kemeny,
Anne Covey, Christopher H. Crane, James Harding,
Jinru Shia, and Eileen M. O’Reilly
78 Carcinoma of the Pancreas,  1342
Ana De Jesus-Acosta, Amol Narang,
Lauren Mauro, Joseph Herman,
Elizabeth M. Jaffee, and Daniel A. Laheru
Section G Genitourinary
79 Cancer of the Kidney,  1361
Megan A. McNamara, Tian Zhang,
Michael R. Harrison, and Daniel J. George
80 Carcinoma of the Bladder,  1382
Angela B. Smith, Arjun V. Balar,
Matthew I. Milowsky, and Ronald C. Chen
81 Prostate Cancer,  1401
William G. Nelson, Emmanuel S.
Antonarakis, H. Ballentine Carter,
Angelo M. DeMarzo and Theodore L. DeWeese
82 Cancer of the Penis,  1433
Jonathan E. Heinlen, Mohammad O. Ramadan,
Kelly Stratton, and Daniel J. Culkin
83 Testicular Cancer,  1442
Terence W. Friedlander and Eric Small
Section H Gynecological
84 Cancers of the Cervix, Vulva, and Vagina,  1468
Anuja Jhingran, Anthony H. Russell,
Michael V. Seiden, Linda R. Duska,
Annekathryn Goodman, Susanna L. Lee,
Subba R. Digumarthy, and Arlan F. Fuller, Jr.
85 Uterine Cancer,  1508
John F. Boggess, Joshua E. Kilgore,
and Arthur-Quan Tran
86 Carcinoma of the Ovaries and Fallopian
Tubes, 1525
Robert L. Coleman, Jinsong Liu, Koji Matsuo,
Premal H. Thaker, Shannon N. Westin,
and Anil K. Sood
87 Gestational Trophoblastic Disease,  1544
Donald P. Goldstein, Ross S. Berkowitz,
and Neil S. Horowitz
88 Cancer of the Breast,  1560
N. Lynn Henry, Payal D. Shah, Irfanullah Haider,
Phoebe E. Freer, Reshma Jagsi,
and Michael S. Sabel
Section I Sarcomas
89 Sarcomas of Bone,  1604
Megan E. Anderson, Steven G. DuBois,
and Mark C. Gebhardt
90 Sarcomas of Soft Tissue,  1655
Brian A. Van Tine
Section J Cancer of Undefined Site of Origin
91 Carcinoma of Unknown Primary,  1694
Gauri Varadhachary and James L. Abbruzzese
Section K Pediatrics
92 Pediatric Solid Tumors,  1703
Jeffrey S. Dome, Carlos Rodriguez-Galindo,
Sheri L. Spunt, and Victor M. Santana
93 Childhood Leukemia,  1748
Stephen P. Hunger, David T. Teachey,
Stephan Grupp, and Richard Aplenc
94 Childhood Lymphoma,  1765
John T. Sandlund and Mihaela Onciu
Section L Hematological
95 Acute Leukemias in Adults,  1783
Frederick R. Appelbaum
96 Myelodysplastic Syndromes,  1798
David P. Steensma and Richard M. Stone
97 Myeloproliferative Neoplasms,  1821
Ayalew Tefferi
98 Chronic Myeloid Leukemia,  1836
Hagop Kantarjian and Jorge Cortes
99 Chronic Lymphocytic Leukemia,  1850
Farrukh T. Awan and John C. Byrd
100 Hairy Cell Leukemia,  1872
Mark B. Geyer, Omar Abdel-Wahab,
Martin S. Tallman, and Jae H. Park
101 Multiple Myeloma and Related Disorders,  1884
S. Vincent Rajkumar and Angela Dispenzieri
 Contents xxxiii

102 Hodgkin Lymphoma,  1911 105 Adult T-Cell Leukemia/Lymphoma,  1965

Nancy Bartlett and Grace Triska Matthew A. Lunning, Neha Mehta-Shah,
and Steven M. Horwitz
103 Non-Hodgkin Lymphomas,  1926
Jeremy S. Abramson Index 1975

104 Cutaneous T-Cell Lymphoma and Cutaneous

B-Cell Lymphoma,  1948
Christiane Querfeld, Steven T. Rosen,
and Madeleine Duvic
This page intentionally left blank

SCIENCE AND CLINICAL ONCOLOGY
A. BIOLOGY AND CANCER
1. Molecular Tools in Cancer
Research
2. Intracellular Signaling
3. Cellular Microenvironment and
Metastases
4. Control of the Cell Cycle
5. Pathophysiology of Cancer
Cell Death
6. Cancer Immunology
7. Stem Cells, Cell Differentiation,
and Cancer
8. Tumor Microenvironment:
Vascular and Extravascular
Compartment
9. Cancer Metabolism
B. GENESIS OF CANCER
10. Environmental Factors
11. DNA Damage Response
Pathways and Cancer
12. Viruses and Human
Cancer
P A R T
I
13. Genetic Factors: Hereditary 22. Lifestyle and Cancer Prevention
Cancer Predisposition
23. Screening and Early Detection
Syndromes
24. Nicotine Dependence: Current
14. Genetic and Epigenetic
Treatments and Future
Alterations in Cancer
Directions
C. DIAGNOSIS OF CANCER
F. TREATMENT
15. Pathology, Biomarkers, and
25. Cancer Pharmacology
Molecular Diagnostics
26. Therapeutic Targeting of
16. Imaging
Cancer Cells: Era of
D. CLINICAL TRIALS Molecularly Targeted Agents
17. Biostatistics and Bioinformatics 27. Basics of Radiation Therapy
in Clinical Trials
28. Hematopoietic Stem Cell
18. Clinical Trial Designs in Transplantation
Oncology
29. Gene Therapy in Oncology
19. Structures Supporting Cancer
Clinical Trials 30. Therapeutic Antibodies and
Immunologic Conjugates
20. Oncology and Health Care
Policy 31. Complementary and Alternative
Medicine
E. PREVENTION AND EARLY
DETECTION
21. Discovery and Characterization
of Cancer Genetic
Susceptibility Alleles
 A. BIOLOGY AND CANCER
1  Molecular Tools in Cancer Research
Mauro W. Costa, Muneer G. Hasham, and Nadia Rosenthal

S UMMARY OF K EY P OI N T S
• Our understanding and treatment of
cancer have always relied heavily on
parallel developments in biologic
research. Molecular biology provides
the basic tools to study genes
involved with cancer growth patterns
and tumor suppression. An
advanced understanding of the
molecular processes governing cell
growth and differentiation has
revolutionized the diagnosis,
prognosis, and treatment of
malignant disorders.
• This introductory chapter relates
basic principles of molecular biology
to emerging perspectives on the
origin and progression of cancer and
explains newly developed laboratory
techniques, including whole-genome
analysis, expression profiling, and
refined genetic manipulation in and
use of genetically diverse animal
models, providing the conceptual
and technical background necessary
to grasp the central principles and
new methods of current cancer
research.

Since the last edition of this book was published, advances in our
understanding of the basic mechanisms of cancer have continued to
inform and refine clinical approaches to prevention and therapy. New
prognostic and predictive markers derived from molecular biology
can now pinpoint specific genetic changes in particular tumors or
detect occult malignant cells in normal tissues, leading to improved
technologies for tumor screening and early detection. Diagnostic
approaches have expanded from morphologic criteria and single-gene
analysis to whole-genome technologies and single-cell genomics
imported from other biologic disciplines. A new systemic vision of
cancer is emerging, in which the importance of individual mutation
has been superseded by an appreciation for higher-order organization
and individual genetic background, disrupted by complex interactions
of disease-associated factors and gene-environmental parameters that
affect tumor cell behavior. Results from these cross-disciplinary
investigations underscore the complexity of carcinogenesis and have
profoundly influenced the design of strategies for both cancer prevention
and advanced cancer therapy.
This overview will serve as a foundation of conceptual and technical
information for understanding the exciting new advances in cancer
research described in subsequent chapters. Since the discovery of
oncogenes, which provided the first concrete evidence of cancer’s
genetic basis, applications of advanced molecular techniques and
instrumentation have yielded new insights into normal cell biology.
A basic fluency in molecular biology and genetics has become a necessary
prerequisite for clinical oncologists because many of the new diagnostic
and prognostic tools currently in use rely on these fundamental
principles of gene, protein, and cell function.
OUR UNSTABLE HEREDITY
Cancer genetics has classically relied on the candidate-gene approach,
detecting acquired or inherited changes in specific genetic loci accu-
mulated in a single cell, which then proliferates to produce a tumor
composed of its identical clonal progeny. During the early steps of
tumor formation, mutations that lead to an intrinsic genetic instability
allow additional deleterious genetic alterations to accumulate. These
genetic changes confer selective advantages on tumor cell clones by
disrupting control of cell proliferation. The identification of specific
mutations that characterize a tumor cell has proved invaluable for
analyzing the neoplastic progression and remission of the disease. The
emergence of cancer cells is a byproduct of the necessity for continuous
cell division and DNA replication to maintain organ functionality
throughout the life cycle.
The highly heterogeneous nature of tumors, each composed of
multiple cell types, led to the formulation of the “cancer stem cell”
hypothesis, which posits that only a subpopulation of cancer cells is
able to maintain self-renewal, unlimited growth, and capacity for
differentiation into other, more specialized cancer cell types. Cancer
stem cells display bona fide stem cell markers, in contrast to other
cancer cells present in the tumor, which do not have tumorigenic
potential. In fact, fewer than 1 in 10,000 cells present in human acute
myeloid leukemia are capable of reinitiating a new tumor when
transplanted into animals. Cancer stem cells have been identified in
many solid tumors in the brain, colon, ovaries, prostate, and pancreas,
suggesting that more effective cancer therapies would target these
self-renewing cells, rather than the tumor as a whole. The cancer stem
cell concept differs from the original clonal evolution hypothesis,
which states that every cell in a tumor mass is capable of self-renewal
and differentiation, and suggests that detecting and targeting subtle
genetic and epigenetic differences that distinguish cancer stem cells
may provide a more effective avenue to intervention in disease
progression.
Heterogeneity can also arise as a result of stochastic mutational
events that lead to cancer progression. Clastogenic insults to the genome,
or genomic instability due to aberrant gene regulation, could lead to
loss of heterozygosity (LOH) of tumor suppressor genes such as TP53,
RB1, or BRCA, and can also lead to tumor heterogeneity and change
in disease progression. Furthermore, activation of DNA or RNA editing
enzymes in tumors could lead to kataegis, a DNA hypermutation
process, and increase tumor heterogeneity. Although there are molecular
biology tools currently available to detect aberrant but stable genomes,
the later processes that lead to genomic instability make diagnosis
and prognosis more challenging.

2
 Molecular Tools in Cancer Research  •  CHAPTER 1 3

DETECTING CANCER MUTATIONS
Methods for mutation detection all rely on the manipulation of DNA,
the basic building block of heredity in the cell. DNA consists of two
long strands of polynucleotides that twist around each other clockwise
in a double helix (Fig. 1.1). Nucleic acid bases attached to the sugar
groups of each strand face each other within the helix, perpendicular
to its axis. These comprise only four bases: the purines adenine and
guanine (A and G) and the pyrimidines cytosine and thymine (C and
T). During assembly of the double helix, stable pairings of nucleotides
from either strand are made between A and T, or between G and C.
Each base pair forms one of the billions of rungs in the long, unbroken
ladder of DNA forming a chromosome.
The functional unit of inherited information in DNA, the gene,
is most often represented by a discrete section of sequence necessary
to encode a particular protein structure. Gene expression is initiated
by forming a copy of the gene, messenger RNA (mRNA), constructed
base by base from the DNA template by a polymerase enzyme. Once
transcribed, an mRNA transcript is modified and the processed product
is transported out of the nucleus. In the cytoplasm, proteins are
then synthesized, or translated, in macromolecular complexes called
ribosomes that read the mRNA sequence and convert the nucleic acid
code, based on three-base segments or codons, into a 20–amino acid
code to form the corresponding protein.
Although these canonic processes drive gene expression in all normal
cells, cancer cells defy the rules. For instance, uracils, which are found

Genes Cell nucleus

containing
23 pairs of
chromosomes

DNA
strand
Chromosomes
Sugar

Cytosine Bases
thymine

Bases

Adenine and
guanine
Phosphate
group P

H
H
C H
H
H C
H
P
H
H
P
H
C
H
H CH2
P P
H
CH2
H
CH2
H
P P
H
CH2
H CH2

Figure 1.1  •  DNA structure. Deoxyribonucleic acid (DNA) is the cell’s genetic material, contained in single compacted strands comprising chromosomes
within the cell nucleus. In the DNA double helix, the two intertwined components of its backbone, composed of sugar (deoxyribose) and phosphate molecules,
are connected by pairs of molecules called bases. The sequence of four bases (guanine, adenine, thymine, and cytosine) in the DNA helix determines the specificity
of genetic information. The bases face inward from the sugar-phosphate backbone and form pairs with complementary bases on the opposing strand for
specific recognition. The arrangement of chemical groups is unique for each base pair, allowing base pairs to be specifically targeted by transcription factors,
polymerases, restriction enzymes, and other DNA-binding proteins. (From http://www.terrapsych.com/dna.jpg.)
4 Part I: Science and Clinical Oncology
on RNA, can be detected in the DNA of cancer cells because of their
high mutation rates. Paradoxically, these deviations from the norm
allow the development of molecular biology tools to better diagnose
and predict tumor progression.
GENERATING DIVERSITY WITH
ALTERNATE SPLICING
In higher organisms, most protein coding gene sequences are interrupted
by stretches of noncoding DNA sequences, called introns. In the
nucleus, these introns are removed after mRNA transcription to produce
a continuous chain of coding sequences, or exons, that subsequently
undergo translation into protein. The splicing process requires absolute
precision because the deletion or addition of a single nucleotide at
the splice junction would throw the three-base coding sequence out
of frame, or lead to exon skipping or addition, creating abnormal
proteins.
The dramatic increase in genetic complexity conferred by alternate
RNA splicing is underscored by the multiple splice patterns of many
medically relevant genes, in which different combinations of exons
are chosen for the final mRNA transcript, such that one gene can
encode many different proteins (Fig. 1.2). The choice of protein isoform
to be expressed from a gene with multiple splicing possibilities is a
decision that can be perturbed in disease. Errors in splicing mechanisms
have been associated with a large group of cancers. These include
mutations in the oncogene p53 in more then 12 different types of
cancer, mutL homolog 1 protein (MLH1) mutation in hereditary
nonpolyposis colorectal cancer, and several transcription factors and
cell signaling and membrane proteins. When mutations in the splicing
site lead to insertion of novel sequences in the mRNA, the encoded
protein can be used as a potential clinical marker, as seen for the
transcription factor NSFR in small cell lung cancer. Owing to their
unique expression in cancer cells, these markers can be further explored
as new cancer-specific therapeutic targets.
GENOMICS OF CANCER
The complete set of DNA sequences carried on all the chromosomes
is known as the genome. Although the general map of the genome
Exon Exon
Gene 1 2
1 2
RNA
Alternative splicing
RNA 1 2 3 4 5 1
Translation
Protein A
Figure 1.2  •  RNA splicing. Alternate splicing produces multiple related proteins, or isoforms, from a single gene. (From Guttmacher AE, Collins F.
Genomic medicine—a primer. N Engl J Med. 2002;347:1512–1520.)
is shared by all members of a species, the recent sequencing of thousands
of individual human genomes has given rise to the new field of
genomics, providing us with new tools to reveal the more subtle
variations that arise between individuals. These variations are critical,
both as a natural engine driving heterogeneity within a species, and
as a source of predisposition to cancer types. The most common forms
of human genetic variations, or alleles, arise as single-nucleotide
polymorphisms (SNPs). Because these allelic dissimilarities are abundant,
inherited, and dispersed throughout the genome, SNPs can be used
to track racial diversity, personal traits, and susceptibility to common
forms of cancer (Fig. 1.3). Commercial entities have developed tools
that can detect thousands of SNPs with relatively little sample material.
Platforms such as MegaMUGA or GigaMUGA can allow mammalian
genetic mapping that can aid in a number of diagnoses and can
distinguish between predictive and prognostic markers.
How do SNPs arise between individuals? One source of variation
in DNA sequence derives from deviations in the strict base-pairing
rule underlying the structure, storage, retrieval, and transfer of genetic
information. The duplicated genetic information in the two strands
of DNA not only permits the repair of a damaged coding sequence
but also forms the basis for the replication of DNA. During cell
division, polymerase enzymes unwind the DNA strands and copy
them, using the base sequences as a template for constructing a new
helix so that the dividing cell passes its entire genetic content on to
its progeny. Errors in this process are rare, and person-to-person
differences comprise only about 0.1% of the human genome. SNPs
are inherited if they occur in the germline. Many genetically inherited
variations occur in regions that do not encode protein or alter the
regulation of nearby genes. Given the disruptive effects even subtle
genetic changes may have on cell function, it is important to distinguish
SNPs that represent true mutations from benign polymorphisms.
Our ability to monitor hundreds of thousands of SNPs simultane-
ously is one of the most important advances in modern medical genetics.
Relatively simple genotyping technologies for SNP detection rely largely
on the polymerase chain reaction (PCR). In procedures that use this
reaction, two chemically synthesized single-stranded DNA fragments,
or primers, are designed to match chromosomal DNA sequences
flanking the segment in which an SNP is positioned. With the addition
of nucleotide building blocks and a heat-stable DNA polymerase, the
Exon Exon Exon
3 4 5
3 4 5
2 4 5 1 2 3 5
Translation Translation
Protein B Protein C
 Molecular Tools in Cancer Research  •  CHAPTER 1 5

Primary tumor Metastasis SNP density

11p15.5
11p15.4
11p15.3
11p15.2
11p15.1
11p14.3
11p14.2
11p14.1
11p13
11p12
Tens to hundreds
11p11.2
of changes between >10 million SNPs 11p11.12
primary and secondary between individuals 11p11.11
tumors 11q11
11q12.1
11q12.2
11q12.3
11q13.1
11q13.2
11q13.3
Primary tumor Metastasis 11q13.4
11q13.5
11q14.1
11q14.2
11q14.3
11q21
11q22.1
11q22.2
11q22.3
11q23.1
11q23.2
11q23.3
11q24.1
11q24.2
11q24.3
11q25

Figure 1.3  •  Determining cancer susceptibility with single-nucleotide polymorphisms (SNPs). Millions of SNPs exist between individuals, as depicted by
the red arrows and the SNP density map of human chromosome 11 (right). By contrast, point mutations, deletions, insertions, and rearrangements between
normal tissues and tumors or between primary and secondary tumors probably number in the tens to hundreds (or potentially thousands), as depicted by the
spectral karyotype image at the bottom of the figure. Because the constitutional genetic polymorphisms are present in all the tissues of the body, it might be
possible to distinguish differences in metastatic versus nonmetastatic tumors and in nontumor tissues before they ever happen to develop a solid tumor. (From
Hunter K. Host genetics influence tumour metastasis. Nat Rev Cancer. 2006;6:141–146.)

primer pairs, or amplicons, initiate synthesis of new DNA strands,
using the chromosomal material as a template. Each successive copying
cycle, initiated by “melting” the resulting double-stranded products
with heat, doubles the number of DNA segments in the reaction (Fig.
1.4). The technique is exceptionally sensitive; millions of identical
DNA copies can be generated in a matter of hours with PCR by using
a single DNA molecule as the starting material.
Other novel methods for large-scale SNP detection include single-
nucleotide primer extension, allele-specific hybridization, oligonucleotide
ligation assay, and invasive signal amplification, which detect poly-
morphisms directly from genomic DNA without the requirement of
PCR amplification. The International HapMap Project was established
with the objective of identifying those variations (commonly thought
to be on the order of 10 million in our genome) in the human popula-
tion. This project is already in its third phase (HapMap3), now
including both SNPs and copy number variations observed in 1184
samples from 11 different human populations. Regardless of the method
used to characterize them, the collective SNPs in a selected genomic
region characterize a haplotype, or specific combination of alleles at
multiple linked genetic loci along a chromosome that are inherited
together.
Even when the SNPs within a given haplotype are not directly
involved in a disease, they provide markers for clonality and for the
loss or rearrangement of specific chromosomal segments in growing
tumors. In the human nucleus, each of the 23 tightly compacted
chromosomes has a characteristic size and structure, and a distinctive
base sequence that carries unique protein coding information. Other
noncoding DNA sequences are used for directing the transcription
of neighboring genes, through complex regulatory circuits involving
protein binding and modification of the DNA itself, or shifting of
its chromosomal packaging. Although genomic instability is generally
considered a consequence of tumor formation rather than the initial
trigger of cancer, the loss, gain, or rearrangement of chromosomal
segments through deletion or translocation is a common form of
neoplastic mutation, as protein coding segments from different genes
are combined or regulatory sequences are brought into new proximity
to genes they do not normally control, as seen in chronic myeloid
leukemia (CML). In CML, recombination events lead to the fusion
of BCR and ABL genes (Philadelphia chromosome). This results in
constitutive activation of the fused gene, leading to loss of proliferative
control in myeloid cells and consequently cancer. Gross changes in
DNA arrangement can be detected by cytogenetic analysis of chro-
mosomal features on metaphase spreads. Although fluorescence in
situ hybridization (FISH) provides greater resolution by localizing
specific chromosomal DNA sequences corresponding to fluorescently
labeled probes (Fig. 1.5), and can be used to track specific alterations
in chromosomal structure where known genes are involved, spectral
karyotyping (SKY) is a powerful and more general tool that could
aid diagnosis of cancer genomes. With each fluorescently labeled
chromosome assigned a specific color, translocations and additions
are revealed as multicolored chromosomes, or large deletions as pieces
of missing chromosomes.
6 Part I: Science and Clinical Oncology

Cycle 1 Cycle 2 Cycle 3 The plethora of data arising from genome-wide association studies
using currently available techniques poses particular challenges to
cancer researchers. Discerning the causal genetic variants among
genotype-phenotype associations requires extensive replication, control
for underlying genetic differences in population cohorts, and consistent
classification of clinical outcomes. New technologies must be met
Separation of strands

Separation of strands

Primers
Sequence
to be
amplified
Primers Heat Heat
Figure 1.4  •  Amplification of DNA by polymerase chain reaction (PCR).
The DNA sequence to be amplified is selected by primers, which are short,
synthetic oligonucleotides that correspond to sequences flanking the DNA to
be amplified. After an excess of primers is added to the DNA, together with
a heat-stable DNA polymerase, the strands of both the genomic DNA and
the primers are separated by heating and allowed to cool. A heat-stable
polymerase elongates the primers on either strand, thus generating two new,
identical double-stranded DNA molecules and doubling the number of DNA
fragments. Each cycle takes just a few minutes and doubles the number of
copies of the original DNA fragment.
Separation of strands
with equivalently sophisticated and rigorous analytic methodologies
for the true genetic cause of cancer to be teased out from our variable
and often unstable heredity.
BUILDING GENE LIBRARIES
The engineering of genes by recombinant DNA technology evolved
from methods initially devised to provide sequences in amounts
sufficient for biochemical analysis. The original protocol involves
clipping the desired segment from the surrounding DNA and inserting
it into a bacterial or viral vector, which is then amplified millions of
times in a host bacterium. Using recombinant DNA technology, genetic
Heat engineering can routinely produce industrial quantities of pure, clinically
useful products in a cost-effective way. For diagnostic purposes, it is
easier and faster to amplify a known genomic DNA sequence directly
from a patient sample with PCR, but the classic approach is still
applied to the construction of recombinant DNA libraries.
To be useful, a DNA library must be as complete as possible, with
recombinant members, or clones, sufficiently numerous to include
all the sequences in an individual genome. For certain kinds of gene-
linkage analysis that require long, uninterrupted stretches of DNA,
special vectors, such as bacterial or yeast artificial chromosomes, can
carry foreign DNA fragments of enormous lengths. Chromosomal
segments represented in genomic DNA libraries can contain the

9 9 22 22

9 der(9) 22 der(22)

Figure 1.5  •  Detection of chromosomal translocations. Fluorescence in situ hybridization (FISH) technology uses a labeled DNA segment as a probe to
search homologous sequences in interphase chromosomes for the t(9;22)(q34;q11) translocation, associated with chronic myeloid leukemia. On the left, patient
nuclei were hybridized with probes for chromosome 9 (labeled with SpectrumRed fluorophore) and chromosome 22 (labeled with SpectrumGreen). (Modified
from Varella-Garcia M. Molecular cytogenetics in solid tumors: laboratorial tool for diagnosis, prognosis, and therapy. Oncologist. 2003;8:45–58.)

structure of an entire gene, including the information that regulates
its expression, and formed the starting material for sequencing of the
human genome.
Many cancer-associated genes were originally identified through
use of partial DNA libraries, which contain only the DNA sequences
transcribed by a particular tissue or type of cell. The starting mate-
rial in this case is mRNA. For cloning purposes, the enzyme reverse
transcriptase can convert mRNA into complementary DNA (cDNA).
The number of clones in a cDNA library is much smaller than
in a genomic library because a cDNA library represents only the
genes expressed by the tissue of interest and contains exclusively the
coding portion of genes. For this particular reason, this technique
has now become obsolete for organisms whose genome has now
been fully sequenced. New advances in PCR chemistry allowed
for the direct cloning of increasingly larger cDNA fragments with
high specificity and low error rates. Highly accurate PCR technol-
ogy, coupled with the constantly evolving generation of genomic
sequence maps in humans and model organisms, has exponentially
expanded the availability of candidate genes to be tested in cancer
biology.
LOSING CONTROL OF THE GENOME
Mutations that lead to oncogenic transformation of a cell invariably
affect the expression of its genetic information that specifies functional
products, either RNA molecules or proteins used for various cellular
functions. The primary level of gene control is the transcription of
DNA into RNA. Gene regulation, or the control of RNA synthesis,
represents a complex process that is itself a frequent target of neoplastic
mutation.
DNA regulatory sequences do not encode a product. However,
without them a cell could not coordinate the expression of the hundreds
of thousands of genes in its nucleus, select only certain genes for
expression, and activate or repress them in response to precise internal
or external signals. These control centers of the genome contain binding
sites for multiple proteins, called transcription factors, which interact
to form regulatory networks controlling gene transcription. Their
function can be altered by signals that induce modifications such as
phosphorylation, or by interactions with other regulators such as steroid
hormones. Many of the cell’s responses to a wide variety of external
stimuli, such as neurotransmitters, antigens, cytokines, and growth
factors, are mediated through transcription factors binding to DNA
regulatory sequences.
Certain regulatory DNA sequences common to many genes are
positioned upstream of the transcription start site (Fig. 1.6). Collectively
called the “promoter” of a gene, these proximal sequences comprise
binding sites for the RNA polymerase and its numerous cofactors.
Whereas the position of the promoter with regard to the transcription
start site is relatively inflexible, other DNA regulatory elements, known
as enhancers, occur in unpredictable locations, often at a considerable
distance from the genes they control. Some transcription factors bind
to particular regions of enhancers and drive their associated genes in
many types of cells, whereas others, active in only a limited variety
of cells, maintain a tissue-specific pattern of gene expression. Enhancers
are often responsible for the aberrant expression of genes induced by
chromosomal translocation associated with specific forms of cancer:
a normally quiescent gene promoting cell growth that is dislocated
to a position near a strong enhancer may be activated inappropriately,
resulting in loss of growth control.
Enhancers and promoters have been assigned specific roles by means
of cell culture assays or in transgenic animals in which putative regula-
tory DNA sequences are linked to test or “reporter” genes, and are
examined for their ability to activate expression of the reporter gene
in response to the appropriate signals. Through assessment of the
effects of deleting, adding, or changing DNA sequences within the
regulatory element, the precise nucleotides that are critical for recogni-
tion by transcription factors can be determined.
Molecular Tools in Cancer Research  •  CHAPTER 1 7
The interaction between protein and DNA is increasingly
used to identify transcription factor binding sites in a regulatory
region. Whereas electrophoretic mobility shift assays (EMSAs), or
DNA footprinting, were once standard techniques for determining
protein-DNA interactions, emerging genome-wide technologies, such
as chromatin immunoprecipitation on microarray chip (ChIP-chip)
and chromatin immunoprecipitation on sequencing (ChIP-seq), are
revolutionizing the way in which we see the interaction of a transcription
factor complex with virtually all of its potential genomic targets in
a particular cell state. These strategies involve the use of candidate
protein–specific antibodies to pull down DNA targets regulated by
them. These targets are further identified with the use of microarray
ChIP-chip or next generation sequencing ChIP-seq technologies
(see Fig. 1.14).
Our appreciation of oncogenic perturbations, by mutation of
regulatory protein coding genes or by loss of controlled signaling by
cell cycle switches or in the target sequences these proteins recognize,
has recently extended to include posttranslational modifications that
control protein activity, such as phosphorylation, ubiquitylation, and
SUMOylation. Tumor-associated changes in these modifications
underscore the multiple levels of control necessary to ensure correct
gene expression that is so central to the normal function of the cell.
EPIGENETICS AND CANCER
Epigenetics refers to general control of gene expression that is inherited
during cell division, although not part of the DNA sequence itself.
Epigenetic regulation involves changes in chromatin, a higher-order
building block of chromosomes that wraps DNA into coils with
scaffolding proteins such as histones. Histones are a necessary com-
ponent of chromosomal compaction, but also play a critical role in
gene accessibility (Fig. 1.7). Active genetic loci are associated with
loosely configured euchromatin, whereas silent loci are condensed in
heterochromatin. The state of chromatin configuration (euchromatin
or heterochromatin) both controls and is controlled by patterns of
histone modifications such as methylation and acetylation on specific
DNA sequences. This pattern relates the underlying genetic information
to its higher-order structure that determines whether a particular gene
regulatory element is available to transcription factors (on or off status).
These epigenetic modifications of the nuclear environment that
determine the accessibility of a gene can persist during cell division,
because inherited epigenetic patterns provide permanent marks for
altered chromatin configuration in daughter cells. The pattern of
modifications generated by the epigenetic code rivals the complexity
of the DNA code itself.
The accessibility of genomic regions can favor mutations. Enzymes
such as the APOBEC family exploit this accessibility to induce C to
U mutation, which is then converted to T or staggered single-strand
breaks. If not rectified, these point mutations or breaks can lead to
hypermutations. Kataegis is an example wherein such hypermutation
occurs on the BRCA locus, generating neoplasia.
Research has linked rearrangement of chromatin and associated
DNA methylation with the inactivation of tumor suppressor genes and
neoplastic transformation. Defects that could lead to cancer involve
perturbations in the “epigenotype” of a particular locus, through the
silencing of normally active genes or activation of normally silent
genes, associated with changes in DNA methylation, histone modifica-
tion, and chromatin proteins (Fig. 1.8). Changes in the number or
density of heterochromatin proteins associated with cancer-related
genes such as EZH2, or of euchromatic proteins such as trithorax in
leukemia, can also be associated with abnormal patterns of methyla-
tion in gene promoter regions and with higher-order chromosomal
structures that are only beginning to be understood. Finally, it is
increasingly evident that interactions among the “epigenome,” the
genome, and the environment are common targets for mutation
and can have profound effects on the gene expression readout of a
cancer cell.
8 Part I: Science and Clinical Oncology

Gene structure
Exon 1 Exon 2 Exon 3
Enhancer Promoter Intron 1 Intron 2

TATAA GT AG GT AG AATAAA

Transcription Gene expression

factors

RNA polymerase
Exon 1 Exon 2 Exon 3

Transcription
Transcription-initiation pre-
complex 5' 3' mRNA
Transcript processing

RNA-clipping enzyme

AAUAAA

5' cap

PolyA tail
AAAA...

Adenosine-adding
Intron lariat enzyme (terminal
transferase)
Splicing
Nucleus AAAA...

Spliceosome

Processed
transcript mRNA
Cytoplasm AAAA...

Translation into protein

Figure 1.6  •  Mammalian gene structure and expression. The DNA sequences that are transcribed as RNA are collectively called the gene and include exons
(expressed sequences) and introns (intervening sequences). Introns invariably begin with the nucleotide sequence GT and end with AG. An AT-rich sequence
in the last exon forms a signal for processing the end of the RNA transcript. Regulatory sequences that make up the promoter and include the TATA box
occur close to the site where transcription starts. Enhancer sequences are located at variable distances from the gene. Gene expression begins with the binding
of multiple protein factors to enhancer sequences and promoter sequences. These factors help form the transcription-initiation complex, which includes the
enzyme RNA polymerase and multiple polymerase-associated proteins. The primary transcript (pre-mRNA) includes both exon and intron sequences. Post-
transcriptional processing begins with changes at both ends of the RNA transcript. At the 5′ end, enzymes add a special nucleotide cap; at the 3′ end, an
enzyme clips the pre-mRNA about 30 base pairs (bp) after the AAUAAA sequence in the last exon. Another enzyme adds a polyA tail, which consists of up
to 200 adenine nucleotides. Next, spliceosomes remove the introns by cutting the RNA at the boundaries between exons and introns. The process of excision
forms lariats of the intron sequences. The spliced mRNA is now mature and can leave the nucleus for protein translation in the cytoplasm. (From Rosenthal
N. Regulation of gene expression. N Engl J Med. 1994;331:931–932.)
 Molecular Tools in Cancer Research  •  CHAPTER 1 9

Nucleosome

DNA

The solenoid

Figure 1.7  •  Chromatin packaging of DNA. The 4 meters of DNA in every human cell must be compressed in the nucleus, reaching compaction ratios
of 1 : 400,000. This is achieved by wrapping the DNA (blue) around histone protein complexes (green), forming nucleosomes connected by a thread of free
linker DNA. Each nucleosome, together with its linker, packages about 200 bp (66 nm) of DNA. The nucleosomes are then coiled into chromatin, a rope of
nucleoprotein about 30 nm thick (bottom left electron micrograph). To allow DNA to be accessed by transcription and replication apparatus, chromatin is relaxed
(bottom right electron micrograph). (Courtesy Jakob Waterborg. www.umkc.edu/sbs/waterborg/chromat/chromatn.html. Copyright 1998 Jakob Waterborg.)

Gene X Gene X
X

Gene Y
X

Gene Y

A Normal B Epigenetic lesions

Figure 1.8  •  Gene accessibility through epigenetics. Illustration depicts known and possible defects in the epigenome that could lead to disease. (A) Gene
X is a transcriptionally active gene with sparse DNA methylation (brown circles), an open chromatin structure, interaction with euchromatin proteins (green
protein complex), and histone modifications such as H3K9 acetylation and H3K4 methylation (green circles). Gene Y is a transcriptionally silent gene with
dense DNA methylation, a closed chromatin structure, interaction with heterochromatin proteins (red protein complex), and histone modifications such as
H3K27 methylation (pink circles). (B) The abnormal cell could switch its epigenotype through the silencing of normally active genes or activation of normally
silent genes, with the attendant changes in DNA methylation, histone modification, and chromatin proteins. In addition, the epigenetic lesion could include
a change in the number or density of heterochromatin proteins in gene X (such as EZH2 in cancer) or euchromatic proteins in gene Y (such as trithorax in
leukemia). There may also be an abnormally dense pattern of methylation in gene promoters (shown in gene X ), and an overall reduction in DNA methylation
(shown in gene Y ) in cancer. The insets show that the higher-order loop configuration may be altered, although such structures are currently only beginning
to be understood.
10 Part I: Science and Clinical Oncology

PROFILING TUMORS underlying tumor progression by following the changes in a tumor

Monitoring global gene expression patterns of cells represents one of
the latest breakthroughs in developing a molecular taxonomy of cancer.
Although classic blotting and probe hybridization techniques (Northern
blot) are still a reliable way to monitor expression of individual genes,
these techniques have limitations, such as unequal hybridization
efficiency of individual probes, sensitivity for low copy or small
transcripts, and difficulty in detecting multiple RNAs simultaneously
or in simultaneously analyzing a large number of targets. For cancer
studies, it is important to be able to compare the expression pattern
of all known RNAs, including noncoding RNAs, between cancer cells
and normal cells. Thus new genome-wide analytic techniques are the
state-of-the-art choice to detect mRNA expression profiles at a single
point in time or cell state. Genome-wide profiling of gene expression
in tumors delivers an unprecedented view into the biologic processes
cell’s transcriptional landscape.
With reliance on two-color fluorescence-based microarray technology
(DNA microarray), simultaneous evaluation of thousands of gene
transcripts and their relative expression can provide a snapshot of the
“transcriptome,” the full complement of RNA transcripts produced
at a specific time during the progression of malignancy.
Transcriptional profiling with microarrays typically involves screens
of mRNA expression from two sources (such as tumor and normal
cells), using cDNA or oligonucleotide libraries that are arranged in
extremely high density on microchips. These are probed with a mixture
of fluorescently tagged cDNAs generated from the tumor and normal
samples, which results in differential staining of each gene spot. The
relative intensity of the two different colors reflects the RNA expres-
sion level of each gene; this is analyzed with a laser confocal scanner
(Fig. 1.9). With microarrays, single genes that constitute diagnostic,

Tumors
Reference Tumor
RNA RNA

Genes

Statistical
cDNA
analysis
Hybridization
of probe to
microarray Multidimensional-scaling plot

A B C

Donor Recipient
paraffin block paraffin block
D E Tissue microarray

Figure 1.9  •  Microarray-based expression profiling of tumor tissue. (A) Reference RNA and tumor RNA are labeled by reverse transcription with different
fluorescent dyes (green for the reference cells and red for the tumor cells) and hybridized to a cDNA microarray containing robotically printed cDNA clones.
(B) The slides are scanned with a confocal laser scanning microscope, and color images are generated with RNA from the tumor and reference cells for each
hybridization. Genes upregulated in the tumors appear red, whereas those with decreased expression appear green. Genes with similar levels of expression in
the two samples appear yellow. Genes of interest are selected on the basis of the differences in the level of expression by known tumor classes (e.g., BRCA1-
mutation–positive and BRCA2-mutation–positive). Statistical analysis determines whether these differences in the gene expression profiles are greater than
would be expected to occur by chance. (C) The differences in the patterns of gene expression between tumor classes can be portrayed in the form of a
color-coded plot, and the relations between tumors can be portrayed in the form of a multidimensional-scaling plot. Tumors with similar gene-expression
profiles cluster close to one another in the multidimensional-scaling plot. (D) Particular genes of interest can be further studied through the use of a large
number of arrayed, paraffin-embedded tumor specimens, referred to as tissue microarrays. (E) Immunohistochemical analyses of hundreds or thousands of
these arrayed biopsy specimens can be performed in order to extend the microarray findings. (From Hedenfalk I, Duggan D, Chen Y, et al. Gene expression
profiles in hereditary breast cancer. N Engl J Med. 2001;344:539–548.)
 Molecular Tools in Cancer Research  •  CHAPTER 1 11

prognostic, or therapeutically relevant markers can be systematically
monitored. Alternatively, the entire set of expressed genes can be
collectively analyzed through use of powerful statistical methods to
classify tumors according to their transcriptional profile. Microarray
analysis has already dramatically improved our ability to explore the
genetic changes associated with cancer etiology and development and
is providing new tools for disease diagnosis and prognostic assessment.
For example, DNA microarray analysis of multiple primary breast
tumor transcriptomes has revealed reproducible signature expression of
70 associated genes. These markers have been recently cleared by the
US Food and Drug Administration (FDA) for PCR–based diagnostics
showing that expression analysis of a relative small gene group can
predict the prognosis of early stage breast cancers. When applied on a
larger scale, these assays can predict response to chemotherapy, or opti-
mize pharmaceutical intervention by targeting therapeutic approaches
to specific patient populations and ultimately to individualized therapy.
A novel high-throughput approach for global transcriptome analysis
has been made possible by advances in strategies that allow mass
sequencing of DNA fragments. With this technique, called RNA-seq,
it is now possible to obtain a comprehensive and unbiased analysis
of all mRNA transcripts present in cells or tissues. (Fig. 1.10). The
technique relies on the generation of small fragments of cDNA from
any RNA sample, followed by sequencing of these expressed tags from
one end (single-end sequencing) or both ends (pair-end sequencing),
resulting in fragments of 30 to 400 base pairs (bp). The resulting
sequences can be then mapped against the known reference genome
or transcriptome of a certain species. Unlike microarray analysis of
preselected gene sets, RNA-seq allows the unbiased identification of
all genes, or even the presence of different isoforms, expressed in the
sample, allowing a comprehensive comparison of transcript levels
between normal and cancer cells.
The technologies just described can also be applied to the analysis
of noncoding RNA species. In addition to the 20,000 protein coding
transcripts used to classify a wide variety of human tumors, hundreds
if not thousands of small, noncoding interference RNA species, with
critical functions in multiple biologic processes, have been discovered;
many of these RNA species are directly or indirectly involved in the
control of cell proliferation. Known as microRNAs (miRNAs), these
short transcripts arise from primary genome-encoded transcripts of
variable sizes that are processed into 70- to 100-nucleotide hairpin-
shaped precursors, which are processed into mature miRNAs of 21 to
23 bp RNA molecules (Fig. 1.11). miRNAs function by base-pairing

2x poly (A) RPKM RPKM

selection
Brain 0.0 0.0

Liver 0.0 0.0

25-bp
reads
Add standards
and shatter RNA Muscle 0.5 50.1

Make cDNA
and sequence Muscle splices

1 kb
Map 25-bp tags RNA-Seq
onto genome graph
Myf5
Myf6
Conservation

25-bpm Uniquely mappable

Calculate splices
transcript
Repeating elements by RepeatMasker
prevalence
Myf6
Conservation 20 kb
Uniquely map.
2 RPKM 1 RPKM 1 RPKM RepeatMasker
A B C

Figure 1.10  •  Methods for high-throughput transcriptome analyses. (A) Schematics of regular protocol for RNA-seq sample preparation, showing poly-A
tail specific mRNA isolation followed by fragmentation of RNA into smaller regions, further used for cDNA conversion. Polymerase chain reaction (PCR)
fragments are then tethered by adaptors, sequenced by synthesis, and aligned to the reference genome or transcriptome to calculate relative prevalence of
mRNAs (RPKM). (B) Target fragments can be used to map exon-intron boundaries and thus infer present and quantify different mRNA isoforms in the
sample of interest, as shown for the muscle specific gene Myf6 in this example. (C) Data generated with this method can also be compared with analysis of
other tissues or samples, allowing assessment of relative quantification of targets, as exemplified here for a highly specific gene (red peaks) for muscle samples.
(From Mortazavi A, Williams BA, et al. Mapping and quantifying mammalian transcriptomes by RNA-seq. Nat Methods. 2008;5:621–628.)
12 Part I: Science and Clinical Oncology

Protein-coding gene MicroRNA gene

Transcription of pri-microRNA
Transcription
of mRNA

Pri-microRNA
OR

Drosha

DGCR8
Processing
Nucleus
of pri-microRNAs
into pre-microRNA

Pre-microRNA Ran-GTP

Exportin 5
Transport of
pre-microRNAs into
the cytoplasm

Processing of
Dicer pre-microRNA into
small RNA duplexes
Loqs/TRBP
Cytoplasm
RISC

||||||||||||||||||||
||||
|||
|||
|||
||| ||||||||||||||||||||||||||||| An
||| |||||||||
||| ||||||
||| |||| |||||
|||| |||||
|||||||||

Delivery of
RISC-microRNA
complex

mRNA degradation Translational repression

Figure 1.11  •  MicroRNA production and gene regulation in animal cells. Mature functional microRNAs of approximately 22 nucleotides are generated
from long primary microRNA (pri-microRNA) transcripts. First, the pri-microRNAs, which usually contain a few hundred to a few thousand base pairs, are
processed in the nucleus into stem-loop precursors (pre-microRNA) of approximately 70 nucleotides by the RNase III endonuclease Drosha and DiGeorge
syndrome critical region gene 8 (DGCR8). The pre-microRNAs are then actively transported into the cytoplasm by exportin 5 and Ran-GTP and further
processed into small RNA duplexes of approximately 22 nucleotides by the Dicer RNase III enzyme and its partner Loqacious (Loqs), a homologue of the
human immunodeficiency virus transactivating response RNA-binding protein. The functional strand of the microRNA duplex is then loaded into the
RNA-induced silencing complex (RISC). Finally, the microRNA guides the RISC to the target messenger RNA (mRNA) target for translational repression or
degradation of mRNA. (Modified from Chen CZ. New Eng J Med. 2005;353:1768–71.)

with target mRNAs to inhibit translation and/or promote mRNA suppression. The usefulness of monitoring the expression of miRNAs
degradation. In the context of cancer, miRNAs may act in concert with in human cancer is just now being explored, but preliminary findings
other effectors such as p53 to inhibit inappropriate cell proliferation. reveal an extraordinary level of diversity in miRNA expression across
A global decrease in miRNA levels is often observed in human cancers, cancers, and the large amount of diagnostic information encoded
indicating that small RNAs may have an intrinsic function in tumor in a relatively small number of miRNAs. Significant technologic
 Molecular Tools in Cancer Research  •  CHAPTER 1 13

advances facilitating the profiling of the miRNA expression patterns
in normal and cancer tissues hint at the unexpected greater reliability of
miRNA expression signatures than the respective signatures of protein
coding genes in classifying cancer types. Along with their potential
diagnostic value, miRNAs are also being tested for their prognostic
use in predicting clinical behaviors of cancer patients.
Because probe specificity in miRNA microarray analysis is prob-
lematic owing to the small target size, hybridization can be performed
first in solution, and then quantified with multicolor flow sorting.
Real-time PCR can also be used to quantify specific miRNA sets, or
to capture a more detailed picture of their changing expression profiles
in tumor progression. Identification of the miRNAs involved in tumor
pathogenesis and elucidation of their action in a specific cancer will
be the next necessary steps for their manipulation in a therapeutic
setting.
Advances in this field have revealed that miRNAs are also involved
in cancer initiation and progression, and specific modulation of
such RNAs may serve as a therapeutic strategy. Inhibition of key
miRNAs using antagomirs (a class of chemically modified anti-miRNA
oligonucleotides) has been effective in suppressing tumor growth in
mouse models. It remains to be seen if these results can be extended to
treatment of cancer in the clinic, but interference with miRNA function
is an attractive new tool for the development of cancer therapies.
CANCER PROTEOME
The term proteome describes the entire complement of proteins expressed
by the genome of a cell, tissue, or organism. More specifically, it is
used to describe the set of all the expressed proteins at a given time
point in a defined setting, such as a tumor. Like RNA transcription,
the synthesis of proteins is a highly regulated process that contributes
to the specific proteome of a particular cell and can be perturbed in
diseases such as cancer.
Advances in protein analytic techniques over the last decades have
progressed to the point that even small numbers of specific proteins
expressed in tissues can be used to predict the prognosis of a cancer.
The improvement of protein-based assays has made it possible to
identify and examine the expression of most proteins, and to envision
large-scale protein analysis on the level of gene-based screens. Various
systematic methodologies have contributed to the current explosion
of information on the proteome. These are now being compared for
their suitability as platforms for the generation of databases on protein
structural features, interaction maps, activity profiles, and regulatory
modifications.
The yeast two-hybrid system has been a popular genetics-based
approach for detecting protein-protein interactions inside a cell (Fig.
1.12). One protein fused to the DNA binding domain (bait) and a

DNA-binding domain
fused to protein A A

A Promoter Reporter gene

Activator region fused to

protein B
B

B Promoter Reporter gene

Figure 1.12  •  Exploring protein-protein interactions with the yeast two-hybrid
system. Two-hybrid technology exploits the fact that transcriptional activators are Activator region fused to
modular in nature. Two physically distinct functional domains are necessary to get protein B
transcription: a DNA-binding domain that binds to the DNA of the promoter and DNA-binding domain
an activation domain that binds to the basal transcription apparatus and activates fused to protein A A B Transcription
transcription. (A) The known gene encoding protein A is cloned into the “bait”
vector, fused to the gene encoding a DNA-binding domain from some transcription
factor. When placed into a yeast system with a reporter gene, this fusion protein
can bind to the reporter gene promoter, but it cannot activate transcription. C Promoter Reporter gene
(B) Separately, a second gene (or a library of cDNA fragments encoding potential
interactors), protein B, is cloned into the “prey” vector, fused to an activation domain
of a different transcription factor. When placed into a yeast strain containing the 96 Bait strains 1 Prey strain
reporter gene, it cannot activate transcription because it has no DNA-binding domain.
(C) When the two vectors are placed into the same yeast, a transcription factor is
formed that can activate the reporter gene if protein B, made by the second plasmid,
binds to protein A. (D) Screening a yeast two-hybrid library. The plate on the left
holds 96 different yeast strains in patches (or colonies), each of which expresses a
different bait protein (top). The plate on the right holds 96 patches, each of the
same yeast strain (prey strain) that expresses a protein fused to an activation domain
(prey). The plate of bait strains and the plate of prey strains are pressed to the same
replica velvet, and the impression is lifted with a plate containing yeast extract
peptone dextrose (YPD) medium. After 1 day of growth on the YPD plate, during
which time the two strains mate to form diploids, the YPD plate is pressed to a Replicate velvet
new replica velvet, and the impression is lifted with a plate containing diploid Diploids
selection medium and an indicator such as X-Gal. Blue patches (dark spots) on the
X-Gal plate indicate that the lacZ reporter is transcribed, suggesting that the prey YPD
interacts with the bait at that location. (C from http://www.nature.com/…/journal/
v403/n6770/full/403601a0_r.html. D from Bartel PL, Fields S, eds. The Yeast Replicate velvet
Two-Hybrid System. New York: Oxford University Press; 1997; Finley RL Jr, Brent
R: Two-hybrid analysis of genetic regulatory networks. Retrieved from http://www. D X-Gal
genetics.wayne.edu/finlab/YTHnetworks.html.)
14 Part I: Science and Clinical Oncology

different protein fused to the activation domain of a transcriptional
activator (prey) are expressed together in yeast cells. If the bait
and prey interact, transcription of a reported gene is induced and
detected typically by a color reaction that reflects the transactiva-
tion of the reporter gene, and by proxy, the interaction of the two
test proteins. The method can also be used for large-scale protein
interactions, determination of RNA-protein interactions, and protein-
ligand binding.
As a complementary proteomics tool, mass spectrometry (MS) is
an accurate mass measurement of charged peptides isolated by two-
dimensional gel electrophoresis, producing a mass-to-charge ratio of
charged samples under vacuum that can be used to determine the
sequence identity of peptides. Combined with a specific proteolytic
cleavage step, mass spectroscopy can be used for peptide mass mapping.
Automation of this process has made mass spectroscopy the analytic
tool of choice for many proteomics projects. For diagnostic purposes,
liquid chromatography and mass spectrometry (LC-MS/MS) have
been combined to detect not only a single–amino acid change in the
whole proteome, but also posttranslational protein modification such
as phosphorylation, SUMOylation, or ubiquitination. These LC-MS/
MS systems, such as the iTRAQ, allow for a more precise and indi-
vidualized diagnosis of cancer.
Monoclonal antibodies (mAbs) have been a cornerstone of protein
analysis in cancer research, and more recently have risen to prominence
as cancer therapeutics based on their exquisite specificity for protein
targets and their potent interference with protein function. Novel
strategies have been developed that target not only antigens highly
expressed in cancer cells but also to enhance the innate immune
response against cancer cells. These antibodies can act via several
mechanisms, including antibody-dependent cellular cytotoxicity
(ADCC), complement-mediated cytotoxicity (CMC), and antibody-
dependent cellular phagocytosis (ADCP) (Fig. 1.13). Laboratory mice
have been the animal model of choice for generating a ready source
of diverse, high-affinity and high-specificity mAbs; however, the use
of rodent antibodies as therapeutic agents has been restricted by the
inherent immunogenicity of mouse proteins in a human setting. The
more recent application of transgenic mouse technology to introduce
variable regions encoded by human sequences into the corresponding

Signal Bispecific
BiTE
perturbation Toxin
cytotoxicity

cytotoxic cells
Direct

Nonrestricted
activation of
Radionuclide

Drug
TrioMab

Tumor cell
CMC death
MAC

inhibitory signaling
Fc-mediated immune
effector engagement

Blockade of
ADCC

Phagocytosis

ADCP

APC Helper T cell Cytotoxic T cell

IC uptake MHC class II presentation MHC class I cross-presentation
Introduction of adaptive immune responses

Antigen BiTE MHC TCR T cell

class I Phagocytic
APC
Monoclonal Immunotoxin MHC Fc receptor Tumor cell
antibody class II
Perforin and
Bispecific Compliment Innate granzymes
CD3 KIR
antibody (C1q) effector

Figure 1.13  •  Mechanisms for antibody-based therapies used against cancer cells. Multiple current approaches involve direct cytotoxicity, Fc-mediated
immune effector engagement, nonrestricted activation of cytotoxic T cells, and blockade of inhibitory signaling. The diverse spectrum of action of these therapies
will allow the inclusion of various anticancer targets in the near future (From Weiner LM, Murray JC, Shuptrine CW. Antibody-based immunotherapy of
cancer. Cell. 2012;148:1081–1084.)
 Molecular Tools in Cancer Research  •  CHAPTER 1 15

mouse immunoglobulin genes has enabled the generation of “human- being developed that have increased specificity toward individualized
ized” therapeutic mAbs with reduced immunogenicity. In addition, cancers.
bispecific antibodies (bsAbs) with dual affinity for tumor antigens, From an epigenetic perspective, new techniques are enabling the
such as TriomAb, have been shown to effectively kill tumor cells by genome-wide characterization of protein-DNA interactions that can
inducing memory T-cell protective immunity. In addition to the uncover novel transcription factor targets, histone modifications,
expected use of mAbs directed at extracellular epitopes (protein regions and DNA methylation patterns within a cancer cell. Combining
recognized by the antibody), evidence from mouse models has raised chromatin immunoprecipitation (ChIP) with microarray (ChIP-chip)
the possibility of using antibodies targeting intracellular epitopes for allows genome-wide screening for the binding position of protein
anticancer therapies. Targeting such antigens would enrich immuno- factors to their gene targets. In ChIP-chip assays or ChIP-seq, a
therapy, allowing the use of tumor-specific intracellular mediators of cross-linking reagent is applied in vivo to proteins associated with
cell survival and proliferation. Numerous mAb-based agents are currently DNA in the nucleus, which then can be coimmunoprecipitated
in trial or in use as therapeutics for cancer, and the potential for with specific antibodies to the protein under analysis. The bound
further optimization of mAbs through genetic engineering promises DNA and appropriate controls are then fluorescently labeled and
to open new avenues for in vivo therapy. applied to microscopic slides for microarray analysis, or directly
A recent advancement in mAb-based cancer therapy is the generation sequenced, rendering a simultaneous profile of all the binding posi-
of chimeric antigen receptor (CAR) T lymphocytes to target tumors tions of specific proteins in the cancer cell’s genome (Fig. 1.14). The
in vivo. These are effector T-lymphocytes engineered to express a mAb global profiling of promoter occupancy of specific cancers, wherein
that recognizes specific groups of cancer cells. The receptors are chimeric, protein-DNA interaction profiles discriminate patients with tumors
composed of engineered molecules from diverse sources. The first from those presenting different clinical outcomes, is a promising
generation of CAR-modified T cells (CAR T cells) showed success in predictive method.
preclinical trials and have entered phase I clinical trials in ovarian After a decade of development, proteomics is still primarily a
cancer, neuroblastoma, and various types of leukemia and lymphoma. basic research activity, yet in the near future this technology is likely
Newer generations of these therapeutic lymphocytes are currently to have a profound impact on medicine. By defining the collective

Analysis and visualization Peak calling Alignment

Chr1:13456-13486
Chr1:24323-24293
2
Chr1:45678-45708 Sequencing
Chr1:54321-54351
Information content

1.5
Chr1:55679-55709
1
etc...
0.5
ChlP-Seq 0
1 2 3 4 5 6 7 8 9 10
Position

Library
construction

Antibody
Binding sites

TF TF

105
4
P < 0.001
log2 enrichment

20Kb

104
Cy3 intensity

0
–1
1000
ChlP-on-chip 200bp
Binding sites

ChlP replicate 1
100
Peak

ChlP replicate 2
Peak
10
10 100 1000 104 105
Cy5 intensity

Binding site identification Array data analysis Genomic arrays

Figure 1.14  •  Methods for unbiased identification of transcription factor binding sites. Chromatin immunoprecipitation on sequencing (ChIP-seq) and
chromatin immunoprecipitation on microarray chip (ChIP-chip) can provide location, isolation, and identification of the DNA sequences occupied by specific
DNA-binding proteins in cells. Proteins capable of DNA interactions are targeted with specific antibodies. DNA and the associated proteins are cross-linked;
DNA is fragmented into 150 to 500 bp and immunoprecipitated. After reversion of the cross-link, DNA is isolated and either mass-sequenced (ChIP-seq)
or used as probes in a genomic array (ChIP-chip), and binding sites occupied by the proteins can be identified in the genome. These binding sites may indicate
functions of various transcriptional regulators and help identify their target genes during development and disease progression. The types of functional elements
identified with these techniques include promoters, enhancers, repressor and silencing elements, insulators, boundary elements, and sequences that control
DNA replication. (From Kim TH, Ren B. Annu Rev Genomics Hum Genet. 2006;7:81–102 and Liu et al. BMC Biol. 2010;8:56.)
16 Part I: Science and Clinical Oncology

Human Drosophila Yeast C. elegans Arabidopsis

Transcriptional Virus-host Metabolic Protein-protein Disease

regulatory network network network interaction network
Alzheimer’s
disease

Hypertension
Pseudohypo-
Atherosclerosis aldosteronism
Asthma

Figure 1.15  •  Interactome networks and human disease. Networks are integrated sources of information obtained from biochemical, molecular, proteomic,
and other high-throughput analyses. Different networks can be obtained for each organism, organ, or cell. In the first instance, central regulatory “nodes”
identify important components in the network. These networks and their data can then be integrated and compared with healthy and disease models, allowing
an integrative view of events that is much more powerful than isolated networks. (Modified from Vidal M, Cusick ME, Barabási A. Interactome networks
and human disease. Cell. 2011;144:986–998.)

protein-protein interactions in a cancer cell (its “interactome”),
functional relationships between disease-promoting genes may be
revealed that provide novel candidates for intervention (Fig. 1.15).
Networks of disorder-gene associations are already being built that
offer a platform for describing all known phenotype and disease-gene
associations, often indicating the common genetic origin of many
diseases. A precise diagnosis of cancer through use of proteomics
can be envisioned, based on highly discriminating patterns of
proteins in easily accessible patient samples. Proteomics informa-
tion also promises to provide sophisticated mathematical models of
the molecular events underlying a process as complex as neoplastic
transformation, which will capture the dynamics of the disease with
unprecedented power.
MODELING CANCER IN VIVO
Once the mechanistic underpinnings of a particular cancer have been
described, creating an animal model to test that mechanism becomes
critical to understanding the pathophysiology and to design therapeutic
strategies for treatment. Advances in manipulation of the mouse genome
have resulted in more sophisticated models of human cancer. These
methodologies can circumvent embryonic death by targeted alteration
of gene expression only after a critical period in development, and
reduce the complexity of gene functional analysis by restricting its
pattern of activation. Inducible gene expression or silencing also allows
acute, as opposed to chronic, effects to be assessed. Although species
differences in tumor susceptibility and disease remission exist between
mouse and man, the tools for genetic manipulation in mouse are
superior to those in other mammals, and useful information about
the function of oncogenes can be gained by targeted expression of
mutant protein products in mouse tissues.
A major hurdle in generation of clinically relevant mouse models
to develop cancer treatments stems from the lack of patient tailoring.
Cancer cells present a highly heterogeneous population that varies
with the genetic makeup of the individual patient. This shortcoming
has been addressed with the advent of patient-specific avatars, also
known as personalized mouse models or patient-derived xenograft
(PDX) models (Fig. 1.16). Implanting patient biopsy specimens into
immunodeficient mice allows growth of the tumor, generating in vivo
precision models without further in vitro manipulation of the tumor
tissue. These models show great promise for designing treatment and
drug tests that should best target the patient-specific tumor. Most
recently, PDX models have been further optimized with the use of
humanized host mice that are modified to contain human immune
systems.
TRANSGENIC MODELS OF CANCER
Integrating an oncogene that causes malignancy into the genome of
a mouse without altering the mouse’s own genes generates a transgenic,
cancer-prone mouse that transmits this trait to its offspring with a
dominant pattern of inheritance. The technology for producing
transgenic mice joins recombinant DNA methodology with standard
techniques that are used today by in vitro fertilization clinics, relying
on the understanding of mammalian reproduction and the development
of protocols to harvest, manipulate, and reimplant eggs and early
embryos (Fig. 1.17). The transgene is constructed so that the gene
product will be expressed under appropriate spatial and temporal
control. In addition to all the standard signals necessary for efficient
transcription and translation of the gene, transgenes contain a promoter,
or regulatory region, that drives transcription in either a ubiquitous
or a tissue-restricted pattern. This requires an extensive knowledge of
genetic regulation in the target cells. A recent advance that circumvents
this requirement involves embedding the transgene inside another
gene locus that is expressed in the desired pattern. Held in a bacterial
artificial chromosome (BAC) for easier manipulation, this long stretch
of DNA surrounding the host gene is likely to carry all the necessary
regulatory information to guarantee a predictable expression pattern
of the introduced transgene.
The transgene DNA is then injected into the male pronucleus of
a fertilized mouse egg, obtained from a female mouse in which
hyperovulation has been hormonally induced. The injected eggs are
cultured to the two-cell stage and then implanted in the oviduct of
another recipient female mouse. Transgenic pups are identified by the
presence of the transgene in their genomic DNA (obtained from the
tip of the tail and analyzed with PCR assay). Typically, several copies
of the transgene are incorporated in a head-to-tail orientation into a
single random site in the mouse genome. About 30% percent of the
resulting pups will have integrated the transgene into their germline
DNA and constitute the founders of the transgenic lines. RNA analysis
of their progeny determines the level of transgene expression, and
whether the transgene is being expressed in the desired location or at
the appropriate time. Given the variability in transgene number and
chromosomal location, transgene expression patterns and levels can
 Molecular Tools in Cancer Research  •  CHAPTER 1 17

A B
Figure 1.16  •  Mouse avatar (PDX) models. (A) Patient-derived xenograft (PDX) mice are generated by implanting patient tumors into immunodeficient/
humanized mice. The tumors can then be propagated for several passages in fresh mice for a number of generations. (B) Usually, after the third generation
the tumors can be isolated and characterized for further study. These mice can potentially be used for patient drug-specific testing and molecular characterization,
therefore allowing for personalization of cancer treatment. (From http://www.the-scientist.com/?articles.view/articleNo/42470/title/My-Mighty-Mouse/.)

diverge considerably among different founder lines carrying the same
transgene.
In general, transgenesis is optimal for modeling oncogenic mutations
that cause a gain of function, producing disease even when they occur
in only one of a gene’s two alleles. For example, an activating mutation
in a growth factor that causes abnormal cell proliferation can be
mimicked by introducing a transgenic version of the mutated growth
factor gene under the control of an appropriate regulatory sequence
for expression in the tissue of interest. The relative susceptibility of
such a transgenic mouse to tumorigenesis can help distinguish between
a primary and secondary role of the mutant factor, and established
lines of these animals can be used for testing new therapeutic
protocols.
CONDITIONAL CONTROL OF
ONCOGENE ACTIVATION
The genetic construction of cancer-prone transgenic mice with the
capacity to induce oncogene expression in vivo provides a new avenue
to modeling the role of oncogenes in tumor generation and mainte-
nance. This technology relies on conditional mutagenesis. Producing
conditional mutations in mice requires a DNA recombinase enzyme
that does not recognize any mouse sequence, but rather targets short,
foreign recognition sequences to catalyze recombination between them.
By strategic placement of these recognition sequences in appropriate
orientations either beside or within a mouse gene, the recombination
results in deletion, insertion, inversion, or translocation of associated
genomic DNA (Fig. 1.18). Two recombinase systems are currently in
use: the Cre-loxP system from bacteriophage P1, and the Flp-FRT
system from yeast. The 34 bp loxP or FRT recognition sequences do
not occur in the mouse genome, and both Cre and Flp recombinases
function autonomously, without the need for cofactors. Cre- or Flp-
mediated recombination is not distance or cell-type dependent, and
can occur in proliferating or differentiated tissues.
The general scheme involves two mouse lines, one carrying the
recombinase either as a transgene driven by inducible regulatory
elements or knocked into one allele of a gene expressed in the desired
tissue. The other mouse line harbors a modified gene target including
recognition sequences. Mating the two lines results in progeny carrying
both the target gene and the recombinase, which interacts with the
target gene only in the desired tissue.
A popular conditional methodology is based on the activation of
nuclear hormone receptors to control gene expression. Two current
systems involve activation of a mammalian estrogen receptor, estrogen
analogue 4-hydroxy-tamoxifen, or an insect hormone receptor with
the corresponding ligand ecdysone. Although several variations on
these hormone-receptor systems are currently in use, the underlying
principle is the same. The Cre recombinase gene, or another regulatory
protein, such as a transcription factor, is fused with the ligand-binding
domain (LBD) from a nuclear hormone receptor protein. The resulting
chimeric transgene is placed under the control of a promoter that
directs expression to the tissue of interest, and transgenic animals are
generated. In the absence of the hormone or an analogue, the fusion
protein accumulates in the desired tissue but is rendered inactive
through its association with resident heat shock proteins. Hormone,
administered either systemically or topically, binds to the LBD moiety
of the fusion protein, dissociates it from the heat shock protein, and
allows the transcriptional regulatory component to find its natural
DNA targets and promote lox-P mediated recombination, or in the
case of an inducible transcription factor, activate expression of the
corresponding genes. If the LBD is fused to a recombinase, administra-
tion of hormone leads to the rearrangement of target sequences. This
reaction is not reversible, but lends additional temporal control over
recombinase-based mutation. If the LBD is fused to a transcription
factor, removal of hormone leads to inactivation of the fusion protein
and gene downregulation.
Another inducible method in use is the tetracycline (tet) regula-
tory system. In the classic design (tTA or tet-off ), a fusion protein
18 Part I: Science and Clinical Oncology

Figure 1.17  •  Generation of transgenic mice. The transgene containing

3' Flanking the DNA sequences necessary for the expression of a functional protein is
Promoter 5' UT Coding region 2' UT
region injected into the male (larger) pronucleus of uncleaved fertilized eggs through
Transgene
a micropipette. The early embryos are then transferred into the reproductive
tract of a mouse rendered “pseudopregnant” by hormonal therapy. The resulting
pups (founders) are tested for incorporation of the transgene by assaying
genomic DNA from their tails. Founder animals that have incorporated the
transgene (+) are mated with nontransgenic mice, and their offspring are mated
with each other to confirm germline integration and to establish a line of
homozygous transgenic mice. Several transgenic lines that have incorporated
Collection of fertilized eggs from different numbers of transgenes at different integration sites (and thus express
a superovulated donor mouse various amounts of the protein of interest) are usually studied. UT, Untranslated.
(From Schuldiner AR. Transgenic animals. N Engl J Med. 1996;334:653–655.)

Cell type specific

promoter Cre loxP Target gene loxP

X
Injection
of transgene into
male pronucleus of
uncleaved fertilized egg

Transfer of early embryos into reproductive

tract of a pseudopregnant mouse Cre Cre

A Special cell type All other cells

CMV-β actin
– promoter
– βgeo 3PA EGFP

+ loxP loxP
+ Cre
–
CMV-β actin
promoter
+ EGFP
Assay of genomic DNA from tails of founder
animals for incorporation of the transgene B loxP

Figure 1.18  •  Conditional mutagenesis schemes. (A) Two mouse lines are
required for conditional gene deletion: first, a conventional transgenic mouse
line with Cre targeted to a specific tissue or cell type; and second, a mouse
strain that embodies a target gene (endogenous gene or transgene) flanked by
two loxP sites in a direct orientation (“floxed gene”). Recombination (excision
and consequently inactivation of the target gene) occurs only in those cells
expressing Cre recombinase. Hence, the target gene remains active in all cells
and tissues that do not express the Cre recombinase. (B) The Z/EG double
reporter system. These transgenic mice constitutively express lacZ under the
Sequential matings to determine control of the cytomegalovirus enhancer/chicken actin promoter. Expression
germline integration is widespread, with notable exceptions being liver and lung tissue. Expression
is observed throughout all embryonic and adult stages. When crossed with a
Cre recombinase-expressing strain, lacZ expression is replaced with enhanced
Study of phenotype green fluorescent protein expression in tissues expressing Cre. This double
reporter system makes it possible to distinguish a lack of reporter expression
from a lack of Cre recombinase expression while providing a means to assess
Cre excision activity in live animals and cells. (A Courtesy Kay-Uwe Wagner,
National Institutes of Health; B from Novak A, Guo C, Yang W, Nagy A,
Lobe CG. Z/EG, a double reporter mouse line that expresses enhanced green
fluorescent protein upon Cre-mediated excision. Genesis. 2000;28:147–155.)

combining a bacterial tet repressor and a viral transactivation domain
drives expression of the target transgene by binding to upstream tet
operator sequences flanking the transgene transcription start site. In the
presence of the antibiotic inducer, the fusion protein is dissociated from
the operator sequences, inactivating the transgene. In a complementary
design, called reverse tetracycline-controlled transactivator (rtTA or
tet-on), structural modification of the tet repressor makes the antibiotic
an active requirement for binding of the fusion protein to the operator
sequences, such that its administration activates transgene expression at
any time during the life span of the mouse, whereas withdrawal results in
downregulation of the gene. It is important that the transgene integrate
into a genomic locus that permits proper tTA or rtTA regulation so that
the system exhibits minimal “intrinsic leakiness” and good antibiotic
responsiveness.
Conditional expression systems have already been developed to
generate hematopoietic, leukemogenic, and lymphomagenic mutations
in the mouse, as well as solid tumors. These inducible cancer models
can be exploited to identify oncogenic signals that influence host-tumor
interactions, to establish the role of a given oncogenic lesion in advanced
tumors, and to evaluate therapies targeted toward cancer-causing
mutations. Potential clinical application of inducible systems include
targeting virally delivered transgene expression to malignant tissues
by the use of specific inducible regulatory elements, restricting the
expression of transgenes exclusively to affected tissues, and increasing
the therapeutic index of the vectors, particularly in the context of
solid tumors. In all cases, a basic knowledge of the specific mutations
involved in the molecular genetics of malignancies is required because
it is often unclear that the causal mutation underlying the genesis of
neoplasia continues to play a central role in the progression to the
fully transformed state. This is particularly important in modeling
cancers characterized by genetic plasticity, wherein drug resistance can
arise subsequent to primary tumor formation.
MODELS OF RECESSIVE GENE MUTATIONS
IN CANCER
In contrast to dominantly acting oncogenes, recessive genetic disorders,
such as loss-of-function mutations in tumor suppressor genes, require
both copies (alleles) of a gene to be inactivated. The methods needed
to produce animal models of recessive genetic disease differ from those
used in studying dominant traits. Gene knockout technology has been
developed to generate mice wherein one allele of an endogenous gene
is removed or altered in a heritable pattern (Fig. 1.19). Gene disruption
or replacement is first engineered in pluripotential cells, termed
embryonic stem cells (ESCs), which are genetically altered by introduc-
tion of a replacement gene that is inactive or mutant.
To reduce random integration of the foreign DNA, the replacement
gene is embedded into a long stretch DNA from its native locus in
the mouse, which targets the recombination event to the homologous
position in the ESC genome. Inclusion of selectable markers along
with the replacement gene allows selection of the cells in which
homologous recombination has taken place. Site-specific recombinase
systems combined with gene targeting techniques in ESCs can also
be used to induce recessive single point mutations or site-specific
chromosomal rearrangements in a tissue- and time-restricted pattern.
In a variation on this theme called knockin, a foreign gene, such as
one encoding a marker or a mutated gene, can be placed in the locus
of an endogenous gene. The engineered ESCs are then microinjected
into the cavity of an intact mouse blastocyst sufficiently early in gestation
that they can, in principle, populate all the tissues of the developing
chimeric embryo. This is rarely the case, so contribution of ESCs to
the resulting animal is most often assessed with use of ESCs and
blastocysts whose genes for coat color differ.
If the ESCs contribute to the germ cells of the founder mouse,
their entire haploid genome can be passed on to subsequent generations.
Through mating together of subsequent progeny of the founder mouse,
both alleles of the mutated gene can be passed to a single animal.
Molecular Tools in Cancer Research  •  CHAPTER 1 19
Overlapping genetic functions can also be defined by crossbreeding
mice with mutations in different genes. In this way it is possible to
study the combinatorial effects of oncogene and tumor suppressor
gene mutations.
Several caveats are important in considering the use of knockout
technology in modeling cancer. Most knockouts generate loss-of-
function (null) germline mutations. Inactivation of widely expressed
genes with multiple functions may have complex phenotypes. Con-
versely, if the functions of two genes overlap, a mutation in one of
the genes may not produce an abnormal phenotype, owing to compensa-
tion by the unaltered partner.
Perhaps the greatest drawback of conventional knockout technology
derives from the disruption of gene function at the earliest stage
of its expression. If the gene has a vital developmental role, the
identification of functions later in development can be occluded.
Therefore, although the generation of a null mutation is an excel-
lent starting point for analysis, it is far from being functionally
exhaustive. For these reasons, conditional mutagenesis is the emerg-
ing method of choice for the elucidation of the gene functions
that exert pleiotropic effects in a variety of cell types and tissues
throughout the life of the animal, which is particularly relevant
for the generation of mouse models of adult-onset diseases such
as cancer.
Use of recombinase-mediated gene mutation as described earlier
for conditional transgenesis, conditional knockout mutations can be
designed to disrupt the function of a target gene in a specific tissue
(spatial control) and/or life stage (temporal control). Depending on
the design of the experiment, recombinase action can delete an entire
gene, remove blocking sequences to induce gene expression, or rearrange
chromosomal segments. With the advent of recent internationally
coordinated systematic mutagenesis programs aiming to place a
conditional inactivating mutation in each of the 20,000 genes in the
mouse genome, the possibilities for modeling cancer are limited only
by a researcher’s choice of the gene loci to test. The constantly evolving
techniques for gene manipulation in vivo constitute a major advance
in cancer research.
Genetically modified mice are of great value in dissecting the
pathogenesis of many tumor types. In some knockout studies, the
phenotype of the mutated gene is anticipated by prior knowledge
of the gene’s function. However, unexpected mutant phenotypes
may help clarify the mechanism of the underlying neoplasia.
Pharmacologic manipulation of transgenic, knockout, diversified
animal models of cancer will prove useful in screening therapeutic
agents with potential for study in clinical trials. Therapy involving
gene or cell replacement can be also tested in genetically engineered
disease models.
EXPLOITING MOUSE DIVERSITY FOR
CANCER RESEARCH
A novel in vivo tool has emerged that aids in understanding the etiology
of cancers, by more accurately reflecting the broad genetic variability
in the human population. Cancer research performed with mice has
largely focused on a few individual highly inbred strains with limited
genetic diversity, which would equate to single individuals in the
population. Yet drugs designed to treat one individual are often not
effective in other patients. The Collaborative Cross (CC) was created
to provide mouse models that better represent the diversity seen in
natural human populations while still retaining the broad power of
genetic analysis seen in mice. The CC resource is a large panel of
recombinant inbred (RI) strains generated by randomly mixing the
genetic diversity of eight extant inbred mouse lines, and can be used
to test the impact of treatments in a diverse genetic pool akin to the
human population (Fig. 1.20). A related resource, the Diversity Outcross
(DO), offers higher mapping resolution by randomized outcrossing
of partially inbred CC strains, which segregates the same allelic variants
but embeds them in a distinct population architecture in which each
20 Part I: Science and Clinical Oncology

Embryonic stem cell

Tumor suppressor gene

5' Homologous 3' Homologous

region region
Intron
Cellular
gene

Embryonic stem
Plasmid
cell culture pgk-neo pgk-tk DNA
Knockout
vector
Homologous
recombination
Cellular gene
replaced

Injection of embryonic stem Implantation of chimeric

cells into host blastocyst blastocyst in foster mother
Selection by neomycin and ganciclovir

Germline offspring Chimeric offspring

Figure 1.19  •  Gene knockout strategies. Embryonic stem cells (upper left panel) contain the tumor suppressor cellular gene (upper right panel), which
consists of exon 1 (olive green, a 5′ noncoding region), an intron, and exon 2 (red, a protein coding region, and yellow, a 3′ noncoding region). A knockout
vector—consisting of a collinear assembly of a DNA flanking segment 5′ to the cellular gene (blue), the phosphoglycerate kinase–bacterial neomycin gene
(pgk-neo, violet), a 3′ segment of the cellular gene (yellow), a DNA flanking segment 3′ to the cellular gene (green), and the phosphoglycerate kinase–viral
thymidine kinase gene (pgk-tk, orange)—is created and introduced into the embryonic–stem cell culture. Double recombination occurs between the cellular gene
and the knockout vector in the 5′ homologous regions and the 3′ homologous regions (dashed lines), resulting in the incorporation of the inactive knockout
vector, including pgk-neo but not pgk-tk, into the cellular genomic locus of the embryonic stem cell. The presence of pgk-neo and the absence of pgk-tk in
these replaced genes will allow survival of these embryonic stem cells after positive-negative selection with neomycin and ganciclovir. The clone of mutant
embryonic stem cells is injected into a host blastocyst, which is implanted into a pseudopregnant foster mother and subsequently develops into a chimeric
offspring (bottom). The contribution of the embryonic stem cells to the germ cells of the chimeric mouse results in germline transmission of the embryonic
stem cell genome to offspring that are heterozygous for the mutated tumor suppressor allele. The heterozygotes are mated to produce mutant, cancer-prone
mice homozygous for tumor suppressor deficiency. (Modified from Mazjoub JA, Muglia LJ. Knockout mice. N Engl J Med. 1996;334:904–906.)

Founder strains Diversity Outbred (DO)
A/J
C57BL/6J
Outbreeding
129S1/SvImJ
NOD/ShiLtJ
NZO/H1LtJ Collaborative Cross (CC)
Inbreeding
CAST/EiJ
PWK/PhJ
WSB/EiJ
A by trial and error find a balance between alleviating the neoplastic
B not only in terms of how quickly a patient is cancer free, but how
Figure 1.20  •  Generation and characteristics of Collaborative Cross (CC)
and Diversity Outbred (DO) mice. (A) Each CC line originates from intercrosses
obtained from eight founder lines. Individual unique independent line is
inbred for at least 15 generations, creating individual CC lines. Together, those
lines represent a much broader genetic variation when compared with the
parental lines and therefore are a better representation of natural populations
but contain a high degree of homozygosity. Random mating from early 144
CC crosses leads to a highly genetic heterogeneous outbred population that
more closely resembles the diversity found in human populations. (B) Images
of mice representing CC lines. (From Centre for Diabetes Research, Harry
Perkins Institute of Medical Research, Nedlands 6009, W.A Australia” and
“School of Medical and Health Sciences, Edith Cowan University, Joondalup
W.A. 6027 Australia.)
animal has a high degree of heterozygosity and carries a unique
combination of alleles.
These diversified mice have been shown to be a powerful tool in
determining the etiology of cancers. In a recent study, analysis of mice
generated by crossing CC strains with a mouse line carrying a mutated
tumor suppressor APCmin showed that colon cancer frequency and
spectrum varied predictably with genetic background. Identification
of these genetic modifiers of cancer genesis or suppression would
inform the design of novel mouse cancer models, potentially yielding
genetic markers that can predict human cancers.
FUTURE VIEW
Recent discoveries that cancer stem cells share essential signaling nodes
with normal stem cells suggest that targeting critical steps in these
pathways may lead to improved alternative cancer therapies. However,
every human tumor is subtly different. We are now fast approaching
a new era in medicine: creating “tailored” treatments to individual
tumors by obtaining integrative “personal omics profiles” (Fig. 1.21).
The generation of novel mouse strains that represent the diversity
seen in human populations will likely lead to a more tailored approach
in understanding cancer diversity and therefore will allow the develop-
ment of more efficient treatments.
Understanding of underlying molecular biologic principles of
malignancy, with pathophysiologic consequences, will generate an
invaluable resource for resolution of complex genetics of tumor
Molecular Tools in Cancer Research  •  CHAPTER 1 21
formation and holds great promise for improved treatment of
human cancer.
Chemotherapy still has numerous side effects. As a number of
oncologists may testify, patients sometimes forgo treatment because
of its toxic nature. In a number of cases of chronic lymphocytic
leukemia, if the symptoms of the disease are “under control,” treatment
is not prescribed. Therefore it is important to find alleviation strategies
and cures that minimize the side effects. The goal should be to cure
the patient without devastating the person. One future avenue, apart
from finding treatment regimens that reduce side effects, is the discovery
of chemotherapies with minimal side effects. The nature of several
generations of chemotherapy was to attack cellular processes, such as
DNA replication, metabolism, and cell division, with brute force, and
growth and not interfering with the normal processes. It may be time
to rethink that premise. We now see examples wherein a less potent
chemotherapy elicits a similar effect on a cancer as a potent one,
although the less potent compound may take longer to achieve its
effect. However, because of its low potency, the side effects are minimal.
Therefore, in the future, the success of a therapy needs to be measured
well the patient is during and after the treatment. With emerging
technologies we will be able to fine-tune current successful therapies
so that treatment is not so burdensome.
In any field of medicine, resistance to the therapy occurs. Evolution-
ary processes show that predation leads to natural selection of species
with mutations that avoid their elimination. Similarly, in cancers,
during the predation—chemotherapy—clones arise that become
resistant to the therapy. With emerging technologies such as single-cell
deep sequencing, we will be able to not only detect the rare subclone
that could give rise to resistance, but also predict the probability of
the development of resistance by sequencing certain markers. For
instance, clones with mutations in DNA repair pathways have a higher
probability for genomic instability, which is a precursor for the rise
of resistant clones.
While we pursue new technologies to diagnose patients and
understand the molecular nature of the cancers, an emerging trend
will be to reexamine previous formulated hypotheses or treatments
that could not be tackled before because of the lack of technologies
or resources. For instance, it has been long thought that genetic variation
plays an important role in cancer treatment. This premise was observed
in human clinical trials, wherein conditions in some populations were
refractory to certain treatments. In the future, we would be able to
predict the extent to which a treatment would either work or fail in
certain population by using diverse mice, and to map alleles that
confer resistance or susceptibility of a tumor before reaching human
clinical trials. Another example is processes that lead to neoplasticity,
such as LOH, which have been studied in cell lines that are homo-
geneous in nature. Attempts to use primary cells were not possible
because of polyclonality of cells and the short life span of tumor cells
in vitro.
With all the aforementioned technologies, we now can, and
need to, reexamine older hypotheses with the appropriate type of
primary cells. We will probably uncover novel processes of cancer
that were masked, and resolve decades-old controversies and
competing hypotheses, leading to better understanding and cures
for cancer.
In the future, fields of medicine will continue to marry and
merge; this has been exemplified in numerous examples, some
mentioned in this chapter—for instance, the use of viruses to deliver
chemotherapy, or the use of engineered T cells to attack cancer. As
emerging molecular tools uncover novel processes in different fields
of medicine, the cross talk between oncology and these other fields
will continue, enabling discovery of new avenues to alleviate or even
cure cancers.
22 Part I: Science and Clinical Oncology

SAMPLE TYPE METHOD ANALYSES

Whole-genome
Variant calling/phasing
sequencing
Heteroallelic and variant
expression
Whole-transcriptome
PBMC sequencing RNA editing
(mRNA and miRNA)
Quantitative differential

Integrated personal OMICS

expression & dynamics

Variant confirmation in
Proteome profiling
RNA and protein

Untargeted proteome Quantitative differential

profiling expression and dynamics

Targeted proteome
Quantitative expression
profiling (cytokines)

Serum Metabolome profiling Dynamics

AutoAntibodyome Differential reactivity

profiling

Medical/lab tests Glucose, HbA1c, CRP,

Telomere length
A

3 4

5
2
Serpina 1
E366K
RNA edits
6

Heteroallelic SNVs
1

Protein-downregulated
7

(HRV vs healthy)
Protein-upregulated
Y

(HRV vs healthy)
RNA-downregulated
8

(HRV vs healthy)
X

RNA-upregulated
(HRV vs healthy)
Indels
9
22

SV-duplications
21

SV-deletions
20

10

Chr. ideogram
19

18
11
17 14 Chr. number
16 12
B 15
14 13

Figure 1.21  •  Integrative personal omics profiles (iPOP). (A) Integrative analysis of iPOP experimental design, indicating tissues and techniques analyzed
in healthy and diseased individuals. (B) Circos plot summarizing iPOP. From outer to inner rings: chromosome ideogram; genomic data (pale blue ring),
structural variants (deletions [blue tiles], duplications [red tiles]), indels (green triangles); transcriptomic data (yellow ring); proteomic data (light purple ring).
(Modified from Chen R, Mias GI, et al. Personal omics profiling reveals dynamic molecular and medical phenotypes. Cell. 2012;148:1293–1307.)

SUGGESTED READINGS
Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter
P. Molecular Biology of the Cell. 4th ed. London, UK:
Taylor and Francis Group; 2002.
Chen CZ. MicroRNAs as oncogenes and tumor suppres-
sors. New Eng. J. Med. 2005;353:1768–1771.
Feinberg AP. Phenotypic plasticity and the epigenetics of
human disease. Nature. 2007;447:433–440.
Frese KK, Tuveson DA. Maximizing mouse cancer models.
Nat Rev Cancer. 2007;7:654–658.
Goh KI, Cusick ME, Valle D, Childs B, Vidal M, Barabasi
AL. The human disease network. Proc Natl Acad Sci
USA. 2007;104:8685–8690.
Molecular Tools in Cancer Research  •  CHAPTER 1 23
Hunter K. Host genetics influence tumour metastasis.
Nat Rev Cancer. 2006;6:141–146.
Malaney P, Nicosia SV, Davé V. One mouse, one patient
paradigm: new avatars of personalized cancer therapy.
Cancer Lett. 2014;344:1–12.
Pecorino L. Molecular Biology of Cancer: Mechanisms,
Targets, and Therapeutics. USA: Oxford University
Press; 2005.
Svenson KL, Gatti DM, Valdar W, Welsh CE, Cheng R,
Chelser EJ, et al. High resolution genetic mapping
using the mouse diversity outbred population. Genetics.
2012;190:437–447.
The Complex Trait Consortium. The Collaborative
Cross, a community resource for the genetic analysis
of complex traits. Nat Genet. 2004;36:1133–1137.
Weinberg RA. Biology of Cancer. Garland Science; 2006.
Rosenthal N, Brown S. The mouse ascending: per-
spectives for human-disease models. Nat Cell Biol.
2007;9:993–999.
Wu J, Smith LT, Plass C, Huang TH. ChIP-chip comes
of age for genome-wide functional analysis. Cancer
Res. 2006;66:6899–6902.
 2  Intracellular Signaling
Aphrothiti J. Hanrahan, Gopa Iyer, and David B. Solit

S UMMARY OF K EY
• Ligand binding and activation of
cell surface and internal receptors
trigger the activation and/or
suppression of signaling cascades
that regulate diverse cellular
processes including cell growth,
proliferation, survival, and invasion,
among others.
• Multiple nodes within these
intracellular signaling networks are
genetically and epigenetically altered
in human cancers, leading to
constitutive pathway activation or
suppression.
• Some cancers are dependent on
genomic alterations in oncogenes or
tumor suppressor genes for their
P OI N T S
growth and survival, a phenomenon
known as oncogene addiction.
• Drugs that selectively target mutated
proteins critical for the maintenance
of the transformed phenotype have
shown unprecedented clinical
activity in genetically defined cancer
subsets.
• Precision medicine refers to the use
of genetic and epigenetic information
unique to an individual cancer
patient to develop treatment
regimens that target the driver
oncogenes and tumor suppressors
responsible for tumor progression.
Potential challenges to the
application of this approach include
the current inability to directly inhibit
some oncogenic proteins (i.e.,
mutant KRAS), the development of
drug resistance, technical hurdles
posed by limited tissue availability
for prospective molecular
characterizing, and intratumoral and
lesion-to-lesion genomic
heterogeneity.
• Routine genomic analysis of tumors
or tumor-derived cell free DNA in
plasma is now a component of
standard care in an increasing
number of cancer types, with the
results used to guide treatment
selection.

The underlying basis of the cancer phenotype is deregulated cell
growth, which stems from two main hallmarks of cancer: uncon-
trolled proliferation, and loss of programmed cell death (enhanced
survival). In normal cells, these processes are tightly controlled
through integration of signaling cascades that translate extracellular
and intracellular cues into specific output responses. These signaling
pathways are often initiated on binding of ligand to the extracellular
domain of a receptor, followed by recruitment of adaptor proteins
or kinases that activate an intracellular cascading network of protein
and lipid intermediaries that ultimately produce a cellular response.
In normal cells, the specificity, amplitude, and duration of signaling
are tightly regulated, and these constraints are often abrogated in
human cancers.
Investigation of the signal transduction pathways that regulate normal
cellular functions has revealed that key components of these networks
are commonly altered in cancer cells by mutation, amplification and
deletion, chromosomal translocation, overexpression, or epigenetic
silencing. These alterations lead to activation or suppression of
signaling cascades that underlie the various hallmarks of the cancer
phenotype. This chapter reviews the major signal transduction cascades,
with a focus on those that are frequently altered in human cancers.
Individual sections highlight signaling intermediaries that have been
validated as drug targets in patients with cancer. Table 2.1 summarizes
actionable gene-level and mutation-level alterations in cancer and
the drugs that are currently approved by the US Food and Drug
Administration (FDA) for treatment, that are recommended standard
of care biomarkers, or that have promising clinical or preclinical
efficacy.1
RECEPTOR TYROSINE KINASE SIGNALING
The receptor tyrosine kinases (RTKs) comprise a family of
transmembrane (TM) cell surface receptors that transduce
extracellular signals internally to promote growth and survival and/
or to regulate other cellular phenotypes.2,3 Members of this protein
family share a similar modular domain structure. Growth factors
bind to the extracellular ligand-binding domain of RTKs and
induce dimerization of two receptor monomers, juxtaposing the
intracellular tyrosine kinase domains of each monomer.4 This results
in transphosphorylation of tyrosine residues within the cytoplasmic
domains of the RTK dimer. Following transphosphorylation, a variety
of intracellular proteins are recruited to the activated RTK through
Src homology 2 (SH2) domains that recognize the phosphotyrosine
plus a specific amino acid sequence motif C-terminal to the tyrosine
residues.5,6
Over 117 SH2 domains have been characterized, each with unique
phosphotyrosine sequence specificities.7 Each domain is part of a larger
adaptor protein involved in transducing extracellular signals to activate,
or in some cases suppress, specific intracellular signaling cascades. Thus
the complement of signaling pathways that a given RTK regulates is
dictated by the profile of phosphorylated tyrosine residues plus flanking
amino acids within their intracellular domains.8,9 However, more than
one adaptor protein can often recognize individual context-dependent
phosphotyrosine motifs within an RTK, underscoring how this system
is designed to provide both specificity and diversity of intracellular
signaling.
Text continued on p. 29

24
 Intracellular Signaling  •  CHAPTER 2 25

Table 2.1  Targeted Therapy for Disease-Specific Alterations of Actionable Oncogenes in Cancer
Gene Variant Cancer Type Drug Evidencea
ABL1 BCR-ABL1 fusion ALL Dasatinib 1
Imatinib 1
CML Dasatinib 1
Imatinib 1
Nilotinib 1
AKT1 E17K Breast AZD5363 3
Ovarian AZD5363 3
All Tumors ARQ 751 4
ALK Fusions NSCLC Alectinib 1
Ceritinib 1
Crizotinib 1
Oncogenic mutations NSCLC Brigatinib 1
Fusions Soft tissue sarcoma Ceritinib 2
Crizotinib 2
L1196M, L1196Q NSCLC Brigatinib 3
R1275Q Embryonal tumor Crizotinib 4
ARAF S214A Histiocytosis Sorafenib 3
S214C NSCLC Sorafenib 3
ATM N2875K, R3008C, truncating mutations Prostate cancer Olaparib 4
BRAF V600D, V600E, V600G, V600K, V600M, V600R Melanoma Cobimetinib + vemurafenib 1
Dabrafenib 1
Dabrafenib + trametinib 1
Vemurafenib 1
Histiocytosis Vemurafenib 2
NSCLC Dabrafenib 2
Dabrafenib + trametinib 2
Vemurafenib 2
Colorectal Binimetinib + cetuximab + 3
encorafenib
Panitumumab + vemurafenib 3
Colorectal Fluorouracil + radiation + 4
trametinib
V600E, V600K Melanoma Trametinib 1
Fusions Ovarian Paclitaxel + selumetinib 3
K601E Melanoma Trametinib 3
L597Q, L597R, L597S, L597V Melanoma Trametinib 3
D594E, D594N, G466V, G469A, G469V, G596C Melanoma Trametinib 4
KIAA1549-BRAF Fusion Soft tissue sarcoma Sorafenib + temsirolimus 4
L597Q, L597V Melanoma BGB659 4
BRCA1 Oncogenic mutations Ovarian Niraparib 1
Rucaparib 1
Olaparib 2
BRCA2 Oncogenic mutations Ovarian Niraparib 1
Rucaparib 1
Olaparib 2
CDK4 Amplification Dedifferentiated liposarcoma Abemaciclib, palbociclib 2
Well-differentiated Abemaciclib, palbociclib 2
liposarcoma
CDKN2A Oncogenic mutations Breast Letrozole + palbociclib 4
Esophagogastric Palbociclib 4
EGFR 729_761del, 729_761indel, L858R NSCLC Afatinib 1
Erlotinib 1
Gefitinib 1
Osimertinib 4
E709_T710delinsD, E709K, G719A, G719C, G719D, G719S, NSCLC Afatinib, erlotinib, gefitinib 1
A763_Y764insFQEA, L747P, A750P, A763_Y764insFQEA,
L833V, L861Q, L861R, S768I, EGFR-KDD
T790M NSCLC Osimertinib 1
762_823ins NSCLC EGF816 4
762_823ins, G719A, L861R, S768I NSCLC AP32788 4

Continued
26 Part I: Science and Clinical Oncology

Table 2.1  Targeted Therapy for Disease-Specific Alterations of Actionable Oncogenes in Cancer—cont’d
Gene Variant Cancer Type Drug Evidencea
ERBB2 Amplification Breast Ado-trastuzumab emtansine 1
Lapatinib 1
Lapatinib + trastuzumab 1
Pertuzumab + trastuzumab 1
Trastuzumab 1
Esophagogastric Trastuzumab 1
Breast Neratinib 3
Oncogenic mutations Breast Neratinib 3
V659E NSCLC Lapatinib 3
E770_K831indel, E770_K831ins NSCLC AP32788 4
ERCC2 Oncogenic mutations Bladder Cisplatin 3
ESR1 Oncogenic mutations Breast AZD9496, fulvestrant 3
D538G, Y537S Breast GDC-0810 4
EZH2 Oncogenic mutations Diffuse large B-cell GSK126 4
lymphoma Tazemetostat 4
FGFR1 Amplification Lung squamous cell AZD4547 3
carcinoma Debio1347 3
BCR-FGFR1 fusion Leukemia Ponatinib 4
FGFR2 Fusions Adrenocortical carcinoma Debio1347 3
JNJ-42756493 3
Bladder Debio1347 3
JNJ-42756493 3
Cholangiocarcinoma BGJ398 3
Debio1347 3
Endometrial Debio1347 3
JNJ-42756493 3
FGFR3 Fusions Adrenocortical carcinoma Debio1347 3
JNJ-42756493 3
Bladder Debio1347 3
JNJ-42756493 3
Glioma Debio1347 3
JNJ-42756493 3
G370C, G380R, K650E, K650M, K650N, K650Q, K650R, K650T, Bladder Debio1347 3
R248C, S249C, S371C, Y373C JNJ-42756493 3
Breast Debio1347 4
FLT3 Y572_Y630ins AML Sorafenib 3
IDH1 R132C, R132G, R132H, R132Q, R132S AML AG-120 3
All tumors BAY1436032 4
CB-839 4
IDH2 R140Q, R172G, R172K, R172M, R172S All liquid tumors AG-221 3
JAK2 PCM1-JAK2 fusion Leukemia Ruxolitinib 3
KIT 449_514mut, 550_592mut, A502_Y503dup, D579del, D820G, GIST Sunitinib 1
E554_K558del, H697Y, K550_W557del, K558delinsNP, L576P, Thymic tumor Sunitinib 2
P551_M552del, V555_L576del, V560D, V560del, V654A
449_514mut, 550_592mut, D419del, D579del, E554_I571del, GIST Imatinib 1
E554_K558del, E554_V559del, F522C, I563_L576del, I653T,
K550_W557del, K558N, K558_E562del, K558_V559del,
K558delinsNP, K642E, L576P, M541L, M552_W557del,
N564_Y578del, N822H, N822Y, P573_D579del, P577_
W582delinsPYD, P838L, Q556_K558del, T417_D419delinsI,
T417_D419delinsRG, T574insTQLPYD, V530I, V555_L576del,
V555_V559del, V559C, V559D, V559G, V559_V560del,
V559del, V560D, V560G, V560del, V569_L576del, W557G,
W557R, W557_K558del, Y553N, Y553_K558del, Y570H, Y578C
449_514mut, 550_592mut, D820G, D820Y, K550_W557del, GIST Regorafenib 1
K558delinsNP, N822K, V560D
D816F, D816Y, D820G, D820Y, L576P, N822I, V559D, V560G, GIST Dasatinib 2
W557_K558del
D816V, D820A, D820G, D820Y, K642E, L576P, V555_L576del, GIST Nilotinib 2
V559C, V559D, V654A, W557_K558del
D820A, D820E, D820G, D820Y, K642E, N505I, P577_D579del, GIST Sorafenib 2
V559D, W557_K558del Thymic tumor Sorafenib 2
K642E, L576P, V559A Melanoma Imatinib 2
 Intracellular Signaling  •  CHAPTER 2 27

Table 2.1  Targeted Therapy for Disease-Specific Alterations of Actionable Oncogenes in Cancer—cont’d
Gene Variant Cancer Type Drug Evidencea
KRAS Wild type Colorectal Cetuximab 1
Panitumumab 1
Regorafenib 1
Cabozantinib + 4
panitumumab
Panitumumab + regorafenib 4
Pembrolizumab 4
Oncogenic mutations All tumors alpelisib + binimetinib 4
Cobimetinib + GDC-0994 4
Colorectal Atezolizumab + cobimetinib 4
Fluorouracil + radiation 4
therapy + trametinib
NSCLC Abemaciclib, PD0325901 + 4
palbociclib, palbociclib,
ribociclib, ribociclib +
trametinib
Binimetinib + erlotinib 4
Binimetinib, selumetinib, 4
trametinib
Docetaxel + trametinib 4
MAP2K1 Oncogenic mutations Histiocytic disorder Cobimetinib, selumetinib, 3
trametinib
Low-grade serous ovarian Cobimetinib, selumetinib, 3
trametinib
Melanoma Cobimetinib, selumetinib, 3
trametinib
NSCLC Cobimetinib, selumetinib, 3
trametinib
MDM2 Amplification Liposarcoma DS-3032b 3
RG7112 3
SAR405838 4
MET 963_D1010splice, 981_1028splice, X1006_splice, X1007_ NSCLC Crizotinib 2
splice, X1008_splice, X1009_splice, X1010_splice, X963_ Cabozantinib 3
splice Capmatinib 3
Amplification NSCLC Crizotinib 2
RCC Cabozantinib 2
D1010H, D1010N, D1010Y NSCLC Crizotinib 2
Cabozatinib 3
Capmatinib 3
MTOR E2014K Bladder Everolimus 3
C1483F, F1888L, L2230V, S2215F, T1977K RCC (clear cell) Everolimus, rapamycin, 4
temsirolimus
NF1 Oncogenic mutations Glioblastoma Trametinib 4
Melanoma Trametinib 4
Neurofibroma Binimetinib 4
Neurofibroma PLX3397 4
NRAS Oncogenic mutations Colorectal Atezolizumab + cobimetinib 3
Melanoma Binimetinib 3
Binimetinib + ribociclib 3
Thyroid Radioiodine uptake therapy 3
+ selumetinib
Colorectal Fluorouracil + radiation 4
therapy + trametinib
NTRK1 Fusions All tumors Larotrectinib 3
Salivary gland Entrectinib 3
NTRK2 Fusions Salivary gland Entrectinib 3
Larotrectinib 3
NTRK3 Fusions Salivary gland Entrectinib 3
Larotrectinib 3

Continued
28 Part I: Science and Clinical Oncology

Table 2.1  Targeted Therapy for Disease-Specific Alterations of Actionable Oncogenes in Cancer—cont’d
Gene Variant Cancer Type Drug Evidencea
PDGFRA FIP1L1-PDGFRA fusion Leukemia Imatinib 1
Fusions Myelodysplasia Imatinib 1
Myeloproliferative Neoplasm Imatinib 1
560_561insER, A633T, C450_K451insMIEWMI, C456_N468del, GIST Imatinib 2
C456_R481del, D568N, D842I, D842_H845del, D842_
M844del, D846Y, E311_K312del, G853D, H650Q, H845Y,
H845_N848delinsP, I843del, N659K, N659R, N659S, N848K,
P577S, Q579R, R748G, R841K, S566_E571delinsR, S584L,
V469A, V536E, V544_L545insAVLVLLVIVIISLI, V561A, V561D,
V561_I562insER, V658A, W559_R560del, Y375_K455del,
Y555C, Y849C, Y849S
D842V GIST Dasatinib 2
PDGFRB Fusions Dermatofibrosarcoma Imatinib 1
Protuberans
Myelodysplasia Imatinib 1
Myeloproliferative neoplasm Imatinib 1
PIK3CA Oncogenic mutations Breast Alpelisib 3
Alpelisib + fulvestrant 3
Buparlisib 3
Buparlisib + fulvestrant 3
Copanlisib 3
Fulvestrant + taselisib 3
GDC-0077 3
Serabelisib 3
Taselisib 3
Alpelisib + everolimus 4
Alpelisib + letrozole, Alpelisib 4
+ letrozole + ribociclib
Alpelisib + LJM716 + 4
trastuzumab
Alpelisib + olaparib, 4
buparlisib + olaparib
AZD5363 + fulvestrant 4
AZD8835 + fulvestrant 4
MLN0128 + serabelisib 4
All tumors ARQ 751 4
AZD5363 + olaparib 4
GDC-0077 4
Endometrial Alpelisib + fulvestrant 4
Buparlisib + fulvestrant 4
Fulvestrant + taselisib 4
Ovarian Alpelisib + fulvestrant 4
Buparlisib + fulvestrant 4
Fulvestrant + taselisib 4
PTCH1 Truncating mutations Embryonal tumor Sonidegib 3
Skin cancer, nonmelanoma Sonidegib 3
Vismodegib 3
PTEN Oncogenic mutations All tumors ARQ 751 4
AZD5363 + olaparib 4
AZD8186 4
Gedatolisib + palbociclib 4
GSK2636771 4
LY3023414 4
Endometrial Olaparib 4
Prostate Enzalutamide + LY3023414 4
RAF1 S257L Lung adenocarcinoma Sorafenib 4
RET Fusions NSCLC Cabozantinib 2
Vandetanib 3
ROS1 Fusions NSCLC Crizotinib 1
D2033N Cabozantinib 3
 Intracellular Signaling  •  CHAPTER 2 29

Table 2.1  Targeted Therapy for Disease-Specific Alterations of Actionable Oncogenes in Cancer—cont’d
Gene Variant Cancer Type Drug Evidencea
TSC1 Oncogenic mutations CNS Everolimus 2
RCC Everolimus 2
TSC2 Oncogenic mutations CNS Everolimus 2

a
Levels of evidence:
• Level 1: FDA-recognized biomarker predictive of response to an FDA-approved drug in this indication
• Level 2: Standard care biomarker predictive of response to an FDA-approved drug in this indication or another indication; including those recommended by NCCN, but
not FDA recognized as standard of care
• Level 3: Evidence of clinical activity in this indication, or another indication
• Level 4: Preclinical or biologic evidence of activity
ALL, Acute lymphoblastic leukemia; AML, acute myeloid leukemia; CML, chronic myelogenous leukemia; CNS, central nervous system; FDA, US Food and Drug Administration;
GIST, gastrointestinal stromal tumor; NCCN, National Comprehensive Cancer Network; NSCLC, non-small cell lung cancer; RCC, renal cell carcinoma.
Data modified from oncokb.org1; Chakravarty D, Gao J, Phillips S Kundra R, Zhang H, Wang J. OncoKB: a precision oncology knowledge base. JCO Precis Oncol. Published
online May 16, 2017.

Recruitment of signaling intermediaries to the plasma membrane
facilitates their interaction with membrane-bound proteins responsible
for stimulating a diverse array of downstream pathways (Fig. 2.1). As
an example, the lipid kinase phosphatidylinositol 3-kinase (PI3 kinase),
described in more detail in a later section, recognizes and binds to a
pattern of phosphorylated tyrosine residues present within multiple
activated RTKs through the SH2 domain located in its p85 regulatory
subunit. Binding of the p85 regulatory subunit in turn results in
activation of its kinase activity. Approximately 20 classes of RTKs
have been defined based on growth factor specificity. This section will
focus on those RTK classes for which specific cancer therapies exist
or are in development.
Epidermal Growth Factor Receptor Signaling
Historically, the growth factors that stimulate RTKs were first
discovered, followed by the structural and functional characterization
of the RTKs themselves.10,11 Epidermal growth factor (EGF) was
initially purified from mouse submaxillary glands in 1962 by Stanley
Cohen and was found to stimulate premature eyelid opening and
incisor eruption, phenotypes that suggested a role for EGF in the
regulation of cellular proliferation.12 In 1978, the epidermal growth
factor receptor (EGFR) was identified as the cell surface binding
site for EGF.13 Over the next several years, tyrosine phosphorylation
was identified in cells, followed by the discovery that the viral Src
oncogene, which induces transformation of cells in vitro, is itself a
tyrosine kinase, underscoring the potential importance of tyrosine
phosphorylation for oncogenesis.14,15 Once the complete sequence
of the EGFR protein was elucidated in the 1980s,16 the amino acid
sequence of the receptor cytoplasmic domain was found to be similar
to Src, suggesting that EGFR also possessed tyrosine phosphorylation
activity. The connection between RTK activation and oncogenesis was
further solidified when the amino acid sequence of EGFR was found
to be homologous to the avian erythroblastosis virus erbB oncogene,
which, when infected into chicken red blood cell precursors, is sufficient
to induce erythroleukemia.17,18 The erbB oncogene encodes a TM
protein that lacks the extracellular ligand binding domain of EGFR
but possesses a cytoplasmic kinase domain that, when expressed in cells,
can signal in a growth factor–independent manner. Subsequent studies
have since identified within human cancers numerous alterations of
EGFR and other RTKs that enhance proliferation without the need
for growth factor stimulation.
The EGFR class of RTKs comprises four receptor proteins encoded
by four genes (in parentheses): EGFR (ERBB1), HER2/Neu (ERBB2),
HER3 (ERBB3), and HER4 (ERBB4). EGFR binds to and is activated
by a number of ligands, including EGF, transforming growth factor–α
(TGF-α), HB-EGF, amphiregulin, betacellulin, epiregulin, and
epigen.19–21 Growth factor binding promotes either homodimerization
or heterodimerization with other HER family members, followed by
transphosphorylation.22 A ligand for HER2 has not yet been identified;
instead, HER2 is activated through heterodimer formation with one
of the other three ligand-bound receptors.23 Notably, HER2 is the
preferred dimerization partner for EGFR, and EGFR-HER2 heterodi-
mers are more stable than EGFR homodimers, remaining at the cell
surface for a longer duration and undergoing endocytosis at a lower
rate than EGFR homodimers.24,25 Furthermore, HER2 reduces the
dissociation rate of EGF from EGFR, allowing for a more sustained
period of EGF-induced signaling.19 The EGFR and HER2 components
of the EGFR-HER2 heterodimer are also more likely to be recycled
back to the cell surface than EGFR homodimers, which are more
readily targeted for degradation.26 In addition, HER2-HER3 het-
erodimers possess the most potent mitogenic activity among the
heterodimer and homodimer HER kinase combinations.27 In contrast
to the other HER kinase family members, HER3 does not have intrinsic
kinase activity and preferentially forms heterodimers with HER2.28
The ligands for HER3 and HER4 are the neuregulins, including
heregulin.
A number of tumor types frequently exhibit alterations within the
EGFR family of RTKs.21 Sustained activation of these pathways can
result in oncogene- or pathway-addicted tumors, and selective HER
kinase inhibitors are now a component of the standard treatment of
several malignancies. Alterations that affect RTK activity include
mutations that result in constitutive activation of the tyrosine kinase;
overexpression of the receptor, often due to gene amplification; and
elevated levels of RTK ligands that stimulate signaling. EGFR mutations
are found in 10% to 25% of non–small cell lung cancers (NSCLCs),
with variation in the frequency of such alterations influenced by
ethnicity and geographic location; in-frame microdeletions in exon
19 and point mutations in exon 21 (most commonly L858R or L861Q)
or exon 18 (G719X) comprise over 80% of these alterations.29–31 In
glioblastoma multiform (GBM), EGFR mutations, indels (including
the EGFRvIII variant in which exons 2 to 7 of the extracellular domain
are deleted, generating a ligand-independent, activated protein),
amplification, splice variants, and rearrangements occur in 57% of
tumors.32–34 However, because of the heterogeneity of GBM tumors,
targeting EGFR is complicated; EGFR alteration is often concurrent
with amplification or mutation of another RTK such as PDGFR,
MET, or FGFR, or the presence of EGFRvIII on extrachromosomal
DNA, or activation of IDH1.35–38 Overexpression of wild-type EGFR
as a result of gene amplification has been observed in NSCLC and
breast, gastric, colorectal, and head and neck cancers, and less commonly
in other tumor types.39–41
Up to 30% of breast cancers display overexpression of HER2,
which is an unfavorable prognostic factor, and therapy for these
30 Part I: Science and Clinical Oncology

Growth Factor Ligand oncogene

tumor suppressor
Receptor Tyrosine Kinase

P Shc
Ras-GDP NF1
P Grb2 Trafficking/
Sos RalGDS RalA/B
proliferation
Ras-GTP PLCε
Vermurafenib
Dabrafenib
Raf p85
P P TIAM1
MEKK1/NF-κB PIK3CA-
p110α
Trametinib P P
Selumetinib MEK
Cobimetinib
CDC42/RAC AKT

SCH772984 P P
DUSPs NF- κB/actin
BVD-523 ERK
DEL-22379 mTORC1
P
p90RSK
translation
NF- κB, Myt-1, GSK3, PP-1

P
Fos/Jun/etc. Proliferation/growth

Negative feedback

Figure 2.1  •  Canonical Ras/MAPK signaling pathway. Ras proteins cycle between GDP-bound inactive and GTP-bound active states. Ras is often activated
in response to ligand-specific binding to its cognate receptor. Ras can also be activated via intracellular cross talk. This schematic depicts the classic Ras/MAPK
signal transduction cascade. Growth factor stimulation induces receptor tyrosine kinase (RTK) dimerization and autophosphorylation of tyrosine residues
located within the intracellular domain of the receptor. These phosphorylated tyrosine residues serve as docking sites for scaffold proteins that facilitate activation
of intracellular signaling cascades. For example, the adaptor protein Grb2, via its SH3 domain, recruits the Ras GEF (guanine-nucleotide exchange factor)
SOS. Colocalization of SOS and Ras facilitates substitution of GTP for GDP and thus Ras activation. Active GTP-bound Ras binds and recruits the Raf (A-,
B-, and C-Raf ) serine/threonine kinases to the plasma membrane and facilitates their activation. Active Raf in turn phosphorylates and activates MEK, which
in turn phosphorylates and activates ERK. ERK phosphorylates substrates in the cytoplasm (p90RSK) and in the nucleus (Jun, Fos, Ets-2, Elk-1, CREB1,
AP-1, ATF-2, among others), which regulate cell proliferation and survival. Ras interacts with more than 20 effector proteins, including the p110α subunit
of PI3 kinase, RasGDS, PLCε, and TIAM1, which in sum control transcription, translation, vesicular trafficking, cell cycle progression, cytoskeletal changes,
metabolic processes, immune inflammatory responses, and survival. Induction of Ras signaling also upregulates negative feedback elements that inhibit the
pathway (e.g., DUSPs and SPROUTYs/SPREDs). Several proteins within the Ras signaling cascade are proto-oncogenes (green) and tumor suppressors (red)
that are mutated, amplified, or deleted in many cancers. A number of selective inhibitors of Ras effectors have been tested as anticancer therapies. Examples
include kinase inhibitors, which selectively target B-Raf and its downstream effectors MEK and ERK (see red boxes).

ERBB2-amplified breast cancers is now distinct from that of breast
cancers with normal HER2 expression levels.21,42 ERBB2 amplification
is also a driving event in gastric and to a lesser extent in bladder,
endometrial, and cervical cancer.43–45 More recently, activating mutations
and in-frame insertions/indels in ERBB2 were found to occur in 1%
to 2% of all cancer patients, most commonly in patients with bladder
cancer.44 Mutations in ERBB2 localize to either the extracellular domain,
where they are presumed to promote dimer formation, or the kinase
domain.46,47 In addition, activating mutations in ERBB3 have also
been identified in bladder, colon, and gastric cancers.48,49
Numerous targeted agents have been developed that selectively
inhibit EGFR-induced signaling (see Table 2.1 and Fig. 2.2).21,50
Cetuximab, a chimeric monoclonal antibody that binds to the
extracellular domain of EGFR and competitively inhibits ligand binding,
thereby preventing receptor activation, is approved for the treatment
of KRAS wild-type colorectal and head and neck cancers.51–56 Pani-
tumumab, another human monoclonal anti-EGFR antibody, is also
approved for KRAS wild-type metastatic colorectal cancer.56
The first-generation reversible EGFR tyrosine kinase inhibitors
gefitinib and erlotinib, as well as the second-generation irreversible
inhibitor afatinib, are FDA approved for the treatment of NSCLC,
with greatest efficacy in patients with EGFR mutations or in-frame
deletions.57–60 A second site mutation in EGFR (T790M) is a common
mechanism of acquired resistance to first generation EGFR inhibitors.
Osimertinib (AZD9291), a third-generation EGFR inhibitor, is highly
active in patients with NSCLC in which resistance is mediated by the
EGFR T790M mutation and is now FDA approved for this indica-
tion.61,62 The development of osimertinib and the fourth-generation
EGFR inhibitor EAI04563 highlights how studies of acquired resistance
can lead to the rational development of more effective kinase inhibitors.
 Intracellular Signaling  •  CHAPTER 2 31

XL147
Buparlisib Growth Factor Ligand
Alpelisib (α-specific) EGFR:Cetuximab
AZD5363 AZD8186 (β-specific) EGFR/HER2:Lapa­nib,
Copanlisib (α/δ-specific) Receptor
Afureser­b Trastuzumab
Idelalisib (δ-specific) Tyrosine
Kinase

EGFR:gefi­nib, erlo­nib,
PIP2/3 P
PIP2 P osimer­nib
AKT P PI3K-
p85
IRS1 EGFR/HER2:lapa­nib,
P P P
PTEN p110α afa­nib, nera­nib
P
Other: ima­nib, alec­nib,
PIP3 Ras-
IκB PDK1 larotrec­nib, etc
P GTP
mTORC2 SGK CDC42/RAC
Bad
P Dual PI3K-mTOR inhibitors:
P RHEB-GDP NF-κB/ac­n
NF-κB dactolisib, voxtalisib
GSK3β apoptosis TSC1
TSC2

RHEB-GTP
P P
Cyclin
D1 mTOR inhibitors:
PRAS40 mTORC1 rapamycin, everolimus,
temsirolimus, AZD8055, RapaLink
degrada­on
P P
4EBP1 p70S6K
P
Protein transla­on S6

FOXO1/4 Arrest/Apoptosis
oncogene
tumor suppressor

prolifera­on and survival

Figure 2.2  •  PI3K/mTOR signaling pathway. The PI3 kinase family proteins are lipid kinases that transduce signals from receptor tyrosine kinases (RTK)
and G protein–coupled receptors to intracellular cascades that control proliferation, survival, and other cellular phenotypes. As an example, growth factor
binding causes receptor dimerization and subsequent phosphorylation of tyrosine residues in the intracellular domain of the receptor. These tyrosine phosphoryla-
tion sites serve as docking sites for the p85 regulatory subunit of PI3 kinase and adaptor proteins such as IRS-1 in the case of signaling induced by the insulin/
IGF1 receptors. This results in allosteric activation of the catalytic subunit of PI3 kinase, which converts PIP2 to PIP3. PIP3 recruits PDK1 and AKT to the
membrane via their pleckstrin homology (PH) domains. Colocalization of PDK1 and AKT results in phosphorylation (on threonine 308) and activation of
AKT. Phosphorylation of AKT on Ser473 by mTORC2 is required for full activation of AKT. Activated AKT phosphorylates several effectors, including
GSK3β, Bad, PRAS40, IκB, the FOXO1/4 transcription factors, and TSC2. AKT phosphorylation of TSC2, which is bound to TSC1, inhibits the GTPase
function of this complex, thereby allowing activation of Rheb and subsequent activation of mTORC1. In turn, mTORC1 phosphorylates p70S6 kinase
(p70S6K) and 4EBP1, an inhibitor of the eIF4E component of the cap-dependent translation initiation complex. p70S6K and mTOR also function to negatively
regulate the pathway by initiating the phosphorylation and inhibition of IRS-1. PI3 kinases can also signal to other effectors such as Rac/CDC42 and the
serum-glucocorticoid kinase (SGK) family to promote cellular survival, motility, and cytoskeletal rearrangement. Many components of the PI3 kinase signaling
pathway are mutationally altered in cancer (oncogenes in green, tumor suppressors in red). A variety of compounds have been developed that selectively inhibit
PI3 kinase signaling components. US Food and Drug Administration (FDA)–approved drugs and novel inhibitors in clinical testing that target RTKs, PI3
kinase, mTOR kinase, and AKT are highlighted in the red boxes.

ERBB2 amplification strongly correlates with HER2 protein
overexpression, and the presence of either marker predicts for trastu-
zumab response in certain cancers.64 Trastuzumab, a humanized antibody
that binds to the extracellular domain of HER2, has been FDA approved
for the treatment of breast65–71 and esophagogastric72 cancers displaying
HER2 overexpression. In patients with breast and gastric cancers,
trastuzumab has modest activity when administered as single-
agent therapy and is most commonly used in combination with
chemotherapy.72–74 The combination of docetaxel, trastuzumab, and
pertuzumab,75 an antibody that binds to a different HER2 epitope
(the dimerization domain) than trastuzumab and results in impaired
dimer formation, is also approved for breast cancer.76–78
Although the introduction of trastuzumab has resulted in a sig-
nificant improvement in the survival of patients with HER2-
overexpressing breast cancers, drug resistance remains a major clinical
problem. Potential resistance mechanisms include concomitant
overexpression of other HER kinase family members and/or ligands,
PTEN loss, and the expression of a truncated HER2 protein lacking
the extracellular antibody binding site.79 Additional HER2-directed
agents include the tyrosine kinase inhibitors lapatinib and neratinib
(see Table 2.1). Lapatinib is FDA approved for use in combination
with capecitabine in patients with HER2-overexpressing advanced or
metastatic breast cancer that has progressed on prior therapy with
trastuzumab and certain classes of chemotherapy.80 The combination
32 Part I: Science and Clinical Oncology
of lapatinib and trastuzumab is also FDA approved in HER2-amplified
breast cancer.81–83 Lapatinib also received accelerated approval for use
in combination with the aromatase inhibitor letrozole.84 Clinical efficacy
has been reported with lapatinib in HER2-mutant NSCLC.85,86 The
irreversible pan-HER kinase inhibitor neratinib has shown promising
clinical activity in patients with ERBB2-amplified and ERBB2-mutant
breast tumors, but also other cancer types.46,87–93
Insulin, Insulin-Like Growth Factor-1 Receptor
Signaling, ALK, and ROS1
The insulin and insulin-like growth factor 1 (IGF1) receptor family
is dysregulated in multiple malignancies.94 The insulin receptor exists
as two isoforms encoded by splice variants of the same gene.95 Each
isoform can dimerize with the other (forming hybrid dimers) or with
itself.96 The IGF1 receptor (IGF1R) can dimerize with either of the
insulin receptor isoforms or with itself, resulting in six different dimer
combinations.96 The insulin receptor is stimulated by insulin or
insulin-like growth factor 2 (IGF2), whereas IGF1R can be activated
by either IGF1 or IGF2. Both of these latter ligands can stimulate
IGF1R in an autocrine fashion or can be elaborated from distant
sites.97,98 Circulating IGF binding proteins have a similar affinity for
IGF1 and 2 as IGF1R does and therefore compete for binding to
both ligands, thus titrating the amount of free ligand available for
IGF1R stimulation.99 IGF binding protein proteases provide an
additional mechanism for controlling ligand levels by increasing the
half-life of free ligand available for receptor binding.100
After ligand binding, IGF1R dimerizes and undergoes transphos-
phorylation, leading to activation of downstream signaling pathways,
including both the Ras-Raf-MAPK and the PI3 kinase-AKT-mTOR
cascades (see individual sections later in the chapter; see also Fig.
2.1).101 Specifically, insulin receptor substrate 1 (IRS1) binds to a
phosphotyrosine motif on IGF1R via its SH2 domains and is phos-
phorylated by IGF1R.102 It subsequently recruits PI3 kinase to the
plasma membrane, which converts phosphatidylinositol-4,5-bisphosphate
(PIP2) to phosphatidylinositol-3,4,5-triphosphate (PIP3), subsequently
resulting in AKT and mTOR pathway activation.
Activating mutations of IGF1R do not appear to be common in
human cancer. However, amplification of the IGF1R gene locus has
been identified in some colon, pancreas, and lung cancers. Sarcomas
often exhibit either increased expression of the IGF1 and IGF2 ligands
or decreased IGFBP-3 expression (Ewing sarcoma), which results in
increased IGF1 levels in the tumor microenvironment.103 Gastrointestinal
stromal tumors (GISTs) lacking c-KIT and platelet-derived growth
factor receptor (PDGFR) mutations also commonly harbor IGF1R
amplification.104 AMG479, a monoclonal human antibody targeting
IGF1R, has shown promising antitumor activity in patients with
Ewing sarcoma.103
Activating kinase domain point mutations and gene rearrange-
ments of the insulin receptor family members anaplastic lymphoma
kinase (ALK) and ROS1 play driving roles in many cancers, most
notably lymphomas, neuroblastoma, NSCLC, and thyroid cancer.105–108
Chromosomal translocations involving ALK and at least 22 5′ fusion
partners have been identified,108 which dictate spatial and temporal
expression of the ALK fusions, and likely their function and tumorigenic
potential.105 In NSCLC, the EML4 gene is the preferred translocation
partner, resulting in the expression of an EML4-ALK fusion protein
in 4% to 6% of patients.109,110 Notably, EML4-ALK fusions are found
in a mutually exclusive pattern with EGFR kinase domain mutations,
suggesting that they have overlapping downstream effects. ROS1 gene
rearrangements are also found in a minority of NSCLC patients with
binding partners including SLC34A2 and CD74.106,111,112 Crizotinib,
an inhibitor of the ALK, ROS1, and MET tyrosine kinases (see Table
2.1), is now FDA approved for use in NSCLC patients with ALK
or ROS1 fusions (see Table 2.1),113–115 although acquired resistance
mutations in ALK (of note, C1156Y and the gatekeeper mutation
L1196M) commonly develop. Newer ALK inhibitors including
alectinib and ceritinib that are either more potent or more selective
for ALK have significant clinical activity in patients with acquired
resistance to crizotinib and are FDA approved for this indication.116,117
In addition, brigatinib, a dual inhibitor of ALK and EGFR, was
granted accelerated FDA approval in 2017 for patients with metastatic
NSCLC and in patients with ALK alterations who progressed on
crizotinib.118
Platelet-Derived Growth Factor Receptor, KIT,
and FLT-3 Signaling
Platelet-derived growth factor (PDGF) is the ligand for PDGFRs,
which stimulate the proliferation and migration of mesenchymal cells,
such as oligodendrocyte precursors, vascular smooth muscle cells, and
pericytes during embryonic development.119 PDGF signaling is also
implicated in organ development, including lung and intestinal epithelial
folding and glomerular capillary tuft formation. Furthermore, PDGFs
promote angiogenesis, wound healing, and erythropoiesis.120 Aberrations
in the PDGFR pathway result in uncontrolled proliferation and
enhanced angiogenesis.
Four isoforms of PDGF have been identified: PDGFA, PDGFB,
PDGFC, and PDGFD.121 These isoforms are activated by proteolytic
cleavage and assemble into five homodimeric or heterodimeric com-
binations that bind to and stimulate either PDGFRα or PDGFRβ.
PDGFRα homodimers inhibit chemotaxis, whereas PDGFRβ
homodimers and α/β heterodimers stimulate chemotaxis within
fibroblasts and smooth muscle cells.122 Angiogenic endothelial cells
recruit PDGFRβ-positive pericytes to cover blood channels and aid
in their maturation and stabilization through secretion of PDGFβ.123
Following dimerization and transphosphorylation, PDGFRs activate
signal transduction pathways through recruitment of adaptor proteins
containing SH2 domains, most notably the Grb2 protein, which in
turn binds the guanine nucleotide exchange factor (GEF) Sos, which
subsequently activates Ras.124,125 In addition, phosphorylated tyrosine
residues serve as docking sites for SH2 domain–containing kinases,
including PI3 kinase, phospholipase-Cγ, and Src, as well as the tyrosine
phosphatase SHP2 and the STAT transcription factor family.125
Alterations in PDGFR signaling in cancer include excess autocrine
secretion of PDGF (glioblastoma, sarcomas), gain-of-function mutations
that cause constitutive tyrosine kinase activation (GISTs),126 transloca-
tion of either the PDGF or PDGFR gene (dermatofibrosarcoma
protruberans, chronic myelomonocytic leukemia, hypereosinophilic
syndrome),127–129 and PDGFR gene amplification (glioblastoma).130
PDGFRα mutations are found in approximately 10% of KIT wild-type
GISTs and are sensitive to imatinib, a tyrosine kinase inhibitor of
KIT, BCR-ABL, and PDGFRs, which is standard of care in this setting
(see Table 2.1).122,126,131 The D842V mutation comprises approximately
two-thirds of PDGFRα activating mutations, and confers resistance
to imatinib. Notably, the second-generation inhibitor dasatinib is
effective in preclinical models of imatinib-resistant GIST.126,132,133
Dermatofibrosarcoma protuberans is a rare, low-grade cutaneous
sarcoma that harbors a chromosome 17;22 translocation that fuses
portions of the COL1A1 (collagen 1A1) gene and PDGFB, resulting
in overexpression of PDGF-β and subsequent stimulation of PDGFR
signaling.122 Twenty other fusions partners have been identified in
PDGFβ rearrangements, including ETV6 and EBF1. Imatinib has
shown significant benefit in patients with recurrent or metastatic
dermatofibrosarcoma protuberans, myelodysplasia, and myeloprolifera-
tive neoplasms and is FDA approved for these indications.134–138
The KIT gene is a member of the type III RTK family, which
includes PDGFR and FLT3 (see later).139–141 It was first identified as
the human homologue of the viral oncogene v-Kit responsible for the
Hardy-Zuckerman IV feline sarcoma virus.142 Mutation of KIT or its
ligand, stem cell factor (SCF),143–146 in mice induces coat color
abnormalities (“white spotting”), anemia, and mast cell deficiencies,
suggesting that it plays a role in hematopoiesis and melanogenesis.147,148
Furthermore, the KIT protein was discovered as a cell surface receptor

in acute myeloid leukemia (AML).149 KIT expression is mainly restricted
to mast cells, hematopoietic cells, germ cells, melanocytes, and the
interstitial cells of Cajal (ICCs) in the gut.150,151 SCF and KIT integrate
signals that lead to mitogen-activated protein kinase (MAPK), PI3K,
and SRC pathway activation, and mediate critical survival and prolifera-
tion cues to distinct hematopoietic lineages, including the bone marrow
and progenitor cells.
Hot-spot mutations in exons 9 and 11 of KIT have been identified
in several tumor types, including GIST, melanomas, and germ cell
tumors.146,152–156 In GIST, 85% of tumors have activating KIT mutations
that drive the transformation of precursors of ICCs.153 Imatinib157–161
and sunitinib158,162,163 inhibit KIT and PDGFR, among other kinases,
and are FDA approved for use in patients with KIT mutant GIST
(see Table 2.1). Regorafenib is approved for patients with imatinib- or
sunitinib-refractory GIST.164 Imatinib has also been shown to induce
tumor regression in patients with KIT-mutant or KIT-amplified
melanoma.165,166 Second site mutations in KIT, typically in exon 17,
are a mechanism of acquired resistance to imatinib therapy in patients
with GIST.167 Novel agents that retain activity in the setting of an
exon 17 KIT mutation are now in clinical testing (clinical trial
NCT02401815).168 New areas of investigation into mutant KIT
therapies in GIST include blocking mutant KIT subcellular localization
to the Golgi150 and combination of FGFR3 and KIT inhibitors to
quench pathway cross talk.169
The FMS-like tyrosine kinase 3 receptor (FLT3), a third member
of the RTK class that includes PDGFR and KIT, is involved in the
development of normal hematopoietic cells. It contains an extracel-
lular region composed of five immunoglobulin (Ig) domains, TM
and juxtamembrane domains, and two cytoplasmic tyrosine kinase
domains that transmit proliferative signals through the RAS/MAPK,
PI3K/AKT, and STAT5 pathways.170 Two main FLT3 alterations are
common in hematopoietic malignancies, namely in approximately
30% of AMLs.171 First, internal tandem duplication (ITD) within
exons 14 and 15 of the FLT3 gene (FLT-ITD) interferes with the
negative regulatory function of the juxtamembrane segment.172 This
duplication results in ligand-independent activation of FLT3 and is
associated with a poor prognosis in patients with AML. Second, kinase
domain mutations at or near D835 in the activation loop of FLT3
disrupt autoinhibitory interactions and render the kinase open and
active.173 The clinical activity of FLT3 inhibitors has been modest to
date, although responses appear to be more common in patients with
FLT3/ITD AML.171 TAK-659, a reversible dual Syk/Flt inhibitor,
showed early clinical activity in numerous lymphoma subtypes and
AML.174,175 Sorafenib has been shown in preclinical in vitro studies,
mouse models, and in a phase I study of AML patients to reduce
leukemia burden and block signaling selectively in FLT3-ITD versus
FLT-wt settings.176 Interesting to note, resistance to FLT3 inhibition in
such patients is associated with selection for secondary mutations within
the tyrosine kinase domain of FLT3, suggesting a central role of FLT3 in
AML pathogenesis.171
Fibroblast Growth Factor Receptor Signaling
Fibroblast growth factor receptors (highly conserved FGFR1, FGFR2,
FGFR3, and FGFR4; and FGFRL1/FGFR5, which lacks a kinase
domain) comprise a family of RTKs that regulate cell proliferation,
differentiation, and migration as well as selective apoptosis during
embryogenesis. The FGFRs are composed of an extracellular ligand-
binding domain, a hydrophobic TM region, and an intracellular tyrosine
kinase domain.177 The extracellular domain is organized into three Ig
domains; differential splicing of the second half of the third Ig domain
dictates tissue-specific expression of the receptor.
Fibroblast growth factors (FGFs) are protein ligands that bind to
the extracellular domain of the FGFRs in combination with specific
heparan sulfate glycosaminoglycans inducing FGFR dimerization and
transphosphorylation of intracellular tyrosine residues. Eighteen FGFs
have been identified, and specificity for FGFRs is based on numerous
Intracellular Signaling  •  CHAPTER 2 33
factors, including tissue-specific FGF ligand and receptor expression, the
presence of cell surface molecules that facilitate the interaction between
individual FGF ligands and receptors, and the differential binding
capability of the ligands themselves for specific FGFRs.178 Subsequent
stimulation of the tyrosine kinase domain leads to phosphorylation
and activation of multiple downstream signaling proteins in the same
manner as described earlier for other RTKs. Unique to the FGFR
signaling complex is FGFR substrate 2 (FRS2), an adaptor protein
that binds to specific phosphotyrosines on the intracellular domain
of active FGFR dimers.179 FRS2 is itself phosphorylated by FGFRs
and serves as a docking site for the Grb2-Sos adaptor complex, which
activates the Ras/Raf/MAPK pathway. Phosphorylated FRS2 also
recruits Grb2-associated binding protein 1 (GAB1), which activates
PI3 kinase. In addition, phospholipase-Cγ binds to phosphorylated
FGFR dimers via an SH2 domain, leading to its activation and the
cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) to form inositol
1,4,5-triphosphate (IP3) and diacylglycerol (DAG).
Germline mutations of the FGFR genes are the basis of a spectrum
of skeletal developmental disorders that are thought to derive from
premature differentiation and growth restriction of chondrocytes
resulting from dysregulated FGFR pathway activation.180,181 FGFR
signaling is dysregulated in cancer by multiple mechanisms includ-
ing mutational or translocation-induced activation of FGFRs, gene
amplification of receptors, and abnormal ligand regulation.182 For
example, autocrine and paracrine FGF ligand secretion with resultant
pathway activation has been reported to occur in a subset of melanomas
and prostate cancers, respectively.183,184 FGFR1 amplification occurs in
approximately 17% of squamous cell lung cancers and 6% of small cell
lung cancers (SCLCs).185 Approximately 10% of diffuse-type, aggressive
gastric cancers display FGFR2 gene amplification, and cell lines with
this amplification show ligand-independent pathway activation and
sensitivity to selective FGFR inhibitors.186,187 Whereas FGFR1 mutations
are rather rare, FGFR2 mutations are found in approximately 10%
of endometrial cancers.188,189 FGFR3 mutations occur in up to 75%
of non–muscle invasive bladder cancers and 15% of patients with
advanced urothelial tumors.44,179,189,190 Activating mutations within
FGFR3 result in constitutive receptor dimerization and subsequent
signaling. Unlike EGFR-activating mutations, which predominantly
affect the tyrosine kinase domain of the receptor, FGFR3 mutations
are commonly located within the extracellular domain (R248, S249)
and TM segment (G370, Y373) and promote ligand-independent
receptor dimerization through formation of an aberrant disulfide bridge
between two receptor monomers.191 Chromosomal rearrangement of
FGFRs have been identified using next-generation sequencing. Up to
15% of multiple myelomas harbor an intergenic 4;14 translocation
between the FGFR3 gene and the Ig heavy chain locus, which places
FGFR3 expression under the highly active heavy chain promoter.192,193
More recently, translocations involving FGFR2 have been reported
in cholangiocarcinoma and more rarely in other cancers, whereas
FGFR3 fusions are most common in glioblastoma and bladder
cancers but also are found rarely in other solid tumor types.194,195
The FGFR3-TACC3 constitutively active fusion protein has been
characterized to induce aneuploidy by disrupting proper chromosomal
segregation.196
Multiple FGFR inhibitors are currently being tested in early-phase
clinical trials, but the majority of these compounds are multitargeted
tyrosine kinase inhibitors, many of which also potently inhibit members
of the VEGFR and PDGFR families. The close structural similarity
between these RTKs has made development of FGFR-selective inhibitors
challenging, although several such drugs are now in early clinical testing,
such as BGJ398, AZD4547, JNJ-42756493, and Debio1347 (see Table
2.1).179,182,197–200 On-target hyperphosphatemia resulting from FGFR1
inhibition is a primary toxicity with this class of agents, suggesting that
the development of isoform-selective FGFR inhibitors may be a more
rational approach for patients whose tumors are driven by mutations
or translocations in FGFR2 and FGFR3. FGFRs are located on the
cell surface and thus may also be susceptible to monoclonal antibody
34 Part I: Science and Clinical Oncology
mediated inhibition similar to trastuzumab-mediated inhibition of
HER2. FGFR ligand traps are also in development.182
RET Signaling
The RET (Rearranged During Transfection) gene encodes three
alternatively spliced isoforms (RET9, RET43, and RET51) which are
TM RTKs that contain four cadherin-like extracellular repeats important
for dimerization, a cysteine-rich juxtamembrane region critical for
ligand binding and conformation, and an intracellular kinase domain.201
RET9 and RET51 are highly conserved in all vertebrates and play
important roles in the normal development and maintenance of many
tissues, including the kidney, spermatogonial stem cells, and the enteric
nervous system.201–203 RET is expressed predominantly on the surface
of neural crest tissues, and glial-derived neurotrophic factors (GDNFs)
such as neurturin, artemin, and persephin serve as ligands for RET.
GDNFs initially bind to their cognate coreceptors, the GDNF receptors
(GFPα1 to GFPα4), on the cell surface, which recruits RET into
lipid raft membrane domains and causes conformational changes via
the cadherin-like moieties, dimer formation, and then subsequent
transphosphorylation of tyrosine residues and kinase activation.204
Y1062 is the common docking site for all three RET isoforms and
serves to recruit many adapters including SHC1, FRS2, IRS1/2, DOK,
and JNK.201 Phosphorylation of Y752 and Y928 binds STAT3, whereas
other phosphorylated residues are recognized by Src, resulting in
activation of focal adhesion kinase (FAK), which promotes cell migration
and metastatic spread. In addition, the MAP kinase, PI3 kinase/AKT,
and phospholipase-Cγ pathways can be activated by RET to promote
cellular proliferation and survival.204
Germline loss-of-function RET mutations occur in Hirschsprung
and CAKUT (congenital anomalies of the kidney and urinary tract)
disease, which causes abnormalities of the developing gut and kidneys,
respectively. Conversely, germline activating RET mutations are the
basis for the multiple endocrine neoplasia type 2 (MEN2) syndromes.
Patients with MEN2 develop familial medullary thyroid carcinomas
and other cancers.205 MEN2A is mainly driven by mutations in six
cysteine resides in the RET extracellular domain (C609, C611, C618,
C620, C630, and C634), whereas the kinase domain mutations M918T
or A883F are associated with MEN2B. Sporadic medullary thyroid
carcinomas are much more common, and up to 60% of such tumors
harbor somatic mutations in RET, notably G691S, which are thought
to be a driver alteration in this disease.206 Furthermore, RET gene
rearrangements with numerous fusion partners, including CCDC6
and NCOA4, termed RET-PTC1 and RET-PTC3, respectively, occur
in 20% to 40% of papillary thyroid carcinomas (PTCs) and often
occur as a consequence of high doses of radiation.207
RET inhibitors have shown significant antitumor activity in patients
with medullary thyroid cancer. Vandetanib, an oral inhibitor of RET,
EGFR, and VEGFR, is FDA approved for the treatment of patients
with advanced medullary thyroid cancer.208 A randomized, placebo-
controlled phase III study of cabozantinib, an oral, multitargeted TKI
that inhibits RET, VEGFR2, and MET, was also recently conducted
in patients with unresectable, locally advanced, or metastatic medullary
thyroid carcinoma.209 This trial documented a statistically significant
improvement in median progression-free survival with cabozantinib
as compared with placebo (11.2 months versus 4.2 months in placebo
arm, P < .0001). More recently, cabozantinib was FDA approved for
the treatment of renal cell carcinomas that progressed on antiangiogenic
therapy, although the activity of cabozantinib in this context may not
be attributable to its inhibition of RET. Several RET inhibitors have,
however, shown promising clinical activity in patients with NSCLC
treated with RET fusions (see Table 2.1).210
Vascular Endothelial Growth Factor Signaling
Six vascular endothelial growth factor (VEGF) ligands have been
identified: VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental
growth factors 1 and 2.211 VEGF-A has four isoforms produced by
alternative gene splicing, with the 165–amino acid length isoform
playing a central role in tumor angiogenesis.212 Specifically, VEGF-A
enhances vascular permeability and stimulates endothelial cell
proliferation, resulting in new blood vessel formation. Vascular
endothelial growth factor receptors (VEGFR1 to VEGFR3) are
RTKs that possess a modular structure consisting of an extracellular
domain with seven Ig-like regions, a TM domain, and an intracellular
tyrosine kinase domain.211 VEGF-A, VEGF-B, and placental growth
factor all bind VEGFR1 (also known as FLT1), but the exact role
of VEGFR1 in tumor angiogenesis has yet to be fully elucidated. In
some settings it may act as a decoy receptor that prevents ligand-
mediated stimulation of VEGFR2 (also known as FLK1/KDR).213
VEGFR2 has been implicated in the development of vasculature
during development and is considered the primary receptor through
which VEGF exerts its angiogenic effects in endothelial cells.213,214
Binding of ligand to VEGFR2 results in receptor dimerization and
transphosphorylation followed by activation of multiple mitogenic
signal transduction cascades.215,216 More recently, VEGF has been
implicated in many angiogenesis-independent roles including regula-
tion of immune cells in the tumor microenvironment, fibroblasts
in the tumor stroma, and cancer stem cells.217 VEGF can also bind
and signal through a class of TM glycoprotein coreceptors called
neuropilins (NRP1 and NRP2), which are found on tumor cells
and can signal along many oncogenic axes including Hedgehog
and JNK.217
The complex network of cross talk among VEGFs, VEGFRs, and
canonic oncogenic pathways makes VEGF and VEGFR critical but
elusive targets in cancer therapy. Targeted therapies that inhibit VEGF
signaling include antibodies that bind circulating ligand and RTK
inhibitors. The humanized monoclonal antibody bevacizumab binds
to free VEGF, thereby preventing its association with VEGFRs. This
antibody has been FDA approved for use in combination with che-
motherapy for patients with several cancers, including metastatic
colorectal218 and nonsquamous NSCLCs.211,219 Bevacizumab also has
activity in patients with glioblastoma220 and metastatic renal cell
carcinoma, where it is often used in combination with interferon-α
(IFN-α).221 In addition, ramucirumab is a VEGFR2-directed antibody
that has received FDA approval with or without chemotherapy in
several cancers.222
Sorafenib, sunitinib, pazopanib, and axitinib are multitargeted
tyrosine kinase inhibitors with nanomolar potency for VEGFR2.
Sunitinib is used in the treatment of patients with metastatic renal
cell carcinoma, GISTs, and pancreatic neuroendocrine tumors.223
Sorafenib has been approved for the treatment of liver and renal cell
cancers.224,225 Although these agents inhibit multiple kinases, their
antitumor effects have been attributed primarily to their antiangiogenic
activity. More recently, the tyrosine kinase inhibitor pazopanib was
approved for the initial treatment of metastatic renal cell carcinoma
and in cytokine-pretreated patients,226 and axitinib227 was approved
in the second-line setting following failure of prior systemic therapy.
Lenvatinib, a multitargeted RTK inhibitor that inhibits VEGFR1,
VEGFR2, and VEGFR3, has received recent FDA approval for both
thyroid cancer (as monotherapy)228 and renal cell carcinoma in combina-
tion with everolimus.229
Despite widespread activity in preclinical models, antiangiogenic
therapies have shown disappointing activity in several tumor types.
A number of resistance mechanisms have been hypothesized to
explain the lack of broader clinical activity, including the activation
of redundant signaling pathways that promote angiogenesis; the
recruitment by tumors of bone marrow–derived endothelial progenitor
cells; increased pericyte density around existing blood vessels, which
enhances vascular growth and survival; and the ability of tumor cells
to invade surrounding stroma to co-opt additional blood supply.230
A better understanding of these resistance mechanisms may lead
to the development of more effective antiangiogenic therapies in
the future.

Hepatocyte Growth Factor Receptor Signaling
The hepatocyte growth factor receptor (HGFR or MET) is encoded
by the MET gene.231,232 Both MET and its ligand hepatocyte growth
factor/scatter factor (HGF/SF) are expressed as immature precursors
that require proteolytic cleavage.233 The MET extracellular domain
consists of an alpha subunit connected by a disulfide bridge to a TM
beta subunit, and contains a Sema domain, a PSI domain, and four
IPT domains.234,235 The intracellular portion of the receptor contains
a juxtamembrane region that harbors a serine residue (Ser 975) that
inhibits RTK activity on phosphorylation, as well as a tyrosine kinase
domain with Y1234 and Y1235 acting as key sites of autophosphoryla-
tion required for activation.235 A tyrosine residue at position 1003,
proximal to the tyrosine kinase domain, serves as an interaction site
for the ubiquitin ligase CBL, which marks the receptor for endocytosis
and degradation.236,237 C-terminal residues Y1349 and Y1356 represent
docking sites for adaptor proteins.238 On binding of HGF/SF to the
extracellular portion of MET, receptor dimerization occurs, followed
by transphosphorylation. A number of adaptor proteins then bind
to phosphorylated tyrosine residues, including Grb2 and GAB1,
phospholipase-C (PLC), and SRC, which promotes the activation of
the MAP kinase and PI3 kinase/AKT signaling pathways.239,240 MET
can also activate RAC1/CDC42 and p21-activated kinase (PAK1), both
of which regulate cytoskeletal proteins and integrin expression and
activation, and thus cell migration.238,239 MET also plays an important
role in driving epithelial-to-mesenchymal transition (EMT) cell migra-
tion during embryo development, and organ regeneration.238,241
Dysregulation of MET signaling can occur through multiple
mechanisms, including activating point mutations (often in the kinase
domain; prevalent in lung cancer), exon skipping events, receptor
overexpression, and upregulation of HGF, which can activate MET in
an autocrine and/or paracrine manner.238,240,242–246 Germline mutations
of MET are found in patients with hereditary papillary renal cell
carcinomas, and MET overexpression is observed in a significant
proportion of sporadic papillary cancers as well as collecting duct
carcinomas.247 Multiple other malignancies exhibit aberrations in
MET signaling, including lung, breast, pancreatic, colon, and gastric
cancers. Amplification of MET is associated with a worse prognosis
in lung and gastric cancers, whereas expression of MET or HGF is
an unfavorable prognostic biomarker in liver, kidney, colorectal, and
gastric cancers.248
Recurrent somatic splice site alterations involving MET exon 14
(METex14) have been identified in lung cancer. These mutations
result in exon skipping, loss of the juxtamembrane CBL E3-ubiquitin
ligase-binding site, diminished receptor turnover, and ultimately,
MET activation.243,249,250 These exon 14 MET mutations are mutually
exclusive with activating mutations in EGFR and KRAS as well as
ALK, ROS1, and RET fusions, and treatment of patients with exon 14
MET splice variants with a MET kinase inhibitor is now considered
to be a standard treatment option (see Table 2.1).210 Several RTKs
have also been shown to activate MET, including EGFR, HER2, and
IGF1R. For example, EGFR activation can stimulate MET signaling,
and resistance to EGFR inhibitors in some lung cancers has been
shown to stem from coactivation of MET in the setting of gene
amplification.251
Inhibitors of MET signaling have been in development for a number
of years. Therapeutic strategies for targeting MET activation in cancer
patients include antibodies that target the extracellular domain of the
receptor, antibodies that bind to and thus sequester circulating HGF,
and small-molecule tyrosine kinase inhibitors that selectively target
MET or are multi-kinase MET inhibitors.248 Durable responses to
crizotinib and cabozantinib, multikinase MET inhibitors, have been
reported in patients with NSCLC with MET splice mutants or MET
amplification (see Table 2.1).252,253 Cabozantinib is also now standard
of care in MET-amplified renal cell carcinoma.254,255 Combination
therapies to combat MET reactivation after EGFR kinase inhibitor
therapy suggest an improvement in progression-free survival.256
Intracellular Signaling  •  CHAPTER 2 35
Tropomyosin Receptor Kinases/Neurotrophic
Tyrosine Kinase
First cloned as an oncogenic fusion partner of the tropomyosin receptor
kinases and subsequently characterized for their role in neural dif-
ferentiation and survival in the peripheral and central nervous systems,
the TRKA/B/C family of RTKs, encoded by the neurotrophic tyrosine
kinase (NTRK) genes 1 to 3 (NTRK1/2/3), respectively, integrate
ligand stimulation from nerve growth factor, brain-derived neurotrophic
factor, neurotrophins 3 to 6 with downstream activation of PI3K,
phospholipase-C (PLC)-gamma, and MAPK signaling.257–261 Chro-
mosomal rearrangements involving the tropomyosin receptor kinases
(TrkA/NTRK1, TrkB/NTRK2, and TrkC/NTRK3) were recently shown
to occur at high frequency in several rare cancer types including
mammary secretory carcinoma of the breast and congenital-infantile
fibrosarcoma.262–264 NTRK fusions also occur at low frequency in a
broad range of more common adult solid tumors.260,265 These NTRK
fusions induce ligand-independent constitutive kinase activity, resulting
in upregulation of canonic downstream signaling pathways involved
in growth and survival. TPM3, LMNA, MPRIP, TRIM24, ETV6, and
PPL, among others, have been identified as fusions partners with the
NTRK genes.266,267 Dramatic and durable clinical responses have recently
been reported in patients with NTRK fusions, with first- and second-
generation TRK inhibitors such as larotrectinib (LOXO-101),
entrectinib, and LOXO-195 (see Table 2.1).268–273 Notably, clinical
activity was observed in both adult and pediatric patients and was
independent of site of tumor origin.264
G PROTEIN–COUPLED RECEPTOR SIGNALING
G protein–coupled receptors (GPCRs) are seven TM domain–containing
proteins that transduce ligand-specific signals across the plasma
membrane to mediate numerous physiologic processes including sensory
perception, immunologic responses, neurotransmission, weight regula-
tion, and cardiovascular activity.274 GPCRs also regulate basic cellular
functions including growth, motility, differentiation, and gene transcrip-
tion. The GPCR family comprises more than 800 receptors, which
are the targets of over 30% of all FDA-approved drugs, although few
to date have found a role as anticancer therapies.275,276 Given that
GPCRs activate many of the signaling cascades that are deregulated
in human cancer, it is not surprising that studies have implicated
GPCRs in cancer initiation and progression.277–279
GPCRs can be categorized into five or six families, depending on
the nomenclature used.280 In a more recent phylogenetic classification,
the five major families are represented by the acronym GRAFS:
Glutamate, Rhodopsin, Adhesion, Frizzled/Taste2, and Secretin.281
The Rhodopsin family of receptors (also referred to as class A GPCRs)
is the largest class, with more than 670 members. Crystallization of
the bovine rhodopsin receptor in 2000 provided the first high-resolution
insight into the structure of GPCRs.282 In general, Rhodopsin family
receptors have short N-termini. Included in this family are the α
group (histamine, dopamine, serotonin, adrenoceptors, and muscarinic,
prostanoid, and cannabinoid receptors), the β group (endothelin,
gonadotropin-releasing hormone, and neuropeptide Y), the γ group
(opioid, somatostatin, and angiotensin), and the δ group (P2RYs,
glycoprotein-binding FSHR/TSHR/LHCGR, PARs, and olfactory
receptors).283
The 15 Secretin receptors (class B) all have conserved cysteines in
the first and second extracellular loop, and most have three cysteine
bridges in the N-termini. This class includes the calcitonin-like,
corticotrophin-releasing hormone, glucagon-like, gastric inhibitory
polypeptide, growth hormone–releasing hormone, adenylate cyclase–
activating polypeptide, parathyroid hormone, secretin, and vasoactive
intestinal peptide receptors.284 The Adhesion family (also included in
class B, according to another classification system) has distinctly long
N-termini, and only 3 of 33 receptors in the family have known
ligands (epidermal growth factor-like module containing mucin-like
36 Part I: Science and Clinical Oncology
receptors EMR2, EMR, EMR4).285 The Glutamate receptor family
(class C) binds ligands in their N-termini, which have a complex
two-domain folded structure bridged by disulfide bonds. Common
receptors in this family of 22 include the glutamate, GABAB, calcium-
sensing, sweet and umami taste (TAS1R1–TAS1R3), and GPCR6
receptors.286,287 The Frizzled receptors (FZD1–FZD10 and SMO; see
later for in depth discussion of signaling) bind Wnt ligands in the
extracellular domain region containing nine conserved cysteine resi-
dues.288 The Taste2 receptors (25 members of T2R, bitter taste) have
varying sequence homologies that likely allow the sensing of thousands
of distinct bitter tastes.289,290 Overall, GPCRs share the seven–TM
domain structure, but have different regions of conservation. All of
the families include orphan receptors, which are related by sequence
and structure but have no identified ligand to date.
All GPCRs have seven α-helical domains that weave through the
plasma membrane and are interconnected by flexible extracellular and
intracellular segments, thus engendering the synonyms serpentine or
heptahelical receptors. The specificity of biologic response initiated by
each GPCR depends on (1) ligand recognition; (2) the distinct structure
of each receptor class and subclass; and (3) ligand-directed binding
of specific cytosolic enzymes and adapters that initiate a plethora of
intracellular signaling cascades (general and cancer-specific examples
are discussed further later). The GPCR Network was created in 2010
to tackle the challenge of delineating the structure and function of
this diverse class of receptors.291
Operationally, ligand binding on the GPCR extracellular surface
induces a conformational change in the receptor, mainly via TM
helices TM3, TM5, and TM6, which creates a deep pocket in the
intracellular face of the receptor.292–294 This cleft enables binding and
activation of heterotrimeric G proteins, which consist of an inactive,
GDP-bound Gα subunit and a Gβγ-subunit dimer, which act as a
molecular switch.295,296 Activated GPCRs promote GDP for GTP
exchange on the Gα nucleotide binding site.297 This GTP-bound Gα
dissociates from Gβγ and the receptor, and then both activated subunits
go on to initiate signaling cascades. There are four members of the
Gα family, Gαs, Gαi/o, Gαq/11, and Gα12/13, which can be further
subtyped and can each stimulate several downstream effectors. Moreover,
each GPCR can couple to multiple Gα family members, thus generating
a complex pattern of intracellular signaling.
Classic GPCR activation of Gαs stimulates adenylyl cyclase, which
generates the second messenger 3′-5′-cyclic adenosine monophosphate
(cAMP).298,299 cAMP activates multiple downstream effectors including
cAMP-gated ion channels; Epac, a GEF for Rap1/2 (which functions
in cell adhesion and junction formation); and protein kinase A
(PKA).274,295,300–303 cAMP binds to PKA regulatory subunits, releasing
catalytic subunits and triggering the activation of cytosolic and
nuclear substrates, including the transcription factor CREB (cAMP
response element binding protein), which can induce proliferation and
differentiation, among other phenotypes, depending on cell origin.304–306
Gαs also activates the Src tyrosine kinase and the GTPase activity of
tubulin.295,300 GPCR-coupled Gαi/o typically works in opposition to
Gαs by inhibiting adenylyl cyclase and decreasing cAMP levels.274,307
In addition, some Gαi/o isoforms can signal to K+ and Ca+ channels,
increase cGMP phosphodiesterases, interact with Rap1GAP1,
and cross talk to the MAP kinase pathway (as described later in
the chapter).274
Members of the Gαq/11 family activate phospholipase-Cβ, which
catalyzes the hydrolysis of PIP2 to yield IP3 and DAG.308 IP3 mobilizes
calcium from intracellular stores, whereas DAG activates some isoforms
of protein kinase C (PKC).309,310 Both Gαq/11 and Gα12/13 activate a
variety of RhoGEFs (p115-RhoGEF, PDZ-RhoGEF, LARG, Lbc,
AKAP-Lbc) and thus regulate Rho activity (mainly RhoA) and its
contribution to actin stress fiber formation, cell shape and polarity, cell
adhesion and migration, gene transcription, and cell cycle progression.311
In addition, Gα12/13 activates the Na+/H− exchanger, inducible nitric
oxide synthase, phospholipase D, E-cadherin, radixin, and protein
phosphatase 5. Gβγ signaling is equally complex, as the active dimer
stimulates G protein–regulated inwardly rectifying K+ channels adenylyl
cyclase (types II, IV, VII), PLCβ, PI3Kγ, Src, and GPCR kinases
(GRKs; see later). It inhibits adenylyl cyclase (type I), some Ca+
channels, and calmodulin; stabilizes Gα in the GDP-bound, inactive
state; and helps specify coupling to the proper Gα member.274,295,296,300
Gα signaling is switched off by a family of GTPase activating
proteins called Regulators of G-protein Signaling, or RGS proteins,
which enhance the intrinsic rate of GTP hydrolysis by greater than
1000-fold.312,313 Some RGS proteins enhance signaling through
RhoGEFs and act as scaffolds for other signaling cascades (e.g., tethering
to Raf and MEK2). The GPCR effectors PKA and PKC also contribute
to receptor inhibition by phosphorylating their cognate-activated
GPCR and thereby uncoupling and inactivating Gαs/Gαq in a classic
negative feedback loop.314,315
GPCRs can also function via G protein–independent mechanisms
by binding to the arrestin family of cytosolic adapter proteins.316,317
GPCR-coupled arrestins have pleiotropic cellular roles including (1)
dampening G-protein signaling by scaffolding enzymes that degrade
G-protein second messengers; (2) desensitizing receptors by binding
GPCR kinase (GRK)–phosphorylated GPCRs and sterically blocking
access to further Gα subunits; (3) mediating GPCR trafficking and
endocytosis to clathrin-coated pits; and (4) acting as a scaffold for
multiple MAP kinase cascades. For example, MEK1 is engaged by a
tethered complex of Raf-1, ERK2, and β-arrestin to facilitate mitogenic
signaling.318,319
GPCRs are well established drug targets for antihistamine, antacid,
cardiovascular, and antipsychotic drugs; pain suppressants; and
antihypertension therapies.275,276 Although less well appreciated as drug
targets in cancer, there is increasing evidence that dysregulated GPCR
signaling contributes to cancer initiation and progression. For example,
in 1986 the wild-type MAS1 gene, which encodes the MAS GPCR,
was reported to induce transformation by coupling to the small G
protein Rac.320,321 Large-scale deep sequencing efforts have revealed
that GPCR mutations occur in approximately 20% of all cancers.
Receptors for thyroid-stimulating hormone (TSHR), Hedgehog
(Smoothened receptor [SMO]), glutamate (GRM), the adhesion family,
lysophosphatidic acid (LPA), and sphingosine-1-phosphate (SIP) are
the most frequent GPCRs altered in cancer.277,312,313,322 G proteins
themselves are also mutated in cancer. In particular, recurrent mutations
in GNAS (which encodes Gαs) have been identified in thyroid and
pituitary tumors, as well as mutations in GNAQ and GNA11 in
melanoma of the eye and skin, respectively.322 Hot spot resides that
disrupt GTPase activity and render the protein constitutively active
have been identified in GNAS at positions R201 and Q227, in GNAQ
at R183, and in GNA11 at Q209. Numerous inhibitors of GPCR
signaling are also being studied in the clinic including BKT-140/
BL-8040 in pancreatic adenocarcinoma and blood cancers (target:
CXCR4 receptor), and CXCR2 ligands (target: CXCR2 receptor).278
Detailed later are a few specific examples of GPCRs that have been
shown to play a role in cancer initiation and/or progression.
A connection between GPCRs and EGFR signaling has been
established in both normal cell physiology and in colon, lung, breast,
ovarian, prostate, and head and neck cancer development.323–326
Ligand-bound GPCRs activate Src, PKC, Ca+ channels, and PKA
intermediaries, which stimulate proteolytic cleavage and release of
membrane-tethered growth factors that bind and thereby transactivate
EGFR. Specifically, estrogen binding to the GPCR GRP30 facilitates
matrix metalloproteinase–2 (MMP2) and MMP9-mediated cleavage of
the growth factor precursor pro-heparin-binding-EGF (pro-HB-EGF),
thus initiating HB-EGF–mediated EGFR transactivation.324 Through
a related mechanism, LPA-, SIP- and thrombin-activated GPCRs
transactivate EGFR in breast cancer cells via growth factor shedding
of tumor necrosis factor–α (TNF-α) through the action of TACE/
ADAM17 zinc-dependent proteases.326 Thrombin-mediated N-terminal
cleavage and activation of proteinase-activated receptor 1 (PAR1), which
can act through EGFR, has been found to promote metastasis and
invasiveness in melanoma, breast, colon, and prostate cancers.278,279

Intriguingly, MMP1 was found to function similarly to thrombin
in activating PAR1 and promoting breast cancer tumorigenesis and
invasion.327,328 This cross talk between GPCRs and EGFR provides
a rationale for the combinatorial use of GPCR agonists/antagonists
and EGFR inhibitors in patients with EGFR-driven lung and
colorectal cancers.
Aberrant GPCR signaling through Gα12/13 also contributes to
tumorigenesis by enhancing cancer cell migration, invasion, angio-
genesis, and metastasis.329 Ligand-activated LPA, PAR1, SIP, throm-
boxane A2 (TP), CXC chemokine (CXCR4), and prostaglandin E2
(PGE2) receptors couple to Gα12/13 and RhoGEFs to hyperactivate
RhoA (see earlier), which elicits these progression-associated phenotypes
in glioma, melanoma, lung, breast, and ovarian cancers.278,279 Over-
expression of Gα12/13 and RhoA in breast, prostate, and colon cancers
also promotes metastasis by decreasing cell adhesion.311 RhoGEF
inhibitors; RhoGTPase inhibitors; inhibitors of prenylation, which
could indirectly impair proper Rho localization (statins, farnesyl/
geranylgeranyl transferase inhibitors); and inhibitors of kinases
downstream of Rho (ROCK, LIMK, MRCK, PAK) have all been
developed and tested in biochemical, cell line, and mouse experiments.
However, a clear benefit to the use of such compounds in cancer
patients has yet to be established.330
Two other prominent examples of dysregulated GPCR signaling
in human cancer are the Hedgehog/Smoothened and Wnt/Frizzled
signaling pathways.277,278,331 Both pathways, along with Notch, also
play critical roles in cell fate and have been implicated in the underlying
pathway cross talk that is key to cancer stem cells.332 Secreted Hedgehog
(Hh) ligands (first identified based on their roles in normal development
and stem cell homeostasis, with Sonic Hedgehog [SHH] being the
most ubiquitous) bind to the 12-pass TM receptor Patched (PTCH),
which relieves its repression of the GPCR Smoothened (SMO).333,334
Activated Smoothened couples to Gαi and Gα12, which regulate the
glioma-associated oncogene homologue (GLI) transcription factors,
which in turn regulate proliferative, survival, and differentiation signals
involving cyclin D1, myc, BCL2 and the Forkhead transcription factors,
to name a few.335,336 Mutations in SHH, PTCH, and SMO are found
in patients with inherited and sporadic basal cell carcinomas337,338 and
ameloblastomas,339 whereas overexpression of Hh ligands has been
shown to result in hyperactivation of the pathway in breast, colon,
prostate, and pancreatic ductal adenocarcinomas331,340 Vismodegib and
sonidegib, both selective Smoothened receptor inhibitors, are FDA
approved for the treatment of advanced basal cell carcinomas (see
Table 2.1).341–344 Several other Hh/SMO inhibitors are currently being
tested in patients with cancer, mostly for advanced and/or metastatic
solid tumors.278,345,346
The secreted Wnt glycolipoprotein ligands activate the single
TM low-density lipoprotein–related coreceptors LRP5/6 and the
GPCR-like TM protein Frizzled (Fz), which are phosphorylated and
likely couple to Gαq and Gαo, respectively, to activate the cytoplasmic
scaffold Dishevelled.347–349 Dishevelled in turn inhibits the β-catenin
degradation complex (which consists of APC, axin, CKIα and GSK-3β,
and the E3 ubiquitin ligase β-TrCP).350 This results in accumulation
of β-catenin, which translocates to the nucleus where it induces TCF/
LEF-mediated transcription of genes important for cell differentiation
and proliferation, including myc, cyclin D1, VEGF, FGF4/18, E-cadherin,
COX-2, and members of the Wnt cascade itself.349,351,352 Noncanonic
Wnt/Fz pathways include signaling to the transcription factors
NFAT via Gαq/i/Gβγ/PLC/PKC/Ca+ (Wnt-calcium pathway) and
AP1 via Rho/Rac/JNK (planar cell polarity pathway).277,353 These
pathways regulate cell polarity and migration and are implicated in
cancer metastasis.353 Aberrations in canonical Wnt signaling promote
tumorigenesis in melanoma and colon, liver, ovarian, and prostate
cancers.354,355 Specifically, loss-of-function mutations or truncations
in APC and AXIN1/2 and gain-of-functions mutations in β-catenin
are found in almost all colorectal cancers, with APC alteration found
in over 85%.349,356 Germline mutations in the APC gene are also
the basis for the inherited cancer predisposition syndrome familial
Intracellular Signaling  •  CHAPTER 2 37
adenomatous polyposis (FAP).357 Efforts to develop selective inhibitors
of Wnt signaling are ongoing and will be aided by current endeavors
to crystallize members of the Wnt cascade.354,358 Of note, nonsteroidal
antiinflammatory drugs (NSAIDs) have shown some promise in
modulating Wnt signaling, likely by inhibiting the Wnt-output
gene COX-2 or by enhancing E-cadherin signaling.349,359 COX-2
inhibitors have shown efficacy in reducing the risk of polyps in patients
with FAP.358,360
CYTOKINE RECEPTOR SIGNALING
Cytokines are protein and glycoprotein ligands secreted by immune
cells; they initiate diverse and often opposing effects based on target
cell lineage. Processes regulated by cytokine signaling include cell
proliferation, differentiation, survival, inflammation, angiogenesis,
antiviral activity, and modulation of immune function. Cytokines
signal in an autocrine and/or paracrine fashion and can be subclassified
by protein structure into four families, which total over 100 members:
hematopoietins, IFNs, chemokines, and the TNF superfamily.
The hematopoietin family consists of interleukins (IL-1 to IL-31),
growth hormone, prolactin, erythropoietin, thrombopoietin, leptin,
granulocyte colony-stimulating factor and granulocyte-macrophage
colony-stimulating factor, and a few others.361 The majority bind to
either class I or II cytokine receptors, which are single TM glycoproteins
that lack kinase activity and diverge in their extracellular domains in
order to specify ligand binding.362,363 Class I receptors function as a
cluster of two or three subunits that each have two sets of conserved
cysteine pairs and a WSXWS motif in their external domains. Class
II receptors lack the WSXWS motif and one of the class I conserved
cysteine pairs, but contain conserved proline, tryptophan, and an
additional two conserved cysteine residues. Ligand binding causes
aggregation of the γ-chain (also called γc, CD132) which is common
to many cytokines, and the β-chain (also known as IL-2Rβ, IL-15Rβ,
or CD122).364,365 In the case of specific cytokines, such as IL-2,
association with a third subunit, the α chain (also called IL-2Rα,
CD25, or Tac antigen), allows for high-affinity ligand binding.366,367
The IFN family members are divided into type I, II, and III
classes.368,369 Most IFNs are type I and can be further subtyped.370 All
type I IFNs bind the type I IFN receptor, which consists of two
subunits (IFNAR1 and IFNAR2). The sole type II IFN, IFN-γ, binds
the type II IFN receptor, which is composed of the IFNGR1 and
IFNGR2 subunits. Both types of IFN receptors belong to the class
II cytokine receptor family. The IFN family also includes a third
branch, the IFN-like molecules, IFN-λ1 (IL-29), IFN-λ2 (IL-28A),
and IFN-λ3 (IL-28B), which display some structural overlap with
ILs and the antiviral properties of IFNs, and bind a distinct receptor
made up of IFNLR1/IL-28Rα and IL-10Rβ.371 Given that cytokine
receptors are promiscuous and bind multiple cytokine ligands, there
is a high degree of redundancy in the output profiles of individual
cytokines. This redundancy serves to amplify and sustain signaling
downstream of these transitory stimuli.
Hematopoietin or IFN binding induces oligomerization of cytokine
receptor subunits and autophosphorylation and activation of the JAK
(Janus activated kinase) family of intracellular tyrosine kinases (TYK2,
JAK1–JAK3), which are constitutively bound to box I and box II
α-helix motifs in the receptor cytoplasmic tail.372 Activated JAK proteins
phosphorylate tyrosine residues on the cytokine receptor chains, which
classically bind the STAT (signal transducer and activator of transcrip-
tion) family of transcription factors (STAT1–STAT6).361,373 Docking
of STATs facilitates their phosphorylation by JAKs, which in turn
causes STAT dimerization, nuclear translocation, and alterations in
gene transcription.374 There is also cross talk between STAT signaling
and the nuclear factor–κB (NF-κB) and SMAD signaling pathways.375
STATs are also activated by RTKs, SRC, and ABL, and STAT activation
also plays a role in transformation initiated by these oncogenes.376
JAK/STAT activity is regulated by several posttranslational modifications
as well as by the SOCS (suppressor of cytokine signaling) and PIAS
38 Part I: Science and Clinical Oncology
(protein inhibitor of activated STAT) proteins.375 In addition to
JAK-STAT signaling, cytokine receptors can transduce messages through
LCK and SYK (Src-family kinases), through BCL-2, and via PI3
kinase/AKT and Ras/Raf mediated upregulation of Fos- and Jun-
dependent transcription.376,377
The 29-member TNF superfamily of receptors (TNFSFR1) and
their corresponding 19 ligands (TNFSF) induce inflammation in
addition to prosurvival and proapoptotic phenotypes.378,379 TNF ligands
are TM proteins that function as membrane-integrated or cleaved,
soluble trimers that bind and activate preformed, single-TM TNF
receptor trimers on the cell surface.380 Conserved cysteine residues in
the TNF receptor external domains dictate ligand binding, and the
presence of death domains (DDs), TRAF-interacting motifs (TIMs),
or neither (decoy) dictate downstream signaling. For example, on
apoptotic stimuli, ligand activation of the TNF-R1 and DR3 receptors
recruits the adaptor TRADD (TNFR-associated death domain) via
DDs, which in turn binds to FADD (Fas-associated protein with
death domain).378,379,381 FADD binds procaspase-8 and procaspase-10
via death effector domains (DEDs), and induces their cleavage to
form active enzymes, which cleave caspase-3 and induce apoptosis.
TRADD binding is also capable of inducing the intrinsic apoptotic
cascade (mitochondrial release of ROS, cytochrome C, and Bax, which
leads to caspase-9 and caspase-3 activation and apoptosis).378,382
Under proliferative stimuli, TNF-α–dependent activation of
TNF-R1/TRADD and TNF-R2 converges on the recruitment of
TRAF2 (TNFR-associated factor 2). TRAF2 binding sequentially
recruits the RIP (receptor interacting protein), TAK1 (TGF-β activated
kinase 1), and IκB kinase (IKK) trimer; this functions to degrade
IκB-α (inhibitor of NF-κB-α) and in turn facilitates the activation
and translocation of NF-κB.378,379 NF-κB in turn induces mediators
of inflammation and cytoprotective phenotypes.383 Alternatively, TAK1
can signal to MKK3/6 (MAP kinase kinase 3/6) and to two MAP
kinases, ERK (proproliferation) and p38α (to activate the transcription
factor AP-1 [activator protein-1]).384 Upstream, TRAF2 can additionally
trigger MEKK1 (MAP/ERK kinase kinase 1), MKK7, and JNK (c-Jun
activating kinase), the recruitment of which also converges to activate
AP-1 and thus regulate proliferation and survival.385–387 There is an
important avenue of cross talk between the TNF-directed NF-κB and
JNK pathways, the balance of which ultimately decides cell fate.388
In cancer, dysregulation of cytokine signaling promotes chronic
inflammatory signals and prevents the immune system from attacking
cancer cells. Unfortunately, because of their pleiotropic effects, which
are often cell type and microenvironment specific, therapeutic strategies
that modulate cytokine signaling have demonstrated only modest
clinical activity in patients with cancer. For example, the recombinant
human IFN-α2a mimetic, Roferon-A, has been shown to inhibit
tumor growth in patients with melanoma and hairy cell leukemia,
but such treatments have now been supplanted by more active thera-
pies.389 Moreover, focal delivery of TNF-α to limbs affected by soft
tissue sarcomas and melanomas through isolated limb perfusion
techniques has been more effective than systemic use, which is
toxic.390,391 Proapoptotic, anti-TRAIL therapies, such as mapatumumab,
are also in clinical testing.381
Downstream components of the cytokine signaling cascades are
also being explored as targets for drug development. Gain-of-function
mutations in the JAK2 (hot spot V617F) and MPL genes, the latter
of which encodes the thrombopoietin receptor, are common in
myeloproliferative neoplasms, and the selective JAK1/2 kinase inhibitor
ruxolitinib has been approved for this indication (see Table 2.1).392–394
STAT3 is frequently hyperactivated in cancer, because it is a downstream
effector of both cytokine receptors and mutated and amplified tyrosine
kinases.376,395 Constitutive activation of STAT5 and STAT6 plays a
critical role in BCR-Abl–driven chronic myelogenous leukemia (CML),
and IL-13–driven lymphomas and leukemias.376 On the basis of these
findings, direct inhibitors of JAK and STAT are currently in develop-
ment.374,396 Inhibition of NF-κB has been shown to induce apoptosis
in leukemias and lymphomas and enhance chemotherapy and radiation
response.383 JNK1 is upregulated in hepatocellular carcinoma and
prostate cancer, whereas p38α activity is either lost (in hepatocellular
carcinoma) or activated in numerous cancers, and targeted inhibitors
are being developed.385,397
SERINE/THREONINE RECEPTOR SIGNALING
Receptor serine/threonine kinases are exemplified by the TGF-β type
I and II receptors.398,399 Ligands for these receptors include the TGF-β
superfamily, which comprises the TGF-β1-3 isoforms, activins, inhibins,
Nodal, and Lefty, and the more distantly related bone morphogenic
proteins (BMPs), growth and differentiation factors (GDFs) and
müllerian inhibitory substance (MIS). TGF-β ligands regulate a diverse
array of physiologic processes including growth, proliferation, survival,
hormone release, and differentiation.398,400 They thus have a central
role in embryonic patterning, tissue development, and morphogenesis.
Signaling mediated by TGF-β, the namesake and most studied ligand
of the superfamily, will be used to exemplify the general structure of
the serine/threonine kinase cascades.
TGF-β is ubiquitously expressed and requires a multistep maturation
and secretion process to be functional and bioavailable. Initially
translated as an immature proprotein, the prodomain (called latency-
associated protein [LAP]) is cleaved and noncovalently bound to the
remaining mature form of TGF-β.401 Covalently bound mature, active
dimers are further bound to latent TGF-β binding protein (LTBP)
such that this complex is sequestered by LTBP binding to the extracel-
lular matrix (ECM) until appropriate signals initiate matrix metal-
loproteinase, plasmin, or thrombin-dependent cleavage of LTBP and
subsequent release of active TGF-β dimers.401,402 TGF-β dimers bind
constitutively active, single TM, homodimeric TGF-β type II receptors
(TβRII). Once bound by ligand, TβRII forms a complex with TGF-β
type I (TβRI) receptor homodimers, thus creating a receptor hetero-
tetramer.398,400 TβRII receptors phosphorylate and activate the intrinsic
kinase activity of TβRI, which in turn phosphorylates serine residues
in the C-terminal–SSXS motif of receptor-activated SMAD proteins
(R-SMAD2 and R-SMAD3 for TGF-β).403 Activated R-SMAD2 and
R-SMAD3 then form a heterotrimer with SMAD4, which then localizes
to the nucleus, where it both binds DNA and partners with other
transcription factors (i.e., Forkhead, homeobox, zinc-finger, AP-Ets,
and bHLH family transcription factors) and cofactors (e.g., p300,
CBP).404,405 These SMAD-containing complexes then target selective
promoter elements with high affinity, leading to the induction or
repression of hundreds of genes, depending on cell context. For example,
TGF-β induces the expression of 4EBP1 and the cyclin-dependent
kinase inhibitors INK4B and p21 and represses Myc to elicit growth
inhibitory effects.406 It also activates DAPK (death-associated protein
kinase), GADD45β (growth arrest and DNA damage-inducible 45β),
and BIM to promote apoptosis and PDGF in smooth muscle cells
to enhance proliferation, among other effects.407 TGF-β also signals
through many non-SMAD effectors including Shc, which can result
in enhancement of Ras/ERK signaling, and TRAF6, which activates
the TAK1/MKK3&6/JNK/p38 cascades.408 TGF-β, through indirect
mediators, can also activate Src, Rho, and PI3 kinase, and through
pathway cross talk the Wnt, Hedgehog, and Notch cascades.409 The
outcome of TGF-β–directed transcriptional responses depends on
access of TGF-β to particular signaling receptors and SMAD complexes,
the availability of transcription factors, and the epigenetic status of
the cell.410
Alterations in TGF-β signaling are common in cancer.407,411,412
For example, mutational inactivation of TβRII is seen in colorectal
cancers with microsatellite instability.413 Mutations and loss of SMAD4
expression are common in colorectal, pancreas, and head and neck
cancers.414,415 Conversely, increased TGF-β expression occurs in breast,
prostate, and colorectal cancers and has been associated with cancer
progression and the development of metastases.407 TGF-β has also
been shown to play a role in maintaining the tumor-initiating cell
or cancer stem cell population in gliomas, leukemias, and breast

cancer.407 It has been proposed that TGF-β signaling promotes
invasion and metastasis by promoting EMT.406 Interesting to note,
in pancreatic ductal adenocarcinoma, wherein loss of SMAD4 is
common, TGF-β can play a novel tumor suppressor role by inducing
a lethal EMT through the lineage and progenitor regulators Klf5
and Sox4.416
Intense efforts are underway to develop therapeutic strategies that
inhibit the tumorigenic properties of TGF-β signaling. These include
the development of selective TGFβRI inhibitors, TGF-β blocking
antibodies, soluble TGF-β antisense therapies, and selective kinase
inhibitors.411,417,418 The development of inhibitors of TGF-β signaling
has, however, been confounded by the ability of TGF-β to both promote
and suppress tumor progression in a context-specific manner.419,420
NOTCH RECEPTOR SIGNALING
The mammalian Notch receptors, Notch1 to Notch4, are single-pass
TM receptors that are functionally unique with regard to receptor
processing and signal activation and transduction.421–424 Notch receptors
are translated as immature receptors that undergo S1 cleavage by
furin-like convertase during Golgi trafficking.425 This generates two
subunits that are retethered at the cell membrane by noncovalent
bonds in the heterodimerization domain.423,426 The extracellular domain
subunit consists of ligand-binding EGF-like repeats, a heterodimer
domain, three negative regulatory LIN12 and Notch repeats (LNRs)
containing numerous cysteines, and a hydrophobic TM-interacting
region. The other subunit contains the TM domain and the intracellular
domain, which has ankyrin repeats and a RAM domain central to
the Notch receptor’s unusual activity as a direct transcription factor,
and a PEST motif important for the quick termination of Notch
activity and ubiquitin-mediated degradation.
Delta-like ligand (DLL1, DLL3, DLL4) and Jagged (JAG1 and
JAG2) are the five TM-protein ligands for the Notch receptors.427 A
Notch receptor on one cell binds to DLL or JAG ligand on an adjacent
cell. This results in a change in Notch receptor conformation, which
facilitates a series of proteolytic cleavages required for receptor activation.
First, the ADAM17/TACE (a disintegrin and metalloprotease-17/
TNF-α converting enzyme) metalloprotease performs S2 cleavage of
the extracellular domain.428,429 This allows subsequent S3 cleavage of
the TM domain by the γ-secretase complex and release of the Notch
intracellular domain (NICD).430 The active NICD translocates to the
nucleus, where it binds to and converts the repressor complex CSL
(CBF1, Suppressor of Hairless, Lag-1) into a transcriptional activator
that recruits coactivators including the Mastermind-like family proteins
and p300. NICD target genes include the HES (hairy enhancer of
split) and HRT/HEY (hair-related transcription factor) transcriptional
repressors, as well as cyclin D1, myc, p21, NF-κB, and the Notch
receptors and ligands themselves, among others.422,426,431 Notch is also
heavily involved in cross talk with other pathways, including RTKs,
PI3 kinase, Ras/ERK, JAK/STAT, Wnt, Hedgehog, TGF-β/SMAD,
and p53. Overall, Notch plays a role in proliferation, cell fate determina-
tion in development, and survival.
Notch’s causal role in tumorigenesis was established on its discovery
in 1991, when a translocation event in T-cell acute lymphoblastic
leukemia or lymphoma (T-ALL) was identified that fused the T-cell
receptor-β promoter/enhancer elements to Notch, generating a
truncated form of Notch1 that was constitutively nuclear and active.432
Activating NOTCH1 mutations have since been found to occur in
approximately 60% of T-ALL patients.433 Subsequent studies have
determined that Notch and its ligands have both oncogenic and tumor
suppressor properties in several hematologic and solid tumor
malignancies, including melanoma, glioblastoma, and breast, lung,
colorectal, and pancreatic cancers, depending on cellular context.423,426
Thus far, efforts to develop inhibitors of Notch signaling have focused
primarily on inhibiting cleavage, and thus activation of Notch.
Specifically, selective γ-secretase inhibitors (GSIs), such as MK-0752,
PF03084014, and BMS-906024, are currently being tested in early-stage
Intracellular Signaling  •  CHAPTER 2 39
clinical trials.422,434–436 Newer avenues of drug development include
monoclonal antibodies targeting Notch receptors or ligands.437
NUCLEAR HORMONE RECEPTOR SIGNALING
The nuclear hormone superfamily is composed of hormone receptors
and orphan receptors (receptors for which no ligand has yet been
identified). The nuclear hormone receptors are characterized structurally
by a ligand binding domain, a DNA binding domain, and a hinge
region that connects the ligand and DNA binding domains.438 Nuclear
hormone receptors are classified into four subtypes based on ligand
specificity: steroid, retinoid X receptor (RXR), monomeric or tethered
orphan receptors, and dimeric orphan receptors.439 The steroid receptor
ligands include estrogen, progesterone, androgen, and growth hormone.
Ligand binding occurs in the cytoplasm and results in receptor
homodimerization followed by nuclear translocation. Once translocated
into the nucleus, the ligand-bound receptor acts as a transcription
factor that modulates the expression of several downstream proteins
through binding to steroid response elements, which are conserved
nucleotide sequences within the regulatory regions of genes.440 In
contrast to the steroid receptors, the RXR receptors form heterodimeric
complexes with other partners, including the retinoic acid receptor,
the thyroid hormone receptor, and vitamin D receptors.
Hormone receptor blockade is a cornerstone in the treatment of
estrogen receptor (ER)– and progesterone receptor–expressing breast
cancers. The selective estrogen receptor modulator (SERM) tamoxifen
competes with estradiol for binding to ER. Notably, tamoxifen binding
results in ER dimerization, nuclear translocation, and receptor binding
to estrogen response elements in the promoter regions of estradiol-target
genes. It is thought that the ER/tamoxifen complex recruits transcrip-
tional corepressors, in contrast to ER/estradiol binding, which recruits
transcriptional coactivators.440,441 Although tamoxifen has antiprolifera-
tive effects in ER-expressing breast cancer cells, it causes hypertrophy
and neoplastic transformation of endometrial tissue, likely as a result
of cell type and context-specific recruitment of transcriptional coactiva-
tors that are differentially expressed in these tissues.440 Recently,
mutations in the ESR1 gene, which encodes the ER, have been shown
to be a common mechanism of acquired resistance to hormonal therapy
in patients with breast cancer.442 Retrospective studies suggest that
patients with ESR1-mutant breast cancer may have more durable
responses to the selective estrogen receptor downregulator (SERD)
fulvestrant than those treated with the aromatase inhibitor exemestane
(see Table 2.1).442,443
Dihydrotestosterone is the primary ligand of the androgen receptor
(AR). AR blockade by antiandrogens such as bicalutamide is a com-
monly used therapeutic modality for patients with locally advanced
or metastatic prostate cancer. Bicalutamide competes with dihydrotes-
tosterone for binding to cytoplasmic AR.444 The mechanism by which
bicalutamide-bound AR inhibits androgen-dependent gene transcription
is unclear but may involve the recruitment of transcriptional corepressors
as well as histone modifications that lead to tighter chromatin binding
and therefore reduced access to promoter regions by transcription
factors. Enzalutamide, a nonsteroidal small-molecule antagonist of
the AR that inhibits AR nuclear translocation and DNA binding is
FDA approved for the treatment of patients with metastatic prostate
cancer that has progressed following treatment with bicalutamide and
medical castration (see Table 2.1).445 Abiraterone acetate, an irreversible
inhibitor of CYP17A1 that enables intratumoral and adrenal androgen
depletion, also has significant clinical activity in patients with castrate-
resistant prostate cancer.446
INTEGRIN RECEPTOR SIGNALING
The integrin receptor family regulates cell adhesion, migration, invasion,
and cell survival.447,448 Integrin receptors are heterodimeric molecules
consisting of combinations of alpha and beta subunits. Each combina-
tion dictates the spectrum of ECM components to which these receptors
40 Part I: Science and Clinical Oncology
bind. Once ligated to the ECM, the receptors recruit multiple proteins
to the cell membrane, including cytoskeletal molecules such as paxillin
and vinculin that form focal adhesions to ECM components.449 Unlike
the RTK family, integrin receptors do not possess intrinsic kinase
activity but rather promote signaling by facilitating the activation of
kinases such as Src or FAK.450 Integrins are also unique in that they
participate in bidirectional signaling. “Inside-out” signaling occurs
when intracellular adapters, of which talin-1 and kindlin-1/2/3 are
the best known activators, trigger a conformational change of the
cytoplasmic tails of the alpha and beta subunits, which is transduced
to the extracellular component of the receptor, resulting in increased
affinity for portions of the ECM.451 Conversely, “outside-in” signaling
involves binding of ligand to integrins, which stimulate the activation
of multiple intracellular signaling pathways.452 Collectively, the term
integrin adhesome is used to describe the site at which integrins initiate
contact with the ECM and other cells and recruit all of the underlying
intracellular machinery (e.g., cytoskeleton, scaffolds, signaling adapters—
over 200 intrinsic and transient components) to generate diverse types
of adhesions (e.g., focal complexes, focal adhesions, fibrillar adhesions,
podosomes, invadopodia).453
Integrins are expressed on cancer cells and have been shown to
promote disease progression.447,450,454 Integrins are also present on
stromal cells, including pericytes (which promote endothelial cell
growth and proliferation and thus angiogenesis) and fibroblasts, where
they influence the surrounding microenvironment and thus indirectly
stimulate tumor growth and proliferation. For example, vascular cell
adhesion molecule 1 (VCAM1) is expressed on pericytes and binds
to the integrin receptor α4β1, which is found on the surface of
endothelial cells, resulting in pericyte recruitment to sites of vascular
maturation.455 Integrin signaling also plays a role in the activation of
matrix metalloproteinase 2,456 which promotes cell invasion, and has
been shown to regulate cyclin D and cyclin-dependent kinase inhibitor
expression, thereby controlling cell cycle progression.457 Finally, integrin
receptor activation can lead to increased secretion of growth factors,
which then stimulate tumor invasion through autocrine and paracrine
mechanisms.456
Tumors that express integrin receptors include melanomas, glio-
blastomas, and breast cancers. In melanoma, the αvβ3 and α5β1
integrin receptors promote vertical growth and metastatic spread to
lymph nodes.458,459 In glioblastoma, αvβ3 and αvβ5 are expressed
mainly at the edge of tumors, suggesting a role in tumor invasion.460
The expression of the α6β4 and αvβ3 integrin receptors in breast
cancer is associated with higher grade and tumor size,461 the development
of bone metastases,462 and decreased survival.463 To date, drug develop-
ment has targeted three integrins—αIIbβ3, α4β1, and α4β7—in the
context of platelet activation and blood clotting, multiple sclerosis,
and inflammatory bowel disease.451 Clinical trials of monoclonal
antibodies that target integrin receptors are underway in several cancer
types. For example, etaracizumab, a humanized monoclonal antibody
targeting αvβ3 integrin, is being tested in solid tumors and has shown
activity in patients with metastatic melanoma.464 Cilengitide, an
inhibitor of the αvβ3 and αvβ5 integrins, inhibited angiogenesis and
tumor cell proliferation in preclinical studies; however, a phase III
trial in combination with temozolomide and radiation in patients
with glioblastoma did not result in improved outcomes compared
with chemoradiation alone.456,465
NON–RECEPTOR TYROSINE
KINASE SIGNALING
SRC Signaling
The SRC family of intracellular, non-RTK proteins is composed of
11 members (SRC, FYN, YES, Blk, Yrk, Frk/Rak, Fgr, Hck, Lck,
Srm, and Lyn).466 They share common structural features including
the so-called SRC-homology (SH) domains 1 to 4. The SH1 domain
includes the kinase domain. Only SRC, FYN, and YES are expressed
ubiquitously, whereas the tissue distribution of the latter six is more
restricted.467 Together, SRC family kinases have pleiotropic roles in
cellular proliferation, apoptosis, differentiation, motility, adhesion,
angiogenesis, and immunity.468,469
SRC is by far the most intensively studied family member and
was the first gene observed to have oncogenic potential.470 Peyton
Rous was awarded the Nobel Prize for a series of experiments showing
that a transmissible factor was present in avian sarcomas capable of
initiating tumors in recipient birds. Five decades later the viral oncogene
v-Src was identified as the oncogenic factor in the Rous sarcoma
virus.471–473 Bishop and Varmus later showed that v-Src was a mutant
form of the cellular proto-oncogene c-Src, and Hunter and colleagues
showed that its transformative capacity was dependent on its tyrosine
kinase activity.14,15,474
SRC is regulated in a number of ways. First, SRC has a myristoylation
site in its N-terminus that is necessary for membrane localization and
that promotes its interaction with nearby membrane-bound effectors.475
The SH2 and SH3 domains facilitate protein-protein interactions and
conformational changes in the protein. Inactive SRC is maintained
in a closed conformation with phosphorylated Y530 (mediated by
CSK, C-terminal SRC, and Csk homology kinases) interacting with
the folded-over SH2 domain.476 The closed, inactive confirmation
of SRC is further stabilized by proline-rich segments of the kinase
domain associating with the SH3 domain.477 SRC activation requires
dephosphorylation of Y530, likely by PTPα/γ/1β (protein tyrosine
phosphatase α/γ/1β) or SHP1/2 (SH-containing phosphatases), which
allows the kinase to assume an open conformation.466,478 Autophos-
phorylation of Y419 in the activation loop of the kinase domain also
promotes full activity,479 whereas binding of FAK and CRK-associated
substrate (CAS) to the SH2 domain induces SRC activation and links
SRC signaling to the regulation of focal adhesion, actin reorganization,
and migratory phenotypes.480,481 SRC is a downstream mediator of
numerous receptor families including RTKs, integrin receptors, hormone
receptors, cytokine receptors, and GCPRs and promotes signaling
through the PI3 kinase/AKT, Ras/MAP kinase, and JAK/STAT cascades,
among others.466,467,469,478,482–486 More than two decades of research
have uncovered numerous SRC substrates, including p85-cortactin,
p110-AFAP1, p130Cas, p125FAK, and p120-catenin.487
Although mutations in SRC are rare in human cancers, SRC is
frequently activated as a consequence of other mutational events in
colorectal, breast, esophageal, gastric, pancreatic, hepatocellular, ovarian,
and lung cancers.466 In colorectal and hepatocellular carcinomas, SRC
activation occurs in the setting of concomitant loss of CSK.488–490 Newer
signaling discoveries have identified roles for SRC in promoting tissue
repair after intestinal inflammatory injury, as seen in inflammatory bowel
diseases and colorectal cancer, via the IL-6 cytokine coreceptor gp130
and YAP.491 Furthermore, norepinephrine-mediated, β-adrenergic/PKA
activation of SRC has recently been shown to enhance tumor cell
migration, invasion, and growth.492 The tyrosine kinase inhibitor
dasatinib, which is used in the treatment of CML and Philadelphia
chromosome–positive acute lymphoblastic leukemia (ALL), inhibits
SRC family kinases, in addition to BCR-ABL, KIT, Ephrin A2 receptor,
and PDGFR (see Table 2.1).493–495 Additional dual SRC/ABL and SRC
selective inhibitors are approved in CML (bosutinib, ponatinib; see
later section on ABL signaling) or are in clinical testing, including
saracatinib, XL-228, KX2-391, and DCC2036. Most have shown
limited single-agent activity and are being developed as combination
therapies.469,478,484–486
ABL Signaling
The ABL gene was first identified in 1980 as the oncogene responsible
for driving the Abelson murine leukemia virus, and later was found
to be part of the translocations found in many types of human leu-
kemias.496 ABL1 and ABL2 isoforms have both overlapping and
divergent functions. ABL is found in an autoinhibited conformation
dictated by clamping of the N-terminal hydrophobic region, SH3

and SH2 domains onto the C-lobe of the kinase domain.497 Inter-
molecular interactions with adaptors such as RIN1 or phosphorylation
by SRC, among others, promote stabilization of the open, active form
of ABL 23842626. The ABL tyrosine kinase is found in both the
cytoplasm and the nucleus, and its function varies based on subcellular
localization.498 Cytoplasmic ABL has been implicated in G1/S check-
point regulation and interaction with actin on C-terminal binding
sites,499 whereas nuclear ABL inhibits binding of the DNA repair
protein Rad51 to sites of DNA damage.500,501 Activated ABL kinases
are now recognized to play an important role in tumor initiation by
disrupting cell polarity and by promoting invasion and metastasis by
regulating invadopodia.502
In cancer, translocation of the ABL gene on chromosome 9 with
the breakpoint cluster region (BCR) gene on chromosome 22 results
in the expression of a BCR-ABL fusion protein.503 This translocation,
the Philadelphia chromosome, is found in almost all CMLs and
represents the pathognomonic molecular lesion in this disease.
BCR-ABL translocations are also found in approximately one-third
of acute lymphoblastic leukemias (ALLs).504,505 In addition, at least
eight other fusion partners of ABL have been discovered.502 The
realization that the proliferation and survival of CML cells is critically
dependent on the ABL fusion protein led to the development of
imatinib, an inhibitor of the ABL tyrosine kinase.505 To date, there
are five FDA-approved inhibitors for CML: imatinib, dasatinib,
bosutinib, nilotinib, and ponatinib (see Table 2.1).506 Moreover, imatinib
and dasatinib are approved for treatment of ALL.507–511 A secondary
mutation in the gatekeeper site T315I is the most common reason
for acquired resistance to ABL kinase inhibitors. Combination of
ATP-competitive and allosteric inhibitors such as GNF-5 may be a
novel strategy to combat resistance.512
RAS/MAP KINASE PATHWAY SIGNALING
First identified over 30 years ago as the oncogenes responsible for the
transforming potential of the Harvey and Kirsten murine sarcoma
retroviruses (Ha-MSV and Ki-MSV), RAS proteins are guanine
nucleotide binding proteins.513–517 Ras proteins have intrinsic GTPase
activity and cycle between inactive GDP- and active GTP-bound
states. GDP/GTP exchange thus allows Ras proteins to function as
binary molecular switches.
In the human genome, there are three RAS genes, which encode
four homologous proteins (HRas, NRas, and the alternative splice
variants KRAS4A and KRAS4B) with highly conserved N-terminal
and variable C-terminal regions. Following stimulation of cells by
serum growth factors, cytokines, hormones, and neurotransmitters,
Ras undergoes a series of C-terminal (C186AAX) posttranslational
modifications that result in its localization to specific membrane
microdomains.518 Membrane localization is required for the transforming
properties of Ras, because mutation of Ras at C186 results in cytosolic
localization and protein inactivation whereas Ras activity can be rescued
by myristoylation, which promotes membrane localization.519–521 GEFs
promote Ras activation by binding to GDP-bound Ras and facilitating
the release of GDP and the binding of GTP. SOS1, RAS-GRF (dual
specificity GEFs for RAS and RAC), and RAS-GRP (stimulated by
DAG/phorbol esters and Ca+) are the mostly highly characterized
RAS-GEFs.522–524 Using RTK stimulation as an example, ligand binding
induces RTK dimerization and autophosphorylation of tyrosine residues
in the receptor cytoplasmic tail. Phosphotyrosine docking sites recruit
scaffold proteins such as SHC and permit interaction with the SH2
domains of adaptor proteins such as Grb2.525 Grb2 in turn recruits
SOS1 via its SRC homology 3 (SH3) domain, thereby positioning
SOS near membrane-anchored RAS (see Fig. 2.1).525–529 SOS1 in turn
activates RAS via its CDC25 homology (RASGEF) domain and
N-terminal RAS exchanger motif (REM or RASGEFN domain).
Ras inactivation is catalyzed by GTPase-activating proteins
(GAPs), which enhance the intrinsic GTPase activity of Ras proteins
by 100,000-fold.530 RAS GAPs, which include p120-RASGAP,
Intracellular Signaling  •  CHAPTER 2 41
neurofibromin (NF1), GAP1IP4BP, and CAPRI, negatively regulate
RAS activity and thus function as tumor suppressors.514,518
Binding of GTP to RAS induces conformational changes in the
switch I (loop 2 residues 30–38) and switch II (helix 2 and loop 4
residues 60–76) domains, which facilitate the association of RAS with
regulators and downstream effectors.531,532 RAS directly interacts with
over 20 effector proteins, of which the RAF kinases, PI3 kinases, and
RALGDS are the best characterized (see Fig. 2.1). The canonic RAS/
RAF/MEK/ERK (classic MAP kinase) cascade is by far the most
extensively characterized RAS effector pathway. This prototype of a
three-tiered kinase signaling cascade exemplifies numerous RAS-
dependent mitogen-activated protein kinase (MAPK) cascades that
respond to diverse signals, including cell stress and cytokine signaling
(see section on cytokines for details of the JNK and p38 pathways).533
The RAF protein family (which represent the top-tier MAPK kinase
kinases [MAPKKKs], or MEK kinases [MEKKs]) is composed of
three differentially expressed isoforms, A-RAF, B-RAF, and C-RAF
(RAF-1).534,535 RAF, via its RAS binding domain (RBD) and cysteine
rich domain (CRD), interacts with GTP-bound Ras.536–539 Binding
of RAF to GTP-bound RAS results in RAF localization to the plasma
membrane and its subsequent phosphorylation and activation.540 The
mechanisms of RAF-1 and B-RAF phosphorylation and activation
are distinct and are derived from the summation of signaling inputs
from small G proteins (RAS, RAC, CDC42, RAP-1), kinases (activating
inputs: SRC/PAK/PKC, inhibitory inputs: PKA/AKT/SGK), isoform
homodimerization and heterodimerization, phosphatases (PP2, PP2A),
scaffolding proteins (KSR, RKIP, HSP90, and so on) and cofactors
(14-3-3), with phosphorylation events regulating critical aspects of
RAF activation.535,541 In brief, RAS binding to RAF-1 releases 14-3-3,
a negative regulator that binds to basally phosphorylated residues
S259 and S261 and sequesters RAF-1 in the cytosol in a closed,
inactive conformation. Liberation from 14-3-3 exposes the PKA/
AKT/SGK-dependent inhibitory phosphorylation on S259, facilitating
its dephosphorylation by protein phosphatase 2A.542–546 Loss of this
inhibitory phosphorylation primes RAF-1 for RAS, PAK, SRC, growth
factor, and integrin-stimulated activating phosphorylations on S338,
Y341, T491, and S494.547–551 A-RAF is activated similarly to RAF-1.
Notably, B-RAF activation requires fewer steps owing to its constitutive
phosphorylation at S445, a site analogous to S338 in RAF-1.541,552
Binding to RAS-GTP stimulates B-RAF phosphorylation at critical
residues in its activation loop, T599 and S602 (analogous to T491
and S494 in RAF-1).553,554 The B-RAF isoform is the most potent
activator of ERK pathway output.535,552,554
Activated Raf proteins bind and phosphorylate MEK1 and MEK2
(mitogen-activated protein kinase/extracellular signal-regulated kinase
kinases 1 and 2, or MAPKK, or MAP2K) on serines 217 and 221.555
Activated MEK, a dual-specificity threonine/tyrosine kinase, in turn
phosphorylates MAPK/ERK-1/2 (mitogen-activated protein kinases/
extracellular signal-regulated kinases 1 and 2) on threonine 183 and
tyrosine 185, inducing a conformational change, activation, and disso-
ciation from MEK.556 Activated ERK then phosphorylates substrates in
the cytosol (e.g., p90RSK) and in the nucleus (such as the transcription
factors ELK-1, ETS-2, FOS, JUN, ATF-2, AP-1, MYC, CREB1), which
promote proliferation and survival.557,558 Raf-1 has also been shown
to mediate suppression of apoptosis through non–MEK-dependent
interactions with ASK1, MST2, and MEKK1/NF-κB.559
RAS/RAF signaling triggers numerous regulatory mechanisms,
including classic negative feedback loops, which serve to attenuate
pathway output.560 The Sprouty and Sprouty-related EVH1-domain-
containing protein (Spred) proteins (encoded by the SPRY1–4 and
SPRED1–4 genes, respectively) inhibit the cascade at the level of RAS
and RAF activation.561–564 The dual-specificity phosphatases (MKPs/
DUSPs) dephosphorylate MAP kinases, including ERK.565,566 In
addition, increased pathway activity induces ERK-dependent negative
phosphorylations on B-RAF (at S151, S750, T401 and T753) and
on RAF-1 (at S29, S43, S289, S296, S301 and S642) that abrogate
interactions with RAS and homodimer and heterodimer formation.567,568
42 Part I: Science and Clinical Oncology
RAS signaling is further regulated by RKIP (RAF kinase-inhibitory
protein), which disrupts the RAF-1/MEK interaction and IMP (impedes
mitogenic-signal propagation), which represses KSR-dependent scaf-
folding of RAF/MEK, among other functions.534,569–571
In its active, GTP-bound state, RAS alternatively binds to the
p110α catalytic subunit of class I PI3 kinases,572,573 causing activation
of its lipid kinase activity and thereby generating PIP3, which in turn
stimulates the proproliferative and prosurvival kinases PDK1 and
AKT (see section on PI3 kinase signaling for details). RAS-dependent
activation of PI3 kinase can further stimulate RAC, a RHO family
GTPase involved in regulation of actin and NF-κB.513,574,575 A third
major class of RAS effectors is the group of GEFs for RALA and RALB,
namely RALGDS, RGL, and RGL2.576–578 These RAL exchange factors
stimulate phospholipase D and the CDC42/RAC-GAP RAL binding
protein 1 (RALBP1) and inhibit Forkhead transcription factors to
regulate transcription, vesicular trafficking, and cell cycle progression.
Several additional RAS effectors have been identified. These include
(1) PLCε, which generates IP3 and DAG and regulates calcium release
and PKC activation; (2) T-cell lymphoma invasion and metastasis-1
(TIAM1), which facilitates actin reorganization via RAC; (3) AF6,
which contributes to cytoskeletal changes; (4) RIN1, which regulates
endocytosis; and (5) RASSF and NORE1, which have been shown
to regulate apoptosis and cell cycle progression.513,518 Many other
nondirect connections allow RAS to affect the cellular microenviron-
ment, metabolic signaling, autophagy, inflammation, and immune
responses. Overall, the complex effects of RAS activation result in a
diversity of context-dependent phenotypes ranging from cell prolifera-
tion to cell death that are influenced by a wide variety of extracellular
stimuli and intricately woven layers of regulation.
The potent oncogenic effects of RAS signaling are highlighted by
the high prevalence of mutations in the RAS genes and its proximal
downstream effectors.518,579–581 RAS mutations are found in approxi-
mately 30% of all human tumors, the majority of which (85%) occur
in the KRAS isoform. KRAS is frequently mutated in pancreatic cancers
(58%), colorectal and biliary tree cancers (33 and 31%), and non–small
cell lung adenocarcinomas (17%). Mutations in HRAS are most
common in low-grade bladder cancers (11%), whereas mutation in
NRAS is a frequent event in melanoma (18%) and biliary tree cancers
(11%). Mutations that lock RAS in its GTP-bound state confer
oncogenic potential. Point (missense) mutations in residues 12, 13,
61 (exons 2 and 3) generate a constitutively active RAS oncoprotein
by abrogating intrinsic GTPase activity and inhibiting GAP binding.582
Other mutations, including those at residues 117 and 146, contribute
to RAS activation by increasing GDP-to-GTP exchange.583,584 Heritable
germline mutations of several RAS/MAPK pathway components have
been shown to be the underlying cause of the so-called RASopathies
neurofibromatosis type 1 (NF1); Noonan, Costello, and cardiofacio-
cutaneous syndromes; and other developmental disorders.579 More
recently, deep sequencing efforts have identified recurrent somatic
mutations in the RAS-GAP NF1 in glioblastoma585 and melanoma,586,587
as well as in A-RAF (hot spot at S214) in histiocytosis and lung
cancer588–590 and in RAF-1 (hot spot S257).588 Mutations in BRAF
are also common in human cancer and typically occur in a mutually
exclusive pattern with RAS mutations. BRAF mutations have been
identified in approximately 8% of all cancers, most notably in melanoma
(50%–60%) and in papillary thyroid (30%–50%), biliary tree (14%),
colorectal (10%), ovarian (12%), and lung cancers (3%) and hairy
cell leukemia (100%).534,535,559,579,591,592 A single valine to glutamic acid
substitution at residue 600 (V600E) accounts for more than 80% of
all BRAF mutations and renders BRAF an active monomer in settings
of low RAS activity, that is sensitive to RAF inhibition. Other non-
V600E BRAF mutants have been recently identified and characterized
to function as RAS-independent dimers that are insensitive to current
RAF inhibitors, such as vemurafenib, that only effectively inhibit
mutant monomers.593 Activating mutations in MEK1 (MAP2K1) and
MEK2 (MAP2K2) are also present in approximately 1% of human
tumors, more prominently in melanoma and lung cancer, and have
emerged as a mechanism of acquired resistance to RAF inhibition.594,595
Hot spots include mutations at MEK1 residues F53, K57, and P124
and at MEK2 residue F57, which is paralogous to MEK1 F53. ERK
amplification and mutation, although equally rare (approximately 1%
of all tumors), may also emerge as mechanisms of resistance to upstream
MAPK inhibition, including alteration at the hot spot residue E322.596
Given the high prevalence of RAS pathway alterations in human
cancers, significant attention has been directed toward the development
of selective inhibitors of this pathway.560,580,597,598 To date, clinically
effective direct inhibitors of oncogenic RAS have yet to be identified.
The inability to directly target RAS has been attributed to the high
affinity of the RAS-GTP interaction. Extensive efforts were thus directed
toward inhibiting the posttranslation modifications required for RAS
activation. Specifically, inhibitors of the enzyme farnesyltransferase,
which regulates RAS localization, were tested in randomized phase III
trials but were found to be inactive.599 The failure of farnesyltransferase
inhibitors in KRAS-mutant tumors was predicted by the preclinical
observation that geranylgeranyl modification can substitute for farne-
sylation in targeting KRAS and NRAS to the plasma membrane.600,601
Small molecules that irreversibly bind to the mutant cysteine in tumors
with G12C K-RAS have been developed.602 Because this class of
compounds is selective for the G12C mutant, they are a promising
novel approach for a subset of patients with KRAS-mutant tumors.
As an alternative, extensive efforts have focused on the development
of selective inhibitors of key kinase effectors of RAS transformation.
The selective RAF inhibitors vemurafenib and dabrafenib induce tumor
regressions in most patients with BRAF V600E mutant melanoma
and are FDA approved for this indication (see Table 2.1).603–612 Notably,
these agents selectively inhibit RAF signaling in BRAF-mutant tumors
and are thus inactive in RAS-mutant tumors.613 Several mechanisms
of resistance to RAF inhibitors have emerged, including BRAF V600E
splice variants or amplification; loss or mutation of NF1; parallel
pathway activation of RTKs; mutation of PI3K/AKT components;
mutations in NRAS, RAF1, and MEK1/2; and amplification of
MITF.592,614 Sorafenib, a multikinase inhibitor of RAF, VEGFR2, and
PDGFRβ, has been shown to have some clinical efficacy in ARAF-
mutant histiocytosis and NSCLC.588,589,615
The selective MEK inhibitor trametinib is also FDA approved
for use in melanomas with BRAF mutation (see Table 2.1).605,616–619
Furthermore, the combination of a RAF and a MEK inhibitor has
been shown to have greater activity than Raf inhibitor monotherapy
in both preclinical models and in patients with melanoma, including
the combinations of vemurafenib plus cobimetinib620 and dabrafenib
plus trametinib.605,619,621,622 MEK inhibition is also emerging as a
strategy to target NRAS-mutant melanoma,623 colorectal624 and thyroid
cancers,625 NF1-mutant melanoma,587 GBM,626 and neuroblastoma.627
MEK inhibition in combination with immunotherapy, chemotherapy,
radiation, and PI3K and CDK4/6 inhibitors is being investigated
in KRAS-mutant tumors including colorectal and lung cancer (see
Table 2.1).624,628 Furthermore, clinical evidence of activity of the MEK
inhibitors cobimetinib, selumetinib, and trametinib has been revealed
in cases and preclinical reports of MEK1-mutant histiocytosis,589
low-grade serous ovarian cancer,629 NSCLC,594 and melanoma.630
Alternative strategies for inhibiting MAP kinase signaling include
HSP90 inhibitors that induce the degradation of RAF1 and mutant
BRAF.631 Drug development of kinase- and dimerization-targeted ERK
inhibitors (SCH772984, BVD-523, DEL-22379) is also underway and
may provide a downstream alternative when upstream RAF or MEK
inhibitors fail.632–635 Combinatorial approaches are also being actively
pursued, given the modest activity of MEK inhibitors in patients
with RAS-mutant tumors and the frequent co-occurrence of RAS and
PI3 kinase pathway alterations in multiple cancer types.636–638 Given
the recent success of immunotherapy in many cancers, preclinical
evidence supports the triple combination of BRAF and MEK inhibitors
with immunotherapy in BRAF V600E mutant melanoma, despite
substantial liver toxicities described with initial trials with combined
treatment of vemurafenib and ipilimumab.639 In addition, elevated

antitumor immune responses in mouse models of triple negative
breast cancer following combined MEK inhibition and immuno-
therapy suggest that hyperactivate MEK signaling may contribute to
immune evasion.640
Given the prominent role of activated ERK in initiating the
transcription and translation of cell cycle components such as cyclin
D1, inhibitors of cell cycle kinase components have emerged as
an alternative strategy to abrogate the output of MAPK signaling.
CDK4, a critical serine/threonine kinase that functions in an active
complex with cyclin D to phosphorylate RB and stimulate E2F
release and S phase entry, is amplified in liposarcoma. Palbociclib,
an inhibitor of CDK4, is FDA approved for use in postmenopausal
women with ER+, HER2− metastatic breast cancer; a second drug,
abemaciclib, has also show efficacy in this same patient population
(see Table 2.1).641–644
PI3 KINASE/AKT/MTOR PATHWAY SIGNALING
The PI3 kinase/AKT/mTOR pathway is a key regulator of growth
factor–mediated proliferation and survival.645 Several extracellular
growth factors stimulate PI3 kinase by binding to their cognate RTKs
or GPCRs.646 Activated PI3 kinase phosphorylates the 3-OH group
of the inositol ring of phosphatidylinositol, catalyzing the conversion
of PIP2 to PIP3. PIP3 then binds to the pleckstrin homology domain
(PH domain) of multiple proteins, facilitating their recruitment to the
plasma membrane and thus regulating their function (see Fig. 2.2).
PI3 kinases are grouped into class I, II, and III kinases based on
their structure and substrate preferences, although class II and III
kinases have been much less studied. Class I PI3 kinases are subdivided
into two groups, IA and IB. Class IA comprises the PIK3CA, PIK3CB,
and PIK3CD genes, which encode the catalytic p110α, p110β, and
p110δ subunits, respectively. These p110 components heterodimerize
with a regulatory subunit (p85), of which there are five isoforms and
splice variants encoded by the PIK3R1-3 genes. Together, the p110/
p85 complex functions primarily in the generation of PIP3.647,648 The
PIK3CG gene encodes the class IB p110γ isoform, which couples
with p101 (PIK3R5) or p87 (PIK3R6) regulatory subunits. Notably,
p110δ/γ catalytic subunits have specialized expression patterns mainly
in leukocytes, whereas p110α/β are ubiquitous.649 Analogous to the
activation of the Ras pathway detailed previously, the p85 regulatory
subunit of PI3 kinase associates with phosphorylated tyrosine residues
located on the intracellular domains of RTKs through an SH2 domain,
resulting in allosteric activation of the p110 catalytic subunit. PI3
kinases can also be activated indirectly through the adaptor protein
Grb2 following its association with the scaffolding protein GAB1.
PI3 kinase pathway activity is negatively regulated by the tumor
suppressor PTEN (phosphatase and tensin homolog), which is a dual
lipid and protein phosphatase that dephosphorylates PIP3, converting
it back to PIP2.650,651
The best characterized effectors of PI3 kinase are the three members
of the serine/threonine kinase AKT family (AKT1, AKT2, and AKT3).
Both AKT and phosphoinositide-dependent kinase 1 (PDK1) are
recruited by PIP3 to the plasma membrane via their PH domain,
where AKT is phosphorylated at Thr308 by PDK1, resulting in its
activation.652–654 Phosphorylation at a second residue, Ser473, by mTOR
complex 2 (mTORC2) further enhances AKT activity.655 Activated
AKT promotes cellular proliferation, survival, and other phenotypes
through activation of multiple downstream effectors. Proliferative
effects are regulated through phosphorylation and inhibition of GSK3β,
which phosphorylates cyclin D1 and marks it for degradation; FOXO4,
a transcription factor that regulates the expression of the CDK inhibitor
p27; and p21, a second CDK inhibitor that, on phosphorylation,
translocates from the nucleus to the cytoplasm, where it regulates cell
survival.656–659 In sum, these actions promote the expression of cyclin
D1, which drives progression of cells through the cell cycle and the
downregulation of cyclin-dependent kinase inhibitors that inhibit cell
cycle progression.
Intracellular Signaling  •  CHAPTER 2 43
AKT-mediated antiapoptotic effects occur through phosphorylation
and inhibition of Bad, which negatively regulates the antiapoptotic
protein Bcl-xL; caspase-9, a proapoptotic protease; and the FOXO1
transcription factor which regulates the expression of proapoptotic
genes.660–662 AKT also controls cell survival by upregulating NF-κB
activity through phosphorylation and activation of Iκb kinase, which
marks Iκb, an inhibitor of NF-κB, for degradation.663,664 Once released
from IκB, NF-κB translocates into the nucleus, where it regulates a
multitude of genes involved in cell survival. AKT also phosphorylates
and activates Mdm2, an E3 ubiquitin ligase that binds to the pro-
apoptotic tumor suppressor p53 and directs it for proteasomal
degradation.665
AKT-independent effectors of PI3 kinase activation have also been
identified and likely play important roles in the development and
progression of some cancers. These include CDC42 and Rac1, which
are involved in motility and reorganization of the cytoskeleton, and
the serum glucocorticoid kinase (SGK) family of serine/threonine
kinases, which promote cell survival.666,667
Many of the canonic functions of AKT with regard to cell growth
and proliferation are mediated through the mTOR pathway. mTOR
is a serine/threonine kinase and a member of the phosphatidylino-
sitol kinase-related kinase family.668 mTOR is a component of two
complexes, the rapamycin-sensitive mTORC1 and the rapamycin-
insensitive mTORC2 complexes.669 Within mTORC1, mTOR
associates with Raptor (regulatory associated protein of mTOR) and
mLST8, whereas the mTORC2 complex consists of mTOR, Rictor
(rapamycin insensitive companion of mTOR), SIN1, and mLST8.670
In addition to its role in activating AKT as described earlier, mTORC2
also controls cytoskeletal changes through regulation of paxillin, Rho,
Rac, and PKCα.671
mTORC1 activation is regulated in part by AKT phosphorylation
of TSC2. TSC2 forms a heterodimeric complex with TSC1 that acts
as a GTPase-activating protein (GAP) for the small GTPase Rheb,
causing accumulation of inactive Rheb-GDP.672,673 TSC2 phosphoryla-
tion results in suppression of this GAP activity and subsequent activation
and accumulation of Rheb-GTP, which then activates mTORC1.
mTORC1 serves as a central nexus for integration of multiple extracel-
lular signals, including oxygen and amino acid levels, growth factors,
and stress. Based on these input signals, mTORC1 activity influences
cellular growth, metabolism, protein synthesis, and cell cycle progres-
sion. One such example is the energy sensing mechanism of the cell
comprised of LKB1 and AMPK.674 Increasing levels of AMP, a marker
of decreased nutrient levels, results in AMPK phosphorylation and
activation by LKB1. AMPK phosphorylates TSC2, which, as described
earlier, inhibits mTORC1 activity, leading to downregulation of protein
synthesis and cell growth in response to low nutrient levels.675 In part,
mTORC1 activation regulates these phenotypes via phosphorylation
and activation of p70S6 kinase and inhibition of 4EBP1. The former
protein stimulates mRNA translation, whereas the latter inhibits
translation of mRNA transcripts with a 5′ cap.676
Both mTORC1 and S6 kinase also participate in a negative feedback
loop in which both proteins activate insulin receptor substrate 1 (IRS1),
which results in inhibition of insulin-mediated PI3 kinase activation.677
AKT can also activate mTORC1 in a TSC2-independent manner via
phosphorylation of PRAS40, a protein that interacts with mTORC1.
The mechanisms responsible for PI3 kinase pathway activation in
cancer are diverse and include activating mutations and amplification
of PIK3CA, AKT1, AKT2, and AKT3, and mTOR; deletion or loss
of PTEN expression or function; mutation in PIK3R1; loss of TSC1
or TSC2 function; RAS mutation; and dysregulated growth factor
receptor and integrin activation, as outlined later.645,648,678 In human
tumors, activation of PI3 kinase is frequently a direct consequence
of dysregulated RTK signaling secondary to mutation, amplification,
or ligand overexpression. For example, ERBB2 amplification in breast
and gastric cancer results in AKT activation.27,678 Similarly, kinase
domain mutations of EGFR induce constitutive AKT activation in
lung cancers and glioblastomas, and AKT activity in these tumors
44 Part I: Science and Clinical Oncology
is critical for EGFR-mediating transformation.637,679 Oncogenic
Ras mutations also activate PI3 kinase, and PI3 kinase activation is
required for Ras-mediated tumorigenesis in genetically engineered
mouse models.572,680–682
Mutations in the PIK3CA gene, which encodes the p110α catalytic
subunit, are frequently observed in tumors of the colon, breast, brain,
stomach, and ovary and other cancers.648,678,683 The most frequent are
E542K and E545K, located in the helical domain (exon 9), and
H1047R (exon 20), located in the kinase domain.684 All three mutants
demonstrate increased lipid kinase activity, induce phosphorylation
of AKT and its downstream effectors, and can transform chicken
embryo fibroblasts.685 Exon 9 helical domain mutations block the
inhibitory interaction between the p85 regulatory subunit and the
p110 subunit and result in constitutive kinase activation.686 Exon 20
catalytic domain mutations result in constitutive kinase activation.678
PIK3CA mutations commonly co-occur with KRAS mutations, and
ERBB2 amplification in colon and breast cancers, respectively, and
expression of mutant PI3 kinase in breast cell lines as well as fibroblasts
causes neoplastic transformation.687 Unlike wild-type p110α which
lacks oncogenic potential, wild-type overexpression of the other three
isoforms can induce transformation of cultured cells.688 PIK3R1 encodes
the p85 regulatory subunit of PI3 kinase. Alterations in within this
gene have also been reported in multiple cancers, including glioblas-
tomas and colorectal, endometrial, and ovarian cancers.585,689 A recurrent
PIK3R1 mutation, PIK3R1(R348∗), has been shown to have neo-
morphic functions resulting in activation of MEK and JNK and
sensitivity to inhibitors of these effector kinases.690 Furthermore, partial
loss of the PIK3R1 gene product p85α was demonstrated to increase
the proportion of p110α/p85 heterodimers bound to active receptors,
indicating that targeting p110α-selective inhibitors may be effective
in the setting of PIK3R1 loss.691
Loss of PTEN function is the most frequently observed PI3 kinase/
AKT pathway alteration in human malignancies and is common in
tumors of the prostate, breast, ovary, lung, colon, and bladder as well
as melanomas and glioblastomas.678 Loss of PTEN function in tumors
is mediated by a diversity of mechanisms including mutation, deletion,
posttranslational modification, and promoter hypermethylation.678
Dysregulated expression of microRNAs that target the 3′-untranslated
region of PTEN has also been shown to induce cell survival and
cisplatin resistance.692
As AKT activation enhances proliferation and suppresses apoptosis,
its activation would be predicted to have strong oncogenic func-
tion. Indeed, AKT was initially identified as a proto-oncogene in
the mouse leukemia virus AKT8.693 A recurring hot spot mutation
in the PH-domain of AKT1 (E17K) occurs with low frequency in
breast, colorectal, bladder, endometrial, and ovarian cancers.694–696
This mutation results in constitutive localization to the plasma
membrane without the need for PIP3 recruitment.684 Amplification
of the AKT2 gene has been reported in ovarian and pancreatic cancers,
and gain-of-function AKT3 mutations have been reported to occur in
melanoma.697
Given the significant proportion of malignancies that harbor muta-
tions in the PI3 kinase/AKT/mTOR pathway, a concerted effort is
ongoing to identify selective inhibitors of various PI3 kinase pathway
components. These agents can be categorized as pan-selective or selec-
tive PI3 kinase inhibitors, dual PI3 kinase/mTOR kinase inhibitors,
AKT inhibitors, and mTOR inhibitors. Both isoform-specific and
pan-selective inhibitors of PI3 kinase are being explored in several
clinical trials across cancers. Most notably, the PI3Kδ inhibitor idelalisib
is FDA approved for non-Hodgkin lymphoma and certain types of
leukemia.698,699 Promising clinical responses have also been reported
with α-selective PI3 kinase inhibitors in patients with several cancer
types, most notably breast cancer (see Table 2.1).700–703 Although clinical
activity with AKT inhibitors has been modest in patients, a pan-cancer
basket trial of the ATP-competitive pan-AKT kinase inhibitor AZD5363
demonstrated significant clinical responses in patients with AKT1 E17K
mutant tumors.704
Temsirolimus and everolimus, analogues of rapamycin that inhibit
the mTORC1 complex, are FDA approved for use in patients with
renal cell carcinomas, and everolimus is FDA approved for the treatment
of patients with pancreatic neuroendocrine tumors (see Table 2.1).705–708
Everolimus also has significant clinical activity in patients with sub-
ependymal giant cell astrocytomas that arise in the setting of tuberous
sclerosis, an inherited cancer-predisposition syndrome resulting from
germline mutations in the TSC1 and TSC2 genes.709,710 In metastatic
renal cell carcinoma, patients that benefited from treatment with
everolimus more commonly harbored TSC1/2 or mTOR mutations
(see Table 2.1).711
A complete response to everolimus has been reported in a patient
with metastatic bladder cancer that harbored loss-of-function mutations
in TSC1 and NF2, suggesting that such agents can induce significant
antitumor responses in genetically selected patients.712 Additional
responses to everolimus were observed in TSC1-mutant tumors but
not to the degree of the complete responder, indicating that coaltered
genes likely confer resistance to mTOR inhibitory therapy even in
the setting of a canonic predictive biomarker of response. In sum,
mTOR inhibitors are likely most effective when used in genetically
defined patient populations, with the majority of patients requiring
combination therapies because of the presence of coaltered genes. As
an example of the latter, everolimus in combination with exemestane
was FDA approved for the treatment of advanced hormone receptor–
positive, HER2− breast cancer.713
Resistance to mTOR inhibition is thought to derive from multiple
mechanisms, including activation of parallel signaling networks such
as the MAP kinase pathway.714 Furthermore, inhibiting mTORC1
can preferentially lead to an increase in mTORC2 signaling, and, as
described earlier, mTORC2 enhances AKT activation via phosphoryla-
tion of the serine 473 residue.715 As an alternative approach, potent
mTOR kinase inhibitors are being developed, including RapaLink-1,
and have shown significant preclinical activity in cell lines resistant
to rapamycin and in in vivo models of glioblastoma.716,717
TRANSLATIONAL IMPLICATIONS
As outlined earlier, mutational and epigenetic alterations induce
constitutive activation of a broad array of signaling pathways in human
tumors. In some instances, the growth and survival of tumor cells
have been shown to be dependent on a single signaling pathway
activated by a mutated oncogene or tumor suppressor, a phenomenon
referred to as oncogene addiction.718 In such instances, targeted inhibitors
of such pathways have demonstrated unprecedented clinical activity
in molecularly defined subsets of patients (see Table 2.1). Examples
include imatinib in patients with CML, erlotinib in patients with
EGFR-mutant NSCLC, and vemurafenib in patients with BRAF-mutant
melanoma.30,505,511,603,612,719 Despite these dramatic successes, the majority
of cancer patients have yet to benefit from this approach. Potential
explanations for this lack of benefit include the redundant regulation
of key downstream mediators of transformation by multiple signaling
pathways, the lack of specificity of the drug for the driver alteration,
and intrinsic and acquired drug resistance due to second site mutations
in the target gene or by other mechanisms. Progress in this field has
also been delayed by the continued practice of performing clinical
trials of targeted inhibitors in unselected patient populations.
Recent advances in sequencing methodology have made it feasible
to now prospectively sequence all patients with advanced cancer with
the goal of identifying potentially “actionable” genomic alterations.720
Such prospective sequencing efforts have highlighted several challenges
that have slowed the application of targeted inhibitors in cancer patients.
As one example, most mutations, even in well-characterized cancer
genes, are likely inert passenger mutations. Furthermore, not all
activating mutations respond similarly to targeted inhibitors. For
example, exon 19 EGFR deletions are sensitive to the EGFR kinase
inhibitor erlotinib, whereas exon 20 insertions are intrinsically
resistant.721,722 Given the large number of somatic mutations present
 Intracellular Signaling  •  CHAPTER 2 45

in each human tumor, and the variable biologic and clinical significance
of individual mutant alleles, there is an urgent need for clinical support
tools that will aid clinicians in interpreting molecular tumor profiling
and guiding treatment selection.
A second challenge is that many oncogenic alterations are rare. To
address this challenge, novel clinical trial designs have been formulated
to test the efficacy of targeted inhibitors in patients with defined
molecular events independent of the primary site of disease. Eligibility
for these “basket” studies is based on the presence of a particular
molecular alteration (e.g., BRAF V600E, AKT1 E17K, or an NTRK
fusion) rather than site of tumor origin.268,273,704,723 Although promising
results from such trials highlight the importance of tumor mutational
profile in dictating drug sensitivity, lineage-specific differences also
play a role in determining clinical response to inhibitors of activated
signaling pathways. As an example, most BRAF V600E melanomas
respond to vemurafenib, whereas colorectal tumors with the same
mutation are intrinsically resistant. Resistance in the latter results from
rapid adaptation of the cancer cell resulting in activation of EGFR.724
This phenomenon of adaptive resistance, defined as a rapid reactivation
of parallel signaling pathways after relief of negative feedback signals,
likely abrogates the effects of selective pathway inhibitors in many
cancer types.592,725,726 Because significant drug development efforts are
currently focused on the development of targeted inhibitors of
oncogene-activated signaling pathways, a detailed understanding of
normal physiologic pathways and their dysregulation in cancer will
be critical for the optimal development and clinical application of
targeted kinase inhibitors.
The complete reference list is available online at
ExpertConsult.com.

KEY REFERENCES
1. Chakravarty D, et al. OncoKB: A Precision Oncol-
ogy Knowledge Base. JCO Precis Oncol. 2017;
1–16.
21. Yarden Y, Pines G. The ERBB network: at last,
cancer therapy meets systems biology. Nat Rev
Cancer. 2012;12:553–563.
34. Brennan CW, et al. The somatic genomic landscape
of glioblastoma. Cell. 2013;155:462–477.
38. Nathanson DA, et al. Targeted therapy resistance
mediated by dynamic regulation of extrachromo-
somal mutant EGFR DNA. Science. 2014;343:
72–76.
43. Cristescu R, et al. Molecular analysis of gastric cancer
identifies subtypes associated with distinct clinical
outcomes. Nat Med. 2015;21:449–456.
44. Cancer Genome Atlas Research, N. Comprehensive
molecular characterization of urothelial bladder
carcinoma. Nature. 2014;507:315–322.
46. Bose R, et al. Activating HER2 mutations in HER2
gene amplification negative breast cancer. Cancer
Discov. 2013;3:224–237.
50. Tebbutt N, Pedersen MW, Johns TG. Targeting the
ERBB family in cancer: couples therapy. Nat Rev
Cancer. 2013;13:663–673.
62. Janne PA, et al. AZD9291 in EGFR inhibitor-
resistant non-small-cell lung cancer. N Engl J Med.
2015;372:1689–1699.
78. Swain SM, et al. Pertuzumab, trastuzumab, and
docetaxel for HER2-positive metastatic breast cancer
(CLEOPATRA study): overall survival results from a
randomised, double-blind, placebo-controlled, phase
3 study. Lancet Oncol. 2013;14:461–471.
103. Gombos A, Metzger-Filho O, Dal Lago L, Awada-
Hussein A. Clinical development of insulin-like
growth factor receptor-1 (IGF-1R) inhibitors:
At the crossroad? Invest New Drugs. 2012;30:
2433–2442.
106. Takeuchi K, et al. RET, ROS1 and ALK fusions in
lung cancer. Nat Med. 2012;18:378–381.
117. Shaw AT, et  al. Alectinib in ALK-positive,
crizotinib-resistant, non-small-cell lung cancer: a
single-group, multicentre, phase 2 trial. Lancet Oncol.
2016;17:234–242.
150. Obata Y, et al. Oncogenic signaling by Kit tyrosine
kinase occurs selectively on the Golgi apparatus
in gastrointestinal stromal tumors. Oncogene.
2017;36:3661–3672.
169. Javidi-Sharifi N, et al. Crosstalk between KIT
and FGFR3 promotes gastrointestinal stromal
tumor cell growth and drug resistance. Cancer Res.
2015;75:880–891.
182. Babina IS, Turner NC. Advances and challenges
in targeting FGFR signalling in cancer. Nat Rev
Cancer. 2017;17:318–332.
196. Singh D, et al. Transforming fusions of FGFR
and TACC genes in human glioblastoma. Science.
2012;337:1231–1235.
210. Jordan EJ, et al. Prospective comprehensive molecular
characterization of lung adenocarcinomas for effi-
cient patient matching to approved and emerging
therapies. Cancer Discov. 2017;7:596–609.
217. Goel HL, Mercurio AM. VEGF targets the tumour
cell. Nat Rev Cancer. 2013;13:871–882.
249. Frampton GM, et al. Activation of MET via diverse
exon 14 splicing alterations occurs in multiple tumor
types and confers clinical sensitivity to MET inhibi-
tors. Cancer Discov. 2015;5:850–859.
252. Paik PK, et al. Response to MET inhibitors in
patients with stage IV lung adenocarcinomas
harboring MET mutations causing exon 14 skipping.
Cancer Discov. 2015;5:842–849.
266. Stransky N, Cerami E, Schalm S, Kim JL, Lengauer
C. The landscape of kinase fusions in cancer. Nat
Commun. 2014;5:4846.
268. Drilon A, et al. A Next-generation TRK kinase
inhibitor overcomes acquired resistance to prior
TRK kinase inhibition in patients with TRK
fusion-positive solid tumors. Cancer Discov. 2017.
278. Lappano R, Maggiolini M. G protein-coupled
receptors: novel targets for drug discovery in cancer.
Nat Rev Drug Discov. 2011;10:47–60.
322. O’Hayre M, et al. The emerging mutational land-
scape of G proteins and G-protein-coupled receptors
in cancer. Nat Rev Cancer. 2013;13:412–424.
334. Briscoe J, Therond PP. The mechanisms of Hedgehog
signalling and its roles in development and disease.
Nat Rev Mol Cell Biol. 2013;14:416–429.
344. Migden MR, et al. Treatment with two different
doses of sonidegib in patients with locally advanced
or metastatic basal cell carcinoma (BOLT): a mul-
ticentre, randomised, double-blind phase 2 trial.
Lancet Oncol. 2015;16:716–728.
394. Verstovsek S, et al. A double-blind, placebo-
controlled trial of ruxolitinib for myelofibrosis.
N Engl J Med. 2012;366:799–807.
396. O’Shea JJ, Holland SM, Staudt LM. JAKs and STATs
in immunity, immunodeficiency, and cancer. N Engl
J Med. 2013;368:161–170.
416. David CJ, et al. TGF-beta tumor suppression through
a lethal EMT. Cell. 2016;164:1015–1030.
421. Takebe N, Nguyen D, Yang SX. Targeting notch
signaling pathway in cancer: clinical development
advances and challenges. Pharmacol Ther. 2014;141:
140–149.
437. Yen WC, et al. Targeting Notch signaling with a
Notch2/Notch3 antagonist (tarextumab) inhibits
tumor growth and decreases tumor-initiating cell
frequency. Clin Cancer Res. 2015;21:2084–2095.
442. Toy W, et al. Activating ESR1 Mutations differen-
tially affect the efficacy of ER antagonists. Cancer
Discov. 2017;7:277–287.
445. Scher HI, et al. Increased survival with enzalutamide
in prostate cancer after chemotherapy. N Engl J Med.
2012;367:1187–1197.
465. Stupp R, et al. Cilengitide combined with stan-
dard treatment for patients with newly diagnosed
glioblastoma with methylated MGMT promoter
(CENTRIC EORTC 26071-22072 study): a
multicentre, randomised, open-label, phase 3 trial.
Lancet Oncol. 2014;15:1100–1108.
492. Armaiz-Pena GN, et al. Src activation by beta-
adrenoreceptors is a key switch for tumour metastasis.
Nat Commun. 2013;4:1403.
506. Hantschel O, Grebien F, Superti-Furga G. The
growing arsenal of ATP-competitive and allosteric
inhibitors of BCR-ABL. Cancer Res. 2012;72:
4890–4895.
586. Cancer Genome Atlas, N. Genomic classification
of cutaneous melanoma. Cell. 2015;161:1681–
1696.
589. Diamond EL, et al. Diverse and targetable kinase
alterations drive histiocytic neoplasms. Cancer Discov.
2016;6:154–165.
592. Lito P, Rosen N, Solit DB. Tumor adaptation
and resistance to RAF inhibitors. Nat Med.
2013;19:1401–1409.
593. Yao Z, et al. BRAF mutants evade ERK-dependent
feedback by different mechanisms that determine
their sensitivity to pharmacologic inhibition. Cancer
Cell. 2015;28:370–383.
595. Van Allen EM, et al. The genetic landscape of clinical
resistance to RAF inhibition in metastatic melanoma.
Cancer Discov. 2014;4:94–109.
602. Ostrem JM, Peters U, Sos ML, Wells JA, Shokat
KM. K-Ras(G12C) inhibitors allosterically control
GTP affinity and effector interactions. Nature.
2013;503:548–551.
619. Long GV, et al. Combined BRAF and MEK inhibi-
tion versus BRAF inhibition alone in melanoma.
N Engl J Med. 2014;371:1877–1888.
632. Herrero A, et al. Small molecule inhibition of ERK
dimerization prevents tumorigenesis by RAS-ERK
pathway oncogenes. Cancer Cell. 2015;28:170–182.
635. Li BT, et al. First-in-class oral ERK1/2 inhibitor
ulixertinib (BVD-523) in patients with advanced
solid tumors: final results of a phase I dose escala-
tion and expansion study. J Clin Oncol. 2017;35:
2508.
639. Hu-Lieskovan S, et al. Improved antitumor activity
of immunotherapy with BRAF and MEK inhibi-
tors in BRAF(V600E) melanoma. Sci Transl Med.
2015;7:279ra241.
46 Part I: Science and Clinical Oncology
644. Beaver JA, et al. FDA approval: palbociclib for the
treatment of postmenopausal patients with estrogen
receptor-positive, HER2-negative metastatic breast
cancer. Clin Cancer Res. 2015;21:4760–4766.
690. Cheung LW, et al. Naturally occurring neomorphic
PIK3R1 mutations activate the MAPK pathway,
dictating therapeutic response to MAPK pathway
inhibitors. Cancer Cell. 2014;26:479–494.
691. Thorpe LM, et al. PI3K-p110alpha mediates the
oncogenic activity induced by loss of the novel tumor
suppressor PI3K-p85alpha. Proc Natl Acad Sci USA.
2017;114:7095–7100.
704. Hyman DM, et al. AKT Inhibition in solid tumors
with AKT1 mutations. J Clin Oncol. 2017;35:
2251–2259.
717. Rodrik-Outmezguine VS, et al. Overcoming mTOR
resistance mutations with a new-generation mTOR
inhibitor. Nature. 2016;534:272–276.
720. Zehir A, et al. Mutational landscape of metastatic
cancer revealed from prospective clinical sequenc-
ing of 10,000 patients. Nat Med. 2017;23:703–
713.

REFERENCES
1. Chakravarty D, et al. OncoKB: A precision oncology
knowledge base. JCO Precis Oncol. 2017;1–16.
2. Robinson DR, Wu YM, Lin SF. The protein tyrosine
kinase family of the human genome. Oncogene.
2000;19:5548–5557.
3. Schlessinger J. Cell signaling by receptor tyrosine
kinases. Cell. 2000;103:211–225.
4. Hubbard SR, Till JH. Protein tyrosine kinase
structure and function. Annu Rev Biochem. 2000;69:
373–398.
5. Kuriyan J, Cowburn D. Modular peptide recognition
domains in eukaryotic signaling. Annu Rev Biophys
Biomol Struct. 1997;26:259–288.
6. Pawson T. Protein modules and signalling networks.
Nature. 1995;373:573–580.
7. Liu BA, et al. The human and mouse complement
of SH2 domain proteins-establishing the boundaries
of phosphotyrosine signaling. Mol Cell. 2006;22:
851–868.
8. Pawson T. Specificity in signal transduction: from
phosphotyrosine-SH2 domain interactions to
complex cellular systems. Cell. 2004;116:191–203.
9. Pawson T, Nash P. Assembly of cell regulatory
systems through protein interaction domains. Science.
2003;300:445–452.
10. Levi-Montalcini R. Effects of mouse tumor trans-
plantation on the nervous system. Ann N Y Acad
Sci. 1952;55:330–344.
11. Gschwind A, Fischer OM, Ullrich A. The discovery
of receptor tyrosine kinases: targets for cancer
therapy. Nat Rev Cancer. 2004;4:361–370.
12. Cohen S. Isolation of a mouse submaxillary gland
protein accelerating incisor eruption and eyelid opening
in the new-born animal. J Biol Chem. 1962;237:
1555–1562.
13. Carpenter G, King L Jr, Cohen S. Epidermal growth
factor stimulates phosphorylation in membrane
preparations in vitro. Nature. 1978;276:409–410.
14. Eckhart W, Hutchinson MA, Hunter T. An activity
phosphorylating tyrosine in polyoma T antigen
immunoprecipitates. Cell. 1979;18:925–933.
15. Hunter T, Sefton BM. Transforming gene product
of Rous sarcoma virus phosphorylates tyrosine. Proc
Natl Acad Sci USA. 1980;77:1311–1315.
16. Ullrich A, et al. Human epidermal growth factor
receptor cDNA sequence and aberrant expression of
the amplified gene in A431 epidermoid carcinoma
cells. Nature. 1984;309:418–425.
17. Yamamoto T, Hihara H, Nishida T, Kawai S,
Toyoshima K. A new avian erythroblastosis virus,
AEV-H, carries erbB gene responsible for the
induction of both erythroblastosis and sarcomas.
Cell. 1983;34:225–232.
18. Downward J, et al. Close similarity of epidermal
growth factor receptor and v-erb-B oncogene protein
sequences. Nature. 1984;307:521–527.
19. Yarden Y, Sliwkowski MX. Untangling the ErbB
signalling network. Nat Rev Mol Cell Biol. 2001;2:
127–137.
20. Jones JT, Akita RW, Sliwkowski MX. Binding
specificities and affinities of egf domains for ErbB
receptors. FEBS Lett. 1999;447:227–231.
21. Yarden Y, Pines G. The ERBB network: at last,
cancer therapy meets systems biology. Nat Rev
Cancer. 2012;12:553–563.
22. Marshall J. Clinical implications of the mechanism of
epidermal growth factor receptor inhibitors. Cancer.
2006;107:1207–1218.
23. Klapper LN, et al. The ErbB-2/HER2 oncoprotein
of human carcinomas may function solely as a shared
coreceptor for multiple stroma-derived growth
factors. Proc Natl Acad Sci USA. 1999;96:4995–5000.
24. Graus-Porta D, Beerli RR, Daly JM, Hynes NE.
ErbB-2, the preferred heterodimerization partner of
all ErbB receptors, is a mediator of lateral signaling.
EMBO J. 1997;16:1647–1655.
25. Zaczek A, Brandt B, Bielawski KP. The diverse signal-
ing network of EGFR, HER2, HER3 and HER4
tyrosine kinase receptors and the consequences for
therapeutic approaches. Histol Histopathol. 2005;20:
1005–1015.
26. Worthylake R, Opresko LK, Wiley HS. ErbB-2
amplification inhibits down-regulation and induces
constitutive activation of both ErbB-2 and epidermal
growth factor receptors. J Biol Chem. 1999;274:
8865–8874.
27. Okines A, Cunningham D, Chau I. Targeting the
human EGFR family in esophagogastric cancer. Nat
Rev Clin Oncol. 2011;8:492–503.
28. Pinkas-Kramarski R, et al. Diversification of Neu
differentiation factor and epidermal growth factor
signaling by combinatorial receptor interactions.
EMBO J. 1996;15:2452–2467.
29. Janne PA, Engelman JA, Johnson BE. Epidermal
growth factor receptor mutations in non-small-cell
lung cancer: implications for treatment and tumor
biology. J Clin Oncol. 2005;23:3227–3234.
30. Pao W, et al. EGF receptor gene mutations are
common in lung cancers from “never smokers” and
are associated with sensitivity of tumors to gefitinib
and erlotinib. Proc Natl Acad Sci USA. 2004;101:
13306–13311.
31. Chen Z, Fillmore CM, Hammerman PS, Kim CF,
Wong KK. Non-small-cell lung cancers: a hetero-
geneous set of diseases. Nat Rev Cancer. 2014;14:
535–546.
32. Wong AJ, et al. Structural alterations of the epidermal
growth factor receptor gene in human gliomas. Proc
Natl Acad Sci USA. 1992;89:2965–2969.
33. Li B, et al. Mutant epidermal growth factor receptor
displays increased signaling through the phospha-
tidylinositol-3 kinase/AKT pathway and promotes
radioresistance in cells of astrocytic origin. Oncogene.
2004;23:4594–4602.
34. Brennan CW, et al. The somatic genomic landscape
of glioblastoma. Cell. 2013;155:462–477.
35. Furnari FB, Cloughesy TF, Cavenee WK, Mischel PS.
Heterogeneity of epidermal growth factor receptor
signalling networks in glioblastoma. Nat Rev Cancer.
2015;15:302–310.
36. Tanaka S, Louis DN, Curry WT, Batchelor TT,
Dietrich J. Diagnostic and therapeutic avenues for
glioblastoma: no longer a dead end? Nat Rev Clin
Oncol. 2013;10:14–26.
37. Francis JM, et al. EGFR variant heterogeneity
in glioblastoma resolved through single-nucleus
sequencing. Cancer Discov. 2014;4:956–971.
38. Nathanson DA, et al. Targeted therapy resistance
mediated by dynamic regulation of extrachromosomal
mutant EGFR DNA. Science. 2014;343:72–76.
39. Krause DS, Van Etten RA. Tyrosine kinases as targets
for cancer therapy. N Engl J Med. 2005;353:172–187.
40. Hirsch FR, et al. Epidermal growth factor receptor in
non-small-cell lung carcinomas: correlation between
gene copy number and protein expression and
impact on prognosis. J Clin Oncol. 2003;21:3798–
3807.
41. Cancer Genome Atlas Research, N. et al. Integrated
genomic characterization of oesophageal carcinoma.
Nature. 2017;541:169–175.
42. Slamon DJ, et al. Human breast cancer: correlation
of relapse and survival with amplification of the
HER-2/neu oncogene. Science. 1987;235:177–182.
43. Cristescu R, et al. Molecular analysis of gastric cancer
identifies subtypes associated with distinct clinical
outcomes. Nat Med. 2015;21:449–456.
44. Cancer Genome Atlas Research, N. Comprehensive
molecular characterization of urothelial bladder
carcinoma. Nature. 2014;507:315–322.
45. Cancer Genome Atlas Research, N. Comprehensive
molecular characterization of gastric adenocarcinoma.
Nature. 2014;513:202–209.
Intracellular Signaling  •  CHAPTER 2 46.e1
46.e1
46. Bose R, et al. Activating HER2 mutations in HER2
gene amplification negative breast cancer. Cancer
Discov. 2013;3:224–237.
47. Greulich H, et al. Functional analysis of receptor
tyrosine kinase mutations in lung cancer identifies
oncogenic extracellular domain mutations of ERBB2.
Proc Natl Acad Sci USA. 2012;109:14476–14481.
48. Jaiswal BS, et al. Oncogenic ERBB3 mutations in
human cancers. Cancer Cell. 2013;23:603–617.
49. Hanrahan AJ, Hyman DM, Sfakianos J, Jones
A, Ramirez R, Johnsen H, et al. Abstract 1101:
Functional genomics of HER2 and HER3 muta-
tions and response to neratinib. AACR Cancer Res.
2015;75:1101.
50. Tebbutt N, Pedersen MW, Johns TG. Targeting the
ERBB family in cancer: couples therapy. Nat Rev
Cancer. 2013;13:663–673.
51. Cunningham D, et al. Cetuximab monotherapy
and cetuximab plus irinotecan in irinotecan-
refractory metastatic colorectal cancer. N Engl J
Med. 2004;351:337–345.
52. Saltz LB, et al. Phase II trial of cetuximab in patients
with refractory colorectal cancer that expresses the epi-
dermal growth factor receptor. J Clin Oncol. 2004;22:
1201–1208.
53. Bonner JA, et al. Radiotherapy plus cetuximab for
squamous-cell carcinoma of the head and neck. N
Engl J Med. 2006;354:567–578.
54. Chung KY, et al. Cetuximab shows activity in
colorectal cancer patients with tumors that do
not express the epidermal growth factor receptor
by immunohistochemistry. J Clin Oncol. 2005;23:
1803–1810.
55. Van Cutsem E, et al. Cetuximab and chemotherapy
as initial treatment for metastatic colorectal cancer.
N Engl J Med. 2009;360:1408–1417.
56. Price TJ, et al. Panitumumab versus cetuximab
in patients with chemotherapy-refractory wild-
type KRAS exon 2 metastatic colorectal cancer
(ASPECCT): a randomised, multicentre, open-label,
non-inferiority phase 3 study. Lancet Oncol. 2014;15:
569–579.
57. Lynch TJ, et al. Activating mutations in the epider-
mal growth factor receptor underlying responsiveness
of non-small-cell lung cancer to gefitinib. N Engl J
Med. 2004;350:2129–2139.
58. Yang JC, et al. Afatinib versus cisplatin-based
chemotherapy for EGFR mutation-positive lung
adenocarcinoma (LUX-Lung 3 and LUX-Lung 6):
analysis of overall survival data from two randomised,
phase 3 trials. Lancet Oncol. 2015;16:141–151.
59. Rosell R, et al. Erlotinib versus standard che-
motherapy as first-line treatment for European
patients with advanced EGFR mutation-positive
non-small-cell lung cancer (EURTAC): a multicentre,
open-label, randomised phase 3 trial. Lancet Oncol.
2012;13:239–246.
60. Maemondo M, et al. Gefitinib or chemotherapy for
non-small-cell lung cancer with mutated EGFR.
N Engl J Med. 2010;362:2380–2388.
61. Skoulidis F, Papadimitrakopoulou VA. Targeting
the gatekeeper: osimertinib in EGFR T790M
mutation-positive non-small cell lung cancer. Clin
Cancer Res. 2017;23:618–622.
62. Janne PA, et al. AZD9291 in EGFR inhibitor-
resistant non-small-cell lung cancer. N Engl J Med.
2015;372:1689–1699.
63. Jia Y, et al. Overcoming EGFR(T790M) and
EGFR(C797S) resistance with mutant-selective
allosteric inhibitors. Nature. 2016;534:129–132.
64. Valabrega G, Montemurro F, Aglietta M. Trastu-
zumab: mechanism of action, resistance and future
perspectives in HER2-overexpressing breast cancer.
Ann Oncol. 2007;18:977–984.
65. Cobleigh MA, et al. Multinational study of the effi-
cacy and safety of humanized anti-HER2 monoclonal
46.e2 Part I: Science and Clinical Oncology
antibody in women who have HER2-overexpressing
metastatic breast cancer that has progressed after
chemotherapy for metastatic disease. J Clin Oncol.
1999;17:2639–2648.
66. Slamon DJ, et al. Use of chemotherapy plus a
monoclonal antibody against HER2 for metastatic
breast cancer that overexpresses HER2. N Engl J
Med. 2001;344:783–792.
67. Piccart-Gebhart MJ, et al. Trastuzumab after adjuvant
chemotherapy in HER2-positive breast cancer. N
Engl J Med. 2005;353:1659–1672.
68. Perez EA, et al. Four-year follow-up of trastuzumab
plus adjuvant chemotherapy for operable human
epidermal growth factor receptor 2-positive breast
cancer: joint analysis of data from NCCTG N9831
and NSABP B-31. J Clin Oncol. 2011;29:3366–
3373.
69. Goldhirsch A, et al. 2 years versus 1 year of adjuvant
trastuzumab for HER2-positive breast cancer
(HERA): an open-label, randomised controlled
trial. Lancet. 2013;382:1021–1028.
70. Junttila TT, Li G, Parsons K, Phillips GL, Sliwkowski
MX. Trastuzumab-DM1 (T-DM1) retains all the
mechanisms of action of trastuzumab and efficiently
inhibits growth of lapatinib insensitive breast cancer.
Breast Cancer Res Treat. 2011;128:347–356.
71. Verma S, et al. Trastuzumab emtansine for HER2-pos-
itive advanced breast cancer. N Engl J Med. 2012;367:
1783–1791.
72. Bang YJ, et al. Trastuzumab in combination with
chemotherapy versus chemotherapy alone for
treatment of HER2-positive advanced gastric or
gastro-oesophageal junction cancer (ToGA): a phase
3, open-label, randomised controlled trial. Lancet.
2010;376:687–697.
73. Romond EH, et al. Trastuzumab plus adjuvant
chemotherapy for operable HER2-positive breast
cancer. N Engl J Med. 2005;353:1673–1684.
74. Nahta R, Esteva FJ. HER-2-targeted therapy:
lessons learned and future directions. Clin Cancer
Res. 2003;9:5078–5084.
75. Franklin MC, et al. Insights into ErbB signaling from
the structure of the ErbB2-pertuzumab complex.
Cancer Cell. 2004;5:317–328.
76. Baselga J, et al. Pertuzumab plus trastuzumab plus
docetaxel for metastatic breast cancer. N Engl J Med.
2012;366:109–119.
77. Gianni L, et al. Efficacy and safety of neoadjuvant
pertuzumab and trastuzumab in women with
locally advanced, inflammatory, or early HER2-
positive breast cancer (NeoSphere): a randomised
multicentre, open-label, phase 2 trial. Lancet Oncol.
2012;13:25–32.
78. Swain SM, et al. Pertuzumab, trastuzumab, and
docetaxel for HER2-positive metastatic breast cancer
(CLEOPATRA study): overall survival results from a
randomised, double-blind, placebo-controlled, phase
3 study. Lancet Oncol. 2013;14:461–471.
79. Shojaei S, Gardaneh M, Rahimi Shamabadi A. Target
points in trastuzumab resistance. Int J Breast Cancer.
2012;2012:761917.
80. Geyer CE, et al. Lapatinib plus capecitabine for
HER2-positive advanced breast cancer. N Engl J
Med. 2006;355:2733–2743.
81. Xia W, et al. Combining lapatinib (GW572016), a
small molecule inhibitor of ErbB1 and ErbB2 tyro-
sine kinases, with therapeutic anti-ErbB2 antibodies
enhances apoptosis of ErbB2-overexpressing breast
cancer cells. Oncogene. 2005;24:6213–6221.
82. Scaltriti M, et al. Lapatinib, a HER2 tyrosine kinase
inhibitor, induces stabilization and accumulation of
HER2 and potentiates trastuzumab-dependent cell
cytotoxicity. Oncogene. 2009;28:803–814.
83. Baselga J, et al. Lapatinib with trastuzumab for
HER2-positive early breast cancer (NeoALTTO):
a randomised, open-label, multicentre, phase 3 trial.
Lancet. 2012;379:633–640.
84. Johnston S, et al. Lapatinib combined with letrozole
versus letrozole and placebo as first-line therapy for
postmenopausal hormone receptor-positive metastatic
breast cancer. J Clin Oncol. 2009;27:5538–5546.
85. Frampton JE. Lapatinib: a review of its use in the
treatment of HER2-overexpressing, trastuzumab-
refractory, advanced or metastatic breast cancer.
Drugs. 2009;69:2125–2148.
86. Serra V, et al. Clinical response to a lapatinib-based
therapy for a Li-Fraumeni syndrome patient with
a novel HER2V659E mutation. Cancer Discov.
2013;3:1238–1244.
87. Wong KK, et al. A phase I study with neratinib
(HKI-272), an irreversible pan ErbB receptor
tyrosine kinase inhibitor, in patients with solid
tumors. Clin Cancer Res. 2009;15:2552–2558.
88. Burstein HJ, et al. Neratinib, an irreversible ErbB
receptor tyrosine kinase inhibitor, in patients with
advanced ErbB2-positive breast cancer. J Clin Oncol.
2010;28:1301–1307.
89. Awada A, et al. Safety and efficacy of neratinib
(HKI-272) plus vinorelbine in the treatment of
patients with ErbB2-positive metastatic breast
cancer pretreated with anti-HER2 therapy. Ann
Oncol. 2013;24:109–116.
90. Chow LW, et al. Combination neratinib (HKI-272)
and paclitaxel therapy in patients with HER2-positive
metastatic breast cancer. Br J Cancer. 2013;108:
1985–1993.
91. Jankowitz RC, et al. Safety and efficacy of neratinib
in combination with weekly paclitaxel and trastu-
zumab in women with metastatic HER2positive
breast cancer: an NSABP Foundation Research
Program phase I study. Cancer Chemother Pharmacol.
2013;72:1205–1212.
92. Saura C, et al. Safety and efficacy of neratinib in com-
bination with capecitabine in patients with metastatic
human epidermal growth factor receptor 2-positive
breast cancer. J Clin Oncol. 2014;32:3626–3633.
93. Hyman D, et al. Abstract PD5-05: Neratinib for
ERBB2 mutant, HER2 non-amplified, metastatic
breast cancer: Preliminary analysis from a multi-
center, open-label, multi-histology phase II basket
trial. Cancer Res. 2016;76:PD5-05-PD05-05.
94. Baserga R, Peruzzi F, Reiss K. The IGF-1 receptor
in cancer biology. Int J Cancer. 2003;107:873–877.
95. Belfiore A, Frasca F, Pandini G, Sciacca L, Vigneri
R. Insulin receptor isoforms and insulin receptor/
insulin-like growth factor receptor hybrids in physiol-
ogy and disease. Endocr Rev. 2009;30:586–623.
96. Benyoucef S, Surinya KH, Hadaschik D, Siddle K.
Characterization of insulin/IGF hybrid receptors:
contributions of the insulin receptor L2 and Fn1
domains and the alternatively spliced exon 11
sequence to ligand binding and receptor activation.
Biochem J. 2007;403:603–613.
97. Jones JI, Clemmons DR. Insulin-like growth factors
and their binding proteins: biological actions. Endocr
Rev. 1995;16:3–34.
98. De Meyts P, Whittaker J. Structural biology of insulin
and IGF1 receptors: implications for drug design.
Nat Rev Drug Discov. 2002;1:769–783.
99. Firth SM, Baxter RC. Cellular actions of the insulin-
like growth factor binding proteins. Endocr Rev.
2002;23:824–854.
100. Cohen P, et al. Prostate-specific antigen (PSA) is an
insulin-like growth factor binding protein-3 protease
found in seminal plasma. J Clin Endocrinol Metab.
1992;75:1046–1053.
101. Nakae J, Kido Y, Accili D. Distinct and overlapping
functions of insulin and IGF-I receptors. Endocr
Rev. 2001;22:818–835.
102. Pollak MN, Schernhammer ES, Hankinson SE.
Insulin-like growth factors and neoplasia. Nat Rev
Cancer. 2004;4:505–518.
103. Gombos A, Metzger-Filho O, Dal Lago L, Awada-
Hussein A. Clinical development of insulin-like
growth factor receptor-1 (IGF-1R) inhibitors: At the
crossroad? Invest New Drugs. 2012;30:2433–2442.
104. Tarn C, et al. Insulin-like growth factor 1 receptor
is a potential therapeutic target for gastrointestinal
stromal tumors. Proc Natl Acad Sci USA. 2008;105:
8387–8392.
105. Hallberg B, Palmer RH. Mechanistic insight into
ALK receptor tyrosine kinase in human cancer
biology. Nat Rev Cancer. 2013;13:685–700.
106. Takeuchi K, et al. RET, ROS1 and ALK fusions in
lung cancer. Nat Med. 2012;18:378–381.
107. Sonnenberg-Riethmacher E, Walter B, Riethmacher
D, Godecke S, Birchmeier C. The c-ros tyrosine
kinase receptor controls regionalization and dif-
ferentiation of epithelial cells in the epididymis.
Genes Dev. 1996;10:1184–1193.
108. Camidge DR, Doebele RC. Treating ALK-positive
lung cancer—early successes and future challenges.
Nat Rev Clin Oncol. 2012;9:268–277.
109. Soda M, et al. Identification of the transforming
EML4-ALK fusion gene in non-small-cell lung
cancer. Nature. 2007;448:561–566.
110. Takeuchi K, et al. Multiplex reverse transcription-
PCR screening for EML4-ALK fusion transcripts.
Clin Cancer Res. 2008;14:6618–6624.
111. Bergethon K, et al. ROS1 rearrangements define
a unique molecular class of lung cancers. J Clin
Oncol. 2012;30:863–870.
112. Davies KD, et al. Identifying and targeting ROS1
gene fusions in non-small cell lung cancer. Clin
Cancer Res. 2012;18:4570–4579.
113. Kwak EL, et al. Anaplastic lymphoma kinase inhibi-
tion in non-small-cell lung cancer. N Engl J Med. 2010;
363:1693–1703.
114. Shaw AT, et al. Crizotinib in ROS1-rearranged
non-small-cell lung cancer. N Engl J Med. 2014;371:
1963–1971.
115. Shaw AT, et al. Crizotinib versus chemotherapy in
advanced ALK-positive lung cancer. N Engl J Med.
2013;368:2385–2394.
116. Ou SH, et al. Alectinib in crizotinib-refractory
ALK-rearranged non-small-cell lung cancer: a phase
II global study. J Clin Oncol. 2016;34:661–668.
117. Shaw AT, et  al. Alectinib in ALK-positive,
crizotinib-resistant, non-small-cell lung cancer: a
single-group, multicentre, phase 2 trial. Lancet Oncol.
2016;17:234–242.
118. Gettinger SN, et al. Activity and safety of brigatinib
in ALK-rearranged non-small-cell lung cancer and
other malignancies: a single-arm, open-label, phase
1/2 trial. Lancet Oncol. 2016;17:1683–1696.
119. Hoch RV, Soriano P. Roles of PDGF in animal
development. Development. 2003;130:4769–4784.
120. Heldin CH, Westermark B. Mechanism of action
and in vivo role of platelet-derived growth factor.
Physiol Rev. 1999;79:1283–1316.
121. Heldin CH, Eriksson U, Ostman A. New members of
the platelet-derived growth factor family of mitogens.
Arch Biochem Biophys. 2002;398:284–290.
122. Ostman A, Heldin CH. PDGF receptors as targets in
tumor treatment. Adv Cancer Res. 2007;97:247–274.
123. Carmeliet P, Jain RK. Molecular mechanisms
and clinical applications of angiogenesis. Nature.
2011;473:298–307.
124. Seger R, Krebs EG. The MAPK signaling cascade.
FASEB J. 1995;9:726–735.
125. Andrae J, Gallini R, Betsholtz C. Role of platelet-
derived growth factors in physiology and medicine.
Genes Dev. 2008;22:1276–1312.
126. Corless CL, et  al. PDGFRA mutations in
gastrointestinal stromal tumors: frequency, spectrum
and in vitro sensitivity to imatinib. J Clin Oncol.
2005;23:5357–5364.
127. Carroll M, Tomasson MH, Barker GF, Golub TR,
Gilliland DG. The TEL/platelet-derived growth
factor beta receptor (PDGF beta R) fusion in
chronic myelomonocytic leukemia is a transforming
protein that self-associates and activates PDGF beta
R kinase-dependent signaling pathways. Proc Natl
Acad Sci USA. 1996;93:14845–14850.
128. McArthur GA. Dermatofibrosarcoma protuberans:
a surgical disease with a molecular savior. Curr Opin
Oncol. 2006;18:341–346.

129. Cools J, et al. A tyrosine kinase created by fusion
of the PDGFRA and FIP1L1 genes as a therapeutic
target of imatinib in idiopathic hypereosinophilic
syndrome. N Engl J Med. 2003;348:1201–1214.
130. Fleming TP, et al. Amplification and/or overexpres-
sion of platelet-derived growth factor receptors and
epidermal growth factor receptor in human glial
tumors. Cancer Res. 1992;52:4550–4553.
131. Iqbal N, Iqbal N. Imatinib: a breakthrough of targeted
therapy in cancer. Chemother Res Pract. 2014;2014:
357027.
132. von Mehren M. Beyond imatinib: second gen-
eration c-KIT inhibitors for the management of
gastrointestinal stromal tumors. Clin Colorectal
Cancer. 2006;6(suppl 1):S30–S34.
133. Dewaele B, et al. Activity of dasatinib, a dual SRC/
ABL kinase inhibitor, and IPI-504, a heat shock
protein 90 inhibitor, against gastrointestinal stromal
tumor-associated PDGFRAD842V mutation. Clin
Cancer Res. 2008;14:5749–5758.
134. McArthur GA, et  al. Molecular and clinical
analysis of locally advanced dermatofibrosarcoma
protuberans treated with imatinib: Imatinib Target
Exploration Consortium Study B2225. J Clin Oncol.
2005;23:866–873.
135. Kerob D, et al. Imatinib mesylate as a preopera-
tive therapy in dermatofibrosarcoma: results of a
multicenter phase II study on 25 patients. Clin
Cancer Res. 2010;16:3288–3295.
136. Apperley JF, et al. Response to imatinib mesylate in
patients with chronic myeloproliferative diseases with
rearrangements of the platelet-derived growth factor
receptor beta. N Engl J Med. 2002;347:481–487.
137. Cheah CY, et al. Patients with myeloid malignancies
bearing PDGFRB fusion genes achieve durable
long-term remissions with imatinib. Blood.
2014;123:3574–3577.
138. Roberts KG, et al. Genetic alterations activating
kinase and cytokine receptor signaling in high-
risk acute lymphoblastic leukemia. Cancer Cell.
2012;22:153–166.
139. Andre C, et al. Sequence analysis of two genomic
regions containing the KIT and the FMS receptor
tyrosine kinase genes. Genomics. 1997;39:216–226.
140. Yarden Y, et al. Human proto-oncogene c-kit:
a new cell surface receptor tyrosine kinase for
an unidentified ligand. EMBO J. 1987;6:3341–
3351.
141. Ashman LK. The biology of stem cell factor and
its receptor C-kit. Int J Biochem Cell Biol. 1999;31:
1037–1051.
142. Besmer P, et al. A new acute transforming feline
retrovirus and relationship of its oncogene v-kit
with the protein kinase gene family. Nature.
1986;320:415–421.
143. Huang E, et al. The hematopoietic growth factor
KL is encoded by the Sl locus and is the ligand of
the c-kit receptor, the gene product of the W locus.
Cell. 1990;63:225–233.
144. Mol CD, et al. Structural basis for the autoinhibition
and STI-571 inhibition of c-Kit tyrosine kinase.
J Biol Chem. 2004;279:31655–31663.
145. Lev S, Yarden Y, Givol D. Dimerization and
activation of the kit receptor by monovalent and
bivalent binding of the stem cell factor. J Biol Chem.
1992;267:15970–15977.
146. Antonescu CR. The GIST paradigm: lessons for other
kinase-driven cancers. J Pathol. 2011;223:251–261.
147. Russell ES. Hereditary anemias of the mouse: a
review for geneticists. Adv Genet. 1979;20:357–459.
148. Dexter TM, Moore MA. In vitro duplication and
“cure” of haemopoietic defects in genetically anaemic
mice. Nature. 1977;269:412–414.
149. Gadd SJ, Ashman LK. A murine monoclonal
antibody specific for a cell-surface antigen expressed
by a subgroup of human myeloid leukaemias. Leuk
Res. 1985;9:1329–1336.
150. Obata Y, et al. Oncogenic signaling by Kit tyrosine
kinase occurs selectively on the Golgi apparatus in
gastrointestinal stromal tumors. Oncogene. 2017;36:
3661–3672.
151. Thomsen L, et al. Interstitial cells of Cajal gener-
ate a rhythmic pacemaker current. Nat Med.
1998;4:848–851.
152. Corless CL, Barnett CM, Heinrich MC. Gastro-
intestinal stromal tumours: origin and molecular
oncology. Nat Rev Cancer. 2011;11:865–878.
153. Hirota S, et al. Gain-of-function mutations of c-kit
in human gastrointestinal stromal tumors. Science.
1998;279:577–580.
154. Kindblom LG, Remotti HE, Aldenborg F, Meis-
Kindblom JM. Gastrointestinal pacemaker cell tumor
(GIPACT): gastrointestinal stromal tumors show
phenotypic characteristics of the interstitial cells of
Cajal. Am J Pathol. 1998;152:1259–1269.
155. Fletcher JA, Rubin BP. KIT mutations in GIST.
Curr Opin Genet Dev. 2007;17:3–7.
156. Bagrodia A, et al. Genetic determinants of cisplatin
resistance in patients with advanced germ cell tumors.
J Clin Oncol. 2016;34:4000–4007.
157. Demetri GD, et al. Efficacy and safety of imatinib
mesylate in advanced gastrointestinal stromal tumors.
N Engl J Med. 2002;347:472–480.
158. Demetri GD, et al. Efficacy and safety of sunitinib
in patients with advanced gastrointestinal stromal
tumour after failure of imatinib: a randomised
controlled trial. Lancet. 2006;368:1329–1338.
159. Verweij J, et al. Progression-free survival in gastro-
intestinal stromal tumours with high-dose imatinib:
randomised trial. Lancet. 2004;364:1127–1134.
160. Zalcberg JR, et al. Outcome of patients with
advanced gastro-intestinal stromal tumours crossing
over to a daily imatinib dose of 800 mg after progres-
sion on 400 mg. Eur J Cancer. 2005;41:1751–1757.
161. Blanke CD, et al. Long-term results from a random-
ized phase II trial of standard- versus higher-dose
imatinib mesylate for patients with unresectable or
metastatic gastrointestinal stromal tumors expressing
KIT. J Clin Oncol. 2008;26:620–625.
162. George S, et al. Clinical evaluation of continuous
daily dosing of sunitinib malate in patients with
advanced gastrointestinal stromal tumour after
imatinib failure. Eur J Cancer. 2009;45:1959–1968.
163. Reichardt P, et al. Clinical outcomes of patients
with advanced gastrointestinal stromal tumors:
safety and efficacy in a worldwide treatment-use
trial of sunitinib. Cancer. 2015;121:1405–1413.
164. Demetri GD, et al. Efficacy and safety of regorafenib
for advanced gastrointestinal stromal tumours
after failure of imatinib and sunitinib (GRID): an
international, multicentre, randomised, placebo-
controlled, phase 3 trial. Lancet. 2013;381:295–302.
165. Carvajal RD, et al. KIT as a therapeutic target in
metastatic melanoma. JAMA. 2011;305:2327–2334.
166. Hodi FS, et al. Imatinib for melanomas harboring
mutationally activated or amplified KIT arising on
mucosal, acral, and chronically sun-damaged skin.
J Clin Oncol. 2013;31:3182–3190.
167. Nishida T, et al. Secondary mutations in the
kinase domain of the KIT gene are predominant
in imatinib-resistant gastrointestinal stromal tumor.
Cancer Sci. 2008;99:799–804.
168. Bollag G. Abstract IA32: Optimizing kinase inhibi-
tors to treat cancer. Cancer Res. 2016;76:IA32.
169. Javidi-Sharifi N, et al. Crosstalk between KIT and
FGFR3 promotes gastrointestinal stromal tumor cell
growth and drug resistance. Cancer Res. 2015;75:
880–891.
170. Stirewalt DL, Radich JP. The role of FLT3 in hae-
matopoietic malignancies. Nat Rev Cancer. 2003;3:
650–665.
171. Leung AY, Man CH, Kwong YL. FLT3 inhibition:
a moving and evolving target in acute myeloid
leukaemia. Leukemia. 2012.
172. Cancer Genome Atlas Research, N. et al. Genomic
and epigenomic landscapes of adult de novo acute
myeloid leukemia. N Engl J Med. 2013;368:
2059–2074.
Intracellular Signaling  •  CHAPTER 2 46.e3
46.e3
173. Antar A, et al. Inhibition of FLT3 in AML: a focus
on sorafenib. Bone Marrow Transplant. 2017;52:
344–351.
174. Yu J, et al. Anti-tumor activity of TAK-659, a dual
inhibitor of SYK and FLT-3 kinases, in AML models.
J Clin Oncol. 2016;34:e14091.
175. Kaplan J, et al. TAK-659, An investigational revers-
ible dual SYK/FLT-3 inhibitor, in patients with
lymphoma: updated results from dose-escalation
and expansion cohorts of a phase 1 study. Hematol
Oncol. 2017;35:72–74.
176. Zhang W, et al. Mutant FLT3: a direct target of
sorafenib in acute myelogenous leukemia. J Natl
Cancer Inst. 2008;100:184–198.
177. Knowles MA. Role of FGFR3 in urothelial cell
carcinoma: biomarker and potential therapeutic
target. World J Urol. 2007;25:581–593.
178. Ornitz DM, et al. Receptor specificity of the fibro-
blast growth factor family. J Biol Chem. 1996;271:
15292–15297.
179. Turner N, Grose R. Fibroblast growth factor signal-
ling: from development to cancer. Nat Rev Cancer.
2010;10:116–129.
180. Ornitz DM, Marie PJ. Fibroblast growth factor
signaling in skeletal development and disease. Genes
Dev. 2015;29:1463–1486.
181. Iyer G, Milowsky MI. Fibroblast growth factor
receptor-3 in urothelial tumorigenesis. Urol Oncol.
2012.
182. Babina IS, Turner NC. Advances and challenges
in targeting FGFR signalling in cancer. Nat Rev
Cancer. 2017;17:318–332.
183. Wang Y, Becker D. Antisense targeting of basic
fibroblast growth factor and fibroblast growth
factor receptor-1 in human melanomas blocks
intratumoral angiogenesis and tumor growth. Nat
Med. 1997;3:887–893.
184. Memarzadeh S, et al. Enhanced paracrine FGF10
expression promotes formation of multifocal pros-
tate adenocarcinoma and an increase in epithelial
androgen receptor. Cancer Cell. 2007;12:572–585.
185. Weiss J, et al. Frequent and focal FGFR1 amplifica-
tion associates with therapeutically tractable FGFR1
dependency in squamous cell lung cancer. Sci Transl
Med. 2010;2:62ra93.
186. Takeda M, et al. AZD2171 shows potent antitumor
activity against gastric cancer over-expressing fibro-
blast growth factor receptor 2/keratinocyte growth
factor receptor. Clin Cancer Res. 2007;13:3051–
3057.
187. Matsumoto K, et al. FGFR2 gene amplification and
clinicopathological features in gastric cancer. Br J
Cancer. 2012;106:727–732.
188. Dutt A, et al. Drug-sensitive FGFR2 mutations in
endometrial carcinoma. Proc Natl Acad Sci USA.
2008;105:8713–8717.
189. Helsten T, et al. The FGFR landscape in cancer:
analysis of 4,853 tumors by next-generation sequenc-
ing. Clin Cancer Res. 2016;22:259–267.
190. Iyer G, et al. Prevalence and co-occurrence of
actionable genomic alterations in high-grade bladder
cancer. J Clin Oncol. 2013;31:3133–3140.
191. Bernard-Pierrot I, et al. Oncogenic properties of the
mutated forms of fibroblast growth factor receptor
3b. Carcinogenesis. 2006;27:740–747.
192. Sibley K, et  al. A molecular study of the
t(4;14) in multiple myeloma. Br J Haematol.
2002;118:514–520.
193. Wu YM, et al. Identification of targetable FGFR
gene fusions in diverse cancers. Cancer Discov.
2013;3:636–647.
194. Ang C. Role of the fibroblast growth factor receptor
axis in cholangiocarcinoma. J Gastroenterol Hepatol.
2015;30:1116–1122.
195. Costa R, et al. FGFR3-TACC3 fusion in solid tumors:
mini review. Oncotarget. 2016;7:55924–55938.
196. Singh D, et al. Transforming fusions of FGFR
and TACC genes in human glioblastoma. Science.
2012;337:1231–1235.
46.e4 Part I: Science and Clinical Oncology
197. Nogova L, et al. Evaluation of BGJ398, a fibroblast
growth factor receptor 1-3 kinase inhibitor, in patients
with advanced solid tumors harboring genetic altera-
tions in fibroblast growth factor receptors: results of
a global phase I, dose-escalation and dose-expansion
study. J Clin Oncol. 2017;35:157–165.
198. Paik PK, et al. A Phase 1b open label multicentre
study of AZD4547 in patients with advanced
squamous cell lung cancers. Clin Cancer Res. 2017.
199. Tabernero J, et al. Phase I dose-escalation study of
JNJ-42756493, an oral pan-fibroblast growth factor
receptor inhibitor, in patients with advanced solid
tumors. J Clin Oncol. 2015;33:3401–3408.
200. Martin Henner Voss CH, Heist RS, Cleary JM,
Meric-Bernstam F, Gandhi L, Ishii N, et al. 1347, an
oral FGFR inhibitor: results from a first-in-human,
phase I dose-escalation study in patients with FGFR
genomically activated advanced solid tumors. J Clin
Oncol. 2017;35(suppl; abstr 2500).
201. Mulligan LM. RET revisited: expanding the
oncogenic portfolio. Nat Rev Cancer. 2014;14:173–
186.
202. Costantini F, Shakya R. GDNF/Ret signaling
and the development of the kidney. Bioessays.
2006;28:117–127.
203. Taraviras S, et al. Signalling by the RET receptor
tyrosine kinase and its role in the development of
the mammalian enteric nervous system. Development.
1999;126:2785–2797.
204. Wells SA Jr, Santoro M. Targeting the RET
pathway in thyroid cancer. Clin Cancer Res.
2009;15:7119–7123.
205. Mulligan LM, et al. Germ-line mutations of the
RET proto-oncogene in multiple endocrine neoplasia
type 2A. Nature. 1993;363:458–460.
206. Lantieri F, Caroli F, Ceccherini I, Griseri P. The
involvement of the RET variant G691S in medullary
thyroid carcinoma enlightened by a meta-analysis
study. Int J Cancer. 2013;132:2808–2819.
207. Romei C, Elisei R. RET/PTC translocations and
clinico-pathological features in human papillary
thyroid carcinoma. Front Endocrinol (Lausanne).
2012;3:54.
208. Wells SA Jr, et al. Vandetanib in patients with locally
advanced or metastatic medullary thyroid cancer:
a randomized, double-blind phase III trial. J Clin
Oncol. 2012;30:134–141.
209. Kurzrock R, et al. Activity of XL184 (Cabozantinib),
an oral tyrosine kinase inhibitor, in patients with
medullary thyroid cancer. J Clin Oncol. 2011;29:
2660–2666.
210. Jordan EJ, et al. Prospective comprehensive molecular
characterization of lung adenocarcinomas for effi-
cient patient matching to approved and emerging
therapies. Cancer Discov. 2017;7:596–609.
211. Ferrara N, Gerber HP, LeCouter J. The biology of
VEGF and its receptors. Nat Med. 2003;9:669–676.
212. Leung DW, Cachianes G, Kuang WJ, Goeddel
DV, Ferrara N. Vascular endothelial growth
factor is a secreted angiogenic mitogen. Science.
1989;246:1306–1309.
213. Ferrara N. Vascular endothelial growth factor: basic
science and clinical progress. Endocr Rev. 2004;25:
581–611.
214. Kowanetz M, Ferrara N. Vascular endothelial growth
factor signaling pathways: therapeutic perspective.
Clin Cancer Res. 2006;12:5018–5022.
215. Olsson AK, Dimberg A, Kreuger J, Claesson-Welsh
L. VEGF receptor signalling - in control of vascular
function. Nat Rev Mol Cell Biol. 2006;7:359–371.
216. Simons M, Gordon E, Claesson-Welsh L. Mecha-
nisms and regulation of endothelial VEGF receptor
signalling. Nat Rev Mol Cell Biol. 2016;17:611–
625.
217. Goel HL, Mercurio AM. VEGF targets the tumour
cell. Nat Rev Cancer. 2013;13:871–882.
218. Hurwitz H, et al. Bevacizumab plus irinotecan,
fluorouracil, and leucovorin for metastatic colorectal
cancer. N Engl J Med. 2004;350:2335–2342.
219. Sandler A, et al. Paclitaxel-carboplatin alone or with
bevacizumab for non-small-cell lung cancer. N Engl
J Med. 2006;355:2542–2550.
220. Vredenburgh JJ, et al. Bevacizumab plus irinotecan
in recurrent glioblastoma multiforme. J Clin Oncol.
2007;25:4722–4729.
221. Escudier B, et al. Bevacizumab plus interferon alfa-2a
for treatment of metastatic renal cell carcinoma: a
randomised, double-blind phase III trial. Lancet.
2007;370:2103–2111.
222. Tabernero J, et al. Ramucirumab versus placebo
in combination with second-line FOLFIRI in
patients with metastatic colorectal carcinoma that
progressed during or after first-line therapy with
bevacizumab, oxaliplatin, and a fluoropyrimidine
(RAISE): a randomised, double-blind, multicentre,
phase 3 study. Lancet Oncol. 2015;16:499–508.
223. Chow LQ, Eckhardt SG. Sunitinib: from
rational design to clinical efficacy. J Clin Oncol.
2007;25:884–896.
224. Kane RC, et al. Sorafenib for the treatment of
unresectable hepatocellular carcinoma. Oncologist.
2009;14:95–100.
225. Escudier B, et al. Sorafenib for treatment of renal
cell carcinoma: final efficacy and safety results of the
phase III treatment approaches in renal cancer global
evaluation trial. J Clin Oncol. 2009;27:3312–3318.
226. Sternberg CN, et al. Pazopanib in locally advanced
or metastatic renal cell carcinoma: results of a
randomized phase III trial. J Clin Oncol. 2010;28:
1061–1068.
227. Rini BI, et al. Comparative effectiveness of axitinib
versus sorafenib in advanced renal cell carcinoma
(AXIS): a randomised phase 3 trial. Lancet. 2011;378:
1931–1939.
228. Yeung KT, Cohen EE. Lenvatinib in advanced,
radioactive iodine-refractory, differentiated thyroid
carcinoma. Clin Cancer Res. 2015;21:5420–5426.
229. Motzer RJ, et al. Lenvatinib, everolimus, and the
combination in patients with metastatic renal cell
carcinoma: a randomised, phase 2, open-label,
multicentre trial. Lancet Oncol. 2015;16:1473–1482.
230. Loges S, Schmidt T, Carmeliet P. Mechanisms of
resistance to anti-angiogenic therapy and develop-
ment of third-generation anti-angiogenic drug
candidates. Genes Cancer. 2010;1:12–25.
231. Bottaro DP, et al. Identification of the hepatocyte
growth factor receptor as the c-met proto-oncogene
product. Science. 1991;251:802–804.
232. Cooper CS, et al. Molecular cloning of a new
transforming gene from a chemically transformed
human cell line. Nature. 1984;311:29–33.
233. Hanna JA, Bordeaux J, Rimm DL, Agarwal S. The
function, proteolytic processing, and histopathology
of Met in cancer. Adv Cancer Res. 2009;103:1–23.
234. Birchmeier C, Birchmeier W, Gherardi E, Vande
Woude GF. Met, metastasis, motility and more.
Nat Rev Mol Cell Biol. 2003;4:915–925.
235. Ho-Yen CM, Jones JL, Kermorgant S. The clinical
and functional significance of c-Met in breast cancer:
a review. Breast Cancer Res. 2015;17:52.
236. Peschard P, et al. Mutation of the c-Cbl TKB domain
binding site on the Met receptor tyrosine kinase
converts it into a transforming protein. Mol Cell.
2001;8:995–1004.
237. Abella JV, et al. Met/Hepatocyte growth factor
receptor ubiquitination suppresses transformation
and is required for Hrs phosphorylation. Mol Cell
Biol. 2005;25:9632–9645.
238. Trusolino L, Bertotti A, Comoglio PM. MET
signalling: principles and functions in development,
organ regeneration and cancer. Nat Rev Mol Cell
Biol. 2010;11:834–848.
239. Furge KA, Zhang YW, Vande Woude GF. Met
receptor tyrosine kinase: enhanced signaling through
adapter proteins. Oncogene. 2000;19:5582–5589.
240. Gherardi E, Birchmeier W, Birchmeier C, Vande
Woude G. Targeting MET in cancer: rationale and
progress. Nat Rev Cancer. 2012;12:89–103.
241. Bladt F, Riethmacher D, Isenmann S, Aguzzi A,
Birchmeier C. Essential role for the c-met receptor
in the migration of myogenic precursor cells into
the limb bud. Nature. 1995;376:768–771.
242. Appleman LJ. MET signaling pathway: a rational
target for cancer therapy. J Clin Oncol. 2011;29:
4837–4838.
243. Awad MM, et al. MET exon 14 mutations in non-
small-cell lung cancer are associated with advanced
age and stage-dependent MET genomic amplification
and c-Met overexpression. J Clin Oncol. 2016;34:
721–730.
244. Ma PC, et al. Functional expression and mutations of
c-Met and its therapeutic inhibition with SU11274
and small interfering RNA in non-small cell lung
cancer. Cancer Res. 2005;65:1479–1488.
245. Ma PC, et al. Expression and mutational analysis of
MET in human solid cancers. Genes Chromosomes
Cancer. 2008;47:1025–1037.
246. Liu X, et al. Next-generation sequencing of pulmo-
nary sarcomatoid carcinoma reveals high frequency
of actionable MET gene mutations. J Clin Oncol.
2016;34:794–802.
247. Schmidt L, et al. Germline and somatic mutations
in the tyrosine kinase domain of the MET proto-
oncogene in papillary renal carcinomas. Nat Genet.
1997;16:68–73.
248. Peters S, Adjei AA. MET: a promising anticancer
therapeutic target. Nat Rev Clin Oncol. 2012;9:
314–326.
249. Frampton GM, et al. Activation of MET via diverse
exon 14 splicing alterations occurs in multiple tumor
types and confers clinical sensitivity to MET inhibi-
tors. Cancer Discov. 2015;5:850–859.
250. Kong-Beltran M, et al. Somatic mutations lead to
an oncogenic deletion of met in lung cancer. Cancer
Res. 2006;66:283–289.
251. Engelman JA, et al. MET amplification leads to
gefitinib resistance in lung cancer by activating
ERBB3 signaling. Science. 2007;316:1039–1043.
252. Paik PK, et al. Response to MET inhibitors in
patients with stage IV lung adenocarcinomas
harboring MET mutations causing exon 14 skipping.
Cancer Discov. 2015;5:842–849.
253. Ou SH, et al. Activity of crizotinib (PF02341066),
a dual mesenchymal-epithelial transition (MET) and
anaplastic lymphoma kinase (ALK) inhibitor, in a
non-small cell lung cancer patient with de novo MET
amplification. J Thorac Oncol. 2011;6:942–946.
254. Choueiri TK, et al. Cabozantinib versus everolimus
in advanced renal cell carcinoma (METEOR): final
results from a randomised, open-label, phase 3 trial.
Lancet Oncol. 2016;17:917–927.
255. Grassi P, et al. Cabozantinib in the treatment of
advanced renal cell carcinoma: design, development,
and potential place in the therapy. Drug Des Devel
Ther. 2016;10:2167–2172.
256. Spigel D, Ervin TJ, Ramlau R, et al. Final efficacy
results from OAM4558g, a randomized phase II
study evaluating MetMAb or placebo in combination
with erlotinib in advanced NSCLC. J Clin Oncol.
2011;29.
257. Klein R, Jing SQ, Nanduri V, O’Rourke E, Barbacid
M. The trk proto-oncogene encodes a receptor for
nerve growth factor. Cell. 1991;65:189–197.
258. Hempstead BL, Martin-Zanca D, Kaplan DR, Parada
LF, Chao MV. High-affinity NGF binding requires
coexpression of the trk proto-oncogene and the
low-affinity NGF receptor. Nature. 1991;350:678–
683.
259. Rolfo C, et al. Entrectinib: a potent new TRK,
ROS1, and ALK inhibitor. Expert Opin Investig
Drugs. 2015;24:1493–1500.
260. Brodeur GM, et al. Trk receptor expression and
inhibition in neuroblastomas. Clin Cancer Res.
2009;15:3244–3250.
261. Nakagawara A. Trk receptor tyrosine kinases: a bridge
between cancer and neural development. Cancer
Lett. 2001;169:107–114.

262. Euhus DM, Timmons CF, Tomlinson GE. ETV6-
NTRK3—Trk-ing the primary event in human
secretory breast cancer. Cancer Cell. 2002;2:347–348.
263. Davis JL, et  al. Infantile NTRK-associated
mesenchymal tumors. Pediatr Dev Pathol. 2017;
1093526617712639.
264. Nagasubramanian R, et al. Infantile fibrosarcoma
with NTRK3-ETV6 fusion successfully treated with
the tropomyosin-related kinase inhibitor LOXO-101.
Pediatr Blood Cancer. 2016;63:1468–1470.
265. Tabbo F, Pizzi M, Kyriakides PW, Ruggeri B, Inghi-
rami G. Oncogenic kinase fusions: an evolving arena
with innovative clinical opportunities. Oncotarget.
2016;7:25064–25086.
266. Stransky N, Cerami E, Schalm S, Kim JL, Lengauer
C. The landscape of kinase fusions in cancer. Nat
Commun. 2014;5:4846.
267. Amatu A, Sartore-Bianchi A, Siena S. NTRK gene
fusions as novel targets of cancer therapy across mul-
tiple tumour types. ESMO Open. 2016;1:e000023.
268. Drilon A, et al. A Next-generation TRK kinase
inhibitor overcomes acquired resistance to prior
TRK kinase inhibition in patients with TRK
fusion-positive solid tumors. Cancer Discov. 2017.
269. Doebele RC, et al. An oncogenic NTRK fusion in
a patient with soft-tissue sarcoma with response to
the tropomyosin-related kinase inhibitor LOXO-101.
Cancer Discov. 2015;5:1049–1057.
270. American Association for Cancer Research. TRK
inhibitor shows early promise. Cancer Discov. 2016;6:
OF4.
271. Farago AF, et al. Durable clinical response to
entrectinib in NTRK1-rearranged non-small cell
lung cancer. J Thorac Oncol. 2015;10:1670–1674.
272. Drilon A, et al. What hides behind the MASC: clini-
cal response and acquired resistance to entrectinib
after ETV6-NTRK3 identification in a mammary
analogue secretory carcinoma (MASC). Ann Oncol.
2016;27:920–926.
273. Hyman DM, et al. The efficacy of larotrectinib
(LOXO-101), a selective tropomyosin receptor kinase
(TRK) inhibitor, in adult and pediatric TRK fusion
cancers. J Clin Oncol. 2017;35:LBA2501.
274. Pierce KL, Premont RT, Lefkowitz RJ. Seven-
transmembrane receptors. Nat Rev Mol Cell Biol.
2002;3:639–650.
275. Jacoby E, Bouhelal R, Gerspacher M, Seuwen K.
The 7 TM G-protein-coupled receptor target family.
ChemMedChem. 2006;1:761–782.
276. Tyndall JD, Sandilya R. GPCR agonists and antago-
nists in the clinic. Med Chem. 2005;1:405–421.
277. Dorsam RT, Gutkind JS. G-protein-coupled receptors
and cancer. Nat Rev Cancer. 2007;7:79–94.
278. Lappano R, Maggiolini M. G protein-coupled
receptors: novel targets for drug discovery in cancer.
Nat Rev Drug Discov. 2011;10:47–60.
279. Spiegelberg BD, Hamm HE. Roles of G-protein-
coupled receptor signaling in cancer biology and gene
transcription. Curr Opin Genet Dev. 2007;17:40–44.
280. Lagerstrom MC, Schioth HB. Structural diversity
of G protein-coupled receptors and significance
for drug discovery. Nat Rev Drug Discov. 2008;7:
339–357.
281. Fredriksson R, Lagerstrom MC, Lundin LG, Schioth
HB. The G-protein-coupled receptors in the human
genome form five main families. Phylogenetic
analysis, paralogon groups, and fingerprints. Mol
Pharmacol. 2003;63:1256–1272.
282. Palczewski K, et al. Crystal structure of rhodopsin:
A G protein-coupled receptor. Science. 2000;289:
739–745.
283. Gloriam DE, Fredriksson R, Schioth HB. The
G protein-coupled receptor subset of the rat
genome. BMC Genomics. 2007;8:338.
284. Harmar AJ. Family-B G-protein-coupled receptors.
Genome Biol. 2001;2:REVIEWS3013.
285. Bjarnadottir TK, et al. The human and mouse
repertoire of the adhesion family of G-protein-
coupled receptors. Genomics. 2004;84:23–33.
286. Kunishima N, et al. Structural basis of glutamate
recognition by a dimeric metabotropic glutamate
receptor. Nature. 2000;407:971–977.
287. Muto T, Tsuchiya D, Morikawa K, Jingami H.
Structures of the extracellular regions of the group
II/III metabotropic glutamate receptors. Proc Natl
Acad Sci USA. 2007;104:3759–3764.
288. Dann CE, et al. Insights into Wnt binding and
signalling from the structures of two Frizzled
cysteine-rich domains. Nature. 2001;412:86–90.
289. Adler E, et al. A novel family of mammalian taste
receptors. Cell. 2000;100:693–702.
290. Matsunami H, Montmayeur JP, Buck LB. A
family of candidate taste receptors in human and
mouse. Nature. 2000;404:601–604.
291. Stevens RC, et al. The GPCR Network: a large-scale
collaboration to determine human GPCR structure
and function. Nat Rev Drug Discov. 2013;12:25–34.
292. Scheerer P, et al. Crystal structure of opsin in
its G-protein-interacting conformation. Nature.
2008;455:497–502.
293. Rasmussen SG, et al. Crystal structure of the beta2
adrenergic receptor-Gs protein complex. Nature.
2011;477:549–555.
294. Audet M, Bouvier M. Restructuring g-protein-
coupled receptor activation. Cell. 2012;151:14–23.
295. Cabrera-Vera TM, et al. Insights into G protein
structure, function, and regulation. Endocr Rev.
2003;24:765–781.
296. Milligan G, Kostenis E. Heterotrimeric G-proteins:
a short history. Br J Pharmacol. 2006;147(suppl
1):S46–S55.
297. Oldham WM, Hamm HE. Heterotrimeric G protein
activation by G-protein-coupled receptors. Nat Rev
Mol Cell Biol. 2008;9:60–71.
298. Rodbell M, Birnbaumer L, Pohl SL, Krans HM. The
glucagon-sensitive adenyl cyclase system in plasma
membranes of rat liver. V. An obligatory role of
guanylnucleotides in glucagon action. J Biol Chem.
1971;246:1877–1882.
299. Hayes JS, Brunton LL, Mayer SE. Selective activation
of particulate cAMP-dependent protein kinase by
isoproterenol and prostaglandin E1. J Biol Chem.
1980;255:5113–5119.
300. Denis C, Sauliere A, Galandrin S, Senard JM, Gales
C. Probing heterotrimeric G protein activation:
applications to biased ligands. Curr Pharm Des.
2012;18:128–144.
301. Edwards HV, Christian F, Baillie GS. cAMP: novel
concepts in compartmentalised signalling. Semin
Cell Dev Biol. 2012;23:181–190.
302. Metrich M, et al. Role of the cAMP-binding protein
Epac in cardiovascular physiology and pathophysiol-
ogy. Pflugers Arch. 2010;459:535–546.
303. Kopperud R, Krakstad C, Selheim F, Doskeland SO.
cAMP effector mechanisms. Novel twists for an ‘old’
signaling system. FEBS Lett. 2003;546:121–126.
304. Walsh DA, Perkins JP, Krebs EG. An adenosine
3′,5′-monophosphate-dependent protein kinase
from rabbit skeletal muscle. J Biol Chem. 1968;243:
3763–3765.
305. Altarejos JY, Montminy M. CREB and the CRTC
co-activators: sensors for hormonal and metabolic
signals. Nat Rev Mol Cell Biol. 2011;12:141–151.
306. Beene DL, Scott JD. A-kinase anchoring proteins
take shape. Curr Opin Cell Biol. 2007;19:192–198.
307. Sunahara RK, Dessauer CW, Gilman AG. Complex-
ity and diversity of mammalian adenylyl cyclases.
Annu Rev Pharmacol Toxicol. 1996;36:461–480.
308. Birnbaumer L. Expansion of signal transduction by
G proteins. The second 15 years or so: from 3 to
16 alpha subunits plus betagamma dimers. Biochim
Biophys Acta. 2007;1768:772–793.
309. Streb H, Irvine RF, Berridge MJ, Schulz I. Release of
Ca2+ from a nonmitochondrial intracellular store in
pancreatic acinar cells by inositol-1,4,5-trisphosphate.
Nature. 1983;306:67–69.
310. Thomas AP, Marks JS, Coll KE, Williamson JR.
Quantitation and early kinetics of inositol lipid
Intracellular Signaling  •  CHAPTER 2 46.e5
46.e5
changes induced by vasopressin in isolated and
cultured hepatocytes. J Biol Chem. 1983;258:
5716–5725.
311. Siehler S. Regulation of RhoGEF proteins by
G12/13-coupled receptors. Br J Pharmacol.
2009;158:41–49.
312. Sjogren B, Blazer LL, Neubig RR. Regulators of
G protein signaling proteins as targets for drug
discovery. Prog Mol Biol Transl Sci. 2010;91:81–119.
313. Hurst JH, Hooks SB. Regulator of G-protein
signaling (RGS) proteins in cancer biology. Biochem
Pharmacol. 2009;78:1289–1297.
314. Daaka Y, Luttrell LM, Lefkowitz RJ. Switching of
the coupling of the beta2-adrenergic receptor to
different G proteins by protein kinase A. Nature.
1997;390:88–91.
315. Pitcher JA, Freedman NJ, Lefkowitz RJ. G protein-
coupled receptor kinases. Annu Rev Biochem.
1998;67:653–692.
316. Gurevich EV, Gurevich VV. Arrestins: ubiquitous
regulators of cellular signaling pathways. Genome
Biol. 2006;7:236.
317. Shenoy SK, Lefkowitz RJ. beta-Arrestin-mediated
receptor trafficking and signal transduction. Trends
Pharmacol Sci. 2011;32:521–533.
318. Luttrell LM, et al. Activation and targeting of extra-
cellular signal-regulated kinases by beta-arrestin scaf-
folds. Proc Natl Acad Sci USA. 2001;98:2449–2454.
319. DeFea KA, et al. Beta-arrestin-dependent endocytosis
of proteinase-activated receptor 2 is required for
intracellular targeting of activated ERK1/2. J Cell
Biol. 2000;148:1267–1281.
320. Young D, Waitches G, Birchmeier C, Fasano O,
Wigler M. Isolation and characterization of a
new cellular oncogene encoding a protein with
multiple potential transmembrane domains. Cell.
1986;45:711–719.
321. Zohn IE, Symons M, Chrzanowska-Wodnicka M,
Westwick JK, Der CJ. Mas oncogene signaling
and transformation require the small GTP-binding
protein Rac. Mol Cell Biol. 1998;18:1225–1235.
322. O’Hayre M, et al. The emerging mutational land-
scape of G proteins and G-protein-coupled receptors
in cancer. Nat Rev Cancer. 2013;13:412–424.
323. Daub H, Wallasch C, Lankenau A, Herrlich A, Ullrich
A. Signal characteristics of G protein-transactivated
EGF receptor. EMBO J. 1997;16:7032–7044.
324. Filardo EJ, Quinn JA, Bland KI, Frackelton AR
Jr. Estrogen-induced activation of Erk-1 and Erk-2
requires the G protein-coupled receptor homolog,
GPR30, and occurs via trans-activation of the
epidermal growth factor receptor through release
of HB-EGF. Mol Endocrinol. 2000;14:1649–
1660.
325. Pierce KL, Luttrell LM, Lefkowitz RJ. New
mechanisms in heptahelical receptor signaling to
mitogen activated protein kinase cascades. Oncogene.
2001;20:1532–1539.
326. Hart S, et al. GPCR-induced migration of breast
carcinoma cells depends on both EGFR signal
transactivation and EGFR-independent pathways.
Biol Chem. 2005;386:845–855.
327. Boire A, et al. PAR1 is a matrix metalloprotease-1
receptor that promotes invasion and tumorigenesis
of breast cancer cells. Cell. 2005;120:303–313.
328. Trivedi V, et al. Platelet matrix metalloprotease-1
mediates thrombogenesis by activating PAR1 at a
cryptic ligand site. Cell. 2009;137:332–343.
329. Sahai E, Marshall CJ. RHO-GTPases and cancer.
Nat Rev Cancer. 2002;2:133–142.
330. Mardilovich K, Olson MF, Baugh M. Targeting Rho
GTPase signaling for cancer therapy. Future Oncol.
2012;8:165–177.
331. Morris JPT, Wang SC, Hebrok M. KRAS, Hedgehog,
Wnt and the twisted developmental biology of
pancreatic ductal adenocarcinoma. Nat Rev Cancer.
2010;10:683–695.
332. Takebe N, Harris PJ, Warren RQ, Ivy SP. Targeting
cancer stem cells by inhibiting Wnt, Notch, and
46.e6 Part I: Science and Clinical Oncology
Hedgehog pathways. Nat Rev Clin Oncol. 2011;8:
97–106.
333. Rubin LL, de Sauvage FJ. Targeting the Hedgehog
pathway in cancer. Nat Rev Drug Discov. 2006;5:
1026–1033.
334. Briscoe J, Therond PP. The mechanisms of Hedgehog
signalling and its roles in development and disease.
Nat Rev Mol Cell Biol. 2013;14:416–429.
335. Kasai K, et al. The G12 family of heterotrimeric G
proteins and Rho GTPase mediate Sonic hedgehog
signalling. Genes Cells. 2004;9:49–58.
336. Riobo NA, Saucy B, Dilizio C, Manning DR. Activa-
tion of heterotrimeric G proteins by Smoothened.
Proc Natl Acad Sci USA. 2006;103:12607–12612.
337. Hahn H, et al. Mutations of the human homolog of
Drosophila patched in the nevoid basal cell carcinoma
syndrome. Cell. 1996;85:841–851.
338. Xie J, et al. Activating Smoothened mutations in spo-
radic basal-cell carcinoma. Nature. 1998;391:90–92.
339. Sweeney RT, et al. Identification of recurrent SMO
and BRAF mutations in ameloblastomas. Nat Genet.
2014;46:722–725.
340. Thayer SP, et al. Hedgehog is an early and late
mediator of pancreatic cancer tumorigenesis. Nature.
2003;425:851–856.
341. Von Hoff DD, et al. Inhibition of the hedgehog
pathway in advanced basal-cell carcinoma. N Engl
J Med. 2009;361:1164–1172.
342. Sekulic A, et al. Efficacy and safety of vismodegib
in advanced basal-cell carcinoma. N Engl J Med.
2012;366:2171–2179.
343. Casey D, et al. FDA approval summary: sonidegib
for locally advanced basal cell carcinoma. Clin Cancer
Res. 2017;23:2377–2381.
344. Migden MR, et al. Treatment with two different
doses of sonidegib in patients with locally advanced
or metastatic basal cell carcinoma (BOLT): a mul-
ticentre, randomised, double-blind phase 2 trial.
Lancet Oncol. 2015;16:716–728.
345. Rimkus TK, Carpenter RL, Qasem S, Chan M,
Lo HW. Targeting the Sonic Hedgehog signaling
pathway: review of Smoothened and GLI inhibitors.
Cancers (Basel). 2016;8.
346. Guha M. Hedgehog inhibitor gets landmark skin
cancer approval, but questions remain for wider
potential. Nat Rev Drug Discov. 2012;11:257–258.
347. Clevers H. Wnt/beta-catenin signaling in develop-
ment and disease. Cell. 2006;127:469–480.
348. Liu T, Liu X, Wang H, Moon RT, Malbon CC.
Activation of rat frizzled-1 promotes Wnt signaling
and differentiation of mouse F9 teratocarcinoma cells
via pathways that require Galpha(q) and Galpha(o)
function. J Biol Chem. 1999;274:33539–33544.
349. Klaus A, Birchmeier W. Wnt signalling and its
impact on development and cancer. Nat Rev Cancer.
2008;8:387–398.
350. MacDonald BT, Tamai K, He X. Wnt/beta-
catenin signaling: components, mechanisms, and
diseases. Dev Cell. 2009;17:9–26.
351. He TC, et al. Identification of c-MYC as a target of
the APC pathway. Science. 1998;281:1509–1512.
352. Riese J, et al. LEF-1, a nuclear factor coordinating
signaling inputs from wingless and decapentaplegic.
Cell. 1997;88:777–787.
353. Lai SL, Chien AJ, Moon RT. Wnt/Fz signaling and
the cytoskeleton: potential roles in tumorigenesis.
Cell Res. 2009;19:532–545.
354. Anastas JN, Moon RT. WNT signalling pathways
as therapeutic targets in cancer. Nat Rev Cancer.
2013;13:11–26.
355. Behrens J, Lustig B. The Wnt connection to
tumorigenesis. Int J Dev Biol. 2004;48:477–487.
356. Ashton-Rickardt PG, et al. High frequency of APC
loss in sporadic colorectal carcinoma due to breaks
clustered in 5q21-22. Oncogene. 1989;4:1169–
1174.
357. Groden J, et al. Identification and characterization
of the familial adenomatous polyposis coli gene.
Cell. 1991;66:589–600.
358. Barker N, Clevers H. Mining the Wnt pathway for
cancer therapeutics. Nat Rev Drug Discov. 2006;5:
997–1014.
359. Dihlmann S, von Knebel Doeberitz M. Wnt/beta-
catenin-pathway as a molecular target for future
anti-cancer therapeutics. Int J Cancer. 2005;113:
515–524.
360. Smigel K. Arthritis drug approved for polyp preven-
tion blazes trail for other prevention trials. J Natl
Cancer Inst. 2000;92:297–299.
361. Haque SJ, Sharma P. Interleukins and STAT signal-
ing. Vitam Horm. 2006;74:165–206.
362. Bazan JF. Structural design and molecular evolution
of a cytokine receptor superfamily. Proc Natl Acad
Sci USA. 1990;87:6934–6938.
363. Bazan JF. Shared architecture of hormone binding
domains in type I and II interferon receptors. Cell.
1990;61:753–754.
364. Kim HP, Imbert J, Leonard WJ. Both integrated
and differential regulation of components of the
IL-2/IL-2 receptor system. Cytokine Growth Factor
Rev. 2006;17:349–366.
365. Liao W, Lin JX, Leonard WJ. IL-2 family cytokines:
new insights into the complex roles of IL-2 as a
broad regulator of T helper cell differentiation. Curr
Opin Immunol. 2011;23:598–604.
366. Waldmann TA. The biology of interleukin-2 and
interleukin-15: implications for cancer therapy and
vaccine design. Nat Rev Immunol. 2006;6:595–601.
367. Malek TR, Castro I. Interleukin-2 receptor signaling:
at the interface between tolerance and immunity.
Immunity. 2010;33:153–165.
368. Gonzalez-Navajas JM, Lee J, David M, Raz E.
Immunomodulatory functions of type I interferons.
Nat Rev Immunol. 2012;12:125–135.
369. Platanias LC. Mechanisms of type-I- and type-II-
interferon-mediated signalling. Nat Rev Immunol.
2005;5:375–386.
370. Pestka S, Krause CD, Walter MR. Interferons,
interferon-like cytokines, and their receptors.
Immunol Rev. 2004;202:8–32.
371. Gad HH, et al. Interferon-lambda is function-
ally an interferon but structurally related to the
interleukin-10 family. J Biol Chem. 2009;284:20869–
20875.
372. Stark GR, Darnell JE Jr. The JAK-STAT pathway
at twenty. Immunity. 2012;36:503–514.
373. Yu H, Pardoll D, Jove R. STATs in cancer inflam-
mation and immunity: a leading role for STAT3.
Nat Rev Cancer. 2009;9:798–809.
374. O’Shea JJ, et al. The JAK-STAT pathway: impact on
human disease and therapeutic intervention. Annu
Rev Med. 2015;66:311–328.
375. Shuai K, Liu B. Regulation of JAK-STAT signal-
ling in the immune system. Nat Rev Immunol.
2003;3:900–911.
376. Yu H, Kortylewski M, Pardoll D. Crosstalk between
cancer and immune cells: role of STAT3 in the
tumour microenvironment. Nat Rev Immunol.
2007;7:41–51.
377. Miyazaki T, et al. Three distinct IL-2 signaling
pathways mediated by bcl-2, c-myc, and lck
cooperate in hematopoietic cell proliferation. Cell.
1995;81:223–231.
378. Aggarwal BB, Gupta SC, Kim JH. Historical
perspectives on tumor necrosis factor and its
superfamily: 25 years later, a golden journey. Blood.
2012;119:651–665.
379. Dempsey PW, Doyle SE, He JQ, Cheng G. The
signaling adaptors and pathways activated by TNF
superfamily. Cytokine Growth Factor Rev. 2003;14:
193–209.
380. De Paepe B, Creus KK, De Bleecker JL. The
tumor necrosis factor superfamily of cytokines in
the inflammatory myopathies: potential targets for
therapy. Clin Dev Immunol. 2012;2012:369432.
381. Mahmood Z, Shukla Y. Death receptors: targets
for cancer therapy. Exp Cell Res. 2010;316:887–
899.
382. Morgan MJ, Liu ZG. Reactive oxygen species in
TNFalpha-induced signaling and cell death. Mol
Cells. 2010;30:1–12.
383. Lin A, Karin M. NF-kappaB in cancer: a marked
target. Semin Cancer Biol. 2003;13:107–114.
384. Coulthard LR, White DE, Jones DL, McDermott
MF, Burchill SA. p38(MAPK): stress responses from
molecular mechanisms to therapeutics. Trends Mol
Med. 2009;15:369–379.
385. Wagner EF, Nebreda AR. Signal integration by JNK
and p38 MAPK pathways in cancer development.
Nat Rev Cancer. 2009;9:537–549.
386. Sabapathy K. Role of the JNK pathway in human
diseases. Prog Mol Biol Transl Sci. 2012;106:145–
169.
387. Varfolomeev EE, Ashkenazi A. Tumor necrosis factor:
an apoptosis JuNKie? Cell. 2004;116:491–497.
388. Wullaert A, Heyninck K, Beyaert R. Mechanisms
of crosstalk between TNF-induced NF-kappaB and
JNK activation in hepatocytes. Biochem Pharmacol.
2006;72:1090–1101.
389. Kuroki M, et al. Biological response modifiers
used in cancer biotherapy. Anticancer Res. 2012;32:
2229–2233.
390. Deroose JP, et al. 20 years experience of TNF-based
isolated limb perfusion for in-transit melanoma
metastases: TNF dose matters. Ann Surg Oncol.
2012;19:627–635.
391. Deroose JP, et al. Long-term results of tumor necrosis
factor alpha- and melphalan-based isolated limb
perfusion in locally advanced extremity soft tissue
sarcomas. J Clin Oncol. 2011;29:4036–4044.
392. Deisseroth A, et al. U.S. Food and Drug Administra-
tion approval: ruxolitinib for the treatment of patients
with intermediate and high-risk myelofibrosis. Clin
Cancer Res. 2012;18:3212–3217.
393. Mascarenhas J, Hoffman R. Ruxolitinib: the
first FDA approved therapy for the treatment of
myelofibrosis. Clin Cancer Res. 2012;18:3008–
3014.
394. Verstovsek S, et al. A double-blind, placebo-
controlled trial of ruxolitinib for myelofibrosis. N
Engl J Med. 2012;366:799–807.
395. Yu H, Lee H, Herrmann A, Buettner R, Jove R.
Revisiting STAT3 signalling in cancer: new and
unexpected biological functions. Nat Rev Cancer.
2014;14:736–746.
396. O’Shea JJ, Holland SM, Staudt LM. JAKs and STATs
in immunity, immunodeficiency, and cancer. N Engl
J Med. 2013;368:161–170.
397. Bubici C, Papa S. JNK signalling in cancer: in need
of new, smarter therapeutic targets. Br J Pharmacol.
2014;171:24–37.
398. Hinck AP. Structural studies of the TGF-betas and
their receptors - insights into evolution of the TGF-
beta superfamily. FEBS Lett. 2012;586:1860–1870.
399. Shi Y, Massague J. Mechanisms of TGF-beta
signaling from cell membrane to the nucleus. Cell.
2003;113:685–700.
400. Zi Z, Chapnick DA, Liu X. Dynamics of TGF-beta/
Smad signaling. FEBS Lett. 2012;586:1921–1928.
401. Annes JP, Munger JS, Rifkin DB. Making
sense of latent TGFbeta activation. J Cell Sci.
2003;116:217–224.
402. Rifkin DB. Latent transforming growth factor-beta
(TGF-beta) binding proteins: orchestrators of TGF-
beta availability. J Biol Chem. 2005;280:7409–7412.
403. Xu P, Liu J, Derynck R. Post-translational regulation
of TGF-beta receptor and Smad signaling. FEBS
Lett. 2012;586:1871–1884.
404. Derynck R, Zhang YE. Smad-dependent and
Smad-independent pathways in TGF-beta family
signalling. Nature. 2003;425:577–584.
405. Itoh S, ten Dijke P. Negative regulation of TGF-beta
receptor/Smad signal transduction. Curr Opin Cell
Biol. 2007;19:176–184.
406. Heldin CH, Vanlandewijck M, Moustakas A.
Regulation of EMT by TGFbeta in cancer. FEBS
Lett. 2012;586:1959–1970.

407. Ikushima H, Miyazono K. TGFbeta signalling: a
complex web in cancer progression. Nat Rev Cancer.
2010;10:415–424.
408. Moustakas A, Heldin CH. The regulation of
TGFbeta signal transduction. Development.
2009;136:3699–3714.
409. Javelaud D, Pierrat MJ, Mauviel A. Crosstalk between
TGF-beta and hedgehog signaling in cancer. FEBS
Lett. 2012;586:2016–2025.
410. Massague J. TGFbeta signalling in context. Nat Rev
Mol Cell Biol. 2012;13:616–630.
411. Neuzillet C, et al. Targeting the TGFbeta pathway
for cancer therapy. Pharmacol Ther. 2015;147:22–31.
412. Massague J. TGFbeta in Cancer. Cell. 2008;134:
215–230.
413. Markowitz S, et al. Inactivation of the type II TGF-
beta receptor in colon cancer cells with microsatellite
instability. Science. 1995;268:1336–1338.
414. Bornstein S, et al. Smad4 loss in mice causes
spontaneous head and neck cancer with increased
genomic instability and inflammation. J Clin Invest.
2009;119:3408–3419.
415. Levy L, Hill CS. Alterations in components of the
TGF-beta superfamily signaling pathways in human
cancer. Cytokine Growth Factor Rev. 2006;17:41–58.
416. David CJ, et al. TGF-beta tumor suppression through
a lethal EMT. Cell. 2016;164:1015–1030.
417. Yingling JM, Blanchard KL, Sawyer JS. Development
of TGF-beta signalling inhibitors for cancer therapy.
Nat Rev Drug Discov. 2004;3:1011–1022.
418. Schlingensiepen KH, et al. Targeted tumor therapy
with the TGF-beta 2 antisense compound AP 12009.
Cytokine Growth Factor Rev. 2006;17:129–139.
419. Roberts AB, Wakefield LM. The two faces of
transforming growth factor beta in carcinogenesis.
Proc Natl Acad Sci USA. 2003;100:8621–8623.
420. Bierie B, Moses HL. Tumour microenvironment:
TGFbeta: the molecular Jekyll and Hyde of cancer.
Nat Rev Cancer. 2006;6:506–520.
421. Takebe N, Nguyen D, Yang SX. Targeting notch
signaling pathway in cancer: clinical develop-
ment advances and challenges. Pharmacol Ther.
2014;141:140–149.
422. Yin L, Velazquez OC, Liu ZJ. Notch signaling:
emerging molecular targets for cancer therapy.
Biochem Pharmacol. 2010;80:690–701.
423. Ranganathan P, Weaver KL, Capobianco AJ.
Notch signalling in solid tumours: a little bit of
everything but not all the time. Nat Rev Cancer.
2011;11:338–351.
424. Roy M, Pear WS, Aster JC. The multifaceted
role of Notch in cancer. Curr Opin Genet Dev.
2007;17:52–59.
425. Blaumueller CM, Qi H, Zagouras P, Artavanis-
Tsakonas S. Intracellular cleavage of Notch leads
to a heterodimeric receptor on the plasma membrane.
Cell. 1997;90:281–291.
426. Shao H, Huang Q, Liu ZJ. Targeting notch signaling
for cancer therapeutic intervention. Adv Pharmacol.
2012;65:191–234.
427. Bray SJ. Notch signalling: a simple pathway becomes
complex. Nat Rev Mol Cell Biol. 2006;7:678–689.
428. Brou C, et al. A novel proteolytic cleavage involved
in Notch signaling: the role of the disintegrin-
metalloprotease TACE. Mol Cell. 2000;5:207–216.
429. Mumm JS, et al. A ligand-induced extracellular
cleavage regulates gamma-secretase-like proteolytic
activation of Notch1. Mol Cell. 2000;5:197–206.
430. Schroeter EH, Kisslinger JA, Kopan R. Notch-1
signalling requires ligand-induced proteolytic release
of intracellular domain. Nature. 1998;393:382–386.
431. Aster JC, Blacklow SC, Pear WS. Notch signal-
ling in T-cell lymphoblastic leukaemia/lymphoma
and other haematological malignancies. J Pathol.
2011;223:262–273.
432. Ellisen LW, et al. TAN-1, the human homolog of the
Drosophila notch gene, is broken by chromosomal
translocations in T lymphoblastic neoplasms. Cell.
1991;66:649–661.
433. Weng AP, et al. Activating mutations of NOTCH1 in
human T cell acute lymphoblastic leukemia. Science.
2004;306:269–271.
434. Krop I, et al. Phase I Pharmacologic and pharma-
codynamic study of the gamma secretase (Notch)
inhibitor MK-0752 in adult patients with advanced
solid tumors. J Clin Oncol. 2012;30:2307–2313.
435. Schott AF, et al. Preclinical and clinical studies of
gamma secretase inhibitors with docetaxel on human
breast tumors. Clin Cancer Res. 2013;19:1512–1524.
436. Gavai AV, et al. Discovery of clinical candidate
BMS-906024: a potent pan-Notch inhibitor for
the treatment of leukemia and solid tumors. ACS
Med Chem Lett. 2015;6:523–527.
437. Yen WC, et al. Targeting Notch signaling with a
Notch2/Notch3 antagonist (tarextumab) inhibits
tumor growth and decreases tumor-initiating cell
frequency. Clin Cancer Res. 2015;21:2084–2095.
438. Kumar R, Thompson EB. The structure of the nuclear
hormone receptors. Steroids. 1999;64:310–319.
439. Mangelsdorf DJ, et al. The nuclear receptor
superfamily: the second decade. Cell. 1995;83:835–
839.
440. Gronemeyer H, Gustafsson JA, Laudet V. Principles
for modulation of the nuclear receptor superfamily.
Nat Rev Drug Discov. 2004;3:950–964.
441. Shang Y, Hu X, DiRenzo J, Lazar MA, Brown M.
Cofactor dynamics and sufficiency in estrogen recep-
tor-regulated transcription. Cell. 2000;103:843–852.
442. Toy W, et al. Activating ESR1 mutations differentially
affect the efficacy of ER antagonists. Cancer Discov.
2017;7:277–287.
443. Fribbens C, et al. Plasma ESR1 mutations and the
treatment of estrogen receptor-positive advanced
breast cancer. J Clin Oncol. 2016;34:2961–2968.
444. Wakeling AE. Steroid antagonists as nuclear receptor
blockers. Cancer Surv. 1992;14:71–85.
445. Scher HI, et al. Increased survival with enzalutamide
in prostate cancer after chemotherapy. N Engl J Med.
2012;367:1187–1197.
446. Ryan CJ, et al. Abiraterone acetate plus prednisone
versus placebo plus prednisone in chemotherapy-
naive men with metastatic castration-resistant
prostate cancer (COU-AA-302): final overall
survival analysis of a randomised, double-blind,
placebo-controlled phase 3 study. Lancet Oncol.
2015;16:152–160.
447. Mitra SK, Schlaepfer DD. Integrin-regulated
FAK-Src signaling in normal and cancer cells. Curr
Opin Cell Biol. 2006;18:516–523.
448. Assoian RK, Klein EA. Growth control by intracel-
lular tension and extracellular stiffness. Trends Cell
Biol. 2008;18:347–352.
449. Pytela R, Pierschbacher MD, Ruoslahti E. Iden-
tification and isolation of a 140 kd cell surface
glycoprotein with properties expected of a fibronectin
receptor. Cell. 1985;40:191–198.
450. Guo W, Giancotti FG. Integrin signalling during
tumour progression. Nat Rev Mol Cell Biol.
2004;5:816–826.
451. Ley K, Rivera-Nieves J, Sandborn WJ, Shattil
S. Integrin-based therapeutics: biological basis,
clinical use and new drugs. Nat Rev Drug Discov.
2016;15:173–183.
452. Legate KR, Wickstrom SA, Fassler R. Genetic and cell
biological analysis of integrin outside-in signaling.
Genes Dev. 2009;23:397–418.
453. Winograd-Katz SE, Fassler R, Geiger B, Legate
KR. The integrin adhesome: from genes and
proteins to human disease. Nat Rev Mol Cell Biol.
2014;15:273–288.
454. Han S, Khuri FR, Roman J. Fibronectin stimulates
non-small cell lung carcinoma cell growth through
activation of Akt/mammalian target of rapamycin/
S6 kinase and inactivation of LKB1/AMP-activated
protein kinase signal pathways. Cancer Res. 2006;66:
315–323.
455. Garmy-Susini B, et al. Integrin alpha4beta1-VCAM-
1-mediated adhesion between endothelial and mural
Intracellular Signaling  •  CHAPTER 2 46.e7
46.e7
cells is required for blood vessel maturation. J Clin
Invest. 2005;115:1542–1551.
456. Desgrosellier JS, Cheresh DA. Integrins in cancer:
biological implications and therapeutic opportuni-
ties. Nat Rev Cancer. 2010;10:9–22.
457. Fournier AK, et al. Rac-dependent cyclin D1 gene
expression regulated by cadherin- and integrin-
mediated adhesion. J Cell Sci. 2008;121:226–233.
458. Hsu MY, et al. Adenoviral gene transfer of beta3
integrin subunit induces conversion from radial to
vertical growth phase in primary human melanoma.
Am J Pathol. 1998;153:1435–1442.
459. Nip J, Shibata H, Loskutoff DJ, Cheresh DA,
Brodt P. Human melanoma cells derived from
lymphatic metastases use integrin alpha v beta 3
to adhere to lymph node vitronectin. J Clin Invest.
1992;90:1406–1413.
460. Bello L, et al. Alpha(v)beta3 and alpha(v)beta5
integrin expression in glioma periphery. Neurosurgery.
2001;49:380–389, discussion 390.
461. Diaz LK, et al. Beta4 integrin subunit gene expression
correlates with tumor size and nuclear grade in early
breast cancer. Mod Pathol. 2005;18:1165–1175.
462. Felding-Habermann B, et al. Integrin activation
controls metastasis in human breast cancer. Proc
Natl Acad Sci USA. 2001;98:1853–1858.
463. Friedrichs K, et al. High expression level of alpha
6 integrin in human breast carcinoma is correlated
with reduced survival. Cancer Res. 1995;55:901–906.
464. Hersey P, et al. A phase II, randomized, open-label
study evaluating the antitumor activity of MEDI-
522, a humanized monoclonal antibody directed
against the human alpha v beta 3 (avb3) integrin,
± dacarbazine (DTIC) in patients with metastatic
melanoma (MM). J Clin Oncol. 2005;23.
465. Stupp R, et al. Cilengitide combined with stan-
dard treatment for patients with newly diagnosed
glioblastoma with methylated MGMT promoter
(CENTRIC EORTC 26071-22072 study): a
multicentre, randomised, open-label, phase 3 trial.
Lancet Oncol. 2014;15:1100–1108.
466. Yeatman TJ. A renaissance for SRC. Nat Rev Cancer.
2004;4:470–480.
467. Thomas SM, Brugge JS. Cellular functions regulated
by Src family kinases. Annu Rev Cell Dev Biol.
1997;13:513–609.
468. Playford MP, Schaller MD. The interplay between
Src and integrins in normal and tumor biology.
Oncogene. 2004;23:7928–7946.
469. Kim LC, Song L, Haura EB. Src kinases as
therapeutic targets for cancer. Nat Rev Clin Oncol.
2009;6:587–595.
470. Vogt PK. Retroviral oncogenes: a historical primer.
Nat Rev Cancer. 2012;12:639–648.
471. Martin GS. Rous sarcoma virus: a function required
for the maintenance of the transformed state. Nature.
1970;227:1021–1023.
472. Rous P. A sarcoma of the fowl transmissible by an
agent separable from the tumor cells. J Exp Med.
1911;13:397–411.
473. Rubin H. Quantitative relations between causative
virus and cell in the Rous no. 1 chicken sarcoma.
Virology. 1955;1:445–473.
474. Stehelin D, Varmus HE, Bishop JM, Vogt PK. DNA
related to the transforming gene(s) of avian sarcoma
viruses is present in normal avian DNA. Nature.
1976;260:170–173.
475. Aleshin A, Finn RS. SRC: a century of science
brought to the clinic. Neoplasia. 2010;12:599–607.
476. Cartwright CA, Eckhart W, Simon S, Kaplan PL.
Cell transformation by pp60c-src mutated in the
carboxy-terminal regulatory domain. Cell. 1987;49:
83–91.
477. Yu H, et al. Solution structure of the SH3 domain
of Src and identification of its ligand-binding site.
Science. 1992;258:1665–1668.
478. Sen B, Johnson FM. Regulation of SRC family kinases
in human cancers. J Signal Transduct. 2011;2011:
865819.
46.e8 Part I: Science and Clinical Oncology
479. Xu W, Harrison SC, Eck MJ. Three-dimensional
structure of the tyrosine kinase c-Src. Nature. 1997;
385:595–602.
480. Sakai R, et al. A novel signaling molecule, p130,
forms stable complexes in vivo with v-Crk and v-Src
in a tyrosine phosphorylation-dependent manner.
EMBO J. 1994;13:3748–3756.
481. Westhoff MA, Serrels B, Fincham VJ, Frame MC,
Carragher NO. SRC-mediated phosphorylation of
focal adhesion kinase couples actin and adhesion
dynamics to survival signaling. Mol Cell Biol. 2004;24:
8113–8133.
482. Parsons JT, Parsons SJ. Src family protein tyrosine
kinases: cooperating with growth factor and adhesion
signaling pathways. Curr Opin Cell Biol. 1997;9:
187–192.
483. Frame MC. Src in cancer: deregulation and conse-
quences for cell behaviour. Biochim Biophys Acta.
2002;1602:114–130.
484. Zhang S, Yu D. Targeting Src family kinases in
anti-cancer therapies: turning promise into triumph.
Trends Pharmacol Sci. 2012;33:122–128.
485. Montero JC, Seoane S, Ocana A, Pandiella A. Inhibi-
tion of SRC family kinases and receptor tyrosine
kinases by dasatinib: possible combinations in solid
tumors. Clin Cancer Res. 2011;17:5546–5552.
486. Puls LN, Eadens M, Messersmith W. Current
status of SRC inhibitors in solid tumor malignan-
cies. Oncologist. 2011;16:566–578.
487. Reynolds AB, et al. SRChing for the substrates of
Src. Oncogene. 2014;33:4537–4547.
488. Masaki T, et al. pp60c-src activation in hepatocellular
carcinoma of humans and LEC rats. Hepatology.
1998;27:1257–1264.
489. Masaki T, et al. Reduced C-terminal Src kinase (Csk)
activities in hepatocellular carcinoma. Hepatology.
1999;29:379–384.
490. Cam WR, et al. Reduced C-terminal Src kinase activ-
ity is correlated inversely with pp60(c-src) activity
in colorectal carcinoma. Cancer. 2001;92:61–70.
491. Taniguchi K, et al. A gp130-Src-YAP module links
inflammation to epithelial regeneration. Nature.
2015;519:57–62.
492. Armaiz-Pena GN, et al. Src activation by beta-
adrenoreceptors is a key switch for tumour metastasis.
Nat Commun. 2013;4:1403.
493. Lindauer M, Hochhaus A. Dasatinib. Recent Results
Cancer Res. 2010;184:83–102.
494. Lombardo LJ, et al. Discovery of N-(2-chloro-
6-methyl-phenyl)-2-(6-(4-(2-hydroxyethyl)-
piperazin-1-yl)-2-methylpyrimidin-4-ylamino)
thiazole-5-carboxamide (BMS-354825), a dual Src/
Abl kinase inhibitor with potent antitumor activity in
preclinical assays. J Med Chem. 2004;47:6658–6661.
495. Chang Q, Jorgensen C, Pawson T, Hedley DW.
Effects of dasatinib on EphA2 receptor tyrosine
kinase activity and downstream signalling in
pancreatic cancer. Br J Cancer. 2008;99:1074–1082.
496. Goff SP, Gilboa E, Witte ON, Baltimore D. Structure
of the Abelson murine leukemia virus genome and
the homologous cellular gene: studies with cloned
viral DNA. Cell. 1980;22:777–785.
497. Hantschel O, et al. A myristoyl/phosphotyrosine
switch regulates c-Abl. Cell. 2003;112:845–857.
498. Schlatterer SD, Acker CM, Davies P. c-Abl
in neurodegenerative disease. J Mol Neurosci.
2011;45:445–452.
499. Daniel R, Cai Y, Wong PM, Chung SW. Deregula-
tion of c-abl mediated cell growth after retroviral
transfer and expression of antisense sequences.
Oncogene. 1995;10:1607–1614.
500. Yuan ZM, et al. Regulation of Rad51 function by
c-Abl in response to DNA damage. J Biol Chem.
1998;273:3799–3802.
501. Sirvent A, Benistant C, Roche S. Cytoplasmic
signalling by the c-Abl tyrosine kinase in normal
and cancer cells. Biol Cell. 2008;100:617–631.
502. Greuber EK, Smith-Pearson P, Wang J, Pendergast
AM. Role of ABL family kinases in cancer: from
leukaemia to solid tumours. Nat Rev Cancer. 2013;13:
559–571.
503. Ren R. Mechanisms of BCR-ABL in the pathogenesis
of chronic myelogenous leukaemia. Nat Rev Cancer.
2005;5:172–183.
504. O’Hare T, Zabriskie MS, Eiring AM, Deininger
MW. Pushing the limits of targeted therapy in
chronic myeloid leukaemia. Nat Rev Cancer.
2012;12:513–526.
505. Druker BJ, et al. Effects of a selective inhibitor of
the Abl tyrosine kinase on the growth of Bcr-Abl
positive cells. Nat Med. 1996;2:561–566.
506. Hantschel O, Grebien F, Superti-Furga G. The
growing arsenal of ATP-competitive and allosteric
inhibitors of BCR-ABL. Cancer Res. 2012;72:
4890–4895.
507. Ottmann O, et  al. Dasatinib induces rapid
hematologic and cytogenetic responses in adult
patients with Philadelphia chromosome positive
acute lymphoblastic leukemia with resistance or
intolerance to imatinib: interim results of a phase
2 study. Blood. 2007;110:2309–2315.
508. Lilly MB, et al. Dasatinib 140 mg once daily versus
70 mg twice daily in patients with Ph-positive acute
lymphoblastic leukemia who failed imatinib: Results
from a phase 3 study. Am J Hematol. 2010;85:
164–170.
509. Druker BJ, et al. Activity of a specific inhibitor of
the BCR-ABL tyrosine kinase in the blast crisis of
chronic myeloid leukemia and acute lymphoblastic
leukemia with the Philadelphia chromosome. N Engl
J Med. 2001;344:1038–1042.
510. Ottmann OG, et al. A phase 2 study of imatinib
in patients with relapsed or refractory Philadelphia
chromosome-positive acute lymphoid leukemias.
Blood. 2002;100:1965–1971.
511. Druker BJ, et al. Five-year follow-up of patients
receiving imatinib for chronic myeloid leukemia.
N Engl J Med. 2006;355:2408–2417.
512. Zhang J, et al. Targeting Bcr-Abl by combining
allosteric with ATP-binding-site inhibitors. Nature.
2010;463:501–506.
513. Downward J. Targeting RAS signalling pathways
in cancer therapy. Nat Rev Cancer. 2003;3:11–22.
514. Malumbres M, Barbacid M. RAS oncogenes: the
first 30 years. Nat Rev Cancer. 2003;3:459–465.
515. Harvey JJ. An unidentified virus which causes
the rapid production of tumours in mice. Nature.
1964;204:1104–1105.
516. Kirsten WH, Mayer LA. Morphologic responses to
a murine erythroblastosis virus. J Natl Cancer Inst.
1967;39:311–335.
517. Shih TY, Papageorge AG, Stokes PE, Weeks MO,
Scolnick EM. Guanine nucleotide-binding and
autophosphorylating activities associated with the
p21src protein of Harvey murine sarcoma virus.
Nature. 1980;287:686–691.
518. Karnoub AE, Weinberg RA. Ras oncogenes: split
personalities. Nat Rev Mol Cell Biol. 2008;9:517–531.
519. Lacal PM, Pennington CY, Lacal JC. Transforming
activity of ras proteins translocated to the plasma
membrane by a myristoylation sequence from the
src gene product. Oncogene. 1988;2:533–537.
520. Mor A, Philips MR. Compartmentalized Ras/MAPK
signaling. Annu Rev Immunol. 2006;24:771–800.
521. Willumsen BM, Christensen A, Hubbert NL,
Papageorge AG, Lowy DR. The p21 ras C-terminus
is required for transformation and membrane associa-
tion. Nature. 1984;310:583–586.
522. Mitin N, Rossman KL, Der CJ. Signaling
interplay in Ras superfamily function. Curr Biol.
2005;15:R563–R574.
523. Vigil D, Cherfils J, Rossman KL, Der CJ. Ras
superfamily GEFs and GAPs: validated and trac-
table targets for cancer therapy? Nat Rev Cancer.
2010;10:842–857.
524. Bourne HR, Sanders DA, McCormick F. The
GTPase superfamily: a conserved switch for diverse
cell functions. Nature. 1990;348:125–132.
525. Gale NW, Kaplan S, Lowenstein EJ, Schlessinger
J, Bar-Sagi D. Grb2 mediates the EGF-dependent
activation of guanine nucleotide exchange on Ras.
Nature. 1993;363:88–92.
526. Buday L, Downward J. Epidermal growth factor
regulates p21ras through the formation of a complex
of receptor, Grb2 adapter protein, and Sos nucleotide
exchange factor. Cell. 1993;73:611–620.
527. Egan SE, et al. Association of Sos Ras exchange
protein with Grb2 is implicated in tyrosine kinase
signal transduction and transformation. Nature.
1993;363:45–51.
528. Li N, et al. Guanine-nucleotide-releasing factor hSos1
binds to Grb2 and links receptor tyrosine kinases
to Ras signalling. Nature. 1993;363:85–88.
529. Rozakis-Adcock M, Fernley R, Wade J, Pawson T,
Bowtell D. The SH2 and SH3 domains of mam-
malian Grb2 couple the EGF receptor to the Ras
activator mSos1. Nature. 1993;363:83–85.
530. Donovan S, Shannon KM, Bollag G. GTPase
activating proteins: critical regulators of intracellular
signaling. Biochim Biophys Acta. 2002;1602:23–45.
531. Wittinghofer A, Pai EF. The structure of Ras protein:
a model for a universal molecular switch. Trends
Biochem Sci. 1991;16:382–387.
532. Ma J, Karplus M. Molecular switch in signal
transduction: reaction paths of the conformational
changes in ras p21. Proc Natl Acad Sci USA. 1997;94:
11905–11910.
533. Chang L, Karin M. Mammalian MAP kinase
signalling cascades. Nature. 2001;410:37–40.
534. Osborne JK, Zaganjor E, Cobb MH. Signal control
through Raf: in sickness and in health. Cell Res.
2012;22:14–22.
535. Beeram M, Patnaik A, Rowinsky EK. Raf: a strategic
target for therapeutic development against cancer.
J Clin Oncol. 2005;23:6771–6790.
536. Moodie SA, Willumsen BM, Weber MJ, Wolfman
A. Complexes of Ras.GTP with Raf-1 and mitogen-
activated protein kinase kinase. Science. 1993;260:
1658–1661.
537. Vojtek AB, Hollenberg SM, Cooper JA. Mammalian
Ras interacts directly with the serine/threonine kinase
Raf. Cell. 1993;74:205–214.
538. Warne PH, Viciana PR, Downward J. Direct
interaction of Ras and the amino-terminal region
of Raf-1 in vitro. Nature. 1993;364:352–355.
539. Zhang XF, et al. Normal and oncogenic p21ras
proteins bind to the amino-terminal regulatory
domain of c-Raf-1. Nature. 1993;364:308–313.
540. Avruch J, et al. Ras activation of the Raf kinase:
tyrosine kinase recruitment of the MAP kinase
cascade. Recent Prog Horm Res. 2001;56:127–155.
541. Marais R, Light Y, Paterson HF, Mason CS, Marshall
CJ. Differential regulation of Raf-1, A-Raf, and
B-Raf by oncogenic ras and tyrosine kinases. J Biol
Chem. 1997;272:4378–4383.
542. Kubicek M, et al. Dephosphorylation of Ser-259
regulates Raf-1 membrane association. J Biol Chem.
2002;277:7913–7919.
543. Zimmermann S, Moelling K. Phosphorylation and
regulation of Raf by Akt (protein kinase B). Science.
1999;286:1741–1744.
544. Dhillon AS, et al. A Raf-1 mutant that dissociates
MEK/extracellular signal-regulated kinase activation
from malignant transformation and differentiation
but not proliferation. Mol Cell Biol. 2003;23:
1983–1993.
545. Dhillon AS, Meikle S, Yazici Z, Eulitz M, Kolch
W. Regulation of Raf-1 activation and signalling
by dephosphorylation. EMBO J. 2002;21:64–
71.
546. Zhang BH, et al. Serum- and glucocorticoid-inducible
kinase SGK phosphorylates and negatively regulates
B-Raf. J Biol Chem. 2001;276:31620–31626.
547. Morrison DK, Heidecker G, Rapp UR,
Copeland TD. Identification of the major phos-
phorylation sites of the Raf-1 kinase. J Biol Chem.
1993;268:17309–17316.

548. Diaz B, et al. Phosphorylation of Raf-1 serine
338-serine 339 is an essential regulatory event for
Ras-dependent activation and biological signaling.
Mol Cell Biol. 1997;17:4509–4516.
549. King AJ, et al. The protein kinase Pak3 positively
regulates Raf-1 activity through phosphorylation of
serine 338. Nature. 1998;396:180–183.
550. Fabian JR, Daar IO, Morrison DK. Critical tyrosine
residues regulate the enzymatic and biological activity
of Raf-1 kinase. Mol Cell Biol. 1993;13:7170–7179.
551. Marais R, Light Y, Paterson HF, Marshall CJ.
Ras recruits Raf-1 to the plasma membrane for
activation by tyrosine phosphorylation. EMBO J.
1995;14:3136–3145.
552. Mason CS, et al. Serine and tyrosine phosphoryla-
tions cooperate in Raf-1, but not B-Raf activation.
EMBO J. 1999;18:2137–2148.
553. Chong H, Lee J, Guan KL. Positive and negative
regulation of Raf kinase activity and function by
phosphorylation. EMBO J. 2001;20:3716–3727.
554. Zhang BH, Guan KL. Activation of B-Raf kinase
requires phosphorylation of the conserved residues
Thr598 and Ser601. EMBO J. 2000;19:5429–5439.
555. Dent P, et al. Activation of mitogen-activated protein
kinase kinase by v-Raf in NIH 3T3 cells and in
vitro. Science. 1992;257:1404–1407.
556. Khokhlatchev AV, et al. Phosphorylation of the MAP
kinase ERK2 promotes its homodimerization and
nuclear translocation. Cell. 1998;93:605–615.
557. Yoon S, Seger R. The extracellular signal-regulated
kinase: multiple substrates regulate diverse cellular
functions. Growth Factors. 2006;24:21–44.
558. Robinson MJ, Cobb MH. Mitogen-activated protein
kinase pathways. Curr Opin Cell Biol. 1997;9:
180–186.
559. Pratilas CA, Solit DB. Therapeutic strategies for
targeting BRAF in human cancer. Rev Recent Clin
Trials. 2007;2:121–134.
560. Pratilas CA, Solit DB. Targeting the mitogen-
activated protein kinase pathway: physiological feed-
back and drug response. Clin Cancer Res. 2010;16:
3329–3334.
561. Kim HJ, Bar-Sagi D. Modulation of signalling by
Sprouty: a developing story. Nat Rev Mol Cell Biol.
2004;5:441–450.
562. Sasaki A, et al. Mammalian Sprouty4 suppresses
Ras-independent ERK activation by binding to Raf1.
Nat Cell Biol. 2003;5:427–432.
563. Hanafusa H, Torii S, Yasunaga T, Nishida E.
Sprouty1 and Sprouty2 provide a control mechanism
for the Ras/MAPK signalling pathway. Nat Cell Biol.
2002;4:850–858.
564. Wakioka T, et al. Spred is a Sprouty-related suppres-
sor of Ras signalling. Nature. 2001;412:647–651.
565. Owens DM, Keyse SM. Differential regulation of
MAP kinase signalling by dual-specificity protein
phosphatases. Oncogene. 2007;26:3203–3213.
566. Keyse SM. Dual-specificity MAP kinase phospha-
tases (MKPs) and cancer. Cancer Metastasis Rev.
2008;27:253–261.
567. Ritt DA, Monson DM, Specht SI, Morrison DK.
Impact of feedback phosphorylation and Raf
heterodimerization on normal and mutant B-Raf
signaling. Mol Cell Biol. 2010;30:806–819.
568. Dougherty MK, et al. Regulation of Raf-1 by direct
feedback phosphorylation. Mol Cell. 2005;17:
215–224.
569. Matheny SA, et al. Ras regulates assembly of
mitogenic signalling complexes through the effector
protein IMP. Nature. 2004;427:256–260.
570. Therrien M, et al. KSR, a novel protein kinase
required for RAS signal transduction. Cell. 1995;83:
879–888.
571. Yeung K, et al. Suppression of Raf-1 kinase activ-
ity and MAP kinase signalling by RKIP. Nature.
1999;401:173–177.
572. Rodriguez-Viciana P, et al. Phosphatidylinositol-3-OH
kinase as a direct target of Ras. Nature. 1994;370:
527–532.
573. Sjolander A, Yamamoto K, Huber BE, Lapetina
EG. Association of p21ras with phosphatidylino-
sitol 3-kinase. Proc Natl Acad Sci USA. 1991;88:
7908–7912.
574. Gille H, Downward J. Multiple ras effector pathways
contribute to G(1) cell cycle progression. J Biol
Chem. 1999;274:22033–22040.
575. Rodriguez-Viciana P, et al. Role of phosphoinositide
3-OH kinase in cell transformation and control of the
actin cytoskeleton by Ras. Cell. 1997;89:457–467.
576. Hofer F, Fields S, Schneider C, Martin GS. Activated
Ras interacts with the Ral guanine nucleotide
dissociation stimulator. Proc Natl Acad Sci USA.
1994;91:11089–11093.
577. Kikuchi A, Demo SD, Ye ZH, Chen YW, Williams
LT. ralGDS family members interact with the effector
loop of ras. Mol Cell Biol. 1994;14(7483–7491):p21.
578. Spaargaren M, Bischoff JR. Identification of
the guanine nucleotide dissociation stimulator
for Ral as a putative effector molecule of R-ras,
H-ras, K-ras, and Rap. Proc Natl Acad Sci USA.
1994;91:12609–12613.
579. Schubbert S, Shannon K, Bollag G. Hyperactive
Ras in developmental disorders and cancer. Nat Rev
Cancer. 2007;7:295–308.
580. Vakiani E, Solit DB. KRAS and BRAF: drug targets
and predictive biomarkers. J Pathol. 2011;223:
219–229.
581. Pylayeva-Gupta Y, Grabocka E, Bar-Sagi D. RAS
oncogenes: weaving a tumorigenic web. Nat Rev
Cancer. 2011;11:761–774.
582. Malumbres M, Pellicer A. RAS pathways to cell
cycle control and cell transformation. Front Biosci.
1998;3:d887–d912.
583. Feig LA, Cooper GM. Relationship among guanine
nucleotide exchange, GTP hydrolysis, and transform-
ing potential of mutated ras proteins. Mol Cell Biol.
1988;8:2472–2478.
584. Chen SY, Huff SY, Lai CC, Der CJ, Powers S.
Ras-15A protein shares highly similar dominant-
negative biological properties with Ras-17N
and forms a stable, guanine-nucleotide resistant
complex with CDC25 exchange factor. Oncogene.
1994;9:2691–2698.
585. Cancer Genome Atlas Research Network. Compre-
hensive genomic characterization defines human glio-
blastoma genes and core pathways. Nature. 2008;455:
1061–1068.
586. Cancer Genome Atlas Network. Genomic clas-
sification of cutaneous melanoma. Cell. 2015;161:
1681–1696.
587. Nissan MH, et al. Loss of NF1 in cutaneous
melanoma is associated with RAS activation and
MEK dependence. Cancer Res. 2014;74:2340–2350.
588. Imielinski M, et al. Oncogenic and sorafenib-sensitive
ARAF mutations in lung adenocarcinoma. J Clin
Invest. 2014;124:1582–1586.
589. Diamond EL, et al. Diverse and targetable kinase
alterations drive histiocytic neoplasms. Cancer Discov.
2016;6:154–165.
590. Nelson DS, et  al. Somatic activating ARAF
mutations in Langerhans cell histiocytosis. Blood.
2014;123:3152–3155.
591. Tiacci E, et al. BRAF mutations in hairy-cell
leukemia. N Engl J Med. 2011;364:2305–2315.
592. Lito P, Rosen N, Solit DB. Tumor adaptation
and resistance to RAF inhibitors. Nat Med.
2013;19:1401–1409.
593. Yao Z, et al. BRAF mutants evade ERK-dependent
feedback by different mechanisms that determine
their sensitivity to pharmacologic inhibition. Cancer
Cell. 2015;28:370–383.
594. Arcila ME, et al. MAP2K1 (MEK1) mutations define
a distinct subset of lung adenocarcinoma associated
with smoking. Clin Cancer Res. 2015;21:1935–
1943.
595. Van Allen EM, et al. The genetic landscape of clinical
resistance to RAF inhibition in metastatic melanoma.
Cancer Discov. 2014;4:94–109.
Intracellular Signaling  •  CHAPTER 2 46.e9
46.e9
596. Brenan L, et al. Phenotypic characterization of
a comprehensive set of MAPK1/ERK2 missense
mutants. Cell Rep. 2016;17:1171–1183.
597. Montagut C, Settleman J. Targeting the RAF-
MEK-ERK pathway in cancer therapy. Cancer Lett.
2009;283:125–134.
598. Santarpia L, Lippman SM, El-Naggar AK. Targeting
the MAPK-RAS-RAF signaling pathway in cancer
therapy. Expert Opin Ther Targets. 2012;16:103–119.
599. Rao S, et al. Phase III double-blind placebo-
controlled study of farnesyl transferase inhibitor
R115777 in patients with refractory advanced
colorectal cancer. J Clin Oncol. 2004;22:3950–3957.
600. Rowell CA, Kowalczyk JJ, Lewis MD, Garcia
AM. Direct demonstration of geranylgeranylation
and farnesylation of Ki-Ras in vivo. J Biol Chem.
1997;272:14093–14097.
601. Whyte DB, et al. K- and N-Ras are geranylgeranyl-
ated in cells treated with farnesyl protein transferase
inhibitors. J Biol Chem. 1997;272:14459–14464.
602. Ostrem JM, Peters U, Sos ML, Wells JA, Shokat
KM. K-Ras(G12C) inhibitors allosterically control
GTP affinity and effector interactions. Nature. 2013;
503:548–551.
603. Flaherty KT, et al. Inhibition of mutated, activated
BRAF in metastatic melanoma. N Engl J Med.
2010;363:809–819.
604. McArthur GA, et al. Safety and efficacy of vemu-
rafenib in BRAF(V600E) and BRAF(V600K)
mutation-positive melanoma (BRIM-3): extended
follow-up of a phase 3, randomised, open-label study.
Lancet Oncol. 2014;15:323–332.
605. Robert C, et al. Improved overall survival in mela-
noma with combined dabrafenib and trametinib.
N Engl J Med. 2015;372:30–39.
606. Long GV, et al. Dabrafenib in patients with
Val600Glu or Val600Lys BRAF-mutant melanoma
metastatic to the brain (BREAK-MB): a multi-
centre, open-label, phase 2 trial. Lancet Oncol.
2012;13:1087–1095.
607. Ascierto PA, et al. Phase II trial (BREAK-2) of
the BRAF inhibitor dabrafenib (GSK2118436) in
patients with metastatic melanoma. J Clin Oncol.
2013;31:3205–3211.
608. McGettigan S. Dabrafenib: A new therapy for use
in BRAF-mutated metastatic melanoma. J Adv Pract
Oncol. 2014;5:211–215.
609. Falchook GS, et al. Dabrafenib in patients with
melanoma, untreated brain metastases, and other
solid tumours: a phase 1 dose-escalation trial. Lancet.
2012;379:1893–1901.
610. Hauschild A, et al. Dabrafenib in BRAF-mutated
metastatic melanoma: a multicentre, open-label, phase
3 randomised controlled trial. Lancet. 2012;380:
358–365.
611. Sosman JA, et al. Survival in BRAF V600-mutant
advanced melanoma treated with vemurafenib.
N Engl J Med. 2012;366:707–714.
612. Chapman PB, et al. Improved survival with vemu-
rafenib in melanoma with BRAF V600E mutation.
N Engl J Med. 2011;364:2507–2516.
613. Joseph EW, et al. The RAF inhibitor PLX4032
inhibits ERK signaling and tumor cell proliferation
in a V600E BRAF-selective manner. Proc Natl Acad
Sci USA. 2010;107:14903–14908.
614. Solit DB, Rosen N. Towards a unified model of
RAF inhibitor resistance. Cancer Discov. 2014;4:27–
30.
615. Wilhelm S, et al. Discovery and development of
sorafenib: a multikinase inhibitor for treating cancer.
Nat Rev Drug Discov. 2006;5:835–844.
616. Infante JR, et al. Safety and efficacy results from
the first-time-in-human study of the oral MEK
1/2 inhibitor GSK1120212. Journal of Clinical
Oncology, 2010 ASCO Annual Meeting Proceedings;
2010:28.
617. Flaherty KT, et al. Improved survival with MEK
inhibition in BRAF-mutated melanoma. N Engl J
Med. 2012;367:107–114.
46.e10 Part I: Science and Clinical Oncology
618. Wright CJ, McCormack PL. Trametinib: first global
approval. Drugs. 2013;73:1245–1254.
619. Long GV, et al. Combined BRAF and MEK inhibi-
tion versus BRAF inhibition alone in melanoma.
N Engl J Med. 2014;371:1877–1888.
620. Larkin J, et  al. Combined vemurafenib and
cobimetinib in BRAF-mutated melanoma. N Engl
J Med. 2014;371:1867–1876.
621. Flaherty KT, et al. Combined BRAF and MEK
Inhibition in Melanoma with BRAF V600 Muta-
tions. N Engl J Med. 2012.
622. Johnson DB, et al. Combined BRAF (dabrafenib)
and MEK inhibition (trametinib) in patients with
BRAFV600-mutant melanoma experiencing progres-
sion with single-agent BRAF inhibitor. J Clin Oncol.
2014;32:3697–3704.
623. Ascierto PA, et al. MEK162 for patients with
advanced melanoma harbouring NRAS or Val600
BRAF mutations: a non-randomised, open-label
phase 2 study. Lancet Oncol. 2013;14:249–256.
624. Bendell JC, et al. Clinical activity and safety of
cobimetinib (cobi) and atezolizumab in colorectal
cancer (CRC). J Clin Oncol. 2016;34:3502.
625. Ho AL, et al. Selumetinib-enhanced radioiodine
uptake in advanced thyroid cancer. N Engl J Med.
2013;368:623–632.
626. Ameratunga M, McArthur G, Gan H, Cher L.
Prolonged disease control with MEK inhibitor in
neurofibromatosis type I-associated glioblastoma.
J Clin Pharm Ther. 2016;41:357–359.
627. Woodfield SE, Zhang L, Scorsone KA, Liu Y, Zage
PE. Binimetinib inhibits MEK and is effective against
neuroblastoma tumor cells with low NF1 expression.
BMC Cancer. 2016;16:172.
628. Tao Z, et al. Coadministration of trametinib and
palbociclib radiosensitizes KRAS-mutant non-small
cell lung cancers in vitro and in vivo. Clin Cancer
Res. 2016;22:122–133.
629. Grisham RN, et al. Extreme outlier analysis identifies
occult mitogen-activated protein kinase pathway
mutations in patients with low-grade serous ovarian
cancer. J Clin Oncol. 2015;33:4099–4105.
630. Shi H, et al. Preexisting MEK1 exon 3 mutations in
V600E/KBRAF melanomas do not confer resistance
to BRAF inhibitors. Cancer Discov. 2012;2:414–424.
631. Grbovic OM, et al. V600E B-Raf requires the
Hsp90 chaperone for stability and is degraded in
response to Hsp90 inhibitors. Proc Natl Acad Sci
USA. 2006;103:57–62.
632. Herrero A, et al. Small molecule inhibition of ERK
dimerization prevents tumorigenesis by RAS-ERK
pathway oncogenes. Cancer Cell. 2015;28:170–
182.
633. Morris EJ, et al. Discovery of a novel ERK inhibi-
tor with activity in models of acquired resistance
to BRAF and MEK inhibitors. Cancer Discov.
2013;3:742–750.
634. Germann U, et al. Abstract 4693: The selective ERK
inhibitor BVD-523 is active in models of MAPK
pathway-dependent cancers, including those with
intrinsic and acquired drug resistance. Cancer Res.
2015;75:4693.
635. Li BT, et al. First-in-class oral ERK1/2 inhibitor
Ulixertinib (BVD-523) in patients with advanced
solid tumors: final results of a phase I dose escalation
and expansion study. J Clin Oncol. 2017;35:2508.
636. Dankort D, et al. Braf(V600E) cooperates with Pten
loss to induce metastatic melanoma. Nat Genet.
2009;41:544–552.
637. Engelman JA, et al. Effective use of PI3K and
MEK inhibitors to treat mutant Kras G12D and
PIK3CA H1047R murine lung cancers. Nat Med.
2008;14:1351–1356.
638. Halilovic E, et al. PIK3CA mutation uncouples
tumor growth and cyclin D1 regulation from
MEK/ERK and mutant KRAS signaling. Cancer
Res. 2010;70:6804–6814.
639. Hu-Lieskovan S, et al. Improved antitumor activity
of immunotherapy with BRAF and MEK inhibitors
in BRAF(V600E) melanoma. Sci Transl Med. 2015;7:
279ra241.
640. Loi S, et al. RAS/MAPK activation is associated
with reduced tumor-infiltrating lymphocytes in
triple-negative breast cancer: therapeutic cooperation
between MEK and PD-1/PD-L1 immune checkpoint
inhibitors. Clin Cancer Res. 2016;22:1499–1509.
641. Barretina J, et al. Subtype-specific genomic alterations
define new targets for soft-tissue sarcoma therapy.
Nat Genet. 2010;42:715–721.
642. Schwartz GK, et al. Phase I study of PD 0332991,
a cyclin-dependent kinase inhibitor, administered
in 3-week cycles (Schedule 2/1). Br J Cancer.
2011;104:1862–1868.
643. Dickson MA, et al. Phase II trial of the CDK4
inhibitor PD0332991 in patients with advanced
CDK4-amplified well-differentiated or dedifferen-
tiated liposarcoma. J Clin Oncol. 2013;31:2024–
2028.
644. Beaver JA, et al. FDA Approval: Palbociclib for the
treatment of postmenopausal patients with estrogen
receptor-positive, HER2-negative metastatic breast
cancer. Clin Cancer Res. 2015;21:4760–4766.
645. Thorpe LM, Yuzugullu H, Zhao JJ. PI3K in cancer:
divergent roles of isoforms, modes of activation and
therapeutic targeting. Nat Rev Cancer. 2015;15:7–24.
646. Vanhaesebroeck B, Stephens L, Hawkins P. PI3K
signalling: the path to discovery and understanding.
Nat Rev Mol Cell Biol. 2012;13:195–203.
647. Hiles ID, et al. Phosphatidylinositol 3-kinase:
structure and expression of the 110 kd catalytic
subunit. Cell. 1992;70:419–429.
648. Hennessy BT, Smith DL, Ram PT, Lu Y, Mills
GB. Exploiting the PI3K/AKT pathway for cancer
drug discovery. Nat Rev Drug Discov. 2005;4:988–
1004.
649. Okkenhaug K, Vanhaesebroeck B. PI3K in lympho-
cyte development, differentiation and activation.
Nat Rev Immunol. 2003;3:317–330.
650. Vivanco I, Sawyers CL. The phosphatidylinositol
3-kinase AKT pathway in human cancer. Nat Rev
Cancer. 2002;2:489–501.
651. Maehama T, Dixon JE. The tumor suppressor,
PTEN/MMAC1, dephosphorylates the lipid second
messenger, phosphatidylinositol 3,4,5-trisphosphate.
J Biol Chem. 1998;273:13375–13378.
652. Alessi DR, et  al. Characterization of a 3-
phosphoinositide-dependent protein kinase which
phosphorylates and activates protein kinase Balpha.
Curr Biol. 1997;7:261–269.
653. Currie RA, et al. Role of phosphatidylinositol
3,4,5-trisphosphate in regulating the activity and
localization of 3-phosphoinositide-dependent protein
kinase-1. Biochem J. 1999;337(Pt 3):575–583.
654. Stokoe D, et al. Dual role of phosphatidylinosi-
tol-3,4,5-trisphosphate in the activation of protein
kinase B. Science. 1997;277:567–570.
655. Bellacosa A, et al. Akt activation by growth factors is
a multiple-step process: the role of the PH domain.
Oncogene. 1998;17:313–325.
656. Diehl JA, Cheng M, Roussel MF, Sherr CJ.
Glycogen synthase kinase-3beta regulates cyclin
D1 proteolysis and subcellular localization. Genes
Dev. 1998;12:3499–3511.
657. Medema RH, Kops GJ, Bos JL, Burgering BM.
AFX-like Forkhead transcription factors mediate
cell-cycle regulation by Ras and PKB through
p27kip1. Nature. 2000;404:782–787.
658. Lawlor MA, Rotwein P. Insulin-like growth factor-
mediated muscle cell survival: central roles for Akt
and cyclin-dependent kinase inhibitor p21. Mol Cell
Biol. 2000;20:8983–8995.
659. Rossig L, et al. Akt-dependent phosphorylation
of p21(Cip1) regulates PCNA binding and
proliferation of endothelial cells. Mol Cell Biol.
2001;21:5644–5657.
660. Datta SR, et al. Akt phosphorylation of BAD couples
survival signals to the cell-intrinsic death machinery.
Cell. 1997;91:231–241.
661. Cardone MH, et al. Regulation of cell death
protease caspase-9 by phosphorylation. Science.
1998;282:1318–1321.
662. Brunet A, et al. Akt promotes cell survival by phos-
phorylating and inhibiting a Forkhead transcription
factor. Cell. 1999;96:857–868.
663. Romashkova JA, Makarov SS. NF-kappaB is a target
of AKT in anti-apoptotic PDGF signalling. Nature.
1999;401:86–90.
664. Kane LP, Shapiro VS, Stokoe D, Weiss A. Induction
of NF-kappaB by the Akt/PKB kinase. Curr Biol.
1999;9:601–604.
665. Mayo LD, Donner DB. A phosphatidylinositol
3-kinase/Akt pathway promotes translocation of
Mdm2 from the cytoplasm to the nucleus. Proc
Natl Acad Sci USA. 2001;98:11598–11603.
666. Han J, et al. Role of substrates and products of
PI 3-kinase in regulating activation of Rac-related
guanosine triphosphatases by Vav. Science. 1998;279:
558–560.
667. Brunet A, et al. Protein kinase SGK mediates
survival signals by phosphorylating the forkhead
transcription factor FKHRL1 (FOXO3a). Mol Cell
Biol. 2001;21:952–965.
668. Zoncu R, Efeyan A, Sabatini DM. mTOR: from
growth signal integration to cancer, diabetes and
ageing. Nat Rev Mol Cell Biol. 2011;12:21–35.
669. Sabatini DM. mTOR and cancer: insights into
a complex relationship. Nat Rev Cancer. 2006;6:
729–734.
670. Loewith R, et al. Two TOR complexes, only one
of which is rapamycin sensitive, have distinct roles
in cell growth control. Mol Cell. 2002;10:457–468.
671. Laplante M, Sabatini DM. mTOR signaling in
growth control and disease. Cell. 2012;149:274–293.
672. Gao X, et al. Tsc tumour suppressor proteins
antagonize amino-acid-TOR signalling. Nat Cell
Biol. 2002;4:699–704.
673. Zhang Y, et al. Rheb is a direct target of the tuberous
sclerosis tumour suppressor proteins. Nat Cell Biol.
2003;5:578–581.
674. Faubert B, Vincent EE, Poffenberger MC, Jones
RG. The AMP-activated protein kinase (AMPK)
and cancer: many faces of a metabolic regulator.
Cancer Lett. 2015;356:165–170.
675. Shaw RJ. LKB1 and AMP-activated protein kinase
control of mTOR signalling and growth. Acta Physiol
(Oxf ). 2009;196:65–80.
676. Hay N, Sonenberg N. Upstream and downstream
of mTOR. Genes Dev. 2004;18:1926–1945.
677. Harrington LS, et al. The TSC1-2 tumor suppressor
controls insulin-PI3K signaling via regulation of IRS
proteins. J Cell Biol. 2004;166:213–223.
678. Courtney KD, Corcoran RB, Engelman JA. The
PI3K pathway as drug target in human cancer.
J Clin Oncol. 2010;28:1075–1083.
679. Mellinghoff IK, et al. Molecular determinants of the
response of glioblastomas to EGFR kinase inhibitors.
N Engl J Med. 2005;353:2012–2024.
680. Almoguera C, et al. Most human carcinomas of the
exocrine pancreas contain mutant c-K-ras genes.
Cell. 1988;53:549–554.
681. Rodriguez-Viciana P, Warne PH, Vanhaesebroeck B,
Waterfield MD, Downward J. Activation of phos-
phoinositide 3-kinase by interaction with Ras and
by point mutation. EMBO J. 1996;15:2442–2451.
682. Gupta S, et al. Binding of ras to phosphoinositide
3-kinase p110alpha is required for ras-driven
tumorigenesis in mice. Cell. 2007;129:957–968.
683. Samuels Y, Velculescu VE. Oncogenic muta-
tions of PIK3CA in human cancers. Cell Cycle.
2004;3:1221–1224.
684. Engelman JA. Targeting PI3K signalling in cancer:
opportunities, challenges and limitations. Nat Rev
Cancer. 2009;9:550–562.
685. Aoki M, Jiang H, Vogt PK. Proteasomal degrada-
tion of the FoxO1 transcriptional regulator in cells
transformed by the P3k and Akt oncoproteins. Proc
Natl Acad Sci USA. 2004;101:13613–13617.

686. Miled N, et al. Mechanism of two classes of cancer
mutations in the phosphoinositide 3-kinase catalytic
subunit. Science. 2007;317:239–242.
687. Oda K, et al. PIK3CA cooperates with other
phosphatidylinositol 3’-kinase pathway mutations to
effect oncogenic transformation. Cancer Res. 2008;68:
8127–8136.
688. Kang S, Denley A, Vanhaesebroeck B, Vogt
PK. Oncogenic transformation induced by the
p110beta, -gamma, and -delta isoforms of class I
phosphoinositide 3-kinase. Proc Natl Acad Sci USA.
2006;103:1289–1294.
689. Philp AJ, et al. The phosphatidylinositol 3′-kinase
p85alpha gene is an oncogene in human ovarian
and colon tumors. Cancer Res. 2001;61:7426–7429.
690. Cheung LW, et al. Naturally occurring neomorphic
PIK3R1 mutations activate the MAPK pathway,
dictating therapeutic response to MAPK pathway
inhibitors. Cancer Cell. 2014;26:479–494.
691. Thorpe LM, et al. PI3K-p110alpha mediates the
oncogenic activity induced by loss of the novel tumor
suppressor PI3K-p85alpha. Proc Natl Acad Sci USA.
2017;114:7095–7100.
692. Yang H, et al. MicroRNA expression profiling in
human ovarian cancer: miR-214 induces cell survival
and cisplatin resistance by targeting PTEN. Cancer
Res. 2008;68:425–433.
693. Staal SP, Hartley JW. Thymic lymphoma induc-
tion by the AKT8 murine retrovirus. J Exp Med.
1988;167:1259–1264.
694. Carpten JD, et al. A transforming mutation in the
pleckstrin homology domain of AKT1 in cancer.
Nature. 2007;448:439–444.
695. Cohen Y, et al. AKT1 pleckstrin homology domain
E17K activating mutation in endometrial carcinoma.
Gynecol Oncol. 2010;116:88–91.
696. Zilberman DE, et al. AKT1 E17 K pleckstrin
homology domain mutation in urothelial carcinoma.
Cancer Genet Cytogenet. 2009;191:34–37.
697. Madhunapantula SV, Robertson GP. Therapeutic
implications of targeting AKT signaling in mela-
noma. Enzyme Res. 2011;2011:327923.
698. Rosenthal A. Small molecule inhibitors in chronic
lymphocytic lymphoma and B cell non-Hodgkin
lymphoma. Curr Hematol Malig Rep. 2017;12:
207–216.
699. Miller BW, et al. FDA approval: idelalisib mono-
therapy for the treatment of patients with follicular
lymphoma and small lymphocytic lymphoma. Clin
Cancer Res. 2015;21:1525–1529.
700. Davies BR, et al. Preclinical pharmacology of
AZD5363, an inhibitor of AKT: pharmacodynamics,
antitumor activity, and correlation of monotherapy
activity with genetic background. Mol Cancer Ther.
2012;11:873–887.
701. Addie M, et  al. Discovery of 4-amino-N-
[(1S)-1-(4-chlorophenyl)-3-hydroxypropyl]-1-
(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-
4-carboxamide (AZD5363), an orally bioavailable,
potent inhibitor of Akt kinases. J Med Chem.
2013;56:2059–2073.
702. Davies BR, et al. Tumors with AKT1E17K mutations
are rational targets for single agent or combination
therapy with AKT INhibitors. Mol Cancer Ther.
2015;14:2441–2451.
703. Hyman DM, et al. Abstract B109: AZD5363, a cata-
lytic pan-Akt inhibitor, in Akt1 E17K mutation posi-
tive advanced solid tumors. Mol Cancer Ther. 2015;14:
B109.
704. Hyman DM, et al. AKT inhibition in solid
tumors with AKT1 mutations. J Clin Oncol.
2017;35:2251–2259.
705. Wander SA, Hennessy BT, Slingerland JM. Next-
generation mTOR inhibitors in clinical oncology:
how pathway complexity informs therapeutic
strategy. J Clin Invest. 2011;121:1231–1241.
706. Hudes G, et al. Temsirolimus, interferon alfa, or
both for advanced renal-cell carcinoma. N Engl J
Med. 2007;356:2271–2281.
707. Motzer RJ, et al. Efficacy of everolimus in advanced
renal cell carcinoma: a double-blind, randomised,
placebo-controlled phase III trial. Lancet. 2008;372:
449–456.
708. Yao JC, et al. Everolimus for advanced pancreatic
neuroendocrine tumors. N Engl J Med. 2011;
364:514–523.
709. Krueger DA, et al. Everolimus for subependymal
giant-cell astrocytomas in tuberous sclerosis. N Engl
J Med. 2010;363:1801–1811.
710. Franz DN, et al. Efficacy and safety of everolimus for
subependymal giant cell astrocytomas associated with
tuberous sclerosis complex (EXIST-1): a multicentre,
randomised, placebo-controlled phase 3 trial. Lancet.
2013;381:125–132.
711. Kwiatkowski DJ, et al. Mutations in TSC1, TSC2,
and MTOR are associated with response to rapalogs
in patients with metastatic renal cell carcinoma. Clin
Cancer Res. 2016;22:2445–2452.
712. Iyer G, et al. Genome sequencing identifies a basis
for everolimus sensitivity. Science. 2012;338:221.
Intracellular Signaling  •  CHAPTER 2 46.e11
46.e11
713. Baselga J, et al. Everolimus in postmenopausal
hormone-receptor-positive advanced breast cancer.
N Engl J Med. 2012;366:520–529.
714. Carracedo A, et al. Inhibition of mTORC1 leads
to MAPK pathway activation through a PI3K-
dependent feedback loop in human cancer. J Clin
Invest. 2008;118:3065–3074.
715. Breuleux M, et al. Increased AKT S473 phosphoryla-
tion after mTORC1 inhibition is rictor dependent
and does not predict tumor cell response to PI3K/
mTOR inhibition. Mol Cancer Ther. 2009;8:
742–753.
716. Fan Q, et al. A kinase inhibitor targeted to mTORC1
drives regression in glioblastoma. Cancer Cell.
2017;31:424–435.
717. Rodrik-Outmezguine VS, et al. Overcoming mTOR
resistance mutations with a new-generation mTOR
inhibitor. Nature. 2016;534:272–276.
718. Weinstein IB, Joe A. Oncogene addiction. Cancer
Res. 2008;68:3077–3080, discussion 3080.
719. Eberhard DA, et al. Mutations in the epidermal
growth factor receptor and in KRAS are predictive
and prognostic indicators in patients with non-small-
cell lung cancer treated with chemotherapy alone
and in combination with erlotinib. J Clin Oncol.
2005;23:5900–5909.
720. Zehir A, et al. Mutational landscape of metastatic
cancer revealed from prospective clinical sequencing
of 10,000 patients. Nat Med. 2017;23:703–713.
721. Wu JY, et al. Lung cancer with epidermal growth
factor receptor exon 20 mutations is associated with
poor gefitinib treatment response. Clin Cancer Res.
2008;14:4877–4882.
722. Yasuda H, Kobayashi S, Costa DB. EGFR exon 20
insertion mutations in non-small-cell lung cancer:
preclinical data and clinical implications. Lancet
Oncol. 2012;13:e23–e31.
723. Hyman DM, et al. Vemurafenib in multiple
nonmelanoma cancers with BRAF V600 mutations.
N Engl J Med. 2015;373:726–736.
724. Prahallad A, et al. Unresponsiveness of colon cancer
to BRAF(V600E) inhibition through feedback
activation of EGFR. Nature. 2012;483:100–103.
725. Camidge DR, Pao W, Sequist LV. Acquired resistance
to TKIs in solid tumours: learning from lung cancer.
Nat Rev Clin Oncol. 2014;11:473–481.
726. Holohan C, Van Schaeybroeck S, Longley DB,
Johnston PG. Cancer drug resistance: an evolv-
ing paradigm. Nat Rev Cancer. 2013;13:714–
726.
46.e12 Part I: Science and Clinical Oncology
SELF-ASSESSMENT REVIEW QUESTIONS
1. Next-generation sequencing has helped reveal numerous unusual
genomic events, including chromosomal translocations. Which
of the following genes is NOT found as oncogenic fusion
partners in chromosomal re-arrangements?
a. ABL
b. PTEN
c. PDGFRβ
d. ALK
e. NTRK
2. Which of the following is true of Hedgehog (SHH) signaling
and targeted therapies?
a. Secreted hedgehog ligands bind Smoothened receptors, which
causes receptor dimerization and autophosphorylation of
intracellular tyrosine residues.
b. Erlotinib and gefitinib are FDA approved Smoothened
receptor inhibitors.
c. Sonic hedgehog (SHH) binds to the receptor Patched
(PTCH), which relieves its repression of Smoothened
(SMO).
d. Mutations in SHH, PTCH, and SMO are found in patients
with small cell lung cancer.
e. Smoothened receptors couple directly to actin.
3. The Ras/MAP kinase and PI3 kinase/mTOR signaling cascades
are parallel but interconnected pathways that are frequently
mutated in human cancer. Which of the following has NOT
been an effective approach in targeting these pathways?
a. Allosteric inhibitors of MEK kinase
b. Small-molecule inhibitors and kinase of mTOR
c. Monoclonal antibodies that bind to EGFR
d. Farnesyltransferase inhibitors of Ras
e. Kinase inhibitors of mutant BRAF
4. Acquired resistance commonly occurs after treatment with
targeted inhibitors. Which of the following are known
mechanisms of resistance?
a. Parallel pathway upregulation
b. Gatekeeper mutation
c. Amplification of specific RTKs
d. Alternative splicing or amplification of the driver oncogene
e. All of the above
5. Which of the following statements is FALSE?
a. Chromosomal rearrangement of BCR and ALK to generate
the Philadelphia chromosome is the driving event in CML.
b. Recurrent somatic splice site alterations involving MET exon
14 (METex14) have been identified in lung cancer.
c. Integrin receptors perform inside-out and outside-in signaling
at the integrin adhesome.
d. ESR1 mutations are a common mechanism of acquired
resistance to hormonal therapy in patients with breast cancer.
e. Extracellular and transmembrane FGFR3 mutations are
recurrent in noninvasive and high-grade invasive bladder
cancer.
ANSWERS
1. (b) PTEN is a tumor suppressor of the AKT pathway and
would not be a likely candidate fusion partner, because
truncating mutations or deletions confer loss of PTEN function.
All of the other genes listed have been found in patients with a
variety of fusions partners. BCR-ABL, the Philadelphia
chromosome, which is found in nearly all cases of chronic
myelogenous leukemias (CML) and one-third of ALLs, is
the classic example of an oncogenic fusion driving cancer.
EML4-ALK fusions play a driving role in non–small cell lung
cancer (NSCLC). COL1A1-PDGFRβ fusions occur in
dermatofibrosarcoma protruberans, a rare sarcoma.
TPM3-TRKA fusions were first identified in colon cancer.
2. (c)  The Smoothened receptor is a GPCR, not a receptor
tyrosine kinase, and therefore it does not get activated by
dimerization and transphosphorylation. Vismodegib and
sonidegib are FDA-approved smoothened receptor inhibitors.
Mutations in SHH, PTCH, and SMO are found in patients
with inherited and sporadic basal cell carcinomas. Smoothened
receptors couple to the G proteins Gαi and Gα12.
3. (d)  No direct inhibitors of Ras are available for clinical use.
Farnesyltransferase inhibitors were tested but were found to be
ineffective in tumors in which Ras mutations are common,
likely because of the ability of geranylgeranyl modifications to
substitute for farnesylation in effectively targeting Ras to the
membrane. New drug development of allosteric inhibitors that
specifically target KRAS G12C is underway. Trametinib is an
FDA-approved allosteric inhibitor of MEK. Rapamycin and its
analogues, as well as novel kinase inhibitors such as RapaLink,
are efficacious mTOR inhibitors. Cetuximab and panitumumab
are anti-EGFR monoclonal antibody therapies. Vemurafenib,
an ATP-competitive inhibitor of BRAF, is approved for the
treatment of patients with BRAF V600E mutant melanoma.
4. (e)  Several mechanisms of resistance to kinase inhibitors have
emerged. For example, patients with BRAF V600E mutations
that are treated with the specific RAF inhibitor vemurafenib
often develop resistance mediated by BRAF V600E splice
variants or amplification; loss or mutation of NF1; parallel
pathway activation of RTKs; mutation of PI3K/AKT
components; mutations in NRAS, RAF1 and MAP2K1/2
(MEK1/2); and amplification of MITF.
5. (a)  Translocation of the ABL gene on chromosome 9 with the
breakpoint cluster region (BCR) gene on chromosome 22 results
in the expression of a BCR-ABL fusion protein, called the
Philadelphia chromosome. This event is the pathognomonic
molecular lesion in almost all chronic myelogenous leukemias
(CML). It is also found in approximately one-third of acute
lymphoblastic leukemias (ALLs). The ABL tyrosine kinase
inhibitor imatinib, as well as four other ABL inhibitors, are
FDA approved in CML; imatinib and dasatinib are approved
for ALL.
 Cellular Microenvironment and Metastases 3 
Erinn B. Rankin and Amato J. Giaccia

S UMMARY OF K EY P OI NT S
• Metastases are responsible for more the distant tissue; dormancy; and blood flow and by tissue-specific
than 90% of all cancer-related reactivation and proliferation in the factors.
deaths. new tissue microenvironment. • Primary tumors possess stem cells
• Gene dysregulation, the tumor • Colonization of metastatic tumor that can recapitulate the tumor from
microenvironment, and host cells cells requires the ability to a single cell, and a subset of these
drive the metastatic spread of tumor metabolically adapt, develop cancer stem cells may inherently
cells. angiogenesis, overcome dormancy, possess altered gene expression
• Metastasis can be subdivided into and proliferate in a foreign tissue. changes with increased metastatic
invasion and migration from the • The formation of a premetastatic potential.
primary tumor; intravasation into niche is essential for the growth of • Antimetastatic therapy will likely
the vasculature; dissemination extravasating metastatic tumor require the targeted inhibition of
and survival in the circulation; cells. many pathways that control
extravasation from the vasculature; • Organ specificity of tumor proliferation, invasion, angiogenesis,
survival and metabolic adaptation in metastases is determined both by and immune evasion.

One of the most important challenges in clinical oncology is the
prevention and treatment of metastatic disease. With advances in
surgical techniques and conventional and targeted therapies, localized
disease is effectively managed in the clinic. However, metastatic disease
is the primary cause of cancer-related deaths. Tumor metastasis involves
tumor cell invasion and migration from the primary tumor, intravasation
into the vasculature, dissemination and survival in the circulation,
extravasation into distant tissues, survival and metabolic adaptation
in the distant tissue, dormancy, reactivation, and overt colonization
to form a new macroscopic tumor at a distant site.1 This process is
highly inefficient; it has been estimated that less than 0.01% of tumor
cells that enter the circulation develop into metastases. Despite this
inefficiency, metastases are responsible for more than 90% of all
cancer-related deaths. Understanding the biology and vulnerabilities
of metastatic tumor cells is of critical importance to improve overall
survival rates in cancer patients. With little evidence to support
mutations in “metastasis genes” as drivers of metastasis, current data
suggest that microenvironmental factors as well as epigenetic changes
may play key roles in metastasis. This chapter describes the cellular
and molecular traits driving tumor metastasis and addresses how the
tumor microenvironment influences this process. Most important, it
discusses how this knowledge can be translated into current and future
cancer therapies.
TUMOR MICROENVIRONMENT AND
METASTASIS
Although cellular intrinsic traits acquired by tumor cells are required
for successful metastatic colonization, cellular and molecular factors
within the tumor microenvironment significantly contribute to meta-
static progression. The tumor microenvironment contains a number
of cell types that promote tumor progression, including cancer stem
cells, angiogenic vascular cells, infiltrating immune cells, and cancer-
associated fibroblasts (CAFs).2 In addition, extracellular factors within
the tumor microenvironment such as hypoxia and extracellular vesicles
promote metastatic phenotypes within these cell types.
Cancer Stem Cells
The traditional view of tumors with a relatively homogeneous
population of tumorigenic cells has been significantly revised with
the isolation and characterization of cancer stem cells. Stem cells are
primal cells that retain the ability to renew themselves through cell
division and can differentiate into a wide range of specialized cell
types. Cells with stem cell properties have been identified in a variety
of solid tumors including colon, breast, head and neck, and pancreatic
tumors, glioblastomas, medulloblastomas, and melanoma.3 This rare
subpopulation of tumor cells exhibits enhanced tumor-initiating
potential: the ability to self-renew and differentiate into multiple
cell types.
The origin of cancer stem cells remains unclear and may differ
among tumor types. It has been hypothesized that cancer stem cells
arise from either transformed resident stem and/or progenitor cells,
or may represent a dedifferentiated epithelial tumor cell. In intestinal
cancer, mouse modeling studies have identified intestinal stem cells as
the cells of origin. For example, intestinal stem cell–specific deletion
of adenomatous polyposis coli (APC) leads to rapid cellular transforma-
tion with uncontrolled cellular growth and solid tumor formation.
In contrast, deletion of APC in short-lived transit-amplifying cells
was not sufficient to induce long-term tumor growth.4 Stem cell
properties can also be acquired by tumors cells through epithelial-
mesenchymal transition (EMT). Induction of EMT in immortalized
human mammary epithelial cells was sufficient to induce the expression
of stem cell markers, enhance self-renewal, and increase the number
of tumor-initiating cells.5 Although the molecular mechanisms that
drive the cancer stem cell phenotype in tumor cells remains largely
unknown, a study showed that the transcription factors Slug and
Sox9 drive EMT and the cancer stem cell phenotype in breast cancer

47
48 Part I: Science and Clinical Oncology

cells.6 Future studies are eagerly awaited to further delineate the

intracellular signaling pathways driving the cancer stem cell phenotype I, TSP-1
ICAM IL-3
ctin, 3, C
in vivo. sel
e D2
, P- io n 48
The role of cancer stem cells in multistage tumor progression, B3 sat n Intr
KF ava atio ava
particularly with respect to metastasis, is poorly defined. Given that PF Intr avas cy sat
tr n ion
metastasis is an infrequent event that is achieved by only a small Ex orma
D
portion of cancer cells that reach distant sites, it has been hypothesized

GF
that cancer stem cells may be responsible for metastatic disease. Evidence Endothelial Pericyte

s, VEGF, CCLI8, E

MMP
to support this hypothesis has been generated in models of breast and

Extravasation

Premetastatic nic
Intravasation
pancreatic cancer. In the MMTV-PyMT model of breast cancer,

s, ALOX5, HGF, T
Invasion

Intravasation
Malanchi and colleagues observed that only the CD90+ CD24+

Invasion
population of cancer stem cells isolated from primary breast tumors Macrophage TME Neutrophil

contained cells with the ability to form metastases in the lung when

MMP
introduced into the tail vein.7 Furthermore, this study identified the

he
extracellular matrix protein, periostin, produced by fibroblasts and Platelet Fibroblast

GF
stromal tumor cells, as a critical factor to maintain the cancer stem on

-B
lati Pre
me
ircu on
cell phenotype and metastatic potential in these cells.7 In human n c
al i sat
i Intr tasta
tic
breast cancers, single-cell analysis of early-stage metastatic cells
av
rviv ava n rin
s CX Inv asati niche
Su Extr vasio asi on
demonstrated that these samples express distinct stemlike, EMT, eg
In t CL on
,in I2,
tin
prosurvival, and dormancy-associated gene expression signatures
GM
lec -CS
-se F, P
compared with metastatic cells from high-burden tissues.8 Moreover,
, P
TGF-B OSTN

transplanted stemlike metastatic cells from low-burden tissues exhibited

tumor-initiating capacity and were able to differentiate into luminal-like
cancer cells.8 Similarly in pancreatic cancer, lineage-tracing studies
showed that circulating pancreatic tumor cells exhibit EMT and cancer
stem cell properties, and initiate tumor formation. A critical role for
inflammation to maintain the ability of cancer stem cells to metastasize
was observed in this study.9 In addition, the extracellular protein
tenascin C (TNC), expressed by stem cell niches and cancer cells,
was also found to promote stem cell signaling and lung metastases in
breast cancer cells.10 Collectively, these data strongly implicate the
tumor microenvironment in promoting the cancer stem cell phenotype
and the initiation of tumor metastasis.
Angiogenic Vascular Cells
Tumor angiogenesis facilitates the hematogenous spread of metastatic
tumors. The aberrant production of proangiogenic factors by tumor
cells results in malformed and irregular tumor blood vessels that often
contain breaks in their lining that facilitate tumor cell intravasation
and dissemination. Endothelial cells and pericytes are important
structural and functional components of the tumor and distant tissue
vasculature, and both have been considered as potential targets in
metastatic therapy.11
Endothelial Cells
Endothelial cells play an active role in tumor cell intravasation,
extravasation, and dormancy (Fig. 3.1). Studies have shown that
metabolic reprogramming of tumor endothelial cells toward a glycolytic
phenotype contributes to the early stages of metastasis by promoting
an abnormal tumor vasculature, tumor intravasation, and dissemination.
Cantelmo and colleagues performed mRNA sequencing analysis of
tumor-associated endothelial cells and discovered that tumor endothelial
cells expressed a hyperglycolytic signature compared with normal
endothelial cells.12 The glycolytic phenotype within tumor-associated
endothelial cells could be inhibited through genetic and pharmacologic
inhibition of the glycolytic enzyme 6-phosphofructo-2-kinase/fructose-
2,6-bisphosphatase-3 (PFKFB3). Moreover, PFKFB3 inhibition in
endothelial cells was sufficient to normalize the tumor vasculature
and significantly reduce tumor cell intravasation and metastasis.12
There are multiple mechanisms by which PFKFB3 inhibition in
endothelial cells may have impaired metastatic dissemination, including
enhancing the integrity of the endothelial cell barrier, improving vessel
maturation and pericyte coverage, and inhibiting cancer cell adhesion
molecules. These findings suggest that targeting glucose metabolism
in tumor endothelial cells may offer therapeutic benefit for anticancer
therapy.
Figure 3.1  •  Multiple cellular components of the tumor microenvironment
(TME) contribute to metastasis. Key cell types and mechanisms of action are
shown.
Adhesion to the endothelium is an initial step in extravasation
that is followed by transendothelial cell migration.13 Tumor cell adhesion
to the endothelium requires the expression of cognate ligands and
receptors by cancer cells and endothelial cells.13 A variety of ligand-
receptor interactions have been shown to contribute to tumor
extravasation, including interactions with selectins, integrins, cadherins,
CD44, and immunoglobulin (Ig) superfamily receptors (for a review,
see Reymond and colleagues13). For example, endothelial cell P- and
E-selectins bind to tumor cells through cell-cell adhesion molecules
such as integrins and CD44.14–19 Studies have suggested a role for the
immunosuppressive cytokine interleukin (IL)-35 in facilitating cancer
cell adhesion to endothelial cells. IL-35 was found to be highly expressed
by human pancreatic cancer cells in which it promoted ICAM1
expression to facilitate endothelial cell adhesion and transendothelial
migration via an ICAM1-fibrogen-ICAM1 bridge.20
Finally, endothelial cells within in the perivascular niche of the
bone marrow have been implicated in promoting tumor cell dormancy.
Similar to hematopoietic stem cells, dormant cancer cells have been
found to be localized within perivascular niches of the bone marrow
microenvironment.21 Within stable microvasculature niches, endothelial
cells promote tumor cell quiescence through the production of
thrombospondin-1 (TSP1)21. In contrast, endothelial cells isolated
from sprouting neovasculature promote tumor proliferation through
the production of tumor-promoting factors including transforming
growth factor–β1 (TGF-β1) and periostin from sprouting endothelial
cell tips.21
Pericytes
Similar to endothelial cells, pericytes are thought to play a protumori-
genic role by supporting blood vessel maturation and function (see
Fig. 3.1). Pericytes promote endothelial cell survival and stabilize the
tumor vasculature.22 In addition, pericytes can directly facilitate distant
metastasis by promoting tumor cell intravasation through the upregula-
tion of the transmembrane receptor endosialin (CD248).23 Studies
have demonstrated that pericytes can also promote tumor cell intravasa-
tion and metastasis through the IL-33–dependent recruitment of
macrophages.24 Therefore it has been proposed that targeting pericytes
alone or in combination with endothelial cells may be an effective
strategy in the treatment of cancer.24,25 However, clinical data indicate

that low pericyte coverage is associated with metastasis and poor
prognosis in a number of cancers.25 A recent study demonstrated that
depletion of pericytes suppressed tumor growth and enhanced metastasis
of murine models of breast cancer.25 Enhanced metastasis was associated
with increased levels of tumor hypoxia, EMT, and Met receptor
activation.25 These findings indicate that pericyte coverage may be
important to stabilize the tumor vasculature and prevent the hypoxic
selection of aggressive tumor cells (see later discussion of hypoxia and
metastasis). These findings raise concerns when considering antipericyte
treatments in cancer therapy, especially in the setting of metastasis.
Immune Cells
Most solid tumors contain immune infiltrates derived from both the
myeloid and lymphoid lineages that can have both positive and negative
effects on metastasis.2 This section focuses on immune cells that
stimulate metastatic progression.
Macrophages
Studies have linked a unique subset of macrophages, termed tumor-
associated macrophages (TAMs), to tumor progression and metastasis.
Clinically, the presence of excessive macrophages is associated with
poor prognosis in the majority of patients with solid tumors including
breast, prostate, cervix, bladder, endometrial, and kidney cancer.26–28
TAMs express a variety of growth factors and cytokines that promote
tumor angiogenesis, invasion, intravasation, metastasis, and immune
suppression (see Fig. 3.1)28,29. For example, macrophages within the
primary tumor contribute to tumor cell invasion and extravasation
by releasing a variety of proteases including serine, cysteine, and
metalloproteases that remodel the extracellular matrix to enhance
tumor cell invasion and migration (for a review, see Cho and col-
leagues2). Perivascular macrophages are thought to play a particularly
important role in tumor cell intravasation by promoting vascular
permeability. In preclinical models of breast cancer, genetic ablation
of vascular endothelial growth factor (VEGF) from macrophages was
sufficient to reduce transient vascular permeability, leading to reduced
numbers of circulating tumor cells.30 Moreover, macrophages can
promote tumor cell invasion through the release of chemokines and
cytokines including CCL18 and epidermal growth factor (EGF; for
a review, see Ruffell and colleagues28).
Many questions remain regarding the factors that control the biologic
activities of TAMs within the tumor microenvironment. TAMs localize
to regions of hypoxia, and thus it has been hypothesized that the
hypoxic microenvironment may influence the unique characteristics
and gene expression profiles of TAMs. Indeed, TAMs express high
levels of the hypoxia inducible transcription factor (HIF) HIF-2, which
has been found to be an independent prognostic factor of outcome.31
When exposed to hypoxia, macrophages upregulate the expression of
mitogenic and proangiogenic cytokines.32 Experimentally, deletion of
myeloid HIF-2 reduced TAM infiltration into tumors and impeded
tumor proliferation and growth.32 Thus, targeting TAMs and CD11b
cells may be a viable mechanism for antimetastatic therapies.
Neutrophils
Neutrophils represent 50% to 75% of the total circulating leukocytes
in blood. They play an important role in inflammation and are early
responders to pathogens including bacteria, fungi, and viruses.
Neutrophils mediate direct antimicrobial activities through the release
of enzymes and toxic factors, the generation of reactive oxygen species,
and the release of nuclear material into extracellular traps called
neutrophil extracellular traps (NETs).33
Although neutrophils can inhibit the metastatic seeding of dis-
seminated tumor cells under some conditions, accumulating evidence
suggests a prometastatic role for neutrophils (see Fig. 3.1).34–37 Study
findings have suggested that that metastatic cancer cells promote the
formation of NETs through the release of G-CSF.37 NETs may promote
metastasis through multiple mechanisms including (1) trapping
Cellular Microenvironment and Metastases  •  CHAPTER 3 49
circulating tumor cells within DNA meshes at distant tissues, (2)
promoting vascular permeability and the premetastatic niche, and (3)
stimulating tumor cell invasion and proliferation at distant tissue
sites.36–38 Important to note, digesting NETs with DNase I is sufficient
to reduce metastasis in multiple metastatic tumor models, suggesting
that therapeutic targeting of NETs may prevent metastasis.36,37
Platelets
Platelets are the second most abundant cell type in the blood and
play an important role in hemostasis, thrombosis, inflammation, and
vascular biology.39 Platelets are among the first cell types that interact
with cancer cells in the circulation.20 Tumor cells release platelet
coagulation factors such as thrombin to promote platelet aggregation
around circulating tumor cells. Indeed, increased blood coagulation
is often observed in cancer patients as a result of elevated levels of
thromboplastin, procoagulant A, and phosphatidylserine produced
by tumor cells.40,41 Platelets facilitate metastasis through multiple
mechanisms (see Fig. 3.1). Platelets are thought to form a physical
barrier around disseminated cancer cells, protecting them from shear
stress and lysis mediated by natural killer cells.42 In addition, platelets
can facilitate tumor cell extravasation by enhancing tumor cell adhesion
to endothelial cells and by promoting endothelial cell permeability.43–46
Finally, platelets have been shown to directly stimulate cancer cell
EMT and invasion through the release of TGF-β.47 Therapeutic
inhibition of platelet binding to cancer cells through the disruption
of α6β1 integrin/ADAM9 receptor may be an effective therapeutic
strategy to target platelet-mediated metastasis in vivo.48
Fibroblasts and Mesenchymal Stem Cells
Accumulating evidence supports a critical role for CAFs in tumor
progression and metastasis (see Fig. 3.1). Clinically it was observed
that epigenetic changes and mutations commonly found in cancer
cells, such as p53 and PTEN mutations, can also be found in cancer-
associated stroma.49,50 These studies provided early evidence to suggest
that alterations in the stroma may contribute to tumor progression.
Gene expression profiling studies of CAFs in human specimens and
murine tumor models revealed an “activated” proinflammatory gene
signature.51 Among the cytokines produced by CAFs, the chemokine
CXCL12 is of particular interest, given its role in driving tumor cell
migration and recruitment of endothelial progenitor cells expressing
the CXCR4 receptor. Consistent with these data, an important role
for CAFs in promoting tumor invasion has been observed in several
murine models of cancer.51 In breast cancer, Hu and colleagues identified
a role for CAFs in the transition of mammary ductal carcinoma in
situ (DCIS) to invasive carcinoma. Coinjection of fibroblasts with
DCIS cells resulted in invasive carcinomas, whereas injection of DCIS
cells alone was only sufficient to induce mammary DCIS.52 CAFs
promote tumor cell invasion by establishing invasion-permissive tracks
in the extracellular matrix through the secretion of factors that induce
matrix remodeling.51,53 CAFs can also support tumor cell survival and
migration through the release of extracellular vesicles carrying annexin
A6/LDL receptor–related protein/thrombospondin 1 (ANXA6/LRP1/
TSP1) complexes.54 In addition to promotion of tumor invasion,
supportive roles for CAFs in the maintenance of cancer stem cell
signaling and the establishment of the premetastatic niche have been
found.51 Further characterization of the distinct subsets of CAFs relevant
to metastasis in various tumor types is needed. In addition, further
elucidation of the interplay between tumors and the stroma is warranted
and may reveal novel strategies in the treatment of metastatic disease.
Studies have suggested an important role for a subset of CAFs,
mesenchymal stem cells (MSCs), in tumor progression and metastasis.
Although MSCs are commonly isolated in CAF preparations, MSCs
are functionally distinct in that they are able to undergo multipotent
differentiation. MSCs have been identified as important components
of the tumor stroma that promote the progression of ovarian, colon,
and pancreatic cancers.55–57 Cancer-associated MSCs have recently
50 Part I: Science and Clinical Oncology
been shown to promote pancreatic cancer cell proliferation, inva-
sion, and transendothelial cell migration through the production
of the cytokine granulocyte-macrophage colony-stimulating factor
(GM-CSF).55 These findings suggest that inhibition of tumor-
MSC cross talk may be a therapeutic strategy for the treatment of
pancreatic cancer.
Extracellular Vesicles
Extracellular vesicles are emerging as key factors that promote metastasis.
Studies have shown that tumor and stromal cells secrete small vesicles
that contain a variety of bioactive molecules such as proteins, lipids,
RNA, and DNA that can promote intercellular signaling and tumor
progression. Extracellular vesicles can be derived from either the
endosome (exosome) or the plasma membrane (microvesicle) to mediate
intercellular signaling with tumor and stromal cells within the local
and distant microenvironments. Clinically, extracellular vesicles and
their cargo have been associated with tumor progression. The concentra-
tions of exosomes have been found to be increased in the peripheral
blood of patients with ovarian, breast, and pancreatic cancers compared
with healthy control patients (for a review, see Becker and colleagues58).
Specific nucleic acid and protein expression signatures within exosomes
have also been associated with tumor progression and metastasis. For
example, exosomal miR-141 expression in the serum of prostate cancer
patients is significantly higher in metastatic prostate patients compared
with patients with localized disease.59 An exosomal protein signature
containing the melanoma-specific protein tyrosinase-related protein-2
(TYRP2), very late antigen 4 (VLA-4), HSP-70, and the MET
oncoprotein has been associated with metastasis in melanoma patients.60
In pancreatic cancer, the level of circulating glypican-1–positive
exosomes correlates with poor patient survival.61
Functionally, extracellular vesicles have been shown to play an
important role in the tumor microenvironment and promote metastasis
through a variety of mechanisms including tumor immune suppression,
invasion, angiogenesis, and the premetastatic niche (for a review, see
Kalluri and colleagues62). For example, melanoma cells promote immune
evasion through the release of FasL-bearing microvesicles that trigger
Fas-dependent apoptosis of lymphoid cells.63 Tumor-derived exosomes
can also directly promote the immunosuppressive phenotype of
myeloid-derived suppressor cells through the activation of STAT3
signaling in these cells.64 Tumor-derived exosomes promote an invasive
phenotype by activating stromal cells to produce matrix metallo­
proteinases (MMPs) such as MMP1.65 In addition, tumor-derived
extracellular vesicles can directly promote the invasive capacity of
nonmetastatic cells at local and distant sites by transferring mRNAs
involved in migration and metastasis.66 Conversely, stromal microvesicles
have also been shown to promote tumor cell invasion and metastasis.
Fibroblast-secreted exosomes promote breast cancer cell migration
through the activation of planar cell polarity (PCP) signaling.67
Astrocyte-mediated transfer of exosomal PTEN-targeting microRNAs
leads to PTEN loss in brain metastatic tumor cells that can support
metastatic outgrowth through mechanisms involving the increased
expression of the chemokine CCL2 and adaptive metastatic outgrowth.68
Exosome-mediated transfer of miR-105 by breast cancer cells promotes
vascular leakiness and metastasis by disrupting endothelial cell tight
junctions.69 Tumor-derived exosomes may also play a key role in
establishing a premetastatic niche at distant sites by activating signaling
within organ-specific cells to recruit bone marrow–derived macrophages,
activate Src phosphorylation and proinflammatory S100 gene expression,
and increase nutrient availability.70–72
The factors that regulate extracellular vesicle formation and content
in cancer remain poorly understood. Ostrowski and colleagues used
an RNA interference screen to identify factors important in exosome
secretion in cancer cells. These studies revealed an important role for
the Rab GTPases Rab27a and Rab27b and the Rab27 effectors Slp4
and Slac2b in exosome secretion.73 Studies have implicated hypoxia
as an important microenvironmental factor that can influence both
exosome content and shedding. The shedding of exosomes from tumor
cells is increased under hypoxic conditions. In breast cancer cells,
hypoxia promotes microvesicle shedding through the HIF-dependent
regulation of the small GTPase RAB22A, a protein that is localized
to budding microvesicles and promotes metastasis.74 Hypoxia also
plays an important role in regulating exosome content and function.
Clinically, the levels of hypoxia-regulated proteins in plasma exosomes
are significantly higher in glioblastoma multiforme (GBM) patients
compared with healthy control patients.75 Moreover, exosomes derived
from hypoxic GBM cells are potent inducers of angiogenesis compared
with exosomes isolated from normoxic GBM cells.75 Given that
extracellular vesicles can be detected in patient biologic fluids and
contribute to cancer progression, there is great interest in use of
exosomes as diagnostic biomarkers and therapeutic targets in cancer.
Hypoxia
Hypoxia is a potent microenvironmental factor driving metastatic
tumor progression. Clinically, hypoxia is associated with metastasis
and poor survival in variety of cancer patients.76–79 Hypoxia selects
for cells with low apoptotic potential and increases genomic instability,
allowing for rapid mutational adaptations.80–82 In addition, hypoxia
directly increases the expression of genes involved in glycolysis,
angiogenesis, cell survival, invasion, immune suppression, the cancer
stem cell phenotype, and metastasis. All of these changes allow cells
to adapt to oxygen-deprived conditions and permit cells to escape
these conditions by establishing new blood supplies or by physically
moving from an oxygen-poor environment to an oxygen-rich
environment.
The primary molecular mediators of hypoxic signaling are HIF-1
and HIF-2. In the presence of oxygen, the alpha subunits of HIF-1
and HIF-2 are rapidly degraded through the cooperative actions of
prolyl hydroxylase (PHD) enzymes PHD1, PHD2, and PHD3, and
the E3 ubiquitin ligase substrate recognition component VHL.83,84
Under hypoxia, HIF-1 and HIF-2 are stabilized and activate the
expression of genes containing hypoxia response elements (HREs).76
Over 200 genes that allow cells to survive and adapt to low oxygen
tensions are activated in response to HIF-1 and HIF-2.
Consistent with the association between hypoxia and metastasis,
HIF-1 and HIF-2 are highly expressed in primary tumors and
metastases. Immunohistochemical analysis of human cancer specimens
indicates that the majority of primary tumors and metastases have
increased HIF-1 and/or HIF-2 protein compared with the normal
adjacent tissue.76–78 Moreover, increased HIF expression is often
associated with increased patient mortality.85 Experimentally, overexpres-
sion of HIF in tumor cells promotes metastasis,86 whereas inactivation
of HIF significantly decreases the metastatic potential of metastatic
tumor cells.87–89 These clinical and experimental findings indicate an
important role for HIF in metastatic tumor progression.
HIFs influence multiple steps within the metastatic cascade
(Fig. 3.2). HIF-1 and HIF-2 activate the early stages of metastasis in
the primary tumor by promoting EMT, the cancer stem cell phenotype,
invasion, and migration (for a review, see Rankin and Giaccia79). HIFs
promote invasion and migration through multiple mechanisms. First,
HIF regulates the E- to N-cadherin switch during EMT phenotype
through the direct activation of E-cadherin repressors including Twist1,
Zeb1/2, Snail.86,90,91 HIF signaling has also been shown to indirectly
promote EMT through the activation of Notch, TGF-β, integrin-linked
kinase (ILK), and the receptor tyrosine kinase AXL (for a review, see
Rankin and Giaccia79). Second, HIF directly upregulates the expression
of multiple factors driving cellular invasion and migration. Most
notably, HIF drives the expression of the extracellular matrix protein
LOX.92 Increased LOX expression correlates with decreased distant
metastasis-free survival and overall survival in patients with breast and
head and neck cancer.92 In addition, LOX activation promotes the
invasive and metastatic potential of breast cancer cells. Erler and
colleagues demonstrated that genetic and pharmacologic inhibition

Intravasation/
Invasion/migration
extravasation
SNAIL AXL ANGPTL44
TWIST CDCP1 L1CAM
ZEB1/2 ZEB1/2 VEGF
MMPs MET UPAR
LOX PLOD1/2 MMPS
CTGF AKAP12 SDF-1/CXCR4
CCR5 RAB222 VCAM1
PGF PLUAR ICAM1
Figure 3.2  •  Hypoxia inducible transcription factor (HIF) signaling regulates multiple steps within the metastatic cascade. The key steps of the metastatic
cascade and target genes by which HIF signaling regulates these processes are shown.
of LOX in metastatic breast cancer is sufficient to prevent hypoxia-
induced cell invasion and metastasis. These findings indicate that
LOX is a promising therapeutic target for the treatment of metastatic
disease.
HIF signaling also facilitates tumor cell intravasation and extravasa-
tion from the vasculature. HIF activity in tumor cells results in the
release of factors that modulate endothelial-endothelial cell and
endothelial-tumor cell interactions. The upregulation of angiopoietin-
like 4 (ANGPTL4) by HIF promotes tumor cell motility and
intravasation of tumor cells through blood vessels.88 Simultaneously,
HIF strengthens tumor cell–endothelial cell interactions through the
activation of L1 cell adhesion molecule (L1CAM).88 Another mechanism
by which HIF promotes tumor cell intravasation and extravasation
is through the activation of genes that control vascular permeability.
Hypoxic induction of VEGF, angiopoietin 2, MMPs, and UPAR
cooperatively act to destabilize the vascular wall and allow for tumor
cell entry.93
Third, HIF activity in the primary tumor is involved in the forma-
tion of the metastatic niche. As mentioned earlier, priming the pre-
metastatic site for the recruitment and survival of metastatic tumor
cells is a critical step in successful metastatic colonization. In breast
cancer cells, HIF activity results in the upregulation and release of
LOX and LOX-like proteins (LOXL2 and LOXL4). These proteins
recruit bone marrow–derived cells (BMDCs) into the lung and prime
the lung for metastatic colonization.87,92,94 BMDCs produce chemokines
that recruit tumor cells and stimulate blood vessel invasion.95–98 Studies
have also demonstrated a role for LOX in the establishment of the
premetastatic niche in the bone wherein tumor-secreted LOX is both
necessary and sufficient to induce osteolytic bone lesions and cortical
bone loss in mice before the arrival of tumor cells.99 Another mechanism
by which HIF signaling controls the directional migration of metastatic
tumor cell sites is through the upregulation of SDF1/CXCR4 signal-
ing.100,101 Stromal cells within target tissue sites produce stromal derived
factor-1 (SDF1), which recruits cancer cells expressing the receptor
CXCR4.29 Although these studies have indicated an important cellular
intrinsic role for hypoxia and HIF signaling in the primary tumor,
future studies are eagerly awaited to elucidate the role of HIF signaling
in tumor support cells.
Finally, HIF promotes the late stages of metastasis at a distant site
by stimulating angiogenesis. As in the primary tumor, angiogenesis
is a critical step for metastatic tumor growth. VEGF-A is a proangiogenic
factor produced by tumor cells that stimulates the recruitment and
Cellular Microenvironment and Metastases  •  CHAPTER 3 51
Premetastatic niche Distant growth
BMDC
Fibroblast
LOX
VEGF HK1/2
LOXL2
ANGPT1/2 LDHA
LOXL4
PDFG PDK1
SDF-1
PIGF CKB
VEGF
PAI-1 ENO1
CXCL12
TIE-2 ALDHA
CCL2
PGK1 GLUT-1/3
EXOSOMES
proliferation of endothelial cells and supports pericytes.29 VEGF-A is
a well-established HIF target and is significantly induced by HIF
signaling in both primary tumors and metastases.102 In summary, HIF
affects multiple aspects of tumor metastasis, indicating that therapeutic
targeting of HIF may be an effective strategy to selectively inhibit
multiple aspects of metastasis.
PATTERNS OF METASTASIS
Seed and Soil Hypothesis
The patterns of colonization cannot solely be explained by the circula-
tory, lymphatic, and transcelomic routes described earlier. The propensity
for certain tumors to seed in particular organs was first discussed as
the “seed and soil” theory by Paget in 1889.103 For example, prostate
cancer often metastasizes to the bones, colon cancer has a tendency
to metastasize to the liver, and the primary site for ovarian metastasis
is the omentum.104 Colonization is an extremely inefficient process
that is heavily dependent on the interactions between “seeding” tumor
cells and the “soil” microenvironment of the secondary site. Many
factors including formation of a premetastatic niche and interactions
between circulating tumor cells and the distant microenvironment
determine patterns of colonization.
Premetastatic Niche
Over the past decade, studies have convincingly shown that formation
of a premetastatic niche is essential for the growth of extravasating
metastatic tumor cells.98 Soluble factors and BMDCs are recruited to
the distant site before the arrival of tumor cells to establish a permissive
environment for malignant colonization. The mechanisms by which
the premetastatic niche is formed remain largely unknown. However,
recent studies have shown that factors secreted by the primary tumor
are directly involved in establishing a permissive ECM environment
and in recruiting BMDCs to the distant site.
BMDCs expressing VEGFR1, c-kit, CD133, and CD134 have been
detected at distant sites and increase angiogenesis at the premetastatic
sites. Targeted inhibition of VEGFR1 prevented niche formation and
subsequent metastatic progression. This tissue preconditioning may thus
represent a key step that could be targeted therapeutically, although
studies with anti-VEGF therapy have failed to show significant benefit
in preventing metastatic growth for long periods of time. The role of
52 Part I: Science and Clinical Oncology

hematopoietic progenitor cells and other BMDCs in tumor progression
is reviewed by Kaplan and colleagues.105
An additional function of the premetastatic niche is to guide
metastases to specific organs. Kaplan and coworkers demonstrated that
injection of secreted factors collected from cancer cells that metastasize
to multiple organs could permit cancer cells that only metastasize
to the lung when grown as subcutaneous tumors in mice to display
widespread metastasis through governing BMDC accumulation.98
Elevated fibronectin expression by fibroblasts and fibroblast-like cells
resident at premetastatic sites seems to be a critical factor in the develop-
ment of the premetastatic niche. The key tumor-secreted factors that
determine metastatic sites and mediate premetastatic niche formation
have yet to be identified, although a role for tumor necrosis factor–α
(TNF-α), TGF-β, and VEGF-α pathways has been demonstrated.106
MMPs may also play an important role in this process. For example,
VEGFR1 signaling has been shown to be required for premetastatic
induction of MMP-9 expression in endothelial cells and macrophages
of the lungs by distant primary tumors.107 This is thought to make
the lung microenvironment more compliant for invasion of metastasiz-
ing cells. This concept is supported by the finding that pericyte
recruitment and angiogenesis are not observed in tumor-bearing mice
with MMP-9 knockout bone marrow cells.108 Furthermore, stromal-
derived MMP-2 and MMP-9 have also been shown to contribute to
establishment and growth of metastases.109 Thus, whereas there is
evidence that MMPs play multiple roles in metastases, clinical trials
with MMP inhibitors have failed to show significant efficacy. In large
part, this has been due to unexpected normal tissue toxicities and
conflicting roles in metastases.
Organ Specificity
The organ distribution of metastases from a primary tumor is not
random. Minn and colleagues used bioluminescence imaging to
reveal patterns of metastasis formation by human breast cancer cells
in mice.110 They also showed that individual cells from the pleural
effusion of a breast cancer patient showed distinct patterns of organ-
specific metastasis.111 Single-cell progenies derived from this population
demonstrated different abilities to metastasize to the bone, lung, or
adrenal medulla. These studies indicated that there are particular
requirements for circulating tumor cells to colonize specific organs.
The factors contributing to tissue specific colonization include cellular
intrinsic factors within the disseminated tumor cells and specific factors
within the tissue microenvironment. Some of the key cellular intrinsic
molecules determining organ-specific metastasis have been identified
and are briefly discussed in the following section. In addition, we are
beginning to unravel the mechanisms by which infiltrating tumor
cells “educate” the normal tissue stroma at distant sites to support
metastatic growth. Elucidation of the complex signaling networks that
exist between tumor cells and their tissue microenvironment offers
new opportunities for targeted therapy against cancer.
Metastases to the Bone
There are two types of bone metastases: osteoblastic and osteolytic.112
Osteoblastic metastases are observed in patients with advanced prostate
cancer. Both the differentiation of osteoblastic precursors and the
activity of osteoblast cells are stimulated by tumor and microenviron-
mental signals such as bone morphogenetic protein (BMP), fibroblast
growth factor receptors (FGFRs), and insulin-like growth factor 1
receptor (IGF1R).113 Runx-2 is a key transcription factor that regulates
the differentiation of osteoblasts and osteoblastic precursor cells, and
represents a potential new target for inhibiting osteoblastic metastases
by preventing osteoblastic precursor differentiation.114 In contrast,
osteolytic metastases are observed in patients with breast cancer or
multiple myeloma, and in these patients interactions between tumor
cells and the bone microenvironment result in bone resorption and
metastatic growth as a result of the unique interplay between osteoblasts
and osteoclasts (Fig. 3.3).112,115 Parathyroid hormone–related peptide
(PTHrP) secreted by the tumor cells stimulates osteoblasts to produce
receptor activator of nuclear factor–κB (RANK) ligand (RANKL).
Consequently, bone-resorbing osteoclast cells are activated by RANKL
when it binds to the RANK receptor. Activated osteoclasts upregulate
MMPs, which degrade the bone matrix–releasing growth factors such
as TGF-β, IGFs, platelet-derived growth factor (PDGF), fibroblast

37+U3
5$1./
5$1./
7XPRUFHOO 2VWHREODVW

Bone
5$1. marrow
niche
2VWHRFODVW

003V
7*)β
%03
,*)V
3'*)
)*)V
Figure 3.3  •  The vicious cycle of osteoclastic bone metastasis. Interactions between the tumor cells and the bone microenvironment create a vicious cycle
of osteolytic metastatic lesion development. Parathyroid hormone–related peptide (PTHrP), secreted by the tumor cells, stimulates osteoblasts to produce
receptor activator of nuclear factor–κB (RANK) ligand (RANKL). Bone-resorbing osteoclast cells are activated by RANKL when it binds to the RANK receptor.
The activated osteoclasts upregulate matrix metalloproteinases (MMPs) that degrade the bone matrix–releasing growth factors such as transforming growth
factor–β (TGF-β), insulin-like growth factors (IGFs), platelet-derived growth factor (PDGF), fibroblast growth factors (FGFs), and bone morphogenetic proteins
(BMPs). These factors stimulate tumor cells to release PTHrP, thus completing the vicious cycle. (Modified from Steeg PS. Tumor metastasis: mechanistic
insights and clinical challenges. Nat Med. 2006;12:895–904.)

growth factors (FGFs), and BMP.112,116,117 These factors stimulate tumor
cells to release PTHrP, thus restarting this pathway of bone resorption.
Gene profiling has identified other important mediators of osteoclastic
bone metastases including CXCR4, MMP-1, CTGF, and osteopontin.118
Tumor cells additionally induce osteoclast formation by overexpressing
ILs such as IL-8 and IL-11, and by downregulating macrophage
colony-stimulating factor.119,120 All of these factors represent new targets
for metastases, although the importance of each factor in osteoclastic
bone metastases requires further clarification.
Metastases to the Brain
Brain metastases are most commonly observed in patients with breast
cancer, lung cancer, and melanoma. Vascular access to the brain is
strictly regulated by the blood-brain barrier, an endothelial layer
surrounding the brain, connected by tight junctions and further lined
by a basement membrane, pericytes, and astrocytes.121 Macromolecules
are not usually able to traverse the blood-brain barrier, and it remains
unclear how tumor cells are able to penetrate the blood-brain barrier.
However, once the tumor cells are within the brain parenchyma, glial
cells permit establishment and growth of metastases by secreting
chemokines, cytokines, and growth factors.122 Other neurotransmitter
hormones in the brain such as norepinephrine have also been reported
to increase tumor cell motility and metastatic spread.123
Little is known about the key factors that determine colonization
of the brain, mostly because there is a lack of good in vivo models of
brain metastasis. Overexpression of Stat3 increases melanoma metastasis
to the brain and increases invasion of the melanoma cells and angio-
genesis, although the pathways modulated by Stat3 signaling require
elucidation.124 The dependence of brain metastases on VEGF has
been demonstrated experimentally in animals through inhibition studies
in which VEGF neutralization reduced brain metastases.125,126
In general, patients with brain metastases have an extremely poor
prognosis. It is of concern that there has been an increase in the
incidence of brain metastases in patients whose systemic disease is
well controlled.127–129 For example, patients with breast tumors that
overexpress Her2 and who are treated with Her2 targeting trastuzumab
(see later discussion) have an incidence of brain metastases twice that
of breast cancer patients who are treated with other agents.128 This is
thought to be because the brain offers a sanctuary for metastatic breast
tumor cells when systemic disease is being controlled.130 The develop-
ment of drugs that can cross the blood-brain barrier and target brain
metastases is of paramount importance in the development of new
targeted therapies to tackle this problem. Currently, the best treatment
for oligometastases to the brain is radiosurgery.
Metastases to the Lung
Pulmonary metastases are frequently observed in patients with sarcoma,
breast cancer, melanoma, gastrointestinal cancer, and kidney cancer.
Because cardiac output from the pulmonary artery circulates through
the lungs, a high incidence of pulmonary metastases in cancer patients
can be expected on the basis of blood flow alone. Metastases therefore
often initiate in pulmonary arterioles and later traverse the basement
membrane into the lung parenchyma. TGF-β and NF-κB facilitate
this process in breast cancer, as does osteopontin in hepatocellular
cancer, and ezrin in osteosarcoma and breast cancer.131–136 In vivo
studies have identified a gene expression signature for lung metastasis
including several membrane-localized and secreted proteins that has
been validated in breast cancer patients.110 Interesting to note, this
group of genes was able to induce lung metastasis when expressed
together but not individually, suggesting essential cooperation between
proteins. Increased expression of antiapoptotic proteins such as Bcl-2
and Bcl-xL is also observed in lung metastases, facilitating survival
and resulting in resistance to therapy.137–141
Adding to the complexity of signaling interactions needed for
successful metastatic colonization in the lung, studies have revealed
Cellular Microenvironment and Metastases  •  CHAPTER 3 53
that interactions between breast cancer stem cells and the lung stroma
are necessary for metastatic colonization. The ECM components TNC
and periostin (POSTN) have recently been shown to be essential
factors required for tumor initiating cells to form metastases in the
lung.7,10,142 Malanchi and colleagues showed that tumor-initiating cells
induce POSTN expression in lung myofibroblasts to initiate coloniza-
tion and maintain their stem cell phenotype.7 Similarly, Oskarsson
and colleagues demonstrated a role for the extracellular protein TNC
in supporting the breast cancer cell stem cell phenotype and metastases
in the lung.10 Interesting to note, previous studies showed that TNC
and POSTN form complexes together with collagen type I and
fibronectin in the ECM. It was shown that POSTN promotes the
incorporation of TNC into the ECM to strengthen the ECM archi-
tecture, suggesting that these factors may act through similar pathways
in promoting cancer stem cell metastasis.143 In support of this notion,
maintenance of the cancer stem cell phenotype by TNC and POSTN
was mediated at least in part through the induction of WNT signaling.
These studies suggest that targeting signaling pathways mediated
through the ECM may be an effective strategy against cancer stem
cell–driven metastasis.
Metastases to the Liver
Liver metastases are observed in patients with breast, lung, and
pancreatic cancers. However, liver metastases are most commonly
found in patients with metastatic colorectal cancer, because the liver
is the first capillary bed encountered by the metastasizing cells. The
circulatory system of the liver, in particular the liver sinusoids, does
not have a barrier limiting macromolecule flux, and it is well perfused
and highly permeable, permitting metastasizing cancer cells to establish
themselves and grow. Thus, tumor cell invasion and survival are key
determinants in metastatic colonization of the liver.144
Tumor cell survival within the liver is dependent on the cells’
ability to metabolically adapt to the local tissue microenvironment.145
The liver is characterized by regions of hypoxia and highly glycolytic
hepatocytes.146 Preclinical studies have revealed that disseminated colon
cancer cells experience acute hypoxia and competition for glycolytic
substrates early after hepatic dissemination.147 To survive, metastatic
colorectal cells release the enzyme creatine kinase brain-type (CKB)
into the extracellular space.147 CKB controls the amount of rapidly
mobilized high-energy phosphates by catalyzing the transfer of a
high-energy phosphate group from adenosine triphosphate (ATP) to
the metabolite creatine, producing phosphocreatine.148 Under hypoxia,
tumor cells import phosphocreatine and use it as a source of high-energy
phosphate that can be transferred to adenosine diphosphate (ADP)
to generate ATP.148 These findings suggest that hepatic hypoxia is a
barrier to disseminated tumor cell survival and that disseminated
tumor cells metabolically adapt to overcome metabolic stress associated
with hypoxia. Consistent with these findings, Dupuy and colleagues
observed that disseminated breast cancer cells in the liver are dependent
on glycolysis for survival. Glycolytic reprogramming in metastatic
breast cancer cells was mediated by the HIF target gene pyruvate
dehydrogenase kinase (PDK1).149 Under hypoxic conditions, PDK1
upregulation represses mitochondrial function by reducing pyruvate
entry into the TCA cycle and promoting the conversion of pyruvate
to lactate, thereby supporting glycolysis through the regeneration of
regenerate NAD.150 Together, these findings highlight the importance
of metabolic plasticity in promoting tumor cell survival within foreign
tissue microenvironments.
CLINICAL RELEVANCE AND APPLICATIONS
The future of cancer therapy lies in the ability to effectively prevent
and treat metastatic disease. Metastases are largely resistant to current
cytotoxic therapies and as a result are responsible for more than 90%
of all cancer-related deaths. Over the past decade, many of the cellular
and molecular constituents that drive metastatic tumor progression
54 Part I: Science and Clinical Oncology

have been defined. Based on these findings, a number of therapeutic
agents targeting key components of the metastatic cascade have been
developed and tested in preclinical as well as clinical settings. Many
of the initial targeted therapies developed for tumor metastasis inhibit
factors driving tumor cell invasion and migration. Preclinical studies
have demonstrated that agents that inhibit MMPs, the receptor tyrosine
kinase AXL, miR-10b, fascin, and exosomes are effective in blocking
the initiation and/or progression of metastatic tumors in mice (see
reviews by Valastyan and Weinberg151 and Rankin and colleagues152).
Of all of these agents, MMP inhibitors have been the most tested in
clinical trials. Disappointingly, these inhibitors failed to increase survival
in patients with advanced cancer and were associated with adverse
side effects.153 Important lessons regarding clinical trial design were
learned from these trials. Because MMP inhibitors and other inhibitors
of invasion are likely to function at the early stages of metastasis, it
is important to thoughtfully identify patient populations that may
benefit most from these types of therapies. In addition, it is likely
that the combination of targeted agents controlling distinct aspects
of metastasis may prove to be the most effective when targeting
metastatic cancer.
An emerging strategy in the treatment of metastasis involves the
therapeutic targeting of cellular and molecular constituents within
the tumor microenvironment. As mentioned earlier, hypoxia and HIF
signaling are critical drivers of both the tumorigenic and metastatic
phenotypes. HIF and hypoxia-induced proteins represent therapeutic
targets that have the potential to be tumor specific because these
proteins are highly elevated in both the primary tumor and metastases
in comparison with normal tissue.154 In addition, targeting HIF is an
attractive strategy for the treatment of metastatic disease because HIF
controls multiple aspects of metastasis including EMT, invasion,
migration, metastatic niche formation, and metastatic tumor growth.
In this regard, a number of small-molecule HIF inhibitors including
digoxin and acriflavine have been identified and have shown success
in preclinical studies by preventing lung metastasis in mice bearing
primary breast tumors.155 Although the efficacy of these inhibitors in
treating established metastatic disease remains unknown, these
compounds do inhibit metastatic xenograft growth after tumor
implantation.88 In addition to targeting HIF directly, a number of
HIF-induced proteins have been targeted for metastatic therapy. One
very promising target is LOX, a hypoxia-induced secreted protein
involved in multiple stages of metastasis.92 LOX contributes to tumor
cell invasion by cross-linking collagens in the ECM, which stimulates
integrin-mediated cell-matrix adhesion and activation of FAK, and
in addition provides a route (“highway”) by which tumor cells may
travel. Furthermore, LOX is involved in the formation and maintenance
of the metastatic niche, allowing metastatic dissemination and growth.
Targeting secreted LOX through antibody or small-molecule inhibition
significantly reduced formation and growth of metastases to the lung,
liver, bone, and brain, in several preclinical studies. Another promising
hypoxia-induced target is CTGF.156 CTGF is an extracellular matrix
protein that exerts a number of prometastatic activities including
migration, invasion, angiogenesis, and anoikis.157 Both genetic and
therapeutic inhibition of CTGF have shown efficacy in primary and
metastatic tumor growth inhibition of preclinical models of pancreatic
cancer.158,159 The results of clinical trials targeting LOX and CTGF
are eagerly awaited.
Among the therapies targeting the cellular components of the
tumor microenvironment, those that target endothelial cells are the
most advanced and have shown success within the clinic.29 Targeting
VEGF signaling on vascular and lymphatic endothelial cells has shown
clinical benefits in patients with metastatic cancer.160 A variety of
VEGF inhibitors have been approved by the US Food and Drug
Administration (FDA) for the treatment of metastasis. The humanized
anti-VEGF-A monoclonal antibody bevacizumab has been approved
as a first-line therapy in combination with 5-fluorouracil for metastatic
colon cancer. In addition, in patients with metastatic non–small cell
lung cancer, bevacizumab increased survival in combination with
chemotherapy.161 In addition to biologic therapies, small-molecule
VEGF receptor inhibitors have also been developed for the treatment
of metastasis. Both sorafenib and sunitinib have been approved by
the FDA for the treatment of metastatic renal cancer. Phase III clinical
trials demonstrated that sorafenib monotherapy resulted in a significant
increase in progression-free survival in this patient population. The
results of future clinical trials targeting additional key cellular com-
ponents of the tumor microenvironment, such as TAMs, are eagerly
awaited.
CONCLUSION
Metastatic disease, not the primary tumor, kills the majority of cancer
patients. For such an important determinant of long-term survival,
progress in understanding the crucial genes and pathways that drive
metastatic progression has been slow. The reasons for this slow progress
have been numerous, including inadequate animal models that reflect
the metastatic process in humans, failure to identify genes that specifi-
cally affect metastatic tumor growth, the complexity of host and
metastatic tumor interactions, and premature clinical trials focusing
on “attractive” gene targets. Recent studies have elucidated many of
the cellular and molecular factors driving metastatic progression. Based
on the findings of these studies, new opportunities to target metastatic
disease have been identified. The future of metastatic therapy looks
very promising, in large part because we understand the mistakes of
the past and are using multiple genomic and proteomic approaches
to target what has for so long seemed to be an intractable problem.
It is only when we are able to attack the problem of metastases that
we will make significant inroads in our war against cancer.
The complete reference list is available online at
ExpertConsult.com.

KEY REFERENCES
1. Oskarsson T, Batlle E, Massague J. Metastatic stem
cells: sources, niches, and vital pathways. Cell Stem
Cell. 2014;14(3):306–321.
2. Hanahan D, Coussens LM. Accessories to the crime:
functions of cells recruited to the tumor microen-
vironment. Cancer Cell. 2012;21(3):309–322.
7. Malanchi I, Santamaria-Martinez A, Susanto E,
et al. Interactions between cancer stem cells and
their niche govern metastatic colonization. Nature.
2012;481(7379):85–89.
8. Lawson DA, Bhakta NR, Kessenbrock K, et al.
Single-cell analysis reveals a stem-cell program
in human metastatic breast cancer cells. Nature.
2015;526(7571):131–135.
13. Reymond N, d’Agua BB, Ridley AJ. Crossing the
endothelial barrier during metastasis. Nat Rev Cancer.
2013;13(12):858–870.
21. Ghajar CM, Peinado H, Mori H, et al. The peri-
vascular niche regulates breast tumour dormancy.
Nat Cell Biol. 2013;15(7):807–817.
24. Yang Y, Andersson P, Hosaka K, et al. The PDGF-
BB-SOX7 axis-modulated IL-33 in pericytes and
stromal cells promotes metastasis through tumour-
associated macrophages. Nat Commun. 2016;7:
11385.
25. Cooke VG, LeBleu VS, Keskin D, et al. Pericyte
depletion results in hypoxia-associated epithelial-
to-mesenchymal transition and metastasis mediated
by met signaling pathway. Cancer Cell. 2012;21(1):
66–81.
28. Ruffell B, Coussens LM. Macrophages and thera-
peutic resistance in cancer. Cancer Cell. 2015;27(4):
462–472.
29. Joyce JA, Pollard JW. Microenvironmental regulation
of metastasis. Nat Rev Cancer. 2009;9(4):239–252.
30. Harney AS, Arwert EN, Entenberg D, et al.
Real-time imaging reveals local, transient vascular
permeability, and tumor cell intravasation stimulated
by TIE2hi macrophage-derived VEGFA. Cancer
Discov. 2015;5(9):932–943.
37. Park J, Wysocki RW, Amoozgar Z, et al. Cancer
cells induce metastasis-supporting neutrophil

extracellular DNA traps. Sci Transl Med. 2016;8(361):
361ra138.
47. Labelle M, Begum S, Hynes RO. Direct signal-
ing between platelets and cancer cells induces an
epithelial-mesenchymal-like transition and promotes
metastasis. Cancer Cell. 2011;20(5):576–590.
54. Leca J, Martinez S, Lac S, et al. Cancer-associated
fibroblast-derived annexin A6+ extracellular vesicles
support pancreatic cancer aggressiveness. J Clin
Invest. 2016;126(11):4140–4156.
58. Becker A, Thakur BK, Weiss JM, Kim HS, Peinado
H, Lyden D. Extracellular vesicles in cancer: cell-to-
cell mediators of metastasis. Cancer Cell. 2016;30(6):
836–848.
60. Peinado H, Aleckovic M, Lavotshkin S, et al. Mela-
noma exosomes educate bone marrow progenitor
cells toward a pro-metastatic phenotype through
MET. Nat Med. 2012;18(6):883–891.
66. Zomer A, Maynard C, Verweij FJ, et al. In vivo
imaging reveals extracellular vesicle-mediated phe-
nocopying of metastatic behavior. Cell. 2015;161(5):
1046–1057.
69. Zhou W, Fong MY, Min Y, et al. Cancer-secreted
miR-105 destroys vascular endothelial barriers
to promote metastasis. Cancer Cell. 2014;25(4):
501–515.
70. Costa-Silva B, Aiello NM, Ocean AJ, et al. Pancreatic
cancer exosomes initiate pre-metastatic niche forma-
tion in the liver. Nat Cell Biol. 2015;17(6):816–826.
71. Hoshino A, Costa-Silva B, Shen TL, et al. Tumour
exosome integrins determine organotropic metastasis.
Nature. 2015;527(7578):329–335.
72. Fong MY, Zhou W, Liu L, et al. Breast-cancer-
secreted miR-122 reprograms glucose metabolism
in premetastatic niche to promote metastasis. Nat
Cell Biol. 2015;17(2):183–194.
79. Rankin EB, Giaccia AJ. Hypoxic control of metas-
tasis. Science. 2016;352(6282):175–180.
83. Jaakkola P, Mole DR, Tian YM, et al. Targeting of
HIF-alpha to the von Hippel-Lindau ubiquitylation
complex by O2-regulated prolyl hydroxylation.
Science. 2001;292(5516):468–472.
84. Ivan M, Kondo K, Yang H, et al. HIFalpha
targeted for VHL-mediated destruction by proline
hydroxylation: implications for O2 sensing. Science.
2001;292(5516):464–468.
85. Semenza GL. Defining the role of hypoxia-inducible
factor 1 in cancer biology and therapeutics. Oncogene.
2010;29(5):625–634.
Cellular Microenvironment and Metastases  •  CHAPTER 3 55
92. Erler JT, Bennewith KL, Nicolau M, et al. Lysyl
oxidase is essential for hypoxia-induced metastasis.
Nature. 2006;440(7088):1222–1226.
93. De Bock K, Mazzone M, Carmeliet P. Antiangiogenic
therapy, hypoxia, and metastasis: risky liaisons, or
not? Nat Rev Clin Oncol. 2011;8(7):393–404.
94. Erler JT, Bennewith KL, Cox TR, et al. Hypoxia-
induced lysyl oxidase is a critical mediator of bone
marrow cell recruitment to form the premetastatic
niche. Cancer Cell. 2009;15(1):35–44.
95. Yang L, DeBusk LM, Fukuda K, et al. Expansion
of myeloid immune suppressor Gr+CD11b+ cells
in tumor-bearing host directly promotes tumor
angiogenesis. Cancer Cell. 2004;6(4):409–421.
96. Lyden D, Hattori K, Dias S, et al. Impaired recruit-
ment of bone-marrow-derived endothelial and hema-
topoietic precursor cells blocks tumor angiogenesis
and growth. Nat Med. 2001;7(11):1194–1201.
97. Gao D, Nolan DJ, Mellick AS, Bambino K,
McDonnell K, Mittal V. Endothelial progenitor
cells control the angiogenic switch in mouse lung
metastasis. Science. 2008;319(5860):195–198.
98. Kaplan RN, Riba RD, Zacharoulis S, et al.
VEGFR1-positive haematopoietic bone marrow
progenitors initiate the pre-metastatic niche. Nature.
2005;438(7069):820–827.
101. Ceradini DJ, Kulkarni AR, Callaghan MJ, et al.
Progenitor cell trafficking is regulated by hypoxic
gradients through HIF-1 induction of SDF-1. Nat
Med. 2004;10(8):858–864.
104. Nieman KM, Kenny HA, Penicka CV, et al.
Adipocytes promote ovarian cancer metastasis and
provide energy for rapid tumor growth. Nat Med.
2011;17(11):1498–1503.
111. Minn AJ, Kang Y, Serganova I, et al. Distinct
organ-specific metastatic potential of individual
breast cancer cells and primary tumors. J Clin Invest.
2005;115(1):44–55.
112. Mundy GR. Metastasis to bone: causes, consequences
and therapeutic opportunities. Nat Rev Cancer.
2002;2(8):584–593.
118. Kang Y, Siegel PM, Shu W, et al. A multigenic
program mediating breast cancer metastasis to bone.
Cancer Cell. 2003;3(6):537–549.
121. Abbott NJ, Ronnback L, Hansson E. Astrocyte-
endothelial interactions at the blood-brain barrier.
Nat Rev Neurosci. 2006;7(1):41–53.
130. Steeg PS. Tumor metastasis: mechanistic insights and
clinical challenges. Nat Med. 2006;12(8):895–904.
134. Ye QH, Qin LX, Forgues M, et al. Predicting hepatitis
B virus-positive metastatic hepatocellular carcinomas
using gene expression profiling and supervised
machine learning. Nat Med. 2003;9(4):416–423.
135. Khanna C, Wan X, Bose S, et al. The membrane-
cytoskeleton linker ezrin is necessary for osteosarcoma
metastasis. Nat Med. 2004;10(2):182–186.
140. Inbal B, Cohen O, Polak-Charcon S, et al. DAP
kinase links the control of apoptosis to metastasis.
Nature. 1997;390(6656):180–184.
147. Loo JM, Scherl A, Nguyen A, et al. Extracellular
metabolic energetics can promote cancer progression.
Cell. 2015;160(3):393–406.
148. Wyss M, Kaddurah-Daouk R. Creatine and creatinine
metabolism. Physiol Rev. 2000;80(3):1107–1213.
149. Dupuy F, Tabaries S, Andrzejewski S, et al. PDK1-
dependent metabolic reprogramming dictates meta-
static potential in breast cancer. Cell Metab. 2015;22(4):
577–589.
150. Papandreou I, Cairns RA, Fontana L, Lim AL,
Denko NC. HIF-1 mediates adaptation to hypoxia
by actively downregulating mitochondrial oxygen
consumption. Cell Metab. 2006;3(3):187–197.
151. Valastyan S, Weinberg RA. Tumor metastasis:
molecular insights and evolving paradigms. Cell.
2011;147(2):275–292.
152. Rankin EB, Fuh KC, Taylor TE, et al. AXL is an
essential factor and therapeutic target for metastatic
ovarian cancer. Cancer Res. 2010;70(19):7570–
7579.
153. Coussens LM, Fingleton B, Matrisian LM. Matrix
metalloproteinase inhibitors and cancer: trials
and tribulations. Science. 2002;295(5564):2387–
2392.
154. Harris AL. Hypoxia—a key regulatory factor in
tumour growth. Nat Rev Cancer. 2002;2(1):38–47.
158. Dornhofer N, Spong S, Bennewith K, et al. Con-
nective tissue growth factor-specific monoclonal
antibody therapy inhibits pancreatic tumor growth
and metastasis. Cancer Res. 2006;66(11):5816–
5827.
159. Aikawa T, Gunn J, Spong SM, Klaus SJ, Korc
M. Connective tissue growth factor-specific
antibody attenuates tumor growth, metastasis,
and angiogenesis in an orthotopic mouse model of
pancreatic cancer. Mol Cancer Ther. 2006;5(5):1108–
1116.

REFERENCES
1. Oskarsson T, Batlle E, Massague J. Metastatic stem
cells: sources, niches, and vital pathways. Cell Stem
Cell. 2014;14(3):306–321.
2. Hanahan D, Coussens LM. Accessories to the crime:
functions of cells recruited to the tumor microen-
vironment. Cancer Cell. 2012;21(3):309–322.
3. Cho RW, Clarke MF. Recent advances in cancer
stem cells. Curr Opin Genet Dev. 2008;18(1):48–53.
4. Barker N, Ridgway RA, van Es JH, et al. Crypt
stem cells as the cells-of-origin of intestinal cancer.
Nature. 2009;457(7229):608–611.
5. Mani SA, Guo W, Liao MJ, et al. The epithelial-
mesenchymal transition generates cells with proper-
ties of stem cells. Cell. 2008;133(4):704–715.
6. Guo W, Keckesova Z, Donaher JL, et al. Slug and
Sox9 cooperatively determine the mammary stem
cell state. Cell. 2012;148(5):1015–1028.
7. Malanchi I, Santamaria-Martinez A, Susanto E,
et al. Interactions between cancer stem cells and
their niche govern metastatic colonization. Nature.
2012;481(7379):85–89.
8. Lawson DA, Bhakta NR, Kessenbrock K, et al.
Single-cell analysis reveals a stem-cell program
in human metastatic breast cancer cells. Nature.
2015;526(7571):131–135.
9. Rhim AD, Mirek ET, Aiello NM, et al. EMT and
dissemination precede pancreatic tumor formation.
Cell. 2012;148(1–2):349–361.
10. Oskarsson T, Acharyya S, Zhang XH, et al. Breast
cancer cells produce tenascin C as a metastatic
niche component to colonize the lungs. Nat Med.
2011;17(7):867–874.
11. Bergers G, Song S, Meyer-Morse N, Bergsland E,
Hanahan D. Benefits of targeting both pericytes and
endothelial cells in the tumor vasculature with kinase
inhibitors. J Clin Invest. 2003;111(9):1287–1295.
12. Cantelmo AR, Conradi LC, Brajic A, et al.
Inhibition of the glycolytic activator PFKFB3 in
endothelium induces tumor vessel normalization,
impairs metastasis, and improves chemotherapy.
Cancer Cell. 2016;30(6):968–985.
13. Reymond N, d’Agua BB, Ridley AJ. Crossing the
endothelial barrier during metastasis. Nat Rev Cancer.
2013;13(12):858–870.
14. Mannori G, Santoro D, Carter L, Corless C, Nelson
RM, Bevilacqua MP. Inhibition of colon carcinoma
cell lung colony formation by a soluble form of
E-selectin. Am J Pathol. 1997;151(1):233–243.
15. Kim YJ, Borsig L, Varki NM, Varki A. P-selectin
deficiency attenuates tumor growth and metastasis.
Proc Natl Acad Sci USA. 1998;95(16):9325–9330.
16. Nesbit M, Herlyn M. Adhesion receptors in
human melanoma progression. Invasion Metastasis.
1994;14(1–6):131–146.
17. Wang H, Fu W, Im JH, et al. Tumor cell alpha3beta1
integrin and vascular laminin-5 mediate pulmonary
arrest and metastasis. J Cell Biol. 2004;164(6):
935–941.
18. Ruoslahti E. Fibronectin and its alpha 5 beta 1
integrin receptor in malignancy. Invasion Metastasis.
1994;14(1–6):87–97.
19. Birch M, Mitchell S, Hart IR. Isolation and
characterization of human melanoma cell variants
expressing high and low levels of CD44. Cancer
Res. 1991;51(24):6660–6667.
20. Huang C, Li N, Li Z, et al. Tumour-derived Interleu-
kin 35 promotes pancreatic ductal adenocarcinoma
cell extravasation and metastasis by inducing ICAM1
expression. Nat Commun. 2017;8:14035.
21. Ghajar CM, Peinado H, Mori H, et al. The peri-
vascular niche regulates breast tumour dormancy.
Nat Cell Biol. 2013;15(7):807–817.
22. Raza A, Franklin MJ, Dudek AZ. Pericytes and
vessel maturation during tumor angiogenesis and
metastasis. Am J Hematol. 2010;85(8):593–598.
23. Viski C, Konig C, Kijewska M, Mogler C, Isacke
CM, Augustin HG. Endosialin-expressing pericytes
Cellular Microenvironment and Metastases  •  CHAPTER 3 55.e1
promote metastatic dissemination. Cancer Res.
2016;76(18):5313–5325.
24. Yang Y, Andersson P, Hosaka K, et al. The PDGF-
BB-SOX7 axis-modulated IL-33 in pericytes and
stromal cells promotes metastasis through tumour-
associated macrophages. Nat Commun. 2016;7:
11385.
25. Cooke VG, LeBleu VS, Keskin D, et al. Pericyte
depletion results in hypoxia-associated epithelial-
to-mesenchymal transition and metastasis mediated
by met signaling pathway. Cancer Cell. 2012;21(1):
66–81.
26. Lewis CE, Pollard JW. Distinct role of macrophages
in different tumor microenvironments. Cancer Res.
2006;66(2):605–612.
27. Talmadge JE, Donkor M, Scholar E. Inflammatory
cell infiltration of tumors: Jekyll or Hyde. Cancer
Metastasis Rev. 2007;26(3–4):373–400.
28. Ruffell B, Coussens LM. Macrophages and thera-
peutic resistance in cancer. Cancer Cell. 2015;27(4):
462–472.
29. Joyce JA, Pollard JW. Microenvironmental regulation
of metastasis. Nat Rev Cancer. 2009;9(4):239–252.
30. Harney AS, Arwert EN, Entenberg D, et al.
Real-time imaging reveals local, transient vascular
permeability, and tumor cell intravasation stimulated
by TIE2hi macrophage-derived VEGFA. Cancer
Discov. 2015;5(9):932–943.
31. Talks KL, Turley H, Gatter KC, et al. The expression
and distribution of the hypoxia-inducible factors
HIF-1alpha and HIF-2alpha in normal human
tissues, cancers, and tumor-associated macrophages.
Am J Pathol. 2000;157(2):411–421.
32. Imtiyaz HZ, Williams EP, Hickey MM, et al.
Hypoxia-inducible factor 2alpha regulates macro-
phage function in mouse models of acute and tumor
inflammation. J Clin Invest. 2010;120(8):2699–2714.
33. Branzk N, Papayannopoulos V. Molecular mecha-
nisms regulating NETosis in infection and disease.
Semin Immunopathol. 2013;35(4):513–530.
34. Granot Z, Henke E, Comen EA, King TA, Norton
L, Benezra R. Tumor entrained neutrophils inhibit
seeding in the premetastatic lung. Cancer Cell.
2011;20(3):300–314.
35. Spiegel A, Brooks MW, Houshyar S, et al. Neu-
trophils suppress intraluminal NK cell-mediated
tumor cell clearance and enhance extravasation
of disseminated carcinoma cells. Cancer Discov.
2016;6(6):630–649.
36. Cools-Lartigue J, Spicer J, McDonald B, et al.
Neutrophil extracellular traps sequester circulating
tumor cells and promote metastasis. J Clin Invest.
2013 Jul 01.
37. Park J, Wysocki RW, Amoozgar Z, et al. Cancer cells
induce metastasis-supporting neutrophil extracellular
DNA traps. Sci Transl Med. 2016;8(361):361ra138.
38. Kolaczkowska E, Jenne CN, Surewaard BG, et al.
Molecular mechanisms of NET formation and
degradation revealed by intravital imaging in the
liver vasculature. Nat Commun. 2015;6:6673.
39. Kaushansky K. Historical review: megakaryopoiesis
and thrombopoiesis. Blood. 2008;111(3):981–
986.
40. Cliffton EE, Grossi CE. The rationale of anticoagu-
lants in the treatment of cancer. J Med. 1974;5(1):
107–113.
41. Fidler IJ. Macrophages and metastasis—a bio-
logical approach to cancer therapy. Cancer Res.
1985;45(10):4714–4726.
42. Nieswandt B, Hafner M, Echtenacher B, Mannel
DN. Lysis of tumor cells by natural killer cells
in mice is impeded by platelets. Cancer Res.
1999;59(6):1295–1300.
43. Camerer E, Qazi AA, Duong DN, Cornelissen
I, Advincula R, Coughlin SR. Platelets, protease-
activated receptors, and fibrinogen in hematogenous
metastasis. Blood. 2004;104(2):397–401.
55.e1
44. Karpatkin S, Pearlstein E, Ambrogio C, Coller BS.
Role of adhesive proteins in platelet tumor interac-
tion in vitro and metastasis formation in vivo. J Clin
Invest. 1988;81(4):1012–1019.
45. Coupland LA, Chong BH, Parish CR. Platelets
and P-selectin control tumor cell metastasis in an
organ-specific manner and independently of NK
cells. Cancer Res. 2012;72(18):4662–4671.
46. Schumacher D, Strilic B, Sivaraj KK, Wettschureck
N, Offermanns S. Platelet-derived nucleotides
promote tumor-cell transendothelial migration and
metastasis via P2Y2 receptor. Cancer Cell. 2013;24(1):
130–137.
47. Labelle M, Begum S, Hynes RO. Direct signal-
ing between platelets and cancer cells induces an
epithelial-mesenchymal-like transition and promotes
metastasis. Cancer Cell. 2011;20(5):576–590.
48. Mammadova-Bach E, Zigrino P, Brucker C, et al.
Platelet integrin alpha6beta1 controls lung metas-
tasis through direct binding to cancer cell-derived
ADAM9. JCI insight. 2016;1(14):e88245.
49. Hu M, Yao J, Cai L, et al. Distinct epigenetic changes
in the stromal cells of breast cancers. Nat Genet.
2005;37(8):899–905.
50. Kurose K, Gilley K, Matsumoto S, Watson PH, Zhou
XP, Eng C. Frequent somatic mutations in PTEN
and TP53 are mutually exclusive in the stroma of
breast carcinomas. Nat Genet. 2002;32(3):355–357.
51. Strell C, Rundqvist H, Ostman A. Fibroblasts—a
key host cell type in tumor initiation, progression,
and metastasis. Ups J Med Sci. 2012;117(2):187–195.
52. Hu M, Yao J, Carroll DK, et al. Regulation of in
situ to invasive breast carcinoma transition. Cancer
Cell. 2008;13(5):394–406.
53. Gaggioli C, Hooper S, Hidalgo-Carcedo C, et al.
Fibroblast-led collective invasion of carcinoma cells
with differing roles for RhoGTPases in leading and
following cells. Nat Cell Biol. 2007;9(12):1392–1400.
54. Leca J, Martinez S, Lac S, et al. Cancer-associated
fibroblast-derived annexin A6+ extracellular vesicles
support pancreatic cancer aggressiveness. J Clin
Invest. 2016;126(11):4140–4156.
55. Waghray M, Yalamanchili M, Dziubinski M,
et al. GM-CSF mediates mesenchymal-epithelial
cross-talk in pancreatic cancer. Cancer Discov.
2016;6(8):886–899.
56. Lin JT, Wang JY, Chen MK, et al. Colon cancer
mesenchymal stem cells modulate the tumorigenicity
of colon cancer through interleukin 6. Exp Cell Res.
2013;319(14):2216–2229.
57. McLean K, Gong Y, Choi Y, et al. Human ovarian
carcinoma-associated mesenchymal stem cells regu-
late cancer stem cells and tumorigenesis via altered
BMP production. J Clin Invest. 2011;121(8):3206–
3219.
58. Becker A, Thakur BK, Weiss JM, Kim HS, Peinado
H, Lyden D. Extracellular vesicles in cancer:
cell-to-cell mediators of metastasis. Cancer Cell.
2016;30(6):836–848.
59. Li Z, Ma YY, Wang J, et al. Exosomal microRNA-141
is upregulated in the serum of prostate cancer
patients. Onco Targets Ther. 2016;9:139–148.
60. Peinado H, Aleckovic M, Lavotshkin S, et al. Mela-
noma exosomes educate bone marrow progenitor
cells toward a pro-metastatic phenotype through
MET. Nat Med. 2012;18(6):883–891.
61. Melo SA, Luecke LB, Kahlert C, et al. Glypican-1
identifies cancer exosomes and detects early pan-
creatic cancer. Nature. 2015;523(7559):177–182.
62. Kalluri R. The biology and function of exosomes
in cancer. J Clin Invest. 2016;126(4):1208–1215.
63. Andreola G, Rivoltini L, Castelli C, et al. Induction
of lymphocyte apoptosis by tumor cell secre-
tion of FasL-bearing microvesicles. J Exp Med.
2002;195(10):1303–1316.
64. Chalmin F, Ladoire S, Mignot G, et al. Membrane-
associated Hsp72 from tumor-derived exosomes
55.e2 Part I: Science and Clinical Oncology
mediates STAT3-dependent immunosuppressive
function of mouse and human myeloid-derived
suppressor cells. J Clin Invest. 2010;120(2):457–471.
65. Atay S, Banskota S, Crow J, Sethi G, Rink L, Godwin
AK. Oncogenic KIT-containing exosomes increase
gastrointestinal stromal tumor cell invasion. Proc
Natl Acad Sci USA. 2014;111(2):711–716.
66. Zomer A, Maynard C, Verweij FJ, et al. In vivo
imaging reveals extracellular vesicle-mediated phe-
nocopying of metastatic behavior. Cell. 2015;161(5):
1046–1057.
67. Luga V, Zhang L, Viloria-Petit AM, et al. Exosomes
mediate stromal mobilization of autocrine Wnt-PCP
signaling in breast cancer cell migration. Cell.
2012;151(7):1542–1556.
68. Zhang L, Zhang S, Yao J, et al. Microenvironment-
induced PTEN loss by exosomal microRNA primes
brain metastasis outgrowth. Nature. 2015;527(7576):
100–104.
69. Zhou W, Fong MY, Min Y, et al. Cancer-secreted
miR-105 destroys vascular endothelial barriers
to promote metastasis. Cancer Cell. 2014;25(4):
501–515.
70. Costa-Silva B, Aiello NM, Ocean AJ, et al. Pancreatic
cancer exosomes initiate pre-metastatic niche forma-
tion in the liver. Nat Cell Biol. 2015;17(6):816–826.
71. Hoshino A, Costa-Silva B, Shen TL, et al. Tumour
exosome integrins determine organotropic metastasis.
Nature. 2015;527(7578):329–335.
72. Fong MY, Zhou W, Liu L, et al. Breast-cancer-
secreted miR-122 reprograms glucose metabolism
in premetastatic niche to promote metastasis. Nat
Cell Biol. 2015;17(2):183–194.
73. Ostrowski M, Carmo NB, Krumeich S, et al. Rab27a
and Rab27b control different steps of the exosome
secretion pathway. Nat Cell Biol. 2010;12(1):19–30,
sup pp 1–13.
74. Wang T, Gilkes DM, Takano N, et al. Hypoxia-
inducible factors and RAB22A mediate formation
of microvesicles that stimulate breast cancer
invasion and metastasis. Proc Natl Acad Sci USA.
2014;111(31):E3234–E3242.
75. Kucharzewska P, Christianson HC, Welch JE, et al.
Exosomes reflect the hypoxic status of glioma cells
and mediate hypoxia-dependent activation of vascular
cells during tumor development. Proc Natl Acad Sci
USA. 2013;110(18):7312–7317.
76. Rankin EB, Giaccia AJ. The role of hypoxia-
inducible factors in tumorigenesis. Cell Death Differ.
2008;15(4):678–685.
77. Schindl M, Schoppmann SF, Samonigg H, et al.
Overexpression of hypoxia-inducible factor 1alpha
is associated with an unfavorable prognosis in
lymph node-positive breast cancer. Clin Cancer
Res. 2002;8(6):1831–1837.
78. Yamamoto Y, Ibusuki M, Okumura Y, et al.
Hypoxia-inducible factor 1alpha is closely linked
to an aggressive phenotype in breast cancer. Breast
Cancer Res Treat. 2008;110(3):465–475.
79. Rankin EB, Giaccia AJ. Hypoxic control of metas-
tasis. Science. 2016;352(6282):175–180.
80. Knowles HJ, Harris AL. Hypoxia and oxidative stress
in breast cancer. Hypoxia and tumourigenesis. Breast
Cancer Res. 2001;3(5):318–322.
81. Reynolds TY, Rockwell S, Glazer PM. Genetic
instability induced by the tumor microenvironment.
Cancer Res. 1996;56(24):5754–5757.
82. Kim CY, Tsai MH, Osmanian C, et al. Selection of
human cervical epithelial cells that possess reduced
apoptotic potential to low-oxygen conditions. Cancer
Res. 1997;57(19):4200–4204.
83. Jaakkola P, Mole DR, Tian YM, et al. Targeting of
HIF-alpha to the von Hippel-Lindau ubiquitylation
complex by O2-regulated prolyl hydroxylation.
Science. 2001;292(5516):468–472.
84. Ivan M, Kondo K, Yang H, et al. HIFalpha
targeted for VHL-mediated destruction by proline
hydroxylation: implications for O2 sensing. Science.
2001;292(5516):464–468.
85. Semenza GL. Defining the role of hypoxia-inducible
factor 1 in cancer biology and therapeutics. Oncogene.
2010;29(5):625–634.
86. Yang MH, Wu MZ, Chiou SH, et al. Direct regula-
tion of TWIST by HIF-1alpha promotes metastasis.
Nat Cell Biol. 2008;10(3):295–305.
87. Wong CC, Gilkes DM, Zhang H, et al. Hypoxia-
inducible factor 1 is a master regulator of breast
cancer metastatic niche formation. Proc Natl Acad
Sci USA. 2011;108(39):16369–16374.
88. Zhang H, Wong CC, Wei H, et al. HIF-1-dependent
expression of angiopoietin-like 4 and L1CAM
mediates vascular metastasis of hypoxic breast cancer
cells to the lungs. Oncogene. 2012;31(14):1757–
1770.
89. Liao D, Corle C, Seagroves TN, Johnson RS.
Hypoxia-inducible factor-1alpha is a key regulator of
metastasis in a transgenic model of cancer initiation
and progression. Cancer Res. 2007;67(2):563–572.
90. Krishnamachary B, Zagzag D, Nagasawa H, et al.
Hypoxia-inducible factor-1-dependent repres-
sion of E-cadherin in von Hippel-Lindau tumor
suppressor-null renal cell carcinoma mediated
by TCF3, ZFHX1A, and ZFHX1B. Cancer Res.
2006;66(5):2725–2731.
91. Imai T, Horiuchi A, Wang C, et al. Hypoxia attenu-
ates the expression of E-cadherin via up-regulation
of SNAIL in ovarian carcinoma cells. Am J Pathol.
2003;163(4):1437–1447.
92. Erler JT, Bennewith KL, Nicolau M, et al. Lysyl
oxidase is essential for hypoxia-induced metastasis.
Nature. 2006;440(7088):1222–1226.
93. De Bock K, Mazzone M, Carmeliet P. Antiangiogenic
therapy, hypoxia, and metastasis: risky liaisons, or
not? Nat Rev Clin Oncol. 2011;8(7):393–404.
94. Erler JT, Bennewith KL, Cox TR, et al. Hypoxia-
induced lysyl oxidase is a critical mediator of bone
marrow cell recruitment to form the premetastatic
niche. Cancer Cell. 2009;15(1):35–44.
95. Yang L, DeBusk LM, Fukuda K, et al. Expansion
of myeloid immune suppressor Gr+CD11b+ cells
in tumor-bearing host directly promotes tumor
angiogenesis. Cancer Cell. 2004;6(4):409–421.
96. Lyden D, Hattori K, Dias S, et al. Impaired recruit-
ment of bone-marrow-derived endothelial and hema-
topoietic precursor cells blocks tumor angiogenesis
and growth. Nat Med. 2001;7(11):1194–1201.
97. Gao D, Nolan DJ, Mellick AS, Bambino K,
McDonnell K, Mittal V. Endothelial progenitor
cells control the angiogenic switch in mouse lung
metastasis. Science. 2008;319(5860):195–198.
98. Kaplan RN, Riba RD, Zacharoulis S, et al.
VEGFR1-positive haematopoietic bone marrow
progenitors initiate the pre-metastatic niche. Nature.
2005;438(7069):820–827.
99. Cox TR, Rumney RM, Schoof EM, et al. The hypoxic
cancer secretome induces pre-metastatic bone lesions
through lysyl oxidase. Nature. 2015;522(7554):
106–110.
100. Staller P, Sulitkova J, Lisztwan J, Moch H, Oakeley
EJ, Krek W. Chemokine receptor CXCR4 down-
regulated by von Hippel-Lindau tumour suppressor
pVHL. Nature. 2003;425(6955):307–311.
101. Ceradini DJ, Kulkarni AR, Callaghan MJ, et al.
Progenitor cell trafficking is regulated by hypoxic
gradients through HIF-1 induction of SDF-1. Nat
Med. 2004;10(8):858–864.
102. Maxwell PH, Dachs GU, Gleadle JM, et al.
Hypoxia-inducible factor-1 modulates gene
expression in solid tumors and influences both
angiogenesis and tumor growth. Proc Natl Acad
Sci USA. 1997;94(15):8104–8109.
103. Paget S. The distribution of secondary growths in
cancer of the breast. 1889. Cancer Metastasis Rev.
1989;8(2):98–101.
104. Nieman KM, Kenny HA, Penicka CV, et al.
Adipocytes promote ovarian cancer metastasis and
provide energy for rapid tumor growth. Nat Med.
2011;17(11):1498–1503.
105. Kaplan RN, Psaila B, Lyden D. Bone marrow cells in
the ‘pre-metastatic niche’: within bone and beyond.
Cancer Metastasis Rev. 2006;25(4):521–529.
106. Hiratsuka S, Watanabe A, Aburatani H, Maru Y.
Tumour-mediated upregulation of chemoattractants
and recruitment of myeloid cells predetermines lung
metastasis. Nat Cell Biol. 2006;8(12):1369–1375.
107. Hiratsuka S, Nakamura K, Iwai S, et al. MMP9
induction by vascular endothelial growth factor
receptor-1 is involved in lung-specific metastasis.
Cancer Cell. 2002;2(4):289–300.
108. Chantrain CF, Shimada H, Jodele S, et al. Stromal
matrix metalloproteinase-9 regulates the vascular
architecture in neuroblastoma by promoting
pericyte recruitment. Cancer Res. 2004;64(5):1675–
1686.
109. Masson V, de la Ballina LR, Munaut C, et al. Con-
tribution of host MMP-2 and MMP-9 to promote
tumor vascularization and invasion of malignant
keratinocytes. FASEB J. 2005;19(2):234–236.
110. Minn AJ, Gupta GP, Siegel PM, et al. Genes that
mediate breast cancer metastasis to lung. Nature.
2005;436(7050):518–524.
111. Minn AJ, Kang Y, Serganova I, et al. Distinct
organ-specific metastatic potential of individual
breast cancer cells and primary tumors. J Clin Invest.
2005;115(1):44–55.
112. Mundy GR. Metastasis to bone: causes, consequences
and therapeutic opportunities. Nat Rev Cancer.
2002;2(8):584–593.
113. Logothetis CJ, Lin SH. Osteoblasts in prostate cancer
metastasis to bone. Nat Rev Cancer. 2005;5(1):21–28.
114. Harada S, Rodan GA. Control of osteoblast
function and regulation of bone mass. Nature.
2003;423(6937):349–355.
115. Kozlow W, Guise TA. Breast cancer metastasis to
bone: mechanisms of osteolysis and implications
for therapy. J Mammary Gland Biol Neoplasia.
2005;10(2):169–180.
116. Kang Y, He W, Tulley S, et al. Breast cancer
bone metastasis mediated by the Smad tumor
suppressor pathway. Proc Natl Acad Sci USA.
2005;102(39):13909–13914.
117. Yin JJ, Selander K, Chirgwin JM, et al. TGF-beta
signaling blockade inhibits PTHrP secretion by
breast cancer cells and bone metastases development.
J Clin Invest. 1999;103(2):197–206.
118. Kang Y, Siegel PM, Shu W, et al. A multigenic
program mediating breast cancer metastasis to bone.
Cancer Cell. 2003;3(6):537–549.
119. Morgan H, Tumber A, Hill PA. Breast cancer cells
induce osteoclast formation by stimulating host
IL-11 production and downregulating granulocyte/
macrophage colony-stimulating factor. Int J Cancer.
2004;109(5):653–660.
120. Boyle WJ, Simonet WS, Lacey DL. Osteo-
clast differentiation and activation. Nature.
2003;423(6937):337–342.
121. Abbott NJ, Ronnback L, Hansson E. Astrocyte-
endothelial interactions at the blood-brain barrier.
Nat Rev Neurosci. 2006;7(1):41–53.
122. Lassman AB, DeAngelis LM. Brain metastases. Neurol
Clin. 2003;21(1):1–23, vii.
123. Entschladen F, Drell TL, Lang K, Joseph J, Zaenker
KS. Neurotransmitters and chemokines regulate
tumor cell migration: potential for a new pharma-
cological approach to inhibit invasion and metastasis
development. Curr Pharm Des. 2005;11(3):403–411.
124. Xie TX, Huang FJ, Aldape KD, et al. Activation of
stat3 in human melanoma promotes brain metastasis.
Cancer Res. 2006;66(6):3188–3196.
125. Kim LS, Huang S, Lu W, Lev DC, Price JE. Vascular
endothelial growth factor expression promotes the
growth of breast cancer brain metastases in nude
mice. Clin Exp Metastasis. 2004;21(2):107–118.
126. Yano S, Shinohara H, Herbst RS, et al. Expression
of vascular endothelial growth factor is necessary but
not sufficient for production and growth of brain
metastasis. Cancer Res. 2000;60(17):4959–4967.

127. Omuro AM, Kris MG, Miller VA, et al. High
incidence of disease recurrence in the brain and
leptomeninges in patients with nonsmall cell lung
carcinoma after response to gefitinib. Cancer. 2005;
103(11):2344–2348.
128. Clayton AJ, Danson S, Jolly S, et al. Incidence of cere-
bral metastases in patients treated with trastuzumab
for metastatic breast cancer. Br J Cancer. 2004;91(4):
639–643.
129. Bendell JC, Domchek SM, Burstein HJ, et al. Central
nervous system metastases in women who receive
trastuzumab-based therapy for metastatic breast
carcinoma. Cancer. 2003;97(12):2972–2977.
130. Steeg PS. Tumor metastasis: mechanistic insights and
clinical challenges. Nat Med. 2006;12(8):895–904.
131. Yu Q, Stamenkovic I. Transforming growth factor-
beta facilitates breast carcinoma metastasis by
promoting tumor cell survival. Clin Exp Metastasis.
2004;21(3):235–242.
132. Siegel PM, Shu W, Cardiff RD, Muller WJ,
Massague J. Transforming growth factor beta signal-
ing impairs Neu-induced mammary tumorigenesis
while promoting pulmonary metastasis. Proc Natl
Acad Sci USA. 2003;100(14):8430–8435.
133. Luo JL, Maeda S, Hsu LC, Yagita H, Karin M.
Inhibition of NF-kappaB in cancer cells converts
inflammation-induced tumor growth mediated by
TNFalpha to TRAIL-mediated tumor regression.
Cancer Cell. 2004;6(3):297–305.
134. Ye QH, Qin LX, Forgues M, et al. Predicting hepatitis
B virus-positive metastatic hepatocellular carcinomas
using gene expression profiling and supervised
machine learning. Nat Med. 2003;9(4):416–423.
135. Khanna C, Wan X, Bose S, et al. The membrane-
cytoskeleton linker ezrin is necessary for osteosarcoma
metastasis. Nat Med. 2004;10(2):182–186.
136. Sarrio D, Rodriguez-Pinilla SM, Dotor A, Calero F,
Hardisson D, Palacios J. Abnormal ezrin localiza-
tion is associated with clinicopathological features
in invasive breast carcinomas. Breast Cancer Res Treat.
2006;98(1):71–79.
137. Ladeda V, Adam AP, Puricelli L, Bal de Kier Joffe E.
Apoptotic cell death in mammary adenocarcinoma
Cellular Microenvironment and Metastases  •  CHAPTER 3 55.e3
cells is prevented by soluble factors present in the
target organ of metastasis. Breast Cancer Res Treat.
2001;69(1):39–51.
138. Martin SS, Ridgeway AG, Pinkas J, et al. A cyto-
skeleton-based functional genetic screen identifies
Bcl-xL as an enhancer of metastasis, but not primary
tumor growth. Oncogene. 2004;23(26):4641–4645.
139. Pinkas J, Martin SS, Leder P. Bcl-2-mediated
cell survival promotes metastasis of EpH4 beta­
MEKDD mammary epithelial cells. Mol Cancer
Res. 2004;2(10):551–556.
140. Inbal B, Cohen O, Polak-Charcon S, et al. DAP
kinase links the control of apoptosis to metastasis.
Nature. 1997;390(6656):180–184.
141. Wong CW, Lee A, Shientag L, et al. Apoptosis: an
early event in metastatic inefficiency. Cancer Res.
2001;61(1):333–338.
142. O’Connell JT, Sugimoto H, Cooke VG, et al. VEGF-A
and tenascin-C produced by S100A4+ stromal cells
are important for metastatic colonization. Proc Natl
Acad Sci USA. 2011;108(38):16002–16007.
143. Kii I, Nishiyama T, Li M, et al. Incorporation of
tenascin-C into the extracellular matrix by periostin
underlies an extracellular meshwork architecture. J
Biol Chem. 2010;285(3):2028–2039.
144. Chambers AF, Groom AC, MacDonald IC. Dis-
semination and growth of cancer cells in metastatic
sites. Nat Rev Cancer. 2002;2(8):563–572.
145. Lehuede C, Dupuy F, Rabinovitch R, Jones RG,
Siegel PM. Metabolic plasticity as a determinant
of tumor growth and metastasis. Cancer Res.
2016;76(18):5201–5208.
146. Jungermann K. Metabolic zonation of liver paren-
chyma. Semin Liver Dis. 1988;8(4):329–341.
147. Loo JM, Scherl A, Nguyen A, et al. Extracellular
metabolic energetics can promote cancer progression.
Cell. 2015;160(3):393–406.
148. Wyss M, Kaddurah-Daouk R. Creatine and creatinine
metabolism. Physiol Rev. 2000;80(3):1107–1213.
149. Dupuy F, Tabaries S, Andrzejewski S, et al. PDK1-
dependent metabolic reprogramming dictates
metastatic potential in breast cancer. Cell Metab.
2015;22(4):577–589.
55.e3
150. Papandreou I, Cairns RA, Fontana L, Lim AL,
Denko NC. HIF-1 mediates adaptation to hypoxia
by actively downregulating mitochondrial oxygen
consumption. Cell Metab. 2006;3(3):187–197.
151. Valastyan S, Weinberg RA. Tumor metastasis:
molecular insights and evolving paradigms. Cell.
2011;147(2):275–292.
152. Rankin EB, Fuh KC, Taylor TE, et al. AXL is an
essential factor and therapeutic target for metastatic
ovarian cancer. Cancer Res. 2010;70(19):7570–7579.
153. Coussens LM, Fingleton B, Matrisian LM. Matrix
metalloproteinase inhibitors and cancer: trials
and tribulations. Science. 2002;295(5564):2387–
2392.
154. Harris AL. Hypoxia—a key regulatory factor in
tumour growth. Nat Rev Cancer. 2002;2(1):38–47.
155. Wong CC, Zhang H, Gilkes DM, et al. Inhibitors
of hypoxia-inducible factor 1 block breast cancer
metastatic niche formation and lung metastasis.
J Mol Med. 2012;90(7):803–815.
156. Higgins DF, Biju MP, Akai Y, Wutz A, Johnson RS,
Haase VH. Hypoxic induction of Ctgf is directly
mediated by Hif-1. Am J Physiol Renal Physiol.
2004;287(6):F1223–F1232.
157. Chu CY, Chang CC, Prakash E, Kuo ML. Con-
nective tissue growth factor (CTGF) and cancer
progression. J Biomed Sci. 2008;15(6):675–685.
158. Dornhofer N, Spong S, Bennewith K, et al. Con-
nective tissue growth factor-specific monoclonal
antibody therapy inhibits pancreatic tumor growth
and metastasis. Cancer Res. 2006;66(11):5816–
5827.
159. Aikawa T, Gunn J, Spong SM, Klaus SJ, Korc
M. Connective tissue growth factor-specific
antibody attenuates tumor growth, metastasis,
and angiogenesis in an orthotopic mouse model of
pancreatic cancer. Mol Cancer Ther. 2006;5(5):1108–
1116.
160. Sleeman J, Schmid A, Thiele W. Tumor lymphatics.
Semin Cancer Biol. 2009;19(5):285–297.
161. Jain RK. Antiangiogenesis strategies revisited: from
starving tumors to alleviating hypoxia. Cancer Cell.
2014;26(5):605–622.
 4  Control of the Cell Cycle
Marcos Malumbres

S UMMARY OF K EY
• Most cells in postnatal tissues are
quiescent. Exceptions include
abundant cells of the hematopoietic
system, skin, and gastrointestinal
mucosa, as well as other minor
progenitor populations in other
tissues.
• Many quiescent cells can reenter
into the cell cycle with the
appropriate stimuli, and the control
of this process is essential for tissue
homeostasis.
• The key challenges for proliferating
cells are to make an accurate copy
of the 3 billion bases of DNA
(S phase) and to segregate the
duplicated chromosomes equally
into daughter cells (mitosis).
• Progression through the cell cycle is
dependent on both extrinsic and
P OI N T S
intrinsic factors, such as growth
factor or cytokine exposure, cell-to-
cell contact, and metabolic
constraints.
• The internal cell cycle machinery is
controlled largely by oscillating levels
of cyclin proteins and by modulation
of cyclin-dependent kinase (Cdk)
activity. One way in which growth
factors regulate cell cycle
progression is by affecting the levels
of the D-type cyclins, Cdk activity,
and the function of the
retinoblastoma protein.
• Cell cycle checkpoints are
surveillance mechanisms that link the
rate of cell cycle transitions to the
timely and accurate completion of
prior dependent events; p53 is a
checkpoint protein that induces cell
cycle arrest, senescence, or death in
response to cellular stress.
• Checkpoints minimize replication
and segregation of damaged DNA,
or the abnormal segregation of
chromosomes to daughter cells, thus
protecting cells against genome
instability.
• Disruption of cell cycle controls is a
hallmark of all malignant cells.
Frequent tumor-associated
alterations include aberrations in
growth factor signaling pathways,
dysregulation of the core cell cycle
machinery, and/or disruption of cell
cycle checkpoint controls.
• Because cell cycle control is
disrupted in virtually all tumor types,
the cell cycle machinery provides
multiple therapeutic opportunities.

Most cells in the adult body are quiescent—that is, they are biochemi-
cally and functionally active but do not divide to generate daughter
cells. However, specific populations retain the ability to proliferate
throughout the adult life span, which is essential for proper tissue
homeostasis. For example, cells of the hematopoietic compartment
and the gut have a high rate of turnover, and active proliferation is
essential for the maintenance of these tissues. On average, about 2
trillion cell divisions occur in an adult human every 24 hours (about
25 million per second). The decision about whether to proliferate is
tightly regulated.1,2 It is influenced by a variety of exogenous signals,
including nutrients and growth factors, as well as inhibitory factors,
and the interaction of the cell with its neighbors and with the underlying
extracellular matrix. Each of these factors stimulates intracellular signal-
ing pathways that can either promote or suppress proliferation. The
cell integrates all of these signals, and if the balance is favorable, the
cell will initiate a series of processes, collectively known as the cell
cycle, that lead to cell division into two daughter cells.
During the past four decades, extensive effort has been placed on
unraveling the basic molecular events that control the cell division
cycle. Studies in a variety of organisms have identified evolutionarily
conserved machinery that regulates eukaryotic cell cycle transitions
through the action of key enzymes, including cyclin-dependent kinases
(Cdks) and other kinases.3 It is essential that proliferating cells copy
their genomes and segregate them to the daughter cells with high
fidelity. Eukaryotic cells therefore have evolved a series of surveillance
pathways, termed cell cycle checkpoints, that monitor for potential
problems during the cell cycle process.4 Human cells are continuously
exposed to external agents (e.g., reactive chemicals and ultraviolet
light) and to internal agents (e.g., byproducts of normal intracellular
metabolism, such as reactive oxygen intermediates) that can induce
DNA damage. A major goal of specific cell cycle checkpoints is to
detect DNA damage and activate cell cycle arrest and DNA repair
mechanisms, thereby maintaining genomic integrity.
Anything that disrupts proper cell cycle progression can lead to
either the reduction or the expansion of a particular cell population.
It is now clear that such changes are a hallmark of tumor cells, which
carry mutations that impair signaling pathways that suppress prolifera-
tion and/or activate pathways that promote proliferation. In addition,
most (if not all) human tumor cells have mutations within key
components of both the cell cycle machinery and checkpoint path-
ways.1,5,6 This characteristic has important clinical implications, because
the presence of these defects can modulate cellular sensitivity to
chemotherapeutic regimens that induce DNA damage or mitotic
catastrophe. This chapter focuses on the mechanics of the cell cycle
and checkpoint signaling pathways and discusses how this knowledge
can lead to the efficient use of current anticancer therapies and to the
development of novel agents.
CELL DIVISION CYCLE
Overview of the Cell Cycle Machinery
Cell division proceeds through a well-defined series of stages
(Fig. 4.1). First, the cell moves from the nonproliferative, quiescent

56
 Control of the Cell Cycle  •  CHAPTER 4 57

G1/S Intra-S G2/M SAC

Microtubule nucleation
DNA replication & repair Cytokinesis
spindle

Cell cycle entry Centrosome duplication Chromosome Chromosome

G1 and separation condensation segregation

G0 G1 S G2 M M
Cyclin D
Cyclin E Cyclin A Cyclin B

Restriction point
Figure 4.1  •  The cell division cycle. One round of cell division requires high-fidelity duplication of deoxyribonucleic acid during the S phase of the cell
cycle and proper segregation of duplicated chromosomes during mitosis, or the M phase. Before and after the S phase and M phase, the cell transits through
“gap” phases, termed G1 and G2. Quiescent (G0) cells require the appropriate mitogenic stimuli for reentry into the cell cycle by inducing D-type cyclins.
These stimuli are required up to a specific moment, known as the restriction point, at which time the cell cycle becomes independent of the mitogenic signals.
The cellular processes required for transition through the different cell cycle phases are controlled by the action of regulatory pathways and mostly are driven
by the expression of specific cyclins and the activation of cyclin-dependent kinases. These transitions are monitored by signaling pathways known as the cell
cycle checkpoints (represented by blue columns) that function to arrest the cell cycle at different phases (G1/S, intra–S phase, G2/M) in the presence of DNA
damage. In addition, the spindle assembly checkpoint (SAC) delays the exit from mitosis in the presence of defective chromosome alignment by inhibiting
the degradation of cyclin B1 and the subsequent inactivation of cyclin-dependent kinases.

(also known as G0) state into the first gap phase, or G1, in which
the cell essentially is readying itself for the cell division process. This
process involves a dramatic upregulation of both transcriptional and
translational programs, not only to yield the proteins required to
regulate cell division but also to essentially double the complement
of macromolecules so that one cell can give rise to two cells without a
loss of cell size. Not surprisingly, this process takes a significant amount
of time (from 8 to 30 hours in cultured cells) and energy.7 Studies
with cultured cells show that mitogenic growth factors are essential for
continued passage through the G1 phase. Specifically, if growth factors
are withdrawn at any point during this phase, the cell will not divide.
However, as it nears the end of the G1 phase, the cell passes through a
key transition point, called the restriction point, whereupon it becomes
growth factor independent and is fully committed to undergoing cell
division.1 The cell then enters the DNA synthesis phase, or S phase,
in which each of the chromosomes is replicated once and only once.
This is followed by a second gap phase, called G2, which lasts 3 to
5 hours, and the cell then initiates mitosis, or the M phase, a rapid
phase (lasting about 1 hour) in which the chromosomes are segregated.
On completion of mitosis, the daughter cells can enter quiescence
or initiate a second round of cell division, depending on the milieu.
Progression throughout the different phases of the cell cycle depends
on the activity of key molecules that drive transcription, translation,
or the structural changes required for cell division (Table 4.1). A large
number of these changes are modulated by protein phosphorylation
and dephosphorylation, but other molecular processes such as
SUMOylation, acetylation, or ubiquitin-dependent protein degradation
are crucial for ordered cell cycle progression. Many of these cell cycle
regulators have been involved in tumor development or may be attractive
targets for cancer therapy and will be introduced in the following
sections.
Cyclin-Dependent Kinases and Their Regulators
The Cdks constitute a large subfamily of highly conserved Ser/Thr
kinases that are defined by their dependence on a regulatory subunit,
called a cyclin.8–11 The first identified human Cdk, called Cdk1
(originally cdc2), was cloned by virtue of its ability to complement a
mutant cdc2 yeast strain.12 Subsequent studies identified additional
human Cdks and determined that they regulate distinct cell cycle
stages; for example, Cdk4 and Cdk6 regulate cell cycle entry, whereas
Cdk2 may have specific roles during the G1-to-S transition and S
phase. Cdk1 is essential in the control of G2 and mitosis and also
may play additional roles in earlier stages. The human genome encodes
about 15 additional Cdks, although the functional relevance of many
of them is still unknown.3,8–11
The activity of these kinases is controlled by multiple regulatory
mechanisms. Cdks act in association with a cyclin subunit that binds
to the conserved PSTAIRE helix within the kinase.13 Cyclin binding
causes a reorientation of residues within the active sites that is essential
for kinase activity.8,13 The associated cyclin also determines the substrate
specificity of the resulting cyclin/Cdk complex. The cyclins are quite
divergent, especially in their N-terminal sequences, but they all share
a highly conserved 100–amino acid sequence, called the cyclin box,
that mediates Cdk binding and activation.9 As their name implies,
cyclins originally were identified as proteins whose expression was
restricted to a particular stage of the cell cycle14 because of cell
cycle–dependent regulation of both cyclin gene transcription and
protein degradation. The human genome encodes more than 25
cyclin-like proteins, yet only four distinct subclasses—D-, E-, A-, and
B-type cyclins—are thought to play key roles in cell cycle regulation
(see Fig. 4.1).9 Each of these classes has a few paralogs (e.g., cyclin
D1, D2, and D3; cyclin E1 and E2; cyclin A1 and A2; and cyclin
B1, B2, and B3). The relative roles of these paralogs are not completely
clear in most cases. Although some functional redundancy may exist,
published evidence suggests differences in regulation, expression pattern,
and substrate specificity.9,15
The activation of cyclin/Cdk complexes requires considerable
posttranslational regulation.8,9,16 First, kinase activation is dependent
on phosphorylation of a threonine residue that is adjacent to the
active site (Thr160 in Cdk2). This phosphorylation is catalyzed by a
kinase, called Cdk-activating kinase (CAK).17,18 In mammalian cells,
phosphorylation occurs after cyclin binding. Although it appears that
58 Part I: Science and Clinical Oncology

Table 4.1  Representative Molecules Involved in Cell Cycle Regulation

Protein Family/
Complex Representative Members Function
KINASES
Cyclin-dependent Heterodimeric complexes formed of a cyclin (A, B, Phosphorylation of multiple proteins to drive progression
kinases D, and E types) and a Cdk (Cdk1, Cdk2, Cdk4, Cdk6); throughout the different phases of the cell cycle
Cdk7 functions as a Cdk-activating kinase
Wee1/Myt1 Wee1, Myt1 Inactivation of Cdks
Aurora-A holoenzyme Aurora-A and its nonkinase activator, Tpx2 Spindle dynamics, chromosome segregation, and cytokinesis
Chromosome passenger Aurora-B (Aurora-C?), Incenp, survivin, borealin Chromosome segregation
complex (CPC)
Polo-like kinases Plk1–Plk5 Centrosome function, chromosome segregation, and cytokinesis
NIMA-related kinases Nek1–Nek11 Centrosome function and mitosis
Haspin Haspin Phosphorylates histone H3 to recruit the CPC
Mastl Mastl Inhibition of PP2A phosphatases
CDK INHIBITORS
INK4 proteins p16INK4a, p15INK4b, p18INK4c, p19INK4d Inhibition of Cdk4 and Cdk6 during G1 progression
Cip/Kip inhibitors p21Cip1, p27Kip1, p57Kip2 Cdk inhibition and other roles in transcription or the cytoskeleton
TRANSCRIPTIONAL CONTROL
Retinoblastoma family pRB, p107, p130 Repression of the transcription of genes required for the cell cycle
E2F transcription factors E2F1–E2F8 Transcription of genes encoding S-phase and mitotic regulators
PHOSPHATASES
Cdc14 Cdc14a, Cdc14b Control of transcription and cell cycle progression
Cdc25 Cdc25a, Cdc25b, and Cdc25c Cdk activation and cell cycle progression
PP1 Multiple complexes with different regulatory Protein dephosphorylation
subunits
PP2A Multiple complexes with different regulatory Protein dephosphorylation; major Cdk-counteracting phosphatase
subunits
UBIQUITIN LIGASES
SCF E3 ubiquitin ligase formed of Rbx1, Cul1, Skp1, and Targets multiple cell cycle regulators (e.g., p27Kip1 or cyclin E) for
an F-box protein (e.g., Skp2 or βTrCP) ubiquitin-dependent degradation during interphase
APC/C E3 ubiquitin ligase composed for multiple subunits Targets multiple cell cycle regulators for ubiquitin-dependent
including Cdc20 or Cdh1 as coactivator molecules degradation during mitosis (cyclin B, securin) and G1 (e.g.,
Aurora-A, Plk1, or Tpx2)
OTHER FUNCTIONS
Kinesins More than 600 proteins including Eg5, CenpE, Microtubule-based motor proteins that hydrolyze ATP to generate
MCAK energy for movement along microtubule fibers
Cohesins and condensins Smc family (1–4), Rad21, Pds5, and SA (Stag) Structure and regulation of DNA
proteins, among others
Kinetochore proteins More than 100 proteins including the CENP Linking the chromosomes to microtubules and regulation of
(centromere-binding proteins) family, and the Knl1, microtubule dynamics
Mis12, Ndc80, and Dam1 complexes, among many
others
Mitotic checkpoint Mad2, Bub3, BubR1, and Cdc20 Inhibit the APC/C until complete bipolar attachment of
complex (MCC) chromosomes to the mitotic spindle

APC/C, Anaphase-promoting complex/cyclosome; ATP, adenosine triphosphate; Cdk, cyclin-dependent kinase; CPC, chromosomal passenger complex; NIMA, never in mitosis,
gene A; SCF, Skp1–Cullin1–F-box.

at least two mammalian CAKs exist, the major CAK is a trimolecular
complex composed of Cdk7, cyclin H, and Mat1. The Cdk7/cyclinH/
Mat1 complex also is required for the control of basal transcription
via regulation of RNA polymerase II function.11,18 Second, when it
is first formed, the cyclin/Cdk complex frequently is subject to inhibi-
tory phosphorylation of Thr14 and Tyr15 residues within the Cdk’s
active site by the Wee1 (Tyr15) and Myt1 (Thr14 and Tyr15) kinases.9,19
Activation of the cyclin/Cdk complex is then dependent on the action
of a dual-specificity phosphatase called Cdc25. Mammalian cells have
three different Cdc25 proteins, called Cdc25a, Cdc25b, and Cdc25c,
which show some specificity for different cyclin/Cdk complexes and
cell cycle stages.20
Cdks are modulated by a series of Cdk inhibitors (CKIs) that play
a key role in restricting the activity of the cyclin/Cdk complexes in
 Control of the Cell Cycle  •  CHAPTER 4 59

response to either external signals or internal stresses.1,21 The CKIs
can be divided into two distinct families based on their biological
properties. The first CKI family is named INK4, based on their roles
as INhibitors of Cdk4. The INK4 family has four members called
p16INK4a, p15INK4b, p18INK4c, and p19INK4d (encoded by the CDKN2A-D
genes in humans). These INK4 proteins specifically prevent the binding
of cyclins to monomeric Cdk4 and Cdk6 but do not inhibit other
Cdks.13,21 The second CKI family is named Cip/Kip and includes
three members: p21Cip1 (also called p21Waf1), p27Kip1, and p57Kip2
(encoded by the CDKN1A-C genes in humans).21 These Cip/Kip
proteins have two major activities. First, they do not bind to monomeric
Cdks but associate with and inhibit the activity of cyclin/Cdk complexes
already formed. Second, Cip/Kip proteins may promote the assembly
of cyclin D/Cdk4/6 complexes without dramatically perturbing its
kinase activity.21,22 This activity is modulated by phosphorylation of
Cip/Kip proteins by Src, Jak2, and Akt kinases,23–26 directly linking
Cdk regulation with the activity of these upstream mitogenic pathways.
In addition to regulating the cell cycle, Cip/Kip proteins play important
roles in apoptosis, transcriptional regulation, cell fate determination,
cell migration, and cytoskeletal dynamics.27,28
Retinoblastoma Proteins and E2F Transcription Factors
The retinoblastoma protein (pRB) was originally identified by virtue
of its association with hereditary retinoblastoma.29 It behaves as a
classic tumor suppressor: affected persons inherit a germline mutation
within one allele of the pRB-encoding gene, RB1, and loss of hetero-
zygosity is seen in all of the tumors. Subsequent studies showed that
the transforming ability of small DNA tumor viruses, including human
papillomavirus, adenovirus, and simian virus, was dependent on the
ability of virally encoded oncoproteins (E7, E1A, and SV40, respec-
tively) to bind and inhibit pRB.30–32 Moreover, the RB1 gene is
inactivated in approximately one-third of all sporadic human tumors.
pRB and the pRB-related proteins p107 and p130, collectively
known as the pocket proteins, are transcriptional repressors whose
major function is to inhibit the expression of cell cycle–related proteins
(see Table 4.1).33 This suppressive activity is largely dependent on the
ability to prevent cell cycle entry through inhibition of the E2F
transcription factors.33,34 The E2F proteins regulate the cell cycle–
dependent transcription of numerous targets, including core components
of the cell cycle control (e.g., cyclin E and cyclin A) and DNA replica-
tion (e.g., Cdc6, Cdt1, and the Mcm proteins) machineries.33–35 pRB
regulates E2F through two distinct mechanisms. First, its association
with E2F is sufficient to block the transcriptional activity of E2F.
Second, the pRB/E2F complex can recruit histone deacetylases to the
promoters of E2F-responsive genes and thereby actively repress their
transcription. Cell cycle entry requires the phosphorylation of pRB
by cyclin/Cdk complexes and the consequent dissociation of pRB
from E2F.1,8,33
Studies have identified eight E2F genes that encode nine different
E2F proteins.34,35 Pocket proteins can regulate a subset of these factors:
E2F1, E2F2, E2F3a, E2F3b, E2F4, and E2F5. These E2F proteins
associate with a dimerization partner, called DP, and the resulting
complexes function primarily as either activators (E2F1, E2F2, and
E2F3a) or repressors (E2F4 and E2F5) of transcription under the
direction of the pocket proteins. Observations have suggested that
several of these factors may act either as positive or negative regulators
of transcription, depending on the cell type or the differentiation
state.34,36–38 Most classic E2F target genes are regulated by the coor-
dinated action of these repressor and activator E2Fs.
Ubiquitin-Dependent Protein Degradation
The original observation that cyclin levels are tightly regulated during
the cell cycle implies that these proteins are regulated not only at the
transcriptional level but also at the protein level. It is now evident
that ubiquitin-mediated protein degradation is a major regulatory
mechanism to ensure ordered transition through the different phases
of the cell division cycle. Ubiquitylation depends on an enzymatic
cascade, in which ubiquitin ligases recruit specific substrates for
modification. About 600 ubiquitin ligases are encoded by the human
genome. Among them, the Skp1–Cullin1–F-box (SCF) and the
anaphase-promoting complex/cyclosome (APC/C) are known for
driving the degradation of cell cycle regulators to accomplish irreversible
cell cycle transitions.39–41 SCF has three core components: a RING
finger protein, called Rbx1, which recruits the E2-ubiquitin conjugate;
a cullin (Cul1); and Skp1 (Fig. 4.2). Skp1 acts to recruit a family of
proteins, called F-box proteins, which determine the target specificity
of the SCF complex. Once SCF binds its substrate, it transfers a
ubiquitin molecule to lysine residues within the target protein to
create a polyubiquitin chain, which targets the substrate to the protea-
some for degradation.42
The APC/C is a much larger complex, but it also contains a RING
finger protein, called Apc11, to recruit the E2-ubiquitin conjugate,

Apc11 E2
E2
Rbx1
Ubiquitin
Ubiquitin
Substrate
Substrate Apc2
P
Cul1 P
Activator
(Cdc20
F-box or Cdh1)
protein

Skp2

SCF ubiquitin ligase APC/C ubiquitin ligase

Figure 4.2  •  Ubiquitin ligases. The Skp1–Cullin1–F-box (SCF) and anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligases play a key role in
enabling forward passage through key cell cycle transitions. These ligases are both large complexes that include three core components: a scaffolding protein
called a cullin in SCF, a protein that recruits the E2 and its associated ubiquitin molecule, and a specificity factor (called the F-box protein in SCF and the
activator in APC/C) that recruits the substrate. SCF and APC/C catalyze polyubiquitination of their substrates, which acts as a signal for substrate degradation
by the 26S proteasome. SCF has numerous substrates whose degradation promotes passage through the early stages of the cell cycle, including the cyclin-
dependent kinase inhibitor p27Kip1, cyclin E, E2F-1, and Cdt1. APC/C is essential for mitotic progression (by promoting degradation of securin and the
mitotic cyclins) and for cell cycle exit (by promoting degradation of multiple cell cycle regulators).
60 Part I: Science and Clinical Oncology
and a core cullin subunit (Apc2). In addition, APC/C is activated by
a cofactor that, in a manner comparable with that of the F-box proteins
of SCF, establishes substrate specificity (see Fig. 4.2).39 Cdc20 is the
mitotic cofactor of APC/C, and it targets several cell cycle regulators
(A- and B-type cyclins, Nek2, and securin) during mitotic entry and
the metaphase-to-anaphase transition. Cdh1 (also known as FZR1 in
mammals) replaces Cdc20 during mitotic exit and is the cofactor
responsible for the elimination of many cell cycle regulators in the
G0 or G1 phase to prevent unscheduled DNA replication.43 Recent
studies have provided new insights into the intricate relationship
between ubiquitylation and the cell division apparatus, including new
roles for atypical ubiquitin chains, new mechanisms of regulation,
and extensive cross talk between ubiquitylation enzymes.44–49
Mitotic Spindle and Mitotic Kinases and Kinesins
In addition to the central role of Cdks in the cell cycle, many other
kinases play critical roles in cell cycle progression.3 Aurora and Polo
kinases were first identified in genetic studies in flies as a result of
their essential role in mitotic progression.50–52 Each of these families
of kinases is represented by a single member in yeast, whereas three
Aurora kinases (Aurora-A, Aurora-B, and Aurora-C) and five Polo-like
kinases (Plk1 through Plk5) exist in humans (see Table 4.1).52,53
Aurora-A participates in several processes required for building a bipolar
spindle, including centrosome separation and microtubule dynamics.
Aurora-A is activated by Tpx2, and the Aurora-A–Tpx2 holoenzyme
may have critical implications in tumor development.51,52,54 Aurora-B,
on the other hand, is part of the chromosome passenger complex
(CPC) that localizes to the kinetochores from prophase to metaphase
and to the central spindle and midbody in cytokinesis. Other com-
ponents of the CPC include INCENP, survivin, and borealin, and
this complex regulates proper microtubule-kinetochore attachment
and promotes biorientation during mitosis.51 Aurora-C may play similar
roles to Aurora-B, although it is mostly expressed in germ cells and
may play a specific role in meiosis and during the first embryonic
cycles.55–57 Plk1 functions as a major regulator of centrosome matura-
tion, mitotic entry, and cytokinesis, whereas Plk4 is a critical regulator
of centriole duplication.50,58–60 The other members of the family, Plk2,
Plk3, and Plk5, are mostly involved in stress responses during interphase
or in neuron biology.3,53
A different group of additional cell cycle kinases with potential
interest in tumor biology is the never in mitosis, gene A (NIMA)–related
kinase family (Nek1 through Nek11). Four of these proteins, Nek2,
Nek6, Nek7, and Nek9, are involved in cell cycle progression, whereas
all other family members are likely to play critical roles in cilia and
centrioles.3,61–63 Nek2, the closest relative to the Aspergillus NIMA
kinase, localizes to the centrosome and plays a role in establishing the
bipolar spindle by initiating the separation of centrosomes and
contributing to microtubule organization at the G2/M transition by
phosphorylating several centrosomal substrates. Nek2 also may play
additional roles in chromosome condensation and the mitotic check-
point. Nek9, Nek6, and Nek7 function in a kinase cascade that
participates in centrosome separation and the formation and/or
maintenance of the mitotic spindle.61,63
A structurally different kinase, Haspin, is involved in the phos-
phorylation of histone H3 on threonine 3 (H3T3).64,65 This kinase
is activated by Cdk1 and Plk1,66 and phosphorylation of H3T3 is
required for proper Aurora-B localization and activity and coupling
of mitotic chromosome structure with transcription.64,65,67
The activity of all these kinases is functionally linked to many
other proteins associated with chromosomes, microtubules, or different
organelles, whose activity is essential for the structural changes associated
with cell cycle progression and chromosome segregation.68 Among
them, microtubule-associated proteins such as kinesin motor proteins
determine the dynamic behavior of the mitotic spindle required for
chromosome movement during mitosis.69,70 During mitosis, micro-
tubules associate with chromosomes through a large protein assembly
known as the kinetochore.71 The position of kinetochores in the
centromeric region of chromosomes is determined by a complex
epigenetic, DNA sequence–independent mechanism.72–74 Kinetochores
regulate the proper bipolar attachment between microtubules and
chromosomes in order to distribute the replicated genome from a
mother cell to its daughters.71,75 Given the relevance of the kinetochore
in genome integrity, the composition of the kinetochore and the
identification of various physical and functional modules within its
substructure is under deep investigation.76,77
Cell Cycle Phosphatases
Cdc14 phosphatases are the major Cdk-counteracting proteins in
yeast. Although two family members exist in mammals, their relevance
in the cell cycle is not well understood.78,79 In eukaryotes, two major
complexes, PP1 and PP2A, account for more than 90% of protein
phosphatase activity. In fact, these enzymes correspond to hundreds
of phosphatase complexes assembled from a few catalytic subunits
(PP1α, PP1β/δ, and PP1γ1/2 for PP1, and the Cα and Cβ isoforms
for PP2A) and a diverse array of regulatory subunits.80 Recent evidence
suggests that these protein families cooperate in the dephosphorylation
of most cell cycle kinase targets, including the retinoblastoma family
or mitotic phosphoproteins.78,81–83 PP1 and PP2A are major phospha-
tases responsible for pRB dephosphorylation during mitotic exit,
although the relative roles of these complexes or the particular
holoenzymes involved are not clear.81,82 Similarly, both PP1 and PP2A
are required for dephosphorylation of hundreds of mitotic proteins
that are phosphorylated by Cdk1, as well as the other mitotic kinases.83,84
Thus it has been suggested that the cell cycle ultimately is regulated
by the dynamic equilibrium between Cdks (and partially by the other
mitotic kinases) and PP1/PP2A activity. In the absence of Cdk activity,
the balance tilts in favor of the phosphatases. When Cdks are activated,
phosphatase activity is overtaken.
Cdk1 is able to directly inhibit PP1 by direct phosphorylation of
the catalytic subunit. The Cdk-dependent inhibition of PP2A, on the
other hand, is not direct; rather, it is mediated by a new kinase known
as Greatwall in flies and Xenopus or Mastl in mammals.85–87 Cdk1
phosphorylates and activates Mastl, which in turn phosphorylates
Arpp-19 and Ensa, two highly related proteins that function as inhibitors
of a particular PP2A holoenzyme encompassing a regulatory subunit
of the B55 family.85–89 Reactivation of PP1 and PP2A phosphatases
is a mandatory step for the exit from mitosis and the transition to
interphase.90–92
Entry Into the Cell Cycle
Because most adult cells are quiescent, the mechanisms that determine
their quiescent state and their reentry into the cell cycle with the
appropriate stimuli are key determinants of tissue homeostasis. In
quiescent cells, several DP/E2F complexes associate with the promoters
of E2F-responsive genes and recruit pRB-family members, along with
their associated histone deacetylases, to actively repress their transcrip-
tion.93,94 This repression machinery therefore prevents the expression
of proteins required for DNA synthesis and chromosome segregation.
In addition, CKIs normally are expressed in quiescent cells, preventing
the activation of Cdks.21 D-type cyclins are present at very low levels
in most quiescent cells, in large part because they are phosphorylated
by an abundant kinase called Gsk3β and then exported to the cytoplasm
for degradation.95
The transcription of D-type cyclins is induced in response to a
wide variety of mitogenic stimuli.96,97 In addition, Gsk3β is inhibited
by mitogens, thus preventing the degradation of these cyclins. D-type
cyclins then associate with Cdk4 and Cdk6, and the resulting complexes
phosphorylate pRB proteins, partially inactivating their transcriptional
suppressor function (Fig. 4.3).98–101 pRB inactivation causes the release
of its associated DP-E2Fs. Repressive E2Fs such as E2F4 and E2F5
dissociate from the DNA, and the free E2F complexes—DP-E2F1,
DP-E2F2, and DP-E2F3—now occupy the promoters and activate
their transcription. DP-E2F targets include many genes encoding
 Control of the Cell Cycle  •  CHAPTER 4 61

Inhibitory Mitogens
growth factors

P P
Cip/Kip p107
INK4 p130 P
P P P
pRb pRb
P
E2F P
1,2,3 E2F
Cdk4/6 Cdk2 4,5
CycD CycE
p107 HDAC
p130 DNA synthesis
E2F E2F and mitotic
4,5 1,2,3
regulators
G0 /G1 G1/S
Transcriptional Transcriptional
repression activation

Figure 4.3  •  Entry into the cell cycle. The pocket proteins—pRB, p107, and p130—regulate a subset of the E2F family of proteins among many other
transcription factors. The pocket proteins bind to these E2Fs during the G0/early G1 phases, block their transcriptional activity, and recruit histone deacetylase
(HDAC), which comprises repressive complexes. Mitogenic signaling leads to the induction of D-type cyclins and the activation of interphase cyclin-dependent
kinase (Cdk) complexes, which phosphorylate the pocket proteins and release their associated E2Fs. This process allows the activating E2Fs to induce the
transcription of genes required for DNA synthesis and mitosis.

essential proteins required for DNA synthesis as well as mitosis.33–35

Through the control of pRB proteins, cyclin D-Cdk4/6 activity is a MCM MCM
central mediator of the reentry of cells into the cell cycle. Not surpris-
ingly, Cdk4/6 activity is frequently hyperactivated in cancer cells, as Cdc6 Cdt1
described later. Recent data suggest that Cdk6 may have kinase-
independent, transcriptional functions, although the exact mechanisms
behind these functions and their relevance in tumor development ORC
G1
deserve further investigation.102,103
Cyclin E is itself an E2F-responsive gene, and this regulation creates
a strong feed-forward loop. Cyclin E binds to Cdk2, and this complex
may promote further pRB inactivation.104,105 Cyclin E–Cdk2 also Cdc6
phosphorylates the CKI p27Kip1 on Thr187.106,107 This action creates MCM MCM
a high-affinity binding site for the SCF ubiquitin ligase bound to the Cdt1
F-box protein Skp2, leading to p27Kip1 degradation during the G1/S
transition.108 Finally, cyclin E-Cdk2 phosphorylates itself on multiple
DDK + Cdk
sites, creating a recognition site for SCF-Fbw7/Cdc4 and thereby dependent
ensuring its own destruction.109,110 The fact that lack of Cdk2 in the S
ORC
mouse does not result in defective mitotic cycles suggests that the
activity of this protein overlaps with other Cdks, with Cdk1 being
the best candidate.111–113 Indeed, Cdk1 is able to bind interphase
cyclins such as cyclin D and cyclin E, and it is sufficient for G1/S
C t
transition, at least in the absence of other interphase Cdks.114–115 Cdk2 1
is however an essential role in meiosis, although the molecular basis d
for this requirement is not fully understood.112,116
Figure 4.4  •  Origin licensing and firing. The origin replication complex
(ORC) associates with replication origins. During the G1 phase, Cdc6 and
DNA Replication Cdt1 are loaded on chromatin, and they in turn load the mini chromosome
maintenance (MCM) complex on chromatin, at which point licensing is
The DNA replication machinery is optimized to ensure that the genome considered complete, and the multiprotein complex is called the prereplication
is copied once—and only once—in each cell cycle.117 This optimization complex (pre-RC). Once cells pass the G1-to-S transition, this complex is
is achieved through a two-step process that first establishes a prereplica- activated to form the preinitiation complex (pre-IC), and DNA replication
tion complex (pre-RC) at each origin of replication, a process that is is initiated. Activation requires both cyclin-dependent kinase (Cdk) and
frequently referred to as origin licensing, and subsequently transforms Ddf4-dependent kinase (DDK) activity. It results in recruitment of numerous
pre-RCs into the preinitiation complex (pre-IC) that activates DNA proteins and activation of the MCM complex, which unwinds the DNA.
Subsequently, core components of the replication machinery, including DNA
replication (Fig. 4.4). These two steps occur at distinct stages of the polymerase α and DNA polymerase ε, are recruited to initiation sites. The
cell cycle to ensure that origins are licensed only once per cell cycle transition from pre-RC to pre-IC results in inhibition of Cdt1 by ubiquitin-
and rereplication cannot occur. Pre-RC formation takes place in the mediated degradation and geminin binding. Origin licensing cannot occur
initial steps of the cell cycle. The first event in this process is the again until activation of anaphase-promoting complex/cyclosome at the end
recruitment of the multiprotein complex called the origin recognition of mitosis allows accumulation of Cdt1.
62 Part I: Science and Clinical Oncology
complex to the origin DNA.118 The origin recognition complex recruits
additional proteins including Cdc6, Cdt1, and finally the mini
chromosome maintenance (MCM) complex, a helicase that is required
to unwind the DNA strands to form the pre-RC. Once cells enter S
phase, the transformation of the pre-RC to the pre-IC requires the
activity of two kinases: a Cdk and the Ddf4-dependent kinase, which
is composed of the Dbf4 regulatory subunit and the Cdc7 kinase.119,120
The action of these kinases allows numerous additional proteins to
associate with the pre-RC and form the pre-IC.121 Assembly of the
pre-IC is thought to trigger DNA unwinding by the MCM complex,
recruitment of the DNA polymerases, and initiation of the replication
process, frequently called “origin firing.”
The transformation of the pre-RC to the pre-IC can occur at
different time points in S phase, depending on whether the origin
fires early or late.117 The system can tolerate this heterogeneity because
the pre-RC is disassembled after firing and cannot reform until the
subsequent cell cycle. This process occurs through several mechanisms.
The MCM complex travels with the replication fork in its role as the
DNA helicase. Some evidence also indicates that phosphorylation of
Orc1 reduces its ability to bind to origins. Finally, and most important,
Cdt1 is prevented from participating in pre-RC formation outside of
the G1 phase in two distinct ways. First, Cdt1 is marked for destruction
by ubiquitination.122 This process is mediated by SCF-Skp2 and
particularly by an E4 ubiquitin ligase that includes Rbx1 (to recruit
the E2-ubiquitin), a cullin (Cul4), Ddb1, and Dtl/Cdt2 (the substrate
specificity factor).123–125 Important to note, this Cul4-Ddb1-Dtl/Cdt2
complex functions independently of Cdt1 phosphorylation. Instead,
Cdt1 is targeted only when proliferative cell nuclear antigen is present
on the DNA, which occurs primarily as a consequence of the initiation
of DNA replication.126 Second, cells possess a protein called geminin
that sequesters Cdt1 and prevents it from participating in pre-RC
formation. Geminin is present specifically in S-, G2-, and early M-phase
cells. The APC/C ubiquitinates geminin and thereby triggers its
destruction during mitotic exit. This action creates a window between
late mitosis and the end of G1 phase (when APC/C-Cdh1 is inactivated)
in which geminin is absent, and therefore Cdt1 is free to participate
in pre-RC formation.127
The A-type cyclins are first transcribed late during the G1 phase
under the control of the E2F transcription factors in a similar manner
to that of cyclin E. Cyclin A associates with both Cdk2 and Cdk1
and acts both during S-phase and G2/M.128,129 At the start of S
phase, cyclin A–Cdk2 enters the nucleus and is specifically localized
at nuclear replication foci, where it is thought to be actively involved
in the firing of replication origins.130 As was described previously,
cyclin A–Cdk2 also is required to phosphorylate E2F-1 and mediate
its degradation, which is required to prevent E2F1 from triggering
apoptosis.131,132
Given the redundancy between different cyclin and Cdk family
members, the specific role of cyclin A in DNA synthesis is unclear.
Studies in mouse models identified a partially overlapping role between
E- and A-type cyclins in the control of DNA synthesis in a cell-type
specific manner.133 Fibroblasts lacking both cyclin A1 and cyclin A2
are able to proceed to S phase, and this is abrogated if E-type cyclins
are deleted. However, A-type cyclins are essential in hematopoietic
stem cells, suggesting cell-type differences probably caused by the
levels of expression of the encoding genes. Although these observations
suggest overlapping functions for E- and A-type cyclins in the activation
of Cdks, a new kinase-independent role as an RNA-binding protein
has been recently proposed for cyclin A2.134 Cyclin A2 binds directly
to the 3′ UTR of Mre11 mRNA in a Cdk-independent manner to
promote its translation. Mre11 is a component of the MRN complex,
composed of Mre11, Rad50, and Nbs1, which plays a central role in
DSB repair and replication fork restart.135 Depletion of cyclin A2
results in a reduction in Mre11 levels and defective repair of replication
errors. Thus cyclin A2 participates in DNA synthesis in a Cdk-
dependent manner while reinforcing stabilization of the key machinery
responsible for repairing DNA lesions caused by replication errors.134
Mitosis
The mitotic machinery is optimized to ensure that the replicated
chromosomes are faithfully segregated to the daughter cells. This
segregation is achieved through the use of a specialized microtubule-
based structure, the mitotic spindle, on which the original chromosomes
and their newly replicated copies, called sister chromatids, align and
then are partitioned to opposite poles of the cell. The mitotic spindle
is a highly dynamic structure that is maintained by many protein
families, including motor molecules and other microtubule-associated
proteins.69,70,136,137 The appropriate side-by-side alignment of the sister
chromatids, termed biorientation, is facilitated by the physical tethering
of the sister chromatids to one another. This process, called cohesion,
actually occurs in S phase in a manner that is coordinated with the
replication process.138–140 Cohesin is mediated by four proteins that
together make up the cohesin complex. Two of these proteins, Smc1
and Smc3, have a long coiled structure with a dimerization domain
at one end that allows them to heterodimerize to form a V-like structure.
Important to note, the remaining ends of Smc1 and Smc3 can associate
with each another to form a functional adenosine triphosphate (ATP)
domain. This domain acts in an ATP-dependent manner to recruit
two additional proteins, Scc1 and Scc3, which form a closed-ring
structure that most likely encircles the chromosomes.141–143 Cohesin
loading onto chromosomes, catalyzed by a separate complex called
kollerin, is thought to be mediated by the entry of DNA into cohesin
rings, whereas dissociation, catalyzed by Wapl and several other cohesin
subunits, is mediated by the subsequent exit of DNA.144 Increasing
evidence indicates that cohesin participates in other cellular processes
that involve DNA looping such as transcriptional regulation. Interesting
to note, mutations in genes encoding cohesin subunits and other
regulators of the complex have been identified in several tumor types.145
The related family of chromosomal proteins, condensins (condensin
I and condensin II), are formed from a conserved pair of Smc proteins
(Smc2 and Smc4) and distinct sets of non-SMC regulatory subunits.146
Condensins participate in a diverse array of chromosomal functions
including chromosomal organization in interphase or the assembly
of mitotic chromosomes.
Mitotic Entry
In addition to its role in DNA replication as discussed earlier, cyclin A/
Cdk complexes are critical mediators of the changes that occur during
the G2 phase in preparation for mitosis.128 Here they are thought to
play a key role in initiating the condensation of chromatin and also
might participate in the activation of the cyclin B/Cdk1 complexes.
Data have suggested that cyclin A2 is also crucial for loading of kinesins
to microtubules, thus contributing to the formation of a functional
spindle.134 Cyclin A2 is destroyed at nuclear envelop breakdown in
an APC/C-Cdc20–dependent manner.147 A-type cyclins are then
substituted with B-type cyclins. Cyclin B1 protein accumulates steadily
through the G2 phase and associates mainly with Cdk1, although it also
may associate with Cdk2.129 The resulting cyclin B1/Cdk1 complex is
mostly sequestered in the cytoplasm, and it is retained in an inactive
form throughout the G2 phase via the inhibitory phosphorylation
of Thr14 and Tyr15 in Cdk1’s active site by the Myt1 and Wee1
kinases.148–150
Activation of cyclin B1–Cdk1 occurs in a highly synchronous
manner during G2-prophase transition (see Fig. 4.1).151 This activation
is mediated by two changes. First, the activities of Myt1 and Wee1
are downregulated at the transition between the G2 and M phases.
Second, a dramatic increase in the activity of the Cdc25 phosphatases
occurs that relieves the inhibitory phosphorylation of Thr14 and
Tyr15. These activity changes are triggered by the phosphorylation
of Myt1, Wee1, Cdc25a, and Cdc25c. Three different kinases are
thought to contribute to this phosphorylation: Polo-like kinase
(Plk1), cyclin A–Cdk1, and cyclin B1–Cdk1 itself. The involvement
of cyclin B1–Cdk1 creates a powerful feed-forward loop; once a
small amount of cyclin B1–Cdk1 is activated, it simultaneously

inactivates its own inhibitors and activates its activators, enabling
a rapid transformation of the entire cyclin B1–Cdk1 pool from the
inactive state to the active state. Once active, cyclin B1–Cdk1 phos-
phorylates components of the centrosomes and initiates a process called
centrosome separation, in which the centrosomes move to opposing
poles of the nascent spindle, an event essential for formation of the
mitotic spindle.68,152
The activation of mitotic kinases leads to dramatic changes in
DNA structure. Largely on the basis of these morphologic changes,
mitosis is divided into five different stages—prophase, prometaphase,
metaphase, anaphase, and telophase (Fig. 4.5)—before separation of
the daughter cells or cytokinesis.
Prophase
Prophase is characterized by condensation of sister chromatids—
essentially packaging into a more compact chromatin structure. This
process involves condensins I and II, which require phosphorylation
by mitotic Cdks.145 During prophase, the nuclear envelope is still
intact; consequently, differences in subcellular localization of the
condensin and Cdk complexes allow only condensin II and cyclin
A-Cdk1 (nuclear), and not condensin I and cyclin B-Cdk1 (cytoplas-
mic), to initiate condensation. In a parallel process called resolution,
the sister chromatids are untangled via the action of topoisomerase
II.141,142 Resolution requires removal of the chromosome arm cohesin
through phosphorylation of Scc3 by Plk1 and histone H3 by the
Aurora-B kinase. Important to note, the cohesin complex at the
centromere is somehow protected from this modification by a protein
called shugosin (Sgo).153–155
The second major event in prophase is the translocation of the
cytoplasmic cyclin B1–Cdk1 to the nucleus, where it phosphorylates
components of the nuclear envelope and triggers its breakdown,68,156
which defines the transition from prophase to prometaphase. To prevent
the activity of phosphatases induced by the mixture of cytoplasmic
and nuclear compartments, Mastl (which is mostly nuclear in prophase)
is exported to the cytoplasm, thus inhibiting the Cdk-counteracting
phosphatase PP2A-B55.68,157 Cdk1 phosphorylates more than 100
proteins and participates in such activities as chromosome condensation,
nuclear envelope breakdown, modifications in the Golgi apparatus,
and formation and dynamics of the mitotic spindle.9,68 Cdk1-deficient
mouse embryos arrest during the first embryonic divisions, indicating
that this kinase is absolutely required for mitosis and cannot be
compensated by other mammalian Cdks.115,158
Prometaphase
During prometaphase, the condensation process is accelerated because
condensin I and cyclin B-Cdk1 now have access to the DNA. The
sister chromatids become attached to spindle microtubules through
the kinetochore, which is assembled onto centromeric DNA.71,72,74–76
Microtubules nucleated from the centrosomes attach to the kinetochore
through a process called search and capture, in which individual
microtubules grow and shrink until they contact and bind the kin-
teochore.73 Typically, one sister chromatid of the pair attaches first,
and this attachment is further stabilized through the recruitment of
additional microtubules from the same pole of the mitotic spindle to
create a kinetochore fiber—that is, highly bundled microtubules bound
to the kinetochore. The sister chromatids oscillate in the cell until
the second sister chromatid is captured by microtubules emanating
from the other pole. These oscillations continue until all of the
chromosomes are properly aligned on the metaphase plate.
Metaphase
Metaphase is defined as the point at which all of the chromosome
pairs are fully condensed, attached to the mitotic spindle, and aligned
at the center—termed the metaphase plate. The pulling of the kineto-
chore fibers toward the poles creates tension through the cohesin
complex at the kinetochores, which indicates that the sister chromatids
have achieved appropriate biorientation. The cell constantly monitors
Control of the Cell Cycle  •  CHAPTER 4 63
the attachments of microtubules to the chromosomes, and possibly
the tension that is generated by microtubules on the kinetochores
ensures that the sister chromatids are properly aligned at the metaphase
plate.159,160 This process is the basis of one of several cell cycle check-
points, called the mitotic spindle assembly checkpoint (SAC), which
is described in more detail in the following sections.
Anaphase
Anaphase is characterized by the segregation of the chromosomes.161
This event is controlled by the mitotic ubiquitin ligase APC/C-Cdc20.
APC/C-Cdc20 ubiquitinates, and thereby triggers the degradation of,
cyclin B1 and a protein called securin.39 Both securin and cyclin B1/
Cdk1 complexes are able to bind and inhibit a protease called sepa-
rase.162,163 APC/C-Cdc20 activity results in the degradation of cyclin
B and securin and the subsequent separase activation. Once released,
separase cleaves the Scc1 component of the cohesin complex, which
opens the cohesin ring, unlinking the sister chromatids and allowing
them to be pulled to opposite poles (see Fig. 4.5). The spindle poles
then move farther apart to ensure that the chromosomes are fully
segregated. The separase-dependent cleavage of Scc1 also is essential
to link segregation of chromatids with the separation of centrioles
during mitotic exit.163 Cyclin B degradation results in the parallel
inhibition of Cdk1 activity, thereby releasing the inhibitory mechanism
that limit PP1 and PP2A activity during the earlier phases of
mitosis.84,90,92 The reactivation of these phosphatases results in the
massive dephosphorylation of mitotic phosphoproteins and results in
the disassembly of the mitotic spindle, chromosome decondensation,
and the reformation of the nuclear envelope.161
During anaphase, Cdh1, which is inhibited by Cdk-dependent
phosphorylation during mitosis, is dephosphorylated and replaces
Cdc20 as the main APC/C activator.39 APC/C-Cdh1 is responsible
for the degradation of multiple cell cycle regulators, including Cdc20.
APC/C-Cdh1 also activates the ubiquitination and degradation of
geminin, allowing accumulation of Cdt1 for origin relicensing in the
subsequent G1 phase, and the mitotic cyclins, allowing loss of Cdk
kinase activity. Loss of Cdh1 does not result in major abnormalities
during mitotic exit but results in earlier entry into the following S
phase because of increased Cdk activity and DNA damage.164,165 Cdh1
therefore is required to prevent unscheduled entry into S phase and
genomic instability.43
Telophase
The reactivation of phosphatases during mitotic exit leads to the
dismantling of the mitotic spindle, leaving two discrete sets of
chromosomes with each nascent daughter cell.161 The elimination
of mitotic phosphoresidues by these phosphatases also results in
DNA decondensation, and the nuclear envelope reforms around
the segregated chromosomes to create two new nuclei, an event that
defines telophase.166
Cytokinesis
Finally, the cell undergoes cytokinesis, or cytoplasmic division. This
process involves formation of a structure containing actin and myosin,
called the contractile ring, on the inner face of the cell membrane.
The position of the contractile ring is carefully controlled. In cultured
cells, the ring typically begins to form in anaphase, and its position
is established by the position of the metaphase plate. As the membrane
grows, the contractile ring contracts steadily to form a constriction,
termed the “cleavage furrow,” which ultimately separates the two nuclei
and forms the two daughter cells.167,168 This process partially depends
on several mitotic kinases such as Aurora-B and Plk1, which are
located at the midbody.51,52,58,169 Plk1 controls the activity of RhoA
guanosine triphosphatases, which are major regulators of the actomyosin
ring required for abscission.58,170 Aurora-B also ensures that abscission
does not occur in the presence of DNA bridges to avoid DNA damage
in a process known as the abscission checkpoint.168,171,172
64 Part I: Science and Clinical Oncology

Cyclin A/B-Cdk1 activity

Interphase

Chromatin begins to condense

Centrosomes move to poles
and mitotic spindle starts to form

NE Chromosomes attach to
breakdown microtubules of spindle
Prophase

Prometaphase

Chromosomes align at metaphase plate

Sister chromatids separate

Chromatin expands
Cytoplasm divides

APC
Metaphase

Cytokinesis
Anaphase

Telophase

Figure 4.5  •  Key stages of mitosis. As the parent cell enters prophase, the chromosomes begin to condense, and multiple proteins associate to form the
kinetochores. The centrosomes segregate to the poles to begin formation of the mitotic spindle. Nuclear envelope (NE) breakdown denotes the start of pro-
metaphase. In this phase, the sister chromatids continue to condense, and they attach to spindle microtubules via their kinetochores. During metaphase, the
sister chromatids align at the metaphase plate and eventually achieve appropriate biorientation. At the onset of anaphase, the sister chromatids separate and
move toward the poles of the spindle. During telophase, the two daughter nuclei are reformed and the daughter cells are finally separated by cytokinesis.
APC/C, Anaphase-promoting complex/cyclosome.
 Control of the Cell Cycle  •  CHAPTER 4 65

CELL CYCLE CHECKPOINTS G1/S Checkpoint

At key transitions during eukaryotic cell cycle progression, signaling
pathways monitor the successful completion of events in one phase
of the cell cycle before proceeding to the next phase. These regulatory
pathways are commonly referred to as cell cycle checkpoints.4 In a
broader context, cell cycle checkpoints are signal transduction pathways
that link the rate of cell cycle phase transitions to the timely and
accurate completion of prior dependent events. Checkpoint surveillance
functions are not confined to monitoring normal cell cycle progression;
they also are activated by both external and internal stress signals.
The checkpoint pathways include sensor proteins that detect these
lesions and simultaneously trigger two processes: they recruit additional
effector complexes to correct the problems and activate signaling
pathways that induce a temporary cell cycle arrest. In certain situations,
which are determined by the cell type and the degree of damage, the
checkpoint pathways eventually can induce permanent cell cycle arrest
(a process called senescence) or apoptosis.
To minimize the possibility of errors, checkpoints exist at the
four different phases of the cell cycle: G1 (to prevent entry into S
phase), intra S, G2 (to prevent mitosis), and M (to avoid mitotic
exit with abnormal segregation of chromosomes) (see Fig. 4.1). In
general, these checkpoints monitor the status and structure of DNA
during cell cycle progression. In particular, cells scan the chromatin
for partially replicated DNA, as well as DNA strand breaks and other
DNA lesions that can result from both extrinsic (e.g., chemicals,
ionizing or ultraviolet radiation) and intrinsic (e.g., byproducts
of intracellular metabolism) DNA-damaging agents. In addition,
checkpoints also monitor the proper structure of chromosomes and
their biorientation to ensure equal distribution between the two
daughter cells.
The molecular pathway that determines cell cycle entry with the
appropriate mitogenic stimuli (previously described) is not considered
a cell cycle checkpoint, strictly speaking. However, several of its
components also are used by a checkpoint that monitors DNA altera-
tions before replication. In G1 cells, double-stranded DNA breaks
(DSBs) are the most common and most deleterious type of DNA
damage. The central components of the DNA damage response (DDR)
are two members of the phosphoinositide 3-kinase–related kinase
family: ataxia telangiectasia mutated (ATM) and ATM- and rad3-related
(ATR).173 ATM originally was identified by virtue of its mutation in
a hereditary syndrome, ataxia-telangiectasia, which is associated with
radiation hypersensitivity and cancer predisposition.174 ATR also is
associated with a hereditary syndrome called Seckel syndrome.175 Early
studies suggested that ATM and ATR played distinct roles in the
response to DSBs (ATM) versus replicative defects and single-stranded
breaks (ATR). However, we now know that the regulation is more
complex; considerable cross talk occurs between ATM and ATR, and
they share many mediators and effectors, but the precise composition
and role of the DDR complexes vary depending on both the type of
damage and the stage of the cell cycle (Fig. 4.6).176
These DSBs are recognized by the multifunctional Mre11-Rad50-
Nbs1 (MRN) complex.135,173 This complex recruits ATM to the site
of damage. The active ATM then recruits proteins to modify the
chromatin at the region of the break and activate repair and signaling.
As a first step in this process, ATM phosphorylates histone H2AX to
form γH2AX, which helps hold the damaged ends together and acts
as a binding platform for additional factors, including Mdc1, 53BP1,
and Brca1, as well as more MRN and ATM. In contrast to the S and
G2 response, no recruitment of ATR to DSB in G1 cells occurs, and

G1 Intra S G2/M

DNA damage Replication- DNA damage

associated
error
DSB DSB

ssDNA
MRN RPA MRN

ATM ATR ATM

γH2AX ATM γH2AX ATR γH2AX

Mediators Mediators Mediators

repair repair repair
machinery machinery machinery

Chk2P Chk2P Chk1P Chk1P Chk2P

Figure 4.6  •  Ataxia telangiectasia mutated/ATM- and rad3-related (ATM/ATR) signaling is activated by DNA damage and replication stress. The cell
constantly monitors the chromatin for lesions, using complex signal transduction pathways that center on the ATM and ATR kinases. The precise mechanism
of response varies according to the type of DNA damage and the cell cycle stage. Double-stranded breaks (DSBs) are the most deleterious form of DNA
damage. DSBs are recognized by the Mre11-Rad50-Nbs1 (MRN) complex that consists of Mre11, Rad50, and Nbs1. This complex recruits ATM to the site
of damage. ATM phosphorylates histone H2AX to form γH2AX, which creates a binding platform for additional proteins that propagate the DNA damage
response and activate repair. For S and G2 phase cells, but not G1 phase cells, ATR also is recruited to the damage site. ATR and/or ATM signal to their
effector kinases (Chk1 and Chk2, respectively) to influence cell cycle progression as described in Fig. 4.7. Errors in DNA replication also can activate the
DNA damage response machinery through the presence of single-stranded DNA (ssDNA), which is a hallmark of the replication fork. The ssDNA is coated
with replication protein A (RPA) and bound by ATR. Active ATR then recruits the DNA damage and repair machinery, including ATM, leading to the
sequential activation of Chk1 and then Chk2.
66 Part I: Science and Clinical Oncology

G1, Intra S, G2/M Intra S G1/S

DNA Replicative Oncogenic
damage stress stress

P P p14ARF
Chk1 and/or Chk1 and
P P
Chk2 Chk2

Phosphorylation of Hdm2
all three Cdc25 proteins
Ubiquitination
P P Degradation
Cdc25a
p53 p53

P Bound and
Cdc25b Oligomerizes to
P inhibited
Cdc25c form active
by 14-3-3
transcription factor

Cdk activation p21Cip1 Pro-apoptotic

genes
Cdk activity

Figure 4.7  •  DNA damage, replicative stress, and oncogenic stress induce cell cycle arrest. DNA damage and replication stress lead to the rapid phosphorylation
and activation of the Chk1 and/or Chk2 kinases. These kinases enforce cell cycle arrest through two mechanisms. Chk1 and Chk2 both phosphorylate the
Cdc25 phosphatases, which triggers their ubiquitination and degradation (Cdc25a) or binding and inhibition by 14-3-3 (Cdc25b and Cdc25c), thereby preventing
activation of cyclin/Cdk kinase complexes. Chk1 and Chk2 also phosphorylate p53 and prevent it from being targeted by Hdm2 for ubiquitin-mediated degrada-
tion. As a result, p53 accumulates and activates transcription of p21Cip1, inhibiting Cdk2 and Cdk1 kinase complexes, or proapoptotic genes. Oncogenic stress
also leads to cell cycle arrest by activating replicative stress and/or inducing transcription or the p14ARF tumor suppressor and suppressing Hdm2-mediated
inhibition of p53. Cdk, Cyclin-dependent kinase.

thus ATM is solely responsible for checkpoint activation. The recruit-
ment of additional ATM amplifies the signal, and ATM acts via
phosphorylation and activation of the effector kinase Chk2.177,178
Chk2 influences the G1 cell cycle arrest via two mechanisms (Fig.
4.7). First, it phosphorylates all three members of the Cdc25 family.
Phospho-Cdc25a is ubiquitinated by SCF-TrCPβ and degraded, whereas
phospho-Cdc25b and phospho-Cdc25c are bound and sequestered
by a cytoplasmic protein called 14-3-3.20,179 This process is a rapid
response that can take effect within minutes after DNA damage, and
it has a widespread effect on cell cycle progression by preventing
activation of Cdks. Second, Chk2 phosphorylates p53, a critical regula-
tor of cell cycle checkpoints.180 In normal, nonstressed cells, p53
protein is maintained at low steady state levels because it has a very
short half-life. This half-life is a result of rapid ubiquitination of p53
by Hdm2 (the human ortholog of murine Mdm2 protein) and its
consequent degradation.181,182 The importance of Mdm2 for mainte-
nance of appropriate p53 levels in vivo is highlighted by the fact that
absence of Mdm2 in knockout mice results in early embryonic lethality
that is rescued by a dual knockout of Mdm2 and p53.183,184 Phosphoryla-
tion of p53 by Chk2 is sufficient to prevent its association with Hdm2/
Mdm2,183 which leads to an accumulation of p53, which functions
as a transcriptional activator; p53 induces expression of many genes
involved in cell cycle arrest, including the CKI p21Cip1.185,186 This
p53-mediated arrest takes longer to develop than does the Cdc25
response (because it requires transcription and protein synthesis), but
it appears to be much more robust. Moreover, in addition to inducing
cell cycle arrest, p53 has the capacity to induce apoptosis through the
transcriptional activation of proapoptotic regulators (e.g., the BH3-only
proteins Puma and Noxa).187
Important to note, p53 also is activated by other stress signals (see
Fig. 4.7). In particular, it is now well established that numerous
oncogenes trigger a stress response (called oncogene-induced stress)
that leads to the activation of p53.188,189 The emerging view is that
this process occurs through two distinct mechanisms. First, oncogene
activation is thought to yield replicative stress that activates p53 via
activation of Chk kinases and phosphorylation of Hdm2/Mdm2 as
just described.190,191 Second, many oncogenes activate transcription
of cell cycle inhibitors such as p16INK4a, p15INK4a, p21CIP1, or p19ARF
in a p53-independent manner.188,192–195 The protein p19ARF is encoded
by the INK4A/ARF (CDKN2A) locus, and it actually shares two
coding exons, which are read in alternate reading frames (hence the
name ARF) with the p16INKa tumor suppressor.194 The ARF protein
product, called p14ARF in humans and p19ARF in mice, binds to Hdm2/
Mdm2 and prevents it from regulating p53.196–199 As with the DDR,
this mechanism frees p53 to activate the transcription or proarrest or
proapoptotic targets.
As an additional DDR in G1 cells, genotoxic agents also inhibit
origin licensing by way of an ATM/ATR-independent process that is
achieved through regulation of Cdt1.200 As described previously, Cdt1
is required for pre-RC formation. In an undamaged cell, Cdt1 is
available during the G1 phase but is inhibited after origin firing by
degradation (mediated by the SCF-Skp2 and Cul4-Ddb1-Dtl/Cdt2
ubiquitin ligases) and geminin binding. As a key feature of this regula-
tory system, Cdt1 is completely resistant to Cul4-Ddb1-Dtl/Cdt2 in
the G1 phase. However, DNA damage allows the Cul4-Ddb1-Dtl/
Cdt2 complex to ubiquitinate Cdt1 and induce its degradation.
Important to note, the degradation of Cdt1 is extremely rapid, occurring
within minutes of the DNA damage. Both Cdt1 and Cdt2 are

phosphorylated in response to DNA damage, which results in the
ubiquitination of Cdt1. This process depends on the activity of the
p97 AAA+-ATPase and its cofactor Ufd1, a complex required for the
extraction of ubiquinated proteins from the chromatin.124,201,202 As a
result, origin licensing is completely blocked until the damage is repaired
and Cdt1 is resynthesized.
Intra–S Phase Checkpoint
One of the major goals of cell cycle checkpoints is to prevent the
deleterious consequences of replicating damaged DNA. Therefore
S-phase cells must respond virtually instantaneously to DNA damage
to halt initiation of new replication forks throughout the S phase.203
The most deleterious type of damage is DSBs. DSBs can occur through
the action of DNA-damaging agents (from either extrinsic or intrinsic
sources) or as a consequence of the replication process itself—for
example, if the replication fork passes through nicked DNA or if
replication stalls at sites of DNA damage.204 The cell senses the damage
in different ways, depending on whether the lesion is associated with
replication. Ultimately, both ATM and ATR are recruited to the site
of damage, but the order of binding is different.176,203 Replication-linked
DSBs are distinguished by the presence of single-stranded DNA
(ssDNA), a hallmark of the replication process. The ssDNA is coated
by replication protein A (RPA) and bound by ATR and its regulator
subunit ATRIP, even during the normal replication process. In response
to DNA damage, the ATR kinase is activated, and it then recruits a
variety of complexes that mediate both repair and checkpoint activation,
including ATM. In contrast, nonreplication-associated DSBs initially
recruit and activate ATM through the MRN-dependent process
described previously for the G1/S checkpoint. However, in S-phase
cells, DSB resection causes the formation of ssDNA (through the
action of the MRN endonuclease), which is then bound by RPA and
ATR/ATRIP.203 Thus in S-phase cells, ATR and ATM jointly orchestrate
the DDR. ATR contributes to the checkpoint response in a similar
manner to ATM: it activates Chk1, which also can phosphorylate the
Cdc25 proteins and p53.205–208
G2 Checkpoint
Whereas the G1/S and intra–S phase checkpoints prevent cells from
unfaithful replication, the G2 checkpoint is required to prevent the
passage of DNA lesions to the two daughter cells during mitosis.173,209,210
DSBs are detected exactly as described previously for the S-phase
nonreplication-associated DSBs. Similarly, the ATR/Chk1 and ATM/
Chk2 pathways enforce arrest through inhibition of G2 and mitotic
Cdk complexes via the rapid removal of the Cdc25 phosphates and
the p53-dependent induction of p21CIP1 to inhibit mitotic Cdk
complexes (cyclin A/B in combination with Cdk1/2 kinases).208,210,211
Ubiquitin ligases also are involved in this process. SCF-βTrCP regulates
the levels of Cdc25, Claspin, and Wee1, whereas APC/C-Cdh1 is
critical for the elimination of Plk1, a kinase essential for checkpoint
recovery.211,212
Spindle Assembly Checkpoint
The SAC acts to ensure that appropriate partitioning of the chromo-
somes occurs during mitosis.159,160 The concept that chromosome
segregation is prevented until all condensed sister chromatid pairs are
aligned at the metaphase plate with the appropriate biorientation has
already been introduced. This process actually is controlled by a signal-
ing network that constitutes the SAC (Fig. 4.8). The core components
of this checkpoint—called Mad1, Mad2, BubR1, Bub1, and Bub3
in humans—originally were identified through screens in yeast for
“mitotic arrest deficient” (MAD) and “budding uninhibited by
benzimidazole” (BUB) mutants.159 Other components of the checkpoint
include the Mps1 kinase and the three subunits of the Rod, Zwich,
and ZW10 (RZZ) complex. During prometaphase, these proteins
Control of the Cell Cycle  •  CHAPTER 4 67
localize to the outer kinetochore and, in the absence of biorientation,
prevent the Cdc20 activator from binding to the APC/C.
The kinetochore is made of approximately 30 scaffold proteins
that link chromatin and the mitotic spindle.71,72,75–77 Additional regula-
tory elements include sensors of microtubule-kinetochore attachment
and a complex signaling pathway that modulates APC/C-Cdc20 activity.
Lack of tension or lack of attachment at the kinetochore results in
stable Mad1/Mad2 complexes that convert an inactive open-Mad2
conformation into a closed-Mad2 conformation that is able to bind
to Cdc20.159,213,214 The Mad2-Cdc20 association triggers the recruitment
of BubR1-Bub3 into an APC/C-inhibitory complex (the mitotic
checkpoint complex [MCC]). Because the closed-Mad2 signal is
diffusible, a single unattached kinetochore is sufficient to form these
complexes and inhibit APC/C-Cdc20 activity. As a result, separase is
inhibited by the high levels of securin and cyclin B/Cdk1 complexes,
being unable to cleave the centromeric cohesin (see Fig. 4.8). Once
all chromosomes are bipolarly attached to the mitotic spindle, the
SAC is satisfied and the Mad1/Mad2 complex is removed from the
kinetochores.71,77 How the checkpoint signaling is inhibited currently
is unclear. Cdc20 is now released from the MCC complex and activates
the APC/C, leading to the rapid ubiquitination and degradation of
cyclin B and securin. Inactivation of these two proteins results in two
major processes. First, lack of securin and cyclin B results in the
activation of separase, a caspase-like protease that cleaves cohesin, the
molecule that holds sister chromatids together at the centromere.
Second, inhibition of Cdk1, due to the lack of cyclin B, leads to the
activation of mitotic phosphatases such as PP1 and PP2A, triggering
mitotic exit as described earlier.161
CELL CYCLE DEREGULATION IN
HUMAN CANCERS
The central relevance of cell cycle regulation in cancer is underscored
by the finding that virtually all human tumors carry mutations in (1)
the basic cell cycle machinery that controls entry into the cell cycle
and (2) checkpoint regulators such as the p53 pathway (Table 4.2)
and by the observation that most human tumors display aberrant
chromosome numbers.1,5,6,173,215 Together, the pRB and p53 pathways
are critical gatekeepers of cell cycle progression and stress response,
and their function has crucial implications in the maintenance of
genome integrity at the level of both structural and numeric aberrations
in chromosomes.216,217
Unscheduled Cell Cycle Entry in Cancer
The alterations in the cell cycle machinery that occur most frequently
include loss or mutation of the pRB tumor suppressor; overexpression
of cyclins, Cdks, and Cdc25 phosphatases; and loss of expression of
CKIs.1 Mutations that affect the pRB pathway have been identified
in most human cancers.1,29,217 The RB1 gene originally was identified
by virtue of its mutation in both familial and sporadic retinoblastoma,
but it is defective in many other tumor types, especially osteosarcoma
and lung cancer.
In tumors that lack RB1 mutations, alterations in other components
of the signaling pathways that regulate pRB frequently are found,
including cyclin overexpression or loss of CKIs. Nearly 50% of inva-
sive breast cancers have elevated cyclin D expression compared with
surrounding normal breast epithelium, and in transgenic mice with
overexpression of human cyclin D1 or cyclin E in mammary gland
cells, mammary adenocarcinomas develop.218–220 Similarly, Cdk4 and
Cdk6 gene amplification occurs in breast cancers, sarcomas, gliomas,
and melanomas, and specific translocations lead to cyclin D or Cdk6
overexpression in hematopoietic disorders.1,221 Modifications of CKIs
that act upstream of pRB activity also commonly are found in human
tumors. The CKI p27Kip1 often is aberrantly expressed in human
breast cancer, and reduced p27Kip1 protein levels are correlated with
more aggressive breast tumors.222–224 Likewise, decreased expression
68 Part I: Science and Clinical Oncology

Prometaphase

No attachment
Pole
“Wait” Cohesion
Unattached
kinetochore Spindle
Low checkpoint
Mad2 proteins Cdc20
Metaphase High
Mad2

Attachment
Active Inactive
APC/C APC/C
Securin
ubiquitination
+ degradation
Anaphase
Securin
Inactive
Active separase Separase

Cohesion
(by Scc1
cleavage)

Figure 4.8  •  The spindle assembly checkpoint (SAC). Improper chromosome alignment on the mitotic spindle, disruption of microtubule dynamics, or
unattached kinetochores maintains the SAC in an active state. Checkpoint signaling is mediated by the Bub1, Bub3, BubR1, and Mad2 proteins, which
localize to kinetochores. These core spindle checkpoint regulators prevent Cdc20 from activating the anaphase-promoting complex/cyclosome (APC/C) and
therefore protect securin and cyclin B, two major APC/C-Cdc20 targets, from ubiquitin-mediated degradation. As a result, securin and cyclin B1-Cdk1 remain
bound to separase, which prevents cleavage of Scc1 and loss of centromeric cohesin. The “wait” signal is inhibited at the end of the metaphase by the appropriate
biorientation of the sister chromatids at the metaphase plate. The sensing mechanism involves detecting attachment or tension at the kinetochores that is
created by the pulling of the spindle fibers toward the poles. Mad2 then dissociates from the attached kinetochore, which allows Cdc20 to activate APC/C,
targeting securin and cyclin B for degradation. This process leads to the inactivation of Cdk1 and the activation of separase, triggering sister chromatid segregation
and mitotic exit.

of the CKI p57Kip2 is found in human bladder cancers. Although
p21Cip1 is not commonly mutated in human cancer, its expression
is strongly reduced in multiple tumors as a consequence of defective
p53 signaling.225 The CKI most frequently affected appears to be
p16INK4a; it was identified as a tumor suppressor that is associated
with familial melanoma, and it is inactivated by point mutation,
deletion, and/or promoter methylation in approximately 30% of all
human tumors.221,226 In contrast, point mutations in p15INK4b, p18INK4c,
and p19INK4d are rare, but promoter methylation and reduced protein
expression of some of these inhibitors have been seen in a variety
of tumor types.1,221,227–229 Germline mutations in p16INK4a predispose
individuals to melanoma, whereas deletion of p15INK4b and p16INK4a is
linked to the pathogenesis of lymphomas, mesotheliomas, and pancreatic
cancers.1,221 Although point mutations in Cdks are not common in
human tumors, a specific mutation of Cdk4 (R24C) that prevents its
inhibition by INK4 proteins has been found in persons with familial
melanoma.230 Knockin mice harboring this mutation show not only
an increased susceptibility to melanoma but also the development
of multiple tumor types, indicating the relevance of this regulatory
circuit in human tumors.231,232 Both Cdc25a and Cdc25b phosphatases
also are overexpressed in more than 30% of primary breast tumors,
40% to 60% of non–small cell lung cancers, 50% of head and neck
tumors, and a significant fraction of non-Hodgkin lymphomas.20,233,234
All these events can result in increased activation of Cdks, defec-
tive pRB signaling, unscheduled cell proliferation, and override of
checkpoint arrest.
Mutations in p53 and Checkpoint Regulators
The most frequently altered cell cycle checkpoint signaling molecule
is the p53 tumor suppressor. The importance of p53-dependent signal-
ing in tumor suppression is underscored by the frequency of mutation
(~50%) in sporadic tumors235 and the finding that germline mutations
of p53 result in Li-Fraumeni syndrome, a highly penetrant familial
cancer syndrome that is associated with significantly increased rates
of brain tumors, breast cancers, and sarcomas.236 In human tumors
that lack p53 gene mutation, p53 function may be disrupted by
alterations in cellular proteins that modulate the levels, localization,
and biochemical activity of p53. For example, in some tumors with
wild-type p53 alleles, Mdm2 gene amplification occurs, resulting in
Mdm2 protein overexpression and subsequent p53 inactivation.237 In
human papillomavirus–induced cervical carcinoma, p53 typically is
not mutated; however, the human papillomavirus E6 protein binds
p53 and targets it for degradation, abrogating p53-dependent
signaling.238
Proteins that reside upstream of p53 (including ATM and Chk2)
also are targeted for mutation in human tumors, and their discovery
and analysis have greatly deepened our insight into DDR signaling
pathways. ATM mutations occur in ataxia-telangiectasia, a disorder
in which patients have increased sensitivity to radiation and an
elevated incidence of leukemias, lymphomas, and breast cancer.174,239
ATM-null mice exhibit growth retardation, neurologic dysfunction,
infertility, defective lymphocyte maturation, and sensitivity to ionizing
 Control of the Cell Cycle  •  CHAPTER 4 69

Table 4.2  Alteration of Cell Cycle Regulators in Human Tumorsa

Tumors Associated With Mutations or Hereditary Syndromes Associated With
Protein (Gene) Altered Expression Germline Mutations
ATM Breast carcinomas, lymphomas, leukemias Ataxia-telangiectasia
Aurora-A (AURKA) Wide array of tumors NR
Bub1 Colon, lung, and pancreatic cancer NR
BubR1 (BUB1B) Wilms tumor, rhabdomyosarcoma, and acute leukemia Mosaic variegated aneuploidy and premature chromatid
separation syndrome
Brca1/2 Breast and ovarian carcinoma Familial breast and ovarian cancer
Cdc25a Carcinomas of the breast, lung, head, and neck and lymphoma NR
Cdc25b Carcinomas of the breast, lung, head, and neck and lymphoma NR
Cdk4 Wide array of cancers Familial melanoma
Cdk6 Wide array of cancers NR
Chk1 Colorectal and endometrial carcinomas NR
Chk2 Carcinomas of the breast, lung, colon, urogenital tract, and testis Li-Fraumeni syndrome
Cyclin D1 Wide array of cancers NR
(CCND1)
Cyclin D2 Lymphoma and carcinomas of the colon, testis, and ovary NR
(CCND2)
Cyclin D3 Lymphoma, pancreatic carcinoma NR
(CCND3)
Cyclin E Wide array of cancers NR
(CCNE1/2)
Mdm2 (HDM2) Soft tissue tumors, osteosarcomas, esophageal carcinomas NR
Mps1 (TTK) Lung, gastric and bladder cancer NR
Mre11 Lymphoma Ataxia-telangiectasia–like disorder
Nbs1 Lymphomas, leukemias Nijmegen breakage syndrome
p15INK4b (CDKN2B) Wide array of cancers NR
p16INK4a (CDKN2A) Wide array of cancers Familial melanoma
p27Kip1 (CDKN1B) Wide array of cancers NR
p53 (TP53) Wide array of cancers Li-Fraumeni syndrome
p57Kip2 (CDKN1C) Bladder carcinomas NR
p130 (RBL2) Wide array of cancers NR
pRB (RB1) Wide array of cancers Familial retinoblastoma
SA1 (STAG2) Bladder, colorectal, glioblastoma, melanoma and Ewing’s sarcoma NR
Tpx2 Lung, bone, and pancreatic cancer NR

a
Only genetic alterations or defects that are present in more than 10% of primary tumors are represented.
NR, Not reported.

radiation.240,241 The DNA double-strand break repair gene Mre11 also
is mutated in persons with an ataxia-telangiectasia–like disorder.242
Mutations of Chk2 and Chk1 also arise in human cancers. Chk2
mutations have been reported in several cancers, including lung cancer,
whereas Chk1 mutations have been observed in human colon and
endometrial cancers.243,244 In addition, heterozygous alteration of Chk2
occurs in a subset of persons with Li-Fraumeni syndrome who lack
p53 gene mutations.245 These findings support the theory that in
human tumors in which p53 is intact, the function of this signaling
pathway might be disrupted by alterations in cellular proteins that
modulate the levels or activity of p53. In addition, the breast cancer
susceptibility tumor suppressors Brca1 and Brca2 are known to
participate in the DDR and repair.246 Similarly, Fanconi anemia proteins,
which originally were identified by virtue of their association with a
recessive development disorder called Fanconi anemia, which is associ-
ated with increased cancer predisposition (particularly acute myeloid
leukemia), also function in the DDR.246 Mutations in the Nbs1 gene,
a component of the MRN complex that sensors damage, are responsible
for Nijmegen breakage syndrome, a rare autosomal-recessive disorder
characterized by chromosome instability, hypersensitivity to ionizing
radiation, and high susceptibility to the development of tumors.135,247
Aneuploidy and Chromosomal Instability
Abnormal chromosome numbers, called aneuploidy, is a frequent feature
of cancer cells.6,215,248 In addition, many cancer cells also display
chromosomal instability or an aberrantly high change in the karyotype.
Chromosomal instability may lead to aneuploidy, but not all aneuploidy
cells display chromosomal instability, because many cancer cells exhibit
stable aneuploidy karyotypes. Aneuploidy has a high rate of frequency
(more than 75%) in human tumors, and about one-quarter of the
genome is affected by whole-arm or whole-chromosome number
alterations.249 Yet it is not clear whether aneuploidy is a cause or
consequence of cancer, and the relative level of chromosomal instability
may have either oncogenic or tumor suppressor properties during
tumor development.6,250–252
70 Part I: Science and Clinical Oncology
In vitro estimates suggest that normal cells missegregate a chromo-
some every hundred cell divisions.215 This rate is dramatically increased
in cells that display chromosomal instability, which missegregate a
chromosome in every one to three cell divisions in vitro.253 These
defects occur for multiple reasons, including aberrant centrosome
numbers, abnormal microtubule-kinetochore attachments, and
imperfect SAC. In particular, there has been considerable speculation
that disruption of the SAC could occur during tumor progres-
sion.252,254,255 Notably, inactivating mutations in BUB1 have been
identified in human colon carcinoma cell lines, which are known to
have a high degree of aneuploidy.256 These mutations facilitate the
transformation of cells that lack the breast cancer susceptibility gene
BRCA2.257 The gene encoding BubR1, BUB1B, is also mutated in
persons with mosaic variegated aneuploidy and the premature chromatid
separation syndrome. Both BUB1 and BUB1B also are silenced by
promoter hypermethylation in specific tumors.258 Additional mutations
and epigenetic alterations have been found in the SAC components
Mad1 and Mad2.258 Most of these alterations are loss-of-function
mutations, suggesting that the SAC is not functional in a variety of
tumors. Moreover, haploinsufficiency of Mad2 has been shown to
cause elevated rates of lung tumor development in mice.259 Interesting
to note, overexpression of Mad2 also results in increased tumor
susceptibility and frequent relapses of chromosomally unstable tumors.260
In fact, Mad2 is also frequently overexpressed in human tumors.
Several other mitotic regulators such as separase, securin, condensins,
Cdc20, or Aurora kinases, as well as the SAC component Mps1, are
included in the overexpression signature that marks chromosomally
unstable cancers.258,261 All these data, together with the recent evidence
gathered in mouse models, suggest that small aberrations, either
overexpression or downregulation, in the levels of mitotic and SAC
regulators may contribute to aneuploidy and/or chromosomal instability
in human tumors.6,255 Because functional disruption of SAC proteins
results in the abrogation of the checkpoint and a failure to arrest in
mitosis in the presence of microtubule poisons such as taxol and
nocodazole, these aberrations also may play a role in the response to
mitotic poisons currently used in the clinic.262
Multiple cell cycle aberrations, including pRB or p53 inactivation,
lead to aberrant levels of mitotic regulators resulting in “oncogene-
induced mitotic stress.”216 The consequences of this aberration in
cancer cell proliferation are possibly limited by additional alterations
in other mitotic regulators. For instance, the cancer-associated upregula-
tion of the SAC component Mad2 can be balanced by upregulation
of its target Cdc20, thus maintaining the balance between SAC and
APC/C activity required for genomic stability.216 In the same line,
mutations in the APC/C component Cdc27 may slow down APC/C
activity, thereby facilitating the correction of chromosome segregation
errors in the presence of the weak SAC frequently found in tumor
cells.263 Tumor cells also accumulate mutations in other genes whose
deregulation may induce chromosomal instability, such as cohesin
subunits and their regulators. However, whether chromosome misseg-
regation is the major contribution of cohesin mutations to cancer
progression is not clear at present.146
Chromosome missegregation not only results in whole-chromosome
aneuploidy but also may generate chromosome breaks and rearrange-
ments. A possible mechanism for DNA damage caused by chromosome
missegregation is based on the defective DNA replication of the
micronuclei present in cells affected by aneuploidy.264 These micronuclei
undergo asynchronous DNA replication, resulting in DNA damage
and pulverization of chromosomes. This process may provide, at least
partially, an explanation for “chromothripsis,” a situation in which
chromosomes undergo massive local DNA breakage and rearrange-
ments.264,265 In addition, chromosomes that missegregate may be
damaged during cytokinesis, resulting in DNA double-strand breaks
that can lead to unbalanced translocations in the daughter cells.266
All these processes provide additional mechanisms by which defective
chromosome segregation may induce genomic aberrations during
tumor development.
THERAPEUTIC MANIPULATION OF CELL
CYCLE CONTROLS
Research during the past several decades has shown that alterations
in cell cycle machinery and checkpoint signaling lead to tumorigenesis.
These findings have important implications for the optimization of
current therapeutic regimens and for the selection of novel cell cycle
targets for the future development of anticancer agents. A leading
goal in cancer research is to identify compounds that will target key
cell cycle controls in a tumor-specific manner.
Targeting Cyclin-Dependent Kinase Activity
Considerable debate has occurred about whether inhibition of Cdk
activity is a rational strategy for anticancer therapies.267 Cdk activity
frequently is elevated in human tumors, but it also is required to
maintain specific cell populations in adults (e.g., the hematopoietic
compartment and gut) that are essential for viability.3 Thus the key
issue is whether tumor cells may require different Cdks for proliferation
or whether sufficient difference in the Cdk activity exists to create a
therapeutic window. During the last few years, the analysis of Cdk
and cyclin mouse models has yielded considerable insight into this
question but also has raised additional questions.268 These mouse
models show that the cell cycle machinery is extremely robust; it
adapts easily to the loss of Cdks or cyclins by using other Cdks or
cyclins to substitute for the missing activity. For example, cells are
able to proliferate without specific interphase Cdks because novel
cyclin/Cdk complexes can be formed that allow the cell cycle to
progress.111–115 This ability raises the possibility that tumor cells will
develop resistance to Cdk-inhibitory drugs rapidly simply by adapting
their cell cycle machinery. On the positive side, studies in cell lines
and mouse models clearly show that tumors can be more dependent
on Cdk activity, or at least a specific Cdk activity, than are normal
tissues. In parallel to these biologic studies, numerous small-molecule
inhibitors have been developed with different specificity profiles against
Cdks (Table 4.3).268–270
Which Cdk should be targeted in each tumor type? Pioneering
studies in the mouse showed that loss of D-type Cdk had little or no
effect on the development and maintenance of many tissues but can
greatly suppress the development of certain tumor types, depending
on the tissue and the identity of the initiating oncogenic lesions.271–274
Mammary gland tumor proliferation dramatically depends on cyclin
D1/Cdk4 complexes, whereas the absence of these molecules does
not alter normal mammary gland development. In a pioneering study
in mouse models, cyclin D1–Cdk4 was shown to be required for
Her2 (ErbB2)- or HRas-driven tumors, whereas it was dispensable
for Myc- or Wnt-induced neoplasias, suggesting that the efficacy of
CKIs may have a strong dependence on the genetic background of
target tumors.272,275,276 Similarly, Cdk4 is required for KRAS-mutant
lung tumors, but it seems dispensable for similar lung tumors with
wild-type KRAS alleles.274 On the other hand, cyclin D3/Cdk6
complexes are critical for Notch1-mutant T-cell leukemias.277 These
studies and parallel efforts in human cancer cell lines led to multiple
clinical trials to test the effect of specific Cdk4/6 inhibitors in a wide
spectrum of tumors.278 The success of some of these trials resulted in
the rapid approval of palbociclib for the treatment of hormone-positive
breast cancer, and other Cdk4/6 inhibitors such as abemaciclib and
ribociclib have showed significant efficacy in advanced clinical trials
in multiple tumor subtypes.279–281
Targeting DNA Damage Response Proteins
In the past decade, there has been a growing appreciation that many
tumor cells carry mutations that disrupt their DDR. This characteristic
is a major factor in establishing the resistance of tumors to chemo-
therapeutic agents, many of which work by causing DNA damage
and triggering apoptosis through induction of DNA damage pathways.

Table 4.3  Cell Cycle Regulators With Therapeutic Interest
Target Representative Small-Molecule Inhibitors
MITOTIC SPINDLE
Tubulin Stabilizing: taxanes, eleutherobins, epothilones, laulimalide,
sarcodictyins, and discodermolide; polymerization inhibitors:
vinca-alkaloids, cryptophycins, halichondrins, estramustine
Eg5 (KSP) ARRY-520, AZD4877, MK-0731, SB-715992 (ispinesib),
SB-743921
KINASES
ATR AZD6738, VX-970, ATR-101
Aurora kinases AT9283, CYC116, GSK1070916A, MLN8237, PF-03814735, VX-680
Cdks (1, 2, 4, 6) AG-024322, EM-1421, LEE-011 (ribociclib), LY2835219
(abemaciclib), P276-00, PD-0332991 (palbociclib)
Chk1 LY2606368, UCN01, CCT245737
Plk1 BI2536, BI6727 (volasertib), GSK461364, NMS-1286937,
TKM-080301
Mps1 (TTK) NMS-P153, BAY1161909, NTRC 0066-0
PHOSPHATASES
Cdc25 ARQ-501, IRC 083864
DNA REPAIR
PARP ABT-888, AZD-2281 (olaparib), AG014699, BMN-673, CEP 9722,
MK-4827 (niraparib)
UBIQUITIN LIGASES
APC/C TAME, APCIN
APC/C, Anaphase-promoting complex/cyclosome; Cdk, cyclin-dependent kinase; ATR, ATM- and rad3-related; PARP, poly (ADP-ribose) polymerase.
Therefore considerable attention has focused on designing cancer
treatments that would be effective in cells with an impaired DDR.
Because it is difficult to restore the function of mutant or missing
proteins, the prevailing strategy is to identify drugs that would synergize
with the defective DDR to selectively kill the tumor cells and not the
normal cells. For example, inhibitors of poly (adenosine diphosphate
[ADP]-ribose) polymerase (PARP) selectively kill cells that lack either
Brca1 or Brca2.282,283 The rationale for this action is that these proteins
provide two alternative repair mechanisms in response to DNA damage:
homologous recombination (Brca1 and Brca2) and base excision repair
(PARP). Therefore loss of one but not both of these pathways can be
tolerated. As a second example, inhibition of Chk1 sensitizes p53
mutant cells to DNA damage.284 Because p53 is mutated in approxi-
mately half of all human tumors and the absence of p53 is a major
predictor of poor response to classic chemotherapeutic agents, consider-
able efforts are being made to develop small-molecular inhibitors of
Chk1. Although the initial model proposed that this effect was due
to the simultaneous abrogation of the G2 (Chk1) and G1 (p53)
checkpoints, new evidence suggests that the toxicity of Chk1 inhibitors
may be due to the generation of replicative stress (RS) in cells, with
the less restrictive S-phase entry due to the lack of p53.285 Chk1
inhibitors therefore might synergize with other mutations that promote
a promiscuous S-phase entry. A similar rationale applies to other
molecules that target the RS response, such as inhibitors of the Chk1-
activating kinase ATR. Indeed, ATR inhibitors display a selective
effect in Myc-driven tumors that display high levels of RS as a con-
sequence of the overexpression of Myc.286 This strategy can be general-
ized to multiple oncogenic alterations that result in cell cycle defects,
causing an increased reliance on ATR checkpoint activity, as recently
shown for different tumor types.287–291
Control of the Cell Cycle  •  CHAPTER 4 71
Preclinical or Clinical Data
In clinical use for solid and hematopoietic tumors
Clinical trials in advanced solid tumors, lymphoma, and
taxane-resistant cancer
Clinical trials in monotherapy or combination with
radiotherapy in solid tumors
Clinical trials in solid tumors and hematopoietic malignancies
Clinical trials in solid tumors and leukemias; Cdk4/6 inhibitors
approved for hormone-positive breast cancer
Clinical trials in combination with DNA damaging agents
Clinical trials in solid tumors and hematopoietic malignancies
Clinical trials in metastatic solid tumors
Preclinical studies and clinical trials in advanced solid tumors
Clinical trials in solid tumors and hematopoietic malignancies;
olaparib and rucaparib are approved for treating ovarian
cancer
Preclinical studies
Targeting the Mitotic Spindle
Microtubule poisons, such as taxol and vinblastine, kill cancer cells
at least partially by exploiting their effects on the mitotic spindle.292
Taxanes, such as docetaxel or paclitaxel, are microtubule-stabilizing
drugs widely used to treat breast and ovarian tumors, non–small cell
lung cancer, and Kaposi sarcoma. Vinca alkaloids, such as vinblastine
or vincristine, are microtubule-destabilizing compounds and have
shown clinical efficacy against a broad range of hematologic malignan-
cies. Both classes of drugs bind tubulin and inhibit microtubule
dynamics, impairing the formation of a functional spindle. The SAC
senses lack of proper attachment of kinetochores and arrests cells in
prometaphase in the presence of these compounds. Thus inhibition
of spindle dynamics results in abnormal chromosome segregation that
frequently results in either aneuploidy or cell death. The low mitotic
index in human tumors suggests that microtubule poisons may have
therapeutic potential in a mitotic-independent manner although this
deserves further exploration.293 About 30 microtubule poisons are
currently in clinical development.
The success of these molecules in the clinic led to the search for
additional compounds that target specific regulators of microtubule
dynamics rather than tubulin itself. In a pioneer screening, monastrol
was identified as an inhibitor of the kinesin Eg5, a protein required
for centrosome separation and the formation of a bipolar spindle.294
New drugs have been characterized that result in similar defects by
inhibiting CenpE, another kinesin with critical roles in microtubule-
to-kinetochore attachment.295–297 Inhibition of Aurora-A or Plk1 also
may be considered to be an antispindle strategy because these kinases,
although involved in other mitotic processes, are essential for centrosome
maturation and separation and the formation of a bipolar spindle.52,295
72 Part I: Science and Clinical Oncology

In general, the inhibition of all these molecules results in an SAC-
dependent arrest that impairs proliferation or viability of targeted
cells. The Plk1 inhibitor volasertib has shown considerable promise
in clinical studies, having reached phase III trials and been granted
the breakthrough therapy designation for its effects in acute myeloid
leukemia.298
Targeting Mitotic Entry and Exit
As indicated in the earlier sections of this chapter, multiple enzymatic
activities are required for mitosis. Cdk1 is the major engine in this
process, and its activity is essential for mitosis. However, it has not
been considered as a major target because its inhibition may have
strong undesired effects.270 Yet some evidence suggests that it may be
an interesting target in specific situations. Cdk1 is specifically required
in cells transformed with Myc but not in cells transformed by other
oncogenes. Inhibition of Cdk1 rapidly downregulates survivin expres-
sion, a protein required to avoid apoptosis in the presence of an excess
of Myc. Cdk1 inhibition therefore results in Myc-dependent apoptosis
and regression of Myc-dependent lymphoma and hepatoblastoma
tumors.299 Cdk1 also is implicated in DNA repair by homologous
recombination, and its inhibition results in impaired Brca1 activity.
Cdk1 inhibition synergizes with PARP inhibitors, and partial inhibition
of Cdk1 therefore may sensitize Brca1/2-proficient cancer cells to
inhibit PARP, suggesting specific applications of Cdk1-targeting
compounds in cancer cells.300 Cdk1 activators such as Cdc25 phos-
phatases are additional targets whose inhibition results in impaired
Cdk activity and cell cycle arrest.20 The therapeutic usefulness of
inhibition of additional mitotic kinases such as Mastl, Nek proteins,
or Haspin, among others, is currently undergoing preclinical
evaluation.3,295,301
Inhibiting mitotic entry or progression or preventing spindle
dynamics may result in different outcomes, including cell death or
the exit from the cell cycle without chromosome segregation.295 In
fact, a rapid exit from mitosis is a major resistance mechanism that
generates viable cells in the presence of mitotic poisons. This finding
led to the evaluation of mitotic exit pathways as new targets for
therapy.302 In fact, genetic ablation of APC/C-Cdc20 completely
prevents mitotic exit, and these mutant cells arrest in mitosis until
they die.90 This action results in complete tumor repression in mouse
models, and a first generation of APC/C inhibitors is currently available
to test this strategy in preclinical studies (see Table 4.3).90,303,304
Targeting the Spindle Assembly Checkpoint
and Aneuploidy
Similarly to what was discussed for the DNA damage checkpoint, a
different concept is provided by the use of SAC inhibitors (checkpoint
abrogation) to increase instability in cancer cells. Checkpoint kinases
such as Mps1 (also known as TTK) and Aurora-B (not considered to
be a “core” checkpoint protein but involved in an error-correction
mechanism) are required for the proper bipolar spindle attachment
of chromosomes.3 Inhibition of these kinases results in rapid exit from
mitosis without chromosome segregation, generating tetraploid or
aneuploid cells. Important to note, inhibition of these molecules
prevents mitotic arrest in the presence of microtubule poisons and
therefore generates aberrant cells.52,305,306 It has been shown that the
reduction in these checkpoint proteins makes tumor cells more sensitive
than untransformed cells to low doses of spindle poisons.307 The efficacy
of several Aurora or Mps1 small-molecule inhibitors currently is being
tested in preclinical or clinical assays (see Table 4.3).52,295,306,308
Aneuploidy is a hallmark of cancer and is now considered as a
highly attractive therapeutic target.215,309 Most tumor cells are aneuploid,
whereas this abnormality is infrequent (although this has not been
precisely established) in wild-type cells. This specificity is especially
interesting given the problems that most therapeutic strategies that
target mitosis have in specifically inhibiting tumor cells. Aneuploid
cells display specific defects in cell cycle kinetics, growth rate, metabo-
lism, and the response to specific stresses.310,311 These defects can be
exploited by targeting specific cellular pathways that protect cells from
the deleterious effects of aneuploidy. For instance, inhibiting the
proteotoxic and metabolic stress pathways specifically reduces the
viability of aneuploidy cells because of the unbalanced load of proteins
in these cells.312 As previously described, targeting the mitotic checkpoint
may increase the levels of aneuploidy in the tumor cells, which opens
the opportunity to combine different types of antimitotic strategies
to further induce chromosomal instability and aneuploidy and inhibit
the pathways that cancer cells use to tolerate this abnormal state.309
SUMMARY
During the past several decades, investigators have uncovered a wealth
of information about the proteins that control the division of human
cells. A key finding is that deregulation of the cell cycle machinery and/
or its checkpoints is a universal alteration in human cancer.1,2 Because
the basic machinery that controls the cell cycle is similar in all cell
types, the hope is that common strategies will be developed against
a wide variety of cancers. Even though several of the currently used
anticancer therapies target nonselective and non–mechanism-based
targets, their effectiveness, albeit limited in many cases, is likely due
to the fact that they ultimately target cell cycle regulatory or DDR
signaling pathways, the status of which is different in normal cells
versus tumor cells. Identification of all the components of the cellular
machinery that control the cell cycle both positively and negatively
is vital to the continued development of anticancer agents that can
preferentially eliminate cancer cells and minimize the toxicity to normal
tissues. The complexity of the human genome makes this task difficult
because many members of the major protein families that regulate the
cell cycle have not been studied thus far. In addition, our knowledge of
the in vivo relevance of these proteins in different tissues is limited. It is
obvious from the mouse models studied that the efficacy of inhibiting
specific cell cycle targets depends on the tumor type and the oncogenic
environment in each specific tumor. As our understanding of cell cycle
regulation and checkpoint signaling improves, the goal is to use this
knowledge in the design of mechanism-based therapeutics that will
bring anticancer therapy to a new level. There can be little doubt of
the value of targeting the cell cycle in drug discovery.
The complete reference list is available online at
ExpertConsult.com.

KEY REFERENCES
1. Malumbres M, Barbacid M. To cycle or not to
cycle: a critical decision in cancer. Nat Rev Cancer.
2001;1:222–231.
2. Hartwell LH, Kastan MB. Cell cycle control and
cancer. Science. 1994;266:1821–1828.
3. Malumbres M. Physiological relevance of cell cycle
kinases. Physiol Rev. 2011;91:973–1007.
4. Hartwell LH, Weinert TA. Checkpoints: controls
that ensure the order of cell cycle events. Science.
1989;246:629–634.
5. Kastan MB, Bartek J. Cell-cycle checkpoints and
cancer. Nature. 2004;432:316–323.
6. Holland AJ, Cleveland DW. Boveri revisited: chro-
mosomal instability, aneuploidy and tumorigenesis.
Nat Rev Mol Cell Biol. 2009;10:478–487.

8. Morgan DO. Cyclin-dependent kinases: engines,
clocks, and microprocessors. Annu Rev Cell Dev Biol.
1997;13:261–291.
9. Malumbres M, Barbacid M. Mammalian
cyclin-dependent kinases. Trends Biochem Sci.
2005;30:630–641.
20. Boutros R, Lobjois V, Ducommun B. CDC25
phosphatases in cancer cells: key players? Good
targets? Nat Rev Cancer. 2007;7:495–507.
32. Dyson NJ. RB1: a prototype tumor suppressor and
an enigma. Genes Dev. 2016;30:1492–1502.
34. Chen HZ, Tsai SY, Leone G. Emerging roles of
E2Fs in cancer: an exit from cell cycle control. Nat
Rev Cancer. 2009;9:785–797.
39. Peters JM. The anaphase promoting complex/
cyclosome: a machine designed to destroy. Nat Rev
Mol Cell Biol. 2006;7:644–656.
40. Silverman JS, Skaar JR, Pagano M. SCF ubiquitin
ligases in the maintenance of genome stability. Trends
Biochem Sci. 2012;37:66–73.
45. Alfieri C, Chang L, Zhang Z, Yang J, Maslen S,
Skehel M, et al. Molecular basis of APC/C regula-
tion by the spindle assembly checkpoint. Nature.
2016;536:431–436.
52. Lens SM, Voest EE, Medema RH. Shared and
separate functions of polo-like kinases and aurora
kinases in cancer. Nat Rev Cancer. 2010;10:825–
841.
70. Cross RA, McAinsh A. Prime movers: the mecha-
nochemistry of mitotic kinesins. Nat Rev Mol Cell
Biol. 2014;15:257–271.
76. Etemad B, Kops GJ. Attachment issues: kinetochore
transformations and spindle checkpoint silencing.
Curr Opin Cell Biol. 2016;39:101–108.
77. Weir JR, Faesen AC, Klare K, Petrovic A, Basilico
F, Fischböck J, et al. Insights from biochemical
reconstitution into the architecture of human
kinetochores. Nature. 2016;537:249–253.
78. Wurzenberger C, Gerlich DW. Phosphatases:
providing safe passage through mitotic exit. Nat
Rev Mol Cell Biol. 2011;12:469–482.
83. Qian J, Winkler C, Bollen M. 4D-networking
by mitotic phosphatases. Curr Opin Cell Biol.
2013;25:697–703.
87. Glover DM. The overlooked greatwall: a new perspec-
tive on mitotic control. Open Biol. 2012;2:120023.
ADDITIONAL RESOURCES
Chromosome aberrations in cancer: http://cgap.nci.nih.gov/
Chromosomes/Mitelman.
Clinical trials: http://www.clinicaltrials.gov/.
p53 Databases: http://www-p53.iarc.fr/; http://p53.free.fr/
Database/p53_database.html.
115. Santamaria D, Barriere C, Cerqueira A, et al. Cdk1 is
sufficient to drive the mammalian cell cycle. Nature.
2007;448:811–815.
117. Bell SP, Dutta A. DNA replication in eukaryotic
cells. Annu Rev Biochem. 2002;71:333–374.
144. Nasmyth K. Cohesin: a catenase with separate entry
and exit gates? Nat Cell Biol. 2011;13:1170–1177.
145. Losada A. Cohesin in cancer: chromosome segrega-
tion and beyond. Nat Rev Cancer. 2014;14:389–
393.
152. Lindqvist A, Rodríguez-Bravo V, Medema RH.
The decision to enter mitosis: feedback and
redundancy in the mitotic entry network. J Cell
Biol. 2009;185:193–202.
159. Musacchio A, Salmon ED. The spindle-assembly
checkpoint in space and time. Nat Rev Mol Cell
Biol. 2007;8:379–393.
160. Sacristan C, Kops GJ. Joined at the hip: kinetochores,
microtubules, and spindle assembly checkpoint
signaling. Trends Cell Biol. 2015;25:21–28.
161. Sullivan M, Morgan DO. Finishing mitosis, one
step at a time. Nat Rev Mol Cell Biol. 2007;8:894–
903.
168. Mierzwa B, Gerlich DW. Cytokinetic abscission:
molecular mechanisms and temporal control. Dev
Cell. 2014;31:525–538.
172. Nähse V, Christ L, Stenmark H, Campsteijn C.
The abscission checkpoint: making it to the final
cut. Trends Cell Biol. 2017;27:1–11.
173. Bartek J, Lukas J. DNA damage checkpoints: from
initiation to recovery or adaptation. Curr Opin Cell
Biol. 2007;19:238–245.
189. Collado M, Serrano M. Senescence in tumours:
evidence from mice and humans. Nat Rev Cancer.
2010;10:51–57.
190. Bartkova J, Rezaei N, Liontos M, et al. Oncogene-
induced senescence is part of the tumorigenesis
barrier imposed by DNA damage checkpoints.
Nature. 2006;444:633–637.
191. Di Micco R, Fumagalli M, Cicalese A, et al.
Oncogene-induced senescence is a DNA damage
response triggered by DNA hyper-replication.
Nature. 2006;444:638–642.
204. Muñoz S, Méndez J. DNA replication stress:
from molecular mechanisms to human disease.
Chromosoma. 2016;doi:10.1007/s00412-016-0573-x.
Control of the Cell Cycle  •  CHAPTER 4 73
209. Branzei D, Foiani M. Regulation of DNA repair
throughout the cell cycle. Nat Rev Mol Cell Biol.
2008;9:297–308.
210. Lobrich M, Jeggo PA. The impact of a negligent
G2/M checkpoint on genomic instability and cancer
induction. Nat Rev Cancer. 2007;7:861–869.
215. Gordon DJ, Resio B, Pellman D. Causes and
consequences of aneuploidy in cancer. Nat Rev
Genet. 2012;13:189–203.
246. Wang W. Emergence of a DNA-damage response
network consisting of Fanconi anaemia and BRCA
proteins. Nat Rev Genet. 2007;8:735–748.
248. Kops GJ, Weaver BA, Cleveland DW. On the road to
cancer: aneuploidy and the mitotic checkpoint. Nat
Rev Cancer. 2005;5:773–785.
265. Stephens PJ, Greenman CD, Fu B, et al. Massive
genomic rearrangement acquired in a single
catastrophic event during cancer development. Cell.
2011;144:27–40.
268. Malumbres M, Barbacid M. Cell cycle, CDKs
and cancer: a changing paradigm. Nat Rev Cancer.
2009;9:153–166.
279. Asghar U, Witkiewicz AK, Turner NC, Knudsen ES.
The history and future of targeting cyclin-dependent
kinases in cancer therapy. Nat Rev Drug Discov.
2015;14:130–146.
280. Finn RS, Martin M, Rugo HS, Jones S, Im
SA, Gelmon K, et al. Palbociclib and Letrozole
in Advanced Breast Cancer. N Engl J Med.
2016;375:1925–1936.
283. Bryant HE, Schultz N, Thomas HD, et al.
Specific killing of BRCA2-deficient tumours with
inhibitors of poly(ADP-ribose) polymerase. Nature.
2005;434:913–917.
284. Garrett MD, Collins I. Anticancer therapy with
checkpoint inhibitors: what, where and when? Trends
Pharmacol Sci. 2011;32:308–316.
292. Dumontet C, Jordan MA. Microtubule-binding
agents: a dynamic field of cancer therapeutics. Nat
Rev Drug Discov. 2010;9:790–803.
306. Dominguez-Brauer C, Thu KL, Mason JM, Blaser
H, Bray MR, Mak TW. Targeting Mitosis in Cancer:
Emerging Strategies. Mol Cell. 2015;60:524–536.
312. Sheltzer JM, Amon A. The aneuploidy paradox:
costs and benefits of an incorrect karyotype. Trends
Genet. 2011;27:446–453.

REFERENCES
1. Malumbres M, Barbacid M. To cycle or not to
cycle: a critical decision in cancer. Nat Rev Cancer.
2001;1:222–231.
2. Hartwell LH, Kastan MB. Cell cycle control and
cancer. Science. 1994;266:1821–1828.
3. Malumbres M. Physiological relevance of cell cycle
kinases. Physiol Rev. 2011;91:973–1007.
4. Hartwell LH, Weinert TA. Checkpoints: controls
that ensure the order of cell cycle events. Science.
1989;246:629–634.
5. Kastan MB, Bartek J. Cell-cycle checkpoints and
cancer. Nature. 2004;432:316–323.
6. Holland AJ, Cleveland DW. Boveri revisited: chro-
mosomal instability, aneuploidy and tumorigenesis.
Nat Rev Mol Cell Biol. 2009;10:478–487.
7. Salazar-Roa M, Malumbres M. Fueling the cell
division cycle. Trends Cell Biol. 2017;27:69–81.
8. Morgan DO. Cyclin-dependent kinases: engines,
clocks, and microprocessors. Annu Rev Cell Dev Biol.
1997;13:261–291.
9. Malumbres M, Barbacid M. Mammalian cyclin-
dependent kinases. Trends Biochem Sci. 2005;30:
630–641.
10. Malumbres M, Harlow E, Hunt T, et al. Cyclin-
dependent kinases: a family portrait. Nat Cell Biol.
2009;11:1275–1276.
11. Malumbres M. Cyclin-dependent kinases. Genome
Biol. 2014;15:122.
12. Lee MG, Nurse P. Complementation used to clone
a human homologue of the fission yeast cell cycle
control gene cdc2. Nature. 1987;327:31–35.
13. Pavletich NP. Mechanisms of cyclin-dependent
kinase regulation: structures of Cdks, their cyclin
activators, and Cip and INK4 inhibitors. J Mol Biol.
1999;287:821–828.
14. Evans T, Rosenthal ET, Youngblom J, et al. Cyclin:
a protein specified by maternal mRNA in sea urchin
eggs that is destroyed at each cleavage division. Cell.
1983;33:389–396.
15. Deshpande A, Sicinski P, Hinds PW. Cyclins and cdks
in development and cancer: a perspective. Oncogene.
2005;24:2909–2915.
16. Russo AA, Jeffrey PD, Pavletich NP. Structural
basis of cyclin-dependent kinase activation by
phosphorylation. Nat Struct Biol. 1996;3:696–700.
17. Lolli G, Johnson LN. CAK-cyclin-dependent
activating kinase: a key kinase in cell cycle control
and a target for drugs? Cell Cycle. 2005;4:572–577.
18. Fisher RP. Secrets of a double agent: CDK7 in
cell-cycle control and transcription. J Cell Sci.
2005;118:5171–5180.
19. Kellogg DR. Wee1-dependent mechanisms required
for coordination of cell growth and cell division.
J Cell Sci. 2003;116:4883–4890.
20. Boutros R, Lobjois V, Ducommun B. CDC25
phosphatases in cancer cells: key players? Good
targets? Nat Rev Cancer. 2007;7:495–507.
21. Sherr CJ, Roberts JM. CDK inhibitors: positive
and negative regulators of G1-phase progression.
Genes Dev. 1999;13:1501–1512.
22. LaBaer J, Garrett MD, Stevenson LF, et al. New
functional activities for the p21 family of CDK
inhibitors. Genes Dev. 1997;11:847–862.
23. Chu I, Sun J, Arnaout A, et al. p27 phosphorylation
by Src regulates inhibition of cyclin E-Cdk2. Cell.
2007;128:281–294.
24. Grimmler M, Wang Y, Mund T, et al. Cdk-
inhibitory activity and stability of p27Kip1 are
directly regulated by oncogenic tyrosine kinases.
Cell. 2007;128:269–280.
25. Larrea MD, Liang J, Da Silva T, et al. Phosphoryla-
tion of p27Kip1 regulates assembly and activation of
cyclin D1-Cdk4. Mol Cell Biol. 2008;28:6462–6472.
26. Jakel H, Weinl C, Hengst L. Phosphorylation
of p27Kip1 by JAK2 directly links cytokine
receptor signaling to cell cycle control. Oncogene.
2011;30:3502–3512.
27. Besson A, Assoian RK, Roberts JM. Regulation of
the cytoskeleton: an oncogenic function for CDK
inhibitors? Nat Rev Cancer. 2004;4:948–955.
28. Besson A, Dowdy SF, Roberts JM. CDK inhibi-
tors: cell cycle regulators and beyond. Dev Cell.
2008;14:159–169.
29. Weinberg RA. The retinoblastoma gene and gene
product. Cancer Surv. 1992;12:43–57.
30. Helt A-M, Galloway DA. Mechanisms by which
DNA tumor virus oncoproteins target the Rb family
of pocket proteins. Carcinogenesis. 2003;24:159–169.
31. DeCaprio JA. How the Rb tumor suppressor
structure and function was revealed by the study of
Adenovirus and SV40. Virology. 2009;384:274–284.
32. Dyson NJ. RB1: a prototype tumor suppressor and
an enigma. Genes Dev. 2016;30:1492–1502.
33. van den Heuvel S, Dyson NJ. Conserved functions
of the pRB and E2F families. Nat Rev Mol Cell Biol.
2008;9:713–724.
34. Chen HZ, Tsai SY, Leone G. Emerging roles of
E2Fs in cancer: an exit from cell cycle control. Nat
Rev Cancer. 2009;9:785–797.
35. Trimarchi JM, Lees JA. Sibling rivalry in the E2F
family. Nat Rev Mol Cell Biol. 2002;3:11–20.
36. Chong JL, Wenzel PL, Saenz-Robles MT, et al.
E2f1-3 switch from activators in progenitor
cells to repressors in differentiating cells. Nature.
2009;462:930–934.
37. Iglesias A, Murga M, Laresgoiti U, et al. Dia-
betes and exocrine pancreatic insufficiency in
E2F1/E2F2 double-mutant mice. J Clin Invest.
2004;113:1398–1407.
38. Infante A, Laresgoiti U, Fernandez-Rueda J, et al.
E2F2 represses cell cycle regulators to maintain
quiescence. Cell Cycle. 2008;7:3915–3927.
39. Peters JM. The anaphase promoting complex/cyclo-
some: a machine designed to destroy. Nat Rev Mol
Cell Biol. 2006;7:644–656.
40. Silverman JS, Skaar JR, Pagano M. SCF ubiquitin
ligases in the maintenance of genome stability. Trends
Biochem Sci. 2012;37:66–73.
41. Skaar JR, Pagano M. Control of cell growth by the
SCF and APC/C ubiquitin ligases. Curr Opin Cell
Biol. 2009;21:816–824.
42. Mocciaro A, Rape M. Emerging regulatory mecha-
nisms in ubiquitin-dependent cell cycle control.
J Cell Sci. 2012;125:255–263.
43. Eguren M, Manchado E, Malumbres M. Non-mitotic
functions of the Anaphase-Promoting Complex.
Semin Cell Dev Biol. 2011;22:572–578.
44. Choudhury R, Bonacci T, Arceci A, Lahiri D, Mills
CA, Kernan JL, et al. APC/C and SCF(cyclin F)
constitute a reciprocal feedback circuit controlling
S-phase entry. Cell Rep. 2016;16:3359–3372.
45. Alfieri C, Chang L, Zhang Z, Yang J, Maslen S,
Skehel M, et al. Molecular basis of APC/C regula-
tion by the spindle assembly checkpoint. Nature.
2016;536:431–436.
46. Zhang S, Chang L, Alfieri C, Zhang Z, Yang J,
Maslen S, et al. Molecular mechanism of APC/C
activation by mitotic phosphorylation. Nature.
2016;533:260–264.
47. Brown NG, VanderLinden R, Watson ER,
Weissmann F, Ordureau A, Wu KP, et al. E3
architectures regulate multiubiquitination and ubiq-
uitin chain elongation by APC/C. Cell. 2016;165:
1440–1453.
48. Qiao R, Weissmann F, Yamaguchi M, Brown NG,
VanderLinden R, Imre R, et al. Mechanism of APC/
CCDC20 activation by mitotic phosphorylation.
Proc Natl Acad Sci USA. 2016;113:E2570–E2578.
49. Fujimitsu K, Grimaldi M, Yamano H. Cyclin-
dependent kinase 1-dependent activation of APC/C
ubiquitin ligase. Science. 2016;352:1121–1124.
50. Archambault V, Glover DM. Polo-like kinases:
conservation and divergence in their functions and
regulation. Nat Rev Mol Cell Biol. 2009;10:265–275.
Control of the Cell Cycle  •  CHAPTER 4 73.e1
73.e1
51. Carmena M, Earnshaw WC. The cellular geog-
raphy of aurora kinases. Nat Rev Mol Cell Biol.
2003;4:842–854.
52. Lens SM, Voest EE, Medema RH. Shared and
separate functions of polo-like kinases and aurora
kinases in cancer. Nat Rev Cancer. 2010;10:825–841.
53. de Carcer G, Manning G, Malumbres M. From Plk1
to Plk5: functional evolution of polo-like kinases.
Cell Cycle. 2011;10:2255–2262.
54. Asteriti IA, Rensen WM, Lindon C, et al. The
Aurora-A/TPX2 complex: a novel oncogenic holo-
enzyme? Biochim Biophys Acta. 2010;1806:230–239.
55. Yang KT, Li SK, Chang CC, et al. Aurora-C kinase
deficiency causes cytokinesis failure in meiosis I and
production of large polyploid oocytes in mice. Mol
Biol Cell. 2010;21:2371–2383.
56. Fernandez-Miranda G, Trakala M, Martin J, et al.
Genetic disruption of aurora B uncovers an essential
role for aurora C during early mammalian develop-
ment. Development. 2011;138:2661–2672.
57. Avo Santos M, van de Werken C, de Vries M, et al.
A role for Aurora C in the chromosomal passenger
complex during human preimplantation embryo
development. Hum Reprod. 2011;26:1868–1881.
58. Petronczki M, Lenart P, Peters JM. Polo on the
rise—from mitotic entry to cytokinesis with
Plk1. Dev Cell. 2008;14:646–659.
59. Bettencourt-Dias M, Rodrigues-Martins A,
Carpenter L, et al. SAK/PLK4 is required for
centriole duplication and flagella development.
Curr Biol. 2005;15:2199–2207.
60. Rodrigues-Martins A, Riparbelli M, Callaini G,
et al. Revisiting the role of the mother centriole in
centriole biogenesis. Science. 2007;316:1046–1050.
61. Fry AM, O’Regan L, Sabir SR, Bayliss R. Cell cycle
regulation by the NEK family of protein kinases.
J Cell Sci. 2012;125:4423–4433.
62. Quarmby LM, Mahjoub MR. Caught Nek-ing: cilia
and centrioles. J Cell Sci. 2005;118:5161–5169.
63. Sdelci S, Bertran MT, Roig J. Nek9, Nek6, Nek7
and the separation of centrosomes. Cell Cycle.
2011;10:3816–3817.
64. Wang F, Dai J, Daum JR, Niedzialkowska E,
Banerjee B, Stukenberg PT, et al. Histone H3 Thr-3
phosphorylation by Haspin positions Aurora B at
centromeres in mitosis. Science. 2010;330:231–235.
65. Kelly AE, Ghenoiu C, Xue JZ, Zierhut C, Kimura H,
Funabiki H. Survivin reads phosphorylated histone
H3 threonine 3 to activate the mitotic kinase Aurora
B. Science. 2010;330:235–239.
66. Moutinho-Santos T, Maiato H. Plk1 puts a (Has)
pin on the mitotic histone code. EMBO Rep.
2014;15:203–204.
67. Varier RA, Outchkourov NS, de Graaf P, van
Schaik FM, Ensing HJ, Wang F, et al. A phospho/
methyl switch at histone H3 regulates TFIID
association with mitotic chromosomes. EMBO J.
2010;29:3967–3978.
68. Alvarez-Fernández M, Malumbres M. Preparing a
cell for nuclear envelope breakdown: spatio-temporal
control of phosphorylation during mitotic entry.
Bioessays. 2014;36:757–765.
69. Wordeman L. How kinesin motor proteins drive
mitotic spindle function: lessons from molecular
assays. Semin Cell Dev Biol. 2010;21:260–268.
70. Cross RA, McAinsh A. Prime movers: the mecha-
nochemistry of mitotic kinesins. Nat Rev Mol Cell
Biol. 2014;15:257–271.
71. Foley EA, Kapoor TM. Microtubule attachment
and spindle assembly checkpoint signalling at the
kinetochore. Nat Rev Mol Cell Biol. 2013;14:25–37.
72. Fukagawa T, Earnshaw WC. The centromere:
chromatin foundation for the kinetochore machinery.
Dev Cell. 2014;30:496–508.
73. Bloom KS. Centromeric heterochromatin: the
primordial segregation machine. Annu Rev Genet.
2014;48:457–484.
73.e2 Part I: Science and Clinical Oncology
74. McKinley KL, Cheeseman IM. The molecular basis
for centromere identity and function. Nat Rev Mol
Cell Biol. 2016;17:16–29.
75. Godek KM, Kabeche L, Compton DA. Regulation
of kinetochore-microtubule attachments through
homeostatic control during mitosis. Nat Rev Mol
Cell Biol. 2015;16:57–64.
76. Etemad B, Kops GJ. Attachment issues: kinetochore
transformations and spindle checkpoint silencing.
Curr Opin Cell Biol. 2016;39:101–108.
77. Weir JR, Faesen AC, Klare K, Petrovic A, Basilico
F, Fischböck J, et al. Insights from biochemical
reconstitution into the architecture of human
kinetochores. Nature. 2016;537:249–253.
78. Wurzenberger C, Gerlich DW. Phosphatases:
providing safe passage through mitotic exit. Nat
Rev Mol Cell Biol. 2011;12:469–482.
79. Mocciaro A, Schiebel E. Cdc14: a highly conserved
family of phosphatases with non-conserved func-
tions? J Cell Sci. 2010;123:2867–2876.
80. Virshup DM, Shenolikar S. From promiscuity to
precision: protein phosphatases get a makeover. Mol
Cell. 2009;33:537–545.
81. Kurimchak A, Grana X. PP2A holoenzymes nega-
tively and positively regulate cell cycle progression
by dephosphorylating pocket proteins and multiple
CDK substrates. Gene. 2012;499:1–7.
82. Kolupaeva V, Janssens V. PP1 and PP2A phos-
phatases–cooperating partners in modulating
retinoblastoma protein activation. FEBS J. 2013;280:
627–643.
83. Qian J, Winkler C, Bollen M. 4D-networking
by mitotic phosphatases. Curr Opin Cell Biol.
2013;25:697–703.
84. Grallert A, Boke E, Hagting A, Hodgson B,
Connolly Y, Griffiths JR, et al. A PP1-PP2A
phosphatase relay controls mitotic progression.
Nature. 2015;517:94–98.
85. Castilho PV, Williams BC, Mochida S, et al.
The M phase kinase Greatwall (Gwl) promotes
inactivation of PP2A/B55delta, a phosphatase
directed against CDK phosphosites. Mol Biol Cell.
2009;20:4777–4789.
86. Vigneron S, Brioudes E, Burgess A, et al. Greatwall
maintains mitosis through regulation of PP2A.
EMBO J. 2009;28:2786–2793.
87. Glover DM. The overlooked greatwall: a new perspec-
tive on mitotic control. Open Biol. 2012;2:120023.
88. Mochida S, Maslen SL, Skehel M, Hunt T.
Greatwall phosphorylates an inhibitor of protein
phosphatase 2A that is essential for mitosis. Science.
2010;330:1670–1673.
89. Gharbi-Ayachi A, Labbe JC, Burgess A, et al. The
substrate of Greatwall kinase, Arpp19, controls
mitosis by inhibiting protein phosphatase 2A. Science.
2010;330:1673–1677.
90. Manchado E, Guillamot M, de Carcer G, et al.
Targeting mitotic exit leads to tumor regression
in vivo: modulation by Cdk1, Mastl, and the
PP2A/B55alpha,delta phosphatase. Cancer Cell.
2010;18:641–654.
91. Schmitz MH, Held M, Janssens V, et al. Live-cell
imaging RNAi screen identifies PP2A-B55alpha
and importin-beta1 as key mitotic exit regulators
in human cells. Nat Cell Biol. 2010;12:886–893.
92. Wu JQ, Guo JY, Tang W, et al. PP1-mediated
dephosphorylation of phosphoproteins at mitotic exit
is controlled by inhibitor-1 and PP1 phosphoryla-
tion. Nat Cell Biol. 2009;11:644–651.
93. Takahashi Y, Rayman JB, Dynlacht BD. Analysis of
promoter binding by the E2F and pRB families in
vivo: distinct E2F proteins mediate activation and
repression. Genes Dev. 2000;14:804–816.
94. Rayman JB, Takahashi Y, Indjeian VB, et al. E2F
mediates cell cycle-dependent transcriptional repres-
sion in vivo by recruitment of an HDAC1/mSin3B
corepressor complex. Genes Dev. 2002;16:933–947.
95. Diehl JA, Cheng M, Roussel MF, Sherr CJ.
Glycogen synthase kinase-3beta regulates cyclin
D1 proteolysis and subcellular localization. Genes
Dev. 1998;12:3499–3511.
96. Klein EA, Assoian RK. Transcriptional regulation of
the cyclin D1 gene at a glance. J Cell Sci. 2008;121:
3853–3857.
97. Yu Q, Ciemerych MA, Sicinski P. Ras and Myc can
drive oncogenic cell proliferation through individual
D-cyclins. Oncogene. 2005;24:7114–7119.
98. Ewen ME, Sluss HK, Sherr CJ, et al. Functional
interactions of the retinoblastoma protein with
mammalian D-type cyclins. Cell. 1993;73:487–497.
99. Connell-Crowley L, Harper JW, Goodrich
DW. Cyclin D1/Cdk4 regulates retinoblastoma
protein-mediated cell cycle arrest by site-specific
phosphorylation. Mol Biol Cell. 1997;8:287–301.
100. Connell-Crowley L, Elledge SJ, Harper JW. G1
cyclin-dependent kinases are sufficient to initiate
DNA synthesis in quiescent human fibroblasts. Curr
Biol. 1998;8:65–68.
101. Kato J, Matsushime H, Hiebert SW, et al. Direct
binding of cyclin D to the retinoblastoma gene
product (pRb) and pRb phosphorylation by the
cyclin D-dependent kinase CDK4. Genes Dev.
1993;7:331–342.
102. Kollmann K, Heller G, Schneckenleithner C, Warsch
W, Scheicher R, Ott RG, et al. A kinase-independent
function of CDK6 links the cell cycle to tumor
angiogenesis. Cancer Cell. 2013;24:167–181.
103. Tigan AS, Bellutti F, Kollmann K, Tebb G, Sexl
V. CDK6-a review of the past and a glimpse into
the future: from cell-cycle control to transcriptional
regulation. Oncogene. 2016;35:3083–3091.
104. Ohtani K, DeGregori J, Nevins JR. Regulation of
the cyclin E gene by transcription factor E2F1. Proc
Natl Acad Sci USA. 1995;92:12146–12150.
105. Geng Y, Eaton EN, Picon M, et al. Regulation of
cyclin E transcription by E2Fs and retinoblastoma
protein. Oncogene. 1996;12:1173–1180.
106. Montagnoli A, Fiore F, Eytan E, et al. Ubiquitination
of p27 is regulated by Cdk-dependent phosphoryla-
tion and trimeric complex formation. Genes Dev.
1999;13:1181–1189.
107. Sheaff RJ, Groudine M, Gordon M, et al. Cyclin
E-CDK2 is a regulator of p27Kip1. Genes Dev.
1997;11:1464–1478.
108. Carrano AC, Eytan E, Hershko A, Pagano M. SKP2
is required for ubiquitin-mediated degradation of the
CDK inhibitor p27. Nat Cell Biol. 1999;1:193–199.
109. Koepp DM, Schaefer LK, Ye X, et al. Phos-
phorylation-dependent ubiquitination of cyclin
E by the SCFFbw7 ubiquitin ligase. Science.
2001;294:173–177.
110. Clurman BE, Sheaff RJ, Thress K, et al. Turnover
of cyclin E by the ubiquitin-proteasome pathway is
regulated by cdk2 binding and cyclin phosphoryla-
tion. Genes Dev. 1996;10:1979–1990.
111. Berthet C, Aleem E, Coppola V, et al. Cdk2 knock-
out mice are viable. Curr Biol. 2003;13:1775–1785.
112. Ortega S, Prieto I, Odajima J, et al. Cyclin-dependent
kinase 2 is essential for meiosis but not for mitotic
cell division in mice. Nat Genet. 2003;35:25–31.
113. Martin A, Odajima J, Hunt SL, et al. Cdk2 is
dispensable for cell cycle inhibition and tumor
suppression mediated by p27(Kip1) and p21(Cip1).
Cancer Cell. 2005;7:591–598.
114. Aleem E, Kiyokawa H, Kaldis P. Cdc2-cyclin E
complexes regulate the G1/S phase transition. Nat
Cell Biol. 2005;7(8):831–836.
115. Santamaria D, Barriere C, Cerqueira A, et al. Cdk1 is
sufficient to drive the mammalian cell cycle. Nature.
2007;448:811–815.
116. Viera A, Rufas JS, Martinez I, et al. CDK2 is required
for proper homologous pairing, recombination and
sex-body formation during male mouse meiosis.
J Cell Sci. 2009;122:2149–2159.
117. Bell SP, Dutta A. DNA replication in eukaryotic
cells. Annu Rev Biochem. 2002;71:333–374.
118. Bell SP, Stillman B. ATP-dependent recognition
of eukaryotic origins of DNA replication by a
multiprotein complex. Nature. 1992;357:128–
134.
119. Nougarede R, Della Seta F, Zarzov P, Schwob E.
Hierarchy of S-phase-promoting factors: yeast Dbf4-
Cdc7 kinase requires prior S-phase cyclin-dependent
kinase activation. Mol Cell Biol. 2000;20:3795–3806.
120. Zou L, Stillman B. Assembly of a complex contain-
ing Cdc45p, replication protein A, and Mcm2p
at replication origins controlled by S-phase cyclin-
dependent kinases and Cdc7p-Dbf4p kinase. Mol
Cell Biol. 2000;20:3086–3096.
121. Walter J, Newport J. Initiation of eukaryotic
DNA replication: origin unwinding and sequential
chromatin association of Cdc45, RPA, and DNA
polymerase alpha. Mol Cell. 2000;5:617–627.
122. Arias EE, Walter JC. Replication-dependent destruc-
tion of Cdt1 limits DNA replication to a single
round per cell cycle in Xenopus egg extracts. Genes
Dev. 2005;19:114–126.
123. Higa LA, Banks D, Wu M, et al. L2DTL/CDT2
interacts with the CUL4/DDB1 complex and PCNA
and regulates CDT1 proteolysis in response to DNA
damage. Cell Cycle. 2006;5:1675–1680.
124. Jin J, Arias EE, Chen J, et al. A family of diverse
Cul4-Ddb1-interacting proteins includes Cdt2,
which is required for S phase destruction of the
replication factor Cdt1. Mol Cell. 2006;23:709–721.
125. Sansam CL, Shepard JL, Lai K, et al. DTL/CDT2
is essential for both CDT1 regulation and the early
G2/M checkpoint. Genes Dev. 2006;20:3117–3129.
126. Arias EE, Walter JC. PCNA functions as a molecular
platform to trigger Cdt1 destruction and prevent
re-replication. Nat Cell Biol. 2006;8:84–90.
127. Sivaprasad U, Machida YJ, Dutta A. APC/C—the
master controller of origin licensing? Cell Div.
2007;2:8.
128. Pagano M, Pepperkok R, Verde F, et al. Cyclin A
is required at two points in the human cell cycle.
EMBO J. 1992;11:961–971.
129. Draetta G, Luca F, Westendorf J, et al. Cdc2 protein
kinase is complexed with both cyclin A and B:
evidence for proteolytic inactivation of MPF. Cell.
1989;56:829–838.
130. Cardoso MC, Leonhardt H, Nadal-Ginard B.
Reversal of terminal differentiation and control of
DNA replication: cyclin A and Cdk2 specifically
localize at subnuclear sites of DNA replication. Cell.
1993;74:979–992.
131. Dynlacht BD, Flores O, Lees JA, Harlow E. Dif-
ferential regulation of E2F transactivation by cyclin/
cdk2 complexes. Genes Dev. 1994;8:1772–1786.
132. Krek W, Xu G, Livingston DM. Cyclin A-kinase
regulation of E2F-1 DNA binding function
underlies suppression of an S phase checkpoint.
Cell. 1995;83:1149–1158.
133. Kalaszczynska I, Geng Y, Iino T, Mizuno S, Choi
Y, Kondratiuk I, et al. Cyclin A is redundant in
fibroblasts but essential in hematopoietic and
embryonic stem cells. Cell. 2009;138:352–365.
134. Kanakkanthara A, Jeganathan KB, Limzerwala JF,
Baker DJ, Hamada M, Nam HJ, et al. Cyclin A2
is an RNA binding protein that controls Mre11
mRNA translation. Science. 2016;353:1549–1552.
135. Stracker TH, Petrini JH. The MRE11 complex:
starting from the ends. Nat Rev Mol Cell Biol.
2011;12:90–103.
136. Glotzer M. The 3Ms of central spindle assembly:
microtubules, motors and MAPs. Nat Rev Mol Cell
Biol. 2009;10:9–20.
137. Hornick JE, Karanjeet K, Collins ES, Hinchcliffe
EH. Kinesins to the core: the role of microtubule-
based motor proteins in building the mitotic spindle
midzone. Semin Cell Dev Biol. 2010;21:290–299.
138. Nasmyth K, Haering CH. The structure and function
of SMC and kleisin complexes. Annu Rev Biochem.
2005;74:595–648.
139. Wood AJ, Severson AF, Meyer BJ. Condensin and
cohesin complexity: the expanding repertoire of
functions. Nat Rev Genet. 2010;11:391–404.

140. Sherwood R, Takahashi TS, Jallepalli PV. Sister
acts: coordinating DNA replication and cohesion
establishment. Genes Dev. 2010;24:2723–2731.
141. Jeppsson K, Kanno T, Shirahige K, Sjögren C. The
maintenance of chromosome structure: positioning
and functioning of SMC complexes. Nat Rev Mol
Cell Biol. 2014;15:601–614.
142. Nasmyth K, Haering CH. Cohesin: its roles and
mechanisms. Annu Rev Genet. 2009;43:525–558.
143. Gligoris T, Löwe J. Structural insights into ring
formation of cohesin and related Smc complexes.
Trends Cell Biol. 2016;26:680–693.
144. Nasmyth K. Cohesin: a catenase with separate entry
and exit gates? Nat Cell Biol. 2011;13:1170–1177.
145. Losada A. Cohesin in cancer: chromosome segrega-
tion and beyond. Nat Rev Cancer. 2014;14:389–393.
146. Hirano T. Condensin-based chromosome organiza-
tion from bacteria to vertebrates. Cell. 2016;164:
847–857.
147. Di Fiore B, Pines J. How cyclin A destruction
escapes the spindle assembly checkpoint. J Cell
Biol. 2010;190:501–509.
148. Lundgren K, Walworth N, Booher R, et al. mik1
and wee1 cooperate in the inhibitory tyrosine
phosphorylation of cdc2. Cell. 1991;64:1111–1122.
149. Parker LL, Piwnica-Worms H. Inactivation of the
p34cdc2-cyclin B complex by the human WEE1
tyrosine kinase. Science. 1992;257:1955–1957.
150. Heald R, McLoughlin M, McKeon F. Human wee1
maintains mitotic timing by protecting the nucleus
from cytoplasmically activated Cdc2 kinase. Cell.
1993;74:463–474.
151. Gavet O, Pines J. Activation of cyclin B1-Cdk1
synchronizes events in the nucleus and the cytoplasm
at mitosis. J Cell Biol. 2010;189:247–259.
152. Lindqvist A, Rodríguez-Bravo V, Medema RH.
The decision to enter mitosis: feedback and
redundancy in the mitotic entry network. J Cell
Biol. 2009;185:193–202.
153. Salic A, Waters JC, Mitchison TJ. Vertebrate
shugoshin links sister centromere cohesion and
kinetochore microtubule stability in mitosis. Cell.
2004;118:567–578.
154. Rivera T, Losada A. Shugoshin and PP2A, shared
duties at the centromere. Bioessays. 2006;28:775–779.
155. Watanabe Y. Shugoshin: guardian spirit at the
centromere. Curr Opin Cell Biol. 2005;17:590–595.
156. Champion L, Linder MI, Kutay U. Cellular
reorganization during mitotic entry. Trends Cell
Biol. 2017;27:26–41.
157. Álvarez-Fernández M, Sánchez-Martínez R, Sanz-
Castillo B, Gan PP, Sanz-Flores M, Trakala M, et al.
Greatwall is essential to prevent mitotic collapse after
nuclear envelope breakdown in mammals. Proc Natl
Acad Sci USA. 2013;110:17374–17379.
158. Diril MK, Ratnacaram CK, Padmakumar VC, Du T,
Wasser M, Coppola V, et al. Cyclin-dependent kinase
1 (Cdk1) is essential for cell division and suppression
of DNA re-replication but not for liver regeneration.
Proc Natl Acad Sci USA. 2012;109:3826–3831.
159. Musacchio A, Salmon ED. The spindle-assembly
checkpoint in space and time. Nat Rev Mol Cell
Biol. 2007;8:379–393.
160. Sacristan C, Kops GJ. Joined at the hip: kinetochores,
microtubules, and spindle assembly checkpoint
signaling. Trends Cell Biol. 2015;25:21–28.
161. Sullivan M, Morgan DO. Finishing mitosis, one step
at a time. Nat Rev Mol Cell Biol. 2007;8:894–903.
162. Gorr IH, Boos D, Stemmann O. Mutual inhibition
of separase and Cdk1 by two-step complex forma-
tion. Mol Cell. 2005;19:135–141.
163. Schockel L, Mockel M, Mayer B, et al. Cleavage of
cohesin rings coordinates the separation of centrioles
and chromatids. Nat Cell Biol. 2011;13:966–
972.
164. Garcia-Higuera I, Manchado E, Dubus P, et al.
Genomic stability and tumour suppression by the
APC/C cofactor Cdh1. Nat Cell Biol. 2008;10:
802–811.
165. Li M, Shin YH, Hou L, et al. The adaptor protein of
the anaphase promoting complex Cdh1 is essential
in maintaining replicative lifespan and in learning
and memory. Nat Cell Biol. 2008;10:1083–1089.
166. Vagnarelli P, Earnshaw WC. Repo-Man-PP1: a link
between chromatin remodelling and nuclear envelope
reassembly. Nucleus. 2012;3:138–142.
167. Green RA, Paluch E, Oegema K. Cytokinesis in
animal cells. Annu Rev Cell Dev Biol. 2012;28:29–58.
168. Mierzwa B, Gerlich DW. Cytokinetic abscission:
molecular mechanisms and temporal control. Dev
Cell. 2014;31:525–538.
169. D’Avino PP, Capalbo L. Regulation of midbody
formation and function by mitotic kinases. Semin
Cell Dev Biol. 2016;53:57–63.
170. Takaki T, Trenz K, Costanzo V, Petronczki M. Polo-
like kinase 1 reaches beyond mitosis–cytokinesis,
DNA damage response, and development. Curr
Opin Cell Biol. 2008;20:650–660.
171. Steigemann P, Wurzenberger C, Schmitz MH, et al.
Aurora B-mediated abscission checkpoint protects
against tetraploidization. Cell. 2009;136:473–484.
172. Nähse V, Christ L, Stenmark H, Campsteijn C.
The abscission checkpoint: making it to the final
cut. Trends Cell Biol. 2017;27:1–11.
173. Bartek J, Lukas J. DNA damage checkpoints: from
initiation to recovery or adaptation. Curr Opin Cell
Biol. 2007;19:238–245.
174. Taylor AM, Harnden DG, Arlett CF, et al. Ataxia
telangiectasia: a human mutation with abnormal
radiation sensitivity. Nature. 1975;258:427–429.
175. Klingseisen A, Jackson AP. Mechanisms and pathways
of growth failure in primordial dwarfism. Genes Dev.
2011;25:2011–2024.
176. Cuadrado M, Martinez-Pastor B, Murga M,
et al. ATM regulates ATR chromatin loading in
response to DNA double-strand breaks. J Exp Med.
2006;203:297–303.
177. Matsuoka S, Huang M, Elledge SJ. Linkage of ATM
to cell cycle regulation by the Chk2 protein kinase.
Science. 1998;282:1893–1897.
178. Matsuoka S, Rotman G, Ogawa A, et al. Ataxia
telangiectasia-mutated phosphorylates Chk2
in vivo and in vitro. Proc Natl Acad Sci USA.
2000;97:10389–10394.
179. Busino L, Donzelli M, Chiesa M, et al. Degradation
of Cdc25A by beta-TrCP during S phase and in
response to DNA damage. Nature. 2003;426:87–91.
180. Hirao A, Kong YY, Matsuoka S, et al. DNA damage-
induced activation of p53 by the checkpoint kinase
Chk2. Science. 2000;287:1824–1827.
181. Manfredi JJ. The Mdm2-p53 relationship evolves:
Mdm2 swings both ways as an oncogene and a
tumor suppressor. Genes Dev. 2010;24:1580–1589.
182. Brown CJ, Cheok CF, Verma CS, Lane DP. Reactiva-
tion of p53: from peptides to small molecules. Trends
Pharmacol Sci. 2011;32:53–62.
183. Montes de Oca Luna R, Wagner DS, Lozano G.
Rescue of early embryonic lethality in mdm2-
deficient mice by deletion of p53. Nature. 1995;378:
203–206.
184. Jones SN, Roe AE, Donehower LA, Bradley A.
Rescue of embryonic lethality in Mdm2-deficient
mice by absence of p53. Nature. 1995;378:206–208.
185. Waldman T, Kinzler KW, Vogelstein B. p21 is
necessary for the p53-mediated G1 arrest in human
cancer cells. Cancer Res. 1995;55:5187–5190.
186. el-Deiry WS, Tokino T, Velculescu VE, et al. WAF1,
a potential mediator of p53 tumor suppression. Cell.
1993;75:817–825.
187. Vousden KH, Lu X. Live or let die: the cell’s response
to p53. Nat Rev Cancer. 2002;2:594–604.
188. Serrano M, Lin AW, McCurrach ME, et al.
Oncogenic ras provokes premature cell senescence
associated with accumulation of p53 and p16INK4a.
Cell. 1997;88:593–602.
189. Collado M, Serrano M. Senescence in tumours:
evidence from mice and humans. Nat Rev Cancer.
2010;10:51–57.
Control of the Cell Cycle  •  CHAPTER 4 73.e3
73.e3
190. Bartkova J, Rezaei N, Liontos M, et al. Oncogene-
induced senescence is part of the tumorigenesis
barrier imposed by DNA damage checkpoints.
Nature. 2006;444:633–637.
191. Di Micco R, Fumagalli M, Cicalese A, et al.
Oncogene-induced senescence is a DNA damage
response triggered by DNA hyper-replication.
Nature. 2006;444:638–642.
192. Palmero I, Pantoja C, Serrano M. p19ARF
links the tumour suppressor p53 to Ras. Nature.
1998;395:125–126.
193. Malumbres M, Perez De Castro I, Hernandez MI,
et al. Cellular response to oncogenic ras involves
induction of the Cdk4 and Cdk6 inhibitor
p15(INK4b). Mol Cell Biol. 2000;20:2915–2925.
194. Sherr CJ. The INK4a/ARF network in tumour
suppression. Nat Rev Mol Cell Biol. 2001;2:731–737.
195. Dimri GP, Itahana K, Acosta M, Campisi J. Regula-
tion of a senescence checkpoint response by the
E2F1 transcription factor and p14(ARF) tumor
suppressor. Mol Cell Biol. 2000;20:273–285.
196. Kamijo T, Weber JD, Zambetti G, et al. Functional
and physical interactions of the ARF tumor sup-
pressor with p53 and Mdm2. Proc Natl Acad Sci
USA. 1998;95:8292–8297.
197. Honda R, Yasuda H. Association of p19(ARF) with
Mdm2 inhibits ubiquitin ligase activity of Mdm2 for
tumor suppressor p53. EMBO J. 1999;18:22–27.
198. Pomerantz J, Schreiber-Agus N, Liegeois NJ, et al.
The Ink4a tumor suppressor gene product, p19Arf,
interacts with MDM2 and neutralizes MDM2’s
inhibition of p53. Cell. 1998;92:713–723.
199. Weber JD, Taylor LJ, Roussel MF, et al. Nucleolar
Arf sequesters Mdm2 and activates p53. Nat Cell
Biol. 1999;1:20–26.
200. Higa LAA, Mihaylov IS, Banks DP, et al. Radiation-
mediated proteolysis of CDT1 by CUL4-ROC1 and
CSN complexes constitutes a new checkpoint. Nat
Cell Biol. 2003;5:1008–1015.
201. O’Connell BC, Harper JW. Ubiquitin proteasome
system (UPS): what can chromatin do for you? Curr
Opin Cell Biol. 2007;19:206–214.
202. Raman M, Havens CG, Walter JC, Harper JW. A
genome-wide screen identifies p97 as an essential
regulator of DNA damage-dependent CDT1
destruction. Mol Cell. 2011;44:72–84.
203. Gottifredi V, Prives C. The S phase checkpoint:
when the crowd meets at the fork. Semin Cell Dev
Biol. 2005;16:355–368.
204. Muñoz S, Méndez J. DNA replication stress:
from molecular mechanisms to human disease.
Chromosoma. 2016;doi:10.1007/s00412-016-0573-x.
205. Furnari B, Blasina A, Boddy MN, et al. Cdc25
inhibited in vivo and in vitro by checkpoint kinases
Cds1 and Chk1. Mol Biol Cell. 1999;10:833–
845.
206. Liu Q, Guntuku S, Cui XS, et al. Chk1 is an essential
kinase that is regulated by Atr and required for
the G(2)/M DNA damage checkpoint. Genes Dev.
2000;14:1448–1459.
207. Flynn RL, Zou L. ATR: a master conductor of
cellular responses to DNA replication stress. Trends
Biochem Sci. 2011;36:133–140.
208. Smith J, Tho LM, Xu N, Gillespie DA. The
ATM-Chk2 and ATR-Chk1 pathways in DNA
damage signaling and cancer. Adv Cancer Res.
2010;108:73–112.
209. Branzei D, Foiani M. Regulation of DNA repair
throughout the cell cycle. Nat Rev Mol Cell Biol.
2008;9:297–308.
210. Lobrich M, Jeggo PA. The impact of a negligent
G2/M checkpoint on genomic instability and cancer
induction. Nat Rev Cancer. 2007;7:861–869.
211. Bassermann F, Pagano M. Dissecting the role
of ubiquitylation in the DNA damage response
checkpoint in G2. Cell Death Differ. 2010;17:78–85.
212. van Vugt MA, Medema RH. Getting in and out of
mitosis with Polo-like kinase-1. Oncogene. 2005;24:
2844–2859.
73.e4 Part I: Science and Clinical Oncology
213. Mapelli M, Massimiliano L, Santaguida S, Musacchio
A. The Mad2 conformational dimer: structure and
implications for the spindle assembly checkpoint.
Cell. 2007;131:730–743.
214. Maresca TJ, Salmon ED. Welcome to a new kind
of tension: translating kinetochore mechanics into a
wait-anaphase signal. J Cell Sci. 2010;123:825–835.
215. Gordon DJ, Resio B, Pellman D. Causes and
consequences of aneuploidy in cancer. Nat Rev
Genet. 2012;13:189–203.
216. Malumbres M. Oncogene-induced mitotic stress: p53
and pRb get mad too. Cancer Cell. 2011;19:691–692.
217. Manning AL, Dyson NJ. RB: mitotic implica-
tions of a tumour suppressor. Nat Rev Cancer.
2012;12:220–226.
218. Weinstat-Saslow D, Merino MJ, Manrow RE, et al.
Overexpression of cyclin D mRNA distinguishes
invasive and in situ breast carcinomas from non-
malignant lesions. Nat Med. 1995;1:1257–1260.
219. Wang TC, Cardiff RD, Zukerberg L, et al. Mammary
hyperplasia and carcinoma in MMTV-cyclin D1
transgenic mice. Nature. 1994;369:669–671.
220. Bortner DM, Rosenberg MP. Induction of mammary
gland hyperplasia and carcinomas in transgenic
mice expressing human cyclin E. Mol Cell Biol.
1997;17:453–459.
221. Ortega S, Malumbres M, Barbacid M. Cyclin
D-dependent kinases, INK4 inhibitors and cancer.
Biochim Biophys Acta. 2002;1602:73–87.
222. Catzavelos C, Bhattacharya N, Ung YC, et al.
Decreased levels of the cell-cycle inhibitor p27Kip1
protein: prognostic implications in primary breast
cancer. Nat Med. 1997;3:227–230.
223. Porter PL, Malone KE, Heagerty PJ, et al. Expression
of cell-cycle regulators p27Kip1 and cyclin E, alone
and in combination, correlate with survival in young
breast cancer patients. Nat Med. 1997;3:222–225.
224. Chu IM, Hengst L, Slingerland JM. The Cdk
inhibitor p27 in human cancer: prognostic potential
and relevance to anticancer therapy. Nat Rev Cancer.
2008;8:253–267.
225. Malumbres M, Carnero A. Cell cycle deregulation:
a common motif in cancer. Prog Cell Cycle Res.
2003;5:5–18.
226. Gil J, Peters G. Regulation of the INK4b-ARF-
INK4a tumour suppressor locus: all for one or one
for all. Nat Rev Mol Cell Biol. 2006;7:667–677.
227. Bartkova J, Thullberg M, Rajpert-De Meyts E,
et al. Cell cycle regulators in testicular cancer: loss
of p18INK4C marks progression from carcinoma
in situ to invasive germ cell tumours. Int J Cancer.
2000;85:370–375.
228. Sanchez-Aguilera A, Delgado J, Camacho FI, et al.
Silencing of the p18INK4c gene by promoter
hypermethylation in Reed-Sternberg cells in Hodgkin
lymphomas. Blood. 2004;103:2351–2357.
229. Morishita A, Masaki T, Yoshiji H, et al. Reduced expres-
sion of cell cycle regulator p18(INK4C) in human
hepatocellular carcinoma. Hepatology. 2004;40:
677–686.
230. Wolfel T, Hauer M, Schneider J, et al. A p16INK4a-
insensitive CDK4 mutant targeted by cytolytic
T lymphocytes in a human melanoma. Science.
1995;269:1281–1284.
231. Sotillo R, Garcia JF, Ortega S, et al. Invasive
melanoma in Cdk4-targeted mice. Proc Natl Acad
Sci USA. 2001;98:13312–13317.
232. Sotillo R, Dubus P, Martin J, et al. Wide spectrum
of tumors in knock-in mice carrying a Cdk4
protein insensitive to INK4 inhibitors. EMBO J.
2001;20:6637–6647.
233. Gasparotto D, Maestro R, Piccinin S, et al. Over-
expression of CDC25A and CDC25B in head and
neck cancers. Cancer Res. 1997;57:2366–2368.
234. Wu W, Fan YH, Kemp BL, et al. Overexpression
of cdc25A and cdc25B is frequent in primary non-
small cell lung cancer but is not associated with
overexpression of c-myc. Cancer Res. 1998;58:4082–
4085.
235. Edlund K, Larsson O, Ameur A, et al. Data-driven
unbiased curation of the TP53 tumor suppressor
gene mutation database and validation by ultradeep
sequencing of human tumors. Proc Natl Acad Sci
USA. 2012;109:9551–9556.
236. Nigro JM, Baker SJ, Preisinger AC, et al. Mutations
in the p53 gene occur in diverse human tumour
types. Nature. 1989;342:705–708.
237. Momand J, Jung D, Wilczynski S, Niland J. The
MDM2 gene amplification database. Nucleic Acids
Res. 1998;26:3453–3459.
238. Scheffner M, Werness BA, Huibregtse JM, et al. The
E6 oncoprotein encoded by human papillomavirus
types 16 and 18 promotes the degradation of p53.
Cell. 1990;63:1129–1136.
239. Khanna KK. Cancer risk and the ATM gene: a con-
tinuing debate. J Natl Cancer Inst. 2000;92:795–802.
240. Barlow C, Hirotsune S, Paylor R, et al. Atm-deficient
mice: a paradigm of ataxia telangiectasia. Cell.
1996;86:159–171.
241. Xu Y, Ashley T, Brainerd EE, et al. Targeted
disruption of ATM leads to growth retardation,
chromosomal fragmentation during meiosis,
immune defects, and thymic lymphoma. Genes
Dev. 1996;10:2411–2422.
242. Petrini JH. The Mre11 complex and ATM: col-
laborating to navigate S phase. Curr Opin Cell Biol.
2000;12:293–296.
243. Bertoni F, Codegoni AM, Furlan D, et al. CHK1
frameshift mutations in genetically unstable colorec-
tal and endometrial cancers. Genes Chromosomes
Cancer. 1999;26:176–180.
244. Matsuoka S, Nakagawa T, Masuda A, et al. Reduced
expression and impaired kinase activity of a Chk2
mutant identified in human lung cancer. Cancer
Res. 2001;61:5362–5365.
245. Bell DW, Varley JM, Szydlo TE, et al. Heterozygous
germ line hCHK2 mutations in Li-Fraumeni
syndrome. Science. 1999;286:2528–2531.
246. Wang W. Emergence of a DNA-damage response
network consisting of Fanconi anaemia and BRCA
proteins. Nat Rev Genet. 2007;8:735–748.
247. di Masi A, Antoccia A. NBS1 Heterozygosity
and Cancer Risk. Curr Genomics. 2008;9:275–
281.
248. Kops GJ, Weaver BA, Cleveland DW. On the road to
cancer: aneuploidy and the mitotic checkpoint. Nat
Rev Cancer. 2005;5:773–785.
249. Beroukhim R, Mermel CH, Porter D, et al. The
landscape of somatic copy-number alteration across
human cancers. Nature. 2010;463:899–905.
250. Weaver BA, Cleveland DW. Aneuploidy: instiga-
tor and inhibitor of tumorigenesis. Cancer Res.
2007;67:10103–10105.
251. Funk LC, Zasadil LM, Weaver BA. Living in
CIN: mitotic infidelity and its consequences for
tumor promotion and suppression. Dev Cell.
2016;39:638–652.
252. Sansregret L, Swanton C. The role of aneuploidy
in cancer evolution. Cold Spring Harb Perspect Med.
2017;7:pii: a028373. doi:10.1101/cshperspect.
a028373.
253. Thompson SL, Compton DA. Examining the link
between chromosomal instability and aneuploidy in
human cells. J Cell Biol. 2008;180:665–672.
254. Ricke RM, van Ree JH, van Deursen JM. Whole
chromosome instability and cancer: a complex
relationship. Trends Genet. 2008;24:457–466.
255. Schvartzman JM, Sotillo R, Benezra R. Mitotic
chromosomal instability and cancer: mouse
modelling of the human disease. Nat Rev Cancer.
2010;10:102–115.
256. Cahill DP, Lengauer C, Yu J, et al. Mutations of
mitotic checkpoint genes in human cancers. Nature.
1998;392:300–303.
257. Lee H, Trainer AH, Friedman LS, et al. Mitotic
checkpoint inactivation fosters transformation in
cells lacking the breast cancer susceptibility gene,
Brca2. Mol Cell. 1999;4:1–10.
258. Perez de Castro I, de Carcer G, Malumbres M. A
census of mitotic cancer genes: new insights into
tumor cell biology and cancer therapy. Carcinogenesis.
2007;28:899–912.
259. Michel LS, Liberal V, Chatterjee A, et al. MAD2
haplo-insufficiency causes premature anaphase and
chromosome instability in mammalian cells. Nature.
2001;409:355–359.
260. Sotillo R, Schvartzman JM, Socci ND, Benezra R.
Mad2-induced chromosome instability leads to lung
tumour relapse after oncogene withdrawal. Nature.
2010;464:436–440.
261. Carter SL, Eklund AC, Kohane IS, et al. A signature
of chromosomal instability inferred from gene expres-
sion profiles predicts clinical outcome in multiple
human cancers. Nat Genet. 2006;38:1043–1048.
262. Yamada HY, Rao CV. Genes that modulate the sen-
sitivity for anti-microtubule drug-mediated chemo-
therapy. Curr Cancer Drug Targets. 2010;10:623–633.
263. Sansregret L, Patterson JO, Dewhurst S, López-
García C, Koch A, McGranahan N, et al. APC/C
dysfunction limits excessive cancer chromosomal
instability. Cancer Discov. 2017;doi:10.1158/2159-
8290.CD-16-0645.
264. Crasta K, Ganem NJ, Dagher R, et al. DNA
breaks and chromosome pulverization from errors
in mitosis. Nature. 2012;482:53–58.
265. Stephens PJ, Greenman CD, Fu B, et al. Massive
genomic rearrangement acquired in a single
catastrophic event during cancer development. Cell.
2011;144:27–40.
266. Janssen A, van der Burg M, Szuhai K, et al. Chromo-
some segregation errors as a cause of DNA damage
and structural chromosome aberrations. Science.
2011;333:1895–1898.
267. Hunt T. You never know: Cdk inhibitors as anti-
cancer drugs. Cell Cycle. 2008;7:3789–3790.
268. Malumbres M, Barbacid M. Cell cycle, CDKs
and cancer: a changing paradigm. Nat Rev Cancer.
2009;9:153–166.
269. Shapiro GI. Cyclin-dependent kinase pathways
as targets for cancer treatment. J Clin Oncol.
2006;24:1770–1783.
270. Malumbres M, Pevarello P, Barbacid M, Bischoff
JR. CDK inhibitors in cancer therapy: what is next?
Trends Pharmacol Sci. 2008;29:16–21.
271. Robles AI, Rodriguez-Puebla ML, Glick AB,
et al. Reduced skin tumor development in cyclin
D1-deficient mice highlights the oncogenic ras
pathway in vivo. Genes Dev. 1998;12:2469–2474.
272. Yu Q, Geng Y, Sicinski P. Specific protection
against breast cancers by cyclin D1 ablation. Nature.
2001;411:1017–1021.
273. Campaner S, Doni M, Hydbring P, et al. Cdk2
suppresses cellular senescence induced by the c-myc
oncogene. Nat Cell Biol. 2010;12:54–59.
274. Puyol M, Martin A, Dubus P, et al. A synthetic
lethal interaction between K-Ras oncogenes and
Cdk4 unveils a therapeutic strategy for non-small
cell lung carcinoma. Cancer Cell. 2010;18:63–73.
275. Landis MW, Pawlyk BS, Li T, et al. Cyclin
D1-dependent kinase activity in murine develop-
ment and mammary tumorigenesis. Cancer Cell.
2006;9:13–22.
276. Yu Q, Sicinska E, Geng Y, et al. Requirement for
CDK4 kinase function in breast cancer. Cancer Cell.
2006;9:23–32.
277. Sawai CM, Freund J, Oh P, Ndiaye-Lobry D, Bretz
JC, Strikoudis A, et al. Therapeutic targeting of the
cyclin D3:CDK4/6 complex in T cell leukemia.
Cancer Cell. 2012;22:452–465.
278. Malumbres M. Cell cycle-based therapies move
forward. Cancer Cell. 2012;22:419–420.
279. Asghar U, Witkiewicz AK, Turner NC, Knudsen ES.
The history and future of targeting cyclin-dependent
kinases in cancer therapy. Nat Rev Drug Discov.
2015;14:130–146.
280. Finn RS, Martin M, Rugo HS, Jones S, Im SA,
Gelmon K, et al. Palbociclib and Letrozole in

Advanced Breast Cancer. N Engl J Med. 2016;375:
1925–1936.
281. Patnaik A, Rosen LS, Tolaney SM, Tolcher AW,
Goldman JW, Gandhi L, et al. Efficacy and safety
of abemaciclib, an inhibitor of CDK4 and CDK6,
for patients with breast cancer, non-small cell lung
cancer, and other solid tumors. Cancer Discov.
2016;6:740–753.
282. Farmer H, McCabe N, Lord CJ, et al. Targeting
the DNA repair defect in BRCA mutant cells as
a therapeutic strategy. Nature. 2005;434:917–
921.
283. Bryant HE, Schultz N, Thomas HD, et al.
Specific killing of BRCA2-deficient tumours with
inhibitors of poly(ADP-ribose) polymerase. Nature.
2005;434:913–917.
284. Garrett MD, Collins I. Anticancer therapy with
checkpoint inhibitors: what, where and when? Trends
Pharmacol Sci. 2011;32:308–316.
285. Toledo LI, Murga M, Fernandez-Capetillo O. Target-
ing ATR and Chk1 kinases for cancer treatment:
a new model for new (and old) drugs. Mol Oncol.
2011;5:368–373.
286. Murga M, Campaner S, Lopez-Contreras AJ, et al.
Exploiting oncogene-induced replicative stress for the
selective killing of Myc-driven tumors. Nat Struct
Mol Biol. 2011;18:1331–1335.
287. Cottini F, Hideshima T, Suzuki R, Tai YT, Bianchini
G, Richardson PG, et al. Synthetic Lethal Approaches
Exploiting DNA Damage in Aggressive Myeloma.
Cancer Discov. 2015;5:972–987.
288. Kwok M, Davies N, Agathanggelou A, Smith E,
Petermann E, Yates E, et al. Synthetic lethality in
chronic lymphocytic leukaemia with DNA damage
response defects by targeting the ATR pathway.
Lancet. 2015;385(suppl 1):S58.
289. Williamson CT, Miller R, Pemberton HN, Jones
SE, Campbell J, Konde A, et al. ATR inhibitors as
a synthetic lethal therapy for tumours deficient in
ARID1A. Nat Commun. 2016;7:13837.
290. Morgado-Palacin I, Day A, Murga M, Lafarga V,
Anton ME, Tubbs A, et al. Targeting the kinase
activities of ATR and ATM exhibits antitumoral
activity in mouse models of MLL-rearranged AML.
Sci Signal. 2016;9:ra91.
291. Karnitz LM, Zou L. Molecular pathways: target-
ing ATR in cancer therapy. Clin Cancer Res.
2015;21:4780–4785.
292. Dumontet C, Jordan MA. Microtubule-binding
agents: a dynamic field of cancer therapeutics. Nat
Rev Drug Discov. 2010;9:790–803.
293. Komlodi-Pasztor E, Sackett D, Wilkerson J, Fojo
T. Mitosis is not a key target of microtubule
agents in patient tumors. Nat Rev Clin Oncol.
2011;8:244–250.
294. Mayer TU, Kapoor TM, Haggarty SJ, et al. Small
molecule inhibitor of mitotic spindle bipolarity
identified in a phenotype-based screen. Science.
1999;286:971–974.
295. Manchado E, Guillamot M, Malumbres M.
Killing cells by targeting mitosis. Cell Death Differ.
2012;19:369–377.
296. Wood KW, Chua P, Sutton D, Jackson JR.
Centromere-associated protein E: a motor that puts
the brakes on the mitotic checkpoint. Clin Cancer
Res. 2008;14:7588–7592.
297. Wood KW, Lad L, Luo L, et al. Antitumor
activity of an allosteric inhibitor of centromere-
associated protein-E. Proc Natl Acad Sci USA.
2010;107:5839–5844.
298. Gjertsen BT, Schöffski P. Discovery and development
of the Polo-like kinase inhibitor volasertib in cancer
therapy. Leukemia. 2015;29:11–19.
299. Goga A, Yang D, Tward AD, et al. Inhibition of
CDK1 as a potential therapy for tumors over-
expressing MYC. Nat Med. 2007;13:820–827.
300. Johnson N, Li YC, Walton ZE, et al. Compromised
CDK1 activity sensitizes BRCA-proficient cancers
to PARP inhibition. Nat Med. 2011;17:875–
882.
Control of the Cell Cycle  •  CHAPTER 4 73.e5
73.e5
301. Higgins JM. Haspin: a newly discovered regulator
of mitotic chromosome behavior. Chromosoma.
2010;119:137–147.
302. Huang HC, Shi J, Orth JD, Mitchison TJ.
Evidence that mitotic exit is a better cancer thera-
peutic target than spindle assembly. Cancer Cell.
2009;16:347–358.
303. Zeng X, Sigoillot F, Gaur S, et al. Pharmacologic
inhibition of the anaphase-promoting complex
induces a spindle checkpoint-dependent mitotic
arrest in the absence of spindle damage. Cancer
Cell. 2010;18:382–395.
304. Sackton KL, Dimova N, Zeng X, Tian W, Zhang M,
Sackton TB, et al. Synergistic blockade of mitotic exit
by two chemical inhibitors of the APC/C. Nature.
2014;514:646–649.
305. Liu X, Winey M. The MPS1 family of protein
kinases. Annu Rev Biochem. 2012;81:561–585.
306. Dominguez-Brauer C, Thu KL, Mason JM, Blaser
H, Bray MR, Mak TW. Targeting mitosis in cancer:
emerging strategies. Mol Cell. 2015;60:524–536.
307. Janssen A, Kops GJ, Medema RH. Elevating the
frequency of chromosome mis-segregation as a
strategy to kill tumor cells. Proc Natl Acad Sci USA.
2009;106:19108–19113.
308. Koch A, Maia A, Janssen A, Medema RH. Molecular
basis underlying resistance to Mps1/TTK inhibitors.
Oncogene. 2016;35:2518–2528.
309. Manchado E, Malumbres M. Targeting aneuploidy
for cancer therapy. Cell. 2011;144:465–466.
310. Williams BR, Prabhu VR, Hunter KE, et al.
Aneuploidy affects proliferation and spontaneous
immortalization in mammalian cells. Science.
2008;322:703–709.
311. Sheltzer JM, Amon A. The aneuploidy paradox:
costs and benefits of an incorrect karyotype. Trends
Genet. 2011;27:446–453.
312. Tang YC, Williams BR, Siegel JJ, Amon A. Iden-
tification of aneuploidy-selective antiproliferation
compounds. Cell. 2011;144:499–512.
 5  Pathophysiology of Cancer Cell Death
Lorenzo Galluzzi, Andreas Linkermann, Oliver Kepp, and Guido Kroemer

S UMMARY OF K EY
• Regulated cell death mainly occurs
via extrinsic apoptosis, intrinsic
apoptosis, necroptosis, and
mitochondrial permeability transition
(MPT)–driven regulated necrosis.
Autophagy operates as a bona fide
cell death mechanism in a few,
mostly developmental, settings.
P OI N T S
• Oncogenesis results from multiple
molecular alterations, one of which
frequently impairs the ability of
cancer cells to die in response to
exogenous or endogenous signals.
• Several oncoproteins and
oncosuppressor proteins regulate,
either directly or in an indirect
fashion, the molecular machinery for
apoptotic or necrotic cell death.
• Targeting deregulated cell death
signaling pathways in cancer
constitutes a clinical reality and
underlies promising approaches for
the development of novel anticancer
regimens.

Perhaps biased by their focus on the cell’s vital functions, biologists
have disregarded the existence of programmed cell death (PCD; see
later for a definition) for a long time. Sporadic observations of PCD
were made throughout the 19th century by scientists such as Carl
Vogt, August Weismann, Ludwig Stieda, Élie Metchnikoff, Walther
Flemming, Sigmund Mayer, and John Beard.1 The concept of PCD
was first theorized in the 1960s, thanks to the work of Sir Richard
Lockshin.2 In 1972 John Kerr, Alastair Currie, and Andrew Wyllie
introduced the term apoptosis (from Greek apo, “from, off, without,”
and ptosis, “falling”) to describe one type of cell death that manifests
with peculiar morphologic traits.3 At that time and for the subsequent
30 years, apoptosis was thought to be the only form of PCD, an
oversimplistic notion that has been invalidated only in this century.4,5
In the middle of the first decade of the century, it indeed became
clear that other subroutines of cell death, notably necrosis, can occur
in a regulated fashion and account for some instances of PCD.6–8
Regulated cell death (RCD) constitutes a conserved mechanism
whose usefulness trespasses evolutionistic barriers.9,10 For instance, in
unicellular organisms that grow in colonies (such as yeast), the controlled
demise of old and damaged cells increases the probability that fit
individuals will survive adverse environmental conditions, and hence
will perpetuate their genes.11 Conversely, in metazoans (including
humans), PCD is critical for embryonic and postembryonic develop-
ment, as well as for the maintenance of adult tissue homeostasis.12 In
line with this notion, defects in the molecular mechanisms that mediate
cell death contribute to the development of a wide array of human
diseases. On one hand, the excessive demise of postmitotic cells
decisively contributes to the pathogenesis of diseases encompassing
ischemia (of the heart, brain, and kidney) and neurodegeneration.
On the other hand, insufficient rates of cell death have been associated
with autoimmune disorders and cancer.13
The first hint that the molecular machinery for cell death is involved
in oncogenesis came in the mid-1980s, when it was found that a
translocation between chromosomes 14 and 18 (t14;18), which is
common in lymphoma patients, leads to the overexpression of the
protein BCL2.14 Subsequent work clarified that BCL2 promotes
lymphomagenesis by inhibiting the programmed demise of excessive
B cells rather than by stimulating their proliferation.15 This was the
first demonstration that disabled cell death constitutes one of the
hallmarks of cancer, irrespective of the histologic origin of malignant
cells.16 During the subsequent two decades, it rapidly became clear
that defects in the signaling pathways leading to cell death not only
contribute to oncogenesis and tumor progression, but also determine,
at least in some instances, the resistance of neoplastic cells to chemo-
therapy and radiotherapy.17
Our understanding of cell death is constantly advancing, and this
knowledge has already been translated into multiple therapeutic
successes. Nevertheless, future investigations will have to provide deeper
insights into the cancer-associated defects of cell death, in all its forms.
FUNDAMENTAL SCIENCE: MECHANISMS OF
CELL DEATH
In cell death research, the attribute “programmed” is used to highlight
the implication of one particular instance of cell death in developmental
programs (and hence its physiologic relevance), whereas the adjective
“regulated” is employed to stress the notion that one particular instance
of cell death can be inhibited by specific pharmacologic or genetic
interventions (and hence is mediated by genetically encoded molecular
mechanisms).4 Thus all instances of PCD are regulated by definition,
but not vice versa. Additional recommendations on how to define
specific cell death subroutines based on morphologic5 or biochemical4
parameters have been formulated in the past few years, and will be
respected throughout this chapter. The Nomenclature Committee on
Cell Death (NCCD) has expressed doubts regarding the causal
implication of specific biochemical processes in RCD.18 Indeed,
inhibiting the mechanisms that until now were considered to be the
cause of RCD (in all its instances) generally delays, but does not
prevent, cell death (at least in mammalian organisms).18 This has
far-reaching conceptual and therapeutic implications, especially for
the development of cytoprotective clinical interventions.18
According to currently accepted models, there are two main types
of cell death: apoptosis and necrosis.19–23 Additional cell death sub-
routines with very specific biochemical traits have been described,
although they often constitute particular cases of apoptosis and
necrosis.4,24,25 Macroautophagy (hereafter referred to as autophagy)
has also been implicated as a potential mechanism of cell death, a
notion that remains highly debated (see later).26–28

74

Apoptosis
For a long time, instances of cell death have been catalogued as
apoptotic based on purely morphologic manifestations, including
cytoplasmic and nuclear shrinkage (pyknosis), nuclear breakdown
(karyorrhexis), and plasma membrane blebbing.5,29 This morphologic
definition, reflecting the original observations by Kerr, Currie, and
Wyllie,3 has been abandoned in favor of a biochemical one.4 Thus,
the term apoptosis is now used to define an instance of RCD that is
precipitated by the activation of caspase 3 (CASP3), implying that it
can be delayed by chemical CASP3 inhibitors as well as by specific
genetic interventions that inactivate CASP3.18 Apoptosis can be triggered
by the activation of either of two distinct but not mutually exclusive
signaling pathways. Thus, whereas extrinsic apoptosis is initiated by an
extracellular stimulus acting on plasma membrane receptors, intrinsic
apoptosis follows the permeabilization of mitochondrial membranes
driven by an intracellular stimulus.4 Of note, the enzymatic activity
of CASP3, caspase 6 (CASP6), and caspase 7 (CASP7)—which are
cumulatively known as executioner caspases—is responsible for multiple
(but not all) classic morphologic and biochemical manifestations of
apoptosis, including karyorrhexis, internucleosomal DNA degradation,
and the exposure of the phospholipid phosphatidylserine on the outer
surface of the plasma membrane.30
Extrinsic apoptosis is frequently elicited by the ligand-induced
activation of plasma membrane proteins of the death receptor family,
such as Fas cell surface death receptor (FAS) and the tumor necrosis
factor (TNF) superfamily member 1A (TNFRSF1A, best known as
TNFR1).31 Alternatively, apoptotic signals can be transduced by the
so-called dependence receptors (such as Patched 1, PTCH1) when
the extracellular concentration of trophic factors, such as Sonic
Hedgehog (SHH), falls below a critical threshold level. This means
that, at odds with death receptors, dependence receptors deliver
proapoptotic signals in the absence rather than in the presence of
their ligands.32 Death receptors undergo spontaneous trimerization
owing to the so-called preligand assembly domain (PLAD).33 Ligand
binding stabilizes this configuration and hence allows for the recruitment
of several proteins at the cytoplasmic tails of death receptors, including
(but in some instances not limited to) pro-caspase 8 (pro-CASP8),
receptor interacting serine/threonine kinase 1 (RIPK1, also known as
RIP1), FAS-associated protein with a death domain (FADD), and
cellular inhibitor of apoptosis proteins (cIAP1 and cIAP2, which are
E3 ubiquitin ligases that also inhibit apoptosis owing to their ability
to interfere with caspase activation).34 This multiprotein platform is
known as death-inducing signaling complex (DISC) and stimulates
the conversion of pro-CASP8 into caspase 8 (CASP8), which proteoliti-
cally activates CASP3 and hence initiates the degradative cascade that
precipitates extrinsic apoptosis.35 Of note, the components of the
signaling pathways that emanate from specific death receptors exhibit
some degree of variation, yet all converge (in apoptosis-permissive
conditions; see later) to the activation of CASP8. The molecular
mechanisms that link dependence receptors to CASP3 activation are
not precisely understood, yet appear to involve the adaptor protein
DRAL and CASP9 (Fig. 5.1).36
Intrinsic apoptosis (also known as mitochondrial apoptosis) can be
activated by a plethora of stimuli, including intracellular damage (to
virtually any of the subcellular compartments) and oncogenic stress
(see later). Cells are equipped with a heterogeneous set of intracellular
sensors that respond to microenvironmental perturbations by activating
signaling pathways for the reestablishment of homeostasis and the
repair of damage, and then, if damage is irreparable, by igniting intrinsic
apoptosis.37 The central step in this cascade is regulated by the integrity
of mitochondrial structure and function. Indeed, if proapoptotic signals
are predominant, mitochondrial membranes get permeabilized, resulting
in the abrupt cessation of adenosine triphosphate (ATP) synthesis and
other metabolic functions, as well as in the spillage of several proteins
that are normally confined in the mitochondrial intermembrane space.37
These proteins include cytochrome c, somatic (CYCS, a semisoluble
Pathophysiology of Cancer Cell Death  •  CHAPTER 5 75
component of the mitochondrial respiratory chain), diablo IAP-binding
mitochondrial protein (DIABLO), and HtrA serine peptidase 2
(HTRA2).38–40 Once in the cytosol, CYCS—together with dATP and
the adaptor protein apoptotic peptidase activating factor 1 (APAF1)—
drives the assembly of the apoptosome, a supramolecular platform
for the activation of CASP9.40 DIABLO and HTRA2 also stimulate
caspase activation, although indirectly, by sequestering and/or degrading
cytosolic caspase inhibitors of the IAP family.38,39 Other mitochondrial
proteins with cytotoxic potential are released in the cytosol during
the course of intrinsic apoptosis, including apoptosis inducing factor,
mitochondria associated 1 (AIFM1, which normally contributes to
the stability and function of respiratory complex I) and endonuclease
G (ENDOG).41,42 Although both AIFM1 and ENDOG can translocate
to the nucleus and mediate large-scale DNA degradation independently
of caspases,42,43 their implication in intrinsic apoptosis has been
dismissed.44,45
According to current models, mitochondrial outer membrane
permeabilization (MOMP) is initiated at the mitochondrial outer
membrane (OM) by the pore-forming activity of proapoptotic
multidomain proteins of the Bcl-2 family.19 These proteins, such as
BCL2 associated X, apoptosis regulator (BAX), and BCL2 antagonist/
killer 1 (BAK1), contain several Bcl-2 homology (BH) domains as
well as a transmembrane domain that allow for their constitutive or
inducible insertion into the OM. MOMP execution by BAK1 and
BAX is regulated by other members of the same protein family. In
particular, antiapoptotic proteins such as BCL2, apoptosis regulator
(BCL2), BCL2-like 1 (BCL2L1, best known as BCL-xL), and MCL1,
BCL2 family apoptosis regulator (MCL1) inhibit MOMP by binding
to BAX and BAK1 and hence by maintaining them in an inactive
conformation.46 Conversely, the so-called BH3-only proteins (small
members of the Bcl-2 family that often contain only the BH3 domain)
can promote the pore-forming activity of BAK1 and BAX by two
different mechanisms. Thus, BH3-only proteins can either stimulate
the conformational activation of BAX and BAK1 in a direct fashion
or competitively displace BAX, BAK1 or other BH3-only proteins
from inhibitory interactions with BCL2, BCL-xL and MCL1. BH3-only
proteins can be regulated at the transcriptional level as well as by
rapid posttranslational modifications (e.g., phosphorylation, proteolytic
processing), de facto constituting sensors of intracellular stress that
directly impinge on the regulation of intrinsic apoptosis.47
Of note, the signaling pathways leading to extrinsic and intrinsic
apoptosis exhibit some degree of cross talk. Thus, whereas in some
cells including lymphocytes (type I cells) the activation of death
receptors leads to pro-CASP3 processing and apoptosis without any
mitochondrial involvement,48 in other cells such as hepatocytes (type
II cells), CASP8 not only activates CASP3 but also mediates the
proteolytic cleavage of the BH3-only protein BH3 interacting domain
death agonist (BID), generating a MOMP-inducing fragment.49 Thus,
in type II cells, MOMP functions as an amplifier of apoptotic signaling
by eliciting the CASP9-mediated activation of CASP3. Interesting to
note, whether cells respond to death receptor ligation in a type I- or
type II-like manner depends on the expression levels of the cIAP-like
protein X-linked inhibitor of apoptosis (XIAP).50 The cross talk between
extrinsic and intrinsic apoptosis is pathophysiologically relevant in
vivo, as demonstrated by the fact that the hepatocytes of Bid−/− mice
are partially protected from FAS-induced apoptosis (see Fig. 5.1).51
Necrosis
Classically, necrosis was defined as an instance of cell death lacking
the peculiar morphological manifestations of apoptosis as well as the
accumulation of cytoplasmic vacuoles that characterize autophagic
cells. Somehow, this was in line with the belief that necrosis would
always proceed in an unregulated fashion and would only terminate
accidental instances of cell death.5 In the 1990s, Vandenabeele’s and
Schulze-Osthoff ’s groups demonstrated that engagement of FAS does
not always lead to cell death via extrinsic apoptosis.52–54 This observation
76 Part I: Science and Clinical Oncology

Figure 5.1  •  Apoptosis. Extrinsic apoptosis is often ignited by the ligation of death receptors such as FAS. This allows for the stabilization of receptor
trimers and for recruitment at their intracellular tails of a multiprotein complex known as death-inducing signaling complex (DISC). Within the DISC,
pro-CASP8 undergoes spatial-proximity induced autoactivation, hence becoming able to cleave multiple substrates including BID and pro-CASP3. As an
alternative, extrinsic apoptosis can be initiated by so-called “dependence receptors,” such as PTCH1, when the concentrations of their ligands (e.g., SHH)
falls below a specific threshold. In this case, the activation of CASP3 proceeds via a molecular mechanism involving CASP9 and the adaptor proteins DRAL
and TUCAN. Intrinsic apoptosis is activated in response to intracellular stress conditions (e.g., DNA damage) and involves a central step of mitochondrial
regulation. Thus, if proapoptotic signals (often relayed by BH3-only proteins) predominate over antiapoptotic ones, mitochondrial membranes lose their
structural integrity because of the pore-forming activity of Bcl-2 family members such as BAK1 and BAX. Consequent mitochondrial outer membrane per-
meabilization (MOMP) allows for the release of mitochondrial proteins into the cytosol, including direct activators of caspases, such as CYCS, as well as
proteins that indirectly facilitate caspase activation, such as DIABLO and HTRA2. Cytosolic CYCS directs the assembly of an APAF1-containing, dATP-dependent
supramolecular platform that catalyzes CASP9 activation, hence initiating a proteolytic cascade that culminates in CASP3 (CASP6 and CASP7) activation.
Extrinsic apoptosis and intrinsic apoptosis exhibit some degree of cross talk. Indeed, in some cell types, CASP8 can convert the BH3-only protein BID into
a MOMP-promoting fragment, further accelerating caspase activation and apoptosis. FADD, FAS-associated protein with a death domain; FASLG, FAS ligand;
IAPs, inhibitor of apoptosis proteins; tBID, truncated BID.

instilled in some researchers the suspicion that, similar to apoptosis,
necrosis also might be orchestrated by a refined molecular machinery;
this ignited an intense wave of research that has not yet come to an
end.6 Important to note, regulated necrosis occurs during mammalian
development, in particular at the bone growth plate (i.e., the zone of
the bone that controls its length), as well as during adult tissue
homeostasis, for instance in the lower regions of intestinal crypts.55,56
Moreover, multiple instances of necrotic RCD have been involved in
the pathophysiology of diseases including viral infection, neurodegenera-
tion, ischemia, and others.8,22,57
Necroptosis
The best-characterized pathway of regulated necrosis, which is known
as necroptosis, can be elicited by the ligation of death receptors in
conditions in which CASP8 is inhibited (either by pharmacologic or
by genetic interventions). In this context, DISC-bound RIPK1 does
not get degraded by CASP8 as it occurs during extrinsic apoptosis,
but recruits and functionally interacts with its homolog receptor
interacting serine/threonine kinase 3 (RIPK3) and mixed-lineage kinase
like (MLKL), generating the so-called necrosome.58–60 Although PGAM
family member 5, mitochondrial serine/threonine protein phosphatase
(PGAM5) has been suggested to contribute to necroptosis downstream
of RIPK1 and RIPK3 by virtue of its capacity to promote mitochondrial
fragmentation,61,62 such a possibility has now been discarded.63,64 Rather,
it is now clear that phosphorylated MLKL can form oligomers that
relocalize to the inner leaflet of the plasma membrane and compromise
its stability, hence precipitating RCD.65–71 Whether phosphorylated
MLKL is sufficient to permeabilize the plasma membrane or whether

additional factors are required for necroptosis execution remains a
matter of debate. RIPK3 activation is tonically inhibited by a het-
erodimer composed of CASP8 and the long isoform of CASP8 and
FADD-like apoptosis regulator (CFLAR), best known as FLIPL, but
the underlying molecular mechanisms remain to be elucidated.72
Moreover, RIPK3 oligomerization and consequent phosphorylation
of MLKL is controlled by the RIP homotypic interacting motif (RHIM)
of RIPK1, the absence of which results in spontaneous necroptosis.73,74
Important to note, stimuli other than death receptor ligands (e.g.,
alkylating DNA damage, viral infection) trigger bona fide RIPK3- and
MLKL-dependent necroptosis (Fig. 5.2).75,76
Mitochondrial Permeability Transition–Driven
Regulated Necrosis
In conditions of intense oxidative stress and/or in the presence of
high cytosolic Ca2+ concentrations, mitochondria experience an
abrupt increase in the permeability to ions and small solutes of the
mitochondrial inner membrane (IM), a process known as mitochondrial
permeability transition (MPT).77,78 MPT results in virtually immediate
Figure 5.2  •  Necroptosis. Necroptosis can be triggered by the ligation of
TNFR1 when caspases (notably CASP8) are inactive (for instance, owing to
the presence of the pan-caspase inhibitor Z-VAD-fmk). In these conditions,
RIPK1 is deubiquitinated and engages in physical and functional interactions
with its homolog RIPK3 as well as with MLKL in the context of a supramo-
lecular entity called necrosome. Phosphorylated MLKL forms oligomers that
precipitate necroptosis by relocalizing at the inner leaflet of the plasma membrane
and favoring its irreversible permeabilization. CYLD, CYLD lysine 63 deu-
biquitinase; FADD, FAS-associated protein with a death domain; CFLARL,
CASP8 and FADD like apoptosis regulator, long isoform; Ub, ubiquitin,
TNF, tumor necrosis factor; TRADD, TNFRSF1A associated via death domain.
Pathophysiology of Cancer Cell Death  •  CHAPTER 5 77
dissipation of the mitochondrial transmembrane potential (Δψm), rapid
cessation of all Δψm-dependent mitochondrial activities (including
ATP synthesis and protein import), and osmotic breakdown of the
organelle.77,78 MPT has been ascribed to the activity of a multiprotein
protein complex that is assembled at the juxtaposition sites between the
OM and the IM, the so-called permeability transition pore complex
(PTPC).79 The main components of the PTPC, including members of
the voltage-dependent anion channel (VDAC) family (located in the
OM) and the adenine nucleotide translocase (ANT) family (located
in the IM), as well as peptidylprolyl isomerase F (PPIF, a protein of
the mitochondrial matrix best known as CYPD), normally mediate
physiologic functions. For instance, ANT catalyzes the exchange of
adenosine diphosphate (ADP) and ATP between the cytosol and the
mitochondrial matrix. In response to cytosolic Ca2+ overload oxidative
stress, the PTPC has been proposed to assume a high-conductance
conformation leading to rapid mitochondrial breakdown.37 So far,
mouse knockout experiments failed to attribute to specific compo-
nents of the PTPC a critical role in the regulation of MPT, perhaps
because of the existence of multiple and (at least partially) redundant
isoforms of these proteins.80–82 One notable exception is represented by
CYPD, whose absence has been shown to limit pathologic cell death
in multiple circumstances, in vitro and in vivo.83,84 Thus MPT-driven
necrosis can be defined biochemically as a form of RCD that can be
retarded by pharmacologic or genetic inhibition of CYPD.18 Recent
findings suggest that the c subunit of the F1FO ATPase (which in
humans is encoded by ATP5G1, ATP5G2, and ATP5G3) may constitute
the long-sought pore-forming component of the PTPC,85 but robust
genetic evidence in support of this hypothesis is missing (Fig. 5.3).77,78
Other Forms of Regulated Cell Death
Several other specific instances of apoptotic and necrotic RCD have
been described throughout the past two decades, most of which
eventually impinge on the core molecular machinery of apoptosis
(CASP3 activation), necroptosis (RIPK3 and MLKL activation), or
MPT-driven regulated necrosis (CYPD activation).23 Of all such specific
instances, we find of particular relevance (because of their peculiar
mechanistic aspects and pathophysiologic implications) ferroptosis,
pyroptosis, and parthanatos.
Figure 5.3  •  MPT-driven regulated necrosis. In response to oxidative stress
or cytosolic Ca2+ overload, the so-called permeability transition pore complex
(PTPC) assumes a high-conductance conformation that allows for the
unregulated entry of solutes and water into the mitochondrial matrix. This
CYPD-dependent process causes a structural and functional breakdown of
the mitochondrial network that rapidly seals the cell fate. Δψm, Mitochondrial
transmembrane potential; MPT, mitochondrial permeability transition; IM,
inner mitochondrial membrane; OM, outer mitochondrial membrane; RN,
regulated necrosis.
78 Part I: Science and Clinical Oncology
Ferroptosis
Ferroptosis is an iron-dependent form of RCD that involves the
depletion of intracellular antioxidants including reduced glutathione
(GSH), and consequent lethal lipid peroxidation.86–88 Ferroptosis is
tonically inhibited by glutathione peroxidase 4 (GPX4), a major
gatekeeper of intracellular redox balance.89,90 Moreover, various
molecules cumulatively known as ferrostatins have been shown to
retard ferroptosis in vitro and in vivo, presumably as they inhibit lipid
peroxidation by lipoxygenases.86,91 Ferroptosis appears to be particularly
relevant for the synchronized demise of renal tubules in the context
of acute kidney injury.92,93
Pyroptosis
For a long time, pyroptosis was defined as a cell death modality
associated with the secretion of interleukin (IL)-1B and IL-18,94 two
key mediators of systemic inflammation.95 Thus, pyroptosis was thought
to originate from, or at least to be associated with, the activation of
so-called inflammatory caspases, that is, caspase 1 (CASP1), caspase
4 (CASP4), caspase 5 (CASP5), or mouse caspase 11 (Casp11, the
murine orthologue of human CASP4 and CASP5).96 Study findings
have demonstrated that inflammatory caspases proteolytically cleave
gasdermin D (GSDMD) to generate an N-terminal fragment that
precipitates pyroptosis by forming pores in the plasma membrane on
interaction with phosphoinositides.97–101 Thus pyroptosis should now
be defined as a GSDMD-dependent instance of RCD.102
Parthanatos
Alkylating DNA damage activates repair mechanisms involving the
NAD+-dependent enzyme poly (ADP-ribose) polymerase 1 (PARP1).103
Catalytically active PARP1 recruits multiple components of the DNA
repair machinery, hence facilitating the recovery of genetic homeo-
stasis.103 However, in the context of irreparable alkylating DNA damage,
PARP1 hyperactivation has lethal consequences for the cell because
(1) it favors a nonrecoverable NAD+ depletion that culminates in
irreversible bioenergetic crisis, and (2) it promotes the selective release
of AIFM1 from mitochondria, thereby unleashing its concealed
nucleolytic activity.104,105 Parthanatos can be defined as a PARP1-
dependent form of RCD.
Autophagy
Autophagy entails the engulfment of intracellular structures (including
organelles, protein aggregates, and portions of cytoplasm) by double-
membraned vacuoles called autophagosomes.106 Autophagosomes
normally fuse with lysosomes, leading to the degradation of their
content by lysosomal hydrolases. Baseline levels of autophagy contribute
to the maintenance of intracellular homeostasis by ensuring the removal
of old and damaged (and hence potentially dangerous) organelles,
notably mitochondria.107,108 In addition, autophagy is upregulated in
response to a wide array of stressful conditions, including nutrient
deprivation, hypoxia, and the presence of xenobiotics, including multiple
anticancer agents.109
For a while, it was thought that the continuative activation of
autophagy would eventually lead to the exhaustion of cellular resources
and cell death. Such an “autophagic cell death” was defined morphologi-
cally by an extensive vacuolization of the cytoplasm, representative of
an elevated number of autophagosomes and autolysosomes (the
organelles that are generated by the autophagosomal-lysosomal
fusion).5,26 However, the association between the accumulation of
autophagosomes and cell death has rarely if ever been proved to be
causal, in particular in settings of stress-induced cancer cell death.
Indeed, the inhibition of autophagy by pharmacologic or genetic
means often accelerates (rather than inhibits) cell death, suggesting
that autophagy constitutes a stress response mechanism that attempts
(but fails) to avoid the cellular demise.110 These results cast doubts
on the appropriateness of the term autophagic cell death, which
inadvertently suggests a cause-effect relationship between these two
phenomena.26 Thus far, autophagy has been demonstrated to mediate
cell death in several developmental scenarios, notably during the
metamorphosis of insects.111,112 Moreover, at least in some instances,
autophagy appears to contribute to the execution of human cancer
cells that succumb to specific experimental cell death inducers in
vitro.113 These observations de facto justify the use of the expression
autophagic cell death under highly selected circumstances.4 Defects of
the autophagic machinery have been associated with a plethora of
human pathophysiologic conditions, including accelerated aging,
neurodegeneration, and cancer.114,115 However, this appears to be more
strictly related to the role that autophagy exerts in the regulation of
intracellular homeostasis rather than as a bona fide cell death mecha-
nism28,107 and hence is not discussed here in further detail.
FUNDAMENTAL SCIENCE: CELL DEATH
AND CANCER
According to classic models, single molecular alterations are per se
unable to fully transform normal cells into highly aggressive cancer
cells. Rather, oncogenesis seems to proceed along with a progressive
increase in genetic instability and with the accumulation of several
molecular defects. Often, if not always, one of these alterations consists
in the interruption of the signaling cascades that ensure the homeostatic
death of continuously proliferating cells.116 As they evolve, premalignant
and malignant cells are indeed subjected to elevated levels of stress,
in part as a result of the overactivation of cell-intrinsic oncogenic
signaling pathways (so-called oncogenic stress), and in part due to
microenvironmental conditions, which are often characterized by
hypoxia and nutrient shortage (especially in rapidly proliferating
neoplasms).117 Thus, in virtually all scenarios, carcinogenesis requires
the (at least partial) suppression of cell death signaling pathways. This
can result from loss-of-function mutations in proteins that transduce
lethal signals or execute cell death (as many oncosuppressor proteins),
but also from gain-of-function alterations in molecules that normally
deliver pro-survival signals (as several oncoproteins). Defects in the
molecular pathways that regulate cell death also are instrumental to
tumors when it comes to resistance to chemotherapy and radiotherapy.17
Important to note, intrinsic alterations are generally insufficient for
a healthy cell to form a clinically manifest aggressive neoplasm, owing
to the existence of a systemic layer of control on cellular homeostasis
mediated by the immune system (so-called “cancer immunosurveil-
lance”).118 For the sake of simplicity, however, the cell-extrinsic regula-
tion of oncogenesis and tumor progression is not discussed in further
detail here.
Oncogenes and Cell Death Regulation
Oncogenes—that is, genes that stimulate malignant transformation—
were originally identified in tumorigenic viruses and then were shown
to exist as inactive variants (or proto-oncogenes), also in the human
genome.119 Proto-oncogenes including MYC and NRAS are involved
in the regulation of mitogenic signals and hence play a critical role
in the control of tissue homeostasis. Per se, proto-oncogenes are not
tumorigenic and must acquire gain-of-function alterations to become
so. These alterations can be as gross as chromosomal translocations
that bring proto-oncogene coding sequences in the proximity of strong
transcriptional regulators (as in the case of MYC, which is often rear-
ranged in lymphoma patients), or as specific as point mutations that
render proto-oncogene products constitutively active (as in the case
of NRAS, which is affected by hyperactivating mutations in 20%
to 25% of all cancers).120,121 By transducing strong and persistent
mitogenic signals, constitutively active MYC and NRAS, in addi-
tion to other oncoproteins such as epidermal growth factor receptor
(EGFR, which is often hyperactivated in lung and colon cancer)
and Abelson tyrosine kinase (ABL, which is fused to BCR on the
t(9;22) translocation in chronic myelogenous leukemia), facilitate the

escape of malignant precursors from the growth control that usually
guarantees tissue homeostasis. As mentioned earlier, however, one
single alteration of this kind is insufficient to convert normal cells
into their malignant counterparts. The hyperactivation of mitogenic
pathways causes consistent functional alterations and drives cells into
a state of constant stress featuring significant metabolic rewiring and
increased generation of reactive oxygen species. Because oncogenic
stress would normally lead to cell death, only premalignant cells in
which the cell death signaling pathways are blocked can progress
toward tumorigenesis.116
Premalignant cells resist oncogenic stress by activating one (or
more) of several pathways that (1) directly counteract the execution
of cell death (because of the deregulation of additional proto-oncogenes)
or (2) facilitate the management of intracellular stress (because of the
elicitation of per se nononcogenic stress response pathways). In general,
this leads to two distinct phenomena of dependence, commonly referred
to as “oncogene addiction” and “non–oncogene addiction.”117 In the
former scenario, not only the tumorigenic phenotype but also the
survival of cancer cells becomes dependent on the hyperactivation of
one or more proto-oncogenes. This has provided a rationale for the
use of multiple oncogene-targeted agents in anticancer therapy, some
of which (such as the BCR-ABL–targeting compound imatinib) have
impressive rates of therapeutic responses.116 In the latter scenario,
cancer cells become dependent on the activation of per se nononcogenic
mechanisms of intracellular stress management, such as those centered
around molecular chaperones of the heat-shock protein (HSP) family.117
Major examples of prosurvival systems that often get hyperactivated
throughout (and contribute to) oncogenesis are provided by antiapop-
totic Bcl-2 family members, the nuclear factor–κB (NF-κB) pathway,
and AKT1.
Antiapoptotic Bcl-2 family members have been shown to regulate
cell death by multiple mechanisms, including the sequestration of
Figure 5.4  •  Bcl-2 family proteins. Human Bcl-2 family proteins. Official gene names (within brackets) and MW are reported. BH, Bcl-2 homology
domain; TM, transmembrane domain.
Pathophysiology of Cancer Cell Death  •  CHAPTER 5 79
their proapoptotic counterparts into inactive complexes, in addition
to Ca2+-related and metabolic effects (Fig. 5.4).46,122,123 After the
discovery that the overexpression of BCL2 as a result of the t(14;18)
translocation underlies multiple instances of lymphomagenesis,14
preclinical and clinical evidence has accumulated to demonstrate that
not only BCL2 but also its antiapoptotic homologues BCL-xL and
MCL1 exert bona fide oncogenic functions.124 The overexpression of
BCL2 shortens the latency period required for transgenic mice
overexpressing MYC in B cells to develop lymphomas.15 Along similar
lines, both BCL-xL and MCL1 have been shown to cooperate with
MYC in murine models of hematopoietic malignant transforma-
tion.125,126 However, consistent with the notion of multistep oncogenesis
described earlier, mice engineered for the overexpression of BCL2,
BCL-xL, or MCL1 in hematopoietic cells exhibit perturbed myelopoieis
or lymphopoieis but are only slightly more prone to the development
of age-related lymphomas than their normal counterparts.125,127,128 Of
note, the protein expression levels of BCL2, BCL-xL, and MCL1 are
elevated in a wide range of human tumors.129
NF-κB is a transcription factor that participates in the cellular
response to a wide array of conditions, including (but not limited to)
cytokine stimulation, infection, and oxidative stress. Under normal
circumstances, NF-κB subunits such as p65RELA and p50NFKB1 are held
in check in the cytoplasm by inhibitory interactions with IκBα. In
the presence of NF-κB–inducing conditions, however, IκBα is
phosphorylated by the so-called IκB kinase (IKK, a multiprotein
complex including two catalytic subunits [IKKα and IKKβ] and one
regulatory component [IKKγ/NEMO]), resulting in its proteosomal
degradation and in the nuclear translocation of NF-κB.130 NF-κB
homodimers and heterodimers control the expression of more than
150 target genes, including genes that code for mitogens, other
transcription factors (including MYC), inhibitors of extrinsic apoptosis
(e.g., the so-called FLIPs), IAPs, and BCL2 and BCL-xL (Fig. 5.5).131
80 Part I: Science and Clinical Oncology
Figure 5.5  •  The nuclear factor–κB (NF-κB) canonic pathway. In the
canonic activation pathway (a “noncanonic” pathway exists but is not described
here for the sake of simplicity), the IκB kinase (IKK) complex (comprising
one regulatory subunit, known as IKKγ or NEMO, and two catalytic subunits,
IKKα and IKKβ) responds to specific signals (including the ligation of death
receptors) or nonspecific stress by phosphorylating IκB, thereby targeting it
for proteosomal degradation. The degradation of IκB unmasks a nuclear
localization signal that allows preformed NF-κB homodimers and heterodimers
to enter the nucleus and bind DNA. In the nucleus, NF-κB dimers control
the expression of genes involved in several distinct pathophysiologic processes,
including innate and adaptive immunity, inflammation, proliferation, and
cell death and survival. P, Phosphate; Ub, ubiquitin.
Thus, the activation of NF-κB orchestrates the response of cells to
stress while delivering a strong prosurvival (and in some cases mitogenic)
signal. Multiple lines of evidence demonstrate that the NF-κB system
exerts bona fide oncogenic functions. First, REL genes are homologues
of the avian reticuloendotheliosis virus v-rel oncogene, which is strictly
required for virus-induced malignant transformation.132,133 Second,
the human leukemia/lymphoma virus type I (HTLV1) can transform
target cells via a mechanism that depends, at least in part, on the
activation of cellular IKK by the viral protein Tax.134 Finally, several
components of the NF-κB pathway get overexpressed or hyperactivated
during malignant transformation. For instance, REL genes are amplified
or affected by gain-of-function mutations in a constituent proportion
of B-cell lymphomas,135 and the IκB-related protein BCL3 (which
stimulates the nuclear translocation and activity of NF-κB dimers)136
is involved in a chromosomal rearrangement, t(14;19), that is found
in some hematologic malignancies.137 Of note, the NF-κB system
appears to play a critical role in the handling of oncogenic stress, as
demonstrated by the fact that multiple oncoproteins including RAS
and BCR-ABL stimulate NF-κB nuclear translocation and transcrip-
tional activity.138,139
One of the hallmarks of cancer cells as originally formulated by
Hanahan and Weinberg in 2000 consists of their ability to circumvent
the absence of extracellular growth factors and de facto emancipate
from homeostatic tissue regulation.140 Several growth factors, including
IL-2, platelet-derived growth factor (PDGF), and insulin-like growth
factor 1 (IGF1), promote proliferation and survival via transmembrane
receptors that activate phosphoinositide-3-kinases (PI3-Ks, also known
as phosphatidylinositol-3 kinases). Active PI3-K generates plasma
membrane-bound phosphatidylinositol-3,4,5-triphosphate, in turn
stimulating the activation of the serine/threonine kinase AKT1 (also
known as protein kinase B [PKB]). By phosphorylating multiple
Figure 5.6  •  AKT1 signaling. Several growth factor receptors are
coupled to phosphoinositide-3-kinase (PI3-K), catalyzing the conversion
of membrane-bound phosphatidylinositol-4,5-diphosphate (PIP2) into
phosphatidylinositol-3,4,5-triphosphate (PIP3). By binding to a pleckstrin
homology (PH) domain, PIP3 recruits AKT1 at the intracellular leaflet of the
plasma membrane, hence allowing for its phosphorylation-dependent activation
by phosphoinositide-dependent kinase 1 (PDK1). Active AKT1 phosphorylates
multiple substrates, thereby transducing multipronged prosurvival signals. For
instance, AKT1 inhibits the BH3-only protein BAD by facilitating its sequestra-
tion by 14-3-3 proteins, stimulates the degradation of the oncosuppressive
transcription factor p53, promotes glucose uptake (hence favoring the
maintenance of bioenergetic such as FKHRL1. Moreover, AKT1 indirectly
activates the mechanistic target of rapamycin (MTOR), hence stimulating
cell growth and proliferation while inhibiting autophagy. By catalyzing the
dephosphorylation of PIP3, phosphatase and tensin homolog (PTEN) function-
ally antagonizes PI3-K, de facto inhibiting AKT1 activation. P, Phosphate.
substrates, AKT1 transduces prosurvival signals via distinct mechanisms.
Among others, AKT1 inhibits the BH3-only protein BCL2-associated
agonist of cell death (BAD) by facilitating its sequestration by chap-
erones of the 14-3-3 protein family, stimulates the degradation of the
oncosuppressor tumor protein p53 (TP53, best known as p53; see
later), promotes glucose uptake and hence favors the maintenance of
bioenergetic homeostasis, and blocks the nuclear translocation of
proapoptotic Forkhead transcription factors such as Forkhead box O3
(FOXO3).141 Moreover, AKT1 indirectly activates mechanistic target
of rapamycin (MTOR), thus stimulating cell growth and proliferation
while inhibiting autophagy (Fig. 5.6).142 Several preclinical and clinical
studies have demonstrated the importance of AKT1 in tumorigenesis.
Transgenic mice expressing constitutively active Akt1 under the control
of a T cell–restricted promoter develop lymphoma early in life.143
Amplification of AKT1 or gain-of-function AKT1 mutations have
been detected in several neoplasms, including (but not limited to)
breast and gastric cancers. In addition, loss-of-function of the oncosup-
pressor PTEN (the phosphatase that directly antagonizes PI3-K and
hence inhibits AKT1, see later) is common across a wide array of
tumors.144 Of note, the inhibition of MTOR limits tumor formation
in Pten+/− mice,145 demonstrating that AKT1-driven oncogenesis relies,
at least in part, on MTOR hyperactivation. This is particularly interest-
ing in view of the fact the MTOR inhibition stimulates autophagy,

which is considered as a bona fide oncosuppressor mechanism during
early tumorigenesis.146
Oncosuppressors and Cell Death Regulation
The existence of tumor suppressor genes (also known as oncosuppressor
genes)—that is, genes whose products actively counteract tumorigenesis
—was first suspected in the 1960s, when Harris and colleagues observed
that highly malignant Ehrlich ascites cells lose the ability to form
tumors on fusion with virtually nontumorigenic cells.147 The molecular
characterization of the first tumor suppressor gene, however, lagged
until the 1980s, when the cDNA encoding the protein product of
the RB1 gene (a negative regulator of cell cycle progression involved
in the development of pediatric retinoblastoma) was isolated and
sequenced.148 Since then, several other oncosuppressor proteins have
been identified, encompassing negative regulators of mitogenic signals,
such as PTEN; proapoptotic Bcl-2 family members such as BAX; and
proteins that couple the management of cellular stress with cell death
induction, such as ATM and p53. Oncosuppressor genes are inactivated
during oncogenesis not only with loss-of-function mutations and
deletions, but also after epigenetic gene silencing. Thus, promoter
hypermethylation at CpG islands or histone deacetylation can turn
off oncosuppressor gene expression, owing to local chromatin remodel-
ing and impaired access by essential transcription factors.149 These
instances are exemplified by the loss of CASP8 expression in pediatric
neuroblastomas or by the loss of p14ARF (an upstream activator of
p53; see later) expression in multiple tumor types.150,151 Irrespective
of the underlying mechanisms, both alleles of one tumor suppressor
gene must be inactivated for the establishment of tumor-permissive
conditions. In this context, germline mutations in oncosuppressor
genes are associated with familial tumor syndromes (as one copy of
the gene is lost in the whole organism), whereas somatic mutations
are frequently detected in sporadic tumors (in this case, both alleles
have been inactivated during tumorigenesis).152
As mentioned earlier, the protein product of PTEN (phosphatase
and tensin homolog) functions as a phosphatase, catalyzing the conver-
sion of phosphatidylinositol-3,4,5-triphosphate into phosphatidylinositol-
4,5-diphosphate and de facto antagonizing the enzymatic activity of
PI3-Ks and negatively regulating AKT1 (see Fig. 5.6).153 PTEN was
discovered in the search for a tumor suppressor on chromosome 10
that is often lost in glioblastoma and breast, endometrial, and prostate
cancer.154 Subsequent preclinical and clinical observations supported
the notion that PTEN loss of function sustains tumorigenesis in many
different organs.155,156 The oncosuppressive functions of PTEN have
been shown to be particularly sensitive to gene dosage, even beyond
the classic concept of haploinsufficiency.157 Of note, the frequency of
PTEN inactivation in human tumors is exceeded only by that of p53
(see later).144
Pro-apoptotic BCL-2 family members, in particular BAX and BAK1,
exert crucial oncosuppressive functions as they precipitate RCD in
response to adverse conditions, including oncogenic stress (see Fig.
5.4). Results from multiple murine models support the notion that
BAX and BAK1 operate in vivo to counteract oncogenesis independent
of p53. For instance, murine cells expressing adenoviral E1A (a prolifera-
tive factor) and dominant negative p53 are unable to form tumors in
mice unless Bax and Bak1 are lost.158 In addition, loss-of-function
mutations in BAX and BAK1 have been detected in many hematopoietic
and solid malignancies.129,159 By acting as stress sensors that activate
intrinsic apoptosis, BH3-only proteins could also exert, at least theoreti-
cally, prominent oncosuppressive functions. This said, evidence
implicating them as bona fide tumor suppressors is scant, and only a
few articles report their loss in cancer patients.160 The reasons underlying
this apparent discrepancy have not yet been clarified, but perhaps are
linked to the fact that whereas BAX and BAK1 operate at the con-
vergence of multiple lethal signaling pathways, single BH3-only proteins
are involved in highly specific signaling cascades and perhaps are rela-
tively redundant among themselves.
Pathophysiology of Cancer Cell Death  •  CHAPTER 5 81
p53 was originally identified in 1979 by two methodologically
distinct approaches: as a cellular protein coimmunoprecipitating with
the large-T antigen in SV40-transformed cells,161,162 and as the target
of antibodies isolated from the serum of immunodeficient mice bearing
human tumors.163,164 Since then, p53 has been subjected to an intense
wave of investigation, leading to the discovery that p53 regulates an
ever-growing list of cellular processes.165 Historically, the first function
ascribed to p53 was that of a stress-responsive transcription factor. In
physiologic conditions, the intracellular levels of p53 are regulated by
MDM2, an E3 ubiquitin ligase that polyubiquitinates p53 and hence
targets it to proteosomal degradation.166 In response to a variety of
adverse conditions, including DNA damage and oncogenic stress,
p53 escapes MDM2-mediated degradation and accumulates in the
cytoplasm. This can be due to posttranslational modifications of p53
that reduce its affinity for MDM2 or to the expression of MDM2
inhibitors such as p14ARF.167 Irrespective of the underlying molecular
mechanisms, stabilized p53 assembles into transcriptionally proficient
tetramers that can regulate the expression of distinct sets of genes,
leading to highly diverse functional outcomes.168 Among the most
common p53-regulated responses are reversible cell cycle arrest,
senescence, and cell death. Cell death is accomplished via the upregula-
tion of multiple genes involved in the execution of apoptosis, including
APAF1, BAX, FAS, and the BH3-only protein BCL2 binding component
3 (BBC3, best known as PUMA),168–171 as well as via transcription-
independent mechanisms whereby cytoplasmic p53 stimulates MOMP
by interacting with both proapoptotic and antiapoptotic members of
the Bcl-2 family or MPT by interacting with CYPD (Fig. 5.7).172–176
The inactivation of p53, be it mutational, due to the overexpression
or overactivation of p53 negative regulators such as MDM2, or resulting
from the inactivation of upstream p53-activating factors such as p14ARF,
is the most common molecular alteration in human cancer, affecting
more than 50% of neoplasms, all confounded. In addition, the p53
status has been shown to influence prognosis and/or therapeutic
outcome in multiple oncologic settings, including (but not limited
to) breast, lung, and colorectal cancer.177 These clinical data reflect a
huge number of preclinical observations demonstrating that p53 exerts
bona fide oncosuppression in vivo.178 Of note, recent research has
focused on physiologic aspects of the p53 biology, unveiling a critical
role for baseline p53 levels in the maintenance of bioenergetic, redox,
and genomic homeostasis.179–182 Thus, p53 appears to exert oncosup-
pressive functions both as it regulates intracellular homeostasis, thereby
preventing malignant transformation, and as it orchestrates the
elimination of potentially tumorigenic cells.
CLINICAL RELEVANCE AND APPLICATIONS
As introduced earlier, genetic, epigenetic, and functional alterations
of the mechanisms that underlie RCD (1) are highly prevalent in
human cancer, (2) are often required for oncogenesis and/or tumor
progression, and (3) underlie many instances of cancer chemoresistance
and radioresistance. The clinical relevance of these alterations is therefore
dual. On one hand, the characterization of the genotype and functional
phenotype of specific neoplasms can provide prognostic and/or predic-
tive information. This allows for the stratification of patients into
precise risk groups and—at least in some instances—for the implementa-
tion of personalized therapeutic regimens.183 On the other hand, a
great number of anticancer agents that specifically target alterations
in cell death–regulating signaling pathways have been developed, and
some of them have successfully entered clinical routines. At odds with
conventional chemotherapeutics, which frequently kill tumor cells
because of their elevated proliferative potential, targeted anticancer
agents act on cancer cell–specific defects and therefore are generally
associated with a reduced incidence and severity of side effects.184
These agents include compounds that interrupt oncogene or nonon-
cogene addiction as well as strategies that attempt to reconstitute the
lost function of tumor suppressors. In most cases, these approaches
lead to the apoptotic or necrotic demise of cancer cells, although in
82 Part I: Science and Clinical Oncology
Figure 5.7  •  The p53 system. In physiologic settings, p53 levels are
maintained under control by MDM2, an E3 ubiquitin ligase that, on polyu-
biquitination, targets it to proteosomal destruction. In response to multiple
stress stimuli (e.g., DNA damage and oncogene activation), p53 is subjected
to posttranslational modifications that reduce its affinity for MDM2. Alter-
natively, stressful conditions result in the upregulation of MDM2 inhibitors
such as p14ARF. In both cases, the cytoplasmic levels of p53 rise, allowing p53
to assemble into tetramers that enter the nucleus and regulate a plethora of
transcriptional responses. Furthermore, a pool of monoubiquitinated p53
persists into the cytoplasm, where it can facilitate apoptosis by directly activating
the proapoptotic protein BAX and/or by inhibiting antiapoptotic members
of the Bcl-2 family, or it can promote mitochondrial permeability transition
(MPT)-driven regulating necrosis by interacting with CYPD upon entering
mitochondria. Ac, Acetyl; Me, methyl; Ne, Nedd8; P, phosphate; Su, SUMO;
Ub, ubiquitin.
some cases the therapeutic benefit results from the activation of cellular
senescence (an irreversible arrest in cell cycle progression).185 Cell
death mechanisms have also attracted attention for the development
of strategies for chemosensitization and radiosensitization. In this case,
restoring the proficiency of cancer cells to undergo stress-induced cell
death is not therapeutic per se but restores the tumor sensitivity to
conventional chemotherapeutics, which is often lost during tumor
progression.186 Of note, the success of targeted anticancer agents appears
to depend, at least in part, on the activation of immune effector
mechanisms,187 which has been demonstrated in multiple settings, in
vitro and in vivo, including models of pure oncogene addiction.188
The list of successful approaches that target deregulated cell death
signaling in cancer cells is continuously growing and encompasses,
among others, the proteosomal inhibitor bortezomib, which blocks
NF-κB activation and is approved by the US Food and Drug Admin-
istration (FDA) for the treatment of multiple myeloma and mantle
cell lymphoma189; so-called BH3 mimetics, that is, small molecules
that inhibit antiapoptotic members of the Bcl-2 protein family, such
as venetoclax (an orally available agent licensed by the FDA for the
treatment of chronic lymphocytic leukemia)190; rapamycin, an MTOR
inhibitor that is approved by the FDA for transplant rejection and is
now being evaluated in combination therapies against multiple
neoplasms191; and p53-reconstituting strategies, aimed at either
reintroducing wild-type p53 in p53-deficient cancer cells (by gene
therapy) or at blocking MDM2 in p53-proficient cells (by pharma-
cologic inhibitors).192,193 Of note, a recombinant adenovirus engineered
to express wild-type p53 (known as gendicine) is the first gene therapy
product approved for clinical use in humans.194 In addition to targeted
approaches, such as the aforementioned, promising results have been
obtained with epigenetic regulators such as suberoylanilide hydroxamic
acid (SAHA, also known as vorinostat), a histone deacetylase inhibitor
that is currently approved by the FDA for the treatment of cutaneous
T-cell lymphoma,195 and azacitidine or decitabine, methyltransferase
inhibitors that are currently used for the therapy of myelodysplastic
syndromes.196
WHAT THE FUTURE HOLDS
Finely manipulating cell death mechanisms in cancer, a strategy that
20 years ago appeared as a distant and hardly reachable goal, now
constitutes a reality with crucial clinical implications. Future investiga-
tions will have to provide further insights into the molecular cascades
that underlie cell death in both physiologic (homeostatic cell death)
and pathologic (in response to oncogenic stress) scenarios, and will
have to unravel the mechanisms through which these are disabled
during oncogenesis and tumor progression. This will be instrumental
not only for the discovery of novel drug targets and hence for the
development of new anticancer agents, but also for the precise identifica-
tion of patients who may respond to particular therapeutic regimens.
Furthermore, strategies for the therapeutic induction of nonapoptotic
RCD modes, including necroptosis and MPT-driven necrosis, or
oncosuppressive signaling networks that operate in multiple ways,
such as mitotic catastrophe,179 should be elaborated. These approaches
will be particularly relevant for the treatment of chemoresistant and
radioresistant cancers, which nearly always exhibit alterations in the
molecular machinery that initiates or executes apoptotic cell death.
Finally, it will be indispensable to understand how distinct cell death
subroutines cross talk (i.e., to which extent they can substitute for
each other in scenarios in which one is specifically disabled by
tumorigenic alterations) and to what extent the immune system
contributes to the therapeutic efficacy of currently used anticancer
regimens. In this context, the induction of immunogenic cell death,
which converts dying tumor cells into a vaccine that is capable of
eliciting a tumor-specific immune response,197–199 may constitute a
particularly interesting therapeutic goal.
The complete reference list is available online at
ExpertConsult.com.

KEY REFERENCES
2. Lockshin RA, Williams CM. Programmed cell death
II. Endocrine potentiation of the breakdown of the
intersegmental muscles of silkmoths. J Insect Physiol.
1964;10:643–649.
3. Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic
biological phenomenon with wide-ranging implica-
tions in tissue kinetics. Br J Cancer. 1972;26(4):
239–257.
4. Galluzzi L, Vitale I, Abrams JM, et al. Molecular
definitions of cell death subroutines: recommenda-
tions of the Nomenclature Committee on Cell Death
2012. Cell Death Differ. 2012;19(1):107–120.
14. Tsujimoto Y, Finger LR, Yunis J, Nowell PC, Croce
CM. Cloning of the chromosome breakpoint of
neoplastic B cells with the t(14;18) chromosome
translocation. Science. 1984;226(4678):1097–1099.
16. Hanahan D, Weinberg RA. Hallmarks of cancer:
the next generation. Cell. 2011;144(5):646–674.
18. Galluzzi L, Bravo-San Pedro JM, Vitale I, et al.
Essential versus accessory aspects of cell death:
recommendations of the NCCD 2015. Cell Death
Differ. 2015;22(1):58–73.
19. Tait SW, Green DR. Mitochondria and cell death:
outer membrane permeabilization and beyond. Nat
Rev Mol Cell Biol. 2010;11(9):621–632.
21. Wallach D, Kang TB, Dillon CP, Green DR.
Programmed necrosis in inflammation: toward
identification of the effector molecules. Science. 2016;
352(6281):aaf2154.
22. Conrad M, Angeli JP, Vandenabeele P, Stockwell
BR. Regulated necrosis: disease relevance and
therapeutic opportunities. Nat Rev Drug Discov.
2016;15(5):348–366.
23. Vanden Berghe T, Linkermann A, Jouan-Lanhouet S,
Walczak H, Vandenabeele P. Regulated necrosis: the
expanding network of non-apoptotic cell death path-
ways. Nat Rev Mol Cell Biol. 2014;15(2):135–147.
40. Li P, Nijhawan D, Budihardjo I, et al. Cytochrome c
and dATP-dependent formation of Apaf-1/caspase-9
complex initiates an apoptotic protease cascade. Cell.
1997;91(4):479–489.
44. Pospisilik JA, Knauf C, Joza N, et al. Targeted
deletion of AIF decreases mitochondrial oxidative
phosphorylation and protects from obesity and
diabetes. Cell. 2007;131(3):476–491.
Pathophysiology of Cancer Cell Death  •  CHAPTER 5 83
51. Luo X, Budihardjo I, Zou H, Slaughter C, Wang
X. Bid, a Bcl2 interacting protein, mediates cyto-
chrome c release from mitochondria in response
to activation of cell surface death receptors. Cell.
1998;94(4):481–490.
53. Vercammen D, Brouckaert G, Denecker G, et al.
Dual signaling of the Fas receptor: initiation of both
apoptotic and necrotic cell death pathways. J Exp
Med. 1998;188(5):919–930.
54. Schulze-Osthoff K, Krammer PH, Droge W. Diver-
gent signalling via APO-1/Fas and the TNF receptor,
two homologous molecules involved in physiological
cell death. EMBO J. 1994;13(19):4587–4596.
57. Linkermann A, Brasen JH, Darding M, et al. Two
independent pathways of regulated necrosis mediate
ischemia-reperfusion injury. Proc Natl Acad Sci USA.
2013;110(29):12024–12029.
72. Oberst A, Dillon CP, Weinlich R, et  al.
Catalytic activity of the caspase-8-FLIP(L) complex
inhibits RIPK3-dependent necrosis. Nature.
2011;471(7338):363–367.
74. Dillon CP, Weinlich R, Rodriguez DA, et al. RIPK1
blocks early postnatal lethality mediated by caspase-8
and RIPK3. Cell. 2014;157(5):1189–1202.
83. Baines CP, Kaiser RA, Purcell NH, et al. Loss of
cyclophilin D reveals a critical role for mitochon-
drial permeability transition in cell death. Nature.
2005;434(7033):658–662.
87. Dixon SJ, Lemberg KM, Lamprecht MR, et al.
Ferroptosis: an iron-dependent form of nonapoptotic
cell death. Cell. 2012;149(5):1060–1072.
93. Linkermann A, Skouta R, Himmerkus N, et al.
Synchronized renal tubular cell death involves fer-
roptosis. Proc Natl Acad Sci USA. 2014;111(47):
16836–16841.
100. Kayagaki N, Stowe IB, Lee BL, et al. Caspase-11
cleaves gasdermin D for non-canonical inflamma-
some signalling. Nature. 2015;526(7575):666–
671.
101. Shi J, Zhao Y, Wang K, et al. Cleavage of GSDMD
by inflammatory caspases determines pyroptotic cell
death. Nature. 2015;526(7575):660–665.
110. Boya P, Gonzalez-Polo RA, Casares N, et al. Inhibi-
tion of macroautophagy triggers apoptosis. Mol Cell
Biol. 2005;25(3):1025–1040.
111. Berry DL, Baehrecke EH. Growth arrest and
autophagy are required for salivary gland cell degra-
dation in Drosophila. Cell. 2007;131(6):1137–1148.
117. Luo J, Solimini NL, Elledge SJ. Principles of cancer
therapy: oncogene and non-oncogene addiction. Cell.
2009;136(5):823–837.
146. Galluzzi L, Pietrocola F, Bravo-San Pedro JM, et al.
Autophagy in malignant transformation and cancer
progression. EMBO J. 2015;34(7):856–880.
161. Lane DP, Crawford LV. T antigen is bound to a
host protein in SV40-transformed cells. Nature.
1979;278(5701):261–263.
162. Linzer DI, Levine AJ. Characterization of a 54K
dalton cellular SV40 tumor antigen present in
SV40-transformed cells and uninfected embryonal
carcinoma cells. Cell. 1979;17(1):43–52.
163. Kress M, May E, Cassingena R, May P. Simian virus
40-transformed cells express new species of proteins
precipitable by anti-simian virus 40 tumor serum.
J Virol. 1979;31(2):472–483.
172. Caelles C, Helmberg A, Karin M. p53-dependent
apoptosis in the absence of transcriptional activation of
p53-target genes. Nature. 1994;370(6486):220–223.
175. Mihara M, Erster S, Zaika A, et al. p53 has a direct
apoptogenic role at the mitochondria. Mol Cell.
2003;11(3):577–590.
176. Vaseva AV, Marchenko ND, Ji K, Tsirka SE,
Holzmann S, Moll UM. p53 opens the mitochon-
drial permeability transition pore to trigger necrosis.
Cell. 2012;149(7):1536–1548.
182. Matoba S, Kang JG, Patino WD, et al. p53
regulates mitochondrial respiration. Science. 2006;
312(5780):1650–1653.
187. Galluzzi L, Buque A, Kepp O, Zitvogel L, Kroemer
G. Immunological effects of conventional chemo-
therapy and targeted anticancer agents. Cancer Cell.
2015;28(6):690–714.
197. Kroemer G, Galluzzi L, Kepp O, Zitvogel L.
Immunogenic cell death in cancer therapy. Annu
Rev Immunol. 2013;31:51–72.
198. Linkermann A, Stockwell BR, Krautwald S, Anders
HJ. Regulated cell death and inflammation: an
auto-amplification loop causes organ failure. Nat
Rev Immunol. 2014;14(11):759–767.

REFERENCES
1. Clarke PG, Clarke S. Nineteenth century research
on naturally occurring cell death and related phe-
nomena. Anat Embryol (Berl). 1996;193(2):81–99.
2. Lockshin RA, Williams CM. Programmed cell death
II. Endocrine potentiation of the breakdown of the
intersegmental muscles of silkmoths. J Insect Physiol.
1964;10:643–649.
3. Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic bio-
logical phenomenon with wide-ranging implications
in tissue kinetics. Br J Cancer. 1972;26(4):239–257.
4. Galluzzi L, Vitale I, Abrams JM, et al. Molecular
definitions of cell death subroutines: recommenda-
tions of the Nomenclature Committee on Cell Death
2012. Cell Death Differ. 2012;19(1):107–120.
5. Kroemer G, Galluzzi L, Vandenabeele P, et al.
Classification of cell death: recommendations of
the Nomenclature Committee on Cell Death 2009.
Cell Death Differ. 2009;16(1):3–11.
6. Vandenabeele P, Galluzzi L, Vanden Berghe T,
Kroemer G. Molecular mechanisms of necroptosis:
an ordered cellular explosion. Nat Rev Mol Cell Biol.
2010;11(10):700–714.
7. Galluzzi L, Kepp O, Krautwald S, Kroemer G,
Linkermann A. Molecular mechanisms of regulated
necrosis. Semin Cell Dev Biol. 2014;35:24–32.
8. Galluzzi L, Vanden Berghe T, Vanlangenakker N,
et al. Programmed necrosis from molecules to health
and disease. Int Rev Cell Mol Biol. 2011;289:1–35.
9. Bender CE, Fitzgerald P, Tait SW, et al. Mitochon-
drial pathway of apoptosis is ancestral in metazoans.
Proc Natl Acad Sci USA. 2012;109(13):4904–4909.
10. Galluzzi L, Kepp O, Kroemer G. Mitochondrial
regulation of cell death: a phylogenetically conserved
control. Microb Cell. 2016;3(3):101–108.
11. Carmona-Gutierrez D, Ruckenstuhl C, Bauer MA,
Eisenberg T, Buttner S, Madeo F. Cell death in yeast:
growing applications of a dying buddy. Cell Death
Differ. 2010;17(5):733–734.
12. Meier P, Finch A, Evan G. Apoptosis in development.
Nature. 2000;407(6805):796–801.
13. Green DR, Kroemer G. The pathophysiology of
mitochondrial cell death. Science. 2004;305(5684):
626–629.
14. Tsujimoto Y, Finger LR, Yunis J, Nowell PC, Croce
CM. Cloning of the chromosome breakpoint of
neoplastic B cells with the t(14;18) chromosome
translocation. Science. 1984;226(4678):1097–1099.
15. Vaux DL, Cory S, Adams JM. Bcl-2 gene pro-
motes haemopoietic cell survival and cooperates
with c-myc to immortalize pre-B cells. Nature.
1988;335(6189):440–442.
16. Hanahan D, Weinberg RA. Hallmarks of cancer:
the next generation. Cell. 2011;144(5):646–674.
17. Igney FH, Krammer PH. Death and anti-death: tumour
resistance to apoptosis. Nat Rev Cancer. 2002;2(4):
277–288.
18. Galluzzi L, Bravo-San Pedro JM, Vitale I, et al.
Essential versus accessory aspects of cell death:
recommendations of the NCCD 2015. Cell Death
Differ. 2015;22(1):58–73.
19. Tait SW, Green DR. Mitochondria and cell death:
outer membrane permeabilization and beyond. Nat
Rev Mol Cell Biol. 2010;11(9):621–632.
20. Tait SW, Green DR. Mitochondrial regulation of
cell death. Cold Spring Harb Perspect Biol. 2013;5(9).
21. Wallach D, Kang TB, Dillon CP, Green DR.
Programmed necrosis in inflammation: toward
identification of the effector molecules. Science.
2016;352(6281):aaf2154.
22. Conrad M, Angeli JP, Vandenabeele P, Stockwell BR.
Regulated necrosis: disease relevance and therapeutic
opportunities. Nat Rev Drug Discov. 2016;15(5):
348–366.
23. Vanden Berghe T, Linkermann A, Jouan-Lanhouet S,
Walczak H, Vandenabeele P. Regulated necrosis: the
expanding network of non-apoptotic cell death path-
ways. Nat Rev Mol Cell Biol. 2014;15(2):135–147.
Pathophysiology of Cancer Cell Death  •  CHAPTER 5 83.e1
24. Green DR, Galluzzi L, Kroemer G. Cell
biology. Metabolic control of cell death. Science.
2014;345(6203):1250256.
25. Galluzzi L, Bravo-San Pedro JM, Kroemer G.
Organelle-specific initiation of cell death. Nat Cell
Biol. 2014;16(8):728–736.
26. Kroemer G, Levine B. Autophagic cell death:
the story of a misnomer. Nat Rev Mol Cell Biol.
2008;9(12):1004–1010.
27. Galluzzi L, Pietrocola F, Levine B, Kroemer G.
Metabolic control of autophagy. Cell. 2014;159(6):
1263–1276.
28. Sica V, Galluzzi L, Bravo-San Pedro JM, Izzo V,
Maiuri MC, Kroemer G. Organelle-specific initiation
of autophagy. Mol Cell. 2015;59(4):522–539.
29. Galluzzi L, Maiuri MC, Vitale I, et al. Cell
death modalities: classification and pathophysi-
ological implications. Cell Death Differ. 2007;14(7):
1237–1243.
30. Sakahira H, Enari M, Nagata S. Cleavage of CAD
inhibitor in CAD activation and DNA degradation
during apoptosis. Nature. 1998;391(6662):96–99.
31. Wajant H. The Fas signaling pathway: more than
a paradigm. Science. 2002;296(5573):1635–1636.
32. Mehlen P, Bredesen DE. Dependence receptors:
from basic research to drug development. Sci Signal.
2011;4(157):mr2.
33. Siegel RM, Frederiksen JK, Zacharias DA, et al.
Fas preassociation required for apoptosis signaling
and dominant inhibition by pathogenic mutations.
Science. 2000;288(5475):2354–2357.
34. Schulze-Osthoff K, Ferrari D, Los M, Wesselborg S,
Peter ME. Apoptosis signaling by death receptors.
Eur J Biochem. 1998;254(3):439–459.
35. Cohen GM. Caspases: the executioners of apoptosis.
Biochem J. 1997;326(Pt 1):1–16.
36. Mille F, Thibert C, Fombonne J, et al. The
Patched dependence receptor triggers apoptosis
through a DRAL-caspase-9 complex. Nat Cell Biol.
2009;11(6):739–746.
37. Kroemer G, Galluzzi L, Brenner C. Mitochondrial
membrane permeabilization in cell death. Physiol
Rev. 2007;87(1):99–163.
38. Chai J, Du C, Wu JW, Kyin S, Wang X, Shi Y. Struc-
tural and biochemical basis of apoptotic activation
by Smac/DIABLO. Nature. 2000;406(6798):855–
862.
39. Hegde R, Srinivasula SM, Zhang Z, et  al.
Identification of Omi/HtrA2 as a mitochondrial
apoptotic serine protease that disrupts inhibitor of
apoptosis protein-caspase interaction. J Biol Chem.
2002;277(1):432–438.
40. Li P, Nijhawan D, Budihardjo I, et al. Cytochrome c
and dATP-dependent formation of Apaf-1/caspase-9
complex initiates an apoptotic protease cascade. Cell.
1997;91(4):479–489.
41. Galluzzi L, Joza N, Tasdemir E, et al. No death
without life: vital functions of apoptotic effectors.
Cell Death Differ. 2008;15(7):1113–1123.
42. Li LY, Luo X, Wang X. Endonuclease G is an
apoptotic DNase when released from mitochondria.
Nature. 2001;412(6842):95–99.
43. Joza N, Susin SA, Daugas E, et al. Essential role
of the mitochondrial apoptosis-inducing factor in
programmed cell death. Nature. 2001;410(6828):
549–554.
44. Pospisilik JA, Knauf C, Joza N, et al. Targeted
deletion of AIF decreases mitochondrial oxidative
phosphorylation and protects from obesity and
diabetes. Cell. 2007;131(3):476–491.
45. David KK, Sasaki M, Yu SW, Dawson TM, Dawson
VL. EndoG is dispensable in embryogenesis and
apoptosis. Cell Death Differ. 2006;13(7):1147–
1155.
46. Youle RJ, Strasser A. The BCL-2 protein family:
opposing activities that mediate cell death. Nat Rev
Mol Cell Biol. 2008;9(1):47–59.
83.e1
47. Willis SN, Adams JM. Life in the balance: how
BH3-only proteins induce apoptosis. Curr Opin
Cell Biol. 2005;17(6):617–625.
48. Barnhart BC, Alappat EC, Peter ME. The CD95
type I/type II model. Semin Immunol. 2003;15(3):
185–193.
49. Li H, Zhu H, Xu CJ, Yuan J. Cleavage of BID by
caspase 8 mediates the mitochondrial damage in the
Fas pathway of apoptosis. Cell. 1998;94(4):491–501.
50. Jost PJ, Grabow S, Gray D, et al. XIAP discriminates
between type I and type II FAS-induced apoptosis.
Nature. 2009;460(7258):1035–1039.
51. Luo X, Budihardjo I, Zou H, Slaughter C, Wang
X. Bid, a Bcl2 interacting protein, mediates cyto-
chrome c release from mitochondria in response
to activation of cell surface death receptors. Cell.
1998;94(4):481–490.
52. Vercammen D, Beyaert R, Denecker G, et al.
Inhibition of caspases increases the sensitivity of
L929 cells to necrosis mediated by tumor necrosis
factor. J Exp Med. 1998;187(9):1477–1485.
53. Vercammen D, Brouckaert G, Denecker G, et al.
Dual signaling of the Fas receptor: initiation of both
apoptotic and necrotic cell death pathways. J Exp
Med. 1998;188(5):919–930.
54. Schulze-Osthoff K, Krammer PH, Droge W. Diver-
gent signalling via APO-1/Fas and the TNF receptor,
two homologous molecules involved in physiological
cell death. EMBO J. 1994;13(19):4587–4596.
55. Barkla DH, Gibson PR. The fate of epithelial
cells in the human large intestine. Pathology.
1999;31(3):230–238.
56. Roach HI, Clarke NM. Physiological cell death of
chondrocytes in vivo is not confined to apoptosis.
New observations on the mammalian growth plate.
J Bone Joint Surg Br. 2000;82(4):601–613.
57. Linkermann A, Brasen JH, Darding M, et al. Two
independent pathways of regulated necrosis mediate
ischemia-reperfusion injury. Proc Natl Acad Sci USA.
2013;110(29):12024–12029.
58. Cho YS, Challa S, Moquin D, et al. Phosphorylation-
driven assembly of the RIP1-RIP3 complex regulates
programmed necrosis and virus-induced inflamma-
tion. Cell. 2009;137(6):1112–1123.
59. He S, Wang L, Miao L, et al. Receptor interacting
protein kinase-3 determines cellular necrotic response
to TNF-alpha. Cell. 2009;137(6):1100–1111.
60. Zhang DW, Shao J, Lin J, et al. RIP3, an energy
metabolism regulator that switches TNF-induced
cell death from apoptosis to necrosis. Science. 2009;
325(5938):332–336.
61. Sun L, Wang H, Wang Z, et al. Mixed lineage kinase
domain-like protein mediates necrosis signaling
downstream of RIP3 kinase. Cell. 2012;148(1–2):
213–227.
62. Wang Z, Jiang H, Chen S, Du F, Wang X. The
mitochondrial phosphatase PGAM5 functions at
the convergence point of multiple necrotic death
pathways. Cell. 2012;148(1–2):228–243.
63. Tait SW, Oberst A, Quarato G, et al. Widespread
mitochondrial depletion via mitophagy does not
compromise necroptosis. Cell Rep. 2013;5(4):878–
885.
64. Moriwaki K, Farias Luz N, Balaji S, et al. The mito-
chondrial phosphatase PGAM5 is dispensable for
necroptosis but promotes inflammasome activation
in macrophages. J Immunol. 2016;196(1):407–415.
65. Xia B, Fang S, Chen X, et al. MLKL forms cation
channels. Cell Res. 2016;26(5):517–528.
66. Quarato G, Guy CS, Grace CR, et al. Sequential
engagement of distinct MLKL phosphatidylino-
sitol-binding sites executes necroptosis. Mol Cell.
2016;61(4):589–601.
67. Cai Z, Jitkaew S, Zhao J, et al. Plasma membrane
translocation of trimerized MLKL protein is
required for TNF-induced necroptosis. Nat Cell
Biol. 2014;16(1):55–65.
83.e2 Part I: Science and Clinical Oncology
68. Chen X, Li W, Ren J, et al. Translocation of mixed
lineage kinase domain-like protein to plasma mem-
brane leads to necrotic cell death. Cell Res. 2014;24(1):
105–121.
69. Hildebrand JM, Tanzer MC, Lucet IS, et al.
Activation of the pseudokinase MLKL unleashes
the four-helix bundle domain to induce membrane
localization and necroptotic cell death. Proc Natl
Acad Sci USA. 2014;111(42):15072–15077.
70. Dondelinger Y, Declercq W, Montessuit S, et al.
MLKL compromises plasma membrane integrity
by binding to phosphatidylinositol phosphates. Cell
Rep. 2014;7(4):971–981.
71. Wang H, Sun L, Su L, et al. Mixed lineage kinase
domain-like protein MLKL causes necrotic mem-
brane disruption upon phosphorylation by RIP3.
Mol Cell. 2014;54(1):133–146.
72. Oberst A, Dillon CP, Weinlich R, et al. Catalytic
activity of the caspase-8-FLIP(L) complex inhibits
RIPK3-dependent necrosis. Nature. 2011;471(7338):
363–367.
73. Orozco S, Yatim N, Werner MR, et al. RIPK1
both positively and negatively regulates RIPK3
oligomerization and necroptosis. Cell Death Differ.
2014;21(10):1511–1521.
74. Dillon CP, Weinlich R, Rodriguez DA, et al. RIPK1
blocks early postnatal lethality mediated by caspase-8
and RIPK3. Cell. 2014;157(5):1189–1202.
75. Zong WX, Ditsworth D, Bauer DE, Wang ZQ,
Thompson CB. Alkylating DNA damage stimulates
a regulated form of necrotic cell death. Genes Dev.
2004;18(11):1272–1282.
76. Nogusa S, Thapa RJ, Dillon CP, et al. RIPK3
activates parallel pathways of MLKL-driven
necroptosis and FADD-mediated apoptosis to
protect against influenza A virus. Cell Host Microbe.
2016;20(1):13–24.
77. Bonora M, Wieckowski MR, Chinopoulos C, et al.
Molecular mechanisms of cell death: central implica-
tion of ATP synthase in mitochondrial permeability
transition. Oncogene. 2015;34(12):1475–1486.
78. Izzo V, Bravo-San Pedro JM, Sica V, Kroemer G,
Galluzzi L. Mitochondrial permeability transition:
new findings and persisting uncertainties. Trends
Cell Biol. 2016.
79. Brenner C, Grimm S. The permeability transition pore
complex in cancer cell death. Oncogene. 2006;25(34):
4744–4756.
80. Baines CP, Kaiser RA, Sheiko T, Craigen WJ,
Molkentin JD. Voltage-dependent anion channels
are dispensable for mitochondrial-dependent cell
death. Nat Cell Biol. 2007;9(5):550–555.
81. Galluzzi L, Kroemer G. Mitochondrial apoptosis
without VDAC. Nat Cell Biol. 2007;9(5):487–
489.
82. Kokoszka JE, Waymire KG, Levy SE, et al. The
ADP/ATP translocator is not essential for the
mitochondrial permeability transition pore. Nature.
2004;427(6973):461–465.
83. Baines CP, Kaiser RA, Purcell NH, et al. Loss of
cyclophilin D reveals a critical role for mitochon-
drial permeability transition in cell death. Nature.
2005;434(7033):658–662.
84. Schinzel AC, Takeuchi O, Huang Z, et al.
Cyclophilin D is a component of mitochondrial
permeability transition and mediates neuronal cell
death after focal cerebral ischemia. Proc Natl Acad
Sci USA. 2005;102(34):12005–12010.
85. Bonora M, Bononi A, De Marchi E, et al. Role of the
c subunit of the FO ATP synthase in mitochondrial
permeability transition. Cell Cycle. 2013;12(4):
674–683.
86. Yang WS, Kim KJ, Gaschler MM, Patel M,
Shchepinov MS, Stockwell BR. Peroxidation of
polyunsaturated fatty acids by lipoxygenases drives
ferroptosis. Proc Natl Acad Sci USA. 2016.
87. Dixon SJ, Lemberg KM, Lamprecht MR, et al.
Ferroptosis: an iron-dependent form of nonapoptotic
cell death. Cell. 2012;149(5):1060–1072.
88. Dixon SJ, Patel DN, Welsch M, et al. Pharmacologi-
cal inhibition of cystine-glutamate exchange induces
endoplasmic reticulum stress and ferroptosis. Elife.
2014;3:e02523.
89. Friedmann Angeli JP, Schneider M, Proneth B,
et al. Inactivation of the ferroptosis regulator Gpx4
triggers acute renal failure in mice. Nat Cell Biol.
2014;16(12):1180–1191.
90. Yang WS, SriRamaratnam R, Welsch ME, et al.
Regulation of ferroptotic cancer cell death by GPX4.
Cell. 2014;156(1–2):317–331.
91. Skouta R, Dixon SJ, Wang J, et al. Ferrostatins inhibit
oxidative lipid damage and cell death in diverse
disease models. J Am Chem Soc. 2014;136(12):
4551–4556.
92. Linkermann A. Nonapoptotic cell death in acute
kidney injury and transplantation. Kidney Int.
2016;89(1):46–57.
93. Linkermann A, Skouta R, Himmerkus N, et al.
Synchronized renal tubular cell death involves fer-
roptosis. Proc Natl Acad Sci USA. 2014;111(47):
16836–16841.
94. Kepp O, Galluzzi L, Zitvogel L, Kroemer G.
Pyroptosis—a cell death modality of its kind? Eur
J Immunol. 2010;40(3):627–630.
95. Zitvogel L, Kepp O, Galluzzi L, Kroemer G. Inflam-
masomes in carcinogenesis and anticancer immune
responses. Nat Immunol. 2012;13(4):343–351.
96. Galluzzi L, Lopez-Soto A, Kumar S, Kroemer G.
Caspases connect cell-death signaling to organismal
homeostasis. Immunity. 2016;44(2):221–231.
97. Sborgi L, Ruhl S, Mulvihill E, et al. GSDMD
membrane pore formation constitutes the mechanism
of pyroptotic cell death. EMBO J. 2016.
98. Liu X, Zhang Z, Ruan J, et al. Inflammasome-
activated gasdermin D causes pyroptosis by forming
membrane pores. Nature. 2016;535(7610):153–158.
99. Aglietti RA, Estevez A, Gupta A, et al. GsdmD
p30 elicited by caspase-11 during pyroptosis forms
pores in membranes. Proc Natl Acad Sci USA.
2016;113(28):7858–7863.
100. Kayagaki N, Stowe IB, Lee BL, et al. Caspase-11
cleaves gasdermin D for non-canonical inflamma-
some signalling. Nature. 2015;526(7575):666–671.
101. Shi J, Zhao Y, Wang K, et al. Cleavage of GSDMD
by inflammatory caspases determines pyroptotic cell
death. Nature. 2015;526(7575):660–665.
102. Lim Y, Kumar S. A single cut to pyroptosis.
Oncotarget. 2015;6(35):36926–36927.
103. Gibson BA, Kraus WL. New insights into the
molecular and cellular functions of poly(ADP-ribose)
and PARPs. Nat Rev Mol Cell Biol. 2012;13(7):411–
424.
104. Andrabi SA, Umanah GK, Chang C, et al. Poly(ADP-
ribose) polymerase-dependent energy depletion
occurs through inhibition of glycolysis. Proc Natl
Acad Sci USA. 2014;111(28):10209–10214.
105. Wang Y, Kim NS, Haince JF, et al. Poly(ADP-ribose)
(PAR) binding to apoptosis-inducing factor is
critical for PAR polymerase-1-dependent cell death
(parthanatos). Sci Signal. 2011;4(167):ra20.
106. Galluzzi L, Bravo-San Pedro JM, Blomgren K,
Kroemer G. Autophagy in acute brain injury. Nat
Rev Neurosci. 2016;17(8):467–484.
107. Green DR, Galluzzi L, Kroemer G. Mitochondria and
the autophagy-inflammation-cell death axis in organ-
ismal aging. Science. 2011;333(6046):1109–1112.
108. Mizushima N, Levine B, Cuervo AM, Klionsky
DJ. Autophagy fights disease through cellular self-
digestion. Nature. 2008;451(7182):1069–1075.
109. Galluzzi L, Bravo-San Pedro JM, Kepp O, Kroemer
G. Regulated cell death and adaptive stress responses.
Cell Mol Life Sci. 2016;73(11–12):2405–2410.
110. Boya P, Gonzalez-Polo RA, Casares N, et al. Inhibi-
tion of macroautophagy triggers apoptosis. Mol Cell
Biol. 2005;25(3):1025–1040.
111. Berry DL, Baehrecke EH. Growth arrest and
autophagy are required for salivary gland cell degra-
dation in Drosophila. Cell. 2007;131(6):1137–1148.
112. Denton D, Shravage B, Simin R, et al. Autophagy,
not apoptosis, is essential for midgut cell death in
Drosophila. Curr Biol. 2009;19(20):1741–1746.
113. Grander D, Kharaziha P, Laane E, Pokrovskaja K,
Panaretakis T. Autophagy as the main means of
cytotoxicity by glucocorticoids in hematological
malignancies. Autophagy. 2009;5(8):1198–1200.
114. Levine B, Kroemer G. Autophagy in the pathogenesis
of disease. Cell. 2008;132(1):27–42.
115. Levine B, Kroemer G. Autophagy in aging, disease
and death: the true identity of a cell death impostor.
Cell Death Differ. 2009;16(1):1–2.
116. Serrano M, Lin AW, McCurrach ME, Beach D,
Lowe SW. Oncogenic ras provokes premature cell
senescence associated with accumulation of p53 and
p16INK4a. Cell. 1997;88(5):593–602.
117. Luo J, Solimini NL, Elledge SJ. Principles of cancer
therapy: oncogene and non-oncogene addiction. Cell.
2009;136(5):823–837.
118. Kroemer G, Senovilla L, Galluzzi L, Andre F, Zitvogel
L. Natural and therapy-induced immunosurveillance
in breast cancer. Nat Med. 2015;21(10):1128–1138.
119. Heisterkamp N, Groffen J, Stephenson JR,
et al. Chromosomal localization of human cel-
lular homologues of two viral oncogenes. Nature.
1982;299(5885):747–749.
120. Meyer N, Penn LZ. Reflecting on 25 years with
MYC. Nat Rev Cancer. 2008;8(12):976–990.
121. Schubbert S, Shannon K, Bollag G. Hyperactive
Ras in developmental disorders and cancer. Nat Rev
Cancer. 2007;7(4):295–308.
122. Rong Y, Distelhorst CW. Bcl-2 protein family
members: versatile regulators of calcium signaling in
cell survival and apoptosis. Annu Rev Physiol. 2008;70:
73–91.
123. Vander Heiden MG, Chandel NS, Schumacker PT,
Thompson CB. Bcl-xL prevents cell death following
growth factor withdrawal by facilitating mitochon-
drial ATP/ADP exchange. Mol Cell. 1999;3(2):
159–167.
124. Rassidakis GZ, Jones D, Lai R, et al. BCL-2 family
proteins in peripheral T-cell lymphomas: correlation
with tumour apoptosis and proliferation. J Pathol.
2003;200(2):240–248.
125. Linden M, Kirchhof N, Carlson C, Van Ness B.
Targeted overexpression of Bcl-XL in B-lymphoid
cells results in lymphoproliferative disease and
plasma cell malignancies. Blood. 2004;103(7):2779–
2786.
126. Brunelle JK, Ryan J, Yecies D, Opferman JT, Letai
A. MCL-1-dependent leukemia cells are more
sensitive to chemotherapy than BCL-2-dependent
counterparts. J Cell Biol. 2009;187(3):429–442.
127. Strasser A, Harris AW, Cory S. E mu-bcl-2 transgene
facilitates spontaneous transformation of early pre-B
and immunoglobulin-secreting cells but not T cells.
Oncogene. 1993;8(1):1–9.
128. Campbell KJ, Bath ML, Turner ML, et al. Elevated
Mcl-1 perturbs lymphopoiesis, promotes transforma-
tion of hematopoietic stem/progenitor cells, and
enhances drug resistance. Blood. 2010;116(17):
3197–3207.
129. Galluzzi L, Morselli E, Kepp O, et al. Mito-
chondrial gateways to cancer. Mol Aspects Med.
2010;31(1):1–20.
130. Perkins ND. Integrating cell-signalling pathways
with NF-kappaB and IKK function. Nat Rev Mol
Cell Biol. 2007;8(1):49–62.
131. Pahl HL. Activators and target genes of Rel/NF-
kappaB transcription factors. Oncogene. 1999;18(49):
6853–6866.
132. Chen IS, Temin HM. Substitution of 5′ helper virus
sequences into non-rel portion of reticuloendothe-
liosis virus strain T suppresses transformation of
chicken spleen cells. Cell. 1982;31(1):111–120.
133. Carrasco D, Rizzo CA, Dorfman K, Bravo R.
The v-rel oncogene promotes malignant T-cell
leukemia/lymphoma in transgenic mice. EMBO J.
1996;15(14):3640–3650.

134. Chu ZL, DiDonato JA, Hawiger J, Ballard DW. The
tax oncoprotein of human T-cell leukemia virus type
1 associates with and persistently activates IkappaB
kinases containing IKKalpha and IKKbeta. J Biol
Chem. 1998;273(26):15891–15894.
135. Rayet B, Gelinas C. Aberrant rel/nfkb genes and
activity in human cancer. Oncogene. 1999;18(49):
6938–6947.
136. Fujita T, Nolan GP, Liou HC, Scott ML, Baltimore
D. The candidate proto-oncogene bcl-3 encodes a
transcriptional coactivator that activates through NF-
kappa B p50 homodimers. Genes Dev. 1993;7(7B):
1354–1363.
137. Michaux L, Mecucci C, Stul M, et al. BCL3
rearrangement and t(14;19)(q32;q13) in lympho-
proliferative disorders. Genes Chromosomes Cancer.
1996;15(1):38–47.
138. Finco TS, Westwick JK, Norris JL, Beg AA, Der CJ,
Baldwin AS Jr. Oncogenic Ha-Ras-induced signaling
activates NF-kappaB transcriptional activity, which
is required for cellular transformation. J Biol Chem.
1997;272(39):24113–24116.
139. Reuther JY, Reuther GW, Cortez D, Pendergast
AM, Baldwin AS Jr. A requirement for NF-kappaB
activation in Bcr-Abl-mediated transformation. Genes
Dev. 1998;12(7):968–981.
140. Hanahan D, Weinberg RA. The hallmarks of cancer.
Cell. 2000;100(1):57–70.
141. Manning BD, Cantley LC. AKT/PKB signaling:
navigating downstream. Cell. 2007;129(7):1261–
1274.
142. Manning BD, Tee AR, Logsdon MN, Blenis J,
Cantley LC. Identification of the tuberous sclerosis
complex-2 tumor suppressor gene product tuberin
as a target of the phosphoinositide 3-kinase/akt
pathway. Mol Cell. 2002;10(1):151–162.
143. Malstrom S, Tili E, Kappes D, Ceci JD, Tsichlis
PN. Tumor induction by an Lck-MyrAkt transgene
is delayed by mechanisms controlling the size of
the thymus. Proc Natl Acad Sci USA. 2001;98(26):
14967–14972.
144. Hollander MC, Blumenthal GM, Dennis PA. PTEN
loss in the continuum of common cancers, rare
syndromes and mouse models. Nat Rev Cancer.
2011;11(4):289–301.
145. Podsypanina K, Lee RT, Politis C, et al. An inhibitor
of mTOR reduces neoplasia and normalizes p70/
S6 kinase activity in Pten+/− mice. Proc Natl Acad
Sci USA. 2001;98(18):10320–10325.
146. Galluzzi L, Pietrocola F, Bravo-San Pedro JM, et al.
Autophagy in malignant transformation and cancer
progression. EMBO J. 2015;34(7):856–880.
147. Harris H, Miller OJ, Klein G, Worst P, Tachibana
T. Suppression of malignancy by cell fusion. Nature.
1969;223(5204):363–368.
148. Friend SH, Bernards R, Rogelj S, et al. A human
DNA segment with properties of the gene that
predisposes to retinoblastoma and osteosarcoma.
Nature. 1986;323(6089):643–646.
149. Baylin SB, Ohm JE. Epigenetic gene silencing in
cancer—a mechanism for early oncogenic pathway
addiction? Nat Rev Cancer. 2006;6(2):107–116.
150. Esteller M, Cordon-Cardo C, Corn PG, et al.
p14ARF silencing by promoter hypermethylation
mediates abnormal intracellular localization of
MDM2. Cancer Res. 2001;61(7):2816–2821.
151. Teitz T, Wei T, Valentine MB, et al. Caspase 8
is deleted or silenced preferentially in childhood
neuroblastomas with amplification of MYCN. Nat
Med. 2000;6(5):529–535.
152. Hansen MF, Cavenee WK. Tumor suppressors: reces-
sive mutations that lead to cancer. Cell. 1988;53(2):
173–174.
153. Stambolic V, Suzuki A, de la Pompa JL, et al.
Negative regulation of PKB/Akt-dependent cell
survival by the tumor suppressor PTEN. Cell.
1998;95(1):29–39.
Pathophysiology of Cancer Cell Death  •  CHAPTER 5 83.e3
154. Li J, Yen C, Liaw D, et al. PTEN, a putative
protein tyrosine phosphatase gene mutated in
human brain, breast, and prostate cancer. Science.
1997;275(5308):1943–1947.
155. Podsypanina K, Ellenson LH, Nemes A, et al.
Mutation of Pten/Mmac1 in mice causes neoplasia
in multiple organ systems. Proc Natl Acad Sci USA.
1999;96(4):1563–1568.
156. Di Cristofano A, Pesce B, Cordon-Cardo C, Pandolfi
PP. Pten is essential for embryonic development
and tumour suppression. Nat Genet. 1998;19(4):
348–355.
157. Alimonti A, Carracedo A, Clohessy JG, et al. Subtle
variations in Pten dose determine cancer susceptibil-
ity. Nat Genet. 2010;42(5):454–458.
158. Degenhardt K, Chen G, Lindsten T, White E. BAX
and BAK mediate p53-independent suppression of
tumorigenesis. Cancer Cell. 2002;2(3):193–203.
159. Rampino N, Yamamoto H, Ionov Y, et al. Somatic
frameshift mutations in the BAX gene in colon
cancers of the microsatellite mutator phenotype.
Science. 1997;275(5302):967–969.
160. Sturm I, Stephan C, Gillissen B, et al. Loss of the
tissue-specific proapoptotic BH3-only protein Nbk/
Bik is a unifying feature of renal cell carcinoma. Cell
Death Differ. 2006;13(4):619–627.
161. Lane DP, Crawford LV. T antigen is bound to a
host protein in SV40-transformed cells. Nature.
1979;278(5701):261–263.
162. Linzer DI, Levine AJ. Characterization of a 54K
dalton cellular SV40 tumor antigen present in
SV40-transformed cells and uninfected embryonal
carcinoma cells. Cell. 1979;17(1):43–52.
163. Kress M, May E, Cassingena R, May P. Simian virus
40-transformed cells express new species of proteins
precipitable by anti-simian virus 40 tumor serum.
J Virol. 1979;31(2):472–483.
164. Rotter V, Witte ON, Coffman R, Baltimore D.
Abelson murine leukemia virus-induced tumors elicit
antibodies against a host cell protein, P50. J Virol.
1980;36(2):547–555.
165. Vousden KH, Prives C. Blinded by the light: the
growing complexity of p53. Cell. 2009;137(3):
413–431.
166. Li M, Brooks CL, Wu-Baer F, Chen D, Baer R,
Gu W. Mono- versus polyubiquitination: dif-
ferential control of p53 fate by Mdm2. Science.
2003;302(5652):1972–1975.
167. Kruse JP, Gu W. Modes of p53 regulation. Cell.
2009;137(4):609–622.
168. Riley T, Sontag E, Chen P, Levine A. Transcriptional
control of human p53-regulated genes. Nat Rev Mol
Cell Biol. 2008;9(5):402–412.
169. Miyashita T, Krajewski S, Krajewska M, et al.
Tumor suppressor p53 is a regulator of bcl-2 and
bax gene expression in vitro and in vivo. Oncogene.
1994;9(6):1799–1805.
170. Moroni MC, Hickman ES, Lazzerini Denchi E,
et al. Apaf-1 is a transcriptional target for E2F and
p53. Nat Cell Biol. 2001;3(6):552–558.
171. Nakano K, Vousden KH. PUMA, a novel
proapoptotic gene, is induced by p53. Mol Cell.
2001;7(3):683–694.
172. Caelles C, Helmberg A, Karin M. p53-dependent
apoptosis in the absence of transcriptional activation of
p53-target genes. Nature. 1994;370(6486):220–223.
173. Chipuk JE, Bouchier-Hayes L, Kuwana T, Newmeyer
DD, Green DR. PUMA couples the nuclear and
cytoplasmic proapoptotic function of p53. Science.
2005;309(5741):1732–1735.
174. Chipuk JE, Kuwana T, Bouchier-Hayes L, et al.
Direct activation of Bax by p53 mediates mito-
chondrial membrane permeabilization and apoptosis.
Science. 2004;303(5660):1010–1014.
175. Mihara M, Erster S, Zaika A, et al. p53 has a direct
apoptogenic role at the mitochondria. Mol Cell.
2003;11(3):577–590.
83.e3
176. Vaseva AV, Marchenko ND, Ji K, Tsirka SE,
Holzmann S, Moll UM. p53 opens the mitochon-
drial permeability transition pore to trigger necrosis.
Cell. 2012;149(7):1536–1548.
177. Cheok CF, Verma CS, Baselga J, Lane DP. Trans-
lating p53 into the clinic. Nat Rev Clin Oncol.
2011;8(1):25–37.
178. Donehower LA, Harvey M, Slagle BL, et al. Mice
deficient for p53 are developmentally normal
but susceptible to spontaneous tumours. Nature.
1992;356(6366):215–221.
179. Vitale I, Galluzzi L, Castedo M, Kroemer G. Mitotic
catastrophe: a mechanism for avoiding genomic insta-
bility. Nat Rev Mol Cell Biol. 2011;12(6):385–392.
180. Sablina AA, Budanov AV, Ilyinskaya GV, Agapova
LS, Kravchenko JE, Chumakov PM. The antioxidant
function of the p53 tumor suppressor. Nat Med.
2005;11(12):1306–1313.
181. Vousden KH, Ryan KM. p53 and metabolism. Nat
Rev Cancer. 2009;9(10):691–700.
182. Matoba S, Kang JG, Patino WD, et al. p53
regulates mitochondrial respiration. Science.
2006;312(5780):1650–1653.
183. Diamandis M, White NM, Yousef GM. Personalized
medicine: marking a new epoch in cancer patient
management. Mol Cancer Res. 2010;8(9):1175–1187.
184. Dancey JE, Chen HX. Strategies for optimizing
combinations of molecularly targeted anticancer
agents. Nat Rev Drug Discov. 2006;5(8):649–659.
185. Nardella C, Clohessy JG, Alimonti A, Pandolfi PP.
Pro-senescence therapy for cancer treatment. Nat
Rev Cancer. 2011;11(7):503–511.
186. Shabbits JA, Hu Y, Mayer LD. Tumor chemosensi-
tization strategies based on apoptosis manipulations.
Mol Cancer Ther. 2003;2(8):805–813.
187. Galluzzi L, Buque A, Kepp O, Zitvogel L, Kroemer
G. Immunological effects of conventional chemo-
therapy and targeted anticancer agents. Cancer Cell.
2015;28(6):690–714.
188. Rakhra K, Bachireddy P, Zabuawala T, et al.
CD4(+) T cells contribute to the remodeling of
the microenvironment required for sustained tumor
regression upon oncogene inactivation. Cancer Cell.
2010;18(5):485–498.
189. Sanchez-Serrano I. Success in translational research:
lessons from the development of bortezomib. Nat
Rev Drug Discov. 2006;5(2):107–114.
190. Roberts AW, Davids MS, Pagel JM, et al. Targeting
BCL2 with venetoclax in relapsed chronic lympho-
cytic leukemia. N Engl J Med. 2016;374(4):311–322.
191. Dancey J. mTOR signaling and drug development
in cancer. Nat Rev Clin Oncol. 2010;7(4):209–219.
192. Vassilev LT, Vu BT, Graves B, et al. In vivo activation
of the p53 pathway by small-molecule antagonists
of MDM2. Science. 2004;303(5659):844–848.
193. Brown CJ, Lain S, Verma CS, Fersht AR, Lane
DP. Awakening guardian angels: drugging the p53
pathway. Nat Rev Cancer. 2009;9(12):862–873.
194. Wilson JM. Gendicine: the first commercial gene
therapy product. Hum Gene Ther. 2005;16(9):
1014–1015.
195. Grant S, Easley C, Kirkpatrick P. Vorinostat. Nat
Rev Drug Discov. 2007;6(1):21–22.
196. Quintas-Cardama A, Santos FP, Garcia-Manero G.
Therapy with azanucleosides for myelodysplastic
syndromes. Nat Rev Clin Oncol. 2010;7(8):433–444.
197. Kroemer G, Galluzzi L, Kepp O, Zitvogel L.
Immunogenic cell death in cancer therapy. Annu
Rev Immunol. 2013;31:51–72.
198. Linkermann A, Stockwell BR, Krautwald S, Anders
HJ. Regulated cell death and inflammation: an
auto-amplification loop causes organ failure. Nat
Rev Immunol. 2014;14(11):759–767.
199. Land WG, Agostinis P, Gasser S, Garg AD,
Linkermann A. Transplantation and damage associ-
ated molecular patterns (DAMPs). Am J Transplant.
2016.
83.e4 Part I: Science and Clinical Oncology
SELF-ASSESSMENT REVIEW QUESTIONS
1. Which one of the following molecular alterations may be
common among lymphoma patients?
a. A translocation between chromosome 14 and chromosome
19, resulting in the overexpression of BAX
b. A loss-of-function point mutation in AKT1
c. An amplification in the chromosomal region 12q13-14,
encompassing MDM2
2. The detection of a point mutation in TP53 that negatively
affects p53-regulated gene transactivation would prompt for:
a. The administration of a hypothetical FDA-approved MDM2
inhibitor
b. The administration of DNA damaging agents
c. The use of gendicine
3. Which one of the following may constitute the basis for the
development of novel anticancer regimens?
a. The screening of a combinatorial chemical library for the
identification of MLKL activators
b. The discovery of a new inhibitor of caspases
c. The identification of a new inhibitor of BAX
4. Which one of the following statements is best supported by
experimental evidence?
a. Death receptors and dependence receptors activate the same
signaling pathways.
b. Death receptors and dependence receptors lead to CASP3
activation.
c. Death receptors and dependence receptors operate in similar
settings.
ANSWERS
1. (c)  BAX overexpression is expected to counteract oncogenesis,
similar to inactivating mutations in AKT1. Conversely, the
amplification of MDM2 is relatively common among tumors,
leading to the functional inactivation of the p53 system.
2. (c)  Increasing the levels of mutant p53 by employing MDM2
inhibitors is expected to be useless in the presence of
inactivating TP53 mutations. Along similar lines, the use of
DNA-damaging agents would presumably yield poor results,
because these compounds most often function by activating
p53. Conversely, gendicine would lead to the restoration of a
functional p53 system, perhaps exerting direct anticancer effects
or rendering cancer cells responsive to chemotherapy.
3. (a)  Inhibition of BAX or caspases appears to be therapeutically
useful in conditions featuring excessive cell death, such as
ischemia or neurodegeneration. Conversely, novel activators of
MLKL that would selectively trigger the necrotic demise of
cancer cells might be very promising, because cancer cells are
often intrinsically resistant to the therapeutic induction of
apoptosis.
4. (b)  Although both death receptors and dependence receptors
trigger extrinsic apoptosis, not only do they respond to different
conditions, but also they engage distinct signaling cascades. Still,
both death receptors and dependence receptors eventually lead
to the proteolytic activation of CASP3.
 6  Cancer Immunology
Diane Tseng, Liora Schultz, Drew Pardoll, and Crystal Mackall

S UMMARY OF K EY
• Cancer is characterized by genetic
and epigenetic instability leading termed editing. Multiple cells and
to both unique and sometimes
common mutations and “ectopic”
overexpression of genes not
normally expressed in the tissue of
origin. Cancer-specific proteins
arising as a result of genetic
mutations appear to provide the
most potent antigens visible to and PD-1, inhibitory receptors on antigens differentially expressed on
the T-cell arm of the immune
system. Because the number of
cancer-specific antigens expressed
varies greatly among individual
patients and among cancer
histologies, cancer immunogenicity
varies substantially.
• As cancers develop, they are
sculpted by “immune pressure” to
eliminate antigens, or diminish the
degree to which antigenic peptides
are processed or presented to the
P OI N T S
immune system, through a process
molecular interactions in the tumor
microenvironment also inhibit
antitumor immune responses,
including regulatory T cells and
myeloid-derived suppressor cells.
Tumor-induced immunosuppression
is also mediated by signaling CTLA-4
T cells.
• Inhibition of CTLA-4 and/or PD-1
signaling on T cells enhances
antitumor immunity by unleashing
naturally induced adaptive antitumor
immune responses that have
undergone active suppression.
Clinical trials using antibodies that
block CTLA-4 have demonstrated
impressive effects in melanoma, and
clinical trials using antibodies that
block PD-1 signaling have
demonstrated impressive effects in
many cancers, including melanoma,
non–small cell lung cancer, renal
cell carcinoma, bladder carcinoma,
head and neck cancer, Hodgkin
lymphoma, Merkel cell carcinoma,
and others.
• Synthetic biology can be used to
engineer immunotherapies toward
cancer versus normal tissues, and
such therapeutics do not require
inherent tumor immunogenicity to
be effective. Examples of such
therapeutics are monoclonal
antibodies, bispecific antibodies,
and T cells engineered to express
chimeric antigen receptors. T cells
expressing a chimeric antigen
receptor targeting CD19 have
demonstrated impressive effects
against B-cell malignancies.

OVERVIEW developments in T-cell checkpoint-blocking antibodies, adoptive

At least two features of the immune system make it a unique therapeutic
tool against cancer. First, the diversity of receptors in the adaptive
immune system (T-cell receptor [TCR] for T cells and antibodies
made by B cells) offers unparalleled capacity for target specificity, far
greater than any synthetic drug library. Second, diverse cell-killing
weaponry from both the innate immune system and cytotoxic T cells
offers the potential to kill any cell, once appropriately recognized.
Central to the concept of successful cancer immunotherapy are the
dual tenets that tumor cells express an antigenic target distinct from
their normal cellular counterparts and that the immune system is
capable of recognizing these antigenic differences.
The notion that the immune system can be used as an anticancer
therapy emerged from experiments in animal models of carcinogen-
induced cancer. It was demonstrated that a number of experimentally
induced tumors could be rejected on transplantation into syngeneic
immunocompetent animals.1–4 Extensive studies by Prehn on the
phenomenon of tumor rejection suggested that the most potent tumor
rejection antigens were unique to the individual tumor.5 These studies
led to the hypothesis that the immune system may be harnessed to
eliminate cancer cells while sparing normal tissue. This chapter reviews
the biology of tumor–immune system interactions and discusses how
scientific insights from immunology have translated into novel strategies
for harnessing the immune response to treat cancer, highlighting recent
therapies with engineered T cells, and tumor vaccines.
THE ANTIGENIC PROFILE THAT
DISTINGUISHES TUMORS FROM
NORMAL TISSUES
Genetic instability, a hallmark of cancer, is a primary generator of
tumor-specific antigens. On average, cancers express between 50 and
1000 missense mutations in coding regions, roughly 20% of which
create neoantigenic peptides presented by at least one of the individual’s
human leukocyte antigen (HLA) alleles and thus recognized by T
cells.6–14 In addition, deletions, amplifications, and chromosomal
rearrangements can result in new genetic sequences resulting from
juxtaposition of coding sequences not normally contiguous in untrans-
formed cells. The vast majority of these mutations occur in intracellular
proteins, and therefore the “neoantigens” they encode would not be
readily targeted by antibodies. However, the major histocompatibility
complex (MHC) presentation system for T-cell recognition renders
peptides derived from all cellular proteins available on the cell surface
as peptide MHC complexes capable of being recognized by T cells.
In accordance with the original findings of Prehn,5 the vast majority
of tumor-specific antigens derived from genetic mutations are unique
to individual tumors. As a result, antigen-specific immunotherapies

84
 Cancer Immunology  •  CHAPTER 6 85

Immune
surveillance
ELIMINATION
Normal Genetic Tumor Resistance
cell alterations cell mechanisms
SURVIVAL
Transformation
progression x
x
x
x Tolerance induction
SURVIVAL

Figure 6.1  •  The balance among immune surveillance, resistance, and tolerance. Transformation of normal cells to cancer cells involves the creation
of neoantigens as a result of mutation as well as upregulation of self-antigens. Successful immune surveillance of tumors based on recognition of these
tumor-specific antigens would lead to tumor elimination at early stages. Clinically relevant tumor survival and progression require that tumors develop resistance
mechanisms that inhibit tumor-specific immune responses to kill tumor cells. Alternatively, if the tumor develops mechanisms to induce immune tolerance
to its antigens, antitumor effector responses do not develop. Finally, tumors could respond to immune pressure from T cells by eliminating mutations that
are efficiently presented by the tumor’s major histocompatibility complex, a process termed immune editing. Evidence is accumulating that tumors actively
develop immune resistance mechanisms as well as immune tolerance mechanisms in order to survive despite displaying antigens capable of recognition by the
immune system. Evidence for immune editing has been demonstrated in experimental carcinogen-induced tumors in mice but not yet in human cancers.

targeted at truly tumor-specific antigens are most commonly patient
specific. However, some tumor-specific mutations are shared (e.g.,
KRAS codon 12 G->A in colon and pancreatic cancers; BRAF V600E
found in melanomas; p53 codon 249 G->T mutation found in
hepatocellular carcinomas)15–18 and could serve as potential immuno-
therapy targets.
Tumors also manifest global alterations in DNA methylation and
chromatin structure resulting in dramatic shifts in gene expression.19
Tumors often overexpress hundreds of genes relative to their normal
counterparts, including genes that are normally completely silent in
their normal cellular counterparts. Overexpressed genes in tumor cells
are attractive targets for both antibody- and cell-based immunotherapies,
because overexpressed genes are shared among many tumors of a given
tissue origin or sometimes multiple tumor types. For example,
mesothelin, which is targeted by T cells from vaccinated pancreatic
cancer patients,20 is highly expressed in virtually all pancreatic cancers,
mesotheliomas, and most ovarian cancers.21,22 Whereas mesothelin is
expressed at low to moderate levels in the pleural mesothelium, it is
not expressed at all in normal pancreatic or ovarian ductal epithelial
cells. Another example of tumor-selective expression of epigenetically
altered genes is the so-called cancer-testis antigens.23 These genes are
expressed almost exclusively in germ cells of the testis and ovaries,
and some appear to encode proteins associated with meiosis.24–26 Many
cancer-testis antigens are recognized by T cells from nonvaccinated
and vaccinated cancer patients.23 A final category of tumor antigen
consists of tissue-specific differentiation antigens shared by tumors of
similar histologic origin. Commonly generated melanoma-reactive T
cells from melanoma patients recognize melanocyte antigens,27 including
tyrosinase, a melanocyte-specific protein required for melanin syn-
thesis.28,29 Although tissue-specific antigens are not truly tumor specific,
they are nonetheless potentially effective targets when the tissue is
dispensable (e.g., prostate cancer or melanoma).
IMMUNE SURVEILLANCE HYPOTHESIS
OF CANCER
The immune surveillance hypothesis, first conceived nearly a half
century ago,30,31 proposed that a fundamental role of the immune
system is to survey the body for tumors as it does for infection with
pathogens, recognizing and eliminating them based on their expression
of tumor-associated antigens (TAAs). In animal models, carcinogen-
induced tumors can be divided into those that grow progressively
(termed progressor tumors) and those that are rejected after an initial
period of growth (termed regressor tumors).2,3 The phenomenon of
regressor tumors was explained based on ongoing immune surveillance
of cancer. A corollary to the original immune surveillance hypothesis
is that progressor tumors in animals (presumed to represent clinically
progressing cancers in humans) fail to be eliminated because they
develop active mechanisms of either immune escape or resistance
(Fig. 6.1).
Genetically manipulated mice have provided clear evidence that
the immune system can eliminate both carcinogen-induced and
spontaneously arising cancers.32–34 When profoundly immunodeficient
mice were treated with carcinogens or crossed onto a cancer-prone
p53 knockout, the incidence of cancers was modestly but significantly
increased relative to nonimmunodeficient counterparts when observed
over an extended period (greater than 1 year). Further, tumors that
arise in immunodeficient animals behave as regressor tumors when
transplanted into immunocompetent animals. These findings are
consistent with a model wherein tumors that arise in immunodeficient
animals would have been eliminated had they arisen in immunocom-
petent animals.
Epidemiologic studies of patients with heritable immunodeficiencies
have also confirmed a significantly increased risk of certain cancers
that are distinct from the epithelial cancers commonly observed in
normal immunocompetent adults.35–37 Although many cancers observed
in immunodeficient individuals are associated with viral infections,
(e.g., Epstein-Barr virus [EBV]–associated lymphomas,38 Kaposi
sarcoma–associated herpesvirus [KSHV]–associated Kaposi sarcoma,39
and human papillomavirus [HPV])–associated cervical cancer40,41),
immunodeficient individuals also demonstrate an increased incidence
of non–pathogen-associated cancers, particularly melanoma.42
IMMUNE HALLMARKS OF CANCER:
AVOIDING IMMUNE DESTRUCTION AND
TUMOR-PROMOTING INFLAMMATION
Avoiding Immune Destruction
Evidence from murine and human tumors demonstrates the capacity
for tumors to induce T-cell tolerance to their antigens.43–46 Tolerance
induction among tumor antigen–specific T cells can be an active
process involving direct antigen recognition or can be associated with
failure of antigen recognition by T cells—that is, the immune system
“ignores” the tumor.47,48 However, tumors do not uniformly tolerize
T cells. Ohashi and colleagues observed that lymphocytic choriomen-
ingitis virus (LCMV) GP33–specific TCR transgenic CD8 T cells
adoptively transferred into mice expressing pancreatic islet cell tumors
that express GP3349 manifested evidence for CD8 T-cell activation
as a result of antigen cross-presentation in the draining lymph nodes.
Despite the activation of tumor-specific T cells, the tumors grew
progressively, indicating that the degree of immune activation induced
by tumor growth was insufficient to ultimately eliminate the tumors.
These results suggest that in some cases T-cell responses are induced
86 Part I: Science and Clinical Oncology

to developing tumors, but if the level of immune activation ultimately
does not “keep up” with tumor progression, the ultimate result is
tumor outgrowth. In the case of the LCMV GP33-specific TCR
transgenic mice, because neither anergic nor deletional tolerance was
observed, animals treated with the dendritic cell (DC) stimulatory
anti-CD40 antibody demonstrated significant slowing of tumor growth.
Thus depending on the mechanism of immune escape at play, agents
that affect the overall activation state of either antigen-presenting cells
or T cells could be used to shift the balance between tumor immune
evasion and tumor immune recognition.
T cells retrieved from patients with cancer tend either to be of
low affinity for their cognate antigen or to recognize antigens that
bind poorly to their presenting HLA (human MHC) molecule, resulting
in inefficient recognition by T cells. Furthermore, tumors commonly
acquire defects in MHC class I expression to avoid immune destruction.
MHC class I proteins display small peptide antigens on the surface
of cells and are key for immune recognition by CD8 T cells. For
example, activated HRAS leads to reduced levels of messenger RNA
(mRNA) transcripts of the transporter associated with antigen processing
(TAP), a key protein in the antigen-processing pathway.50 Repression
of proteins involved in the antigen presentation pathway are linked
to overexpressed epidermal growth factor receptor (EGFR) or HPV
E7 oncoprotein.51,52 MHC class I downregulation can also result from
MHC gene mutations, defects in other genes in the antigen presentation
pathway such as ERAP1 and ERAP2, and epigenetic regulation of
TAP genes.53–55
Tumor-Promoting Inflammation in the Tumor
Microenvironment
The tumor microenvironment is replete with suppressive mechanisms
that dampen antitumor immune immunity (Fig. 6.2). The inhibitory
cells, molecules, and signaling pathways found in the tumor micro-
environment are not unique to tumors, but rather compose an array
of physiologic mechanisms evolved to regulate immune responses to
self-antigens and to downmodulate immune and inflammatory responses
to foreign antigens so that collateral tissue damage is limited. Tumors
co-opt and upregulate mechanisms for resistance to immune attack,
and much activity in the field of immuno-oncology is focused on
delineating these pathways and developing therapeutics capable of
reversing the effects of these mediators.
Regulatory T Cells and Cancer
Regulatory T cells (Tregs) maintain tolerance to self-antigens, down-
regulate immune responses to pathogens,56,57 and induce tolerance
to tumor antigens.58,59 CD4+ Tregs are characterized by expression
of Foxp3, a central master regulatory transcription factor.60,61 Other
Tregs produce inhibitory cytokines such as interleukin (IL)-10 and

Maturation
Inhibitory cytokines ↑STAT3
Tumor (VEGF, IL-10, TGF-β) DC/MΦ

PD-L1
PD-L1/2

B7-H3 LAG-3
PD-1
IDO B7-H4
↑STAT3
TDO CD4 T cell
Adenosine
Treg
A2aR IFNγ
PD-L1
IL-10, TGF-β
PD-L1
PD-1

LAG-3
Antitumor
↑STAT3 CD8 killer cell Tim-3
PD-L2
MDSC
IDO BTLA

Figure 6.2  •  The immune microenvironment of the tumor is generally inhibitory to antitumor immune responses. Activation of oncogenic pathways
such as STAT3 in the tumor leads to a cascade of molecular and cellular processes in the tumor microenvironment that block the killing function of innate
immune effectors such as natural killer (NK) cells and granulocytes and block dendritic cell (DC) maturation and activation/effector function of Th1 T cells
and CD8 cytotoxic T cells. This inhibitory microenvironment is organized by both cytokines, such as interleukin (IL)-10, IL-6, and transforming growth
factor–β (TGF-β), and multiple cell membrane coinhibitory molecules such as PD-L1, PD-L2, B7-H3, and B7-H4 that interact with their cognate checkpoint
receptors expressed at high levels on T cells in the microenvironment. Myeloid-derived suppressor cells (MDSC) produce nitric oxide (NO) that inhibits T
cells, and both tumor cells and myeloid cells produce tryptophan dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO), which deplete tryptophan.
Regulatory T cells (Treg) also accumulate in the tumor microenvironment, further blunting antitumor T-cell responses via production of inhibitory cytokines.
Tumors also produce adenosine as a byproduct of cell death, which inhibits immune responses and enhances local Treg generation and suppressive function
via interaction with the adenosine A2a receptor. BTLA, B- and T-lymphocyte attenuator; IFN-γ, interferon-γ; VEGF, vascular endothelial growth factor.

transforming growth factor–β (TGF-β). In addition, a recently described
IL-12 family “hybrid” cytokine, IL-35, consisting of the alpha subunit of
IL-12 and the beta subunit of IL-27, is produced by Tregs and suppresses
antitumor immunity.62 Numerous murine studies have demonstrated
that Tregs expand in animals with cancer and limit the potency of
antitumor immune responses.63 It is now appreciated that treatment with
low-dose cyclophosphamide is a relatively simple and reasonably effective
way to temporarily eliminate cycling Tregs.64–67 In addition, antibodies
neutralizing IL-35 limited tumor growth in multiple preclinical mouse
models, but their use alone was not sufficient to eliminate tumors.62
As new cell membrane molecules that define Tregs are identified, the
capacity to block regulatory T-cell activity with antibodies to these
molecules presents new opportunities for immunotherapeutic strategies
to break tolerance to tumor antigens. Evidence for a role of Tregs
in suppressing antitumor immunity in humans includes an extensive
study correlating Treg number in resected ovarian cancers with clinical
outcome. Patients with greater numbers of CD4+CD25hiFoxp3+ cells
had a worse outcome.58 A number of clinical trials have been performed
using a toxin-conjugated IL-2 reagent (denileukin diftitox) that would
bind CD25 and selectively kill Tregs. Clinical efficacy of this agent was
described in phase III studies of recurrent or refractory cutaneous T-cell
lymphoma, and the drug was approved by the US Food and Drug
Administration (FDA) for this indication, but the agent is currently not
available owing to the manufacturer’s decision to prioritize development
of a newer improved formulation of the drug.68
Immature Myeloid Cells and Tumor-Associated Macrophages
Immature myeloid cells (iMCs)69,70 are a heterogeneous class of cells
that include myeloid-derived suppressor cells (MDSCs)71–74 as well as
tumor-associated macrophages (TAMs). In mice, iMCs and MDSCs
are characterized by coexpression of CD11b (considered a macrophage
marker) and Gr1 (considered a granulocyte marker) while expressing
low or no MHC class II or the CD86 costimulatory molecule. In
humans, MDSCs are most commonly described as CD33+ but lack
MHC class II expression, as well as markers of mature macrophages,
DCs, or granulocytes. A number of molecular species produced by
tumors tend to drive iMC and MDSC accumulation, including IL-6,
colony stimulating factor 1 (CSF-1), IL-10, and gangliosides. IL-6
and IL-10 are potent inducers of STAT3 signaling, which has been
shown to be important in iMC and MDSC persistence and activity.
Macrophages have been divided into M1 type (Th1/interferon [IFN]
instructed) and M2 type (Th2/IL-4 and IL-13 instructed). M1
macrophages are characterized by expression of genes encoding the
nitric oxide (NO)–producing iNOS-2 enzyme and the cytokine IL-12,
which amplifies Th1 responses. M1 macrophage activation is thought
to be one of the mediators of Th1-orchestrated antitumor immunity,
whereas M2 macrophages are thought to be tumor promoting.
Macrophage infiltration into tumors has been largely associated
with poor patient prognosis in multiple tumor types. Therapeutic
strategies for targeting TAMs include inhibition of macrophage
recruitment to the tumor or promotion of their antitumor properties
or effector function. Strategies for preventing macrophage accumulation
in tumors include disruption of the CCL2/CCR2 axis in phase I
clinical trials in patients with advanced-phase solid cancers.75,76 The
CSF-1/CSF-1R pathway is also being evaluated as a therapeutic strategy
to target tumor TAMs, and agents targeting these pathways have been
shown to reduce TAM accumulation in tumors or repolarize them
into an antitumor phenotype in early-phase clinical trials.77 Strategies
to promote phagocytosis and antigen presentation by TAMs are very
promising—for example, by disrupting the CD47-SIRPalpha axis,
which has been shown to be effective in preclinical models and is
being tested in ongoing clinical trials.78–80
Immature Dendritic Cells
DCs found within the tumor microenvironment typically have an
immature, unactivated phenotype characterized by low levels of
proinflammatory cytokine production, CD86, and surface MHC class
Cancer Immunology  •  CHAPTER 6 87
II expression (Fig. 6.3). Immature DCs can present antigens to T
cells, but without costimulatory molecules, induce T-cell tolerance,81,82
whereas in the context of microbial ligands or endogenous “danger
signals,” DCs are activated to present antigen together with costimula-
tory signals that result in an effector T-cell phenotype. A major
inhibitory signaling pathway induced in tumor infiltrating DC is the
STAT3 pathway, which, when activated, strongly antagonizes Toll-like
receptor (TLR) and CD40-mediated DC activation. Tumor-derived
factors such as IL-10, IL-6, and vascular endothelial growth factor
(VEGF) (in part induced by STAT3 signaling in the tumor cell) can
induce STAT3 activation in DCs, and such mechanisms shift tumor-
specific T cells from a state of activation to tolerance (Fig. 6.4).
Numerous cancer immunotherapies seek to induce activation and
maturation of DCs, such as engaging CD40.
Immune Inhibitory Molecules Expressed in the Tumor
Microenvironment
A large number of immune inhibitory molecules are expressed in the
microenvironment of tumors, spanning enzymes that metabolize certain
amino acids on which lymphocytes are highly dependent, enzymes
that produce immune-inhibitory products, cytokines that inhibit
antitumor immune responses by acting on immune effector cells to
either inhibit their tumor-lytic activity, and membrane-bound ligands
that bind to inhibitory receptors on lymphocytes (so-called checkpoints).
Myeloid cells in the tumor microenvironment express a number of
enzymes whose metabolic activity ultimately results in inhibition of
T-cell responses. These include production of reactive oxygen species
(ROS) and reactive nitrogen species (RNS). NO production by iMCs
and MDSCs as a result of arginase and inducible nitric oxide synthase
(iNOS) activity has been well documented, and inhibition of this
pathway with a number of drugs can mitigate the inhibitory effects
of iMCs and MDSCs. ROS, including H2O2, have been reported to
block T-cell function associated with the downmodulation of the zeta
(ζ) chain of the TCR signaling complex,83 a phenomenon well rec-
ognized in T cells from cancer patients and associated with generalized
T-cell unresponsiveness.
Another mediator of T-cell unresponsiveness associated with cancer
is the production of indoleamine 2,3-dioxygenase (IDO).84 IDO appears
to be produced by DCs either within tumors or in tumor-draining
lymph nodes. It is interesting to note that IDO in DCs has been
reported to be induced via backward signaling by B7-1/2 on ligation
with CTLA-4.85,86 The major IDO-producing DC is thought to be
either a plasmacytoid dendritic cell (PDC) or a PDC-related cell that
is B220+87; however, IDO has been subsequently shown to be expressed
by multiple cell types in the immune microenvironment, including
tumor cells themselves.88 IDO appears to inhibit T-cell responses through
catabolism of tryptophan. Activated T cells are highly dependent on
tryptophan and are therefore sensitive to tryptophan depletion. Thus
Munn and Mellor have proposed a bystander mechanism, whereby
DCs in the local environment deplete tryptophan via IDO upregulation,
thereby inducing metabolic apoptosis in locally activated T cells.84 IDO
has two isoforms, IDO-1 and IDO-2, encoded by distinct genes. The
role of IDO-2 in human cancer is still unclear; a major IDO-2 poly-
morphism in humans encodes an inactive enzyme. Epacadostat is an
oral inhibitor of IDO-1 and is being tested in combination with
pembrolizumab (anti-PD-1) in patients with advanced melanoma,
which has demonstrated promising results in phase I/II clinical trials.89
Transforming Growth Factor–β: A Major Inhibitory Cytokine
in the Tumor Microenvironment
TGF-β is a major inhibitory cytokine implicated in blunting antitumor
immune responses. TGF-β is produced by a variety of cell types,
including tumor cells, and has pleiotropic physiologic effects. For
most normal epithelial cells, TGF-β is a potent inhibitor of cell prolifera-
tion, causing cell cycle arrest in the G1 stage.90 In many cancer cells,
88 Part I: Science and Clinical Oncology

GM-CSF

IL-4, FLT-3L

Bone marrow Dendritic cell

progenitor progenitor
Antigen uptake/Processing
Microbial infection
No danger danger signals
signals
Exogenous Endogenous
LPS TNF-
CpG CD40L
Tolerizing DC Activated DC

Moderate MHC II
Costimulatory molecules

High MHC II
Costimulatory molecules

Figure 6.3  •  A dendritic cell (DC) can either activate adaptive immunity or tolerize T cells depending on its state of maturation. DC progenitors
develop from hematopoietic (bone marrow–derived) progenitors under the influence of various cytokines, particularly granulocyte-macrophage colony-stimulating
factor (GM-CSF). Under circumstances of microbial infection, specific pathogen-associated molecular patterns (PAMPs) engage pattern recognition receptors
(PRRs), as well as endogenous proinflammatory cytokines and “danger”-associated molecules (DAMPs), inducing DC maturation. PAMPs include unmethylated
CpG tracks of DNA characteristic of DNA viruses and bacteria; uncapped RNA, characteristic of RNA viruses; flagellin, the major protein component of
bacterial flagella; and lipopolysaccharide (LPS), a major bacterial cell wall component. DC maturation leads to upregulation of costimulatory molecules, major
histocompatibility complex (MHC), and certain cytokines, as well as decreased expression of coinhibitory molecules, allowing them to efficiently activate
antigen-specific T cells to effector cells (right side of figure). In the absence of these danger signals, DCs follow a default pathway (left side of figure) in which
they become tolerizing DCs that present antigen to T cells in the absence of costimulatory signals and with an excess of coinhibitory signals. This represents
a steady state pathway for continuous presentation of self-antigens. The consequence is that these T cells are turned off (anergy) or deleted, inducing tolerance.
IL-4, Interleukin-4; TNF-α, tumor necrosis factor–α.

LN
Activation
Activated of tumor-
DCs specific
T cells
Danger

A
Inhibition of
danger LN Anergy/
signals
Tolerizing Deletion of
DCs tumor-
specific
Danger T cells

B
Figure 6.4  •  Inhibition of dendritic cell (DC) activation in the tumor microenvironment can shift tumor-specific immune responses from activation
to tolerance. Based on the scenario presented in Fig. 6.3, if a tumor is able to produce factors that inhibit local DCs from becoming activated in response to
the endogenous danger signals associated with tissue invasion, it could shift tumor-specific T cells from a state of activation (A) to one of tumor-specific tolerance
(B). LN, Lymph node.
 Cancer Immunology  •  CHAPTER 6 89

however, mutations in the TGF-β pathway confer resistance to cell
cycle inhibition, allowing uncontrolled proliferation, and promote
tissue invasion through induction of matrix metalloproteinases. In
vivo, TGF-β directly stimulates angiogenesis; this stimulation can be
blocked by anti-TGF-β antibodies.91 Elevated serum TGF-β levels
are associated with poor prognosis in a number of malignancies,
including prostate cancer,92 lung cancer,93 gastric cancer,94 and bladder
cancer.92 Several inhibitors of the TGF-β pathway have been tested
in phase I clinical trials, including fresolimumab, a human
anti-TGF-β1/2/3 monoclonal antibody (mAb), and TβM1, a human
anti-TGFβ1 mAb. These molecules were shown to be safe, but data
on efficacy have been limited or not yet reported.95,96
COINHIBITORY LIGANDS AND RECEPTORS
THAT DOWNMODULATE TUMOR IMMUNITY
Major molecules successfully targeted in clinical cancer immunotherapy
are the growing class of ligand-receptor pairs, commonly referred to
as immune checkpoints. In considering the mechanism(s) of action of
inhibitors of various checkpoints, it is critical to appreciate the diversity
of immune functions that they regulate. For example, the two immune
checkpoint receptors that were first targeted in the context of clinical
cancer immunotherapy, CTLA-4 (CD152) and PD-1 (CD279), regulate
immune responses at very different levels and by very different
mechanisms (Fig. 6.5). The clinical activity of blocking antibodies
for each of these receptors implies that antitumor immunity can be
enhanced at multiple levels and that combinatorial strategies can be
intelligently designed, guided by mechanistic considerations and
preclinical models. This section focuses particular attention on the
CTLA-4 and PD-1 pathways because they were the two checkpoints
whose inhibition has revolutionized clinical cancer immunotherapy.
CTLA-4 Checkpoint: A Global Regulator of
T-Cell Activation
CTLA-4 is expressed exclusively on T cells, where it primarily regulates
the amplitude of the early stages of T-cell activation, by counteracting
the activity of the T-cell costimulatory receptor CD28.97–99 CD28
does not affect T-cell activation unless the TCR is first engaged by
cognate antigen. Once antigen recognition occurs, CD28 signaling
strongly amplifies the TCR signal to activate T cells. CD28 and CTLA-4
share identical ligands: CD80 (B7-1) and CD86 (B7-2).100–104 Because
CTLA-4 has higher affinity for both ligands, its expression on the
surface of T cells dampens the activation of T cells both by outcompet-
ing CD28 in binding CD80 and CD86 and by actively delivering
inhibitory signals to the T cell.105–110 The specific signaling pathways
by which CTLA-4 blocks T-cell activation are still under investigation,
although a number of studies have suggested that activation of the
phosphatases SHP2 and PP2A are important in counteracting kinase
signals induced by TCR and CD28.98 However, CTLA-4 also confers
“signaling-independent” T-cell inhibition through sequestration of
CD80 and CD86 from CD28 engagement, as well as active removal
from the antigen-presenting cell surface.111 The central role of CTLA-4
in maintaining T-cell activation in check is dramatically demonstrated
by the systemic immune hyperactivation phenotype of CTLA-4
knockout mice.112,113
Even though CTLA-4 is expressed by activated CD8 killer T cells,
the major physiologic role of CTLA-4 appears to be through distinct
effects on the two major subsets of CD4 T cells: downmodulation of
helper T-cell activity and enhancement of regulatory T-cell suppressive
activity.97,114,115 CTLA-4 blockade results in a broad enhancement of
immune responses dependent on helper T cells, and conversely, CTLA-4
engagement on Tregs enhances their suppressive function. CTLA-4

B7-1/2 CD28 B7-1/2 CD28

T cell still in
+ secondary +
APC APC
lymphoid tissue

Signal 1 Signal 1
–
CTLA-4
Activation of naïve Antigen-experienced
or resting T cells T cell

B7-1/2 CD28
Tissue
+ Traffic to or
DC
periphery tumor
Signal 1 Signal 1
–

PD-L1 PD-1
Activation of naïve Antigen-experienced
or resting T cells Inflammation T cell

Figure 6.5  •  CTLA-4 and PD-1 checkpoints act to regulate different elements of the T-cell response. Naïve T cells and resting T cells express little
CTLA-4 or PD-1 on their surface. On initial T-cell activation via triggering of the T-cell receptor (TCR) by cognate peptide/major histocompatibility complex
(MHC) complexes together with engagement of CD28 by B7-1 and/or B7-2, CTLA-4, which is stored preformed in intracellular vesicles, rapidly migrates
to the cell surface while the T cell is still engaged with its antigen-presenting cell (APC; in general, a dendritic cell), usually in the secondary lymphoid tissue.
The greater the TCR stimulus, the more CTLA-4 is expressed on the surface. Inhibitory interaction of CTLA-4 with B7-1 and B7-2 results in a counterregulatory
signal that downmodulates the ultimate amplitude of T-cell activation (top row). In contrast, the PD-1 checkpoint primarily operates in the periphery
(bottom row). PD-1 is induced more slowly than CTLA-4 when T cells become activated. Inflammatory signals, such as interferon-γ, at sites of effector T-cell
function (in the tissue or in tumors) induce PD-L1 (and PD-L2) expression, which downmodulates tissue immune responses, thereby protecting tissues from
collateral damage.
90 Part I: Science and Clinical Oncology
is a target gene of the transcription factor Foxp3,116,117 the expression
of which determines the Treg lineage,118,119 and Tregs therefore express
CTLA-4 constitutively. Although the mechanism by which CTLA-4
enhances the inhibitory function of Tregs is not known, Treg-specific
CTLA-4 knockout or blockade significantly inhibits their ability to
regulate both autoimmunity and antitumor immunity.114,115 Thus in
considering the mechanism of action for CTLA-4 blockade, both
enhancement of effector CD4 T-cell activity and inhibition of Treg-
dependent immune suppression are likely important factors.
PD-1 Checkpoint: A Pathway That Functions Within
the Tumor Microenvironment
In contrast to CTLA-4, the major role of PD-1 is to limit the activity
of T cells in the peripheral tissues at the time of an inflammatory
response to infection and to limit autoimmunity (see Fig. 6.5).120–126
This translates to a major immune resistance mechanism within the
tumor microenvironment.127–129 PD-1 expression is induced when T
cells become activated.121 When engaged by one of its ligands, PD-1
inhibits kinases involved in T-cell activation via the phosphatase
SHP2.120 Because PD-1 engagement inhibits the TCR stop signal,
this pathway may also modify the duration of T-cell/APC or T-cell/
target cell contact.130 PD-1 is expressed on a large proportion of
tumor-infiltrating lymphocytes (TILs) from many different tumor
types.131,132 Similar to CTLA-4, PD-1 is highly expressed on Tregs,
where it may enhance their proliferation in the presence of ligand.133
Because many tumors are highly infiltrated with Tregs, PD-1 pathway
blockade may also enhance antitumor responses by diminishing the
number and/or suppressive activity of intratumoral Tregs. Increased
PD-1 expression on CD8 TILs may reflect an anergic or exhausted
state, as has been suggested by decreased cytokine production by
PD-1+ versus PD-1− TILs from melanomas.131 PD-1 is also expressed
on other non–T lymphocyte subsets, including B cells, natural killer
(NK) cells, and macrophages.134,135 Thus whereas PD-1 blockade is
typically viewed as enhancing the activity of effector T cells in tissues
and in the tumor microenvironment, it likely also enhances NK activity
in tumors and tissues and may also enhance antibody production
either indirectly or through direct effects on PD-1+ B cells.136 PD-1
has also been shown to be a marker of dysfunctional macrophages
and monocytes.137
The two ligands for PD-1 are B7-H1/PD-L1 (CD274) and B7-DC/
PD-L2 (CD273).120,138–140 These B7 family members share 37%
sequence homology and arose via gene duplication, positioning them
within 100 kb of each other in the genome.140 An unexpected molecular
interaction between PD-L1 and CD80 was discovered,141 whereby
CD80 expressed on T cells (and possibly antigen-presenting cells) can
potentially behave as a receptor rather than a ligand, delivering inhibi-
tory signals when engaged by B7-H1142,143; the relevance of this
interaction in tumor immune resistance has not yet been determined.
PD-L1 has also been proposed to function as a receptor to transmit
survival signals to cancer cells and T cells, although the downstream
pathways mediating this function are not understood.144,145 Understand-
ing the role of these various interactions in given cancer settings is
highly relevant for selection of both antibodies and recombinant ligands
for use in the clinic.
Just as PD-1 is highly expressed on TILs from many cancers,
PD-1 ligands are commonly upregulated on many different human
tumors.128,146 On solid tumors, the major PD-1 ligand to be expressed
is B7-H1/PD-L1.128,147,148 In addition to tumor cells, B7-H1/PD-L1
is commonly expressed on myeloid cells in the tumor microenviron-
ment such as macrophages and DCs.149–151 Although most of the
analyses of PD-1 ligand expression has focused on B7-H1/PD-L1,
B7-DC/PD-L2 has also been reported to be upregulated on a number
of tumors. B7-DC/PD-L2 is highly upregulated on certain B-cell
lymphomas such as primary mediastinal, follicular cell B-cell lym-
phoma and Hodgkin disease.152 Upregulation in these lymphomas
is commonly associated with gene amplification or rearrangement
to the CIITA locus, which is highly transcriptionally active in B-cell
lymphomas.153
Two general mechanisms for regulation of B7-H1/PD-L1 expression
have emerged: innate (tumor cell intrinsic) and adaptive (tumor cell
extrinsic) (Fig. 6.6). In the innate (tumor cell intrinsic) mechanism,
oncogenes drive expression of PD-L1. For some tumors such as
glioblastoma, deletion or silencing of phosphatase and tensin homolog
(PTEN) has been shown to drive PD-L1 expression, implicating the
PI3K-AKT pathway.154 In addition, constitutive anaplastic lymphoma
kinase (ALK) signaling, observed in certain lymphomas and occasionally
in lung cancer, has been reported to drive PD-L1/B7-H1 expression
via STAT3 signaling.155 In Hodgkin disease, genomic gains in chromo-
some 9p24 are frequently observed, a region containing the oncogene
JAK2, as well as PD-L1 and PD-L2. PD-L1 expression in tumors can
also be driven by IFNs, which illustrates an adaptive mechanism
(tumor cell extrinsic) for its regulation. This hypothesis is supported
by work from studies in melanoma demonstrating a high correlation
between cell surface PD-L1/B7-H1 expression on tumor cells and
lymphocytic infiltration and intratumoral IFN-γ expression. This
correlation was seen not only among tumors but within individual
B7-H1/PD-L1+ tumors at the regional level, in which regions of
lymphocyte infiltration were exactly the regions where B7-H1/PD-L1
was expressed on both tumor cells and infiltrating leukocytes.129
Additional Checkpoints Participate in Tumor
Immune Resistance and Tolerance
Successful clinical outcomes of CTLA-4 and PD-1 pathway targeting
have garnered interest in a number of additional checkpoints
(Fig. 6.7). In addition to defined lymphocyte inhibitory receptors, a
number of B7 family inhibitory ligands—in particular B7-H3 (CD276)
and B7-H4—do not yet have defined receptors, but murine knockout
experiments support an inhibitory role for both these molecules,156
and they are upregulated on tumor cells or tumor-infiltrating cells.157
Lymphocyte activation gene-3 (Lag-3), T cell immunoglobulin-3 (Tim-3),
and adenosine A2a receptor (A2aR) are also associated with inhibition
of lymphocyte activity. Antibody targeting of these receptors, either
alone or in combination with a second immune checkpoint blocker,
has been shown to enhance antitumor immunity in animal models
of cancer. Because many tumors express multiple inhibitory ligands
and TILs express multiple inhibitory receptors, dual or triple blockade
of immune checkpoints could further augment antitumor reactivity.
Human blocking antibodies specific for a number of these “second-
generation” inhibitory receptors are under development.
Lag-3 was first discovered as a CD4 homologue expressed on
activated CD4 T cells, CD8 T cells, and a subset of NK cells.158
Lag-3 has been shown to play a role in enhancing Treg function,159,160
and subsequently to inhibit CD8 effector function.161 Lag-3 binds to
MHCII, which is upregulated on some epithelial cancers (typically
in response to IFN-γ) but is also expressed on tumor-infiltrating
macrophages and DCs. Another Lag-3 ligand that has been recently
proposed is LSECtin, a member of the DC-SIGN family.162 Lag-3 is one
of a number of immune checkpoint receptors coordinately upregulated
on both Tregs and anergic T cells, and simultaneous blockade can result
in enhanced reversal of this anergic state relative to blockade of either
receptor. In particular, PD-1 and Lag-3 are commonly coexpressed on
anergic or exhausted T cells.163,164 Dual blockade of Lag-3 and PD-1
could synergize to reverse exhaustion among tumor-specific CD8 T
cells. Phase I/IIb clinical trials are underway examining the safety
and efficacy of anti-Lag-3 antibodies alone or in combination with
nivolumab for the treatment of B-cell malignancies or solid tumors.
Tim-3, first discovered to be expressed on IFN-γ expressing CD4
cells and CD8 T cells, binds to the ligand galectin-9, which is
upregulated in a number of cancer types such as breast cancer.
Additional ligands for Tim-3 have also been identified, including
CEACAM1, HMGB1, and phosphatidyl serine.165–167 In preclinical
models, Tim-3 is often coexpressed with PD-1 on a subset of T cells
 Cancer Immunology  •  CHAPTER 6 91

Innate (tumor cell intrinsic) resistance

MHC-pep

TCR

Constitutive tumor signaling

induces PD-L1 on tumor cells
Oncogenic
pathway
(STAT3, Akt) PD-L1 PD-1 T Cell

Tumor

Adaptive resistance
T cell-induced
PD-L1 upregulation

T Cell T Cell

Tumor Tumor IFN-γ

Figure 6.6  •  Two mechanisms for PD-L1 induction on tumors: innate and adaptive. PD-L1 can be constitutively expressed on tumors as a consequence
of oncogene-driven transcriptional activation. Alternatively, PD-L1 can be induced in an adaptive fashion when there are the right inflammatory cytokines in
the tumor microenvironment consequent to an ongoing immune response to the tumor. This mechanism of tumor resistance to immune attack is co-opted
from physiologic PD-L1 expression for tissue protection in the setting of antimicrobial immune responses. Innate and adaptive mechanisms of PD-L1 expression
on tumors can coexist. The adaptive resistance mechanism implies that PD-L1 expression is a “marker” of endogenous antitumor immunity. IFN-γ, Interferon-γ;
MHC, major histocompatibility complex; TCR, T-cell receptor.

that are highly dysfunctional,168 and blocking Tim-3 with anti-Tim-3
antibodies has been shown to enhance antitumor immunity.157
Coordinate blockade of PD-1 and Tim3 in preclinical models was
reported to enhance antitumor responses and tumor rejection under
circumstances in which only modest effects from blockade of each
individual molecule were observed.169–171 It is interesting to note that
Tim-3 has been shown to be upregulated after anti-PD-1 therapy,
both in preclinical mouse models of lung cancer and in patients,
supporting upregulated Tim-3 as a mechanism of resistance to anti-PD-1
therapy.172 Anti-Tim-3 antibodies are currently being studied in phase
I clinical trials for patients with advanced solid tumors.
A2aR binds adenosine and inhibits T-cell responses, in part by
driving CD4 T cells to express Foxp3 and develop into Tregs.173
Knockout of this receptor results in enhanced and sometimes pathologic
inflammatory responses to infection. This receptor is particularly
relevant in tumor immunity because the rate of cell death in tumors
from cell turnover is high and dying cells release adenosine. In addition,
Tregs express high levels of the exoenzymes CD39, which converts
extracellular adenosine triphosphate (ATP) to adenosine monophosphate
(AMP), and CD73, which converts AMP to adenosine.174 A2aR
engagement by adenosine to drive T cells to become Tregs can produce
a self-amplifying loop within the tumor, as illustrated by the evidence
that tumors grow more slowly in A2aR knockout mice and tumor
vaccines are more effective against established tumors in these mice.175
A2aR can be inhibited either by antibodies that block adenosine
binding or by adenosine analogues, some of which are fairly specific
for A2aR. Currently, an oral small-molecule inhibitor of A2aR CPI-444
is being tested in advanced cancers as monotherapy and in combination
with atezolizumab in phase I clinical trials (ClinicalTrials.gov ID:
NCT02655822). Another oral small molecule inhibitor of A2aR,
PBF-509, is being tested in phase I clinical trials for patients with
non–small cell lung cancer as monotherapy and in combination with
the PD-1 antibody PDR001 (ClinicalTrial.gov ID: NCT02403193).
IMPLICATIONS FOR CANCER
IMMUNOTHERAPY
Recent successes in clinical immunotherapy of cancer have emerged
directly from a basic understanding of molecular and cellular immunol-
ogy applied to the cancer setting. Recent advances have been made
in three major areas of cellular cancer immunotherapy that seek to
use T cells as the antitumor weapon: checkpoint blockade, cellular
therapy, and cancer vaccination.
Checkpoint Blockade
Therapeutic strategies targeting the CTLA-4 and PD-1 immune
checkpoints have changed paradigms in cancer therapy by emphasizing
the importance of targeting the immune system rather than the tumor
and offering the possibility of cure in some patients. CTLA-4 was
the first immune checkpoint receptor to be clinically targeted in human
metastatic melanoma.176 Antibodies targeting PD-1 or its ligand PD-L1
have subsequently revolutionized treatment of a variety of human
cancers, including lung cancer, melanoma, kidney and bladder cancer,
Hodgkin disease, head and neck cancer, mismatch-repair deficient
cancers, and Merkel cell cancers.177–183
Encouraged by murine studies demonstrating antitumor effects of
CTLA-4 blockade, human anti-CTLA-4 antibodies were developed
and one antibody, ipilimumab, was shown to produce a roughly 10%
objective response rate in melanoma according to standard RECIST
(Response Evaluation Criteria in Solid Tumors) clinical criteria.184
Anti-CTLA-4 produced significant immune-related toxicity in 25%
92 Part I: Science and Clinical Oncology

Antigen-presenting cell
or tumor cell T Cell
CD28
+

B7.1/B7.2 CTLA-4
–
?
+
PD-L1/PD/L2 PD-1
–
ICOS-L ICOS
+

B7-H3 ?
–
B7-H4 ?
–
LIGHT

HVEM BTLA
+ –

MHC/pep TCR Signal 1

LAG-3
–
4-1BBL 4-1BB
+
OX40L OX40
+
LIGHT LIGHT-R
+
PS/galectin9 Tim-1/ Tim-3
–
CD200R CD200
–
CD40 CD40L
+

Cytokines (IL-1, IL-6, IL-10, IL-12, IL-18)

Figure 6.7  •  Multiple costimulatory and coinhibitory ligand-receptor interactions ultimately determine the amplitude of T-cell activation and the
potency of effector T-cell responses in tissue and tumor. B7 family ligands and CD28 family receptors are shown in purple and TNF/TNFR family
ligand-receptor pairs and shown in blue. There are additional inhibitory ligand-receptor pairs that do not fit into either of these families. Some of the receptors
for B7 family members are not yet discovered. Although TNF/TNFR interactions are usually one-on-one pairs, B7 family ligands often interact with multiple
receptors. HVEM is a TNFR family member; in addition to its interaction with the TNF family member LIGHT, it also interacts with the inhibitory receptor
BTLA, which is a member of the CD28 family. Additional signals of activation or inhibition are contributed by cytokines. BTLA, B- and T-lymphocyte
attenuator; HVEM, herpesvirus entry mediator; ICOS, inducible T-Cell COStimulator; IL, interleukin; MHC, major histocompatibility complex; PS, phos-
phatidylserine; TCR, T-cell receptor; TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor.

to 30% of patients, commonly involving the skin, liver, or colon.
Subsequent clinical development of ipilimumab culminated in two
successful phase III trials in patients with advanced melanoma and
FDA approval in 2011.185,186 Since these studies, ipilimumab has also
been shown to prolong survival in patients with stage III melanoma
in the adjuvant setting following surgery.187 In addition, ipilimumab
is being investigated in the neoadjuvant setting in the context of
regionally advanced but surgically operable melanoma.188
In the case of PD-1, the basic discoveries that this pathway
downmodulated immune responses in the tissue, together with
upregulation of PD-1 ligands in human tumors, motivated clinical
testing in the mid-2000s. Despite the fact that most of the patients
in the initial phase I trial had end-stage disease, a number of dramatic
clinical responses were observed in multiple cancer types. This led to
larger clinical trials of both anti-PD-1 and anti-PD-L1, culminating
in the demonstration of major clinical efficacy in multiple tumor
types.189,190 The durability of the responses, even after cessation of
treatment, far exceeds that of chemotherapy or tyrosine kinase inhibitors.
These findings reinforce the notion that a “reeducated” immune system
not only has memory, but also can adapt to maneuvers by the tumor
to develop resistance. Just as predicted from the mouse knockout
studies, toxicity from anti-PD-1 or anti-PD-L1 therapy is lower than
from anti-CTLA-4 therapy and also of different distribution than
anti-CTLA-4 therapy (colitis is most common with anti-CTLA-4
therapy, and pneumonitis is more common with anti-PD-1).
A number of interesting clinical insights have emerged from the
clinical experience with checkpoint inhibitors, which call for novel
approaches to monitoring the safety and efficacy of these drugs. The
ipilimumab experience illustrated that the kinetics of clinical responses
to anti-CTLA-4 (i.e., tumor shrinkage) tends to be slower than

chemotherapy or tyrosine kinase inhibitors, and in some patients
tumor regression is proceeded by apparent progression as assessed
with computed tomography (CT) or magnetic resonance imaging
(MRI) scans. This finding suggests a mechanism of action in which
ultimate tumor regression is preceded by immune activation and tumor
infiltration of activated lymphocytes. In addition, although formal
regressions are induced in a relatively small proportion of patients
and complete responses as assessed with CT or MRI are rare (<5%),
roughly 20% of patients treated with only four doses of ipilimumab
remained alive more than 4 years after therapy (as opposed to approxi-
mately 5% in the control group). These clinical findings led to the
development of “immune response criteria” to help clinicians evaluate
the efficacy of this new class of immunotherapy in their patients.191
These agents also changed paradigms for dose selection. For example,
efficacy and toxicity do not always correlate, so immunotherapies may
not achieve a maximum tolerated dose, and this measurement may
be less relevant for selecting a recommended dose in phase I trials to
be used in phase II trials.192 These novel clinical paradigms as well as
the unique immune toxicity profiles encountered with these agents
highlight the importance of immune monitoring of patients on clinical
trials to advance the understanding of the mechanistic basis of response
and resistance and to pave the way for improving these therapies.
Since 2010, there has been tremendous energy focused on under-
standing the molecular basis of response and resistance to CTLA-4
and PD-1 checkpoint pathway blockade. Because PD-L1 was the
major ligand for PD-1 and mediated T-cell suppression, it was
hypothesized that PD-L1 expression in tumor cells or tumor antigen-
presenting cells may predict clinical responsiveness to PD-1 blockade.
Consistent with this, PD-L1 expression in tumor tissue in melanoma,
non–small cell lung cancer, and renal cell carcinoma correlates with
response to anti-PD1 therapy.193–196 Expression of PD-L1 in tumor-
infiltrating immune cells also correlated with response to anti-PD-L1
therapy.197 However, PD-L1 is an imperfect biomarker given its
heterogeneous and dynamic expression combined with the observation
that tumors with negative PD-L1 expression may still respond to
therapy. In patients with melanoma treated with ipilimumab (anti-
CTLA4) and nivolumab (anti-PD-1), clinical responses were observed
in patients with both high and low PD-L1 expression.198,199 This may
be due to variability in PD-L1 expression in different parts of the
tumor as well as the dynamic nature of PD-L1 expression.
The burden of cancer neoantigens has also been examined as a
predictor of CTLA-4 and PD-1 checkpoint pathway blockade. This
was initially described in a preclinical mouse sarcoma model, which
demonstrated that tumor neoantigen-specific T cells were activated
by treatment with anti-CTLA-4 and anti-PD-1.200 In patients with
non–small cell lung cancer treated with pembrolizumab (anti-PD-1),
higher nonsynonymous mutation burden and higher neoantigen burden
were associated with clinical response.201 In patients with melanoma
treated with CTLA-4 blockade, a neoantigen signature was identified
that correlated with response to therapy.202 Similarly, colorectal cancers
with mismatch-repair deficiency had higher objective response rates to
pembrolizumab (anti-PD-1) treatment compared with mismatch-repair
proficient colorectal cancers, and colorectal cancers with mismatch-repair
deficiency also manifested higher somatic mutation loads compared with
mismatch-repair proficient colorectal cancers.182 Neoantigen intratumoral
heterogeneity has also demonstrated that response to PD-1 blockade in
lung cancer and CTLA-4 blockade in melanoma were associated with
higher clonal neoantigens.203 Together, these data support the notion
that tumor neoantigen burden correlates with response to checkpoint
blockade. It is important to note, however, that there is broad overlap in
mutational and neoantigen load between responders and nonresponders,
limiting the clinically applicability of these measures as a predictor of
response. Moreover, quantifying mutational burden in tumors may not
necessarily correlate with immunogenic neoantigen burden.
The role of tumor-infiltrating T cells in shaping response to check-
point blockade therapy is an active area of investigation. In metastatic
melanoma, patients responding to pembrolizumab (anti-PD-1) typically
Cancer Immunology  •  CHAPTER 6 93
demonstrate higher numbers of intratumoral CD8 T cells and a
more clonal TCR repertoire in their pretreatment tumor samples.204
T-cell clonotype was also examined after anti-CTLA-4 treatment
in patients with metastatic castrate-resistant prostate cancer and in
metastatic melanoma. This study demonstrated that maintenance of
high-frequency T-cell clones over the course of treatment, but not
the absolute number of T-cell clonotypes, was associated with clinical
response to anti-CTLA-4 blockade.205 These studies demonstrate the
importance of T-cell infiltration and clonal expansion, but illustrate
that there is still much to be learned regarding qualitative aspects
of T-cell responsiveness, such as antigen specificity and downstream
programs associated with an antitumor response. Recently, response
to anti-PD-1 therapy in melanoma patients was found to correlate
with a measure of T-cell reinvigoration in the peripheral blood after
correcting for tumor burden.206 This may be a simple blood-based
immunologic metric that can be assessed as soon as 3 to 6 weeks
after treatment, but it remains to be seen whether this is predictive,
reproducible, and relevant to other tumor types.
Recently several studies have examined tumor intrinsic mechanisms
of poor T-cell infiltration in cancer. Epigenetic silencing has been
shown to be a mechanism for suppressing Th1 chemokines CXCL9
and CXCL10 in human ovarian cancer. Epigenetic reprogramming
targeting EZH2 or DNMT1 can lead to upregulation of T cell–
attracting chemokines and increase efficacy of anti-PD-1 therapy.207
In addition to epigenetic silencing, another tumor-intrinsic mechanism
associated with poor T-cell trafficking to tumors is the WNT-β-catenin
pathway. In melanoma, activation of WNT/β-catenin is associated
with the absence of a T-cell gene expression signature. In mice, β-catenin
was found to negatively regulate CCL4 expression, limiting DC
recruitment and T-cell activation. Intratumoral injection of DCs in
this model was effective in overcoming resistance to anti-CTLA-4
and anti-PD-1 therapy.208 Understanding the tumor-intrinsic mecha-
nisms responsible for poor T-cell recruitment to tumors may guide
rationally designed strategies for overcoming resistance.
Genomic and transcriptomic characteristics of response have been
examined in detail in metastatic melanoma. This approach found that
BRCA2 mutations were enriched in patients responding to anti-PD-1
therapies. A transcriptional signature of innate PD-1 resistance (IPRES)
was described, demonstrating that the upregulation of genes involved
in mesenchymal transition, angiogenesis, and wound healing correlated
with lack of response to anti-PD-1 therapy.209 This raises the hypothesis
that modulation of pathways involved in the IPRES signature may
be a strategy for overcoming resistance to anti-PD-1 therapy. Beyond
molecular correlates of response described in the tumor and tumor micro-
environment, the gut microbiome has also been shown to shape response
to checkpoint blockade. In murine melanoma studies, administration of
Bifidobacterium improved tumor response to anti-PD-L1 therapy,210 and
response to anti-CTLA-4 treatment to melanoma correlated with the
presence of B. thetaiotaomicron or B. fragilis.211 These results demonstrate
that both host and environmental factors shape responses to CTLA-4
and PD-1 checkpoint pathway blockade in patients.
Additional insights from cancer immunology are paving the way
to combinatorial therapies—for instance, combining checkpoint
blockade with additional strategies that improve the efficacy of this
approach. Combination of anti-PD-1 blockade (nivolumab) with anti-
CTLA-4 blockade (ipilimumab) in metastatic melanoma has already
demonstrated improved response rates, but was also associated with
increased toxicity.199 In the future, a comprehensive personalized assay
incorporating genetic information from the tumor with information on
relevant target molecules in the tumor immune microenvironment may
enable biomarker-based prediction of which immune pathways should
be targeted in a specific patient. At this point, multiple strategies for
improving clinical responses to checkpoint immunotherapy are being
investigated, including strategies targeting the tumor or the tumor
microenvironment or other factors such as the microbiome. Examples
of tumor-targeting strategies include immune-stimulating radiation
strategies, chemotherapeutic agents that activate the IFN signaling
94 Part I: Science and Clinical Oncology
pathway or recruit antigen-presenting cells to the tumor, receptor
tyrosine kinase inhibitors that increase T-cell accumulation in the
tumor,212 antibody-drug conjugates that promote antigen presentation
in tumors,213 or epigenetic modifiers that promote T-cell trafficking to
tumors.207 Examples of tumor microenvironment–targeting strategies
include activation of DC and antigen presentation (e.g., with STING
agonist, CpG vaccine, neoantigen vaccine, oncolytic virus), inhibition
of TAMs or MDSCs (e.g., targeting phosphoinositide 3-kinase gamma
[PI3Kγ] in myeloid cells,214 inhibiting their recruitment to tumors, pro-
moting phagocytosis and antigen presentation), inhibition of Tregs (e.g.,
with denileukin diftitox, neutralizing antibodies to IL-35, or low-dose
cyclophosphamide), or activation of effector T cells (such as targeting
additional immune-suppressive checkpoints or activating tumor necrosis
factor [TNF] receptors). The historical segregation of immunotherapy
and small-molecule “tumor-targeted” therapy is being replaced by
integrative therapeutic approaches that leverage the effects of signaling
modulators directly on the immune system apart from their effects on the
tumor itself.
Immunotherapy Using Adoptive T-Cell Strategies
Tumor-Infiltrating Lymphocytes
Adoptive cell transfer employs ex vivo manipulation and transfer of
naturally occurring or genetically engineered immune effector cells.
TILs are a naturally occurring, polyclonal lymphocyte population
found within the tumor environment, primarily composed of T cells,
a fraction of which express TCRs targeting unique or shared TAAs.
TILs can be isolated from neoplastic lesions and the respective sup-
pressive microenvironment, selected for tumor specificity and expanded
ex vivo. After lymphodepletion, reinfusion of expanded TILs has yielded
promising results with tumor-specific cytolysis and T-cell persistence
in malignant melanoma.215 One benefit of TIL therapy is the broad
repertoire of TCRs that target both defined and undefined tumor
antigens. This contrasts with TCR and chimeric antigen receptor
(CAR) methods in which tumor targeting is focused on a single defined
tumor antigen.215 Unfortunately, in settings beyond malignant mela-
noma, TIL therapy has demonstrated limited clinical efficacy. Alternative
adoptive T-cell therapy strategies that rely on genetic modifications
of patient-derived T cells driving tumor specificity and enhancing
T-cell cytotoxicity are coming into favor.
Genetically Engineered Adoptive T-Cell Strategies
Adoptive immunotherapy using genetically engineered T cells seeks
to induce expression of novel genes in cytotoxic T cells that facilitate
tumor recognition, enhance T-cell activation, induce tumor-specific
cytotoxicity, and/or augment immune memory. The two most common
genetic modifications used are expression of transgenes encoding a
tumor-specific TCR or a CAR. To achieve stable, reliable, prolonged
transgene expression, most commonly lentiviral or retroviral transduc-
tion methods are used.216,217 Electroporation or nanoparticle-mediated
delivery of transposon and transposases can also achieve stable transgene
expression at lower cost with nanoparticle technology, introducing
the possibility for in vivo CAR delivery.218–220 Delivery of RNA can
alternatively be used if transient transgene expression is desirable.221
New platforms such as megaTAL222 and CRISPR/Cas9223 are available
to permit gene editing, gene deletion, or more precise gene integration.
In contrast to past experiences with genetic manipulation of hema-
topoietic stem cells, retroviral-mediated insertional oncogenesis of T
cells has not occurred,224 although it remains a theoretical risk that
may vary among different delivery systems.
Genetically Modified T-Cell Receptors for Adoptive
Cellular Therapy
T cells are naturally armed with a widely variable repertoire of TCRs
designed to recognize short contiguous amino acid sequences derived
from processed intracellular proteins, presented within the target cell
surface MHC complex. Binding of a specific TCR to its cognate
antigen initiates T-cell activation and function.225 In cancer patients,
T cells expressing tumor-specific TCRs comprise only a small fraction
of the total T-cell pool and are often tolerized, or demonstrate low-
affinity binding to tumor-derived peptides. T cells with higher affinity
TCRs specific to TAAs can be isolated directly from tumor-bearing
patients or from humanized mice immunized against tumor-specific
antigen. On isolation and identification of tumor-specific TCRs and
their respective variable alpha (α) and beta (β) chains, T cells can be
genetically engineered to clonally express a tumor-specific TCR.226
Genetic engineering can efficiently express a transgenic TCR in T
cells with high frequency and allow for mutagenesis to derive high-
affinity variants of native tumor-reactive TCRs.227–229 In addition,
non-TCR elements can be coexpressed within the T cell to enhance
functionality beyond what could be accomplished by a nonengineered
T cell. Adoptive cellular immunotherapy with transgenic TCRs seeks
to provide the host with a larger pool of functional T cells capable of
efficiently recognizing tumor-specific antigens and eradicating tumor.
One practical challenge of TCR therapy is MHC restriction of
these receptors, which permits targeting of intracellular antigens but
also restricts binding of any particular TCR to a specific HLA allele.
Most clinical trials to date have focused on antigens presented within
HLA-A*02, an allele expressed in approximately 40% of Caucasians, to
demonstrate proof of principle for the approach, leaving most patients
ineligible for studies involving engineered TCRs because of the absence
of the HLA-A*02 allele. Because each TCR represents a novel receptor,
targeting the same antigen in patients with different MHC alleles requires
substantial additional preclinical and clinical development.
Clinical experience with engineered TCRs was initiated in malignant
melanoma, but applications have since been expanded broadly to both
hematologic and solid malignancies. The first clinical trial that
demonstrated efficacy of TCR therapy used an HLA-A*02 restricted
TCR targeting the melanoma-associated antigen MART-1, administered
after lymphodepletion.230 Fourteen of 15 patients achieved cellular
engraftment at 1 month, with two patients demonstrating objective
tumor regression and prolonged persistence of modified cells for more
than 1 year.230 The response rate in 2 of 15 patients (13%) was inferior
to response rates with use of alternative therapeutic modalities but
supported further exploration and optimization.230 In contrast to the
experience with MART-1, early clinical trials using a TCR with
specificity for NY-ESO-1, a cancer-testis antigen, demonstrated robust
clinical responses in synovial sarcoma and melanoma.231,232 A human-
derived affinity-enhanced TCR targeting the NY-ESO–derived antigen
presented within HLA-A*02 yielded objective clinical responses in
61% of patients (11 of 18) with synovial sarcoma and 55% of patients
(11 of 20) with melanoma. This promising work supported the potential
of TCR therapy in solid tumors and led the FDA to grant breakthrough
therapy designation for affinity-enhanced TCRs targeting NY-ESO-1
in synovial sarcoma in February 2016. This same NY-ESO-1–specific
TCR was studied in advanced multiple myeloma following autologous
stem cell transplantation and yielded an 80% clinical response rate
and demonstrated that T cells, lentivirally transduced to express TCR,
expanded and persisted in vivo, trafficked to the bone marrow, and
exhibited tumor-specific cytotoxicity. Currently, promising TCR product
candidates directed at other TAAs including HPV-16 E6 and E7
oncoproteins for the treatment of HPV-associated epithelial malignan-
cies and the PRAME cancer testes antigen for the treatment of acute
myeloid leukemia (AML)233,234 are under study.
Important lessons regarding the potential for TCR-mediated toxicity
were garnered from early studies of this approach. A phase I/II study
of TCR therapy targeting the MAGE-A3/A9 cancer testes antigens
demonstrated unexpected fatal neurotoxicity. The transgenic TCR
was later found to broadly bind to peptides derived from MAGE-A2,
MAGE-A6, and MAGE-A12, and MAGE-A12 expression was identified
on brain parenchyma.235 A study of MAGE-A3 targeting TCRs in
patients with myeloma and melanoma revealed unanticipated fatal
cardiac toxicity236,237 in the first two treated patients. Autopsy revealed

gross myocardial damage and evidence of histopathologic T-cell infil-
tration. Although MAGE-A3 expression was not found on cardiac
myocytes, unanticipated cross-reactivity of the engineered TCR was
found to peptides derived from titin, an unrelated protein found on
striated muscle. These experiences highlight the risks of first-in-human
targeted therapies and remind us of the experimental nature of the field.
Chimeric Antigen Receptors for Adoptive Cellular Therapy
CARs are artificial tumor-specific receptors generated most often from
the fusion of an antibody-derived binding domain to T-cell–derived
signaling domains.238 In contrast to TCRs, antibody binding does not
require antigen presentation within an endogenous MHC complex,
and CARs can therefore recognize surface antigens in the face of
tumor MHC downregulation. Antibodies have variable regions
composed of a heavy and a light chain that dictate antigen specificity.
Genetically crafting a receptor to include a single-chain variable
fragment (scFv) coding for both heavy and light chain variable fragments
allows CARs to preserve the antigen specificity of the parental antibody.
T cells require both an activation signal and a costimulatory signal
to induce activation. On encountering a cell that solely stimulates
CD3ζ in the absence of costimulation, the T cell will become anergic
to that cell and will be inert upon subsequent encounters. First-
generation CARs linked scFvs to the CD3ζ activation signal alone
without the inclusion of a costimulatory domain and did not achieve
therapeutic benefit.216 Because most tumors do not express ample
levels of costimulation signals, second-generation CARs have been
developed to include additional costimulatory domains (most commonly
CD28 or 4-1BB)238 and have demonstrated remarkable efficacy. It is
now evident that costimulatory signals are not equivalent and influence
the persistence and efficacy of the CAR T-cell product. Inclusion of
CD28 potentially confers greater initial cytotoxicity; however, CARs
incorporating CD28 signaling endodomains do not demonstrate
long-term, high-level CAR persistence.239 In contrast, CARs with
4-1BB costimulatory signals often demonstrate persistence, and such
CARs are not infrequently found in patients more than 2 years after
CAR infusion.240
Third-generation CARs, which incorporate multiple costimulatory
signals, have yet to confer superiority over second-generation CARs.241
The inclusion of more costimulation may in fact influence the exhaus-
tion status of the T cell, thereby restricting maximal long-term immune
effect. Numerous additional genetic modifications are undergoing
testing in CAR T cells, including expression of modifiers designed to
protect the T cell from the tumor inhibitory microenvironment, such
as modifying CARs to express molecules designed to prevent signaling
via PD-1, or to augment signaling of activating cytokine transgenes
or chemokine receptors.242 The elegance of the CAR is that on binding
of a single scFv, multiple signals can be directly stimulated, facilitating
maximal activation and tumor-specific cytolysis.243 An additional benefit
of the CAR platform is the potential generalizability to any tumor
for which an antibody targeting a tumor-specific antigen has been
developed. In practice, the efficacy of CARs derived from mAbs varies
significantly, and therefore substantial preclinical optimization is
typically required to generate an effective CAR for a particular target.
Clinical Translation of Chimeric Antigen Receptor Therapy
Hematologic malignancies
The first clinical report demonstrating CAR efficacy described dramatic
tumor regression in a patient with advanced follicular lymphoma
following administration of a second-generation CD19-28z CAR.244
Subsequently, three patients with chronic lymphocytic leukemia (CLL)
treated with CD19-BB.z-CARs achieved 1000-fold CAR T-cell expan-
sion and CAR persistence for at least 6 months, some of which conferred
a memory phenotype by flow cytometry.245–247 Subsequent work in
CLL demonstrated the importance of lymphodepletion before adoptive
transfer of CAR T cells, and the researchers concluded that disease
response and persistence were inversely correlated with tumor burden.248
This early work in lymphoma paved the way for CAR T-cell trials in
Cancer Immunology  •  CHAPTER 6 95
B-cell acute lymphoblastic leukemia (B-ALL) that demonstrated not
only a high feasibility of generating CAR T cells in this setting but
also that B-ALL is exquisitely sensitive to CD19-CAR T-cell therapy.
Several groups from different institutions have now demonstrated
that CD19-targeting CARs can induce striking remissions in adults
and children with relapsed or refractory B-ALL despite significant
interinstitutional heterogeneity in CAR constructs, preparative regimens,
and vector platforms.238,240,249–251 All groups report remarkable complete
remission rates ranging from 70% to 90% in B-ALL, which are
markedly superior to historic complete remission rates of 20% to
30% when standard salvage chemotherapy is used in patients with
relapsed or refractory ALL.251 This work led to FDA approval of
CTL019, CD19-targeted CAR on August 2017.
Several insights have been gained from parallel yet varying strategies
pursued by the different institutions. It is now clear that in vivo CAR
T-cell expansion correlates with clinical response250 and that diminished
persistence of CAR T cells is associated with a higher rate of disease
relapse.252 CAR costimulatory domains were shown to critically affect
the T-cell capacity for long-term persistence, with endogenous 4-1BB
mediating superior persistence as compared with CAR T cells with
CD28 costimulation.240,251 Different relapse patterns following CAR
therapy were identified with CD19 negative relapse due to tumor
antigen remodeling under the pressure of targeted CAR being the
most common.240,249,250,253 Additional to surface antigen remodeling,
tumors can undergo lineage reprogramming and relapse with myeloid
disease.254
Beyond the anticipated on-target–off-tumor B-cell aplasia expe-
rienced following CD19-CAR therapy, cytokine release syndrome
(CRS) and neurotoxicity are the two most common ominous toxici-
ties observed. CRS is a clinical syndrome characterized by fever and
hypotension in context of high levels of proinflammatory cytokines
that typically develops within 1 to 2 weeks after treatment and can
range from a mild syndrome with constitutional symptoms to a
severe life-threatening syndrome necessitating intensive care unit–level
support. Dramatic cytokine elevations including IL-6, IL-10, IFN-γ,
and TNF-α correlate with CRS severity.255 IL-6 has been implicated
in propagating the clinical syndrome, and treatment with tocilizumab,
an anti-IL6R mAb, has been shown to effectively and promptly reverse
the syndrome.256 Corticosteroids provide an alternative treatment for
CRS, although uncontrolled trials suggest that they may constrain the
antitumor immune response more than IL-6 neutralizing strategies.257
Neurologic toxicities following CAR therapy can occur in the
presence or absence of CRS, suggesting that the mechanisms may
differ and cytokine dysregulation alone may not fully explain CAR-
mediated neurotoxicity.256 Symptoms include headaches, confusion,
delirium, hallucinations, facial nerve palsies, apraxia, ataxia, dysmetria,
dysphagia, seizures, and altered consciousness that can require airway
protection with ventilator support.258 Neurotoxicity has not been shown
to directly correlate with central nervous system disease, but CAR T cells
and elevated cytokine levels have been identified in the cerebrospinal
fluid of patients experiencing neurotoxicity.258 This syndrome is most
commonly reversible; however, rarely patients have developed rapidly
fatal progression of neurotoxicity and cerebral edema following use of
CD19-CARs, with the precise pathophysiology not fully elucidated at
this time. Preclinical primate studies259 and clinical scrutiny are critical
to understanding and preventing this devastating neurologic sequela.
Solid tumors
Despite several clinical trials targeting numerous antigens in a variety
of solid cancers, outcomes using CAR therapy in solid tumors have
been less encouraging than in hematologic tumors. Barriers implicated
in restraining outcomes include a dearth of antigens with conserved,
specific tumor expression that spare vital tissues, impaired T-cell
trafficking, immunosuppression within the tumor microenvironment,260
and challenges related to CAR engineering that affect the potency
and exhaustion propensity of some CARs versus others.239 The most
advanced published solid tumor experiences have been with GD2-CARs
96 Part I: Science and Clinical Oncology

in neuroblastoma and sarcoma and Her2.28.z-CARs in Her2+ sarcomas.
First-generation GD2-CARs mediated long-term complete responses
in 2 of 11 patients with neuroblastoma.261,262 Subsequent investigation
with a third-generation GD2-CAR263 resulted in early signs of exhaus-
tion attributed to tonic CAR signaling and limited expansion, per-
sistence, or clinical response.239 Recent work using a Her2.28.z-CAR
in Her2+ sarcomas resulted in 4 of 17 treated patients achieving stable
disease for 12 weeks to 14 months, and CAR T cells persisted at least
6 weeks at low levels in the peripheral blood.264 A recent case report
of a patient with metastatic glioblastoma multiforme who had a dramatic
response to regional delivery of a CAR targeting IL13Rα2265 provides
additional data suggesting that IL13Rα2 may be a promising target
for CAR-based targeting of brain tumors.265 Although results have
not been robust in solid tumors to date, these early demonstrations
of efficacy support the potential of CARs to change outcomes in
otherwise devastating solid tumors.
Cancer Vaccines
Cancer vaccines include preventive and treatment strategies. Preventive
strategies such as the routine administration of HPV vaccination to
prevent HPV-driven cervical cancer and hepatitis B vaccination to
prevent hepatocellular carcinoma have proven highly effective, safe,
and feasible. The first successful treatment vaccine to yield clinical
benefit in a randomized phase III cancer vaccine trial was a putative
DC vaccine, sipuleucel-T, used in patients with advanced hormone-
resistant prostate cancer.266 This vaccine is based on the concept that
optimal T-cell activation requires antigen processing and presentation
by DCs, with the capacity to concomitantly deliver strong costimulatory
signals in the form of membrane ligands and secreted cytokines.
Sipuleucel-T is a patient-specific vaccine produced by transiently
incubating the patient’s own peripheral blood mononuclear cells with
a fusion protein consisting of prostatic acid phosphatase (PAP; a
prostate/prostate cancer–specific antigen) linked to the DC growth
and differentiation factor GM-CSF. A 4-month overall survival benefit
relative to the control arm (uncultured peripheral blood mononuclear
cells without PAP/GM-CSF fusion protein) in the absence of objective
tumor regressions or effect on time to progression emphasizes a
developing clinical paradigm in immunotherapy—that immunotherapy
can potentially provide overall survival benefits that are not reflected
in progression-free survival or objective response rate (ORR). The
survival benefit of sipuleucel-T ultimately led to FDA approval in
2010. Recently there has been a revival of interest in the field of
therapeutic cancer vaccinations, and efforts to develop personalized
neoantigen vaccines are underway.
CONCLUSIONS
In this chapter we have discussed several key principles of cancer immu-
nology, including tumor associated antigens, the immune surveillance
hypothesis, and the immune hallmarks of cancer. We also introduced
multiple mechanisms by which cancer and the immune system shape
each other. These include downregulation of tumor antigen presentation,
development of a tumor microenvironment suppressing antitumor
immunity, and expression of coinhibitory ligands and receptors that
negatively regulate antitumor immunity. In the last section, we review
three classes of cancer immunotherapy: checkpoint blockade, cellular
therapy, and cancer vaccination. These strategies are currently being
tested across cancer types that vary in their cancer immunogenic-
ity, which likely reflects quantitative neoantigen burden as well as
qualitative properties such as antigen binding to HLA, the nature of
T-cell recognition of these antigen/HLA complexes, and qualitative
distinctions within the immunosuppressive tumor microenvironment.
Current models hold that checkpoint blockade is more effective for
immunogenic tumors, whereas engineered immunotherapeutics such
as T cells engineered to express CARs may mediate similar antitumor
effects regardless of the level of inherent tumor immunogenicity. Cancer
vaccines offer a promising approach for shaping and focusing an
antitumor immune response, but there is still much to be understood
regarding how to identify the types of antigens to target, the number
of antigens, and how to produce the product in a streamlined manner.
In reality, there is broad overlap of tumor immunogenicity across
tumor types, and tumors of the same histologic type also vary in
their immunogenicity. Therefore advances in clinical immunotherapy
aimed at augmenting clinical responses while minimizing toxicity
requires a deeper understanding of the molecular determinants of
antitumor immunity.
The complete reference list is available online at
ExpertConsult.com.

KEY REFERENCES
177. Reck M, Rodriguez-Abreu D, Robinson AG,
et al. Pembrolizumab versus chemotherapy for
PD-L1-positive non-small-cell lung cancer. N Engl
J Med. 2016;375(19):1823–1833.
179. Motzer RJ, Escudier B, McDermott DF, et al.
Nivolumab versus everolimus in advanced renal-cell
carcinoma. N Engl J Med. 2015;373(19):1803–1813.
181. Ferris RL, Blumenschein G Jr, Fayette J, et al.
Nivolumab for recurrent squamous-cell carcinoma
of the head and neck. N Engl J Med. 2016;375(19):
1856–1867.
182. Le DT, Uram JN, Wang H, et al. PD-1 Blockade
in tumors with mismatch-repair deficiency. N Engl
J Med. 2015;372(26):2509–2520.
186. Robert C, Thomas L, Bondarenko I, et al. Ipili-
mumab plus dacarbazine for previously untreated
metastatic melanoma. N Engl J Med. 2011;364(26):
2517–2526.
190. Topalian SL, Hodi FS, Brahmer JR, et al. Safety,
activity, and immune correlates of anti-PD-1
antibody in cancer. N Engl J Med. 2012;366(26):
2443–2454.
195. Garon EB, Rizvi NA, Hui R, et al. Pembrolizumab
for the treatment of non-small-cell lung cancer. N
Engl J Med. 2015;372(21):2018–2028.
198. Wolchok JD, Kluger H, Callahan MK, et al.
Nivolumab plus ipilimumab in advanced melanoma.
N Engl J Med. 2013;369(2):122–133.
200. Gubin MM, Zhang X, Schuster H, et al. Checkpoint
blockade cancer immunotherapy targets tumour-
specific mutant antigens. Nature. 2014;515(7528):
577–581.
201. Rizvi NA, Hellmann MD, Snyder A, et al. Cancer
immunology. Mutational landscape determines
sensitivity to PD-1 blockade in non-small cell lung
cancer. Science. 2015;348(6230):124–128.
202. Snyder A, Makarov V, Merghoub T, et al. Genetic
basis for clinical response to CTLA-4 blockade
in melanoma. N Engl J Med. 2014;371(23):
2189–2199.
204. Tumeh PC, Harview CL, Yearley JH, et al. PD-1
blockade induces responses by inhibiting adaptive
immune resistance. Nature. 2014;515(7528):568–
571.
207. Peng D, Kryczek I, Nagarsheth N, et al. Epigenetic
silencing of TH1-type chemokines shapes tumour
immunity and immunotherapy. Nature. 2015;
527(7577):249–253.
231. Robbins PF, Morgan RA, Feldman SA, et al. Tumor
regression in patients with metastatic synovial cell
sarcoma and melanoma using genetically engineered
lymphocytes reactive with NY-ESO-1. J Clin Oncol.
2011;29(7):917–924.
236. Cameron BJ, Gerry AB, Dukes J, et al. Identifica-
tion of a Titin-derived HLA-A1-presented peptide
as a cross-reactive target for engineered MAGE
A3-directed T cells. Sci Transl Med. 2013;5(197):
197ra103.
239. Long AH, Haso WM, Shern JF, et al. 4-1BB
costimulation ameliorates T cell exhaustion induced
by tonic signaling of chimeric antigen receptors. Nat
Med. 2015;21(6):581–590.
240. Maude SL, Frey N, Shaw PA, et al. Chimeric antigen
receptor T cells for sustained remissions in leukemia.
N Engl J Med. 2014;371(16):1507–1517.
244. Kochenderfer JN, Wilson WH, Janik JE, et al.
Eradication of B-lineage cells and regression of
lymphoma in a patient treated with autologous
T cells genetically engineered to recognize CD19.
Blood. 2010;116(20):4099–4102.
250. Lee DW, Kochenderfer JN, Stetler-Stevenson M,
et al. T cells expressing CD19 chimeric antigen
receptors for acute lymphoblastic leukaemia in
children and young adults: a phase 1 dose-escalation
trial. Lancet. 2015;385(9967):517–528.
257. Davila ML, Riviere I, Wang X, et al. Efficacy and
toxicity management of 19-28z CAR T cell therapy
in B cell acute lymphoblastic leukemia. Sci Transl
Med. 2014;6(224):224ra225.
266. Kantoff PW, Higano CS, Shore ND, et al. Sipuleucel-
T immunotherapy for castration-resistant prostate
cancer. N Engl J Med. 2010;363(5):411–422.

REFERENCES
1. Baldwin RW. Immunity to methylcholanthrene-
induced tumours in inbred rats following atrophy
and regression of the implanted tumours. Br J
Cancer. 1955;9(4):652–657.
2. Foley EJ. Antigenic properties of methylcholanthrene-
induced tumors in mice of the strain of origin.
Cancer Res. 1953;13(12):835–837.
3. Gross L. Intradermal immunization of C3H mice
against a sarcoma that originated in an animal of
the same line. Cancer Res. 1943;3(5):326–333.
4. Old LJ, Carswell EA, Boyse EA, Clarke DA.
Antigenic properties of chemically induced tumors.
Ann N Y Acad Sci. 1962;101(1):80–&.
5. Prehn RT, Main JM. Immunity to methylcho-
lanthrene-induced sarcomas. J Natl Cancer Inst.
1957;18(6):769–778.
6. Bronner CE, Baker SM, Morrison PT, et al. Muta-
tion in the DNA mismatch repair gene homologue
hMLH1 is associated with hereditary non-polyposis
colon cancer. Nature. 1994;368(6468):258–261.
7. Fearon ER, Vogelstein B. A genetic model for
colorectal tumorigenesis. Cell. 1990;61(5):759–
767.
8. Fishel R, Lescoe MK, Rao MRS, et al. The human
mutator gene homolog Msh2 and its association
with hereditary nonpolyposis colon-cancer. Cell.
1993;75(5):1027–1038.
9. Gowen LC, Avrutskaya AV, Latour AM, Koller
BH, Leadon SA. BRCA1 required for transcription-
coupled repair of oxidative DNA damage (Retracted
article. See vol 300, pg 1657, June 13 2003). Science.
1998;281(5379):1009–1012.
10. Leach FS, Nicolaides NC, Papadopoulos N, et al.
Mutations of a Muts homolog in hereditary non-
polyposis colorectal-cancer. Cell. 1993;75(6):1215–
1225.
11. Lu X, Lane DP. Differential induction of tran-
scriptionally active P53 following UV or ionizing-
radiation—defects in chromosome instability
syndromes. Cell. 1993;75(4):765–778.
12. Papadopoulos N, Nicolaides NC, Wei YF, et al.
Mutation of a mutL homolog in hereditary colon
cancer. Science. 1994;263(5153):1625–1629.
13. Sharan SK, Morimatsu M, Albrecht U, et al.
Embryonic lethality and radiation hypersensitivity
mediated by Rad51 in mice lacking Brca2. Nature.
1997;386(6627):804–810.
14. Sjoblom T, Jones S, Wood LD, et al. The consensus
coding sequences of human breast and colorectal
cancers. Science. 2006;314(5797):268–274.
15. Almoguera C, Shibata D, Forrester K, Martin J,
Arnheim N, Perucho M. Most human carcinomas
of the exocrine pancreas contain mutant c-K-ras
genes. Cell. 1988;53(4):549–554.
16. Bressac B, Kew M, Wands J, Ozturk M. Selective
G to T mutations of p53 gene in hepatocellular
carcinoma from southern Africa. Nature. 1991;
350(6317):429–431.
17. Davies H, Bignell GR, Cox C, et al. Mutations of
the BRAF gene in human cancer. Nature. 2002;
417(6892):949–954.
18. Forrester K, Almoguera C, Han K, Grizzle WE,
Perucho M. Detection of high incidence of K-ras
oncogenes during human colon tumorigenesis.
Nature. 1987;327(6120):298–303.
19. Jones PA, Baylin SB. The epigenomics of cancer.
Cell. 2007;128(4):683–692.
20. Thomas AM, Santarsiero LM, Lutz ER, et al.
Mesothelin-specific CD8(+) T cell responses
provide evidence of in vivo cross-priming by
antigen-presenting cells in vaccinated pancreatic
cancer patients. J Exp Med. 2004;200(3):297–306.
21. Argani P, Iacobuzio-Donahue C, Ryu B, et al.
Mesothelin is overexpressed in the vast majority of
ductal adenocarcinomas of the pancreas: identifica-
tion of a new pancreatic cancer marker by serial
analysis of gene expression (SAGE). Clin Cancer
Res. 2001;7(12):3862–3868.
22. Chang K, Pastan I. Molecular cloning of mesothelin,
a differentiation antigen present on mesothelium,
mesotheliomas, and ovarian cancers. Proc Natl Acad
Sci USA. 1996;93(1):136–140.
23. Van Der Bruggen P, Zhang Y, Chaux P, et al.
Tumor-specific shared antigenic peptides recog-
nized by human T cells. Immunol Rev. 2002;188:
51–64.
24. Madsen B, Tarsounas M, Burchell JM, Hall D,
Poulsom R, Taylor-Papadimitriou J. PLU-1, a tran-
scriptional repressor and putative testis-cancer antigen,
has a specific expression and localisation pattern
during meiosis. Chromosoma. 2003;112(3):124–
132.
25. Osterlund C, Tohonen V, Forslund KO, Nordqvist K.
Mage-b4, a novel melanoma antigen (MAGE) gene
specifically expressed during germ cell differentiation.
Cancer Res. 2000;60(4):1054–1061.
26. Tureci O, Sahin U, Zwick C, Koslowski M, Seitz G,
Pfreundschuh M. Identification of a meiosis-specific
protein as a member of the class of cancer/testis
antigens. Proc Natl Acad Sci USA. 1998;95(9):
5211–5216.
27. Kawakami Y, Robbins PF, Wang RF, Parkhurst M,
Kang XQ, Rosenberg SA. The use of melanosomal
proteins in the immunotherapy of melanoma.
J Immunother. 1998;21(4):237–246.
28. Brichard V, Vanpel A, Wolfel T, et al. The Tyrosinase
gene codes for an antigen recognized by autologous
cytolytic T-lymphocytes on HLA-A2 melanomas.
J Exp Med. 1993;178(2):489–495.
29. Topalian SL, Rivoltini L, Mancini M, et al. Human
CD4+ T cells specifically recognize a shared mela-
noma-associated antigen encoded by the tyrosinase
gene. Proc Natl Acad Sci USA. 1994;91(20):9461–
9465.
30. Burnet FM. The concept of immunological surveil-
lance. Prog Exp Tumor Res. 1970;13:1–27.
31. Thomas L. In: Lawrence HS, ed. Discussion of
Cellular and Humoral Aspects of the Hypersensitive
States. New York: Hoeber-Harper; 1959.
32. Dunn GP, Koebel CM, Schreiber RD. Interferons,
immunity and cancer immunoediting. Nat Rev
Immunol. 2006;6(11):836–848.
33. Fenner JE, Starr R, Cornish AL, et al. Suppressor
of cytokine signaling 1 regulates the immune
response to infection by a unique inhibition of
type I interferon activity. Nat Immunol. 2006;7(1):
33–39.
34. Shankaran V, Ikeda H, Bruce AT, et al. IFNgamma
and lymphocytes prevent primary tumour develop-
ment and shape tumour immunogenicity. Nature.
2001;410(6832):1107–1111.
35. Gaidano G, Dalla-Favera R. Biologic aspects of
human immunodeficiency virus-related lymphoma.
Curr Opin Oncol. 1992;4(5):900–906.
36. Penn I. Tumors of the immunocompromised patient.
Annu Rev Med. 1988;39:63–73.
37. Knowles DM. In: Frizzera G, ed. Neoplastic
Hematopathology. Baltimore: Williams & Wilkins;
1992:459–495.
38. Heslop HE, Ng CY, Li C, et al. Long-term
restoration of immunity against Epstein-Barr virus
infection by adoptive transfer of gene-modified
virus-specific T lymphocytes. Nat Med. 1996;2(5):
551–555.
39. Mesri EA, Cesarman E, Arvanitakis L, et al.
Human herpesvirus-8/Kaposi’s sarcoma–associated
herpesvirus is a new transmissible virus that infects
B cells. J Exp Med. 1996;183(5):2385–2390.
40. Beaudenon S, Kremsdorf D, Obalek S, et al. Plurality
of genital human papillomaviruses: characterization
of two new types with distinct biological properties.
Virology. 1987;161(2):374–384.
Cancer Immunology  •  CHAPTER 6 96.e1
41. Boshart M, Gissmann L, Ikenberg H, Kleinheinz
A, Scheurlen W, zur Hausen H. A new type of
papillomavirus DNA, its presence in genital cancer
biopsies and in cell lines derived from cervical cancer.
EMBO J. 1984;3(5):1151–1157.
42. Euvrard S, Kanitakis J, Pouteil-Noble C, Claudy
A, Touraine JL. Skin cancers in organ transplant
recipients. Ann Transplant. 1997;2(4):28–32.
43. Bogen B. Peripheral T cell tolerance as a tumor escape
mechanism: deletion of CD4+ T cells specific for
a monoclonal immunoglobulin idiotype secreted
by a plasmacytoma. Eur J Immunol. 1996;26(11):
2671–2679.
44. Bogen B, Munthe L, Sollien A, et al. Naive CD4+
T cells confer idiotype-specific tumor resistance in
the absence of antibodies. Eur J Immunol. 1995;
25(11):3079–3086.
45. Sotomayor EM, Borrello I, Rattis FM, et al. Cross-
presentation of tumor antigens by bone marrow-
derived antigen-presenting cells is the dominant
mechanism in the induction of T-cell tolerance
during B-cell lymphoma progression. Blood. 2001;
98(4):1070–1077.
46. Staveley-O’Carroll K, Sotomayor E, Montgomery
J, et al. Induction of antigen-specific T cell anergy:
an early event in the course of tumor progression.
Proc Natl Acad Sci USA. 1998;95(3):1178–1183.
47. Speiser DE, Miranda R, Zakarian A, et al. Self
antigens expressed by solid tumors Do not efficiently
stimulate naive or activated T cells: implications
for immunotherapy. J Exp Med. 1997;186(5):645–
653.
48. Wick M, Dubey P, Koeppen H, et al. Antigenic
cancer cells grow progressively in immune hosts
without evidence for T cell exhaustion or systemic
anergy. J Exp Med. 1997;186(2):229–238.
49. Nguyen LT, Elford AR, Murakami K, et al. Tumor
growth enhances cross-presentation leading to limited
T cell activation without tolerance. J Exp Med.
2002;195(4):423–435.
50. Seliger B, Harders C, Lohmann S, et al. Down-
regulation of the MHC class I antigen-processing
machinery after oncogenic transformation of murine
fibroblasts. Eur J Immunol. 1998;28(1):122–133.
51. Concha-Benavente F, Srivastava RM, Ferrone S,
Ferris RL. EGFR-mediated tumor immunoescape:
the imbalance between phosphorylated STAT1 and
phosphorylated STAT3. Oncoimmunology. 2013;
2(12):e27215.
52. Georgopoulos NT, Proffitt JL, Blair GE. Transcrip-
tional regulation of the major histocompatibility
complex (MHC) class I heavy chain, TAP1 and
LMP2 genes by the human papillomavirus (HPV)
type 6b, 16 and 18 E7 oncoproteins. Oncogene.
2000;19(42):4930–4935.
53. Setiadi AF, Omilusik K, David MD, et al. Epigenetic
enhancement of antigen processing and presentation
promotes immune recognition of tumors. Cancer
Res. 2008;68(23):9601–9607.
54. Khan AN, Gregorie CJ, Tomasi TB. Histone
deacetylase inhibitors induce TAP, LMP, Tapasin
genes and MHC class I antigen presentation by
melanoma cells. Cancer Immunol Immunother.
2008;57(5):647–654.
55. Fruci D, Giacomini P, Nicotra MR, et al. Altered
expression of endoplasmic reticulum aminopeptidases
ERAP1 and ERAP2 in transformed non-lymphoid
human tissues. J Cell Physiol. 2008;216(3):742–
749.
56. Sakaguchi S, Ono M, Setoguchi R, et al. Foxp3+
CD25+ CD4+ natural regulatory T cells in dominant
self-tolerance and autoimmune disease. Immunol
Rev. 2006;212:8–27.
57. Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda
M. Immunologic self-tolerance maintained by acti-
vated T cells expressing IL-2 receptor alpha-chains
96.e2 Part I: Science and Clinical Oncology
(CD25). Breakdown of a single mechanism of
self-tolerance causes various autoimmune diseases.
J Immunol. 1995;155(3):1151–1164.
58. Curiel TJ, Coukos G, Zou L, et al. Specific recruit-
ment of regulatory T cells in ovarian carcinoma
fosters immune privilege and predicts reduced
survival. Nat Med. 2004;10(9):942–949.
59. Liyanage UK, Moore TT, Joo HG, et al. Prevalence
of regulatory T cells is increased in peripheral blood
and tumor microenvironment of patients with
pancreas or breast adenocarcinoma. J Immunol.
2002;169(5):2756–2761.
60. Williams LM, Rudensky AY. Maintenance of the
Foxp3-dependent developmental program in mature
regulatory T cells requires continued expression of
Foxp3. Nat Immunol. 2007;8(3):277–284.
61. Zheng Y, Rudensky AY. Foxp3 in control of
the regulatory T cell lineage. Nat Immunol.
2007;8(5):457–462.
62. Turnis ME, Sawant DV, Szymczak-Workman AL,
et al. Interleukin-35 limits anti-tumor immu-
nity. Immunity. 2016;44(2):316–329.
63. Sutmuller RP, van Duivenvoorde LM, van Elsas
A, et al. Synergism of cytotoxic T lymphocyte-
associated antigen 4 blockade and depletion of
CD25(+) regulatory T cells in antitumor therapy
reveals alternative pathways for suppression of
autoreactive cytotoxic T lymphocyte responses.
J Exp Med. 2001;194(6):823–832.
64. Berd D, Maguire HC Jr, Mastrangelo MJ. Induc-
tion of cell-mediated immunity to autologous
melanoma cells and regression of metastases after
treatment with a melanoma cell vaccine preceded by
cyclophosphamide. Cancer Res. 1986;46(5):2572–
2577.
65. Berd D, Mastrangelo MJ, Engstrom PF, Paul A,
Maguire H. Augmentation of the human immune
response by cyclophosphamide. Cancer Res. 1982;
42(11):4862–4866.
66. Ercolini AM, Ladle BH, Manning EA, et al. Recruit-
ment of latent pools of high-avidity CD8(+) T cells
to the antitumor immune response. J Exp Med.
2005;201(10):1591–1602.
67. North RJ. Cyclophosphamide-facilitated adoptive
immunotherapy of an established tumor depends
on elimination of tumor-induced suppressor T cells.
J Exp Med. 1982;155(4):1063–1074.
68. Prince HM, Duvic M, Martin A, et al. Phase III
placebo-controlled trial of denileukin diftitox for
patients with cutaneous T-cell lymphoma. J Clin
Oncol. 2010;28(11):1870–1877.
69. Kusmartsev S, Gabrilovich DI. Role of immature
myeloid cells in mechanisms of immune evasion in
cancer. Cancer Immunol Immunother. 2006;55(3):237–
245.
70. Young MR, Petruzzelli GJ, Kolesiak K, Achille N,
Lathers DM, Gabrilovich DI. Human squamous
cell carcinomas of the head and neck chemoattract
immune suppressive CD34(+) progenitor cells. Hum
Immunol. 2001;62(4):332–341.
71. Bronte V, Apolloni E, Cabrelle A, et al. Identification
of a CD11b(+)/Gr-1(+)/CD31(+) myeloid progeni-
tor capable of activating or suppressing CD8(+) T
cells. Blood. 2000;96(12):3838–3846.
72. Bronte V, Serafini P, De Santo C, et al. IL-4-induced
arginase 1 suppresses alloreactive T cells in tumor-
bearing mice. J Immunol. 2003;170(1):270–278.
73. Mazzoni A, Bronte V, Visintin A, et al. Myeloid
suppressor lines inhibit T cell responses by
an NO-dependent mechanism. J Immunol.
2002;168(2):689–695.
74. Zea AH, Rodriguez PC, Atkins MB, et al. Arginase-
producing myeloid suppressor cells in renal cell
carcinoma patients: a mechanism of tumor evasion.
Cancer Res. 2005;65(8):3044–3048.
75. Moisan F, Francisco EB, Brozovic A, et al. Enhance-
ment of paclitaxel and carboplatin therapies by CCL2
blockade in ovarian cancers. Mol Oncol. 2014;8(7):
1231–1239.
76. Nywening TM, Wang-Gillam A, Sanford DE,
et al. Targeting tumour-associated macrophages
with CCR2 inhibition in combination with FOL-
FIRINOX in patients with borderline resectable
and locally advanced pancreatic cancer: a single-
centre, open-label, dose-finding, non-randomised,
phase 1b trial. Lancet Oncol. 2016;17(5):651–
662.
77. Ries CH, Cannarile MA, Hoves S, et al. Targeting
tumor-associated macrophages with anti-CSF-1R
antibody reveals a strategy for cancer therapy. Cancer
Cell. 2014;25(6):846–859.
78. Tseng D, Volkmer JP, Willingham SB, et al. Anti-
CD47 antibody-mediated phagocytosis of cancer by
macrophages primes an effective antitumor T-cell
response. Proc Natl Acad Sci USA. 2013;110(27):
11103–11108.
79. Willingham SB, Volkmer JP, Gentles AJ, et al.
The CD47-signal regulatory protein alpha (SIRPa)
interaction is a therapeutic target for human solid
tumors. Proc Natl Acad Sci USA. 2012;109(17):
6662–6667.
80. Majeti R, Chao MP, Alizadeh AA, et al. CD47 is an
adverse prognostic factor and therapeutic antibody
target on human acute myeloid leukemia stem cells.
Cell. 2009;138(2):286–299.
81. Bonifaz L, Bonnyay D, Mahnke K, Rivera M,
Nussenzweig MC, Steinman RM. Efficient
targeting of protein antigen to the dendritic cell
receptor DEC-205 in the steady state leads to
antigen presentation on major histocompatibility
complex class I products and peripheral CD8+ T
cell tolerance. J Exp Med. 2002;196(12):1627–1638.
82. Steinman RM, Hawiger D, Nussenzweig MC.
Tolerogenic dendritic cells. Annu Rev Immunol.
2003;21:685–711.
83. Schmielau J, Finn OJ. Activated granulocytes and
granulocyte-derived hydrogen peroxide are the
underlying mechanism of suppression of t-cell
function in advanced cancer patients. Cancer Res.
2001;61(12):4756–4760.
84. Munn DH, Sharma MD, Lee JR, et al. Potential
regulatory function of human dendritic cells express-
ing indoleamine 2,3-dioxygenase. Science. 2002;297
(5588):1867–1870.
85. Baban B, Hansen AM, Chandler PR, et al. A minor
population of splenic dendritic cells expressing CD19
mediates IDO-dependent T cell suppression via type
I IFN signaling following B7 ligation. Int Immunol.
2005;17(7):909–919.
86. Mellor AL, Chandler P, Baban B, et al. Specific
subsets of murine dendritic cells acquire potent T
cell regulatory functions following CTLA4-mediated
induction of indoleamine 2,3 dioxygenase. Int
Immunol. 2004;16(10):1391–1401.
87. Munn DH, Sharma MD, Hou D, et al. Expression
of indoleamine 2,3-dioxygenase by plasmacytoid
dendritic cells in tumor-draining lymph nodes.
J Clin Invest. 2004;114(2):280–290.
88. Uyttenhove C, Pilotte L, Theate I, et al. Evidence
for a tumoral immune resistance mechanism
based on tryptophan degradation by indoleamine
2,3-dioxygenase. Nat Med. 2003;9(10):1269–
1274.
89. Gangadhar T. Epacadostat plus pembrolizumab in
patients with advanced melanoma and select solid
tumors: updated phase 1 results from ECHO-202
/ KEYNOTE-037. Ann Oncol. 2016;27(suppl 6).
90. Blobe GC, Schiemann WP, Lodish HF. Role of
transforming growth factor beta in human disease.
N Engl J Med. 2000;342(18):1350–1358.
91. Pepper MS. Transforming growth factor-beta:
vasculogenesis, angiogenesis, and vessel wall integrity.
Cytokine Growth Factor Rev. 1997;8(1):21–43.
92. Shariat SF, Kim JH, Andrews B, et al. Preoperative
plasma levels of transforming growth factor beta(1)
strongly predict clinical outcome in patients with
bladder carcinoma. Cancer. 2001;92(12):2985–
2992.
93. Hasegawa Y, Takanashi S, Kanehira Y, Tsushima T,
Imai T, Okumura K. Transforming growth factor-
beta1 level correlates with angiogenesis, tumor
progression, and prognosis in patients with nonsmall
cell lung carcinoma. Cancer. 2001;91(5):964–
971.
94. Saito H, Tsujitani S, Oka S, et al. The expression
of transforming growth factor-beta1 is significantly
correlated with the expression of vascular endothelial
growth factor and poor prognosis of patients with
advanced gastric carcinoma. Cancer. 1999;86(8):
1455–1462.
95. Morris JC, Tan AR, Olencki TE, et al. Phase
I study of GC1008 (fresolimumab): a human
anti-transforming growth factor-beta (TGFbeta)
monoclonal antibody in patients with advanced
malignant melanoma or renal cell carcinoma. PLoS
ONE. 2014;9(3):e90353.
96. Cohn A, Lahn MM, Williams KE, et al. A phase
I dose-escalation study to a predefined dose of a
transforming growth factor-beta1 monoclonal
antibody (TbetaM1) in patients with metastatic
cancer. Int J Oncol. 2014;45(6):2221–2231.
97. Lenschow DJ, Walunas TL, Bluestone JA. CD28/B7
system of T cell costimulation. Annu Rev Immunol.
1996;14:233–258.
98. Rudd CE, Taylor A, Schneider H. CD28 and
CTLA-4 coreceptor expression and signal transduc-
tion. Immunol Rev. 2009;229(1):12–26.
99. Schwartz RH. Costimulation of T lymphocytes: the
role of CD28, CTLA-4, and B7/BB1 in interleukin-2
production and immunotherapy. Cell. 1992;71(7):
1065–1068.
100. Azuma M, Ito D, Yagita H, et al. B70 antigen is
a second ligand for CTLA-4 and CD28. Nature.
1993;366(6450):76–79.
101. Freeman GJ, Gribben JG, Boussiotis VA, et al.
Cloning of B7-2: a CTLA-4 counter-receptor that
costimulates human T cell proliferation. Science.
1993;262(5135):909–911.
102. Hathcock KS, Laszlo G, Dickler HB, Bradshaw J,
Linsley P, Hodes RJ. Identification of an alternative
CTLA-4 ligand costimulatory for T cell activation.
Science. 1993;262(5135):905–907.
103. Linsley PS, Brady W, Urnes M, Grosmaire LS, Damle
NK, Ledbetter JA. CTLA-4 is a second receptor
for the B cell activation antigen B7. J Exp Med.
1991;174(3):561–569.
104. Linsley PS, Clark EA, Ledbetter JA. T-cell antigen
CD28 mediates adhesion with B cells by interacting
with activation antigen B7/BB-1. Proc Natl Acad Sci
USA. 1990;87(13):5031–5035.
105. Egen JG, Allison JP. Cytotoxic T lymphocyte
antigen-4 accumulation in the immunological
synapse is regulated by TCR signal strength.
Immunity. 2002;16(1):23–35.
106. Linsley PS, Greene JL, Brady W, Bajorath J, Ledbet-
ter JA, Peach R. Human B7-1 (CD80) and B7-2
(CD86) bind with similar avidities but distinct
kinetics to CD28 and CTLA-4 receptors. Immunity.
1994;1(9):793–801.
107. Parry RV, Chemnitz JM, Frauwirth KA, et al.
CTLA-4 and PD-1 receptors inhibit T-cell activation
by distinct mechanisms. Mol Cell Biol. 2005;25(21):
9543–9553.
108. Riley JL, Mao M, Kobayashi S, et al. Modulation
of TCR-induced transcriptional profiles by ligation
of CD28, ICOS, and CTLA-4 receptors. Proc Natl
Acad Sci USA. 2002;99(18):11790–11795.
109. Schneider H, Downey J, Smith A, et al. Reversal
of the TCR stop signal by CTLA-4. Science.
2006;313(5795):1972–1975.
110. Schneider H, Mandelbrot DA, Greenwald RJ,
et al. Cutting edge: CTLA-4 (CD152) differen-
tially regulates mitogen-activated protein kinases
(extracellular signal-regulated kinase and c-Jun
N-terminal kinase) in CD4+ T cells from receptor/
ligand-deficient mice. J Immunol. 2002;169(7):3475–
3479.

111. Qureshi OS, Zheng Y, Nakamura K, et al. Trans-
endocytosis of CD80 and CD86: a molecular basis
for the cell-extrinsic function of CTLA-4. Science.
2011;332(6029):600–603.
112. Tivol EA, Borriello F, Schweitzer AN, Lynch WP,
Bluestone JA, Sharpe AH. Loss of CTLA-4 leads to
massive lymphoproliferation and fatal multiorgan
tissue destruction, revealing a critical negative regula-
tory role of CTLA-4. Immunity. 1995;3(5):541–
547.
113. Waterhouse P, Penninger JM, Timms E, et al.
Lymphoproliferative disorders with early lethality
in mice deficient in Ctla-4. Science. 1995;270(5238):
985–988.
114. Peggs KS, Quezada SA, Chambers CA, Korman AJ,
Allison JP. Blockade of CTLA-4 on both effector
and regulatory T cell compartments contributes to
the antitumor activity of anti-CTLA-4 antibodies.
J Exp Med. 2009;206(8):1717–1725.
115. Wing K, Onishi Y, Prieto-Martin P, et al. CTLA-4
control over Foxp3+ regulatory T cell function.
Science. 2008;322(5899):271–275.
116. Fontenot JD, Gavin MA, Rudensky AY. Foxp3
programs the development and function of
CD4+CD25+ regulatory T cells. Nat Immunol.
2003;4(4):330–336.
117. Hori S, Nomura T, Sakaguchi S. Control of regula-
tory T cell development by the transcription factor
Foxp3. Science. 2003;299(5609):1057–1061.
118. Gavin MA, Rasmussen JP, Fontenot JD, et al.
Foxp3-dependent programme of regulatory T-
cell differentiation. Nature. 2007;445(7129):771–
775.
119. Hill JA, Feuerer M, Tash K, et  al. Foxp3
transcription-factor-dependent and -independent
regulation of the regulatory T cell transcriptional
signature. Immunity. 2007;27(5):786–800.
120. Freeman GJ, Long AJ, Iwai Y, et al. Engagement of
the PD-1 immunoinhibitory receptor by a novel B7
family member leads to negative regulation of lym-
phocyte activation. J Exp Med. 2000;192(7):1027–
1034.
121. Ishida Y, Agata Y, Shibahara K, Honjo T. Induced
expression of PD-1, a novel member of the immu-
noglobulin gene superfamily, upon programmed cell
death. EMBO J. 1992;11(11):3887–3895.
122. Keir ME, Butte MJ, Freeman GJ, Sharpe AH. PD-1
and its ligands in tolerance and immunity. Annu
Rev Immunol. 2008;26:677–704.
123. Keir ME, Liang SC, Guleria I, et al. Tissue expression
of PD-L1 mediates peripheral T cell tolerance. J Exp
Med. 2006;203(4):883–895.
124. Nishimura H, Nose M, Hiai H, Minato N, Honjo
T. Development of lupus-like autoimmune diseases
by disruption of the PD-1 gene encoding an
ITIM motif-carrying immunoreceptor. Immunity.
1999;11(2):141–151.
125. Nishimura H, Okazaki T, Tanaka Y, et  al.
Autoimmune dilated cardiomyopathy in PD-1
receptor-deficient mice. Science. 2001;291(5502):
319–322.
126. Okazaki T, Honjo T. PD-1 and PD-1 ligands:
from discovery to clinical application. Int Immunol.
2007;19(7):813–824.
127. Blank C, Brown I, Peterson AC, et al. PD-L1/B7H-1
inhibits the effector phase of tumor rejection by T
cell receptor (TCR) transgenic CD8+ T cells. Cancer
Res. 2004;64(3):1140–1145.
128. Dong H, Strome SE, Salomao DR, et al. Tumor-
associated B7-H1 promotes T-cell apoptosis: a
potential mechanism of immune evasion. Nat Med.
2002;8(8):793–800.
129. Taube JMAR, Xu H, Sharma R, McMiller T,
Klein A, Pardoll DM, et al. B7-H1 expression
co-localizes with inflammatory infiltrates in benign
and malignant melanocytic lesions: implications for
immunotherapy. Sci Transl Med. 2012;in press.
130. Fife BT, Pauken KE, Eagar TN, et al. Interactions
between PD-1 and PD-L1 promote tolerance by
blocking the TCR-induced stop signal. Nat Immunol.
2009;10(11):1185–1192.
131. Ahmadzadeh M, Johnson LA, Heemskerk B, et al.
Tumor antigen-specific CD8 T cells infiltrating the
tumor express high levels of PD-1 and are function-
ally impaired. Blood. 2009;114(8):1537–1544.
132. Sfanos KS, Bruno TC, Meeker AK, De Marzo AM,
Isaacs WB, Drake CG. Human prostate-infiltrating
CD8+ T lymphocytes are oligoclonal and PD-1.
Prostate. 2009;69(15):1694–1703.
133. Francisco LM, Salinas VH, Brown KE, et al. PD-
L1 regulates the development, maintenance, and
function of induced regulatory T cells. J Exp Med.
2009;206(13):3015–3029.
134. Fanoni D, Tavecchio S, Recalcati S, et al. New mono-
clonal antibodies against B-cell antigens: possible
new strategies for diagnosis of primary cutaneous
B-cell lymphomas. Immunol Lett. 2011;134(2):157–
160.
135. Terme M, Ullrich E, Aymeric L, et al. IL-18 induces
PD-1-dependent immunosuppression in cancer.
Cancer Res. 2011;71(16):5393–5399.
136. Velu V, Titanji K, Zhu B, et al. Enhancing SIV-
specific immunity in vivo by PD-1 blockade. Nature.
2009;458(7235):206–210.
137. Huang X, Venet F, Wang YL, et al. PD-1 expression
by macrophages plays a pathologic role in altering
microbial clearance and the innate inflamma-
tory response to sepsis. Proc Natl Acad Sci USA.
2009;106(15):6303–6308.
138. Dong H, Zhu G, Tamada K, Chen L. B7-H1, a
third member of the B7 family, co-stimulates T-cell
proliferation and interleukin-10 secretion. Nat Med.
1999;5(12):1365–1369.
139. Latchman Y, Wood CR, Chernova T, et al. PD-L2
is a second ligand for PD-1 and inhibits T cell
activation. Nat Immunol. 2001;2(3):261–268.
140. Tseng SY, Otsuji M, Gorski K, et al. B7-DC, a new
dendritic cell molecule with potent costimulatory
properties for T cells. J Exp Med. 2001;193(7):
839–846.
141. Butte MJ, Keir ME, Phamduy TB, Sharpe AH,
Freeman GJ. Programmed death-1 ligand 1 interacts
specifically with the B7-1 costimulatory molecule
to inhibit T cell responses. Immunity. 2007;27(1):
111–122.
142. Park JJ, Omiya R, Matsumura Y, et al. B7-H1/
CD80 interaction is required for the induction and
maintenance of peripheral T-cell tolerance. Blood.
2010;116(8):1291–1298.
143. Paterson AM, Brown KE, Keir ME, et al. The
programmed death-1 ligand 1:B7-1 pathway restrains
diabetogenic effector T cells in vivo. J Immunol.
2011;187(3):1097–1105.
144. Dong H, Strome SE, Matteson EL, et  al.
Costimulating aberrant T cell responses by B7-H1
autoantibodies in rheumatoid arthritis. J Clin Invest.
2003;111(3):363–370.
145. Azuma T, Yao S, Zhu G, Flies AS, Flies SJ, Chen
L. B7-H1 is a ubiquitous antiapoptotic receptor on
cancer cells. Blood. 2008;111(7):3635–3643.
146. Zou W, Chen L. Inhibitory B7-family molecules in
the tumour microenvironment. Nat Rev Immunol.
2008;8(6):467–477.
147. Brown JA, Dorfman DM, Ma FR, et al. Blockade
of programmed death-1 ligands on dendritic cells
enhances T cell activation and cytokine production.
J Immunol. 2003;170(3):1257–1266.
148. Konishi J, Yamazaki K, Azuma M, Kinoshita I,
Dosaka-Akita H, Nishimura M. B7-H1 expression
on non-small cell lung cancer cells and its relation-
ship with tumor-infiltrating lymphocytes and their
PD-1 expression. Clin Cancer Res. 2004;10(15):
5094–5100.
149. Curiel TJ, Wei S, Dong H, et al. Blockade of B7-H1
improves myeloid dendritic cell-mediated antitumor
immunity. Nat Med. 2003;9(5):562–567.
150. Kuang DM, Zhao Q, Peng C, et al. Activated
monocytes in peritumoral stroma of hepatocellular
Cancer Immunology  •  CHAPTER 6 96.e3
carcinoma foster immune privilege and disease pro-
gression through PD-L1. J Exp Med. 2009;206(6):
1327–1337.
151. Liu Y, Zeng B, Zhang Z, Zhang Y, Yang R. B7-H1
on myeloid-derived suppressor cells in immune
suppression by a mouse model of ovarian cancer.
Clin Immunol. 2008;129(3):471–481.
152. Hino R, Kabashima K, Kato Y, et al. Tumor cell
expression of programmed cell death-1 ligand 1 is a
prognostic factor for malignant melanoma. Cancer.
2010;116(7):1757–1766.
153. Steidl C, Shah SP, Woolcock BW, et al. MHC class
II transactivator CIITA is a recurrent gene fusion
partner in lymphoid cancers. Nature. 2011;471
(7338):377–381.
154. Parsa AT, Waldron JS, Panner A, et al. Loss of
tumor suppressor PTEN function increases B7-H1
expression and immunoresistance in glioma. Nat
Med. 2007;13(1):84–88.
155. Marzec M, Zhang Q, Goradia A, et al. Oncogenic
kinase NPM/ALK induces through STAT3 expres-
sion of immunosuppressive protein CD274 (PD-L1,
B7-H1). Proc Natl Acad Sci USA. 2008;105(52):
20852–20857.
156. Yi KH, Chen L. Fine tuning the immune response
through B7-H3 and B7-H4. Immunol Rev.
2009;229(1):145–151.
157. Ngiow SF, von Scheidt B, Akiba H, Yagita H, Teng
MW, Smyth MJ. Anti-TIM3 antibody promotes T
cell IFN-gamma-mediated antitumor immunity and
suppresses established tumors. Cancer Res. 2011;71
(10):3540–3551.
158. Triebel F, Jitsukawa S, Baixeras E, et al. LAG-3, a
novel lymphocyte activation gene closely related to
CD4. J Exp Med. 1990;171(5):1393–1405.
159. Goldberg MV, Drake CG. LAG-3 in Cancer
Immunotherapy. Curr Top Microbiol Immunol.
2011;344:269–278.
160. Huang CT, Workman CJ, Flies D, et al. Role of
LAG-3 in regulatory T cells. Immunity. 2004;21(4):
503–513.
161. Grosso JF, Kelleher CC, Harris TJ, et al. LAG-3
regulates CD8+ T cell accumulation and
effector function in murine self- and tumor-
tolerance systems. J Clin Invest. 2007;117(11):3383–
3392.
162. Xu F, Liu J, Liu D, et al. LSECtin expressed on
melanoma cells promotes tumor progression by
inhibiting antitumor T-cell responses. Cancer Res.
2014;74(13):3418–3428.
163. Blackburn SD, Shin H, Haining WN, et al.
Coregulation of CD8+ T cell exhaustion by multiple
inhibitory receptors during chronic viral infection.
Nat Immunol. 2009;10(1):29–37.
164. Grosso JF, Goldberg MV, Getnet D, et al. Function-
ally distinct LAG-3 and PD-1 subsets on activated
and chronically stimulated CD8 T cells. J Immunol.
2009;182(11):6659–6669.
165. Santiago C, Ballesteros A, Tami C, Martinez-Munoz
L, Kaplan GG, Casasnovas JM. Structures of T Cell
immunoglobulin mucin receptors 1 and 2 reveal
mechanisms for regulation of immune responses
by the TIM receptor family. Immunity. 2007;26(3):
299–310.
166. Chiba S, Baghdadi M, Akiba H, et al. Tumor-
infiltrating DCs suppress nucleic acid-mediated
innate immune responses through interactions
between the receptor TIM-3 and the alarmin
HMGB1. Nat Immunol. 2012;13(9):832–842.
167. Huang YH, Zhu C, Kondo Y, et al. CEACAM1
regulates TIM-3-mediated tolerance and exhaus-
tion. Nature. 2015;517(7534):386–390.
168. Zhu C, Anderson AC, Schubart A, et al. The Tim-3
ligand galectin-9 negatively regulates T helper type
1 immunity. Nat Immunol. 2005;6(12):1245–
1252.
169. Baitsch L, Legat A, Barba L, et al. Extended
co-expression of inhibitory receptors by human
CD8 T-cells depending on differentiation,
96.e4 Part I: Science and Clinical Oncology
antigen-specificity and anatomical localization.
PLoS ONE. 2012;7(2):e30852.
170. Fourcade J, Sun Z, Benallaoua M, et  al.
Upregulation of Tim-3 and PD-1 expression is
associated with tumor antigen-specific CD8+ T
cell dysfunction in melanoma patients. J Exp Med.
2010;207(10):2175–2186.
171. Sakuishi K, Apetoh L, Sullivan JM, Blazar BR,
Kuchroo VK, Anderson AC. Targeting Tim-3
and PD-1 pathways to reverse T cell exhaustion
and restore anti-tumor immunity. J Exp Med.
2010;207(10):2187–2194.
172. Koyama S, Akbay EA, Li YY, et al. Adaptive resistance
to therapeutic PD-1 blockade is associated with
upregulation of alternative immune checkpoints.
Nat Commun. 2016;7:10501.
173. Zarek PE, Huang CT, Lutz ER, et al. A2A receptor
signaling promotes peripheral tolerance by inducing
T-cell anergy and the generation of adaptive regula-
tory T cells. Blood. 2008;111(1):251–259.
174. Deaglio S, Dwyer KM, Gao W, et al. Adenosine
generation catalyzed by CD39 and CD73 expressed
on regulatory T cells mediates immune suppression.
J Exp Med. 2007;204(6):1257–1265.
175. Waickman AT, Alme A, Senaldi L, Zarek PE, Horton
M, Powell JD. Enhancement of tumor immuno-
therapy by deletion of the A2A adenosine receptor.
Cancer Immunol Immunother. 2012;61(6):917–926.
176. Hodi FS, O’Day SJ, McDermott DF, et al. Improved
survival with ipilimumab in patients with metastatic
melanoma. N Engl J Med. 2010;363(8):711–723.
177. Reck M, Rodriguez-Abreu D, Robinson AG,
et al. Pembrolizumab versus chemotherapy for
PD-L1-positive non-small-cell lung cancer. N Engl
J Med. 2016;375(19):1823–1833.
178. Robert C, Schachter J, Long GV, et al. Pembroli-
zumab versus ipilimumab in advanced melanoma.
N Engl J Med. 2015;372(26):2521–2532.
179. Motzer RJ, Escudier B, McDermott DF, et al.
Nivolumab versus everolimus in advanced renal-cell
carcinoma. N Engl J Med. 2015;373(19):1803–1813.
180. Rosenberg JE, Hoffman-Censits J, Powles T, et al.
Atezolizumab in patients with locally advanced
and metastatic urothelial carcinoma who have
progressed following treatment with platinum-based
chemotherapy: a single-arm, multicentre, phase 2
trial. Lancet. 2016;387(10031):1909–1920.
181. Ferris RL, Blumenschein G Jr, Fayette J,
et al. Nivolumab for recurrent squamous-cell
carcinoma of the head and neck. N Engl J Med.
2016;375(19):1856–1867.
182. Le DT, Uram JN, Wang H, et al. PD-1 Blockade
in tumors with mismatch-repair deficiency. N Engl
J Med. 2015;372(26):2509–2520.
183. Nghiem PT, Bhatia S, Lipson EJ, et al. PD-1 Block-
ade with pembrolizumab in advanced Merkel-cell
carcinoma. N Engl J Med. 2016;374(26):2542–2552.
184. Phan GQ, Yang JC, Sherry RM, et al. Cancer
regression and autoimmunity induced by cytotoxic T
lymphocyte-associated antigen 4 blockade in patients
with metastatic melanoma. Proc Natl Acad Sci USA.
2003;100(14):8372–8377.
185. Hodi FS, O’Day SJ, McDermott DF, et al. Improved
survival with ipilimumab in patients with metastatic
melanoma. N Engl J Med. 2010;363(8):711–723.
186. Robert C, Thomas L, Bondarenko I, et al. Ipilimumab
plus dacarbazine for previously untreated metastatic
melanoma. N Engl J Med. 2011;364(26):2517–2526.
187. Eggermont AM, Chiarion-Sileni V, Grob JJ,
et al. Prolonged survival in stage III melanoma
with ipilimumab adjuvant therapy. N Engl J Med.
2016;375(19):1845–1855.
188. Tarhini AA, Edington H, Butterfield LH, et al.
Immune monitoring of the circulation and the
tumor microenvironment in patients with region-
ally advanced melanoma receiving neoadjuvant
ipilimumab. PLoS ONE. 2014;9(2):e87705.
189. Brahmer JR, Tykodi SS, Chow LQ, et  al.
Safety and activity of anti-PD-L1 antibody in
patients with advanced cancer. N Engl J Med.
2012;366(26):2455–2465.
190. Topalian SL, Hodi FS, Brahmer JR, et al. Safety,
activity, and immune correlates of anti-PD-1 antibody
in cancer. N Engl J Med. 2012;366(26):2443–2454.
191. Hoos A, Eggermont AM, Janetzki S, et al. Improved
endpoints for cancer immunotherapy trials. J Natl
Cancer Inst. 2010;102(18):1388–1397.
192. Kohrt HE, Tumeh PC, Benson D, et al. Immuno-
dynamics: a cancer immunotherapy trials network
review of immune monitoring in immuno-oncology
clinical trials. J Immunother Cancer. 2016;4:15.
193. Brahmer JR, Tykodi SS, Chow LQ, et  al.
Safety and activity of anti-PD-L1 antibody in
patients with advanced cancer. N Engl J Med.
2012;366(26):2455–2465.
194. Topalian SL, Hodi FS, Brahmer JR, et al. Safety,
activity, and immune correlates of anti-PD-1 antibody
in cancer. N Engl J Med. 2012;366(26):2443–2454.
195. Garon EB, Rizvi NA, Hui R, et al. Pembrolizumab
for the treatment of non-small-cell lung cancer.
N Engl J Med. 2015;372(21):2018–2028.
196. Taube JM, Klein A, Brahmer JR, et al. Association
of PD-1, PD-1 ligands, and other features of the
tumor immune microenvironment with response to
anti-PD-1 therapy. Clin Cancer Res. 2014;20(19):
5064–5074.
197. Herbst RS, Soria JC, Kowanetz M, et al. Predic-
tive correlates of response to the anti-PD-L1
antibody MPDL3280A in cancer patients. Nature.
2014;515(7528):563–567.
198. Wolchok JD, Kluger H, Callahan MK, et al.
Nivolumab plus ipilimumab in advanced melanoma.
N Engl J Med. 2013;369(2):122–133.
199. Postow MA, Chesney J, Pavlick AC, et al. Nivolumab
and ipilimumab versus ipilimumab in untreated
melanoma. N Engl J Med. 2015;372(21):2006–
2017.
200. Gubin MM, Zhang X, Schuster H, et  al.
Checkpoint blockade cancer immunotherapy
targets tumour-specific mutant antigens. Nature.
2014;515(7528):577–581.
201. Rizvi NA, Hellmann MD, Snyder A, et al. Cancer
immunology. Mutational landscape determines
sensitivity to PD-1 blockade in non-small cell lung
cancer. Science. 2015;348(6230):124–128.
202. Snyder A, Makarov V, Merghoub T, et al. Genetic
basis for clinical response to CTLA-4 blockade in
melanoma. N Engl J Med. 2014;371(23):2189–2199.
203. McGranahan N, Furness AJ, Rosenthal R, et al.
Clonal neoantigens elicit T cell immunoreactivity
and sensitivity to immune checkpoint blockade.
Science. 2016;351(6280):1463–1469.
204. Tumeh PC, Harview CL, Yearley JH, et al. PD-1
blockade induces responses by inhibiting adaptive
immune resistance. Nature. 2014;515(7528):
568–571.
205. Cha E, Klinger M, Hou Y, et al. Improved survival
with T cell clonotype stability after anti-CTLA-4 treat-
ment in cancer patients. Sci Transl Med. 2014;6(238):
238ra270.
206. Huang AC, Postow MA, Orlowski RJ, et al. T-cell
invigoration to tumour burden ratio associated with
anti-PD-1 response. Nature. Apr 10 2017.
207. Peng D, Kryczek I, Nagarsheth N, et al. Epigen-
etic silencing of TH1-type chemokines shapes
tumour immunity and immunotherapy. Nature.
2015;527(7577):249–253.
208. Spranger S, Bao R, Gajewski TF. Melanoma-intrinsic
beta-catenin signalling prevents anti-tumour immu-
nity. Nature. 2015;523(7559):231–235.
209. Hugo W, Zaretsky JM, Sun L, et al. Genomic
and transcriptomic features of response to anti-
PD-1 therapy in metastatic melanoma. Cell.
2016;165(1):35–44.
210. Sivan A, Corrales L, Hubert N, et al. Commensal Bifi-
dobacterium promotes antitumor immunity and facil-
itates anti-PD-L1 efficacy. Science. 2015;350(6264):
1084–1089.
211. Vetizou M, Pitt JM, Daillere R, et al. Anticancer
immunotherapy by CTLA-4 blockade relies on the
gut microbiota. Science. 2015;350(6264):1079–1084.
212. Ebert PJ, Cheung J, Yang Y, et al. MAP kinase
inhibition promotes T cell and anti-tumor activity
in combination with PD-L1 checkpoint blockade.
Immunity. 2016;44(3):609–621.
213. Muller P, Kreuzaler M, Khan T, et al. Trastuzumab
emtansine (T-DM1) renders HER2+ breast cancer
highly susceptible to CTLA-4/PD-1 blockade. Sci
Transl Med. 2015;7(315):315ra188.
214. De Henau O, Rausch M, Winkler D, et al. Over-
coming resistance to checkpoint blockade therapy
by targeting PI3Kgamma in myeloid cells. Nature.
2016;539(7629):443–447.
215. Wu R, Forget MA, Chacon J, et al. Adoptive
T-cell therapy using autologous tumor-infiltrating
lymphocytes for metastatic melanoma: current status
and future outlook. Cancer J. 2012;18(2):160–175.
216. Sadelain M, Riviere I, Brentjens R. Targeting tumours
with genetically enhanced T lymphocytes. Nat Rev
Cancer. 2003;3(1):35–45.
217. Thomas S, Hart DP, Xue SA, Cesco-Gaspere M,
Stauss HJ. T-cell receptor gene therapy for cancer:
the progress to date and future objectives. Expert
Opin Biol Ther. 2007;7(8):1207–1218.
218. Kebriaei P, Singh H, Huls MH, et al. Phase I trials
using Sleeping Beauty to generate CD19-specific
CAR T cells. J Clin Invest. 2016;126(9):3363–3376.
219. Singh H, Huls H, Kebriaei P, Cooper LJ. A new
approach to gene therapy using Sleeping Beauty to
genetically modify clinical-grade T cells to target
CD19. Immunol Rev. 2014;257(1):181–190.
220. Smith TT, Stephan SB, Moffett HF, et al. In situ
programming of leukaemia-specific T cells using
synthetic DNA nanocarriers. Nat Nanotechnol. Apr
17 2017.
221. Schutsky K, Song DG, Lynn R, et al. Rigorous
optimization and validation of potent RNA CAR
T cell therapy for the treatment of common epithelial
cancers expressing folate receptor. Oncotarget.
2015;6(30):28911–28928.
222. Sather BD, Romano Ibarra GS, Sommer K,
et al. Efficient modification of CCR5 in primary
human hematopoietic cells using a megaTAL
nuclease and AAV donor template. Sci Transl Med.
2015;7(307):307ra156.
223. Ren J, Liu X, Fang C, Jiang S, June CH, Zhao Y.
Multiplex genome editing to generate universal CAR
T cells resistant to PD1 inhibition. Clin Cancer Res.
Nov 04 2016.
224. Scholler J, Brady TL, Binder-Scholl G, et al. Decade-
long safety and function of retroviral-modified
chimeric antigen receptor T cells. Sci Transl Med.
2012;4(132):132ra153.
225. Han A, Glanville J, Hansmann L, Davis MM.
Linking T-cell receptor sequence to functional
phenotype at the single-cell level. Nat Biotechnol.
2014;32(7):684–692.
226. Schmitt TM, Ragnarsson GB, Greenberg PD. T
cell receptor gene therapy for cancer. Hum Gene
Ther. 2009;20(11):1240–1248.
227. Zhao Y, Bennett AD, Zheng Z, et al. High-affinity
TCRs generated by phage display provide CD4+
T cells with the ability to recognize and kill tumor
cell lines. J Immunol. 2007;179(9):5845–5854.
228. Varela-Rohena A, Molloy PE, Dunn SM, et al.
Control of HIV-1 immune escape by CD8 T cells
expressing enhanced T-cell receptor. Nat Med.
2008;14(12):1390–1395.
229. Amir AL, van der Steen DM, van Loenen MM,
et al. PRAME-specific Allo-HLA-restricted T
cells with potent antitumor reactivity useful for
therapeutic T-cell receptor gene transfer. Clin Cancer
Res. 2011;17(17):5615–5625.
230. Morgan RA, Dudley ME, Wunderlich JR, et al. Cancer
regression in patients after transfer of genetically
engineered lymphocytes. Science. 2006;314(5796):
126–129.

231. Robbins PF, Morgan RA, Feldman SA, et al. Tumor
regression in patients with metastatic synovial cell
sarcoma and melanoma using genetically engineered
lymphocytes reactive with NY-ESO-1. J Clin Oncol.
2011;29(7):917–924.
232. Robbins PF, Kassim SH, Tran TL, et al. A pilot
trial using lymphocytes genetically engineered with
an NY-ESO-1-reactive T-cell receptor: long-term
follow-up and correlates with response. Clin Cancer
Res. 2015;21(5):1019–1027.
233. Ding K, Wang XM, Fu R, Ruan EB, Liu H, Shao
ZH. PRAME gene expression in acute leukemia
and its clinical significance. Cancer Biol Med.
2012;9(1):73–76.
234. Goswami M, Hensel N, Smith BD, et al. Expres-
sion of putative targets of immunotherapy in acute
myeloid leukemia and healthy tissues. Leukemia.
2014;28(5):1167–1170.
235. Morgan RA, Chinnasamy N, Abate-Daga D, et al.
Cancer regression and neurological toxicity following
anti-MAGE-A3 TCR gene therapy. J Immunother.
2013;36(2):133–151.
236. Cameron BJ, Gerry AB, Dukes J, et al. Identifica-
tion of a Titin-derived HLA-A1-presented peptide
as a cross-reactive target for engineered MAGE
A3-directed T cells. Sci Transl Med. 2013;5(197):
197ra103.
237. Linette GP, Stadtmauer EA, Maus MV, et al.
Cardiovascular toxicity and titin cross-reactivity of
affinity-enhanced T cells in myeloma and melanoma.
Blood. 2013;122(6):863–871.
238. Brentjens RJ, Santos E, Nikhamin Y, et al.
Genetically targeted T cells eradicate systemic acute
lymphoblastic leukemia xenografts. Clin Cancer Res.
2007;13(18 Pt 1):5426–5435.
239. Long AH, Haso WM, Shern JF, et al. 4-1BB
costimulation ameliorates T cell exhaustion induced
by tonic signaling of chimeric antigen receptors. Nat
Med. 2015;21(6):581–590.
240. Maude SL, Frey N, Shaw PA, et al. Chimeric antigen
receptor T cells for sustained remissions in leukemia.
N Engl J Med. 2014;371(16):1507–1517.
241. Sadelain M, Brentjens R, Riviere I. The basic
principles of chimeric antigen receptor design.
Cancer Discov. 2013;3(4):388–398.
242. Pegram HJ, Park JH, Brentjens RJ. CD28z CARs
and armored CARs. Cancer J. 2014;20(2):127–
133.
243. Brentjens RJ, Curran KJ. Novel cellular therapies
for leukemia: CAR-modified T cells targeted to the
CD19 antigen. Hematology Am Soc Hematol Educ
Program. 2012;2012:143–151.
244. Kochenderfer JN, Wilson WH, Janik JE, et al.
Eradication of B-lineage cells and regression of
lymphoma in a patient treated with autologous
T cells genetically engineered to recognize CD19.
Blood. 2010;116(20):4099–4102.
245. Porter DL, Levine BL, Kalos M, Bagg A, June
CH. Chimeric antigen receptor-modified T cells
in chronic lymphoid leukemia. N Engl J Med.
2011;365(8):725–733.
246. Kalos M, Levine BL, Porter DL, et al. T cells
with chimeric antigen receptors have potent
antitumor effects and can establish memory in
patients with advanced leukemia. Sci Transl Med.
2011;3(95):95ra73.
247. Porter DL, Kalos M, Zheng Z, Levine B, June
C. Chimeric antigen receptor therapy for B-cell
malignancies. J Cancer. 2011;2:331–332.
248. Brentjens RJ, Riviere I, Park JH, et al. Safety and
persistence of adoptively transferred autologous
CD19-targeted T cells in patients with relapsed or
chemotherapy refractory B-cell leukemias. Blood.
2011;118(18):4817–4828.
249. Grupp SA, Kalos M, Barrett D, et al. Chimeric
antigen receptor-modified T cells for acute lymphoid
leukemia. N Engl J Med. 2013;368(16):1509–
1518.
250. Lee DW, Kochenderfer JN, Stetler-Stevenson M,
et al. T cells expressing CD19 chimeric antigen
receptors for acute lymphoblastic leukaemia in
children and young adults: a phase 1 dose-escalation
trial. Lancet. 2015;385(9967):517–528.
251. Davila ML, Sauter C, Brentjens R. CD19-targeted T
cells for hematologic malignancies: clinical experience
to date. Cancer J. 2015;21(6):470–474.
252. Ghoneim HE, Zamora AE, Thomas PG, Youngblood
BA. Cell-intrinsic barriers of T cell-based immuno-
therapy. Trends Mol Med. Nov 4 2016.
253. Sotillo E, Barrett DM, Black KL, et al. Convergence
of acquired mutations and alternative splicing of
CD19 enables resistance to CART-19 immuno-
therapy. Cancer Discov. 2015;5(12):1282–1295.
254. Jacoby E, Nguyen SM, Fountaine TJ, et al. CD19
CAR immune pressure induces B-precursor acute
lymphoblastic leukaemia lineage switch exposing
Cancer Immunology  •  CHAPTER 6 96.e5
inherent leukaemic plasticity. Nat Commun. 2016;7:
12320.
255. Bonifant CL, Jackson HJ, Brentjens RJ, Curran KJ.
Toxicity and management in CAR T-cell therapy.
Mol Ther Oncolytics. 2016;3:16011.
256. Lee DW, Gardner R, Porter DL, et al. Current con-
cepts in the diagnosis and management of cytokine
release syndrome. Blood. 2014;124(2):188–195.
257. Davila ML, Riviere I, Wang X, et al. Efficacy and
toxicity management of 19-28z CAR T cell therapy
in B cell acute lymphoblastic leukemia. Sci Transl
Med. 2014;6(224):224ra225.
258. Brudno JN, Kochenderfer JN. Toxicities of chimeric
antigen receptor T cells: recognition and manage-
ment. Blood. 2016;127(26):3321–3330.
259. Taraseviciute A, Kean L, Jensen M. Creation of
the first non-human primate model that faithfully
recapitulates chimeric antigen receptor (CAR) T
cell-mediated cytokine release syndrome (CRS) and
neurologic toxicity following B cell-directed CAR-T
cell therapy. paper presented at: Blood 2016.
260. Newick K, Moon E, Albelda SM. Chimeric antigen
receptor T-cell therapy for solid tumors. Mol Ther
Oncolytics. 2016;3:16006.
261. Pule MA, Savoldo B, Myers GD, et al. Virus-
specific T cells engineered to coexpress tumor-
specific receptors: persistence and antitumor
activity in individuals with neuroblastoma. Nat
Med. 2008;14(11):1264–1270.
262. Louis CU, Savoldo B, Dotti G, et al. Antitumor
activity and long-term fate of chimeric antigen
receptor-positive T cells in patients with neuro-
blastoma. Blood. 2011;118(23):6050–6056.
263. Savoldo B, Ramos CA, Liu E, et al. CD28 costimula-
tion improves expansion and persistence of chimeric
antigen receptor-modified T cells in lymphoma
patients. J Clin Invest. 2011;121(5):1822–1826.
264. Ahmed N, Brawley VS, Hegde M, et al. Human
epidermal growth factor receptor 2 (HER2)–specific
chimeric antigen receptor-modified T cells for the
immunotherapy of HER2-positive sarcoma. J Clin
Oncol. 2015;33(15):1688–1696.
265. Brown CE, Alizadeh D, Starr R, et al. Regression of
glioblastoma after chimeric antigen receptor T-cell
therapy. N Engl J Med. 2016;375(26):2561–2569.
266. Kantoff PW, Higano CS, Shore ND, et al. Sipuleucel-
T immunotherapy for castration-resistant prostate
cancer. N Engl J Med. 2010;363(5):411–422.
 Stem Cells, Cell Differentiation, and Cancer 7 
Piero Dalerba, Maximilian Diehn, Irving L. Weissman, and Michael F. Clarke

S UMMARY OF K EY P OI NT S
• Many tumors originate in organs and
tissues that undergo a continuous
process of cell turnover, which is
sustained by a minority population of
stem cells (e.g., the colon, breast,
lung, prostate, brain, and bone
marrow).
• Stem cells have four fundamental
properties: the ability to give rise to
new stem cells with intact and
unlimited growth potential
(self-renewal), the ability to give rise
to a progeny of specialized cells
(differentiation), the ability to migrate
into new tissue locations and
establish tissue growth (migration
and tissue repair), and the ability to
balance the previous three properties
according to a genetic program that
places constraints on their numeric
expansion (homeostatic control).
• In many tissues, stem cells are the
only long-lived cell population. This
observation suggests that early
transforming events, either genetic
mutations or epigenetic
modifications, are likely to
accumulate in stem cells.
• In addition to oncogenes that control
cell survival and proliferation, there is
a class of oncogenes that regulates
self-renewal. In some cancers, tumor
growth might be sustained by
progenitor cells, which naturally do
not self-renew but have aberrantly
acquired this ability during disease
progression, as a result of mutations
that either activate genes required
for self-renewal, or inactivate genes
that disable it.
• Experimental data suggest that in
many forms of human cancer (e.g.,
leukemias, brain gliomas, breast,
colon, head and neck, bladder, and
prostate carcinomas), only a specific,
phenotypically distinct, subset of
cancer cells is able to form tumors
when serially transplanted in mice.
• To result in cure, therapies must
eradicate self-renewing cancer cells
(cancer stem cells). The ability to
identify these cells should allow the
identification of new diagnostic
markers and therapeutic targets.
• Studies on the gene-expression
profile of cancer stem cell
populations helped identify tumor
subtypes with differential response
to antitumor drugs, providing first
evidence for the clinical usefulness
of cancer stem cell research,
especially in guiding treatment
decisions.

Many tumors originate from tissues that naturally undergo a continuous
process of cell turnover. In such tissues, cell maturation is arranged
according to a hierarchic system, in which a minority population of
stem cells is able to perpetuate itself through a process called self-renewal
while also giving rise to several stages of intermediate progenitors and,
eventually, to terminally differentiated cells (Fig. 7.1). As opposed to
stem cells, intermediate progenitors and terminally differentiated cells
have a limited expansion potential and are unable to self-renew.1,2
Because stem cells are frequently found in small numbers, they must
be isolated and tested prospectively to define their molecular and
biochemical properties. Among the stem cell populations that have
been best characterized are those that give rise to the lymphohema-
topoietic system, known as hematopoietic stem cells (HSCs), which
have been purified from both mice and humans.3–7 HSCs have
important applications in cancer therapy, especially in clinical settings
where bone marrow transplantation is used to regenerate the hema-
topoietic system after myeloablative treatments.6 The ability to isolate
HSCs with high degrees of purity enables the execution of tumor-free
autologous bone-marrow transplants in cancer patients.8,9
Understanding the biology of the normal tissues from which tumors
originate, and especially of their stem cell populations, can provide
important insights into cancer biology. Several aspects of stem cell
biology are relevant to cancer.10,11 First, many cancer cells share with
normal stem cells the capacity to self-renew, and emerging evidence
suggests that similar molecular pathways underpin this property in
the two cell types.12–17 Second, because stem cells are long-lived, often
the only long-lived cells of normal adult tissues, they also represent
the most likely target population in which transforming events, either
genetic mutations or epigenetic modifications, progressively accumulate,
at least in the initial phases of the disease.2 Third, increasing evidence
suggests that many tumor tissues, akin to normal ones, contain a
minority “cancer stem cell” population with extensive proliferative
potential, which drives tumor growth and metastasis, and which can
undergo differentiation, sustaining the formation of a heterogeneous
progeny of specialized cell types.18–21 Finally, many physiologic properties
of stem cells are mirrored in cancer cells and contribute to define
their behavior. For example, because stem cells are long-lived and
necessary to maintain tissue homeostasis over the lifetime of an organ-
ism, they are frequently endowed with high levels of enzymatic systems
that protect them from environmental hazards. These very same
enzymatic systems endow specific subsets of cancer cells with resistance
to antitumor agents and might contribute to treatment failure.
PROPERTIES OF NORMAL STEM CELLS
HSCs are the most studied and best understood type of adult stem
cells and serve as a model for stem cells in other tissues.22,23 Hema-
topoiesis is a tightly regulated process in which a small pool of HSCs
give rise to an increasingly diversified population of oligolineage
intermediates, which in turn form the full repertoire of mature elements
of the lymphohematopoietic system (e.g., erythrocytes, platelets,
granulocytes, macrophages, and lymphocytes; see Fig. 7.1).24,25 These

97
98 Part I: Science and Clinical Oncology

HSC Self-renewal Phenotype in humans:

CD34+ CD38- CD90+
Differentiation

MPPs
Phenotype in humans:
CD34+ CD38- CD90-

Common
myeloid
progenitor
CMP
Megakaryocyte
Common
erythroid Granulocyte lymphoid
progenitor macrophage progenitor
progenitor CLP
MEP GMP

Erythrocyte Granulocyte Monocyte Dendritic cell NK-cell T-cell B-cell

Megakaryocyte progenitors progenitors progenitors progenitors progenitors progenitors progenitors

Platelets Erythrocytes Macrophages NK cells B-cells

Granulocytes Dendritic cells T-cells

Figure 7.1  •  Developmental hierarchy of the hematopoietic system. All mature blood cell types arise from a small subset of immature and multipotent
cells, which contains two distinct cell types: the hematopoietic stem cells (HSCs) and the multipotent progenitors (MPPs). Both HSCs and MPPs are capable
of multilineage reconstitution of the hematopoietic tissues in lethally irradiated mice, but only the HSCs are capable of self-renewal for the lifetime of the
animals. In contrast, MPPs, although able to give rise to large numbers of mature blood cells, can do so for only a limited time, usually a few months. (Diagram
modified from Seita J, Weissman IL. Hematopoietic stem cell: self-renewal versus differentiation. Wiley Interdiscip Rev Syst Biol Med. 2010;2:640–653.)

mature cell types perform a diverse set of functions, ensuring tissue
oxygenation, blood coagulation, and immunity. HSCs have four
fundamental properties: self-renewal, differentiation, migration, and
homeostatic control. First, HSCs need to self-renew to maintain the
stem cell pool. Self-renewal is not synonymous with proliferation.
Self-renewal is a cell division in which one or both of the daughter
cells remain undifferentiated and maintain the identity of a stem cell,
endowed with unlimited growth potential. Second, HSCs must undergo
differentiation: they must generate a progeny of cells that expand and
progressively specialize in order to replenish the various populations
of mature cells that are damaged, aged, or otherwise lost. Third, HSCs
are able to enter the circulation and migrate from one blood-forming
site to another (e.g., from the marrow of one bone to that of another)
to replace HSCs that have been lost and maintain a constant output
of blood cells.26 Finally, HSCs must balance self-renewal, differentiation,
migration, and tissue repair according to a strict genetic program,
which ensures homeostatic control of their numbers and which regulates
their expansion in response to various types of stress, such as bleeding
or infection.27–29
In mice, HSCs represent less than 0.05% of bone marrow cells and
are admixed with a heterogeneous pool of other immature progeni-
tors. This pool includes a specific subset of immature multipotent
progenitors (MPPs), which are able to differentiate into the full variety
of hematopoietic lineages but lack self-renewal capacity.30 These popula-
tions form a hierarchy in which HSCs give rise to MPPs, which in
turn give rise to oligolineage progenitors and the various mature cell
types of the blood and lymphoid tissues (see Fig. 7.1).6,25 As HSCs
mature to become MPPs, they increase their proliferation rate but lose
the ability to self-renew. Only HSCs can reconstitute hematopoiesis
in the long-term, for the lifetime of the animal, whereas MPPs can
reconstitute hematopoiesis for a short period of time, usually less than
16 weeks.31
A similar hierarchy is mirrored also in human blood tissues, wherein
HSCs and MPPs are characterized by a CD34+CD38− phenotype and
by the lack of mature blood cell lineage markers (Lin−), but can be
separated based on the differential expression of CD90 in HSCs (see
Fig. 7.1).32 More generally, the hierarchic organization of hematopoietic
tissues can be successfully applied to model the cellular composition
and developmental dynamics of many adult mammalian tissues, such
as the brain, mammary gland, skin, and intestinal epithelium.33–37
GENETIC REGULATION OF SELF-RENEWAL
The long-term survival of a tissue, either normal or neoplastic, is
dependent on its capacity to self-renew, whereas its overall size is
determined by the balance between the rates of cell proliferation and
cell death (or removal) across its various components.38 In normal
tissues, stem cell numbers are under tight genetic regulation, resulting
in the long-term maintenance of a constant tissue size.27–29 In contrast,
tumor tissues have escaped this homeostatic regulation. Within a
tumor, the number of cells with the ability to self-renew is constantly
expanding, resulting in continuous tissue growth. It is not surprising
therefore that many known oncogenes are able to expand stem cell
numbers, either by protecting them from apoptosis or by inhibiting
their differentiation. For example, enforced expression of Bcl2 results
in an expansion of HSCs,39 whereas enforced expression of c-myb and

c-myc prevents hematopoietic cell differentiation along the erythroid
lineage.40,41
Because the size of both normal and cancer tissues is dependent
on the number of cells able to self-renew, it is also not surprising that
a specific subset of oncogenes and/or tumor suppressor genes might
directly activate and/or disable self-renewal pathways.10,42 Indeed, the
basic molecular machinery that ensures the unlimited growth capacity
of most cancer cells (e.g., the telomerase enzymatic complex) is
fundamental to ensure self-renewal of most normal stem cells.12–14
The capacity to modulate self-renewal, however, is not a necessary
attribute of all oncogenes and/or tumor suppressor genes: enforced
expression of Bcl2, for instance, although able to expand the number
of self-renewing HSCs, is unable per se to endow self-renewal properties
on more mature cell types, such as MPPs.39
Among cancer genes with direct control over self-renewal func-
tions, the best example is probably the Bmi1 oncogene, which can
cooperate with c-myc to induce B-cell lymphomas.43 Bmi1 is a member
of the Polycomb repressor complex 1 (PRC1), a multiprotein tran-
scriptional repression complex involved in the epigenetic regulation
of developmental and differentiation processes. In the hematopoietic
system, Bmi1 expression is highest in HSCs and gradually decreases
as they differentiate into MPPs and oligolineage progenitors.44 In
mice genetically deficient for Bmi1 (Bmi1−/−) the number of HSCs
is markedly reduced at birth, and their transplantation in lethally
irradiated animals is able to reconstitute hematopoiesis, but only in a
transient and self-limiting fashion, indicating a cell autonomous defect
of self-renewal.15 Lack of Bmi1 expression causes similar phenotypes
across many types of adult stem cells, including neural and mammary
stem cells,45,46 and can be phenocopied by the overexpression of PRC1
inhibitors, such as the histone deubiquitinase Usp16.47 Important to
note, lack of Bmi1 expression is also associated with upregulation of
tumor suppressors, such as the cyclin-dependent kinase inhibitors
encoded by the alternative transcripts of the Cdkn2a locus (i.e., the
p16Ink4a and p19Arf proteins), and with overexpression of transcription
factors that promote differentiation, such as selected members of the
Hox family.15 This suggests that, in stem cells, the function of Bmi1
is to prevent the expression of a cascade of genes whose coordinated
activity can lead to loss of self-renewal capacity. Remarkably, in a
mouse model of leukemia initiated in Bmi1−/− mice, leukemic cells
are initially able to expand and cause the death of their primary hosts,
but are unable to sustain long-term growth when transplanted into
secondary syngeneic animals, where they eventually arrest, differentiate,
and undergo apoptosis.16 Infection of Bmi1−/− leukemic cells with a
retrovirus encoding for Bmi1 completely rescues this defect, allowing
leukemic cells to grow indefinitely and be serially passaged in mice.16
Similar observations have also been made in human breast cancer,
where downregulation of BMI1 by means of enforced expression of
microRNA-200c associates with reduced expansion and impaired
tumorigenicity of cancer cells.17 Once more, this defect can be rescued
by enforced expression of a modified version of the BMI1 mRNA,
insensitive to the inhibitory action of microRNA-200c.17 Interesting to
note, human BMI1 appears to induce telomerase activity.48 Again, in
many human model systems, cancer cells lacking telomerase, although
capable of substantial short-term proliferation and numeric expansion,
progressively exhaust their growth capacity and irreversibly arrest.14
These studies reinforce the concept that in order to endow cancer cells
with unlimited growth potential, a property frequently referred to as
“immortality,”38 constitutive activation of proliferation pathways is not
sufficient. It is also necessary to ensure the activation of self-renewal
pathways, either directly (i.e., by activating genes that positively
regulate them) or indirectly (i.e., by inactivating genes that negatively
regulate them).
Many other signaling pathways implicated in oncogenesis are known
to play central roles in stem cell biology, tissue morphogenesis and
development. Classic examples among them are the Wnt, Notch, and
Sonic hedgehog (SHH) pathways, all of which appear to regulate self-
renewal functions in many normal tissues.
Stem Cells, Cell Differentiation, and Cancer  •  CHAPTER 7 99
The Wnt pathway was first implicated in a mouse model of breast
cancer, in which aberrant expression of Wnt1, caused by insertion of
the mouse mammary tumor virus (MMTV) close to the Wnt1 gene,
resulted in mammary tumors.49,50 Wnt1 belongs to a large family
of secreted proteins that bind to receptors of both the Frizzled and
low-density lipoprotein receptor–related protein (Lrp) families, result-
ing in activation of β-catenin.51 In the absence of Wnt stimulation,
β-catenin is degraded by the adenomatous polyposis coli (Apc), glycogen
synthase kinase-3β (Gsk3β), and Axin protein complex.52 Wnt/β-catenin
signaling plays a pivotal role in the self-renewal of many normal
stem cells.53 Activation of β-catenin signaling by Wnt proteins allows
expansion of stem/progenitor cells, both in vitro and in vivo, and
across different tissues, including the bone marrow, skin, mammary
gland, and small intestinal epithelium.54–57 Constitutive activation
of the β-catenin pathway is oncogenic58,59 and is almost invariably
observed in colon cancer, most frequently by inactivating mutations
of human APC.60 Current evidence suggests that β-catenin signaling is
key for cancer cells to maintain a stem/progenitor cell phenotype.20,61
In colon cancer cells, inhibition of the β-catenin pathway induces
expression of p21cip1/waf1, a cell-cycle inhibitor, followed by prolifera-
tion arrest and acquisition of a differentiated phenotype.61 Enforced
expression of c-myc, an oncogene whose transcription is activated by
β-catenin, inhibits p21cip1/waf1 expression and allows cancer cells to
continue proliferating in the absence of β-catenin signaling, revealing
yet another role for a classic oncogene, c-myc, in the regulation of cell
differentiation.61
The Notch family of receptors, first identified as regulators of wing
patterning in the Drosophila fruit fly, controls development and dif-
ferentiation in many tissues, and across many animal species.62 In
vitro stimulation of HSCs with selected Notch ligands (i.e., Jagged-1,
Delta) transiently increases the activity of stem/progenitor cells, both
in vitro and in vivo, suggesting that Notch activation promotes
expansion of either HSCs or MPPs.63,64 Constitutive activation of the
Notch pathway is endowed with powerful oncogenic effects in both
mouse and human cells. The mouse oncogene int-3 encodes a con-
stitutively active, truncated variant of the Notch-4 receptor.65 In humans,
aberrant activation of NOTCH1, either by point mutation or chro-
mosomal translocation, is a common occurrence in T-cell acute
lymphoblastic leukemia (T-ALL).66,67 Important to note, inhibition
of NOTCH-1 signaling can induce apoptosis in T-ALL cell lines, and
is now being explored as a promising therapeutic strategy.68
The SHH pathway provides yet another example of a pathway with
key roles in tissue development, stem cell homeostasis, and oncogenesis.
Like the Wnt factors, SHH is a secreted molecule and a powerful
morphogen.69 SHH acts as a ligand for receptors encoded by members
of the Patched gene family69 and activates signaling circuitries that
regulate self-renewal in selected types of stem cells, such as bladder
stem cells.70 Germline mutations in the SHH gene cause aberrations
of embryo development in Drosophila and holoprosencephaly in
humans.42 With regard to human cancer, germline mutations in
Patched-1 (PTCH1) cause Gorlin or basal cell nevus syndrome (BCNS),
whereas sporadic mutations in PTCH1 are observed in the majority
of sporadic basal cell carcinomas (BCCs) and a substantial fraction of
medulloblastomas.42 Most importantly, pharmacologic inhibition of the
SHH pathway has shown powerful antitumor activity in clinical trials,
against both human BCCs and medulloblastomas harboring PTCH1
mutations, and is rapidly changing treatment guidelines for these
diseases.71–74
In conclusion, mouse genetic studies have contributed to the
discovery of several gene families involved in either the positive or
negative regulation of self-renewal properties in normal stem cell
populations. Normal stem cells are characterized by the high expression
of genes required to enable self renewal, and by the low expression
of genes known to disable it. The observation that, in cancer cells,
transforming mutations often target the same gene families, either by
constitutively activating genes that promote self-renewal (Bmi1, Wnt,
Notch, SHH) or by inactivating genes that contribute to restrict it
100 Part I: Science and Clinical Oncology

(Cdkn2a), suggests that stem cells and cancer cells depend on a common
set of signaling pathways to control their numbers and stimulate their
growth. The therapeutic success obtained with SHH inhibitors
demonstrates how the study of stem cells and their self-renewal pathways
can lead to novel and powerful antitumor treatments.
TARGET CELLS FOR MALIGNANT
TRANSFORMATION
If oncogenic mutations often target signaling pathways that regulate
self-renewal, then are stem cells the targets of neoplastic transformation?
Several lines of evidence suggest that stem cells might be involved in
the initial phases of cancer development. First, the fact that multiple
and sequential mutations are necessary for a cell to become cancerous75,76
suggests that, in many cases, mutations might need, at first, to
accumulate in a stem cell. Because mature cells often have a limited
life span, it is unlikely that the full repertoire of mutations necessary
to achieve malignant transformation might occur during their relatively
short life. Second, many tumors arise in tissues that are known to
contain stem cell populations, intrinsically endowed with the ability
to self-renew. Because cancer cells must undergo self-renewal to achieve
unlimited growth, stem cells might be more easily poised to undergo
early malignant transformation steps as compared with more mature
cells, which naturally lack this fundamental property and would need
to aberrantly activate self-renewal pathways to become malignant.
However, even if the first set of initiating events is likely to target a
stem cell population, it is possible that, as part of disease progression,
the aberrant progeny of mutated stem cells might acquire additional
changes and eventually gain the capacity to self-renew. In this scenario,
the malignant clone would acquire a second population of self-renewing
cells, whose phenotypic identity might resemble that of a progenitor
rather than a stem cell (Fig. 7.2).2

Mature cells
Normal stem cell Progenitor cells

Loss of homeostatic controls due to:

“Early” mutations - inhibition of apoptosis
- increased proliferation
1-5 - escape from innate immune surveillance
- escape from adaptive immune surveillance
Aberrant and/or incomplete
differentiation
Pre-cancerous
lesion 1-5
1-2 1-5
1 1-5 1-5
1-5
1-4 1-5 1-5
1-3 1-5 1-5
Early stage
disease 1-5 1-5
1-5
(e.g., chronic phase
of CML)

“Late” mutations Aberrant acquisition of Blast transformation and

6-7 self-renewal capacity by accelerated disease
progenitor cells
disease progression
1-7
1-7
1-6 1-7 1-7
1-7
1-7 1-7
1-7 1-7 1-7
1-7 1-7
1-7

Figure 7.2  •  Target cells for neoplastic transformation. In many tissues, stem cells are the only cells capable of self-renewal, which confers them a long
life-span and unlimited expansion capacity. As a result of this unique property, stem cells are ideal targets for neoplastic transformation, because they can
accumulate several mutations over time and are naturally endowed with one of the hallmark features of cancer cells (i.e., immortality). On the other hand,
for other cell types to become the cells of origin of a malignant tumor, they must first acquire the ability to self-renew. Current evidence suggests that during
the natural history of human tumors such as chronic myelogenous leukemia (CML), both events can take place. During the initial stages of the disease, a first
set of “early” mutations (1–5) accumulates in the stem-cells, causing loss of normal homeostatic controls on tissue expansion. Subsequently, during the advanced
stages of the disease, a second set of “late” mutations (6–7) causes aberrant acquisition of self-renewal by more mature progenitor compartments of the
neoplastic clone, resulting in the onset of a second population of cancer cells with unlimited growth potential. (Diagram modified from Rossi DJ, Jamieson
CH, Weissman IL. Stem cells and the pathways to aging and cancer. Cell. 2008;132:681–696.)

Experimental evidence in support of a stem cell origin of cancer,
at least during early initiation phases, is provided by elegant experiments
in a mouse model of glioblastoma, caused by conditional deletion of
the Neurofibromatosis-1 (Nf1), Tp53, and Pten tumor suppressor genes.77
In this model, combined deletion of the three genes in brain tissues
leads to rapid development of high-grade gliomas with 100% penetrance.
This effect, however, can be observed only when the deletion is specifi-
cally targeted to cell compartments that include stem cell populations,
either by conditional knockout of the tumor suppressor genes in cells
expressing Nestin (a marker associated with neural stem or progenitor
cells), or by somatic ablation of the tumor suppressor genes in the
mouse brain’s subventricular zone (SVZ), where neural stem cells
reside, achieved by stereotactic injection of an adenovirus encoding
the Cre recombinase in mice genetically engineered to carry Nf1,
Tp53, and Pten alleles between loxP sites. Most remarkably, somatic
ablation of the same tumor suppressors in other areas of the brain,
such as the striatum, is unable to generate any tumor. Moreover,
tumors generated from adenovirus injection in the SVZ are able to
disseminate throughout the brain parenchyma, following natural
migration pathways of neural stem cells, and exhibiting phenotypic
hallmarks of multilineage differentiation along the astrocytic, neuronal,
and oligodendrocytic lineages. Conceptually similar observations have
also been made in mouse models of intestinal cancer.78
Although it is likely that a first set of early-transforming mutations
might accumulate in the stem cell compartment, it is also possible that
more mature, downstream progenitors that originated as the progeny
of mutated stem cells might acquire a second set of late mutations,
which might constitute the ultimate transforming event giving rise
to cancer.79 In this scenario, mutated stem cells might represent a
“reservoir” population of precancerous cells, and fully transformed
progenitors might sustain the growth of the full-blown neoplastic
mass (see Fig. 7.2).80,81 This concept is supported by studies on normal
hematopoietic development and hematologic malignancies, in which
the developmental hierarchy of the different cellular compartments is
best understood, and in which phenotypic differences between HSCs
and MPPs allow a careful dissection of their relative contributions.
For example, in mice, simultaneous deletion of the Trp53 gene and
the Cdkn2a locus, which encodes for the p16Ink4a and p19Arf tumor
suppressor proteins, leads to abnormal and selective acquisition of
self-renewal properties by MPPs, which remain capable of producing
the various differentiated cell types, but also acquire self-renewal and
become capable of supporting the production of blood tissues on serial
transplantation, in essence behaving as stem cells.82 This observation
indicates that genetic mutations commonly observed in human cancer
might be responsible for a progressive and pathologic “unleashing”
of self-renewal properties in cells that are usually not endowed
with them.
Similar observations have been made also for different forms of
human leukemia, such as acute myelogenous leukemia (AML) and
chronic myelogenous leukemia (CML). One of the most frequent
mutations in AML is the t(8;21) translocation, which results in the
expression of a chimeric AML1/ETO transcript.83 Marrow samples
from patients with t(8;21) AML treated at Hiroshima Hospital, when
collected at clinical remission, contained HSCs (CD34+CD90+
CD38−Lin−) that had up to 90% incidence of the chimeric AML1-ETO
transcript.84 When these mutated HSCs were analyzed in vitro for
their differentiation capacity, they gave rise to normal myeloerythroid
progenies, showing that the mutation, alone and by itself, was insuf-
ficient to confer a fully transformed phenotype. Remarkably, marrow
samples from the same patients, when collected at relapse, contained
high numbers of cells with an MPP phenotype (CD34+CD90−
CD38−Lin−) that gave rise to leukemic “blast” colonies in vitro.84 This
observation is common to many forms of AML85 and is associated
with the expansion of a pathologic population of MPPs that have
abnormally gained the capacity to self-renew.86 Taken together, these
observations suggest that, in human AML, a first set of mutations
(e.g., AML1/ETO) might initially accumulate in stem cells, and that
Stem Cells, Cell Differentiation, and Cancer  •  CHAPTER 7 101
subsequent mutations in either the stem cells or their progeny might
lead to overt leukemia.2 Indeed, recent studies indicate that, within
the same AML patient, cells that appear to behave as phenotypically
normal HSCs are often carriers of a subset of the somatic mutations
found in the leukemic blasts.80 Interesting to note, such shared muta-
tions often target genes encoding for epigenetic modifiers involved
in the regulation of DNA methylation and hydroxymethylation
(DNMT3A, TET2), whereas mutations restricted to leukemic blasts
often target classic oncogenes (FLT3, KRAS).81
A similar scenario is observed in patients with CML, wherein
leukemic cells are characterized by the t(9;22) BCR/ABL chromosomal
translocation. In the initial “chronic phase” of human CML, the
leukemic population undergoes a multilineage differentiation process,
similar to that sustained by normal HSCs.87,88 At this initial stage,
β-catenin signaling is active in HSCs but not in their descendant
progenitor cells. However, when CML progresses to “myeloid blast
crisis”, patients develop an AML-like disease. In this second stage,
the leukemic clone acquires a second, phenotypically distinct self-
renewing population, whose surface marker profile corresponds to
that of a granulocyte-macrophage progenitor cell (GMP) and which
is able to transplant the disease to immunodeficient mice.89 This
second population is characterized by de novo aberrant activation of
the β-catenin pathway, often secondary to a pathologic missplicing
of the mRNA encoding for the GSK3β inhibitory enzyme.90 Thus
disease progression in human CML appears to result from aberrant
activation of self-renewal pathways in a progenitor cell population or,
more likely, failure to shut down these pathways during differentiation
processes. Once again, these studies suggest that, in human CML, early
mutational events (e.g., BCR/ABL) accumulate in HSCs during the
chronic phase of the disease, whereas during blast crisis a second set
of mutations leads to the rise of a second self-renewing population,
aberrantly originated from a progenitor cell, not an HSC (see Fig. 7.2).
Most remarkably, a conceptually analogous picture is also emerging
with regard to different forms of human lymphoid malignancy.91 In
the case of chronic lymphocytic leukemia (CLL), for example, a subset
of the somatic mutations associated with the transformed B-cell clone
are frequently found in the CD34+CD19− (i.e., stem/progenitor)
fraction of circulating hematopoietic cells.92 This finding is theoretically
important because it introduces the possibility that therapies aimed
at eradicating B-cell populations (e.g., anti-CD20 monoclonal antibod-
ies, anti-CD19 bispecific antibodies, T cells engineered with anti-CD19
chimeric antigen receptors), although capable of inducing profound
clinical responses, might be unable to fully eradicate the preneoplastic
cells that give rise to such disorders. In this scenario, treated patients
might develop late recurrences, in which both the mutational repertoire
and phenotypic traits of the relapsed clone could share only a limited
overlap with that of the original disease, as commonly observed in
the case of follicular lymphomas (FLs) that “transform” into diffuse
large B-cell lymphomas (DLBCLs).93
Taken together, these observations support the notion that “early”
oncogenic mutations might accumulate in stem cells and that during
disease progression additional “late” mutations can accumulate in
their transformed progenies, leading to aberrant acquisition of self-
renewal in more differentiated cell types.
EVIDENCE FOR CANCER STEM CELLS
It has long been known that tumor tissues contain heterogeneous
populations of cancer cells, characterized by different phenotypes.94
This diversity can arise in part from accumulation of sequential and/
or divergent mutations caused by genetic instability, and in part by
the variable action of environmental factors (Fig. 7.3A). In addition,
however, a tumor can also be viewed as an aberrant organ, sustained in
its growth by a pathologic stem cell population, which originates from
genetic mutations and/or epigenetic modifications leading to increased
cell proliferation and tissue expansion. It is also possible that, in this
aberrant organ, the pathologic stem cell population could undergo
102 Part I: Science and Clinical Oncology

Genetic
mutation

on
ati
Genetic

nti
mutation

e
fer
Dif
Environmental Environmental
modulation modulation

A B

Figure 7.3  •  Two models of cancer cell heterogeneity. (A) In the traditional model, heterogeneity is explained by differences in the repertoire of genetic
mutations (magenta cells) and by the variable effects of microenvironmental factors (red cells). In this model, all cancer cells are assumed to have the intrinsic
ability to self-renew and form new tumors (circular arrows). (B) In the “cancer stem cell” model, cell heterogeneity is caused not only by genetic mutations
and environmental factors, but also by differentiation. In this case, only a subset of cancer cells, the cancer stem cells (CSCs), are capable of self-renewal and
the formation of new tumors (orange cells). As observed for normal stem cells in healthy tissues, CSCs can give rise to more CSCs with the capacity to self-renew,
as well as differentiated cancer cells that lack this ability (various shades of green cells). CSCs can also acquire additional mutations (magenta cells) and are subject
to the action of environmental stimuli, both of which add to the tumor tissue’s diversity (red cells). Genetic mutations can sometimes perturb differentiation
processes, leading to aberrant differentiated phenotypes (blue cells). (Diagram modified from Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer,
and cancer stem cells. Nature 2001;414:105-11.)

an abnormal multilineage differentiation process that is reminiscent
(a “caricature”) of the one observed in normal tissues, giving rise to
multiple, phenotypically diverse populations of cancer cells, some of
which, although genetically identical, might differ in their epigenetic
status and their ability to self-renew (Fig. 7.3B).10,11 Several lines of
evidence indicate that, indeed, multilineage differentiation does occur
in cancer tissues and contributes substantially to cellular heterogeneity,95
in addition to genetic instability and environmental factors, which
undoubtedly contribute to the observed variability.96 Indeed, it is well
documented that the cell composition of cancer tissues frequently
mirrors that of their normal counterparts in terms of differentiated
cell types.95 A classic example is the frequent presence of abundant
mucus-producing gobletlike cells in human colon carcinomas (Fig.
7.4A–B). Perhaps one of the most striking examples of abnormal
differentiation in cancer is observed in some germ cell tumors, such as
teratomas and teratocarcinomas, in which mature organlike structures,
such as teeth, skin, and hair, can be found simultaneously admixed
within the tumor mass. Remarkably, within many of these tumors,
only a minority of cancer cells express immature cell markers such
as α-fetoprotein (Fig. 7.4D). The terminally differentiated cancer
cells that form the teeth and hair in the tumors are, most likely,
unable to proliferate and therefore contribute substantially to
the long term expansion and metastasis of the malignant tissues.
On the contrary, the minority population of immature cancer cells
expressing α-fetoprotein is likely to be the source of long-term tumor
growth, as well as represent the progenitor population from which the
other, more mature populations arise. Preliminary data on teratoma
formation by human embryonic stem cells (hESCs) lend support to
this working model.97
If a tumor is viewed as an abnormal organ, then the principles of
stem cell biology can be applied to model its physiology and clinical
behavior.10,11 Historically, this concept was first explored using clono-
genic assays, in which single-cell suspensions of cancer cells were tested
in limiting dilution for their capacity to form colonies, either in vitro
or in vivo.98 Indeed, in vivo clonogenic assays had been previously
used to provide the first experimental evidence of HSCs, because they
showed that only a minority of normal bone marrow cells, when
transplanted in irradiated mice, were able to engraft and give rise to
blood-forming colonies in the spleen.99 When applied to tumors,
clonogenic assays showed that only a minority of cancer cells, ranging
from 1 : 100 to 1 : 10,000, were able to form colonies.98 These results
were reproduced across many tumor types and suggested the existence
of minority populations of stemlike cancer cells, but researchers were
unable to formally prove this hypothesis because they could not rule
out stochastic effects in cell survival and engraftment.100
To prove that tumor tissues contain a specific subset of cancer cells
that is preferentially responsible for sustaining tumor growth, it is
necessary to isolate different populations of cancer cells in parallel
and compare their properties side by side, in a prospective assay—for
example, a transplantation assay in which cancer cells are tested for
their tumorigenic capacity.101 This type of experiment was first per-
formed in human AML, where it was shown that, in some cases, the
“tumor-initiating” capacity was enriched in a phenotypic subset of
the leukemic clone. In these studies, the subset of leukemic cells that
displayed the highest capacity for establishing human AML in
immunodeficient mice was the population characterized by the
CD34+CD38− phenotype.86,102,103 As previously discussed, the human
CD34+CD38− population corresponds to a very immature subset of
hematopoietic cells that contains HSCs (i.e., CD90+CD34+CD38−)
and MPPs (i.e., CD90−CD34+CD38−),32 both of which are involved
in the natural history of these leukemic disorders.84,86 Although
characterized by the highest frequency of tumorigenic cells, the
CD34+CD38− population is not the only self-renewing population
observed across all cases of AML.104 As observed for CML during
blast crisis, a second tumorigenic population, characterized by a more
mature, GMP-like phenotype (CD34+CD38+CD110−CD45RA+), can
be frequently detected also in AML. This second population is hierarchi-
cally related to the first (i.e., originates as the progeny of CD34+CD38−
cells, but not vice versa) and is characterized by a much lower frequency
of tumorigenic cells.86
Tumorigenic and nontumorigenic subsets of cancer cells have been
isolated also from epithelial solid tumors, such as breast and colon
carcinomas.18,19 As in the case of leukemias, tumorigenic cancer cells
could be distinguished from nontumorigenic ones based on their dif-
ferential expression of surface markers. In breast cancer, the tumorigenic
cell population was characterized as CD44+CD24−/low,18 whereas in colon
cancer it was characterized as EpCAMhighCD44+CD166+.19 In both
experimental systems, as few as 100 to 200 cells from the tumorigenic
 Stem Cells, Cell Differentiation, and Cancer  •  CHAPTER 7 103

A B

C D

E F

Figure 7.4  •  Clinical evidence in support of the “cancer stem cell” (CSC) model. When analyzed histologically, cancer tissues frequently recapitulate the
cell composition of their normal counterparts, including the presence of mature, differentiated cells. (A) Normal colon epithelia contain large numbers of
differentiated mucus-secreting goblet cells, which can be easily visualized by immunohistochemistry against the Mucin-2 (MUC2) antigen (brown staining).
(B) Colon cancer tissues frequently display a cellular composition similar to that of normal epithelia, characterized by large numbers of abnormal goblet cells.
Similar observations can been made in other forms of solid cancer, including teratocarcinomas. (C) A pretreatment abdominal computed tomography (CT)
scan of a patient with metastatic teratocarcinoma of the testis demonstrates a large retroperitoneal mass (arrowheads). (D) A biopsy specimen of the testicular
tumor analyzed with immunohistochemistry reveals expression of the immature marker α-fetoprotein by only rare cancer cells (brown cells, arrows). (E) A CT
scan after four cycles of platinum-based chemotherapy reveals persistence of a large retroperitoneal mass (arrowhead). (F) A biopsy of the residual mass reveals
only mature teratoma tissues and no cells expressing α-fetoprotein. This patient remained disease-free for more than 10 years, suggesting that, in some patients,
therapies that selectively eliminate a minority CSC population could be curative.

population were able to consistently form tumors when injected in
immunodeficient mice, whereas as many as 104 cells from other cell
populations within the same tumors repeatedly failed to do so. In both
cases, cancer tissues originating from the tumorigenic subset contained
the full repertoire of cell populations observed in the parent tissues,
including the tumorigenic subset itself and other nontumorigenic
populations. Important to note, these cancer tissues could be serially
passaged in mice, each time with similar results. Lineage-tracing
experiments performed in colon cancer showed that monoclonal
cancer tissues, originated by injection of a single tumorigenic cancer
cell bearing the EpCAMhighCD44+ phenotype and infected with a
reporter lentivirus encoding for the enhanced green fluorescence protein
(EGFP), reproduced the full cellular diversity of the parent tissues,
including the presence of EGFP+EpCAMlowCD44− nontumorigenic
cancer cells, and a variety of multiple EGFP+ differentiated cell types,
including MUC2+ goblet cells.95 These experiments provide formal
proof that (1) nontumorigenic populations originate as the progeny of
tumorigenic ones and (2) tumorigenic cells can undergo multilineage
differentiation processes, akin to those that allow normal stem cells to
sustain the generation of complex and diverse tissues. Conceptually
similar results have also been obtained from lineage-tracing experiments
in transgenic mouse cancer models105,106 and from single-cell RNA-
sequencing experiments in human primary oligodendrogliomas.107
Taken together, these data indicate the presence of a cellular
hierarchy within many cancer tissues, in which only a fraction of the
cells have the ability to engraft in mice. These cells can expand
indefinitely and differentiate into multiple lineages of specialized cell
types, thus showing two hallmark properties of stem cells: self-renewal
and differentiation. Based on the fulfillment of these two properties,
the tumorigenic subset of cancer cells is often called the “cancer stem
104 Part I: Science and Clinical Oncology
Box 7.1. ALTERNATIVE MODELS OF CANCER CELL HETEROGENEITY
An alternative explanation for the fact that different subsets of cancer
cells display different tumorigenic capacity when transplanted in
immunodeficient mice could be that all cancer cells are actually
tumorigenic, but only a specific subset, with a specific phenotype, is
able to engraft in the mouse. The ability of purified cell populations to
engraft in immunodeficient mice is dependent on many external
variables, such as the purification and transplantation procedures, the
degree of mouse immunodeficiency, the capacity of the cells to resist
nutrient deprivation and induce neoangiogenesis, and the degree of
cross talk between secreted growth factors and their receptors across
mice and humans.145 Despite this, multiple lines of evidence argue in
support of the existence of CSCs across many forms of cancer. For
example, lineage-tracing experiments have shown that human tumors
engrafted in mice do recapitulate the cell composition and phenotypic
diversity of the primary tumors from which they have been derived,
and contain both tumorigenic and nontumorigenic populations.95 This
finding indicates that the mouse environment is compatible with
multilineage differentiation processes and with survival of human
nontumorigenic cancer cells. Most importantly, identical results can be
reproduced using mouse-in-mouse tumors, with regard to in vivo
lineage tracing experiments105,106,146 and the existence of both
tumorigenic and nontumorigenic cancer cell populations.20,147 In the
cell” subset. It is important to remember, however, that this is an
operational definition, introduced to identify a subgroup of cancer
cells characterized by unique functional properties, but which, as
observed in leukemia, might not always necessarily correspond to the
stem cell population of their respective normal tissues. In colon
carcinomas, for instance, CSCs display a surface marker phenotype
(EpCAMhighCD44+CD166+) that is characteristic of cells found at the
bottom of normal colon crypts, and that defines a cell compartment
probably encompassing both stem and MPP subsets.95,108–110 Because
of our limited understanding of differentiation hierarchies in both
normal breast and normal colon epithelia, it is currently impossible
to dissect with precision whether in breast and colon cancer CSC
activity is found in stem cells, in downstream MPPs, or in both, and
this remains the subject of active investigations.
CSCs have also been identified in many other human tumors,
including glioblastomas and several types of epithelial cancer, such as
head and neck, pancreas, prostate, and bladder carcinomas.111–115 CD44
can be used for the isolation of CSCs across many epithelial carcinomas.
This is in agreement with the notion that CD44 is often expressed
heterogeneously within normal epithelia, usually in a gradient, with
the highest levels being observed at the most basal layers, where normal
stem or progenitor cells are usually thought to reside. Important to
note, histologic analysis of well-differentiated head-and-neck carcinomas
showed that, in these tumors, CD44 expression is often inversely
correlated to expression of mature cell markers, such as involucrin
(IVL), and positively associated with expression of BMI1, a key element
of the self-renewal machinery in many types of stem cells.112 Box 7.1
discusses alternative models for cancer cell heterogeneity.
CLINICAL IMPLICATIONS OF CANCER
STEM CELLS
The existence of CSCs has profound implications for clinical oncology.
One of them is concerned with the prediction of patient outcomes
at the time of diagnosis. If tumor tissues vary in CSC content, then
tumors containing a higher fraction of CSCs might be characterized
by a more aggressive biology and worse clinical outcomes. Indeed, a
number of studies have shown that tumors whose bulk gene expression
profiles are similar to those of purified CSCs are associated with
case of mouse tumors, transplantation experiments are performed into
syngeneic hosts, thus ruling out confounding effects from cross-species
barriers, either biologic or immunologic; lineage-tracing experiments
are performed in genetically engineered mice, in which primary tumors
can be studied in situ, in the original location where they arise, thus
ruling out transplantation artifacts. Among the advantages of
lineage-tracing experiments performed in transgenic mouse models is
the possibility to perform intravital imaging on primary tumors and
follow the clonal growth dynamics of individual cancer cells in a
time-lapse fashion. This is usually achieved by performing experiments
in genetically engineered mice that enable the “tagging” of individual
cancer cells with distinct fluorescent reporters. The results emerging
from this generation of in vivo imaging experiments show that, within
the same tumor, some cells are able to grow indefinitely, whereas
others appear to gradually exhaust their proliferation capacity.146
Finally, the differences observed among cancer cells in the expression
of lineage-restricted differentiation antigens do not appear to be
caused by clonal differences in their repertoire of genetic mutations, as
demonstrated by single-cell RNA-sequencing experiments in human
primary oligodendrogliomas.107 Taken together, these findings support
the idea that, as predicted by CSC models, tumors can retain elements
of the hierarchic organization of their parent tissues.
poor outcomes, likely reflecting a higher CSC content and a more
immature differentiation state. This was first shown for breast cancer,
using a 186-gene signature called the “invasiveness gene signature”
(IGS), obtained by comparing the gene expression profile of breast
CSCs with that of normal breast epithelial cells.116 The IGS was used
to stratify breast cancer patients with early-stage disease in different
groups, based on the similarity of their whole tumor’s gene expression
profile with the IGS. Remarkably, high similarity to the IGS was
associated with reduced overall and metastasis-free survival. Conceptu-
ally similar findings have also been observed across a variety of other
cancer types, including leukemia, head and neck, bladder, and colon
cancer.95,104,117–120
More recently, a new generation of studies explored whether the
gene-expression profile of CSCs can be used to guide the discovery of
clinically actionable biomarkers (e.g., markers able to predict tumor
response to chemotherapy and guide treatment decisions). In colon
cancer, for example, a bioinformatics analysis designed to discover
biomarkers of colon epithelial differentiation (i.e., markers whose
expression was inversely related to that of CSC markers, such as
CD166) led to the identification of a novel subtype of clinically
aggressive, poorly differentiated intestinal malignancies, characterized
by the lack of expression of the caudal-type homeobox transcription
factor 2 (CDX2), a master transcription factor that is necessary for
the correct multilineage differentiation of colon epithelial cells.121
Human CDX2neg colon tumors are associated with an increased risk
of relapse and reduced survival rates, irrespective of their clinical
stage or pathologic grade. Most importantly, however, patients with
CDX2neg colon tumors also appear to derive substantial benefit from
early treatment with adjuvant chemotherapy, in both stage II and stage
III disease.121 This observation is very important because, although
colon cancer patients with stage III disease are routinely treated with
adjuvant chemotherapy, patients with stage II disease are usually not,
as most of them are cured by surgery alone, and clinicians do not
have a reliable way to identify the small patient subgroup in which
the hazards of chemotherapy can be offset by a reduction in the risk
of relapse. The identification of CDX2 as a predictive biomarker
of benefit from adjuvant chemotherapy therefore has the potential
to change the treatment algorithms for stage II colon cancer, and
represents the first example of a finding related to CSC biology that

can be translated into clinical practice. A similar set of findings has also
been made in the case of AML, wherein the gene-expression profile
of leukemic cells with CSC properties was used to generate a 17-gene
“leukemia stem cell” (LSC17) signature that can be used to identify
AML patients who are unlikely to benefit from standard treatments,
such as induction chemotherapy followed by allogeneic hematopoietic
stem cell transplantation (HSCT), and who should be prioritized
for enrollment in clinical studies aimed at testing new therapeutic
approaches.122 Taken together, these studies highlight the prognostic and
predictive significance of CSCs in the natural history of human tumors,
and underscore their relevance in the clinical management of cancer
patients.
The existence of CSCs has important implications also for the
clinical evaluation of antitumor therapies. Historically, most antitumor
agents have been developed based on their ability to reduce tumor
size. Tumor shrinkage is dependent on the elimination of large numbers
of cancer cells, but not necessarily of the CSCs, which can constitute
a minority of the cancer tissue. This implies that CSCs might be
spared even when tumors appear to be responding. Indeed, in diseases
such as metastatic lung or breast cancer, although standard treatments
frequently shrink tumors, tumors rapidly recur and only limited impact
on patient survival is usually observed.123,124 Because anticancer therapies
are often screened based on their ability to shrink tumors rapidly,
agents that selectively target CSCs could be overlooked. Indeed, in
the initial phases of the screen, CSC-specific agents might cause only
a modest reduction in tumor growth. However, if complete elimination
of CSCs could be achieved, these treatments could permanently
eradicate tumor tissues. Perhaps the best clinical evidence for this
concept comes from patients with teratocarcinoma. Platinum-based
chemotherapy is curative in the majority of these patients.125 Many
patients, however, are left with residual masses (see Fig. 7.4E), which
usually undergo surgical resection. In most cases, histologic analysis
of residual tumor tissues reveals that cancer cells expressing α-fetoprotein
have been eliminated, leaving only differentiated cancer cells in a
mature teratoma (see Fig. 7.4F). Patients with mature teratomas rarely
develop metastases and frequently achieve long-term cure, suggesting
that selective elimination of immature cancer cell populations can be
sufficient to permanently abolish tumor growth. Conceptually similar
observations have also been made in experimental mouse models of
glioblastoma.126
The lack of strong associations between tumor shrinkage and tumor
eradication led to the hypothesis that CSCs may be intrinsically resistant
to chemotherapy and radiotherapy. This hypothesis is supported by
the observation that normal adult stem cells, such as HSCs, are fre-
quently found in a G0 quiescent state, which reduces their sensitivity
to antiproliferative agents.127 If the same is true for CSCs, then these
cells may be significantly more resistant to cytotoxic agents than their
nontumorigenic progeny. In support of this concept, for instance, is
the observation that the antitumor drug cytosine arabinoside (ara-C),
although capable of killing the majority of AML blast cells, selectively
spares the CD34+CD38−CD123+ subset of leukemic CSCs.127 Similarly,
it has been shown that CSCs can be resistant to both chemotherapy
and radiotherapy in many types of solid tumors, and several mechanisms
mediating this resistance have been uncovered. For example, glioblas-
toma CSCs have been shown to be radioresistant compared with their
nontumorigenic counterparts.128 This resistance is mediated, in part,
by enhanced activation of DNA repair pathways and rapid repair of
radiation-induced DNA damage. Similar observations have been made
also for breast CSCs, in this case as the result of enhanced free radical
scavenging, which prevents radiation-induced DNA damage.129,130 In
some tumors, CSCs might also be able to selectively evade elimination
by cytotoxic T-cell effectors of the adaptive immune system. In head
and neck squamous cell carcinomas (HNSCCs), for example, CD44+
cancer cells, which are enriched in CSCs, can sometimes over-express
PD-L1, a costimulatory molecule able to dampen the activation of
cytotoxic T cells by engaging a cognate receptor (PD-1) on their
surface.131 Because inhibitors of the PD-1/PD-L1 pathway have emerged
Stem Cells, Cell Differentiation, and Cancer  •  CHAPTER 7 105
as powerful antitumor drugs, future studies will need to address to
what extent their efficacy is related to their capacity to enable T-cell
elimination of CSCs.132
Overall, preclinical studies have suggested that CSCs might hold
the key to understanding many aspects of cancer resistance to antitumor
therapies in human patients. However, to formally address this question,
CSCs must be enumerated before and after therapy, thus requiring
repeated in vivo tumor sampling. This kind of analysis can be performed
in the case of patients undergoing neoadjuvant treatment regimens,
because they receive chemotherapy and/or radiotherapy immediately
before surgery. Remarkably, neoadjuvant treatment of breast cancer
with standard chemotherapy has been shown to cause an increase in
the percentage content of CSCs in vivo.133 Given the variety of resistance
mechanisms displayed by CSCs across different tumor types and
individual patients, characterization of the dominant resistance
mechanisms in a tumor’s CSCs may become integral to the future
development of personalized therapies.
Therapies specifically designed to target CSCs are still in their
infancy but are making rapid progress. One promising avenue of
research is targeting stem cell pathways, such as the wnt, Notch, and
SHH pathways. The remarkable clinical results observed in BCCs
and medulloblastomas with inhibition of the SHH pathway support
the use of this approach.
Another promising target is CD47, a cell surface receptor that is
overexpressed by CSCs in many tumor types.134 In normal physiology,
CD47 mediates a “don’t eat me” signal that prevents normal cells,
such as healthy red blood cells, from being eliminated by macro-
phages.135 CD47 thus acts as an inhibitor of the normal physiologic
pathways that mediate the removal of aged and/or damaged cells by
macrophage scavenging, a process also known as “programmed cell
removal”.136,137 CD47 is upregulated by HSCs when they enter the
bloodstream to recirculate, thus protecting them from phagocytosis.138
Targeted inhibition of CD47 leads to eradication of established tumors
in many in vivo experimental models, including leukemias, lymphomas,
and various forms of solid cancer.134,139–141
These observations suggest that, as part of neoplastic transformation,
cancer cells commonly disable pathways that are meant to ensure
homeostatic control of cell numbers by means of active cell elimination.
These pathways include “suicide” or “programmed cell death” pathways
(i.e., elimination by apoptosis) and “programmed cell removal” pathways
(i.e., elimination by phagocytosis).136,137 Although several molecular
mechanisms are known to mediate the escape of cancer cells from
apoptosis (e.g., Bcl2 overexpression, p53 inactivation), only one is
currently known to inhibit programmed cell removal: overexpression
of CD47. Future investigations will shed new light on the molecular
regulation of CD47 and reveal other molecules involved in programmed
cell removal, such as those mediating positive “eat me” (i.e., opsoniza-
tion) signals.142,143 These investigations will provide new molecular
targets for therapeutic interventions.
In summary, a new generation of antitumor therapies specifically
targeting CSCs is currently under development and is about to enter
clinical experimentation. Ultimately, well-designed trials of CSC-
directed therapies will provide the final testing ground for the CSC
hypothesis and its promise to improve patient lives.
FUTURE IMPLICATIONS OF CANCER
STEM CELLS
The identification of CSCs across many tumor types is key to the
future development of clinical oncology, especially for the discovery
of new predictive biomarkers and the design of novel antitumor agents.
The study of CSCs, however, is technically challenging. CSCs often
represent a minority of the cancer cell populations and need to be
purified from primary tissues if their functional properties are to be
evaluated in the context of their original tissue microenvironment.
Future studies will need to devise new experimental platforms to
address this challenge, such as single-cell genomics technologies, able
106 Part I: Science and Clinical Oncology

to perform precise measurements on small biopsy samples and with
single-cell resolution.95
The ability to identify and quantify CSCs should improve our
ability to evaluate the curative potential of both traditional and
investigational therapies. Although cancer cell lines are useful experi-
mental tools, especially suited for the in vitro dissection of individual
biochemical pathways, they have often proven unreliable when used
to model the clinical efficacy of antitumor drugs.144 Human xenografts
established in immunodeficient mice recapitulate more accurately the
morphology and phenotypic diversity of the patients’ original tumors,
including their three-dimensional architecture and the presence of
both tumorigenic and nontumorigenic cell populations.19 Xenograft
models may help evaluate the differential effect of antitumor drugs
on different cancer cell populations. New antitumor agents could
therefore be tested for their ability to eliminate CSCs in vivo, across
multiple xenografts, and those with the greatest activity could be
selected for evaluation in clinical trials, hopefully paving the way to
a new generation of more effective cancer therapies.
ACKNOWLEDGMENTS
The authors have been supported by grants and scholarships from
the National Institutes of Health (NIH), the National Cancer Institute
(NCI), the Department of Defense (DoD), the California Institute
for Regenerative Medicine (CIRM), the Ludwig Institute for Cancer
Research, the Thomas and Stacey Siebel Foundation, the Damon
Runyon Cancer Research Foundation (Island Outreach Foundation),
the Schaefer Research Scholars Program of Columbia University, the
Breast Cancer Research Foundation (BCRF), the Adenoid Cystic
Carcinoma Research Foundation (ACCRF), the Edward Mallinckrodt
Jr. Foundation, the Sydney Kimmel Foundation for Cancer Research,
the Doris Duke Charitable Foundation, and the Ellison Medical
Foundation.
P.D., M.D., I.L.W and M.F.C. are listed as a co-inventors on
several patents and patent applications related to the contents of this
chapter. Such patents are owned by academic institutions (University
of Michigan, Stanford University, Columbia University) and some of
them have been licensed to various biotechnology companies that
develop anti-tumor treatments, including: OncoMed Pharmaceuticals
Inc., Quanticel Pharmaceuticals Inc. (now a fully owned subsidiary
of Celgene) and Forty Seven Inc. As a result of the licensing agreements
negotiated by the aforementioned academic institutions, the authors
have received royalties from and/or stock in the aforementioned
companies. M.D. received research support from Varian Medical
Systems. I.L.W. is a founder and member of the board of directors
of Forty Seven Inc.
The complete reference list is available online at
ExpertConsult.com.

KEY REFERENCES
1. Weissman IL. Stem cells: units of development,
units of regeneration, and units in evolution. Cell.
2000;100(1):157–168.
2. Weissman I. Stem cell research: paths to cancer thera-
pies and regenerative medicine. JAMA. 2005;294(11):
1359–1366.
3. Spangrude GJ, Heimfeld S, Weissman IL. Purifica-
tion and characterization of mouse hematopoietic
stem cells. Science. 1988;241(4861):58–62.
4. Baum CM, Weissman IL, Tsukamoto AS, Buckle
AM, Peault B. Isolation of a candidate human
hematopoietic stem-cell population. Proc Natl Acad
Sci USA. 1992;89(7):2804–2808.
6. Weissman IL, Shizuru JA. The origins of the
identification and isolation of hematopoietic stem
cells, and their capability to induce donor-specific
transplantation tolerance and treat autoimmune
diseases. Blood. 2008;112(9):3543–3553.
7. Chen JY, Miyanishi M, Wang SK, et al. Hoxb5
marks long-term haematopoietic stem cells and
reveals a homogenous perivascular niche. Nature.
2016;530(7589):223–227.
10. Reya T, Morrison SJ, Clarke MF, Weissman IL.
Stem cells, cancer, and cancer stem cells. Nature.
2001;414(6859):105–111.
11. Dalerba P, Cho RW, Clarke MF. Cancer stem cells:
models and concepts. Annu Rev Med. 2007;58:
267–284.
12. Morrison SJ, Prowse KR, Ho P, Weissman IL. Telom-
erase activity in hematopoietic cells is associated
with self-renewal potential. Immunity. 1996;5(3):
207–216.
14. Dalerba P, Guiducci C, Poliani PL, et al. Recon-
stitution of human telomerase reverse transcriptase
expression rescues colorectal carcinoma cells from
in vitro senescence: evidence against immortality
as a constitutive trait of tumor cells. Cancer Res.
2005;65(6):2321–2329.
15. Park IK, Qian D, Kiel M, et al. Bmi-1 is required for
maintenance of adult self-renewing haematopoietic
stem cells. Nature. 2003;423(6937):302–305.
16. Lessard J, Sauvageau G. Bmi-1 determines the
proliferative capacity of normal and leukaemic stem
cells. Nature. 2003;423(6937):255–260.
17. Shimono Y, Zabala M, Cho RW, et al. Downregula-
tion of miRNA-200c links breast cancer stem cells
with normal stem cells. Cell. 2009;138(3):592–603.
18. Al-Hajj M, Wicha MS, Benito-Hernandez A,
Morrison SJ, Clarke MF. Prospective identification
of tumorigenic breast cancer cells. Proc Natl Acad
Sci USA. 2003;100(7):3983–3988.
19. Dalerba P, Dylla SJ, Park IK, et al. Phenotypic charac-
terization of human colorectal cancer stem cells. Proc
Natl Acad Sci USA. 2007;104(24):10158–10163.
20. Malanchi I, Peinado H, Kassen D, et al. Cutaneous
cancer stem cell maintenance is dependent on beta-
catenin signalling. Nature. 2008;452(7187):650–
653.
26. Wright DE, Wagers AJ, Gulati AP, Johnson
FL, Weissman IL. Physiological migration of
hematopoietic stem and progenitor cells. Science.
2001;294(5548):1933–1936.
30. Morrison SJ, Wandycz AM, Hemmati HD, Wright
DE, Weissman IL. Identification of a lineage of
multipotent hematopoietic progenitors. Development.
1997;124(10):1929–1939.
32. Majeti R, Park CY, Weissman IL. Identification of a
hierarchy of multipotent hematopoietic progenitors
in human cord blood. Cell Stem Cell. 2007;1(6):
635–645.
33. Uchida N, Buck DW, He D, et al. Direct isolation
of human central nervous system stem cells. Proc
Natl Acad Sci USA. 2000;97(26):14720–14725.
54. Zeng YA, Nusse R. Wnt proteins are self-renewal
factors for mammary stem cells and promote their
long-term expansion in culture. Cell Stem Cell.
2010;6(6):568–577.
71. Rudin CM, Hann CL, Laterra J, et al. Treatment
of medulloblastoma with hedgehog pathway
inhibitor GDC-0449. N Engl J Med. 2009;361(12):
1173–1178.
73. Sekulic A, Migden MR, Oro AE, et al. Efficacy
and safety of vismodegib in advanced basal-cell
carcinoma. N Engl J Med. 2012;366(23):2171–2179.
77. Alcantara Llaguno S, Chen J, Kwon CH, et al.
Malignant astrocytomas originate from neural stem/
progenitor cells in a somatic tumor suppressor mouse
model. Cancer Cell. 2009;15(1):45–56.
81. Corces-Zimmerman MR, Hong WJ, Weissman IL,
Medeiros BC, Majeti R. Preleukemic mutations in
human acute myeloid leukemia affect epigenetic
regulators and persist in remission. Proc Natl Acad
Sci USA. 2014;111(7):2548–2553.
82. Akala OO, Park IK, Qian D, Pihalja M, Becker MW,
Clarke MF. Long-term haematopoietic reconstitution
by Trp53−/−p16Ink4a−/−p19Arf−/− multipotent
progenitors. Nature. 2008;453(7192):228–232.
84. Miyamoto T, Weissman IL, Akashi K. AML1/
ETO-expressing nonleukemic stem cells in acute
myelogenous leukemia with 8;21 chromosomal
translocation. Proc Natl Acad Sci USA. 2000;97(13):
7521–7526.
86. Goardon N, Marchi E, Atzberger A, et al. Coex-
istence of LMPP-like and GMP-like leukemia
stem cells in acute myeloid leukemia. Cancer Cell.
2011;19(1):138–152.
87. Fialkow PJ, Jacobson RJ, Papayannopoulou T.
Chronic myelocytic leukemia: clonal origin in a
stem cell common to the granulocyte, erythrocyte,
platelet and monocyte/macrophage. Am J Med.
1977;63(1):125–130.
89. Jamieson CH, Ailles LE, Dylla SJ, et al. Granulocyte-
macrophage progenitors as candidate leukemic stem
cells in blast-crisis CML. N Engl J Med. 2004;351(7):
657–667.
90. Abrahamsson AE, Geron I, Gotlib J, et al. Glycogen
synthase kinase 3beta missplicing contributes to
leukemia stem cell generation. Proc Natl Acad Sci
USA. 2009;106(10):3925–3929.
91. Kikushige Y, Ishikawa F, Miyamoto T, et al. Self-
renewing hematopoietic stem cell is the primary
target in pathogenesis of human chronic lymphocytic
leukemia. Cancer Cell. 2011;20(2):246–259.
92. Damm F, Mylonas E, Cosson A, et al. Acquired
initiating mutations in early hematopoietic cells
of CLL patients. Cancer Discov. 2014;4(9):1088–
1101.
95. Dalerba P, Kalisky T, Sahoo D, et al. Single-cell dissec-
tion of transcriptional heterogeneity in human colon
tumors. Nat Biotechnol. 2011;29(12):1120–1127.
97. Tang C, Lee AS, Volkmer JP, et al. An antibody
against SSEA-5 glycan on human pluripotent stem

cells enables removal of teratoma-forming cells. Nat
Biotechnol. 2011;29(9):829–834.
103. Bonnet D, Dick JE. Human acute myeloid leu-
kemia is organized as a hierarchy that originates
from a primitive hematopoietic cell. Nat Med.
1997;3(7):730–737.
105. Driessens G, Beck B, Caauwe A, Simons BD,
Blanpain C. Defining the mode of tumour growth
by clonal analysis. Nature. 2012;488(7412):527–
530.
106. Schepers AG, Snippert HJ, Stange DE, et al. Lineage
tracing reveals Lgr5+ stem cell activity in mouse
intestinal adenomas. Science. 2012;337(6095):730–
735.
107. Tirosh I, Venteicher AS, Hebert C, et al. Single-cell
RNA-seq supports a developmental hierarchy in
human oligodendroglioma. Nature. 2016;539(7628):
309–313.
112. Prince ME, Sivanandan R, Kaczorowski A, et al.
Identification of a subpopulation of cells with cancer
Stem Cells, Cell Differentiation, and Cancer  •  CHAPTER 7 107
stem cell properties in head and neck squamous cell
carcinoma. Proc Natl Acad Sci USA. 2007;104(3):
973–978.
116. Liu R, Wang X, Chen GY, et al. The prognostic role
of a gene signature from tumorigenic breast-cancer
cells. N Engl J Med. 2007;356(3):217–226.
121. Dalerba P, Sahoo D, Paik S, et al. CDX2 as a
Prognostic Biomarker in Stage II and Stage III
Colon Cancer. N Engl J Med. 2016;374(3):211–222.
122. Ng SW, Mitchell A, Kennedy JA, et al. A 17-gene
stemness score for rapid determination of risk in
acute leukaemia. Nature. 2016;540(7633):433–
437.
126. Chen J, Li Y, Yu TS, et al. A restricted cell population
propagates glioblastoma growth after chemotherapy.
Nature. 2012;488(7412):522–526.
128. Bao S, Wu Q, McLendon RE, et al. Glioma
stem cells promote radioresistance by preferential
activation of the DNA damage response. Nature.
2006;444(7120):756–760.
130. Diehn M, Cho RW, Lobo NA, et al. Association of
reactive oxygen species levels and radioresistance in
cancer stem cells. Nature. 2009;458(7239):780–783.
131. Lee Y, Shin JH, Longmire M, et al. CD44+ cells in
head and neck squamous cell carcinoma suppress
T-cell-mediated immunity by selective constitutive
and inducible expression of PD-L1. Clin Cancer Res.
2016;22(14):3571–3581.
134. Willingham SB, Volkmer JP, Gentles AJ, et al. The
CD47-signal regulatory protein alpha (SIRPa) inter-
action is a therapeutic target for human solid tumors.
Proc Natl Acad Sci USA. 2012;109(17):6662–6667.
138. Jaiswal S, Jamieson CH, Pang WW, et al. CD47
is upregulated on circulating hematopoietic stem
cells and leukemia cells to avoid phagocytosis. Cell.
2009;138(2):271–285.
146. Zomer A, Ellenbroek SI, Ritsma L, Beerling E,
Vrisekoop N, Van Rheenen J. Intravital imaging
of cancer stem cell plasticity in mammary tumors.
Stem Cells. 2013;31(3):602–606.

REFERENCES
1. Weissman IL. Stem cells: units of development,
units of regeneration, and units in evolution. Cell.
2000;100(1):157–168.
2. Weissman I. Stem cell research: paths to cancer thera-
pies and regenerative medicine. JAMA. 2005;294(11):
1359–1366.
3. Spangrude GJ, Heimfeld S, Weissman IL. Purifica-
tion and characterization of mouse hematopoietic
stem cells. Science. 1988;241(4861):58–62.
4. Baum CM, Weissman IL, Tsukamoto AS, Buckle
AM, Peault B. Isolation of a candidate human
hematopoietic stem-cell population. Proc Natl Acad
Sci USA. 1992;89(7):2804–2808.
5. Morrison SJ, Weissman IL. The long-term
repopulating subset of hematopoietic stem cells is
deterministic and isolatable by phenotype. Immunity.
1994;1(8):661–673.
6. Weissman IL, Shizuru JA. The origins of the
identification and isolation of hematopoietic stem
cells, and their capability to induce donor-specific
transplantation tolerance and treat autoimmune
diseases. Blood. 2008;112(9):3543–3553.
7. Chen JY, Miyanishi M, Wang SK, et al. Hoxb5
marks long-term haematopoietic stem cells and
reveals a homogenous perivascular niche. Nature.
2016;530(7589):223–227.
8. Negrin RS, Atkinson K, Leemhuis T, et al. Trans-
plantation of highly purified CD34+Thy-1+ hema-
topoietic stem cells in patients with metastatic breast
cancer. Biol Blood Marrow Transplant. 2000;6(3):
262–271.
9. Muller AM, Kohrt HE, Cha S, et al. Long-term
outcome of patients with metastatic breast cancer
treated with high-dose chemotherapy and transplan-
tation of purified autologous hematopoietic stem
cells. Biol Blood Marrow Transplant. 2012;18(1):
125–133.
10. Reya T, Morrison SJ, Clarke MF, Weissman IL.
Stem cells, cancer, and cancer stem cells. Nature.
2001;414(6859):105–111.
11. Dalerba P, Cho RW, Clarke MF. Cancer stem cells:
models and concepts. Annu Rev Med. 2007;58:
267–284.
12. Morrison SJ, Prowse KR, Ho P, Weissman IL.
Telomerase activity in hematopoietic cells is
associated with self-renewal potential. Immunity.
1996;5(3):207–216.
13. Allsopp RC, Morin GB, DePinho R, Harley CB,
Weissman IL. Telomerase is required to slow telo-
mere shortening and extend replicative lifespan of
HSCs during serial transplantation. Blood. 2003;
102(2):517–520.
14. Dalerba P, Guiducci C, Poliani PL, et al. Recon-
stitution of human telomerase reverse transcriptase
expression rescues colorectal carcinoma cells from
in vitro senescence: evidence against immortality
as a constitutive trait of tumor cells. Cancer Res.
2005;65(6):2321–2329.
15. Park IK, Qian D, Kiel M, et al. Bmi-1 is required for
maintenance of adult self-renewing haematopoietic
stem cells. Nature. 2003;423(6937):302–305.
16. Lessard J, Sauvageau G. Bmi-1 determines the
proliferative capacity of normal and leukaemic stem
cells. Nature. 2003;423(6937):255–260.
17. Shimono Y, Zabala M, Cho RW, et al. Downregula-
tion of miRNA-200c links breast cancer stem cells
with normal stem cells. Cell. 2009;138(3):592–603.
18. Al-Hajj M, Wicha MS, Benito-Hernandez A,
Morrison SJ, Clarke MF. Prospective identification
of tumorigenic breast cancer cells. Proc Natl Acad
Sci USA. 2003;100(7):3983–3988.
19. Dalerba P, Dylla SJ, Park IK, et al. Phenotypic charac-
terization of human colorectal cancer stem cells. Proc
Natl Acad Sci USA. 2007;104(24):10158–10163.
20. Malanchi I, Peinado H, Kassen D, et al. Cutaneous
cancer stem cell maintenance is dependent on beta-
catenin signalling. Nature. 2008;452(7187):650–653.
Stem Cells, Cell Differentiation, and Cancer  •  CHAPTER 7 107.e1
21. Liu H, Patel MR, Prescher JA, et al. Cancer stem
cells from human breast tumors are involved in
spontaneous metastases in orthotopic mouse models.
Proc Natl Acad Sci USA. 2010;107(42):18115–
18120.
22. Weissman IL. Translating stem and progenitor cell
biology to the clinic: barriers and opportunities.
Science. 2000;287(5457):1442–1446.
23. Terskikh AV, Easterday MC, Li L, et al. From
hematopoiesis to neuropoiesis: evidence of overlap-
ping genetic programs. Proc Natl Acad Sci USA.
2001;98(14):7934–7939.
24. Kondo M, Wagers AJ, Manz MG, et al. Biology of
hematopoietic stem cells and progenitors: implica-
tions for clinical application. Annu Rev Immunol.
2003;21:759–806.
25. Seita J, Weissman IL. Hematopoietic stem cell:
self-renewal versus differentiation. Wiley Interdiscip
Rev Syst Biol Med. 2010;2(6):640–653.
26. Wright DE, Wagers AJ, Gulati AP, Johnson
FL, Weissman IL. Physiological migration of
hematopoietic stem and progenitor cells. Science.
2001;294(5548):1933–1936.
27. Phillips RL, Reinhart AJ, Van Zant G. Genetic
control of murine hematopoietic stem cell pool
sizes and cycling kinetics. Proc Natl Acad Sci USA.
1992;89(23):11607–11611.
28. Muller-Sieburg CE, Cho RH, Sieburg HB,
Kupriyanov S, Riblet R. Genetic control of hema-
topoietic stem cell frequency in mice is mostly cell
autonomous. Blood. 2000;95(7):2446–2448.
29. Morrison SJ, Qian D, Jerabek L, et al. A genetic
determinant that specifically regulates the frequency
of hematopoietic stem cells. J Immunol. 2002;168(2):
635–642.
30. Morrison SJ, Wandycz AM, Hemmati HD, Wright
DE, Weissman IL. Identification of a lineage of
multipotent hematopoietic progenitors. Development.
1997;124(10):1929–1939.
31. Kiel MJ, Yilmaz OH, Iwashita T, Terhorst C,
Morrison SJ. SLAM family receptors distinguish
hematopoietic stem and progenitor cells and reveal
endothelial niches for stem cells. Cell. 2005;121(7):
1109–1121.
32. Majeti R, Park CY, Weissman IL. Identification
of a hierarchy of multipotent hematopoietic
progenitors in human cord blood. Cell Stem Cell.
2007;1(6):635–645.
33. Uchida N, Buck DW, He D, et al. Direct isolation
of human central nervous system stem cells. Proc
Natl Acad Sci USA. 2000;97(26):14720–14725.
34. Stingl J, Eirew P, Ricketson I, et al. Purification and
unique properties of mammary epithelial stem cells.
Nature. 2006;439(7079):993–997.
35. Shackleton M, Vaillant F, Simpson KJ, et al. Genera-
tion of a functional mammary gland from a single
stem cell. Nature. 2006;439(7072):84–88.
36. Yui S, Nakamura T, Sato T, et al. Functional engraft-
ment of colon epithelium expanded in vitro from a
single adult Lgr5(+) stem cell. Nat Med. 2012;18(4):
618–623.
37. Fuchs E. Skin stem cells: rising to the surface. J Cell
Biol. 2008;180(2):273–284.
38. Hanahan D, Weinberg RA. Hallmarks of cancer:
the next generation. Cell. 2011;144(5):646–674.
39. Domen J, Cheshier SH, Weissman IL. The role of
apoptosis in the regulation of hematopoietic stem
cells: Overexpression of Bcl-2 increases both their
number and repopulation potential. J Exp Med.
2000;191(2):253–264.
40. Prochownik EV, Kukowska J. Deregulated expression
of c-myc by murine erythroleukaemia cells prevents
differentiation. Nature. 1986;322(6082):848–850.
41. Clarke MF, Kukowska-Latallo JF, Westin E, Smith M,
Prochownik EV. Constitutive expression of a c-myb
cDNA blocks Friend murine erythroleukemia cell
differentiation. Mol Cell Biol. 1988;8(2):884–892.
107.e1
42. Taipale J, Beachy PA. The Hedgehog and Wnt signal-
ling pathways in cancer. Nature. 2001;411(6835):
349–354.
43. van Lohuizen M, Verbeek S, Scheijen B, Wientjens
E, van der Gulden H, Berns A. Identification of
cooperating oncogenes in E mu-myc transgenic mice
by provirus tagging. Cell. 1991;65(5):737–752.
44. Akashi K, He X, Chen J, et al. Transcriptional
accessibility for genes of multiple tissues and hema-
topoietic lineages is hierarchically controlled during
early hematopoiesis. Blood. 2003;101(2):383–389.
45. Molofsky AV, Pardal R, Iwashita T, Park IK, Clarke
MF, Morrison SJ. Bmi-1 dependence distinguishes
neural stem cell self-renewal from progenitor
proliferation. Nature. 2003;425(6961):962–967.
46. Pietersen AM, Evers B, Prasad AA, et al. Bmi1 regu-
lates stem cells and proliferation and differentiation
of committed cells in mammary epithelium. Curr
Biol. 2008;18(14):1094–1099.
47. Adorno M, Sikandar S, Mitra SS, et al. Usp16
contributes to somatic stem-cell defects in Down’s
syndrome. Nature. 2013;501(7467):380–384.
48. Dimri GP, Martinez JL, Jacobs JJ, et al. The Bmi-1
oncogene induces telomerase activity and immortal-
izes human mammary epithelial cells. Cancer Res.
2002;62(16):4736–4745.
49. Nusse R, Brown A, Papkoff J, et al. A new nomen-
clature for int-1 and related genes: the Wnt gene
family. Cell. 1991;64(2):231.
50. Tsukamoto AS, Grosschedl R, Guzman RC, Parslow
T, Varmus HE. Expression of the int-1 gene in
transgenic mice is associated with mammary gland
hyperplasia and adenocarcinomas in male and female
mice. Cell. 1988;55(4):619–625.
51. Cadigan KM, Nusse R. Wnt signaling: a common
theme in animal development. Genes Dev. 1997;11(24):
3286–3305.
52. Spink KE, Polakis P, Weis WI. Structural basis of
the Axin-adenomatous polyposis coli interaction.
EMBO J. 2000;19(10):2270–2279.
53. Reya T, Clevers H. Wnt signalling in stem cells and
cancer. Nature. 2005;434(7035):843–850.
54. Zeng YA, Nusse R. Wnt proteins are self-renewal
factors for mammary stem cells and promote their long-
term expansion in culture. Cell Stem Cell. 2010;6(6):
568–577.
55. Reya T, Duncan AW, Ailles L, et al. A role for Wnt
signalling in self-renewal of haematopoietic stem
cells. Nature. 2003;423(6938):409–414.
56. Korinek V, Barker N, Moerer P, et al. Deple-
tion of epithelial stem-cell compartments in the
small intestine of mice lacking Tcf-4. Nat Genet.
1998;19(4):379–383.
57. Kishimoto J, Burgeson RE, Morgan BA. Wnt
signaling maintains the hair-inducing activity of the
dermal papilla. Genes Dev. 2000;14(10):1181–1185.
58. Gat U, DasGupta R, Degenstein L, Fuchs E. De
Novo hair follicle morphogenesis and hair tumors
in mice expressing a truncated beta-catenin in skin.
Cell. 1998;95(5):605–614.
59. Chan EF, Gat U, McNiff JM, Fuchs E. A common
human skin tumour is caused by activating mutations
in beta-catenin. Nat Genet. 1999;21(4):410–413.
60. Segditsas S, Tomlinson I. Colorectal cancer and
genetic alterations in the Wnt pathway. Oncogene.
2006;25(57):7531–7537.
61. van de Wetering M, Sancho E, Verweij C, et al.
The beta-catenin/TCF-4 complex imposes a crypt
progenitor phenotype on colorectal cancer cells. Cell.
2002;111(2):241–250.
62. Artavanis-Tsakonas S, Rand MD, Lake RJ. Notch
signaling: cell fate control and signal integration in
development. Science. 1999;284(5415):770–776.
63. Varnum-Finney B, Xu L, Brashem-Stein C, et al.
Pluripotent, cytokine-dependent, hematopoietic
stem cells are immortalized by constitutive Notch1
signaling. Nat Med. 2000;6(11):1278–1281.
107.e2 Part I: Science and Clinical Oncology
64. Duncan AW, Rattis FM, DiMascio LN, et al.
Integration of Notch and Wnt signaling in hema-
topoietic stem cell maintenance. Nat Immunol.
2005;6(3):314–322.
65. Gallahan D, Callahan R. The mouse mammary
tumor associated gene INT3 is a unique member
of the NOTCH gene family (NOTCH4). Oncogene.
1997;14(16):1883–1890.
66. Ellisen LW, Bird J, West DC, et al. TAN-1, the
human homolog of the Drosophila notch gene,
is broken by chromosomal translocations in T
lymphoblastic neoplasms. Cell. 1991;66(4):649–661.
67. Weng AP, Ferrando AA, Lee W, et al. Activating muta-
tions of NOTCH1 in human T cell acute lympho-
blastic leukemia. Science. 2004;306(5694):269–271.
68. Samon JB, Castillo-Martin M, Hadler M, et al.
Preclinical analysis of the gamma-secretase inhibitor
PF-03084014 in combination with glucocorticoids
in T-cell acute lymphoblastic leukemia. Mol Cancer
Ther. 2012;11(7):1565–1575.
69. Lum L, Beachy PA. The Hedgehog response
network: sensors, switches, and routers. Science.
2004;304(5678):1755–1759.
70. Shin K, Lee J, Guo N, et al. Hedgehog/Wnt feedback
supports regenerative proliferation of epithelial stem
cells in bladder. Nature. 2011;472(7341):110–114.
71. Rudin CM, Hann CL, Laterra J, et al. Treat-
ment of medulloblastoma with hedgehog
pathway inhibitor GDC-0449. N Engl J Med.
2009;361(12):1173–1178.
72. Von Hoff DD, LoRusso PM, Rudin CM, et al. Inhi-
bition of the hedgehog pathway in advanced basal-cell
carcinoma. N Engl J Med. 2009;361(12):1164–1172.
73. Sekulic A, Migden MR, Oro AE, et al. Efficacy
and safety of vismodegib in advanced basal-cell
carcinoma. N Engl J Med. 2012;366(23):2171–2179.
74. Tang JY, Mackay-Wiggan JM, Aszterbaum M,
et al. Inhibiting the hedgehog pathway in patients
with the basal-cell nevus syndrome. N Engl J Med.
2012;366(23):2180–2188.
75. Knudson AG Jr, Strong LC, Anderson DE. Hered-
ity and cancer in man. Prog Med Genet. 1973;9:
113–158.
76. Fearon ER, Vogelstein B. A genetic model for
colorectal tumorigenesis. Cell. 1990;61(5):759–767.
77. Alcantara Llaguno S, Chen J, Kwon CH, et al.
Malignant astrocytomas originate from neural stem/
progenitor cells in a somatic tumor suppressor mouse
model. Cancer Cell. 2009;15(1):45–56.
78. Barker N, Ridgway RA, van Es JH, et al. Crypt
stem cells as the cells-of-origin of intestinal cancer.
Nature. 2009;457(7229):608–611.
79. Rossi DJ, Jamieson CH, Weissman IL. Stems
cells and the pathways to aging and cancer. Cell.
2008;132(4):681–696.
80. Jan M, Snyder TM, Corces-Zimmerman MR, et al.
Clonal evolution of preleukemic hematopoietic stem
cells precedes human acute myeloid leukemia. Sci
Transl Med. 2012;4(149):149ra118.
81. Corces-Zimmerman MR, Hong WJ, Weissman IL,
Medeiros BC, Majeti R. Preleukemic mutations in
human acute myeloid leukemia affect epigenetic
regulators and persist in remission. Proc Natl Acad
Sci USA. 2014;111(7):2548–2553.
82. Akala OO, Park IK, Qian D, Pihalja M, Becker MW,
Clarke MF. Long-term haematopoietic reconstitution
by Trp53−/−p16Ink4a−/−p19Arf−/− multipotent
progenitors. Nature. 2008;453(7192):228–232.
83. Peterson LF, Boyapati A, Ahn EY, et al. Acute
myeloid leukemia with the 8q22;21q22 transloca-
tion: secondary mutational events and alternative
t(8;21) transcripts. Blood. 2007;110(3):799–805.
84. Miyamoto T, Weissman IL, Akashi K. AML1/
ETO-expressing nonleukemic stem cells in acute
myelogenous leukemia with 8;21 chromosomal
translocation. Proc Natl Acad Sci USA. 2000;97(13):
7521–7526.
85. Blair A, Hogge DE, Ailles LE, Lansdorp PM,
Sutherland HJ. Lack of expression of Thy-1 (CD90)
on acute myeloid leukemia cells with long-term prolif-
erative ability in vitro and in vivo. Blood. 1997;89(9):
3104–3112.
86. Goardon N, Marchi E, Atzberger A, et al. Coex-
istence of LMPP-like and GMP-like leukemia
stem cells in acute myeloid leukemia. Cancer Cell.
2011;19(1):138–152.
87. Fialkow PJ, Jacobson RJ, Papayannopoulou T.
Chronic myelocytic leukemia: clonal origin in a
stem cell common to the granulocyte, erythrocyte,
platelet and monocyte/macrophage. Am J Med.
1977;63(1):125–130.
88. Takahashi N, Miura I, Saitoh K, Miura AB. Lineage
involvement of stem cells bearing the Philadelphia
chromosome in chronic myeloid leukemia in
the chronic phase as shown by a combination of
fluorescence-activated cell sorting and fluorescence in
situ hybridization. Blood. 1998;92(12):4758–4763.
89. Jamieson CH, Ailles LE, Dylla SJ, et al. Granulocyte-
macrophage progenitors as candidate leukemic stem
cells in blast-crisis CML. N Engl J Med. 2004;351(7):
657–667.
90. Abrahamsson AE, Geron I, Gotlib J, et al. Glycogen
synthase kinase 3beta missplicing contributes to
leukemia stem cell generation. Proc Natl Acad Sci
USA. 2009;106(10):3925–3929.
91. Kikushige Y, Ishikawa F, Miyamoto T, et al. Self-
renewing hematopoietic stem cell is the primary
target in pathogenesis of human chronic lymphocytic
leukemia. Cancer Cell. 2011;20(2):246–259.
92. Damm F, Mylonas E, Cosson A, et al. Acquired
initiating mutations in early hematopoietic cells of
CLL patients. Cancer Discov. 2014;4(9):1088–1101.
93. Pasqualucci L, Khiabanian H, Fangazio M, et al.
Genetics of follicular lymphoma transformation.
Cell Rep. 2014;6(1):130–140.
94. Heppner GH. Tumor heterogeneity. Cancer Res.
1984;44(6):2259–2265.
95. Dalerba P, Kalisky T, Sahoo D, et al. Single-cell dissec-
tion of transcriptional heterogeneity in human colon
tumors. Nat Biotechnol. 2011;29(12):1120–1127.
96. Gerlinger M, Rowan AJ, Horswell S, et al.
Intratumor heterogeneity and branched evolution
revealed by multiregion sequencing. N Engl J Med.
2012;366(10):883–892.
97. Tang C, Lee AS, Volkmer JP, et al. An antibody
against SSEA-5 glycan on human pluripotent stem
cells enables removal of teratoma-forming cells. Nat
Biotechnol. 2011;29(9):829–834.
98. Hamburger AW, Salmon SE. Primary bioassay of
human tumor stem cells. Science. 1977;197(4302):
461–463.
99. Till JE, Mc CE. A direct measurement of the radia-
tion sensitivity of normal mouse bone marrow cells.
Radiat Res. 1961;14:213–222.
100. Weisenthal LM, Lippman ME. Clonogenic and
nonclonogenic in vitro chemosensitivity assays.
Cancer Treat Rep. 1985;69(6):615–632.
101. Clarke MF. A self-renewal assay for cancer stem
cells. Cancer Chemother Pharmacol. 2005;56(suppl 1):
64–68.
102. Lapidot T, Sirard C, Vormoor J, et al. A cell initiating
human acute myeloid leukaemia after transplantation
into SCID mice. Nature. 1994;367(6464):645–648.
103. Bonnet D, Dick JE. Human acute myeloid leukemia
is organized as a hierarchy that originates from a
primitive hematopoietic cell. Nat Med. 1997;3(7):
730–737.
104. Eppert K, Takenaka K, Lechman ER, et al.
Stem cell gene expression programs influence
clinical outcome in human leukemia. Nat Med.
2011;17(9):1086–1093.
105. Driessens G, Beck B, Caauwe A, Simons BD,
Blanpain C. Defining the mode of tumour growth
by clonal analysis. Nature. 2012;488(7412):527–
530.
106. Schepers AG, Snippert HJ, Stange DE, et al. Lineage
tracing reveals Lgr5+ stem cell activity in mouse intes-
tinal adenomas. Science. 2012;337(6095):730–735.
107. Tirosh I, Venteicher AS, Hebert C, et al. Single-cell
RNA-seq supports a developmental hierarchy in
human oligodendroglioma. Nature. 2016;539(7628):
309–313.
108. Wielenga VJ, Smits R, Korinek V, et al. Expression of
CD44 in Apc and Tcf mutant mice implies regulation
by the WNT pathway. Am J Pathol. 1999;154(2):
515–523.
109. Jiao YF, Nakamura S, Sugai T, Yamada N, Habano
W. Serrated adenoma of the colorectum undergoes
a proliferation versus differentiation process: new
conceptual interpretation of morphogenesis. Oncol-
ogy. 2008;74(3–4):127–134.
110. Levin TG, Powell AE, Davies PS, et al. Charac-
terization of the intestinal cancer stem cell marker
CD166 in the human and mouse gastrointestinal
tract. Gastroenterology. 2010;139(6):2072–2082,
e2075.
111. Singh SK, Hawkins C, Clarke ID, et al. Identification
of human brain tumour initiating cells. Nature.
2004;432(7015):396–401.
112. Prince ME, Sivanandan R, Kaczorowski A, et al.
Identification of a subpopulation of cells with cancer
stem cell properties in head and neck squamous cell
carcinoma. Proc Natl Acad Sci USA. 2007;104(3):
973–978.
113. Chan KS, Espinosa I, Chao M, et al. Identification,
molecular characterization, clinical prognosis, and
therapeutic targeting of human bladder tumor-
initiating cells. Proc Natl Acad Sci USA. 2009;106(33):
14016–14021.
114. Li C, Heidt DG, Dalerba P, et al. Identifica-
tion of pancreatic cancer stem cells. Cancer Res.
2007;67(3):1030–1037.
115. Patrawala L, Calhoun T, Schneider-Broussard
R, et al. Highly purified CD44+ prostate cancer
cells from xenograft human tumors are enriched
in tumorigenic and metastatic progenitor cells.
Oncogene. 2006;25(12):1696–1708.
116. Liu R, Wang X, Chen GY, et al. The prognostic role
of a gene signature from tumorigenic breast-cancer
cells. N Engl J Med. 2007;356(3):217–226.
117. Gentles AJ, Plevritis SK, Majeti R, Alizadeh AA.
Association of a leukemic stem cell gene expression
signature with clinical outcomes in acute myeloid
leukemia. JAMA. 2010;304(24):2706–2715.
118. Joshua B, Kaplan MJ, Doweck I, et al. Frequency of
cells expressing CD44, a head and neck cancer stem
cell marker: correlation with tumor aggressiveness.
Head Neck. 2012;34(1):42–49.
119. Merlos-Suarez A, Barriga FM, Jung P, et al. The
intestinal stem cell signature identifies colorectal
cancer stem cells and predicts disease relapse. Cell
Stem Cell. 2011;8:511–524.
120. Volkmer JP, Sahoo D, Chin RK, et al. Three
differentiation states risk-stratify bladder cancer
into distinct subtypes. Proc Natl Acad Sci USA.
2012;109(6):2078–2083.
121. Dalerba P, Sahoo D, Paik S, et al. CDX2 as a
prognostic biomarker in stage II and stage III colon
cancer. N Engl J Med. 2016;374(3):211–222.
122. Ng SW, Mitchell A, Kennedy JA, et al. A 17-gene
stemness score for rapid determination of risk in
acute leukaemia. Nature. 2016;540(7633):433–437.
123. Maemondo M, Inoue A, Kobayashi K, et al. Gefitinib
or chemotherapy for non–small-cell lung cancer
with mutated EGFR. N Engl J Med. 2010;362(25):
2380–2388.
124. O’Shaughnessy J, Osborne C, Pippen JE, et al. Ini-
parib plus chemotherapy in metastatic triple-negative
breast cancer. N Engl J Med. 2011;364(3):205–214.
125. Williams SD, Birch R, Einhorn LH, Irwin L,
Greco FA, Loehrer PJ. Treatment of disseminated
germ-cell tumors with cisplatin, bleomycin, and
either vinblastine or etoposide. N Engl J Med.
1987;316(23):1435–1440.
126. Chen J, Li Y, Yu TS, et al. A restricted cell population
propagates glioblastoma growth after chemotherapy.
Nature. 2012;488(7412):522–526.

127. Guzman ML, Neering SJ, Upchurch D, et al. Nuclear
factor-kappaB is constitutively activated in primitive
human acute myelogenous leukemia cells. Blood.
2001;98(8):2301–2307.
128. Bao S, Wu Q, McLendon RE, et al. Glioma
stem cells promote radioresistance by preferential
activation of the DNA damage response. Nature.
2006;444(7120):756–760.
129. Phillips TM, McBride WH, Pajonk F. The response
of CD24(−/low)/CD44+ breast cancer-initiating
cells to radiation. J Natl Cancer Inst. 2006;98(24):
1777–1785.
130. Diehn M, Cho RW, Lobo NA, et al. Association of
reactive oxygen species levels and radioresistance in
cancer stem cells. Nature. 2009;458(7239):780–783.
131. Lee Y, Shin JH, Longmire M, et al. CD44+ cells in
head and neck squamous cell carcinoma suppress
T-cell-mediated immunity by selective constitutive
and inducible expression of PD-L1. Clin Cancer Res.
2016;22(14):3571–3581.
132. Topalian SL, Taube JM, Anders RA, Pardoll DM.
Mechanism-driven biomarkers to guide immune
checkpoint blockade in cancer therapy. Nat Rev
Cancer. 2016;16(5):275–287.
133. Li X, Lewis MT, Huang J, et al. Intrinsic resistance
of tumorigenic breast cancer cells to chemotherapy.
J Natl Cancer Inst. 2008;100(9):672–679.
Stem Cells, Cell Differentiation, and Cancer  •  CHAPTER 7 107.e3
134. Willingham SB, Volkmer JP, Gentles AJ, et al. The
CD47-signal regulatory protein alpha (SIRPa) inter-
action is a therapeutic target for human solid tumors.
Proc Natl Acad Sci USA. 2012;109(17):6662–
6667.
135. Oldenborg PA, Zheleznyak A, Fang YF, Lagenaur
CF, Gresham HD, Lindberg FP. Role of CD47
as a marker of self on red blood cells. Science.
2000;288(5473):2051–2054.
136. Lagasse E, Weissman IL. bcl-2 inhibits apoptosis
of neutrophils but not their engulfment by mac-
rophages. J Exp Med. 1994;179(3):1047–1052.
137. Chao MP, Majeti R, Weissman IL. Programmed cell
removal: a new obstacle in the road to developing
cancer. Nat Rev Cancer. 2011;12(1):58–67.
138. Jaiswal S, Jamieson CH, Pang WW, et al. CD47
is upregulated on circulating hematopoietic stem
cells and leukemia cells to avoid phagocytosis. Cell.
2009;138(2):271–285.
139. Majeti R, Chao MP, Alizadeh AA, et al. CD47 is an
adverse prognostic factor and therapeutic antibody
target on human acute myeloid leukemia stem cells.
Cell. 2009;138(2):286–299.
140. Chao MP, Alizadeh AA, Tang C, et al. Thera-
peutic antibody targeting of CD47 eliminates
human acute lymphoblastic leukemia. Cancer Res.
2011;71(4):1374–1384.
107.e3
141. Chao MP, Alizadeh AA, Tang C, et al. Anti-CD47
antibody synergizes with rituximab to promote
phagocytosis and eradicate non-Hodgkin lymphoma.
Cell. 2010;142(5):699–713.
142. Chao MP, Jaiswal S, Weissman-Tsukamoto R, et al.
Calreticulin is the dominant pro-phagocytic signal
on multiple human cancers and is counterbalanced
by CD47. Sci Transl Med. 2010;2(63):63ra94.
143. Feng M, Chen JY, Weissman-Tsukamoto R, et al.
Macrophages eat cancer cells using their own
calreticulin as a guide: roles of TLR and Btk. Proc
Natl Acad Sci USA. 2015;112(7):2145–2150.
144. Brown JM. NCI’s anticancer drug screening program
may not be selecting for clinically active compounds.
Oncol Res. 1997;9(5):213–215.
145. Quintana E, Shackleton M, Sabel MS, Fullen
DR, Johnson TM, Morrison SJ. Efficient tumour
formation by single human melanoma cells. Nature.
2008;456(7222):593–598.
146. Zomer A, Ellenbroek SI, Ritsma L, Beerling E,
Vrisekoop N, Van Rheenen J. Intravital imaging
of cancer stem cell plasticity in mammary tumors.
Stem Cells. 2013;31(3):602–606.
147. Cho RW, Wang X, Diehn M, et al. Isolation and
molecular characterization of cancer stem cells in
MMTV-Wnt-1 murine breast tumors. Stem Cells.
2008;26(2):364–371.
107.e4 Part I: Science and Clinical Oncology
SELF-ASSESSMENT REVIEW QUESTIONS
1. What is self-renewal?
a. The capacity of selected cells to naturally undergo a process
of full epigenetic reprogramming, which causes loss of
differentiation identity and reversal to a totipotent stem
cell state
b. A fundamental property of stem cells, which consists of the
capacity to divide and give rise to functionally identical stem
cells, with intact long-term expansion, proliferation, and
differentiation potential
c. The capacity possessed by stem cells to induce, when
damaged, the dedifferentiation of neighboring daughter cells,
thus creating new stem cells and renewing the stem cell pool
d. A unique property possessed by mesenchymal stem cells,
defined by the capacity to actively migrate out of a specific
tissue and relocate into a new one, where they can change
their epigenetic identity and acquire the function of other
types of resident stem cells
2. What is the definition of cancer stem cells?
a. Normal hematopoietic stem cells that have migrated inside
the tumor mass as a result of the strong inflammatory
processes usually observed in cancer
b. Embryonic stem cells that have been artificially transformed
in vitro by means of transduction with retroviral vectors
encoding the NRAS oncogene and endowed with high
tumorigenic capacity in immunodeficient mice
c. A subpopulation of cancer cells, often a minority subset,
identified by a specific surface marker phenotype and
defined by the capacity to self-renew and differentiate,
as assessed with serial transplantation experiments in
immunodeficient mice
d. Fusion hybrids between mesenchymal stem cells and cancer
cells, endowed with tumorigenic capacity and high
metastatic potential in immunodeficient mice
3. To what kind of normal cell type do cancer stem cells
correspond, in terms of surface marker phenotype?
a. They are always characterized by a surface marker profile
found exclusively in normal stem cells.
b. They are always characterized by a surface marker profile
found exclusively in a specific lineage of terminally
differentiated, effector cells.
c. Their surface marker profile is usually typical of immature or
progenitor cells and can correspond to that of stem cells
and/or immature multipotent, oligopotent, or lineage-
restricted progenitors, depending on the tumor subtype and
the disease stage.
d. Their surface marker profile is usually typical of terminally
differentiated effector cells and can correspond to that of a
specialized cellular lineage, depending on the tumor subtype
and the disease stage.
4. Which important implication might cancer stem cells have for
the future development of novel diagnostic and/or prognostic
assays in cancer patients?
a. The analysis by gene-expression array of a bulk tumor’s
transcriptional profile, and the identification of a high degree
of similarity with a cancer stem cell gene-expression
signature, could be used to identify patients characterized by
a more aggressive disease, associated with poor prognosis and
worse clinical outcomes.
b. The presence of a low percentage of cancer stem cells
within cancer tissues (<30%), as evaluated with
immunohistochemistry for CD44, could be used to
discriminate between benign and malignant tumors across
different tissue types (e.g., between a teratoma and a
teratocarcinoma, a benign and a malignant mole, and an
adenoma and a carcinoma).
c. The development of a diagnostic bone scan test, based on
immunoscintigraphy using anti-CD44 monoclonal
antibodies labeled with radioactive isotopes, could aid in the
early identification of bone metastases.
d. The development of a diagnostic assay based on PCR
(polymerase chain reaction) and aimed at measuring CD44
mRNA expression could aid in the early identification of
lymph node metastases.
5. Which important implication might cancer stem cells have for
the future development of antitumor drugs?
a. The development of novel antitumor drugs could be based
on finding agents able to arrest differentiation processes
within the tumor mass.
b. To overcome tumor resistance to radiotherapy, new
radiosensitizing agents should be able to selectively protect
nontumorigenic cancer cells during irradiation, thus creating
a darwinian competition between cancer stem cells and their
nontumorigenic progeny.
c. The development of novel antitumor drugs could be based
exclusively on in vitro experiments performed on cell lines
similar to cancer stem cells, thus eliminating the need for in
vivo animal models.
d. To permanently eradicate cancer tissues and abolish the risk
of tumor relapse, novel antitumor agents must be able to
eliminate self-renewing cancer stem cells.
ANSWERS
1. (b)  Self-renewal is a defining and intrinsic property of all types
of stem cells. As opposed to most other cell types, which
progressively exhaust their long-term expansion potential during
sequential rounds of cell division (i.e., they undergo senescence),
stem cells are able to maintain their capacity for unlimited
proliferation (i.e., they self-renew). The capacity to self-renew is
dependent on the capacity of stem cells to retain over time a
specific epigenetic identity, rather than to change or transform it.
2. (c)  The term cancer stem cells (CSCs) is an operational
definition (i.e., it is based on the experimental fulfillment of a
specific set of functional properties). It identifies a specific
subset of cancer cells that are able to self-renew (i.e., to form
tumors that can be serially passaged in vivo) and to differentiate
(i.e., to form tumors that also contain other types of
nontumorigenic cancer cells). These cells are usually identified
based on a specific repertoire of surface markers and frequently
represent a minority subset of the neoplastic clone. Although
they display stem cell properties (i.e., self-renewal and
differentiation), their molecular and phenotypic identity does
not necessarily correspond to that of normal stem cells.
3. (c)  It is important to remember that cancer stem cells is an
operational definition (see explanation of question 2). As such,
this term does not necessarily define a cell population whose
phenotypic or molecular identity is always the same as that of
normal stem cell populations. As a general rule, the surface
marker phenotype of cancer stem cells corresponds to that of
immature progenitor cells and can encompass both stem cells
and various types of multipotent, oligopotent, or lineage-
restricted progenitors, depending on the tumor subtype and the
stage of the disease.
4. (a)  Several studies have shown that malignant tumors
characterized by a gene-expression profile similar to that of
cancer stem cells (CSCs) are associated with worse clinical
outcomes. A high degree of similarity between a whole tumor’s
gene-expression profile and that of CSCs is likely the
consequence of a high CSC content in the tumor. In many
epithelial carcinomas, CD44 can be used, in combination with

a panel of other markers, to isolate a subpopulation of cancer
cells enriched in CSCs. This is likely because, within many
epithelial tissues, CD44 is expressed in a gradient, with the
highest levels observed in the most basal layers, where immature
stem and progenitor cells usually reside. CD44, however, is not
specific to epithelial stem and progenitor cells and is expressed
abundantly across many other cell types, including stromal and
immune cells (e.g., fibroblasts, endothelial cells, monocytes,
lymphocytes). Expression of CD44 cannot be used to purify
and/or identify CSCs across all tumors, especially nonepithelial
tumors. Even within epithelial tumors, expression of CD44
alone (e.g., without careful exclusion of the contribution of
stromal cells) cannot be used to accurately define CSCs.
5. (d)  Being able to self-renew, cancer stem cells (CSCs) are able
to propagate themselves indefinitely. If not eliminated, they
Stem Cells, Cell Differentiation, and Cancer  •  CHAPTER 7 107.e5
107.e5
hold the potential to lead to tumor relapse. To permanently
eradicate tumor tissues, antitumor therapies must be able to kill
or otherwise eliminate CSCs. From a theoretical perspective, a
possible strategy to deplete tumor tissues of CSCs could be to
promote their differentiation into terminally mature cells, not to
inhibit or prevent it. As a general rule, nontumorigenic cancer
cells are the progeny of CSCs, not a different genetic clone of
the cancer tissue. Thus nontumorigenic cancer cells originate
from CSCs and are not in “Darwinian competition” with them.
The dynamic equilibrium between CSCs and other cellular
components of cancer tissues is extremely difficult to model in
vitro using traditional cell lines grown as two-dimensional
monolayers. Currently, CSCs can be best, if not exclusively,
studied using solid tissue xenografts in animal models.
 8  Tumor Microenvironment: Vascular and
Extravascular Compartment
Rakesh K. Jain, John D. Martin, Vikash P. Chauhan, and Dan G. Duda

S UMMARY OF K EY P OI N T S
• A solid tumor is an organ composed
of neoplastic cells and stromal cells
including immune cells nourished by
vasculature made of endothelial
cells—all embedded in an
extracellular matrix. The interactions
among these cells, their surrounding
matrix, and their local
microenvironment control the
expression of various genes. The
products encoded by these genes, in
turn, control the pathophysiologic
characteristics of the tumor. Tumor
pathophysiology governs not only
tumor growth, invasion, and
metastasis but also the response to
various therapies.
• Tumor vasculature is made of host
vessels co-opted by tumor cells and
by new vessels formed by the
processes of vasculogenesis and
angiogenesis. A constellation of
positive and negative regulators of
angiogenesis governs the process delivery.
of neovascularization.
• Tumor vessels are abnormal in terms
of their organization, structure, and
function. These abnormalities
contribute to heterogeneity in
vascular permeability, blood flow,
and the microenvironment.
• Tumor interstitial matrix is formed of
proteins secreted by host and tumor
cells and by those leaked from the
nascent blood vessels.
• Tumor interstitium is heterogeneous,
with some regions fairly permeable
and others difficult to penetrate.
Modification of collagen and
hyaluronan in the matrix can improve
penetration of large-molecular-weight
therapeutics.
• Solid components of tumors—cancer
cells, stromal cells, and matrix
molecules—mechanically compress
blood and lymphatic vessels,
resulting in reduced perfusion that
limits oxygen and drug supply.
Reducing these constituents
decompresses blood vessels verification of biomarkers predictive
to enhance perfusion and drug
• Interstitial hypertension results from
vessel leakiness, lack of functional
lymphatics, and compression of
blood and lymphatic vessels by
proliferating cancer cells. Interstitial
hypertension cannot compress leaky
vessels, because the intravascular
and extravascular fluid pressures
equilibrate.
• Judicious application of angiogenic
therapy can normalize the tumor
vessels and make them more
efficient for delivery of oxygen (a
known radiosensitizer) and drugs.
Indeed, clinical studies indicate that
antiangiogenic agents can increase
perfusion and oxygenation in tumors,
and that these effects are associated
with improved response.
• Twelve antiangiogenic agents have
been approved for cancer. Two main
goals to improve patient outcomes
with antiangiogenic agents are the
development of therapies that fortify
vessels without pruning and
of response. Additionally, the
potential of combining antiangiogenic
and solid stress–alleviating therapies
with immunotherapies should be
confirmed in clinical trials.

A solid tumor is an organ composed of neoplastic cells and host
stromal cells including resident and transiting immune cells nourished
by the vasculature made of endothelial cells (ECs)—all embedded in
an extracellular matrix (Fig. 8.1). The interactions among these cells
and between these cells, their surrounding matrix, and their local
microenvironment, control the expression of various genes. The products
encoded by these genes, in turn, control the pathophysiologic char-
acteristics of the tumor. Tumor pathophysiology governs not only
tumor growth, invasion, and metastasis but also the response to various
therapies. In this chapter we will discuss various pathophysiologic
parameters that characterize the vascular and extravascular compartments
of a tumor and the mechanisms governing the formation and function
of these compartments.
VASCULAR COMPARTMENT
Neoplastic cells, like normal cells, need oxygen and other nutrients
for their survival and growth. Every normal cell in the human body
is located within 100 to 200 µm of a blood capillary so that it can
receive oxygen and other nutrients by the process of diffusion. Likewise,
cells undergoing neoplastic transformation depend on nearby capillaries
for growth. These preneoplastic (i.e., hyperplastic or dysplastic) cells
can grow as a spherical or ellipsoidal cellular aggregate. Once the size
of the cellular aggregate reaches the diffusion limit for critical nutrients
and oxygen, however, the aggregate as a whole can become dormant.
Indeed, human tumors can remain dormant for many years because
of a balance between neoplastic cell proliferation and apoptosis.

108
 Tumor Microenvironment: Vascular and Extravascular Compartment  •  CHAPTER 8 109

However, once they have access to new blood vessels, they may grow
and metastasize. What triggers the growth of new vessels? What
molecular and cellular mechanisms are involved? How do these vessels
compare with normal vessels with respect to structure and function?
Can we prevent or delay tumor progression only by interfering with
the neovascularization process?
Blood vessel
Basement
membrane
Interstitial matrix and fluid
Cancer cells Host cells
Figure 8.1  •  Schematic representation of a solid tumor. The key components
include cancer cells, host cells, and vasculature made of endothelial cells—all
embedded in a matrix bathed in interstitial fluid. Arrows indicate interactions
between the components. (Modified from Jain RK. Angiogenesis and lym-
phangiogenesis in tumors: insights from intravital microscopy. Cold Spring
Harbor Symp Quant Biol [The Cardiovascular System]. 2002;67:239–248.)
New Vessel Formation
It has been known for nearly a century that the vascular system is
associated with tumor growth in animals and humans.1 Powerful
insights into the neovascularization of transplanted tumors using the
transparent window techniques were developed in the 1940s.2–5 The
possibility that tumors produce an “angiogenic” substance was suggested
in 1968.6 The hypothesis that blocking angiogenesis should block
tumor growth and metastasis was proposed shortly thereafter in 1971.7
The concept that a tissue acquires angiogenic capacity during neoplastic
transformation—and, by extension, that antiangiogenesis could be
used to prevent cancer—was put forward in 1978.8 The first antian-
giogenic agent approved for cancer patients was bevacizumab, an
antibody specific to vascular endothelial growth factor (VEGF), on
the basis of the increased survival seen in metastatic colorectal cancer
patients with the combination of bevacizumab with standard chemo-
therapy in a pivotal randomized placebo-controlled phase III trial.9
At present, various antiangiogenesis and proangiogenesis strategies are
being evaluated clinically to prevent or treat a large number of diseases,
including cancer.10–13 Both normal and pathologic angiogenic processes
are governed by the net balance between proangiogenic and antian-
giogenic factors.14,15 This balance is spatially and temporally regulated
under physiologic conditions, so that the “angiogenic switch” is “on”
when needed (e.g., during embryonic development, wound healing,
formation of the corpus luteum) and “off ” at other times. During
neoplastic transformation and tumor progression, this regulation is
deranged, and blood vessels form ectopically to support a growing
tumor mass.
Cellular Mechanisms
At least four cellular mechanisms are involved in the vascularization
of tumors: co-option, intussusception, sprouting (angiogenesis), and
vasculogenesis (Fig. 8.2).12 Tumor cells can co-opt and grow around
existing vessels to form “perivascular” cuffs. However, as stated earlier,
these cuffs cannot grow beyond the diffusion limit of critical nutrients

Endothelial precursor

Intussusceptive
growth
Angiogenic sprouting

Figure 8.2  •  Cellular mechanisms of vascularization in tumors. At least four mechanisms are involved: (1) intussusception, wherein tumor vessels enlarge
and an interstitial tissue column grows in the enlarged lumen, expanding the network; (2) vasculogenesis, wherein endothelial precursor cells mobilized from
the bone marrow or peripheral blood contribute to the endothelial lining of tumor vessels; (3) “sprouting” angiogenesis, wherein the existing vascular network
expands by forming sprouts or bridges; and (4) co-option (not shown), wherein tumor cells grow around existing vessels to form “perivascular” cuffs. (Modified
from Jain RK, Carmeliet PF. Angiogenesis in cancer and other diseases. Nature. 2000;407:249–257.)
110 Part I: Science and Clinical Oncology
and may actually cause the collapse of the vessels owing to growth
pressure (referred to as “solid stress”). Alternatively, an existing vessel
may enlarge in response to the growth factors released by tumors, and
an interstitial tissue column may grow in the enlarged lumen and
partition the lumen to form an expanded vascular network. This mode
of intussusceptive microvascular growth has been observed during
tumor growth, wound healing, and gene therapy.16–19 “Sprouting”
angiogenesis is perhaps the most widely studied mechanism of vessel
formation. During sprouting angiogenesis, the existing vessels become
leaky in response to growth factors released by normal cells or cancer
cells; the basement membrane and the interstitial matrix dissolve;
pericytes dissociate from the vessel; ECs migrate and proliferate to
form an array or sprout; a lumen is formed in the sprout (a process
referred to as canalization); branches and loops are formed by confluence
and anastomoses of sprouts to permit blood flow; and finally, these
immature vessels are invested with basement membrane and pericytes.
During physiologic angiogenesis, these vessels differentiate into mature
arterioles, capillaries, and venules, whereas in tumors they remain
largely immature.5,12,13,20 During embryonic development, a primitive
vascular plexus is formed from endothelial precursor cells (EPCs, also
known as angioblasts) by a process referred to as vasculogenesis. In
adults, EPCs—mobilized from bone marrow niches into the peripheral
blood circulation—can also contribute to neovascularization (a process
referred to as postnatal vasculogenesis) in tumors and other tissues.21–23
In addition, reports have demonstrated that a fraction of ECs in tumor
vessels may be derived via transdifferentiation from cancer cells,24 or
from cancer stem-like cells (e.g., in glioblastomas [GBMs]).12,25–27 In
this larger context, the term vasculogenesis might be invoked to describe
neovascularization in tumors from tumor stem cells.28 Tumor vascu-
logenesis may also occur from EPCs of non–bone marrow origin that
are recruited from adjacent tissues and/or circulation.29,30 The current
challenge is to discern the relative contribution of each of these
mechanisms of neovascularization during tumor growth, and during
and after treatment of tumors.28,31
Molecular Mechanisms
Various proangiogenic and antiangiogenic molecules that orchestrate
different steps in vessel formation, along with their functions, are
listed in Table 8.1. VEGF is currently considered the most critical
proangiogenic molecule. Originally discovered in 1983 as the vascular
permeability factor and cloned in 1989, VEGF increases vascular
permeability, promotes migration and proliferation of ECs, serves as
an EC survival factor, can mobilize EPC populations from the bone
marrow, and is known to upregulate leukocyte adhesion molecules
on ECs.16,23,32–34 During tumor progression, or with treatment, the
number of distinct angiogenic molecules produced by a tumor can
increase.35–37 Thus, after VEGF signaling is blocked, a tumor might
rely on other, alternative angiogenic molecules (e.g., basic fibroblast
growth factor [bFGF], stromal-derived factor 1α [SDF1α], placental-
derived growth factor [PlGF], or interleukin-8 [IL-8]).38 Other positive
regulators of angiogenesis include the angiopoietins that are involved
in stabilizing vessels and controlling vascular permeability; various
proteases involved in dissolving and remodeling matrix and releasing
growth factors; and organ-specific angiogenic stimulators (e.g., endocrine
gland VEGF).20,39,40 Angiogenesis inhibitors include endogenous soluble
receptors of various proangiogenic ligands (e.g., sVEGFR1/sFLT1)
and molecules that downregulate the expression of stimulators (e.g.,
interferons) or that interfere with the release of the stimulators or
binding with their receptors (e.g., platelet factor 4). Thrombospondins
are among the first and best characterized endogenous inhibitors that
interfere with the growth, adhesion, migration, and survival of ECs.14
Other endogenous inhibitors include fragments of various plasma or
matrix proteins (e.g., angiostatin, a fragment of plasminogen; endostatin,
a fragment of collagen XVIII; tumstatin, a fragment of collagen IV).41–43
Neither the mechanisms of action of the matrix-derived inhibitors
nor their physiologic role is well understood.44 The generation of
proangiogenic and antiangiogenic molecules can be triggered by
metabolic stress (e.g., low Po2, low pH, or hypoglycemia), mechanical
stress (e.g., shear stress, solid stress), immune or inflammatory cells
that have infiltrated the tissue, and genetic mutations (e.g., activation
of oncogenes or deletion of suppressor genes that control the production
of angiogenesis regulators).14,15,45–48 These molecules can emanate from
cancer cells, ECs, stromal cells, blood, and extracellular matrix (Fig.
8.3).49–52 Because the normal host cells differ among organs, the detailed
mechanisms of angiogenesis might depend on the specific host-tumor
interactions operating within a given tissue.53–60 Furthermore, because
the tumor microenvironment is likely to change during tumor growth,
regression, and relapse, profiles of proangiogenic and antiangiogenic
molecules are likely to change with time and space.61,62 The challenge
currently is to develop a unified conceptual framework to describe
the temporal and spatial profiles of this increasingly diverse array of
angiogenesis regulators with the aim of developing effective therapeutic
strategies.38,63–65
Vascular Architecture
In a normal tissue, blood flows from an artery to arterioles to capillaries
to venules to a vein. Although the tumor vasculature originates from
these host vessels and the mechanisms of angiogenesis are similar, its
organization may differ dramatically, depending on the tumor type, its
location, and whether it is growing, regressing, or relapsing.16,66–69 In
general, tumor vessels are dilated, saccular, tortuous, and chaotic in their
patterns of interconnection.70 For example, whereas normal vasculature
is characterized by dichotomous branching, tumor vasculature has
many trifurcations and branches with uneven diameters.71,72 The
fractal dimensions and minimum path lengths of tumor vasculature
are different from those of normal host vasculature.66–68 The molecular
mechanisms of this abnormal vascular architecture are not understood,
but it seems reasonable to hypothesize that the imbalance of VEGF
and angiopoietins is a key contributor.20,73 In mice, “normalization”
of the tumor vasculature observed during treatment with therapies
that reduce VEGF (e.g., hormone withdrawal from a hormone-
dependent tumor), interfere with VEGF signaling (e.g., treatment
with anti-VEGF or anti-VEGFR2 antibody; Fig. 8.4), or mimic an
antiangiogenic cocktail (e.g., trastuzumab [Herceptin] treatment of
a HER2-overexpressing tumor) is in concert with this molecular
hypothesis.62,64,74–77 Mechanical stress generated by proliferating cancer
and stromal cells and their excessive interstitial matrix production also
can lead to the partially compressed or totally collapsed vessels often
found in tumors (Fig. 8.5A–B).65,78,79 The decompression of blood
vessels observed after induction of apoptosis in perivascular cells or
stromal cells, or enzymatic degradation of matrix hyaluronan, supports
this mechanical hypothesis (Fig. 8.5C).79–82 Perhaps the combination of
both molecular and mechanical factors renders the tumor vasculature
abnormal, and thus both types of factors must be taken into account
in designing novel strategies for cancer treatment.
Blood Flow and Microcirculation
Blood flow in a vascular network, whether normal or abnormal, is
governed by the arteriovenous pressure difference and flow resistance.
Flow resistance is a function of the vascular architecture (referred to as
geometric resistance) and of the blood viscosity (rheology, referred to
as viscous resistance).70 Abnormalities in both vasculature and viscosity
increase the resistance to blood flow in tumors.72,83–85 As a result, overall
perfusion rates (blood flow rate per unit volume) in tumors are lower
than in many normal tissues.86,87 Both macroscopically and microscopi-
cally, tumor blood flow is temporally and spatially chaotic. Macroscopi-
cally, four spatial regions can be recognized in a tumor (Fig. 8.6):
1. An avascular necrotic region
2. A seminecrotic region
3. A stabilized microcirculation region
4. An advancing front88,89
 Tumor Microenvironment: Vascular and Extravascular Compartment  •  CHAPTER 8 111

Table 8.1  Angiogenesis Activators and Inhibitorsa

Activators Function Inhibitors Function
VEGF family membersb,c Stimulate angiogenesis and VEGFR1; soluble VEGFR1; Sink for VEGF, VEGF-B, PlGF
vasculogenesis, permeability, soluble neuropilin-1 (NRP-1)
leukocyte adhesion
VEGFRc, NRP-1, NRP-2 Integrate angiogenic and survival Ang 2b,c Antagonist of Ang 1
signals
EG-VEGF Stimulate growth of endothelial cells TSP-1,2 Inhibit endothelial migration,
derived from endocrine glands growth, adhesion, and survival
Ang 1 and Tie 2b,c Stabilize vessels Angiostatin and related Inhibit endothelial migration and
plasminogen kringles survival
PDGF-BB and receptors Recruit smooth muscle cells Endostatin (collagen XVIII Inhibit endothelial survival and
fragment) migration
TGF-β1d, endoglin, TGF-β Stimulate extracellular matrix Tumstatin (collagen IV Inhibit endothelial protein
receptors production fragment) synthesis
FGF, HGF, MCP-1 Stimulate angio/arteriogenesis Vasostatin; calreticulin Inhibit endothelial growth
Integrins αvβ3, αvβ5, α5β1 Receptors for matrix macromolecules Platelet factor 4 Inhibit binding of bFGF and VEGF
and proteinases
VE-cadherin; PECAM (CD31) Endothelial junctional molecules Tissue inhibitors of MMP Suppress pathologic angiogenesis
(TIMPs); MMP inhibitors; PEX
Ephrinsc Regulate arterial and venous Meth-1; Meth-2 Inhibitors containing MMP-, TSP-,
specification and disintegrin domains
Plasminogen activators, MMPs Remodel matrix, release growth factor IFN-α, IFN-β, IFN-γ; IP-10, Inhibit endothelial migration;
IL-4, IL-12, IL-18 downregulate bFGF
PAI-1 Stabilize nascent vessels Prothrombin kringle-2; Suppress endothelial growth
antithrombin III fragment
NOS; COX-2 Stimulate angiogenesis and 16 kD-prolactin Inhibit bFGF/VEGF
vasodilation
AC133 Regulate angioblast differentiation VEGI Modulate cell growth
Chemokinesd Pleiotropic role in angiogenesis Fragment of SPARC Inhibit endothelial binding and
activity of VEGF
Id1/Id3 Inhibit differentiation Osteopontin fragment Interfere with integrin signaling
Maspin, canstatin, Protease inhibitor
proliferin-related protein, Mechanisms unknown
restin

a
Selected list updated from Carmeliet P, Jain RK. Molecular mechanisms and clinical applications of angiogenesis. Nature. 2011;473:298–30712; for complete function and
references, see supplementary information (http://steele.mgh.harvard.edu).
b
Also present in or affecting nonendothelial cells.
c
See Jain.20
d
Opposite effect in some contexts.
COX-2, Cyclooxygenase-2; EG-VEGF, endocrine gland–derived vascular endothelial growth factor; bFGF, basic fibroblast growth factor; HGF, hepatocyte growth factor; IFN,
interferon; IL, interleukin; IP, interferon-inducible protein; MCP-1, monocyte chemotactic protein-1; MMP, metalloproteinase; NRP-1, neuropilin-1; NOS, nitric oxide synthase; PAI-1,
plasminogen activator inhibitor-1; PDGF-BB, platelet-derived growth factor-BB; PECAM, platelet endothelial cell adhesion molecule; PEX, hemopexin fragment of MMP-2; PlGF,
placental growth factor; SPARC, secreted protein acidic and rich in cysteine; TGF, transforming growth factor; TIMP, tissue inhibitor of metalloproteinase; TSP, thrombospondin;
VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; VEGI, vascular endothelial growth inhibitor. See text for explanation of
abbreviations.

At the microscopic level, in normal tissues, erythrocyte velocity is
dependent on vessel diameter, but there is no such dependence in
most tumors.55,60,90 Furthermore, the average erythrocyte velocity can
be an order of magnitude lower in some tumors as compared with
that of normal host tissue (Fig. 8.7).60 In a given vessel within a tumor,
blood flow fluctuates with time and can reverse its direction.88,90,91 In
addition to the elevated geometric and viscous (rheologic) resistance,
other molecular and mechanical factors contribute to this spatial and
temporal heterogeneity. These include imbalance between proangiogenic
and antiangiogenic molecules, solid stress generated by proliferating
tumor cells and matrix production, vascular remodeling by intussuscep-
tion, and coupling between luminal and interstitial fluid pressure
(IFP) via hyperpermeability of tumor vessels.6,17,65,66,78–80,92–94 As will
be discussed later, this heterogeneity contributes to both acute and
chronic hypoxia in tumors—a major cause of resistance to radiation
and other therapies. Increased perfusion also correlates with a positive
response to radiotherapy or chemotherapy and patient survival.95,96
Considerable effort has gone into increasing tumor blood flow for
improving radiation therapy, or decreasing tumor perfusion in the
case of hyperthermia. This has been difficult to achieve reproducibly,
because tumor vasculature consists of both vessels co-opted from the
preexisting host vasculature and vessels resulting from the angiogenic
response of host vessels to cancer cells. The former are invested in
normal contractile perivascular cells, whereas the latter lack these
perivascular cells or these cells are abnormal.49,70,97 Presumably as a
result, efforts to increase the tumor blood flow with pharmacologic
or physical agents have not always been reproducible or success-
ful.49,70,86,97 On the other hand, the strategy of decreasing or shutting
112 Part I: Science and Clinical Oncology

A B

C D E
100 µm 100 µm 50 µm

Figure 8.3  •  Tumor induction of host promoter activity in stromal cells. The expression of vascular endothelial growth factor (VEGF) in host cells can
be examined through use of transgenic mice expressing a green fluorescent protein (GFP) under the control of the VEGF promoter. (A) A murine mammary
carcinoma xenograft shows host cell VEGF expression mainly at the periphery of the tumor after 1 week. (B) After 2 weeks, the VEGF-expressing host cells
have infiltrated the tumor. (C) A GFP-expressing layer of host cells can be seen at the tumor-host interface. (D–E) The VEGF-expressing host cells colocalize
with the angiogenic tumor vessels. ([A–B] From Fukumura D, Xavier R, Sugiura T, et al. Tumor induction of VEGF promoter activity in stromal cells. Cell.
1998;94:715–725. [C–E] From Brown EB, Campbell RB, Tsuzuki Y, et al. In vivo measurement of gene expression, angiogenesis and physiological function
in tumors using multiphoton laser scanning microscopy. Nat Med. 2001;7:1069.)

down the tumor blood flow—by “stealing” blood away from the
“passive component” of the tumor vasculature with vasodilators, through
vascular targeting, or by means of intravascular coagulation—has shown
promise in experimental systems.86,87,98–100 It also appears that judiciously
applied antiangiogenic therapy could “normalize” the abnormal tumor
microcirculation by pruning the immature vessels (see Fig. 8.4),
thus rendering the remaining vasculature more responsive to
vasoactive agents.101
VASCULAR PERMEABILITY
Once a blood-borne molecule has reached an exchange vessel, its
extravasation occurs by diffusion, convection, and, to some extent,
presumably by transcytosis.101,102 The effective permeability, P, of
a molecule depends on the size, shape, charge, and flexibility of
the molecule, and on the size, shape, charge, and dynamics of the
transvascular transport pathway. In normal vessels, these pathways
include diffusion along the EC membrane (for lipophilic solutes),
trans-EC diffusion, interendothelial junctions (<7 nm), open or closed
fenestrations (<10 nm), and transendothelial channels (including vesicles
or vesicovacuolar channels).16,103 Some of these anatomic pathways
may be lined with glycocalyx on EC, thus effectively reducing the
size of the pathway. A basement membrane may further retard
the movement of molecules. Ultrastructure studies show widened
interendothelial junctions, increased numbers of fenestrations, vesicles,
and vesicovacuolar channels in tumor vessels, and a lack of normal
basement membrane and pericytes.16,52,97,103,104 In concert with these
ultrastructural findings, both vascular permeability and hydraulic
conductivity (a measure of water movement by pressure gradient)
of tumors, in general, are significantly higher than those for various
normal tissues.60,105–109 Furthermore, unlike normal vessels in the
surrounding tissue, tumor vessels partially lose molecular size–based
selectivity in permeability to different molecules.110 Despite increased
overall permeability, not all blood vessels of a tumor are leaky (Fig.
8.8A). Even the leaky vessels have a finite pore size that is tumor
dependent (Fig. 8.8B), and ultrastructural studies have shown that
the larger pore size in tumors represents wide interendothelial junc-
tions.57,104 Thus permeability is inversely related to molecular size
because of steric and hydrodynamic hindrance from these pores, and
very large molecules, such as nanoparticles, do not penetrate tumors
efficiently.111,112 Molecular shape also influences penetration of these
pores, such that rod-shaped molecules have a higher permeability than
spherical molecules.113 Similarly, positively charged molecules have a
higher affinity for the negatively charged glycocalyx in the pores of these
angiogenic tumor vessels, reducing this hindrance and giving them a
higher permeability.114–117 Not only do the vascular permeability and
pore size vary from one tumor to the next (see Fig. 8.8A–C), but within
the same tumor they vary spatially and temporally, as they do during
 Tumor Microenvironment: Vascular and Extravascular Compartment  •  CHAPTER 8 113
Tumor Normalized
Normal
Inadequate
Figure 8.4  •  Normalization of tumor vasculature. Normal vessels are well
organized with even diameters. In contrast, tumor vessels are tortuous, with
increased vessel diameter, length, density, and permeability. Antiangiogenic
therapies “normalize” the tumor vascular network and could ultimately reduce
the vasculature to the point at which it provides inadequate support for oxygen
and drug delivery. (Modified from Jain RK. Normalizing tumor vasculature
with anti-angiogenic therapy: a new paradigm for combination therapy. Nat
Med. 2001;7:987–989; Jain RK. Angiogenesis and lymphangiogenesis in
tumors: insights from intravital microscopy. Cold Spring Harbor Symp Quant
Biol [The Cardiovascular System]. 2002;67:239–248; Jain RK, Carmeliet PF.
Vessels of death or life. Sci Am. 2001;285:38.)
tumor growth, regression, and relapse.57,61,62 The local microenvironment
plays an important role in controlling vascular permeability (see Fig.
8.8D). For example, a human glioma (HGL21) has fairly leaky vessels
when grown subcutaneously in immunodeficient mice, but it exhibits
blood-brain barrier properties in the brain.60,73 Such site-dependent
differences for other tumors have been observed in other orthotopic
sites.55,58,59 One possible explanation is that the host-tumor interactions
control the production and secretion of cytokines associated with
permeability increase (e.g., VEGF) and decrease (e.g., angiopoietin
1).20,40,73,118,119 A better understanding of the molecular mechanisms
of permeability regulation in tumors is likely to yield strategies for
improved delivery of molecular medicine to tumors.
Movement of Cells Across Vessel Walls
Both cancer cells and immune cells frequently move across the walls
of blood vessels—the former in the process of metastasis, and the
latter during immune response or cell-based immunotherapy. Both
transendothelial (through ECs) and periendothelial (between ECs)
pathways have been proposed as a route for intravasation and
extravasation of cells. Very little is known about intravasation except
that tumors might shed more than a million cells per gram per day,
and most of these are not clonogenic.120–123 We have demonstrated
that metastatic cells could bring their own stroma from the primary
tumor in the form of heterotypic cell clumps.124 More is known about
the molecular and cellular mechanisms of extravasation.125 When a
cell enters a blood vessel, it can continue to move with the flowing
blood, collide with the vessel wall, adhere transiently or stably, and
finally extravasate. These interactions are governed by both local
hydrodynamic forces and adhesive forces. The former are determined
by the vessel diameter and fluid velocity, and the latter by the expression,
strength, and kinetics of bond formation between adhesion molecules
and by the surface area of contact.126–130 Deformability of cells affects
both types of forces.131 In addition, cancer cells may grow intravascularly
at the distant site (e.g., in the lungs).132
Rolling of endogenous leukocytes is generally low in tumor vessels,
whereas stable adhesion (≥30 seconds) is comparable between normal
vessels and tumor vessels.133 On the other hand, both rolling and
stable adhesion are nearly zero in angiogenic vessels induced in collagen
gels by bFGF or VEGF, two of the most potent angiogenic factors.134
Whether this observation is due to a low flux of leukocytes into
angiogenic vessels and/or to downregulation of adhesion molecules
in these immature vessels is currently not known. The age of the
animal also plays an important role in leukocyte-endothelial interac-
tions.135 Further insight into the types of cells that adhere to tumor
vessels comes from studies on the localization of IL-2–activated natural
killer (A-NK) cells in normal and tumor tissues in mice in which
positron emission tomography was used.136,137 After systemic injection,
these cells localized primarily in the lungs immediately after injection
and could not be detected in the tumor.136 Increased rigidity caused
by IL-2 activation might contribute to the mechanical entrapment of
these cells in the lung microcirculation.138,139 Constitutive expression
of certain adhesion molecules in the lung vasculature might also facilitate
their retention in the lungs.125 One approach to reducing lung entrap-
ment is to reduce the rigidity of these cells.112,117 Alternatively, the
lung can be circumvented by injecting A-NK cells directly into the
blood supply of tumors. In this case, A-NK cells, both xenogeneic
and syngeneic, adhered to some blood vessels in three different tumor
models via CD18 and very large antigen 4 (VLA-4) on the A-NK
cells and intercellular adhesion molecule 1 (ICAM-1), vascular cell
adhesion molecule 1 (VCAM-1), and E-selectin on the activated
endothelium of angiogenic vessels.34,137,140–143 These molecules can be
upregulated by tumor necrosis factor-α (TNF-α) and a protein of 90
kd molecular weight (p90) that is secreted by some neoplastic cells
and downregulated by transforming growth factor-β (TGF-β)—also,
presumably, secreted by cancer cells.56,126,144–148 Surprisingly, the
proangiogenic VEGF also can upregulate these molecules, whereas
another proangiogenic molecule, bFGF, can downregulate these
molecules.34,52,62,77,149 On the other hand, inflammatory cells such as
monocytes and macrophages or neutrophils are also recruited by VEGF
and may play important roles in promoting matrix remodeling and
angiogenesis.150 The challenge currently is to decrease nonspecific
entrapment of immune cells in normal vessels and to increase their
delivery to tumor vessels to improve various cell-based therapies,
including gene therapy.
EXTRAVASCULAR COMPARTMENT
Composition and Origin
The extravascular compartment of a solid tumor consists of neoplastic
cells (parenchyma) and host cells (e.g., inflammatory cells, fibroblasts)
residing in an interstitial matrix bathed by the interstitial fluid (see
Fig. 8.1). Depending on the tumor type and its stage of differentiation,
neoplastic cells might be dispersed in the matrix as individual cells
(e.g., lymphomas, melanomas) or as clumps, sheets, or nests (e.g.,
carcinomas). More than 80% of tumors are carcinomas arising from
114 Part I: Science and Clinical Oncology

DEPLETE CANCER CELLS DEPLETE FIBROBLASTS

Cancer cell
Fibroblast
Open blood vessel
Partially open blood vessel
Collapsed vessel
Collagen
Stretched Collagen
HA
Compressed HA

DEPLETE COLLAGEN DEPLETE HYALURONAN

Intact Deformed

Untreated Solid stress alleviated

C Collagen Perfused vessel

Figure 8.5  •  Causes and remedies of vascular compression in tumors. (A) Middle, In solid tumors, proliferating cancer cells and activated fibroblasts
deform the interstitial matrix, resulting in stretched collagen fibers, compressed hyaluronan, and deformed cells—all storing solid stress, a type of mechanical
force transmitted by solid tissue components. This stress compresses intratumor blood and lymphatic vessels. Potential strategies to alleviate solid stress and
decompress vessels involve depleting these components. Depleting cancer cells (top left) or fibroblasts (top right) relaxes collagen fibers, hyaluronan, and the
remaining cells, alleviating solid stress. Depleting collagen (bottom left) alleviates the stress that was held within these fibers while also relaxing stretched/
activated fibroblasts and compressed cancer cells within nodules. Finally, depleting hyaluronan (bottom right) alleviates the stored compressive stress, allowing
nearby components to decompress. (For simplification of the schematic, other stromal cells, such as pericytes, macrophages, and various immune cells that
might also control production of collagen or hyaluronan, are not shown. Lymphatic vessels are also not shown for the same reason.) (B) Image of an intact
(left) and deformed (right) soft tissue sarcoma freshly excised from a patient. The intact tumor is free of external loads. Cutting through the long axis of the
tumor allows the tissue to deform, which demonstrates the existence of solid stress held within tumor tissue. (C) Intravital microscopy images of a murine
breast tumor before (left) and after (right) treatment to reduce solid stress. The treatment reduces the density of collagen fibers (blue) leading to a homogenous
distribution of perfused vessels (green). ([A–B] From Stylianopoulos T, Martin JD, Chauhan VP, et al. Causes, consequences, and remedies for growth-induced
solid stress in murine and human tumors. Proc Natl Acad Sci U S A. 2012;109:15101–15108. [C] From Chauhan VP, Martin JD, Liu H, et al. Angiotensin
inhibition enhances drug delivery and potentiates chemotherapy by decompressing tumour blood vessels. Nat Commun. 2013;4:2516.)
 Tumor Microenvironment: Vascular and Extravascular Compartment  •  CHAPTER 8 115

e
ur

H
ss

rp
re

y
er
n
la
p O ia l p

io

liv
lu
on

at
l

de
ce
tit

er
si

rs
fu

tra

lif

g
te

ru
r

o
2
Tumor region

Pe

Ex

Pr
In

D
Proliferative ++ + ++ <7.4 ++ ++
Quiescent + +++ + ~ 6.5-7 +/– +
Necrotic – +++ – ~ 7-8 – +/–

Figure 8.6  •  The tumor microenvironment is heterogeneous with proliferative, quiescent, and necrotic regions. These regions can be characterized in
terms of various physiologic parameters. Decreasing magnitude of these parameters is indicated as +++, ++, +, +/−. and −. (From Jain RK, Forbes NS. Can
engineered bacteria help control cancer? Proc Natl Acad Sci U S A. 2001;98:14748–14750.)

Vmax (mm/s)
epithelial cells. The remaining include sarcomas arising from mesen-
chymal cells (e.g., bone or muscle cells), lymphomas arising from
Arterioles 35 Venules lymphoid tissue, leukemias arising from hematopoietic cells, and
hemangiomas arising from ECs. In a poorly differentiated carcinoma,
30 the cancer cells might be packed loosely in clumps, whereas in a
well-differentiated carcinoma, the cells might be connected with
25
intercellular junctions and tightly packed in a nest enveloped by a
20 basement membrane. With tumor progression, cancer cells can invade
the basement membrane and spread to other regions.16 These various
15 types of normal host cells must migrate into the tumor from normal
tissue. Inflammatory cells might enter the tumor via blood vessels or
10 might infiltrate from the adjacent tissue or lymphatics.125 Adaptive
immune cells recruit innate immune response cells based on signals
5 from the stroma.151 Macrophages, which are of the innate response,
are plastic in that they can either fight tumor growth or support it
when exposed to certain cytokines.152 Other host cells, such as
50 25 0 25 50 fibroblasts, might proliferate and migrate from the adjacent connective
A Pial vessel diameter (µm) tissue.20,49,153 The interstitial subcompartment of a tumor is bounded
by the walls of the blood vessels on one side and by the membranes
of cancer and stromal cells on the other. In normal tissues, the blood
0.8 vessels are surrounded by a basement membrane, which, as discussed
MCalV U87
0.7
earlier, is defective in tumors.20 In addition, functional lymphatics
might be confined to the tumor margin.154,155 The interstitial space
0.6 of tumors, like that of normal tissues, is composed of a collagen and
RBC velocity (mm/s)

elastin fiber network that provides structural support to the tissue.

0.5
0.4
0.3
0.2
0.1
0.0
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70
B Tumor vessel diameter (µm)
Figure 8.7  •  Blood velocity as a function of vessel diameter. (A) Normal
pial vessels. (B) MCaIV (mammary carcinoma) and U87 (glioma) tumors
xenografted on the pial surface. The measured tumor blood velocities are an
order of magnitude lower than the velocities in the normal host tissue and
are not related to the vessel density. (From Yuan F, Salehi HA, Boucher Y,
et al. Vascular permeability and microcirculation of gliomas and mammary
carcinomas transplanted in rat and mouse cranial windows. Cancer Res.
1994;54:4564–4568.)
Interdispersed in this cross-linked structure are the interstitial fluid
and macromolecular constituents (polysaccharides, hyaluronan, and
proteoglycans), which form a hydrophilic gel. Compared with our
understanding of blood vessel formation, our understanding of stroma
generation is minimal. Dvorak proposed that the extravasated plasma
protein fibrinogen, a key component of the tumor interstitial fluid,
clots to form fibrin, which serves as a major component of the pro-
visional stroma. This provisional stroma eventually is replaced by
more mature connective tissue stroma. The tumor interstitial fluid
also contains several other Arg-Gly-Asp (RGD)–containing proteins,
including fibronectin, vitronectin, osteopontin, thrombospondin,
decorin, and tenacin.16 These proteins are present in both free and
bound forms. Their Arg-Gly-Asp sequence provides a binding site for
adhesion that assists in the migration of various cells, including stromal
cells. In addition to extravasating from the leaky tumor vessels, these
proteins, along with collagen and various proteoglycans, are also
synthesized by the stromal cells, albeit in a form that differs from that
in the plasma or normal tissues.16 Tumor interstitial fluid also can
contain various growth factors that facilitate stroma formation. For
example, in vitro studies have suggested that platelet-derived growth
factor–BB (PDGF-BB) is involved in the recruitment of fibroblasts
116 Part I: Science and Clinical Oncology

2,500
Dorsal window

2,000

Tracer size (nm)

1,500

1,000

500

ST-12

ST-8
Shionogi

HCaI

LS174T

MCaIV
A B

6.0
Cranial window 2,500
MCaIV HCaI
5.0
2,000
Permeability (nm10−7 cm/sec)

Tracer size (nm)

4.0
1,500

3.0
1,000

2.0
500

1.0
0
Cranial

Cranial
Sc

Sc
0.0
C MCaIV U87 HGL21 LS174T D
Figure 8.8  •  Heterogeneous permeability of tumor vessels. Vessel permeability varies spatially within a given tumor, between tumors of identical type
implanted in different host organ environments, and between different tumor types in the same organ environment. (A) Vessels within a given tumor are
leaky in some areas and relatively impermeable in others. (B–C) Tumors of different types grown in the same environment show variations in both vessel pore
size and permeability. Pore sizes are measured by the largest tracer particle able to permeate the vessel wall. (D) MCaIV and HCaI tumors implanted in two
different sites (subcutaneous and cranial) have vessels with different pore sizes. For both tumor types, larger pore sizes are observed in the subcutaneous (Sc)
tumors compared with the cranial tumors. ([A and C] From Yuan F, Leunig M, Huang SK, et al. Microvascular permeability and interstitial penetration of
sterically stabilized [stealth] liposomes in a human tumor xenograft. Cancer Res. 1994;54:3352–3356. [B and D] From Hobbs SK, Monsky WL, Yuan F, et al.
Regulation of transport pathways in tumor vessels: role of tumor type and microenvironment. Proc Natl Acad Sci U S A. 1998;95:4607–4612.)

to tumors, and TGF-β induces the production of collagen and other
matrix molecules in tumors.20,156 With the increasing interest in use
of the fragments of matrix constituents for controlling angiogenesis,
our understanding of the molecular and cellular mechanisms of stroma
generation in tumors will increase.44
Interstitial Transport
Once a molecule has extravasated, its movement through the interstitial
space occurs by diffusion and convection.102,157 Diffusion is proportional
to the concentration gradient in the interstitium, and convection
is proportional to the interstitial fluid velocity, which, in turn, is
proportional to the fluid pressure gradient in the interstitium. Just as
the interstitial diffusion coefficient D (cm2/s) relates the diffusive flux
to the concentration gradient, the interstitial hydraulic conductivity
K (cm2/mm Hg/s) relates the interstitial velocity to the pressure
gradient.134 Values of these transport coefficients are governed by the
structure and composition of the interstitial compartment and by the
physicochemical properties of the solute molecule.158–168 The value of
K for a human colon carcinoma xenograft (LS174T), measured by
means of two different methods, was found to be higher than that
of a hepatoma, which, in turn, was higher than that of the normal
liver.168–170 With use of fluorescence recovery after photobleaching,
D of various molecules in tumors was found to be about one-third
that in water and higher than the values in the host tissue (Fig.
8.9A).160,171 Collagen content and structure have a significant effect
on D in tumors.162,166,169,172–176 This is surprising because hyaluronan
and proteoglycans, not collagen, account for most of the resistance
to transport in normal tissues. Because collagen is produced by host
cells (e.g., fibroblasts), the penetrability into a tumor depends on the
 −5
10

−6
10
2 −1
Diffusion coefficient, D, cm s

−7
10

−8
10

PBS
−9
10 U87 DC
U87 CW
Mu89 CW
Mu89 DC
−10
10

0.1 1 10 100

Radius, RH, nm
A

Control injection Collagenase injection

Initial virus
distribution
Specific antibody

Initial cell
infection

1 sec 10 sec 100 sec

First viral
replication

Secondary
cell infection
B C Nonspecific antibody

Figure 8.9  •  Interstitial transport in tumors. Transport of molecules through a tumor is affected by several factors. (A) Diffusivity of a molecule in a tumor
decreases with increasing molecular weight and is dependent on the host-tumor interaction. The diffusivity of macromolecules is lower in subcutaneous tumors
in dorsal chamber (DC; U87 glioblastoma [purple circles] and Mu89 melanoma [blue circles]) than in the same tumors grown in cranial windows (CW; U87
[yellow squares] and Mu89 [green diamonds]), and both are less than in phosphate-buffered saline (PBS [red triangles]). (B) A representative model of improvement
in oncolytic viral distribution and tumor cell infection by collagenase treatment. After direct intratumor injection, viral spread (red area) is limited by fibrillar
collagen (red lines) and results in a cluster of infected cells (light green). The collagen network also restricts the distribution of subsequent viral progeny, and
tumor cell infection beyond the initial injection site is not achieved. In contrast, injection of virus together with collagenase results in a more diffuse distribution
of viral particles and a greater number of initially infected cells (light green). Viral particles released by these cells have greater access to neighboring uninfected
cells. This process results in more widespread secondary infection (dark green) and ultimately greater therapeutic efficacy. (C) Interstitial transport is also
reduced by binding. The fluorescence of a photobleached spot recovers much more slowly with a specific antibody than with a nonspecific antibody. The
binding of the specific antibody hinders the transport of the molecule into the photobleached spot, slowing the fluorescence recovery. ([A] From Pluen A,
Boucher Y, Ramanujan S, et al. Role of tumor-host interactions in interstitial diffusion of macromolecules: cranial vs. subcutaneous tumors. Proc Natl Acad
Sci U S A. 2001;98:4628–4633. [B] From McKee TD, Grandi P, Mok W, et al. Degradation of fibrillar collagen in a human melanoma xenograft improves
the efficacy of an oncolytic herpes simplex virus vector. Cancer Res. 2006;66:2509–2513. [C] Modified from Berk DA, Yuan F, Leunig M, Jain RK. Direct in
vivo measurement of targeted binding in a human tumor xenograft. Proc Natl Acad Sci U S A. 1997;94:1785–1790.)
118 Part I: Science and Clinical Oncology

host-tumor interaction (see Fig. 8.9A). Thus, agents that interfere with
collagen synthesis and/or organization (e.g., relaxin, losartan, bacterial
collagenase) might increase interstitial transport in tumors159,172,173 (see
Fig. 8.9B). The time constant for a molecule with diffusion coefficient
D to diffuse across a distance L is approximately L2/4D. For diffusion
of immunoglobulin G (IgG) in tumors, this time constant is on the
order of 1 hour for a 100-µm distance, days for a 1-mm distance,
and months for a 1-cm distance. So for a 1-mm distance in tumor,
diffusional transport would take days, and for a 1-cm distance in tumor,
it would take months. Furthermore, the path through the interstitium
is tortuous over large length scales, greatly increasing the distance a
molecule must travel.161 If the central vessels have collapsed completely
owing to cellular proliferation and interstitial matrix rearrangement,
the reduced delivery of macromolecules by blood flow would make
diffusion the primary mechanism of delivery to this necrotic center.6,80
Binding of a low- or high-molecular-weight drug to plasma proteins
and various tissue components could further retard their transport in
tumors.171,177–183 The role of binding is clearly illustrated in Fig. 8.9C,
which compares the rate of fluorescence recovery of a photobleached
spot in tumor tissue injected with a nonspecific versus a specific IgG.
In addition to the heterogeneity of D in tumors, the most unexpected
result of these photobleaching studies was the large extent (30%–40%)
of nonspecific binding.171 These results collectively suggest that the
interstitial compartment of a tumor can be a formidable barrier to
the uniform delivery of therapeutic macromolecules (e.g., antibodies,
genes) in tumors, and strategies are needed to modify this barrier.
Lymphangiogenesis and Lymphatic Transport
In most normal tissues, extravasated plasma and macromolecules are
taken up by the lymphatics and returned to the blood circulation. It
is widely accepted that lymphatic vessels are present in the tumor
margin and the peritumoral tissue (Fig. 8.10A). Indeed, invasion of
peritumoral lymphatics is considered to be a poor prognostic factor
for several tumors (e.g., breast, colorectal, and endometrial cancers),
and lymphatic metastasis is a major cause of morbidity and mortality
for others (e.g., melanoma, head and neck cancer, lung cancer, and
cervical cancer). The hotly debated issue for nearly a century has been
whether anatomically defined lymphatic vessels are present within
solid tumors and, if so, whether they function (see Fig. 8.10A).184,185
Currently available immunohistochemical markers stain for structures
in some tumors that resemble lymphatic vessels. Because many of
these markers lack specificity, however, it is not clear whether they
stain functional lymphatic vessels, ECs from remnant lymphatic vessels,
or some other structures (e.g., preferential fluid channels).155,169,186,187
Mechanical solid stress induced by proliferating tumor cells and matrix
production compresses and impairs lymphatic vessels that are co-opted
or formed within a tumor.6,79,81 The impaired lymphatic vessels, in
turn, contribute to the interstitial hypertension characteristic of animal
and human tumors (to be discussed shortly). Embryonic lymphatic
vessels originate primarily from blood vessels according to the following
process (see Fig. 8.10B).188–190
1. In the early embryo, ECs of the cardinal vein express lymphatic
vascular endothelial receptor 1 (LYVE1) and VEGFR3, molecules
observed primarily (but not exclusively) on lymphatic vessels in
normal adult tissues.
2. An as yet unknown signal triggers the expression of the homeobox
gene Prox1 so that the protein is displayed in a polarized fashion
in the ECs of the cardinal vein. This marks the first stage of
commitment to the lymphatic lineage.
3. These LYVE1+VEGFR3+Prox1+ cells then start to bud, again in a
polarized fashion.
4. At this stage, these early lymphatic ECs start expressing secondary
lymphoid chemokine and increased levels of VEGFR3, markers
of mature lymphatic ECs.
5. They then begin to form the lymphatic system.
Members of the angiopoietin family (Ang-2) and its receptor (Tie-2)
and podoplanin are presumably involved in the maturation and
patterning of these nascent lymphatic vessels.191 The hematopoietic
signaling pathway, Syk/SLP-76, contributes to the separation of
lymphatics from blood vessels. The molecules involved in angiogenesis
are also involved in lymphangiogenesis. For example, VEGF-C and
VEGF-D can induce both angiogenesis and lymphangiogenesis and
are associated with lymphogenic metastasis in a variety of tumors.154
Their receptor VEGFR3 is present in both lymphatic and selected
vascular endothelium. Nonetheless, sprouting angiogenesis does not
occur during the initial growth of lymph node metastases.192 As is the
case with vascular angiogenesis, other positive and negative regulators
(e.g., angiopoietins) and other receptors (e.g., chemokine receptors
and neuropilins) could be involved in lymphangiogenesis, and as
discussed already, mechanisms analogous to co-option, intussusception,
sprouting, and vasculogenesis might operate in lymphatic growth.12,191
Similar to the organ-specific angiogenic molecule (EG-VEGF) and
EPCs, there could be organ-specific lymphangiogenic molecules and
lymphatic EPCs that contribute to tumor-associated lymphangiogen-
esis.23,39,189 Moreover, the proteolytic processing of lymphangiogenic
molecules and the phenotype and function of the resulting lymphatics
might depend not only on the tumor type but also on the host organ
in which the tumor is growing.77,187–189
The mechanical and/or molecular signals that could trigger the
lymphangiogenic switch are unknown. Because lymphatic vessels help
maintain the balance of fluid in tissues, hydrostatic pressure is a probable
trigger. Whether the hyperplasia and the increased density of lymphatic
vessels seen in the tumor margins are a response to elevated hydrostatic
pressure in tumors and whether the newly formed lymphatics are able to
remain open and carry cancer cells are open questions. Techniques such

Figure 8.10  •  (A) Schematic of lymphatics in tumors. It is widely accepted that peritumoral lymphatics exist and that metastasis can occur via these
lymphatic vessels. Evidence shows that structures within tumors that stain for lymphatic markers are not functional. Lower left inset, Molecular players in
lymphangiogenesis. (B) Mechanisms of lymphatic vessel formation and separation from blood vessels. (C) The tumor is modeled as a sphere of radius R,
embedded in a body fluid (e.g, peritoneal cavity, pleural cavity) or host tissue. The interstitial fluid oozing from the tumor periphery carries therapeutics (e.g,
monoclonal antibodies), growth factors (e.g, vascular endothelial growth factor [VEGF]–A, VEGF-B, VEGF-C, and VEGF-D), and cells (e.g., metastatic
cells) into the surrounding fluid or tissue. The result may be higher fluxes of cancer cells and growth factors from the tumor into the surrounding tissue or
body fluid. The growth factors (including VEGF-A, VEGF-B, VEGF-C, and VEGF-D) can induce angiogenesis and lymphangiogenesis, and the cancer cells
can contribute to metastatic dissemination via the lymphatic or blood vessels. Fluid seeping from the tumor surface can also cause edema (e.g., around brain
tumors) and ascites formation (e.g., ovarian cancer). Antiangiogenic therapy can “normalize” the tumor vasculature, leading to lower interstitial fluid pressure
(IFP) at the center, less steep IFP gradients, and lower fluid flow rates at the tumor margin, thus potentially reducing peritumoral edema, ascites formation,
and lymphatic metastasis. Furthermore, the normalized vessels may be more resistant to cancer cell intravasation, a prerequisite for hematogenous metastasis.
([A] From Jain RK, Fenton BT. Intratumoral lymphatic vessels: a case of mistaken identity or malfunction? J Natl Cancer Inst. 2002;94:417–421. [B] From
Jain RK, Padera TP. Lymphatics make the break. Science. 2003;299:209–210. Illustration: Katherine Sutliff. Reprinted with permission from AAAS. [C] From
Jain RK, Tong RT, Munn LL. Effect of vascular normalization by anti-angiogenic therapy on interstitial hypertension, peritumor edema and lymphatic
metastasis: insights from a mathematical model. Cancer Res. 2007;67:2729–2735).
 Tumor Microenvironment: Vascular and Extravascular Compartment  •  CHAPTER 8 119

Mock transduced tumor VEGF-C overexpression

Tumor cell VEGFR-3

Tumor cell

VEGF-C

Normal
lymphatic Increased
VEGFR-3 vessel lymph flow

Dilated
e lymphatic vessel
ad
b lock
R-3
GF
VEGF-C is blocked VE
VEGF-C overexpression
by anti-VEGFR-3 Ab
Increased tumor cell arrival Afferent
in the lymph node lymphatic vessel
Tumor cell VEGF-C
VEGFR-3 Tumor cell Subcapsular sinus

Lymph node
cortex
Apoptotic and
Lymphatic hyperplasia proliferation rate
Lymphocyte
is blocked Anti-VEGFR-3 the same as control
A

Embryonic vein Metastatic cancer cell

Step 1 LYVE-1
VEGFR-3 Lymphatic
Hyperplastic vessel
lymphatic
vessel Tumor-generated growth factors
(VEGF-A, VEGF-C…)

tic Step 2 Blo

ha od
p
Lym

Prox-1

VEGFR-3 Uniformly Gradient

Step 3 high IFP of IFP
except at
VEGFR-3 margin
Prox-1
SLC Early
lymphocytic
endothelial cells
LYVE-1

Step 4 VEGFR-1
VEGFR-2
Prox-1
Tie-2
Tie-2 High IFV Lower IFV
Nrp-2
Nrp-2 at margin at margin
CD31
SLC
CD34
etc...
etc...

IFP
Step 5 Lymphocytes
with Syk and
SLP-76

? C Untreated tumor Normalized tumor

LEPCs ?
?Syk
?SLP-76
B
120 Part I: Science and Clinical Oncology

Table 8.2  Interstitial Fluid Pressure (in Millimeters 12

of Mercury) in Normal and Neoplastic

Interstitial pressure (mm Hg)

Tissues in Patients 10

Tissue Type N Mean Range References 8

Normal skin 5 0.4 −1.0 to 3.0 208 6
Normal breast 8 0.0 −0.5 to 3.0 208
Head and neck 19 13.2 4.0–33.0 207 4
carcinomas
2
Cervical carcinomas 12 15.7 10.0–26.0 205
Cervical carcinomas 102 19.0b −3.0 to 48.0 214 0
Lung carcinomas 26 10.0 1.0–27.0 124 0.0 0.4 1.2 2.0
Metastatic melanomas 10 21.7 0.0–60.0 209 A Depth (mm)
Metastatic melanomas 12 14.3 2.0–41.0 204
Breast carcinomas 13 29.0 5.0–53.0 211
Breast carcinomas 8 15.0 4.0–33.0 208
HER2− breast 70 14.0 0.0–43.0 273
carcinomasa
Brain tumorsa 17 7.0 2.0–15.0 171
Brain tumorsa 11 2.0 −0.5 to 8.0 213
Colorectal liver 8 21.0 6.0–45.0 208
metastasis
Lymphomas 6 4.6 1.0–12.5 209
Renal cell carcinoma 1 38.0 — 208
Pancreatic ductal 4 11.8 6.1–16.6 322
adenocarcinoma P

a
t
Patients were treated with antiedema therapy.
b
Interstitial fluid pressure given is a median value.

as microlymphangiography, reagents that block signaling of VEGF-C
and VEGF-D, and lymphangiogenic factors yet to be discovered will
allow us to answer these important questions.4,187,189,193–202
Interstitial Hypertension
Unlike normal tissues, in which the IFP is around 0 mm Hg, both
animal and human tumors exhibit interstitial hypertension (Table
8.2).154,157,170,203–214 The tumor IFP begins to increase as soon as the
host vessels become leaky in response to permeability factors such as
VEGF, and thus IFP can be lowered by antibodies against VEGF or
VEGFR2.215–217 The IFP increases with tumor size in some tumors
and remains independent of tumor size in others.203–205,207,208 Three
mechanisms contribute to interstitial hypertension in tumors. In normal
tissues, the lymphatics maintain the fluid homeostasis; thus the lack
of functional lymphatics in tumors is a key contributor. Indeed, DiResta
and colleagues were able to lower the IFP by placing “artificial lymphat-
ics” in tumors.218 The second contributor is the high permeability of
tumor vessels. As a result, the hydrostatic and oncotic (colloid osmotic)
pressures become almost equal between the intravascular and extra-
vascular spaces.206,219 At least two pieces of evidence support this
hypothesis. First, reducing permeability by blocking VEGF signaling
lowers IFP.216,217,220–223 Second, IFP increases and decreases with the
microvascular pressure (MVP) within seconds.224–226 The two mecha-
nisms described thus far can explain interstitial hypertension only up
to 20 to 30 mm Hg, the MVP of most exchange vessels (the hydrostatic
pressure within the lumina of capillaries) in human bodies, but IFPs
as high as 94 mm Hg have been measured in human tumors.205 Because
MVP is the driving force for IFP in tumors, these tumors must have
a high MVP. Indeed, this is the case.206 There are two possible explana-
tions for elevated MVP in tumors: the tumor vessels have reduced
B
Figure 8.11  •  Interstitial fluid pressure (IFP) profile in a tumor and its
potential application. (A) IFP is elevated and nearly uniform in the bulk of
the tumor and drops precipitously in the tumor margin. (B) This elevated
pressure can be exploited to determine the location of a tumor precisely. ([A]
Modified from Boucher Y, Baxter LT, Jain RK. Interstitial pressure gradients
in tissue-isolated and subcutaneous tumors: implications for therapy. Cancer
Res. 1990;50:4478–4484. [B] From Jain RK, Boucher Y, Stacey-Clear A, et al.
Method for locating tumors prior to needle biopsy. US patent 19955396897.
1995.)
arterial resistance, so the MVP becomes closer to arterial pressure;
and/or the tumor vessels have increased venous resistance because of
compression and tortuousity, so the whole intratumor vascular network
is under hypertension. Indirect evidence for the latter comes from
the decrease in IFP after decompression of tumor vessels by taxol-
induced apoptosis of perivascular cells.80
The elevated pressure can compromise the tumor microcirculation
and delivery of therapeutics in three ways:
1. Reduced transmural pressure gradients due to equilibrium between
MVP and IFP reduce convection across tumor vessel walls and
thus compromise the transport of macromolecules.206,225
2. Because IFP is nearly uniform throughout a tumor and drops
precipitously in the tumor margin, the interstitial fluid oozes out
of the tumor into the surrounding normal tissue, carrying mac-
romolecules with it (Fig. 8.11).203,227
3. Finally, transmural coupling between IFP and MVP due to high
permeability of tumor vessels can lead to blood flow stasis in tumors
without physically occluding the vessels.92–94
 Tumor Microenvironment: Vascular and Extravascular Compartment  •  CHAPTER 8 121

The enhanced permeability can also facilitate lymphatic metastasis
(see Fig. 8.10C). Thus, decreasing vascular permeability might restore
the transmural pressure gradients and potentially resume or reestablish
blood flow in the nonperfused regions of tumors. Some direct and
indirect antiangiogenic therapies might “normalize” the tumor vas-
culature through this mechanism (see Fig. 8.4).101 This normalization
can also reduce the number of cells shed into peritumor lymphatics
and alleviate peritumor edema (see Fig. 8.10C).74,228
METABOLIC MICROENVIRONMENT
Hypoxia
A key function of the vasculature is to provide adequate levels of
nutrients and oxygen to the parenchymal cells and to remove waste
products. Based on the anatomy of the capillary bed and a mathemati-
cal model of oxygen diffusion and consumption, the Nobel laureate
August Krogh introduced the concept of a diffusion limit for oxygen
of 100 to 200 µm nearly a century ago.229 This unit of tissue—a
single capillary surrounded by a 100- to 200-µm-radius cylinder—is
referred to as a “Krogh cylinder” in physiology. Nearly 50 years later,
Thomlinson and Gray identified similar “cords” in human lung cancer
and found necrotic cells beyond 180 mm away from blood vessels,
presumably as a result of lack of oxygen.230 This is referred to as
“chronic hypoxia” or “diffusion-limited” hypoxia. Although various
hypoxia markers and microelectrodes have suggested these gradients,
the first direct measurements of these perivascular gradients, along
with Po2 and blood flow rate of the same vessels, became possible only
with the development of phosphorescence quenching microscopy (Fig.
8.12A).231,232 As discussed previously, blood flow in tumor vessels is
intermittent, and thus some regions of a tumor are starved for oxygen
periodically. The resulting hypoxia is referred to as “acute hypoxia” or
“perfusion-limited hypoxia.”233,234 A necessary consequence of intermit-
tent blood flow is the resumption of blood flow after shutdown, and
the resulting production of free radicals can lead to “reperfusion injury”
or “reoxygenation injury,” applying additional selection pressure on
cancer cells.
Low pH
Another consequence of the abnormal microcirculation of the tumor
is low extracellular pH. There are at least two sources of H+ ions in
tumors—lactic acid and carbonic acid.235 The former results from
glycolysis and the latter from conversion of CO2 and H2O via carbonic
anhydrase. The intracellular pH of cancer cells remains neutral or
alkaline (pH 7.4), however, despite the acidic extracellular pH. Because
carbonic anhydrase-9 (CAIX), various glucose transporters (GLUT1,

7.4 14
pH
7.3 pO2 (mm Hg)
12

7.2
10
pO2 (mm Hg)

7.1
pH

8
7.0
6
6.9
4
6.8

6.7 2

6.6 0
0 50 100 150 200 250 300 350 400
A Distance (µm)

30

7.2
pO2 (mm Hg)

20
pH

7.0

pH
pO2 (mm Hg)
6.8 10
0 200 400
B
Figure 8.12  •  (A) pH and Po2 as a function of distance from a blood vessel in a tumor. The tumor environment becomes progressively more hypoxic
and acidic farther away from a blood vessel. (B) Lack of correlation between pH and Po2 in tumors. (From Helmlinger G, Yuan F, Dellian M, et al. Interstitial
pH and Po2 gradients in solid tumors in vivo: high-resolution measurements reveal a lack of correlation. Nat Med. 1997;3:177.)
122 Part I: Science and Clinical Oncology
GLUT3), and enzymes in the glycolytic pathway are upregulated
by hypoxia, one would expect low extracellular pH and hypoxia to
track each other and to colocalize with regions of low blood flow.236
Surprisingly, there is a lack of spatial correlation among these parameters
(see Fig. 8.12B), a discovery made possible by developments in optical
techniques that permit the simultaneous high-resolution mapping of
multiple physiologic parameters.231 A potential explanation for this
lack of concordance is that some perfused tumor vessels carry hypoxic
blood.231 Thus, although they might not be able to deliver adequate
oxygen to the surrounding cells, they may be able to carry away the
waste products (e.g., lactic acid).
Molecular, Cellular, and Therapeutic Consequences
The abnormal microenvironment promotes the malignant tumor
progression. Indeed, hypoxia is a validated independent negative
prognostic biomarker for many cancers, and endogenous markers of
acidosis are potential negative biomarkers.237–240 Hypoxia and acidosis
select for aggressive cancer cells and promote a vicious cycle of
angiogenesis, desmoplasia, and inflammation and immunosuppression
that leads to exacerbated abnormal metabolism.241–244 Increased hypoxia
predicts the likelihood of metastasis.245
Hypoxia and acidosis indicate poor delivery of blood, nutrients,
and blood-borne therapies to tumors, and they also directly promote
resistance to radiotherapy, chemotherapy, and immunotherapy. The
presence of oxygen during irradiation makes the damage to DNA
produced by radiation-induced free radicals permanent, whereas such
damage can be repaired under hypoxic conditions.246 Therefore, hypoxia
in solid tumors significantly reduces their radiation sensitivity. In
addition, hypoxia and low extracellular pH adversely affect the cel-
lular uptake and cytotoxicity of certain therapeutics, although there
are several drugs whose efficacy increases in such conditions.246–249
Finally, hypoxia induces immune suppression and reduces the efficacy
of immune therapies.250–253,253a As a result, for nearly half a century
considerable preclinical and clinical efforts have been focused on
alleviating hypoxia by improving tumor perfusion with various therapies,
including:
• Mild hyperthermia or drugs
• Increased oxygen content of the blood (e.g., via hyperbaric
oxygenation)
• Increased hemoglobin and hematocrit (e.g., via erythropoietin)
• Radiation sensitizers
Unfortunately, these strategies’ success in clinical studies has been
limited for multiple reasons. One common theme of these strategies
is that they rely on the abnormal tumor vasculature. As a result, these
strategies are incapable of increasing Po2 in all regions of tumors to
optimal levels and/or delivering radiation sensitizers or chemotherapeutic
drugs to all regions of a tumor at therapeutically effective levels.
Currently, three broad strategies are emerging:
1. Use of hypoxia and/or low pH to selectively activate drugs in the
tumor or to attract anaerobic bacteria
2. Dissection of hypoxia-induced pathways to identify novel targets
for drug development
3. Normalization of tumor vasculature and/or reduction of solid stress
to alleviate hypoxia254,255
The first strategy led to the development of agents such as tira-
pazamine and to the rejuvenation of interest in bacteriolytic therapy;
both approaches are still being tested in clinical trials.89,246 The second
strategy has revealed several molecular determinants of the physiologic
and pathophysiologic responses to hypoxia.236 The balance between
hypoxia-induced apoptosis and necrosis on one hand, and the increased
resistance to cell death mediated by various hypoxia-induced pathways
on the other, determines whether a tumor can survive and even grow
under hypoxic conditions. Ultimately, hypoxia might select for tumor
cells that are more malignant, more invasive and genetically unstable,
and less susceptible to apoptosis, thus rendering them resistant to
various therapies. Therefore several molecules in the hypoxia-induced
pathways are now being targeted in the development of diagnostic
and therapeutic agents. Nonetheless, the first and second strategies,
although exploiting hypoxia, are dependent on the inadequate vas-
culature for delivery of the therapeutic agents and are thus most
amenable to tumors featuring intermittent rather than diffusion-limited
hypoxia. The third strategy involving normalization of tumor vessel
function by antiangiogenic and anti–solid stress therapy may reduce
intermittent and diffusion-limited hypoxia within the tumor micro-
environment and synergize with other strategies.256
Hypoxia-induced pathways include genes involved in the following
processes:
• Oxygen delivery (e.g., heme oxygenase 1, erythropoietin)
• Glycolysis and glucose uptake (e.g., GLUT1 and GLUT3,
hexokinase-1 and hexokinase-2)
• pH control (e.g., carbonic anhydrase-9 and carbonic anhydrase-12)
• Stress-response pathways (e.g., growth arrest- and DNA damage-
induced gene GADD153)
• Growth factor signaling (e.g., IL-6, IL-8, insulin-like growth factor-2
[IGF-2])
• PDGF-β
• Angiogenesis (e.g., VEGF-A, VEGFR1, Ang-2, Tie-2, FGF-3,
TGF-β, nitric oxide synthase [NOS], cyclooxygenase-2 [COX-2],
hepatocyte growth factor [HGF])
• Transcription (e.g., HIF-1α and HIF-2α, JUN, FOS, nuclear
factor–κB [NF-κB])
• Apoptosis (e.g., BCL-interacting killer [BIK], annexin V, 19-kd
interacting protein-3 [NIP3], NIP3-like protein X [NIX])
• Growth inhibition signaling factors (e.g., p21, p27, GADD153)
• Invasion and metastasis (e.g., metalloproteinases [MMPs], MMP-13,
plasminogen activator inhibitor-1 [PAI-1])236
Of the various molecules involved in sensing and responding to
hypoxia, HIF-1α has received the most attention. This transcription
factor is upregulated in several human tumors.236 Regulated by a
proline hydroxylase, HIF-1α can activate the genes for desmoplasia,
angiogenesis, vasodilation, glycolysis, and erythrocyte production by
binding to the hypoxia-response element.243,257,258 Surprisingly, teratomas
arising from HIF-1α−/− embryonic stem cells grow more rapidly despite
lower levels of VEGF and angiogenesis.52,259 This counterintuitive
finding could be a result of the ability of HIF-1α−/− cells to survive
under hypoxic conditions instead of undergoing apoptosis.49 Interesting
to note, other HIF-1α−/− cancer cells lead to slowly growing tumors.
As a result, molecular therapies that target HIF-1α or hypoxia-response
element are under intensive investigation for cancer detection and
treatment.260
CLINICAL RELEVANCE OF APPROACHES TO
ALLEVIATE HYPOXIA
Two major problems currently plague the nonsurgical treatment of
malignant solid tumors. First, physiologic barriers within tumors
impede the delivery of therapeutics and oxygen (a key sensitizer to
ionizing radiation and other therapies) at effective concentrations to
all cancer cells.261,262 Second, inherent or acquired resistance result-
ing from genetic and epigenetic mechanisms, which themselves are
promoted by hypoxia, reduces the effectiveness of both conventional
and novel therapies.263,264 Clinical imaging studies are consistent
with the hypothesis that patients whose disease responds to therapies
with increased perfusion and oxygenation have better outcomes (Fig.
8.13A–B).96,265–268 Therefore cancer researchers must strive to optimize
strategies that decrease hypoxia in tumor lesions. How can we exploit
the pathophysiology of tumors to overcome hypoxia for better disease
management?
 Tumor Microenvironment: Vascular and Extravascular Compartment  •  CHAPTER 8 123

100 1
P = .028 D
80
.8 In ecre
cr
In ea ase
Overall Survival (%)

De cre sed d p

Overall Survival
60

cr ase hy erfu
.6
ea d p
se pe oxi sion
40

Dose
.4
d rfu a
P = 0.035 hy s
20
po ion
.2
∆ MTT ≤ 0.76 (n = 7)
xia
0 0.76 < ∆ MTT ≤ 1.05 (n = 6)
0 200 400 600 800 ∆ MTT > 1.05 (n = 6)
0
Time (days)
No effect on vessels
Perfusion increased (n = 20)
0 6 12 18 24 30 36
Follow-up time (months)
A Perfusion stable or decreased (n = 20)
B C Length of treatment

Mature vessels
Supply large volumes of tissue
Untreated Normalized
Well Normalization Normalization
Vessel dense
perfused including through
vessel vessel
pruning Combination
fortification only
of vessel
fortification
and stromal
High extent of Enha
Enhanced reprogramming
normalization
oxy
xy
yg
oxygenation

Sparse Dense
perfusion perfusion
Ex Leaves Grants
prucess regions all
nin ive without regions
Vessel sparse g Poorly
flow flow
perfused
Solid tumors Stromal
pretreatment reprogramming

Insufficient Dimi
Diminished
vascular oxygenation Proangiogenesis
network or
stromal destruction

Leaky vessels
Supply small volumes of tissue
blood perfusion recruited
pericytes
vessels oxygenation pericytes
Hypoxia Normoxia
D E
Figure 8.13  •  Vascular “normalization” in cancer patients. (A) Glioblastoma patients who responded to antiangiogenic therapy with increased perfusion
and oxygenation (blue) had longer overall survival compared with those who did not (orange). These data support the normalization hypothesis as well as the
need to test whether perfusion and oxygenation are predictive biomarkers of response in large, prospective trials. (B) Non–small cell lung cancer patients who
responded to antiangiogenic therapy with reduced (green) or stable (red) mean transit time—the absolute value of this quantity is inversely proportional to
perfusion—had longer overall survival than patients with increased mean transit time (gray). (C) In patients, a tumor vessel “normalization window” of
enhanced perfusion and oxygenation depends on the dose and the length of treatment. (D) Preclinical and clinical studies support the hypothesis that the
therapeutic effect of antiangiogenic therapies is best when vascular density is high and normalization fortifies rather than prunes vessels. The left quadrants
depict tumor vessels (red) before antiangiogenic therapy. The right quadrants depict possible outcomes of antiangiogenic therapy. Tumors with low pretreatment
vessel density do not respond to antiangiogenic therapy (bottom left to right quadrant), as the increase in vessel function from the recruitment of pericytes (teal)
cannot overcome the paucity of vessels. In contrast, tumors with high baseline vascularity that recruit pericytes have better outcomes (top left quadrant to top
right quadrant) than tumors with excessive pruning (top left quadrant to bottom right quadrant). (E) Combinations of normalizing therapies that increase vessel
maturity without pruning with solid stress alleviating agents that reprogram rather than destroy stroma might be optimal for alleviating hypoxia. Tumors
(black) usually feature both leaky vessels and sparsely perfused regions, thereby leaving the tissue hypoxic (white). Reengineering the tumor microenvironment
could promote normoxia (blue) and lead to improved treatment outcomes. Normalization strategies that induce pruning lead to sparse perfusion (orange),
whereas proangiogenic and stromal destruction strategies increase vessel leakiness (red). Fortification of vessels with pericytes (green) or reprogramming the
stroma to alleviate solid stress (magenta) avoid detrimental effects, and the combination (greenish blue) might be ideal to alleviate hypoxia in most cases. ([A]
From Batchelor TT, Gerstner ER, Emblem KE, et al. Improved tumor oxygenation and survival in glioblastoma patients who show increased blood perfusion
after cediranib and chemoradiation. Proc Natl Acad Sci U S A. 2013;110:19059–19064. [B] From Heist RS, Duda DG, Sahani DV, et al. Improved tumor
vascularization after anti-VEGF therapy with carboplatin and nab-paclitaxel associates with survival in lung cancer. Proc Natl Acad Sci U S A. 2015;112:1547–1552.
[C–E] From Martin JD, Fukumura D, Duda DG, et al. Reengineering the tumor microenvironment to alleviate hypoxia and overcome cancer heterogeneity.
Cold Spring Harb Perspect Med. 2016;6:a027094.)

Vascular Normalization Through types of cancers including some metastatic settings.10 Studies in a
Antiangiogenic Therapy range of these tumor types have largely confirmed the hypotheses that
anti-VEGF/VEGFR therapy has antivascular effects and can induce
The broad success of antiangiogenic therapy in phase III clinical trials vascular normalization.221,223,267–274 How these effects on the vasculature
across various tumor types provides substantial data to generate new relate to hypoxia and patient survival is dependent on the tumor type
hypotheses regarding strategies to eliminate hypoxia. There are 12 and a topic of continued research.
VEGF-blocking antiangiogenic agents (bevacizumab, regorafenib, Tumor types that are highly sensitive to antiangiogenic therapy
ramucirumab, sorafenib, sunitinib, pazopanib, axitinib, vandetanib, are either highly VEGF dependent (i.e., clear cell renal carcinoma,
lenvatinib, nintedanib, carbozantinib, and aflibercept) that are approved ovarian cancer, cervical cancer, and thyroid cancer) or highly vascularized
by either the Food and Drug Administration or the European Medicines (i.e., neuroendocrine cancers and hepatocellular carcinoma [HCC]).10
Agency.10,12,269 These agents have been approved for treatment of 10 These types of tumors have a “tumor vessel” phenotype, which consists
124 Part I: Science and Clinical Oncology
of vessels among cancer cells without significant intervening stroma.275
Clinical studies in these tumors indicated that blockade of VEGF has
antivascular and normalizing effects, which are identified as potential
biomarkers of response.274,276–278 Preclinical and clinical studies also
demonstrated that there is a dose- and treatment duration–dependent
window in which VEGF blockade increases oxygen delivery by normal-
izing before excessively pruning vessels (see Fig. 8.13C).268,272,279,280
Nonetheless, it remains unclear whether these antivascular and normal-
izing effects lead to increased oxygen delivery or not. Indeed, blocking
VEGF in highly dependent tumors might be sufficient to elicit a
strong antitumor response even while concurrently increasing hypoxia,
but increased hypoxia eventually leads to resistance, as several studies
in liver cancer have demonstrated.281–284 Thus, efforts must be made
to maintain and improve the high response rates of antiangiogenic
therapy in these sensitive tumors while avoiding increasing hypoxia
in order to extend overall survival.
Other tumor types that range from partially to mostly resistant to
antiangiogenic therapy, such as colorectal and breast cancer, respectively,
often have a stromal vessel phenotype characterized by dense stroma
encapsulating avascular cancer cell nests.275 Pancreatic ductal adeno-
carcinoma, which is a highly desmoplastic tumor type, is resistant to
antiangiogenic therapy.10 In this phenotype, VEGF blockade destroys
the stroma including blood vessels without reducing tumor size even
transiently.275 In the long term, destruction of the stroma could increase
disease progression.285–287 Stromal vessel phenotype tumors have a
lower vessel density than tumor vessel phenotype tumors.275 Less than
half of the vessels in desmoplastic preclinical breast and pancreatic
tumor models are patent and perfused because of solid stress.288,289
Thus, stromal vessel phenotype tumors are usually hypovascular and
hypoperfused. Because VEGF blockade can increase existing vessel
function but it cannot create new vessels, it might only be effective
in tumors that have a relatively high microvessel density and when
the treatment induces pericyte recruitment (vessel maturation) without
pruning (see Fig. 8.13D).242,258,273,290–292 In addition to preclinical and
clinical tissue biomarker studies, the findings of clinical imaging studies
indicating that patients with hypoxic tumors are unresponsive to VEGF
blockade are consistent with this hypothesis.293–295 A small clinical
study has suggested that a cytotoxic agent that likely reduces solid
stress could be more efficient at increasing oxygenation than VEGF
blockade in breast cancer.296
Mathematical modeling and preliminary clinical studies indicate
that combining antiangiogenic therapies with solid stress–alleviating
agents in treating stromal vessel phenotype or antiangiogenic therapy–
resistant tumors might increase oxygen delivery further (see Fig.
8.13E).297,298 Metronomic chemotherapy alone could normalize vessels
by modulating angiogenesis pathways while alleviating solid stress by
killing cancer cells.299
To an even larger extent than in stromal vessel phenotype tumors,
antiangiogenic therapies are ineffective against early metastases.300
Lymph node metastases lack angiogenesis and thus do not respond
to antiangiogenic therapies.192 Similarly, preclinical evidence indicates
that lung metastases co-opt the existing vasculature without relying
on sprouting angiogenesis.301 When cancer cells are co-opting existing
vasculature, they are nearly mature and normally functioning vessels,
so normalizing therapy is unlikely to affect tumor progression. Fur-
thermore, these invading cells are insensitive to chemotherapy, so new
cytotoxic agents must be developed to target these cells.302–307
Biomarkers of Response to Antiangiogenic Therapy
As with many targeted therapies, the survival benefit of cancer patients
after antiangiogenic therapies remains modest and restricted to as yet
unidentified subsets of tumors. Unfortunately, unlike with many
anticancer targeted therapies, there are no validated biomarkers of
patient selection, response, or resistance for antiangiogenic agents.38,308
If biomarkers could be found to identify patients more likely to benefit
from these drugs, the survival benefit in patients on anti-VEGF drugs
would be improved. Unlike preclinical models, the phase III bevaci-
zumab experience in metastatic colorectal cancer patients did not
identify P53, KRAS, or BRAF status, VEGF or thrombospondin-2
(TSP2) expression, or microvascular density at baseline as predictive
markers of response.309,310 Similarly, circulating VEGF levels—although
of potential prognostic biomarker value—have unclear predictive
biomarker value.38,277 In collaboration with our clinical colleagues, we
have been examining these questions in over 30 multidisciplinary
trials by measuring tissue, circulating, and imaging biomarkers at
various time points during and after treatment, and correlating these
with treatment outcomes. Several promising biomarkers have emerged.
One is the level of circulating sVEGFR1/sFLT1 (an endogenous blocker
of VEGF and PlGF) as a biomarker of intrinsic resistance to anti-VEGF
therapy. Locally advanced rectal carcinoma patients who had high
plasma sVEGFR1 levels before treatment were less likely to benefit—or
experience toxicities—from bevacizumab therapy combined with
chemoradiation.223,311 Later the same association was found for newly
diagnosed GBM, triple-negative breast cancer, non–small cell lung
cancer, HCC, and metastatic colorectal carcinoma patients treated
with anti-VEGF agents.267,273,312–315 Of interest, a retrospective analysis
in two randomized phase III trials showed that a genetic variation in
the VEGFR1 gene correlates with increased VEGFR1 expression and
poor outcome of bevacizumab treatment in metastatic renal cell
carcinoma and pancreatic ductal adenocarcinoma patients.257 A second
biomarker is the SDF1α/CXCR4 axis as a potential evasive pathway.
Circulating levels of the chemokine SDF1α increase in patients at
the time of evasion from anti-VEGF therapies. This was the case in
rectal carcinoma patients treated with bevacizumab, in GBM patients
with cediranib, in HCC patients with sunitinib, and in sarcoma patients
with sorafenib.220,271,276,314,316 Interesting to note, the sources of SDF1α
are different in each of these diseases, and so is the role of the SDF1α/
CXCR4 pathway.317 For example, in GBM this pathway appears to
facilitate invasion of cancer cells and co-option of host vessels by
invading cancer cells. These biomarker candidates need to be tested
prospectively.
Toxicity of Antiangiogenic Therapy
Experimental studies showed that in 11 of the 17 healthy organs
studied, VEGF blockade significantly decreased the number of normal
capillaries.318 In cancer patients, most anti-VEGF agents often induce
proteinuria, hypertension, thyroid-stimulating hormone elevation, and
gastrointestinal toxicity, but agent-specific toxicities have also been
reported.11,319 In addition, the long-term effects of antiangiogenic
therapies in patients with less advanced lesions remain to be established.
Solid Stress Alleviation Through Stromal
Reprogramming
Solid stress comprises the forces generated by proliferating and contract-
ing cancer and stromal cells that are stored as strain energy and applied
by the extracellular matrix and surrounding tissue.289,320–322,322a
Hyaluronan and collagen collaborate to transmit solid stress to compress-
ing vessels, suggesting that vessel compression is a problem in stromal
vessel phenotype tumors.288,323 Nonetheless, vessels are compressed in
small dysplastic lesions, suggesting that even vessels in tumor vessel
phenotype tumors might be compressed.324 Targeting solid stress by
depleting the stroma could aggravate tumor aggressiveness.285,287,325
Thus, stromal reprogramming is favored, as activated fibroblasts that
produce and maintain desmoplasia are deactivated, leading to vessel
decompression and increased oxygenation.288,326,327 Retrospective studies
each surveying hundreds of patients with non–small cell lung cancer,
pancreatic ductal adenocarcinoma, and renal cell carcinoma have
supported the hypothesis that repurposing angiotensin system inhibitors
from hypertension to reprogramming the stroma of cancer can increase
patient survival.328–332 Preliminary clinical data indicate that angiotensin
system inhibition in patients decompresses vessels, which is consistent
 Tumor Microenvironment: Vascular and Extravascular Compartment  •  CHAPTER 8 125

with preclinical data.333 In addition, metastases are desmoplastic and
preclinical data indicate that angiotensin system inhibition could inhibit
metastasis through inhibition of monocyte recruitment.334,335 Clinical
trials are ongoing to test whether stromal reprogramming can increase
survival.335a Stromal depleting agents have had mixed success clinically;
agents targeting lysyl oxidase homolog 2 and Hedgehog pathway
signaling have failed, whereas one agent targeting hyaluronidase has
succeeded in a phase II trial.326,336–338
Perspective
The major directions for the immediate future are developing therapies
that favor pericyte recruitment over pruning, understanding how to
combine antiangiogenic therapies with solid stress–alleviating agents,
and identifying the first biomarkers for anti-VEGF therapy.38 Achieving
these goals would allow optimization of treatment protocols and
reduction of the adverse effects, and better integration of these drugs
with emerging immunotherapies.
First, identifying the vascular “normalization window” in patients
would allow synergistic combinations with chemotherapy, radiotherapy,
and immunotherapy.253a Second, understanding the mechanisms of
vessel pruning and cancer cell apoptosis induced by anti-VEGF therapy,
and tumor escape from it, may allow further sensitization of tumor
cells to cytotoxic therapies. Third, characterization of the effect of
anti-VEGF therapy on bone marrow–derived cells’ contribution to
tumor growth and relapse would allow judicious and more effective
approaches to therapy involving these cells. Finally, clarification of
other mechanisms involving the immune system or the stromal and
interstitial matrix compartments would contribute to the establishment
of more efficacious combinatorial strategies.
In this respect, new biomarkers and improved imaging techniques
will play a major role in monitoring effects and stratifying patients,
with the ultimate goal of individualized therapy.
CONCLUSION
The successes of the anti-VEGF agents and development of solid
stress–alleviating agents have raised great hope and have taught us
important lessons about the significance of the target, timing, and
dosage of each agent.38,76
• According to the results of the phase III trials completed to date,
bevacizumab can increase median overall survival in certain cancers
when combined with standard chemotherapy, but not when used
as monotherapy.
• Anti-VEGF therapy with bevacizumab can increase overall survival
and/or progression-free survival in advanced colorectal carcinoma,
non–small cell lung carcinoma, breast carcinoma, renal cell carci-
noma, ovarian cancer, and cervical cancer when combined with
cytotoxic agents or immunotherapy. Aflibercept (“VEGF Trap,” an
sFLT1 chimera) and ramucirumab combinations with chemotherapy
resulted in improved survival in metastatic colorectal cancer and
lung cancer (for ramucirumab).
• Improved survival has been observed with broad-spectrum multitar-
geted tyrosine kinase inhibitors (e.g., sorafenib, sunitinib, pazopanib,
axitinib, vandetanib, and lenvatinib) when used as monotherapy.
Until recently, VEGF receptor–selective tyrosine kinase inhibitors
had repeatedly failed to confer an advantage when combined with
chemotherapy. However, a study in lung cancer showed increased
overall survival with nintedanib with chemotherapy.
• Anti-VEGF therapy can prune and normalize tumor vascular
structure and function.
• There is an urgent need to identify biomarkers to guide anti-VEGF
therapy and combination therapies using anti-VEGF agents.
Antiangiogenic agents are now expected to make a difference in
cancer patients with a wide variety of tumor types. With the advent
of specific and potent new agents—approved or in the process of
being approved—oncologists have a variety of direct and indirect
antiangiogenic agents to choose from when designing therapy protocols.
Determining whether the regimens used in the successful trials are
optimal, however, and whether antiangiogenic agents will work in
patients outside the rigorous inclusion criteria used for those trials,
will be critical for deciding the standard of care for different malignan-
cies. Establishing the most advantageous combinations will require a
better understanding of the mechanisms of action of each anti-VEGF
agent and the sensitivity of each tumor type, as well as development
of robust biomarkers and imaging techniques to guide patient selection
and protocol design. A deeper understanding of the mechanisms of
antitumor activity of specific and multitargeted antiangiogenic agents
in patients, how these agents can best be combined with other treatment
approaches such as chemotherapy, radiation therapy, and immuno-
therapy, and how optimization of these effects can be monitored
clinically should contribute to significantly improved cancer treatment
and extend survival of cancer patients in the near future, in addition
to enhancing the prospects for development of curative treatments
for different cancers in the more distant future.
ACKNOWLEDGMENTS
We would like to thank Kevin Kozak for his input on updating this
chapter. This chapter is an updated and expanded version of a chapter
by R. K. Jain entitled Molecular Pathophysiology of Tumors (in Perez
CA, Brady LW, Halperin EC, Schmidt-Ullrich R, eds: Principles and
Practice of Radiation Therapy. New York: Lippincott, Williams &
Wilkins; 2003:163–179) and a review article by J. D. Martin, D.
Fukumura, D. G. Duda, Y. Boucher, and R. K. Jain entitled Reengineer-
ing the Tumor Microenvironment to Alleviate Hypoxia and Overcome
Cancer Heterogeneity (in Cold Spring Harbor Perspectives in Medicine,
December 2016, Volume 6, Issue 12). The work summarized here
has been supported continuously by the National Cancer Institute
since 1980 with funding to R. K. Jain.
The complete reference list is available online at
ExpertConsult.com.

KEY REFERENCES
4. Jain RK, Munn LL, Fukumura D. Dissecting tumour
pathophysiology using intravital microscopy. Nat
Rev Cancer. 2002;2:266–276.
6. Helmlinger G, Netti PA, Lichtenbeld HC, et al.
Solid stress inhibits the growth of multicellular
tumor spheroids. Nat Biotechnol. 1997;15:778–
783.
7. Folkman J. Tumor angiogenesis: therapeutic implica-
tions. N Engl J Med. 1971;285:1182–1186.
8. Gullino PM. Angiogenesis and oncogenesis. J Natl
Cancer Inst. 1978;61:639–643.
9. Hurwitz H, Fehrenbacher L, Novotny W, et al.
Bevacizumab plus irinotecan, fluorouracil, and
leucovorin for metastatic colorectal cancer. N Engl
J Med. 2004;350:2335–2342.
12. Carmeliet P, Jain RK. Molecular mechanisms
and clinical applications of angiogenesis. Nature.
2011;473:298–307.
15. Hanahan D, Weinberg RA. Hallmarks of cancer:
the next generation. Cell. 2011;144:646–674.
16. Dvorak HF. Vascular permeability factor/vascular
endothelial growth factor: a critical cytokine in tumor
angiogenesis and a potential target for diagnosis and
therapy. J Clin Oncol. 2002;20:4368–4380.
20. Jain RK. Molecular regulation of vessel maturation.
Nat Med. 2003;9:685–693.
33. Ferrara N, Gerber HP, LeCouter J. The biology of
VEGF and its receptors. Nat Med. 2003;9:669–676.
34. Melder RJ, Koenig GC, Witwer BP, et al. During
angiogenesis, vascular endothelial growth factor
and basic fibroblast growth factor regulate natural
killer cell adhesion to tumor endothelium. Nat Med.
1996;2:992–997.
126 Part I: Science and Clinical Oncology
36. Fidler IJ. Angiogenic heterogeneity: regulation of
neoplastic angiogenesis by the organ microenviron-
ment. J Natl Cancer Inst. 2001;93:1040–1041.
38. Jain RK, Duda DG, Willett CG, et al. Biomarkers
of response and resistance to antiangiogenic therapy.
Nat Rev Clin Oncol. 2009;6:327–338.
49. Brown EB, Campbell RB, Tsuzuki Y, et al. In
vivo measurement of gene expression, angiogen-
esis and physiological function in tumors using
multiphoton laser scanning microscopy. Nat Med.
2001;7:864–868.
57. Hobbs SK, Monsky WL, Yuan F, et al. Regulation of
transport pathways in tumor vessels: role of tumor
type and microenvironment. Proc Natl Acad Sci USA.
1998;95:4607–4612.
61. Izumi Y, Xu L, di Tomaso E, et al. Tumour biology:
Herceptin acts as an anti-angiogenic cocktail. Nature.
2002;416:279–280.
63. Jain RK. Normalization of tumor vasculature: an
emerging concept in antiangiogenic therapy. Science.
2005;307:58–62.
64. Jain RK. Taming vessels to treat cancer. Sci Am.
2008;298:56–63.
70. Jain RK. Determinants of tumor blood flow: a review.
Cancer Res. 1988;48:2641–2658.
74. Goel S, Duda DG, Xu L, et al. Normalization of
the vasculature for treatment of cancer and other
diseases. Physiol Rev. 2011;91:1071–1121.
79. Stylianopoulos T, Martin JD, Chauhan VP, et al.
Causes, consequences and remedies for growth-
induced solid stress in murine and human tumors.
Proc Natl Acad Sci USA. 2012;109(38):15101–15108.
81. Padera TP, Stoll BR, Tooredman JB, et al. Pathology:
cancer cells compress intratumour vessels. Nature.
2004;427:695.
96. Sorensen AG, Emblem KE, Polaskova P, et al.
Increased survival of glioblastoma patients who
respond to antiangiogenic therapy with elevated
blood perfusion. Cancer Res. 2012;72:402–407.
101. Jain RK. Normalizing tumor vasculature with anti-
angiogenic therapy: a new paradigm for combination
therapy. Nat Med. 2001;7:987–989.
112. Chauhan VP, Stylianopoulos T, Martin JD, et al.
Normalization of tumour blood vessels improves
the delivery of nanomedicines in a size-dependent
manner. Nat Nanotechnol. 2012;7:383–388.
119. Yuan F, Chen Y, Dellian M, et al. Time-dependent
vascular regression and permeability changes in
established human tumor xenografts induced by
an anti-vascular endothelial growth factor/vascular
permeability factor antibody. Proc Natl Acad Sci
USA. 1996;93:14765–14770.
124. Duda DG, Duyverman AM, Kohno M, et al.
Malignant cells facilitate lung metastasis by
bringing their own soil. Proc Natl Acad Sci USA.
2010;107:21677–21682.
150. Hanahan D, Coussens LM. Accessories to the
crime: functions of cells recruited to the tumor
microenvironment. Cancer Cell. 2012;21:309–322.
153. Fukumura D, Xavier R, Sugiura T, et al. Tumor
induction of VEGF promoter activity in stromal
cells. Cell. 1998;94:715–725.
155. Padera TP, Kadambi A, di Tomaso E, et al. Lymphatic
metastasis in the absence of functional intratumor
lymphatics. Science. 2002;296:1883–1886.
157. Jain RK. Transport of molecules in the tumor inter-
stitium: a review. Cancer Res. 1987;47:3039–3051.
159. Brown E, McKee T, diTomaso E, et al. Dynamic
imaging of collagen and its modulation in tumors
in vivo using second-harmonic generation. Nat Med.
2003;9:796–800.
165. Nugent LJ, Jain RK. Extravascular diffusion
in normal and neoplastic tissues. Cancer Res.
1984;44:238–244.
166. Pluen A, Boucher Y, Ramanujan S, et al. Role of
tumor-host interactions in interstitial diffusion of
macromolecules: cranial vs. subcutaneous tumors.
Proc Natl Acad Sci USA. 2001;98:4628–4633.
172. Diop-Frimpong B, Chauhan VP, Krane S, et al.
Losartan inhibits collagen I synthesis and improves
the distribution and efficacy of nanotherapeutics
in tumors. Proc Natl Acad Sci USA. 2011;108:
2909–2914.
206. Boucher Y, Jain RK. Microvascular pressure is the
principal driving force for interstitial hypertension
in solid tumors: implications for vascular collapse.
Cancer Res. 1992;52:5110–5114.
217. Tong RT, Boucher Y, Kozin SV, et al. Vascular
normalization by vascular endothelial growth factor
receptor 2 blockade induces a pressure gradient across
the vasculature and improves drug penetration in
tumors. Cancer Res. 2004;64:3731–3736.
221. Willett CG, Boucher Y, di Tomaso E, et al.
Direct evidence that the VEGF-specific antibody
bevacizumab has antivascular effects in human rectal
cancer. Nat Med. 2004;10:145–147.
222. Willett CG, Boucher Y, Duda DG, et al. Surrogate
markers for antiangiogenic therapy and dose-limiting
toxicities for bevacizumab with radiation and
chemotherapy: continued experience of a phase I
trial in rectal cancer patients. J Clin Oncol. 2005;23:
8136–8139.
223. Willett CG, Duda DG, di Tomaso E, et al. Efficacy,
safety, and biomarkers of neoadjuvant bevacizumab,
radiation therapy, and fluorouracil in rectal cancer:
a multidisciplinary phase II study. J Clin Oncol.
2009;27:3020–3026.
225. Netti PA, Hamberg LM, Babich JW, et al. Enhance-
ment of fluid filtration across tumor vessels: implica-
tion for delivery of macromolecules. Proc Natl Acad
Sci USA. 1999;96:3137–3142.
229. Krogh A. The Anatomy and Physiology of Capillaries.
New York: Yale University Press; 1922.
230. Thomlinson RH, Gray LH. The histological structure
of some human lung cancers and the possible
implications for radiotherapy. Br J Cancer. 1955;9:
539–549.
231. Helmlinger G, Yuan F, Dellian M, et al. Interstitial
pH and pO2 gradients in solid tumors in vivo:
high-resolution measurements reveal a lack of
correlation. Nat Med. 1997;3:177–182.
236. Harris AL. Hypoxia: a key regulatory factor in
tumour growth. Nat Rev Cancer. 2002;2:38–47.
258. Mazzone M, Dettori D, Leite de Oliveira R, et al.
Heterozygous deficiency of PHD2 restores tumor
oxygenation and inhibits metastasis via endothelial
normalization. Cell. 2009;136:839–851.
288. Chauhan VP, Martin JD, Liu H, et al. Angiotensin
inhibition enhances drug delivery and potentiates
chemotherapy by decompressing tumour blood
vessels. Nat Commun. 2013;4:2516.
323. Chauhan VP, Boucher Y, Ferrone CR, et al. Compres-
sion of pancreatic tumor blood vessels by hyaluronan
is caused by solid stress and not interstitial fluid
pressure. Cancer Cell. 2014;26:14–15.

REFERENCES
1. Goldman E. The growth of malignant disease in
man and the lower animals with special reference
to the vascular system. Lancet. 1907;2:1236–1240.
2. Algire GH, Chalkley HW. Vascular reactions of
normal and malignant tissues in vivo. I. Vascular
reactions of mice to wounds and to normal and neo-
plastic transplants. J Natl Cancer Inst. 1945;6:73–85.
3. Ide AG, Baker NH, Warren SL. Vascularization of the
Brown-Pearce rabbit epithelioma transplant as seen
in the transplant ear chamber. Am J Radiol. 1939;42:
891–899.
4. Jain RK, Munn LL, Fukumura D. Dissecting tumour
pathophysiology using intravital microscopy. Nat
Rev Cancer. 2002;2:266–276.
5. Jain RK, Schlenger K, Hockel M, et al. Quantitative
angiogenesis assays: progress and problems. Nat Med.
1997;3:1203–1208.
6. Helmlinger G, Netti PA, Lichtenbeld HC, et al.
Solid stress inhibits the growth of multicellular tumor
spheroids. Nat Biotechnol. 1997;15:778–783.
7. Folkman J. Tumor angiogenesis: therapeutic implica-
tions. N Engl J Med. 1971;285:1182–1186.
8. Gullino PM. Angiogenesis and oncogenesis. J Natl
Cancer Inst. 1978;61:639–643.
9. Hurwitz H, Fehrenbacher L, Novotny W, et al.
Bevacizumab plus irinotecan, fluorouracil, and
leucovorin for metastatic colorectal cancer. N Engl
J Med. 2004;350:2335–2342.
10. Jayson GC, Kerbel R, Ellis LM, et al. Antiangiogenic
therapy in oncology: current status and future direc-
tions. Lancet. 2016.
11. Carmeliet P. Angiogenesis in life, disease and
medicine. Nature. 2005;438:932–936.
12. Carmeliet P, Jain RK. Molecular mechanisms
and clinical applications of angiogenesis. Nature.
2011;473:298–307.
13. Jain RK, Carmeliet PF. Vessels of death or life. Sci
Am. 2001;285:38–45.
14. Bouck N, Stellmach V, Hsu SC. How tumors become
angiogenic. Adv Cancer Res. 1996;135–174.
15. Hanahan D, Weinberg RA. Hallmarks of cancer:
the next generation. Cell. 2011;144:646–674.
16. Dvorak HF. Vascular permeability factor/vascular
endothelial growth factor: a critical cytokine in tumor
angiogenesis and a potential target for diagnosis and
therapy. J Clin Oncol. 2002;20:4368–4380.
17. Patan S, Munn LL, Jain RK. Intussusceptive micro-
vascular growth in a human colon adenocarcinoma
xenograft: a novel mechanism of tumor angiogenesis.
Microvasc Res. 1996;51:260–272.
18. Patan S, Munn LL, Tanda S, et al. Vascular mor-
phogenesis and remodeling in a model of tissue
repair: blood vessel formation and growth in the
ovarian pedicle after ovariectomy. Circ Res. 2001;89:
723–731.
19. Patan S, Tanda S, Roberge S, et al. Vascular morpho-
genesis and remodeling in a human tumor xenograft:
blood vessel formation and growth after ovariectomy
and tumor implantation. Circ Res. 2001;89:732–739.
20. Jain RK. Molecular regulation of vessel maturation.
Nat Med. 2003;9:685–693.
21. Duda DG, Cohen KS, Kozin SV, et al. Evidence for
incorporation of bone marrow-derived endothelial
cells into perfused blood vessels in tumors. Blood.
2006;107:2774–2776.
22. Isner JM. Myocardial gene therapy. Nature.
2002;415:234.
23. Rafii S, Lyden D, Benezra R, et al. Vascular and
haematopoietic stem cells: novel targets for anti-
angiogenesis therapy? Nat Rev Cancer. 2002;2:
826–835.
24. Sajithlal GB, McGuire TF, Lu J, et al. Endothelial-
like cells derived directly from human tumor
xenografts. Int J Cancer. 2010;127:2268–2278.
25. Ricci-Vitiani L, Pallini R, Biffoni M, et al. Tumour
vascularization via endothelial differentiation of glio-
blastoma stem-like cells. Nature. 2010;468:824–828.
Tumor Microenvironment: Vascular and Extravascular Compartment  •  CHAPTER 8 126.e1
26. Soda Y, Marumoto T, Friedmann-Morvinski D,
et al. Transdifferentiation of glioblastoma cells into
vascular endothelial cells. Proc Natl Acad Sci USA.
2011;108:4274–4280.
27. Wang R, Chadalavada K, Wilshire J, et al. Glioblas-
toma stem-like cells give rise to tumour endothelium.
Nature. 2010;468:829–833.
28. Kozin SV, Duda DG, Munn LL, et al. Neovascular-
ization after irradiation: what is the source of newly
formed vessels in recurring tumors? J Natl Cancer
Inst. 2012;104:899–905.
29. Ergun S, Hohn HP, Kilic N, et al. Endothelial and
hematopoietic progenitor cells (EPCs and HPCs):
hand in hand fate determining partners for cancer
cells. Stem Cell Rev. 2008;4:169–177.
30. Aicher A, Rentsch M, Sasaki K, et al. Nonbone
marrow-derived circulating progenitor cells contrib-
ute to postnatal neovascularization following tissue
ischemia. Circ Res. 2007;100:581–589.
31. Kozin SV, Duda DG, Munn LL, et al. Is vasculogen-
esis crucial for the regrowth of irradiated tumours?
Nat Rev Cancer. 2011;11:532.
32. Dvorak HF, Nagy JA, Feng D, et al. Tumor
Architecture and Targeted Delivery. In: Abrams PG,
Fritzberg AR, eds. Radioimmunotherapy of Cancer.
New York: Marcel Dekker, Inc.; 2000:107–135.
33. Ferrara N, Gerber HP, LeCouter J. The biology of
VEGF and its receptors. Nat Med. 2003;9:669–676.
34. Melder RJ, Koenig GC, Witwer BP, et al. During
angiogenesis, vascular endothelial growth factor
and basic fibroblast growth factor regulate natural
killer cell adhesion to tumor endothelium. Nat Med.
1996;2:992–997.
35. Casanovas O, Hicklin DJ, Bergers G, et al. Drug
resistance by evasion of antiangiogenic targeting of
VEGF signaling in late-stage pancreatic islet tumors.
Cancer Cell. 2005;8:299–309.
36. Fidler IJ. Angiogenic heterogeneity: regulation of
neoplastic angiogenesis by the organ microenviron-
ment. J Natl Cancer Inst. 2001;93:1040–1041.
37. Yoshiji H, Harris SR, Thorgeirsson UP. Vascular
endothelial growth factor is essential for initial but
not continued in vivo growth of human breast
carcinoma cells. Cancer Res. 1997;57:3924–3928.
38. Jain RK, Duda DG, Willett CG, et al. Biomarkers
of response and resistance to antiangiogenic therapy.
Nat Rev Clin Oncol. 2009;6:327–338.
39. LeCouter J, Ferrara N. EG-VEGF and Bv8: a novel
family of tissue-selective mediators of angiogenesis,
endothelial phenotype, and function. Trends Car-
diovasc Med. 2003;13:276–282.
40. Yancopoulos GD, Davis S, Gale NW, et al. Vascular-
specific growth factors and blood vessel formation.
Nature. 2000;407:242–248.
41. Maeshima Y, Sudhakar A, Lively JC, et al. Tumstatin,
an endothelial cell-specific inhibitor of protein
synthesis. Science. 2002;295:140–143.
42. O’Reilly MS, Boehm T, Shing Y, et al. Endostatin:
an endogenous inhibitor of angiogenesis and tumor
growth. Cell. 1997;88:277–285.
43. O’Reilly MS, Holmgren L, Shing Y, et al. Angio-
statin: a novel angiogenesis inhibitor that mediates
the suppression of metastases by a Lewis lung
carcinoma. Cell. 1994;79:315–328.
44. Kalluri R. Basement membranes: structure, assembly
and role in tumour angiogenesis. Nat Rev Cancer.
2003;3:422–433.
45. Fukumura D, Xu L, Chen Y, et al. Hypoxia and
acidosis independently up-regulate vascular endo-
thelial growth factor transcription in brain tumors
in vivo. Cancer Res. 2001;61:6020–6024.
46. Garkavtsev I, Kozin SV, Chernova O, et al. The
candidate tumour suppressor protein ING4 regulates
brain tumour growth and angiogenesis. Nature.
2004;428:328–332.
47. Kerbel RS. Tumor angiogenesis. N Engl J Med.
2008;358:2039–2049.
126.e1
48. Xu L, Fukumura D, Jain RK. Acidic extracellular pH
induces vascular endothelial growth factor (VEGF)
in human glioblastoma cells via ERK1/2 MAPK
signaling pathway: mechanism of low pH-induced
VEGF. J Biol Chem. 2002;277:11368–11374.
49. Brown EB, Campbell RB, Tsuzuki Y, et al. In
vivo measurement of gene expression, angiogen-
esis and physiological function in tumors using
multiphoton laser scanning microscopy. Nat Med.
2001;7:864–868.
50. Fukumura D, Jain RK. Role of nitric oxide in
angiogenesis and microcirculation in tumors. Cancer
Metastasis Rev. 1998;17:77–89.
51. Helmlinger G, Endo M, Ferrara N, et  al.
Formation of endothelial cell networks. Nature.
2000;405:139–141.
52. Tsuzuki Y, Fukumura D, Oosthuyse B, et al. Vascular
endothelial growth factor (VEGF) modulation by
targeting hypoxia-inducible factor-1alpha → hypoxia
response element → VEGF cascade differentially
regulates vascular response and growth rate in
tumors. Cancer Res. 2000;60:6248–6252.
53. Blouw B, Song H, Tihan T, et al. The hypoxic
response of tumors is dependent on their micro-
environment. Cancer Cell. 2003;4:133–146.
54. Dellian M, Helmlinger G, Yuan F, et al. Fluorescence
ratio imaging of interstitial pH in solid tumours:
effect of glucose on spatial and temporal gradients.
Br J Cancer. 1996;74:1206–1215.
55. Fukumura D, Yuan F, Monsky WL, et al. Effect of host
microenvironment on the microcirculation of human
colon adenocarcinoma. Am J Pathol. 1997;151:
679–688.
56. Gohongi T, Fukumura D, Boucher Y, et al. Tumor-
host interactions in the gallbladder suppress distal
angiogenesis and tumor growth: involvement of
transforming growth factor beta1. Nat Med. 1999;5:
1203–1208.
57. Hobbs SK, Monsky WL, Yuan F, et al. Regulation of
transport pathways in tumor vessels: role of tumor
type and microenvironment. Proc Natl Acad Sci USA.
1998;95:4607–4612.
58. Monsky WL, Mouta Carreira C, Tsuzuki Y, et al.
Role of host microenvironment in angiogenesis and
microvascular functions in human breast cancer
xenografts: mammary fat pad versus cranial tumors.
Clin Cancer Res. 2002;8:1008–1013.
59. Tsuzuki Y, Mouta Carreira C, Bockhorn M, et al.
Pancreas microenvironment promotes VEGF expres-
sion and tumor growth: novel window models for
pancreatic tumor angiogenesis and microcirculation.
Lab Invest. 2001;81:1439–1451.
60. Yuan F, Salehi HA, Boucher Y, et al. Vascular
permeability and microcirculation of gliomas and
mammary carcinomas transplanted in rat and
mouse cranial windows. Cancer Res. 1994;54:4564–
4568.
61. Izumi Y, Xu L, di Tomaso E, et al. Tumour biology:
Herceptin acts as an anti-angiogenic cocktail. Nature.
2002;416:279–280.
62. Jain RK, Safabakhsh N, Sckell A, et al. Endothelial
cell death, angiogenesis, and microvascular function
after castration in an androgen-dependent tumor:
role of vascular endothelial growth factor. Proc Natl
Acad Sci USA. 1998;95:10820–10825.
63. Jain RK. Normalization of tumor vasculature: an
emerging concept in antiangiogenic therapy. Science.
2005;307:58–62.
64. Jain RK. Taming vessels to treat cancer. Sci Am.
2008;298:56–63.
65. Ramanujan S, Koenig GC, Padera TP, et al. Local
imbalance of proangiogenic and antiangiogenic
factors: a potential mechanism of focal necrosis
and dormancy in tumors. Cancer Res. 2000;60:
1442–1448.
66. Baish JW, Jain RK. Fractals and cancer. Cancer Res.
2000;60:3683–3688.
126.e2 Part I: Science and Clinical Oncology
67. Gazit Y, Berk DA, Leunig M, et al. Scale-invariant
behavior and vascular network formation in normal
and tumor-tissue. Phys Rev Lett. 1995;75:2428–
2431.
68. Gazit Y, Baish JW, Safabakhsh N, et al. Fractal charac-
teristics of tumor vascular architecture during tumor
growth and regression. Microcirculation. 1997;4:
395–402.
69. McDonald DM, Choyke PL. Imaging of angio-
genesis: from microscope to clinic. Nat Med.
2003;9:713–725.
70. Jain RK. Determinants of tumor blood flow: a review.
Cancer Res. 1988;48:2641–2658.
71. Less JR, Posner MC, Skalak TC, et al. Geometric
resistance and microvascular network architecture
of human colorectal carcinoma. Microcirculation.
1997;4:25–33.
72. Less JR, Skalak TC, Sevick EM, et al. Microvascular
architecture in a mammary carcinoma: branch-
ing patterns and vessel dimensions. Cancer Res.
1991;51:265–273.
73. Jain RK, Munn LL. Leaky vessels? Call Angl1! Nat
Med. 2000;6:131–132.
74. Goel S, Duda DG, Xu L, et al. Normalization of
the vasculature for treatment of cancer and other
diseases. Physiol Rev. 2011;91:1071–1121.
75. Goel S, Fukumura D, Jain RK. Normalization of
the tumor vasculature through oncogenic inhibition:
an emerging paradigm in tumor biology. Proc Natl
Acad Sci USA. 2012;doi:10.1073/pnas.1203794109.
76. Huang Y, Yuan J, Righi E, et al. Vascular normalizing
dose of antiangiogenic treatment reprograms the
immunosuppressive tumor microenvironment and
enhances immunotherapy. Proc Natl Acad Sci USA.
2012;109:17561–17566.
77. Kadambi A, Mouta Carreira C, Yun CO, et al.
Vascular endothelial growth factor (VEGF)-C
differentially affects tumor vascular function and
leukocyte recruitment: role of VEGF-receptor 2 and
host VEGF-A. Cancer Res. 2001;61:2404–2408.
78. Koike C, McKee TD, Pluen A, et al. Solid stress
facilitates spheroid formation: potential involvement
of hyaluronan. Br J Cancer. 2002;86:947–953.
79. Stylianopoulos T, Martin JD, Chauhan VP, et al.
Causes, consequences and remedies for growth-
induced solid stress in murine and human tumors.
Proc Natl Acad Sci USA. 2012;109(38):15101–
15108.
80. Griffon-Etienne G, Boucher Y, Brekken C, et al.
Taxane-induced apoptosis decompresses blood
vessels and lowers interstitial fluid pressure in solid
tumors: clinical implications. Cancer Res. 1999;59:
3776–3782.
81. Padera TP, Stoll BR, Tooredman JB, et al. Pathology:
cancer cells compress intratumour vessels. Nature.
2004;427:695.
82. Jacobetz MA, Chan DS, Neesse A, et  al.
Hyaluronan impairs vascular function and drug
delivery in a mouse model of pancreatic cancer.
Gut. 2013;62(1):112–120.
83. Sevick EM, Jain RK. Geometric resistance to blood
flow in solid tumors perfused ex vivo: effects of
tumor size and perfusion pressure. Cancer Res.
1989;49:3506–3512.
84. Sevick EM, Jain RK. Viscous resistance to blood flow
in solid tumors: effect of hematocrit on intratumor
blood viscosity. Cancer Res. 1989;49:3513–3519.
85. Sevick EM, Jain RK. Effect of red blood cell rigidity
on tumor blood flow: increase in viscous resistance
during hyperglycemia. Cancer Res. 1991;51:
2727–2730.
86. Jain RK, Ward-Hartley KA: Tumor blood flow: char-
acterization, modifications and role in hyperthermia.
IEEE Transactions in Sonics and Ultrasonics; Special
Issue on Hyperthermia, SU-31:504-526, 1984.
87. Vaupel P, Kallinowski F, Okunieff P. Blood flow,
oxygen and nutrient supply, and metabolic micro-
environment of human tumors: a review. Cancer
Res. 1989;49:6449–6465.
88. Endrich B, Reinhold HS, Gross JF, et al. Tissue
perfusion inhomogeneity during early tumor growth
in rats. J Natl Cancer Inst. 1979;62:387–395.
89. Jain RK, Forbes NS. Can engineered bacteria
help control cancer? Proc Natl Acad Sci USA.
2001;98:14748–14750.
90. Leunig M, Yuan F, Menger MD, et al. Angiogenesis,
microvascular architecture, microhemodynamics,
and interstitial fluid pressure during early growth
of human adenocarcinoma LS174T in SCID mice.
Cancer Res. 1992;52:6553–6560.
91. Brizel DM, Klitzman B, Cook JM, et al. A com-
parison of tumor and normal tissue microvascular
hematocrits and red cell fluxes in a rat window
chamber model. Int J Radiat Oncol Biol Phys. 1993;25:
269–276.
92. Baish JW, Netti PA, Jain RK. Transmural coupling
of fluid flow in microcirculatory network and inter-
stitium in tumors. Microvasc Res. 1997;53:128–141.
93. Netti PA, Roberge S, Boucher Y, et al. Effect of
transvascular fluid exchange on pressure-flow
relationship in tumors: a proposed mechanism
for tumor blood flow heterogeneity. Microvasc Res.
1996;52:27–46.
94. Mollica F, Jain RK, Netti PA. A model for temporal
heterogeneities of tumor blood flow. Microvasc Res.
2003;65:56–60.
95. Park MS, Klotz E, Kim MJ, et al. Perfusion CT:
noninvasive surrogate marker for stratification of
pancreatic cancer response to concurrent chemo-
and radiation therapy. Radiology. 2009;250:110–
117.
96. Sorensen AG, Emblem KE, Polaskova P, et al.
Increased survival of glioblastoma patients who
respond to antiangiogenic therapy with elevated
blood perfusion. Cancer Res. 2012;72:402–407.
97. Morikawa S, Baluk P, Kaidoh T, et al. Abnormalities
in pericytes on blood vessels and endothelial sprouts
in tumors. Am J Pathol. 2002;160:985–1000.
98. Dolmans DE, Kadambi A, Hill JS, et al. Vascular
accumulation of a novel photosensitizer, MV6401,
causes selective thrombosis in tumor vessels after pho-
todynamic therapy. Cancer Res. 2002;62:2151–2156.
99. Huang X, Molema G, King S, et al. Tumor infarction
in mice by antibody-directed targeting of tissue factor
to tumor vasculature. Science. 1997;275:547–550.
100. Ruoslahti E. Specialization of tumor vasculature.
Nat Rev Cancer. 2002;2:83–90.
101. Jain RK. Normalizing tumor vasculature with anti-
angiogenic therapy: a new paradigm for combination
therapy. Nat Med. 2001;7:987–989.
102. Chauhan VP, Stylianopoulos T, Boucher Y, et al.
Delivery of molecular and nanoscale medicine to
tumors: transport barriers and strategies. Annu Rev
Chem Biomol Eng. 2011;2:281–298.
103. Jain RK. Transport of molecules across tumor
vasculature. Cancer Metastasis Rev. 1987;6:559–593.
104. Hashizume H, Baluk P, Morikawa S, et al. Openings
between defective endothelial cells explain tumor
vessel leakiness. Am J Pathol. 2000;156:1363–1380.
105. Endo M, Jain RK, Witwer B, et al. Water channel
(aquaporin 1) expression and distribution in mammary
carcinomas and glioblastomas. Microvasc Res. 1999;
58:89–98.
106. Gerlowski LE, Jain RK. Microvascular permeability
of normal and neoplastic tissues. Microvasc Res.
1986;31:288–305.
107. Lichtenbeld HC, Yuan F, Michel CC, et al. Perfu-
sion of single tumor microvessels: application to
vascular permeability measurement. Microcirculation.
1996;3:349–357.
108. Sevick EM, Jain RK. Measurement of capillary
filtration coefficient in a solid tumor. Cancer Res.
1991;51:1352–1355.
109. Yuan F, Leunig M, Berk DA, et al. Microvascular
permeability of albumin, vascular surface area, and
vascular volume measured in human adenocarcinoma
LS174T using dorsal chamber in SCID mice.
Microvasc Res. 1993;45:269–289.
110. Yuan F, Dellian M, Fukumura D, et al. Vascular
permeability in a human tumor xenograft: molecular
size dependence and cutoff size. Cancer Res. 1995;55:
3752–3756.
111. Dreher MR, Liu W, Michelich CR, et al. Tumor
vascular permeability, accumulation, and penetration
of macromolecular drug carriers. J Natl Cancer Inst.
2006;98:335–344.
112. Chauhan VP, Stylianopoulos T, Martin JD, et al.
Normalization of tumour blood vessels improves
the delivery of nanomedicines in a size-dependent
manner. Nat Nanotechnol. 2012;7:383–388.
113. Chauhan VP, Popovic Z, Chen O, et al. Fluorescent
nanorods and nanospheres for real-time in vivo
probing of nanoparticle shape-dependent tumor
penetration. Angew Chem Int Ed Engl. 2011;50:
11417–11420.
114. Campbell RB, Fukumura D, Brown EB, et al. Cat-
ionic charge determines the distribution of liposomes
between the vascular and extravascular compartments
of tumors. Cancer Res. 2002;62:6831–6836.
115. Dellian M, Yuan F, Trubetskoy VS, et al. Vascular
permeability in a human tumour xenograft: molecu-
lar charge dependence. Br J Cancer. 2000;82:1513–
1518.
116. Stylianopoulos T, Soteriou K, Fukumura D, et al.
Cationic nanoparticles have superior transvascular
flux into solid tumors: insights from a mathematical
model. Ann Biomed Eng. 2012.
117. Thurston G, McLean JW, Rizen M, et al. Cationic
liposomes target angiogenic endothelial cells in
tumors and chronic inflammation in mice. J Clin
Invest. 1998;101:1401–1413.
118. Monsky WL, Fukumura D, Gohongi T, et al.
Augmentation of transvascular transport of macro-
molecules and nanoparticles in tumors using vascular
endothelial growth factor. Cancer Res. 1999;59:
4129–4135.
119. Yuan F, Chen Y, Dellian M, et al. Time-dependent
vascular regression and permeability changes in
established human tumor xenografts induced by
an anti-vascular endothelial growth factor/vascular
permeability factor antibody. Proc Natl Acad Sci
USA. 1996;93:14765–14770.
120. Bockhorn M, Roberge S, Sousa C, et al. Differential
gene expression in metastasizing cells shed from
kidney tumors. Cancer Res. 2004;64:2469–2473.
121. Chang YS, di Tomaso E, McDonald DM, et al.
Mosaic blood vessels in tumors: Frequency of cancer
cells in contact with flowing blood. Proc Natl Acad
Sci USA. 2000;97:14608–14613.
122. Gullino PM. Techniques in tumor pathophysiology.
In: Busch H, ed. Methods in Cancer Research. New
York: Academic Press; 1970:45–92.
123. Swartz MA, Kristensen CA, Melder RJ, et al. Cells
shed from tumours show reduced clonogenicity,
resistance to apoptosis, and in vivo tumorigenicity.
Br J Cancer. 1999;81:756–759.
124. Duda DG, Duyverman AM, Kohno M, et al.
Malignant cells facilitate lung metastasis by bringing
their own soil. Proc Natl Acad Sci USA. 2010;107:
21677–21682.
125. Jain RK, Koenig GC, Dellian M, et al. Leukocyte-
endothelial adhesion and angiogenesis in tumors.
Cancer Metastasis Rev. 1996;15:195–204.
126. Melder RJ, Munn LL, Yamada S, et al. Selectin- and
integrin-mediated T-lymphocyte rolling and arrest
on TNF-alpha-activated endothelium: augmentation
by erythrocytes. Biophys J. 1995;69:2131–2138.
127. Melder RJ, Yuan J, Munn LL, et al. Erythrocytes
enhance lymphocyte rolling and arrest in vivo.
Microvasc Res. 2000;59:316–322.
128. Migliorini C, Qian Y, Chen H, et al. Red blood
cells augment leukocyte rolling in a virtual blood
vessel. Biophys J. 2002;83:1834–1841.
129. Munn LL, Melder RJ, Jain RK. Role of erythrocytes
in leukocyte-endothelial interactions: mathemati-
cal model and experimental validation. Biophys J.
1996;71:466–478.

130. Yuan J, Melder RJ, Jain RK, et al. Lateral view flow
system for studies of cell adhesion and deformation
under flow conditions. Biotechniques. 2001;30:
388–394.
131. Melder RJ, Kristensen CA, Munn LL, et al. Modula-
tion of A-NK cell rigidity: In vitro characterization
and in vivo implications for cell delivery. Biorheology.
2001;38:151–159.
132. Al-Mehdi AB, Tozawa K, Fisher AB, et al. Intra-
vascular origin of metastasis from the proliferation
of endothelium-attached tumor cells: a new model
for metastasis. Nat Med. 2000;6:100–102.
133. Fukumura D, Salehi HA, Witwer B, et al. Tumor
necrosis factor alpha-induced leukocyte adhesion
in normal and tumor vessels: effect of tumor type,
transplantation site, and host strain. Cancer Res.
1995;55:4824–4829.
134. Dellian M, Witwer BP, Salehi HA, et al. Quantitation
and physiological characterization of angiogenic
vessels in mice—effect of basic fibroblast growth
factor vascular endothelial growth factor vascular
permeability factor, and host microenvironment.
Am J Pathol. 1996;149:59–71.
135. Yamada S, Mayadas TN, Yuan F, et al. Rolling in
P-selectin-deficient mice is reduced but not elimi-
nated in the dorsal skin. Blood. 1995;86:3487–3492.
136. Melder RJ, Brownell AL, Shoup TM, et al. Imaging
of activated natural killer cells in mice by positron
emission tomography: preferential uptake in tumors.
Cancer Res. 1993;53:5867–5871.
137. Melder RJ, Jain RK. Reduction of rigidity in human
activated natural killer cells by thioglycollate treat-
ment. J Immunol Methods. 1994;175:69–77.
138. Melder RJ, Jain RK. Kinetics of interleukin-2 induced
changes in rigidity of human natural killer cells. Cell
Biophys. 1992;20:161–176.
139. Sasaki A, Jain RK, Maghazachi AA, et al. Low
deformability of lymphokine-activated killer cells
as a possible determinant of in vivo distribution.
Cancer Res. 1989;49:3742–3746.
140. Melder RJ, Salehi HA, Jain RK. Interaction of
activated natural killer cells with normal and tumor
vessels in cranial windows in mice. Microvasc Res.
1995;50:35–44.
141. Munn LL, Melder RJ, Jain RK. Analysis of cell flux
in the parallel plate flow chamber: implications for
cell capture studies. Biophys J. 1994;67:889–895.
142. Munn LL, Koenig GC, Jain RK, et al. Kinetics of adhe-
sion molecule expression and spatial organization using
targeted sampling fluorometry. Biotechniques. 1995;
19:622–631.
143. Sasaki A, Melder RJ, Whiteside TL, et al. Preferential
localization of human adherent lymphokine-activated
killer cells in tumor microcirculation. J Natl Cancer
Inst. 1991;83:433–437.
144. Melder RJ, Koenig GC, Munn LL, et al. Adhesion
of activated natural killer cells to tumor necrosis
factor-alpha-treated endothelium under physiological
flow conditions. Nat Immun. 1996;15:154–163.
145. Gamble JR, Khew-Goodall Y, Vadas MA. Trans-
forming growth factor-beta inhibits E-selectin
expression on human endothelial cells. J Immunol.
1993;150:4494–4503.
146. Gamble JR, Vadas MA. Endothelial adhesiveness
for blood neutrophils is inhibited by transforming
growth factor-beta. Science. 1988;242:97–99.
147. Gamble JR, Vadas MA. Endothelial cell adhesiveness
for human T lymphocytes is inhibited by transform-
ing growth factor-beta 1. J Immunol. 1991;146:
1149–1154.
148. Jallal B, Powell J, Zachwieja J, et al. Suppression of
tumor growth in vivo by local and systemic 90K
level increase. Cancer Res. 1995;55:3223–3227.
149. Detmar M, Brown LF, Schon MP, et al. Increased
microvascular density and enhanced leukocyte rolling
and adhesion in the skin of VEGF transgenic mice.
J Invest Dermatol. 1998;111:1–6.
150. Hanahan D, Coussens LM. Accessories to the
crime: functions of cells recruited to the tumor
Tumor Microenvironment: Vascular and Extravascular Compartment  •  CHAPTER 8 126.e3
microenvironment. Cancer Cell. 2012;21:309–
322.
151. DeNardo DG, Andreu P, Coussens LM. Interactions
between lymphocytes and myeloid cells regulate pro-
versus anti-tumor immunity. Cancer and Metastasis
Reviews. 2010;29:309–316.
152. Coussens LM, Zitvogel L, Palucka AK. Neutralizing
tumor-promoting chronic inflammation: a magic
bullet? Science. 2013;339:286–291.
153. Fukumura D, Xavier R, Sugiura T, et al. Tumor
induction of VEGF promoter activity in stromal
cells. Cell. 1998;94:715–725.
154. Jain RK, Fenton BT. Intratumoral lymphatic vessels:
a case of mistaken identity or malfunction? J Natl
Cancer Inst. 2002;94:417–421.
155. Padera TP, Kadambi A, di Tomaso E, et al. Lymphatic
metastasis in the absence of functional intratumor
lymphatics. Science. 2002;296:1883–1886.
156. Elenbaas B, Weinberg RA. Heterotypic signaling
between epithelial tumor cells and fibroblasts in car-
cinoma formation. Exp Cell Res. 2001;264:169–184.
157. Jain RK. Transport of molecules in the tumor inter-
stitium: a review. Cancer Res. 1987;47:3039–3051.
158. Berk DA, Yuan F, Leunig M, et al. Fluorescence
photobleaching with spatial Fourier analysis:
measurement of diffusion in light-scattering media.
Biophys J. 1993;65:2428–2436.
159. Brown E, McKee T, diTomaso E, et al. Dynamic
imaging of collagen and its modulation in tumors
in vivo using second-harmonic generation. Nat Med.
2003;9:796–800.
160. Chary SR, Jain RK. Direct measurement of interstitial
convection and diffusion of albumin in normal and
neoplastic tissues by fluorescence photobleaching.
Proc Natl Acad Sci USA. 1989;86:5385–5389.
161. Chauhan VP, Lanning RM, Diop-Frimpong B, et al.
Multiscale measurements distinguish cellular and
interstitial hindrances to diffusion in vivo. Biophys
J. 2009;97:330–336.
162. Davies CL, Berk DA, Pluen A, et al. Comparison of
IgG diffusion and extracellular matrix composition
in rhabdomyosarcomas grown in mice versus in vitro
as spheroids reveals the role of host stromal cells.
Br J Cancer. 2002;86:1639–1644.
163. Johnson EM, Berk DA, Jain RK, et al. Diffusion
and partitioning of proteins in charged agarose gels.
Biophys J. 1995;68:1561–1568.
164. Johnson EM, Berk DA, Jain RK, et al. Hindered
diffusion in agarose gels: test of effective medium
model. Biophys J. 1996;70:1017–1023.
165. Nugent LJ, Jain RK. Extravascular diffusion in
normal and neoplastic tissues. Cancer Res. 1984;44:
238–244.
166. Pluen A, Boucher Y, Ramanujan S, et al. Role of
tumor-host interactions in interstitial diffusion of
macromolecules: cranial vs. subcutaneous tumors.
Proc Natl Acad Sci USA. 2001;98:4628–4633.
167. Pluen A, Netti PA, Jain RK, et al. Diffusion of
macromolecules in agarose gels: comparison of linear
and globular configurations. Biophys J. 1999;77:
542–552.
168. Swabb EA, Wei J, Gullino PM. Diffusion and
convection in normal and neoplastic tissues. Cancer
Res. 1974;34:2814–2822.
169. Boucher Y, Brekken C, Netti PA, et al. Intratumoral
infusion of fluid: estimation of hydraulic conductivity
and implications for the delivery of therapeutic
agents. Br J Cancer. 1998;78:1442–1448.
170. Znati CA, Rosenstein M, McKee TD, et al. Irradia-
tion reduces interstitial fluid transport and increases
the collagen content in tumors. Clin Cancer Res.
2003;9:5508–5513.
171. Berk DA, Yuan F, Leunig M, et al. Direct in vivo
measurement of targeted binding in a human
tumor xenograft. Proc Natl Acad Sci USA. 1997;94:
1785–1790.
172. Diop-Frimpong B, Chauhan VP, Krane S, et al.
Losartan inhibits collagen I synthesis and improves
the distribution and efficacy of nanotherapeutics
126.e3
in tumors. Proc Natl Acad Sci USA. 2011;108:
2909–2914.
173. McKee TD, Grandi P, Mok W, et al. Degradation
of fibrillar collagen in a human melanoma xenograft
improves the efficacy of an oncolytic herpes simplex
virus vector. Cancer Res. 2006;66:2509–2513.
174. Netti PA, Berk DA, Swartz MA, et al. Role of
extracellular matrix assembly in interstitial transport
in solid tumors. Cancer Res. 2000;60:2497–2503.
175. Ramanujan S, Pluen A, McKee TD, et al. Diffusion
and convection in collagen gels: implications for trans-
port in the tumor interstitium. Biophys J. 2002;83:
1650–1660.
176. Stylianopoulos T, Poh MZ, Insin N, et al. Diffusion
of particles in the extracellular matrix: the effect
of repulsive electrostatic interactions. Biophys J.
2010;99:1342–1349.
177. Baxter LT, Jain RK. Transport of fluid and macro-
molecules in tumors. I. Role of interstitial pressure
and convection. Microvasc Res. 1989;37:77–104.
178. Baxter LT, Jain RK. Transport of fluid and mac-
romolecules in tumors. III. Role of binding and
metabolism. Microvasc Res. 1991;41:5–23.
179. Juweid M, Neumann R, Paik C, et al. Micro-
pharmacology of monoclonal antibodies in solid
tumors: direct experimental evidence for a binding
site barrier. Cancer Res. 1992;52:5144–5153.
180. Kaufman EN, Jain RK. Quantification of transport
and binding parameters using fluorescence recovery
after photobleaching. Potential for in vivo applica-
tions. Biophys J. 1990;58:873–885.
181. Kaufman EN, Jain RK. Measurement of mass
transport and reaction parameters in bulk solution
using photobleaching: Reaction limited binding
regime. Biophys J. 1991;60:596–610.
182. Kaufman EN, Jain RK. Effect of bivalent interaction
upon apparent antibody affinity: experimental confir-
mation of theory using fluorescence photobleaching
and implications for antibody binding assays. Cancer
Res. 1992;52:4157–4167.
183. Kaufman EN, Jain RK. In vitro measurement and
screening of monoclonal antibody affinity using
fluorescence photobleaching. J Immunol Methods.
1992;155:1–17.
184. Gullino PM. Extracellular compartments of solid
tumors. In: Becker FF, ed. Cancer. New York:
Plenum; 1975:327–354.
185. Leu AJ, Berk DA, Lymboussaki A, et al. Absence
of functional lymphatics within a murine sarcoma:
a molecular and functional evaluation. Cancer Res.
2000;60:4324–4327.
186. Carreira CM, Nasser SM, di Tomaso E, et al. LYVE-1
is not restricted to the lymph vessels: Expression in
normal liver blood sinusoids and down-regulation
in human liver cancer and cirrhosis. Cancer Res.
2001;61:8079–8084.
187. Hoshida T, Isaka N, Hagendoorn J, et al. Imaging
steps of lymphatic metastasis reveals that vascular
endothelial growth factor-C increases metastasis
by increasing delivery of cancer cells to lymph
nodes: therapeutic implications. Cancer Res.
2006;66:8065–8075.
188. Alitalo K, Carmeliet P. Molecular mechanisms of
lymphangiogenesis in health and disease. Cancer
Cell. 2002;1:219–227.
189. Jain RK, Padera TP. Development. Lymphatics make
the break. Science. 2003;299:209–210.
190. Oliver G, Detmar M. The rediscovery of the
lymphatic system: old and new insights into the
development and biological function of the lym-
phatic vasculature. Genes Dev. 2002;16:773–783.
191. Gale NW, Thurston G, Hackett SF, et al. Angio-
poietin-2 is required for postnatal angiogenesis and
lymphatic patterning, and only the latter role is
rescued by angiopoietin-1. Development Cell. 2002;3:
411–423.
192. Jeong HS, Jones D, Liao S, et al. Investigation of the
lack of angiogenesis in the formation of lymph node
metastases. J Natl Cancer Inst. 2015;107:djv155.
126.e4 Part I: Science and Clinical Oncology
193. Jain RK, Padera TP. Prevention and treatment
of lymphatic metastasis by antilymphangiogenic
therapy. J Natl Cancer Inst. 2002;94:785–787.
194. Padera TP, Stoll BR, So PT, et al. Conventional
and high-speed intravital multiphoton laser scanning
microscopy of microvasculature, lymphatics, and leu-
kocyte-endothelial interactions. Mol Imaging. 2002;
1:9–15.
195. Leu AJ, Berk DA, Yuan F, et al. Flow velocity in
the superficial lymphatic network of the mouse tail.
Am J Physiol. 1994;267:H1507–H1513.
196. Berk DA, Swartz MA, Leu AJ, et al. Transport
in lymphatic capillaries. II. Microscopic velocity
measurement with fluorescence photobleaching. Am
J Physiol. 1996;270:H330–H337.
197. Swartz MA, Berk DA, Jain RK. Transport in lym-
phatic capillaries. I. Macroscopic measurements using
residence time distribution theory. Am J Physiol.
1996;270:H324–H329.
198. Jeltsch M, Kaipainen A, Joukov V, et al. Hyperplasia
of lymphatic vessels in VEGF-C transgenic mice.
Science. 1997;276:1423–1425.
199. Isaka N, Padera TP, Hagendoorn J, et al. Peritumor
lymphatics induced by vascular endothelial growth fac-
tor-C exhibit abnormal function. Cancer Res. 2004;64:
4400–4404.
200. Fukumura D, Duda DG, Munn LL, et al. Tumor
microvasculature and microenvironment: novel
insights through intravital imaging in pre-clinical
models. Microcirculation. 2010;17:206–225.
201. Liao S, Cheng G, Conner DA, et al. Impaired
lymphatic contraction associated with immuno-
suppression. Proc Natl Acad Sci USA. 2011;108:
18784–18789.
202. Munn LL, Padera TP. Imaging the lymphatic system.
Microvasc Res. 2014;96:55–63.
203. Boucher Y, Baxter LT, Jain RK. Interstitial pressure
gradients in tissue-isolated and subcutaneous tumors:
implications for therapy. Cancer Res. 1990;50:
4478–4484.
204. Boucher Y, Kirkwood JM, Opacic D, et al. Interstitial
hypertension in superficial metastatic melanomas in
humans. Cancer Res. 1991;51:6691–6694.
205. Roh HD, Boucher Y, Kalnicki S, et al. Interstitial
hypertension in carcinoma of uterine cervix in
patients: possible correlation with tumor oxygenation
and radiation response. Cancer Res. 1991;51:6695–
6698.
206. Boucher Y, Jain RK. Microvascular pressure is the
principal driving force for interstitial hypertension
in solid tumors: implications for vascular collapse.
Cancer Res. 1992;52:5110–5114.
207. Gutmann R, Leunig M, Feyh J, et al. Interstitial
hypertension in head and neck tumors in
patients: correlation with tumor size. Cancer Res.
1992;52:1993–1995.
208. Less JR, Posner MC, Boucher Y, et al. Interstitial
hypertension in human breast and colorectal tumors.
Cancer Res. 1992;52:6371–6374.
209. Curti BD, Urba WJ, Alvord WG, et al. Interstitial
pressure of subcutaneous nodules in melanoma
and lymphoma patients: changes during treatment.
Cancer Res. 1993;53:2204–2207.
210. Arbit E, Lee J, DiResta GR. Interstitial hypertension
in human brain tumors: possible role in peritumoral
edema formulation. In: Nagai H, Kamiya K, Ishi
S, eds. Intracranial Pressure. Tokyo: Spirnger-Verlag;
1994:604–619.
211. Nathanson SD, Nelson L. Interstitial fluid pressure
in breast-cancer, benign breast conditions, and
breast parenchyma. Ann Surg Oncol. 1994;1:333–
338.
212. Boucher Y, Lee I, Jain RK. Lack of general correla-
tion between interstitial fluid pressure and oxygen
partial pressure in solid tumors. Microvasc Res.
1995;50:175–182.
213. Boucher Y, Salehi H, Witwer B, et al. Interstitial
fluid pressure in intracranial tumours in patients
and in rodents. Br J Cancer. 1997;75:829–836.
214. Milosevic M, Fyles A, Hedley D, et al. Interstitial
fluid pressure predicts survival in patients with cervix
cancer independent of clinical prognostic factors and
tumor: oxygen measurements. Cancer Res. 2001;
61:6400–6405.
215. Boucher Y, Leunig M, Jain RK. Tumor angio-
genesis and interstitial hypertension. Cancer Res.
1996;56:4264–4266.
216. Lee CG, Heijn M, di Tomaso E, et al. Anti-vascular
endothelial growth factor treatment augments tumor
radiation response under normoxic or hypoxic
conditions. Cancer Res. 2000;60:5565–5570.
217. Tong RT, Boucher Y, Kozin SV, et al. Vascular
normalization by vascular endothelial growth factor
receptor 2 blockade induces a pressure gradient across
the vasculature and improves drug penetration in
tumors. Cancer Res. 2004;64:3731–3736.
218. DiResta GR, Lee J, Healey JH, et al. Artificial
lymphatic system”: a new approach to reduce inter-
stitial hypertension and increase blood flow, pH and
pO(2) in solid tumors. Ann Biomed Eng. 2000;28:
543–555.
219. Stohrer M, Boucher Y, Stangassinger M, et al.
Oncotic pressure in solid tumors is elevated. Cancer
Res. 2000;60:4251–4255.
220. Raut CP, Boucher Y, Duda DG, et al. Effects of
sorafenib on intra-tumoral interstitial fluid pressure
and circulating biomarkers in patients with refrac-
tory sarcomas (NCI protocol 6948). PLoS ONE.
2012;7:e26331.
221. Willett CG, Boucher Y, di Tomaso E, et al.
Direct evidence that the VEGF-specific antibody
bevacizumab has antivascular effects in human rectal
cancer. Nat Med. 2004;10:145–147.
222. Willett CG, Boucher Y, Duda DG, et al. Surrogate
markers for antiangiogenic therapy and dose-limiting
toxicities for bevacizumab with radiation and
chemotherapy: continued experience of a phase I
trial in rectal cancer patients. J Clin Oncol. 2005;23:
8136–8139.
223. Willett CG, Duda DG, di Tomaso E, et al. Efficacy,
safety, and biomarkers of neoadjuvant bevacizumab,
radiation therapy, and fluorouracil in rectal cancer:
a multidisciplinary phase II study. J Clin Oncol.
2009;27:3020–3026.
224. Netti PA, Baxter LT, Boucher Y, et al. Time-
dependent behavior of interstitial fluid pressure in
solid tumors: implications for drug delivery. Cancer
Res. 1995;55:5451–5458.
225. Netti PA, Hamberg LM, Babich JW, et al. Enhance-
ment of fluid filtration across tumor vessels: implica-
tion for delivery of macromolecules. Proc Natl Acad
Sci USA. 1999;96:3137–3142.
226. Zlotecki RA, Boucher Y, Lee I, et al. Effect of angio-
tensin II induced hypertension on tumor blood flow
and interstitial fluid pressure. Cancer Res. 1993;53:
2466–2468.
227. Butler TP, Grantham FH, Gullino PM. Bulk transfer
of fluid in interstitial compartment of mammary
tumors. Cancer Res. 1975;35:3084–3088.
228. Jain RK, Tong RT, Munn LL. Effect of vascular
normalization by antiangiogenic therapy on
interstitial hypertension, peritumor edema, and
lymphatic metastasis: insights from a mathematical
model. Cancer Res. 2007;67:2729–2735.
229. Krogh A. The Anatomy and Physiology of Capillaries.
New York: Yale University Press; 1922.
230. Thomlinson RH, Gray LH. The histological
structure of some human lung cancers and the
possible implications for radiotherapy. Br J Cancer.
1955;9:539–549.
231. Helmlinger G, Yuan F, Dellian M, et al. Interstitial
pH and pO2 gradients in solid tumors in vivo:
high-resolution measurements reveal a lack of
correlation. Nat Med. 1997;3:177–182.
232. Torres-Filho IP, Leunig M, Yuan F, et al. Noninvasive
measurement of microvascular and interstitial oxygen
profiles in a human tumor in SCID mice. Proc Natl
Acad Sci USA. 1994;91:2081–2085.
233. Brown JM, Giaccia AJ. The unique physiology of
solid tumors: opportunities (and problems) for cancer
therapy. Cancer Res. 1998;58:1408–1416.
234. Dewhirst MW. Concepts of oxygen transport at
the microcirculatory level. Semin Radiat Oncol.
1998;8:143–150.
235. Helmlinger G, Sckell A, Dellian M, et al. Acid
production in glycolysis-impaired tumors provides
new insights into tumor metabolism. Clin Cancer
Res. 2002;8:1284–1291.
236. Harris AL. Hypoxia: a key regulatory factor in
tumour growth. Nat Rev Cancer. 2002;2:38–
47.
237. Nordsmark M, Bentzen SM, Rudat V, et al. Prog-
nostic value of tumor oxygenation in 397 head and
neck tumors after primary radiation therapy. An
international multi-center study. Radiother Oncol.
2005;77:18–24.
238. Giatromanolaki A, Koukourakis MI, Sivridis E,
et al. Expression of hypoxia-inducible carbonic
anhydrase-9 relates to angiogenic pathways and
independently to poor outcome in non-small cell
lung cancer. Cancer Res. 2001;61:7992–7998.
239. Koukourakis M, Giatromanolaki A, Sivridis E, et al.
Lactate dehydrogenase-5 (LDH-5) overexpression
in non-small-cell lung cancer tissues is linked to
tumour hypoxia, angiogenic factor production and
poor prognosis. Br J Cancer. 2003;89:877–885.
240. Vaupel P, Mayer A. Hypoxia in tumors: pathogenesis-
related classification, characterization of hypoxia
subtypes, and associated biological and clinical impli-
cations. Oxygen Transport to Tissue XXXVI. 2014;
(Springer):19–24.
241. Estrella V, Chen T, Lloyd M, et al. Acidity generated
by the tumor microenvironment drives local invasion.
Cancer Res. 2013;73:1524–1535.
242. Martin JD, Fukumura D, Duda DG, et al.
Reengineering the tumor microenvironment to
alleviate hypoxia and overcome cancer heterogeneity.
Cold Spring Harbor Perspectives in Medicine. 2016;
a027094.
243. Spivak-Kroizman TR, Hostetter G, Posner R, et al.
Hypoxia triggers hedgehog-mediated tumor-stromal
interactions in pancreatic cancer. Cancer Res. 2013;73:
3235–3247.
244. Wenes M, Shang M, Di Matteo M, et al. Macrophage
metabolism controls tumor blood vessel morphogen-
esis and metastasis. Cell Metab. 2016;24:701–715.
245. Brizel DM, Scully SP, Harrelson JM, et al. Tumor
oxygenation predicts for the likelihood of distant
metastases in human soft tissue sarcoma. Cancer
Res. 1996;56:941–943.
246. Brown JM. The hypoxic cell: a target for selective
cancer therapy. Eighteenth Bruce F. Cain Memorial
Award lecture. Cancer Res. 1999;59:5863–5870.
247. Cowan DS, Tannock IF. Factors that influence the
penetration of methotrexate through solid tissue.
Int J Cancer. 2001;91:120–125.
248. Kozin SV, Boucher Y, Hicklin DJ, et al. Vascular
endothelial growth factor receptor-2-blocking anti-
body potentiates radiation-induced long-term control
of human tumor xenografts. Cancer Res. 2001;61:
39–44.
249. Vukovic V, Tannock IF. Influence of low pH
on cytotoxicity of paclitaxel, mitoxantrone and
topotecan. Br J Cancer. 1997;75:1167–1172.
250. Clever D, Roychoudhuri R, Constantinides MG,
et al. Oxygen sensing by T cells establishes an
immunologically tolerant metastatic niche. Cell.
2016;166:1117–1131, e14.
251. Noman MZ, Desantis G, Janji B, et al. PD-L1 is
a novel direct target of HIF-1α, and its blockade
under hypoxia enhanced MDSC-mediated T cell
activation. J Exp Med. 2014;211:781–790.
252. El-Kenawi AE, Ibrahim-Hashim AA, Luddy KA,
et al. Extracellular acidosis alters polarization of
macrophages. Cancer Res. 2015;75:3213.
253. Noman MZ, Hasmim M, Messai Y, et al. Hypoxia: a
key player in antitumor immune response. A review

in the theme: cellular responses to hypoxia. Am J
Physiol Cell Physiol. 2015;309:C569–C579.
253a.  Fukumura D, Kloepper J, Amoozgar Z, Duda DG,
Jain RK. Enhancing cancer immunotherapy using
antiangiogenics: opportunities and challenges. Nat
Rev Clin Oncol. 2018;15:325–340.
254. Martin JD, Fukumura D, Duda DG, et al. Reen-
gineering the tumor microenvironment to alleviate
hypoxia and overcome cancer heterogeneity. Cold
Spring Harbor Perspectives in Medicine. 2016;6:
a027094.
255. Jain RK. Normalizing tumor microenvironment to
treat cancer: bench to bedside to biomarkers. J Clin
Oncol. 2013;31:2205–2218.
256. Winkler F, Kozin SV, Tong RT, et al. Kinetics of
vascular normalization by VEGFR2 blockade governs
brain tumor response to radiation: role of oxygen-
ation, angiopoietin-1, and matrix metalloproteinases.
Cancer Cell. 2004;6:553–563.
257. Leite de Oliveira R, Deschoemaeker S, Henze AT,
et al. Gene-targeting of phd2 improves tumor
response to chemotherapy and prevents side-toxicity.
Cancer Cell. 2012;22:263–277.
258. Mazzone M, Dettori D, Leite de Oliveira R, et al.
Heterozygous deficiency of PHD2 restores tumor
oxygenation and inhibits metastasis via endothelial
normalization. Cell. 2009;136:839–851.
259. Carmeliet P, Dor Y, Herbert JM, et al. Role of HIF-
1alpha in hypoxia-mediated apoptosis, cell prolifera-
tion and tumour angiogenesis. Nature. 1998;394:
485–490.
260. Harris AL. Hypoxia—a key regulatory factor in
tumour growth. Nat Rev Cancer. 2002;2:38–47.
261. Jain RK. Barriers to drug delivery in solid tumors.
Sci Am. 1994;271:58–65.
262. Jain RK. The next frontier of molecular medicine:
delivery of therapeutics. Nat Med. 1998;4:655–657.
263. Thienpont B, Steinbacher J, Zhao H, et al. Tumour
hypoxia causes DNA hypermethylation by reducing
TET activity. Nature. 2016.
264. McCormick F. New-age drug meets resistance.
Nature. 2001;412:281–282.
265. Batchelor TT, Gerstner ER, Emblem KE, et al.
Improved tumor oxygenation and survival in
glioblastoma patients who show increased blood
perfusion after cediranib and chemoradiation. Proc
Natl Acad Sci USA. 2013;110:19059–19064.
266. Emblem KE, Mouridsen K, Bjornerud A, et al.
Vessel architectural imaging identifies cancer patient
responders to anti-angiogenic therapy. Nat Med.
2013;19:1178–1183.
267. Heist RS, Duda DG, Sahani DV, et al. Improved
tumor vascularization after anti-VEGF therapy
with carboplatin and nab-paclitaxel associates with
survival in lung cancer. Proc Natl Acad Sci USA.
2015;112:1547–1552.
268. Sorensen AG, Batchelor TT, Zhang WT, et al.
A “vascular normalization index” as potential
mechanistic biomarker to predict survival after a
single dose of cediranib in recurrent glioblastoma
patients. Cancer Res. 2009;69:5296–5300.
269. Jain RK, Duda DG, Clark JW, et al. Lessons from
phase III clinical trials on anti-VEGF therapy for
cancer. Nat Clin Pract Oncol. 2006;3:24–40.
270. Carmeliet P, Jain RK. Principles and mechanisms of
vessel normalization for cancer and other angiogenic
diseases. Nat Rev Drug Discov. 2011;10:417–427.
271. Batchelor TT, Duda DG, di Tomaso E, et al. Phase
II study of cediranib, an oral pan-vascular endothelial
growth factor receptor tyrosine kinase inhibitor, in
patients with recurrent glioblastoma. J Clin Oncol.
2010;28:2817–2823.
272. Batchelor TT, Sorensen AG, di Tomaso E, et al.
AZD2171, a pan-VEGF receptor tyrosine kinase
inhibitor, normalizes tumor vasculature and alleviates
edema in glioblastoma patients. Cancer Cell. 2007;11:
83–95.
273. Tolaney SM, Boucher Y, Duda DG, et al. Role of
vascular density and normalization in response to
Tumor Microenvironment: Vascular and Extravascular Compartment  •  CHAPTER 8 126.e5
neoadjuvant bevacizumab and chemotherapy in breast
cancer patients. Proc Natl Acad Sci USA. 2015;112:
14325–14330.
274. Vasudev NS, Reynolds AR. Anti-angiogenic therapy
for cancer: current progress, unresolved questions and
future directions. Angiogenesis. 2014;17:471–494.
275. Smith NR, Baker D, Farren M, et al. Tumor
stromal architecture can define the intrinsic tumor
response to VEGF-targeted therapy. Clin Cancer
Res. 2013;19:6943–6956.
276. Zhu AX, Sahani DV, Duda DG, et al. Efficacy, safety,
and potential biomarkers of sunitinib monotherapy
in advanced hepatocellular carcinoma: a phase II
study. J Clin Oncol. 2009;27:3027–3035.
277. Zhu AX, Duda DG, Sahani DV, et al. HCC and
angiogenesis: possible targets and future directions.
Nat Rev Clin Oncol. 2011;8:292–301.
278. Vasudev N, Goh V, Juttla J, et al. Changes in tumour
vessel density upon treatment with anti-angiogenic
agents: relationship with response and resistance to
therapy. Br J Cancer. 2013;109:1230–1242.
279. Huang Y, Yuan J, Righi E, et al. Vascular normalizing
doses of antiangiogenic treatment reprogram the
immunosuppressive tumor microenvironment and
enhance immunotherapy. Proc Natl Acad Sci USA.
2012;109:17561–17566.
280. Batchelor TT, Duda DG, di Tomaso E, et al. Phase
II study of cediranib, an oral pan-vascular endothelial
growth factor receptor tyrosine kinase inhibitor, in
patients with recurrent glioblastoma. J Clin Oncol.
2010;28:2817–2823.
281. Frentzas S, Simoneau E, Bridgeman VL, et al. Vessel
co-option mediates resistance to anti-angiogenic
therapy in liver metastases. Nat Med. 2016.
282. Chen Y, Huang Y, Reiberger T, et al. Differential
effects of sorafenib on liver versus tumor fibrosis
mediated by stromal-derived factor 1 alpha/C-X-C
receptor type 4 axis and myeloid differentiation
antigen-positive myeloid cell infiltration in mice.
Hepatology. 2014;59:1435–1447.
283. Chen Y, Ramjiawan RR, Reiberger T, et al. CXCR4
inhibition in tumor microenvironment facilitates
anti-programmed death receptor-1 immunotherapy
in sorafenib-treated hepatocellular carcinoma in mice.
Hepatology. 2015;61:1591–1602.
284. Rahbari NN, Kedrin D, Incio J, et al. Anti-VEGF
therapy induces ECM remodeling and mechani-
cal barriers to therapy in colorectal cancer liver
metastases. Sci Transl Med. 2016;8:360ra135.
285. Rhim AD, Oberstein PE, Thomas DH, et al.
Stromal elements act to restrain, rather than support,
pancreatic ductal adenocarcinoma. Cancer Cell.
2014;25:735–747.
286. Ozdemir BC, Pentcheva-Hoang T, Carstens JL, et al.
Depletion of carcinoma-associated fibroblasts and
fibrosis induces immunosuppression and accelerates
pancreas cancer with reduced survival. Cancer Cell.
2014;25:719–734.
287. Cooke VG, LeBleu VS, Keskin D, et al. Pericyte
depletion results in hypoxia-associated epithelial-
to-mesenchymal transition and metastasis mediated
by met signaling pathway. Cancer Cell. 2012;21:
66–81.
288. Chauhan VP, Martin JD, Liu H, et al. Angiotensin
inhibition enhances drug delivery and potentiates
chemotherapy by decompressing tumour blood
vessels. Nat Commun. 2013;4:2516.
289. Stylianopoulos T, Martin JD, Chauhan VP, et al.
Causes, consequences, and remedies for growth-
induced solid stress in murine and human tumors.
Proc Natl Acad Sci USA. 2012;109:15101–15108.
290. Goel S, Gupta N, Walcott BP, et al. Effects of
vascular-endothelial protein tyrosine phosphatase
inhibition on breast cancer vasculature and metastatic
progression. J Natl Cancer Inst. 2013;105:1188–
1201.
291. Maes H, Kuchnio A, Peric A, et al. Tumor vessel
normalization by chloroquine independent of
autophagy. Cancer Cell. 2014;26:190–206.
126.e5
292. Patenaude A, Woerher S, Umlandt P, et al. A novel
population of local pericyte precursor cells in tumor
stroma that require Notch signaling for differentia-
tion. Microvasc Res. 2015;101:38–47.
293. Ueda S, Roblyer D, Cerussi A, et al. Baseline tumor
oxygen saturation correlates with a pathologic com-
plete response in breast cancer patients undergoing
neoadjuvant chemotherapy. Cancer Res. 2012;72:
4318–4328.
294. Ueda S, Kuji I, Shigekawa T, et al. Optical imaging
for monitoring tumor oxygenation response after
initiation of single-agent bevacizumab followed by
cytotoxic chemotherapy in breast cancer patients.
PLoS ONE. 2014;9:e98715.
295. Quintela-Fandino M, Lluch A, Manso LM, et al.
18F-fluoromisonidazole PET and activity of neo-
adjuvant nintedanib in early HER2-negative breast
cancer: a window-of-opportunity randomized trial.
American Association for Cancer Research:clincanres.
2016;0738.2016.
296. Ueda S, Saeki T, Takeuchi H, et al. In vivo imaging
of eribulin-induced reoxygenation in advanced breast
cancer patients: a comparison to bevacizumab. Br J
Cancer. 2016.
297. Stylianopoulos T, Jain RK. Combining two strate-
gies to improve perfusion and drug delivery in
solid tumors. Proc Natl Acad Sci USA. 2013;110:
18632–18637.
298. Emblem KE, Gerstner ER, Sorensen AG, et al.
Matrix-depleting anti-hypertensives decompress
tumor blood vessels and improve perfusion in
patients with glioblastomas receiving anti-angiogenic
therapy. AACR Annual Meeting:abstr. 2016;3975.
299. Mpekris F, Baish JW, Stylianopoulos T, et al. Role of
vascular normalization in benefit from metronomic
chemotherapy: insights from a mathematical
model. Proc Natl Acad Sci USA. 2017;114:1994–
1999.
300. Sledge GW. Anti-vascular endothelial growth factor
therapy in breast cancer: game over? J Clin Oncol.
2015;33:133–135.
301. Szabo V, Bugyik E, Dezso K, et al. Mechanism
of tumour vascularization in experimental lung
metastases. J Pathol. 2015;235:384–396.
302. Matus DQ, Lohmer LL, Kelley LC, et al. Invasive
cell fate requires G1 cell-cycle arrest and histone
deacetylase-mediated changes in gene expression.
Dev Cell. 2015;35:162–174.
303. Cheng G, Tse J, Jain RK, et al. Micro-environmental
mechanical stress controls tumor spheroid size and
morphology by suppressing proliferation and induc-
ing apoptosis in cancer cells. PLoS ONE. 2009;4:
e4632.
304. Desmaison A, Frongia C, Grenier K, et al. Mechani-
cal stress impairs mitosis progression in multi-cellular
tumor spheroids. PLoS ONE. 2013;8:e80447.
305. Delarue M, Montel F, Vignjevic D, et al. Com-
pressive stress inhibits proliferation in tumor
spheroids through a volume limitation. Biophys J.
2014;107:1821–1828.
306. Fischer KR, Durrans A, Lee S, et al. Epithelial-to-
mesenchymal transition is not required for lung
metastasis but contributes to chemoresistance.
Nature. 2015;527:472–476.
307. Zheng X, Carstens JL, Kim J, et al. Epithelial-to-
mesenchymal transition is dispensable for metastasis
but induces chemoresistance in pancreatic cancer.
Nature. 2015;527:525–530.
308. Lambrechts D, Lenz HJ, de Haas S, et al. Markers of
response for the antiangiogenic agent bevacizumab.
J Clin Oncol. 2013;31:1219–1230.
309. Ince WL, Jubb AM, Holden SN, et al. Association
of k-ras, b-raf, and p53 status with the treatment
effect of bevacizumab. J Natl Cancer Inst. 2005;97:
981–989.
310. Jubb AM, Hurwitz HI, Bai W, et al. Impact of
vascular endothelial growth factor-A expression,
thrombospondin-2 expression, and microvessel
density on the treatment effect of bevacizumab
126.e6 Part I: Science and Clinical Oncology
in metastatic colorectal cancer. J Clin Oncol.
2006;24:217–227.
311. Duda DG, Willett CG, Ancukiewicz M, et al. Plasma
soluble VEGFR-1 is a potential dual biomarker
of response and toxicity for bevacizumab with
chemoradiation in locally advanced rectal cancer.
Oncologist. 2010;15:577–583.
312. Gerstner ER, Emblem KE, Chi AS, et al. Effects of
cediranib, a VEGF signaling inhibitor, in combina-
tion with chemoradiation on tumor blood flow and
survival in newly diagnosed glioblastoma. J Clin
Oncol. 2012;30(suppl; abstr 2009).
313. Meyerhardt JA, Ancukiewicz M, Abrams TA,
et al. Phase I study of cetuximab, irinotecan, and
vandetanib (ZD6474) as therapy for patients with
previously treated metastatic colorectal cancer. PLoS
ONE. 2012;7:e38231.
314. Tolaney SM, Duda DG, Boucher Y, et al. A phase
II study of preoperative (preop) bevacizumab (bev)
followed by dose-dense (dd) doxorubicin (A)/
cyclophosphamide (C)/paclitaxel (T) in combination
with bev in HER2-negative operable breast cancer
(BC). J Clin Oncol. 2012;30(suppl; abstr 1026).
315. Zhu AX, Ancukiewicz M, Supko JG, et al. Clinical,
pharmacodynamic (PD), and pharmacokinetic (PK)
evaluation of cediranib in advanced hepatocellular
carcinoma (HCC): A phase II study (CTEP 7147).
J Clin Oncol. 2012;30(suppl; abstr 4112).
316. Xu L, Duda DG, di Tomaso E, et al. Direct evi-
dence that bevacizumab, an anti-VEGF antibody,
up-regulates SDF1alpha, CXCR4, CXCL6, and
neuropilin 1 in tumors from patients with rectal
cancer. Cancer Res. 2009;69:7905–7910.
317. Duda DG, Kozin SV, Kirkpatrick ND, et al.
CXCL12 (SDF1alpha)-CXCR4/CXCR7 pathway
inhibition: an emerging sensitizer for anticancer
therapies? Clin Cancer Res. 2011;17:2074–2080.
318. Kamba T, Tam BY, Hashizume H, et al. VEGF-
dependent plasticity of fenestrated capillaries in the
normal adult microvasculature. Am J Physiol Heart
Circ Physiol. 2006;290:H560–H576.
319. Verheul HM, Pinedo HM. Possible molecular
mechanisms involved in the toxicity of angiogenesis
inhibition. Nat Rev Cancer. 2007;7:475–485.
320. Stylianopoulos T, Martin JD, Snuderl M, et al.
Coevolution of solid stress and interstitial fluid
pressure in tumors during progression: implications
for vascular collapse. Cancer Res. 2013;73:3833–
3841.
321. Jain RK, Martin JD. Stylianopoulos T: The role of
mechanical forces in tumor growth and therapy.
Annu Rev Biomed Eng. 2014;16:321–346.
322. Nia HT, Liu H, Seano G, et al. Solid stress and elastic
energy as measures of tumour mechanopathology.
Nature Biomedical Engineering. 2016;1:0004.
322a.  Stylianopoulos T, Munn LL, Jain RK. Reengineering
the physical microenvironment of tumors to improve
drug delivery and efficacy: from math modeling to
bench to bedside. Trends Cancer. 2018;4:292–319.
323. Chauhan VP, Boucher Y, Ferrone CR, et al. Compres-
sion of pancreatic tumor blood vessels by hyaluronan
is caused by solid stress and not interstitial fluid
pressure. Cancer Cell. 2014;26:14–15.
324. Hagendoorn J, Tong R, Fukumura D, et al. Onset
of abnormal blood and lymphatic vessel function
and interstitial hypertension in early stages of
carcinogenesis. Cancer Res. 2006;66:3360–3364.
325. Lee JJ, Perera RM, Wang H, et al. Stromal
response to Hedgehog signaling restrains pancre-
atic cancer progression. Proc Natl Acad Sci USA.
2014;111:E3091–E3100.
326. Whatcott CJ, Han H, Von Hoff DD. Orchestrating
the tumor microenvironment to improve survival
for patients with pancreatic cancer: normalization,
not destruction. The Cancer Journal. 2015;21:299–
306.
327. Sherman MH, Yu RT, Engle DD, et al. Vitamin D
receptor-mediated stromal reprogramming suppresses
pancreatitis and enhances pancreatic cancer therapy.
Cell. 2014;159:80–93.
328. Menter AR, Carroll N, Delate T, et al: Effect of
angiotensin system inhibitors on survival in patients
receiving chemotherapy for advanced non-small
cell lung cancer, 50th Annual Meeting of ASCO.
Chicago, IL, 2014.
329. Keizman D, Huang P, Eisenberger MA, et al.
Angiotensin system inhibitors and outcome of
sunitinib treatment in patients with metastatic renal
cell carcinoma: a retrospective examination. Eur J
Cancer. 2011;47:1955–1961.
330. Nakai Y, Isayama H, Ijichi H, et al. Inhibition
of renin-angiotensin system affects prognosis of
advanced pancreatic cancer receiving gemcitabine.
Br J Cancer. 2010;103:1644–1648.
331. Nakai Y, Isayama H, Sasaki T, et al. The inhibition
of renin-angiotensin system in advanced pancreatic
cancer: an exploratory analysis in 349 patients.
J Cancer Res Clin Oncol. 2015;141:933–939.
332. Wilop S, von Hobe S, Crysandt M, et al. Impact
of angiotensin I converting enzyme inhibitors and
angiotensin II type 1 receptor blockers on survival
in patients with advanced non-small-cell lung
cancer undergoing first-line platinum-based che-
motherapy. J Cancer Res Clin Oncol. 2009;135:1429–
1435.
333. Emblem KE, Gerstner ER, Sorensen G, et al.
Matrix-depleting anti-hypertensives decompress
tumor blood vessels and improve perfusion in
patients with glioblastomas receiving anti-angiogenic
therapy. Cancer Res. 2016;76:3975.
334. Regan DP, Guth AM, Hansen RJ, et al. Abstract
A161: Angiotensin II type 1 receptor antagonism
suppresses tumor metastasis through inhibition of
CCL2-CCR2 mediated monocyte recruitment.
Cancer Immunol Res. 2016;4:A161.
335. Whatcott CJ, Diep CH, Jiang P, et al. Desmoplasia in
primary tumors and metastatic lesions of pancreatic
cancer. Clin Cancer Res. 2015;21:3561–3568.
335a.  Murphy JE, Wo JYL, Ryan DP, et al. Potentially
curative combination of TGF-b1 inhibitor losartan
and FOLFIRINOX (FFX) for locally advanced
pancreatic cancer (LAPC): R0 resection rates and
preliminary survival data from a prospective phase
II study. ASCO. 2018;Abstract #222259 (2018).
336. Benson A, Bendell J, Wainberg Z, et al. 618PDA
phase 2 randomized, double-blind, placebo controlled
study of simtuzumab or placebo in combination with
gemcitabine for the first line treatment of pancreatic
adenocarcinoma. Ann Oncol. 2014;25:iv211.
337. Catenacci DV, Junttila MR, Karrison T, et al.
Randomized Phase Ib/II study of gemcitabine
plus placebo or vismodegib, a hedgehog pathway
inhibitor, in patients with metastatic pancreatic
cancer. J Clin Oncol. 2015;33:4284–4292.
338. Hingorani SR, Harris WP, Beck JT, et al. Phase
Ib study of PEGylated recombinant human hyal-
uronidase and gemcitabine in patients with advanced
pancreatic cancer. Clin Cancer Res. 2016.
 Tumor Microenvironment: Vascular and Extravascular Compartment  •  CHAPTER 8 126.e7
126.e7

SELF-ASSESSMENT QUESTIONS ANSWERS

1. Malignant tumors are 1. (c) Malignant tumors are organlike structures composed of
a. conglomerates of genetically mutated cancer cells. malignant cells and host-derived stromal cells (recruited from
b. conglomerates of genetically mutated cancer cells and surrounding tissue and from blood circulation) in variable
stromal cells recruited from blood circulation. proportions depending on tumor type, stage, and site.
c. organlike structures composed of extracellular matrices 2. (e) Solid components of tumors, including cancer cells, stromal
containing genetically mutated cancer cells, host-derived cells, and matrix molecules, lead to elevated mechanical solid
stromal cells recruited from surrounding tissues and from stress. These solid tissue forces can greatly exceed the
blood circulation and extracellular matrices. microvascular pressure, and thus solid components of tumors
2. Vascular compression in tumors limits perfusion and is caused compress blood vessels. Interstitial hypertension is caused by the
by leakiness of tumor blood vessels, as in physiological edema
a. interstitial hypertension associated with uid retained in during in inflammation. Thus interstitial fluid pressure (fluid
tumors. stress) in tumors cannot exceed the microvascular pressure and
b. solid stress produced from cancer and stromal cells as they these fluid pressures completely oppose each other, such that
proliferate. interstitial hypertension cannot lead to vascular compression.
c. transmission of solid stress by matrix molecules. 3. (b) sVEGFR1/sFLT1 is an endogenous blocker of VEGF and
d. a and b placental-derived growth factor and has been proposed as a
e. b and c predictive biomarker of intrinsic resistance to anti-VEGF
3. Plasma soluble VEGFR1 is an endogenous vascular endothelial therapy. Our group reported that patients with locally advanced
growth factor (VEGF) inhibitor and is currently explored in rectal carcinoma who had high plasma sVEGFR1 levels prior to
trials of antiangiogenic agents as a treatment were less likely to benefit—or experience toxicities—
a. pharmacodynamic biomarker. from bevacizumab therapy combined with chemoradiation. We
b. predictive biomarker. later found the same association for newly diagnosed patients
c. prognostic biomarker. with glioblastoma multiforme, triple-negative breast cancer,
d. surrogate biomarker. hepatocellular carcinoma, and metastatic colorectal carcinoma
4. Interstitial hypertension is a hallmark of solid tumors and who were treated with anti-VEGF agents.
results from 4. (d) The blood vasculature of malignant tumors is abnormal and
a. vessel leakiness. hyperpermeable, and the lymphatic (drainage) system is
b. lack of functional lymphatics. disrupted within tumors. Along with the pressure created by
c. compression of vessels. proliferating cancer cells, these factors result to an increase in
d. all of the above. interstitial fluid pressure from approximately 0 mm Hg
e. none of the above. (normal) to levels that can reach 94 mm Hg.
5. Low vascular perfusion in solid tumors leads to 5. (e) Reduced perfusion, caused by vascular compression and
a. hypoxia. interstitial hypertension, limits the supply of oxygen and drugs
b. poor response to chemotherapy. to tumors. This limitation promotes hypoxia and inadequate
c. reduced metastasis. drug delivery in tumors, both leading to drug resistance. Higher
d. all of the above. perfusion correlates with lengthened survival in patients, and
e. a and b. agents in preclinical development that improve perfusion lead to
enhanced therapeutic outcomes. Metastasis thus far is not
affected by agents that increase perfusion.
 Cancer Metabolism 9 
Michael A. Reid, Sydney M. Sanderson, and Jason W. Locasale

S UMMARY OF K EY
• Metabolism supports biosynthesis,
energetics, and cell signaling
including chromatin state and
epigenetics.
• A common feature of each element
of malignancy is the necessity to
adapt metabolic functions.
• Altered metabolism in key metabolic
pathways and networks is being
P OI NT S
exploited clinically for diagnostics their environment, germline genetics,
and therapeutics. liver physiology, the microbiome,
• Old and new therapies target cancer and other influences including diet
metabolism, and the repurposing of and exercise.
metabolic drugs for cancer therapy
is attractive and ongoing.
• Future work in the area of cancer
metabolism will incorporate the
interaction between tumor cells and

Cancers are diseases in which cells acquire mutations that result in
proliferation that occurs outside the context of normal tissue develop-
ment. These somatic mutations interact with their environment to
result in cell transformation. This environment is determined by
numerous factors including, but not exclusively, host genetics, lifestyle
including diet, host physiology, the state of the immune system, and
the microenvironment of the tumor cells. At the nexus of this interaction
between genes and environment is cellular metabolism.
Metabolism involves a set of interconnected chemical reactions
mediated by enzymes. At the cellular level, nutrients including glucose,
lipids, and amino acids that are derived from their constituent dietary
macronutrients—carbohydrates, fats, and proteins—are taken up and
then transformed through a series of interconnected chemical reactions
that comprise the metabolic network (Fig. 9.1). These nutrients are
stored in their electron-rich reduced forms and through a process
known as catabolism are oxidized. The release of the electrons from
these oxidative processes generates energy that is used to sustain all
elements of life. Metabolism also allows for the conversion of intermedi-
ate products of catabolism to materials used as structural components
or building blocks for the production of proteins, nucleic acids, and
cell membranes. This is termed anabolism or anabolic metabolism.
Metabolism also can communicate directly to other elements of cellular
machinery by generating molecules that directly react with and alter
the activity of proteins and nucleic acids.
A common feature of each element of malignancy is the necessity
to adapt each of these metabolic functions to support the demands
of uncontrolled proliferation. Indeed, differential metabolic require-
ments manifest during each cancer-associated process such as anoikis,
invasion, and metastasis. This metabolic programming comes from
both the oncogenic signaling that drives distinct metabolic processes
and the environmental factors such as the tissue vasculature and the
nutrient content of serum. Furthermore, metabolic enzymes, by virtue
of their design and particularly their binding pocket, which encapsulates
small molecules, are readily druggable. In addition, metabolism may
be manipulated by lifestyle factors such as diet and exercise. Thus
there is tremendous opportunity to exploit metabolism for cancer
prevention and therapy.
This chapter discusses the fundamentals of metabolic reprogramming
and adaptation in cancer cells and outlines both the principles that
lead to this altered metabolism and the specific requirements that
result. Particular attention will be paid to key metabolic pathways
and networks and to areas that have direct clinical application such
as current antimetabolite chemotherapy agents and ongoing drug
development efforts. Our hope is to give the reader a broad overview
of this rapidly progressing area of cancer biology and medical
oncology.
FUNDAMENTAL SCIENCE
Warburg Effect
In conditions with sufficient oxygen, terminally differentiated cells
usually metabolize glucose through three highly regulated sequences
of enzymatic processes: glycolysis, the tricarboxylic acid (TCA) cycle,
and the electron transport chain (ETC).1,2 On complete oxidation,
one molecule of glucose can ultimately generate approximately 36
units of the energy-storing molecule adenosine 5′-triphosphate (ATP)
and other energy-rich metabolic currency such as reduced nicotinamide
adenine dinucleotide (NADH).1,2 These processes are compartmental-
ized: glycolysis in the cytosol, and the TCA cycle and ETC in the
mitochondria. Under conditions of oxygen limitation such as exercise
in muscle, the end product of glycolysis (pyruvate) is diverted away
from the mitochondria to produce the fermentation product lactate.2
This is known as anaerobic glycolysis and generates only two net ATP
molecules per molecule of glucose while consuming NADH (produces
oxidized nicotinamide adenine dinucleotide [NAD+]), and is a common
form of glucose metabolism for unicellular organisms such as certain
yeasts and parasites.3,4 Although this may seem less efficient for mam-
malian cells, the rate of ATP production via glycolysis can be much
faster than the time it takes for mitochondrial respiration; therefore
in some cases greater amounts of ATP can be generated from glycolysis
in a given amount of time in the presence of abundant glucose.5,6
One of the first documented points of evidence for an altered
molecular property in tumors was the seminal observation made by
Otto Warburg and colleagues in 1924 regarding the tumor cell–specific
fate of glucose (Fig. 9.2). The essential features of what came to be
defined as the “Warburg effect” were the observations that neoplastic
cells consumed large amounts of glucose and fermented it into lactate.7

127
128 Part I: Science and Clinical Oncology

and fibroblasts use aerobic glycolysis for normal function.12,13 Nonethe-

less, the Warburg effect in tumors is generally broad and has been
exploited clinically for many years through imaging of glucose uptake
Dietary sources Roles (discussed later in further detail) for diagnosis and monitoring of
–Carbohydrates 1 Energy treatment progress in numerous cancers including lymphomas, breast
–Fats – Oxidation cancer, non–small cell lung cancer (NSCLC), esophageal cancer, and
–Protein 2 Biosynthesis colorectal cancer.14
– Reduction Several hypotheses as to why cancer cells favor what seems like a
wasteful process have been proposed.15 Warburg and colleagues eventu-
3 Signalling
Digestion ally concluded it was due to defective mitochondria,16 but many cancer
cells in fact use diverse mitochondrial metabolic functions for growth
advantages, which are discussed in detail later. One proposal for why
cancer cells undergo the Warburg effect is the need for rapid production
Macronutrients of ATP, and as mentioned previously this can be achieved by enhanced
–Glucose glycolysis. In support of this, it has been demonstrated that cells
–Lipids upregulate aerobic glycolysis when challenged with conditions that
–Amino acids require generation of large amounts of ATP while oxidative phosphoryla-
tion remains constant.17 However, mathematical calculations indicate
that the amount of ATP required for cell growth and division may
Cellular be less than the amount required for normal cell survival, suggesting
uptake
that ATP may not be a limiting factor in cancer cell proliferation.18
Another model hypothesizes that the Warburg effect is important for
Central carbon generating biomass in the form of donating carbons for biosynthetic
metabolism processes. For example, the enzyme phosphoglycerate dehydrogenase
(PHGDH) diverts glucose carbons away from glycolysis into the serine
biosynthetic pathway,19 which is important for one-carbon metabolism
Figure 9.1  •  Overview of macronutrient metabolism. Macronutrients, and nucleotide synthesis (discussed later), but it remains unclear if
carbohydrates, fats, and proteins are digested into constituent macronutrients or how this would promote biomass production because the majority
glucose, lipids, and amino acids. The cellar uptake of these macronutrients serves of glucose carbons are excreted as lactate during the Warburg effect.20
three main roles: energy production, biosynthesis, and cellular signaling. Energy
production is generally oxidative, and biosynthesis is generally reductive. An intriguing hypothesis suggests that the acidic microenvironment
created by lactate secretion promotes tumorigenesis. This is supported
by studies showing that increased acidity promotes cancer cell invasive-
ness,21,22 but the Warburg effect occurs at an early time point during
18FDG-PET Glucose tumorigenesis before invasion, which calls into question whether this
can be the main function. Another model reasons that cancer cells
undergo aerobic glycolysis for the purpose of cellular signaling such
as the opening of chromatin, although it is difficult to conduct experi-
ments that conclusively demonstrate this.15
Although the exact reason why cells favor the Warburg effect remains
elusive, there has been considerable effort toward exploiting it for
± Oxygen cancer treatment by generating new therapeutic targets. It is clearly
required for certain tumors, and furthermore, glucose uptake and
catabolism are driven by enzymes encoded by oncogenes commonly
mutated or overexpressed in many tumor types such as PI3K, KRas,
and HIF1α, suggesting that tumors select for aerobic glycolysis.23
Future work will focus on elucidating exactly how the Warburg effect
promotes tumorigenesis and better defining the specificity of targetable
Liver metastasis nodes for new cancer therapeutic strategies.
(Source: Wikipedia)

Amino Acid Metabolism

Lactate
Figure 9.2  •  The Warburg effect. In the presence or absence of oxygen,
cancer cells take in large amounts of glucose and preferentially ferment the
final product of glycolysis, pyruvate, into lactate. Fluorine-18 fluorodeoxy-
glucose–positron emission tomography (18F-FDG–PET) is commonly used
as an imaging modality to monitor tumors.
It was also found that this occurred even in the presence of abundant
oxygen; thus this phenomenon is commonly referred to as aerobic
glycolysis, and Warburg hypothesized that killing cancer cells would
require depleting both glucose and oxygen.8 These initial findings
have been conclusively validated by genetic and pharmacologic
studies,9,10 but not all cancers undergo or require aerobic glycolysis.11
Moreover, nontransformed proliferative cell types such as lymphocytes
In addition to glucose, cancer cells rely on other nutrients to meet
increased metabolic demands. Several amino acids have been shown
to play important roles for various cancer cell functions. Similar to
glucose, amino acid uptake through cell membrane transport channels
and catabolism can be driven by oncogenes.23–26 Cancer cells can also
obtain amino acids via de novo synthesis, breakdown of the serum
peptide albumin after micropinocytosis,27 and autophagic digestion
of cellular proteins.28,29 Of the 20 common amino acids, nine are
essential—meaning that they are obtained from dietary sources.
However, certain nonessential amino acids are considered to be
conditionally essential in cases in which rapid catabolism outcompetes
the local rate of generation.30
There have been substantial efforts in studying cancer cell gluta-
mine metabolism as both a carbon and a nitrogen source. Glutamine
levels in serum are the highest of all amino acids,31 and glutamine is
important for several cellular functions. Glutamine maintains redox

balance by contributing to glutathione (GSH) synthesis and participates
in anabolic metabolism such as lipid biosynthesis through reductive
carboxylation.32,33 Glutamine also maintains nitrogen pools for the
synthesis of nonessential amino acids and nucleotides.34 Of particular
importance is that glutamine-derived glutamate is converted into
α-ketoglutarate (αKG), which can maintain the flow of the TCA
cycle leading to ATP production via the ETC.35 This is known as
anaplerosis and is critical for continuing mitochondrial respiration.
There is strong evidence that this occurs in cell culture, but reports
using stable isotope labeling in mice have indicated that glutamine may
not be a major source of αKG in all tumors.36 However, numerous
studies have demonstrated the importance of glutamine in certain
tumors; inhibition of glutamine metabolism prevents tumor growth
in models of breast cancer,37 liver cancer,38 kidney cancer,39 and T-cell
acute lymphoblastic leukemia.40
Another fundamental metabolic process is one-carbon metabolism,
which involves the amino acids serine, glycine, and methionine.
One-carbon metabolism that consists of the folate and methionine
cycles is a master integrator of nutritional status. Conversion of serine
to glycine via serine hydroxymethyltransferase (SHMT) generates
one-carbon molecules that are passed to folate moieties.41 These folate
moieties undergo further chemical reactions that lead to the transfer
of carbon atoms to downstream metabolic pathways. A major output
of the folate cycle is the generation of nucleotides, which are essential
for proliferating cells. Serine can be taken up by cells through transport-
ers or synthesized de novo from glucose or gluconeogenic intermedi-
ates.42 In addition to contributing to the generation of nucleotides,
serine coupled to the folate cycle is necessary for other biomass such
as phospholipids and the generation of redox factors such as NADPH,
a molecule used for reductive biosynthesis and restoration of gluta-
thionine, which is used to protect against oxidative stress.43 Serine
further contributes to redox balance by ligating with homocysteine
to begin the transsulfuration pathway for cysteine synthesis, which is
required for GSH production.44 Highlighting the importance of serine
biosynthesis for certain cancers was the discovery that PHGDH, the
gene encoding the enzyme that catalyzes the committed step, is amplified
in melanoma and breast cancer.19 Moreover, PHGDH inhibitors have
shown promise in reducing tumor growth in mouse models.45–47 Also
of note, one-carbon units are used to methylate homocysteine to
generate methionine, which can have a major effect on protein regula-
tion and epigenetics (discussed in more detail later).48 In many cases,
the majority of one-carbon units used to recycle methionine come
from serine.49 In all, carbon units derived from serine, glycine, and
other one-carbon donors such as choline, betaine, sarcosine, and
histidine are used to produce biosynthetic components, maintain redox
status, and provide the substrates for methylation reactions.43,44
Other amino acids important for tumorigenesis include arginine,
tryptophan, and the branched-chain amino acids leucine, isoleucine,
and valine. Arginine can act as nitrogen donor for nitric oxide (NO)
signaling, as a fuel for the urea cycle, or to maintain pools of nonessential
amino acids, and tumors lacking the argininosuccinate synthase enzyme
highly depend on exogenous arginine for growth.50 Tryptophan
catabolism by tumors has been shown to create an immunosuppressive
tumor microenvironment.51,52 Branched-chain amino acids contribute
to biosynthesis of alanine and glutamine, and high levels are found
in the serum of patients with early-stage pancreatic cancer.53 In addition,
leucine and arginine are potent activators of mammalian target of
rapamycin (mTOR), which is a regulator of protein translation and
metabolism and cell growth and often dysregulated in cancers.54
Mitochondrial Metabolism
Mitochondria are organelles that play an important role in cell survival
and proliferation.1,2 Mitochondrial metabolism is important for the
function of the TCA cycle; ETC, fatty acid oxidation (FAO); synthesis
of amino acids, lipids, and nucleotides; and production of ATP and
antioxidants.55 One consequence of mitochondrial metabolism is the
Cancer Metabolism  •  CHAPTER 9 129
Glucose Lactate
Pyruvate
Pyruvate
TCA
Acetyl-coA cycle
Energy
Lipid (Oxidative
Amino acids phosphorylation)
TCA Biosynthesis
cycle (Nucleic acids,
amino acids)
Signalling
(e.g. Reactive oxygen
Epigenetics
(Histone/DNA species, HIFs)
methylation)
Figure 9.3  •  Mitochondrial metabolism. Mitochondria serve at the center
of macronutrient metabolism. Glucose, lipids, and amino acids enter the
mitochondria and are oxidized through the tricarboxylic acid (TCA) cycle.
The TCA cycle provides several functions, noted on the right. HIF, Hypoxia-
inducible factor.
generation of reactive oxygen species (ROS). This led to the proposal
that mitochondria may promote DNA damage via high amounts of
ROS accumulation, which could contribute to genome instability.56
Although potentially damaging to cells at high levels, at lower concentra-
tions ROS have been shown to act as signaling molecules that can
promote proliferation and may be essential for tumorigenicity.57 In
all, mitochondria promote cell growth through generation of energy,
biomass, and signaling cascades (Fig. 9.3).
Warburg and others originally hypothesized that tumor cells
harbored dysfunctional mitochondria, which is how it was reasoned
that the TCA cycle and ETC were not used for tumor cell glucose
metabolism.16 However, subsequent studies found that tumor cells
could undergo oxidative metabolism, and in fact the mitochondria
play a critical role in cancer cell metabolism. For example, it was
reported in the 1950s that oxidation of the fatty acid (FA) palmitate
occurred in a manner similar to that in healthy liver cells.58 In addition,
enzymes of the TCA cycle such as isocitrate dehydrogenase (IDH)
have similar expression levels in cancer cell and normal cell mitochon-
dria.59 Numerous studies have corroborated these findings, and several
reports have provided genetic evidence that mitochondrial metabolism
is necessary for tumorigenesis.57,60 Indeed, the current paradigm in
the cancer metabolism field is that cancer cells actively use and require
both glycolysis and mitochondrial metabolism.61
There is developing interest in targeting tumor mitochondrial
processes for cancer therapy.61 As mentioned, the Warburg effect
can generate sufficient amounts of ATP to fuel cancer cells in the
absence of mitochondrial metabolism. Thus targeting mitochondrial
ATP production may not be a viable therapeutic strategy. However,
certain subtypes of cancer have been shown to be highly reliant on
mitochondrial-dependent ATP production via oxidative phosphoryla-
tion.62,63 Moreover, the tumor microenvironment may have limited
glucose availability, which would reduce the glycolytic capacity of
the cell and make it more dependent on mitochondrial metabolism
(discussed later in detail). A major pitfall for targeting these processes
is the fact that a majority of normal cells require functional mito-
chondrial metabolism for survival. Thus tumor-specific delivery and
uptake of therapeutics or the targeting of biosynthetic precursors
selectively required in cancer cells would be essential for reducing
healthy cell toxicity. Potential candidates include the antidiabetes
130 Part I: Science and Clinical Oncology
drug metformin and certain glutaminase inhibitors (discussed later
in detail).
Lipid Metabolism
Although given less attention than glucose or amino acids, lipid
metabolism provides important signaling intermediates and biomass
and energy resources to cancer cells. There are numerous species of
lipids, or FAs, and all contain a hydrocarbon chain and terminal
carboxyl group.1,2 The synthesis and oxidation of FAs contribute to
numerous cellular growth and survival functions such as membrane
formation, signaling molecules, and energy sources. Similar to other
nutrients that support proliferation, lipid metabolism is upregulated
in cancer cells.64 This is largely because cells must double their lipid
membrane in order to divide, resulting in a massive demand for
lipid and phospholipid precursors. Building blocks for membrane
production and lipid energy sources come from exogenous sources
such as diet and/or are synthesized de novo by cells. Cancer cells
preferentially synthesize lipids locally, and early studies revealed a shift
toward FA synthesis in neoplastic cells.65
De novo synthesis of FAs is coupled to glucose and glutamine
metabolism. The TCA cycle intermediate citrate is the starting material
for a majority of local FA production.66 As mentioned, citrate can be
generated by glucose-derived pyruvate or, in high–Warburg effect
cells, glutamine-derived αKG. Several enzymes including ATP citrate
lyase (ACLY), acetyl-coenzyme A (CoA) carboxylase (ACC), and fatty
acid synthase (FASN) are required to metabolize citrate into bioactive
FAs.67 ACLY converts citrate to oxaloacetate and acetyl-CoA, and
acetyl-CoA is the major precursor for all lipids in the cell. The
importance of ACLY for tumor growth is demonstrated by studies
showing that loss of ACLY reduced tumor growth in mouse models.68–70
ACC catalyzes the committed step in FA synthesis via carboxylation
of acetyl-CoA to malonyl-CoA. ACC is a highly regulated enzyme,
and its activity is negatively regulated by the tumor suppressor LKB1
via AMPK-dependent phosphorylation.71 ACC has been shown to be
required for cancer cell growth and survival,72–74 but additional complex-
ity comes from other studies that have indicated a tumor suppressive
role for ACC.75 FASN is commonly upregulated in several cancer
types, and its expression correlates with poor prognosis.76 FASN
condensates one acetyl-CoA and seven malonyl-CoA molecules to
generate the initial product of FA synthesis, palmitate, which is then
chemically altered to fuel pools of a diverse array of lipids including
those involved in membrane formation and signaling molecules.76
In addition to relying on lipids for biomass, cancer cells can oxidize
FAs for energy production. The synthesis and oxidation of FAs are
mutually exclusive, and several mechanisms for this regulation have
been described in cancer cells. In a process known as β-oxidation,
cellular FAs such as palmitate are catabolized in peroxisomes and
mitochondria to generate energy and reducing molecules. Carnitine
palmitoyltransferase 1 (CPT1) catalyzes the rate-limiting step for
importing FAs into the mitochondria, where it is catabolized to generate
acetyl-CoA. The 16-carbon palmitate ultimately generates eight
acetyl-CoA molecules, which condense with oxaloacetate to drive the
TCA cycle.77 Thus one molecule of palmitate can produce more energy
than fully oxidized glucose. The brain isoform of CPT1 (CPT1C)
was identified as a potential oncogene for its role in promoting cancer
cell survival under metabolic stress.78 β-Oxidation has been shown to
be particularly important for cancer cells that have lost attachment
to the basal membrane,79 and targeting β-oxidation was sufficient to
reduce tumor growth in a patient-derived triple-negative breast cancer
(TNBC) xenograft model.80
Because cancer cells may selectively rely on de novo FA synthesis,
many efforts have been made toward targeting these pathways. Owing
to its upregulation in numerous cancers, FASN in particular has been
a popular target for drug development efforts, with several inhibitors
evaluated in preclinical studies showing strong antitumor efficacy.76
Unfortunately, harsh side effects such as detrimental weight loss have
been observed in preclinical models.81 Other enzymes of targetable
interest include CPT1, ACLY, and ACC, with results similar to those
with FASN inhibition.82 An interesting strategy for using cancer cell
lipid dependence against tumors is loading drugs into liposomal
nanoparticles, which has been shown to increase tumor-specific drug
delivery.83 As is the case with targeting most metabolic processes, the
inherent challenge is overcoming toxic side effects to normal cells.
Metabolism and Epigenetics
Epigenetics, or more specifically chromatin state, encompasses changes
in gene expression that are not the result of mutations in DNA
sequences.84 Chromatin accessibility can in part determine gene
expression and is regulated by posttranslational modifications of the
chromatin-bound histone proteins including methylation, acetylation,
ubiquitination, and phosphorylation in addition to methylation of
the DNA base cytosine.85 Changes in epigenetics and chromatin have
emerged as drivers of cancer initiation and progression86,87 and can
function similarly to the amplification of oncogenes or the deletion
of tumor suppressor genes.88,89 From a therapeutic perspective, targeting
epigenetics is appealing for several reasons, including the druggability
of the enzymes that carry out modifications to chromatin and DNA
and the fact that many of the effects are reversible, as opposed to the
irreversible changes of genetic mutations.
Histone acetylation and DNA and histone methylation are among
the best studied modifications. DNA methylation occurs at the fifth
position of cytosine bases and is commonly observed in promoter
regions of genes.86 Histone acetylation and methylation occur on
lysine (K) residues. Acetylation generally leads to active chromatin,
whereas histone methylation is associated with both active and inactive
chromatin.90 For example, methylation of histone H3K4 leads to
enhanced gene expression, whereas methylation of H3K9 or H3K27
is associated with gene suppression. Underlying many epigenetic states
is cellular metabolism because certain metabolites provide substrates
for posttranslational modifications and essential cofactors for epigenetic
machinery.91,92
Methionine-derived S-adenosyl methionine (SAM) is a methyl
donor for several methyl transferase enzymes, including DNA and
histone methyl transferases (Fig. 9.4A). Therefore changes in one-carbon
metabolism can have dramatic effects on epigenetics.93 Indeed, dietary
restriction of methionine reduces levels of H3K4 methylation.48
Moreover, limiting glycine input reduces SAM levels and has a similar
effect on H3K4 methylation.94 Similarly, SAM levels also affect DNA
methylation during osteoclast differentiation.95 Acetyl-CoA derived
from glycolysis and β-oxidation is the primary acetyl donor for histone
acetylation reactions carried out by histone acetyltransferases (Fig.
9.4B). Previous studies have demonstrated that acetyl-CoA availability
largely regulates histone acetylation,96 and histone acetylation depends
on ACLY activity.97 Histone deacetylases remove acetyl groups from
histones, and some require NAD+ as a cofactor.98 Therefore the NAD+/
NADH ratio in addition to the levels of acetyl-CoA, and NAD+,
which can be increased in high Warburg cells, may have an effect on
epigenetics and gene expression.
A major breakthrough in understanding of the connection between
cancer metabolism and epigenetics was the discovery of mutations
in the IDH genes IDH1 and IDH2 in leukemias and gliomas.99,100
The enzymes IDH1 and IDH2 normally convert citrate to αKG
in the mitochondria and cytosol, respectively. Mutations in IDH1
and IHD2 occur in the active site, and loss of αKG production by
IDH-mutant enzymes was originally attributed to loss-of-function.101
Subsequent studies found the IDH mutants to be gain-of-function
mutations, where αKG was converted to 2-hydroxyglutarate (2HG)
at high levels, resulting in a neomorphic enzymatic activity.102 Because
αKG is an essential cofactor and 2HG is an inhibitor of many DNA
and histone demethylases, IDH-mutant cells show broad DNA and
histone hypermethylation, and supplementation with αKG attenuates
these changes.103,104 Of note, pharmaceutical targeting of IDH1 and
 Cancer Metabolism  •  CHAPTER 9 131

Methionine Glutamine Glucose

Succinate
SAM Fumarate
2HG
αKG

H/D MT H/D DM Mutant

Methyl IDH1/2
K K Methyl K K K K
Methyl

C C C

Figure 9.4  •  Metabolism and epigenetics. (A) Methionine- and S-adenosyl methionine (SAM)–derived methyl moieties are attached to lysine (K) residues
of DNA binding histone proteins and/or cytosine (C) DNA bases by histone or DNA methyltransferases (H/D MT). Cytosine methylation generally attenuates
transcription, whereas histone methylation may activate or suppress transcription. Removal of methyl groups is carried out by histone or DNA demethylase
(H/D DM), and some require glutamine and/or glucose-derived α-ketoglutarate (αKG) as a cofactor. H/D DM can be inhibited by loss of αKG, by accumulation
of succinate and fumarate, or by mutant IDH–produced 2-hydroxyglutarate (2HG). (B) Acetyl-CoA generated from glucose and/or fatty acids such as palmitate
is attached to histone lysine residues by histone acetyltransferase (HAT) enzymes to enhance transcription. HATs require oxidized nicotinamide adenine
dinucleotide (NAD+) as a cofactor.

IDH2 has shown promising results in leukemia and glioma models.105,106
Similarly, the loss of glutamine, which can produce αKG, results in
increased histone methylation of stem cells107 and alters drug responses
in tumors; targeting the methyltransferases or supplementing with
αKG resensitized cancer cells to the drug.108 Other metabolites besides
mutant IDH–derived 2HG can inhibit demethylase activity. Cancer
cells with loss-of-function mutations in the TCA cycle enzymes suc-
cinate dehydrogenase and fumarate hydratase accumulate succinate
and fumarate, respectively, and display global DNA and histone
hypermethylation109 (Fig. 9.4A). Thus future cancer treatments may
target epigenetic machinery or use dietary supplements in combination
with establish chemotherapeutics and new agents that target cancer
metabolism.
Nutrient Heterogeneity and Tumor
Microenvironment
Many studies conducted in cell culture are performed on single cell
types in the presence of abundant nutrients. In physiologic environ-
ments, tumor cells are surrounded by numerous other cell types
including fibroblasts, endothelial cells, and lymphocytes, resulting in
competition for local nutrients110 (Fig. 9.5). In addition, tumors often
contain incomplete and unorganized vasculature, which can lead to
regions of nutrient and oxygen limitation.111 Although tumor cells
require an abundance of nutrients to maintain rapid growth and
proliferation, they paradoxically persist in these harsh environments.
Consequently, cells that adapted to low nutrients and oxygen are
among the most difficult to kill and present major challenges for
many current cancer therapies.108 Thus in order to develop better
therapeutic strategies, it is important to understand the tumor
microenvironment and mechanisms of cellular adaptation to nutrient-
deficient environments.
Hypoxia, or low oxygen, is one of the most studied properties of
the tumor microenvironment. Oxygen levels in healthy tissue are
around 5%, whereas regions of solid tumors can have oxygen levels
below 0.5%.112 The ETC can function in oxygen levels as low as
0.5%,113 allowing tumor cells to mostly undergo normal cellular
functions, but they may gain survival advantages based on signaling
changes in response to oxygen stress due to changes in redox balance.
For example, the hypoxia-inducible factor (HIF) proteins are activated
by hypoxia and promote downstream transcriptional events to adapt
to limited oxygen availability.114,115 This promotes survival in response
to therapeutically induced stress such as radiation, and targeting HIF
in combination with radiation therapy has shown promise.116 HIF
proteins have also been implicated in maintaining cancer initiating
or cancer stem cells that can repopulate tumors, and these cells are
commonly found in hypoxic niches.117,118
In addition to oxygen, tumor cells often encounter low levels of
nutrients in the microenvironment (see Fig. 9.5). A study conducted
with paired pancreatic cancer biopsies found pancreatic tumors are
generally nutrient poor compared with adjacent normal tissues.119 It
was shown that many amino acids were depleted, including glutamine
and serine. Studies have found that cancer cells can survive glutamine
limitation through induction of cell cycle arrest or alteration in glucose
usage.120–122 Similarly, cells adapt to serine deprivation by inhibiting
cell division.123 In addition, a subtype of breast and melanoma cells
with amplifications of PHGDH can compensate for the loss of
exogenous serine by upregulating de novo serine biosynthesis from
glucose.19 Yet glucose availability itself is reduced in the microenviron-
ment of some tumors.119 Further research will undoubtedly identify
other survival pathways and redundancies used by tumors to survive.
One goal of understanding these mechanisms is to target them in
combination with established therapeutics.
The tumor microenvironment can play a major role in metabolism of
tumors, depending on the tissue of origin. Highlighting this is a study
that showed metabolism within the same tumor types was drastically
different depending on the surrounding tissue.36 Heterogeneity of
nutrient availability and use can occur within a single tumor. For
example, nutrient availability drastically differs in core regions of solid
tumors compared with the peripheral regions.108 Furthermore, a study
of a non–small cell lung carcinoma patient cohort in which stable-
isotope labeling was used found evidence of Warburg and non-Warburg
utilizing cells within a single tumor, and these metabolic phenotypes
largely differed from patient to patient.124 These results indicate that
multiple types of therapeutics targeting metabolism may be necessary
for treatment of a single tumor.
Tumor cell metabolism can alter the microenvironment to promote
tumorigenesis and reduce immune activity. As mentioned previously,
132 Part I: Science and Clinical Oncology
Tumor
Epithelial
cells
Amino
acids Tumor cells
Glucose
Fibroblasts
Suppresive
macrophage
Lactate
FAs Macrophage
Blood
vessel
Epithelial
cells
Nutrient and oxygen availability
Figure 9.5  •  Nutrient heterogeneity and tumor microenvironment. Tumors contain numerous cell types including tumor cells, fibroblasts, epithelial cells,
macrophages, and T cells, which all compete for local nutrients. Tumor cell metabolism such as enhanced lactate secretion and glucose consumption can
suppress tumor-infiltrating immune cells. Cells furthest from the vasculature encounter nutrient- and oxygen-poor environments, which also alters their
characteristics and metabolism. FAs, Fatty acids.
highly glycolytic cells convert the majority of their glucose carbons
to lactate and secrete those carbons out of the cells. Lactate secretion
is coupled to the cotransport of H+, and CO2 generated by the ETC
perfuses out of the cell before conversion to H+ and HCO3− in the
extracellular space.125 Consequently, the accumulation of H+ reduces
the local pH, creating an acidic microenvironment around tumors.
Furthermore, studies have shown that acidified environments activate
matrix metalloproteases, which degrade the extracellular matrix and
promote tumor invasiveness.126,127 Moreover, lactate polarizes tumor-
associated macrophages into an immunosuppressive state.128 In addition
to innate immune cells, adaptive immune cells such as T cells require
specific metabolic functions in order to facilitate their protective
effects. The ability of T cells to transition from a naïve (inactive)
state to an effector (active) state to a memory state is determined by
their metabolism.12 Circulating T cells are normally in nutrient- and
oxygen-rich environments. Upon entering tumors, T cells are met
with challenging metabolic conditions that can affect their activity
(see Fig. 9.5). T cells require large amounts of glucose for activation,
and undergo the Warburg effect to a similar degree as many cancer
cells.12 The absence of abundant glucose can lead to T-cell inaction
or death.129,130 Indeed, much of the immunosuppression found in
the tumor microenvironment is attributed to the inability of T cells
to obtain sufficient nutrients.131 In addition, tumor cells upregulate
indolamine 2′3′-dioxygenase (IDO) to promote tryptophan catabolism,
which induces cell death in effector T cells.132 In addition to nutrient
stress, oxygen availability also plays a major role in affecting T-cell
function. Hypoxia-induced HIF activation transcriptionally upregulates
PD-1 and CTLA4, which act to suppress immune activity.133,134 HIF also
promotes expression of T regulatory cells (Tregs), which downregulate
the immune response.135 Because of this, T cells can be tumor suppres-
sive or tumor promoting.136 Immunotherapy focusing on ex vivo and
in vivo stimulation of T cells is a promising avenue for management of
tumors, but overcoming the tumor microenvironment will be critical to
its success.
Blood
vessel
Active Inactive
T-cells T-cells
Nutrient and oxygen availability
Obesity and Cancer
Obesity is a health crisis in the developed world, with estimates of
over 30% of adults in the United States classified as obese. This is
thought to be a major if not the leading source of human mortality.137
Although associated more commonly with type II diabetes and car-
diovascular disease, accumulating evidence demonstrates that obese
individuals are more likely to develop cancer. Obesity has been shown
to increase risk of numerous cancers including breast, prostate,
pancreatic, and colon cancers,138 with some estimates concluding that
up to one in five cancers are directly attributable to obesity.139 Obesity
is characterized by excessive amounts of adipose tissue resulting in
excessive nutrient storage in the form of lipids. Adipose tissue is
important for energy storage, cell signaling through hormone secretion,
and inflammatory responses through cytokine secretion, all of which
are altered in the obese state.140 It has also been shown in ovarian
cancer that FAs produced by local adipose tissue fueled tumor cell
metastasis.141 Thus it is likely that fat depots in themselves can promote
tumorigenesis.142
The major regulator of glucose homeostasis is the hormone insulin,
which mediates insulin receptor (IR)–initiated cell signaling cascades.
Insulin is released by pancreatic β-cells in response to higher blood
glucose levels and signals the uptake of glucose by peripheral tissues.
On activation, IR promotes the activation of signaling pathways
including PI3K, AKT, and mTOR, which promote glucose uptake,
anabolic metabolism, and cell growth.143,144 Because increased insulin
levels and insulin resistance are hallmarks of obesity and the PI3K/
AKT/mTOR signaling axis is the most frequently altered signaling
cascade, it is possible that tumor cells may use insulin signaling resulting
from obesity-associated hyperinsulinemia to enhance their own glucose
uptake and growth. In support of this are studies demonstrating
increased IR expression in certain liver, breast, and lung cancers.145–147
Similar yet distinct hormones upregulated during obesity are insulin-like
growth factors 1 and 2 (IGF1 and IGF2). IGF1 binds the insulin-like
 Cancer Metabolism  •  CHAPTER 9 133

growth factor receptor (IGFR), leading to similar downstream signaling
cascades as insulin and IR. High levels of IGF1 have been shown to
increase the risk of breast cancer.148 On the other hand, IGF2 can
bind both IR and IGFR149 and reportedly promotes tumor growth
in models of breast cancer.150 Other secreted factors altered in response
to obesity that may contribute to tumorigenesis include adiponectin
and leptin.151–153 Overall, obesity can lead to changes in circulating
signaling molecules that directly enhance the tumorigenic capabilities
of neoplastic tissues. The mechanisms, however, remain controversial
and complicated, with multifactorial effects occurring through signaling,
inflammation, and likely changes in tumor cell autonomous metabolism,
although this effect has not yet been well established.
CLINICAL RELEVANCE AND APPLICATIONS
Antimetabolite Chemotherapy
Antifolates
Folates, commonly supplemented in the diet in their more stable
oxidized form as folic acid, are B9 vitamins that are essential for major
biosynthetic reactions such as amino acid metabolism and cell prolifera-
tion.44 For instance, 5-methyl-tetrahydrofolate (5-methyl-THF) is used
along with vitamin B12 to generate methionine, as was previously
discussed.154 In these folate-mediated reactions, dihydrofolate (DHF)
is produced and is then reduced by dihydrofolate reductase (DHFR)
to regenerate THF cofactor pools.
There is a selective dependence on folates for cancer cell proliferation,
and a class of drugs known as antifolates is a group of commonly
used chemotherapy agents. Methotrexate, a potent and well-characterized
polyglutamatable antifolate compound, mimics folic acid but has an
additional methyl group at the N10 position; this allows the compound
to have an exceptionally high affinity for DHFR, but the subtle
structural methyl modification antagonizes the enzyme’s ability to
reduce DHF155,156 (Fig. 9.6). However, methotrexate can exhibit a
toxicity profile against normal proliferating tissues such as bone marrow
and intestinal tissue157; to increase the therapeutic window, usually
high doses of methotrexate are administered followed by low doses
of leucovorin (5-formyl-THF) to competitively inhibit intracellular
transport of methotrexate in tissues with close proximity to blood
vessels (referred to as the “leucovorin rescue”).158,159 Methotrexate
remains a primary treatment option for lymphoma, osteosarcoma,
and leukemias.155 Other polyglutamatable antifolates that inhibit DHFR
activity include pralatrexate and pemetrexed; the nonpolyglutamatable
antifolates that act on DHFR include trimetrexate, piritrexim, and
talotrexin.160 Pemetrexed remains the first-line treatment for NSCLC
as a dual treatment with cisplatin (a DNA cross-linking compound),161
and remains under clinical investigation for the treatment of various
other cancers.162
Nucleotide Synthesis Inhibitors
Another class of antimetabolites includes compounds that directly
interfere with DNA or RNA synthesis. The original and most well-
known of these compounds is 5-fluorouracil (5-FU), a pyrimidine
analogue that bears structural similarity to uracil but with a fluoride
atom at the 5C position of its ring.163 The enzyme thymidylate synthase
(TS) converts uracil to thymine via a methylation reaction at the 5C
carbon position but is unable to execute this methylation of 5-FU

O BASE O O O

HO P O
Riboneucleotide O P O 5' Thymidylate H 3C
BASE
O
reductase OH O NH synthase NH
O- 4' 1'

3' 2' N O N O
OH OH H H
O OH
Riboneucleotide Uracil Thymine
Deoxyribonucleotide Cell membrane

NH2 F
N O
N O HN NH
HO
O F Nucleus
O 5-FU
Gemcitabine
OH F
NH2
Mitochondria
O COOH N N

NH2 N HO N O
H O
N
N N COOH
CH3 Cytosol OH
Decitabine
H 2N N N
Methotrexate
NH2 NH2
CH3
N N

N O Methyltransferase N O
Dihydrofolate Tetrahydrofolate H H
Dihydrofolate
reductase Cytosine 5-methyl cytosine

Figure 9.6  •  Antimetabolite therapies. Current antimetabolite therapies inhibit synthesis of essential compounds needed for efficient DNA, RNA, and
protein synthesis. Methotrexate differs from folic acid by a key methyl group (circled in red) and acts on the metabolic enzyme dihydrofolate reductase (DHFR)
to inhibit the production of tetrahydrofolate pools. Gemcitabine inhibits the activity of ribonucleotide reductase, and 5-fluorouracil (5-FU) inhibits the production
of thymine by thymidylate synthase because of the presence of a fluoride atom (circled in red); both of these inhibitory activities interfere with DNA synthesis
and elongation. Decitabine inhibits the methylation activity of DNA methyltransferases (DNMTs), thereby preventing DNA methylation and subsequent
gene repression.
134 Part I: Science and Clinical Oncology
owing to the presence of the fluoride atom, effectively inhibiting TS.
5-FU remains a commonly used agent clinically; it is regularly used
as a frontline agent for the treatment of colorectal cancer and is
approved for other tissue types such as breast cancer.
Gemcitabine (2′,2′-difluorodeoxycytidine [dFdC]) is another popular
pyrimidine analogue used as a chemotherapeutic agent. Gemcitabine
is a deoxycytidine analogue that has fluoride atoms in place of the
hydrogen atoms at the 2C position of its ring.164 Gemcitabine (both
alone and in combination with other agents such as cisplatin) has
been approved for the treatment of numerous cancers including breast,
ovarian, and bladder cancers, NSCLC, and pancreatic cancer, and it
is being investigated as an adjuvant to 5-FU therapy for metastatic
colorectal cancer.165
Finally, the pyrimidine analogue decitabine (also referred to as
aza-dCyd) is converted by deoxycytidine kinase to aza-dCTP and is
rapidly incorporated into DNA. This analogue prevents the epigenetic
modification of DNA methylation. Normally, cytosine residues of
DNA can be methylated to prevent transcription; aza-dCTP inhibits
DNA methyltransferases, thereby inducing enhanced gene expression.166
Decitabine is approved for the treatment of myelodysplastic syndromes,
including acute myeloid leukemia (AML) in elderly cancer patients.167
Current Metabolic Drug Targets
IDH1/2
As mentioned, IDH enzymes frequently exhibit heterozygous gain-
of-function mutations in cancer, particularly in glioma (70%)100 and
AML (15%–20%),168 resulting in an ability of the IDH enzymes to
further catalyze the conversion of αKG to 2HG.102,169 Two IDH
inhibitors, AG-221 and AG-120, have been clinically investigated for
the treatment of AML.170 The initial phase I and II clinical trials
successfully demonstrated limited toxicity in addition to proof of
concept, with nearly 40% of patients responding to treatment (Table
9.1). Accelerated approval by the US Food and Drug Administration
(FDA) of these compounds was completed in early 2017.
Glutaminase Inhibitors
Glutaminase is an enzyme that catalyzes the production of glutamate
from the amino acid glutamine, which then feeds into the TCA cycle.171
This enzyme was initially targeted with the small-molecule inhibitor
6-diazo-5-oxy-l-norleucine (DON).172 However, a small-molecule
compound has been found to selectively inhibit glutaminase; this
Table 9.1  Metabolic Drugs Currently in Clinical Trials as of Late 2016
Company and Location Agent
Agios and Celgene AG-221, AG-120, AG-881
Polaris Group, San Diego, CA ADI-PEG 20
Cornerstone Pharmaceuticals, CPI-613
Cranbury, NJ
Calithera Biosciences CB-839
3-V Biosciences TVB-2640
Novartis, Basel, Switzerland IDH305
Forma Therapeutics, Watertown, MA FT-2102
Bayer, Leverkusen, Germany BAY-1436032
Advanced Cancer Therapeutics, PFK-158
Louisville, KY
Aeglea BioTherapeutics, Austin, TX AEB-1102
Listed are examples of metabolic drug targets currently under clinical trial investigation, some of which are discussed in this text.
AML, Acute myeloid leukemia; HCC, hepatocellular carcinoma; IDH, isocitrate dehydrogenase; MDS, myelodysplastic syndrome; PFKFB3, 6-phosphofructo-2-kinase; RCC, renal
cell carcinoma.
Modified from Garber K. (2016) Nature Biotechnology, ref. 170. Pending adaptation approval.
compound, CB-839, has been shown to exhibit antiproliferative activity
against TNBC cell lines and in xenograft murine models,37 and has
successfully moved through phase I clinical trials for both TNBC and
renal cell carcinoma.170 Of note, this small-molecule agent does not
act on the glutaminase isoform found in the liver, which is the main
isoform believed to be responsible for circulating glutamate,173 allowing
for a better toxicity profile.
Fatty Acid Synthase Inhibitors
FASN plays an essential role in lipogenesis by catalyzing the NADPH-
dependent conversion of acetyl-CoA and malonyl-CoA to palmitic
acid, which acts as a precursor to long-chain FAs174 (Fig. 9.7). Cancer
cells have been shown to be dependent on FA synthesis for processes
including membrane biosynthesis and energy metabolism and typically
exhibit a high expression of FASN.175 A small-molecule inhibitor of
FASN, TVB-1366, was found to induce apoptosis in breast and prostate
cancer cell lines, which was shown to be mediated by the disruption
of membrane lipid distribution and localization of membrane-bound
proteins.176 More recently, another small-molecule FASN inhibitor
(TVB-2640) has been investigated in phase I clinical trials for the
treatment of KRAS-positive lung cancer, and as a combinatorial
treatment in breast cancer170; however, as previously mentioned, toxicity
appears to be a limiting factor in its further development.
Indolamine 2,3-Dioxygenase Inhibitors
IDO regulates metabolism of the amino acid tryptophan via the
kynurenine pathway.177 Interesting to note, IDO was found to mediate
the ability of tumors to escape recognition by immune cells by sup-
pressing T cells, activating Treg cells, and promoting inflammatory
and angiogenic processes.178–180 IDO expression was also shown to be
associated with resistance to paclitaxel in patients with serous ovarian
cancer.181 Numerous IDO inhibitors are under preclinical investigation
as novel cancer treatments. 1-Methyl-dl-tryptophan (both the d and
the l isomers) acts as a tryptophan mimetic and has been shown to
effectively inhibit IDO activity in human dendritic cells.182 In addition,
D-1MT (but not L-1MT) was shown to induce T-cell activation and
to prolong survival in combination with paclitaxel in a murine model
of breast cancer; this difference in efficacy was found to be due to
D-1MT’s activity on the IDO2 isomer, which is expressed in only a
subset of tissues that express IDO1.183 D-1MT (also known as
indoximod) is one of four IDO inhibitors under clinical investigation;
the others include INCB024360 (a hydroxyamidine small-molecule
Target Indications Status
IDH1 and IDH2 AML, MDS, solid tumors Phase III
Pegylated arginine deiminase HCC, others Phase III in HCC missed
primary endpoint
Pyruvate dehydrogenase AML, MDS, solid tumors Phase II
Glutaminase 1 RCC, breast cancer Phase I/II
Fatty acid synthase Ovarian, breast, lung Phase II pending
IDH1 Advanced cancers Phase I
IDH1 AML, MDS Phase I
IDH1 Solid tumors Phase I
PFKFB3 Solid tumors Phase I
Modified human arginase AML, MDS, solid tumors Phase I
 Cancer Metabolism  •  CHAPTER 9 135

H+
H +
Gln Lactate Glucose H+ H+ H+ H2O Folic acid Ser Asn Arg Choline
H+ + +
HCO3 CO2
MCT1 MCT4 GLUT1/GLUT4 NEH1 CAs

Gln Glucose Na+ Folate/nucleotide Ser Asn Arg

GLS1 metabolism
HKs PPP
TKTL1 Folic acid Choline
Glu G6P F6P X5P R5P Gln
Glycolysis CK
Nicotinamide PFKFB3
F6P F2.6BP P-choline
NAMPT 5.10-CH2 THF
F1.6BP IMP UMP TYMS
Amino acid Sphingomyelin
NADH metabolism
GAPDH RNR THF DHF
Fatty acids PHGDH
3PG 3PHP SER dNTPs DHFR Isoprenoids
PGAM1
Asp Cholesterol
Fatty acyl-CoA 2PG Lipidogenesis
HMGCR
OAA Acetyl-CoA HMG-CoA Mevalonate
CPT1 PEP
ACLY
PKM2 OAA ACC
Fatty acyl- LDHA
carnitine Lactate Pyruvate Malate Citrate Malonyl-CoA FASN Fatty acids Glycerol
MGLL

MAG
NADH
Cl H+
OAA PC Pyruvate Malate H+ H + H+
Cytosol
PDK1 NAD+
Fatty acyl- Q10
carnitine
Acetyl-CoA
H+

Pol
OAA Citrate H+ H+
OXPHOS H+ Nucleus
β-oxidation
Malate Cyt c
D-isocitrate H+
Krebs cycle H+ H+
IDH ADP H+
ADP ADP
α-KG Glu
H+ H + H+
H+
GLUD1 ATP F1FO
ATP ATPase
ATP
Mitochondrial
matrix
Intermembrane space

Figure 9.7  •  Current metabolic targets in cancer therapy. Current drug targets in cancer therapy include numerous metabolic targets involved in multiple
mitochondrial and cytosolic energetic pathways. Targets labeled in red are under preclinical investigation, targets labeled in blue are in early stages of clinical
trials, and targets labeled in green are in advanced stages of clinical trial investigation. This diagram depicts targets described in this text and additional targets
not covered in this chapter (for detailed review, see ref. 193). ACC, Acetyl-CoA carboxylase; ACLY, ATP citrate lyase; ADP, adenosine diphosphate; ATP, adenosine
triphosphate; αKG, α-ketoglutarate; Arg, arginine; Asn, asparagine; Asp, aspartate; CA, carbonic anhydrase; 5,10-CH2-THF, 5,10-methylene tetrahydrofolate;
CI, complex I; CK, choline kinase; CPT1, carnitine O-palmitoyltransferase 1; Cyt c, cytochrome c; DHF, dihydrofolate; DHFR, dihydrofolate reductase; dNTP,
deoxynucleotide triphosphate; F1,6BP, fructose-1,6-bisphosphate; F2,6BP, fructose-2,6-bisphosphate; F6P, fructose-6-phosphate; FASN, fatty acid synthase;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Gln, glutamine; GLS1, glutaminase 1; Glu, glutamate; GLUD1, glutamate dehydrogenase 1; GLUT,
glucose transporter; G6P, glucose-6-phosphate; HK, hexokinase; HMG-CoA, 3-hydroxy-3-methyl-glutaryl coenzyme A; HMGCR, HMG-CoA reductase; IDH,
isocitrate dehydrogenase; IMP, inosine monophosphate; LDHA, lactate dehydrogenase A; MAG, monoacylglycerol; MCT, monocarboxylate transporter; MGLL,
monoglyceride lipase; NAD+, nicotinamide adenine dinucleotide (oxidixed); NADH, nicotinamide adenine dinucleotide (reduced); NAMPT, nicotinamide
phosphoribosyltransferase; NHE1, Na+/H+ exchanger 1; OAA, oxaloacetate; OXPHOS, oxidative phosphorylation; PC, pyruvate carboxylase; PDK1, pyruvate
dehydrogenase kinase 1; PEP, phosphoenolpyruvate; PFKFB3, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3; 2PG, 2-phosphoglycerate; 3PG,
3-phosphoglycerate; PGAM1, phosphoglycerate mutase 1; PHGDH, phosphoglycerate dehydrogenase; 3PHP, 3-phosphohydroxypyruvate; PKM2, pyruvate
kinase, muscle, M2 isoform; Pol, DNA polymerase; PPP, pentose phosphate pathway; Q10, coenzyme Q10; R5P, ribose-5-phosphate; RNR, ribonucleotide
reductase; Ser, serine; THF, tetrahydrofolate; TKTL1, transketolase-like protein 1; TYMS, thymidylate synthase; UMP, uridine monophosphate; X5P, xylulose
5-phosphate. (Modified from Galluzi L. (2013) Nature Reviews: Drug Discovery.)
136 Part I: Science and Clinical Oncology
compound), NLG919, and an IDO peptide vaccine. All of these
compounds have shown limited toxicity thus far and are being pursued
both as monotherapies and in combination with other leading
chemotherapies.184,185
Ornithine Decarboxylase Inhibitors
Ornithine decarboxylase (ODC) catalyzes the initiation of de novo
polyamine synthesis and is frequently upregulated in cancer as a
consequence of events such as Myc activation,186 ultraviolet (UV)
radiation exposure,187 and methylthioadenosine phosphorylase (MTAP)
deletion.188 This increase in ODC activity provides an abundant
polyamine pool for cell proliferation,189 and promotes signal transduc-
tion by the activation of MAP kinases.190 An FDA-approved ODC
inhibitor, difluoromethylornithine (DFMO, or eflornithine), has been
shown to effectively reduce polyamine levels, ultimately resulting in
reduced tumor vascularity191 and metastasis192 in transgenic murine
models. DFMO has been evaluated for the prevention of numerous
cancers193 and is currently under clinical investigation for the treatment
of Myc-deregulated cancers such as childhood neuroblastoma194;
however, early clinical trials investigating the efficacy of DFMO as a
general chemotherapeutic agent failed to demonstrate a significant
outcome, and the drug was found to induce hearing loss in some
patients.195 Nevertheless, newfound understanding of methionine
metabolism in cancer may provide better rationale for further investigat-
ing these compounds.
Repurposing Common Metabolic Agents
Non–cancer-treating drugs that have been traditionally used to treat
various disorders are currently being investigated as repurposed che-
motherapeutic agents owing to their relatively reduced toxicity, increased
tolerability, and lower cost.196,197 In addition, these FDA-approved
compounds have the potential to enter phase III clinical trials more
readily than novel drugs because their safety profiles have already been
well established. The following section summarizes some of the current
drugs that are being investigated as repurposed cancer treatments.
Metformin
Metformin is the leading therapy prescribed for the management of
type II diabetes.198 This compound has been shown to reduce hepatic
gluconeogenesis199 and increase insulin sensitivity,200 thereby increasing
peripheral glucose uptake and reducing circulating levels of glucose.
Although the exact mechanism of action that mediates its efficacy
remains unknown,201 metformin has been observed to inhibit complex
I (NADH dehydrogenase) of the mitochondrial respiratory chain,202
inhibit mitochondrial glycerophosphate dehydrogenase,199 and activate
AMPK signaling through these mechanisms in certain settings.203
Metformin also inhibits insulin production and IGF signaling,204 which
can also result in antineoplastic effects.
Substantial evidence for the direct effect of metformin on tumor
cell autonomous metabolism is also apparent. Because, as mentioned,
mitochondria have pleiotropic functions in maintaining tumorigenesis,
disruptions to mitochondria by metformin have been observed to
have global effects, altering mitochondrial metabolism in tumor cells.
For instance, although it was originally believed that metformin exerted
its antitumor effects primarily via its ability to activate AMPK signaling,
it was found that cancer cell lines that demonstrated high sensitivity
to metformin tended to exhibit mitochondrial mutations in complex
I genes; this sensitivity was abolished by the exogenous expression of
an enzyme (NDI1, derived from yeast) that enables cells to undergo
oxidative phosphorylation without a functional complex I,205 implying
an AMPK-independent antineoplastic mechanism. Similar results were
found in murine models, in which tumors of NDI1 transgenic mice
failed to show sensitivity to metformin compared with tumors harbored
by mice that did not express NDH1.202 This is also consistent with
metabolic profiles that are observed in the biopsies of ovarian tumors
obtained from patients taking metformin, in which the level of
metformin accumulation within tumors was found to correlate with
the degree of mitochondrial metabolic dysfunction including accumula-
tion of both NADH and ROS.206
Both preclinical and epidemiologic studies have demonstrated the
antitumor potential of metformin. Some meta-analyses have found
an approximately 30% reduced cancer incidence among type II diabetic
metformin users compared with nonusers.207 Dozens of clinical trials
examining metformin as either a monotherapy or in combination
with various chemotherapies are currently ongoing, especially as a
repurposed treatment for breast, endometrial, pancreatic, and prostate
cancers.208
Statins
Inhibitors of hydroxymethylglutaryl coenzyme A (HMG-CoA)
reductase, an enzyme in the mevalonate pathway—commonly referred
to as statins—are a class of cholesterol-lowering drugs that are used
clinically as a preventative measure against and as a treatment for
cardiovascular disease.209,210 A handful of statins are currently FDA
approved, including lovastatin (the most commonly prescribed),
simvastatin, fluvastatin, atorvastatin, rosuvastatin, and pravastatin.
Statins are known to inhibit lipogenesis via inhibition of the mevalonate
pathway.211 Overall, preclinical studies have demonstrated that statins
are able to effectively induce cell death in various types of human
cancer cells.211 Numerous clinical trials have investigated the efficacy
of statins in the prevention and/or treatment of various types of cancer,
yet the majority of these trials have shown inconclusive results.212 A
meta-analysis found that statin treatment was negatively correlated
with cancer incidence, but this effect was mostly limited to lung and
prostate cancer and was concluded to be nonsignificant.213 It is believed
that the inconclusive results found in the large number of observational
investigations are due to multiple confounding factors that differed
among studies, including timing of when treatment was initiated,
dosage regimen, and heterogeneity of other risk factors (such as obesity
and smoking). However, the use of statins in the prevention and
treatment of cancer still remains an attractive prospect and continues
to be under investigation.
Aspirin
The nonsteroidal antiinflammatory drug (NSAID) aspirin, a cyclo-
oxygenase (COX) inhibitor that reduces prostaglandin and thromboxane
production, is an over-the-counter medication used to relieve pain
and reduce fever. Aspirin is also commonly recommended by physicians
to reduce the risk of heart attack and stroke and the recurrence of
pulmonary embolism and deep vein thrombosis, because of its ability
to reduce blood clotting.214 Interesting to note, evidence appears to
also link long-term aspirin use to protection against cancer development
and mortality, particularly colorectal cancer.215 An observational study
investigated aspirin use in 135,965 women and men over the course
of 32 years and found that regular low-dose (weekly administration
of 0.5 to 1.5 standard tablets) aspirin over the course of 6 years reduced
overall cancer risk by 3%, with a 15% reduction in gastrointestinal
cancers.216 Although it has been demonstrated that aspirin can activate
AMPK signaling,217 it is believed that aspirin may exert its antitumor
effects via both off-target (COX-independent) and on-target (COX-
dependent) mechanisms.218 For instance, COX enzymes have been
found to play a significant role in the development of intestinal tumors
in animal models.219 Aspirin may also reduce the incidence of tumor
metastasis via its antiplatelet activity220 because platelets have been
shown to promote secondary lesion development by shielding tumor
cells from immune responses.221 Nevertheless, more work is needed
to understand the molecular mechanisms that may contribute to the
antitumor effects of aspirin.
Vitamins C and D
Evidence suggests that some commonly used compounds contained
in the diet and used as nutritional supplements, particularly vitamins
C and D, may play a much more substantial role in combating cancer

progression than was originally believed. For instance, a recently
published preclinical study found that deletion of the vitamin D
receptor accelerated tumor growth and promoted metastasis in a murine
model of breast cancer,222 implying that vitamin D could act as a
negative regulator of tumor progression. Another study found that in
colorectal cancer cell lines exhibiting mutations in either BRAF or
KRAS, vitamin C was able to induce oxidative stress in cells that
could inactivate GAPDH (among many other effects), thereby prevent-
ing cancer cells from undergoing glycolysis; this finding extended to
mouse models, in which vitamin C administration significantly impaired
tumor growth.223 Interesting to note, studies have also demonstrated
that vitamin C can boost the efficacy of DNA methyltransferase
inhibitors, such as 5-azacytidine and 5-aza-2′-deoxycytidine, by
enhancing the catalytic activity of the TET family of DNA demethylases,
which oxidize methylated cytosine residues to hydroxymethyl cyto-
sine.224,225 Given the tolerability of these vitamins, these preclinical
findings are an exciting new field of investigation, and these compounds
remain under clinical investigation as an adjuvant therapy.226
Diet and Exercise
An emerging topic of investigation is the role of environmental factors
that mediate metabolism, such as diet and exercise, in the develop-
ment and progression of cancer. It has been estimated that diet could
account for roughly 35% of the risk factors associated with cancer
development,227 making dietary intervention an attractive approach
to cancer prevention. Dietary intervention as an adjuvant therapy
in cancer treatment has also gained considerable attention. Because
of the differential nutrient requirements of cancer cells, there is a
possibility of inducing antitumor effects through dietary manipulation.
For instance, under fasting conditions, the brain is capable of using
ketone bodies in the absence of sufficient glucose, whereas tumors
(such as glioblastoma multiforme) remain reliant on an abundant
glucose supply.228,229 This led to the investigation of a high-fat,
low-carbohydrate (“ketogenic”) diet as a viable adjuvant treatment
in glioma, in which increased ketone bodies and reduced glucose in
circulation leads to increased FAO and ultimately induces a “fasting”
state in the patient.230,231 It has been shown in preclinical studies that
a ketogenic diet combined with the targeted therapy temozolomide
significantly prolonged survival in a murine model of glioblastoma
multiforme.232 After a handful of case study reports investigating the
use of a ketogenic diet to treat glioma,233,234 there are a number of
ongoing phase II clinical trials examining the efficacy of this dietary
intervention as an adjuvant therapy (NCT01754350, NCT01535911,
NCT01865162). Similarly, periods of caloric restriction or fasting have
been shown to slow tumor growth and sensitize tumors originating
from various organs to targeted therapies.235,236 Evidence also sug-
gests that deprivation of certain nutrients has antitumor properties;
one preclinical study found that depletion of the amino acid serine
was sufficient to induce the activation of stress pathways and reduce
proliferation in tumors lacking p53.123
As detailed previously, obesity has been shown to be a prominent
risk factor in the development of cancer.237 A significant field of research
is currently investigating the role of energy expenditure in reducing
tumor development and recurrence. Physical activity has been linked
to prolonged disease-free survival and quality of life among cancer
patients,238 and studies have observed a positive effect of exercise on
the reduction of proangiogenic239 and hypoxic240 factors, resulting in
reduced tumor vascularity. A role for physical activity in inhibiting or
reversing tumor growth is a promising current area of investigation.
Metabolic Biomarkers and Diagnostics
Fluorine-18 Fluorodeoxyglucose–Positron
Emission Tomography
With the discovery of the Warburg effect, it became possible for
glucose uptake to be used as a type of biomarker for tumor location.
Cancer Metabolism  •  CHAPTER 9 137
Fluorine-18 fluorodeoxyglucose–positron emission tomography
(18F-FDG–PET) is a technique in which a radioactively labeled glucose
analogue is used to measure glucose uptake, allowing for the anatomic
localization of tumors when coupled with computed tomography
(CT). The glucose analogue 18F-FDG is phosphorylated by hexokinase
similarly to glucose, but is not further metabolized, resulting in the
accumulation of 18F-FDG in regions with high glucose uptake rates
such as the liver, the brain, and the majority of tumors.241 This allows
not only for the initial identification of tumors, but also for tumor
monitoring.
However, this technique exhibits variable sensitivity in detecting
primary tumors localized to different regions; for instance, 18F-FDG–
PET has relatively low sensitivity, about 68%, for primary breast
tumors that are less than 2 cm wide,242 making it an insufficient
technique for initial breast cancer detection. Interesting to note, this
sensitivity jumps to almost 90% for recurrent breast tumors, and is
nearly 100% for metastatic tumors originating from breast tumors,
so it is frequently used for screening cancer recurrence and metastasis
after initial remission.243 It has also become an established standard
in the initial detection of numerous types of cancer including lym-
phoma,244 cervical cancer,245 and lung cancer.246 Important to note,
there does not appear to be a substantial benefit of using 18F-FDG–PET
in the characterization of urologic cancers owing to high signal-to-noise
ratios.247 Another limitation of this technique that warrants consid-
eration is that inflammatory processes that involve the activation of
macrophages and fibroblasts can also induce an increase in the
FDG-PET signal.248
Other Positron Emission Tomography Agents
Various radiolabeled tracers other than 18F-FDG have been investigated
for use in PET imaging in cancer diagnostics for tumors in which
18
F-FDG–PET imaging is insufficient. For example, acetate is produced
as a result of FA synthesis, and the FA synthase enzyme has been
shown to be upregulated in malignant prostate tumors.249 Therefore
carbon-11 (11C)–labeled acetate has been explored as a potential
PET tracer in the detection of recurrent prostate cancer, with an
observed sensitivity of roughly 75%.250 Similarly, 11C-choline was
found to exhibit relatively little accumulation in urine, making it a
more advantageous tracer for prostate cancer251; choline is essential for
cellular membrane synthesis and has also been shown to accumulate
in malignant tumors.252 As mentioned earlier, another hallmark of
tumor biology is the occurrence of hypoxia because proximity to the
vasculature decreases with increasing tumor density.253 The radiotracer
18
F-fluoromisonidazole (18FMISO) is reduced in hypoxic conditions,
resulting in intracellular accumulation; this PET tracer has been investi-
gated in the imaging of breast cancer, glioma, lung cancer, and head and
neck cancers.254
CONCLUSIONS AND FUTURE OUTLOOK
Over recent years, the metabolic reprogramming and adaptation that
occur in cancer cells to support uncontrolled proliferation have been
extensively studied, and their pervasiveness well established. As a result,
new drug targets and other therapeutic strategies that affect this
programming are actively being pursued. Challenges in defining the
cancer contexts (i.e., which patients to give these drugs to) and thera-
peutic windows limit further translation into the clinic of these advances
in basic science. In addition, agents that target metabolism such as
pemetrexed and 5-FU are widely used clinically as effective chemo-
therapeutic agents; however, in these cases the specificity of these
compounds for particular patients, although apparent clinically, is
poorly understood. That is, standard chemotherapy given within a
cancer patient population results in widely disparate clinical outcomes
ranging from complete resistance to full objective responses. Now
with newfound insight into the molecular and cellular basis of metabolic
reprogramming and adaptation, it may be possible to obtain further
specificity in precisely defining a priori for these outcomes.
138 Part I: Science and Clinical Oncology

Although much of the recent surge of interest in cancer metabolism
has focused on the cell autonomous alterations to metabolic pathways
in cancer cells, it is now more appreciated that these tumor cells
interact with their environment, which includes their microenviron-
ment, tissue of origin, germline genetics, liver physiology, microbiome,
and other influences including diet and exercise. Whether and how
variation in these factors can affect metabolic pathways in tumors
remains a highly active area of research. For example, dietary variables
such as caloric or total protein intake can readily be controlled if it
is understood how these factors could influence cancer outcomes in
ways similar to chemotherapies. Future work in the area of cancer
metabolism will undoubtedly need to incorporate these factors into
future study design.
The complete reference list is available online at
ExpertConsult.com.

KEY REFERENCES
8. Warburg O, Wind F, Negelein E. The metabolism
of tumors in the body. J Gen Physiol. 1927;8(6):
519–530.
9. Fantin VR, St-Pierre J, Leder P. Attenuation
of LDH-A expression uncovers a link between
glycolysis, mitochondrial physiology, and tumor
maintenance. Cancer Cell. 2006;9(6):425–434.
10. Shim H, Chun YS, Lewis BC, Dang CV. A unique
glucose-dependent apoptotic pathway induced
by c-Myc. Proc Natl Acad Sci USA. 1998;95(4):
1511–1516.
15. Liberti MV, Locasale JW. The Warburg effect:
how does it benefit cancer cells? Trends Biochem
Sci. 2016;41(3):211–218.
18. Locasale JW, Cantley LC. Metabolic flux and the
regulation of mammalian cell growth. Cell Metab.
2011;14(4):443–451.
19. Locasale JW, Grassian AR, Melman T, et al.
Phosphoglycerate dehydrogenase diverts glycolytic
flux and contributes to oncogenesis. Nat Genet.
2011;43(9):869–874.
23. Boroughs LK, DeBerardinis RJ. Metabolic pathways
promoting cancer cell survival and growth. Nat Cell
Biol. 2015;17(4):351–359.
24. Wise DR, DeBerardinis RJ, Mancuso A, et al.
Myc regulates a transcriptional program that
stimulates mitochondrial glutaminolysis and leads
to glutamine addiction. Proc Natl Acad Sci USA.
2008;105(48):18782–18787.
27. Commisso C, Davidson SM, Soydaner-Azeloglu
RG, et al. Macropinocytosis of protein is an amino
acid supply route in Ras-transformed cells. Nature.
2013;497(7451):633–637.
28. Degenhardt K, Mathew R, Beaudoin B, et al.
Autophagy promotes tumor cell survival and restricts
necrosis, inflammation, and tumorigenesis. Cancer
Cell. 2006;10(1):51–64.
30. Altman BJ, Stine ZE, Dang CV. From Krebs to
clinic: glutamine metabolism to cancer therapy. Nat
Rev Cancer. 2016;16(10):619–634.
34. DeBerardinis RJ, Mancuso A, Daikhin E, et al.
Beyond aerobic glycolysis: transformed cells can
engage in glutamine metabolism that exceeds the
requirement for protein and nucleotide synthesis. Proc
Natl Acad Sci USA. 2007;104(49):19345–19350.
36. Davidson SM, Papagiannakopoulos T, Olenchock
BA, et al. Environment impacts the metabolic
dependencies of Ras-driven non–small cell lung
cancer. Cell Metab. 2016;23(3):517–528.
38. Xiang Y, Stine ZE, Xia J, et al. Targeted inhibition
of tumor-specific glutaminase diminishes cell-
autonomous tumorigenesis. J Clin Invest. 2015;125(6):
2293–2306.
44. Locasale JW. Serine, glycine and one-carbon units:
cancer metabolism in full circle. Nat Rev Cancer. 2013;
13(8):572–583.
48. Mentch SJ, Mehrmohamadi M, Huang L, et al.
Histone methylation dynamics and gene regulation
occur through the sensing of one-carbon metabolism.
Cell Metab. 2015;22(5):861–873.
54. Laplante M, Sabatini DM. mTOR signaling in growth
control and disease. Cell. 2012;149(2):274–293.
63. Haq R, Shoag J, Andreu-Perez P, et al. Oncogenic
BRAF regulates oxidative metabolism via PGC1alpha
and MITF. Cancer Cell. 2013;23(3):302–315.
69. Hatzivassiliou G, Zhao F, Bauer DE, et al. ATP
citrate lyase inhibition can suppress tumor cell
growth. Cancer Cell. 2005;8(4):311–321.
71. Shaw RJ, Kosmatka M, Bardeesy N, et al. The tumor
suppressor LKB1 kinase directly activates AMP-
activated kinase and regulates apoptosis in response
to energy stress. Proc Natl Acad Sci USA. 2004;
101(10):3329–3335.
77. Carracedo A, Cantley LC, Pandolfi PP. Cancer
metabolism: fatty acid oxidation in the limelight. Nat
Rev Cancer. 2013;13(4):227–232.
91. Gao X, Reid MA, Kong M, Locasale JW. Metabolic
interactions with cancer epigenetics. Mol Aspects Med.
2016.
97. Wellen KE, Hatzivassiliou G, Sachdeva UM, Bui
TV, Cross JR, Thompson CB. ATP-citrate lyase links
cellular metabolism to histone acetylation. Science.
2009;324(5930):1076–1080.
100. Yan H, Parsons DW, Jin G, et al. IDH1 and IDH2
mutations in gliomas. N Engl J Med. 2009;360(8):
765–773.
102. Dang L, White DW, Gross S, et al. Cancer-associated
IDH1 mutations produce 2-hydroxyglutarate. Nature.
2009;462(7274):739–744.
104. Lu C, Ward PS, Kapoor GS, et al. IDH mutation
impairs histone demethylation and results in a block
to cell differentiation. Nature. 2012;483(7390):
474–478.
108. Pan M, Reid MA, Lowman XH, et al. Regional
glutamine deficiency in tumours promotes dedif-
ferentiation through inhibition of histone demeth-
ylation. Nat Cell Biol. 2016;18(10):1090–1101.
109. Xiao M, Yang H, Xu W, et al. Inhibition of alpha-
KG-dependent histone and DNA demethylases
by fumarate and succinate that are accumulated
in mutations of FH and SDH tumor suppressors.
Genes Dev. 2012;26(12):1326–1338.
115. Nakazawa MS, Keith B, Simon MC. Oxygen avail-
ability and metabolic adaptations. Nat Rev Cancer.
2016;16(10):663–673.
119. Kamphorst JJ, Nofal M, Commisso C, et al. Human
pancreatic cancer tumors are nutrient poor and
tumor cells actively scavenge extracellular protein.
Cancer Res. 2015;75(3):544–553.
120. Reid MA, Wang WI, Rosales KR, Welliver MX, Pan
M, Kong M. The B55alpha subunit of PP2A drives
a p53-dependent metabolic adaptation to glutamine
deprivation. Mol Cell. 2013;50(2):200–211.
123. Maddocks OD, Berkers CR, Mason SM, et al. Serine
starvation induces stress and p53-dependent metabolic
remodelling in cancer cells. Nature. 2013;493(7433):
542–546.
124. Hensley CT, Faubert B, Yuan Q, et al. Metabolic het-
erogeneity in human lung tumors. Cell. 2016;164(4):
681–694.
128. Colegio OR, Chu NQ, Szabo AL, et al. Functional
polarization of tumour-associated macrophages by
tumour-derived lactic acid. Nature. 2014;513(7519):
559–563.
138. Bhaskaran K, Douglas I, Forbes H, dos-Santos-
Silva I, Leon DA, Smeeth L. Body-mass index
and risk of 22 specific cancers: a population-based
cohort study of 5.24 million UK adults. Lancet.
2014;384(9945):755–765.
141. Nieman KM, Kenny HA, Penicka CV, et al.
Adipocytes promote ovarian cancer metastasis and
provide energy for rapid tumor growth. Nat Med.
2011;17(11):1498–1503.
144. Ruderman NB, Kapeller R, White MF, Cantley LC.
Activation of phosphatidylinositol 3-kinase by insulin.
Proc Natl Acad Sci USA. 1990;87(4):1411–1415.
160. Gonen N, Assaraf YG. Antifolates in cancer therapy:
structure, activity and mechanisms of drug resistance.
Drug Resist Updat. 2012;15(4):183–210.
170. Garber K. Cancer anabolic metabolism inhibitors
move into clinic. Nat Biotechnol. 2016;34(8):794–
795.
185. Galluzzi L, Kepp O, Vander Heiden MG, Kroemer
G. Metabolic targets for cancer therapy. Nat Rev
Drug Discov. 2013;12(11):829–846.
193. Gerner EW, Meyskens FL Jr. Polyamines and cancer:
old molecules, new understanding. Nat Rev Cancer.
2004;4(10):781–792.
197. Ashburn TT, Thor KB. Drug repositioning: identify-
ing and developing new uses for existing drugs. Nat
Rev Drug Discov. 2004;3(8):673–683.
202. Wheaton WW, Weinberg SE, Hamanaka RB, et al.
Metformin inhibits mitochondrial complex I of
cancer cells to reduce tumorigenesis. Elife. 2014;3:
e02242.
206. Liu X, Romero IL, Litchfield LM, Lengyel E,
Locasale JW. Metformin targets central carbon
metabolism and reveals mitochondrial requirements
in human cancers. Cell Metab. 2016;24(5):728–739.
217. Hawley SA, Fullerton MD, Ross FA, et al. The ancient
drug salicylate directly activates AMP-activated
protein kinase. Science. 2012;336(6083):918–922.
220. Rothwell PM, Wilson M, Price JF, Belch JF,
Meade TW, Mehta Z. Effect of daily aspirin on
risk of cancer metastasis: a study of incident cancers
during randomised controlled trials. Lancet. 2012;
379(9826):1591–1601.
235. Lee C, Raffaghello L, Brandhorst S, et al. Fasting cycles
retard growth of tumors and sensitize a range of cancer
cell types to chemotherapy. Sci Transl Med. 2012;
4(124):124ra127.
236. Levine ME, Suarez JA, Brandhorst S, et al. Low
protein intake is associated with a major reduction
in IGF-1, cancer, and overall mortality in the 65
and younger but not older population. Cell Metab.
2014;19(3):407–417.
237. Khandekar MJ, Cohen P, Spiegelman BM. Molecular
mechanisms of cancer development in obesity. Nat
Rev Cancer. 2011;11(12):886–895.
240. Betof AS, Lascola CD, Weitzel D, et al. Modulation
of murine breast tumor vascularity, hypoxia and
chemotherapeutic response by exercise. J Natl Cancer
Inst. 2015;107(5).

REFERENCES
1. Berg JM, Tymoczko JL, Stryer L, Stryer L. Biochem-
istry. 6th ed. New York: WH Freeman; 2007.
2. Lehninger AL, Nelson DL, Cox MM. Lehninger
Principles of Biochemistry. 6th ed. New York: W.H.
Freeman; 2013.
3. Rolland F, Winderickx J, Thevelein JM. Glucose-
sensing and -signalling mechanisms in yeast. FEMS
Yeast Res. 2002;2(2):183–201.
4. Sherman IW. Malaria: Parasite Biology, Pathogenesis,
and Protection. Washington, DC: ASM Press; 1998.
5. Pfeiffer T, Schuster S, Bonhoeffer S. Cooperation
and competition in the evolution of ATP-producing
pathways. Science. 2001;292(5516):504–507.
6. Shestov AA, Liu X, Ser Z, et al. Quantitative
determinants of aerobic glycolysis identify flux
through the enzyme GAPDH as a limiting step. Elife.
2014;3.
7. Warburg O, Posener K, Negelein E. On the
metabolism of carcinoma cells. Biochem Z. 1924;152:
309–344.
8. Warburg O, Wind F, Negelein E. The metabolism
of tumors in the body. J Gen Physiol. 1927;8(6):
519–530.
9. Fantin VR, St-Pierre J, Leder P. Attenuation of
LDH-A expression uncovers a link between glycolysis,
mitochondrial physiology, and tumor maintenance.
Cancer Cell. 2006;9(6):425–434.
10. Shim H, Chun YS, Lewis BC, Dang CV. A unique
glucose-dependent apoptotic pathway induced
by c-Myc. Proc Natl Acad Sci USA. 1998;95(4):
1511–1516.
11. Le A, Stine ZE, Nguyen C, et al. Tumorigenicity
of hypoxic respiring cancer cells revealed by a
hypoxia-cell cycle dual reporter. Proc Natl Acad Sci
USA. 2014;111(34):12486–12491.
12. Gerriets VA, Kishton RJ, Nichols AG, et al. Metabolic
programming and PDHK1 control CD4+ T cell
subsets and inflammation. J Clin Invest. 2015;125(1):
194–207.
13. Vincent AS, Phan TT, Mukhopadhyay A, Lim
HY, Halliwell B, Wong KP. Human skin keloid
fibroblasts display bioenergetics of cancer cells. J
Invest Dermatol. 2008;128(3):702–709.
14. Ben-Haim S, Ell P. 18F-FDG PET and PET/CT
in the evaluation of cancer treatment response. J
Nucl Med. 2009;50(1):88–99.
15. Liberti MV, Locasale JW. The Warburg effect:
how does it benefit cancer cells? Trends Biochem
Sci. 2016;41(3):211–218.
16. Warburg O. On the origin of cancer cells. Science.
1956;123(3191):309–314.
17. Slavov N, Budnik BA, Schwab D, Airoldi EM,
van Oudenaarden A. Constant growth rate can be
supported by decreasing energy flux and increasing
aerobic glycolysis. Cell Rep. 2014;7(3):705–714.
18. Locasale JW, Cantley LC. Metabolic flux and the
regulation of mammalian cell growth. Cell Metab.
2011;14(4):443–451.
19. Locasale JW, Grassian AR, Melman T, et al. Phos-
phoglycerate dehydrogenase diverts glycolytic flux
and contributes to oncogenesis. Nat Genet. 2011;
43(9):869–874.
20. Lunt SY, Vander Heiden MG. Aerobic glycolysis:
meeting the metabolic requirements of cell prolifera-
tion. Annu Rev Cell Dev Biol. 2011;27:441–464.
21. Gatenby RA, Gawlinski ET. A reaction-diffusion
model of cancer invasion. Cancer Res. 1996;56(24):
5745–5753.
22. Estrella V, Chen T, Lloyd M, et al. Acidity generated
by the tumor microenvironment drives local invasion.
Cancer Res. 2013;73(5):1524–1535.
23. Boroughs LK, DeBerardinis RJ. Metabolic pathways
promoting cancer cell survival and growth. Nat Cell
Biol. 2015;17(4):351–359.
24. Wise DR, DeBerardinis RJ, Mancuso A, et al.
Myc regulates a transcriptional program that
stimulates mitochondrial glutaminolysis and leads
to glutamine addiction. Proc Natl Acad Sci USA.
2008;105(48):18782–18787.
25. Gao P, Tchernyshyov I, Chang TC, et al. c-Myc
suppression of miR-23a/b enhances mitochondrial
glutaminase expression and glutamine metabolism.
Nature. 2009;458(7239):762–765.
26. Son J, Lyssiotis CA, Ying H, et al. Glutamine supports
pancreatic cancer growth through a KRAS-regulated
metabolic pathway. Nature. 2013;496(7443):
101–105.
27. Commisso C, Davidson SM, Soydaner-Azeloglu
RG, et al. Macropinocytosis of protein is an amino
acid supply route in Ras-transformed cells. Nature.
2013;497(7451):633–637.
28. Degenhardt K, Mathew R, Beaudoin B, et al.
Autophagy promotes tumor cell survival and restricts
necrosis, inflammation, and tumorigenesis. Cancer
Cell. 2006;10(1):51–64.
29. Yang S, Wang X, Contino G, et al. Pancreatic cancers
require autophagy for tumor growth. Genes Dev. 2011;
25(7):717–729.
30. Altman BJ, Stine ZE, Dang CV. From Krebs to
clinic: glutamine metabolism to cancer therapy. Nat
Rev Cancer. 2016;16(10):619–634.
31. Bergstrom J, Furst P, Noree LO, Vinnars E. Intracel-
lular free amino acid concentration in human muscle
tissue. J Appl Physiol. 1974;36(6):693–697.
32. Welbourne TC. Ammonia production and glutamine
incorporation into glutathione in the functioning
rat kidney. Can J Biochem. 1979;57(3):233–
237.
33. Jiang L, Shestov AA, Swain P, et al. Reductive carbox-
ylation supports redox homeostasis during anchorage-
independent growth. Nature. 2016;532(7598):
255–258.
34. DeBerardinis RJ, Mancuso A, Daikhin E, et al.
Beyond aerobic glycolysis: transformed cells can
engage in glutamine metabolism that exceeds the
requirement for protein and nucleotide synthesis.
Proc Natl Acad Sci USA. 2007;104(49):19345–
19350.
35. DeBerardinis RJ, Lum JJ, Hatzivassiliou G, Thompson
CB. The biology of cancer: metabolic reprogramming
fuels cell growth and proliferation. Cell Metab. 2008;
7(1):11–20.
36. Davidson SM, Papagiannakopoulos T, Olenchock
BA, et al. Environment impacts the metabolic
dependencies of ras-driven non–small cell lung
cancer. Cell Metab. 2016;23(3):517–528.
37. Gross MI, Demo SD, Dennison JB, et al. Antitumor
activity of the glutaminase inhibitor CB-839 in triple-
negative breast cancer. Mol Cancer Ther. 2014;13(4):
890–901.
38. Xiang Y, Stine ZE, Xia J, et al. Targeted inhibition
of tumor-specific glutaminase diminishes cell-
autonomous tumorigenesis. J Clin Invest. 2015;125(6):
2293–2306.
39. Kung HN, Marks JR, Chi JT. Glutamine synthe-
tase is a genetic determinant of cell type-specific
glutamine independence in breast epithelia. PLoS
Genet. 2011;7(8):e1002229.
40. Herranz D, Ambesi-Impiombato A, Sudderth J, et al.
Metabolic reprogramming induces resistance to anti-
NOTCH1 therapies in T cell acute lymphoblastic
leukemia. Nat Med. 2015;21(10):1182–1189.
41. MacFarlane AJ, Liu X, Perry CA, et al. Cytoplasmic
serine hydroxymethyltransferase regulates the meta-
bolic partitioning of methylenetetrahydrofolate but
is not essential in mice. J Biol Chem. 2008;283(38):
25846–25853.
42. Kalhan SC, Uppal SO, Moorman JL, et al. Metabolic
and genomic response to dietary isocaloric protein
restriction in the rat. J Biol Chem. 2011;286(7):
5266–5277.
43. Yang M, Vousden KH. Serine and one-carbon
metabolism in cancer. Nat Rev Cancer. 2016;16(10):
650–662.
Cancer Metabolism  •  CHAPTER 9 138.e1
138.e1
44. Locasale JW. Serine, glycine and one-carbon units:
cancer metabolism in full circle. Nat Rev Cancer.
2013;13(8):572–583.
45. Mullarky E, Lucki NC, Beheshti Zavareh R, et al.
Identification of a small molecule inhibitor of
3-phosphoglycerate dehydrogenase to target serine
biosynthesis in cancers. Proc Natl Acad Sci USA.
2016;113(7):1778–1783.
46. Pacold ME, Brimacombe KR, Chan SH, et al. A
PHGDH inhibitor reveals coordination of serine
synthesis and one-carbon unit fate. Nat Chem Biol.
2016;12(6):452–458.
47. Wang Q, Liberti MV, Liu P, et al. Rational design
of selective allosteric inhibitors of PHGDH and
serine synthesis with anti-tumor activity. Cell Chem
Biol. 2017;24(1):55–65.
48. Mentch SJ, Mehrmohamadi M, Huang L, et al.
Histone methylation dynamics and gene regulation
occur through the sensing of one-carbon metabolism.
Cell Metab. 2015;22(5):861–873.
49. Davis SR, Stacpoole PW, Williamson J, et al. Tracer-
derived total and folate-dependent homocysteine
remethylation and synthesis rates in humans indicate
that serine is the main one-carbon donor. Am J
Physiol Endocrinol Metab. 2004;286(2):E272–E279.
50. Dillon BJ, Prieto VG, Curley SA, et al. Incidence
and distribution of argininosuccinate synthetase
deficiency in human cancers: a method for identify-
ing cancers sensitive to arginine deprivation. Cancer.
2004;100(4):826–833.
51. Grohmann U, Bronte V. Control of immune
response by amino acid metabolism. Immunol Rev.
2010;236:243–264.
52. Mellor AL, Munn DH. IDO expression by dendritic
cells: tolerance and tryptophan catabolism. Nat Rev
Immunol. 2004;4(10):762–774.
53. Mayers JR, Wu C, Clish CB, et al. Elevation of
circulating branched-chain amino acids is an early
event in human pancreatic adenocarcinoma develop-
ment. Nat Med. 2014;20(10):1193–1198.
54. Laplante M, Sabatini DM. mTOR signaling in growth
control and disease. Cell. 2012;149(2):274–293.
55. Wallace DC. Mitochondria and cancer. Nat Rev
Cancer. 2012;12(10):685–698.
56. Ames BN, Shigenaga MK, Hagen TM. Oxidants, anti-
oxidants, and the degenerative diseases of aging. Proc
Natl Acad Sci USA. 1993;90(17):7915–7922.
57. Weinberg F, Hamanaka R, Wheaton WW, et al.
Mitochondrial metabolism and ROS generation are
essential for Kras-mediated tumorigenicity. Proc Natl
Acad Sci USA. 2010;107(19):8788–8793.
58. Weinhouse S, Millington RH, Wenner CE.
Metabolism of neoplastic tissue. I. The oxidation of
carbohydrate and fatty acids in transplanted tumors.
Cancer Res. 1951;11(11):845–850.
59. Wenner CE, Spirtes MA, Weinhouse S. Metabolism
of neoplastic tissue. II. A survey of enzymes of the
citric acid cycle in transplanted tumors. Cancer Res.
1952;12(1):44–49.
60. Fogal V, Richardson AD, Karmali PP, Scheffler
IE, Smith JW, Ruoslahti E. Mitochondrial p32
protein is a critical regulator of tumor metabolism
via maintenance of oxidative phosphorylation. Mol
Cell Biol. 2010;30(6):1303–1318.
61. Weinberg SE, Chandel NS. Targeting mitochondria
metabolism for cancer therapy. Nat Chem Biol.
2015;11(1):9–15.
62. Vazquez F, Lim JH, Chim H, et al. PGC1alpha
expression defines a subset of human melanoma
tumors with increased mitochondrial capacity
and resistance to oxidative stress. Cancer Cell.
2013;23(3):287–301.
63. Haq R, Shoag J, Andreu-Perez P, et al. Oncogenic
BRAF regulates oxidative metabolism via PGC1alpha
and MITF. Cancer Cell. 2013;23(3):302–315.
64. Ookhtens M, Kannan R, Lyon I, Baker N. Liver and
adipose tissue contributions to newly formed fatty acids
138.e2 Part I: Science and Clinical Oncology
in an ascites tumor. Am J Physiol. 1984;247(1 Pt 2):
R146–R153.
65. Medes G, Thomas A, Weinhouse S. Metabolism of
neoplastic tissue. IV. A study of lipid synthesis in neo-
plastic tissue slices in vitro. Cancer Res. 1953;13(1):
27–29.
66. Wakil SJ, Stoops JK, Joshi VC. Fatty acid synthesis
and its regulation. Annu Rev Biochem. 1983;52:
537–579.
67. Currie E, Schulze A, Zechner R, Walther TC, Farese
RV Jr. Cellular fatty acid metabolism and cancer.
Cell Metab. 2013;18(2):153–161.
68. Bauer DE, Hatzivassiliou G, Zhao F, Andreadis C,
Thompson CB. ATP citrate lyase is an important
component of cell growth and transformation.
Oncogene. 2005;24(41):6314–6322.
69. Hatzivassiliou G, Zhao F, Bauer DE, et al. ATP
citrate lyase inhibition can suppress tumor cell
growth. Cancer Cell. 2005;8(4):311–321.
70. Migita T, Narita T, Nomura K, et al. ATP citrate
lyase: activation and therapeutic implications in
non–small cell lung cancer. Cancer Res. 2008;
68(20):8547–8554.
71. Shaw RJ, Kosmatka M, Bardeesy N, et al. The
tumor suppressor LKB1 kinase directly activates
AMP-activated kinase and regulates apoptosis in
response to energy stress. Proc Natl Acad Sci USA.
2004;101(10):3329–3335.
72. Chajes V, Cambot M, Moreau K, Lenoir GM,
Joulin V. Acetyl-CoA carboxylase alpha is essential to
breast cancer cell survival. Cancer Res. 2006;66(10):
5287–5294.
73. Beckers A, Organe S, Timmermans L, et al. Chemical
inhibition of acetyl-CoA carboxylase induces growth
arrest and cytotoxicity selectively in cancer cells.
Cancer Res. 2007;67(17):8180–8187.
74. Jeon SM, Chandel NS, Hay N. AMPK regulates
NADPH homeostasis to promote tumour cell sur-
vival during energy stress. Nature. 2012;485(7400):
661–665.
75. Pizer ES, Thupari J, Han WF, et al. Malonyl-
coenzyme-A is a potential mediator of cytotoxicity
induced by fatty-acid synthase inhibition in human
breast cancer cells and xenografts. Cancer Res.
2000;60(2):213–218.
76. Menendez JA, Lupu R. Fatty acid synthase and the
lipogenic phenotype in cancer pathogenesis. Nat Rev
Cancer. 2007;7(10):763–777.
77. Carracedo A, Cantley LC, Pandolfi PP. Cancer
metabolism: fatty acid oxidation in the limelight. Nat
Rev Cancer. 2013;13(4):227–232.
78. Zaugg K, Yao Y, Reilly PT, et al. Carnitine palmi-
toyltransferase 1C promotes cell survival and tumor
growth under conditions of metabolic stress. Genes
Dev. 2011;25(10):1041–1051.
79. Schafer ZT, Grassian AR, Song L, et al. Anti-
oxidant and oncogene rescue of metabolic defects
caused by loss of matrix attachment. Nature.
2009;461(7260):109–113.
80. Camarda R, Zhou AY, Kohnz RA, et al. Inhibition
of fatty acid oxidation as a therapy for MYC-
overexpressing triple-negative breast cancer. Nat Med.
2016;22(4):427–432.
81. Loftus TM, Jaworsky DE, Frehywot GL, et al.
Reduced food intake and body weight in mice
treated with fatty acid synthase inhibitors. Science.
2000;288(5475):2379–2381.
82. Rohrig F, Schulze A. The multifaceted roles of fatty
acid synthesis in cancer. Nat Rev Cancer. 2016;16(11):
732–749.
83. Graeser R, Bornmann C, Esser N, et al. Anti-
metastatic effects of liposomal gemcitabine and
empty liposomes in an orthotopic mouse model
of pancreatic cancer. Pancreas. 2009;38(3):330–337.
84. Morgan HD, Santos F, Green K, Dean W, Reik W.
Epigenetic reprogramming in mammals. Hum Mol
Genet. 2005;14(Spec1):R47–R58.
85. Dawson MA, Kouzarides T. Cancer epigenetics: from
mechanism to therapy. Cell. 2012;150(1):12–27.
86. Sharma S, Kelly TK, Jones PA. Epigenetics in cancer.
Carcinogenesis. 2010;31(1):27–36.
87. Suva ML, Riggi N, Bernstein BE. Epigenetic
reprogramming in cancer. Science. 2013;339(6127):
1567–1570.
88. Ptashne M. How eukaryotic transcriptional activators
work. Nature. 1988;335(6192):683–689.
89. Ullrich A, Schlessinger J. Signal transduction
by receptors with tyrosine kinase activity. Cell.
1990;61(2):203–212.
90. Yun J, Johnson JL, Hanigan CL, Locasale JW.
Interactions between epigenetics and metabolism
in cancers. Front Oncol. 2012;2:163.
91. Gao X, Reid MA, Kong M, Locasale JW. Metabolic
interactions with cancer epigenetics. Mol Aspects Med.
2016.
92. Kinnaird A, Zhao S, Wellen KE, Michelakis ED.
Metabolic control of epigenetics in cancer. Nat Rev
Cancer. 2016;16(11):694–707.
93. Mentch SJ, Locasale JW. One-carbon metabolism
and epigenetics: understanding the specificity. Ann
N Y Acad Sci. 2016;1363:91–98.
94. Shyh-Chang N, Locasale JW, Lyssiotis CA,
et al. Influence of threonine metabolism on S-
adenosylmethionine and histone methylation. Science.
2013;339(6116):222–226.
95. Nishikawa K, Iwamoto Y, Kobayashi Y, et al.
DNA methyltransferase 3a regulates osteoclast
differentiation by coupling to an S-adenosylme-
thionine-producing metabolic pathway. Nat Med.
2015;21(3):281–287.
96. Cai L, Sutter BM, Li B, Tu BP. Acetyl-CoA induces cell
growth and proliferation by promoting the acetylation
of histones at growth genes. Mol Cell. 2011;42(4):
426–437.
97. Wellen KE, Hatzivassiliou G, Sachdeva UM, Bui
TV, Cross JR, Thompson CB. ATP-citrate lyase links
cellular metabolism to histone acetylation. Science.
2009;324(5930):1076–1080.
98. Chalkiadaki A, Guarente L. The multifaceted func-
tions of sirtuins in cancer. Nat Rev Cancer. 2015;
15(10):608–624.
99. Mardis ER, Ding L, Dooling DJ, et al. Recurring
mutations found by sequencing an acute myeloid
leukemia genome. N Engl J Med. 2009;361(11):
1058–1066.
100. Yan H, Parsons DW, Jin G, et al. IDH1 and IDH2
mutations in gliomas. N Engl J Med. 2009;360(8):
765–773.
101. Zhao S, Lin Y, Xu W, et al. Glioma-derived mutations
in IDH1 dominantly inhibit IDH1 catalytic activity
and induce HIF-1alpha. Science. 2009;324(5924):
261–265.
102. Dang L, White DW, Gross S, et al. Cancer-associated
IDH1 mutations produce 2-hydroxyglutarate. Nature.
2009;462(7274):739–744.
103. Xu W, Yang H, Liu Y, et al. Oncometabolite
2-hydroxyglutarate is a competitive inhibitor of
alpha-ketoglutarate-dependent dioxygenases. Cancer
Cell. 2011;19(1):17–30.
104. Lu C, Ward PS, Kapoor GS, et al. IDH mutation
impairs histone demethylation and results in a block
to cell differentiation. Nature. 2012;483(7390):
474–478.
105. Wang F, Travins J, DeLaBarre B, et al. Targeted
inhibition of mutant IDH2 in leukemia cells induces
cellular differentiation. Science. 2013;340(6132):
622–626.
106. Rohle D, Popovici-Muller J, Palaskas N, et al.
An inhibitor of mutant IDH1 delays growth and
promotes differentiation of glioma cells. Science.
2013;340(6132):626–630.
107. Carey BW, Finley LW, Cross JR, Allis CD, Thompson
CB. Intracellular alpha-ketoglutarate maintains
the pluripotency of embryonic stem cells. Nature.
2015;518(7539):413–416.
108. Pan M, Reid MA, Lowman XH, et al. Regional
glutamine deficiency in tumours promotes
dedifferentiation through inhibition of histone
demethylation. Nat Cell Biol. 2016;18(10):1090–
1101.
109. Xiao M, Yang H, Xu W, et al. Inhibition of alpha-
KG-dependent histone and DNA demethylases
by fumarate and succinate that are accumulated
in mutations of FH and SDH tumor suppressors.
Genes Dev. 2012;26(12):1326–1338.
110. Hanahan D, Coussens LM. Accessories to the crime:
functions of cells recruited to the tumor microen-
vironment. Cancer Cell. 2012;21(3):309–322.
111. Less JR, Skalak TC, Sevick EM, Jain RK. Micro-
vascular architecture in a mammary carcinoma:
branching patterns and vessel dimensions. Cancer
Res. 1991;51(1):265–273.
112. McKeown SR. Defining normoxia, physoxia and
hypoxia in tumours-implications for treatment
response. Br J Radiol. 2014;87(1035):20130676.
113. Rumsey WL, Schlosser C, Nuutinen EM, Robiolio
M, Wilson DF. Cellular energetics and the oxygen
dependence of respiration in cardiac myocytes
isolated from adult rat. J Biol Chem. 1990;265(26):
15392–15402.
114. Rankin EB, Giaccia AJ. The role of hypoxia-
inducible factors in tumorigenesis. Cell Death Differ.
2008;15(4):678–685.
115. Nakazawa MS, Keith B, Simon MC. Oxygen avail-
ability and metabolic adaptations. Nat Rev Cancer.
2016;16(10):663–673.
116. Semenza GL. Targeting HIF-1 for cancer therapy.
Nat Rev Cancer. 2003;3(10):721–732.
117. Zeng W, Wan R, Zheng Y, Singh SR, Wei Y.
Hypoxia, stem cells and bone tumor. Cancer Lett.
2011;313(2):129–136.
118. Lee KE, Simon MC. From stem cells to cancer
stem cells: HIF takes the stage. Curr Opin Cell Biol.
2012;24(2):232–235.
119. Kamphorst JJ, Nofal M, Commisso C, et al. Human
pancreatic cancer tumors are nutrient poor and
tumor cells actively scavenge extracellular protein.
Cancer Res. 2015;75(3):544–553.
120. Reid MA, Wang WI, Rosales KR, Welliver MX, Pan
M, Kong M. The B55alpha subunit of PP2A drives
a p53-dependent metabolic adaptation to glutamine
deprivation. Mol Cell. 2013;50(2):200–211.
121. Reid MA, Lowman XH, Pan M, et al. IKKbeta
promotes metabolic adaptation to glutamine
deprivation via phosphorylation and inhibition of
PFKFB3. Genes Dev. 2016;30(16):1837–1851.
122. Tran TQ, Lowman XH, Reid MA, et al. Tumor-
associated mutant p53 promotes cancer cell survival
upon glutamine deprivation through p21 induction.
Oncogene. 2016.
123. Maddocks OD, Berkers CR, Mason SM, et al.
Serine starvation induces stress and p53-dependent
metabolic remodelling in cancer cells. Nature.
2013;493(7433):542–546.
124. Hensley CT, Faubert B, Yuan Q, et al. Metabolic het-
erogeneity in human lung tumors. Cell. 2016;164(4):
681–694.
125. Swietach P, Vaughan-Jones RD, Harris AL. Regula-
tion of tumor pH and the role of carbonic anhydrase
9. Cancer Metastasis Rev. 2007;26(2):299–310.
126. Martinez-Zaguilan R, Seftor EA, Seftor RE, Chu
YW, Gillies RJ, Hendrix MJ. Acidic pH enhances
the invasive behavior of human melanoma cells.
Clin Exp Metastasis. 1996;14(2):176–186.
127. Rothberg JM, Bailey KM, Wojtkowiak JW, et al.
Acid-mediated tumor proteolysis: contribution of cys-
teine cathepsins. Neoplasia. 2013;15(10):1125–1137.
128. Colegio OR, Chu NQ, Szabo AL, et al. Functional
polarization of tumour-associated macrophages by
tumour-derived lactic acid. Nature. 2014;513(7519):
559–563.
129. Wahl DR, Byersdorfer CA, Ferrara JL, Opipari
AW Jr, Glick GD. Distinct metabolic programs in
activated T cells: opportunities for selective immu-
nomodulation. Immunol Rev. 2012;249(1):104–115.
130. Ron-Harel N, Sharpe AH, Haigis MC. Mitochon-
drial metabolism in T cell activation and senescence:

a mini-review. Gerontology. 2015;61(2):131–
138.
131. Mellor AL, Munn DH. Creating immune privilege:
active local suppression that benefits friends, but
protects foes. Nat Rev Immunol. 2008;8(1):74–80.
132. Fallarino F, Grohmann U, Vacca C, et al. T cell
apoptosis by tryptophan catabolism. Cell Death
Differ. 2002;9(10):1069–1077.
133. Fooksman DR, Vardhana S, Vasiliver-Shamis G, et al.
Functional anatomy of T cell activation and synapse
formation. Annu Rev Immunol. 2010;28:79–105.
134. Patsoukis N, Bardhan K, Chatterjee P, et al. PD-1
alters T-cell metabolic reprogramming by inhibiting
glycolysis and promoting lipolysis and fatty acid
oxidation. Nat Commun. 2015;6:6692.
135. Clambey ET, McNamee EN, Westrich JA, et al.
Hypoxia-inducible factor-1 alpha-dependent induc-
tion of FoxP3 drives regulatory T-cell abundance
and function during inflammatory hypoxia of the
mucosa. Proc Natl Acad Sci USA. 2012;109(41):
E2784–E2793.
136. Kouidhi S, Noman MZ, Kieda C, Elgaaied AB,
Chouaib S. Intrinsic and tumor microenvironment-
induced metabolism adaptations of t cells and impact
on their differentiation and function. Front Immunol.
2016;7:114.
137. Ogden CL, Carroll MD, Kit BK, Flegal KM. Preva-
lence of childhood and adult obesity in the United
States, 2011-2012. JAMA. 2014;311(8):806–814.
138. Bhaskaran K, Douglas I, Forbes H, dos Santos-
Silva I, Leon DA, Smeeth L. Body-mass index
and risk of 22 specific cancers: a population-based
cohort study of 5.24 million UK adults. Lancet.
2014;384(9945):755–765.
139. Calle EE, Kaaks R. Overweight, obesity and cancer:
epidemiological evidence and proposed mechanisms.
Nat Rev Cancer. 2004;4(8):579–591.
140. Font-Burgada J, Sun B, Karin M. Obesity and
cancer: the oil that feeds the flame. Cell Metab.
2016;23(1):48–62.
141. Nieman KM, Kenny HA, Penicka CV, et al.
Adipocytes promote ovarian cancer metastasis and
provide energy for rapid tumor growth. Nat Med.
2011;17(11):1498–1503.
142. Nieman KM, Romero IL, Van Houten B,
Lengyel E. Adipose tissue and adipocytes support
tumorigenesis and metastasis. Biochim Biophys Acta.
2013;1831(10):1533–1541.
143. White MF, Kahn CR. The insulin signaling system.
J Biol Chem. 1994;269(1):1–4.
144. Ruderman NB, Kapeller R, White MF, Cantley LC.
Activation of phosphatidylinositol 3-kinase by insulin.
Proc Natl Acad Sci USA. 1990;87(4):1411–1415.
145. Flannery CA, Saleh FL, Choe GH, et al. Differential
expression of IR-A, IR-B and IGF-1R in endometrial
physiology and distinct signature in adenocarcinoma.
J Clin Endocrinol Metab. 2016;101(7):2883–2891.
146. Huang J, Morehouse C, Streicher K, et al. Altered
expression of insulin receptor isoforms in breast
cancer. PLoS ONE. 2011;6(10):e26177.
147. Jiang L, Zhu W, Streicher K, et al. Increased IR-A/
IR-B ratio in non–small cell lung cancers associ-
ates with lower epithelial-mesenchymal transition
signature and longer survival in squamous cell lung
carcinoma. BMC Cancer. 2014;14:131.
148. Renehan AG, Egger M, Minder C, O’Dwyer
ST, Shalet SM, Zwahlen M. IGF-I, IGF binding
protein-3 and breast cancer risk: comparison of 3
meta-analyses. Int J Cancer. 2005;115(6):1006–1007,
author reply 1008.
149. Alvino CL, Ong SC, McNeil KA, et al. Understand-
ing the mechanism of insulin and insulin-like growth
factor (IGF) receptor activation by IGF-II. PLoS
ONE. 2011;6(11):e27488.
150. Pierre-Eugene C, Pagesy P, Nguyen TT, et al. Effect
of insulin analogues on insulin/IGF1 hybrid recep-
tors: increased activation by glargine but not by its
metabolites M1 and M2. PLoS ONE. 2012;7(7):
e41992.
151. Dalamaga M, Diakopoulos KN, Mantzoros CS. The
role of adiponectin in cancer: a review of current
evidence. Endocr Rev. 2012;33(4):547–594.
152. Uddin S, Hussain AR, Siraj AK, Khan OS, Bavi PP,
Al-Kuraya KS. Role of leptin and its receptors in
the pathogenesis of thyroid cancer. Int J Clin Exp
Pathol. 2011;4(7):637–643.
153. Garofalo C, Surmacz E. Leptin and cancer. J Cell
Physiol. 2006;207(1):12–22.
154. Fox JT, Stover PJ. Folate-mediated one-carbon
metabolism. Vitam Horm. 2008;79:1–44.
155. Visentin M, Zhao R, Goldman ID. The antifolates.
Hematol Oncol Clin North Am. 2012;26(3):629–
648, ix.
156. Fabre I, Fabre G, Goldman ID. Polyglutamylation,
an important element in methotrexate cytotoxicity
and selectivity in tumor versus murine granulocytic
progenitor cells in vitro. Cancer Res. 1984;44(8):
3190–3195.
157. Koizumi S, Ueno Y, Ohno I, Ichihara T, Tamaru Y,
Matsukawa H. Reversal of methotrexate cytotoxicity
to human bone marrow cells and leukemic K562 cells
by leucovorin: methotrexate polyglutamates forma-
tion as a possible important factor. Jpn J Cancer Res.
1990;81(11):1162–1167.
158. Pratt CB, Roberts D, Shanks EC, Warmath EL.
Clinical trials and pharmacokinetics of intermit-
tent high-dose methotrexate—“leucovorin rescue”
for children with malignant tumors. Cancer Res.
1974;34(12):3326–3331.
159. Howard SC, McCormick J, Pui CH, Buddington
RK, Harvey RD. Preventing and managing
toxicities of high-dose methotrexate. Oncologist.
2016;21(12):1471–1482.
160. Gonen N, Assaraf YG. Antifolates in cancer therapy:
structure, activity and mechanisms of drug resistance.
Drug Resist Updat. 2012;15(4):183–210.
161. Cohen MH, Justice R, Pazdur R. Approval summary:
pemetrexed in the initial treatment of advanced/
metastatic non–small cell lung cancer. Oncologist.
2009;14(9):930–935.
162. Jarmula A. Antifolate inhibitors of thymidylate
synthase as anticancer drugs. Mini Rev Med Chem.
2010;10(13):1211–1222.
163. Longley DB, Harkin DP, Johnston PG. 5-
Fluorouracil: mechanisms of action and clinical
strategies. Nat Rev Cancer. 2003;3(5):330–338.
164. Ewald B, Sampath D, Plunkett W. Nucleoside
analogs: molecular mechanisms signaling cell
death. Oncogene. 2008;27(50):6522–6537.
165. Elnaggar M, Giovannetti E, Peters GJ. Molecular
targets of gemcitabine action: rationale for develop-
ment of novel drugs and drug combinations. Curr
Pharm Des. 2012;18(19):2811–2829.
166. Stresemann C, Lyko F. Modes of action of the
DNA methyltransferase inhibitors azacytidine and
decitabine. Int J Cancer. 2008;123(1):8–13.
167. Malik P, Cashen AF. Decitabine in the treatment of
acute myeloid leukemia in elderly patients. Cancer
Manag Res. 2014;6:53–61.
168. Abbas S, Lugthart S, Kavelaars FG, et al. Acquired
mutations in the genes encoding IDH1 and IDH2
both are recurrent aberrations in acute myeloid
leukemia: prevalence and prognostic value. Blood.
2010;116(12):2122–2126.
169. Ward PS, Patel J, Wise DR, et al. The common
feature of leukemia-associated IDH1 and IDH2
mutations is a neomorphic enzyme activity convert-
ing alpha-ketoglutarate to 2-hydroxyglutarate. Cancer
Cell. 2010;17(3):225–234.
170. Garber K. Cancer anabolic metabolism inhibitors
move into clinic. Nat Biotechnol. 2016;34(8):
794–795.
171. Wang JB, Erickson JW, Fuji R, et al. Targeting
mitochondrial glutaminase activity inhibits onco-
genic transformation. Cancer Cell. 2010;18(3):207–
219.
172. Pinkus LM. Glutamine binding sites. Methods
Enzymol. 1977;46:414–427.
Cancer Metabolism  •  CHAPTER 9 138.e3
138.e3
173. Aledo JC, Gomez-Fabre PM, Olalla L, Marquez J.
Identification of two human glutaminase loci and
tissue-specific expression of the two related genes.
Mamm Genome. 2000;11(12):1107–1110.
174. Maier T, Jenni S, Ban N. Architecture of mammalian
fatty acid synthase at 4.5 A resolution. Science.
2006;311(5765):1258–1262.
175. Sebastiani V, Botti C, Di Tondo U, et al. Tissue
microarray analysis of FAS, Bcl-2, Bcl-x, ER, PgR,
Hsp60, p53 and Her2-neu in breast carcinoma.
Anticancer Res. 2006;26(4B):2983–2987.
176. Ventura R, Mordec K, Waszczuk J, et al. Inhibition
of de novo palmitate synthesis by fatty acid synthase
induces apoptosis in tumor cells by remodeling cell
membranes, inhibiting signaling pathways, and repro-
gramming gene expression. EBioMedicine. 2015;2(8):
808–824.
177. Moffett JR, Namboodiri MA. Tryptophan and the
immune response. Immunol Cell Biol. 2003;81(4):
247–265.
178. Prendergast GC, Smith C, Thomas S, et al.
Indoleamine 2,3-dioxygenase pathways of pathogenic
inflammation and immune escape in cancer. Cancer
Immunol Immunother. 2014;63(7):721–735.
179. Uyttenhove C, Pilotte L, Theate I, et al. Evidence
for a tumoral immune resistance mechanism
based on tryptophan degradation by indoleamine
2,3-dioxygenase. Nat Med. 2003;9(10):1269–1274.
180. Munn DH. Indoleamine 2,3-dioxygenase, Tregs and
cancer. Curr Med Chem. 2011;18(15):2240–2246.
181. Okamoto A, Nikaido T, Ochiai K, et al. Indoleamine
2,3-dioxygenase serves as a marker of poor prognosis
in gene expression profiles of serous ovarian cancer
cells. Clin Cancer Res. 2005;11(16):6030–6039.
182. Hou DY, Muller AJ, Sharma MD, et al. Inhibition
of indoleamine 2,3-dioxygenase in dendritic cells
by stereoisomers of 1-methyl-tryptophan correlates
with antitumor responses. Cancer Res. 2007;67(2):
792–801.
183. Metz R, Duhadaway JB, Kamasani U, Laury-Kleintop
L, Muller AJ, Prendergast GC. Novel tryptophan
catabolic enzyme IDO2 is the preferred biochemical
target of the antitumor indoleamine 2,3-dioxygenase
inhibitory compound D-1-methyl-tryptophan.
Cancer Res. 2007;67(15):7082–7087.
184. Moon YW, Hajjar J, Hwu P, Naing A. Targeting the
indoleamine 2,3-dioxygenase pathway in cancer. J
Immunother Cancer. 2015;3:51.
185. Galluzzi L, Kepp O, Vander Heiden MG, Kroemer
G. Metabolic targets for cancer therapy. Nat Rev
Drug Discov. 2013;12(11):829–846.
186. Wagner JP, Seidler FJ, Slotkin TA. Presynaptic input
regulates development of beta-adrenergic control of
rat brain ornithine decarboxylase: effects of 6-hydroxy-
dopamine or propranolol. Brain Res Bull. 1991;26(6):
885–890.
187. Ahmad N, Gilliam AC, Katiyar SK, O’Brien
TG, Mukhtar H. A definitive role of ornithine
decarboxylase in photocarcinogenesis. Am J Pathol.
2001;159(3):885–892.
188. Subhi AL, Diegelman P, Porter CW, et al. Methyl-
thioadenosine phosphorylase regulates ornithine
decarboxylase by production of downstream metabo-
lites. J Biol Chem. 2003;278(50):49868–49873.
189. Oredsson SM. Polyamine dependence of normal
cell-cycle progression. Biochem Soc Trans. 2003;31(2):
366–370.
190. Feith DJ, Bol DK, Carboni JM, et al. Induction of
ornithine decarboxylase activity is a necessary step
for mitogen-activated protein kinase kinase-induced
skin tumorigenesis. Cancer Res. 2005;65(2):572–578.
191. Lan L, Trempus C, Gilmour SK. Inhibition of
ornithine decarboxylase (ODC) decreases tumor
vascularization and reverses spontaneous tumors in
ODC/Ras transgenic mice. Cancer Res. 2000;60(20):
5696–5703.
192. Wheeler DL, Ness KJ, Oberley TD, Verma AK.
Inhibition of the development of metastatic squa-
mous cell carcinoma in protein kinase C epsilon
138.e4 Part I: Science and Clinical Oncology
transgenic mice by alpha-difluoromethylornithine
accompanied by marked hair follicle degeneration
and hair loss. Cancer Res. 2003;63(12):3037–3042.
193. Gerner EW, Meyskens FL Jr. Polyamines and cancer:
old molecules, new understanding. Nat Rev Cancer.
2004;4(10):781–792.
194. Bassiri H, Benavides A, Haber M, Gilmour SK,
Norris MD, Hogarty MD. Translational development
of difluoromethylornithine (DFMO) for the treat-
ment of neuroblastoma. Transl Pediatr. 2015;4(3):
226–238.
195. Schweitzer VG. Ototoxicity of chemotherapeutic
agents. Otolaryngol Clin North Am. 1993;26(5):
759–789.
196. Jiao M, Liu G, Xue Y, Ding C. Computational drug
repositioning for cancer therapeutics. Curr Top Med
Chem. 2015;15(8):767–775.
197. Ashburn TT, Thor KB. Drug repositioning: identify-
ing and developing new uses for existing drugs. Nat
Rev Drug Discov. 2004;3(8):673–683.
198. Inzucchi SE, Bergenstal RM, Buse JB, et al. Manage-
ment of hyperglycaemia in type 2 diabetes, 2015:
a patient-centred approach. Update to a position
statement of the American Diabetes Association and
the European Association for the Study of Diabetes.
Diabetologia. 2015;58(3):429–442.
199. Madiraju AK, Erion DM, Rahimi Y, et al. Met-
formin suppresses gluconeogenesis by inhibiting
mitochondrial glycerophosphate dehydrogenase.
Nature. 2014;510(7506):542–546.
200. Matthaei S, Greten H. Evidence that metformin
ameliorates cellular insulin-resistance by potentiating
insulin-induced translocation of glucose transporters
to the plasma membrane. Diabete Metab. 1991;17(1
Pt 2):150–158.
201. Pernicova I, Korbonits M. Metformin—mode of
action and clinical implications for diabetes and
cancer. Nat Rev Endocrinol. 2014;10(3):143–
156.
202. Wheaton WW, Weinberg SE, Hamanaka RB,
et al. Metformin inhibits mitochondrial complex
I of cancer cells to reduce tumorigenesis. Elife.
2014;3:e02242.
203. Zhou G, Myers R, Li Y, et al. Role of AMP-activated
protein kinase in mechanism of metformin action.
J Clin Invest. 2001;108(8):1167–1174.
204. Xie Y, Wang JL, Ji M, et al. Regulation of insulin-like
growth factor signaling by metformin in endometrial
cancer cells. Oncol Lett. 2014;8(5):1993–1999.
205. Birsoy K, Possemato R, Lorbeer FK, et al. Metabolic
determinants of cancer cell sensitivity to glucose
limitation and biguanides. Nature. 2014;508(7494):
108–112.
206. Liu X, Romero IL, Litchfield LM, Lengyel E,
Locasale JW. Metformin targets central carbon
metabolism and reveals mitochondrial requirements
in human cancers. Cell Metab. 2016;24(5):728–739.
207. Libby G, Donnelly LA, Donnan PT, Alessi DR,
Morris AD, Evans JM. New users of metformin
are at low risk of incident cancer: a cohort study
among people with type 2 diabetes. Diabetes Care.
2009;32(9):1620–1625.
208. Chae YK, Arya A, Malecek MK, et al. Repurposing
metformin for cancer treatment: current clinical
studies. Oncotarget. 2016;7(26):40767–40780.
209. Newby LK, Kristinsson A, Bhapkar MV, et al. Early
statin initiation and outcomes in patients with
acute coronary syndromes. JAMA. 2002;287(23):
3087–3095.
210. Nissen SE, Nicholls SJ, Sipahi I, et al. Effect of
very high-intensity statin therapy on regression
of coronary atherosclerosis: the ASTEROID trial.
JAMA. 2006;295(13):1556–1565.
211. Chan KK, Oza AM, Siu LL. The statins as anticancer
agents. Clin Cancer Res. 2003;9(1):10–19.
212. Boudreau DM, Yu O, Johnson J. Statin use and
cancer risk: a comprehensive review. Expert Opin
Drug Saf. 2010;9(4):603–621.
213. Ahn J, Lim U, Weinstein SJ, et al. Prediagnostic
total and high-density lipoprotein cholesterol and
risk of cancer. Cancer Epidemiol Biomarkers Prev.
2009;18(11):2814–2821.
214. Jankowski J, Attwood S, deCaestecker J, Barr H.
Aspirin in the primary prevention of vascular disease.
Lancet. 2009;374(9693):877–878, author reply 879.
215. Rothwell PM, Price JF, Fowkes FG, et al. Short-term
effects of daily aspirin on cancer incidence, mortality,
and non-vascular death: analysis of the time course
of risks and benefits in 51 randomised controlled
trials. Lancet. 2012;379(9826):1602–1612.
216. Cao Y, Nishihara R, Wu K, et al. Population-wide
impact of long-term use of aspirin and the risk for
cancer. JAMA Oncol. 2016;2(6):762–769.
217. Hawley SA, Fullerton MD, Ross FA, et al. The ancient
drug salicylate directly activates AMP-activated
protein kinase. Science. 2012;336(6083):918–922.
218. Thun MJ, Jacobs EJ, Patrono C. The role of aspirin
in cancer prevention. Nat Rev Clin Oncol. 2012;9(5):
259–267.
219. Oshima M, Taketo MM. COX selectivity and
animal models for colon cancer. Curr Pharm Des.
2002;8(12):1021–1034.
220. Rothwell PM, Wilson M, Price JF, Belch JF, Meade
TW, Mehta Z. Effect of daily aspirin on risk of cancer
metastasis: a study of incident cancers during ran-
domised controlled trials. Lancet. 2012;379(9826):
1591–1601.
221. Gay LJ, Felding-Habermann B. Contribution of plate-
lets to tumour metastasis. Nat Rev Cancer. 2011;11(2):
123–134.
222. Williams JD, Aggarwal A, Swami S, et al. Tumor
autonomous effects of vitamin D deficiency promote
breast cancer metastasis. Endocrinology. 2016;157(4):
1341–1347.
223. Yun J, Mullarky E, Lu C, et al. Vitamin C selectively
kills KRAS and BRAF mutant colorectal cancer cells
by targeting GAPDH. Science. 2015;350(6266):
1391–1396.
224. Sajadian SO, Tripura C, Samani FS, et al. Vitamin
C enhances epigenetic modifications induced by 5-
azacytidine and cell cycle arrest in the hepatocellular
carcinoma cell lines HLE and Huh7. Clin Epigenetics.
2016;8:46.
225. Liu M, Ohtani H, Zhou W, et al. Vitamin C increases
viral mimicry induced by 5-aza-2′-deoxycytidine. Proc
Natl Acad Sci USA. 2016;113(37):10238–10244.
226. High-Dose Vitamin C (PDQ(R)): Health Profes-
sional Version. PDQ Cancer Information Summaries.
Bethesda (MD) 2002.
227. Irigaray P, Newby JA, Clapp R, et al. Lifestyle-related
factors and environmental agents causing cancer:
an overview. Biomed Pharmacother. 2007;61(10):
640–658.
228. Morris AA. Cerebral ketone body metabolism.
J Inherit Metab Dis. 2005;28(2):109–121.
229. Maurer GD, Brucker DP, Bahr O, et al. Differential
utilization of ketone bodies by neurons and glioma
cell lines: a rationale for ketogenic diet as experi-
mental glioma therapy. BMC Cancer. 2011;11:315.
230. Veech RL, Chance B, Kashiwaya Y, Lardy HA, Cahill
GF Jr. Ketone bodies, potential therapeutic uses.
IUBMB Life. 2001;51(4):241–247.
231. Lee C, Longo VD. Fasting vs dietary restriction
in cellular protection and cancer treatment: from
model organisms to patients. Oncogene. 2011;30(30):
3305–3316.
232. Scheck AC, Abdelwahab MG, Fenton KE, Stafford
P. The ketogenic diet for the treatment of glioma:
insights from genetic profiling. Epilepsy Res. 2012;
100(3):327–337.
233. Nebeling LC, Miraldi F, Shurin SB, Lerner E.
Effects of a ketogenic diet on tumor metabolism
and nutritional status in pediatric oncology patients:
two case reports. J Am Coll Nutr. 1995;14(2):202–
208.
234. Zuccoli G, Marcello N, Pisanello A, et al. Metabolic
management of glioblastoma multiforme using
standard therapy together with a restricted ketogenic
diet: Case Report. Nutr Metab (Lond). 2010;7:33.
235. Lee C, Raffaghello L, Brandhorst S, et al. Fasting
cycles retard growth of tumors and sensitize a range
of cancer cell types to chemotherapy. Sci Transl Med.
2012;4(124):124ra127.
236. Levine ME, Suarez JA, Brandhorst S, et al. Low
protein intake is associated with a major reduction
in IGF-1, cancer, and overall mortality in the 65
and younger but not older population. Cell Metab.
2014;19(3):407–417.
237. Khandekar MJ, Cohen P, Spiegelman BM. Molecular
mechanisms of cancer development in obesity. Nat
Rev Cancer. 2011;11(12):886–895.
238. Courneya KS. Exercise in cancer survivors: an over-
view of research. Med Sci Sports Exerc. 2003;35(11):
1846–1852.
239. Jones LW, Fels DR, West M, et al. Modulation of
circulating angiogenic factors and tumor biology by
aerobic training in breast cancer patients receiving
neoadjuvant chemotherapy. Cancer Prev Res (Phila).
2013;6(9):925–937.
240. Betof AS, Lascola CD, Weitzel D, et al. Modulation
of murine breast tumor vascularity, hypoxia and
chemotherapeutic response by exercise. J Natl Cancer
Inst. 2015;107(5).
241. Berger A. How does it work? Positron emission
tomography. BMJ. 2003;326(7404):1449.
242. Yang SK, Cho N, Moon WK. The role of PET/
CT for evaluating breast cancer. Korean J Radiol.
2007;8(5):429–437.
243. Eubank WB, Mankoff DA. Evolving role of positron
emission tomography in breast cancer imaging. Semin
Nucl Med. 2005;35(2):84–99.
244. D’Souza M, Jaimini A, Bansal A, et al. FDG-PET/CT
in lymphoma. Indian J Radiol Imaging. 2013;23(4):
354–365.
245. Nakamoto Y, Saga T, Fujii S. Positron emission
tomography application for gynecologic tumors.
Int J Gynecol Cancer. 2005;15(5):701–709.
246. Ferreira D, Oliveira A, Barroso A, Conde A, Parente
B. Positron emission tomography: indications in lung
cancer - prospective experience of a department. Rev
Port Pneumol. 2007;13(1):35–51.
247. Bouchelouche K, Oehr P. Positron emission tomogra-
phy and positron emission tomography/computerized
tomography of urological malignancies: an update
review. J Urol. 2008;179(1):34–45.
248. Wu C, Li F, Niu G, Chen X. PET imaging of inflam-
mation biomarkers. Theranostics. 2013;3(7):448–466.
249. Liu Y. Fatty acid oxidation is a dominant bioenergetic
pathway in prostate cancer. Prostate Cancer Prostatic
Dis. 2006;9(3):230–234.
250. Oyama N, Akino H, Kanamaru H, et al. 11C-acetate
PET imaging of prostate cancer. J Nucl Med. 2002;
43(2):181–186.
251. Farsad M, Schiavina R, Castellucci P, et al. Detection
and localization of prostate cancer: correlation of (11)
C-choline PET/CT with histopathologic step-section
analysis. J Nucl Med. 2005;46(10):1642–1649.
252. Janardhan S, Srivani P, Sastry GN. Choline kinase:
an important target for cancer. Curr Med Chem.
2006;13(10):1169–1186.
253. Brown JM. Tumor hypoxia in cancer therapy. Methods
Enzymol. 2007;435:297–321.
254. Laking G, Price P. Radionuclide imaging of perfu-
sion and hypoxia. Eur J Nucl Med Mol Imaging.
2010;37(suppl 1):S20–S29.

S UMMARY OF K EY P OI NT S
Current Concepts in
Carcinogenesis
• A key paradigm in environmental
carcinogenesis—that of
gene-environment interactions, from historical observational studies,
a concept used to describe the
complex interplay between individual
or population genetics and
responses to chemical agents— • Next-generation approaches to
has undergone an expansive
transformation into a more
contemporary understanding of the
human “exposome.” The exposome
is the cumulative lifelong burden of
disease-contributing stressors,
including exogenous and
endogenous agents of all types, as
well as an individual’s genetic
susceptibility and “omics” response
profile (genomics, epigenomics,
transcriptomics, proteomics,
metabolomics) and their microbiome.
• A classic sequential model of
initiation, promotion, conversion, and
progression has increasingly evolved
into less linear models that may no
longer classify carcinogens as
initiators or promoters per se. It may
be more important to understand the
contribution of environmental agents
to the various hallmarks of cancer:
genomic instability, resisting cell
death, deregulating cellular
energetics, sustaining proliferative
signals, evading growth suppressors,
avoiding immune destruction,
enabling replicative immortality,
promoting tumor inflammation,
activating invasion and metastasis,
and inducing angiogenesis.
Identification of Human
Carcinogens
• A traditional paradigm classified
carcinogenic agents as
environmental, lifestyle related, or
occupational in origin, but these
B. GENESIS OF CANCER
Environmental Factors
Steven R. Patierno
classifications are blurred by
examples of complex interplay
among all three types of exposure.
• Carcinogen identification has evolved
to occupational cohort epidemiology,
to broad-scale screening and testing
in experimental animals.
carcinogen identification use
exposome approaches of integrating
sophisticated individual exposure
monitoring and biosensing with
highly sensitive “omics” approaches
to detect early perturbation of
disease-relevant signaling pathways.
• Most human cancers probably result
from the interaction of several
exogenous carcinogenic influences
(often unidentified, but many are
dietary or lifestyle related in origin)
along with intrinsic factors (e.g.,
inherited genes, hormones,
inflammation and oxidative stress,
immune status, energy balance).
Role of Environmental Agents in
the Etiology of Human Cancer
• Although the causes of most
individual human cancers are not
identifiable, there is incontrovertible
evidence that certain chemical
agents, radiation, and certain
biologic agents are contributors to
the overall incidence of human
cancer.
• Consumption of tobacco products,
especially cigarette smoking, is
responsible for nearly 30% of all
cancers.
• Chemical carcinogens include
polycyclic aromatic hydrocarbons
(PAHs), aromatic amines, benzene,
aflatoxins, tobacco chemicals, and
chemotherapeutic agents. Metal
carcinogens are largely associated
with occupational exposures and
10 
include specific forms of arsenic,
nickel, and hexavalent chromium.
• Radiation carcinogens include
ultraviolet radiation, ionizing
radiation, and radon.
• Fibers such as asbestos and certain
dusts are etiologic agents in lung
cancers and mesothelioma.
• Many components in the diet can
influence the development of
cancer through carcinogenic or
anticarcinogenic mechanisms.
Exposure Biomarkers,
Susceptibility Factors, and
Prevention
• The identification of molecular
biologic markers of exposure, effect,
and susceptibility (reflecting early
events that link exposure to adverse
outcome, but before detection of
clinical disease) will help further our
understanding of human
carcinogenesis.
• In addition to genetic polymorphisms
indicative of susceptibility,
biomarkers of disease-relevant
exposure will increasingly rely on
highly sensitive, multiplatform, and
multidimensional analysis of the
impact of potential carcinogenic
agents on epigenomics,
transcriptomics, proteomics,
metabolomics, and the microbiome.
• Primary, secondary, and tertiary
cancer prevention approaches will
be greatly facilitated by the
development of noninvasive
biomarkers that identify high-risk
individuals and by identification of
the disease-consequential
perturbations in molecular signaling
that may also be targets for
chemoprevention or cancer
interception.
139
140 Part I: Science and Clinical Oncology
Direct causes of most individual human cancers will not be identifiable,
but there is incontrovertible evidence that certain chemical agents,
radiation, and certain biologic agents are contributors to the overall
incidence of cancer. A traditional paradigm has classified carcinogenic
agents as environmental or lifestyle related or occupational in origin,
but these classifications are blurred by examples of complex interplay
among all three “sources” of exposure.1 For example, cigarette smoking
is likely responsible for 25% to 30% of human cancer: to smoke or
not to smoke can be thought of as a lifestyle choice, but the act of
smoking also gives rise to secondhand (environmental) tobacco smoke
exposure, and smoking is pervasive in some occupational settings.
Obesity is now also recognized as a major risk factor for certain cancers.2
Obesity is etiologically related to energy balance (lifestyle) and genetics,
but certain environmental agents may act as “obesogens” and may
indirectly contribute to cancer risk. Certain occupational chemical
agents may also be released into the general environment, or unknow-
ingly carried home to affect lifestyle, and occupation itself can be
considered a lifestyle choice in some parts of the globe. A more recent
paradigm in environmental carcinogenesis—gene-environment interac-
tions,3 a concept used to describe the complex interplay between
individual or population genetics and responses to chemical agents—has
also undergone an expansive transformation into a more contemporary
understanding of the human “exposome.”4–6 The exposome is the
cumulative lifelong burden of disease-contributing stressors including
exogenous and endogenous agents of all types, as well as an individual’s
“omics” profile (genomics, epigenomics, transcriptomics, proteomics,
metabolomics), and his or her microbiome. Yet another traditional
model of environmental carcinogenesis, that of the classic sequential
model of initiation, promotion, conversion, and progression (history
reviewed succinctly in Weiss7), has also increasingly been supplanted
by less linear models that no longer classify carcinogens as initiators
or promoters per se. Instead, the concepts of epigenomic and tran-
scriptional reprogramming, including alternative RNA splicing, in
response to microenvironmental shifts in allostatic load (cellular
physiologic stress) are increasingly understood as drivers of early-stage
carcinogenesis through the expansion of death-resistant cells.8–11 Thus
a more apt approach may be to understand the contribution of chemical
agents to the various hallmarks of cancer12,13: genomic instability,
resisting cell death, deregulating cellular energetics, sustaining prolifera-
tive signals, evading growth suppressors, avoiding immune destruction,
enabling replicative immortality, promoting tumor inflammation,
activating invasion and metastasis, and inducing angiogenesis. These
evolving new thought paradigms hold promise for improved exposure
monitoring, more accurate identification and earlier detection of
disease-consequential exposures, and increasingly effective primary,
secondary, and tertiary public health and clinical cancer prevention
and interception measures.
EVOLVING MODELS FOR CHEMICAL
CARCINOGENESIS
Fig. 10.1 gives a linear representation of the traditional paradigm for
progressive carcinogenesis. This model is useful in that it depicts what
is thought to be the monoclonal nature of cancer (originating from
a single cell) and the concept of clonal evolution and expansion. It
evokes a chemical- or radiation-provoked initiating event, followed
by promotion of clonal expansion to form a preneoplastic lesion. In
this model, it is thought that a single cell of a preneoplastic lesion
undergoes “conversion,” which theoretically equates to progeny cells
acquiring enough cumulative genotypic and phenotypic alterations
to express a malignant phenotype. Malignant cells, defined pathologi-
cally as “invasive,” can then undergo progression (volume expansion
due to increased net cell proliferation) to a clinically relevant cancer
with metastatic potential. This pictorial sequence is often punctuated
with reference to mutagenic activation of oncogenes, inactivation of
tumor suppressor genes, and, more recently, epigenetic alterations.
Such a linear model is particularly illustrative of protective points of
intervention beginning with avoidance of disease-consequential exposure
and chemoprevention (primary prevention), early diagnosis and cancer
interception (secondary prevention), and prevention of relapse and
second cancers (tertiary prevention). It also helps give temporal perspec-
tive to the carcinogenic “lag” period, the presumptive period of time
between an initiation event and clinical detection of a tumor.
A consequence of this sequential model is that it tends to narrowly
relegate environmental carcinogens as mutagenic “initiating” agents,
and certain other chemical agents as nonmutagenic “promoting” agents,
and does not account for the increasing weight of evidence that the
process of early carcinogenesis at the cellular level is highly nonlinear
and better understood as a perturbation of molecular homeostasis
resulting in accelerated evolution toward acquisition of any of the 10
hallmarks of cancer.12,13 As shown in Fig. 10.2, these hallmarks are
best graphically portrayed in circular fashion because they do not
necessarily follow a temporal sequence. This model elaborates our
understanding of oncogenesis as a disruption of highly interconnected
and complex signaling networks that intersect cytostasis and differentia-
tion circuitry, cell viability circuitry, proliferation circuitry, and motility
circuitry. It necessitates broadening our understanding of mechanisms
of action in environmental carcinogenesis to include the potential
ability of chemical carcinogens to provoke genomic instability, sustained
proliferative signaling, acquisition of death resistance, evasion of growth
suppressors, enabling of replicative immortality, and/or activated cellular
motility and invasiveness.
The “hallmarks of cancer” model also underscores the possible
impact of environmental agents on deregulation of cellular energetics,
avoidance of immune destruction by emerging tumor cells, promotion
of tumor inflammation, and provocation of nonneoplastic additional
cells in the tumor microenvironment, such as stromal cells, to contribute
to the survival, development, and emergence of an early precancerous
cell. One illustrative example of this complexity emerges from increased
understanding of the role of cell death in cancer and carcinogene-
sis.10,11,13–15 Studies of genotoxin-treated cells in culture have drawn
attention to the importance of an overlooked outcome in earlier
attempts to measure mutagenesis as a surrogate marker for carcinogenic
potential: the frequent emergence of death-resistant subclones of cells
out of which an occasional cell carrying a mutated selectable gene
could be detected as a low-frequency event.14,16 This carcinogen-
provoked early acquisition of resistance to apoptosis has challenged
the perspective that active cell death processes necessarily protect against
cancer and carcinogenesis. To the contrary, the facile ability of cells
to circumvent cell death when under stress actually renders the existence
of such cell death signaling pathways as a risk factor for carcinogenesis.15
Indeed, it is now appreciated that additional modes of cell death,
such as autophagy, can mediate either tumor cell survival or death;
and even necrosis, often a consequence of toxic exposure, has been
shown to be potentially proinflammatory and tumor promoting.15,17
In 2015 a series of review articles were published by an international
consortium of scientists, collectively called the Halifax Project, which
conducted detailed assessments of the potential of environmental
chemicals to contribute causatively to the process of carcinogenesis
by affecting each “hallmark” of cancer.18–25
HISTORY OF ENVIRONMENTAL
CARCINOGENESIS AND SUPPORT FOR
THE ROLE OF ENVIRONMENTAL AGENTS IN
THE ETIOLOGY OF HUMAN CANCERS
The methods and concepts for identifying chemical agents potentially
carcinogenic to humans have evolved over time and continue to
evolve as both the general public and regulatory agencies grapple
with the fact that humans live in a chemical-laden society, which
generally contributes to improved global health and increased longev-
ity. In addition to consumption of and exposure to tobacco smoke,
itself containing more than 30 potentially carcinogenic chemicals26–28
 Environmental Factors  •  CHAPTER 10 141

Sequence Leading to Neoplasia Interventions and Strategies

Viruses
Radiation
Chemicals
Procarcinogens Direct carcinogens Avoid Primary
exposure prevention

Initiation

Ultimate carcinogens Prevent

formation of
Genetic and
carcinogens
epigenetic
changes

Initiated Block
Chemo
cell interactions
prevention
with genome
Selective
Promotion clonal
expansion
Preneoplastic Suppress
lesion growth

Genetic and
epigenetic
Conversion changes
Early Secondary
diagnosis prevention
Malignant
tumor

Surgery
Radiation
Cell Therapy
therapy
heterogeneity Chemotherapy

Progression
Prevent relapse
Clinical Tertiary
and secondary
cancer prevention
tumors

Figure 10.1  •  Linear representation of a sequence leading to neoplasia and points of public health intervention strategies for prevention of multistage
carcinogenesis.

(Box 10.1) delivered directly by inhalation of a chronically injurious
toxic fume, humans are awash in a sea of low-level exposure to both
natural and anthropomorphic chemicals. For example, polycyclic
aromatic hydrocarbons (PAHs), a broad class of chemicals including
some with carcinogenic potential, are released into the environment
by both anthropomorphic fuel combustion and natural volcanic
eruptions and forest fires, as well as consumed through smoking
and eating flame-cooked meat. Such exposure complexity neces-
sitates improved differentiation of “any” exposure from “disease-
consequential” exposures, which itself represents a next-generation
evolution of carcinogen identification from historical observational
studies, to occupational cohort epidemiology, to broad-scale screen-
ing and testing in experimental animals, to exposome approaches of
integrating sophisticated individual exposure monitoring with highly
sensitive “omics” approaches to detect perturbation of disease-relevant
signaling pathways.
The carcinogenic effects of a sizable number of environmental or
industrial chemicals were first described observationally in humans.
The influences of occupation and lifestyle in cancer occurrence trace
back at least to the 16th century when Ramazzini, in 1700, noted
that nuns showed a higher frequency of breast cancer than was observed
among other women. Also in that century, Paracelsus and Agricola
described Bergkrankheiten (lung cancer) in German miners, probably
caused by uranium and its decay product radon. In 1761, Hill
observationally associated the use of tobacco snuff with cancer in the
nasal passage, and in 1775 Pott noted the occurrence of soot-related
scrotal cancer among chimney sweeps. In 1895, Rehn published
evidence that occupational exposure to aromatic amines was associated
with bladder cancer, and Unna in 1894 associated sunlight exposure
with skin cancer.29–34
These observational associations gradually gave way to more proac-
tive approaches to attempt to identify potential carcinogens both
computationally (retrospective epidemiology, usually of occupationally
exposed or catastrophe-exposed cohorts) and by animal experimentation.
However, it was not until the early 20th century that animal models
for chemical carcinogenesis were developed, beginning in 1915 when
142 Part I: Science and Clinical Oncology
Figure 10.2  •  Hallmarks of cancer and points of potential carcinogen
impact.
Yamagiwa and Ichikawa demonstrated the production of skin tumors
after topical application of crude coal tar to the ears of rabbits.35 These
early studies gradually led to the standardization of animal carcinogenesis
bioassays, with the goal of testing chemical class prototypes for car-
cinogenic potential, ostensibly before human exposure.
Contrary to experiences in earlier centuries, with the advent of
animal bioassay programs, evidence of carcinogenicity in experimental
animals has sometimes preceded evidence obtained from epidemiologic
studies or case reports. Although the term carcinogen means “giving
rise to carcinomas,” loosely referring to malignancies in general, more
operational definitions are used for carcinogens in animal bioassays.
In this context, a carcinogen may be defined as an agent whose
administration to previously untreated cells or animals leads to a statisti-
cally significant increased incidence of malignant neoplasms, compared
with the incidence in appropriate untreated control cells or animals.
Data from such studies, combined with epidemiologic and mechanistic
studies, are employed by regulatory agencies to try to protect humans
from known chemical, radiation, and infectious hazards. From a
mechanism of action (MOA) perspective, the regulatory thrust has
been to determine whether a carcinogenic agent was mutagenic or
nonmutagenic; the latter involved recognition that chronic tissue
toxicity itself, in the absence of DNA damage, was potentially carci-
nogenic. Synthetic and naturally occurring chemicals comprise the
largest group of known human carcinogens. More than 100 chemicals,
chemical mixtures, biologic agents, physical agents, or industrial
processes have been classified as Class 1 human carcinogens (Table
10.1) by the International Agency for Research on Cancer (IARC),
and more than 300 chemicals have been designated as probable (Class
2A) or possible (Class 2B) human carcinogens.36 For perspective, these
numbers of potential carcinogenic hazards derive from an environmental
milieu of nearly 10 million chemicals.
Substantial growth has occurred in our understanding of how these
extrinsic factors (chemicals, radiation, infectious agents) interact with
intrinsic factors (e.g., genetics of inherited genes, “omics” profile, diet,
body mass index (BMI), hormones, immune status, and microbiome)
to determine overall susceptibility and risk. Indeed, a key concept
Box 10.1.  HEALTH EFFECTS AND CONTROL OF
SMOKING
A series of reports from the US Surgeon General since 1964 have
argued that cigarette smoking is the most significant source of
preventable morbidity and premature mortality in high-income
countries. An estimated annual excess mortality of 400,000 is attributed
to cigarette smoking in the United States. These deaths are the result of
coronary heart disease, cancer, and various respiratory diseases.40
Cancers associated with smoking or smokeless tobacco use include
those of the lung, oral cavity, esophagus, pharynx, larynx, and bladder.
Additional associations have been reported for cancers of the pancreas,
kidney, stomach, nasopharynx, and cervix.12 The overall increase in risk
of disease among smokers compared with nonsmokers is about
tenfold for lung cancer, sixfold for chronic obstructive pulmonary
disease, and twofold for myocardial infarction. The combined effect of
smoking-related diseases on the average life expectancy of smokers is
a reduction of 5 to 8 years. On a worldwide basis, an estimated 6
million deaths per year are attributed to tobacco use, with upward of
80% of these deaths in low- and middle-income countries. If current
use trends continue unabated, estimates as high as 8 million deaths
due to tobacco use annually could be expected by the year 2030, and a
total of 1 billion this century.41
The addictive properties of tobacco smoking, due primarily to its
nicotine content, cause both physiologic and psychologic dependence.
Withdrawal symptoms can be severe and include irritability,
aggressiveness, hostility, depression, difficulty concentrating, and a
craving for tobacco. These pharmacologic factors are reinforced by
social factors such as peer pressure, emulation of family role models,
and cultural influences. Because most smokers begin smoking, and
often form lifelong smoking habits, in their teenage years, preventive
educational measures should be focused on this age group.
Smoking control measures use various strategies: cessation
programs, clinical or community interventions, governmental or
private-sector regulations, taxation of tobacco products, warning labels,
and smoking prevention programs. Although the majority of
ex-smokers have achieved abstinence without extensive personal
assistance from organized cessation programs, other control measures
(e.g., national health education programs, physician counseling, and
indoor smoking regulations) and family pressure are important
influences contributing to smoking cessation. Smoking prevention
programs, particularly in schools, and government taxation have been
somewhat effective in reducing the initiation of smoking among
children and adolescents in developing countries. These approaches
will need to be applied more vigorously, especially in low- to middle-
income countries, to stem the expansion of smoking worldwide. New
challenges also are arising with the creation of new and emerging
tobacco products such as dissolvable tobacco products, e-cigarettes,
and hookah tobacco. The Family Smoking Prevention and Tobacco
Control Act (2009) in the United States has given the US Food and
Drug Administration broad authority to regulate the manufacturing,
distribution, and marketing of these and more traditional tobacco
products to protect public health.
underpinning the role of the environment in cancer risk relates to the
impact of geography on lifestyle adoption and adaptation and cancer
susceptibility, and derives from the following series of epidemiologic
observations:
• Although the overall incidence of cancer development is reasonably
constant among countries, incidences of specific cancer types can
vary up to several hundredfold.
• Large differences in tumor incidence exist within populations of a
single country.
 Environmental Factors  •  CHAPTER 10 143

Table 10.1  Agents and Processes Considered Carcinogenic in Humans (Class 1) by the International
Agency for Research on Cancer
Common Organ or Tissue Common Organ or Tissue
Agent or Process Sites of Cancer Agent or Process Sites of Cancer
AMBIENT AND DIETARY EXPOSURE Mineral oils (untreated and Skin, scrotum
Acetaldehyde (from alcoholic Upper digestive tract mildly treated)
beverages) Mustard gas Lung, larynx and pharynx
Aflatoxins Liver 2-Naphthylamine Urinary bladder
Areca nut Oral cavity Nickel and nickel compounds Lung, nasal sinus
Aristolochic acid (from plants) Renal pelvis and ureter Painting Lung
Arsenic and arsenic compounds Lung, skin 2,3,4,7,8-Pentachlorodibenzofuran Soft tissue sarcoma, non-Hodgkin
Erionite Pleura, peritoneum lymphoma, cancer of the lung
Polychlorinated biphenyl (PCB-126) Liver, bile duct, lymphoid tissue
CULTURAL HABITS
Rubber industry Urinary bladder, leukemia
Alcoholic beverages Oral cavity, pharynx, larynx,
Shale oils Skin, scrotum
esophagus, liver
Silica, crystalline Lung
Betel quid with tobacco Oral cavity
Soots Skin, scrotum, lung
Tobacco products, smokeless Oral cavity
Strong inorganic acid mists Larynx
Tobacco smoke Respiratory tract, urinary bladder,
renal pelvis, pancreas Talc containing asbestiform fibers Lung
Salted fish, Chinese style Nasopharynx Toluidine Urinary bladder
Solar radiation Skin Underground mining with Lung
exposure to radon
OCCUPATIONAL EXPOSURES Vinyl chloride Liver, lung, gastrointestinal tract,
Aluminum production Lung, urinary bladder brain
4-Aminobiphenyl Urinary bladder Wood dust Nasal cavities, paranasal sinuses
Asbestos Lung, pleura, peritoneum, larynx, THERAPEUTIC AGENTS
gastrointestinal tract
Analgesics mixtures containing Renal, urinary bladder
Auramine production Urinary bladder
phenacetin
Benzene Leukemia
Azathioprine Leukemia
Benzidine Urinary bladder
N,N-bis(2-chloroethyl)-2- Urinary bladder
Benzo(a)pyrene Lung naphthylamine
Beryllium Lung Busulphan Lymphohematopoietic tissue
Boot and shoe manufacture Nasal sinus 1,4-Butanediol dimethanesulfonate Leukemia
and repair
Chlorambucil Leukemia
Cadmium Lung
Chlornaphazine Urinary bladder
Chromium (VI) compounds Lung
Cyclophosphamide Urinary bladder, leukemia
Coal combustion (indoors) Lung
Cyclosporin Lymphoma
Coal gasification Lung, urinary bladder, scrotum
Diethylstilbestrol Breast, cervix
Coal-tar pitches Skin, scrotum, lung
Estrogen replacement therapy Endometrium, breast
Coal tars Skin, lung
Estrogens, nonsteroidal Cervix and vagina, breast,
Coke production Skin, scrotum, lung, urinary endometrium, testes
bladder
Estrogens, steroidal Endometrium, breast
Dioxin Multiple sites
Etoposide Lymphohematopoietic tissue
Ethylene oxide Lymphatic, hematopoietic
Melphalan Leukemia
Fission products Multiple sites
8-Methoxypsoralen plus UV Skin
Formaldehyde Liver radiation
Furniture and cabinet making Nasal sinus MOCA (Methylene Urinary bladder
Hematite mining Lung bis[2-chloroaniline])
Ionizing radiation (all types) Multiple sites MOPP combination therapy Leukemia
Iron and steel founding Lung Oral contraceptives (combined) Liver
Isopropyl alcohol manufacture Nasal sinus Oral contraceptives (sequential) Endometrium
(strong acid process) Phenacetin Renal pelvis, ureter
Leather dust Nasal sinus Semustine (methyl-CCNU) Leukemia
Magenta, manufacture of Urinary bladder Sulfur mustard Lung
Methyl ether Lung Tamoxifen Endometrium

Continued
144 Part I: Science and Clinical Oncology
Table 10.1  Agents and Processes Considered Carcinogenic in Humans (Class 1) by the International
Agency for Research on Cancer—cont’d
Common Organ or Tissue
Agent or Process Sites of Cancer
Thiotepa Leukemia
Treosulfan Leukemia
INFECTIOUS AGENTS
Clonorchis sinensis Liver, bile duct
Epstein-Barr virus Lymphoma
Helicobacter pylori Stomach
Hepatitis B virus Liver
Hepatitis C virus Liver
Data from the International Agency for Research on Cancer (IARC). IARC Monographs on the Evaluation of Carcinogenic Risk to Humans. Vols. 1–104. Lyon, France: IARC;
1970–2012. An updated listing of the overall evaluation of carcinogenicity to humans can be accessed at http://monographs.iarc.fr under the heading “Classifications.” (Note: For
examples of carcinogenic ionizing radiations, see Table 10.3.)
• Migrant populations assume the cancer incidence of their new
environment within one to two generations.
• Cancer rates within a population can change rapidly.37
It should be noted, however, that a major aspect of geography-
influenced carcinogenesis is related to exposure to unique dietary and
lifestyle behaviors and infectious agents, and the possible additive or
synergistic interactions between infectious agents and diet. For example,
the incidence of cancer of the esophagus, the sixth most common
cause of death from cancer worldwide30 but the third most common
in developing countries after stomach and lung cancer, varies from
less than 5 per 100,000 persons per year in Central America and
Western Africa to more than 50 per 100,000 persons per year in parts
of China and central Asia. Putative causes for such “hot spots” have
been identified, such as consumption of hot yerba maté in Uruguay,
Brazil, and northeast Argentina; however, the causative factors in other
high-risk areas remain unknown. Other cancers demonstrating unique
geographic distribution include biliary cancer (cholangiocarcinoma)
associated with liver fluke infection in some Asian (especially Thailand)
and African countries; gastric adenocarcinoma associated with virulent
strains of Helicobacter pylori infection in South Korea, Japan, and
parts of China; and hepatocellular carcinoma (HCC) associated with
hepatitis B virus (HBV) and aflatoxin exposure in parts of western
Africa and eastern China.38–40 Other connections between geography
and lifestyle/environment are documented by studies of shifting cancer
organ-site rates over generations following immigration, such as the
decrease in stomach cancer rates and increase in prostate cancer rates
in offspring of Japanese immigrants to North America.41
Chemical Biology of Carcinogenesis
Chemical carcinogens comprise a diverse array of chemical structures,
including both organic and inorganic compounds, the one common
characteristic being that either the chemical itself, or its derivatives,
are reactive species capable of interacting with biomolecules. Because
the innate reactivity of such compounds also tends to make them
unstable, relatively few carcinogens are direct acting. Instead, most
carcinogens require metabolic activation to reactive species, often inside
of the target cells. Metabolic pathways can be strongly influenced by
a variety of extrinsic and intrinsic factors and are important determinants
of both interindividual and target organ susceptibilities to carcinogens.
Because the mammalian species, including humans, developed in a
complex milieu of naturally occurring toxic agents, a panoply of innate
mechanisms of extracellular and intracellular resistance to such toxic
insults developed, ranging from circulating and intracellular sulfhydryl-
rich peptides and antioxidants to extensive DNA repair pathways (see
Common Organ or Tissue
Agent or Process Sites of Cancer
Human immunodeficiency Kaposi sarcoma
virus type 1
Human papillomavirus types Cervix
16, 18, others
Human T-cell lymphotropic Adult T-cell leukemia/lymphoma
virus type I
Kaposi sarcoma herpesvirus Kaposi sarcoma
Opisthorchis viverrini Liver (cholangiocarcinoma)
Schistosoma haematobium Urinary bladder
Chapter 11). These must be overcome before toxic and carcinogenic
properties are manifest, underscoring the importance of dose to
toxicology.
Once formed intracellularly, the reactive intermediates can inter-
act with both protective and vulnerable molecular targets related
to carcinogenesis. As described earlier, in classic linear models of
carcinogenesis, mutagenicity was considered a necessary property
of carcinogenic “initiators.” DNA was considered the principal
molecular target of carcinogens, and it was presumed that nearly
all carcinogens interacted with DNA to produce genetic lesions
(DNA damage) that can result in mutation of critical cellular genes,
including oncogenes and tumor suppressor genes. More recently, the
potential interactions of reactive chemicals with signaling pathways
governing the various hallmarks of cancer, and the cell’s molecular
responses to those interactions, have come to be recognized as part
of the early carcinogenic process. One such example is the potential
connection between compounds known as environmental estrogens,
chronic receptor-mediated growth signaling, and hormonally related
cancers42–44 (Box 10.2). Other critical examples, discussed later, include
the nonmutagenic impact of chemically reactive species on gene
regulation through epigenomic, metabolomic, and/or transcriptional
reprogramming.
EXPOSURE BIOMARKERS AND ASSESSING
HUMAN EXPOSURE
Increased understanding of the mechanistic basis of carcinogenesis
provides opportunities for the identification of molecular biologic
markers that reflect both disease-relevant exposure and/or events
occurring between exposure and clinical disease. It is hoped that the
use of biologic markers will help define the roles of environmental
agents (particularly at low levels and in complex mixtures) in the
etiology of human cancer. These molecular biologic markers can be
classified into three major categories:
1. Markers of exposure reflecting a biologically effective dose of a
carcinogen
2. Markers of effect indicating a disease-relevant biologic response to
an exposure
3. Markers of susceptibility that characterize the inherent susceptibility
of an individual to a carcinogenic agent45–49
The detectable persistent interaction of a carcinogen with macro-
molecules was first demonstrated by Miller and Miller in 1947, who
showed azo dye residues bound to liver proteins of treated rats. Later
studies indicated the importance of carcinogen modification of DNA

Box 10.2.  ENVIRONMENTAL ESTROGENS AND
BREAST CANCER
Women with a high lifetime exposure to estrogen, such as that
occurring with early onset of menarche and late menopause, may be at
higher risk for breast cancer. In obese menopausal women, adipose
tissue becomes the major source of estrogens, and higher blood
estrogen levels among postmenopausal women have shown that
higher levels are associated with increased risk of subsequent breast
cancer.42 These observations provoke an important question: do
xenoestrogens (synthetic chemicals that can act like estrogens) or
other environmental factors such as phytoestrogens (plant estrogens)
play a role in the risk for breast cancer? There is clear evidence that
these molecules may interfere with the interactions of human
estrogens with their receptors, and perturb signaling pathways that not
only promote proliferative signaling, but possibly even early resistance
to antiestrogens.63 However, thus far no epidemiologic “smoking gun”
has been identified. The Long Island Breast Cancer Study was unable to
identify any environmental factors that could be responsible for the
elevated incidence of breast cancer on Long Island or other locations43
and no evidence emerged for organochlorine pesticides, which have
long been known to have weak estrogenic activities. Bisphenol A, an
industrial chemical present in some polycarbonate plastics used in
bottles and infant food packaging, has come under recent scrutiny as a
xenoestrogen.44 Consumer advocacy and industry action rather than
regulatory mandates seem to be reducing exposures to bisphenol
A at this time. Some studies have shown that women who have diets
high in phytoestrogens have lower rates of breast cancer. These
phytoestrogens may actually oppose the actions of natural estrogen
and sometimes lead to lower serum estrogen levels. The role of early
life exposures as risk factors for late-in-life events, potentially via
epigenomic effects, is emerging as a key area of investigation in
environmental carcinogenesis.42
(either directly or after metabolic activation) in the cancer process.
The measurement of carcinogen metabolites, carcinogen-DNA adducts,
or carcinogen-protein adducts in human tissues or fluids provides the
basis for development and application of individual exposure monitor-
ing, molecular dosimetry research, and the rapidly expanding field of
“molecular” cancer epidemiology. The potential advantage of this
approach is that more accurate assessments of individual or group
dose may be achieved than through estimates of carcinogen exposure
in the environment or workplace.
Carcinogen-DNA and carcinogen-protein adducts have been
detected in tissues from a variety of human populations with known
or suspected carcinogen exposure. PAH-DNA adducts are elevated
in white blood cells from persons occupationally exposed to airborne
PAHs, including coke oven workers, foundry workers, and aluminum
plant workers, and in lung tissue from heavy smokers. DNA isolated
from exfoliated bladder epithelial cells of cigarette smokers has been
shown to contain 4-aminobiphenyl adducts. Alkylation damage is
detected in DNA from esophageal tissue of persons with documented
dietary exposure to nitrosamines. Aflatoxin adducts are found in hepatic
DNA after dietary exposure to this mycotoxin. Cisplatin-DNA
intrastrand adducts have been measured in white blood cells of patients
undergoing cancer chemotherapy.
Carcinogen-DNA adducts excreted in urine provide a potentially
noninvasive means for quantifying DNA damage. Urinary concentration
of aflatoxin-guanine adducts is strongly correlated with dietary aflatoxin
intake. In addition to their use as indicators of previous carcinogen
exposure, DNA adducts have the potential for use as direct indicators
of future cancer risk. The first prospective test of this application
appeared in 1992, when detectable levels of aflatoxin-guanine adducts
Environmental Factors  •  CHAPTER 10 145
and several other aflatoxin metabolites in urine were shown to be
predictive of the development of liver cancer.
Carcinogen-protein adducts have also been studied as biomarkers
of human exposure. Alkylation damage in hemoglobin has been used
as an exposure index in risk assessment analyses of persons occupation-
ally exposed to ethylene oxide. It has been found that 4-aminobiphenyl
adducts in hemoglobin are highly specific (and sensitive) markers of
exposure to cigarette smoke. The level of 4-aminobiphenyl hemoglobin
adducts is related to the number of cigarettes smoked, the type of
tobacco smoked, and the metabolic phenotype of the smoker. The
levels of these adducts drop markedly after smoking cessation.37
Unreacted carcinogen metabolites excreted in urine also are
used to monitor human exposure to and uptake of carcinogens and
related compounds. Concentrations of hydroxylated PAHs (e.g.,
1-hydroxypyrene) in urine are elevated in smokers, in patients after
topical treatment with coal tar, in road pavers, in coke oven workers,
in aluminum plant workers, and in persons ingesting PAHs from food.
In occupational settings, the concentration of urinary 1-hydroxypyrene
is highly correlated with estimated or measured concentrations of
airborne PAHs.
These noninvasive approaches to exposure assessment have potential
applications as biomonitoring tools, but at present they have major
limitations, and by themselves do not address the key question of
parsing out disease-relevant exposure. In all of these studies, significant
differences in adduct or metabolite levels are often observed between
persons with similar carcinogen exposure, likely due to individual
biologic variability, exposure misclassification, and confounding variables
such as diet, physical activity, smoking, or personal environment. It
is hoped that the exposome approach, at the intersection between
high-sensitivity biomarker detection and perturbation of disease-relevant
signaling pathways, may improve capabilities for detection of meaningful
exposures as a function of individual susceptibility.
Carcin-Omics and the Exposome Approach to
Environmental Carcinogenesis
The concept of the cumulative lifelong human exposome is the most
recent integrative approach to understanding the complex interplay
among natural and anthropogenic chemicals, the environment-lifestyle-
occupational continuum of exposures, and the response of host cells
to exposure.4–6 This includes the intersection with diet and energy
balance, the interplay between extrinsic and intrinsic exposures in the
context of cellular physiologic stress (illustrated by the conundrum of
the necessity of oxygen for life in juxtaposition with the toxic conse-
quences of oxidative stress), and the concept of disease-consequential
exposure, especially in the context of dose. The importance of dose
is critical, a steady holdover from the 16th century alchemist and
father of toxicology, Paracelsus: “All things are poison and there is
nothing without poison; the right dose differentiates a poison from
a remedy” or “the dose makes the poison.” Herein lies the central
conceptual challenge of exposome research: to drive innovation in
increasingly sensitive methods to monitor and quantify individual
environmental and internal human exposures, and to combine those
data with leading-edge methodologies to detect subclinical perturba-
tions in disease-relevant signaling pathways that directly contribute to
disease evolution.
In traditional risk assessment paradigms, exposure science focused
on providing an exposure estimate that would then be benchmarked
against algorithm-driven regulatory guidance values in order to
maximally protect public health. More recently, exposure science has
shifted toward development and application of advanced analytic
methods including remote and on-person sensors, mechanistic and
computational exposure biology, and advanced “systems-omics” analysis
as next-generation biomarkers for meaningful exposure. These
approaches may revolutionize conventional testing strategies and risk
assessment, particularly as larger numbers of chemicals are being
detected in biologic samples at lower and lower levels. Systems level
146 Part I: Science and Clinical Oncology
“omics” approaches to biomarkers are shedding light on early biologic
effects of external and intrinsic stressors on adverse health outcomes,
including tobacco, diet, occupational exposures, and environmental
pollutants. As described by Escher and colleagues,50 “a mechanistic
understanding of the causal links between exposure and adverse effects
on human health and the environment can be improved by integrating
the exposome approach with the adverse outcome pathway (AOP)
concept that structures and organizes the sequence of biologic events
from an initial molecular interaction of a chemical with a biologic
target to an adverse outcome. Complementing exposome research
with the AOP concept may facilitate a mechanistic understanding of
stress-induced adverse effects, examine the relative contributions from
various components of the exposome, determine the primary risk
drivers in complex mixtures, and promote an integrative assessment
of chemical risks for both human and environmental health.” Similarly,
additional model constructs for understanding the potential impact
of environmental carcinogens point out that different chemicals in
chemical mixtures might affect different target cells (somatic or stem
cell) with different but complementary procarcinogenic effects on
signaling pathways governing the nexus between cell senescence and
immortality.18
Some of these concepts were also recently highlighted by a Working
Group convened by the National Institute of Environmental Health
Sciences (NIEHS) to give guidance on addressing the challenges of
“low-dose” carcinogenesis.51 The Working Group concluded, “The
scientific community will need to acknowledge limitations of animal-
based models in predicting human responses; evaluate biologic events
leading to carcinogenesis both spatially and temporally; examine the
overlap between measurable cancer hallmarks and characteristics of
carcinogens; incorporate epigenetic biomarkers, in silico modelling,
high-performance computing and high-resolution imaging, microbiome,
metabolomics, and transcriptomics into future research efforts; and
build molecular annotations of network perturbations. The restructuring
of many existing regulatory frameworks will require adequate testing
of relevant environmental mixtures to build a critical mass of evidence
on which to base policy decisions.”
Epigenomic Exposure Profiling
The epigenome is a principal determinant of cellular transcriptional
programming, and perturbations in the epigenomic are increasingly
understood as instrumental in cancer development.52–60 In general,
“epigenetics” refers to heritable changes in gene expression that are
not caused by alterations in the primary DNA sequence but are
associated with altered DNA methylation, multiple histone modifica-
tions, and tight regulation of multiple species of noncoding RNAs.
These factors, functionally characterized as epigenetic chromatin writers,
readers, or erasers, govern the dynamics of chromatin conformation
and transcription, and their complex net function can be perturbed
by both genotoxic and nongenotoxin carcinogens. A number of studies
have identified carcinogen-induced, rapid alterations in epigenomic
“marks,” some of which are also present in tumors from that tissue,
and smoking-associated DNA methylation changes in buccal cells
have been associated with DNA methylation changes in smoking-related
epithelial cancers.
Studies have shown that environmental estrogens can disrupt
estrogen receptor (ER) signaling, alter DNA and histone methylation,
and reprogram the epigenome.61,62 Some of this epigenomic reprogram-
ming may occur as a result of adverse exposure to environmental
estrogens during development, and possible disruption of normal
mammary gland development, resulting in changes in disease susceptibil-
ity across the life course. The mechanism of disruption can involve
perturbation of genomic signaling through altered binding of ligand-
associated ER with its DNA binding site, and/or nongenomic signaling
of membrane-bound ER resulting in activation of growth-regulating
or growth-deregulating kinase cascades. A study suggested that
environmental estrogens such as bisphenol A activates growth-promoting
proliferation and resistance to therapeutic drugs that target the epidermal
growth factor receptor (EGFR) pathway.63 Likewise, exposure of the
developing prostate to endocrine-disrupting chemicals can increase
risk of adult prostate cancer, and this has been associated with epig-
enomic disruption via phosphatidylinositol 3-kinase (PI3-K) effects
on histone methyltransferase.55
A complex interplay between genetic (mutational) and epigenetic
contributions to carcinogenesis is highlighted by multidimensional
analysis of the genome, epigenome, and transcriptome of glioblastoma.53
Mutated EGFR appears to promote epigenomic remodeling and
transcriptome reprogramming in EGFR-dependent glioblastoma
multiforme (GBM) pathogenesis. Interesting to note, the MOA of
carcinogenic forms of arsenic and nickel seems to involve epigenetic
modification of chromatin structure and function,58,59 in addition to
genotoxic and actions. Epigenetic dysregulation through changes in
the methylome and histone modification has also been observed in
virus-associated neoplasms.54
Important to note, the epigenome is also a potential target for
chemoprevention; for example, inhibitors of bromodomain proteins
that serve as epigenetic “readers” inhibit in vitro neoplastic transforma-
tion by 12-O-tetradecanoylphorbol-13-acetate (TPA), ostensibly by
inhibiting TPA-induced expression of prosurvival genes.57 Likewise,
a number of anticarcinogenic phytochemicals have a profound
effect on DNA methylation, histone modification, and regulation of
noncoding RNAs.60
Transcriptomic Exposure Profiling
Next-generation gene-chip and deep RNA sequencing technologies,
deployed against both in vivo and in vitro exposures for cross-platform
concordance, are providing insights into early molecular changes
induced by carcinogens, which could contribute to the development
of biomarkers for detection of disease-relevant exposure, early disease
detection, and prevention.64–66 For example, (S)-N′-nitrosonornicotine
([S]-NNN), the major form of NNN in tobacco products, is an oral
cavity and esophageal carcinogen in rats. Differences in the expression
of 40 to 70 genes involved in immune regulation, inflammation, and
cancer were detected in treated rats and cultured oral keratinocytes,
and interrogation of TCGA data sets showed that several of the genes
that were deregulated by (S)-NNN in rat tissues are also altered in
esophageal and head and neck tumors. Likewise, changes in global
gene expression were detected, after carcinogen treatment and before
the appearance of squamous cell carcinoma tumors, in a mouse
tongue model of human oral cavity and esophageal squamous cell
carcinoma through use of the carcinogen 4-nitroquinoline 1-oxide
(4-NQO). Gene ontology and pathway analyses have shown that
transcriptional networks affecting cell cycle progression, migration
and invasion, and cellular energetics were early events after expo-
sure and that changes in these pathways were also present in the
actual tumors.
Another study illustrates the potential application of transcriptomics
to parse out the individual effects of constituents of complex mixtures,
or even metabolites of the same parent carcinogen. Benzo(a)pyrene
(BaP) is produced from incomplete combustion of organic compounds
and also is present in cigarette smoke. The transcriptomic biologic
effects of BaP, its genotoxic metabolite BPDE, and an array of intermedi-
ary metabolites were studied in Hep2 cells, revealing activation, at
very early exposure time points, of several transcription factor networks
affecting metabolism, oxidative stress, cell proliferation, cell energetics,
DNA repair, and apoptotic pathways. These gene expression pattern
changes were compared with a signature set of approximately 9000
gene expressions derived from HCC patients. This approach showed
that exposure of cells to BaP, but not TCDD, deregulated metastatic
markers via nongenotoxic and genotoxic mechanisms, activated
inflammatory pathways, and repressed expression of genes involved
in cholesterol and fatty acid biosynthesis. Other studies on critical
RNA regulatory functions have suggested that disruption of cancer-
related microRNA-mRNA pairings may be an early sentinel of
disease-related exposure. Taken together, such studies suggest that a

transcriptomics approach may identify pathways that change as a
result of exposure, which may also contribute to the evolution of key
hallmarks of cancer.
Metabolomic Exposure Profiling
Metabolite profiling is being increasingly employed in the study of
carcinogenesis as a systems-level approach and means of identifying
biomarkers for exposure that are predictive of risk and diagnostic of
meaningful, disease-relevant exposure.67–71 This includes metabolomic
profiling of exposed target cells (in vitro and in vivo) and profiling
of plasma and urine of exposed experimental animals and humans.
For example, smoking-related biomarkers for lung cancer risk and
early-stage lung cancer are needed to enhance early detection strategies
and to provide a science base for tobacco product regulation. In an
untargeted metabolomics approach using ultra-high-performance
liquid chromatography–quadrupole time-of-flight mass spectrometry
(UHPLC-Q-TOF MS), blood was collected from human subjects
before and after they smoked a single cigarette. Sex- and race-related
dynamic metabolomic changes were detected in plasma, and novel
biomarkers affected by cigarette smoking were identified, including
a network of 12 molecules involved in cancer and carcinogenesis.
Similarly, a nuclear magnetic resonance (NMR)-based metabolomic
study of sera from a rat model of gastric carcinogenesis revealed
early perturbation of metabolic networks associated with oxidative
stress. Choline phosphorylation, fatty acid degradation, amino acid
metabolism, and glycosis were disturbed during gastric carcinogenesis.
Likewise, a series of metabolomics case-control studies of prostate cancer
revealed patterns predictive of disease risk and progression, including
early disruption of pathways associated with abnormal cell growth,
intensive cell proliferation, and dysregulation of lipid metabolism.
Microbiome
One of the most surprising recent developments related to carcinogenesis
is the emerging importance of the microbiome as a major environmental
factor influencing cancer susceptibility and the carcinogenic process.72–78
An individual’s microbiome is a complex function of genetics, lifestyle,
and culture including diet, and other exogenous factors including
antibiotic and antiinflammatory therapy. The relationship between the
microbiome and host health is complex and can include prevention or
promotion of carcinogenesis, modulation of the inflammatory response
of the host, influence on the production of anticarcinogenic metabolic
products such as butyrate, or even the production of carcinogenic
metabolic products or of toxins that disrupt the cell cycle. Although
many of the studies conducted thus far have addressed the role of the
microbiome in gastrointestinal and colorectal cancers, in which protein
residues and fat-stimulated bile acids are metabolized by the microbiota
to inflammatory and/or carcinogenic metabolites, there is increasing
evidence for its role in modulating the evolution of pancreatic, lung,
colorectal cancer, oral squamous cell carcinoma, and HCC.
Chemicals
Following are brief depictions of several chemical and physical agents
of environmental concern, which are illustrative of the roles of these
agents in the etiology of human cancers. A deeper understanding of
the mechanistic basis for the actions of these carcinogenic agents will
allow for more effective means to identify other carcinogens in our
environment and to develop preventive strategies to interrupt, block,
intercept, or reverse the neoplastic process.
Polycyclic Aromatic Hydrocarbons
The English surgeon Percivall Pott was among the first to document
the association of an environmental agent with cancer.29,30 During the
late 18th century, he determined that the unusually high incidence of
scrotal cancer among chimney sweeps was due to their occupational
exposure to soot and tar. It was not until the 20th century that
animal testing showed that the active carcinogens in soot and coal
Environmental Factors  •  CHAPTER 10 147
tar were PAHs,31 with most of the carcinogenic activity attributed to
the PAH BaP.
Humans are exposed to PAHs from a variety of sources that include
occupation, smoking, diet, and air.32 PAHs are readily absorbed into
the body through the skin, lungs, and gastrointestinal tract. Occupa-
tional and medicinal exposures constitute the highest levels of human
PAH exposure (small groups within the population), whereas diet
and smoking are the major sources of exposure to PAHs in the general
population. An excess of lung cancer has been demonstrated among
persons with substantial inhalation exposure to PAHs, including roofers
and pavers, coke oven workers, certain steel and iron manufacturing
workers, and aluminum production workers.33 In addition, several
studies have suggested that workers highly exposed through inhalation
also might be at increased risk of cancer at sites other than skin and
lung, with suggestive increases in risk in the bladder, pancreas, and
upper gastrointestinal tract.
Several biochemical pathways are involved in the metabolism of
PAHs and of BaP in particular.31 One major pathway results in highly
reactive 7,8-dihydrodiol-9,10-epoxide-BaP, thought to produce DNA
adducts leading to cancers of the esophagus, forestomach, intestine,
lungs, and mammary gland in rodents.
Aromatic Amines
The occurrence of bladder cancer among dye industry workers was
reported in 1895 by the German physician Ludwig Rehn, who suggested
a causal relationship. With the rapid expansion of the chemical industry
during and after World War I, increased risk of bladder cancer was
observed among workers employed in chemical manufacturing and
textile dyeing.34 An industry-wide study of workers exposed to dyes
in England and Wales demonstrated strong increased risks of bladder
cancer among men exposed to 1-naphthylamine, benzidine, 2-
naphthylamine, or mixed dyes, with 2-napthyamine implicated at the
highest hazard level.36 Additional studies in the dye industry identified
auramine O and magenta as human bladder carcinogens. Increased
risk of bladder cancer among rubber workers and in the electric cable
industry has been attributed to the naphthylamine added to rubber
as an antioxidant.
Aromatic amines are metabolized and excreted through a process
involving acetylation by N-acetyltransferase.8 Genetic variation in one
of the genes, NAT2, encoding this enzyme produces either rapid or
slow metabolic phenotypes in humans. Analysis of the NAT2 phenotypes
of patients with bladder cancer from the dye industry indicates that
persons showing the slow phenotype could be more susceptible to
bladder cancer caused by aromatic amines. This finding is consistent
with a meta-analysis indicating that NAT2 slows acetylation status is
associated with an increased risk of bladder cancer in the general
population.79,80
Benzene
Exposure to benzene was suspected to be the cause of leukemia in a
number of individual cases and case series reported worldwide between
1928 and 1976.29 Case-control studies indicated increased risks of
nonlymphocytic leukemia among workers in Sweden exposed to
petroleum products containing benzene and of lymphomas among
workers in New York State who were exposed to benzene. Prospective
studies conducted in the rubber industry provide the most convincing
evidence for an association between benzene exposure and leukemia.
Most of the excess leukemia in this industry is found among rubber
workers exposed to solvents, including benzene.
Aflatoxins
The hepatotoxic effect of aflatoxins was first recognized when aflatoxin-
contaminated feed was inadvertently fed to poultry. Subsequent animal
studies demonstrated the carcinogenic potential of the aflatoxins,
particularly aflatoxin B1. The aflatoxins are produced by the fungal
strains Aspergillus flavus and Aspergillus parasiticus. Grains and foodstuffs
for human consumption such as corn, peanuts, and rice can become
148 Part I: Science and Clinical Oncology

contaminated with aflatoxin during growth or storage. The considerable
variation in levels of human exposure to aflatoxin worldwide is
determined by climate and by the preventive measures used to protect
susceptible foods from mold contamination and growth.81
Dietary aflatoxin is correlated with high liver cancer rates in
sub-Saharan Africa, the Philippines, Mozambique, and Asia. The
cocarcinogenic role of HBV infection and dietary aflatoxin in liver
cancer has been the focus of several studies. In a prospective study
conducted in Guangxi Province, China, the incidence of liver cancer
in regions of high and low aflatoxin contamination were compared,
and HBV infection status was determined.82 A strong interaction
between aflatoxin exposure and HBV-positive status was observed
for relative risk of liver cancer. Among HBV-positive individuals, the
incidence of liver cancer was 649 per 100,000 in the high-aflatoxin
region and 66 per 100,000 in the low-aflatoxin region, whereas
among HBV-negative individuals, the incidence of liver cancer was
99 per 100,000 and less than 1 per 100,000 in high- or low-aflatoxin
regions, respectively. The association of urinary aflatoxin-DNA adducts
with risk of liver cancer also has been demonstrated in a prospective
epidemiologic study.45
Tobacco Chemicals
Tobacco use causes more cancer deaths worldwide than any other
human activity. Cigarette smoking is associated with cancers of the
lung, oral cavity, pharynx, larynx, esophagus, bladder, renal pelvis,
and pancreas (see Box 10.1). The use of smokeless tobacco (chewing
tobacco or snuff ) leads to cancer of the oral cavity, indicating that
although combustion enhances the carcinogenic properties of tobacco,
it is not required for cancer induction.
Although the carcinogenic properties of tobacco tar were first
demonstrated experimentally during the 1920s, evidence of a human
cancer risk from the use of tobacco did not appear until 1939, when
Muller and colleagues, as reviewed by Doll,26 reported an association
between tobacco use and lung carcinoma in Germany. Subsequent
epidemiologic studies conducted in the United States and the United
Kingdom confirmed this causal relationship but were met with consider-
able societal resistance because tobacco use was considered a pleasurable
personal habit. At that time the addictive characteristics of nicotine, a
major constituent of tobacco, was unrecognized. Societal acceptance
of a causal association with lung cancer was advanced by the first reports
from the Royal College of Physicians in the United Kingdom (1962)
and from the Surgeon General in the United States (1964) regarding
the risks of tobacco use. By contrast, the tobacco industry has steadfastly
resisted attempts to educate the public regarding the health hazards of
tobacco use and has continued to market cigarettes aggressively, par-
ticularly in developing countries. Epidemic increases in the incidence
of lung cancer can be anticipated in countries where tobacco use is
surging. It is estimated that more than 1 million new cases per year of
lung cancer will occur in China in the 21st century.26–28,81,83
More than 3000 chemicals have been identified in cigarette smoke,
of which at least 30 are known to be carcinogenic in animals (Table
10.2). The gas phase of tobacco smoke contains several carcinogenic
or tumor-promoting compounds, including dimethylnitrosamine,
dialkylnitrosamines, vinyl chloride, acrolein, and benzene. The

Table 10.2  Potential Carcinogenic Agents in Tobacco Smoke

Mainstream Smoke Mainstream Smoke
Compounds Compounds (Per Cigarette) Compounds Compounds (Per Cigarette)
POLYCYCLIC AROMATIC HYDROCARBONS 4-(Methylnitrosamine)-1- 0.08–0.77 µg
Benzo(a)anthracene 20–70 ng (3-pyridyl)-1-butanone
Benzo(b)fluoranthene 4–22 ng N-nitrosoanabasine 0.14–4.6 µg
Benzo(f)fluoranthene 6–21 ng AROMATIC AMINES
Benzo(k)fluoranthene 6–12 ng 2-Toluidine 30–200 ng
Benzo(a)pyrene 20–40 ng 2-Naphthylamine 1–22 ng
Chrysene 40–60 ng 4-Aminobiphenyl 2–5 ng
Dibenz(a,h)anthracene 4 ng ALDEHYDES
Dibenzo(a,i)pyrene 1.7–3.2 ng
Formaldehyde 70–100 µg
Dibenzo(a-1)pyrene Detectable
Acetaldehyde 18–1400 ng
Indenol(1,2,3-c,d)pyrene 4–20 ng
Crotonaldehyde 10–20 µg
5-Methylchrysene 0.6 ng
MISCELLANEOUS ORGANIC COMPOUNDS
AZA-ARENES
Benzene 12–48 µg
Quinoline 1–2 µg
Acrylonitrile 3.2–15 µg
Dibenz(a,h)acridine 0.1 µg
2-Nitropropane 0.73–1.21 µg
Dibenz(a,j)acridine 3–10 ng
Ethylcarbamate 20–38 ng
7H-dibenzo(c,g)carbazole 0.7 ng
Vinyl chloride 1–16 ng
N-NITROSAMINES INORGANIC COMPOUNDS
N-nitrosodimethylamine 0.1–180 ng
Hydrazine 24–43 ng
N-nitrosoethylmethylamine 3–13 ng
Arsenic 40–120 ng
N-nitrosodiethylamine 0–25 ng
Nickel 0–600 ng
N-nitrosopyrrolidine 1.5–110 ng
Chromium 4–70 ng
N-nitrosodiethanolamine 0–36 ng
Cadmium 41–62 ng
N-nitrosonornicotine 0.12–2.7 µg
Polonium-210 0.03–1.0 pCi

From U.S. Department of Health and Human Services. Reducing the Health Consequences of Smoking: 25 Years of Progress. Washington, DC: US Department of Health and
Human Services; 1989.

particulate phase contains carcinogenic and cocarcinogenic PAHs,
methylated PAHs, heterocyclic hydrocarbons, chlorinated hydrocarbons,
phenols, catechols, and metals. There is a strong interactive effect
observed when certain mixtures of these compounds are assayed for
carcinogenic potential.
Secondhand smoke contains many of the same carcinogens
that are inhaled by smokers. It is now known that secondhand
smoke causes lung cancer in adults who do not themselves smoke.13
Nonsmokers exposed to secondhand smoke at home or work are at
a 20% to 30% increased risk for the development of lung cancer.28
Chemotherapeutic Agents
The systemic toxicity of sulfur mustard gas among soldiers exposed
during World War I led to investigations of the cytotoxic MOA of
nitrogen mustard compounds and subsequent use as antineoplastic
drugs in the 1940s. Other types of drugs were also developed at that
time for use in the treatment of cancer, including the antibiotic
actinomycin A and the antimetabolite methotrexate. In later decades
a variety of alkylating agents were introduced (e.g., chlorambucil,
cyclophosphamide, bis-chloroethylnitrosourea, busulfan, and cisplatin),
along with antimetabolites (e.g., 5-fluorouracil and 6-mercaptopurine),
antibiotics (e.g., Adriamycin, bleomycin, and daunomycin), and mitotic
inhibitors (e.g., vincristine and vinblastine) as antineoplastics.
As early as 1948, the carcinogenic properties of the anticancer
drug 4-aminostilbene and its metabolites were reported, leading to the
institution of carcinogenesis bioassays for new anticancer drugs under
development by the National Cancer Institute. The appearance of
frank second malignancies among patients treated with chemotherapy
was reported during the 1970s. The successful treatment of Hodgkin
disease with multiagent chemotherapy is associated with the long-term
complication of acute myeloid leukemia (AML) and non-Hodgkin
lymphoma. Increased risk of AML among patients treated for non-
Hodgkin lymphoma, ovarian cancer, multiple myeloma, or small
cell carcinoma of the lung also has been attributed to antineoplastic
therapy. The risk of AML is most strongly associated with the alkyl-
ating antineoplastics—particularly cyclophosphamide, melphalan,
busulfan, treosulfan, and semustine (methyl-CCNU)—or with
combination chemotherapies that include alkylating agents. Ovarian
cancer patients receiving chemotherapy alone (including melphalan,
thiotepa, chlorambucil, cyclophosphamide, Adriamycin, cisplatinum,
alone or in combination) had a relative risk of 12.0 for leukemia
compared with patients treated with surgery alone (reviewed in De Vita
and Chu84).
Radiation Carcinogenesis
Ultraviolet Radiation
Solar ultraviolet (UV) radiation is the major physical carcinogen in
our environment and the primary cause of skin cancer in humans.
New basal cell carcinoma or squamous cell carcinoma of the skin will
develop in more than 800,000 persons each year in the United States,
making nonmelanoma skin cancer the most common cancer.7 The
incidence of both nonmelanoma and melanoma skin cancer among
light-skinned individuals is increasing at a rate of 3% to 5% per year
in the United States. This increase has been attributed to changing
lifestyle and leisure habits during the past five decades, resulting in
an increase in the number of people receiving greater exposure to
sunlight.
Geography plays a role in the incidence of nonmelanoma skin
cancer, which shows a generally increasing trend with decreasing latitude
among persons with similar skin types.7 Nonmelanoma skin cancers
and the premalignant skin neoplasm actinic keratosis, particularly on
solar-exposed skin areas, are also associated with cumulative lifetime
UV exposure estimated from outdoor occupations and activities. Light
skin complexion, ease of sun burning (skin type), and light hair color
are known to enhance the risk of nonmelanoma skin cancer, whereas
Environmental Factors  •  CHAPTER 10 149
pigmentation of the skin, either constitutive or induced (as in tanning),
plays an important role in protecting skin from the carcinogenic
effects of UV radiation.
Increased levels of DNA photodamage are detected in the normal
epidermis of individuals after exposure to solar UV radiation, and
analysis of mutational spectra in human nonmelanoma skin cancer
DNA shows that mutations specific for UV radiation (dipyrimidine
mutations) often are present. Additional potential contributing effects
include immune suppression and the molecular and cellular conse-
quences of chronic tissue damage and repair. Animal studies confirm
the carcinogenic effects of UV radiation and indicate that the ultraviolet
B (UVB) portion (280 to 320 nm) of the solar spectrum is primarily
responsible for the carcinogenic properties of sunlight. This wave
band encompasses the long-wavelength end of the absorbance spectrum
of DNA and has been shown to cause mutations in mammalian cells
(reviewed in D’Orazio and colleagues85 and Shah and colleagues86).
Ionizing Radiation
The discovery and manipulation of ionizing radiation in the early
20th century led to detrimental health effects among many researchers.
Toxicity, radiation burns, and cancer were observed among handlers
of radioactive materials. The deaths of Marie Curie and Thomas Edison’s
assistant from cancer have been attributed to severe radiation exposure.
The use of radium in luminous paint during the 1930s led to a high
incidence of osteosarcoma among dial painters who inadvertently
ingested radium when shaping their brush tips with the tongue.1 By
the 1940s, an elevated incidence of leukemia was observed among
radiologists. After World War II, excessive rates of leukemia were
observed among atomic bomb survivors and among patients treated
with x-rays.
Epidemiologic studies of populations exposed to high doses of
ionizing radiation have indicated increased risks for a variety of cancers,
depending on the type of radiation and route of exposure (Table
10.3). Among atomic bomb blast survivors, excessive rates of leukemias
appeared within several years of exposure, whereas excessive rates of
cancers of the breast, lung, esophagus, thyroid, colon, bladder, and
ovary, as well as multiple myeloma, appeared only 20 to 25 years later.
In contrast, populations exposed to nuclear weapon fallout show only
an excessive risk of thyroid cancer due to radioactive iodine. Almost
all data on carcinogenic risks of ionizing radiation derive from extremely
high-exposure scenarios, making it difficult to extrapolate risk estimates
to low-dose or ambient exposure levels.
Heavy exposure to x-rays for diagnostic or therapeutic procedures
has been associated with increased risk of the following types of cancers:
leukemia after in utero exposure; breast cancer after repeated chest
exposure; leukemia, lung, stomach, and esophagus after spinal exposure;
and thyroid, skin, and neck after scalp or thymus exposure.5 The use
of cobalt-60 x-ray treatment for cervical cancer is associated with
leukemia and cancers of the stomach, rectum, bladder, vagina, buccal
cavity, nasopharynx, and lung (reviewed in Robertson and colleagues87).
Radon
Radon gas is encountered in hard rock mining for iron, tin, fluorspar,
and uranium. The radioactive decay of radon and its products produces
alpha particles. The earliest reports defining an association between
lung cancer and mining described the high rate of lung cancers among
uranium miners in the Schneeberg region of Czechoslovakia in the
late 19th century. Many potential causes were proposed, including
radon inhalation. Studies of lung cancer mortality among Colorado
uranium miners demonstrated dose-related increases in lung cancer
risk in miners with protracted exposure to radon. In addition, small
cell undifferentiated carcinomas predominated in miners with high
exposure to radon, in contrast to the typical distribution of pulmonary
cancer pathology in the general US population. An elevated risk of
lung cancer also has been reported for iron ore miners in England,
France, and Sweden; however, the proportion of risk attributable to
radon in these populations is more difficult to assess.
150 Part I: Science and Clinical Oncology
Table 10.3  Examples of Radiation-Induced Cancers
Sources of Exposure Exposure Circumstances
EXPLOSIONS OF NUCLEAR WEAPONS
Blast Atomic bombing survivors in Hiroshima and Nagasaki
Fallout Populations exposed through atmospheric testing,
including Marshall Islanders, veterans in the Pacific,
general population in Nevada, Utah
DIAGNOSTIC PROCEDURES
X-rays Children exposed in utero
Thorotrast Cerebral and limb angiography; of biliary passages
Fluoroscopic x-rays Monitoring of lung infections in patients with tuberculosis
THERAPEUTIC PROCEDURES
X-rays Postpartum mastitis
X-rays Ankylosing spondylitis
Cobalt-60 Treatment for cancer of the cervix
X-rays Treatment of benign head and neck conditions
Radium-224 Ankylosing spondylitis, bone tuberculosis
PROFESSIONAL EXPOSURES
X-rays Early radiologists
Radon Uranium, hard-rock miners
X-rays, γ-rays, neutrons Nuclear industry
Radium isotopes Radium dial painters
Modified from Higginson J, Muir CS, Munoz N. Human Cancer: Epidemiology and Environmental Causes. Cambridge Monographs on Cancer Research. Cambridge: Cambridge
University Press; 1992.
Analysis of lung cancer mortality and smoking in Colorado uranium
miners suggests a greater than additive mortality rate for cumulative
radon exposure and cumulative cigarette smoking. In other words,
the increased risk of lung cancer among miners compared with nonmin-
ers is larger when comparing smokers than when comparing nonsmok-
ers. Interesting to note, among atomic bomb survivors, cigarette
smoking and radiation exposure have an additive effect only for lung
cancer risk. This anomaly has been attributed to the different exposure
patterns experienced by atomic bomb survivors (acute) and uranium
miners (chronic) (reviewed in Robertson and colleagues87 and Jostes88).
Metals
Arsenic
Medicinal use of inorganic arsenic was associated with skin cancers
in the early 20th century. More recently, excessive rates of skin cancer
have been observed in populations exposed to drinking water con-
taminated with arsenic, whereas excessive rates of lung cancer have
been found in populations with occupational exposure to inorganic
arsenic compounds. An increased risk of lung cancer of 6- to 14-fold
was reported in gold miners in Rhodesia, where the ore contains
arsenic. Chronic arsenism also was prevalent among these miners.
Several studies in Japan, Sweden, and the United States have docu-
mented excessive rates of lung cancers among workers involved in
copper smelting. Inorganic arsenic is a byproduct of the smelting
process and also is used as a hardener. Two large retrospective studies
of copper smelters have shown that lung cancer mortality is related
to estimated arsenic exposure.
Another source of occupational and potential environmental
exposure is the manufacture and use of arsenical pesticides. An early
study of mortality among workers at a factory manufacturing arsenical
Cancer Types
Leukemia, breast, lung, thyroid, stomach, colon, multiple
myeloma, esophagus, ovary
Thyroid
Leukemia
Liver
Breast
Breast
Leukemia, lung, stomach, esophagus
Leukemia, stomach, rectum, bladder, vagina, female genital,
lung, buccal cavity, nasopharynx, esophagus
Thyroid, skin, central nervous system
Bone sarcoma
Skin, leukemia
Lung cancer
Multiple myeloma
Bone, head sarcoma
sheep dip in Wales found an excess of skin and lung cancers, particularly
among persons directly involved in the chemical processes. Case-control
studies in two US plants manufacturing arsenical pesticides found
arsenic dose–related increases in the risk of lung cancer. Reports of
skin and lung cancers among vineyard workers with exposure to arsenic
fungicides and pesticides appeared during the late 1950s. An autopsy
series of 82 vineyard workers exposed in Germany found 61 deaths
from cancer, including 44 respiratory tract cancers; many skin cancers
and Bowen disease also were reported.
The most common route of exposure to arsenic worldwide is through
drinking water, with estimates of more than 50 million people using
well water containing excess arsenic levels—20 million in Bangladesh
alone. Chronic arsenic exposure has been linked to cancer of the
bladder, lungs, skin, and kidney (reviewed in IARC Working Group
on the Evaluation of Carcinogenic Risks to Humans89 and Bustaffa
and colleagues90).
Nickel and Chromium
Carcinogenic associations for nickel and chromium are generally limited
to occupational inhalation exposure to either specific particulate forms
or acid mists of these metals. The nickel refining industry was established
in South Wales around 1900. During the subsequent 40 years, evidence
accumulated for increased rates of nasal cancer and, later, lung cancer
among workers in nickel refineries, particularly among process workers
in the refinery. Cancer risk for nickel began to decline when occupa-
tional safety measures were introduced in the industry from the 1930s
to 1950s.91,92
Several reports of lung cancer in chromate industry workers appeared
in Germany during the 1930s. Subsequent extensive epidemiologic
investigations examined this association in workers involved in chromate
production, chromate pigment manufacture, and chrome plating in

England and Baltimore. These studies suggest that inhalation of specific
forms of particulate hexavalent chromium compounds, or of acid
mists, were related to excessive rates of lung cancer. Experimental
investigations indicate that highly toxic or cytotoxic doses of hexavalent
chromium salts of chromium are genotoxic and carcinogenic. There
is evidence of a potential high-dose threshold effect related to the
high capacity of body fluids to reduce hexavalent chromium to trivalent
chromium. In contrast to hexavalent chromium, trivalent chromium
is a human essential element and is not carcinogenic (reviewed in
Nickens and colleagues14 and IARC Working Group on the Evaluation
of Carcinogenic Risks to Humans91).
Fibers
Asbestos
The appearance of lung cancer in patients exposed to asbestosis was
first reported in the 1930s. Early epidemiologic studies were limited by
various deficiencies in exposure assessment, but gradually studies showed
increased lung cancer risk in workers with potential exposure to asbestos
during mining, milling, manufacturing, insulating, and shipbuilding.
The association between asbestos exposure and mesothelioma of the
lung, a relatively rare cancer, was reported in the early 1960s and
was confirmed among insulation workers, asbestos manufacturing
workers, and other populations exposed through their occupations.
Further studies showed that the risk of mesothelioma was not limited
to the workers but extended to members of their household and to
residents living in the vicinity of asbestos-related industries. There is
also evidence of a synergistic effect of asbestos and smoking for lung
cancer (reviewed in O’Reilly and colleagues93).
DIETARY FACTORS IN HUMAN
CARCINOGENESIS
Most of the known human carcinogens discussed in the foregoing
sections have been identified from occupational and iatrogenic exposures
at high doses to small cohorts; however, with the exception of tobacco,
such agents are rather minor contributors to the current overall cancer
burden. This fuels an overarching assumption that most human cancers
may result from interactions of several or more carcinogenic influ-
ences, with many modifying factors, the most significant thought to
be diet and other lifestyle habits. Many epidemiologic studies have
indicated that general increases in consumption of fiber-rich cereals,
fruits, and vegetables and decreased consumption of fat-rich foods and
excessive alcohol will serve as prudent approaches to reducing overall
cancer risk.
Two major dietary influences are thought be fat and calorie con-
sumption. Fat consumption is most strongly associated with the
hormone-dependent cancers (i.e., breast [see Box 10.2], ovary, and
endometrium in women and prostate in men) and gastrointestinal
cancers (i.e., gallbladder, colon, and rectum in both sexes). It is not
clear to what degree these relationships are causal or if they relate to
the type of fat (saturated, unsaturated, polyunsaturated) or the overall
caloric content of the diet. Fats can promote tumor development
directly and also are a major source of calories. Animals fed high-fat
diets consistently demonstrate enhanced tumorigenic outcomes.
Conversely, it has been recognized for decades that caloric restriction
has a very powerful, general inhibitory effect on carcinogenesis in
many induced and spontaneous laboratory animal tumor models. The
human behavioral changes required to effect comparable population-
wide reductions in fat and/or calorie consumption, however, pose
formidable challenges.
The increasing prevalence of overweight and obese individuals
worldwide, combined with mounting evidence for a clear association
between obesity and cancer risk, presents a major public health concern.
Analysis has indicated that more than 900 million adults are overweight
and almost 400 million are obese (BMI ≥30 kg/m2).16 In addition to
the noncancer health risks associated with obesity, current evidence
Environmental Factors  •  CHAPTER 10 151
suggests that overall cancer mortality increases by 10% for each 5 kg/
m2 increase in BMI. The IARC concluded that the existing evidence
base supports an association between obesity and cancers of the liver,
colon, postmenopausal breast, endometrium, kidney, and esophagus.36
A recent meta-analysis of prospective studies found strong associations
between increased BMI and esophageal adenocarcinoma, thyroid cancer,
kidney cancer, and colon cancer among men and associations between
increased BMI and endometrial cancer, gallbladder cancer, kidney
cancer, and esophageal cancer among women.
The mechanism by which obesity increases cancer risk is unclear
at this time, but many hypotheses have been advanced, including
increased storage and release of lipophilic carcinogens in and from
fatty tissue; lack of excretion of carcinogens due to decreased physical
activity; and altered gene expression related to inflammation or disrup-
tion in hormonal homeostasis and energy balance. A molecular link
has been found between cholesterol metabolism and breast cancer
risk, related to increased levels of circulating estrogenic metabolites.94–97
The risk of colon cancer is strongly associated with consumption
of red meat and animal fat. This association has been observed in
several international correlative studies and case-control studies. Prospec-
tive studies of colon cancer risk demonstrate an association with
consumption of red meat and animal fat (but not with vegetable fat),
independent of total energy intake. The increased risk associated with
animal fat primarily is due to meat intake as opposed to dairy product
intake. Other studies that have addressed cooking practices have found
that consumption of fried foods and barbecued, broiled, or smoked
meats is associated with an increased risk of colorectal cancer. Several
hypotheses have been proposed to explain the strong association of
colon cancer risk with red meat and animal fat. Diets high in meat
fat increase the incidence of colon cancers in rats and also increase
the excretion of primary bile acids, which are converted to secondary
bile acids by bacterial metabolism. Some secondary bile acids (e.g.,
deoxycholic and lithocholic acid) are colon tumor promoters in animal
models. Moreover, the cooking of meat, particularly by broiling or
frying and pyrolysis of amino acids and proteins, produces at least
two major classes of carcinogens—PAHs and heterocyclic aromatic
amines—that are capable of producing genotoxic and mutagenic
damage. Heterocyclic amines, including quinolines, quinoxalines,
pyridines, and carbolines, cause colon cancer, mammary cancer, liver
cancer, prostate cancer, and lymphoma in experimental animals.
Case-control studies of heterocyclic amine intake have found an
increased risk for colon cancer among those who had the highest
levels of heterocyclic amine intake.98–100
Numerous studies have identified many other dietary components
as having either carcinogenic or opposing anticarcinogenic potential.23
Such food-derived anticarcinogens consist of both nutrient (e.g.,
vitamins and minerals) and nonnutrient components, many of which
function as antioxidants. Inverse epidemiologic associations between
risk of cancer at several sites and ingestion of fruits and vegetables
have been observed.24 These associations might be linked to β-carotene,
other carotenoids, folate, fiber, vitamin C, and other antioxidants,
and to other components of the fruits and vegetables. A deeper
understanding of the role of dietary factors in human carcinogenesis,
coupled with effective behavioral adaptations, will likely have a sig-
nificant impact on disease incidence.49,101
GENETIC POLYMORPHISMS AND
HUMAN SUSCEPTIBILITY
Another major area of research in molecular cancer epidemiology is
the study of individual and population-wide genetic polymorphisms
that affect cancer susceptibility, particularly as they affect metabolic
and DNA repair.102 Many cytochrome P450 metabolic enzymes are
known to be involved in the activation of specific human carcinogens,
and some have been linked to increased cancer risk. Inducible CYP1A1
activity is higher in cultured lymphocytes from persons with lung
cancer than in control subjects. Genetic polymorphisms in the CYP1A1
152 Part I: Science and Clinical Oncology
structural gene have been associated with lung cancer risk in Asian
populations. Hepatic arylamine N-acetyltransferase correlates with
individual differences in susceptibility to bladder cancer in humans.28
A series of genetic polymorphisms of this enzyme exists in humans,
resulting in slow- and rapid-acetylator phenotypes. The rapid-acetylator
phenotype seems to protect aromatic amine-exposed individuals from
bladder cancer. These results are attributed to the competition of
N-acetylation of arylamines against the formation of reactive arylamine
metabolites that reach the bladder.
An important mechanism that protects the genome from genotoxic
effects of carcinogens is the repair of cellular DNA. The rare inherited
disorder xeroderma pigmentosum (XP) is characterized by various
levels of DNA repair deficiencies, and persons with XP show an
unusually high incidence of multiple skin cancers and rarely live beyond
early adulthood in the absence of protective measures against sunlight.103
The median age at onset for nonmelanoma skin cancers among persons
with XP is approximately 8 years, compared with about 60 years in
the general population.
Several other inherited diseases show altered cellular response to
DNA damage. Ataxia telangiectasia, Bloom syndrome, and Fanconi
anemia are autosomal-recessive genetic disorders characterized by
chromosomal instability, with malignancies developing in patients
more frequently and at a younger age than occurs in the general
population. Heterozygous relatives of persons with ataxia telangiectasia
and those with Fanconi anemia also are at moderately increased risk
of cancer compared with unrelated persons.104
Although these are rare diseases, they suggest that moderate or
even small deficiencies in DNA repair may affect susceptibility to
cancer, and many studies have documented significant variability in
DNA repair proficiency among disease-free individuals.
PUBLIC HEALTH APPROACHES TO CANCER
PREVENTION AND INTERCEPTION
The optimal way for dealing with virtually all diseases, including
cancer, is prevention. Thus the challenge to public health professionals
is to devise and implement preventive measures against cancer. As
presented in Fig. 10.1, the prevention of cancer can take several forms.
Primary prevention targets healthy persons and can be achieved by
avoiding exposure to risk factors. Another approach is to stimulate
the defense mechanisms of the host to interfere with the carcinogenic
process. Secondary prevention measures use early detection and
intervention (cancer interception) before pathologic conditions are
clinically apparent. The success of these strategies will be greatly
facilitated by exposome approaches (see earlier) to the development
of noninvasive biomarkers that identify disease-relevant exposures, as
well as high-risk individuals. Finally, tertiary prevention is aimed at
minimizing the effect of existing disease and consequent disability by
avoiding development of new cancers and complications or relapses
after therapy for initial malignancies.
The use of chemical or dietary interventions to alter the susceptibility
of humans to the actions of carcinogens and to retard, block, or reverse
carcinogenesis can be applied to all levels of prevention and has been
termed chemoprevention. These strategies have proved to be extremely
effective in laboratory animals, but increasing evidence indicates that
chemoprevention against cancer also works in humans. The field of
cancer chemoprevention did not receive significant attention until
the early 1970s, when Wattenberg demonstrated that dietary antioxi-
dants could protect against tumor formation. The experimental
observations that seemingly innocuous preservatives found in the
Western diet could dramatically protect against diverse carcinogens
at distal sites sparked the development of chemoprevention as a viable
strategy for the reduction of human cancers. More than 20 classes of
discrete chemicals have been shown to be effective inhibitors of
experimental carcinogenesis. Investigations have established significant
site- and agent-specific effects in the prevention of invasive skin, upper
aerodigestive tract, colorectal, and breast cancers in high-risk groups.
Cancer chemoprevention could be especially valuable in populations
at high risk of certain neoplasms, particularly as improved molecular
techniques allow for the identification of such high-risk individuals.
Preventive strategies can involve interventions with specific nutrients,
nonnutrients, and antiinflammatory drugs in select populations at
high risk of cancer, and lifestyle changes that include altered nutrition
and social habits.105–108 Finally, increased attention is currently being
applied to cancer “interception,” which is focused on identifying the
earliest stages of carcinogenesis and applying therapeutic interventions
to prevent that early cancer from progressing.109
SUMMARY
A key paradigm in environmental carcinogenesis—gene-environment
interactions, a concept used to describe the complex interplay between
individual or population genetics and responses to chemical agents—has
also undergone an expansive transformation into a more contemporary
understanding of the human exposome. The exposome is the cumulative
lifelong burden of disease-contributing stressors including exogenous
and endogenous agents of all types, in addition to an individual’s
“omics” profile (genomics, epigenomics, transcriptomics, proteomics,
metabolomics) and his or her microbiome. A classic sequential model
of initiation, promotion, conversion, and progression has increasingly
evolved into less linear models that no longer classify carcinogens as
initiators or promoters per se. It may be more important to understand
the contribution of chemical agents to the various hallmarks of cancer:
genomic instability, resisting cell death, deregulating cellular energetics,
sustaining proliferative signals, evading growth suppressors, avoiding
immune destruction, enabling replicative immortality, promoting
tumor inflammation, activating invasion and metastasis, and inducing
angiogenesis.
A traditional paradigm has classified carcinogenic agents as envi-
ronmental or lifestyle related or occupational in origin, but these
classifications are blurred by many examples of complex interplay
among all three types of exposure. Carcinogen identification has
evolved from historic observational studies, to occupational cohort
epidemiology, to broad-scale screening and testing in experimental
animals. Next-generation approaches to carcinogen identification use
exposome approaches of integrating sophisticated individual exposure
monitoring and biosensing with highly sensitive “omics” approaches
to detect early perturbation of disease-relevant signaling pathways.
Most human cancers probably result from the interaction of several
carcinogenic influences (often unidentified, but many dietary or lifestyle
related in origin) along with intrinsic factors (e.g., inherited genes,
hormones, inflammation and oxidative stress, immune status, energy
balance).
Although the causes of most individual human cancers are not
identifiable, there is incontrovertible evidence that certain chemical
agents, radiation, and certain biologic agents are contributors to the
overall incidence of human cancer. Consumption of tobacco products,
especially cigarette smoking, is responsible for nearly 30% of all cancers.
Chemical carcinogens include aromatic amines, benzene, aflatoxins,
tobacco chemicals, and chemotherapeutic agents. Metal carcinogens
include specific forms of arsenic, nickel, and hexavalent chromium,
largely associated with occupational exposures. Radiation carcinogens
include UV radiation, ionizing radiation, and radon. Fibers such as
asbestos and certain dusts are etiologic agents in lung cancers and
mesothelioma. Many components in the diet can influence the
development of cancer through carcinogenic or anticarcinogenic
mechanisms.
The identification of molecular biologic markers of exposure,
effect, and susceptibility (reflecting early events that link exposure
to adverse outcome, but before clinical disease) will help further our
understanding of human carcinogenesis. In addition to genetic poly-
morphisms indicative of susceptibility, biomarkers of disease-relevant
exposure will increasingly rely on highly sensitive, multiplatform, and
multidimensional analysis of the impact of potential carcinogenic

agents on epigenomics, transcriptomics, proteomics, metabolomics,
and the microbiome. Primary, secondary, and tertiary cancer preven-
tion approaches will be greatly facilitated by the development of
noninvasive biomarkers that identify high-risk individuals, and by
identification of the disease-consequential perturbations in molecular
KEY REFERENCES
1. Doll R, Peto R. The causes of cancer: quantitative
estimates of avoidable risks of cancer in the United
States today. J Natl Cancer Inst. 1981;66:1191–1308.
3. Hunter DJ. Gene-environment interactions in
human diseases. Nat Rev Genet. 2005;6:287–298.
4. Wild CP. Complementing the genome with an
“exposome”: the outstanding challenge of envi-
ronmental exposure measurement in molecular
epidemiology. Cancer Epidemiol Biomarkers Prev.
2005;14:1847–1850.
5. Rappaport SM. Implications of the exposome for
exposure science. J Exp Sci Environ Epidemiol.
2011;21:5–9.
7. Weiss RA. Multistage carcinogenesis. Br J Cancer.
2004;91:1981–1982.
8. Chappell G, Pogribny IP, Guyton KZ, Rusyn
I. Epigenetic alterations induced by genotoxic
occupational and environmental human chemical
carcinogens: a systematic review. Mutat Res Rev Mutat
Res. 2016;768:27–45.
9. Shkreta L, Bell B, Revil T, Venables JP, Prinos P,
Elela SA, et al. Cancer-associated perturbations in
alternative pre-messenger RNA splicing. Cancer Treat
Res. 2013;158:41–94.
10. Reed JC. Dysregulation of apoptosis in cancer.
J Clin Oncol. 1999;17:2941053.
13. Hanahan D, Weinberg RA. Hallmarks of cancer:
the next generation. Cell. 2011;144:646–674.
14. Nickens KP, Patierno SR, Ceryak S. Chromium
genotoxicity: a double-edged sword. Chem Biol
Interact. 2010;188:276–288.
15. Liu X, He Y, Li F, Huang Q, Kato TA, Hall RP,
et al. Redefining the roles of apoptotic factors in
carcinogenesis. Mol Cell Oncol. 2015;3:e1054550.
16. Pritchard DE, Ceryak S, Ramsey KE, O’Brien TJ,
Ha J, Fornsaglio JL, et al. Resistance to apoptosis,
increased growth potential, and altered gene
expression in cell that survived genotoxic hexavalent
chromium exposure. Mol Cell Biochem. 2005;279:
169–181.
18. Carnero A, et al. Disruptive chemicals, senescence
and immortality. Carcinogenesis. 2015;36:S19–S37.
19. Goodson WH 3rd, et al. Assessing the carcinogenic
potential of low-dose exposures to chemical mixtures
in the environment: the challenge ahead. Carcino-
genesis. 2015;36:S254–S296.
20. Narayanan KB, et al. Disruptive environmental
chemicals and cellular mechanisms that confer
resistance to cell death. Carcinogenesis. 2015;36(suppl
1):S89–S110.
21. Hu Z, et al. Assessing the carcinogenic potential
of low-dose exposure to chemical mixtures in the
environment: focus on the cancer hallmark of
tumor angiogenesis. Carcinogenesis. 2015;36(suppl
1):S184–S202.
signaling that may also be targets for chemoprevention or cancer
interception.
The complete reference list is available online at
ExpertConsult.com.
22. Casey SC, et al. The effect of environmental chemi-
cal on tumor microenvironment. Carcinogenesis.
2015;36(suppl 1):S160–S183.
23. Thompson PA. Environmental immune disrup-
tors, inflammation and cancer risk. Carcinogenesis.
2015;36(suppl 1):S232–S253.
24. Robey RB, et al. Metabolic reprogramming and dysreg-
ulated metabolism: cause, consequence and/or enabler
of environmental carcinogenesis? Carcinogenesis.
2015;36(suppl):S203–S231.
25. Langie SA, et al. Causes of genomic instability: the
effect of low dose chemical exposures in modern
society. Carcinogenesis. 2015;36(suppl):S61–S88.
26. Doll R. Uncovering the effects of smoking: historical
perspective. Stat Methods Med Res. 1998;7:87–117.
28. Centers for Disease Control and Prevention. Smoking-
attributable mortality, years of potential life lost,
and productivity losses—United States, 2000–2004.
MMWR Morb Mortal Wkly Rep. 2008;57(45):
1226–1228.
29. Alderson M. Occupational Cancer. London:
Butterworth; 1986.
33. Higginson J, Muir CS, Munoz N. Human Cancer:
Epidemiology and Environmental Causes. Cambridge
Monographs on Cancer Research. Cambridge:
Cambridge University Press; 1992.
36. International Agency for Research on Cancer. IARC
Monographs on the Evaluation of Carcinogenic Risk
to Humans. Vol. 1–104. Lyon, France: IARC Press;
1970–2012.
37. Rothman N, Hainaut P, Schulte PA, eds. Molecular
Epidemiology: Principles and Practices. IARC Sci Publ
No. 163. Lyon: IARC Press; 2012.
38. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer
statistics, 2002. CA Cancer J Clin. 2005;55:74–108.
40. Munoz N, Day N. Esophageal Cancer. In:
Schottenfeld D, Fraumeni JF, eds. Cancer Epidemiol-
ogy and Prevention. New York: Oxford University
Press; 1996:681–706.
42. Institute of Medicine. Breast Cancer and the Environ-
ment: A Life Course Approach. Washington, DC:
Institute of Medicine; 2011.
49. World Cancer Research Fund/American Institute for
Cancer Research. Food, Nutrition and the Prevention
of Cancer: A Global Perspective. Washington, DC:
AICR; 1997.
50. Escher BI, et al. From the exposome to a mechanistic
understanding of chemical-induced adverse effects.
Environ Int. 2017;99:97–106.
51. Miller MF, et al. Low-dose mixture hypothesis of
carcinogenesis workshop: scientific underpinning and
research recommendations. Environ Health Perspect.
2017;125(2):163–169.
62. Knower KC, To SQ, Leung YK, Ho SM, Clyne CD.
Endocrine disruption of the epigenome: a breast
Environmental Factors  •  CHAPTER 10 153
cancer link. Endocr Relat Cancer. 2014;21:T33–
T55.
71. Hsu PC, Lan RS, Brasky TM, Marian C,
Cheema AK, Ressom HW, et al. Metabolomic
profiles of current cigarette smokers. Mol Carcinog.
2017;56(2):594–606. doi:10.1002/mc.22519. [Epub
2016 Aug 22].
72. Pope JL, Tomkovich S, Yang Y, Jobin C. Microbiota
as a mediator of cancer progression and therapy.
Transl Res. 2017;179:139–154.
73. Spor A, Koren O, Ley R. Unravelling the effects
of the environment and host genotype on the gut
microbiome. Nat Rev Microbiol. 2011;9:279–290.
77. Pevsner-Fischer M, Tuganbaev T, Meijer M, et al.
Role of the microbiome in non-gastrointestinal
cancers. World J Clin Oncol. 2016;73:200–213.
78. Schwabe RF, Jobin C. The microbiome and cancer.
Nat Rev Cancer. 2013;13:800–812.
82. Kensler T, Qian GS, Chen JG, Groopman JD.
Translational strategies for cancer prevention in
liver. Nat Rev Cancer. 2003;3:321–329.
83. World Health Organization. WHO Report on the
Global Tobacco Epidemic, 2011: Warning About the
Dangers of Tobacco. Geneva: World Health Organiza-
tion; 2011.
84. DeVita VT Jr, Chu E. A history of cancer chemo-
therapy. Cancer Res. 2008;21:8643–8653.
85. D’Orazio J, Jarrett S, Amaro-Ortiz A, Scott
T. UV Radiation and the Skin. Int J Mol Sci.
2013;14:12222–12248.
86. Shah DJ, Sachs RK, Wilson DJ. Radiation-induced
cancer: a modern view. Br J Radiol. 2012;85:
1166–1173.
87. Robertson A, Allen J, Laney R, Curnow A. The
cellular and molecular carcinogenic effects of radon
exposure: a review. Int J Mol Sci. 2013;14:14024–
14063.
89. IARC Working Group on the Evaluation of
Carcinogenic Risks to Humans. Arsenic, metals,
fibres and dusts. IARC Monogr Eval Carcinog Risks
Hum. 2012;100:11–465.
91. IARC Working Group on the Evaluation of Carci-
nogenic Risks to Humans. Chromium, nickel and
welding. IARC Monogr Eval Carcinog Risks Hum.
1990;49:1–648.
94. Basen-Engquit K, Chang M. Obesity and cancer
risk: recent review and evidence. Curr Oncol Rep.
2011;13:71–76.
101. Ames BN. Dietary carcinogens and anticarcinogens.
Science. 1983;221:1256–1264.
102. Vineis P, ed. Metabolic Polymorphisms and Susceptibil-
ity in Cancer. IARC Sci Publ No. 148. Lyon: IARC
Press; 1999.
109. Blackburn EH. Cancer Interception. Cancer Prev
Res (Phila). 2011;4:787–792.

REFERENCES
1. Doll R, Peto R. The causes of cancer: quantitative
estimates of avoidable risks of cancer in the United
States today. J Natl Cancer Inst. 1981;66:1191–1308.
2. Arnold M, Leitzmann M, Freisling H, Bray F,
Romieu I, Renehan A, et al. Obesity and cancer:
an update of the global impact. Cancer Epidemiol.
2016;41:8–15.
3. Hunter DJ. Gene-environment interactions in
human diseases. Nat Rev Genet. 2005;6:287–298.
4. Wild CP. Complementing the genome with an “expo-
some”: the outstanding challenge of environmental
exposure measurement in molecular epidemiology.
Cancer Epidemiol Biomarkers Prev. 2005;14:
1847–1850.
5. Rappaport SM. Implications of the exposome for
exposure science. J Exp Sci Environ Epidemiol.
2011;21:5–9.
6. Wild CP, Scalbert A, Herceg Z. Measuring the expo-
some: a powerful basis for evaluating environmental
exposures and cancer risk. Environ Mol Mutagen.
2013;54:480–499.
7. Weiss RA. Multistage carcinogenesis. Br J Cancer.
2004;91:1981–1982.
8. Chappell G, Pogribny IP, Guyton KZ, Rusyn
I. Epigenetic alterations induced by genotoxic
occupational and environmental human chemical
carcinogens: a systematic review. Mutat Res Rev Mutat
Res. 2016;768:27–45.
9. Shkreta L, Bell B, Revil T, Venables JP, Prinos P,
Elela SA, et al. Cancer-associated perturbations in
alternative pre-messenger RNA splicing. Cancer Treat
Res. 2013;158:41–94.
10. Reed JC. Dysregulation of apoptosis in cancer.
J Clin Oncol. 1999;17:2941053.
11. Tang D, Lotze MT, Kang R, Zeh HJ. Apop-
tosis promotes early tumorigenesis. Oncogene.
2011;30:1851–1854.
12. Hanahan D, Weinberg RA. Hallmarks of cancer.
Cell. 2000;100:57–70.
13. Hanahan D, Weinberg RA. Hallmarks of cancer:
the next generation. Cell. 2011;144:646–674.
14. Nickens KP, Patierno SR, Ceryak S. Chromium
genotoxicity: a double-edged sword. Chem Biol
Interact. 2010;188:276–288.
15. Liu X, He Y, Li F, Huang Q, Kato TA, Hall RP,
et al. Redefining the roles of apoptotic factors in
carcinogenesis. Mol Cell Oncol. 2015;3:e1054550.
16. Pritchard DE, Ceryak S, Ramsey KE, O’Brien
TJ, Ha J, Fornsaglio JL, et al. Resistance to
apoptosis, increased growth potential, and altered
gene expression in cell that survived genotoxic
hexavalent chromium exposure. Mol Cell Biochem.
2005;279:169–181.
17. Mathew R, Karantza-Wadsworth V, White E.
Role of Autophagy in cancer. Nat Rev Cancer.
2007;7:961–967.
18. Carnero A, et al. Disruptive chemicals, senescence
and immortality. Carcinogenesis. 2015;36:S19–S37.
19. Goodson WH 3rd, et al. Assessing the carcinogenic
potential of low-dose exposures to chemical mixtures
in the environment: the challenge ahead. Carcino-
genesis. 2015;36:S254–S296.
20. Narayanan KB, et al. Disruptive environmental
chemicals and cellular mechanisms that confer resis-
tance to cell death. Carcinogenesis. 2015;36(suppl1):
S89–S110.
21. Hu Z, et al. Assessing the carcinogenic potential
of low-dose exposure to chemical mixtures in the
environment: focus on the cancer hallmark of
tumor angiogenesis. Carcinogenesis. 2015;36(suppl):
S184–S202.
22. Casey SC, et al. The effect of environmental chemi-
cal on tumor microenvironment. Carcinogenesis.
2015;36(suppl):S160–S183.
23. Thompson PA. Environmental immune disrup-
tors, inflammation and cancer risk. Carcinogenesis.
2015;36(suppl):S232–S253.
24. Robey RB, et al. Metabolic reprogramming and dysreg-
ulated metabolism: cause, consequence and/or enabler
of environmental carcinogenesis? Carcinogenesis.
2015;36(suppl):S203–S231.
25. Langie SA, et al. Causes of genomic instability: the
effect of low dose chemical exposures in modern
society. Carcinogenesis. 2015;36(suppl):S61–S88.
26. Doll R. Uncovering the effects of smoking: historical
perspective. Stat Methods Med Res. 1998;7:87–
117.
27. U.S. Department of Health and Human Services.
The Health Consequences of Involuntary Exposure
to Tobacco Smoke: A Report of the Surgeon General.
Atlanta: U.S. Department of Health and Human
Services, Centers for Disease Control and Preven-
tion, Coordinating Center for Health Promotion,
National Center for Chronic Disease Prevention and
Health Promotion, Office on Smoking and Health;
2006.
28. Centers for Disease Control and Prevention.
Smoking-attributable mortality, years of potential life
lost, and productivity losses—United States, 2000–
2004. MMWR Morb Mortal Wkly Rep. 2008;57(45):
1226–1228.
29. Alderson M. Occupational Cancer. London:
Butterworth; 1986.
30. Waldron HA. A brief history of scrotal cancer. Br
J Ind Med. 1983;40:390–401.
31. Dipple A, Moschel RC, Bigger CAH. Polynuclear
aromatic carcinogens. In: Searle CE, ed. Chemical
Carcinogens, Vol. 2 (ACS Monograph 182). Wash-
ington, DC: American Chemical Society; 1984:41.
32. Sontag JM. Carcinogens in Industry and the Environ-
ment. New York: Marcel Dekker; 1981.
33. Higginson J, Muir CS, Munoz N. Human Cancer:
Epidemiology and Environmental Causes. Cambridge
Monographs on Cancer Research. Cambridge:
Cambridge University Press; 1992.
34. Case RAM, Hosker ME, McDonald DB, Pearson JT.
Tumours of the urinary bladder in workers engaged
in the manufacture and use of certain dyestuff
intermediates in the British chemical industry.
Br J Prev Soc Med. 1954;11:75.
35. Fujiki H. Gist of Dr. Katsusaburo Yamagiwa’s papers
entitled “Experimental study on the pathogenesis
of epithelial tumors” (I to VI reports). Cancer Sci.
2014;105:143–149.
36. International Agency for Research on Cancer. IARC
Monographs on the Evaluation of Carcinogenic Risk
to Humans. Vol. 1–104. Lyon, France: IARC Press;
1970–2012.
37. Rothman N, Hainaut P, Schulte PA, eds. Molecular
Epidemiology: Principles and Practices. IARC Sci Publ
No. 163. Lyon: IARC Press; 2012.
38. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer
statistics, 2002. CA Cancer J Clin. 2005;55:74–108.
39. Islami F, Kamangar F, Aghcheli K, et al. Epidemio-
logic features of upper gastrointestinal tract cancers in
northeastern Iran. Br J Cancer. 2004;5:1402–1406.
40. Munoz N, Day N. Esophageal cancer. In:
Schottenfeld D, Fraumeni JF, eds. Cancer Epidemiol-
ogy and Prevention. New York: Oxford University
Press; 1996:681–706.
41. Tominaga S. Cancer incidence in Japanese in Japan,
Hawaii, and western United States. Natl Cancer Inst
Monogr. 1985;69:83–92.
42. Institute of Medicine. Breast Cancer and the Environ-
ment: A Life Course Approach. Washington, DC:
Institute of Medicine; 2011.
43. Winn DM. Science and society: the long island
breast cancer study project. Nat Rev Cancer. 2005;5:
986–994.
44. Fernandez SV, Russo J. Estrogen and xenoestrogens
in breast cancer. Toxicol Pathol. 2010;38:1–10.
45. Ross RK, Yuan JM, Yu MC, et al. Urinary aflatoxin
biomarkers and risk of hepatocellular carcinoma.
Lancet. 1992;339:943–946.
Environmental Factors  •  CHAPTER 10 153.e1
46. Groopman JD, Kensler TW. The light at the end of
the tunnel for chemical-specific biomarkers: daylight
or headlight? Carcinogenesis. 1999;20:1–11.
47. Strickland P, Kang D, Sithisarankul P. Polycyclic
aromatic hydrocarbon metabolites in urine as
biomarkers of exposure and effect. Environ Health
Perspect. 1996;104(suppl 5):927–932.
48. Huff J. Chemicals causally associated with cancers
in humans and in laboratory animals. In: Waalkes
MP, Ward JM, eds. Carcinogenesis. New York: Raven
Press; 1994.
49. World Cancer Research Fund/American Institute for
Cancer Research. Food, Nutrition and the Prevention
of Cancer: A Global Perspective. Washington, DC:
AICR; 1997.
50. Escher BI, et al. From the exposome to a mechanistic
understanding of chemical-induced adverse effects.
Environ Int. 2017;99:97–106.
51. Miller MF, et al. Low-dose mixture hypothesis of
carcinogenesis workshop: scientific underpinning and
research recommendations. Environ Health Perspect.
2017;125(2):163–169.
52. Brien GL, Valerio DG, Armstrong SA. Exploiting
the epigenome to control cancer-promoting gene-
expression programs. Cancer Cell. 2016;29:464–476.
53. Liu F, Hon GC, Villa GR, Turner KM, Ikegama S,
Yang H, et al. EGFR Mutation promotes glioblas-
toma through epigenome and transcription factor
network remodeling. Mol Cell. 2015;307–318.
54. Minaroits J, Demcsak A, Banati F, Niller HH. Epi-
genetic dysregulation in virus-associated neoplasm.
Adv Exp Med Biol. 2016;879:71–90.
55. Wang Q, Trevino LS, Wong RL, Medvedovic M,
Chen J, Ho SM, et al. Reprogramming of the
epigenome by MLL1 links environmental exposures
to prostate cancer risk. Mol Endocrinol. 2016;30:
856–871.
56. Teschendorff AE, Yang Z, Wong A, Pipinikas CP,
Jones A, Anjum S, et al. Correlation of smoking-
associated DNA methylation changes in buccal cells
with DNA methylation changes in epithelial cancer.
JAMA Oncol. 2015;1:476–485.
57. Zhang C, Su ZY, Wang L, Shu L, Yang Y, Guo Y,
et al. Epigenetic blockade of neoplastic transforma-
tion by bromodomain and extra-terminal (BET)
domain protein inhibitor JQ-1. Biochem Pharmacol.
2016;117:35–45.
58. Eckstein M, Eleazer R, Rea M, Fondufe-Mittendorf
Y. Epigenomic reprogramming in inorganic
arsenic-mediated gene expression patterns during
carcinogenesis. Rev Environ Health. 2016; Oct 4.
59. Sun H, Shamy M, Costa M. Nickel and epigenetic
silencing. Genes (Basel). 2013;25:583–595.
60. Li W, Guo Y, Zhang C, Wu R, Yang AY, Gaspar
J, et al. Dietary phytochemicals and cancer che-
moprevention: a perspective on oxidative stress,
inflammation and epigenetics. Chem Res Toxicol.
2016;29:2071–2095.
61. Trevino LS, Wang Q, Walker CL. Hypothesis: activa-
tion of rapid signaling by environmental estrogens
and epigenetic reprogramming in breast cancer.
Reprod Toxicol. 2015;54:136–140.
62. Knower KC, To SQ, Leung YK, Ho SM, Clyne CD.
Endocrine disruption of the epigenome: a breast
cancer link. Endocr Relat Cancer. 2014;21:T33–T55.
63. Sauer SJ, Tapley M, Shah M, Save AV, Lyerly HK,
Patierno SR, et al. Bisphenol A activates EGFR and
ERK promoting proliferation, tumor spheroid forma-
tion and resistance to EGFR pathway inhibition
in estrogen receptor-negative inflammatory breast
cancer cells. Carcinogenesis. 2017;38(3):252–260.
64. Tang XH, et al. Gene-expression profiling signatures
for the diagnosis and prevention of oral cavity car-
cinogenesis – genome-wide analysis using RNA-seq
technology. Oncotarget. 2015;6:24424–24435.
65. Souza T, et al. New insights into BaP-induced toxic-
ity: role of major metabolites in transcriptomics and
153.e2 Part I: Science and Clinical Oncology
contribution to hepatocarcinogenesis. Arch Toxicol.
2016;90:1449–1458.
66. Shi J, et al. Distinct response of the hepatic transcrip-
tome to Aflatoxin B1 hepatocellular carcinogenesis
and resistance in rats. Sci Rep. 2016;6:31898.
67. Cook JA, et al. Mass spectrometry–based metabo-
lomics identifies longitudinal urinary metabolite
profiles predictive of radiation-induced cancer.
Cancer Res. 2016;76:1569–1577.
68. Xie G, et al. Dysregulated hepatic bile acids
collaboratively promote liver carcinogenesis. Int
J Cancer. 2016;139(8):1764–1775. doi:10.1002/
ijc.30219. [Epub 2016 Jun 17].
69. Gu J, et al. Metabolomic analysis reveals altered
metabolic pathways in a rat model of gastric car-
cinogenesis. Oncotarget. 2016;7(37):60053–60073.
doi:10.18632/oncotarget.11049.
70. Kuligowski J, Sanjuan-Herráez D, Vázquez-Sánchez
MA, Brunet-Vega A, Pericay C, Ramírez-Lázaro MJ,
et al. Metabolomic Analysis of gastric cancer progres-
sion within the Correa’s cascade using ultraperfor-
mance liquid chromatography–mass spectrometry.
J Proteome Res. 2016;15(8):2729–2738. doi:10.1021/
acs.jproteome.6b00281. [Epub 2016 Jul 20].
71. Hsu PC, Lan RS, Brasky TM, Marian C,
Cheema AK, Ressom HW, et al. Metabolomic
profiles of current cigarette smokers. Mol Carcinog.
2017;56(2):594–606. doi:10.1002/mc.22519. [Epub
2016 Aug 22].
72. Pope JL, Tomkovich S, Yang Y, Jobin C. Microbiota
as a mediator of cancer progression and therapy.
Transl Res. 2017;179:139–154.
73. Spor A, Koren O, Ley R. Unravelling the effects
of the environment and host genotype on the gut
microbiome. Nat Rev Microbiol. 2011;9:279–290.
74. Ussar S, Griffin NW, Bezy O, et al. Interactions
between gut microbiota, host genetics and diet
modulate the predisposition to obesity and metabolic
syndrome. Cell Metab. 2015;22:516–530.
75. Borges-Canha M, Portela-Cidade JP, Dinis-Ribeiro
M, Leite-Moreira AF, Pimental-Nunes P. Role of
colonic microbiota in colorectal carcinogenesis: a
systematic review. Rev Esp Enferm Dig. 2015;107:
659–671.
76. Jobin C. Colorectal cancer: looking for answers in
the microbiota. Cancer Discov. 2013;3:384–387.
77. Pevsner-Fischer M, Tuganbaev T, Meijer M, et al.
Role of the microbiome in non-gastrointestinal
cancers. World J Clin Oncol. 2016;73:200–213.
78. Schwabe RF, Jobin C. The microbiome and cancer.
Nat Rev Cancer. 2013;13:800–812.
79. Hein DW. Molecular genetics and function of NAT1
and NAT2: role in aromatic amine metabolism and
carcinogenesis. Mutat Res. 2002;506(507):65–
77.
80. Marcus PM, Vineis P, Rothman N. NAT2 slow
acetylation and bladder cancer risk: a meta-analysis
of 22 case-control studies conducted in the general
population. Pharmacogenetics. 2000;10:115–122.
81. Peto R, Lopez AD. Future worldwide health effects of
current smoking patterns. In: Koop CE, Pearson CE,
Schwartz MR, eds. Critical Issues in Global Health.
San Francisco: Jossey-Bass; 2001.
82. Kensler T, Qian GS, Chen JG, Groopman JD.
Translational strategies for cancer prevention in
liver. Nat Rev Cancer. 2003;3:321–329.
83. World Health Organization. WHO Report on the
Global Tobacco Epidemic, 2011: Warning About the
Dangers of Tobacco. Geneva: World Health Organiza-
tion; 2011.
84. DeVita VT Jr, Chu E. A history of cancer chemo-
therapy. Cancer Res. 2008;21:8643–8653.
85. D’Orazio J, Jarrett S, Amaro-Ortiz A, Scott
T. UV radiation and the skin. Int J Mol Sci.
2013;14:12222–12248.
86. Shah DJ, Sachs RK, Wilson DJ. Radiation-induced
cancer: a modern view. Br J Radiol. 2012;85:
1166–1173.
87. Robertson A, Allen J, Laney R, Curnow A. The
cellular and molecular carcinogenic effects of radon
exposure: a review. Int J Mol Sci. 2013;14:14024–
14063.
88. Jostes RF. Genetic, cytogenetic, and carcinogenic
effects of radon: a review. Mutat Res. 1996;340:
125–139.
89. IARC Working Group on the Evaluation of
Carcinogenic Risks to Humans. Arsenic, metals,
fibres and dusts. IARC Monogr Eval Carcinog Risks
Hum. 2012;100:11–465.
90. Bustaffa E, Stoccoro A, Biachi F, Migliore L.
Genotoxic and epigenetic mechanisms in arsenic
carcinogenicity. Arch Toxicol. 2004;88:1043–1067.
91. IARC Working Group on the Evaluation of Carci-
nogenic Risks to Humans. Chromium, nickel and
welding. IARC Monogr Eval Carcinog Risks Hum.
1990;49:1–648.
92. Lu H, Shi X, Costa M, Huang C. Carcinogenic
effect of nickel compounds. Mol Cell Biochem.
2005;279:45–67.
93. O’Reilly KM, McLaughlin AM, Beckett WS, Sime
PJ. Asbestos-related lung disease. Am Fam Physician.
2007;75:683–688.
94. Basen-Engquit K, Chang M. Obesity and cancer
risk: recent review and evidence. Curr Oncol Rep.
2011;13:71–76.
95. Vainio H, Bianchini F, eds. IARC Handbook of Cancer
Prevention. Vol. 6. Weight control and physical
activity. Lyon, France: IARC Press; 2002.
96. Renehan AG, Tyson M, Egger M, et al. Body-mass
index and incidence of cancer: a systematic review
and meta-analysis of prospective observational
studies. Lancet. 2008;371:569–578.
97. Roberts DL, Dive C, Renehan AG. Biological
mechanisms linking obesity and cancer risk: new
perspectives. Annu Rev Med. 2010;61:301–316.
98. Willett WC, Stampfer MJ, Colditz GA, et al.
Relation of meat, fat, and fiber intake to risk of
colon cancer in a prospective study of women. N
Engl J Med. 1990;323:1664–1672.
99. Knize MG, Kulp KS, Salmon CP, et al. Factors affect-
ing human heterocyclic amine intake and the metab-
olism of PhIP. Mutat Res. 2002;506(507):153–162.
100. Butler LM, Sinha R, Millikan RC, et al. Heterocyclic
amines, meat intake, and association with colon
cancer in a population-based study. Am J Epidemiol.
2003;157:434–445.
101. Ames BN. Dietary carcinogens and anticarcinogens.
Science. 1983;221:1256–1264.
102. Vineis P, ed. Metabolic Polymorphisms and Susceptibil-
ity in Cancer. IARC Sci Publ No. Lyon: IARC Press;
1999:148.
103. Moriwaki S, Kraemer KH. Xeroderma pig-
mentosum—bridging a gap between clinic and
laboratory. Photodermatol Photoimmunol Photomed.
2001;17:47–54.
104. Friedberg EC, Walker GC, Siede W. DNA Repair
and Mutagenesis. Washington, DC: ASM Press; 1995.
105. Kelloff GJ, Sigman CC, Greenwald P. Cancer
chemoprevention: progress and promise. Eur J
Cancer. 1999;35:2031–2038.
106. Lippman SM, Hong WK. Cancer prevention by
delay (commentary). Clin Cancer Res. 2002;8:305–
313.
107. Kelloff GJ, Lippman SM, Dannenberg AJ, et al.
Progress in chemoprevention drug development: the
promise of molecular biomarkers for prevention of
intraepithelial neoplasia and cancer—a plan to move
forward. Clin Cancer Res. 2006;12:3661–3697.
108. Colditz GS, Wolin KY, Gehlert S. Applying what
we know to accelerate cancer prevention. Sci Transl
Med. 2012;4(127):127rv4.
109. Blackburn EH. Cancer interception. Cancer Prev
Res (Phila). 2011;4:787–792.
 11  DNA Damage Response Pathways
and Cancer
James M. Ford and Michael B. Kastan

S UMMARY OF K EY
• DNA repair and the cellular response
to DNA damage are critical for
maintaining genomic stability.
• Defects in DNA repair or the
response to DNA damage
encountered from endogenous or
external sources results in an
increased rate of genetic mutations,
often leading to the development of
cancer.
• Inherited mutations in DNA damage
response pathway genes often result
in cancer susceptibility.
• The major active pathways for number of rare, recessive
DNA repair in humans are nucleotide
excision repair, base excision ataxia-telangiectasia, Nijmegen
repair, mismatch DNA repair,
translesional DNA synthesis, and
homologous recombination or
nonhomologous end joining
processes for double-strand break
repair.
• Inherited defects in nucleotide
excision repair lead to the skin
cancer–prone syndrome xeroderma
pigmentosum, Cockayne syndrome,
and trichothiodystrophy.
P OI N T S
• Inherited defects in base excision
repair can result in colorectal cancer
susceptibility.
• Inherited defects in mismatch repair
result in Lynch syndrome (hereditary
nonpolyposis colorectal cancer
syndrome), leading to increased
incidence of gastrointestinal cancers,
endometrial cancer, and other
malignancies.
• Biallelic inherited defects in DNA
double-strand break repair and
response pathways underlie a
cancer-prone disorders, including
breakage syndrome, Bloom
syndrome, Werner syndrome,
Rothmund-Thomson syndrome, and
Fanconi anemia.
• Persons with autosomal dominant
Li-Fraumeni syndrome, who are
highly prone to cancer because of
inherited monoallelic p53 mutations,
and with breast-ovarian cancer
syndrome, who are highly prone to
cancer because of inherited
mutations of the BRCA1 and BRCA2
genes, exhibit defects in multiple
DNA repair and DNA damage
response pathways.
• Because most cancer therapeutic
agents that are currently used
damage DNA, understanding how
normal cells and tumor cells respond
to and repair DNA damage is an
important aspect of understanding
cancer therapeutic responses and
toxicities of treatment.
• The presence of mutations in DNA
damage response and repair
pathways in tumors presents
opportunities for developing
therapeutics that target alternative
damage response pathways and can
be selectively lethal to the mutated
tumor cells, an approach called
synthetic lethality.
• Genomic instability, particularly that
conferred by underlying DNA repair
defects, can lead to enhanced
responses to immune checkpoint
inhibitors owing to increased
expression of mutant neoantigens.

Cancer is a disease caused by the accumulation over time of changes
to the normal DNA sequence resulting in alterations, loss, amplification,
or changes in expression of genes important for normal cellular functions
and growth properties, including proto-oncogenes and tumor suppressor
genes. Nearly all cancers are clonal in origin—that is, they originate
from a single progenitor cell rather than a group of cells. The develop-
ment of cancer in a particular cell type or tissue is caused by a series
of specific mutations, each of which could be caused by DNA replication
errors or unrepaired endogenous or exogenous DNA damage or could
be the result of inherited mutations. For the most common cancers,
multiple genetic events occur in many different genes during the
process of carcinogenesis, suggesting that an early and perhaps necessary
event in the cancer process is an underlying defect in mechanisms to
maintain genomic stability.1–4 In fact, alterations in the specific genes
required for recognizing, processing, and responding to DNA damage
may result in an enhanced rate of accumulation of additional mutations,
recombinational events, chromosomal abnormalities, and gene amplifica-
tion.5 Whether such a “mutator phenotype” is a required event for
human tumorigenesis remains a controversial issue,6,7 but cellular
responses to DNA damage are clearly important determinants of both
cancer development and outcomes following cancer treatments.
Cancer cells must be able to tolerate increased amounts of unrepaired
DNA damage associated with genomic instability, and therefore
inactivate DNA damage–inducible signaling and checkpoint pathways.4
Therefore, DNA repair and the DNA damage response are essential
not only for the basic processes of transcription and replication necessary
for cellular survival, but also for maintaining genomic stability and
avoiding the development of malignancies. This chapter reviews the
major DNA repair mechanisms active in mammalian cells, and our
understanding of DNA damage signaling pathways that integrate with
other cellular processes regulating transcription, replication, cell division
and apoptosis in response to DNA damage. The relevance of these
mechanisms to cancer is explored by focusing on several human cancer
predisposition syndromes caused by underlying defects in DNA damage
processing. Dysregulation of the DNA damage response is also associated
with increased sensitivity or resistance of tumors to various classes of

154

Table 11.1  Human DNA Repair Pathways
Pathway Type of Approximate
DNA Repair DNA Damage No. of Genes
Nucleotide Bulky or helix-distorting 37
excision repair DNA adducts, for example,
ultraviolet photoproducts
and carcinogen adducts
Base excision Oxidative DNA damage; 40
repair spontaneous depurination
Mismatch repair Mispaired nucleotides 26
1–15 nucleotide
insertion-deletion loops
Homologous Double-strand DNA 20
recombination breaks, DNA cross-links
Nonhomologous Double-strand DNA 10
end joining breaks
therapeutics, and can be further exploited. Finally, new opportunities
for cancer treatment will be discussed based on targeting DNA repair
or DNA repair deficiencies and genomic instability.
Studies of cancer genetics have defined three general groups of
genes involved in the development of human cancers: oncogenes,
tumor suppressor genes, and DNA damage repair and response genes.
The latter set of genes is particularly important to hereditary cancer
susceptibility owing to their direct involvement in maintaining genomic
stability. Much of what we know regarding cancer genes in sporadic
tumors comes from the study of relatively rare inherited cancer
syndromes caused by mutations passed along in the germline DNA
of families and predisposing to the development of cancers, often at
a very young age and at a high incidence, in affected carriers. Individuals
who inherit a germline mutation in genes involved in or required for
DNA repair usually are at increased risk for the development of cancer
because of the enhanced frequency of mutations and increased genomic
instability. Susceptibility to cancer may also be affected by environmental
factors and multiple low-penetrance modifier genes.
Many converging lines of experimental evidence reveal the complex-
ity of the cellular responses to DNA damage and their role in malignant
transformation.8 A number of interrelated biochemical pathways exist
that influence the following actions: (1) the metabolism of potentially
mutagenic or carcinogenic agents, (2) the efficiency and manner by
which damaged DNA is recognized and repaired, (3) cell cycle progres-
sion and the coordination of DNA replication and cell division relative
to the repair of lesions, and (4) the decision point determining survival
or the active induction of programmed death of cells carrying different
types and amounts of DNA damage. Many cellular pathways have
evolved that require hundreds of gene products for the repair of DNA
damage, and involve excision of damaged DNA bases, joining of
broken DNA strands, or replication bypass of DNA lesions (Table
11.1). The central role of DNA damage responses in neoplastic
transformation has been highlighted by the discovery that mutations
in numerous classes of genes required for DNA repair and the
maintenance of genomic integrity result in a predisposition to the
development of many malignancies.9 In fact, a number of rare, inherited
disorders have been described that appear to be caused by defects in
the repair of DNA lesions (Table 11.2), and many of these are associated
with an increased risk of the development of cancers.10
TYPES OF DNA DAMAGE
DNA undergoes many types of spontaneous modifications, and it
also can react with physical and chemical agents, some of which are
endogenous products of normal cellular metabolism (e.g., reactive
DNA Damage Response Pathways and Cancer  •  CHAPTER 11 155
oxygen species), whereas others, including ionizing radiation and
ultraviolet (UV) light, are threats from the external environment (Fig.
11.1). One pronounced example is exposure to genotoxic compounds
in cigarette smoke, which currently are responsible for the most common
cancers in Western countries. Most active chemotherapeutic agents
function by damaging DNA through alkylation, cross-linking, and
other means, and mechanisms to repair these lesions determine the
sensitivity and resultant resistance of a tumor to such treatments.
Damage to DNA can cause genetic mutations, and these mutations
can lead to the development of cancer. A complex set of cellular surveil-
lance and repair mechanisms has evolved to reverse or limit potentially
deleterious DNA damage. Some of these DNA repair systems are so
important that life cannot be sustained without them. An increasing
number of human hereditary diseases that are characterized by severe
developmental problems or a predisposition to cancer have been linked
to deficiencies in DNA repair (see Table 11.2).
CONSEQUENCES OF DNA DAMAGE
The results of DNA damage are diverse and usually adverse. Acute
effects arise from disturbed DNA metabolism, triggering cell cycle
arrest or cell death. Long-term effects result from irreversible mutations
contributing to oncogenesis and inherited genetic disorders. Many
lesions block transcription, which has elicited the development of a
dedicated repair system, transcription-coupled repair (TCR), which
displaces or removes the stalled RNA polymerase and ensures preferential
repair of lesions within the transcribed strand of expressed genes.11–13
Transcriptional stress due to DNA lesions that block RNA polymerase,
and DNA strand breaks caused by DNA damage or stalled replication
forks, constitute two major signals for DNA damage–inducible
responses, including apoptosis,14–16 through both p53-dependent and
p53-independent mechanisms.17
Lesions also may interfere with DNA replication. A class of more
than 17 specialized DNA polymerases that are devoted to overcoming
damage-induced replication stress has been discovered.18–20 These special
polymerases take over temporarily from the stalled replicative DNA
polymerases. Although translesion polymerases protect the genome,
this solution to replication blocks comes at the expense of a higher
replicative error rate, and mutations in some of these polymerases
cause cancer susceptibility.10 Therefore, detection of DNA lesions may
occur by blocked transcription, replication, or specialized sensors.
DNA DAMAGE RESPONSE PATHWAYS
DNA damage checkpoints initially were defined as regulatory pathways
that control the ability of cells to arrest the cell cycle in response to
DNA damage, perhaps allowing time for repair or other cellular
functions.21 However, in addition to controlling cell cycle arrest, proteins
involved in these pathways have been shown to control the activation
of DNA repair pathways,22–26 the movement of DNA repair proteins
to sites of DNA damage,27–32 and activation of transcriptional
responses.33–35 When damage is too significant, a cell may opt for the
ultimate mode of rescue by initiating apoptosis to benefit the organism
at the expense of losing a cell.36–38 As the DNA damage response
pathway has been better defined at a molecular level, it has been seen
as a network of interacting pathways that together execute the response.4
Initial recognition of DNA damage occurs by a variety of damage-
specific DNA binding proteins that, either by themselves or together
with complexes of associated proteins not directly involved in DNA
repair, signal the DNA damage response.39 Initiation of DNA damage
signaling pathways often is carried out by the phosphoinositide-3-kinase-
related proteins which include ataxia-telangiectasia mutated (ATM)
and ATM-Rad3-related (ATR) proteins, DNA-PK, the checkpoint
kinases CHEK1 and CHEK2, and others.8,40–45 ATM plays a critical
role in DNA damage signaling originating at double-strand breaks
(DSBs), whereas ATR responds to single-strand DNA regions. Many
of these protein kinases are themselves targets for phosphorylation
156 Part I: Science and Clinical Oncology

Table 11.2  Human Genetic Diseases Involving Defects in DNA Damage Response Pathways
Syndrome
Xeroderma pigmentosum
Cockayne syndrome
Trichothiodystrophy
Hereditary nonpolyposis
colorectal cancer (Lynch
syndrome)
Ataxia-telangiectasia
Ataxia-telangiectasia–like
disease
Nijmegen breakage
syndrome
Bloom syndrome
Werner syndrome
Rothmund-Thomson
syndrome
Fanconi anemia
Li-Fraumeni syndrome
Li-Fraumeni–like syndrome
Breast-ovarian cancer
syndrome
Gene(s) Biologic Functions Clinical Features Hypersensitivities
XPA-XPG Nucleotide excision repair Sunlight hypersensitivity UVR, chemical carcinogens
XPV Translesional DNA synthesis Neurologic defects; skin cancers
CSA, CSB Transcription-coupled repair Growth retardation UVR, chemical carcinogens
XPB, XPD Mental retardation
Reactive oxygen species
XPG Premature aging; sunlight
hypersensitivity
XPB, XPD, Nucleotide excision repair; Sulfur-deficient brittle hair; dry, UVR
TTDA transcription scaly skin; mental and physical
retardation; sunlight sensitivity
MLH1, MSH2, Mismatch repair 6-Thioguanine and cisplatin
MSH6, PMS2 resistance. Colorectal, endometrial,
gastric, bile duct cancers
ATM DNA damage-responsive Cerebellar ataxia; telangiectasia; Ionizing radiation
kinase immunodeficiency; lymphomas
MRE11 Double-strand break repair Similar to ataxia-telangiectasia
NBS1 Double-strand break repair Microcephaly; immunodeficiency; Ionizing radiation
lymphomas; neuroblastoma;
rhabdomyosarcoma
BLM DNA helicase; homologous Sunlight hypersensitivity; growth UVR, hydroxyurea
recombination at stalled retardation; leukemias, lymphomas;
replication forks? breast and intestinal cancers
WRN DNA helicase; homologous Premature aging; atherosclerosis; 4-NQO, camptothecin;
recombination?; translesional soft tissue sarcomas; melanoma, hydroxyurea
synthesis? thyroid cancer
RECQL4 DNA helicase Growth deficiency; sunlight UVR
sensitivity; osteogenic sarcomas;
squamous cell carcinomas
FANCA–G Interstrand cross-link repair Growth retardation Bifunctional alkylating agents
BRCA2 Homologous recombination Anatomic defects; bone marrow Ionizing radiation
failure; myeloid leukemia;
squamous cell cancers
p53 Apoptosis; cell cycle Breast cancer; brain cancers; ?
checkpoints; nucleotide adrenocortical carcinoma; leukemia;
excision repair bone and soft tissue sarcomas
CHEK2 DNA damage responsive kinase Similar to Li-Fraumeni syndrome
BRCA1, Double-strand break repair Breast cancer, ovarian cancer Ionizing radiation, cisplatin,
BRCA2 PARP inhibitors

PARP, Poly (ADP-ribose) polymerase; UVR, ultraviolet radiation.

and activation; they then further target downstream gene products
critical to oncogenesis such as p53 and BRCA1.40,46–48 The ultimate
targets of this highly regulated DNA damage response include
mechanisms for DNA repair, and although much of DNA repair is
constitutive, a number of regulatory connections between the DNA
damage response pathway and DNA repair have emerged.8 In mammals,
a large number of genes involved in DNA repair are transcriptionally
upregulated in response to DNA damage, suggesting that many facets
of repair are inducible. In fact, the p53 tumor suppressor gene is a
central mediator of the DNA damage–inducible transcriptional response
in humans, and p53-mutant mammalian cells are deficient in several
aspects of DNA repair.22–26,35,49 Therefore the mammalian DNA
damage–inducible response pathway is highly regulated, and fine-tuned
to determine if a particular cell type proceeds to a cell cycle checkpoint
and DNA repair, or cell death, after a significant damage insult. Defects
at any level of these pathways can alter repair and result in carcinogenesis
(Fig. 11.2).
TYPES OF DNA REPAIR AND THEIR
CONTRIBUTION TO CANCER
DNA repair may be defined as those cellular responses associated with the
restoration of the normal nucleotide sequence after events that damage
or alter the genome.50 Given the wide variety of DNA damage a cell
encounters, it is not surprising that there is an equally large number of
repair systems available to handle these insults. Indeed, many are broadly
overlapping and interacting, with several sharing certain strategies and
even specific gene products. In humans, a great deal has been learned
regarding DNA repair from the often rare, autosomal recessive hereditary
syndromes associated with defects in DNA repair genes.10
 DNA Damage Response Pathways and Cancer  •  CHAPTER 11 157

DNA damage type Endogenous Environmental

Spontaneous base changes Chemical mutagens
Replication errors Cytotoxic agents
Oxygen radicals UV and ionizing radiation

DNA lesions Base-pair mismatches Abasic sites DNA adducts Pyrimidine dimers Strand breaks
Insertion and deletions Oxidized DNA Cross-links
Strand breaks

Signaling pathways Protein kinases

p53 BRCA1 BRCA2 Fanconi anemia
ATM, ATR, Chk1, Chk2 complex

DNA repair pathways Mismatch Base excision Nucleotide excision Double-strand

repair repair repair break repair

Cellular responses to Cell cycle Upregulated Apoptosis Translesional

persisting damage checkpoints repair synthesis

Mutation
Consequences Cell survival Cell death
Malignant transformation

Figure 11.1  •  Cellular responses to DNA damage. Different types of DNA damage cause a variety of different types of lesions, and these in turn are dealt
with by a variety of DNA repair mechanisms and signal various cellular response pathways. The outcome of DNA damage may be cell survival of a normal
cell, cell death, or mutagenesis, possibly leading toward malignant transformation.

Nucleotide Excision Repair A unique problem arises if a bulky lesion is encountered by a

The most versatile and ubiquitous mechanisms for DNA repair are
those in which the damaged or incorrect part of a DNA strand is
excised and the resulting gap is filled through repair replication with
use of the complementary strand as a template. The redundancy of
genetic information provided by the duplex DNA structure is essential
to the maintenance of the genome by this “cut and patch” mode
known as excision repair. Each DNA strand can serve as a template
for replication-based repair of the other strand. Nucleotide excision
repair (NER) functions to remove many types of lesions, including
bulky base adducts of chemical carcinogens, intrastrand cross-links
(ICLs), and UV-induced cyclobutane pyrimidine dimers (CPDs) and
6-4 photoproducts.51 Such lesions may serve as structural blocks to
transcription and replication due to distortion of the helical conforma-
tion of DNA, and they also may result in mutations if translesional
replication occurs or if they are not repaired correctly. The sequential
steps for NER are (1) recognition of the damaged site, (2) incision
of the damaged DNA strand near the site of the defect, (3) removal
of a stretch of the affected strand containing the lesion, (4) repair
replication to replace the excised region with a corresponding stretch
of normal nucleotides with use of the complementary strand as a
template, and (5) ligation to join the repair patch at its 3′ end to the
contiguous parental DNA strand.52,53 This excision repair pathway
can remove DNA damage from sites throughout the genome and is
termed global genomic repair (GGR). The majority of human NER
genes have been identified and cloned, and many have been shown to
be mutated in hereditary NER-deficient, cancer-prone diseases.26,49,54
translocating RNA polymerase making messenger RNA before repair
enzymes have removed the damage and restored intact DNA. The
polymerase may be arrested at the site of the lesion and prevent access
to the damage by repair enzymes. Furthermore, the arrest of transcrip-
tion in human cells is a strong signal for p53 activation and can trigger
apoptosis.17 In this situation, a dedicated excision repair pathway
known as transcription-coupled repair comes to the rescue to displace
the RNA polymerase, and then efficiently repairs the blocking lesion
so that transcription may resume and the cell may survive.55
It has become apparent that the GGR subpathway of NER is
damage inducible and highly regulated by both transcriptional and
posttranslational mechanisms after DNA damage, in concert with
damage-inducible cell cycle checkpoints and apoptosis.8,49 In fact, the
p53 gene, central to maintaining genomic stability in human cells, is
required for efficient GGR of UV light– and carcinogen–induced
DNA damage, and functions as a DNA damage–activated transcription
factor that directly regulates the expression of several NER damage
recognition genes.24–26,35 Similarly, several other important cancer-related
genes have been shown to transcriptionally regulate the DNA damage
recognition NER genes XPC and DDB2, including BRCA1 and
E2F1.56–58 Therefore the GGR pathway of NER appears relevant to
suppressing DNA damage–induced malignancy, and highly regulated
by genes involved in tumor suppression.
Despite the plethora of mechanisms for DNA repair described
herein, it is likely that the cellular replication machinery will nevertheless
encounter lesions in the DNA template strand that block replication
during each cell cycle. The solution adapted by cells is DNA damage
158 Part I: Science and Clinical Oncology
Structure distortion 3' 5'
Damage recognition XPC/hHR23B XPE(p48/p127)
RPA XPA
Incision
TFIIH
ERCC1
XPG XPF
RPA XPA
Excision
27-29 n
Repair replication RFC
PCNA PoI
RPA
Rejoining Ligase I
Figure 11.2  •  Mechanism for human nucleotide excision repair (NER).
Ultraviolet irradiation–induced adducts in genomic DNA are recognized by
the XPE and XPC/hHR23B protein heteroduplexes that recruit the XPA/
RPA complex and the larger TFIIH protein complex. Dimers that occur in
the transcribed strand of an expressed gene result in a blocked RNA polymerase
II molecule, which, together with the CSA and CSB gene products, serves to
recruit the downstream repair machinery. The TFIIH complex contains helicases
including XPB and XPD that unwind the DNA and allow the other repair
proteins access for incision and excision of the damaged DNA oligonucleotide.
After excision, repair replication based on the normal DNA template and
ligation of the newly synthesized DNA sequence occurs. In total, more than
25 proteins participate in NER.
tolerance, in which DNA is synthesized past the damaged bases and
subsequently excised after the replication fork has passed—a process
known as translesional synthesis (TLS) and carried out by a class of
specialized error-prone DNA polymerases.18,19,59,60 These Y-family DNA
polymerases take over temporarily from the stalled replicative DNA
polymerases. They have more flexible base-pairing properties that
permit translesion DNA synthesis, with each polymerase probably
designed for a specific category of injury. Although translesion
polymerases protect the genome, this solution to replication blocks
comes at the expense of a higher error rate. For instance, inherited
defects in polymerase eta (pol η), encoded for by the XPV/POLH/
RAD30 gene, which specializes in relatively error-free bypassing of
UV-induced CPDs, cause a variant form of the skin cancer–prone
disorder Xeroderma pigmentosum (XP).61,62
Human Nucleotide Excision Repair–Deficient
Syndromes and Cancer
A direct correlation between unrepaired DNA damage and carcino-
genesis in humans was first established when James Cleaver found
that the cancer-prone hereditary disease XP involved a defect in the
repair of DNA lesions produced by UV light.63 Since then, at least
four syndromes have been attributed to inborn errors in NER: XP,
Cockayne syndrome (CS), trichothiodystrophy (TTD), and ultraviolet-
sensitive syndrome (UVSS), all characterized by exquisite sun sensitivity.
XP is a rare, autosomal recessive disease in which homozygous
individuals display several characteristics: (1) extreme sensitivity of
the skin to sun exposure evident by 1 year of age, (2) pigmentation
abnormalities and premalignant lesions in sun-exposed skin, (3) increases
up to 4000-fold in incidence of skin cancers (predominantly squamous
and basal cell carcinomas, but also melanomas) and ocular neoplasms,
occurring three to five decades earlier than in the general population,
and (4) a 10- to 20-fold increased incidence of internal cancers in
non–sun-exposed sites.10,64,65 Overall, lifespan is reduced by approxi-
mately 30 years in patients with XP, and many die due to malignancies.66
Approximately 20% of patients with XP also display progressive
neurologic degeneration, characterized by peripheral neuropathy,
sensorineural deafness, progressive mental retardation, and cerebellar
and pyramidal tract involvement.67 XP occurs worldwide, in all ethnic
groups, and with a frequency varying from 1 to 10 patients per million.
The biochemical defect in cells from most individuals with XP is
in NER,63 although in a small number of cases (termed XP-variants),
excision repair appears normal, and a defect exists in bypass replication
at unrepaired lesions as a result of a mutation in the pol η TLS gene
(XPV).68 Complementation analysis via fusion of cells from different
patients has demonstrated genetic heterogeneity within XP and provided
evidence for the existence of at least seven excision-deficient comple-
mentation groups, termed XP-A to XP-G, in addition to XP-variant.10
CS is an autosomal recessive disease that is associated with defec-
tive TCR of UV-damaged and oxidative-damaged DNA.69–71 It is
characterized by cutaneous photosensitivity, cachectic dwarfism,
skeletal abnormalities, retinal degeneration, cataracts, severe mental
retardation, and neurologic degeneration characterized by primary
demyelination.67,72 In contrast to patients with XP, those with CS
are not at increased risk for developing skin cancers. The average
lifespan of individuals with CS is only 12 years, with most patients
dying from infectious or renal complications rather than cancer.73 CS
is characterized by the existence of at least three complementation
groups. Several patients have been described in XP groups B, D,
and G who share the DNA repair defects and clinical features of CS
together with the cutaneous manifestations of XP.74,75 One model for
the severe developmental problems in CS is that endogenous oxidative
damage in metabolically active cells, including neurons, is blocking
transcription. The lack of TCR could then lead to apoptosis of these
essential cells. Alternatively, CS could be a “transcription disease”
caused by defects in TFIIH-associated gene products and resulting
in inadequate gene expression.
TTD is an autosomal recessive condition sharing many of the
signs and symptoms of CS, with the additional hallmark of brittle,
sulfur-deficient hair and nails, due to reduced cysteine content in the
component proteins. As with CS, several of the responsible genes
implicated are XPB and XPD, but there is a third complementation
group, TTD-A, in which mutations of a small 8-kDa TFIIH stabilizing
factor GRF2H5 has been identified.76
Analysis of the specific abnormalities in NER displayed by the
various genetic complementation groups of XP, CS, TTD, and UVSS
allow correlations to be drawn with their heterogeneous clinical features.
Specifically, only those subgroups of patients who display a defect in
GGR are at significantly increased risk for developing UV-induced
malignancies. In contrast, the neurologic symptoms and developmental
abnormalities associated with other complementation groups of XP
and CS are found only in those groups that are defective in TCR.
The fact that the TFIIH complex, containing the XPB and XPD
proteins, is common to both core NER and transcriptional initiation,
supports the suggestion that the clinical phenotype of patients with
defects in TCR may actually be due to abnormalities in transcription
rather than in repair itself.77

Although these observations may explain the molecular basis for
many of the clinical characteristics of XP and CS, they present an
apparent paradox with regard to these patients’ cancer risk. Many
currently recognized oncogenes and tumor suppressor genes are known
to possess important cellular functions and to be actively expressed
in normal cells. Because CS cells are defective in the repair of actively
expressed genes, it would be reasonable to expect that these patients
would acquire mutations in genes leading to transformation more
readily than normal patients. However, this is not supported by the
clinical picture. It has been demonstrated that defects in TCR specifically
activate DNA damage–induced apoptosis,14,15 which may eliminate
potentially mutagenic, premalignant cells.
Base Excision Repair
A major source of DNA damage to cellular genomes arises from normal
metabolism in the cytoplasmic environment through hydrolysis and
exposure to reactive metabolites that cause oxidation and alkylation of
DNA. The repair system primarily involved in identifying and removing
such lesions, as well as in dealing with the spontaneous loss of purines
from DNA, is the base excision repair (BER) pathway.78,79 The essential
nature of BER for viability is highlighted by the fact that although a
number of BER proteins have been discovered, only recently has a single
human hereditary disease been identified that appears to result from
a mutation in a gene unique to this pathway.80–83 BER is initiated by
one of a set of lesion-specific glycosylases that recognize the altered or
inappropriate base and cleave it from its sugar moiety in the DNA (Fig.
11.3). Different DNA glycosylases remove different kinds of damage,
conferring specificity to the process. AP-endonuclease (APE1) then
cleaves the phosphodiester backbone 5′ to the AP site, resulting in a
BER NER MMR
Base damage Pyrimidine dimer Mismatch
Recognition, incision, excision
Repair patch synthesis
Ligation
Figure 11.3  •  Excision repair pathways for DNA damage. The three main
excision repair pathways in human cells—base excision repair (BER), nucleotide
excision repair (NER), and mismatch repair (MMR)—proceed through similar
steps to restore the normal DNA sequence. After recognition of altered DNA
bases, using lesion-specific glycosylases for BER, XP proteins for NER, and
MutS homologs for MMR, incision of DNA is achieved by endonucleases,
and displacement or degradation of single-stranded sections of DNA that
contain the damage by enzymes with helicase and exonuclease activity. Repair
replication of the resulting DNA gap and strand ligation results in the repaired
double-stranded DNA molecule. The many repair enzymes involved in each
specific step are tightly coupled and may be regulated or inducible by DNA
damage response pathways.
DNA Damage Response Pathways and Cancer  •  CHAPTER 11 159
single-strand DNA break intermediate. In some cases, bifunctional
glycosylases, which also cleave the phosphodiester bond adjacent to
the damaged base, preclude the need for endonuclease activity of
APE1 and instead require polynucleotide kinase 3′-phosphatase for
single-strand break end-processing. In the short-patch pathway, DNA
polymerase (POLB) displaces the AP site and adds a nucleotide. Last,
ligase (LIG3) forms a phosphodiester bond to complete repair. In the
long-patch pathway, DNA polymerase (POLB, POLD, or POLE)
displaces and adds more than one nucleotide, flap structure-specific
endonuclease (FEN1) removes the displaced nucleotides, and ligase
(LIG1) completes repair. Proliferating cell nuclear antigen (PCNA)
is required for POLD function and acts as a scaffolding protein. Poly
(ADP-ribose) polymerase (PARP) binds to and recruits essential media-
tors to single-strand break intermediates. The major oxidized purine
lesion is 8-oxo-7,8-dihydroguanine (8-oxoG), which is abundant and
has strong mutagenic properties. Oxidized pyrimidines include thymine
glycol, 5-hydroxycytosine, and formamidopyrimidines. Oxidized bases,
including both 8-oxoG and thymine glycol, share the property of
blocking DNA replication and transcription, and must be repaired
efficiently if genomic stability is to be maintained.84,85
In mammalian cells, the gene functions responsible for the strand
incision steps of BER include the glycosylases hNTH1, which removes
oxidized pyrimidines; hOGG1, which targets oxidized purines; and
MYH, which removes adenines mispaired with an 8-oxoG, together
with APE1.86 Given the mutagenic and cytotoxic potential of the
classes of DNA damage that are BER substrates, it seems likely that
altered activity in these pathways would result in enhanced cancer
risk. The most direct evidence for a role for BER in cancer comes
from the discovery that germline mutations in the MYH gene, involved
in processing 8-oxoG lesions, is associated with recessive inheritance
of a predisposition to develop multiple colorectal adenomas (polyposis)
and colon cancers.80–82 Tumors from affected individuals exhibit excess
transversions of a guanine-cytosine pair to a thymine-adenine pair in
the APC gene, itself associated with colon carcinogenesis and causative
of familial adenomatous polyposis. Therefore, biallelic inherited
mutations in MYH result in a polyposis-like syndrome termed MYH-
associated polyposis (MAP). Patients with MAP tend to develop tens
to hundreds of polyps by the age of 40, and nearly 50% have colon
cancer at presentation.83,87
A great deal of effort has gone into attempts to identify functional
polymorphic genetic variation in DNA repair genes that modulate
activity and affect individual cancer risk. To date, genetic epidemiology
studies of single-nucleotide polymorphisms (SNPs), particularly in
BER genes, have been inconsistent.88 However, meta-analyses have
identified modest associated increased lung cancer risk with OGG1
and XRCC1 SNPs.89 Continued advances in SNP maps and high-
throughput genotyping methods will facilitate the analysis of multiple
polymorphisms within multiple genes through use of haplotypes;
with larger sample sizes, perhaps future studies will reveal clearer
risk associations.
Mismatch Repair
Mismatch repair (MMR) is another example of an excision repair
mechanism that uses a similar strategy for genomic maintenance (see
Fig. 11.3). MMR is a process that corrects mismatched nucleotides
in the otherwise complementary paired DNA strands, arising from
DNA replication errors and recombination, as well as from some
types of base modifications.90–92 This repair mode also can deal with
small loops of single-stranded DNA at sites of insertions or deletions
in the duplex DNA structure. The importance of this repair mechanism
in maintaining genetic stability is illustrated by the observation that
its absence results in a large increase in the frequency of spontaneously
occurring mutations, particularly in microsatellite sequences of highly
repetitive DNA.93 Some of these spontaneous mutations arise from
mistakes introduced during DNA replication, in spite of the operation
of a “proofreading” system that also helps to ensure the high fidelity
160 Part I: Science and Clinical Oncology
of replication. In humans, genetic defects in several MMR genes have
been linked to Lynch syndrome (hereditary nonpolyposis colorectal
cancer) and to sporadic cancers that exhibit instability in regions of
DNA containing short repetitive sequences of nucleotides, a feature
known as microsatellite instability (MSI).
As with other modes of excision repair, four principal steps are
required for MMR: (1) mismatch recognition, (2) recruitment of
additional MMR factors, (3) identification of the newly synthesized
DNA strand containing the mismatched nucleotides, followed by
their excision, and (4) resynthesis of the excised tract and ligation.
The biochemical workings of this pathway are best understood in
bacteria, but a similar set of events occurs in human cells. Based on
functional homologies to their bacterial counterparts, and sequence
homology to corresponding yeast genes, a number of human genes
have been cloned that participate in MMR, including those homologous
to the bacterial MutS mismatch recognition protein (hMSH2, hMSH3,
and hMSH6) and to the bacterial MutL gene (hMLH1 and hPMS2).94–96
In humans, heterodimers of the MSH2/6 proteins recognize single
base-pair mismatches and short insertion-deletion loops, whereas
MSH2/3 dimers recognize longer loops. Heterodimeric complexes of
MLH1/PMS2 and MLH1/PMS1 interact with the MSH complexes
and replication factors, for strand discrimination and DNA excision.
Similar to NER and BER, additional proteins are then recruited for
repair replication based on the original DNA template.
The MMR system also may interact with DNA damage because of
certain alkylators and intercalating agents that assume similar structural
alterations in DNA as mismatches and actually result in erroneous or
futile MMR cycles and ultimately in apoptosis. Thus, intact MMR
may confer chemosensitivity to these chemotherapeutic agents, and
MMR-defective tumors may exhibit resistance to certain drugs.97
Human Mismatch Repair Deficiency and Cancer
Lynch syndrome is the most common hereditary colorectal cancer
predisposition syndrome.98,99 Lynch syndrome is an autosomal domi-
nant inherited condition with an incidence of 1 : 1000 in the general
population. It accounts for approximately 3% of all colorectal cancers
patients; patients with Lynch syndrome are also at elevated risk for
cancers of the endometrium, ovary, stomach, small bowel, and other
sites. Patients with Lynch syndrome have an 80% lifetime risk for
colorectal cancer and a 50% lifetime risk for endometrial cancer. The
discovery of MSI—that is, the frequent alteration in the tract lengths
of certain short repetitive nucleotide sequences—in some hereditary
colorectal cancers provided the first indication that the etiology of these
cancers might involve a problem in the MMR system.100 The finding
of germline MMR gene defects in patients with Lynch syndrome
established that these defects are the cause of the enhanced incidence
of cancer.94–96 Germline mutations in MLH1 and MSH2 together
account for more than half of all cases of Lynch syndrome. Defects
in MSH6 cause a late-onset Lynch syndrome phenotype, and less
penetrant mutations in PMS2 can also result in Lynch syndrome. No
strong genotype-phenotype correlations have been observed to date,
but mutations in the MSH2 gene do appear to be associated with more
extracolonic manifestations than are mutations in the MLH1 gene.
MSI has been identified as a source of the genomic instability
driving tumorigenesis in a number of sporadic tumor types, in addition
to those that arise in the context of inherited germline mutations of
MMR genes.100,101 For example, up to 20% of sporadic colon cancers
exhibit MSI, particularly in the ascending colon and in individuals
younger than 50 years, the majority due to epigenetic silencing of
MLH1 gene expression by promoter hypermethylation.102,103 Whether
through genetic or epigenetic inactivation, loss of MMR results in
the elevated rate of mutations, particularly at microsatellite sequences,
several of which occur in the coding sequences of other genes often
found mutated in cancers, including TGF beta type II receptor, BAX,
and the MMR genes MSH3 and MSH6 themselves. Therefore, clear
genetic evidence demonstrates that the phenotype of genetic instability
Box 11.1.  GENOMIC INSTABILITY AND
RESPONSE TO IMMUNE CHECKPOINT
BLOCKADE
One of the most exciting advances in cancer therapeutics has been the
use of immune checkpoint inhibitors in a wide range of tumors,
including classically “immunogenic” cancers such as melanoma and
renal cell cancer,111 but also many others. For most cancers, predictive
biomarkers to, for example, anti-PD-(L)1 agents remain poorly
understood. However, it has recently been appreciated that the overall
tumor mutational burden correlates with clinical benefit of anti-PD-1
and anti-CTLA4 agents, particularly for patients with conditions
involving high somatic mutation rates such as non–small cell lung
cancer and melanoma and those with DNA repair deficiencies.112 It has
been postulated that the load of neoantigens resulting from DNA
mutations in coding regions leads to immunotherapy responses and
may be a biomarker.113 A survey of mutational load in various tumors
showed that among the very highest are microsatellite instability–high
(MSI-H) colorectal cancers and POLE mutant hypermutator colorectal
and endometrial cancers.114 This is consistent with the long-standing
observation that Lynch syndrome tumors exhibit a high level of
tumor-infiltrating lymphocytes (TILs).115 A remarkable clinical study
showed that DNA mismatch repair–deficient MSI-H colorectal and
endometrial cancers achieve excellent and durable responses to
immune checkpoint inhibitors, whereas microsatellite-stable (MSS)
tumors showed no responses.116 Immunohistochemistry showed
greater densities of CD8+ TILs and PD-L1 expression in MSI-H than MSS
tumors in this study, and these were associated with responses. Based
on these results, the checkpoint inhibitor pembrolizumab received
breakthrough therapy designation for MSI-H advanced colorectal
cancers. Trials examining this approach for adjuvant treatment of early
colorectal cancer and any cancers with an MSI phenotype are
underway. Ultimately, there appears to be a clear link among DNA
repair deficiency, mutational landscape, predicted neoantigen load, and
clinical activity of immune checkpoint inhibitors. The role of somatic
tumor mutational load and spectrum in priming the immune system in
tumors is a fascinating new approach to linking personalized cancer
genomic profiling and immunotherapy.
associated with defects in MMR results in the genotype of tumors
that arise because of these defects.104 Intriguingly, the survival of patients
with MSI-associated colorectal cancer is better than in those with
more typical tumors exhibiting chromosomal instability.105–107 These
tumors also demonstrate different sources of genomic instability,
resulting in distinct biologic and pathologic characteristics.108 Whether
their more favorable outcome reflects differences in clinical behavior,
responsiveness to therapy, or both remains to be fully determined.
Given the readily available phenotypic assays for Lynch syndrome
(MSI and immunohistochemistry for the four causative gene products),
“universal” screening is now performed on all colon and endometrial
cancers at many centers, leading to cost-effective identification of
Lynch syndrome families and hopefully improved clinical outcomes.109,110
Recently, very interesting findings have been reported regarding
treatment of MSI tumors that targets their genomic instability phe-
notype (Box 11.1).
Double-Strand Break Repair
DSBs in DNA induced by ionizing radiation, chemicals, and replication
across single-strand breaks and during repair of interstrand DNA cross-
links usually are dealt with through the recombination machinery. An
unrepaired DSB is a highly lethal event, and even a single occurrence
in the entire genome is thought to be sufficient to signal cell cycle
checkpoints that prevent attempted DNA synthesis or cell division
 DNA Damage Response Pathways and Cancer  •  CHAPTER 11 161

Nonhomologous Homologous
end-joining recombination

DSB DSB

RAD51
MRE11
End alignment Resection
NBS1
Ku80 Ku80 RAD52

DNA-PKcs

DNA-PKcs
Ku70 Ku70 RAD52
BRCA1
XRCC4 RAD51 Strand invasion
BRCA2 Branch migration
Ligase IV
RAD54

RAD51

Ligation
Junction resolution

Figure 11.4  •  Mechanisms for DNA double-strand break (DSB) repair. The repair of DNA DSBs is carried out by two mechanisms. Left, The rapid, but
error-prone, nonhomologous end-joining that directly seals breaks but may result in the gain or loss of several nucleotides owing to short areas of microhomologies
used for annealing before ligation. Exposed DNA ends are recognized by the Ku70/80 heterodimer that recruits the DNA-dependent protein kinase catalytic
subunit and other proteins assisting in strand alignment. The XRCC4-ligase IV heteroduplex joins the breaks. Right, The high-fidelity, homologous recombination
of sister chromatids at sites of DSBs is the less prominent mode in mammalian cells. This DNA repair pathway is mediated by RAD51-associated proteins,
including RAD52, which recognizes single-strand DNA ends and together with other proteins results in short nuclease-mediated resection. RAD51 then forms
a nucleoprotein filament on the exposed strand, and probably with BRCA2 and other proteins, promotes strand invasion and displacement at homologous
sequences. Thus, the undamaged sister molecule acts as a template for the resynthesis of the missing nucleotides.

until repair has been completed, or apoptosis, if improperly repaired.
DSBs also pose problems during mitosis because intact chromosomes
are a prerequisite for proper chromosome segregation during cell divi-
sion. Thus these lesions often induce various sorts of chromosomal
aberrations, including aneuploidy, deletions, and chromosomal
translocations—all of which are associated with carcinogenesis. Genetic
recombination is the principal mechanism for dealing with DSBs
that involve homologous stretches of nucleotides at the ends to be
joined. If no such homology is present, however, another system exists
for nonhomologous end joining (NHEJ), which is more error-prone
(Fig. 11.4). Whereas NHEJ is the more predominant mechanism for
DSBR in humans, homologous recombination (HR) is specifically
required for repair of DSBs formed at collapsed replication forks,
and for ICL repair together with NER proteins. Recent studies have
identified a cascade of protein kinases involved in signaling cellular
processes in response to DSBs. Many of these have been found to
be defective in cancer-prone disorders exhibiting genomic instability,
such as the ATM protein45 and the CHEK2 protein kinase.117–119 A
major target for these kinase activities is the p53 tumor suppressor
gene. This protein, when phosphorylated, is activated and involved
in G1 arrest and apoptosis after ionizing radiation40,41,120–123 and,
when found mutated in the germline, results in the Li-Fraumeni
cancer susceptibility syndrome. Many other enzymes involved in the
actual DNA transactions necessary for DSB repair have been found
in cancer-prone disorders, including MRE11 (AT-like disorder), NBS1
(Nijmegen breakage syndrome), BRCA1 and BRCA2 (breast-ovarian
cancer syndrome), and the RecQ-like helicases (Werner, Bloom, and
Rothmund-Thomson syndromes).124
Ataxia-Telangiectasia
Ataxia-telangiectasia (AT) is a disease of pleiotropic abnormalities,
including cerebellar degeneration, cancer predisposition, progressive
pulmonary dysfunction, and profound radiosensitivity. A single gene,
ATM, is responsible for the multiple and surprisingly diverse symptoms
of this disease. Over 10% of AT patients develop cancer at an early
age, primarily leukemias and lymphomas. AT is an autosomal recessive
disease with an incidence of nearly 1 in 100,000 live births. Persons
who are heterozygous for AT mutations, about 1% of the general
population, may have an increased predisposition to cancer, in particular
breast cancers, especially in those individuals expressing an ATM with
missense mutations resulting in a dominant-negative effect on their
wild-type ATM gene.125–127 The ATM gene product is a central signaling
protein in the DNA damage response, and cells lacking ATM fail to
execute many critical cellular responses to DNA damage. For example,
the ATM gene product is a key element in delaying the initiation of
DNA replication after DNA damage resulting in strand breaks. In
fact, lack of ATM function leads to abnormalities in all major DNA
damage–induced cell cycle checkpoints, G1, S, and G2/M.41 In addition,
ATM directly phosphorylates the NBS1 protein, resulting in its
162 Part I: Science and Clinical Oncology
association with the MRE11 and RAD50 gene products, which together
are required for both NHEJ and HR of DSBs.128 Inherited germline
mutations of the NBS1 and MRE11 genes, themselves, result in clinical
variants of AT, termed Nijmegen breakage syndrome and AT-like disorder,
respectively.128,129 Therefore, ATM, NBS1, and MRE11 are central to
DNA damage response pathways critical for regulating recombination
and repair following DSBs. Defects in many of the component proteins
in the pathway result in genomic instability and a predisposition to
cancer. Interesting to note, ATM has also recently been implicated in
cellular processes other than DNA damage responses, including insulin
signaling and mitochondrial function.130,131 Thus, although only a
single gene is mutated in AT, loss of ATM results in a pleiotropic
disease phenotype in patients because of its multifaceted functions.
p53 Gene and Li-Fraumeni Syndrome
The discovery of the p53 tumor suppressor gene over 35 years ago
inspired widespread investigations with the aim of understanding the
basic biology behind its role in maintaining genomic stability and the
cellular response to DNA damage. The p53 gene is one of the most
commonly mutated genes in human cancers,132 and its product is a
multifunctional protein that regulates many physiologic processes,
including cell cycle checkpoints, apoptosis, and DNA repair.26,121,133–135
The primary role of p53 in tumor suppression has been attributed to
its function as a transcription factor, regulating expression of several
hundred different cellular genes,136 although it also exhibits transcription-
independent activities. Indeed, p53 appears to act as a central “node”
that lies at the intersection of upstream signaling cascades induced
by DNA damage and cellular stress responses, and downstream DNA
repair and DNA damage response pathways. In response to a variety
of genotoxic stimuli, p53 protein is induced by both increased mRNA
translation137,138 and protein stabilization through a series of post-
translational modifications.136 The induced protein then binds to DNA
in a sequence-specific manner, resulting in transcriptional regulation
of downstream target genes that contain a consensus p53 response
element in their promoter or intronic segments. These p53 target
genes include those important for cell cycle checkpoints, such as p21139;
apoptosis, such as BAX and PERP140; and DNA repair, such as the
DDB2 and XPC genes required for NER.24,25,35
Patients with the rare autosomal dominant Li-Fraumeni syndrome
(LFS) are at increased risk for developing a number of common tumors
at an early age because of an inherited germline defect in one allele
of the p53 gene, including soft tissue sarcomas and osteosarcomas,
breast cancer, brain tumors, lymphomas, leukemia, and adrenocortical
carcinomas. Mutations in the p53 tumor suppressor gene account for
70% to 85% of classic LFS cases.141–143 Although the heterozygote
carriers of a defective p53 allele do not appear to have clinical problems
or somatic DNA repair defects, when the second allele has been mutated
or lost, the absence of functional p53 results in severe problems for
the cell and substantial cancer susceptibility for the host. First, the
p53-controlled pathway of apoptosis is disengaged, so severely damaged
cells will survive and be at risk for carcinogenic transformation because
of their genomic instability. That genomic instability derives from the
fact that p53 is also an important regulator of cell cycle checkpoints.
Finally, p53 serves an important regulatory function in DNA repair
and recombination, and in its absence some important mutagenic
lesions are simply not repaired, leading to genomic instability and the
consequent development of tumors.
BRCA1, BRCA2, and Breast-Ovarian
Cancer Susceptibility
Hereditary breast cancer includes a broad group of hereditary predisposi-
tion conditions in which breast cancer is a component tumor and
accounts for approximately 5% to 10% of all breast cancer cases.
Hereditary syndromes of breast and ovarian cancer susceptibility have
been particularly associated with germline mutations of two genes,
BRCA1 and BRCA2, in addition to rare cases caused by mutations
in the p53 gene in persons with LFS, the PTEN gene in persons with
Cowden disease, and carriers with mutations in ATM, CHEK2, PALB2,
and others.144,145
The BRCA1 and BRCA2 genes are large and complex. Many
hundreds of different germline mutations have been detected in each.
Strong evidence suggests that BRCA1 and BRCA2 are centrally involved
in various aspects of DNA repair and DNA damage response pathways.
For example, BRCA1 is phosphorylated after exposure to DNA-
damaging agents by ATM, ATR, and Chk2; associates with a number
of DNA repair proteins including ATM, RAD51, and the RAD50-
MRE11-NBS1 protein complex following DNA damage; and localizes
to nuclear foci with these proteins after treatment with ionizing
radiation.29,146,147 The association of BRCA1 with RAD51, an enzyme
involved in the coordination of recombination, suggests its involvement
in DSB repair and implicates BRCA1 in HR.148,149 Other studies
suggest that BRCA1 may regulate cellular processes through tran-
scriptional coactivation. BRCA1 has been shown to transcriptionally
regulate the NER genes XPC and DDB2 and affect GGR of UV- and
cisplatin-induced DNA damage,56,150,151 and to transcriptionally regulate
BER genes and affect BER of oxidative DNA damage151–153 Chromo-
somal instability also is characteristic of breast tumors that harbor
BRCA2 mutations, probably because of defective recombination-
mediated DSB repair. BRCA2 has been shown to bind to the RAD51
protein, an enzyme involved in the coordination of recombination,
and together they colocalize to nuclear sites containing DNA strand
breaks caused by ionizing radiation. Structural studies of BRCA2
DNA binding domains suggest that it may facilitate interactions of
RAD51 with single-stranded DNA during recombination.154,155 The
genomic instability associated with mutations of BRCA1 and BRCA2,
therefore, may be due in part to the intact but error-prone NHEJ
repair pathway.156 See Box 11.2 for a discussion of DNA repair and
cancer treatment.
Fanconi Anemia, Cancer, and Interstrand
Cross-link Repair
The centrality and interwoven nature of DNA repair pathways for
genomic stability have been highlighted by findings regarding Fanconi
anemia, a rare, autosomal recessive disease that confers an increased
risk of acute myeloid leukemia, squamous-cell carcinomas of the head
and neck and esophagus, gynecologic carcinomas, and liver tumors
at a young age.175,176 At least 19 subtypes of Fanconi anemia have
been determined by complementation analyses, and germline mutations
in genes have been identified for most (FANCA to FANCT).177,178,179
The proteins encoded by these genes all cooperate in a common DNA
repair pathway involved in interstrand cross-link repair and processing
of arrested replication forks.180,181 Germline homozygous inactivating
mutations of the BRCA2 gene result in the FANCD1 group.182 A
“core complex” of these proteins consists of subunits of a nuclear E3
ligase, required for the monoubiquitination of the downstream
FANCD2 protein, which itself links Fanconi anemia proteins to BRCA1
in the response to DNA damage by colocalizing to nuclear sites occupied
by RAD51 and BRCA2.183 It has long been appreciated that cells
from Fanconi anemia patients are hypersensitive to DNA cross-linking
agents, such as mitomycin C and cisplatin, in addition to being modestly
sensitive to ionizing radiation. Therefore the Fanconi anemia proteins
appear to function at an interface between several DNA repair and
DNA damage response pathways. Monoallelic germline mutation of
many FANC genes (such as PALB2/FANCN, RAD51C/FANCO, BRIP1/
FANCJ) has been shown to result in a modest increase in breast,
ovarian, and pancreatic cancers.
CONCLUSIONS AND FUTURE DIRECTIONS
Molecular biology and genetic research have provided ample evidence
to support the long-standing prediction that genomic instability is

Box 11.2.  TARGETING DNA REPAIR FOR CANCER TREATMENT
As discussed in this chapter, many genes implicated in the
development of cancer play roles in DNA repair and in maintenance
of genomic stability.4 The mechanism of action for most cancer
chemotherapeutic drugs, as well as radiation therapy, is thought to be
through DNA damage. Therefore, developing inhibitors of DNA damage
response pathways is an attractive approach for making tumor cells
more sensitive to chemotherapy or radiation therapy. Small-molecule
inhibitors of the ATM, ATR, and DNA-PK kinases are currently being
developed and studied for their ability to enhance therapeutic
responses.157 Furthermore, it seems logical that cancers with germline
or acquired somatic defects in DNA repair occurring during the
tumorigenic process would also be particularly susceptible to the
cytotoxic effects of DNA damaging therapeutic agents. However, for
most common cancers, the clinical experience suggests otherwise.
It is likely that the frequently concurrent inactivation of cell cycle
checkpoints and apoptotic processes during tumorigenesis obscures
the effect of DNA repair defects; therefore the response of clinical
tumors to various treatments remains very difficult to predict even
with genetic information. Nevertheless, several examples have emerged
in which a detailed understanding of the DNA repair defects present
in a particular tumor type allows for the rational selection of certain
treatment approaches likely to be more effective.158
A primary example relates to the function of the BRCA1 gene, which
is involved in several types of DNA repair, including DSB repair, NER,
BER, and DNA cross-link repair.49,56,153,159 Germline mutations in the
BRCA1 gene predispose individuals to a very high risk for developing
breast and ovarian cancers, as well as to a lesser degree pancreatic and
prostate cancers, and somatic inactivation of BRCA1 activity has also
been observed in sporadic breast and ovarian cancers because of
promoter methylation. Clinical and experimental data suggest that
breast and ovarian tumors deficient in BRCA1 function are particularly
sensitive to the chemotherapeutic drug cisplatin, which causes DNA
damage repaired through the cross-link repair pathways, and ionizing
radiation, which causes double-strand breaks (DSBs).158,160 Indeed,
tumors beyond those associated with BRCA1/2 mutations exhibiting
defective homologous recombination (HRD), as determined by various
DNA-based measures of genomic instability such as loss of
heterozygosity (LOH), telomeric allelic imbalance, and large-scale state
transitions, also are relatively more sensitive to platinum agents and
potentially other DNA repair–targeted agents as discussed later.161
Strategies to target the three major proteins that trigger the DNA
damage response signaling pathways—ATM, ATR, and DNA-PK—have
identified small-molecule inhibitors with provocative preclinical activity
in a variety of tumor types, particularly when in combination with
classic DNA-damaging chemotherapy or ionizing radiation. A number
of these are currently in phase I trials.157
Another very exciting finding relates to the selective activity of
inhibitors of poly (ADP-ribose) polymerase (PARP) in tumors deficient
for BRCA1/2.162,163 PARP1 is the first described member of a large family
of enzymes that can detect and repair DNA base damage. After DNA
damage, PARP1 is recruited and binds to the damaged DNA with a
subsequent increase in catalytic activity that results in the formation
of polymer chains through use of the substrate NAD+, resulting in
recruitment of the BER machinery to the site of the DNA damage and
relaxation of the chromatin structure to facilitate repair.164 Two primary
therapeutic strategies employing PARP inhibitors in the treatment of
DNA Damage Response Pathways and Cancer  •  CHAPTER 11 163
cancer are currently under investigation; the first is the use of PARP
inhibitors as sensitizers to DNA-damaging chemotherapy or radiation,
and the second approach exploits specific genetic characteristics of
certain cancers that leave them vulnerable to DNA damage via the
mechanism of chemical “synthetic lethality.”165 Because many effective
cytotoxic chemotherapies and ionizing radiotherapy exert their
antitumor effect through production of DNA damage, it was
hypothesized that interference with cellular DNA repair through use of
PARP inhibitors may mitigate repair of this injury, resulting in decreased
treatment resistance. Indeed, preclinical and clinical studies have
demonstrated potentiation or synergy of PARP inhibitors with
DNA-damaging radiotherapy or chemotherapeutic drugs such as
methylating agents, platinums, and topoisomerase inhibitors.58,151,166
Even more compelling was the application of the idea of synthetic
lethality from basic experimental genetics to cancer therapeutics.167–169
A synthetic lethal interaction describes the scenario in which a
mutation in either of two genes alone has no phenotypic effect, but
the combination of both mutations results in death of the cell. Tumors
arising in patients with a germline BRCA1/2 mutation are associated
with reduced DNA repair capacity owing to the loss of BRCA function
and are hypothesized to rely more heavily on alternate compensatory
DNA repair processes for survival. PARP is required for the repair of
oxidative DNA damage–associated single-strand DNA breaks in its
central role in base excision repair, and if PARP is inhibited, repair-
associated breaks result in replication fork-mediated DSB formation,
which require BRCA1/2-associated recombination to resolve. Thus, in
tumors lacking intact BRCA function owing to loss of its second allele,
inhibition of PARP with a small molecule is postulated to be the second
hit that renders the cell incapable of survival and mimics the situation
of genetic absence of PARP. It has also become evident that PARP
trapping on DNA single-strand breaks causes replication fork stalling
and collapse and leads to DSBs.170 However, in the host’s somatic
tissues heterozygous for a BRCA mutation and maintaining normal
BRCA protein function, PARP inhibitors are thought to have little or no
effect. This concept of chemical synthetic lethality with use of PARP
inhibitors in DNA repair–deficient cancers has generated tremendous
enthusiasm and fueled the rapid development of clinical investigation
in this area, particularly in BRCA mutation–associated breast and
ovarian cancer, and phenotypically related triple-negative breast
cancer.165,171 Early clinical trials of several PARP inhibitors showed
durable antitumor responses in patients with advanced germline
BRCA1/2-mutated ovarian, breast, and castration-resistant prostate
cancer, resulting in clinical registration of olaparib (Lynparza;
AstraZeneca) and rucaparib (Clovis).157,172 Additional potent and
selective PARP inhibitors are in late-phase monotherapy and
combination clinical trial development, including niraparib (Tesaro),
talazoparib (Pfizer), and veliparib (AbbVie). Of particular interest will be
the activity of these agents in tumors with mutations in DSB repair
pathway genes other than BRCA1/2 (e.g., ATM, PALB2, BRIP1) or even
HRD without identified causative gene mutations, being discovered
through increasing tumor genomic profiling efforts. Finally, as with all
new therapeutic advances in cancer, tumor resistance soon arises. A
fascinating mechanism for resistance to PARP inhibitors for BRCA1/2-
mutant tumors is genetic reversion events that restore the normal
coding sequence, often through small deletions of the mutated
sequence, and result in improved DNA repair.173,174
164 Part I: Science and Clinical Oncology
a major factor driving the onset and progression of carcinogenesis.4
Overlapping and interacting mechanisms for DNA repair and the
cellular response to DNA damage are critical components for the
maintenance of genomic stability. Alterations in these pathways often
are early events in the multistep acquisition of genetic mutations
leading to cancer development. In addition, mutations in DNA damage
repair and signaling pathways are common in human tumors and offer
KEY REFERENCES
4. Hanahan D, Weinberg RA. Hallmarks of cancer:
the next generation. Cell. 2011;144:646–674.
6. Loeb LA, Bielas JH, Beckman RA. Cancers exhibit
a mutator phenotype: clinical implications. Cancer
Res. 2008;68:3551–3557, discussion 7.
7. Bodmer W, Bielas JH, Beckman RA. Genetic
instability is not a requirement for tumor develop-
ment. Cancer Res. 2008;68:3558–3560, discussion
60–1.
8. Ciccia A, Elledge SJ. The DNA damage response:
making it safe to play with knives. Mol Cell. 2010;40:
179–204.
9. Hoeijmakers JH. DNA damage, aging, and cancer.
N Engl J Med. 2009;361:1475–1485.
10. Ford JM, Hanawalt PC. Role of DNA excision
repair gene defects in the etiology of cancer. Curr
Top Microbiol Immunol. 1997;221:47–70.
13. Mellon I, Spivak G, Hanawalt PC. Selective removal
of transcription-blocking DNA damage from the
transcribed strand of the mammalian DHFR gene.
Cell. 1987;51:241–249.
15. Ljungman M, Zhang F. Blockage of RNA polymerase
as a possible trigger for UV light-induced apoptosis.
Oncogene. 1996;13:823–831.
16. Nelson WG, Kastan MB. DNA strand breaks: the DNA
template alterations that trigger p53-dependent DNA
damage response pathways. Mol Cell Biol. 1994;14:
1815–1823.
18. Sale JE, Lehmann AR, Woodgate R. Y-family DNA
polymerases and their role in tolerance of cellular
DNA damage. Nat Rev Mol Cell Biol. 2012;13:
141–152.
21. Hartwell LH, Kastan MB. Cell cycle control and
cancer. Science. 1994;266:1821–1828.
22. Ford JM, Hanawalt PC. Li-Fraumeni syndrome
fibroblasts homozygous for p53 mutations are
deficient in global DNA repair but exhibit normal
transcription-coupled repair and enhanced UV resis-
tance. Proc Natl Acad Sci USA. 1995;92:8876–8880.
25. Adimoolam S, Ford JM. p53 and DNA damage-
inducible expression of the xeroderma pigmentosum
group C gene. Proc Natl Acad Sci USA. 2002;19:
12985–12990.
27. Scully R, Chen J, Ochs RL, et al. Dynamic
changes of BRCA1 subnuclear location and phos-
phorylation state are initiated by DNA damage.
Cell. 1997;90:425–435.
30. Lim DS, Kim ST, Xu B, et al. ATM phosphorylates
p95/nbs1 in an S-phase checkpoint pathway. Nature.
2000;404:613–617.
32. Fitch ME, Nakajima S, Yasui A, Ford JM. In vivo
recruitment of XPC to UV-induced cyclobutane
pyrimidine dimers by the DDB2 gene product.
J Biol Chem. 2003;278:46906–46910.
opportunities for development of more selective and more effective
anticancer therapies. Continued exploration of the DNA damage
response will prove important for our improved understanding of cancer
etiology, prevention, genetic susceptibility, diagnosis, and treatment.
The complete reference list is available online at
ExpertConsult.com.
37. Lowe SW, Ruley HE, Jacks T, Housman DE.
p53-dependent apoptosis modulates the cytotoxicity
of anticancer agents. Cell. 1993;74:957–967.
40. Canman CE, Lim DS, Cimprich KA, et al. Activation
of the ATM kinase by ionizing radiation and phos-
phorylation of p53. Science. 1998;281:1677–1679.
41. Kastan MB, Lim DS. The many substrates and
functions of ATM. Nat Rev Mol Cell Biol. 2000;1:
179–186.
47. Xu B, O’Donnell AH, Kim ST, Kastan MB. Phos-
phorylation of serine 1387 in Brca1 is specifically
required for the Atm-mediated S-phase checkpoint
after ionizing irradiation. Cancer Res. 2002;62:
4588–4591.
48. Bakkenist CJ, Kastan MB. DNA damage activates
ATM through intermolecular autophosphorylation
and dimer dissociation. Nature. 2003;421:499–506.
51. Spivak G. Nucleotide excision repair in humans.
DNA Repair (Amst). 2015;36:13–18.
53. de Laat WL, Jaspers NG, Hoeijmakers JH. Molecular
mechanism of nucleotide excision repair. Genes Dev.
1999;13:768–785.
55. Spivak G, Ganesan AK. The complex choreography
of transcription-coupled repair. DNA Repair (Amst).
2014;19:64–70.
56. Hartman AR, Ford JM. BRCA1 induces DNA
damage recognition factors and enhances nucleotide
excision repair. Nat Genet. 2002;32:180–184.
61. Masutani C, Kusumoto R, Yamada A, et al. The
XPV (xeroderma pigmentosum variant) gene encodes
human DNA polymerase eta. Nature. 1999;399:
700–704.
63. Cleaver JE. Defective repair replication of DNA
in xeroderma pigmentosum. Nature. 1968;218:
652–656.
80. Al-Tassan N, Chmiel NH, Maynard J, et al.
Inherited variants of MYH associated with somatic
G:C→T:A mutations in colorectal tumors. Nat
Genet. 2002;30:227–232.
81. Jones S, Emmerson P, Maynard J, et al. Biallelic
germline mutations in MYH predispose to multiple
colorectal adenoma and somatic G:C→T:A muta-
tions. Hum Mol Genet. 2002;11:2961–2967.
83. Cheadle JP, Sampson JR. MUTYH-associated
polyposis—from defect in base excision repair to
clinical genetic testing. DNA Repair (Amst). 2007;6:
274–279.
85. Bohr VA. Repair of oxidative DNA damage in nuclear
and mitochondrial DNA, and some changes with aging
in mammalian cells. Free Radic Biol Med. 2002;32:
804–812.
96. Papadopoulos N, Nicolaides NC, Wei YF, et al.
Mutation of a mutL homolog in hereditary colon
cancer. Science. 1994;263:1625–1629.
99. Lynch HT, de la Chapelle A. Hereditary colorectal
cancer. N Engl J Med. 2003;348:919–932.
105. Gryfe R, Kim H, Hsieh ET, et al. Tumor micro-
satellite instability and clinical outcome in young
patients with colorectal cancer. N Engl J Med. 2000;
342:69–77.
109. Hampel H, Frankel WL, Martin E, et al. Feasibility
of screening for Lynch syndrome among patients with
colorectal cancer. J Clin Oncol. 2008;26:5783–5788.
110. Ladabaum U, Wang G, Terdiman J, et al. Strategies
to identify the Lynch syndrome among patients with
colorectal cancer: a cost-effectiveness analysis. Ann
Intern Med. 2011;155:69–79.
113. Lee V, Murphy A, Le DT, Diaz LA Jr. Mismatch
repair deficiency and response to immune checkpoint
blockade. Oncologist. 2016;21:1200–1211.
114. Vogelstein B, Papadopoulos N, Velculescu VE,
Zhou S, Diaz LA Jr, Kinzler KW. Cancer genome
landscapes. Science. 2013;339:1546–1558.
116. Le DT, Uram JN, Wang H, et al. PD-1 blockade
in tumors with mismatch-repair deficiency. N Engl
J Med. 2015;372:2509–2520.
132. Hollstein M, Sidranksky D, Vogelstein B, Harris
CC. p53 mutations in human cancers. Science. 1991;
253:49–53.
135. Sengupta S, Harris CC. p53: traffic cop at the
crossroads of DNA repair and recombination. Nat
Rev Mol Cell Biol. 2005;6:44–55.
144. Kurian AW, Hare EE, Mills MA, et al. Clinical
evaluation of a multiple-gene sequencing panel
for hereditary cancer risk assessment. J Clin Oncol.
2014;32:2001–2009.
146. Xu B, Kim S, Kastan MB. Involvement of Brca1 in
S-phase and G(2)-phase checkpoints after ionizing
irradiation. Mol Cell Biol. 2001;21:3445–3450.
153. Alli E, Sharma VB, Sunderesakumar P, Ford JM.
Defective repair of oxidative DNA damage in
triple-negative breast cancer confers sensitivity to
inhibition of poly(ADP-ribose) polymerase. Cancer
Res. 2009;69:3589–3596.
157. Brown JS, O’Carrigan B, Jackson SP, Yap TA.
Targeting DNA repair in cancer: beyond PARP
inhibitors. Cancer Discov. 2017;7:20–37.
158. Lord CJ, Ashworth A. The DNA damage response
and cancer therapy. Nature. 2012;481:287–294.
163. Farmer H, McCabe N, Lord CJ, et al. Targeting
the DNA repair defect in BRCA mutant cells as a
therapeutic strategy. Nature. 2005;434:917–921.
177. D’Andrea AD. Susceptibility pathways in Fanconi’s
anemia and breast cancer. N Engl J Med. 2010;
362:1909–1919.

REFERENCES
1. Gray J, Druker B. Genomics: the breast cancer
landscape. Nature. 2012;486:328–329.
2. Comprehensive molecular characterization of human
colon and rectal cancer. Nature. 2012;487:330–337.
3. Nik-Zainal S, Davies H, Staaf J, et al. Landscape
of somatic mutations in 560 breast cancer whole-
genome sequences. Nature. 2016;534:47–54.
4. Hanahan D, Weinberg RA. Hallmarks of cancer:
the next generation. Cell. 2011;144:646–674.
5. Loeb LA, Loeb KR, Anderson JP. Multiple mutations
and cancer. Proc Natl Acad Sci USA. 2003;100:
776–781.
6. Loeb LA, Bielas JH, Beckman RA. Cancers exhibit
a mutator phenotype: clinical implications. Cancer
Res. 2008;68:3551–3557, discussion 7.
7. Bodmer W, Bielas JH, Beckman RA. Genetic
instability is not a requirement for tumor develop-
ment. Cancer Res. 2008;68:3558–3560, discussion
60–1.
8. Ciccia A, Elledge SJ. The DNA damage response:
making it safe to play with knives. Mol Cell. 2010;40:
179–204.
9. Hoeijmakers JH. DNA damage, aging, and cancer.
N Engl J Med. 2009;361:1475–1485.
10. Ford JM, Hanawalt PC. Role of DNA excision
repair gene defects in the etiology of cancer. Curr
Top Microbiol Immunol. 1997;221:47–70.
11. Bohr VA, Smith CA, Okumoto DS, Hanawalt PC.
DNA repair in an active gene: removal of pyrimidine
dimers from the DHFR gene of CHO cells is much
more efficient than in the genome overall. Cell.
1985;40:359–369.
12. Mellon I, Bohr VA, Smith CA, Hanawalt PC.
Preferential DNA repair of an active gene in human
cells. Proc Natl Acad Sci USA. 1986;83:8878–8882.
13. Mellon I, Spivak G, Hanawalt PC. Selective removal
of transcription-blocking DNA damage from the
transcribed strand of the mammalian DHFR gene.
Cell. 1987;51:241–249.
14. Yamaizumi M, Sugano T. UV-induced nuclear
accumulation of p53 is evoked through DNA
damage of actively transcribed genes independent
of the cell cycle. Oncogene. 1994;9:2775–2784.
15. Ljungman M, Zhang F. Blockage of RNA polymerase
as a possible trigger for UV light-induced apoptosis.
Oncogene. 1996;13:823–831.
16. Nelson WG, Kastan MB. DNA strand breaks: the
DNA template alterations that trigger p53-dependent
DNA damage response pathways. Mol Cell Biol.
1994;14:1815–1823.
17. Ljungman M, Lane DP. Transcription—guarding the
genome by sensing DNA damage. Nat Rev Cancer.
2004;4:727–737.
18. Sale JE, Lehmann AR, Woodgate R. Y-family DNA
polymerases and their role in tolerance of cellular
DNA damage. Nat Rev Mol Cell Biol. 2012;13:
141–152.
19. Wickramasinghe CM, Arzouk H, Frey A, Maiter
A, Sale JE. Contributions of the specialised DNA
polymerases to replication of structured DNA. DNA
Repair (Amst). 2015;29:83–90.
20. Wood RD, Doublie S. DNA polymerase theta
(POLQ), double-strand break repair, and cancer.
DNA Repair (Amst). 2016;44:22–32.
21. Hartwell LH, Kastan MB. Cell cycle control and
cancer. Science. 1994;266:1821–1828.
22. Ford JM, Hanawalt PC. Li-Fraumeni syndrome
fibroblasts homozygous for p53 mutations are
deficient in global DNA repair but exhibit normal
transcription-coupled repair and enhanced UV resis-
tance. Proc Natl Acad Sci USA. 1995;92:8876–8880.
23. Ford JM, Hanawalt PC. Expression of wild-type p53
is required for efficient global genomic nucleotide
excision repair in UV-irradiated human fibroblasts.
J Biol Chem. 1997;272:28073–28080.
24. Hwang BJ, Ford JM, Hanawalt PC, Chu G. Expres-
sion of the p48 xeroderma pigmentosum gene is
DNA Damage Response Pathways and Cancer  •  CHAPTER 11 164.e1
p53-dependent and is involved in global genomic
repair. Proc Natl Acad Sci USA. 1999;96:424–428.
25. Adimoolam S, Ford JM. p53 and DNA damage-
inducible expression of the xeroderma pigmen-
tosum group C gene. Proc Natl Acad Sci USA.
2002;19:12985–12990.
26. Adimoolam S, Ford JM. p53 and regulation of DNA
damage recognition during nucleotide excision repair.
DNA Repair (Amst). 2003;2:947–954.
27. Scully R, Chen J, Ochs RL, et al. Dynamic
changes of BRCA1 subnuclear location and phos-
phorylation state are initiated by DNA damage.
Cell. 1997;90:425–435.
28. Scully R, Chen J, Plug A, et al. Association of
BRCA1 with Rad51 in mitotic and meiotic cells.
Cell. 1997;88:265–275.
29. Cortez D, Wang Y, Qin J, Elledge SJ. Requirement
of ATM-dependent phosphorylation of brca1 in
the DNA damage response to double-strand breaks.
Science. 1999;286:1162–1166.
30. Lim DS, Kim ST, Xu B, et al. ATM phosphorylates
p95/nbs1 in an S-phase checkpoint pathway. Nature.
2000;404:613–617.
31. Fitch ME, Cross IV, Ford JM. p53 responsive
nucleotide excision repair gene products p48 and
XPC, but not p53, localize to sites of UV-irradiation-
induced DNA damage, in vivo. Carcinogenesis.
2003;24:843–850.
32. Fitch ME, Nakajima S, Yasui A, Ford JM. In vivo
recruitment of XPC to UV-induced cyclobutane
pyrimidine dimers by the DDB2 gene product.
J Biol Chem. 2003;278:46906–46910.
33. Smith ML, Fornace AJ Jr. Mammalian DNA damage-
inducible genes associated with growth arrest and
apoptosis. Mutat Res. 1996;340:109–124.
34. Fornace AJ Jr, Amundson SA, Bittner M, et al. The
complexity of radiation stress responses: analysis by
informatics and functional genomics approaches.
Gene Expr. 1999;7:387–400.
35. Hastak K, Adimoolam S, Trinklein ND, Myers
RM, Ford JM. Identification of a functional in
vivo p53 response element in the coding sequence
of the xeroderma pigmentosum group C gene. Genes
Cancer. 2012;2:1–10.
36. Lowe SW, Schmitt EM, Smith SW, Osborne BA,
Jacks T. p53 is required for radiation-induced apopto-
sis in mouse thymocytes. Nature. 1993;362:847–849.
37. Lowe SW, Ruley HE, Jacks T, Housman DE.
p53-dependent apoptosis modulates the cytotoxicity
of anticancer agents. Cell. 1993;74:957–967.
38. Clarke AR, Purdie CA, Harrison DJ, et al. Thy-
mocyte apoptosis induced by p53-dependent and
independent pathways. Nature. 1993;362:849–852.
39. Cline SD, Hanawalt PC. Who’s on first in the cellular
response to DNA damage? Nat Rev Mol Cell Biol.
2003;4:361–373.
40. Canman CE, Lim DS, Cimprich KA, et al. Activation
of the ATM kinase by ionizing radiation and phos-
phorylation of p53. Science. 1998;281:1677–1679.
41. Kastan MB, Lim DS. The many substrates and
functions of ATM. Nat Rev Mol Cell Biol. 2000;1:
179–186.
42. Shieh SY, Ahn J, Tamai K, Taya Y, Prives C.
The human homologs of checkpoint kinases
Chk1 and Cds1 (Chk2) phosphorylate p53 at
multiple DNA damage-inducible sites. Genes Dev.
2000;14:289–300.
43. Matsuoka S, Huang M, Elledge SJ. Linkage of ATM
to cell cycle regulation by the Chk2 protein kinase.
Science. 1998;282:1893–1897.
44. Liu Q, Guntuku S, Cui XS, et al. Chk1 is an essential
kinase that is regulated by Atr and required for
the G(2)/M DNA damage checkpoint. Genes Dev.
2000;14:1448–1459.
45. Shiloh Y. ATM and related protein kinases: safe-
guarding genome integrity. Nat Rev Cancer. 2003;
3:155–168.
164.e1
46. Tibbetts RS, Brumbaugh KM, Williams JM, et al.
A role for ATR in the DNA damage-induced
phosphorylation of. Genes Dev. 1999;13(152–7):
p53.
47. Xu B, O’Donnell AH, Kim ST, Kastan MB.
Phosphorylation of serine 1387 in Brca1 is spe-
cifically required for the Atm-mediated S-phase
checkpoint after ionizing irradiation. Cancer Res.
2002;62:4588–4591.
48. Bakkenist CJ, Kastan MB. DNA damage activates
ATM through intermolecular autophosphorylation
and dimer dissociation. Nature. 2003;421:499–506.
49. Ford JM. Regulation of DNA damage recognition
and nucleotide excision repair: another role for p53.
Mutat Res. 2005;577:195–202.
50. Friedberg EC, Walker G, Siede W, Wood RD, Schultz
R, Ellenburger T. DNA Repair and Mutagenesis. New
York: ASM Press; 2006.
51. Spivak G. Nucleotide excision repair in humans.
DNA Repair (Amst). 2015;36:13–18.
52. Wood RD. Nucleotide excision repair in mammalian
cells. J Biol Chem. 1997;272:23465–23468.
53. de Laat WL, Jaspers NG, Hoeijmakers JH. Molecular
mechanism of nucleotide excision repair. Genes Dev.
1999;13:768–785.
54. Wood RD, Mitchell M, Sgouros J, Lindahl T. Human
DNA repair genes. Science. 2001;291:1284–1289.
55. Spivak G, Ganesan AK. The complex choreography
of transcription-coupled repair. DNA Repair (Amst).
2014;19:64–70.
56. Hartman AR, Ford JM. BRCA1 induces DNA
damage recognition factors and enhances nucleotide
excision repair. Nat Genet. 2002;32:180–184.
57. Lin PS, McPherson LA, Chen AY, Sage J, Ford
JM. The role of the retinoblastoma/E2F1 tumor
suppressor pathway in the lesion recognition step
of nucleotide excision repair. DNA Repair (Amst).
2009;8:795–802.
58. Alli E, Sharma VB, Hartman AR, Lin PS, McPherson
L, Ford JM. Enhanced sensitivity to cisplatin and
gemcitabine in Brca1-deficient murine mammary
epithelial cells. BMC Pharmacol. 2011;11:7.
59. Friedberg EC, Wagner R, Radman M. Specialized
DNA polymerases, cellular survival, and the genesis
of mutations. Science. 2002;296:1627–1630.
60. Kunkel TA. Considering the cancer consequences
of altered DNA polymerase function. Cancer Cell.
2003;3:105–110.
61. Masutani C, Kusumoto R, Yamada A, et al. The
XPV (xeroderma pigmentosum variant) gene encodes
human DNA polymerase eta. Nature. 1999;399:
700–704.
62. Johnson RE, Kondratick CM, Prakash S, Prakash L.
hRAD30 mutations in the variant form of xeroderma
pigmentosum. Science. 1999;285:263–265.
63. Cleaver JE. Defective repair replication of DNA
in xeroderma pigmentosum. Nature. 1968;218:
652–656.
64. Kraemer KH, Lee MM, Scotto J. DNA repair
protects against cutaneous and internal neoplasia:
evidence from xeroderma pigmentosum. Carcino-
genesis. 1984;5:511–514.
65. Cleaver JE, Kraemer KH. Xeroderma pigmentosum.
In: Scriver CR, Beudet AL, Sly WS, Valle D, eds.
Metabolic Basis of Inherited Disease. New York:
McGraw-Hill; 1989:2949–2971.
66. Kraemer KH, Slor H. Xeroderma pigmentosum.
Clin Dermatol. 1985;3:33–69.
67. Robbins JH. Xeroderma pigmentosum: defective
DNA repair causes skin cancer and neurodegenera-
tion. JAMA. 1988;260:384–388.
68. Wang YC, Maher VM, Mitchell DL, McCormick
JJ. Evidence from mutation spectra that the UV
hypermutability of xeroderma pigmentosum variant
cells reflects abnormal, error-prone replication on a
template containing photoproducts. Mol Cell Biol.
1993;13:4276–4283.
164.e2 Part I: Science and Clinical Oncology
69. Schmickel RD, Chu EHY, Trosko JE. Cockayne
syndrome: a cellular sensitivity to ultraviolet light.
Pediatrics. 1977;60:135–139.
70. Venema J, Mullenders LHF, Natarajan AT, Van
Zeeland AA, Mayne LV. The genetic defect in Cock-
ayne syndrome is associated with a defect in repair of
UV-induced DNA damage in transcriptionally active
DNA. Proc Natl Acad Sci USA. 1990;87:4707–4711.
71. Cooper PK, Nouspikel T, Clarkson SG, Leadon SA.
Defective transcription-coupled repair of oxidative
base damage in Cockayne syndrome patients from
XP group G. Science. 1997;275:990–993.
72. Timme TL, Moses RE. Review: diseases with
DNA damage-processing defects. Am J Med Sci.
1988;295:40–48.
73. Nance MA, Berry SA. Cockayne Syndrome: review
of 140 cases. Am J Med Gen. 1992;42:68–84.
74. Robbins JH, Kraemer KH, Lutzner MA, Festoff BW,
Coon HG. Xeroderma pigmentosum: an inherited
disease with sun sensitivity, multiple cutaneous
neoplasms and abnormal repair. Ann Intern Med.
1974;80:221–228.
75. Vermeulen W, Scott RJ, Rodgers S, et al. Clinical
heterogeneity within xeroderma pigmentosum
associated with mutations in the DNA repair and
trasncription gene ERCC3. Am J Hum Genet.
1994;54:191–200.
76. Cleaver JE, Lam ET, Revet I. Disorders of nucleotide
excision repair: the genetic and molecular basis of
heterogeneity. Nat Rev Genet. 2009;10:756–768.
77. Lehmann AR. The xeroderma pigmentosum group D
(XPD) gene: one gene, two functions, three diseases.
Genes Dev. 2001;15:15–23.
78. Demple B, Harrison L. Repair of oxidative damage to
DNA: enzymology and biology. Annu Rev Biochem.
1994;63:915–948.
79. Seeberg E, Eide L, Bjoras M. The base excision repair
pathway. Trends Biochem Sci. 1995;20:391–397.
80. Al-Tassan N, Chmiel NH, Maynard J, et al.
Inherited variants of MYH associated with somatic
G:C→T:A mutations in colorectal tumors. Nat
Genet. 2002;30:227–232.
81. Jones S, Emmerson P, Maynard J, et al. Biallelic
germline mutations in MYH predispose to multiple
colorectal adenoma and somatic G:C→T:A muta-
tions. Hum Mol Genet. 2002;11:2961–2967.
82. Halford SE, Rowan AJ, Lipton L, et al. Germline
mutations but not somatic changes at the MYH locus
contribute to the pathogenesis of unselected colorec-
tal cancers. Am J Pathol. 2003;162:1545–1548.
83. Cheadle JP, Sampson JR. MUTYH-associated
polyposis—from defect in base excision repair
to clinical genetic testing. DNA Repair (Amst).
2007;6:274–279.
84. Le Page F, Kwoh EE, Avrutskaya A, et al. Transcrip-
tion-coupled repair of 8-oxoguanine: requirement
for XPG, TFIIH, and CSB and implications for
Cockayne syndrome. Cell. 2000;101:159–171.
85. Bohr VA. Repair of oxidative DNA damage in
nuclear and mitochondrial DNA, and some changes
with aging in mammalian cells. Free Radic Biol Med.
2002;32:804–812.
86. Morland I, Rolseth V, Luna L, Rognes T, Bjoras
M, Seeberg E. Human DNA glycosylases of the
bacterial Fpg/MutM superfamily: an alternative
pathway for the repair of 8-oxoguanine and other
oxidation products in DNA. Nucleic Acids Res.
2002;30:4926–4936.
87. Goodenberger M, Lindor NM. Lynch syndrome
and MYH-associated polyposis: review and testing
strategy. J Clin Gastroenterol. 2011;45:488–500.
88. Kiyohara C, Takayama K, Nakanishi Y. Association
of genetic polymorphisms in the base excision repair
pathway with lung cancer risk: a meta-analysis. Lung
Cancer. 2006;54:267–283.
89. Hung RJ, Hall J, Brennan P, Boffetta P. Genetic
polymorphisms in the base excision repair pathway
and cancer risk: a HuGE review. Am J Epidemiol.
2005;162:925–942.
90. Kolodner R. Biochemistry and genetics of eukaryotic
mismatch repair. Genes Dev. 1996;10:1433–1442.
91. Kolodner RD, Marsischky GT. Eukaryotic DNA
mismatch repair. Curr Opin Genet Dev. 1999;9:
89–96.
92. Jiricny J, Nystrom-Lahti M. Mismatch repair
defects in cancer. Curr Opin Genet Dev. 2000;10:
157–161.
93. Peltomaki P. Role of DNA mismatch repair defects
in the pathogenesis of human cancer. J Clin Oncol.
2003;21:1174–1179.
94. Fishel R, Lescoe MK, Rao MRS, et al. The human
mutator gene homolog MSH2 and its association
with hereditary nonpolyposis colon cancer. Cell.
1993;75:1027–1038.
95. Hemminki A, Peltomaki P, Mecklin JP, et al.
Loss of the wild type MLH1 gene is a feature of
hereditary nonpolyposis colorectal cancer. Nat Genet.
1994;8:405–410.
96. Papadopoulos N, Nicolaides NC, Wei YF, et al.
Mutation of a mutL homolog in hereditary colon
cancer. Science. 1994;263:1625–1629.
97. Karran P, Bignami M. DNA damage tolerance,
mismatch repair and genome instability. Bioessays.
1994;16:833–839.
98. Lynch HT, Smyrk T. Hereditary nonpolyposis
colorectal cancer (Lynch syndrome). An updated
review. Cancer. 1996;78:1149–1167.
99. Lynch HT, de la Chapelle A. Hereditary colorectal
cancer. N Engl J Med. 2003;348:919–932.
100. Parsons R, Li GM, Longley MJ, et al. Hypermutabil-
ity and mismatch repair deficiency in RER+ tumor
cells. Cell. 1993;75:1227–1236.
101. Thibodeau SN, Bren G, Schaid D. Microsatellite
instability in cancer of the proximal colon. Science.
1993;260:816–819.
102. Kuismanen SA, Holmberg MT, Salovaara R, et al.
Epigenetic phenotypes distinguish microsatellite-
stable and -unstable colorectal cancers. Proc Natl
Acad Sci USA. 1999;96:12661–12666.
103. Nakagawa H, Nuovo GJ, Zervos EE, et al.
Age-related hypermethylation of the 5′ region of
MLH1 in normal colonic mucosa is associated with
microsatellite-unstable colorectal cancer develop-
ment. Cancer Res. 2001;61:6991–6995.
104. Ford JM, Whittemore AS. Predicting and
preventing hereditary colorectal cancer. JAMA.
2006;296:1521–1523.
105. Gryfe R, Kim H, Hsieh ET, et al. Tumor micro-
satellite instability and clinical outcome in young
patients with colorectal cancer. N Engl J Med.
2000;342:69–77.
106. Hemminki A, Mecklin JP, Jarvinen H, Aaltonen LA,
Joensuu H. Microsatellite instability is a favorable
prognostic indicator in patients with colorectal
cancer receiving chemotherapy. Gastroenterology.
2000;119:921–928.
107. Samowitz WS, Curtin K, Ma KN, et al. Microsatellite
instability in sporadic colon cancer is associated with
an improved prognosis at the population level. Cancer
Epidemiol Biomarkers Prev. 2001;10:917–923.
108. Ji H, Kumm J, Zhang M, et al. Molecular inver-
sion probe analysis of gene copy alterations reveals
distinct categories of colorectal carcinoma. Cancer
Res. 2006;66:7910–7919.
109. Hampel H, Frankel WL, Martin E, et al. Feasibility
of screening for Lynch syndrome among patients with
colorectal cancer. J Clin Oncol. 2008;26:5783–5788.
110. Ladabaum U, Wang G, Terdiman J, et al. Strategies
to identify the Lynch syndrome among patients with
colorectal cancer: a cost-effectiveness analysis. Ann
Intern Med. 2011;155:69–79.
111. Hodi FS, O’Day SJ, McDermott DF, et al. Improved
survival with ipilimumab in patients with metastatic
melanoma. N Engl J Med. 2010;363:711–723.
112. Chabanon RM, Pedrero M, Lefebvre C, Marabelle
A, Soria JC, Postel-Vinay S. Mutational landscape
and sensitivity to immune checkpoint blockers. Clin
Cancer Res. 2016;22:4309–4321.
113. Lee V, Murphy A, Le DT, Diaz LA Jr. Mismatch
repair deficiency and response to immune checkpoint
blockade. Oncologist. 2016;21:1200–1211.
114. Vogelstein B, Papadopoulos N, Velculescu VE,
Zhou S, Diaz LA Jr, Kinzler KW. Cancer genome
landscapes. Science. 2013;339:1546–1558.
115. Kim H, Jen J, Vogelstein B, Hamilton SR. Clinical
and pathological characteristics of sporadic colorectal
carcinomas with DNA replication errors in micro-
satellite sequences. Am J Pathol. 1994;145:148–156.
116. Le DT, Uram JN, Wang H, et al. PD-1 Blockade
in tumors with mismatch-repair deficiency. N Engl
J Med. 2015;372:2509–2520.
117. Bell DW, Varley JM, Szydlo TE, et al. Heterozygous
germ line hCHK2 mutations in Li-Fraumeni
syndrome. Science. 1999;286:2528–2531.
118. Meijers-Heijboer H, van den Ouweland A, Klijn J,
et al. Low-penetrance susceptibility to breast cancer
due to CHEK2(*)1100delC in noncarriers of BRCA1
or BRCA2 mutations. Nat Genet. 2002;31:55–59.
119. Vahteristo P, Bartkova J, Eerola H, et al. A CHEK2
genetic variant contributing to a substantial frac-
tion of familial breast cancer. Am J Hum Genet.
2002;71:432–438.
120. Kastan MB, Onyekwere O, Sidransky D, Vogelstein
B, Craig RW. Participation of p53 protein in the
cellular response to DNA damage. Cancer Res.
1991;51:6304–6311.
121. Kuerbitz SJ, Plunkett BS, Walsh WV, Kastan MB.
Wild-type p53 is a cell cycle checkpoint determinant
following irradiation. Proc Natl Acad Sci USA.
1992;89:7491–7495.
122. Khanna KK, Keating KE, Kozlov S, et al. ATM
associates with and phosphorylates p53: mapping the
region of interaction. Nat Genet. 1998;20:398–400.
123. Banin S, Moyal L, Shieh S, et al. Enhanced phos-
phorylation of p53 by ATM in response to DNA
damage. Science. 1998;281:1674–1677.
124. O’Driscoll M, Jeggo PA. The role of double-strand
break repair—insights from human genetics. Nat
Rev Genet. 2006;7:45–54.
125. Gatti RA, Tward A, Concannon P. Cancer risk
in ATM heterozygotes: a model of phenotypic
and mechanistic differences between missense
and truncating mutations. Mol Genet Metab.
1999;68:419–423.
126. Dork T, Bendix R, Bremer M, et al. Spectrum of
ATM gene mutations in a hospital-based series
of unselected breast cancer patients. Cancer Res.
2001;61:7608–7615.
127. Scott SP, Bendix R, Chen P, Clark R, Dork T, Lavin
MF. Missense mutations but not allelic variants alter
the function of ATM by dominant interference in
patients with breast cancer. Proc Natl Acad Sci USA.
2002;99:925–930.
128. Carney JP, Maser RS, Olivares H, et al. The
hMre11/hRad50 protein complex and Nijmegen
breakage syndrome: linkage of double-strand break
repair to the cellular DNA damage response. Cell.
1998;93:477–486.
129. Stewart GS, Maser RS, Stankovic T, et al. The DNA
double-strand break repair gene hMRE11 is mutated
in individuals with an ataxia-telangiectasia-like
disorder. Cell. 1999;99:577–587.
130. Schneider JG, Finck BN, Ren J, et al. ATM-
dependent suppression of stress signaling reduces
vascular disease in metabolic syndrome. Cell Metab.
2006;4:377–389.
131. Valentin-Vega YA, Maclean KH, Tait-Mulder J, et al.
Mitochondrial dysfunction in ataxia-telangiectasia.
Blood. 2012;119:1490–1500.
132. Hollstein M, Sidranksky D, Vogelstein B, Harris
CC. p53 mutations in human cancers. Science.
1991;253:49–53.
133. Kastan MB, Radin AI, Kuerbitz SJ, et al. Levels of
p53 protein increase with maturation in human
hematopoietic cells. Cancer Res. 1991;51:4279–4286.
134. Kastan MB, Zhan Q, El-Deiry WS, et al. A mam-
malian cell cycle checkpoint pathway utilizing p53

and GADD45 is defective in ataxia-telangiectasia.
Cell. 1992;71:587–597.
135. Sengupta S, Harris CC. p53: traffic cop at the
crossroads of DNA repair and recombination. Nat
Rev Mol Cell Biol. 2005;6:44–55.
136. Oren M. Regulation of the p53 tumor suppressor
protein. J Biol Chem. 1999;274:36031–36034.
137. Takagi M, Absalon MJ, McLure KG, Kastan MB.
Regulation of p53 translation and induction after
DNA damage by ribosomal protein L26 and
nucleolin. Cell. 2005;123:49–63.
138. Chen J, Kastan MB. 5′-3′-UTR interactions regulate
p53 mRNA translation and provide a target for
modulating p53 induction after DNA damage. Genes
Dev. 2010;24:2146–2156.
139. El-Deiry WS, Tokino T, Velculescu VE, et al. WAF1,
a potential mediator of p53 tumor suppression. Cell.
1993;75:817–825.
140. Vousden KH, Lu X. Live or let die: the cell’s response
to p53. Nat Rev Cancer. 2002;2:594–604.
141. Malkin D, Li FP, Strong LC, et al. Germ line
p53 mutations in a familial syndrome of breast
cancer, sarcomas and other neoplasms. Science.
1990;250:1233–1238.
142. Srivastava S, Zou ZQ, Pirollo K, Blattner W, Chang
EH. Germ-line transmission of a mutated p53 gene
in a cancer-prone family with Li-Fraumeni syndrome.
Nature. 1990;348:747–749.
143. Frebourg T, Barbier N, Yan YX, et al. Germ-line
p53 mutations in 15 families with Li-Fraumeni
syndrome. Am J Hum Genet. 1995;56:608–615.
144. Kurian AW, Hare EE, Mills MA, et al. Clinical
evaluation of a multiple-gene sequencing panel
for hereditary cancer risk assessment. J Clin Oncol.
2014;32:2001–2009.
145. Kurian AW, Kingham KE, Ford JM. Next-generation
sequencing for hereditary breast and gynecologic
cancer risk assessment. Curr Opin Obstet Gynecol.
2015;27:23–33.
146. Xu B, Kim S, Kastan MB. Involvement of Brca1
in S-phase and G(2)-phase checkpoints after
ionizing irradiation. Mol Cell Biol. 2001;21:
3445–3450.
147. Wang Y, Cortez D, Yazdi P, Neff N, Elledge SJ, Qin
J. BASC, a super complex of BRCA1-associated
proteins involved in the recognition and repair
of aberrant DNA structures. Genes Dev. 2000;
14:927–939.
148. Moynahan ME, Chiu JW, Koller BH, Jasin M. Brca1
controls homology-directed DNA repair. Mol Cell.
1999;4:511–518.
149. Roy R, Chun J, Powell SN. BRCA1 and BRCA2:
different roles in a common pathway of genome
protection. Nat Rev Cancer. 2012;12:68–78.
150. Harkin DP, Bean JM, Miklos D, et al. Induction
of GADD45 and JNK/SAPK-dependent apoptosis
following inducible expression of BRCA1. Cell.
1999;97:575–586.
DNA Damage Response Pathways and Cancer  •  CHAPTER 11 164.e3
151. Hastak K, Alli E, Ford JM. Synergistic chemo-
sensitivity of triple-negative breast cancer cell
lines to poly(ADP-Ribose) polymerase inhibition,
gemcitabine, and cisplatin. Cancer Res. 2010;70:
7970–7980.
152. Saha T, Rih JK, Roy R, Ballal R, Rosen EM.
Transcriptional regulation of the base excision
repair pathway by BRCA1. J Biol Chem. 2010;285:
19092–19105.
153. Alli E, Sharma VB, Sunderesakumar P, Ford JM.
Defective repair of oxidative DNA damage in
triple-negative breast cancer confers sensitivity to
inhibition of poly(ADP-ribose) polymerase. Cancer
Res. 2009;69:3589–3596.
154. Pellegrini L, Yu DS, Lo T, et al. Insights into DNA
recombination from the structure of a RAD51-
BRCA2 complex. Nature. 2002;420:287–293.
155. Yang H, Jeffrey PD, Miller J, et al. BRCA2 func-
tion in DNA binding and recombination from a
BRCA2-DSS1-ssDNA structure. Science. 2002;297:
1837–1848.
156. Venkitaraman AR. Cancer susceptibility and the
functions of BRCA1 and BRCA2. Cell. 2002;108:
171–182.
157. Brown JS, O’Carrigan B, Jackson SP, Yap TA.
Targeting DNA Repair in Cancer: Beyond PARP
Inhibitors. Cancer Discov. 2017;7:20–37.
158. Lord CJ, Ashworth A. The DNA damage response
and cancer therapy. Nature. 2012;481:287–294.
159. Hartman AR, Ford JM. BRCA1 and p53: compensa-
tory roles in DNA repair. J Mol Med. 2003.
160. Telli ML, Jensen KC, Vinayak S, et al. Phase II
Study of gemcitabine, carboplatin, and iniparib
as neoadjuvant therapy for triple-negative and
BRCA1/2 mutation-associated breast cancer with
assessment of a tumor-based measure of genomic
instability: PrECOG 0105. J Clin Oncol. 2015;33:
1895–1901.
161. Telli ML, Timms KM, Reid J, et al. Homologous
recombination deficiency (HRD) score predicts
response to platinum-containing neoadjuvant
chemotherapy in patients with triple-negative breast
cancer. Clin Cancer Res. 2016;22:3764–3773.
162. Bryant HE, Schultz N, Thomas HD, et al. Specific
killing of BRCA2-deficient tumours with inhibitors
of poly(ADP-ribose) polymerase. Nature. 2005;434:
913–917.
163. Farmer H, McCabe N, Lord CJ, et al. Targeting
the DNA repair defect in BRCA mutant cells
as a therapeutic strategy. Nature. 2005;434:917–921.
164. Durkacz BW, Omidiji O, Gray DA, Shall S. (ADP-
ribose)n participates in DNA excision repair. Nature.
1980;283:593–596.
165. Telli ML, Ford JM. PARP inhibitors in breast cancer.
Clin Adv Hematol Oncol. 2010;8:629–635.
166. Javle M, Curtin NJ. The role of PARP in DNA
repair and its therapeutic exploitation. Br J Cancer.
2011;105:1114–1122.
164.e3
167. Lucchesi JC. Synthetic lethality and semi-lethality
among functionally related mutants of Drosophila
melanogaster. Genetics. 1968;59:37–44.
168. Hartwell LH, Szankasi P, Roberts CJ, Murray
AW, Friend SH. Integrating genetic approaches
into the discovery of anticancer drugs. Science.
1997;278:1064–1068.
169. Kaelin WG Jr. The concept of synthetic lethality in
the context of anticancer therapy. Nat Rev Cancer.
2005;5:689–698.
170. Murai J, Huang SY, Das BB, et al. Trapping of
PARP1 and PARP2 by Clinical PARP Inhibi-
tors. Cancer Res. 2012;72:5588–5599.
171. Fong PC, Boss DS, Yap TA, et al. Inhibition of
poly(ADP-ribose) polymerase in tumors from BRCA
mutation carriers. N Engl J Med. 2009;361:123–134.
172. Kaufman B, Shapira-Frommer R, Schmutzler
RK, et al. Olaparib monotherapy in patients with
advanced cancer and a germline BRCA1/2 mutation.
J Clin Oncol. 2015;33:244–250.
173. Bouwman P, Jonkers J. Molecular pathways: how can
BRCA-mutated tumors become resistant to PARP
inhibitors? Clin Cancer Res. 2014;20:540–547.
174. Afghahi A, Timms KM, Vinayak S, et al. Tumor
BRCA1 reversion mutation arising during neoadju-
vant platinum-based chemotherapy in triple-negative
breast cancer is associated with therapy resistance.
Clin Cancer Res. 2017;23(13):3365–3370.
175. Alter BP. Cancer in Fanconi anemia, 1927-2001.
Cancer. 2003;97:425–440.
176. D’Andrea AD, Grompe M. The Fanconi anaemia/
BRCA pathway. Nat Rev Cancer. 2003;3:23–34.
177. D’Andrea AD. Susceptibility pathways in Fanconi’s
anemia and breast cancer. N Engl J Med. 2010;
362:1909–1919.
178. Kennedy RD, D’Andrea AD. DNA repair pathways
in clinical practice: lessons from pediatric cancer
susceptibility syndromes. J Clin Oncol. 2006;24:
3799–3808.
179. Ceccaldi R, Sarangi P, D’Andrea AD. The Fanconi
anaemia pathway: new players and new functions.
Nat Rev Mol Cell Biol. 2016;17:337–349.
180. Moldovan GL, D’Andrea AD. To the rescue: the
Fanconi anemia genome stability pathway salvages
replication forks. Cancer Cell. 2012;22:5–6.
181. Kim H, D’Andrea AD. Regulation of DNA cross-link
repair by the Fanconi anemia/BRCA pathway. Genes
Dev. 2012;26:1393–1408.
182. Howlett NG, Taniguchi T, Olson S, et al. Biallelic
inactivation of BRCA2 in Fanconi anemia. Science.
2002;297:606–609.
183. Kim H, Yang K, Dejsuphong D, D’Andrea AD.
Regulation of Rev1 by the Fanconi anemia core
complex. Nat Struct Mol Biol. 2012;19:164–170.
164.e4 Part I: Science and Clinical Oncology

SELF-ASSESSMENT REVIEW QUESTIONS ANSWERS

1. True or false? Hereditary defects in DNA repair genes are 1. (a)
common in cancer. 2. (d)
a. True 3. (d)
b. False 4. (b)
2. DNA mismatch repair targets which lesions? 5. (e)
a. UV photoproducts 6. (e)
b. Oxidative base damage 7. (d)
c. DNA strand breaks
d. Replication errors
3. Lynch syndrome is associated with enhanced risk for which of
the following malignancies?
a. Colorectal cancer
b. Endometrial cancer
c. Cervical cancer
d. Colorectal and endometrial cancers
e. Colorectal, endometrial, and cervical cancers
4. How common are BRCA1 and BRCA2 mutations in the
general population?
a. 1 in 10,000
b. 1 in 400
c. 1 in 40
d. 1 in 4
5. Microsatellite instability is a feature of which malignancies?
a. All colon cancers in patients with Lynch syndrome
b. Twenty percent of all colon cancers
c. Endometrial cancers in patients with Lynch syndrome
d. Half of breast cancers in patients with Lynch syndrome
e. All of the above
6. Breast cancers that arise in BRCA1 mutation carriers are
specifically sensitive to which therapies?
a. Cisplatin
b. PARP inhibitors
c. Taxanes
d. Bevacizumab
e. a and b
f. All of the above
7. MSI in colon cancer is:
a. A good prognostic marker
b. A poor prognostic marker
c. A predictive marker for drug response
d. a and c
e. b and c
f. None of the above
 Viruses and Human Cancer 12 
Paul F. Lambert and Bill Sugden

S UMMARY OF K EY
• More than 15% of human cancers
are known today to be caused by
viruses.
• A hallmark of virally induced cancers
is that they are associated with
persistent viral infections.
• Although some viruses encode
oncogenes that directly contribute to
the cancers they cause, other
viruses are thought to result in
P OI NT S
cancer indirectly by causing chronic
destruction of the target organ from
which the cancer arises.
• The etiologic role of viruses in
cancer is established through a
combination of epidemiologic and
molecular evidence.
• In many cases, the cancers caused
by the virus represent dead-end
streets for the virus—that is, the
virus is no longer able to replicate in
the cancer.
• Virally induced cancers can be
prevented through the use of
effective prophylactic vaccines.
• Viral genes expressed in associated
cancers represent targets for
therapeutic vaccines and
viral-specific anticancer drugs.

Viruses cause cancers in people. More than 15% of all human cancers
are thought to have a viral etiology,1 and this fraction is likely to grow
as we investigate additional cancers for a potential viral cause and
identify new human viruses. Identifying a human cancer as having
a viral etiology has substantive consequences both for its treatment
and its prevention. The known virally caused human cancers often
express virally encoded products in the tumor cells. These viral
products are potential targets for antiviral, tumor-specific therapies.
Some viral infections can now be prevented with vaccines; some now
can be cured with antiviral drugs; therefore it may be possible to
eliminate the human cancers that require viral contributions for their
development.
For example, we now can realistically look forward to the elimination
of many or most cases of primary hepatocellular carcinoma (HCC)
and to most cases of cervical carcinoma. This happy eventuality is
thanks to the development and eventual use of vaccines effective
against, respectively, hepatitis B virus (HBV) and most tumorigenic
strains of human papillomaviruses (HPVs), and potent antiviral drugs
active against hepatitis C virus (HCV).
The search for human tumor viruses has been propelled by a long
appreciation that viruses can cause cancers in birds and rodents. Viruses
were isolated as filterable extracts from avian tumors in the first decade
of the last century and were shown to induce tumors in susceptible
animals.2,3 Parallel findings were made in mice in the 1940s.4 These
animal tumor viruses were in the retrovirus family and led researchers
to look for retroviruses as human tumor viruses in the 1960s and
1970s. However, most of the human tumor viruses subsequently
identified are in different virus families and do not conform to some
of the expectations derived from the study of highly oncogenic animal
retroviruses. For example, highly oncogenic tumor viruses induce
tumors in animals rapidly. The inoculation of Rous sarcoma virus
into the wing web of newborn chicks can induce fatal sarcomas within
2 weeks in 100% of susceptible animals.5 In addition, highly oncogenic
tumor viruses are oncogenic because they have acquired and express
potent derivatives of cellular proto-oncogenes. In fact, many of the
known human proto-oncogenes were first identified as homologues
of the oncogenes transduced by the highly oncogenic animal retrovi-
ruses.6 Known human tumor viruses, however, usually do not induce
cancers rapidly; often 15 to 50 years will elapse between the primary
infection and tumor development. Nor do human tumor viruses express
cellularly derived oncogenes; rather, some of them have evolved to
inhibit cellular tumor suppressor genes. These differences between
highly oncogenic animal viruses and human tumor viruses probably
have contributed to the delay in our recognition that viruses do cause
cancers in people.
A second obstacle in our recognition that viruses can be tumorigenic
in their human host is that we lack convincing animal models in
which to test these viruses directly. For all practical purposes, all
known human tumor viruses infect only people. In addition, we now
know that human viruses found not to be tumorigenic in people
are tumorigenic when experimentally introduced into test animals.
For example, human adenovirus 12, which causes only respiratory
infections in people, is highly oncogenic when inoculated into newborn
hamsters.7 The lack of an experimentally tractable animal host for
human tumor viruses has required multiple lines of evidence to affirm
that a given virus can contribute to a given human cancer. In particular,
epidemiologic findings have been combined with genetic and molecular
analyses in cell culture to identify human tumor viruses. Experi-
ments with mice transgenic for viral genes also have supported these
identifications.
This chapter introduces the seven known viruses that cause human
tumors, the tumors with which they are associated, data that support
these associations, and models to explain the viral contributions to
these tumors. These viruses are presented in the order of their discovery,
because early findings often have provided insights for analyses of
subsequently identified viruses. Finally, the chapter outlines the types
of virus-specific therapies that are possible to develop and the likelihood
of developing vaccines for human tumor viruses in order to limit or
eliminate specific human cancers.
EPSTEIN-BARR VIRUS
Epstein-Barr virus (EBV) was identified through the insight and
advocacy of Denis Burkitt. As a young surgeon, having identified
Burkitt lymphoma as a new disease entity, he analyzed the geographic
and climatic distribution of this childhood lymphoma, determined

165
166 Part I: Science and Clinical Oncology
that it overlapped with that of malaria, and postulated that it had an
infectious etiology.8 At his urging and with his proffered biopsy samples,
Tony Epstein and colleagues identified EBV in Burkitt lymphoma–
derived cells.9 To do so, they developed the expertise to propagate
these cells in culture.10 Cell lines derived from EBV-positive Burkitt
lymphomas proved to be powerful tools in associating EBV with
different human diseases. Different EBV-positive cell lines express
different viral antigens and thereby have served as test samples for
patients’ expression of antibodies to EBV-encoded antigens.
The analyses of these antibodies by serologic methods led the
Henles in Philadelphia to propose EBV as the cause of infectious
mononucleosis.11,12 A colleague in their laboratory who had lacked
antibodies to EBV-encoded antigens had developed those antibodies
on presentation with infectious mononucleosis. The etiologic role for
EBV in this “self-limiting lymphoproliferation” was subsequently
established by careful, prospective epidemiologic studies in which
serologic analysis was used to demonstrate that only immunologically
naïve people were at risk of the development of infectious mononucleo-
sis; with its development, they would first express antibodies to
EBV-encoded antigens of the immunoglobulin (Ig) M class, and only
later to those of the IgG class.13 Thus about 85% of infectious
mononucleosis cases were shown to arise from a primary infection
with EBV. Serologic studies also allowed the Henles to propose that
nasopharyngeal carcinoma (NPC) might be caused by EBV because
patients with NPC were characterized by having atypically high titers
to EBV-associated antigens.14 However, the data that linked EBV
causally to Burkitt lymphoma and NPC by the early 1970s were only
“guilt by association.” Although EBV caused most infectious mono-
nucleosis on primary infection, serologic findings also had demonstrated
that children in the parts of Africa in which Burkitt lymphoma is
endemic and adults in the parts of China in which NPC is prevalent
all had been infected with EBV—that is, they were “EBV seropositive”
long before these cancers developed.
The serologic analyses of EBV in the 1960s and 1970s illustrate
a conundrum for viruses and human cancers: “How can many people
be infected with a given virus, and yet how can that virus contribute
to tumor development in only a few infected subjects after long
periods?” This apparent paradox applies, in fact, to most cancers
associated with human tumor viruses and explains a major reluctance
to consider viruses as etiologic agents for human cancers. The World
Health Organization (WHO), without resolving this conundrum,
sponsored a prospective epidemiologic survey in Uganda to assay
42,000 youngsters serologically for evidence for or against the contribu-
tion of EBV causally to Burkitt lymphoma. The region studied had
a high incidence of this cancer. Samples of blood were collected from
children, their serum was stored, and for children later identified as
having Burkitt lymphoma, blood samples were collected again and
their titers to EBV antigens were determined. In this prospective
survey, Burkitt lymphoma developed in 14 youngsters during the 5
years they were monitored, and those in whom the lymphoma developed
had, before tumor development, on average a 3.4-fold higher titer of
antibodies to one class of EBV antigens than did the children in
whom the lymphoma did not develop.15 That is, for children in the
portions of the world in which EBV-associated Burkitt lymphoma is
endemic, a high titer of antibodies to a given set of EBV-encoded
antigens represents a 30-fold risk factor for the development of Burkitt
lymphoma.15
Do these findings prove that EBV causes Burkitt lymphoma? No;
proof in such cases for which direct experiments are not feasible
ultimately comes from the accretion of supporting findings in the
absence of confounding data. However, the WHO study did make
it unlikely that EBV is a passenger virus that merely replicates well
in tumor cells, because the antibody titers were elevated 7 to 54
months before tumor detection.15 Similar prospective surveys were
carried out in China, which identified antibodies of the IgA class to
the same set of EBV antigens as a risk factor for the development
of NPC.16
The association of EBV with Burkitt lymphoma and NPC and
the demonstration that EBV causes the bulk of infectious mononucleosis
have led researchers to consider other diseases with which EBV might
be associated. During the past 20 years, EBV also has been linked to
posttransplant lymphoproliferative disease (PTLD)17; oral hairy leu-
koplakia18; approximately one-third to one-half of cases of Hodgkin
disease19,20; one-third of AIDS-related diffuse large B-cell lymphomas
(DLBCLs)21; and one-tenth of gastric carcinomas.22 These linkages
have been made not only through serologic findings but also by
molecular genetic analyses that render the linkages more robust. The
latter analyses have been made possible by the elucidation of the
molecular virology of EBV in cell culture.23
EBV is a herpesvirus; it has a double-stranded DNA of 165,000
to 170,000 base pairs24 and encodes more than 100 genes,25 with 25
of these being pre-microRNAs (miRNAs), which encode up to 50
miRNAs26–29 (Fig. 12.1). As with other herpesviruses, EBV has two
distinct phases to its life cycle. It can infect cells, express a small subset
of its genes (see Fig. 12.1), and cohabit with the cell without killing
it (its “latent” phase). EBV also can emerge from its latency, express
all or most of its genes, amplify its DNA, assemble progeny virions,
and kill its host cell by lysis (its “lytic” phase). Unlike neurotropic
herpesviruses such as herpes simplex virus type 1 (HSV-1) and varicella-
zoster virus, EBV in its latent phase need not be maintained in a
nonproliferating host cell. Rather, it has the capacity to both initiate
and maintain proliferation in at least the B lymphocytes it infects in
cell culture and at early stages of primary infection in vivo.30,31 Although
it is the ability of EBV to support proliferation and survival of its
infected host cell that likely renders it oncogenic, the first step in its
life cycle is to infect a host’s B cells successfully. As with other her-
pesviruses, EBV has evolved a powerful mechanism to inhibit newly
infected B cells from being recognized by the host’s immune response.
EBV encodes at least 44 miRNAs that can target a suite of cellular
mRNAs for degradation. Studies using newly infected B cells and
autologous T cells have shown that viral miRNAs target mRNAs
encoding IL12B, resulting in suppression of type 1 helper T cell (Th1)
differentiation. These viral miRNAs also control gene expression of
HLA class II and three lysosomal enzymes important for proteolysis
and epitope presentation to CD4+ T cells.32 Parallel analyses have
shown that the EBV’s miRNAs also inhibit recognition of infected
cells by cytotoxic CD8+ T cells.33
Clearly EBV’s success as a pathogen reflects, in part, its ability
to inhibit its host from recognizing and killing the cells that the
virus infects.
EBV efficiently induces and maintains proliferation of infected B
cells. In genetic experiments in which two viral genes, EBNA2 and
LMP1 (see Fig. 12.1), are expressed conditionally within the context
of the virus, each gene product when assayed alone needs to function
for infected B cells to continue to proliferate.34,35 These observations
are particularly telling because they help to explain the multistep
evolution of Burkitt lymphoma. Many other genetic analyses have
shown that four additional viral genes—EBNA1, EBNA3a, EBNA3b,
and EBNA3c (see Fig. 12.1)—affect some facet of cell proliferation
or survival.36 EBNA1 is essential for the viral genome to be maintained
as a plasmid in cells.37 Its inhibition leads to the loss of the viral DNA
from tumor cells and to their death.38 EBNA3a acts at the stage of
initiation of proliferation.39 EBNA3b is a tumor suppressor.40 EBNA3a
and EBNA3c inhibit p16INK4 and p14ARF through complex, distinct
binding to promoter elements41,42 A seventh viral gene, LMP2a, can
substitute for the signaling provided by a functional B-cell receptor
(BCR) to support the survival of murine and human B cells that lack
functional BCRs.43–45 This activity of LMP2a likely underlies one
contribution of EBV to the tumor cells in Hodgkin disease that fail
to express functional BCRs.46 Two additional genes of EBV are expressed
as nontranslated, small RNAs, termed “EBERs.” They contribute to
the efficiency with which EBV induces and maintains proliferation
of B lymphocytes infected in culture47; they also induce type I
interferon48 and mediate other activities that potentially contribute
 Viruses and Human Cancer  •  CHAPTER 12 167

BARF0

14
RT

m R- BA RT T2 3
RT2

1
m iR- -BA
m iR- -BA AR RT
-BA

m iR- BAR RT 19 0
miR

iR

iR iR-B BA
m
HI/A
BAM

12
m miR R-B AR RT 1
T1
-B -BA RT T9 0
m
F5

1
BAL BALF
4

m iR- BA
i

T7 8
BALF3

B
A
AR R
BamHI Cp

18
i

T
2
LF

iR
1
LF

58
F1 LMP2A/B
BIL

B9
BamHI Wp
i n LMP-1p
ed
ART17
ART6

ART16

let
ART5
ART15

De

N
miR-B
miR-B

ART1
ART4
miR-B

A C
ART3
miR-B

EBERs
miR-B

W
LF3
miR-B
miR-B

W
miR-B

I TR W
d oriP W
V
W EBNA-LP
X
T W
b W
c W

D W
W
EBV W
G 165 kbp oriLyt
Y
H
EBNA-2
B
F

BHLF1
K Q
R U Qp
Z e P
EBNA-1 O
E

-1
L M a

F1
S

HR
-B
iR
m
2
EBNA-3C LF
BF
EBNA-3A
EBNA-3B LF1 -2 F1
BF HR
F1 R
R -B F 1-3 BH
mi R
-BH
R
mi

Green pre-miRNAs: Encode miRNA on one arm of the pre-miRNA

Purple pre-miRNAs: Encode miRNAs on both arms of pre-miRNA
(as shown in miRBase release 22)
Figure 12.1  •  Map of the Epstein-Barr virus (EBV) genome. The genome of the B95-8 strain of EBV in its circular double-stranded DNA form of
165 kbp is depicted as it is found in latently infected B cells. The genome is a linear DNA within the viral particle and is circularized on infection at its
terminal repeat (TR) elements found at the 5′ and 3′ ends of the linear molecule. The EBV genome encodes approximately 100 genes. Shown as white boxes
are the exons for coding segments of the viral genes expressed in B cells infected in vitro, including EBNA1, EBNA2, EBNA3A/B/C, EBNA-LP, LMP1, and
LMPA/B. The EBERs denoted by the white box are two small RNAs encoded by EBV. Dashed lines represent primary transcripts originating from viral promoters
denoted with a lowercase p. Also shown are the positions of the origins of replication, oriP and oriLyt, that support the latent and lytic replication of the viral
genome, respectively. See reference 23 for details of EBV’s genome and life cycle.

to tumor phenotypes.49 The EBERs usually are highly expressed in proliferate and detectably express only LMP2a (see Fig. 12.1), a viral
EBV-positive tumor cells and thus provide a convenient, sensitive gene product not directly required for cellular proliferation.56,57
assay for identifying these tumor cells.50,51 A failure of this robust immune response to EBV’s transforming
All of the transforming genes of EBV that are expressed as proteins proteins contributes to PTLD. The infected proliferating B cells in
are recognized by the host’s T-cell response.52 EBNA1, which can be the these immunosuppressed patients are of donor origin and express all
only viral protein detected in some EBV-positive tumors,53 is recognized of EBV’s transforming proteins, as well as the EBERs.58 Two types of
in an atypical response involving CD4+ T cells and its endogenous successful treatments for PTLD demonstrate the critical role of the
major histocompatibility complex class II processing.54,55 The host’s patient’s immune response in failing to limit this “iatrogenic” tumor.
cytotoxic response is sufficiently robust that patients recovering from First, if immunosuppression can be reduced for the patient such that
infectious mononucleosis lack B cells that detectably express RNAs the transplant is still tolerated, PTLD may regress. Second, several
that encode these transforming genes. The surviving EBV-infected investigative groups have amplified the donor’s T cells that are cytotoxic
B cells are in distinct, differentiated states in which they no longer for EBV’s transforming proteins before bone marrow transplantation.
168 Part I: Science and Clinical Oncology
Treatment of patients with PTLD who have these syngenic, specific
T-killer cells has cured the disease.59 These encouraging findings
underscore the important role of the immune response in limiting
survival of EBV-infected cells. The need to immunosuppress recipients
of solid organ transplants increases their risk of developing PTLD. A
controversy has arisen about the efficacy of treating recipients with
drugs that inhibit EBV’s lytic cycle to prevent PTLD. If the virus
that causes PTLD is derived from its release following transplantation,
then blocking that release could diminish the incidence of this
lymphoproliferation. A large retrospective study of patients at high
risk, those who are EBV seronegative and receive organs from seroposi-
tive donors, has shown that prophylaxis with drugs that target viral
DNA synthesis during the lytic cycle (acyclovir, ganciclovir, valacyclovir,
and valganciclovir) do not decrease the incidence of PTLD, which
ranges from 1% to 8%.60 It is therefore essential to monitor such
high-risk recipients for the potential development of PTLD after
transplantation.
Two startling features of tumor cells freshly isolated from Burkitt
lymphoma biopsy specimens help to explain the evolution of this
tumor when considered in the context of EBV’s transforming genes
and the immune response to them. These tumor cells express only
EBNA1 among the required viral transforming proteins, along with
the EBERs and miRNAs, yet they proliferate.53 They also display a
chromosomal translocation between one of three human Ig loci and
the c-myc proto-oncogene.61,62 These translocations are likely fostered
by expression of the activation-induced cytidine deaminase gene, which
is essential for Ig class switching.63 The juxtaposition of an Ig locus
to c-myc drives expression of the proto-oncogene in these B cells as
it does in murine plasmacytomas that display similar translocations.64
These observations can be arranged to provide a satisfying though
necessarily speculative model for the genesis of Burkitt lymphoma.
First, EBV infects young children living in regions of central Africa
in which malaria is endemic.65 Malaria is a T-cell immunosuppressive
disease that decreases the children’s ability to limit proliferation of
infected cells66 and increases their viral loads.67 Although the youngsters
with severe EBV infections have increased antibody titers to viral
antigens, they have more proliferating B cells and are at increased risk
for the chromosomal translocations, fostered by the recombination
mechanism, that occur in B cells and use signals at Ig loci.68 A rare
Ig/c-myc translocation provides the cell some undefined selective
advantages. In addition, multiple viral genes including LMP1, EBNA2,
EBNA3a, and EBNA3c are shut off and the cell proliferates, acquires
additional mutations (often mutations inactivating p53), and evolves
rapidly into a Burkitt lymphoma.69
Two recent findings affect this speculative model for Burkitt
lymphoma and have implications for the other EBV-associated cancers
as well. Studies of the replication of EBV’s plasmid genome have
revealed unexpectedly that only 84% of the viral DNAs on average
are synthesized each cell cycle.70 This defect in DNA synthesis occurs
in all cell types tested and indicates that EBV DNA must be lost from
proliferating cells.70 Proliferating populations of cells can remain EBV
positive only if the daughter cells that retain EBV genomes are provided
with selective advantages, such that they outgrow their sisters that
lose EBV. All EBV-associated tumors maintain EBV DNA as plasmids
in most of the tumor cells; therefore EBV must provide all of its
associated tumors one or more selective advantages. This profound
insight means that EBV provides lymphomas such as AIDS-related
DLBCL with selective advantages, even though the EBV-negative and
EBV-positive forms of the disease appear similar,21 or that EBV provides
selective advantages to NPC tumors in vivo even though these tumors
typically lose EBV on explantation into cell culture. EBV, when found
as a plasmid in tumor cells, cannot merely be a passenger.
This insight has been tested with two forms of Burkitt lymphomas
and PTLD. Tumor cells were engineered to force the loss of their
EBV plasmids. All of these tumors died by apoptosis on the loss of
EBV.38 Viral miRNAs rescued the Burkitt lymphomas that expressed
the fewest viral genes.71 These observations underscore the role of
EBV in maintaining its associated tumors and the importance of the
miRNAs it encodes in oncogenesis.
Treatments for Burkitt lymphoma have evolved to favor high-dose,
short-term chemotherapies that can yield greater than 90% 5-year
survival rates for children with localized disease.72 This high rate has
not been achieved, however, in some centers in Africa, where the rate
of event-free survival over 12 months has been found to be only 33%
for patients at all stages of the disease.73
All EBV-associated cancers contain EBV DNA and express EBNA1,
EBERs, and all or a subset of EBV’s miRNAs (see Fig. 12.1).58 We
know less about the genesis of these other tumors, but findings with
NPC provide evidence for an unexpected contribution of EBV to its
etiology. Chan and colleagues have shown that EBV infects cells that
already can be distinguished as being “preneoplastic” in the evolution
of NPC.74 This finding might lead one to think that EBV is merely
a passenger in this tumor. However, the fact that 100% of NPC
tumors are infected with EBV and that most tumor cells retain viral
plasmid genomes means that EBV must provide these tumor cells
with selective advantages so that the cells with EBV outgrow those
that inevitably lose the plasmid. It is not known what this selective
advantage is, but it is reasonable to hypothesize that EBV could provide
these cells with proliferative or survival signals, as it does with Burkitt
lymphoma cells. Viral gene expression in NPC biopsies correlates
with decreased expression of host cell genes involved in antigen display,
which likely facilitates the growth of these cells in vivo as well.75 Some
of EBV’s miRNAs are particularly well expressed in biopsies of NPCs
relative to their levels in Burkitt lymphomas and cells infected in
vitro.76,77 The viral miRNAs are thus likely candidates for providing
NPC tumor cells with selective advantages that act in vivo.
The many clinical and basic scientific studies of EBV and its
associated cancers can be extracted to yield some lessons for tumor
viruses in general. First, the viral genomic nucleic acid remains in
tumor cells and expresses one or more viral genes. Second, the virus
contributes information to infected cells, which provides them with
a selective advantage both in evolving toward and sustaining tumor
cells. However, this information is not sufficient for tumor formation.
Additional, multiple, rare events must occur in the infected cells for
them to evolve into tumors. These additional essential events explain
both why the associated tumors develop in only a fraction of the
people infected with a given tumor and why the tumors usually develop
only after long delays. That tumor viruses are retained in their associated
tumors and help sustain these tumors indicates that targeting the
transforming genes of tumor viruses is a rational approach to the
treatment of these tumors therapeutically.
HEPATITIS B VIRUS
HBV was identified by virtue of being recognized as an antigen in
the sera of donors detected by antibodies in sera of other infected
donors.78 Thoughtful analyses by Blumberg correlated the presence
of the antigen with hepatitis, a correlation strengthened by the
seroconversion of a laboratory member who contracted hepatitis.78
By the late 1960s, blood donated to blood banks was screened for
the antigen, positive samples were removed, and only negative samples
were used for transfusions. This early insightful intervention led to a
significant reduction in transfusion-associated hepatitis.79 Blumberg
also demonstrated a striking association between HBV antibodies to
its antigens, and HCC.78 These early findings have been built on to
demonstrate that HBV does cause HCC, which is either the fifth or
sixth most common cancer in people today. Of the approximately
500,000 new cases of HCC in the world each year, HBV is estimated
to cause between 50% and 70% of them.80,81 Most of the rest of these
cases are attributable to HCV, a member of the Flaviviridae family.
Two types of data have established HBV’s causal role in HCC. A
prospective survey of 22,707 male civil servants in Taiwan was initiated
at the end of 1975.82 Of these subjects, 3454 were found to be positive
for HBV’s surface antigen (HBsAg), indicating that they were

chronically infected with HBV. The entire group of 22,707 members
was observed on average for 8.9 years. By the end of 1986, HCC had
developed in 152 of the 3454 HBsAg-positive men, whereas it had
developed in only 9 of the 19,253 HBsAg-negative men. The relative
risk of the HBsAg-positive cohort for the development of HCC was
therefore 100 times greater than that for the HBsAg-negative group.83
This prospective epidemiologic study provides robust data that the
presence of HBV is strongly associated with the development of HCC.
The second type of data demonstrating that HBV can cause HCC
has been derived by removing HBV from a population and determining
whether the incidence of HCC declines. Taiwan began vaccinating
children in 1984, first with a plasma-derived antigen and eventually
with a recombinant antigen. Between 1984 and 1994, the incidence
of infection as monitored by the presence of HBsAg had dropped
from 9.8% to 1.3% among children 12 years or younger.84 Similar
findings have been made in the Gambia, where children vaccinated
during their first year develop into only 10% as many chronically
infected 9-year-olds as do unvaccinated children.85 HCC is a cancer
that peaks between 50 and 60 years of age and rarely occurs in children
from 6 to 14 years of age. The incidence of HCC in this latter popula-
tion in Taiwan dropped from 0.64 per 100,000 per year when averaged
from 1981 to 1990 to 0.36 per 100,000 per year when averaged
between 1990 and 1994, a finding that is statistically significant (P
< .01).86 This decline presumably reflects the eightfold reduction of
chronic HBV infection in children at risk for the development of
HCC. Removal of a virus from a population with a subsequent decrease
in an associated cancer in that population provides compelling evidence
that the virus contributes causally to the cancer. We can expect that
a decline of HCC in the vaccinated adult population will be more
striking in decades to come.
Although it is clear that HBV causes HCC in people, it is not
clear how it does so. Researchers now invoke two distinct contributions,
direct or indirect, to explain HBV’s oncogenesis. HBV encodes one
gene, pX (Fig. 12.2), which can affect viral and cellular transcription
and has been proposed to contribute directly to oncogenesis. HCC
in general evolves in patients with marked liver cirrhosis. HBV can
contribute to that cirrhosis by providing targets for T-cell killing and
thereby could contribute indirectly to oncogenesis.
Molecular virology studies of HBV have illuminated the viral life
cycle but have yet to identify its mode of oncogenesis.87 HBV is a
small, enveloped virus with a double-stranded DNA genome, one
strand of which is incomplete. The complete viral duplex DNA is
3.2 kilobase pairs (kbp) in length (see Fig. 12.2), serves as a template
for transcription by RNA polymerase II, and is replicated via reverse
transcription of a greater than full-length RNA transcript of approxi-
mately 3.4 kb. All members of the Hepadnaviridae family preferentially
infect hepatocytes. This tropism is apparently mediated by a cellular
receptor expressed in hepatocytes and by viral transcription that is
controlled in part by cellular transcription factors principally expressed
in hepatocytes. Unlike most DNA viruses, HBV undergoes its complete
life cycle to yield progeny virions, which exit from hepatocytes via
secretory pathways without killing the host cell. This anomalous
behavior of hepadnaviruses means that, in the absence of an exogenous
function of the host, an infected hepatocyte could survive, carry out
its normal functions, and release large amounts of infectious HBV
for long periods. Accordingly, some chronically infected people have
large amounts of HBV in their sera.
Mammalian hepadnaviruses encode pX, which is not found in the
avian species; only the mammalian members are known to cause HCC
in their hosts. This correlation has focused interest on pX as being likely
to contribute to the oncogenesis of mammalian hepadnaviruses. It is
difficult to gauge pX’s potential role in the oncogenesis of HBV; the
literature includes much information about it, yet no ready synthesis
of this information explains such a role. Most HCC tumor biopsies
retain viral sequences encoding pX,88 but few express the protein
detectably.89 It has been proposed that pX associates with p53 and
inhibits its activation of apoptosis90; however, pX is not detected
Viruses and Human Cancer  •  CHAPTER 12 169
Pre-S1
Pre-S2
S gene
HBV
3.2 kbp
C gene
P gene
Pre-C
X gene
Figure 12.2  •  Map of the hepatitis B virus (HBV) genome. The 3200 bp
genome of HBV is a circular double-stranded DNA in infected cells (shown
in black). The genome in the viral particle is partially double-stranded DNA
because of an incomplete extension of the plus strand by the viral DNA
polymerase P. Shown as colored boxes are the coding segments for the structural
(blue) and nonstructural (orange) viral genes. The arrowheads represent the
sites at which translation of the viral proteins initiates. These viral proteins
are translated from multiple messenger RNAs: one for Pre-S1, Pre-S2, and S;
one for Pre-S2 and S; one for core and P; and one for x. See reference 378
for details of HBV’s genome and life cycle.
in HCC biopsy specimens,89 and between 30% and 90% of such
biopsy specimens have mutations in p53.88,89 The viral protein pX in
studies in cell culture can bind one subunit of RNA polymerase II,
as well as transcription factor IIB.91,92 It also associates with Smad4,
an integral member of the transforming growth factor–β (TGF-β)
signaling pathway, to foster this pathway’s signaling.93 How these
different transcriptional activities of pX might contribute to the evolu-
tion of HCC is unclear. A study has reported that pX can induce
markers for “stemness” in hepatocytes by activating β-catenin and
epigenetic upregulation of miR-181,94 leading to the hypothesis
that pX may contribute to the induction of cancer stem cells in the
context of HCC.
HBV is often integrated in HCC tumors,95 and it has been proposed
that integration of the viral DNA could affect transcription of nearby
cellular genes. This suggestion has been strengthened by the recognition
that the woodchuck member of the Hepadnaviridae family contributes
to HCC via insertional mutagenesis.96 However, HBV has not been
found to integrate at sites that can be interpreted to affect its onco-
genesis. In addition, HBV DNAs cloned from HCC biopsy specimens
have been tested and found not to score as enhancer sequences in
hepatoma cells in culture.97
The hypothesis that HBV contributes to the development of HCC
indirectly by inducing rounds of cirrhosis and subsequent liver
regeneration is appealing. HBV infection can be acute or chronic; it
appears that acute infection correlates with a robust cytotoxic T-cell
response to all viral antigens, whereas chronic infection correlates with
a weak T-cell response.98 These cytotoxic responses do lead to the
death of hepatocytes; however, experiments in mice transgenic for
HBV genes and in infected chimpanzees indicate that a potent
noncytotoxic mechanism also exists for limiting viral expression in
infected hepatocytes.99,100 In these experiments, immune cells release
γ-interferon, which by some means inhibits viral gene expression and
promotes loss of viral DNA from infected cells.99,100 The administration
of interleukin (IL)-18 limits viral replication efficiently in a transgenic
170 Part I: Science and Clinical Oncology
mouse model by inducing production of both type 1 and type 2
interferons.101 Chronic but not acute infection correlates with the
eventual development of HCC. How these two modes of eliminating
infected cells would be balanced to yield long-term or chronic infection,
the resulting cirrhosis, and the concomitant hepatocellular regeneration
required for the accumulation of mutations predisposing to HCC is
not known.
A role for cytotoxic T cells in the evolution of HCC has been
modeled in mice transgenic for HBV surface proteins (see Fig. 12.2).
These mice are tolerant to these viral antigens, but when the mice
are reconstituted with syngeneic, nontransgenic bone marrow and
subsequently challenged with syngeneic, immune, nontransgenic
splenocytes, cirrhosis develops, and they maintain cytotoxic T cells
specific for HBsAg.102 These animals have long-term liver damage,
and by 18 to 20 months of age, HCC develops.
Although vaccination against HBV is effective at preventing infection
and subsequent disease, there are still approximately 250 million people
in the world chronically infected with it and at risk for developing
cirrhosis and HCC. This risk translates into approximately 700,000
people dying each year, so 15% to 25% of the chronically infected
will eventually die from it.103 Current treatments for the chronically
infected include long-term use of pegylated interferon-α and nucleoside
analogues such as tenofovir.104
These treatments are not cures, and reduce detection of viral antigens
in only 10% of patients. Important to note, they do not eliminate
the viral DNA resident in hepatocyte nuclei, which sustains the chronic
infection. Much necessary work is now focused on developing effective
treatments for eliminating HBV from infected people.
HUMAN PAPILLOMAVIRUSES
Cervical cancer is caused by HPVs. Approximately 200 genotypes of
HPVs have been identified to date. Most HPV genotypes infect
squamous epithelia lining the skin; a subset are mucosotropic and
infect stratified, squamous epithelia lining the anogenital tract and
oral cavity. A subset of these mucosotropic HPVs, the so-called “high-
risk” HPVs, are associated with more than 99% of human cervical
cancers, other anogenital cancers, and a subset of squamous carcinomas
of the head and neck, particularly those of the oropharynx. HPV-16
and HPV-18, the high-risk HPVs most common in cancers, are present
in more than 85% of human cervical carcinomas. The mucosotropic
HPVs are transmitted sexually and represent the most frequently
detected sexually transmitted disease. The association of specific
mucosotropic HPVs with human cancers was first recognized in the
1980s when zur Hausen and associates at the German Cancer Research
Institute in Heidelberg detected the presence of then-novel HPV
genotypes in human cervical cancers and in cell lines derived from
such cancers.105,106 In these cell lines, which include HeLa cells, the
HPV DNA often is integrated into the host genome, and only a
subset of viral genes, E6 and E7, is expressed.107,108 This discovery led
to the hypothesis that HPV E6 and E7 genes contribute to cervical
cancer, a premise now well supported by experimental research.
An association of papillomaviruses with cancer was first demon-
strated in the early 1930s with the recognition that a subcellular,
transmittable (i.e., infectious) agent causes squamous carcinomas in
cottontail rabbits.109 The agent was later identified to be a virus,
cottontail rabbit papillomavirus (CRPV), that induces warts in the
rabbits. In a subset of these infected rabbits, cancers develop at the
original site of the CRPV infection. Other animal papillomaviruses
induce frank cancer. Bovine papillomaviruses (BPVs), which represent
a class of papillomaviruses that induce fibropapillomas and are character-
ized by hyperplasia of both the dermal fibroblasts and epidermal
epithelial cells, can induce epithelial tumors of the alimentary canal
in cows.110 Such tumors are thought to arise when animals ingest
bracken fern, which contains a potent chemical carcinogen,
quercetin.111–113 Thus papillomaviruses and chemical carcinogens act
together to induce tumors in cattle.
The study of papillomaviruses in the laboratory began in earnest
in the late 1970s when Doug Lowy, Peter Howley, and their colleagues
at the National Institutes of Health in Bethesda discovered that BPV-1
infects and transforms a mouse fibroblast cell line, C127, in cell
culture.114–116 The parental C127 cells, although immortalized, are
contact inhibited. Infection by BPV-1 or transfection of C127 cells
with a bacterial recombinant plasmid containing the entire BPV-1
genome yields foci of cells that are no longer contact inhibited.
Transformed C127 cells harbor the viral genome as a nuclear plasmid
and express early viral genes. The viral gene products E5 (E referring
to early and 5 referring to the fifth largest translational open reading
frame [ORF]), E6, and E7117–122 contribute to this transformation
(Fig. 12.3). Thus by the time HPVs were recognized as potential etio-
logic agents in human cervical cancers in the mid-1980s, a wealth of
information pointing to the transforming potential of animal papil-
lomaviruses in tissue culture had been established.
The seminal studies in the early to mid-1980s of zur Hausen and
colleagues, who identified HPV DNA in cell lines derived from cervical
cancers, checkmated a long-argued role for HSV-2 in human cervical
cancer. The posited role for HSV-2 in cervical cancer arose from
findings that patients with cervical cancer often had antibodies to
HSV-2, another sexually transmitted agent. However, their tumor
cells lacked HSV-2 DNA, and today it is accepted that HSV-2 does
not contribute causally to cervical cancer. This early error provides
an important lesson to researchers and epidemiologists trying to identify
biologic agents that contribute to cancer. Proof by today’s standards
requires a “smoking gun” (in this case, the gun includes the viral
genome and its expression of viral genes in cancer cells).
LCR
E6
L1
E7
HPV
8 kbp
E1
L2 E2
E5 E4
Figure 12.3  •  Map of the human papillomavirus (HPV) genome.
Indicated by the circle is the approximately 7900 bp circular double-stranded
DNA genome of HPV-16 as found in viral particles and infected cells. Shown
by the boxes outside the circle are the various translational open reading frames
(ORFs) that encode the viral proteins, including the early (E) and late (L)
ORFs. Most early ORFs (those highlighted in orange) are found expressed
throughout the viral life cycle within stratified squamous epithelia, whereas
the late ORFs, which encode the capsid proteins (highlighted in blue), and
E4 (highlighted in green), are selectively expressed in the productively infected,
terminally differentiated epithelial cells. Note that among the early ORFs are
E6 and E7, the two ORFs encoding like-named oncoproteins commonly
found expressed in HPV-associated anogenital and oral cancers. RNA synthesis
of papillomaviruses is complex, yielding many potential messenger RNAs, all
of which terminate at polyadenylation sites located at the end of the E5 and
L1 open reading frames. The long control region (LCR) encodes multiple
cis-acting elements that regulate viral transcription and synthesis of viral DNA.
See reference 379 for details of the HPV genome and life cycle.

In 1985, both the zur Hausen and Howley laboratories reported
that HPV DNAs (see Fig. 12.3) were integrated in chromosomal
DNAs in cell lines derived from cervical cancers.107,108 This finding
initially led to speculation that HPVs contribute to cervical cancer
by integrating in or nearby to disrupt the function of cellular genes
that protect against cancer (i.e., tumor suppressor genes) or activate
the cellular genes that can promote cancers (i.e., proto-oncogenes),
akin to the mechanism of oncogenesis by certain oncogenic avian and
rodent retroviruses. However, a role of HPV as an insertional mutagen
in cancer is not consistent with its different sites of integration in
different cancers, which rarely are found to be near known or suspected
tumor suppressor genes or proto-oncogenes. Rather, it is likely that
the integration of the HPV genome leads to the selective, and in some
cases, upregulated expression of two viral genes, E6 and E7 (see Fig.
12.3), which encode gene products that directly contribute to cancer.
The mechanism for this upregulation remains poorly understood but
may reflect (1) derepression of E6 and E7 expression from the viral
promoter resulting from the disruption of a viral transcription factor,
E2, that can repress their transcription123; (2) an increase in the stability
of the E6 and E7 mRNAs resulting from the disruption on integration
of an mRNA instability element present in the 3′ end of the E6 and
E7 mRNAs124; or (3) increased transcriptional initiation from the
viral promoter directing expression of E6 and E7 after integration of
the viral DNA. The recognition of the increased expression of E6 and
E7 in cervical carcinomas, coupled with the knowledge that E6 and
E7 contribute to the transforming potential of BPV-1 in mouse
fibroblasts, provided the impetus to examine the tumorigenic activities
of these two viral genes.
Evidence for a critical role of increased expression of HPV E6 and
E7 in the genesis of cervical cancers comes from multiple studies: (1)
cervical epithelial cells harboring integrated HPV-16 DNA have a
selective growth advantage over cells harboring normal extrachromo-
somal viral genomes, and this growth advantage correlates with the
increased expression of E6 and E7125; (2) E6 and E7 bind and inactivate
the tumor suppressor gene products p53 and retinoblastoma protein
(pRB), respectively126,127; (3) p53 and pRB are wild type in cell lines
derived from HPV-positive cervical cancers, whereas they are mutated
in HPV-negative cervical cancer–derived cell lines128; and (4) the
expression of the E6 and E7 viral genes is required for survival of
cervical cancer–derived cell lines,129–134 and in the case of E7, main-
tenance of a tumor phenotype in the context of mouse models for
HPV-driven cervical carcinogenesis is required.135,136 Together, these
observations strongly support the hypothesis that E6 and E7 contribute
causally to human cervical cancers at least by blocking the functions
of cellular tumor suppressors.
The E6 and E7 genes from the high-risk HPVs are transforming
in tissue culture. They act independently or synergistically to immortal-
ize multiple cell types, including human foreskin keratinocytes, cervical
epithelial cells, or mammary epithelial cells.137–142 In addition, E7
cooperates with an activated ras to transform baby rat kidney or human
cervical epithelial cells.143–145 The oncogenic properties of high-risk
HPV E6 and E7 in vivo have been validated through the characterization
of HPV transgenic mice in which expression of E6 and E7, as well
as a third viral oncogene, E5, predisposes animals to epithelial cancers
of the skin, cervix, anus, and head and neck region.146–156
E6 and E7 are best known for their ability to associate with the
cellular tumor suppressors p53 and pRB, respectively.126,127 The discovery
of these interactions represents a major advance in our understanding
of the mechanisms of oncogenesis: the inhibition of tumor suppressors
predisposes cells to evolve into tumors. E6 induces degradation of
p53 via recruitment of a ubiquitin ligase, E6AP.157,158 E6 inhibits p53
protein’s transcriptional regulatory activities in tissue culture cells.159,160
Association of E7 with pRB also promotes the degradation of pRB161,162
and disrupts the capacity of pRB to bind and functionally inactivate
the cellular E2F transcription factors.145,163 Although these abilities of
E6 and E7 to inactivate p53 and pRB, respectively, likely play an
important role in their oncogenic potentials, it is important to recognize
Viruses and Human Cancer  •  CHAPTER 12 171
that E6 and E7 both can bind additional cellular factors, and these
interactions also may contribute to HPV-associated carcinogenesis.
Studies in HPV-16 transgenic mice strongly support this premise.164–167
Which of these many interactions contribute to their oncogenic
potential largely remains to be determined.
E6 proteins from multiple papillomaviruses bind to cellular proteins
other than p53 and E6AP. These other interacting partners include
p300168,169; paxillin170,171; E6 target protein 1 (E6TP1)172; interferon
regulatory factor 3 (IRF3)173; E6 binding protein 1 (E6BP1)174; Bak175;
protein kinase PKN176; myc177; the mammalian homologue of Drosophila
discs large tumor suppressor gene product (DLG)178,179; Scribble180;
MAGI1181; and MUPP1.182 In addition, E6 can induce expression of
telomerase activity at least in part through its interaction with multiple
isoforms of NFX1,183–186 a property that correlates with the immortal-
izing potential of E6. In many cases, including but not limited to
p53, the proteins E6 interacts with are targeted for proteasomal degrada-
tion largely through E6’s recruitment of a ubiquitin ligase, E6AP. The
importance of E6AP in E6-mediated cervical carcinogenesis has been
clearly demonstrated.187
In addition to binding and degrading pRB, E7 can bind to other
cellular proteins, p107 and p130, that are related to pRB protein188
and interact with different members of the E2F family of transcription
factors.189,190 The inactivation of the pocket proteins drives carcino-
genesis in the head and neck region191 but is insufficient to do so in
the cervix.192 E7 also is argued to associate with cyclins188,193–195 and
to inactivate the cyclin-associated kinase inhibitors p21 and p27.196–198
A role for E7’s inactivation of p21 in cervical carcinogenesis has been
demonstrated.166 Thus E7 can associate with and/or alter the activities
of multiple cellular factors that themselves interact normally and thereby
contribute to the normal regulation of the cell cycle. Still other
interactions have been identified between E7 and cellular factors,
including the S4 subunit of the 26 S proteasome199; Mi2beta, a
component of the NuRD histone deacetylase (HDAC) complex200;
the Forkhead domain transcription factor MPP2201; the transcription
factor AP1202; insulin-like growth factor-binding protein 3203; TATA
box-binding protein (TBP)204,205; TBP-associated factor-110206; and
a novel human DnaJ protein, hTid1.207 Recently, E7 has been shown
to alter proteins that modify chromatin,200,208–210 raising the possibility
that E7 causes global changes in host gene expression. It remains
unclear whether any of these targets of E7 contribute to E7’s oncogenic
potential.
Appreciation of the role of HPV is growing, not only in anogenital
cancers such as cervical cancer, but also in head and neck cancers of
the oral cavity and in skin cancers.211–214 The multiple interactions of
E6 and E7 with cellular proteins that have regulatory functions is
consistent with the contribution of E6 and E7 to cancers through
multiple mechanisms. Studies in tissue culture strongly support the
hypothesis that continued expression of E6 and E7 is required for the
continued growth of cervical cancer cells.129–133 Studies in mouse models
also support this premise.135,136 These studies point to the potential
value of developing drugs that interfere with the expression or function
of these viral oncogenes as means for treating HPV-associated cancers.
Cervical cancers take decades to arise in most patients after initial
infection with high-risk HPVs. During this time, the HPV must
persist in the patient. Strategies that can interfere with viral persistence
may prove effective in preventing the development of cancer. E6 and
E7 are important to the replicative phase of the HPV life cycle and
therefore for viral persistence, indicating that they are also appropriate
targets for antiviral and antitumor drug development. Recent studies
have used organotypic tissue culturing to recapitulate the life cycle of
HPVs in fully differentiating, stratified squamous epithelial cells. These
studies have implicated E7 in reprogramming cells within the terminally
differentiating compartment of the epithelia to support the amplification
of the viral DNA genome, likely through its inactivation of pRB.215
The inactivation of p53 by E6 may be necessary for viral replication
by inhibiting cellular stress responses elicited by E7’s inactivation of
pRB.216 At least two more HPV proteins, E1 and E2 (see Fig. 12.3),
172 Part I: Science and Clinical Oncology
contribute to the replication of the viral genome.217 E1 and E2 bind
to an origin-of-DNA replication on the viral genome.218 E1 is a DNA
helicase that unwinds the viral double-stranded DNA genome at its
origin and, together with E2, recruits cellular DNA replication proteins
that then synthesize the viral DNA.218–220 Thus E1 and E2 both represent
potentially useful targets for intervening in the viral life cycle.
So far, we have focused on the role of certain mucosotropic HPVs
in causing human cancer. There is a growing appreciation that certain
cutaneous HPVs may also give rise to skin cancers. This is most clearly
established for patients with the genetic disease epidermodysplasia
verruciformis (EV). EV patients, who carry homozygous mutations
in one of two genes, EVER1 and EVER2, identified so far, are highly
susceptible to warts caused by certain cutaneous HPVs (e.g., HPV-5,
HPV-8), and these warts can progress to squamous cell carcinoma,
particularly at sun-exposed sites.221 A recently discovered mouse
papillomavirus that causes cutaneous warts in laboratory mice has
been shown to induce warts and squamous cell carcinoma in mice
treated with ultraviolet irradiation).222 Interesting to note, the E6
protein of this virus shares the ability of EV-associated HPV-8 E6
proteins to inactivate the Notch and TGF-β signaling pathways.1,223
This mouse papillomavirus may therefore provide an informative
animal model for understanding the role of cutaneous HPVs in skin
cancer, the most common human cancer.
One desirable long-term public health strategy for dealing with
this and other human tumor viruses is the generation of an effective
prophylactic vaccine to prevent initial infection. Such vaccines are
now available and have been shown not only to efficiently prevent
HPV infection but also to reduce significantly the risk of developing
HPV-associated neoplastic disease.
HUMAN T-CELL LEUKEMIA VIRUS TYPE I
Adult T-cell leukemia or lymphoma (ATLL), a tumor of CD4+ T
cells, is caused by human T-cell leukemia virus type I (HTLV-I), the
only retrovirus now accepted as being oncogenic in people. HTLV-I
is found worldwide, with approximately 10 to 20 million people
estimated to be infected today. This number is likely an overestimate
because of a failure to distinguish serologically between HTLV-I and
HTLV-II.224 HTLV-I is particularly prevalent in restricted sites,
including southern Japan, the Caribbean, and West Central Africa.
ATLL is prevalent in areas in which HTLV-I is common, and it is
estimated that ATLL will develop in as many as 5% of HTLV-I–positive
carriers in their lifetime.225 HTLV-I also causes a progressive, paralytic
myelopathy termed HTLV-I–associated myelopathy or tropical spastic
paraparesis. This neurologic disorder appears to arise preferentially in
patients whose human leukocyte antigen (HLA) haplotypes fail to
limit viral load.226
The data that link HTLV-I causally to ATLL are varied. ATLL
can occur in familial clusters. It is characterized by an average age at
onset of 56 years and proves rapidly fatal, with 50% of patients dying
within 6 months of diagnosis.225 In general, patients have antibodies
to HTLV-I–encoded proteins,227 and in 88 of 88 primary biopsy
specimens of ATLL examined, all had single copies of integrated
HTLV-I proviruses.228 The presence of the provirus of HTLV-I in all
of the tumors makes it likely that infection with the virus is an early,
contributing event in the evolution of the tumor. The presence of
ATLL in familial clusters is consistent with the routes of transmission
of HTLV-I. HTLV-I is passed from male to female via semen and
from mother to child by breast milk. The latter route has been
demonstrated prospectively. Encouraging carrier mothers to refrain
from breastfeeding has decreased transmission of HTLV-I to their
children by 80%.229 It appears highly likely that this form of public
health intervention will lead to a corresponding decrease in ATLL in
Japan in the future. Such a decrease would constitute formal proof
of the oncogenic role of HTLV-I in ATLL.
HTLV-I clearly differs from the highly oncogenic animal retroviruses
that encode oncogenes derived from cellular proto-oncogenes. HTLV-I
5' LTR 3' LTR
gag pol
pro env
tax
rex
Figure 12.4  •  Map of the human T-cell leukemia virus I (HTLV-I)
genome. Indicated by the line drawing is the approximately 9000 bp DNA
proviral form of the HTLV-I genome as it is found integrated into the host
genome. The genome as present in the viral particle consists of two copies of
a single-stranded positive-strand RNA. Shown are the long terminal repeats
(LTRs, in green) flanking the unique region that contains the translational
open reading frames (boxes) for structural (blue) and nonstructural (orange)
viral proteins. The LTRs contain cis-acting elements required for transcription
and replication of the viral genome. The structural genes are transcribed late
in the viral life cycle from the 5′ LTR, whereas the nonstructural proteins are
transcribed early from the 5′ LTR from different, spliced transcripts. See reference
380 for details of the genome and life cycle of HTLV-I.
is a complex retrovirus that encodes multiple ORFs in addition to
the gag, pol, and env genes common to simple retroviruses (Fig. 12.4).
However, none of these additional viral genes is obviously related to
known proto-oncogenes, and all are thought to affect the viral life
cycle either directly or indirectly by affecting the host cell.230,231 It is
accepted that one viral protein, Tax (see Fig. 12.4), which regulates
both viral and cellular gene expression, is a major contribution of
HTLV-I to leukemogenesis.232 It is also evident that viral infection
precedes onset of ATLL by 50 years or more, that many cells are
infected, and that this clonal tumor develops in only a minority of
infected people. These combined observations indicate that multiple
rare events in an HTLV-I–infected CD4+ T cell must occur for that
cell to evolve into ATLL.
Multiple approaches demonstrate that Tax can transform cells in
culture and be oncogenic in animal models. The introduction of a
vector expressing Tax into established, adherent rodent cells can
transform them to grow in an anchorage-independent fashion.231 Strains
of rodent cells can be transformed with Tax in combination with the
ras oncogene to yield cells tumorigenic in nude mice.233 Tax also has
been recombined into herpesvirus saimiri and introduced into resting
human T cells. These infected cells can proliferate and yield infected,
immortalized progeny, whereas the Tax-negative parental virus cannot
do so.234 In accordance with these data, variants of HTLV-I from
which the Tax gene has been deleted no longer can immortalize human
T cells in culture.235 These results in culture are paralleled and bolstered
by others in transgenic animal models. Expression of Tax from the
HTLV-I long terminal repeat (LTR; the viral promoter) in transgenic
mice leads to mesenchymal tumors.236 When its expression is directed
to lymphoid cells, leukemias develop in the transgenic animals.237
However, the exact means by which Tax transforms cells in culture
or is oncogenic in animal models is not obvious.
Tax can be considered a paradigm for viral proteins that affect
host cells in that it has multiple, distinct functions not found in any
one cellular protein. Apparently HTLV-I during its evolution has
assimilated multiple cellular activities in this one gene product. Tax
can be viewed as having at least three kinds of activities: it activates
transcription via nuclear factor–κB (NF-κB) and CBP, the cyclic
adenosine monophosphate response element-binding (CREB) protein;
it inhibits transcription, perhaps through binding HDAC1, and it
inhibits several tumor suppressor gene products.231,238–242 Tax potently
activates transcription from HTLV-I’s own LTR243 and activates the

promoters for IL-2, IL-2 receptor α chain, and c-fos.244–246 This
transcriptional activation obviously could contribute to proliferation
of an infected T cell. It also can activate the promoter for Bcl-xL and
thereby help to inhibit apoptosis.247 Tax can positively regulate transcrip-
tion by binding CREB and CBP, as well as some members of the
NF-κB family.248,249 Tax not only binds members of the NF-κB family
directly, but it also can activate NF-κB’s homing to the nucleus by
binding to IκBα and promoting its degradation.250,251 Some of Tax’s
protein-protein associations have been functionally validated through
chromatin immunoprecipitations that have documented the binding
of Tax, CREB, and CBP to HTLV-I’s LTR in intact, HTLV-I–trans-
formed T cells.252
Tax also can inhibit transcription. It has been shown to inhibit
both expression of the β-DNA polymerase gene239 and some promoters
regulated by CBP/p300.253 It has been proposed that the latter inhibition
occurs by Tax’s binding to CBP and effectively sequestering CBP such
that it is unavailable to bind other DNA-binding transcriptional factors.
It also has been shown that Tax binds HDAC1 as measured by co-
immunoprecipitations.236 Were Tax to tether HDAC1 to a promoter,
the resulting localized histone deacetylation presumably would lead
to its decreased support of transcription.254
A third activity of Tax is its inhibition of some cellular tumor
suppressors. Tax has been shown to bind to and inhibit the function
of p16INK4A.242 p16INK4A binds cyclin-dependent kinase 4 (CDK4), a
kinase that, when activated, can phosphorylate pRb, yielding release
of E2F transcription factors and promotion of the G1 to S transition
of the cell cycle. Tax, on binding p16INK4A, can increase the kinase
activity of CDK4 kinase.242 p16INK4A often is found to be mutationally
inactivated in tumors. Consistent with Tax’s functionally inactivating
p16INK4A, p16INK4A was shown to be wild type in sequence in two
HTLV-I–infected T-cell lines in which Tax is expressed, but deleted
in four uninfected T-cell lines.242 Tax also appears to bind cyclin D3
and thereby may foster, by a second means, the activity of CDK4
and CDK6.240 Tax’s binding to p16INK4A and cyclin D3 would inhibit
control of the cell cycle and promote cell proliferation. Tax also can
inhibit the transcriptional activity of p53 by an NF-κB–dependent
mechanism that culminates in the phosphorylation of p53 as certain
residues.241 Such an inhibition of p53 is likely to limit its induction
of apoptosis and increase the rate of survival of HTLV-I–infected,
proliferating T cells.
The multiple activities of Tax likely contribute to HTLV-I’s associ-
ated leukemogenesis but must be insufficient for the development of
ATLL. Multiple T cells are initially infected by HTLV-I, but over the
course of the 50 to 60 years of its development, only one infected
cell and its progeny give rise to the tumor. The additional genetic and
epigenetic events necessary for this evolution are not known. It also
is clear that the expression of viral genes in infected cells is surprisingly
low; measurements of RNAs encoding Tax indicate that between 0.1%
and 10% of freshly harvested, infected T cells express any Tax message
in vivo.255 The fact that Tax expression is absent in approximately
60% of ATLL indicates that a role of this viral gene in cancer develop-
ment must be transient in nature,256 and this finding has led to
additional studies, described later, that have identified another HTLV-I
gene that is potentially as important, if not more important, in ATLL.
One of the hallmarks of ATLL is that, whereas the 5′ untranslated
region (UTR) of HTLV-I that drives expression of Tax is often disrupted
in ATLL, the 3′ UTR is not disrupted.256 Analysis of HTLV-I transcripts
led to the realization that the viral genome gives rise to antisense
transcripts,257 one arising from the 3′ UTR that is spliced, and the
other arising from within the Tax gene that is unspliced.258 Both
encode for highly related proteins referred to as HTLV-I bZIP factor
(HBZ). Important to note, HBZ is found expressed in all ATLLs.259
The two forms of HBZ, which differ by only nine amino acids at
their N-terminus, contain three domains: an N-terminal activation
domain, a central domain, and basic ZIP domain in the C-terminus.
The version of HBZ arising from the spliced mRNA, sHBZ, is more
abundant than that arising from the unspliced mRNA, usHBZ. HBZ
Viruses and Human Cancer  •  CHAPTER 12 173
originally was discovered to inhibit expression of the sense mRNAs,
including those that encode Tax,260 through its interaction with CREB
proteins261 and p300/CBP.262 Of relevance to ATLL, knockdown of
expression of HBZ in ATLL-derived cell lines led to suppression of
proliferation.259 Overexpression of the spliced HBZ could induce the
proliferation of ATLL cells, whereas overexpression of usHBZ could
not induce such proliferation.258 In the context of transgenic mice,
expression of the HBZ gene from the CD4 promoter/enhancer led to
increases in the frequency of CD4+ T cells, systemic inflammation,
and the spontaneous development of T-cell lymphomas after a long
latent period in more than a third of the mice,263 all hallmarks of
human patients infected with HTLV-I. The HBZ protein has recently
been found to activate E2F transcription factor 1 (E2F1)–dependent
transcription by targeting pRb/(E2F1) leading to dysregulation of the
cell cycle. This activity is much like that of the HPV E7 oncoprotein
complex which activates the transcription of genes under E2F1
control that are critical for DNA replication and cell cycle progres-
sion.264 These studies highlight the potential importance of antisense
transcripts arising from human retroviruses, contributing to their
pathogenicity.
A genome-wide analysis of ATLL patients revealed reactivating
mutations in several cellular genes including phospholipase-Cγ1, protein
kinase Cβ, caspase recruitment domain-containing protein 11, discs
large homolog 1, vav guanine nucleotide exchange factor 1, and cluster
of differentiation 28, in that order of prevalence, and show functional
overlap with the Tax interactome, perhaps explaining how ATLL
becomes independent of Tax.265
HUMAN HEPATITIS C VIRUS
Human HCV is accepted as an etiologic agent for HCC along with
HBV. Approximately half of the cases in the United States266 and 25%
globally267 are ascribed to HCV. Infection with HCV constitutes a
twentyfold risk for men in Taiwan of developing HCC.268 HCV
represents the most common chronic viral infection among bloodborne
pathogens in the United States, where the rate of infection during
the period from 1988 to 1994 was estimated to be 1.8%.269 The
WHO estimates the worldwide infection rate at 3%, yielding more
than 170 million infected persons.270 The rates of infection vary widely,
with rates as low as 0.6% in Germany to as high as 22% in Egypt.269
Infection is thought to arise primarily from the use of shared needles
among intravenous drug users, blood transfusions, and contaminated
syringes in medical settings in developing countries.269 The establishment
of sensitive tests for identifying contaminated blood and blood products
fortunately has now greatly reduced the rate of infection in the general
population in developed countries. As with people chronically infected
with HBV, HCC in HCV-positive individuals correlates with chronic
hepatitis and cirrhosis. Di Bisceglie271 has estimated that cirrhosis
develops in 20% of people chronically infected with HCV per decade,
and by two decades, HCC develops in 2% to 7% of chronically
infected people. The hyperproliferative state induced by chronic hepatitis
and cirrhosis is argued to lead to an accumulation of genetic changes
and contribute to the onset of liver cancer. What specifically HCV
contributes to this scheme is not known. Fortunately, potent antiviral
drugs that can clear infection have been developed, so the incidence
of HCV-caused HCC should decrease rapidly.
HCV is a flavivirus, indicating that it is unique among the human
tumor viruses for having RNA as its only genetic material (Fig. 12.5).
It was identified in 1989 during a search for the causal agent of non-A,
non-B hepatitis.272 This flavivirus contains a 9.6-kb, positive-stranded
RNA as its genome, which encodes a single translation product of
approximately 3000 amino acids (see Fig. 12.5). This polyprotein is
cleaved by both cellularly and virally encoded proteases to yield at
least 10 proteins.273 Inhibitors of these viral proteases are being
developed, which do reduce the levels of HCV RNA in the plasma
of treated patients for short times.274,275 The study of HCV has been
daunting for at least two reasons. Its sequence varies in infected people
174 Part I: Science and Clinical Oncology
2A 4A 5C
p22 gp35 gp70 p21 p70 p27 p56 p66
C E1 E2/NS 1 2B NS3 4B 5A 5B
5' 3'
Figure 12.5  Map of the hepatitis C virus (HCV) genome. The
9500-nucleotide-long positive-strand RNA of HCV is shown in black. It
encodes a single 3000–amino acid polyprotein that is proteolytically cleaved
into mature proteins (indicated on the map by labeled boxes with alternative
names indicated above the boxes) by virally encoded proteases (NS3/4A and
possibly 2B). At the 5′ and 3′ ends are short noncoding regions (NCRs) that
contain signals for replication of the viral genome. The 5′ NCR also contains
an internal ribosome entry site (IRES). Structural proteins are shown in blue.
Nonstructural proteins, including proteases, helicases, and RNA polymerase,
are shown in orange. See reference 381 for details of HCV’s genome and
life cycle.
such that there are six recognized genotypes with multiple subtypes
among patients and a spectrum of quasispecies within any one infected
individual.276 The different genotypes vary substantially in the success
with which they can be treated.277 Members of these quasispecies
within one patient vary by 1% to 2% in their sequence.278 In addition,
until quite recently, no cell culture for HCV has been available.
Researchers have made heroic efforts to overcome this hurdle and
have met with partial success.
In 1999 Lohmann and colleagues279 described the replication of a
subgenomic derivative of HCV in a cell line derived from a human
HCC. These subgenomic replicons were difficult to establish, but once
established, they exhibited bona fide characteristics of flaviviral nucleic
acid replication. In each cell, 1000 to 5000 molecules of plus-strand
RNA were present; minus-strand RNA was present at 10% to 20%
of the level of the plus-strand RNA; and this viral RNA replication
was insensitive to treatment of cells with actinomycin D.279 This mode
of nucleic acid replication in cells would allow the elucidation of the
cis and trans elements it requires, but with an unexpected twist. The
efficient replication of the subgenomic derivatives of HCV requires
mutations in at least two viral genes that enhance replication in cells
but abrogate infectivity of intact HCV RNA.280 Chimpanzees are the
only nonhuman host for HCV. The parental strain of HCV used to
derive the subgenomic replicons is infectious in this primate host,
whereas a derivative of it, which contains the mutations required for
the efficient replication of the subgenomic replicons, is not infectious
in this primate host.280 This finding must limit the conclusions to be
drawn from the analyses of replication of these mutated subgenomic
replicons in cells. This finding also indicates that the generation
of the many sequence variants that constitute the quasispecies
within any one infected patient may contribute to the successful
replication of HCV.
Earlier attempts at developing cell cultures that support the
replication of HCV have been substantively advanced by the
identification of an isolate of HCV genotype 2a, JFH1, which replicates
in a human HCC–derived cell line after transfection of its RNA.281,282
A chimeric HCV genome consisting of a portion of this isolate and
a portion of a different isolate of genotype 2a strain has been found
to be infectious in cell culture, and the virus derived in cell culture
has been found to be infectious in chimpanzees and in mice reconstituted
with human hepatocytes.283 These findings will now pave the way to
dissect HCV’s replicative functions molecularly. They have facilitated
the identification and testing of drugs targeted to inhibit HCV’s
replication, such as the inhibitors of its encoded proteases.
A second important insight has been confirmed from the analyses
of HCV propagated in cell culture and animal models. Virus isolated
from infected animals is more infectious on a “per-particle basis” than
virus isolated from cell culture; this increased specific infectivity
correlates with a lower buoyant density of the virus isolated from
animals.283 Although the cellular receptors used by HCV to bind to
and enter cells have not yet been fully elucidated, it is clear that HCV
particles on binding serum molecules such as high-density lipoprotein
become more infectious.284 This binding would lower their buoyant
density and mediate binding to a cellular receptor such as the human
scavenger receptor class B type I.284,285 Virus grown in cell culture
would not be exposed to these serum molecules and thus would be
less infectious.
One promising path to study HCV infections in cell culture is to
generate hepatocytes by inducing their differentiation from either
human embryonic stem cells (hESCs) or induced pluripotent stem
cells (IPSCs). Wu and colleagues286 have generated hepatocytes through
differentiation of both types of stem cells in culture and infected these
hepatocyte-like cells with derivatives of HCV isolated from cell culture
and with serum isolates. Both forms of the virus yielded successful
infections, each of which secreted core antigens.286 The success of
infecting these hepatocyte-like cells with serum isolates of genotypes
1A and 1B is particularly desirable because other culture systems do
not support infection by serum isolates in general and those of genotypes
1A and 1B in particular. The general application of hESCs and IPSCs
after their differentiation to hepatocyte-like cells for study of infection
by HCV will depend both on the efficiency of this differentiation
and the practicality of carrying out this differentiation with large
numbers of precursors.
Evidence for a direct role of HCV in HCC has come from studies
of the HCV Core protein. The HCV Core protein can cooperate
with an activated form of the ras oncogene to transform primary
rodent cells in tissue culture.287 HCC develops in mice transgenic for
the HCV Core protein.288 Several potential mechanisms have been
invoked to explain the transforming potential of HCV Core protein.
It has been found to enhance cell proliferation through the stimulation
of mitogen-activated protein kinase.289,290 In addition, HCV Core
protein can inactivate a transcription factor, lZIP, and this inactivation
correlates with transformation in rodent cells.291 More recently, the
Core protein has been found to activate STAT3, and this activation
may contribute to its transforming potential.292 Whether the HCV
Core protein contributes directly to human HCC is uncertain. Neither
replication of chimeric HCV in cell culture nor infection of chimeric
mice populated with human hepatocytes will allow ready testing of
the possible role of the Core protein in HCV’s oncogenesis.
HCV infection alone is not sufficient to give rise to HCC, indicating
that other events must occur that contribute to this cancer. Exome
sequence analyses have begun to identify changes in cellular genes
that contribute to HCC. One such study293 identified mutations in
a number of known cancer-related genes such as p53 and CTNNB1,
which previously had been identified as being mutated in HCC, as
well as mutations in ARID2, DMXL1, and NLRP1. The mutations
in ARID2 were all found to disrupt the coding for its gene product,
which is consistent with ARID2 being a tumor suppressor in HCC.
ARID2 is a subunit of the polybromo- and BRG1-associated factor
chromatin remodeling complex, which facilitates ligand-dependent
transcriptional activation by nuclear receptors. Interesting to note,
mutations in ARID2 selectively arise in HCV-associated HCC and
are infrequently observed in HBV-associated HCC.293 The significance
of this finding remains unclear.
Potent inhibitors of HCV have been developed that target different
essential steps in viral replication. The first generation of these drugs
inhibits a protease encoded by HCV and is effective in patients at
picomolar concentrations.294 This and other new inhibitors achieve
sustained virologic responses, defined as “undetectable HCV RNA in
the serum at a 24-week follow-up,” in greater than 95% of patients
and are active against most or all genotypes of the virus.294 In the
United States, approximately 10,000 deaths are attributed to HCV
each year, and today infection with HCV is the leading indication
for liver transplantation.269 It is likely that these new treatments will
limit or abrogate HCV in patients with HCC before they are recipients
of liver transplants. Such treatment should increase the long-term
success of this transplant therapy.

KAPOSI SARCOMA HERPESVIRUS
The identification of Kaposi sarcoma herpesvirus (KSHV) represents
the culmination of much scientific detective work. Kaposi sarcoma
(KS) was known before the AIDS epidemic,296,297 but it increased
markedly among HIV-positive people. Researchers therefore looked
for molecular evidence of an infectious agent present in KS lesions.
In 1994, Chang and colleagues298 used a newly developed enrichment
procedure based on polymerase chain reaction to identify DNA
sequences present in KS but absent in normal cells. They identified
a new herpesvirus, KSHV (also termed human herpesvirus 8 [HHV-8]),
which is related to EBV and herpesvirus saimiri.298 Retrospective studies
indicate that KSHV was prevalent in different parts of the world
before the spread of HIV and that in Africa, for example, the prevalence
of KSHV has not changed. What has changed there is the incidence
of KS, so that in regions where it was formerly infrequent, the
incidence of KS rose threefold between 1988 and 1996.299 Similarly,
the frequency of infection with KSHV of certain cohorts in the United
States has not altered with the advent of HIV,300 but the incidence
of KS has changed.301 KSHV also has been detected in one class of
lymphomas termed body cavity–based lymphomas or primary effusion
lymphomas (PELs)302 and in an atypical lymphoproliferative disorder
termed multicentric Castleman disease.303
KSHV can be transmitted sexually, as evidenced by its prevalence
in a population being related to the number of sex partners of its
members.304 Its most frequent route of transmission is via saliva.305
KSHV also is transmitted among intravenous drug users, with 30% of
this group being infected in parts of China.306 Its prevalence worldwide
is clearly nonuniform, with a low prevalence in the United States and
Northern Europe, intermediate levels around the Mediterranean, and
high levels in the Middle East and in sub-Saharan Africa.306 Although
KS was a rather rare tumor before the AIDS epidemic, it has evolved to
become common in areas of the world in which people are coinfected
with KSHV and HIV. The immunosuppressive role of HIV makes it
likely that immunosuppression, along with KSHV, contributes to the
development of KS.307 Treating HIV-positive people with highly active
antiretroviral therapy (HAART) not only decreases a person’s load of
HIV but also decreases the incidence of KS and, importantly, leads to
a regression of the tumor in patients with KS.308 These observations
indicate that immunosuppression not only likely contributes to the
development but also the maintenance of KS, along with infection
with KSHV. HAART also has led to a decrease in the incidence of
overall AIDS-related non-Hodgkin lymphomas, as well as an increased
tolerance for their treatment with chemotherapy.308,309
KSHV is now accepted as contributing causally to KS, PEL, and
multicentric Castleman disease. For example, a prospective study in
HIV-infected adults has identified a significant elevation in titers
of antibodies against KSHV in those in whom KS develops.310 KS
and PEL also display intriguing features likely to reflect their viral
etiology. Single-cell assays of early KS lesions have detected KSHV in
a minority of cells that surround their vascular spaces, whereas in the
more advanced, nodular lesions, more than 90% of the spindle cells
characteristic of these lesions are KSHV-antigen positive.311 The viral
antigen-positive cells also stain with antibodies against VEGF receptor
3, indicating that they are lymphatic or proliferating endothelial cells.311
That only a subset of the cells characteristic of early KS lesions appear
to be infected with KSHV likely indicates that infected cells affect
the development of their neighbors. This possibility is supported by
the multiple cellular homologues of cytokines and receptors encoded
by KSHV that may allow infected cells to interact with adjacent,
uninfected cells.312,313 PEL cells are B cells in origin, and in six of seven
cases examined, they have nongermline Ig mRNAs, indicating that
they are likely derived from B cells that have encountered antigen.314
However, they often fail to express B-cell activation antigens.315 PEL
cells usually but not always are coinfected with EBV.302,315 The role
of EBV in the etiology of this lymphoma is not yet determined,
but for cases in which EBV is retained as a plasmid in the PELs,
Viruses and Human Cancer  •  CHAPTER 12 175
it must be providing the tumor cells with one or more selective
advantages.
KSHV encodes many genes with clear homologies to cellular genes,
some of which are candidates for contributing to viral oncogenesis
(Fig. 12.6). Three categories of these cellular homologues are particularly
likely to be important for tumor development: cyclins; inhibitors of
apoptosis; and cytokines and receptors. KSHV encodes its own cyclin.
Cellular cyclins bind specific, dependent kinases (CdKs) whose activities
promote progression through the cell cycle and are controlled by
cellular inhibitors, including p16INK4A, p21CIP1, and p27Kip1. The
KSHV-encoded cyclin can promote cellular proliferation in part by
overcoming these cellular inhibitors in its complex with CdK6.316
KSHV also encodes its own inhibitors of apoptosis, viral Bcl-2
and viral FLIP (an inhibitor of cellular FLICE, a protein that mediates
Fas ligand-induced cell death) (see Fig. 12.6). The viral Bcl-2 shares
its limited sequence homology with cellular Bcl-2 in the regions critical
for inhibiting apoptosis and fails to dimerize with cellular Bcl-2 and
Bcl-xL, thus avoiding regulation by potential cell-binding partners.317
It can inhibit the apoptosis induced by efficient expression of KSHV’s
cyclin.318 Viral FLIP can inhibit apoptosis mediated by the Fas pathway
and induced by cytotoxic T lymphocytes.319 Viral FLIP also activates
NF-κB signaling by binding to its inhibitor, IKKγ,320,321 which fosters
expression of cytokines in infected cells.316
Finally, KSHV encodes homologues of cytokines, cytokine receptors,
and an intriguing protein that stabilizes cellular cytokine mRNAs (see
Fig. 12.6). Its viral IL-6 may promote proliferation of PEL cells,
although they appear to be dependent on cellular IL-6 and not viral
IL-6 for their continued growth.322 This dependence may be facilitated
by KSHV’s Kaposin gene, which is synthesized by an atypical trans-
lational initiation to yield a protein formed from 23 residue repeats.
Kaposin activates a protein kinase to stabilize mRNAs that encode
cellular cytokines, including IL-6.323 KSHV also encodes a G protein–
coupled receptor, viral GPCR, which is likely to be pivotal for the
development of KS. The expression of viral GPCR in endothelial cells
in mice leads to KS-like tumors, whereas the individual expression of
KSHV cyclin, KSHV Bcl-2, or KSHV FLIP does not lead to such
tumors.324 What is particularly exciting is that cells inoculated into
mice that express viral GPCR promote tumor formation by coinoculated
cells that individually express viral cyclin or viral FLIP, or both.324
These findings support a model in which viral GPCR contributes to
the development of KS by affecting neighboring cells not infected by
KSHV. The model is also supported by the findings that viral GPCR
induces expression of the cellular vascular endothelial growth factor
(VEGF) receptor 2 in endothelial cells that proliferate in the presence
of VEGF and that viral GPCR induces expression of VEGF.325 Clearly,
these observations define an autostimulatory loop in which KSHV
GPCR alone can maintain proliferation of endothelial cells. They are
consistent with findings in which mouse bone marrow cells transfected
with recombinant KSHV grow as “KS-like” tumors in nude mice.326
The inhibition of expression of viral GPCR in these KSHV-positive
cells inhibits tumor formation when the engineered cells are transplanted
into recipients.326 These data indicate that although the viral GPCR
may be expressed only in a minority of infected endothelial cells, it
makes an essential contribution to oncogenicity by KSHV.
KSHV limits its host’s immune response. The virus suppresses
Toll-like receptor 4 (TLR4), which decreases the infected cell’s innate
immune response.327 It inhibits expression of HLA-1 molecules, which
would help to avoid recognition of infected cells by cytotoxic T cells328
but would increase the likelihood of their being recognized by NK
cells. KSHV infection also limits expression of receptors on NK cells
needed to recognize their infected targets,329 showing that it uses
multiple avenues to evade the immune response. This immune evasion
also likely contributes to its oncogenicity.
Recent studies of cell lines derived from PEL and human endothelial
cells are providing the foundation for understanding infections by
KSHV in vivo. PEL cells maintain KSHV DNA extrachromosomally
and consistently express the viral protein LANA-1 (see Fig. 12.6),
176 Part I: Science and Clinical Oncology

TR

GPCR

LANA1

vCYC vIL-6

miR-K12-9a/b

miR -K122-11
vFLIP

miR -K1 -8
miR -K1 2-7
miRK12-10
2

mi -K -6
miRR-K112-5
miR-K12-1

miR -K12

-K 2-4
miR -K112-3
-K1 2-2
2-1
K12 -
miR

miR
KSHV
160 kbp

vBcl2

LANA2

ORF50

pre-miRNAs encode miRNAs on both arms of pre-miRNA

(as shown in miRBase release 22)
Figure 12.6  •  Map of the Kaposi sarcoma herpesvirus (KSHV) genome. The genome of KSHV consists of approximately 160 kbp of linear double-
stranded DNA in the viral particle. The viral genome becomes circularized on infection via the terminal repeats found at the 5′ and 3′ ends of the linear
genome. The terminal repeats also contain the origin of plasmid replication used during the latent phase of the viral life cycle. Shown by the white boxes are
positions of the viral genes expressed during latency and/or thought to contribute to oncogenesis by KSHV. The arrows represent the positions from which
the RNAs encoding the labeled proteins are expressed. Several of these primary transcripts are polycistronic. See reference 382 for details of KSHV’s genome
and life cycle.

which is required for viral plasmid replication.330,331 The KSHV genomes
are found in clusters and partition randomly to daughter cells.331a
This unprecedented mechanism provides an appealing target for antiviral
therapy. In general, these cells support the latent phase of KSHV’s
life cycle and also express the viral cyclin and the viral inhibitor of
cellular FLICE, FLIP, but few other viral proteins. Some PEL cell
lines support an inefficient spontaneous conversion to the lytic phase
of the viral cycle, which can be further induced by treatment of cells
with tetradecanoyl phorbol acetate or introduction of a plasmid
including ORF50 (see Fig. 12.6), a viral inducer of the lytic cycle.332
The released virus is infectious in human dermal microvascular
endothelial cells (DMVECs).333,334 These DMVEC cultures can be
passaged to yield populations in which all cells are infected, express
LANA-1, assume a spindle-cell morphology, and can proliferate
indefinitely.333 The spindle shape is characteristic of cells in KS lesions
in vivo. Staining of these infected DMVEC-derived spindle cells
indicates that 5% to 10% of them spontaneously support early stages
of KSHV’s lytic cycle and 1% to 2% express genes diagnostic of the
late stages of the viral life cycle.333 If endothelial cells infected in vivo
by KSHV display a similar distribution of cells supporting the latent,
early, and late lytic phases of the viral life cycle, then the enigma of
the oncogenic contribution of viral genes expressed only during the
lytic cycle may be resolved. A sustained, spontaneous conversion of
5% to 10% of KSHV-infected cells to support the viral lytic cycle
might allow enough infected cells to express enough KSHV GPCR
to promote the bystander-dependent tumor evolution proposed by
Bais and colleagues.325
KS is the most common malignancy among HIV-positive people.
Although there are currently no effective antiviral therapies directed
against latent infection with KSHV, multiple trials aimed at inhibiting
its lytic cycle appear to have achieved a decrease in the incidence of
KS.335 Treatment with acyclovir is ineffective, but treatment with
ganciclovir reduced the incidence of KS from 40% to 90% in various
trials.335 Similar treatments also were partially effective in limiting the
incidence of PEL and Castleman disease. Whether this inhibition of
KSHV’s lytic cycle is limiting disease by preventing infection of cells
that evolve into tumors or preventing lytically infected cells from
contributing to a necessary tumor microenvironment is not known.
MERKEL CELL POLYOMAVIRUS
The association of viruses with AIDS-related malignancies drove
Chang and colleagues to search for a virus associated with a second
AIDS-related malignancy, Merkel cell carcinoma (MCC). This rare but

highly aggressive skin cancer arises in elderly and in immunosuppressed
and immunodeficient patients, often in sun-exposed regions of the
body.336 In the general population, more than a third of patients with
MCC will die of their cancer337; in immunocompromised patients, the
percentage rises to greater than 50%. MCC is distinguished by a unique
pattern of expression of neuroendocrine and epithelial-specific markers
expressed in Merkel cells, which are specialized mechanoreceptors
cells within epithelia that transduce mechanical stimuli from the skin
(reviewed in references 338–341). Feng and colleagues342 used a state of
the art “digital transcriptome subtraction” (DTS) approach to identify
nonhuman sequences present in MCCs. DTS involves deep sequencing
of complementary DNAs followed by in silico subtraction of host
transcripts to divulge nonhuman sequences present in a sample.342 Using
DTS, Feng and colleagues342 identified nonhuman sequences in four
MCC samples that had similarity but were not identical to the large
tumor antigen genes of previously identified human polyomaviruses,
which led them to a full-length clone of a novel polyomavirus that
they called Merkel cell polyomavirus (MCPyV). MCPyV is present
in approximately 80% of cases of MCC.343–345 Like other human
polyomaviruses, MCPyV has a circular double-stranded DNA genome
containing a well-conserved replication origin and transcriptional control
region and translational ORFs encoding for small and large tumor
antigens on one strand, and capsid proteins VP1, VP2, and VP3 on
the other strand. Serologic studies indicate that most of the human
population is exposed to MCPyV,346–350 likely in early childhood.346,347
The virus has been detected in a number of different cell types, including
non-MCC cells of the skin.351–354 The identification of this ubiquitous
novel human polyomavirus in the skin led other groups to screen for
other human polyomaviruses in the skin using a technique for cloning
small circular DNAs called rolling circle amplification. This screening led
to the identification of many additional novel human polyomaviruses,
most of which have not been linked to any specific disease.
In their analyses of MCC, Shuda and colleagues345 made another
important observation, akin to that made by zur Hausen in the context
of HPV’s role in cervical cancer: the MCPyV genome is found clon-
ally integrated into the host genome in the cancers. Importantly,
these integrated copies of MCPyV carried mutations in the large
tumor antigen gene that resulted in expression of truncated large
tumor antigen gene products (these mutations did not alter the
small tumor antigen gene, which is also expressed in MCC). The
truncated large tumor antigen proteins expressed in MCC retain
the domain within the N-terminal half of the full-length protein
that mediates interaction with the cellular tumor suppressor, pRb,
but they are missing part or all of the highly conserved adenosine
triphosphatase/DNA helicase domain required for viral DNA
replication. This finding led to the hypothesis that in MCC, there
is selection for replication-defective MCPyV genomes that retain an
ability to inactivate pRb. Consistent with this hypothesis, knockdown
of expression of the MCPyV tumor antigens (both large and small
tumor antigens) in MCC-derived cell lines led to cell growth arrest
and death.355 Follow-up studies by Shuda and colleagues,356 however,
led to the further discovery that specifically knocking down expression
of MCPyV small tumor antigen, although not affecting expression
of MCPyV large tumor antigen, led to growth arrest, although not
death, of MCPyV-positive MCC cell lines, leading to the revised
hypothesis that both small and large tumor antigens contribute to
MCC. MCPyV small tumor antigen was found to transform cells
at least in part by acting downstream of the mammalian target of
rapamycin signaling pathway to preserve eukaryotic translation
initiation factor 4E–binding protein 1 hyperphosphorylation, resulting
in dysregulated cap-dependent translation.356 That continued expression
of MCPyV large tumor antigen is specifically necessary for the survival
of MCPyV-positive MCC cells and appears to correlate with large
tumor antigen, causing an increase in levels of expression of survivin.357
A drug that inhibits survivin was shown to induce programmed cell
death of MCPyV-positive MCC cells in tissue culture and prolong
the survival of immunodeficient mice in which these tumor-forming
Viruses and Human Cancer  •  CHAPTER 12 177
MCC cells have been engrafted,357 providing a potential new therapeutic
approach for treating patients with this deadly cancer.
Several transgenic mouse models have been developed in which
the MCPyV tumor antigens are targeted in their expression to different
cell types. In one case, the region of MCPyV present in an MCC
encoding for small tumor antigen and a truncated form of large tumor
antigen was placed under the control of the ROSA26 allele carrying,
upstream of the MCPyV sequences, a strong transcriptional stop
sequence flanked by Lox P sites, allowing for tissue-specific expression
by crossing to mice expressing Cre in a tissue-specific manner. Once
MCPyV genes were turned on in the epidermis, the mice developed
hyperplastic epithelium and spontaneously developed papillomas on
the rear torso.2,358 Both small tumor antigen and the truncated large
tumor antigen were found to be expressed in the epithelium and
tumors. Two other models were generated in which just small tumor
antigen was expressed in the epidermis. In neither of these models
were MCCs observed.3,359 However, in a more recent study, cellular
aggregates with histology and marker expression mimicking that of
human intraepidermal MCC were observed in embryos when investiga-
tors coexpressed in the epidermis small tumor antigen and the cell
fate determinant, atonal bHLH transcription factor 1 (Atoh1),
selectively found to be expressed in Merkel cells.4,360,361
TREATMENT AND PREVENTION OF
VIRAL TUMORS
Tumors associated causally with viruses are treated variously; surgical
resections, when appropriate, are used in combination with chemo-
therapy and/or radiation therapy. These treatments are disappointing
in that overall survival rates are often low and thus far have not been
able to capitalize on any specific antiviral therapies. Current survival
rates after different therapies obviously vary with the malignancy, its
stage, and the setting in which it is treated. For example, youngsters
with Burkitt lymphoma have a 4-year overall survival rate of 65%.362
Approximately 45% of patients with NPC remain disease free after
treatment for 10 years, but this value is highly dependent on the stage
at which the NPC was diagnosed.179 In the United States, 26% of
HBV-positive patients with HCC that has been surgically resected
have a 5-year, local disease-free survival rate compared with 38% for
the analogous HCV-positive group.363 A retrospective study of German
patients with a mix of HBV- and HCV-associated HCC found a
median survival rate of 7 years.364 The 5-year median survival rate for
patients with cervical carcinoma ranges from 65%365 to 75% to 90%366
in different studies. The median survival rate for patients who have
been diagnosed with ATLL is only 10 months.367 The overall survival
rates of patients with KS who are HIV positive have changed with
the introduction of HAARTs. Before this therapy was available, median
overall survival was 13 months; with HAART, it surpasses 28 months.368
The median survival for patients with PEL has been described as
“dismal,” but case reports indicate that some combination therapies
are helpful.369 These actuarial findings indicate that the development
of antiviral therapies directed at the viral gene products that maintain
tumor phenotypes is a highly desirable goal. It also obviously is desirable
to develop vaccines to prevent infections by tumor viruses. The
development of effective vaccines that can prevent an initial infection
or induce the elimination of viral persistence is being pursued for all
human tumor viruses. Success has been achieved for two of them.
Hepatitis B Virus Vaccine
Although four serotypes of HBV exist, highly effective vaccines for
HBV have been developed that consist only of its surface antigen,
HBsAg. Today this subunit vaccine is usually synthesized with a
recombinant DNA expressed in yeast. Vaccines for HBV began to
be used in the early 1980s, and since 2001, 129 countries throughout
the world have routinely vaccinated infants and/or adolescents against
HBV.370 These vaccinations are effective. The fraction of children
178 Part I: Science and Clinical Oncology

infected with HBV has been reported to have declined between
7.5-fold and 10-fold in Taiwan and the Gambia, respectively.84,85 This
decline has been paralleled by a detectable drop in the frequency of
HCC in children in Taiwan, an age group for which this viral tumor
is rare.86
The current HBV vaccines apparently are free of unwanted adverse
effects but can select for variants of HBV resistant to the neutralizing
antibodies they elicit.370 One domain of the HBsAg in particular
elicits neutralizing antibodies efficiently, and mutations in it can allow
for viral escape. The frequency of these escape mutants in populations
of vaccinated children has risen significantly.371 One means to overcome
the selection for such escape mutants would be to generate vaccines
for HBV that include more than the HBsAg as an immunogen; such
vaccines are now being developed.
Human Papillomavirus Vaccine
The second family of human tumor viruses for which an effective
prophylactic vaccine can now be envisioned are the HPVs associated
with cervical cancer, other anogenital cancers, and certain head and
neck cancers, in particular HPV-16 and HPV-18. These two viruses
account for approximately 85% of cervical cancers worldwide, and
in a recent study, 16% of new infections.372 Two highly effective
vaccines that prevent infections by HPV-16 and HPV-18 have been
approved for use in the United States and other countries. These
vaccines are based on the production of viruslike particles (VLPs)
composed of the major capsid protein of HPV-16 and HPV-18, L1,
which self-assembles into icosahedrons (see Fig. 12.3). These L1-based
VLPs induce neutralizing antibodies in vaccinated individuals. Preclini-
cal studies with CRPV and canine oral papillomavirus demonstrated
the effectiveness of VLP-based vaccines in protecting animals from
CRPV and canine oral papillomavirus infection.373,374 Phases I and II
clinical trials likewise demonstrated the effectiveness of HPV-16
VLP-based vaccines in inducing neutralizing antibodies specific for
HVP-16 and in preventing HPV-16 infections.375,376 One issue of
relevance to the effectiveness of these vaccines is the specificity of
immune responses invoked by VLP-based vaccines for specific HPV
genotypes. Approximately a dozen HPV genotypes are associated with
anogenital cancers. VLP-based vaccines induce protective immunity
that is highly specific for the genotype from which the VLP is generated.
This specificity led to the development of polyvalent vaccines composed
of a mixture of VLPs generated with L1 proteins from multiple
mucosotropic HPV genotypes. One of the available vaccines, Gardasil,
is a multivalent vaccine composed of HPV-16, HPV-18, HPV-6, and
HPV-11 VLPs. HPV-6 and HPV-11 are low-risk mucosotropic HPVs
that induce genital warts but do not contribute to cervical cancer.
The other available vaccine, Cervarix, is composed of just HPV-16
and HPV-18 VLPs. It remains to be seen whether, once a population
is protected from HPV-16– and HPV-18–induced cancers, other HPV
genotypes arise to induce a greater incidence of cervical cancers than
they currently cause. For this reason, the development of vaccines
that provide protection against a wider variety of high-risk HPVs is
now being considered. One approach is to add L1 VLPs specific for
other genotypes to the current vaccines. Alternatively, incorporation
of the minor capsid protein L2 may be of benefit, because neutralizing
antibodies induced against L2 tend to be more generally effective at
neutralizing multiple genotypes.377 In this regard, a stand-alone L2
vaccine might be effective. Concern exists about whether the HPVs,
particularly HPV-16 and HPV-18, will evolve to become resistant to
VLP-based vaccines. Assuming the high effectiveness of the vaccines
currently under development and their worldwide distribution, and
given the long latency of cervical cancer, the WHO estimates that
prophylactic HPV vaccines will lead to a reduction of deaths due to
cervical cancer no earlier than 2040. Given that HPVs also cause
other anogenital cancers, as well as head and neck cancers that affect
both men and women, the available vaccines are now being considered
for use in vaccinating both young men and young women in the
United States and elsewhere in the world.
The complete reference list is available online at
ExpertConsult.com.

KEY REFERENCES
1. Parkin DP, Pisani P, Munoz N, Ferlay J. The global
health burden of infection associated cancers. Cancer
Surv. 1999;33:5–33.
3. Rous P. A sarcoma of the fowl transmissible by an agent
separable from the tumor cells. J Exp Med. 1911;13:
397–411.
4. Gross L. “Spontaneous” leukemia developing in
CH3 mice following inoculation in infancy, with
AK-leukemic extracts, or AK-embryos. Proc Soc Exp
Biol Med. 1951;76:27–32.
5. Payne LNAHGP. Diseases of Poultry. 9th ed. Ames,
Iowa, USA: Iowa State University Press; 1991.
6. Cooper GM. Oncogenes. 2nd ed. Boston: Jones and
Bartlett Publishers; 1995.
7. Trentin JJ, Yabe Y, Taylor G. The quest for human
cancer viruses. Science. 1962;137:835–849.
8. Burkitt D. A children’s cancer dependent upon
climatic factors. Nature. 1962;194:232–234.
9. Epstein M, Achong BG, Barr YM. Virus particles
in cultured lymphoblasts from Burkitt’s lymphoma.
Lancet. 1964;1:702–703.
10. Epstein M, Barr YM. Cultivation in vitro of human
lymphoblasts from Burkitt’s lymphoma. Lancet.
1964;1:252–253.
11. Henle G, Henle W, Diehl V. Relation of Burkitt’s
tumor-associated herpes-type virus to infectious
mononucleosis. Proc Natl Acad Sci USA. 1968;59:
94–101.
15. de-The G, Geser A, Day NE. Epidemiological
evidence for causal relationship between Epstein-Barr
virus and Burkitt’s lymphoma from Ugandan
prospective study. Nature. 1978;274:756–761.
23. Kieff E, Rickinson AB. Epstein Barr virus and its
replication. In: Fields Virology. 2001:2511–2573.
29. Pfeffer S, Zavolan M, Grasser FA, et al. Identi-
fication of virus-encoded microRNAs. Science.
2004;304:734–736.
37. Humme S, Reisbach G, Feederle R, et al. The
EBV nuclear antigen 1 (EBNA1) enhances B cell
immortalization several thousandfold. Proc Natl Acad
Sci USA. 2003;100:10989–10994.
47. Yajima M, Kanda T, Takada K. Critical role of
Epstein-Barr virus (EBV)-encoded RNA in efficient
EBV-induced B-lymphocyte growth transformation.
J Virol. 2005;79:4298–4307.
61. Taub R, Kirsch I, Morton C, et al. Translocation
of the c-myc gene into the immunoglobulin heavy
chain locus in human Burkitt lymphoma and
murine plasmacytoma cells. Proc Natl Acad Sci
USA. 1982;79:7837–7841.
70. Nanbo A, Sugden A, Sugden B. The coupling of
synthesis and partitioning of EBV’s plasmid replicon
is revealed in live cells. EMBO J. 2007;26:4252–
4262.
78. Blumberg BS. Australia antigen and the biology of
hepatitis B. Science. 1977;197:17–25.
82. Beasley RP, Hwang LY, Lin CC, Chien CS.
Hepatocellular carcinoma and hepatitis B virus. A
prospective study of 22 707 men in Taiwan. Lancet.
1981;2:1129–1133.
86. Chang MH, Chen CJ, Lai MS, et al. Universal
hepatitis B vaccination in Taiwan and the incidence
of hepatocellular carcinoma in children. Taiwan
Childhood Hepatoma Study Group. N Engl J Med.
1997;336:1855–1859.
89. Henkler F, Waseem N, Golding MH, et al. Mutant
p53 but not hepatitis B virus X protein is present
in hepatitis B virus-related human hepatocellular
carcinoma. Cancer Res. 1995;55:6084–6091.
98. Chisari FV, Ferrari C. Hepatitis B virus immuno-
pathogenesis. Annu Rev Immunol. 1995;13:29–60.
100. Guidotti LG, Rochford R, Chung J, et al. Viral
clearance without destruction of infected cells
during acute HBV infection. Science. 1999;284:825–
829.
106. Durst M, Gissmann L, Ikenberg H, zur Hausen H.
A papillomavirus DNA from a cervical carcinoma
and its prevalence in cancer biopsy samples from
different geographic regions. Proc Natl Acad Sci USA.
1983;80:3812–3815.
107. Schwarz E, Freese UK, Gissmann L, et al. Structure
and transcription of human papillomavirus sequences
in cervical carcinoma cells. Nature. 1985;314:
111–114.
109. Shope RE. A transmissible tumor-like condition in
rabbits. J Exp Med. 1932;56:793–802.
116. Lowy DR, Dvoretzky I, Shober R, et al. In vitro
tumorigenic transformation by a defined sub-
genomic fragment of bovine papilloma virus DNA.
Nature. 1980;287:72–74.

126. Dyson N, Howley PM, Munger K, Harlow E. The
human papillomavirus-16 E7 oncoprotein is able to
bind to the retinoblastoma gene product. Science.
1989;243:934–937.
127. Werness BA, Levine AJ, Howley PM. Association of
human papillomavirus types 16 and 18 E6 proteins
with p53. Science. 1990;248:76–79.
130. Goodwin EC, DiMaio D. Repression of human
papillomavirus oncogenes in HeLa cervical carcinoma
cells causes the orderly reactivation of dormant
tumor suppressor pathways. Proc Natl Acad Sci
USA. 2000;97:12513–12518.
145. Phelps WC, Yee CL, Munger K, Howley PM. The
human papillomavirus type 16 E7 gene encodes
transactivation and transformation functions similar
to those of adenovirus E1A. Cell. 1988;53:539–
547.
146. Arbeit JM, Howley PM, Hanahan D. Chronic
estrogen-induced cervical and vaginal squamous
carcinogenesis in human papillomavirus type 16
transgenic mice. Proc Natl Acad Sci USA. 1996;93:
2930–2935.
191. Shin MK, Pitot HC, Lambert PF. Pocket proteins
suppress head and neck cancer. Cancer Res. 2012;72:
1280–1289.
228. Yoshida M, Seiki M, Yamaguchi K, Takatsuki K.
Monoclonal integration of human T-cell leukemia
provirus in all primary tumors of adult T-cell
leukemia suggests causative role of human T-cell
Viruses and Human Cancer  •  CHAPTER 12 179
leukemia virus in the disease. Proc Natl Acad Sci
USA. 1984;81:2534–2537.
235. Ross TM, Pettiford SM, Green PL. The tax gene of
human T-cell leukemia virus type 2 is essential for trans-
formation of human T lymphocytes. J Virol. 1996;70:
5194–5202.
256. Matsuoka M, Jeang KT. Human T-cell leukaemia
virus type 1 (HTLV-I) infectivity and cellular
transformation. Nat Rev Cancer. 2007;7:270–280.
272. Choo QL, Kuo G, Weiner AJ, et al. Isolation of a
cDNA clone derived from a blood-borne non-A, non-B
viral hepatitis genome. Science. 1989;244:359–362.
279. Lohmann V, Korner F, Koch J, et al. Replication of
subgenomic hepatitis C virus RNAs in a hepatoma
cell line. Science. 1999;285:110–113.
281. Wakita T, Pietschmann T, Kato T, et al. Production
of infectious hepatitis C virus in tissue culture from
a cloned viral genome. Nat Med. 2005;11:791–796.
298. Chang Y, Cesarman E, Pessin MS, et al. Identifica-
tion of herpesvirus-like DNA sequences in AIDS-
associated Kaposi’s sarcoma. Science. 1994;266:
1865–1869.
302. Cesarman E, Chang Y, Moore PS, et al. Kaposi’s
sarcoma-associated herpesvirus-like DNA sequences
in AIDS-related body-cavity-based lymphomas.
N Engl J Med. 1995;332:1186–1191.
313. Moore PS, Chang Y. Kaposi’s sarcoma-associated
herpesvirus-encoded oncogenes and oncogenesis.
J Natl Cancer Inst Monogr. 1998;65–71.
345. Shuda M, Feng H, Kwun HJ, et al. T antigen
mutations are a human tumor-specific signature
for Merkel cell polyomavirus. Proc Natl Acad Sci
USA. 2008;105:16272–16277.
351. Feng H, Shuda M, Chang Y, Moore PS. Clonal
integration of a polyomavirus in human Merkel
cell carcinoma. Science. 2008;319:1096–1100.
356. Shuda M, Kwun HJ, Feng H, et al. Human Merkel
cell polyomavirus small T antigen is an oncoprotein
targeting the 4E-BP1 translation regulator. J Clin
Invest. 2011;121:3623–3634.
357. Arora R, Shuda M, Guastafierro A, et al. Survivin
is a therapeutic target in Merkel cell carcinoma.
Sci Transl Med. 2012;4:133ra156.
373. Christensen ND, Reed CA, Cladel NM, et al. Immu-
nization with viruslike particles induces long-term
protection of rabbits against challenge with cot-
tontail rabbit papillomavirus. J Virol. 1996;70:960–
965.
374. Suzich JA, Ghim SJ, Palmer-Hill FJ, et al. Systemic
immunization with papillomavirus L1 protein
completely prevents the development of viral mucosal
papillomas. Proc Natl Acad Sci USA. 1995;92:
11553–11557.
376. Koutsky LA, Ault KA, Wheeler CM, et al. Proof
of Principle Study, I. A controlled trial of a human
papillomavirus type 16 vaccine. NEJM. 2002;347:
1645–1651.

REFERENCES
1. Parkin DP, Pisani P, Munoz N, Ferlay J. The global
health burden of infection associated cancers. Cancer
Surv. 1999;33:5–33.
2. Ellerman VaOB. Experimentelle Leukamie bei
Huhnern. Zentralbl Bakteriol Parasitenkd Infectionskr
Hyg Abt Orig. 1908;46:595–609.
3. Rous P. A sarcoma of the fowl transmissible by an agent
separable from the tumor cells. J Exp Med. 1911;13:
397–411.
4. Gross L. “Spontaneous” leukemia developing in
CH3 mice following inoculation in infancy, with
AK-leukemic extracts, or AK-embryos. Proc Soc Exp
Biol Med. 1951;76:27–32.
5. Payne LNAHGP. Diseases of Poultry. 9th ed. Ames,
Iowa, USA: Iowa State University Press; 1991.
6. Cooper GM. Oncogenes. 2nd ed. Boston: Jones and
Bartlett Publishers; 1995.
7. Trentin JJ, Yabe Y, Taylor G. The quest for human
cancer viruses. Science. 1962;137:835–849.
8. Burkitt D. A children’s cancer dependent upon
climatic factors. Nature. 1962;194:232–234.
9. Epstein M, Achong BG, Barr YM. Virus particles
in cultured lymphoblasts from Burkitt’s lymphoma.
Lancet. 1964;1:702–703.
10. Epstein M, Barr YM. Cultivation in vitro of human lym-
phoblasts from Burkitt’s lymphoma. Lancet. 1964;1:
252–253.
11. Henle G, Henle W, Diehl V. Relation of Burkitt’s
tumor-associated herpes-type virus to infectious
mononucleosis. Proc Natl Acad Sci USA. 1968;59:
94–101.
12. Niederman JC, McCollum RW, Henle G, Henle W.
Infectious mononucleosis: clinical manifestations in
relation to EB virus antibodies. JAMA. 1968;203.
13. Evans AS, Niederman JC. Epstein Barr virus. In:
Evans AS, ed. Viral Infections of Humans. 3rd ed.
New York: Plenum Press; 1989:265–292.
14. Henle W, Henle G, Ho HC, et al. Antibodies to
Epstein-Barr virus in nasopharyngeal carcinoma,
other head and neck neoplasms, and control groups.
J Natl Cancer Inst. 1970;44:225–231.
15. de-Thé G, Geser A, Day NE. Epidemiological
evidence for causal relationship between Epstein-
Barr virus and Burkitt’s lymphoma from Ugandan
prospective study. Nature. 1978;274:756–761.
16. Zeng Y, Zhang LG, Wu YC, et al. Prospective studies
on nasopharyngeal carcinoma in Epstein-Barr virus
IgA/VCA antibody-positive persons in Wuzhou City,
China. Int J Cancer. 1985;36:545–547.
17. Crawford DH, Thomas JA, Janossy G, et al. Epstein
Barr virus nuclear antigen positive lymphoma
after cyclosporin A treatment in patient with renal
allograft. Lancet. 1980;1:1355–1356.
18. Greenspan JS, Greenspan D, Lennette ET, et al.
Replication of Epstein-Barr virus within the epithelial
cells of oral “hairy” leukoplakia, an AIDS-associated
lesion. N Engl J Med. 1985;313:1564–1571.
19. Evans AS, Gutensohn NM. A population-based
case-control study of EBV and other viral antibodies
among persons with Hodgkin’s disease and their
siblings. Int J Cancer. 1984;34:149–157.
20. Weiss LM, Strickler JG, Warnke RA, et al. Epstein-
Barr viral DNA in tissues of Hodgkin’s disease. Am
J Pathol. 1987;129:86–91.
21. Chadburn A, Chiu A, Lee JY, et al. Immunophe-
notypic analysis of AIDS-related diffuse large B-cell
lymphoma and clinical implications in patients from
AIDS Malignancies Consortium clinical trials 010
and 034. J Clin Oncol. 2009;27:5039–5048.
22. Imai S, Koizumi S, Sugiura M, et al. Gastric
carcinoma: monoclonal epithelial malignant cells
expressing Epstein-Barr virus latent infection protein.
Proc Natl Acad Sci USA. 1994;91:9131–9135.
23. Kieff E, Rickinson AB. Epstein Barr virus and its
replication. In: Knipe DM, Howley PM, Griffin
DE, et  al, eds. Fields Virology. Philadelphia:
Lippincott Williams & Wilkins; 2001:2511–2573.
Viruses and Human Cancer  •  CHAPTER 12 179.e1
24. Bloss TA, Sugden B. Optimal lengths for DNAs
encapsidated by Epstein-Barr virus. J Virol. 1994;68:
8217–8222.
25. Baer R, Bankier AT, Biggin MD, et al. DNA
sequence and expression of the B95-8 Epstein-Barr
virus genome. Nature. 1984;310:207–211.
26. Cai X, Schafer A, Lu S, et al. Epstein-Barr virus
microRNAs are evolutionarily conserved and dif-
ferentially expressed. PLoS Pathog. 2006;2:e23.
27. Chen SJ, Chen GH, Chen YH, et al. Charac-
terization of Epstein-Barr virus miRNAome in
nasopharyngeal carcinoma by deep sequencing.
PLoS ONE. 2010;5(9):pii: e12745.
28. Grundhoff A, Sullivan CS, Ganem D. A combined
computational and microarray-based approach
identifies novel microRNAs encoded by human
gamma-herpesviruses. RNA. 2006;12:733–750.
29. Pfeffer S, Zavolan M, Grasser FA, et al. Identi-
fication of virus-encoded microRNAs. Science.
2004;304:734–736.
30. Henle W, Diehl V, Kohn G, et al. Herpes-type
virus and chromosome marker in normal leukocytes
after growth with irradiated Burkitt cells. Science.
1967;157:1064–1065.
31. Robinson JE, Smith D, Niederman J. Plasmacytic
differentiation of circulating Epstein-Barr virus–
infected B lymphocytes during acute infectious
mononucleosis. J Exp Med. 1981;153:235–244.
32. Tagawa T, Albanese M, Bouvet M, et al. Epstein-Barr
viral miRNAs inhibit antiviral CD4+ T cell responses
targeting IL-12 and peptide processing. J Exp Med.
2016;213(10):2065–2080.
33. Albanese M, Tagawa T, Bouvet M, et al. Epstein-Barr
virus microRNAs reduce immune surveillance by
virus-specific CD8+ T cells. Proc Natl Acad Sci USA.
2016;113(42):E6467–E6475.
34. Kempkes B, Spitkovsky D, Jansen-Durr P, et al. B-cell
proliferation and induction of early G1-regulating
proteins by Epstein-Barr virus mutants conditional
for EBNA2. EMBO J. 1995b;14:88–96.
35. Kilger E, Kieser A, Baumann M, Hammerschmidt
W. Epstein-Barr virus-mediated B-cell proliferation is
dependent upon latent membrane protein 1, which
simulates an activated CD40 receptor. EMBO J.
1998;17:1700–1709.
36. Bornkamm GW, Hammerschmidt W. Molecular
virology of Epstein-Barr virus. Philos Trans R Soc
Lond B Biol Sci. 2001;356:437–459.
37. Humme S, Reisbach G, Feederle R, et al. The
EBV nuclear antigen 1 (EBNA1) enhances B cell
immortalization several thousandfold. Proc Natl Acad
Sci USA. 2003;100:10989–10994.
38. Vereide DT, Sugden B. Lymphomas differ in their
dependence on Epstein-Barr virus. Blood. 2011;117:
1977–1985.
39. Kempkes B, Pich D, Zeidler R, et al. Immortalization
of human B lymphocytes by a plasmid containing
71 kilobase pairs of Epstein-Barr virus DNA. J Virol.
1995a;69:231–238.
40. White RE, Ramer PC, Naresh KN, et al. EBNA3B-
deficient EBV promotes B cell lymphomagenesis in
humanized mice and is found in human tumors.
J Clin Invest. 2012;122:1487–1502.
41. Wang A, Welch R, Zhao B, et al. Epstein-Barr virus
nuclear antigen 3 (EBNA3) Proteins regulate EBNA2
binding to distinct RBPJ genomic sites. J Virol.
2015;90(6):2906–2919.
42. Maruo S, Zhao B, Johannsen E, et al. Epstein-
Barr virus nuclear antigens 3C and 3A maintain
lymphoblastoid cell growth by repressing p16INK4A
and p14ARF expression. Proc Natl Acad Sci USA.
2011;108:1919–1924.
43. Caldwell RG, Wilson JB, Anderson SJ, Longnecker
R. Epstein-Barr virus LMP2A drives B cell devel-
opment and survival in the absence of normal
B cell receptor signals. Immunity. 1998;9:405–
411.
44. Casola S, Otipoby KL, Alimzhanov M, et al. B cell
receptor signal strength determines B cell fate. Nat
Immunol. 2004;5:317–327.
45. Mancao C, Altmann M, Jungnickel B, Hammerschmidt
W. Rescue of “crippled” germinal center B cells from
apoptosis by Epstein-Barr virus. Blood. 2005;106:
4339–4344.
46. Kuppers R, Schwering I, Brauninger A, et al. Biology
of Hodgkin’s lymphoma. Ann Oncol. 2002;13(suppl
1):11–18.
47. Yajima M, Kanda T, Takada K. Critical role of
Epstein-Barr virus (EBV)-encoded RNA in efficient
EBV-induced B-lymphocyte growth transformation.
J Virol. 2005;79:4298–4307.
48. Samanta M, Iwakiri D, Kanda T, et al. EB virus-
encoded RNAs are recognized by RIG-I and
activate signaling to induce type I IFN. EMBO J.
2006;25:4207–4214.
49. Nanbo A, Takada K. The role of Epstein-Barr virus-
encoded small RNAs (EBERs) in oncogenesis. Rev
Med Virol. 2002;12:321–326.
50. Howe JG, Steitz JA. Localization of Epstein-Barr
virus-encoded small RNAs by in situ hybridization.
Proc Natl Acad Sci USA. 1986;83:9006–9010.
51. Wu TC, Mann RB, Epstein JI, et al. Abundant
expression of EBER1 small nuclear RNA in
nasopharyngeal carcinoma. A morphologically
distinctive target for detection of Epstein-Barr virus
in formalin-fixed paraffin-embedded carcinoma
specimens. Am J Pathol. 1991;138:1461–1469.
52. Moss DJ, Burrows SR, Silins SL, et al. The immunol-
ogy of Epstein-Barr virus infection. Philos Trans R
Soc Lond B Biol Sci. 2001;356:475–488.
53. Rowe M, Rowe DT, Gregory CD, et al. Differences
in B cell growth phenotype reflect novel patterns of
Epstein-Barr virus latent gene expression in Burkitt’s
lymphoma cells. EMBO J. 1987;6:2743–2751.
54. Heller KN, Upshaw J, Seyoum B, et al. Distinct
memory CD4+ T-cell subsets mediate immune
recognition of Epstein Barr virus nuclear antigen 1 in
healthy virus carriers. Blood. 2007;109:1138–1146.
55. Paludan C, Schmid D, Landthaler M, et al. Endog-
enous MHC class II processing of a viral nuclear
antigen after autophagy. Science. 2005;307:593–
596.
56. Babcock GJ, Decker LL, Volk M, Thorley-Lawson
DA. EBV persistence in memory B cells in vivo.
Immunity. 1998;9:395–404.
57. Qu L, Rowe DT. Epstein-Barr virus latent gene
expression in uncultured peripheral blood lympho-
cytes. J Virol. 1992;66:3715–3724.
58. Crawford DH. Biology and disease associations of
Epstein-Barr virus. Philos Trans R Soc Lond B Biol
Sci. 2001;356:461–473.
59. Aguilar LK, Rooney CM, Heslop HE. Lympho-
proliferative disorders involving Epstein-Barr virus
after hemopoietic stem cell transplantation. Curr
Opin Oncol. 1999;11:96–101.
60. AlDabbagh MA, Gitman MR, Kumar D, et al. The
role of antiviral prophylaxis for the prevention of
Epstein-Barr virus–associated posttransplant lympho-
proliferative disease in solid organ transplant recipi-
ents: a systematic review. Am J Transplant. 2017;17(3):
770–781.
61. Taub R, Kirsch I, Morton C, et al. Translocation
of the c-myc gene into the immunoglobulin heavy
chain locus in human Burkitt lymphoma and murine
plasmacytoma cells. Proc Natl Acad Sci USA. 1982;
79:7837–7841.
62. Zech L, Haglund U, Nilsson K, Klein G. Charac-
teristic chromosomal abnormalities in biopsies and
lymphoid-cell lines from patients with Burkitt and
non-Burkitt lymphomas. Int J Cancer. 1976;17:
47–56.
63. Ramiro AR, Nussenzweig MC, Nussenzweig A.
Switching on chromosomal translocations. Cancer
Res. 2006;66:7837–7839.
179.e2 Part I: Science and Clinical Oncology
64. Stanton LW, Watt R, Marcu KB. Translocation,
breakage and truncated transcripts of c-myc onco-
gene in murine plasmacytomas. Nature. 1983;303:
401–406.
65. Morrow RH, Kisuule A, Pike MC, Smith PG.
Burkitt’s lymphoma in the Mengo Districts of
Uganda: epidemiologic features and their relationship
to malaria. J Natl Cancer Inst. 1976;56:479–483.
66. Whittle HC, Brown J, Marsh K, et al. T-cell control
of Epstein-Barr virus-infected B cells is lost during
P. falciparum malaria. Nature. 1984;312:449–450.
67. Moormann AM, Chelimo K, Sumba OP, et al.
Exposure to holoendemic malaria results in elevated
Epstein-Barr virus loads in children. J Infect Dis.
2005;191:1233–1238.
68. Lam KM, Syed N, Whittle H, Crawford DH.
Circulating Epstein-Barr virus–carrying B cells in
acute malaria. Lancet. 1991;337:876–878.
69. Gaidano G, Ballerini P, Gong JZ, et al. p53
mutations in human lymphoid malignancies:
association with Burkitt lymphoma and chronic
lymphocytic leukemia. Proc Natl Acad Sci USA.
1991;88:5413–5417.
70. Nanbo A, Sugden A, Sugden B. The coupling of syn-
thesis and partitioning of EBV’s plasmid replicon is
revealed in live cells. EMBO J. 2007;26:4252–4262.
71. Vereide DT, Seto E, Chiu YF, et al. Epstein-Barr virus
maintains lymphomas via its miRNAs. Oncogene.
2014;33(10):1258–1264.
72. Ferry JA. Burkitt’s lymphoma: clinicopathologic fea-
tures and differential diagnosis. Oncologist. 2006;11:
375–383.
73. Hesseling P, Broadhead R, Mansvelt E, et al. The
2000 Burkitt lymphoma trial in Malawi. Pediatr
Blood Cancer. 2005;44:245–250.
74. Chan AS, To KF, Lo KW, et al. High frequency of
chromosome 3p deletion in histologically normal
nasopharyngeal epithelia from southern Chinese.
Cancer Res. 2000;60:5365–5370.
75. Sengupta S, den Boon JA, Chen IH, et al. Genome-
wide expression profiling reveals EBV-associated inhi-
bition of MHC class I expression in nasopharyngeal
carcinoma. Cancer Res. 2006;66:7999–8006.
76. Cosmopoulos K, Pegtel M, Hawkins J, et al.
Comprehensive profiling of Epstein-Barr virus
microRNAs in nasopharyngeal carcinoma. J Virol.
2009;83:2357–2367.
77. Pratt ZL, Kuzembayeva M, Sengupta S, Sugden B.
The microRNAs of Epstein-Barr virus are expressed
at dramatically differing levels among cell lines.
Virology. 2009;386:387–397.
78. Blumberg BS. Australia antigen and the biology of
hepatitis B. Science. 1977;197:17–25.
79. Senior JR, Sutnick AI, Goeser E, et al. Reduction of
post-transfusion hepatitis by exclusion of Australia
antigen from donor blood in an urban public
hospital. Am J Med Sci. 1974;267:171–177.
80. Monto A, Wright TL. The epidemiology and preven-
tion of hepatocellular carcinoma. Semin Oncol. 2001;
28:441–449.
81. Wild CP, Hall AJ. Primary prevention of hepatocel-
lular carcinoma in developing countries. Mutat Res.
2000;462:381–393.
82. Beasley RP, Hwang LY, Lin CC, Chien CS.
Hepatocellular carcinoma and hepatitis B virus. A
prospective study of 22 707 men in Taiwan. Lancet.
1981;2:1129–1133.
83. Beasley RP. Hepatitis B virus. The major etiology
of hepatocellular carcinoma. Cancer. 1988;61:
1942–1956.
84. Chen HL, Chang MH, Ni YH, et al. Seroepide-
miology of hepatitis B virus infection in children:
ten years of mass vaccination in Taiwan. JAMA.
1996;276:906–908.
85. Montesano R. Hepatitis B immunization and
hepatocellular carcinoma: the Gambia Hepatitis
Intervention Study. J Med Virol. 2002;67:444–446.
86. Chang MH, Chen CJ, Lai MS, et al. Universal
hepatitis B vaccination in Taiwan and the incidence
of hepatocellular carcinoma in children. Taiwan
Childhood Hepatoma Study Group. N Engl J Med.
1997;336:1855–1859.
87. Seeger C, Mason WS. Hepatitis B virus biology.
Microbiol Mol Biol Rev. 2000;64:51–68.
88. Unsal H, Yakicier C, Marcais C, et al. Genetic
heterogeneity of hepatocellular carcinoma. Proc
Natl Acad Sci USA. 1994;91:822–826.
89. Henkler F, Waseem N, Golding MH, et al. Mutant
p53 but not hepatitis B virus X protein is present
in hepatitis B virus-related human hepatocellular
carcinoma. Cancer Res. 1995;55:6084–6091.
90. Wang XW, Gibson MK, Vermeulen W, et al. Abroga-
tion of p53-induced apoptosis by the hepatitis B
virus X gene. Cancer Res. 1995;55:6012–6016.
91. Haviv I, Shamay M, Doitsh G, Shaul Y. Hepatitis B
virus pX targets TFIIB in transcription coactivation.
Mol Cell Biol. 1998;18:1562–1569.
92. Lin Y, Nomura T, Cheong J, et al. Hepatitis B
virus X protein is a transcriptional modulator that
communicates with transcription factor IIB and
the RNA polymerase II subunit 5. J Biol Chem.
1997;272:7132–7139.
93. Lee DK, Park SH, Yi Y, et al. The hepatitis B virus
encoded oncoprotein pX amplifies TGF-beta family
signaling through direct interaction with Smad4:
potential mechanism of hepatitis B virus-induced
liver fibrosis. Genes Dev. 2001;15:455–466.
94. Arzumanyan A, Friedman T, Ng IO, et al.
Does the hepatitis B antigen HBx promote the
appearance of liver cancer stem cells? Cancer Res.
2011;71:3701–3708.
95. Buendia MA. Hepatitis B viruses and hepatocellular
carcinoma. Adv Cancer Res. 1992;59:167–226.
96. Fourel G, Couturier J, Wei Y, et al. Evidence for
long-range oncogene activation by hepadnavirus
insertion. EMBO J. 1994;13:2526–2534.
97. Pineau P, Marchio A, Mattei MG, et al. Extensive
analysis of duplicated-inverted hepatitis B virus
integrations in human hepatocellular carcinoma.
J Gen Virol. 1998;79(Pt 3):591–600.
98. Chisari FV, Ferrari C. Hepatitis B virus immuno-
pathogenesis. Annu Rev Immunol. 1995;13:29–60.
99. Guidotti LG, Ando K, Hobbs MV, et al. Cytotoxic T
lymphocytes inhibit hepatitis B virus gene expression
by a noncytolytic mechanism in transgenic mice.
Proc Natl Acad Sci USA. 1994;91:3764–3768.
100. Guidotti LG, Rochford R, Chung J, et al. Viral
clearance without destruction of infected cells
during acute HBV infection. Science. 1999;284:825–
829.
101. Kimura K, Kakimi K, Wieland S, et al. Interleukin-18
inhibits hepatitis B virus replication in the livers of
transgenic mice. J Virol. 2002;76:10702–10707.
102. Nakamoto Y, Guidotti LG, Kuhlen CV, et al.
Immune pathogenesis of hepatocellular carcinoma.
J Exp Med. 1998;188:341–350.
103. Nelson NP, Easterbrook PJ, McMahon BJ. Epide-
miology of hepatitis B virus infection and impact of
vaccination on disease. Clin Liver Dis. 2016;20(4):
607–628.
104. Testoni B, Durantel D, Zoulim F. Novel targets for
hepatitis B virus therapy. Liver Int. 2017;37(suppl
1):33–39.
105. Boshart M, Gissmann L, Ikenberg H, et al. A new
type of papillomavirus DNA, its presence in genital
cancer biopsies and in cell lines derived from cervical
cancer. EMBO J. 1984;3:1151–1157.
106. Durst M, Gissmann L, Ikenberg H, zur Hausen H.
A papillomavirus DNA from a cervical carcinoma
and its prevalence in cancer biopsy samples from
different geographic regions. Proc Natl Acad Sci USA.
1983;80:3812–3815.
107. Schwarz E, Freese UK, Gissmann L, et al. Struc-
ture and transcription of human papillomavirus
sequences in cervical carcinoma cells. Nature.
1985;314:111–114.
108. Yee C, Krishnan HI, Baker CC, et al. Presence and
expression of human papillomavirus sequences in
human cervical carcinoma cell lines. Am J Pathol.
1985;119:361–366.
109. Shope RE. A Transmissible tumor-like condition
in rabbits. J Exp Med. 1932;56:793–802.
110. Jarrett WF, Murphy J, O’Neil BW, Laird HM.
Virus-induced papillomas of the alimentary tract
of cattle. Int J Cancer. 1978;22:323–328.
111. Campo MS, Moar MH, Sartirana ML, et al. The
presence of bovine papillomavirus type 4 DNA is not
required for the progression to, or the maintenance
of, the malignant state in cancers of the alimentary
canal in cattle. EMBO J. 1985;4:1819–1825.
112. Jarrett WF, McNeil PE, Grimshaw WT, et al. High
incidence area of cattle cancer with a possible interac-
tion between an environmental carcinogen and a
papilloma virus. Nature. 1978;274:215–217.
113. Pennie WD, Campo MS. Synergism between
bovine papillomavirus type 4 and the flavonoid
quercetin in cell transformation in vitro. Virology.
1992;190:861–865.
114. Dvoretzky I, Shober R, Chattopadhyay SK, Lowy
DR. A quantitative in vitro focus assay for bovine
papilloma virus. Virology. 1980;103:369–375.
115. Law MF, Lowy DR, Dvoretzky I, Howley PM. Mouse
cells transformed by bovine papillomavirus contain
only extrachromosomal viral DNA sequences.
Proc Natl Acad Sci USA. 1981;78:2727–2731.
116. Lowy DR, Dvoretzky I, Shober R, et al. In vitro
tumorigenic transformation by a defined sub-
genomic fragment of bovine papilloma virus DNA.
Nature. 1980;287:72–74.
117. Androphy EJ, Schiller JT, Lowy DR. Identification of
the protein encoded by the E6 transforming gene of
bovine papillomavirus. Science. 1985;230:442–445.
118. Nakabayashi Y, Chattopadhyay SK, Lowy DR. The
transforming function of bovine papillomavirus
DNA. Proc Natl Acad Sci USA. 1983;80:5832–5836.
119. Sarver N, Rabson MS, Yang YC, et al. Localiza-
tion and analysis of bovine papillomavirus type
1 transforming functions. J Virol. 1984;52:377–
388.
120. Schiller JT, Vass WC, Vousden KH, Lowy DR. E5
open reading frame of bovine papillomavirus type 1
encodes a transforming gene. J Virol. 1986;57:1–6.
121. Schlegel R, Wade GM, Rabson MS, Yang YC. The
E5 transforming gene of bovine papillomavirus
encodes a small, hydrophobic polypeptide. Science.
1986;233:464–467.
122. Yang YC, Okayama H, Howley PM. Bovine papil-
lomavirus contains multiple transforming genes. Proc
Natl Acad Sci USA. 1985;82:1030–1034.
123. Thierry F, Yaniv M. The BPV1-E2 trans-acting
protein can be either an activator or a repressor of
the HPV18 regulatory region. EMBO J. 1987;6:
3391–3397.
124. Jeon S, Lambert PF. Integration of human papil-
lomavirus type 16 DNA into the human genome
leads to increased stability of E6 and E7 mRNAs:
implications for cervical carcinogenesis. Proc Natl
Acad Sci USA. 1995;92:1654–1658.
125. Jeon S, Allen-Hoffmann BL, Lambert PF. Integration
of human papillomavirus type 16 into the human
genome correlates with a selective growth advantage
of cells. J Virol. 1995;69:2989–2997.
126. Dyson N, Howley PM, Munger K, Harlow E. The
human papilloma virus-16 E7 oncoprotein is able
to bind to the retinoblastoma gene product. Science.
1989;243:934–937.
127. Werness BA, Levine AJ, Howley PM. Association of
human papillomavirus types 16 and 18 E6 proteins
with p53. Science. 1990;248:76–79.
128. Scheffner M, Munger K, Byrne JC, Howley PM.
The state of the p53 and retinoblastoma genes in
human cervical carcinoma cell lines. Proc Natl Acad
Sci USA. 1991;88:5523–5527.
129. Francis DA, Schmid SI, Howley PM. Repression
of the integrated papillomavirus E6/E7 promoter is
required for growth suppression of cervical cancer
cells. J Virol. 2000;74:2679–2686.

130. Goodwin EC, DiMaio D. Repression of human papil-
lomavirus oncogenes in HeLa cervical carcinoma cells
causes the orderly reactivation of dormant tumor sup-
pressor pathways. Proc Natl Acad Sci USA. 2000;97:
12513–12518.
131. Goodwin EC, Yang E, Lee CJ, et al. Rapid induction
of senescence in human cervical carcinoma cells.
Proc Natl Acad Sci USA. 2000;97:10978–10983.
132. Hwang ES, Riese DJ 2nd, Settleman J, et al.
Inhibition of cervical carcinoma cell line prolifera-
tion by the introduction of a bovine papillomavirus
regulatory gene. J Virol. 1993;67:3720–3729.
133. Nishimura A, Ono T, Ishimoto A, et al. Mechanisms
of human papillomavirus E2-mediated repression of
viral oncogene expression and cervical cancer cell
growth inhibition. J Virol. 2000;74:3752–3760.
134. Wells SI, Francis DA, Karpova AY, et al. Papil-
lomavirus E2 induces senescence in HPV-positive
cells via pRB- and p21(CIP)-dependent pathways.
EMBO J. 2000;19:5762–5771.
135. Jabbar SF, Abrams L, Glick A, Lambert PF. Persis-
tence of high-grade cervical dysplasia and cervical
cancer requires the continuous expression of the
human papillomavirus type 16 E7 oncogene. Cancer
Res. 2009;69:4407–4414.
136. Jabbar SF, Park S, Schweizer J, et al. Cervical cancers
require the continuous expression of the human
papillomavirus type 16 E7 oncoprotein even in the
presence of the viral E6 oncoprotein. Cancer Res.
2012;72(16):4008–4016.
137. Band V, Zajchowski D, Kulesa V, Sager R. Human
papilloma virus DNAs immortalize normal human
mammary epithelial cells and reduce their growth
factor requirements. Proc Natl Acad Sci USA. 1990;87:
463–467.
138. Durst M, Dzarlieva-Petrusevska RT, Boukamp P,
et al. Molecular and cytogenetic analysis of immortal-
ized human primary keratinocytes obtained after
transfection with human papillomavirus type 16
DNA. Oncogene. 1987;1:251–256.
139. Hawley-Nelson P, Vousden KH, Hubbert NL,
et al. HPV16 E6 and E7 proteins cooperate to
immortalize human foreskin keratinocytes. EMBO
J. 1989;8:3905–3910.
140. Schlegel R, Phelps WC, Zhang YL, Barbosa M.
Quantitative keratinocyte assay detects two biological
activities of human papillomavirus DNA and identi-
fies viral types associated with cervical carcinoma.
EMBO J. 1988;7:3181–3187.
141. Kaur P, McDougall JK. HPV-18 immortalization of
human keratinocytes. Virology. 1989;173:302–310.
142. Woodworth CD, Bowden PE, Doniger J, et al. Char-
acterization of normal human exocervical epithelial
cells immortalized in vitro by papillomavirus types
16 and 18 DNA. Cancer Res. 1988;48:4620–4628.
143. Crook T, Storey A, Almond N, et al. Human
papillomavirus type 16 cooperates with activated
ras and fos oncogenes in the hormone-dependent
transformation of primary mouse cells. Proc Natl
Acad Sci USA. 1988;85:8820–8824.
144. Matlashewski G, Schneider J, Banks L, et al.
Human papillomavirus type 16 DNA cooperates
with activated ras in transforming primary cells.
EMBO J. 1987;6:1741–1746.
145. Phelps WC, Yee CL, Munger K, Howley PM. The
human papillomavirus type 16 E7 gene encodes
transactivation and transformation functions similar
to those of adenovirus E1A. Cell. 1988;53:539–547.
146. Arbeit JM, Howley PM, Hanahan D. Chronic
estrogen-induced cervical and vaginal squamous
carcinogenesis in human papillomavirus type 16
transgenic mice. Proc Natl Acad Sci USA. 1996;93:
2930–2935.
147. Elson DA, Riley RR, Lacey A, et al. Sensitivity
of the cervical transformation zone to estrogen-
induced squamous carcinogenesis. Cancer Res.
2000;60:1267–1275.
148. Greenhalgh DA, Wang XJ, Rothnagel JA, et al.
Transgenic mice expressing targeted HPV-18 E6
Viruses and Human Cancer  •  CHAPTER 12 179.e3
and E7 oncogenes in the epidermis develop verrucous
lesions and spontaneous, rasHa-activated papillomas.
Cell Growth Differ. 1994;5:667–675.
149. Herber R, Liem A, Pitot H, Lambert PF. Squamous
epithelial hyperplasia and carcinoma in mice
transgenic for the human papillomavirus type 16
E7 oncogene. J Virol. 1996;70:1873–1881.
150. Lambert PF, Pan H, Pitot HC, et al. Epidermal
cancer associated with expression of human papil-
lomavirus type 16 E6 and E7 oncogenes in the
skin of transgenic mice. Proc Natl Acad Sci USA.
1993;90:5583–5587.
151. Pan H, Griep AE. Altered cell cycle regulation in
the lens of HPV-16 E6 or E7 transgenic mice:
implications for tumor suppressor gene function
in development. Genes Dev. 1994;8:1285–1299.
152. Song S, Liem A, Miller JA, Lambert PF. Human
papillomavirus types 16 E6 and E7 contribute dif-
ferently to carcinogenesis. Virology. 2000;267:141–
150.
153. Song S, Pitot HC, Lambert PF. The human papillo-
mavirus type 16 E6 gene alone is sufficient to induce
carcinomas in transgenic animals. J Virol. 1999;73:
5887–5893.
154. Jabbar S, Strati K, Shin MK, et al. Human papil-
lomavirus type 16 E6 and E7 oncoproteins act
synergistically to cause head and neck cancer in
mice. Virology. 2010;407:60–67.
155. Riley RR, Duensing S, Brake T, et al. Dissection
of human papillomavirus E6 and E7 function in
transgenic mouse models of cervical carcinogenesis.
Cancer Res. 2003;63:4862–4871.
156. Thomas MK, Pitot HC, Liem A, Lambert PF.
Dominant role of HPV16 E7 in anal carcinogenesis.
Virology. 2011;421:114–118.
157. Huibregtse JM, Scheffner M, Howley PM. A cellular
protein mediates association of p53 with the E6
oncoprotein of human papillomavirus types 16 or
18. EMBO J. 1991;10:4129–4135.
158. Scheffner M, Huibregtse JM, Vierstra RD, Howley
PM. The HPV-16 E6 and E6-AP complex functions
as a ubiquitin-protein ligase in the ubiquitination
of p53. Cell. 1993;75:495–505.
159. Lechner MS, Mack DH, Finicle AB, et al. Human
papillomavirus E6 proteins bind p53 in vivo and
abrogate p53-mediated repression of transcription.
EMBO J. 1992;11:3045–3052.
160. Mietz JA, Unger T, Huibregtse JM, Howley
PM. The transcriptional transactivation function
of wild-type p53 is inhibited by SV40 large
T-antigen and by HPV-16 E6 oncoprotein. EMBO
J. 1992;11:5013–5020.
161. Boyer SN, Wazer DE, Band V. E7 protein of human
papilloma virus-16 induces degradation of retino-
blastoma protein through the ubiquitin-proteasome
pathway. Cancer Res. 1996;56:4620–4624.
162. Jones DL, Thompson DA, Munger K. Destabilization
of the RB tumor suppressor protein and stabilization
of p53 contribute to HPV type 16 E7-induced
apoptosis. Virology. 1997;239:97–107.
163. Chellappan S, Kraus VB, Kroger B, et al. Adenovirus
E1A, simian virus 40 tumor antigen, and human
papillomavirus E7 protein share the capacity to
disrupt the interaction between transcription factor
E2F and the retinoblastoma gene product. Proc Natl
Acad Sci USA. 1992;89:4549–4553.
164. Balsitis S, Dick F, Dyson N, Lambert PF. Critical
roles for non-pRb targets of human papillomavirus
type 16 E7 in cervical carcinogenesis. Cancer Res.
2006;66:9393–9400.
165. Shai A, Brake T, Somoza C, Lambert PF. The human
papillomavirus E6 oncogene dysregulates the cell cycle
and contributes to cervical carcinogenesis through
two independent activities. Cancer Res. 2007;67:
1626–1635.
166. Shin MK, Balsitis S, Brake T, Lambert PF. Human
papillomavirus E7 oncoprotein overrides the tumor
suppressor activity of p21Cip1 in cervical carcino-
genesis. Cancer Res. 2009;69:5656–5663.
167. Simonson SJ, Difilippantonio MJ, Lambert PF.
Two distinct activities contribute to human papil-
lomavirus 16 E6’s oncogenic potential. Cancer Res.
2005;65:8266–8273.
168. Patel D, Huang SM, Baglia LA, McCance DJ. The
E6 protein of human papillomavirus type 16 binds
to and inhibits co-activation by CBP and p300.
EMBO J. 1999;18:5061–5072.
169. Zimmermann H, Degenkolbe R, Bernard HU,
O’Connor MJ. The human papillomavirus type
16 E6 oncoprotein can down-regulate p53 activity
by targeting the transcriptional coactivator CBP/
p300. J Virol. 1999;73:6209–6219.
170. Tong X, Howley PM. The bovine papillomavirus
E6 oncoprotein interacts with paxillin and disrupts
the actin cytoskeleton. Proc Natl Acad Sci USA.
1997;94:4412–4417.
171. Vande Pol SB, Brown MC, Turner CE. Association
of bovine papillomavirus type 1 E6 oncoprotein
with the focal adhesion protein paxillin through a
conserved protein interaction motif. Oncogene. 1998;
16:43–52.
172. Gao Q, Srinivasan S, Boyer SN, et al. The E6
oncoproteins of high-risk papillomaviruses bind to
a novel putative GAP protein, E6TP1, and target it
for degradation. Mol Cell Biol. 1999;19:733–744.
173. Ronco LV, Karpova AY, Vidal M, Howley PM.
Human papillomavirus 16 E6 oncoprotein binds
to interferon regulatory factor-3 and inhibits its tran-
scriptional activity. Genes Dev. 1998;12:2061–2072.
174. Chen JJ, Reid CE, Band V, Androphy EJ. Interaction
of papillomavirus E6 oncoproteins with a putative
calcium-binding protein. Science. 1995;269:529–531.
175. Thomas M, Banks L. Human papillomavirus (HPV)
E6 interactions with Bak are conserved amongst E6
proteins from high and low risk HPV types. J Gen
Virol. 1999;80(Pt 6):1513–1517.
176. Gao Q, Kumar A, Srinivasan S, et al. PKN binds
and phosphorylates human papillomavirus E6
oncoprotein. J Biol Chem. 2000;275:14824–14830.
177. Gross-Mesilaty S, Reinstein E, Bercovich B,
et al. Basal and human papillomavirus E6
oncoprotein-induced degradation of Myc proteins
by the ubiquitin pathway. Proc Natl Acad Sci USA.
1998;95:8058–8063.
178. Kiyono T, Hiraiwa A, Fujita M, et al. Binding of
high-risk human papillomavirus E6 oncoproteins
to the human homologue of the Drosophila discs
large tumor suppressor protein. Proc Natl Acad Sci
USA. 1997;94:11612–11616.
179. Lee SS, Weiss RS, Javier RT. Binding of human virus
oncoproteins to hDlg/SAP97, a mammalian homolog
of the Drosphilia discs large tumor suppressor protein.
Proc Natl Acad Sci U S A. 1997;94:6670–6675.
180. Nakagawa S, Huibregtse JM. Human scribble
(Vartul) is targeted for ubiquitin-mediated degrada-
tion by the high-risk papillomavirus E6 proteins and
the E6AP ubiquitin-protein ligase. Mol Cell Biol.
2000;20:8244–8253.
181. Glaunsinger BA, Lee SS, Thomas M, et al.
Interactions of the PDZ-protein MAGI-1 with
adenovirus E4-ORF1 and high-risk papillomavirus
E6 oncoproteins. Oncogene. 2000;19:5270–5280.
182. Lee SS, Glaunsinger B, Mantovani F, et al. Multi-
PDZ domain protein MUPP1 is a cellular target
for both adenovirus E4-ORF1 and high-risk papil-
lomavirus type 18 E6 oncoproteins. J Virol. 2000;74:
9680–9693.
183. Gewin L, Myers H, Kiyono T, Galloway DA.
Identification of a novel telomerase repressor that
interacts with the human papillomavirus type-16
E6/E6-AP complex. Genes Dev. 2004;18:2269–2282.
184. Xu M, Luo W, Elzi DJ, et al. NFX1 interacts with
mSin3A/histone deacetylase to repress hTERT
transcription in keratinocytes. Mol Cell Biol.
2008;28:4819–4828.
185. Katzenellenbogen RA, Vliet-Gregg P, Xu M, Galloway
DA. NFX1-123 increases hTERT expression and
telomerase activity posttranscriptionally in human
179.e4 Part I: Science and Clinical Oncology
papillomavirus type 16 E6 keratinocytes. J Virol.
2009;83:6446–6456.
186. Katzenellenbogen RA, Vliet-Gregg P, Xu M, Gal-
loway DA. Cytoplasmic poly(A) binding proteins
regulate telomerase activity and cell growth in human
papillomavirus type 16 E6-expressing keratinocytes.
J Virol. 2010;84:12934–12944.
187. Shai A, Pitot HC, Lambert PF. E6-associated
protein is required for human papillomavirus type
16 E6 to cause cervical cancer in mice. Cancer Res.
2010;70:5064–5073.
188. Dyson N, Guida P, Munger K, Harlow E. Homolo-
gous sequences in adenovirus E1A and human
papillomavirus E7 proteins mediate interaction with
the same set of cellular proteins. J Virol. 1992;66:
6893–6902.
189. Classon M, Salama S, Gorka C, et al. Combi-
natorial roles for pRB, p107, and p130 in E2F-
mediated cell cycle control. Proc Natl Acad Sci USA.
2000;97:10820–10825.
190. Dyson N, Dembski M, Fattaey A, et al. Analysis
of p107-associated proteins: p107 associates with
a form of E2F that differs from pRB-associated
E2F-1. J Virol. 1993;67:7641–7647.
191. Shin MK, Pitot HC, Lambert PF. Pocket proteins
suppress head and neck cancer. Cancer Res. 2012;72:
1280–1289.
192. Shin MK, Sage J, Lambert PF. Inactivating all three
rb family pocket proteins is insufficient to initiate
cervical cancer. Cancer Res. 2012;72(20):5418–5427.
193. Arroyo M, Bagchi S, Raychaudhuri P. Association
of the human papillomavirus type 16 E7 protein
with the S-phase-specific E2F-cyclin A complex.
Mol Cell Biol. 1993;13:6537–6546.
194. McIntyre MC, Ruesch MN, Laimins LA. Human
papillomavirus E7 oncoproteins bind a single form of
cyclin E in a complex with cdk2 and p107. Virology.
1996;215:73–82.
195. Tommasino M, Adamczewski JP, Carlotti F, et al.
HPV16 E7 protein associates with the protein kinase
p33CDK2 and cyclin A. Oncogene. 1993;8:195–202.
196. Funk JO, Waga S, Harry JB, et al. Inhibition of CDK
activity and PCNA-dependent DNA replication by
p21 is blocked by interaction with the HPV-16 E7
oncoprotein. Genes Dev. 1997;11:2090–2100.
197. Jones DL, Alani RM, Munger K. The human
papillomavirus E7 oncoprotein can uncouple
cellular differentiation and proliferation in human
keratinocytes by abrogating p21Cip1-mediated
inhibition of cdk2. Genes Dev. 1997;11:2101–2111.
198. Yan X, Shah W, Jing L, et al. High-risk human
papillomavirus type 18 E7 caused p27 elevation
and cytoplasmic localization. Cancer Biol Ther.
2010;9:728–735.
199. Berezutskaya E, Bagchi S. The human papillomavirus
E7 oncoprotein functionally interacts with the S4
subunit of the 26 S proteasome. J Biol Chem.
1997;272:30135–30140.
200. Brehm A, Nielsen SJ, Miska EA, et al. The E7
oncoprotein associates with Mi2 and histone
deacetylase activity to promote cell growth. EMBO
J. 1999;18:2449–2458.
201. Luscher-Firzlaff JM, Westendorf JM, Zwicker J, et al.
Interaction of the fork head domain transcription
factor MPP2 with the human papilloma virus 16
E7 protein: enhancement of transformation and
transactivation. Oncogene. 1999;18:5620–5630.
202. Antinore MJ, Birrer MJ, Patel D, et al. The human
papillomavirus type 16 E7 gene product interacts
with and trans-activates the AP1 family of transcrip-
tion factors. EMBO J. 1996;15:1950–1960.
203. Mannhardt B, Weinzimer SA, Wagner M, et al.
Human papillomavirus type 16 E7 oncoprotein
binds and inactivates growth-inhibitory insulin-like
growth factor binding protein 3. Mol Cell Biol.
2000;20:6483–6495.
204. Massimi P, Pim D, Banks L. Human papillomavirus
type 16 E7 binds to the conserved carboxy-terminal
region of the TATA box binding protein and this
contributes to E7 transforming activity. J Gen Virol.
1997;78(Pt 10):2607–2613.
205. Phillips AC, Vousden KH. Analysis of the interaction
between human papillomavirus type 16 E7 and the
TATA-binding protein, TBP. J Gen Virol. 1997;78(Pt
4):905–909.
206. Mazzarelli JM, Atkins GB, Geisberg JV, Ricciardi RP.
The viral oncoproteins Ad5 E1A, HPV16 E7 and
SV40 TAg bind a common region of the TBP-asso-
ciated factor-110. Oncogene. 1995;11:1859–1864.
207. Schilling B, De-Medina T, Syken J, et al. A novel
human DnaJ protein, hTid-1, a homolog of the
Drosophila tumor suppressor protein Tid56, can
interact with the human papillomavirus type 16
E7 oncoprotein. Virology. 1998;247:74–85.
208. Davies R, Hicks R, Crook T, et al. Human papil-
lomavirus type 16 E7 associates with a histone H1
kinase and with p107 through sequences necessary
for transformation. J Virol. 1993;67:2521–2528.
209. McLaughlin-Drubin ME, Crum CP, Munger K.
Human papillomavirus E7 oncoprotein induces
KDM6A and KDM6B histone demethylase expres-
sion and causes epigenetic reprogramming. Proc Natl
Acad Sci USA. 2011;108:2130–2135.
210. Zhang B, Laribee RN, Klemsz MJ, Roman A.
Human papillomavirus type 16 E7 protein increases
acetylation of histone H3 in human foreskin
keratinocytes. Virology. 2004;329:189–198.
211. Andl T, Kahn T, Pfuhl A, et al. Etiological involve-
ment of oncogenic human papillomavirus in tonsillar
squamous cell carcinomas lacking retinoblastoma
cell cycle control. Cancer Res. 1998;58:5–13.
212. Biliris KA, Koumantakis E, Dokianakis DN, et al.
Human papillomavirus infection of non-melanoma
skin cancers in immunocompetent hosts. Cancer
Lett. 2000;161:83–88.
213. Gillison ML, Koch WM, Capone RB, et al. Evidence
for a causal association between human papilloma-
virus and a subset of head and neck cancers [see
comments]. J Natl Cancer Inst. 2000;92:709–720.
214. Kiviat NB. Papillomaviruses in non-melanoma skin
cancer: epidemiological aspects. Semin Cancer Biol.
1999;9:397–403.
215. Flores ER, Allen-Hoffmann BL, Lee D, Lambert PF.
The human papillomavirus type 16 E7 oncogene
is required for the productive stage of the viral life
cycle. J Virol. 2000;74:6622–6631.
216. Park RB, Androphy EJ. Genetic analysis of high-risk
e6 in episomal maintenance of human papillomavirus
genomes in primary human keratinocytes. J Virol.
2002;76.
217. Ustav M, Stenlund A. Transient replication of
BPV-1 requires two viral polypeptides encoded
by the E1 and E2 open reading frames. EMBO J.
1991;10:449–457.
218. Mohr IJ, Clark R, Sun S, et al. Targeting the E1
replication protein to the papillomavirus origin
of replication by complex formation with the E2
transactivator. Science. 1990;250:1694–1699.
219. Sedman J, Stenlund A. Co-operative interaction
between the initiator E1 and the transcriptional
activator E2 is required for replicator specific DNA
replication of bovine papillomavirus in vivo and in
vitro. EMBO J. 1995;14:6218–6228.
220. Yang L, Mohr I, Fouts E, et  al. The E1
protein of bovine papilloma virus 1 is an ATP-
dependent DNA helicase. Proc Natl Acad Sci USA.
1993;90:5086–5090.
221. Akgul B, Cook JC, Storey A. HPV-associated skin
disease. J Pathol. 2006;208(2):165–175. doi:10.1002/
path.1893. [Epub 2005/12/20]; PubMed PMID
16362995.
222. Uberoi A, Yoshida S, Frazer IH, Pitot H, Lambert
PF. Role of ultraviolet radiation in papillomavirus-
induced disease. PLoS Pathog. 2016;12(5):e1005664.
doi:10.1371/journal.ppat.1005664. [Epub 2016/06/
01]; PMCID: 4887022.
223. Meyers JM, Uberoi Grace M, Lambert PF, Munger
K. Cutaneous HPV8 and MmuPV1 E6 proteins
target the NOTCH and TGF-beta tumor suppressors
to inhibit differentiation and sustain keratinocyte
proliferation. PLoS Pathog. 2017;13(1):e1006171.
doi:10.1371/journal.ppat.1006171. [Epub 2017/01/
21]; PubMed PMID 28107544. PMCID 5287491.
224. Madeleine MM, Wiktor SZ, Goedert JJ, et al.
HTLV-I and HTLV-II world-wide distribution:
reanalysis of 4,832 immunoblot results. Int J Cancer.
1993;54:255–260.
225. Tajima K. Malignant lymphomas in Japan: epidemio-
logical analysis of adult T-cell leukemia/lymphoma
(ATL). Cancer Metastasis Rev. 1988;7:223–241.
226. Jeffery KJ, Usuku K, Hall SE, et al. HLA
alleles determine human T-lymphotropic virus-I
(HTLV-I) proviral load and the risk of HTLV-I-
associated myelopathy. Proc Natl Acad Sci USA.
1999;96:3848–3853.
227. Hinuma Y, Nagata K, Hanaoka M, et al. Adult
T-cell leukemia: antigen in an ATL cell line and
detection of antibodies to the antigen in human
sera. Proc Natl Acad Sci USA. 1981;78:6476–6480.
228. Yoshida M, Seiki M, Yamaguchi K, Takatsuki K.
Monoclonal integration of human T-cell leukemia
provirus in all primary tumors of adult T-cell
leukemia suggests causative role of human T-cell
leukemia virus in the disease. Proc Natl Acad Sci
USA. 1984;81:2534–2537.
229. Hino S, Katamine S, Miyata H, et al. Primary
prevention of HTLV-I in Japan. Leukemia.
1997;11(suppl 3):57–59.
230. Albrecht B, Lairmore MD. Critical role of human
T-lymphotropic virus type 1 accessory proteins in
viral replication and pathogenesis. Microbiol Mol
Biol Rev. 2002;66:396–406.
231. Yoshida M. Multiple viral strategies of HTLV-I
for dysregulation of cell growth control. Annu Rev
Immunol. 2001;19:475–496.
232. Tanaka A, Takahashi C, Yamaoka S, et al. Oncogenic
transformation by the tax gene of human T-cell
leukemia virus type I in vitro. Proc Natl Acad Sci
USA. 1990;87:1071–1075.
233. Pozzatti R, Vogel J, Jay G. The human T-lympho-
tropic virus type I tax gene can cooperate with the
ras oncogene to induce neoplastic transformation
of cells. Mol Cell Biol. 1990;10:413–417.
234. Grassmann R, Berchtold S, Radant I, et al. Role of
human T-cell leukemia virus type 1 X region proteins
in immortalization of primary human lymphocytes
in culture. J Virol. 1992;66:4570–4575.
235. Ross TM, Pettiford SM, Green PL. The tax gene of
human T-cell leukemia virus type 2 is essential for
transformation of human T lymphocytes. J Virol.
1996;70:5194–5202.
236. Nerenberg M, Hinrichs SH, Reynolds RK, et al.
The tat gene of human T-lymphotropic virus type
1 induces mesenchymal tumors in transgenic mice.
Science. 1987;237:1324–1329.
237. Grossman WJ, Kimata JT, Wong FH, et al. Develop-
ment of leukemia in mice transgenic for the tax
gene of human T-cell leukemia virus type I. Proc
Natl Acad Sci USA. 1995;92:1057–1061.
238. Ego T, Ariumi Y, Shimotohno K. The interaction of
HTLV-I Tax with HDAC1 negatively regulates the
viral gene expression. Oncogene. 2002;21:7241–7246.
239. Jeang KT, Widen SG, Semmes OJT, Wilson SH.
HTLV-I trans-activator protein, tax, is a trans-
repressor of the human beta-polymerase gene.
Science. 1990;247:1082–1084.
240. Neuveut C, Low KG, Maldarelli F, et al. Human
T-cell leukemia virus type 1 Tax and cell cycle
progression: role of cyclin D-cdk and p110Rb.
Mol Cell Biol. 1998;18:3620–3632.
241. Pise-Masison CA, Mahieux R, Jiang H, et al.
Inactivation of p53 by human T-cell lymphotropic
virus type 1 Tax requires activation of the NF-kappaB
pathway and is dependent on p53 phosphorylation.
Mol Cell Biol. 2000;20:3377–3386.
242. Suzuki T, Kitao S, Matsushime H, Yoshida M.
HTLV-I Tax protein interacts with cyclin-dependent

kinase inhibitor p16INK4A and counteracts its
inhibitory activity towards CDK4. EMBO J. 1996;15:
1607–1614.
243. Felber BK, Paskalis H, Kleinman-Ewing C, et al. The
pX protein of HTLV-I is a transcriptional activator of
its long terminal repeats. Science. 1985;229:675–679.
244. Cross SL, Feinberg MB, Wolf JB, et al. Regulation
of the human interleukin-2 receptor alpha chain
promoter: activation of a nonfunctional promoter
by the transactivator gene of HTLV-I. Cell. 1987;49:
47–56.
245. Fujii M, Sassone-Corsi P, Verma IM. c-fos promoter
trans-activation by the tax1 protein of human T-cell
leukemia virus type I. Proc Natl Acad Sci USA. 1988;85:
8526–8530.
246. Siekevitz M, Feinberg MB, Holbrook N, et al.
Activation of interleukin 2 and interleukin 2 receptor
(Tac) promoter expression by the trans-activator (tat)
gene product of human T-cell leukemia virus, type
I. Proc Natl Acad Sci USA. 1987;84:5389–5393.
247. Tsukahara T, Kannagi M, Ohashi T, et al. Induction
of Bcl-x(L) expression by human T-cell leukemia virus
type 1 Tax through NF-kappaB in apoptosis-resistant
T-cell transfectants with Tax. J Virol. 1999;73:
7981–7987.
248. Kwok RP, Laurance ME, Lundblad JR, et al. Control
of cAMP-regulated enhancers by the viral transactiva-
tor Tax through CREB and the co-activator CBP.
Nature. 1996;380:642–646.
249. Suzuki T, Hirai H, Fujisawa J, et al. A trans-activator
Tax of human T-cell leukemia virus type 1 binds to
NF-kappa B p50 and serum response factor (SRF)
and associates with enhancer DNAs of the NF-kappa
B site and CArG box. Oncogene. 1993;8:2391–2397.
250. Maggirwar SB, Harhaj E, Sun SC. Activation of
NF-kappa B/Rel by Tax involves degradation of
I kappa B alpha and is blocked by a proteasome
inhibitor. Oncogene. 1995;11:993–998.
251. Suzuki T, Hirai H, Murakami T, Yoshida M. Tax
protein of HTLV-I destabilizes the complexes of NF-
kappa B and I kappa B-alpha and induces nuclear
translocation of NF-kappa B for transcriptional
activation. Oncogene. 1995;10:1199–1207.
252. Lemasson I, Polakowski NJ, Laybourn PJ, Nyborg
JK. Transcription factor binding and histone
modifications on the integrated proviral promoter
in human T-cell leukemia virus-I-infected T-cells.
J Biol Chem. 2002;277:49459–49465.
253. Suzuki T, Uchida-Toita M, Yoshida M. Tax protein
of HTLV-I inhibits CBP/p300-mediated transcrip-
tion by interfering with recruitment of CBP/p300
onto DNA element of E-box or p53 binding site.
Oncogene. 1999;18:4137–4143.
254. Knoepfler PS, Eisenman RN. Sin meets NuRD and
other tails of repression. Cell. 1999;99:447–450.
255. Kinoshita T, Shimoyama M, Tobinai K, et al. Detec-
tion of mRNA for the tax1/rex1 gene of human
T-cell leukemia virus type I in fresh peripheral blood
mononuclear cells of adult T-cell leukemia patients
and viral carriers by using the polymerase chain reac-
tion. Proc Natl Acad Sci USA. 1989;86:5620–5624.
256. Matsuoka M, Jeang KT. Human T-cell leukaemia
virus type 1 (HTLV-I) infectivity and cellular
transformation. Nat Rev Cancer. 2007;7:270–280.
257. Larocca D, Chao LA, Seto MH, Brunck TK. Human
T-cell leukemia virus minus strand transcription
in infected T-cells. Biochem Biophys Res Commun.
1989;163:1006–1013.
258. Yoshida M, Satou Y, Yasunaga J, et al. Transcriptional
control of spliced and unspliced human T-cell
leukemia virus type 1 bZIP factor (HBZ) gene.
J Virol. 2008;82:9359–9368.
259. Satou Y, Yasunaga J, Yoshida M, Matsuoka M.
HTLV-I basic leucine zipper factor gene mRNA
supports proliferation of adult T cell leukemia cells.
Proc Natl Acad Sci USA. 2006;103:720–725.
260. Gaudray G, Gachon F, Basbous J, et al. The comple-
mentary strand of the human T-cell leukemia virus
type 1 RNA genome encodes a bZIP transcription
Viruses and Human Cancer  •  CHAPTER 12 179.e5
factor that down-regulates viral transcription. J Virol.
2002;76:12813–12822.
261. Lemasson I, Lewis MR, Polakowski N, et al. Human
T-cell leukemia virus type 1 (HTLV-I) bZIP protein
interacts with the cellular transcription factor CREB
to inhibit HTLV-I transcription. J Virol. 2007;81:
1543–1553.
262. Clerc I, Polakowski N, Andre-Arpin C, et al. An
interaction between the human T cell leukemia
virus type 1 basic leucine zipper factor (HBZ) and
the KIX domain of p300/CBP contributes to the
down-regulation of tax-dependent viral transcrip-
tion by HBZ. J Biol Chem. 2008;283:23903–
23913.
263. Satou Y, Yasunaga J, Zhao T, et al. HTLV-I bZIP
factor induces T-cell lymphoma and systemic inflam-
mation in vivo. PLoS Pathog. 2011;7:e1001274.
264. Kawatsuki A, Yasunaga JI, Mitobe Y, Green PL,
Matsuoka M. HTLV-1 bZIP factor protein targets
the Rb/E2F-1 pathway to promote prolifera-
tion and apoptosis of primary CD4 (+) T cells.
Oncogene. 2016;35(34):4509–4517. doi:10.1038/
onc.2015.510. [Epub 2016 Jan 25].
265. Kataoka K, Nagata Y, Kitanaka A, et al. Integrated
molecular analysis of adult T cell leukemia/lym-
phoma. Nat Genet. 2015;47:1304–1315.
266. Yao F, Terrault N. Hepatitis C and hepatocellular
carcinoma. Curr Treat Options Oncol. 2001;2:473–
483.
267. Perz JF, Armstrong GL, Farrington LA, et al. The
contributions of hepatitis B virus and hepatitis C
virus infections to cirrhosis and primary liver cancer
worldwide. J Hepatol. 2006;45:529–538.
268. Sun CA, Wu DM, Lin CC, et al. Incidence and
cofactors of hepatitis C virus–related hepatocellular
carcinoma: a prospective study of 12,008 men in
Taiwan. Am J Epidemiol. 2003;157:674–682.
269. Yen T, Keefe EB, Ahmed A. The epidemiology of
hepatitis C virus infection. J Clin Gastroenterol. 2003;
36:47–53.
270. WHO. WHO Global Surveillance and control
of hepatitis C. Report of a WHO Consultation
organized in collaboration with the viral Hepatitis
Prevention Board. J Viral Hepat. 1999;6:35–47.
271. Di Bisceglie AM. Hepatitis C and hepatocellular
carcinoma. Hepatology. 1997;26:34S–38S.
272. Choo QL, Kuo G, Weiner AJ, et al. Isolation of a
cDNA clone derived from a blood-borne non-A,
non-B viral hepatitis genome. Science. 1989;244:
359–362.
273. Rosenberg S. Recent advances in the molecular
biology of hepatitis C virus. J Mol Biol. 2001;313:
451–464.
274. Lamarre D, Anderson PC, Bailey M, et al. An NS3
protease inhibitor with antiviral effects in humans
infected with hepatitis C virus. Nature. 2003;426:
186–189.
275. Reiser M, Hinrichsen H, Benhamou Y, et al. Antiviral
efficacy of NS3-serine protease inhibitor BILN-2061
in patients with chronic genotype 2 and 3 hepatitis
C. Hepatology. 2005;41:832–835.
276. Smith DB, Pathirana S, Davidson F, et al. The
origin of hepatitis C virus genotypes. J Gen Virol.
1997;78(Pt 2):321–328.
277. Lange CM, Moradpour D, Doehring A, et al. Impact
of donor and recipient IL28B rs12979860 genotypes
on hepatitis C virus liver graft reinfection. J Hepatol.
2011;55:322–327.
278. Bukh J, Miller RH, Purcell RH. Genetic heterogene-
ity of hepatitis C virus: quasispecies and genotypes.
Semin Liver Dis. 1995;15:41–63.
279. Lohmann V, Korner F, Koch J, et al. Replication of
subgenomic hepatitis C virus RNAs in a hepatoma
cell line. Science. 1999;285:110–113.
280. Bukh J, Pietschmann T, Lohmann V, et al. Muta-
tions that permit efficient replication of hepatitis
C virus RNA in Huh-7 cells prevent productive
replication in chimpanzees. Proc Natl Acad Sci USA.
2002;99:14416–14421.
281. Wakita T, Pietschmann T, Kato T, et al. Production
of infectious hepatitis C virus in tissue culture from
a cloned viral genome. Nat Med. 2005;11:791–796.
282. Zhong J, Gastaminza P, Cheng G, et al. Robust
hepatitis C virus infection in vitro. Proc Natl Acad
Sci USA. 2005;102:9294–9299.
283. Lindenbach BD, Meuleman P, Ploss A, et al. Cell
culture-grown hepatitis C virus is infectious in vivo
and can be recultured in vitro. Proc Natl Acad Sci
USA. 2006;103:3805–3809.
284. Bartosch B, Cosset FL. Cell entry of hepatitis C
virus. Virology. 2006;348:1–12.
285. Bartosch B, Verney G, Dreux M, et al. An interplay
between hypervariable region 1 of the hepatitis C
virus E2 glycoprotein, the scavenger receptor BI, and
high-density lipoprotein promotes both enhancement
of infection and protection against neutralizing
antibodies. J Virol. 2005;79:8217–8229.
286. Wu X, Robotham JM, Lee E, et al. Productive
hepatitis C virus infection of stem cell-derived
hepatocytes reveals a critical transition to viral
permissiveness during differentiation. PLoS Pathog.
2012;8:e1002617.
287. Ray R, Lagging L, Meyer K, Ray R. Hepatitis C
virus core protein cooperates with ras and transforms
primary rat embryo fibroblasts to tumorigenic
phenotype. J Virol. 1996;70:4438–4443.
288. Moriya K, Fujie H, Shintani Y, et al. The core protein
of hepatitis C virus induces hepatocellular carcinoma
in transgenic mice. Nat Med. 1998;4:1065–1067.
289. Aoki H, Hayashi J, Moriyama M, et al. Hepatitis
C virus core protein interacts with the 14-3-3
protein and activates the kinase Raf-1. J Virol.
2000;74:1736–1741.
290. Hayashi J, Aoki H, Kajino K, et al. Hepatitis C virus
core protein activates the MAPK/ERK cascade syn-
ergistically with tumor promoter TPA, but not with
EGF or TGFalpha. Hepatology. 2000;32:958–961.
291. Jin D, Wang H, Zhou Y, et al. Hepatitis C virus core
protein-induced loss of LZIP function correlates with
cellular transformation. EMBO J. 2000;19:729–740.
292. Yoshida T, Hanada T, Tokuhisa T, et al. Activation
of STAT3 by the hepatitis C virus core protein leads
to cellular transformation. J Exp Med. 2002;196:
641–653.
293. Li M, Zhao H, Zhang X, et al. Inactivating mutations
of the chromatin remodeling gene ARID2 in hepa-
tocellular carcinoma. Nat Genet. 2011;43:828–829.
294. Schinazi RF, Asselah T. From HCV to HBV Cure.
Liver Int. 2017;37(suppl 1):73–80.
295. Deleted in review.
296. Cook-Mozaffari P, Newton R, Beral V, Burkitt DP.
The geographical distribution of Kaposi’s sarcoma
and of lymphomas in Africa before the AIDS
epidemic. Br J Cancer. 1998;78:1521–1528.
297. Geddes M, Franceschi S, Barchielli A, et al. Kaposi’s
sarcoma in Italy before and after the AIDS epidemic.
Br J Cancer. 1994;69:333–336.
298. Chang Y, Cesarman E, Pessin MS, et al. Identification
of herpesvirus-like DNA sequences in AIDS-associ-
ated Kaposi’s sarcoma. Science. 1994;266:1865–1869.
299. Dedicoat M, Newton R. Review of the distribution
of Kaposi’s sarcoma-associated herpesvirus (KSHV)
in Africa in relation to the incidence of Kaposi’s
sarcoma. Br J Cancer. 2003;88:1–3.
300. Osmond DH, Buchbinder S, Cheng A, et al. Preva-
lence of Kaposi sarcoma-associated herpesvirus infec-
tion in homosexual men at beginning of and during
the HIV epidemic. JAMA. 2002;287:221–225.
301. Jacobson LP, Jenkins FJ, Springer G, et al. Interaction
of human immunodeficiency virus type 1 and human
herpesvirus type 8 infections on the incidence of
Kaposi’s sarcoma. J Infect Dis. 2000;181:1940–1949.
302. Cesarman E, Chang Y, Moore PS, et al. Kaposi’s
sarcoma-associated herpesvirus-like DNA sequences
in AIDS-related body-cavity-based lymphomas.
N Engl J Med. 1995;332:1186–1191.
303. Soulier J, Grollet L, Oksenhendler E, et al.
Kaposi’s sarcoma-associated herpesvirus-like DNA
179.e6 Part I: Science and Clinical Oncology
sequences in multicentric Castleman’s disease. Blood.
1995;86:1276–1280.
304. Martin JN. Diagnosis and epidemiology of human
herpesvirus 8 infection. Semin Hematol. 2003;40:
133–142.
305. Uldrick TS, Whitby D. Update on KSHV epidemiol-
ogy, Kaposi sarcoma pathogenesis, and treatment of
Kaposi sarcoma. Cancer Lett. 2011;305:150–162.
306. Zhang T, Shao X, Chen Y, et al. Human herpes-
virus 8 seroprevalence, China. Emerg Infect Dis.
2012;18:150–152.
307. Engels EA, Biggar RJ, Hall HI, et al. Cancer risk
in people infected with human immunodeficiency
virus in the United States. Int J Cancer. 2008;123:
187–194.
308. Scadden DT. AIDS-related malignancies. Annu Rev
Med. 2003;54:285–303.
309. Bower M, Palmieri C, Dhillon T. AIDS-related
malignancies: changing epidemiology and the impact
of highly active antiretroviral therapy. Curr Opin
Infect Dis. 2006;19:14–19.
310. Newton R, Carpenter L, Casabonne D, et al. A
prospective study of Kaposi’s sarcoma–associated
herpesvirus and Epstein-Barr virus in adults with
human immunodeficiency virus-1. Br J Cancer.
2006;94:1504–1509.
311. Dupin N, Fisher C, Kellam P, et al. Distribution
of human herpesvirus-8 latently infected cells in
Kaposi’s sarcoma, multicentric Castleman’s disease,
and primary effusion lymphoma. Proc Natl Acad Sci
USA. 1999;96:4546–4551.
312. Jenner RG, Boshoff C. The molecular pathology of
Kaposi’s sarcoma–associated herpesvirus. Biochim
Biophys Acta. 2002;1602:1–22.
313. Moore PS, Chang Y. Kaposi’s sarcoma-associated
herpesvirus-encoded oncogenes and oncogenesis.
J Natl Cancer Inst Monogr. 1998;65–71.
314. Matolcsy A, Nador RG, Cesarman E, Knowles DM.
Immunoglobulin VH gene mutational analysis sug-
gests that primary effusion lymphomas derive from
different stages of B cell maturation. Am J Pathol.
1998;153:1609–1614.
315. Nador RG, Cesarman E, Chadburn A, et al. Primary
effusion lymphoma: a distinct clinicopathologic
entity associated with the Kaposi’s sarcoma–associ-
ated herpes virus. Blood. 1996;88:645–656.
316. Mesri EA, Cesarman E, Boshoff C. Kaposi’s sarcoma
and its associated herpesvirus. Nat Rev Cancer.
2010;10:707–719.
317. Cheng EH, Nicholas J, Bellows DS, et al. A Bcl-2
homolog encoded by Kaposi sarcoma–associated
virus, human herpesvirus 8, inhibits apoptosis but
does not heterodimerize with Bax or Bak. Proc Natl
Acad Sci USA. 1997;94:690–694.
318. Ojala PM, Yamamoto K, Castanos-Velez E, et al. The
apoptotic v-cyclin-CDK6 complex phosphorylates
and inactivates Bcl-2. Nat Cell Biol. 2000;2:819–825.
319. Djerbi M, Screpanti V, Catrina AI, et al. The inhibi-
tor of death receptor signaling, FLICE-inhibitory
protein defines a new class of tumor progression
factors. J Exp Med. 1999;190:1025–1032.
320. Bagneris C, Ageichik AV, Cronin N, et al. Crystal
structure of a vFlip-IKKgamma complex: insights
into viral activation of the IKK signalosome. Mol
Cell. 2008;30:620–631.
321. Liu L, Eby MT, Rathore N, et al. The human
herpes virus 8–encoded viral FLICE inhibitory
protein physically associates with and persistently
activates the Ikappa B kinase complex. J Biol Chem.
2002;277:13745–13751.
322. Asou H, Said JW, Yang R, et al. Mechanisms of
growth control of Kaposi’s sarcoma–associated herpes
virus-associated primary effusion lymphoma cells.
Blood. 1998;91:2475–2481.
323. McCormick C, Ganem D. The kaposin B protein of
KSHV activates the p38/MK2 pathway and stabilizes
cytokine mRNAs. Science. 2005;307:739–741.
324. Montaner S, Sodhi A, Molinolo A, et al. Endothelial
infection with KSHV genes in vivo reveals that
vGPCR initiates Kaposi’s sarcomagenesis and can
promote the tumorigenic potential of viral latent
genes. Cancer Cell. 2003;3:23–36.
325. Bais C, Van Geelen A, Eroles P, et al. Kaposi’s
sarcoma associated herpesvirus G protein-coupled
receptor immortalizes human endothelial cells by
activation of the VEGF receptor-2/ KDR. Cancer
Cell. 2003;3:131–143.
326. Mutlu AD, Cavallin LE, Vincent L, et al. In
vivo-restricted and reversible malignancy induced
by human herpesvirus-8 KSHV: a cell and animal
model of virally induced Kaposi’s sarcoma. Cancer
Cell. 2007;11:245–258.
327. Lagos D, Vart RJ, Gratrix F, et al. Toll-like receptor
4 mediates innate immunity to Kaposi sarcoma
herpesvirus. Cell Host Microbe. 2008;4:470–483.
328. Ishido S, Wang C, Lee BS, et al. Downregulation of
major histocompatibility complex class I molecules
by Kaposi’s sarcoma-associated herpesvirus K3 and
K5 proteins. J Virol. 2000;74:5300–5309.
329. Dupuy S, Lambert M, Zucman D, et al. Human
Herpesvirus 8 (HHV8) sequentially shapes the NK
cell repertoire during the course of asymptomatic
infection and Kaposi sarcoma. PLoS Pathog.
2012;8:e1002486.
330. Ballestas ME, Kaye KM. Kaposi’s sarcoma-associated
herpesvirus latency-associated nuclear antigen 1
mediates episome persistence through cis-acting
terminal repeat (TR) sequence and specifically binds
TR DNA. J Virol. 2001;75:3250–3258.
331. Hu J, Garber AC, Renne R. The latency-associated
nuclear antigen of Kaposi’s sarcoma-associated
herpesvirus supports latent DNA replication in
dividing cells. J Virol. 2002;76:11677–11687.
331a. Chiu YF, Sugden AU, Fox K, et al. Kaposi’s
sarcoma-associated herpesvirus stably clusters its
genomes across generations to maintain itself extra-
chromosomally. J Cell Biol. 2017;216(9):2745–2758.
332. Gradoville L, Gerlach J, Grogan E, et al. Kaposi’s
sarcoma-associated herpesvirus open reading frame
50/Rta protein activates the entire viral lytic cycle in
the HH-B2 primary effusion lymphoma cell line.
J Virol. 2000;74:6207–6212.
333. Ciufo DM, Cannon JS, Poole LJ, et al. Spindle
cell conversion by Kaposi’s sarcoma–associated
herpesvirus: formation of colonies and plaques
with mixed lytic and latent gene expression in
infected primary dermal microvascular endothelial
cell cultures. J Virol. 2001;75:5614–5626.
334. Moses AV, Fish KN, Ruhl R, et al. Long-term infec-
tion and transformation of dermal microvascular
endothelial cells by human herpesvirus 8. J Virol.
1999;73:6892–6902.
335. Andrei G, Snoeck R. Kaposi’s sarcoma-associated
herpesvirus: the role of lytic replication in targeted
therapy. Curr Opin Infect Dis. 2015;28(6):611–624.
336. Moll I, Bladt U, Jung EG. Presence of Merkel cells in
sun-exposed and not sun-exposed skin: a quantitative
study. Arch Dermatol Res. 1990;282:213–216.
337. Hodgson NC. Merkel cell carcinoma: changing
incidence trends. J Surg Oncol. 2005;89:1–4.
338. Halata Z, Grim M, Bauman KI. Friedrich Sigmund
Merkel and his “Merkel cell,” morphology, develop-
ment, and physiology: review and new results. Anat
Rec A Discov Mol Cell Evol Biol. 2003;271:225–239.
339. Lucarz A, Brand G. Current considerations about
Merkel cells. Eur J Cell Biol. 2007;86:243–251.
340. Moll I, Roessler M, Brandner JM, et al. Human
Merkel cells—aspects of cell biology, distribution
and functions. Eur J Cell Biol. 2005;84:259–271.
341. Sidhu GS, Chandra P, Cassai ND. Merkel cells,
normal and neoplastic: an update. Ultrastruct Pathol.
2005;29:287–294.
342. Feng H, Taylor JL, Benos PV, et al. Human tran-
scriptome subtraction by using short sequence tags to
search for tumor viruses in conjunctival carcinoma.
J Virol. 2007;81:11332–11340.
343. Becker JC, Houben R, Ugurel S, et al. MC
polyomavirus is frequently present in Merkel cell
carcinoma of European patients. J Invest Dermatol.
2009;129:248–250.
344. Kassem A, Schopflin A, Diaz C, et al. Frequent
detection of Merkel cell polyomavirus in human
Merkel cell carcinomas and identification of a unique
deletion in the VP1 gene. Cancer Res. 2008;68:
5009–5013.
345. Shuda M, Feng H, Kwun HJ, et al. T antigen
mutations are a human tumor-specific signature
for Merkel cell polyomavirus. Proc Natl Acad Sci
USA. 2008;105:16272–16277.
346. Carter JJ, Paulson KG, Wipf GC, et al. Association
of Merkel cell polyomavirus-specific antibodies
with Merkel cell carcinoma. J Natl Cancer Inst.
2009;101:1510–1522.
347. Kean JM, Rao S, Wang M, Garcea RL. Seroepide-
miology of human polyomaviruses. PLoS Pathog.
2009;5:e1000363.
348. Pastrana DV, Tolstov YL, Becker JC, et al. Quantita-
tion of human seroresponsiveness to Merkel cell
polyomavirus. PLoS Pathog. 2009;5:e1000578.
349. Tolstov YL, Pastrana DV, Feng H, et al. Human
Merkel cell polyomavirus infection II. MCV is a
common human infection that can be detected by
conformational capsid epitope immunoassays. Int
J Cancer. 2009;125:1250–1256.
350. Touze A, Gaitan J, Arnold F, et al. Generation of
Merkel cell polyomavirus (MCV)-like particles and
their application to detection of MCV antibodies.
J Clin Microbiol. 2010;48:1767–1770.
351. Feng H, Shuda M, Chang Y, Moore PS. Clonal
integration of a polyomavirus in human Merkel
cell carcinoma. Science. 2008;319:1096–1100.
352. Foulongne V, Kluger N, Dereure O, et al. Merkel
cell polyomavirus in cutaneous swabs. Emerg Infect
Dis. 2010;16:685–687.
353. Schowalter RM, Pastrana DV, Pumphrey KA,
et al. Merkel cell polyomavirus and two previously
unknown polyomaviruses are chronically shed from
human skin. Cell Host Microbe. 2010;7:509–515.
354. Wieland U, Mauch C, Kreuter A, et al. Merkel cell
polyomavirus DNA in persons without Merkel cell
carcinoma. Emerg Infect Dis. 2009;15:1496–1498.
355. Houben R, Schrama D, Alb M, et al. Comparable
expression and phosphorylation of the retinoblastoma
protein in Merkel cell polyoma virus-positive and neg-
ative Merkel cell carcinoma. Int J Cancer. 2010;126:
796–798.
356. Shuda M, Kwun HJ, Feng H, et al. Human Merkel
cell polyomavirus small T antigen is an oncoprotein
targeting the 4E-BP1 translation regulator. J Clin
Invest. 2011;121:3623–3634.
357. Arora R, Shuda M, Guastafierro A, et al. Survivin
is a therapeutic target in Merkel cell carcinoma. Sci
Transl Med. 2012;4:133ra156.
358. Spurgeon ME, Cheng J, Bronson RT, Lambert PF,
DeCaprio JA. Tumorigenic activity of merkel cell
polyomavirus T antigens expressed in the stratified
epithelium of mice. Cancer Res. 2015;75(6):1068–
1079. doi:10.1158/0008-5472.CAN-14-2425. [Epub
2015/01/18]; PubMed PMID: 25596282. PMCID:
4359959.
359. Verhaegen ME, Mangelberger D, Harms PW,
Vozheiko TD, Weick JW, Wilbert DM, et al.
Merkel cell polyomavirus small T antigen is
oncogenic in transgenic mice. J Invest Dermatol.
2015;135(5):1415–1424. doi:10.1038/jid.2014.446.
[Epub 2014 Oct 14]; PMID: 25313532.
360. Shuda M, Guastafierro A, Geng X, Shuda Y, Ostrowski
SM, Lukianov S, et al. Merkel cell polyomavirus
small T antigen induces cancer and embryonic
Merkel cell proliferation in a transgenic mouse
model. PLoS ONE. 2015;10(11):e0142329. doi:
10.1371/journal.pone.0142329. PMID: 26544690.
eCollection 2015.
361. Verhaegen ME, Mangelberger D, Harms PW, Eberl
M, Wilbert DM, Meireles J, et al. Merkel cell
polyomavirus small T antigen initiates Merkel cell
carcinoma-like tumor development in mice. Cancer

Res. 2017;doi:10.1158/0008-5472.CAN-17-0035.
[Epub ahead of print]; PMID: 28512245.
362. Cairo MS, Sposto R, Perkins SL, et al. Burkitt’s and
Burkitt-like lymphoma in children and adolescents:
a review of the Children’s Cancer Group experience.
Br J Haematol. 2003;120:660–670.
363. Ahmad SA, Bilimoria MM, Wang X, et al. Hepatitis
B or C virus serology as a prognostic factor in
patients with hepatocellular carcinoma. J Gastrointest
Surg. 2001;5:468–476.
364. Rabe C, Pilz T, Klostermann C, et al. Clinical
characteristics and outcome of a cohort of 101
patients with hepatocellular carcinoma. World J
Gastroenterol. 2001;7:208–215.
365. Wang H, Chia KS, Du WB, et al. Population-based
survival for cervical cancer in Singapore, 1968-1992.
Am J Obstet Gynecol. 2003;188:324–329.
366. Donato DM. Surgical management of stage
IB-IIA cervical carcinoma. Semin Surg Oncol.
1999;16:232–235.
367. Yamaguchi K, Watanabe T. Human T lymphotropic
virus type-I and adult T-cell leukemia in Japan. Int
J Hematol. 2002;76(suppl 2):240–245.
368. Tam HK, Zhang ZF, Jacobson LP, et al. Effect of
highly active antiretroviral therapy on survival among
HIV-infected men with Kaposi sarcoma or non-
Hodgkin lymphoma. Int J Cancer. 2002;98:916–922.
Viruses and Human Cancer  •  CHAPTER 12 179.e7
369. Ghosh SK, Wood C, Boise LH, et al. Potentiation
of TRAIL-induced apoptosis in primary effusion
lymphoma through azidothymidine-mediated inhibi-
tion of NF-kappa B. Blood. 2003;101:2321–2327.
370. Rizzetto M, Zanetti AR. Progress in the prevention
and control of viral hepatitis type B: closing remarks.
J Med Virol. 2002;67:463–466.
371. Zuckerman JN, Zuckerman AJ. Current topics in
hepatitis B. J Infect. 2000;41:130–136.
372. Winer RL, Lee SK, Hughes JP, et al. Genital human
papillomavirus infection: incidence and risk factors
in a cohort of female university students. Am J
Epidemiol. 2003;157:218–226.
373. Christensen ND, Reed CA, Cladel NM, et al. Immu-
nization with viruslike particles induces long-term
protection of rabbits against challenge with cot-
tontail rabbit papillomavirus. J Virol. 1996;70:960–
965.
374. Suzich JA, Ghim SJ, Palmer-Hill FJ, et  al.
Systemic immunization with papillomavirus L1
protein completely prevents the development of
viral mucosal papillomas. Proc Natl Acad Sci USA.
1995;92:11553–11557.
375. Harro CD, Pang YY, Roden RB, et al. Safety and
immunogenicity trial in adult volunteers of a human
papillomavirus 16 L1 virus-like particle vaccine.
J Natl Cancer Inst. 2001;93:284–292.
376. Koutsky LA, Ault KA, Wheeler CM, et al. Proof of
principle study, I. A controlled trial of a human papillo-
mavirus type 16 vaccine. NEJM. 2002;347:1645–1651.
377. Roden RB, Yutzy WHT, Fallon R, et al. Minor
capsid protein of human genital papillomaviruses
contains subdominant, cross-neutralizing epitopes.
Virology. 2000;270:254–257.
378. Ganem D, Schneider RJ. Hepadnaviridae: the viruses
and their replication. In: Knipe DM, Howley PM,
Griffin DE, et al, eds. Fields Virology. Philadelphia,
PA: Lippincott Williams & Wilkins; 2001.
379. Howley PM, Lowy DR. Papillomaviruses and
their replication. In: Knipe DM, Howley PM, eds.
Fields Virology. 4th ed. Philadelphia, PA: Lippincott
Williams & Wilkins; 2001:2197–2229.
380. Green PL, Chen SY. Human T-cell leukemia virus
types 1 and 2. In: Knipe DM, Howley PM, eds.
Fields Virology. 4th ed. Philadelphia, PA: Lippincott
Williams & Wilkins; 2001.
381. Major M, Rehermann B, Feinstone SM. Hepatitis
C viruses. In: Knipe DM, Howley PM, eds. Fields
Virology. Philadelphia: Lippincott Williams &
Wilkins.; 2001:1127–1161.
382. Moore P, Chang Y. Kaposi’s sarcoma–associ-
ated herpesvirus. In: Knipe DM, ed. Virology.
Philadelphia, PA: Lippincott Williams and Wilkins;
2001:2803–2833.
 13  Genetic Factors: Hereditary Cancer
Predisposition Syndromes
Michael F. Walsh, Karen Cadoo, Erin E. Salo-Mullen,
Marianne Dubard-Gault, Zsofia K. Stadler, and Kenneth Offit

S UMMARY OF K EY
• The discovery of inherited mutations
of genes associated with increased
risk for cancer provides important
clinical opportunities for early
detection and prevention of common
and rare forms of human
malignancies.
• Syndromes of cancer predisposition
often involve multiple organ systems,
affect paired organs with bilateral or
multifocal tumors, and have onset leading role in the integration of
at an earlier age compared with
nonfamilial tumors. The diagnosis of
particular cancer predisposition
P OI N T S
syndromes can usually be confirmed
with molecular genetic testing of
patients who have hereditary
malignancies. Genetic testing can
then be extended to relatives as a
predictive test to guide their
preventive management.
• Medical, surgical, and radiation
oncologists, genetic counselors, and
allied professionals are playing a
genetic testing into the practice of
preventive oncology. Recently,
genomic analysis has been applied
to tumors to discover targets for
therapy. Because tumor genomic
analysis will also include a
comparison with the germline, the
need for genetic counseling for both
cancer and noncancer disease risks
will be increased. This chapter
reviews both common and more
recently described familial cancer
syndromes, with an emphasis on the
clinical application of cancer genetic
and genomic analysis in the
management of patients who have or
are at risk for cancer.

During the past decade, the availability of clinical testing for inherited
mutations of cancer predisposition genes has had a major impact on
the practice of clinical oncology.1–3 As these genes were identified and
characterized, guidelines for the responsible clinical translation of this
information were developed by medical and surgical subspecialty
societies, such as the American Society of Clinical Oncology (updated
in 2016),4 the American Association for Cancer Research Pediatric
Cancer Working Group,5–8 and other organizations.9–13 These guidelines
emphasized that in the process of offering a predictive genetic test to
a patient or family that is affected by cancer, the provider and the
individual who is being tested must be prepared to deal with all the
medical, psychological, and social consequences of a positive, negative,
or ambiguous result. These guidelines define the form and content
of genetic counseling as a component of cancer risk assessment and
management.
A selected set of syndromes of cancer predisposition is reviewed
in this chapter (Tables 13.1 and 13.2). Detailed discussions of breast,
colon, gastric, gynecologic, pancreatic, prostate, and renal cancer
susceptibility are found in the chapters that discuss these tumors.
Whether offered by a physician, genetic counselor, or other health
care professional, genetic testing for inherited cancer risk requires
careful informed consent. The elements of informed consent for genetic
testing are summarized in Box 13.1. With the advent of genomic
screening of tumors, gene panel testing, and genomic screening in
the germline, these elements of informed consent must also include
the eventuality of “incidental” findings associated with risk of cancer,
noncancer diseases, and nonpaternity.14,15
Mutations (i.e., pathogenic variants) in genes whose alterations
result in susceptibility to cancer may be inherited, whereby a family
history usually includes multiple individuals diagnosed with cancer,
or may occur de novo in families in which the history of cancer may
be unremarkable. In many instances, the same genes recurrently mutated
in specific sporadic cancers, if mutated in the germline, are also
susceptibility genes for the same types of tumors. This phenomenon
reflects the genotype-phenotype relationships that arise as a result of
aberration of particular biologic pathways and thus define the patterns
that characterize hereditary cancer syndromes. Increasingly, specific
pathologic hallmarks have been associated with cancer predisposition
syndromes (see Table 13.1). It is now widely accepted that cancer
pathogenesis derives from inherited or acquired alterations in a set of
“driver” genes that are associated with abnormal cellular function,
resulting in uncontrolled cell division and/or loss of the normal fidelity
of DNA repair and replication. This process results in accumulation
of further mutations characterized as “passengers” in the neoplastic
process which contribute to tumor survival.16 With the advent of
massively parallel sequencing, progress in the understanding of tumor
genetics has catalogued these “driver” and “passenger” mutations,
illuminating biologic pathways and facilitating development of therapies
targeted toward these pathways. At the same time, the definition of
pathways perturbed in malignant transformation and progression has
provided novel biomarkers of utility for disease prognosis and, as is
the focus here, disease susceptibility.
The syndromes of cancer susceptibility included in this chapter
are those that are most commonly encountered in oncologic
practice, as well as several recently defined entities, some of which
have been associated with targeted therapies. The most common of
these syndromes, predisposing to cancers of the breast, ovary, colon,
prostate, and pancreas, affect tens of thousands of Americans who
are diagnosed with these cancers each year in the United States and
Text continued on p. 194

180
 Genetic Factors: Hereditary Cancer Predisposition Syndromes  •  CHAPTER 13 181

Table 13.1  Pathologic Hallmarks of Selected Cancer Predisposition Syndromes

Neoplastic or Preneoplastic Pathologic Conditions Associated Genetic Syndromes (Gene)
Atypical rhabdoid tumor, schwannomatosis Rhabdoid tumor predisposition syndrome (SMARCB1)
Wilms tumor Li-Fraumeni syndrome (TP53), (WT1),(BWS), Simpson-Golabi-
Behmel syndrome, Beckwith-Wiedemann syndrome, Fanconi
anemia compound heterozygotes (see Fanconi anemia)
Ductal breast cancer with estrogen receptor, progesterone receptor, and Hereditary breast and ovarian cancer syndrome (BRCA1)
HER2-neu negativity (“triple negative”)
Lobular breast cancer Hereditary diffuse gastric cancer syndrome (CDH1)
High-grade serous (papillary serous) ovarian cancer Hereditary breast and ovarian cancer syndrome (see Table 13.2)
Hepatoblastoma Familial adenomatous polyposis (APC), Beckwith-Wiedemann
syndrome, Simpson-Golabi-Behmel syndrome, Sotos syndrome
Hereditary paraganglioma-pheochromocytoma Hereditary paraganglioma-pheochromocytoma syndromes (SDHB,
SDHC, SDHD, SDHAF2)
Hypodiploid leukemia Li-Fraumeni syndrome (TP53)
Clear cell or endometrioid ovarian cancers Lynch syndrome (MLH1, MSH2, MSH6, PMS2, EPCAM)
Ovarian sex cord tumors with annular tubules (SCTATs) Peutz-Jeghers syndrome (STK11)
Clear cell or endometrioid endometrial cancers Lynch syndrome (MLH1, MSH2, MSH6, PMS2, EPCAM)
Uterine leiomyomas Hereditary leiomyoma renal cell carcinoma syndrome (FH)
Adenoma malignum of the cervix Peutz-Jeghers syndrome (STK11)
Colorectal cancer with tumor infiltrating lymphocytes, Crohnlike lymphocytic Lynch syndrome (MLH1, MSH2, MSH6, PMS2, EPCAM)
reaction, mucinous or signet ring cells, medullary growth pattern
Lauren diffuse type, signet ring cell, or linitus plastica gastric cancer Hereditary diffuse gastric cancer syndrome (CDH1)
Leiomyosarcoma Li-Fraumeni syndrome (TP53), retinoblastoma (RB1)
Hamartomatous gastrointestinal polyps Peutz-Jeghers syndrome (STK11); juvenile polyposis syndrome
(BMPR1A, SMAD4) ; Cowden syndrome (PTEN)
Hypermutable tumors Constitutional mismatch repair deficiency, Lynch syndrome (MLH1
MSH2 MSH6 PMS2 EPCAM)
Paget breast tumor Li-Fraumeni syndrome (TP53)
Pancreatic neuroendocrine tumors Multiple endocrine neoplasia type 1 (MEN1) ; von Hippel-Lindau
disease (VHL)
Renal papillary carcinoma type I Hereditary papillary renal cancer syndrome (MET)
Renal papillary carcinoma type II, collecting duct, tubulopapillary Hereditary leiomyoma renal cell carcinoma syndrome (FH)
Renal clear cell carcinoma von Hippel-Lindau disease (VHL)
Renal oncocytoma Birt-Hogg-Dube syndrome (FLCN); tuberous sclerosis complex
(TSC1)
Renal angiomyolipoma Tuberous sclerosis complex (TSC1)
Renal chromophobe Birt-Hogg-Dube syndrome (FLCN)
Renal medullary cancer Sickle cell trait
Sertoli cell tumors of the testes Peutz-Jeghers syndrome (STK11);
Small cell carcinoma of the ovary–hypercalcemic type (SCCOHT) Rhabdoid tumor predisposition syndrome (SMARCA4)
Medullary thyroid cancer Multiple endocrine neoplasia type 2 (RET)
Meningioma Neurofibromatosis type 2 (NF2), Gorlin syndrome (PTCH1)
Cribriform-morula variant of papillary thyroid carcinoma Familial adenomatous polyposis (APC)
Follicular thyroid cancer Cowden syndrome (PTEN)
Medulloblastoma Turcot syndrome variant of familial adenomatous polyposis (APC);
Gorlin syndrome (PTCH1), Fanconi anemia (FANC complex genes),
Glioblastoma Turcot syndrome variant of Lynch syndrome (MLH1, MSH2, MSH6,
nPMS2)
Basal cell carcinoma Xeroderma pigmentosum (XPA,B,C,D; DDB2, ERCC4, ERCC5, POLH);
Gorlin syndrome (PTCH1)
Schwannomatosis Neurofibromatosis type 2; rhabdoid predisposition syndrome
(SMARCB1, LZTR1)
Sebaceous adenoma, sebaceous carcinoma, keratoacanthoma Muir Torre syndrome variant of Lynch syndrome (MLH1, MSH2)
Fibrofolliculomas Birt-Hogg-Dube syndrome (FLCN)
Facial tricholemmomas Cowden syndrome (PTEN)
Table 13.2  Cancer Susceptibility Genes, Syndromes, and Management Considerations
Genes and Details Syndrome Penetrance Associated Neoplasms Management Considerations Other Notes
231,233,422,423
APC Familial High Colorectal lesions (adenomatous Colonoscopy and total colectomy based Genotype-phenotype correlations
Tumor suppressor adenomatous polyposis and cancer) on polyp burden between classic FAP and AFAP: Mutations
Dominant inheritance polyposis (FAP) Gastric and duodenal or small bowel Upper endoscopy on the 5′ and 3′ ends of the gene
Attenuated FAP lesions (adenomatous polyps and cancer) Consideration of small bowel visualization correlate with AFAP
(AFAP) Desmoid tumors Thyroid examination and consideration of High de novo mutation rate (~20%–25%)
Variant: Turcot Thyroid (typically papillary) ultrasound examination Extracolonic features: Congenital
182 Part I: Science and Clinical Oncology

syndrome Hepatoblastoma (patients usually ≤5 yr Consideration of childhood liver hypertrophy of the retinal pigment
old) palpation, abdominal ultrasound epithelium (CHRPE); osteomas,
Pancreatic examination, AFP measurement supernumerary teeth; desmoids,
medulloblastoma (Turcot syndrome) (investigational) epidermoid cysts; gastric fundic gland
polyps
Particularly cribriform-morula variant of
papillary thyroid carcinoma
Chemoprevention not a replacement for
colonoscopy and colectomy
APC*I1307K Moderate Colorectal Colonoscopy based on family history or Ashkenazi Jewish founder mutation
(c.3920T>A)265,424,425 similar to first-degree relative risk Not associated with polyposis
ATM5,84,110,421,426–429 Ataxia Moderate Monoallelic carriers: Monoallelic carriers: Breast screening with Monoallelic carriers: Limited evidence for
telangiectasia (AT) (monoallelic/ Breast, possibly pancreas annual mammogram and breast magnetic pancreas or prostate cancer
heterozygous Biallelic carriers: resonance imaging (MRI) starting at age Risk of autosomal recessive AT condition
carriers) Leukemia, lymphoma 40 yr or per family historya in biallelic offspring of heterozygous
Rare: High Biallelic carriers: AT specialist for carriers
(biallelic) multidisciplinary management: screening AT: Childhood cerebellar ataxia,
complete blood count (CBC) with telangiectasias of the conjunctivae,
differential; consider bone marrow biopsy, immunodeficiency, sensitivity to
AFP measurement radiation
Immunology: Monitoring immunoglobulin
levels per immunologist recommendation
Dermatology: annual skin examination
Pulmonary: Baseline and as-needed
pulmonary function tests
Gastroenterology and nutrition: Baseline
and as-needed swallowing function and
nutritional management
Endocrine: Annual diabetes screen
Neurology: Supportive medications
Orthopedics: Annual scoliosis evaluation
Dental: Biannual examination
BAP1411–413,430 BAP1 tumor Undetermined Atypical Spitz tumors Eye examinations Limited evidence for possible other
Possible tumor predisposition Uveal melanoma Dermatologic examinations associated tumors: non–small cell lung
suppressor syndrome Malignant mesothelioma Consideration of abdominal ultrasound adenocarcinoma, breast cancer,
Dominant inheritance Cutaneous melanoma examination and/or MRI of the kidneys cholangiocarcinoma, meningioma,
Clear cell renal cell carcinoma neuroendocrine carcinoma
Basal cell carcinoma
BLM5,431–436 Bloom syndrome Low or Biallelic: Myelodysplasia Biallelic carriers: Bloom syndrome Monoallelic carriers: Limited evidence for
undetermined Variety of epithelial carcinomas specialist for management: risk of colorectal and breast cancer
(monoallelic/ (gastrointestinal, genitourinary), CBC every 3–4 mo, avoidance of radiation, Bloom syndrome: Chromosome breakage
heterozygous lymphoid, hematopoietic, sarcomas, CNS, breast MRI or ultrasound starting at 18 yr syndrome with growth deficiency,
carriers) and others of age, annual colonoscopy starting at erythematous facial skin lesion,
Rare: High age 15 yr, renal ultrasound examination at immunodeficiency, frequent infections
(biallelic) diagnosis and every 3 mo through age Founder mutation in Ashkenazi Jewish
8 yr to assess for Wilms tumor, HPV population
vaccine per AAP guidelines
Dermatology: Annual skin examination,
limit sun exposure
Pulmonary: Pulmonary function tests
Gastroenterology and nutrition: Baseline
and as-needed swallowing function
evaluation and nutritional management
Endocrine: Annual fasting blood sugar
and TSH level
Orthopedics: Annual scoliosis evaluation
Dental: Biannual examination
BMPR1A and Juvenile polyposis High Gastrointestinal lesions (juvenile/ Colonoscopy based on polyp burden “Juvenile” refers to polyp histology, not
SMAD4423,437–443 syndrome hamartomatous polyps and cancer— Upper endoscopy age of onset
SMAD4: Tumor colorectal, stomach, small bowel) Surgical management based on polyp SMAD4 mutations associated with HHT
suppressor burden SMAD4 mutations associated with
Dominant inheritance Hereditary hemorrhagic telangiectasia massive gastric polyposis
(HHT) screening Limited evidence for association with
pancreas
BRCA1 and Hereditary breast High Breast (BRCA1: often triple-negative Breast screening with annual Founder mutations in certain
BRCA25,91,101,127,137,444–449 and ovarian tumor) mammogram and breast MRI starting at populations—Ashkenazi Jewish,
Tumor suppressor cancer syndrome Male breast age 25–30 yr Icelandic, and others
Dominant inheritance (HBOC) Ovarian (epithelial; high-grade serous), Male self- and clinical-breast examination Autosomal recessive Fanconi anemia
fallopian tube, primary peritoneal Optional risk-reducing mastectomy with biallelic germline mutations
Prostate (high Gleason score) Risk-reducing BSO BRCA1/2 heterozygous mutations
Pancreatic (exocrine) PSA measurement and digital rectal detected in childhood through tumor
examination normal sequencing should be reviewed
Consideration of eligibility and pros and with clinical genetics team and treating
cons of chemoprevention with tamoxifen oncology team for importance
Consideration of eligibility for treatment pertaining to care of relatives and
with PARP inhibitors potentially molecularly based therapy
Investigational-based pancreas screening

Continued
Genetic Factors: Hereditary Cancer Predisposition Syndromes  •  CHAPTER 13 183
Table 13.2  Cancer Susceptibility Genes, Syndromes, and Management Considerations—cont’d
Genes and Details Syndrome Penetrance Associated Neoplasms Management Considerations Other Notes
184 Part I: Science and Clinical Oncology

BRIP184,113,450 Moderate Ovarian (heterozygous carriers) Consider risk-reducing BSO at age Risk of autosomal recessive Fanconi
Possible tumor (monoallelic/ 50–55 yr or as per family historyb anemia in biallelic offspring of
suppressor heterozygous heterozygous carriers
carriers)
Rare: High
(biallelic)
CDH1269,271,451 Hereditary diffuse High Diffuse gastric cancer Prophylactic total gastrectomy Upper endoscopy not proven to be an
Tumor suppressor gastric cancer Lobular breast cancer Breast screening with annual effective method of screening or
Dominant inheritance syndrome mammogram and breast MRI detecting diffuse gastric cancer
Optional risk-reducing mastectomy Insufficient evidence for colorectal
cancer
CDKN2A293,452,453 Familial atypical High Melanoma and dysplastic nevi Dermatologic examination Risk of melanoma may be independent
Tumor suppressor multiple mole Pancreas Investigational-based pancreatic of genetic test result
CDK4 oncogene melanoma screening CDKN2a (p16) founder mutation in
Dominant inheritance (FAMMM) Netherlands
syndrome
CHEK25,63,65,84,109,425 Moderate Breast Breast screening with annual Genotype-phenotype: Different risks for
Tumor suppressor Colon mammogram and breast MRI starting at truncating mutations vs missense
Dominant inheritance age 40 yr or per family historya mutations
Colonoscopy based on family history or Limited evidence for association with
similar to first-degree relative risk prostate cancer
DICER1454–459 High Pleuropulmonary blastoma (PPB) No guidelines have been established Other features: Ciliary body
Dominant inheritance Ovarian sex cord-stromal tumors Consideration of annual physical medulloepithelioma; botryoid-type
(Sertoli-Leydig cell tumor, juvenile examination with targeted systems review embryonal rhabdomyosarcoma of the
granulose cell tumor, gynandroblastoma) and possible imaging cervix or other sites; nasal
Cystic nephroma chondromesenchymal hamartoma; renal
Thyroid gland neoplasia (cancer, sarcoma; pituitary blastoma;
multinodular goiter, adenomas) pineoblastoma
Early-onset disease—before age 40 yr
Maternal fetal medicine care when lung
cysts are identified prenatally
Papillary or follicular thyroid cancer
GREM1 (SCG5- Hereditary mixed High Colorectal lesions (polyps of mixed Colonoscopy and surgical management Only a 40-kb duplication upstream of
GREM1)262,460,461 polyposis histologies and cancer) based on polyp burden GREM1 (spanning 3′ end of SCG5 and
Dominant inheritance syndrome region upstream of GREM1) implicated in
disease; identified in Ashkenazi Jewish
population
ETV6/CEBPA/ Familial leukemia/ High ALL, AML, MDS History and physical examination Other syndromes predisposing to
PAX5/RUNX1 MDS Medical history: Prior cytopenias, bleeding leukemia include LFS (TP53), CMMRD
IKAROS/SAMD9/ history, nononcologic manifestations (MLH1, MSH2, MSH6, PMS2), T21, Bloom
SAMD9L462–465 Family history: (BLM), Nijmegen (NBN), ataxia
Dominant Inheritance Review and document types of cancer telangiectasia (ATM), NF1, Noonan:
and leukemia, ages at cancer or leukemia PTPN11, CBL (RAS-activating syndromes),
onset Fanconi anemia (FANCA-E, BRCA, RAD51D
Include history of antecedent cytopenias [autosomal recessive]), telomere
and/or bleeding syndromes (TERT, TERC, DKC), severe
Physical examination: congenital neutropenia (ELANE, HAX1),
Signs of leukemia, lymphoma Diamond-Blackfan syndrome (RPS19,
Other syndrome specific findings, RPLS, RPL11), Shwachman-Diamond
including signs of solid tumors syndrome (SBDS), GATA2, monosomy 7
CBC
Manual differential
Reticulocyte count
Blood smear with morphology
Bone marrow evaluation
Aspirate and biopsy
Morphology
Cytogenetics
Patient and family education about signs
and symptoms of cancer, including
leukemia
HSCT consultation
Consider HLA typing and genetic testing
of potential familial donors
Discuss enrollment in registries or other
research studies

Continued
Genetic Factors: Hereditary Cancer Predisposition Syndromes  •  CHAPTER 13 185
Table 13.2  Cancer Susceptibility Genes, Syndromes, and Management Considerations—cont’d
Genes and Details Syndrome Penetrance Associated Neoplasms Management Considerations Other Notes
Fanconi anemia Fanconi anemia High: Myelodysplastic syndrome Fanconi anemia specialist for Increased chromosome breakage in
(multiple genes—at Biallelic for Acute myeloid leukemia management lymphocytes tested with diepoxybutane
least 20)5,466–472 the recessive Squamous cell carcinoma of head and HSCT and mitomycin C (does not identify
Recessive inheritance genes and neck, esophagus, vulva Early detection and surgical management carriers)
(most genes) monoallelic Genitourinary (cervix, Wilms tumor, of solid tumors: oral/ otolaryngology/ Physical abnormalities (not in all
Dominant inheritance for RAD51, neuroblastoma) ear-nose-throat examination; gynecologic patients): Short stature, abnormal skin
(RAD51 gene) FANCB, BRCA2, Other solid tumors: Liver, brain, skin, examination. pigmentation (café au lait or
X-linked inheritance PALB2, BRCA1 breast, gastrointestinal HPV prevention hypopigmentation or
186 Part I: Science and Clinical Oncology

(FANCB gene) Moderate: Associated cancers for BRCA2, PALB2, Management for BRCA2, PALB2, BRIP1, and hyperpigmentation), skeletal
Monoallelic BRIP1, and RAD51C carriers RAD51C carriers (discussed above and malformations of the upper and lower
for BRIP1, below) limbs (thumbs, radii, hands, ulnae),
RAD51C microcephaly, developmental delay, and
ophthalmic, genitourinary, cardiac,
gastrointestinal, CNS anomalies
Progressive bone marrow failure (not in
all patients): Pancytopenia,
thrombocytopenia, leucopenia
Extreme toxicities from chemotherapy or
radiation
Genes and complement groups:
BRCA2 FA-D1
BRIP1 FA-J
PALB2 FA-N
RAD51 FA-R
RAD51C FA-O
BRCA1 FANCES
XRCC2 FANCU
FH359,374,398,459,473–476 Hereditary High Cutaneous leiomyomata Dermatologic and gynecologic Fumarate hydratase enzyme assay might
Tumor suppressor leiomyomatosis Uterine leiomyomata examination—evaluate for changes be helpful in some situations
Dominant inheritance and renal cell Renal tumors (type 2 papillary, tubule- suggestive of leiomyosarcoma Risk for uterine leiomyosarcoma is
cancer (HLRCC) papillary, collecting duct carcinomas; Medication or resection of leiomyomata unclear
unilateral, solitary lesions) Imaging for and surgical management of Biallelic FH mutations cause a recessive
renal tumors consider starting at age 8 disorder known as fumarate hydratase
deficiency—metabolic disorder, profound
developmental delay, seizures, fumaric
aciduria
FLCN398,477–480 Birt-Hogg-Dube High Cutaneous (fibrofolliculomas, No consensus at this time Fibrofolliculomas are specific to BHD
Tumor suppressor (BHD) syndrome trichodiscomas/ angiofibromas, Dermatologic care Lung cysts are multiple and bilateral
Dominant inheritance perifollicular fibromas, acrochordons) Imaging for and surgical management Spontaneous pneumothorax
Pulmonary cysts (nephron-sparing) of renal tumors Renal tumors are multifocal, bilateral,
Renal tumors (oncocytoma, slow growing
chromophobe, oncocytic hybrid tumors) Other features: Parotid lesions, oral
papules, thyroid lesions
Unclear data regarding colon cancer
Possible genotype-phenotype
correlations
KIT (C-Kit)406,481–484
Oncogene
Dominant inheritance
MEN18,485–488
Tumor suppressor
Dominant inheritance
MET (c-Met)398,489,490
Oncogene
Dominant inheritance
MLH1, MSH2,
MSH6, PMS2,
EPCAM167,173,175,177,180,182,
188,421,491–504
Tumor suppressor
Dominant inheritance
High Gastrointestinal stromal tumors (GISTs) No consensus at this time Diffuse interstitial cell of Cajal
Imaging and endoscopy might be hyperplasia (ICCH)
considered Skin hyperpigmentation or
hypopigmentation
Mast-cell disorders
Related gene: PDGFRA
Multiple High Gastroenteropancreatic (GEP) tract well- Biochemical testing Primary hyperparathyroidism,
endocrine differentiated endocrine tumors Imaging hypercalcemia, oligomenorrhea or
neoplasia type 1 Pituitary tumors (prolactinoma) Surgical management amenorrhea, galactorrhea, Zollinger-
(MEN1) Parathyroid tumors Various medications Ellison syndrome (gastrinoma),
Carcinoid tumors insulinoma, glucagonoma, vasoactive
Adrenocortical tumors intestinal peptide (VIP)–secreting tumor
(VIPomas)
Other features: Skin (angiofibromas,
collagenomas), lipomas, CNS lesions
(meningioma, ependymoma),
leiomyomas
Hereditary High Papillary renal cancer (multifocal, Imaging
papillary renal bilateral, type 1 papillary) Surgical management
carcinoma (HPRC)
Lynch syndrome High Colorectal Colonoscopy every 1–2 yr starting at age Tumor analyses with
(formerly: Endometrial 20–25 yr immunohistochemical (IHC) staining,
hereditary Ovarian (epithelial) Consideration of prophylactic colectomy microsatellite instability (MSI) analysis,
non-polyposis Stomach Hysterectomy and risk-reducing BSO somatic tumor genetic analysis, BRAF
colorectal cancer Small bowel Consideration of upper endoscopy V600E somatic analysis, MLH1 promoter
[HNPCC]) Upper urinary tract (renal pelvis, ureter) Consideration of urinalysis or urine hypermethylation analysis
Variant: Muir-Torre Pancreas cytology Amsterdam Criteria I and II; Revised
syndrome Hepatobiliary tract Consideration of eligibility for treatment Bethesda Guidelines
Variant: Turcot Sebaceous neoplasms (Muir-Torre with cancer immunotherapy with Recent recommendations for universal
syndrome syndrome) checkpoint blockade screening of colorectal and endometrial
Variant: Glioblastoma (Turcot syndrome) Consideration of aspirin for cancers
Constitutional chemoprevention Genotype-phenotype correlations
mismatch repair CMMR-D recessive syndrome emerging but not significant enough to
deficiency MRI of brain every 6 mo starting at alter management
syndrome diagnosis Limited evidence for moderate risk of
[CMMR-D] Whole-body MRI starting at age 6 once a breast and prostate cancer
(biallelic year Autosomal recessive inheritance—
mutations; CBC every 6 mo biallelic (homozygote or compound
recessive Abdominal ultrasound examination every heterozygote) with childhood onset of
inheritance) 6 mo severe CMMR-D syndrome—café au lait
Upper GI endoscopy, VCE, macules, solid tumors, hematologic
ileocolonoscopy—start at 4–6 yr then cancers
every 12 mo
Gynecologic examination, transvaginal
ultrasound, urine cytology, dipstick
starting at age 20 then every 12 mo
Continued
Genetic Factors: Hereditary Cancer Predisposition Syndromes  •  CHAPTER 13 187
Table 13.2  Cancer Susceptibility Genes, Syndromes, and Management Considerations—cont’d
188 Part I: Science and Clinical Oncology

Genes and Details Syndrome Penetrance Associated Neoplasms Management Considerations Other Notes
MUTYH236,247,505–508 MUTYH-associated High Colorectal lesions (adenomatous Colonoscopy and total colectomy based Phenotypically similar to AFAP
Recessive inheritance polyposis (MAP) polyposis and cancer; serrated and on polyp burden Two Northern European founder
hyperplastic polyps also described) Upper endoscopy mutations
Stomach Controversy regarding risk in
Duodenal lesions (adenomatous polyps, heterozygous carriers (see below)
cancer)
MUTYH425,506,509,510 Moderate Colorectal Colonoscopy based on family history or Controversy regarding risk
Monoallelic/ similar to risk with first-degree relative
heterozygous carrier
NBN5,84,421,429,511,512 Nijmegen Moderate Monoallelic carrier: Monoallelic carriers: Breast screening with Heterozygous carrier breast cancer risk;
breakage (monoallelic/ Breast annual mammogram and breast MRI limited evidence for prostate cancer
syndrome (NBS) heterozygous Biallelic carrier: starting age at age 40 yr or as per family Slavic founder mutation
carriers) Lymphoma historya Risk of autosomal recessive condition in
Rare: High Biallelic carriers: NBS specialist for offspring of heterozygous carriers
(biallelic) multidisciplinary management (NBS)
Pediatric autosomal recessive syndrome NBS: Chromosomal breakage,
Hematology-oncology: History and microcephaly, dysmorphic features,
physical examination, annual CBC, immunodeficiency, lymphomas
metabolic profile and lactate
dehydrogenase, avoid excessive radiation,
HPV vaccine per AAP guidelines
Dermatology: Annual skin examination
Pulmonary: Baseline pulmonary function
tests with follow-up as needed, aggressive
treatment of recurrent infections
Gastroenterology and nutrition: Baseline
and as-needed swallowing function
evaluation and nutritional management
Endocrine: Monitor growth, assess female
patients for ovarian failure
Neurology: Developmental assessment
and early intervention if needed
Ophthalmology: Annual examination
Orthopedics: Baseline assessment for
anomalies and as needed
Dental: Biannual examination
NF1482,513–517 Neurofibromatosis High Cutaneous neurofibromas NF1 specialist for management Café au lait macules, axillary and inguinal
Tumor suppressor type 1 (moderate Plexiform neurofibromas Physical examination; ophthalmologic freckling, iris Lisch nodules, learning
Dominant inheritance risk for breast Optic nerve and other CNS gliomas examination; imaging; surgical disabilities, scoliosis, tibial dysplasia,
cancer) Malignant peripheral nerve sheath management vasculopathy
tumors Breast screening with annual
Breast (moderate risk) mammogram and breast MRI as per
Others: GIST, leukemia family historya
Children with NF1 should have
ophthalmic assessments every 6 to 12 mo
from birth to 8 yr; one baseline
assessment of color vision and visual
fields should be undertaken when the
child is developmentally able
Assess with history and clinical
examination annually for typical signs of
MPNST: any nondermal neurofibroma
with rapid growth, loss of neurologic
function, or increasing pain or change in
consistency
Assess for risk of JMML in NF1 in children
with juvenile xanthogranulomas
Baseline whole-body MRI should be
considered between ages 16 and 20 yr to
assess internal tumor burden to
determine adult follow-up regimen if
signs of MPNST are present
MRI with or without FDG-PET is
recommended
NF2/SMARCB1/ Neurofibromatosis High Acoustic neuromas Annual history and physical examination
LZTR1/SMARCE1518 type 2 Schwannomatosis (including audiology with measurement
Dominant inheritance Meningiomas of pure-tone thresholds and Word
Leukemia Recognition Scores)
Atypical rhabdoid tumor Annual (consider twice yearly in first year
after diagnosis or signs of rapid growth)
brain MRI starting at 10 yr of age;
screening may begin earlier in patients
with high-risk genotypes or symptomatic
diagnoses
If baseline imaging shows no
characteristic sites of involvement, reduce
frequency of screening to every 2 yr
Protocols should include high-resolution
(1- to 3-mm section thickness) imaging
through the internal auditory meatus,
preferably in at least two orthogonal
planes
Surveillance spinal MRI is recommended
at 24- to 36-month intervals beginning at
10 yr of age
Whole-body MRI may be performed

Continued
Genetic Factors: Hereditary Cancer Predisposition Syndromes  •  CHAPTER 13 189
Table 13.2  Cancer Susceptibility Genes, Syndromes, and Management Considerations—cont’d
Genes and Details Syndrome Penetrance Associated Neoplasms Management Considerations Other Notes
PALB217,49–51,84,448,519 High Breast Breast screening with annual Insufficient evidence for ovarian cancer
Tumor suppressor Limited evidence for pancreatic cancer mammogram and breast MRI starting at Risk of autosomal recessive Fanconi
190 Part I: Science and Clinical Oncology

Dominant inheritance age 30 yr or per family historya anemia in biallelic offspring of
Optional risk-reducing mastectomy based heterozygous carriers
on family history
Investigational-based pancreas screening
POLD1, Polymerase High Colorectal lesions (polyps and cancer) Colonoscopy and surgical management Disease-causing mutations occur in the
POLE260,261,520–523 proofreading- based on polyp burden exonuclease domain
Dominant inheritance associated Limited data regarding extracolonic
polyposis (PPAP) cancer risk
PRSS1, SPINK1, Hereditary High (PRSS1) Pancreas Pancreatitis management Pancreatitis—recurrent, acute, chronic
CFTR, CTRC261,524–526 pancreatitis Variable throughout lifetime
Various modes of (SPINK1, CFTR, More severe disease if biallelic mutations
inheritance CTRC) are present
Biallelic (homozygote or compound
heterozygote) CFTR mutations associated
with cystic fibrosis
PTCH, SUFU527–530 Gorlin syndrome/ High Jaw (odontogenic) keratocysts Referral to a specialist for management Other features: Congenital skeletal
Tumor suppressor nevoid basal cell Basal cell carcinomas Physical examination anomalies, cleft lip and palate, cerebral
Dominant inheritance carcinoma Medulloblastoma (PNET—desmoplastic) Sun exposure avoidance and at least and falx calcifications, macrocephaly with
syndrome annual dermatology examination frontal bossing, polydactyly, intellectual
Surgical management disability, lymphomesenteric or pleural
cysts, palmar and plantar pits, cardiac
fibromas, ovarian fibromas, ocular
abnormalities
Related gene: SUFU
PTEN158,459,531–540 Cowden High Breast Breast screening with annual Major and minor criteria (tumors,
Tumor suppressor syndrome/PTEN Thyroid (most often follicular) mammogram and breast MRI physical and dermatologic or
Dominant inheritance hamartoma tumor Endometrial Optional risk-reducing mastectomy mucocutaneous findings, developmental
syndrome Colorectal lesions (hamartomas, Thyroid examination and ultrasound disability)
ganglioneuromas, cancer) Endometrial screening with random Macrocephaly
Genitourinary tumors (renal cell biopsy and ultrasound Bannayan-Riley-Ruvalcaba syndrome
carcinoma) Optional hysterectomy PTEN-related Proteus syndrome
Colonoscopy Autism spectrum disorder
Renal imaging Lhermitte-Duclos disease
RAD51C and Moderate Ovarian Consider risk-reducing BSO at age Risk of autosomal recessive Fanconi
RAD51D43,84,114,541,542 Rare: High 50–55 yr or per family historyb anemia in biallelic RAD51C offspring of
Tumor suppressor (biallelic heterozygous carriers
RAD51C)
RB1543–551 Retinoblastoma High Retinoblastoma (trilateral disease = Ophthalmologic examination and Knudson’s two-hit hypothesis
Tumor suppressor (RB) co-occurrence of pineoblastoma) imaging; care by a multidisciplinary team Usually bilateral or multifocal disease
Dominant inheritance Bone or soft tissue sarcomas Examination schedule for carriers: Mosaic forms, therefore analysis of both
Melanoma Birth to 8 weeks: Non-sedated tumor tissue and blood is important
Others examination every 2 weeks RNA studies may also be recommended
8 weeks to 12 mo: EUA monthly Low-penetrance mutations identified
12–24 mo: EUA every 2 mo Parent-of-origin effect in some families
24–36 mo: EUA every 3 mo
36–48 mo: EUA every 4 mo
48–60 mo: EUA every 6 mo
5–7 yr: Examination under sedation every
6 mo
Surveillance for trilateral RB
Brain MRI at the time of RB diagnosis;
some centers recommend brain MRI every
6 mo until 5 yr old
Surveillance for second primary tumors
Education regarding second primary
tumor risks, and close attention to any
new signs or symptoms
Skin examination by a pediatrician during
well-child visits, to be continued annually
by the primary care physician or
dermatologist for melanoma from age 18
Some consider annual WBMR annually
after age 8, but no consensus
Radiation avoidance
RET8,332,334,552,553 Multiple High Medullary thyroid carcinoma (MTC) Prophylactic thyroidectomy (varies from Genotype-phenotype correlations
Oncogene endocrine (multifocal, bilateral) infancy to 5 yr of age or older depending MEN2A: MTC in early-adulthood
Dominant inheritance neoplasia type 2 and on specific RET pathogenic mutation; FMTC: MTC in middle age
Three subtypes: primary C-cell hyperplasia genotype-phenotype correlations) MEN2B: MTC in early-childhood mucosal
• MEN2A (Sipple PCC (bilateral) (MEN2A and MEN2B) Surgical management of identified neuromas (lips, tongue), dysmorphic
syndrome) Parathyroid adenoma or hyperplasia disease features (large lips, “marfanoid” body
• Familial (hyperparathyroidism, hypercalcemia, Serum calcitonin concentration habitus), gastrointestinal
medullary renal stones) (MEN2A) Consideration of eligibility for treatment ganglioneuromatosis
thyroid with kinase inhibitors RET gene also associated with
carcinoma Biochemical screening and imaging for Hirschsprung disease
(FMTC) pheochromocytoma and parathyroid
• MEN2B abnormalities

Continued
Genetic Factors: Hereditary Cancer Predisposition Syndromes  •  CHAPTER 13 191
Table 13.2  Cancer Susceptibility Genes, Syndromes, and Management Considerations—cont’d
Genes and Details Syndrome Penetrance Associated Neoplasms Management Considerations Other Notes
SDHB, SDHC, Hereditary High Paraganglioma (PGL)—sympathetic and Biochemical screening, physical Secretory and nonsecretory PGL
SDHD, SDHA, paraganglioma- (particularly parasympathetic examination, and imaging High blood pressure, headache, anxiety,
SDHAF2313,482,554–558 PCC syndrome SDHB and PCC Blood pressure at all medical visits profuse sweating, palpitations, pallor
Tumor suppressors SDHD) GISTs starting at age 6–8 SDHB: High risk of malignant
192 Part I: Science and Clinical Oncology

Dominant inheritance Renal clear cell carcinoma Plasma methoxytyramine starting at age transformation
Papillary thyroid carcinoma (unclear data) 6–8 yr then annually SDHD: Parent-of-origin effect—disease
Plasma free or 24-hour fractionated causing when paternally inherited
metanephrines starting at age 6–8 yr then SDHAF2: Possible parent-of-origin effect
annually (paternal inheritance)
Optional serum chromogranin starting at
6–8 yr then annually
Whole-body MRI skull base to pelvis at
6–8 yr then biennially
Optional: Neck MRI with or without
contrast agent starting at age 6–8 then
biennially
CBC with RBC indices start at age 6-8 then
annual
SMARCB1, SMARCA47 Rhabdoid tumor High Rhabdoid tumor Brain MRI every 3 mo to age 5 Ovarian and fallopian tumor removal to
Epigenetic regulator predisposition Small cell carcinoma of the ovary Abdomen: Consider whole-body MRI to be considered in carriers before puberty
Dominant inheritance syndrome hypercalcemic type (SCCOHT) age 5, ultrasound every 3 mo
Schwannomatosis
STK11 (LKB1)438,559–564 Peutz-Jeghers High Gastrointestinal lesions (PJS-type Colonoscopy Mucocutaneous hyperpigmentation of
Tumor suppressor syndrome (PJS) hamartomatous polyps; colorectal, Upper endoscopy with small bowel mouth, lips, nose, eyes, genitalia, fingers;
Dominant inheritance stomach, small bowel cancer) visualization may fade after puberty
Breast Breast screening with annual Females: Sex cord tumors with annular
Pancreas mammogram and breast MRI tubules (SCTAT); adenoma malignum of
Gynecologic (ovarian, cervix, uterus) Investigational-based pancreatic the cervix
Testes (Sertoli cell) screening
Lung Gynecologic examination
Testicular examination
TP53565–573 Li-Fraumeni High Soft tissue and bone sarcomas Breast screening with annual breast MRI Classic LFS criteria
Tumor suppressor syndrome (LFS) Breast and mammogram beginning in the 20s Chompret criteria
Dominant inheritance Brain and 30s, respectively Consider genetic testing in BRCA-
Adrenocortical carcinoma Optional risk-reducing mastectomy negative isolated early-onset breast
Leukemia Physical examination including cancer (age <30)
Others: Gastrointestinal, genitourinary, dermatologic and neurologic Breast cancers more likely to be estrogen,
lung, lymphomas, thyroid, neuroblastoma, examinations progesterone, HER2/neu receptor
skin Colonoscopy positive
Investigational-based whole-body MRI
Consideration of radiation avoidance, if
clinically appropriate
 TSC1 and TSC2574–583 Tuberous sclerosis High Kidney: TSC specialist for management Skin (hypomelanotic macules, facial
Tumor suppressor complex (TSC) Renal cell carcinomas, angiomyolipomas Imaging angiofibromas, shagreen patches,
Dominant inheritance (benign and malignant), Dermatologic, dental, ophthalmologic cephalic plaques, ungual fibromas)
Epithelial cysts, examinations; EEG, echocardiogram, ECG Brain (cortical dysplasias, subependymal
oncocytoma (benign adenomatous Consideration of eligibility for treatment nodules and subependymal giant cell
hamartoma) with mTOR inhibitors astrocytomas, seizures, intellectual
Surgical management disability or developmental delay, autism
spectrum disorder, psychiatric illness)
Heart (rhabdomyomas, arrhythmias)
Lungs (lymphangioleiomyomatosis)
Genotype-phenotype correlations:
Higher risk of renal cell carcinoma in
TSC2
Possible association with NETs
VHL379,380,398,558,584–589 von Hippel-Lindau High Clear cell renal cell carcinoma Eye examination including retina starting Hemangioblastomas of the brain, spinal
Tumor suppressor disease PCC, paragangliomas at birth then annually cord, and retina; renal cysts; pancreatic
Dominant inheritance Pancreatic NETs Blood pressure at all medical visits cysts; endolymphatic sac tumors;
starting at age 2 epididymal and broad ligament cysts
Plasma free metanephrines or 24-hour Genotype-phenotype correlations: VHL
urine fractionated metanephrines starting type 1, type 2A, type 2B, type 2C with
at age 2 then annually different risks of PCC or renal cell
Audiogram starting at age 5 then carcinoma
biennially
MRI of brain with and without contrast
agent and MRI of spine with contrast
starting at age 8 then biennially
MRI of abdomen starting at age 10 then
annually (for RCC and pancreatic NET
screen)

Leukemia and lymphoma predisposition syndromes are not listed. Please refer to dedicated chapters.
The majority of childhood onset conditions are not listed. Please refer to Lindor NM, McMaster ML, Lindor CJ. Concise handbook of familial cancer susceptibility syndromes, 2nd ed. J Natl Cancer Inst Monogr. 2008;(38):1-93.
PMID:18559331
a
Earlier initiation of breast cancer surveillance may be warranted in the presence of a significant family history of breast cancer.
b
Earlier consideration of risk-reducing salpingo-oophorectomy may be warranted in the presence of a clear family history of ovarian cancer (>1 case).
AAP, American Academy of Pediatrics; ALL, acute lymphoblastic leukemia; AML, acute myeloblastic leukemia; BSO, bilateral salpingo-oophorectomy; CBC, complete blood count; CNS, central nervous system; ECG,
electroencephalogram; EEG, electroencephalogram; EUA, examination under anesthesia; FDG-PET, fluorodeoxyglucose–positron emission tomography; GI, gastrointestinal; GIST, gastrointestinal stromal tumor; HPV, human
papillomavirus; HSCT, hematopoietic stem cell transplantation; JMML, juvenile myelo monocytic leukemia; MDS, myelodysplasia; MPNST, malignant peripheral nerve sheath tumors; MRI, magnetic resonance imaging; NET,
neuroendocrine tumor; PARP, poly (ADP-ribose) polymerase; PCC, pheochromocytoma; PSA, prostate-specific antigen; RCC, renal cell carcinoma; TSH, thyroid-stimulating hormone; VCE, video capsule endoscopy; WBMR, whole
body MRI.
Genetic Factors: Hereditary Cancer Predisposition Syndromes  •  CHAPTER 13 193
194 Part I: Science and Clinical Oncology
Box 13.1.  ELEMENTS OF INFORMED CONSENT
FOR GENETIC CANCER TESTING
1. What the test is intended to do—that is, determine whether a
mutation can be detected in a specific cancer susceptibility gene
2. What can be learned from both a positive and negative test result,
including information on the magnitude of health risks associated
with a positive test result, as well as the risks that may remain even
after a negative test result
3. The possibility that no additional risk information will be obtained
after testing or that the test will result in a finding of unknown
significance (e.g., a polymorphism) that might require further
studies
4. The options for approximation of risk without genetic testing—for
example, using empiric risk tables for breast cancer given differing
family histories
5. The risk of passing a mutation on to children, including options for
assisted reproduction (e.g., preimplantation genetics, if discussion
is appropriate)
6. The importance of notification of family members that they might
share a hereditary risk for cancer, with every effort made to assist in
contacting family members and providing them access to
counseling and testing
7. The medical options and limited proof of efficacy for surveillance and
cancer prevention for individuals with a positive test result, as well as
the accepted recommendations for cancer screening even if
genetic testing results are negative
8. The technical accuracy of the test—that is, the sensitivity and
specificity of the analytic methodology
9. The risks of psychologic distress and family disruption whether a
mutation is found or not found
10. The risk of employment and/or insurance discrimination after
disclosure of genetic test results and the level of confidentiality of
results compared with other medical tests and procedures
11. The risks that nonrelatedness of family members will be discovered
and how this information will be disclosed (or not disclosed)
12. The fees and costs of testing, including the laboratory test and the
associated consultation with the health professional who is
providing pretest education, results disclosure, and follow-up, and
the costs of preventive procedures, which might not be covered by
third-party payers
Modified from Offit K. Clinical Cancer Genetics: Risk Counseling and Management.
New York: Wiley-Liss; 1998, with permission.
result in an increased risk for a second neoplasm for millions of cancer
survivors.
Inherited cancer predisposition can be considered as a spectrum,
arising from single or combined low-, moderate-, and high-risk genetic
variants for which the timing of disease onset is likely modified by
the type of genetic variant and its effect on normal cellular function
and the responses to environmental factors. In general, highly penetrant
hereditary cancer syndromes account for about 5% to 10% of most
types of cancer and are caused by rare genetic variants. We are now
also aware of dozens of “moderate-penetrance” genes that in general
confer a modest degree of cancer risk, with a relative risk (RR) ranging
between 2 and 5 (Table 13.3).17 Although screening for such moderate-
penetrance genes was previously not routinely undertaken, with the
availability of next-generation sequencing, screening for mutations in
many genes simultaneously, often in multigene panels, has become
readily available. The value of screening for moderate-penetrance genes
remains controversial because neither the clinical validity, the accuracy
with which a genetic test predicts the development of cancer, nor the
clinical utility, the degree to which the use of the genetic test informs
clinical decision making and leads to improved health outcomes, has
been clearly proven.18 In addition to the high- and moderate-penetrance
cancer genes, for nearly all the common malignancies, hundreds of
additional genetic loci have been identified, largely via genome-wide
association studies, with each genetic variant, usually single-nucleotide
polymorphisms (SNPs), being associated only a modest increased risk
of cancer (RR, ~1.1–1.5).19 Given the limited clinical validity and
utility of SNPs with such small effect sizes, clinical testing for individual
risk loci is currently performed, although research efforts to devise
polygenic risk models are underway20 and have already been incor-
porated into models of risk stratification for BRCA mutation–carrying
males and females.21,22 In addition, these frequent but low-penetrance
variants identified through genome-wide association studies23 are
thought to explain a portion of the excess familiality of cancer. To
account for the missing familiarity that remains, sequencing of protein
coding regions of the genome or the entire genome itself has led to
the discovery of novel cancer susceptibility genes.2,3 Together, the
knowledge of rare and common variations has the potential to inform
the clinician about the combined effect of genetic factors that lead
to cancer, extending beyond risks conferred by single genes. Ultimately,
to assess a person’s risk of cancer, one will need to combine genetic
factors within the context of environmental factors, which also may
include exposures to cancer therapies.
COMMON SYNDROMES OF CANCER
PREDISPOSITION
The major syndromes of cancer predisposition that affect adults include
breast, ovarian, colon, prostate, gastric, and pancreatic cancer, as well
as a number of other less common but equally important cancer
predispositions. Some of these syndromes, including multiple endocrine
neoplasia type 1 (MEN1), retinoblastoma, melanoma, von Hippel-
Lindau (VHL) disease, and familial pheochromocytoma and para-
ganglioma, are described elsewhere in this text. Comprehensive reviews
of these cancer predispositions are offered elsewhere, including Li-
Fraumeni syndrome,24 MEN1,25,26 retinoblastoma,27 melanoma,28,29
and VHL disease.30,31 Some syndromes such as neurofibromatosis also
have cancer predispositions.32–34 A detailed outline of the elements of
setting up a comprehensive cancer genetic and genomics counseling
service has also been published.1,35
Breast and Ovarian Cancer Syndromes
Clinical Features
Although only about 18,000 cases of breast cancer each year are
associated with an obvious hereditary predisposition, primary cancers
developed in more than 200,000 breast cancer survivors in the United
States as a result of a hereditary predisposition, and these survivors
remain at risk for secondary cancers.36,37 Genetic testing has emerged
as one of the most important indicators of risk factors, pointing to a
need for intensified screening for breast cancer.38 When detected at
an early stage, more than 90% of breast cancers are curable. These
statistics underscore the rationale for the use of genetics in clinical
oncology. We have previously reviewed in detail the management of
women and men who are at hereditary risk for breast cancer,39 and
this review is summarized and updated here.
From 1 in 150 to 1 in 800 individuals in the population carry a
genetic susceptibility to breast cancer,40–42 and the prevalence is much
higher in certain ethnic groups. Syndromes of breast cancer susceptibility
are linked to mutations of BRCA1 and BRCA2, in addition to a
smaller number of cases with germline mutations of ATM, PALB2,
PTEN, p53, CHEK2, STK11, CDH1, and rarer syndromes (Tables
13.4 and 13.5).43 Cowden syndrome (CS) was initially described as a
dominant inheritance of multiple hamartomatous lesions, including
papillomas of the lips and mucous membranes and acral keratoses of
the skin.44 This disease was ultimately linked to germline mutations of
PTEN and is discussed separately later. In persons with Li-Fraumeni
 Table 13.3  Selected Moderate-Penetrance Genes and Associated Malignancies
Breast Ovary Colorectal Pancreas Melanoma Prostate Renal Other
APC*I1307K X
RR, 2.17
(95% CI, 1.6–2.9)a
ATM X X Autosomal recessive
RR, 2.8 (90% CI, 2.2–3.7) Ataxia telangiectasia
BARD1 (ID) (ID)
BRIP1 X Autosomal recessive
RR 11.2 (95% CI, 3.2–34.1) Fanconi anemia
in case control;
RR, 3.41 (95% CI, 2.1–5.5) in
segregation analysis
CHEK2 X (ID) X (X)
Truncating: 1100delC:
RR, 3.0 (90% CI 2.6–3.5); RR, 1.88 (95% CI, 1.3–2.7);
Missense (I157T): I157T:
RR, 1.58 (95% CI, 1.4–1.8) RR, 1.56 (95% CI, 1.3–1.8)
MAX X (Pheochromocytoma,
paraganglioma)
MITF X (X)
MRE11A (ID) (ID)
MUTYH X
(monoallelic) RR, 1.17 (95% CI, 1.0–1.3)
NBN (X) Autosomal recessive Nijmegen
c.657del5: breakage syndrome
RR, 2.7 (90% CI 1.9–3.7)
NF1 X High penetrance
Neurofibromatosis type 1
RAD50 (ID) (ID)
RAD51C, X RAD51C:
RAD51D RAD51C: RR, 5.2 (95% CI, Autosomal recessive Fanconi
1.1–24); RAD51D: RR, 12 Anemia
(95% CI, 1.5–90)
TMEM127 X (Pheochromocytoma)

Risk estimates listed assume no family history of the implicated cancer.

a
Risk estimate for APC*I1307K is based on Ashkenazi Jewish population.
(X): To date, limited evidence to support the association and the strength of the association is unclear.
(ID): Insufficient data. Occurrence documented, but association not clearly supported by evidence.
CI, Confidence interval; RR, average relative risk.
Data from Tung N, Domchek SM, Stadler Z, et al. Counselling framework for moderate-penetrance cancer-susceptibility mutations. Nat Rev Clin Oncol. 2016;13(9):581-8; Ma X, Zhang B, Zheng W. Genetic variants associated
with colorectal cancer risk: comprehensive research synopsis, meta-analysis, and epidemiological evidence. Gut. 2014;63(2):326-36; and Liang J, Lin C, HuF, et al. APC polymorphisms and the risk of colorectal neoplasia: a HuGE
review and meta-analysis. Am J Epidemiol. 2013;177(11):1169-79.
Genetic Factors: Hereditary Cancer Predisposition Syndromes  •  CHAPTER 13 195
196 Part I: Science and Clinical Oncology

Table 13.4  Genes Associated With Hereditary Breast Cancer (BC) Predisposition
Relative Risk BC Risk by
Gene Syndrome of BC Age 80 Yr Associated Cancers
HIGH PENETRANCE
BRCA136,87,590–593 HBOC ~15–30 70% Ovarian, other36,75,444
BRCA236,87,591–593 HBOC ~10–20 70%36,75,444 Ovarian, pancreatic, prostate, other
p53594–596 Li-Fraumeni syndrome 10017 50% by 60 yr597,598 Soft tissue sarcoma, osteosarcoma,
brain tumors, adrenocortical
carcinoma, leukemia, other
PTEN599–601 Cowden syndrome No reliable estimate17 70%–80%158,602 Thyroid (follicular and rarely
papillary) endometrial,
genitourinary, other
Bannayan-Riley-Ruvalcaba
syndrome
Proteus
Proteus-like syndrome
STK1147,289 Peutz-Jeghers syndrome No reliable estimate17 30% by age 6047 Small intestine, colorectal, uterine,
testicular and ovarian sex chord
tumors, other
CDH137,270,603 Hereditary diffuse gastric ~3.25 39% Lobular breast, diffuse gastric,
carcinoma other
LOWER OR MODERATE PENETRANCE
ATM (heterozygote)604–606 Ataxia-telangiectasia in ~3 3084 Undefined in heterozygotes
homozygotes
CHK2 (CHEK2)63,64,607,608 Li-Fraumeni variant 1.5-3 20%–30%84 Undefined

PALB2609 None known 5 ~4049,84 Undefined in heterozygotes

HBOC, Hereditary breast and ovarian cancer syndrome.

Table 13.5  Genes Associated With Hereditary Ovarian Cancer (OC) Predisposition
Relative Risk OC Risk by
Gene Syndrome of OC Age 80 Yr Associated Cancers
HIGH PENETRANCE
BRCA1 Hereditary breast ovarian cancer syndrome ~50 ~40%36,75,444 Breast, other
BRCA2 Hereditary breast ovarian cancer syndrome ~8 11%– Breast, pancreas, prostate, other
26%36,75,444
MLH1 Lynch syndrome ~4 ~20%83,610,611 Colon
MSH2 Uterine
MSH6 Stomach
PMS2 Small intestine
EPCAM Urinary tract
Pancreatic
Possible other sites
LOWER OR MODERATE PENETRANCE
RAD51C None ~5 ~6% Undefined84
Autosomal recessive Fanconi anemia
RAD51D None ~12 ~14% Undefined84
Autosomal recessive Fanconi anemia

syndrome, early-onset breast cancer occurs with soft tissue sarcomas,
osteosarcoma, leukemia (particularly hypodiploid acute lymphoblastic
leukemia), brain tumors, adrenal cortical tumors, and other cancers.
Rarely, atypical breast-ovarian kindred may be found to have a germline
p53 mutation.45 Although rare, women with Peutz-Jeghers syndrome,
associated with germline mutations in the STK11 gene, are at increased
risk for breast cancer as well as a variety of other malignancies.46,47 More
recently, mutations in PALB2, partner and localizer of BRCA2, have
been implicated in both familial breast and pancreas cancer risk.48–51
Early linkage studies suggested that in about 50% of breast cancer
kindreds, the cancer was linked to BRCA1; in 30%, it was linked to
BRCA2; and in the remainder, it was linked to other either identified
or unidentified genes.52 In up to two-thirds of families with both male
and female breast cancer, the cancers were due to BRCA2, whereas
 Genetic Factors: Hereditary Cancer Predisposition Syndromes  •  CHAPTER 13 197
more than 80% of families with both breast and ovarian cancer harbored
mutations in BRCA1.53 BRCA1-linked breast cancers are associated
with a basal-like subtype and higher mitotic indices.54 They are more
likely to be of higher grade, are more frequently estrogen and pro-
gesterone receptor negative,55,56 and demonstrate a basal-like pheno-
type.57 They display a more “aggressive” phenotype, including a higher
proportion of cells in S phase, and other indices.54,56,58–60
Multiple moderate-penetrance breast cancer susceptibility genes
have also been identified (see Table 13.4).17,62–66 In Northern European
families, specific mutations of CHEK2 are associated with familial
breast cancer, with pathogenic CHEK2 variants being present in ~1%
of Caucasians of European descent.64 Although the common European
mutation in CHEK2 is rare in persons in North America,67 analysis
of women for CHEK2 founder mutations stratified for family history
of breast cancer demonstrated that carriers with a positive family
history had a greater than 25% lifetime risk for breast cancer.68
Important to note, degree of breast cancer risk in women with mutations
in CHEK2 appears to be dependent on both the nature of the specific
mutation (truncating versus missense) and the strength of the family
breast cancer history. Carriers of mutations in ATM, another moderate-
penetrance breast cancer susceptibility gene, have an approximately
twofold elevated breast cancer risk.62,69,70
Multiple, common, lower-penetrance genes are likely to account
for a significant component of currently unexplained familial breast
cancer risk.71 A host of putative lower-penetrance gene mutations
have been identified through whole-genome association studies,72,73
although the clinical relevance of these associations remains unclear.
In a large cohort of breast cancer patients referred for genetic testing,
approximately 10% had a related germline mutation.74
Compared with the 10% breast cancer risk for women in the
general population, estimates of the breast cancer risk that is conferred
by a common high-penetrance susceptibility gene ranged from 67%
to 69% by age 70 years based on epidemiologic analyses.40,41 This was
confirmed in a prospective study that defined breast cancer risk by
age 80 of 72% and 69% for BRCA1 and BRCA2 carriers, respectively.75
This study also reported a contralateral breast cancer risk, up to 20
years after diagnosis, of 40% and 26% for BRCA1 and BRCA2,
respectively.75 In the setting of a mutation in PALB2, the breast cancer
risk to age 80 is approximately 45%, whereas for those with mutations
in ATM and CHEK2 it is less than 30%.17
Epithelial ovarian carcinoma can be delineated into six distinct
histologic subtypes—high-grade serous, carcinosarcoma, clear cell,
endometrioid (International Federation of Gynecology and Obstetrics
[FIGO] grades 1–3), mucinous, and low-grade serous—with differing
manifestations, prognoses, and molecular and immunohistochemistry
profiles.76–78 The high-grade serous subtype comprises the majority of
cases of ovarian cancer.77 Mutations in BRCA1 and BRCA2 show a
strong association with the high-grade serous carcinoma of ovary and
fallopian tube or peritoneal origin, such that germline testing for
BRCA1 and BRCA2 together can have up to a 25% detection rate in
this histologic subtype.79–82 Germline testing for BRCA1 and BRCA2
is standard of care for all patients with a high-grade serous ovarian
cancer diagnosis. Mutations in the mismatch repair (MMR) genes
are also associated with ovarian cancer risk,83 usually endometrioid
histology; this is further discussed in the later section on Lynch
syndrome (LS). Similar to breast cancer, a number of more moderate-
penetrance ovarian cancer susceptibility genes have also been identified.
BRIP1, RAD51C, and RAD51D are associated with approximately a
5-fold to 12-fold risk of ovarian cancer84 (see Table 13.5). Earlier data
suggested an association with BRIP1 and breast cancer, but a large
population-based study demonstrated that truncating mutations in
this gene were not associated with a substantial increase in breast
cancer risk.85
In a study of close to 2000 women with ovarian cancer, 18% of
patients had a germline mutation in a gene associated with ovarian
cancer risk.86 The majority of patients in this study had high-grade
serous histology (78%), but all histologic subtypes were represented.
As expected, mutations in BRCA1 and BRCA2 accounted for the bulk
of inherited risk, with mutations in BRIP1, RAD51C, RAD51D, and
the MMR genes accounting for 3% cumulatively. Compared with a
population risk for ovarian cancer of approximately 1.5%, mutations
in BRCA1 and BRCA2 are associated with a risk of 44% and 17%
by age 80, respectively.75 For BRIP1, RAD51C, and RAD51D, the
cumulative lifetime risk by age 80 is 4% to 14%.84
The role of ascertainment and other possible biases in deriving
cancer risk estimates, as well as risks for cancer of the prostate, colon,
pancreas, and other sites in BRCA mutation carriers has been reviewed.87
In addition to Fanconi anemia, persons with compound BRCA2
mutations may experience childhood medulloblastomas.88 Heterozygous
mutations in BRCA1 and BRCA2 harbored by pediatric cancer patients
have been detected through tumor normal sequencing; however, it is
not clear at this point if these are cancers associated with the familial
syndrome or simply true and unrelated, and this is an active area of
research.89–91
Genetics
BRCA1 and BRCA2 are both important for DNA repair, in particular
homologous recombination, which enables repair of double-stranded
DNA breaks. BRCA1 is a large gene, spanning more than 100,000
bases of genomic DNA with 22 coding and 2 noncoding exons.
BRCA2 is also large, consisting of 27 exons across 70 kb of genomic
DNA. Both genes, by coincidence, have a large exon 11. An update
of reported mutations is accessible through the Internet at http://
research.nhgri.nih.gov/bic/.
Most BRCA1 mutations cause premature truncation of the peptide
by frameshift or nonsense sequence changes. Large germline rear-
rangements also occur in BRCA1 and BRCA2.92,93 Five percent to
10% of BRCA mutations that are missense are problematic because
they are of unknown clinical significance. The proportion of these
variants that are of unknown significance was as high as 10% to 23%
in some series, posing counseling challenges.94,95 The role of BRCA1
and BRCA2 in DNA damage response and homologous recombination
is reviewed elsewhere.96
Founder BRCA mutations have been documented in genetically
isolated populations. In North American families, the most common
founder mutations occur in persons of Ashkenazi Jewish origin. These
mutations include a two-base-pair deletion in codon 23 of BRCA1,
termed 185delAG (c.68_69delAG); another mutation in BRCA1,
5382insC (c.5266dupC); and the 6174delT (c.5946delT) mutation
in BRCA2.96–99 About 1 in 40 Ashkenazi Jews harbor one of the
common BRCA1 or BRCA2 mutations,98,100,101 a relatively high carrier
frequency for an inherited cancer predisposition syndrome. Other
mutations in BRCA1 and BRCA2 occur in the Ashkenazim; 16 of
737 Ashkenazi Jews who were tested in a clinic-based ascertainment
had a nonfounder mutation (2%).53 In another study of Ashkenazi
individuals with a personal history of breast or ovarian cancer who
had previously been shown not to have a founder mutation, 3 of 70
(4.3%) had a deleterious nonfounder mutation.102 This very low
nonfounder BRCA rate in Ashkenazim was also confirmed again in
a 2017 study.103 Founder mutations in populations other than the
Ashkenazim have also been observed.104–106
Other Genes
Protein truncating variants in PALB2, CHEK2, and ATM have been
associated with increased breast cancer risk as outlined earlier. The risk
associated varies per study and variant. A Finnish founder mutation in
PALB2 c.1592delT is associated with higher than population risk of
breast cancer,107 but risk estimates are lower than those reported with
other PALB2 mutations. Most of the data for CHEK2 and increased
breast cancer risk relates to the truncating c.1100delC variant found
in Northern Europeans.63,64,108 The missense variant CHEK2 I157T is
associated with a very modest increased risk (RR, 1.58; 95% confidence
interval [CI], 1.42–1.75),109 with some laboratories not reporting
this variant as being pathogenic. The autosomal recessive neurologic
198 Part I: Science and Clinical Oncology
disorder ataxia telangiectasia is associated with compound heterozygote
mutations in ATM, a gene involved with DNA double-strand break
repair and cell cycle control. Although this is a rare event, monoallelic
ATM mutations are relatively common, occurring in approximately
3% of Caucasians. Women with heterozygous ATM mutations have
a moderate, approximately double population risk of breast cancer as
noted earlier.62,110,111 BRIP1, BRCA1 interacting protein C-terminal
helicase 1 gene, is part of the DNA repair machinery. A founder
mutation, BRIP1, c.2040_2041insTT was associated with increased
risk of ovarian cancer in the Icelandic population.112 Subsequently,
case-control series reported increased ovarian cancer risk for other
truncating BRIP1 mutations.113 RAD51C and RAD51D are part of
the Fanconi-BRCA homologous recombination repair pathway and
have been associated with a modest increased risk of ovarian cancer.114
Clinical Management
Three elements of breast surveillance that are recommended to women
with increased risk of breast cancer are self-examination, clinician
examination, and imaging. The evidence base underlying these recom-
mendations has been reviewed,39,115 and updated guidelines are
available.116
Increasingly, breast cancer screening including mammography and
magnetic resonance imaging (MRI) together has been shown to have
greatest sensitivity.117 For women who are at the highest hereditary
risk for breast cancer, whose breasts are difficult to examine, or who
have had biopsy results showing atypia, it might be appropriate to
discuss the option of removing the healthy breasts as a preventive
measure (i.e., prophylactic mastectomy). Retrospective studies and
reviews document the well-established risk-reducing role of surgery
in high-risk patients.118–120 Prospective and combined consortium
studies have also shown the efficacy of this approach.64,121,122
Because of their antiestrogen properties, tamoxifen, raloxifene, and
aromatase inhibitors have been shown to decrease breast cancer rates
in persons who are at increased risk.123–125 Two studies on the impact
of tamoxifen on subsequent breast cancer risk in BRCA1 and BRCA2
mutation carriers have shown conflicting conclusions,126,127 although
only one of these studies was sufficiently powered to reach significant
results. Tamoxifen was confirmed to decrease contralateral breast cancer
risk in a follow-up of one of these studies of BRCA mutation carriers.128
Providers are encouraged to discuss breast cancer chemoprevention
with women who are at increased risk of breast cancer.129 There are
no studies demonstrating a mortality benefit for this approach, and
a careful discussion of potential benefit versus toxicity is required.
Small trials have demonstrated the ability of ultrasound with Doppler
and CA125 to reveal early-stage ovarian cancers in BRCA mutation
carriers.130,131 However, the efficacy of this approach has not been proven
in large, prospective studies,132 and the US Food and Drug Administra-
tion (FDA) has recommended against screening for ovarian cancer
in all women, including those at high risk.133 Therefore prophylactic
removal of the ovaries and fallopian tubes is recommended to women
with strong family histories of ovarian cancer, in families linked to
BRCA1 or BRCA2, or to women who are considering hysterectomy
in the setting of a germline mutation associated with LS. Studies
examining specimens from prophylactic salpingo-oophorectomy in
BRCA1 and BRCA2 mutation carriers have implicated the fimbriated
part of the fallopian tube as harboring precursor lesions to high-grade
serous epithelial ovarian cancer.84,134–136 Thus prophylactic salpingo-
oophorectomy is recommended because such surgeries not only decrease
the incidence of subsequent breast and ovarian cancer but also reveal
occult early-stage pelvic neoplasms,136–138 decreasing mortality.139 These
studies have also confirmed that high-grade serous carcinoma can still
occur after prophylactic oophorectomy,140,141 albeit at much reduced
rates. For women with high-penetrance risk genes, risk-reducing
prophylactic oophorectomy is recommended when childbearing is
complete or at approximately age 40.11 Given the lack of an adequate
screening modality, difficulty in diagnosis, and resultant late stage
at presentation and the associated mortality with invasive ovarian
cancer, risk-reducing surgery is also recommended for carriers of the
more modest-risk mutations in genes BRIP1, RAD51C, and RAD51D.
However, given the later age of onset of ovarian cancer compared
with BRCA1 and BRCA2, risk-reducing surgery may be delayed until
patients in this group are perimenopausal or postmenopausal.
Combination oral contraceptives that contain estrogen and high-dose
progestin result in a time-dependent, protective effect against ovarian
cancer in some but not all studies of BRCA mutation carriers.142–144
There remains the concern of a small increased risk of breast cancer
due to oral contraceptives in this group, particularly in persons with
BRCA1 mutations.145
Germline BRCA1 and BRCA2 mutation–associated tumors have
defective DNA repair mechanisms because of the loss of function of
the remaining normal copy of the gene. This inability of the tumor
to repair its DNA effectively can be exploited by therapies that disrupt
alternative DNA repair pathways, such that the tumor cell accumulates
so much DNA damage that it can no longer survive.146,147 Inhibitors
of the base excision repair enzyme poly (ADP-ribose) polymerase
(PARP inhibitors) use this principle of synthetic lethality and are
FDA approved for women with recurrent ovarian cancer in a number
of clinical settings.148–150 Although benefit has been demonstrated in
all patients with ovarian cancer, it is substantially greater in patients
with a germline or somatic BRCA1/2 mutation.148,149 A recent random-
ized trial has also demonstrated that patients with metastatic breast
cancer and germline BRCA1/2 mutations had significant progression-
free survival benefit with olaparib.151
Cowden Syndrome
Clinical Features
CS is an autosomal dominant disorder characterized by multiple
hamartomas with a high risk of benign and malignant tumors of the
thyroid, breast, and endometrium. Consensus criteria for CS establish
three diagnostic categories.152
Pathognomonic criteria include adult Lhermitte-Duclos disease
(LDD) (defined as presence of a cerebellar dysplastic gangliocytoma)153
and mucocutaneous lesions, facial trichilemmomas, acral keratoses,
papillomatous lesions, and mucosal lesions. Major criteria include breast
cancer, thyroid cancer (especially follicular histology), macrocephaly,154
and endometrial carcinoma. Minor criteria include other thyroid
lesions (e.g., goiter), mental retardation, hamartomatous intestinal
polyps, fibrocystic breast disease, lipomas, fibromas, and genitourinary
tumors (e.g., uterine fibroids and renal cell carcinoma) or genitourinary
malformation. The operational diagnosis of CS is made if an individual
meets any one of the following criteria: pathognomonic mucocutaneous
lesions alone if there are six or more facial papules, of which three
or more must be trichilemmoma, or cutaneous facial papules and
oral mucosal papillomatosis; or oral mucosal papillomatosis and acral
keratoses; or six or more palmoplantar keratoses. Alternatively, the
individual may fulfill two major criteria, but one must include either
macrocephaly or LDD. Alternatively, the individual may have one
major and three minor criteria or four minor criteria.
The palmar and plantar hyperkeratotic pits usually become evident
later in childhood. Subcutaneous lipomas and cutaneous hemangiomas
are seen in persons with CS with low frequency.155 An increased
risk of early-onset male breast cancer has been noted in mutation
carriers.156
Cumulative lifetime risks for female breast cancer are 81% to
85.2%157,158; for LDD, 32% (CI, 19%–49%)157; for thyroid cancer,
21%–35.2%,157,158; for endometrial cancer, 19%–28.2%157,158; and
for renal cancer, 15%–33.6% (CI, 6%–32%).157,158 The lifetime risks
for colorectal cancer (CRC) are 9% to 16%,157,158 and the risk for
melanoma is 6%.158
Genetics
The CS-linked PTEN was mapped to 10q22-23.159 Bannayan-Riley-
Ruvalcaba syndrome, characterized by macrocephaly, intestinal polyps,
 Genetic Factors: Hereditary Cancer Predisposition Syndromes  •  CHAPTER 13 199
lipomas, and pigmented penile macules, is also caused by germline
mutations in PTEN.160
PTEN acts as a tumor suppressor by mediating cell cycle arrest,
apoptosis, or both.161 Full sequencing and deletion/duplication analysis
are available clinically, and promoter analysis is available on a research
basis. Heterozygous germline mutations in PTEN cause most cases
of CS. Nonsense, missense, and frameshift mutations that are predicted
to disrupt normal PTEN function have been identified in some
families,159 including mutations that disrupt the protein tyrosine/
dual-specificity phosphatase domain. Initially it was shown that PTEN
mutations can be detected in about 80% of patients with CS.160 More
recently, the mutation detection rate in persons meeting CS criteria
was found to be 34%, raising the possibility that current aspects of
testing criteria are too broad.162
Risk Management Recommendations
Persons with known germline PTEN mutations should undergo
appropriate cancer screening.116,163 In view of recent work outlining
cancer risks in persons with CS, some investigators have suggested
that National Comprehensive Cancer Network (NCCN) management
guidelines be modified to include renal cancer screening using biannual
renal imaging from age 40 years or 5 years earlier than the earliest
kidney cancer diagnosis in the family, and endometrial sampling in
adulthood or 5 years earlier than the earliest endometrial cancer
diagnosis in the family.158 Female patients with CS should have breast
self-examination training and education starting at age 18 years.
Beginning at age 25 years, clinical breast examinations should be
performed every 6 to 12 months, and annual mammography and
MRI screening should start at age 30 years or 5 years before the earliest
age of breast cancer diagnosis in the family.158 Men should perform
monthly breast self-examination. Female patients should receive
endometrial cancer screening beginning around age 30 years or 5
years before the earliest age of endometrial cancer diagnosis in the
family.158 Persons with CS should undergo biannual colonoscopy from
age 40 years.158 Both men and women should have a comprehensive
annual physical examination starting at diagnosis with screening for
skin and thyroid lesions, including a baseline and annual thyroid
ultrasound and dermatologic examination.158 Finally, preimplantation
genetic testing for CS can be performed if a mutation is described in
a parent.
Common Colon Cancer Predisposition Syndromes
High-penetrance cancer susceptibility syndromes account for about
5% of CRC cases. The most common syndrome is LS, with familial
adenomatous polyposis (FAP) constituting a rarer familial syndrome.
Genetic epidemiologic analyses suggest a common susceptibility allele
for both colon cancer and adenomatous polyps that accounts for at
least 15% and possibly half of cases.164,165
Lynch Syndrome
A constellation of colon and endometrial cancers became known as
Lynch syndrome, formerly referred to as hereditary nonpolyposis colon
cancer (HNPCC).166 LS is an autosomal dominant syndrome caused
by mutations in one of four MMR genes (MLH1, MSh2, MSH6,
PMS2), or a gene located nearby (EPCAM), with resultant errors in
DNA replication yielding a microsatellite instability (MSI) phenotype.
LS accounts for 3% of all CRCs.167
Clinical features
LS-associated CRC is characterized by an accelerated progression of
the adenoma-to-carcinoma sequence, with a predominance of right-sided
colorectal tumors with pathology demonstrating poorly differentiated
adenocarcinoma with mucinous and signet ring cell features, a Crohnlike
reaction, and tumor-infiltrating lymphocytes, in addition to presentation
with metachronous or synchronous colorectal tumors. Patients with
LS have a 50% to 80% lifetime risk of colon cancer, with a median
age of diagnosis of 44 years in the proband and 61 years in mutation
carrier relatives.168–171 A 40% to 60% endometrial cancer risk by age
70 years has been reported,172–175 with a median age at onset from the
late 40s to early 50s,176 as compared with a 3% risk for endometrial
cancer in the general population. LS also confers a high lifetime risk
for ovarian cancer (12%–15%).177 Five additional tumor sites dem-
onstrated increased observed/expected (O/E) ratios in LS kindreds:
cancers of the stomach (O/E = 4.1), small intestine (O/E = 25), ureter
(O/E = 22), and kidney (O/E = 3.2).178 More recently, adrenocortical
neoplasm have also been implicated in LS and, although still contro-
versial, some studies also have demonstrated a modest increased risk
of prostate and breast cancer in patients with LS.179–181
At a 1991 meeting in Amsterdam, the International Collabora-
tive Group on LS defined the syndrome as (1) histologically verified
CRC in three or more relatives, including a first-degree relative of
the other two; (2) CRC involving at least two generations; and (3)
one or more CRCs diagnosed before age 50 years. The subsequent
“Amsterdam II criteria” for LS were redefined to include extracolonic
LS-associated cancers.182 In 1996, the “Bethesda criteria” delineated
individuals at risk for LS for whom molecular genetic analysis may
be considered.183 A revised set of Bethesda Guidelines was developed
to identify subjects who are at high risk of having a germline MMR
gene mutation.184 Multivariate logistic regression risk models using
personal and family medical histories estimate the probability of
carrying an LS mutation.185–187 In the most common LS-associated
cancers, including CRC and endometrial cancers, the need for tumor
screening for LS has traditionally been based on the age at cancer
diagnosis or on the strength of the personal and family history, as
outlined in the Revised Bethesda Guidelines.188 However, per the most
recent guidelines from EGAPP (Evaluation of Genomic Applications
in Practice and Prevention Working Group) and NCCN, screening
of all colorectal tumors (or at least all colorectal tumors in patients
younger than 70 years at diagnosis or those older than 70 years who
meet Revised Bethesda Guidelines) and endometrial tumors via MSI
or MMR protein immunohistochemical staining analysis is now
recommended.189,190
Genetics
About 45% to 70% of LS families harbor mutations in one of the
following four genes: MSH2, MLH1, MSH6, and PMS2.191–195 In
addition, germline deletions of the 3′ region of the EPCAM gene,
just upstream of MSH2, also result in LS, through silencing of the
MSH2 gene.196 Mutations of MSH2 and MLH1 were far more
frequent than the others, accounting for about 30% each of families
meeting Amsterdam criteria for LS. More than 75% of mutations in
MSH2 and MLH1 were inactivating insertions, deletions, alterations
in premessenger RNA splicing signals, and nonsense mutations. Of
120 mutations surveyed, 23% were missense mutations.197 Muta-
tions of MSH6 less frequently result in the “replication error repair”
phenotype but account for a significant number of familial colon cancer
families.198 The replication error repair phenotype is commonly detected
as MSI with use of polymerase chain reaction screening of tumors
with microsatellite markers. The MSI phenotype is present in about
80% of LS-associated colon cancers199 and in about 15% of sporadic
colon tumors,200 as well as in other tumors associated with LS (e.g.,
uterine and gastric cancers). MSI results in a genome-wide increased
mutation rate, causing mutations in oncogenes, tumor suppressors, and
microsatellite regions.188
For patients with an abnormal screening test result (MSI-high or
MMR-deficient tumor), various algorithms have been developed to
help guide subsequent evaluations, which may include germline genetic
testing or further tumor analyses, such as MLH1 promoter hyper-
methylation and/or BRAF*V600E somatic mutation in MLH1/PMS2
protein deficient cases.189,190 In older patients with CRC, hypermeth-
ylation of the MLH1 promoter, or presence of a BRAF*V600E somatic
mutation, may account for the lack of MLH1 protein expression.201
This epigenetic (nonhereditary) mechanism of MLH1 promoter
200 Part I: Science and Clinical Oncology
hypermethylation is responsible for most of the remaining patients
whose tumors are characterized by defective DNA MMR.202
Biallelic mutations in the same MMR genes result in constitutional
MMR deficiency, which classically manifests as childhood-onset cancers,
in particular hematologic malignancies, brain tumors, and early-onset
CRCs and café au lait macules similar to those seen in neurofibromatosis
type 1,203,204 although a milder phenotype can exist in persons with
biallelic mutations in PMS2 as evidenced by reports of first cancer
diagnoses in mutation carriers in the third and fourth decades.205,206
Clinical management
For probands diagnosed with LS, observational data have shown that
surveillance colonoscopy lowers CRC incidence and mortality by more
than 50%207 and improves patient survival.208 Persons with LS are
advised to undergo colonoscopic surveillance every 1 to 2 years,
preferably annually, starting at age 20 to 25 years. Some guidelines
suggest that for MSH6 mutation carriers, screening colonoscopy may
be delayed to the age 30 years.209,210 A baseline upper endoscopy
should be performed, but the optimal subsequent screening interval
has yet to be established. Prophylactic subtotal colectomy sparing the
rectum should be considered after the first CRC diagnosis, given the
high rate of metachronous CRCs.211 No benefit has as yet been
demonstrated with other screening strategies directed toward the
LS-associated malignancies.190 Endometrial cancer screening entails
endometrial biopsy at age 30 to 35 years. Annual urinalysis with
cytologic evaluation or urinalysis for microhematuria may also be
considered, especially in MSH2 carriers, in whom urothelial risk is
highest, but minimal data support the efficacy of this screening test
in early urothelial tumor detection. A retrospective study of prophylactic
bilateral oophorectomy and hysterectomy showed protection from
uterine and endometrial cancer,212 although the estimates were
influenced by a retrospective study design.213 Nonetheless, a combination
of risk-reducing bilateral oophorectomy and hysterectomy is a reasonable
option after childbearing or at the time of colorectal resection for a
CRC occurring in women with LS.
Identification of LS at the time of CRC diagnosis can affect clinical
management. Subtotal (versus segmental) colectomy can reduce the
increased risk of metachronous CRC in persons with LS by 31% for
every additional 10 cm of bowel removed.214 Tumors that demonstrate
the MSI-H phenotype may not benefit from 5-fluorouracil–based
chemotherapy and have improved stage-independent survival compared
with proficient-MMR CRC.215–218
More recently, presence of MMR deficiency, a nearly universal
finding in LS-associated tumors, predicted for response to immune
checkpoint blockade. Specifically, in patients with advanced MMR-
deficient CRC, a response rate of 62% was seen, as compared with
none in MMR-proficient tumors, to pembrolizumab, an anti–
programmed death 1 immune checkpoint inhibitor.219 In further
evaluation of immune checkpoint blockade across 12 different
MMR-deficient malignancies, a comparable high response rate was
observed, including response in LS patients.220 Checkpoint blockade
has also been shown to be of benefit for children with biallelic MMR
deficiency.221 Future studies are evaluating the role of immunotherapy
in patients with early-stage MMR-deficient tumors.
Polyposis Syndromes
Familial adenomatous polyposis
Clinical features.  FAP (adenomatous polyposis coli) manifests with
hundreds to thousands of adenomatous polyps at a young age. The
hallmark of FAP is the development of more than 100 adenomatous
colonic polyps in the teenage years with a resultant risk of CRC of
approximately 90% by age 45 years.222,223 Colon cancer is therefore
inevitable if the colon is not removed; cancer occurs at an average age
of 39 years. Flexible sigmoidoscopy at an early age establishes the
diagnosis, and prophylactic colectomy is performed in the teen years.
Patients remain at risk for primary adenomas and carcinomas of the
duodenum and rectum, as well as thyroid carcinoma, hepatoblastoma,
and other hepatopancreatic tumors.224 Gastric fundic gland polyps
occur and are often dysplastic,225 although an increased risk of gastric
cancer has yet to be definitely demonstrated in Western populations.226
Duodenal polyps are also prevalent, with a lifetime risk of duodenal
cancer, particularly ampullary cancer, of 5% to 12%.227,228 Other
FAP-associated cancers are listed in Table 13.2. Benign extraintestinal
manifestations of FAP include desmoid tumors, osteomas of the jaw
and dental anomalies, congenital hypertrophy of the retinal pigment
epithelium (CHRPE), lipomas, fibromas, sebaceous and epidermoid
cysts, and nasopharyngeal angiofibromas.228
Genetics.  FAP is an autosomal dominant syndrome that occurs
in 1 of every 7000 to 38,000 individuals and arises from germline
mutations in the APC gene.228–230 Twenty-five percent of mutations
are de novo,231 whereas a small fraction of cases result from somatic
mosaicism.232 Genotype-phenotype correlations have been described,233
particularly for CHRPE and the number of polyps observed. An
attenuated form of FAP is associated with mutations at the extreme
5′ and 3′ ends of the gene.234,235 Up to 30% of patients with multiple
adenomas (15–100 adenomas) who test negative for APC mutations
carry biallelic mutations in MUTYH,236 discussed in detail later in
this chapter.
Clinical management. FAP may manifest as childhood malig-
nancy, and thus the diagnosis is important as early as possible in
a proband’s lifetime, even prenatally via preimplantation genetic
testing if in vitro fertilization was used.237 Children are at risk for
hepatoblastoma for the first 5 years of life and are screened accord-
ingly. Annual flexible sigmoidoscopy for CRC screening begins at
age 10 to 12 years. Once polyps are identified, annual colonoscopic
surveillance is recommended. Prophylactic proctocolectomy, usually
in the late teens, is the treatment of choice.190 Sulindac, a nonsteroidal
antiinflammatory drug, and selective cyclooxygenase-2 inhibitors
reduce adenoma size and number and may be considered for che-
moprevention to delay but not preclude surgical intervention.238–240
Patients require lifelong surveillance for extracolonic tumors, including
tumors of the upper gastrointestinal tract and the ileal pouch (if
proctocolectomy is performed).241 The burden and histologic features
of duodenal polyps determines the need for endoscopic versus surgical
treatment.242 Additional screening measures are directed toward the
FAP-associated cancers listed in Table 13.2.190 Unaffected individu-
als with wild-type APC who have an affected family member with
a known mutation are screened in the same way as the general
population.243
Attenuated familial adenomatous polyposis
Clinical features. Attenuated familial adenomatous polyposis
(AFAP) is an FAP variant characterized by oligopolyposis (<100 colonic
adenomas) and a CRC onset 10 to 20 years later than in patients
with FAP, although the precise lifetime risk of CRC is not well
defined.244 AFAP may display malignant and benign manifestations
similar to those of FAP.245
Genetics.  Like FAP, AFAP is an autosomal dominant cancer
syndrome caused by germline mutations in the APC gene, with defects
that tend to localize in the 3′ or 5′ regions of the gene.
Clinical management. Right-sided colon lesions are frequent,
making colonoscopy the preferred CRC screening modality. Colonoscopy
begins in the late teens on a 1- to 2-year basis, with surgical intervention
considered with advanced polyp number, size, or histology.190 Surgical
approach (e.g., colectomy versus proctocolectomy) is determined by
the specific APC mutation, clinical presentation, patient preference,
and postulated adherence to postoperative screening.242 Other screening
measures in AFAP are directed toward the associated malignancies
listed in Table 13.2.190
MUTYH-associated polyposis
Clinical features.  Some AFAP probands have MUTYH-associated
polyposis (MAP) on the basis of biallelic germline mutations in the
base excision repair gene MUTYH.236,246,247 MAP is an oligopolyposis
 Genetic Factors: Hereditary Cancer Predisposition Syndromes  •  CHAPTER 13 201
with a CRC risk of at least 50% by age 48 years.232,248,249 Colonic
polyps may be adenomatous, sessile-serrated, and/or hyperplastic.
The full spectrum of disease is still being defined, and extracolonic
manifestations, benign and malignant, may resemble FAP to LS.250
A search for MUTYH mutations should be pursued in patients with
polyposis who have tested negative for APC mutations.236 In this
scenario, the detection rate for biallelic MUTYH mutations may be
17% to 27%.251
Genetics.  MUTYH mutations have a frequency of 1% to 2% in
the general population. Conflicting data exist as to whether monoal-
lelic MUTYH mutation confers increased risk of CRC.252,253 MAP
is considered an autosomal recessive syndrome; parents of affected
homozygote or compound heterozygote mutation carriers have been
reported to be unaffected.254 However, both monoallelic and biallelic
germline mutations in MUTYH have been associated with multiple
adenomas.247,254 Biallelic carriers have a higher frequency of CRC than
do monoallelic patients.255 Patients with multiple adenomas (more
than 15) without APC mutations are more likely to have biallelic
MUTYH mutations than are control subjects.247,256 Combining APC
and MUTYH sequencing increases the yield of diagnosis from 34.4%
to 41.3% in polyposis CRC.257 The mutations Y165C and G382D
are recurring in Caucasian Europeans, making up 86% of biallelic
mutations in three series.247,254,255 In 358 early-onset polyposis cases
that were unselected for APC mutation status, two cases carried bial-
lelic MUTYH mutations, and eight MUTYH mutation heterozygotes
were identified. No MUTYH mutations were detected in 354 control
subjects, leading the authors to conclude that biallelic MUTYH muta-
tions might account for up to 3% of early-onset CRC. Patients in
this series did not exhibit profuse polyposis, consistent with previous
reports, and distally located tumors were prevalent.258,259
Management.  CRC screening in biallelic mutation carriers includes
colonoscopy beginning at age 25 to 30 years and repeated every 3 to
5 years until polyps are detected, at which point the surveillance
interval is decreased to every 1 to 2 years. Prophylactic colectomy is
undertaken depending on patient age, disease location, and polyp
burden. Other screening measures in persons with MAP are directed
toward the associated malignancies listed in Table 13.2.190 Surgical
options for MUTYH mutation carriers depend on polyp distribution
and burden and may include ileorectal anastomosis for younger patients
with few rectal adenomas and a milder family history or with AFAP
or total proctocolectomy with the creation of an ileal pouch and anal
anastomosis for more diffuse polyposis.
Other hereditary predisposition to colon cancer
With use of a combination of whole-exome sequencing and linkage
analysis in individuals with colorectal adenomas without mutations
in known polyposis genes, germline mutations in DNA polymerase
ε (POLE) and δ (POLD1) have been identified.260 These genetic changes
are located in the proofreading (exonuclease) domain of the POLE
and POLD1 genes and result in deficient proofreading repair during
DNA replication. In addition to multiple adenomas and early-onset
CRC, the phenotypic features probably also include brain tumors
and, in POLD1 mutation carriers, endometrial cancer.261
Hereditary mixed polyposis syndrome is characterized by serrated
adenomas, juvenile polyps, adenomas, and CRC, and maps to chromo-
some 15q13-q14. Recently this syndrome has been associated with
mutations in BMPR1A in a family from Singapore, and in GREM1
in an Ashkenazi Jewish family.262,263
Low-penetrance colon cancer susceptibility alleles include the APC
I1307K polymorphism, CHEK2 gene mutations, and monoallelic
MUTYH (see Table 13.3), each associated with an approximate twofold
increased risk of CRC.264,265 Homozygous mutations in BUB1B
predispose a healthy adult to gastrointestinal polyps and CRC.
Identification of BUB1B variants requires karyotype analysis of dividing
cells.266 Peutz-Jeghers syndrome (STK11) and juvenile polyposis
(SMAD4, BMPR1A) are associated with hamartomatous polyposis as
well as an increased CRC risk.
Hereditary Diffuse Gastric Cancer
Clinical Features
Hereditary diffuse gastric cancer (HDGC) is characterized by autosomal
dominant susceptibility to diffuse gastric cancer (DGC) and lobular
breast cancer (LBC). Penetrance for small foci of in situ or invasive
cancer is virtually complete; however, the lifetime risk of clinically
significant gastric cancer is estimated to be 80%.267 The ages of diagnosis
of DGC in CDH1 mutation carriers ranges from 14 to 85 years of
age,37 with an average of 38 years.268 Unfortunately, endoscopic surveil-
lance for DGC is inadequate, and therefore germline CDH1 mutation
carriers are advised to undergo prophylactic total gastrectomies to
remove their risk of DGC. Female carriers also have a 60% lifetime
risk of LBC.267
Genetics
HDGC was initially found to be due to germline mutations in CDH1
in a large Maori kindred, with multiple family members affected with
DGC.269 Since then, 30% and 50% of HDGC families around the
world and in North America have been found to carry pathogenic
germline variants in CDH1, respectively.270–272 These differences likely
relate to overall increases in gastric cancer in the lower-frequency
areas. Pathology is characterized by pagetoid spread of signet ring cells
beneath a normal mucosa.273 The International Gastric Cancer Linkage
Consortium has updated testing criteria for HDGC267 to include (1)
two gastric cancer cases in a family, with one confirmed DGC diagnosed
in a person younger than 50 years, (2) three confirmed DGC cases
in first- or second-degree relatives independent of age, (3) a case of
DGC diagnosed before age 40 years, or (4) a personal or family history
of DGC and LBC, with one diagnosed before age 50 years. Because
of the early age at onset, similar to that of FAP, and the estimated
10% 5-year survival rate after DGC diagnosis, persons as young as
13 years and able to give informed consent or assent should be
considered for genetic counseling.
Clinical Management
CDH1 mutation carriers should be referred to a multidisciplinary
team, including a geneticist, a gastroenterologist, a surgeon, and a
nutritionist experienced in HDGC for discussion and counseling
regarding the recommendation for total prophylactic gastrectomy. If
total prophylactic gastrectomy is refused or delayed, surveillance
gastroscopies should be undertaken on an annual basis, preferably
in a center experienced with HDGC, where there should be sufficient
time for inspection, and a minimum of 30 biopsy specimens should
be taken from the five gastric zones: the antrum, transitional zone,
body, fundus, and cardia.267 Gastrectomy has a significant effect on
quality of life and should be undertaken only after extensive genetic
counseling. The long-term morbidity that can result from gastrectomy
includes weight loss, lactose intolerance, fat malabsorption and
steatorrhea, dumping syndrome, bacterial overgrowth, postprandial
fullness, and vitamin deficiencies.274 Female carriers should be
referred to a high-risk breast clinic and advised regarding monthly
breast self-examinations starting at age 35 years, an annual
mammogram and breast MRI, and a biannual clinical breast
examination. It is currently not well established whether cancer
develops at any other sites with increased frequency with CDH1
mutations.
Pancreatic Adenocarcinoma
Predisposition Syndromes
Clinical Features
Approximately 53,000 new cases of pancreatic adenocarcinoma (PAC)
are expected to be diagnosed in the United States in 2017.275 Familial
clustering of PAC was demonstrated in 5% to 10% of these cases,
whereas 2% could be attributed to mutations in a highly penetrant
mendelian susceptibility gene.276
202 Part I: Science and Clinical Oncology
Genetics
Several hereditary syndromes predispose to PAC (see Table 13.2). The
hereditary breast and ovarian cancer susceptibility gene BRCA2 has
been associated with sporadic and familial PAC; 7% of patients with
sporadic PAC were identified with mutations in BRCA2.277 A BRCA2
gene mutation has been identified in up to 19% of cases with familial
PAC,278,279 and families carrying a BRCA2 gene mutation have a 3.5-fold
to 7-fold increased risk of PAC.280,281 BRCA1 gene mutations likely
also predispose to PAC.282–284 A recent study demonstrated an increased
prevalence of mutations in BRCA2 and BRCA1 in a cohort of Ashkenazi
origin with familial PAC.284 In persons with Fanconi anemia, mutations
in multiple causative genes including PALB2, which complexes with
BRCA2 to become part of the FA-DNA repair pathway, predispose
to PAC.51,276,285–287
FAP confers an RR of 4.5 for PAC,238 whereas LS probands have
an approximately ninefold increased risk of PAC.288 Persons with
Peutz-Jeghers syndrome have an RR of 132 for PAC.289 Familial atypical
multiple mole melanoma syndrome, associated with germline mutations
in CDKN2A/p16, has an RR of 13 to 22 for PAC.290 Hereditary
pancreatitis (PRSS1 or SPINK1) confers a 53- to 87-fold increased
risk for PAC that is compounded by smoking.291,292
Clinical Management
Screening modalities for PAC include endoscopic ultrasound, magnetic
resonance cholangiopancreatography (MRCP), computed tomography
(CT), and serial serologic monitoring for tumor markers. Screening
studies to date have not demonstrated a decrease in mortality. However,
a European study of a large cohort of CDKN2A mutation carriers
suggested that surveillance with annual MRI, MRCP, and/or endoscopic
ultrasound of the pancreas was relatively successful in detecting most
PACs at a resectable stage.293 Participation in clinical trials assessing
the efficacy of PAC screening in high-risk individuals is encouraged.
Carney Complex
Clinical Features
Carney complex (CNC) is a multiple neoplasia syndrome that is
referred to by acronyms such as NAME294 and LAMB syndrome
in the medical genetics literature.163,295 As described by Carney in
1985,163 the complex is characterized by myxomas, skin pigment
abnormalities, endocrine tumors, and schwannomas. The median
age at diagnosis is 20 years, with spotty skin pigmentation and heart
myxomas being the most common initial clinical manifestations.296
The skin abnormalities involve the lips, the conjunctiva and inner or
outer canthi, and the mucosa of the vagina or penis. Atrial myxomas
are by far of greatest clinical concern, because they usually result in a
decreased life span and account for the major causes of mortality in
affected individuals; cardiac myxoma can cause stroke and death.297
Cardiac myxomas occur in 32% of mutation carriers.298 Ductal breast
adenoma has been described as part of CNC, as well as myxoid
fibroadenomas and other findings on breast imaging.299 In one study
of 338 persons diagnosed with CNC, 34 of 194 women (17.5%) had
breast myxomas, often bilateral.297 Large-cell calcifying Sertoli cell
tumor, seen in 41% of male mutation carriers,298 detected as early as
2 years of age, has been associated with infertility due to hormonal
imbalance.300
Primary pigmented nodular adrenocortical disease occurs most
frequently in association with CNC in 60% to 95% of patients,298,301
and 14% have Cushing syndrome.301 Thus the diagnosis of primary
pigmented nodular adrenocortical disease should prompt screening
for CNC. Other organs that are involved in CNC are the thyroid
gland and ovaries. Thyroid gland tumors occur in 25% and include
follicular hyperplasia and follicular adenoma; thyroid cancer, follicular
and papillary carcinoma, occurs in 2.5%.298 Ultrasonography is useful
for screening and diagnosis of these lesions.302 Ovarian serous cyst-
adenomas have been described in the literature, and ovarian lesions
occur in 14%,298 suggesting that pelvic ultrasound examination may
be indicated as a part of the evaluation of women with CNC.303
Diagnostic criteria for CNC297 require two or more of the manifesta-
tions or one major manifestation and one of the minor criteria. Major
criteria are skin pigmentary abnormalities (multiple lentigines of the
face, blue nevus, or epithelioid blue nevus); myxoma (cutaneous or
mucosal myxomatosis); cardiac myxoma; endocrine tumors or overactiv-
ity (primary pigmented nodular adrenocortical disease, a micronodular
form of adrenal hyperplasia, growth hormone–producing pituitary
adenoma, large-cell calcifying Sertoli cell tumor, or thyroid adenoma
or carcinoma); psammomatous melanotic schwannoma; thyroid
carcinoma or multiple thyroid nodules on ultrasound in a young
patient; multiple breast ductal adenomas; and osteochondromyxoma.
To make the diagnosis, an individual must have two of the components
listed or one of the manifestations in addition to an affected first-degree
relative or have an inactivating mutation of the PRKAR1A gene. A
malignant neoplasm is a relatively uncommon finding in affected
patients.
Genetics
Mutations in PRKAR1A (17q23-q24) are identified in about 40% of
persons with CNC.304 PRKAR1A codes for the RI-α subunit of PKA,
a critical cellular component of a number of cyclic nucleotide–dependent
signaling pathways.305 PRKAR1-α frameshift mutations cause haplo-
insufficiency of R1-α and manifest as CNC. As predicted by the
Knudson “two-hit” hypothesis, loss of heterozygosity of the normal
allele supports the model that R1-α may have tumor suppressor function
in the target tissues. The most common mutation in patients with
CNC, a two–base-pair deletion (TGdel) in exon 5 of PRKAR1, has
also been found de novo in several kindreds, suggestive of a mutational
hot spot in the gene.306 About 17% of diagnosed individuals harbor
de novo mutations.
Clinical Management
Recommendations include annual echocardiography, annual measure-
ment of urinary free cortisol, testicular sonogram in males at their
initial visit, thyroid ultrasound at the initial visit and as needed
thereafter, pelvic ultrasound examination in female patients at their
initial visit, breast imaging, spine MRI, pituitary MRI, and adrenal
CT scan.306 Children should undergo echocardiography during the
first 6 months of life and annually thereafter to detect potentially
lethal myxomas. Children with large-cell calcifying Sertoli cell tumor
may require monitoring of growth rate and pubertal status. Bone age
determination and further laboratory evaluation might be necessary
if gynecomastia is present.297
Hereditary Paraganglioma-Pheochromocytoma
Syndromes
Clinical Features
Paragangliomas are tumors of the paraganglia that are derived from
neuroectodermal and mesodermal origins in the parasympathetic and
sympathetic axis and thus can exist from the base of the skull to the
pelvis. These tumors are called chemodectomas in the head and neck
and are derived from nonchromaffin chemoreceptors in the carotid,
aortic, jugular, or vagal bodies. These tumors are usually benign, and
also can be symptomatic because of size and compression effects of
surrounding local structures. Of 30 cases reviewed in one series, about
half were bilateral, with a family history of chemodectoma in about
one-third of these cases.307,308 Most of the familial cases presented
with multiple tumors. The remarkable feature about hereditary
chemodectomas is the evidence of imprinting; chemodectomas develop
in children of affected fathers, but not in the children of affected
mothers.309 Pheochromocytomas are paragangliomas that are located
in the adrenal medulla and or anywhere along the parasympathetic
and sympathetic tracts. Pheochromocytomas are usually benign tumors
and can be active (secreting hormones) or inactive depending on their
 Genetic Factors: Hereditary Cancer Predisposition Syndromes  •  CHAPTER 13 203
sympathetic or parasympathetic origin. The paragangliomas and
pheochromocytomas can be single or multiple; the presence of multiple,
bilateral, recurrent, and early (in patients younger than 40 years)
tumors or a family history of such tumors should prompt early referral
for genetic assessment.
Genetics
Given the oxygen sensor function of the carotid body, a common site
for chemodectomas, in paraganglioma syndromes, loss-of-function
mutation in all four of the subunits of the mitochondrial succinate
dehydrogenase (SDH) enzyme complex encoded by the genes SDHA,
SDHB, SDHC, and SDHD have been implicated,310 in addition to a
gene encoding a mitochondrial protein, SDH5.311 Mutations in SDHD
exhibit autosomal dominant inheritance, with an imprinting mecha-
nism310; germline mutations in SDHAF2,311 which encodes the
mitochondrial protein SDH5 that is required for flavination of the
SDHA subunit of SDH, have also been associated with disease and
have been shown to have imprinted parent-of-origin effects. In contrast,
SDHB and SDHC mutations do not appear to be imprinted genes.
Although maternally derived SDHD cases have been reported, further
analysis revealed that a paternal mutation on an 11p15.5 allele was
also necessary to result in paraganglioma.312 SDHD mutations give
rise to more frequent head and neck tumors, whereas SDHB mutations
exhibit a greater propensity for malignancy and also are associated
with renal cell cancers (RCCs).313
Of 271 paraganglioma and/or pheochromocytoma cases unselected
for family history, germline mutations were identified in 66 (24%),
with SDHD and SDHB mutations accounting for 23 cases. Of the
remaining 43 germline mutation cases, 30 were attributed to VHL
disease mutations, and 13 were caused by mutations in the RET
gene.314 Given the high frequency of germline mutations in paragan-
glioma cases, diagnosis should prompt consideration of genetic testing.315
Recently a Dutch founder mutation in SDHD, c.274G>T, p.Asp92Tyr,
was found to account for 69.1% of all Dutch carriers of a mutation
in an SDH gene; the second most commonly affected gene was
SDHAF2, indicating the importance of considering regional differences
in mutation frequencies when formulating a testing strategy.316
Clinical Management
Treatment for paraganglioma is surgical. Minimal morbidity occurs
with small tumors, but once the size is greater than 5 cm, cranial
nerve loss and baroreceptor failure may be postoperative sequelae.
Screening for pheochromocytoma is by serum and/or urinary meta-
nephrines. Screening for renal cancers is unproven, but abdominal/
pelvic imaging seems appropriate.
A SELECTION OF CANCER PREDISPOSITION
SYNDROMES WITH TARGETED THERAPIES
Multiple Endocrine Neoplasia Type 2
Clinical Features
Medullary thyroid carcinoma runs in families about 25% of the time,
as a syndrome of site-specific familial medullary carcinoma of the
thyroid (FMCT) or as multiple endocrine neoplasia type 2 (MEN2).317
Cases usually manifest in the third and fourth decades and are often
bilateral and multifocal. MEN2A is characterized by predisposition
to medullary thyroid carcinoma, pheochromocytoma, and parathyroid
disease.318 Patients with MEN2B have an earlier age of onset than do
patients with MEN2A and are characterized by enlarged, nodular
lips, a marfanoid habitus, ganglioneuromatosis of the intestine, and
other abnormalities.319 This phenotype also includes medullary thyroid
carcinoma, which may be more aggressive, and pheochromocytoma.
Parathyroid disease is less common than in MEN2A.
Familial papillary thyroid cancer is distinct from FMCT and is
associated with an increased incidence of CRC.320
Genetics
In 1993 it was observed that RET gene mutations were associated
with MEN2A, MEN2B, and FMCT.321,322 Specific RET gene mutations
have been associated with MEN2A, MEN2B, and FMCT.318,321,323–325
RET testing of sporadic cases of medullary carcinoma of the thyroid
will yield a relatively low (5%) rate of diagnosis of MEN2A in the
absence of a family history of the disease, C-cell hyperplasia, or
multifocality.322,326 Nonetheless, RET testing is more sensitive than
traditional biochemical screening. Asymptomatic children with RET
mutations and normal plasma calcitonin levels had small foci of
medullary carcinoma of the thyroid at time of “prophylactic” surgery.327
These findings were confirmed in a large study in the United States.328
A study of 477 MEN2A families showed an association between
codon 634 mutations and pheochromocytoma and between mutations
at codons 768 and 804 and FMCT, whereas codon 918 mutation is
MEN2B-specific. Rare families with both MEN2 and Hirschsprung
disease have MEN2-specific codon mutations.329 A 611 codon mutation
is associated with a mild form of FMCT with slow progression. The
classic M918T mutation in exon 16 is found in MEN2B, and a less
common mutation in RET codon 883 has also been reported.330,331
Clinical Management
RET testing has been established as the gold standard for MEN2A
screening, and prophylactic thyroidectomy has been established as
the primary preventive intervention. Guidelines from the American
Thyroid Association for the management of medullary thyroid cancer
reflect the known genotype-phenotype correlations in relation to the
ages at which they recommend childhood prophylactic surgeries.332
Heterozygotes for RET mutations in the setting of MEN2A are screened
with abdominal ultrasound and CT, as well as 24-hour urine studies
through the adult years, at least to age 35 years. Plasma screening for
the pheochromocytomas has been suggested (see the section on VHL
disease). The treatment of choice for patients with MEN2A or MEN2B
with a unilateral pheochromocytoma is unilateral resection, because
substantial morbidity and mortality are associated with the addisonian
state after bilateral adrenalectomy.333 RET kinase inhibitors have been
in clinical trials to evaluate their efficacy in treatment of metastatic
medullary thyroid cancer in sporadic and familial cases of medullary
thyroid cancer.334–338 The FDA has approved the use of the RET
kinase, vascular endothelial growth factor (VEGF) receptor, and
epidermal growth factor receptor (EGFR) inhibitor vandetanib to
treat adult patients with metastatic medullary thyroid cancer. This
approval emerged from the results of a randomized, double-blind
phase III trial that showed improvement in progression-free survival
after once-daily administration of the oral agent in this setting.334
Gorlin Syndrome and Nevoid Basal Cell
Carcinoma Syndrome
Basal cell carcinomas (BCCs) are the most common malignancy in
humans, with three quarters of a million cases each year. In a small
subset of families, BCCs occur at an early age and in great numbers.
The nevoid basal cell carcinoma syndrome (NBCCS) consists of
multiple BCCs and usually manifests after puberty, accompanied by
odontogenic jaw cysts, congenital skeletal abnormalities, ectopic
calcification of the falx cerebri, and characteristic pits in the skin of
the palms and soles.339,340
A hallmark of the syndrome is the increased susceptibility of the
skin to the damaging and tumor-inducing effects of ionizing radiation.341
Multiple BCCs have developed within 6 to 36 months after radiation
therapy. Unlike Bloom syndrome or ataxia telangiectasia, there is no
in vitro evidence of chromosome fragility.
One estimate of the prevalence of the syndrome, 1 in 57,000,
comes from a study of a population of 4 million in northwest
England.342 As described by Gorlin339 in 1987, penetrance for the
syndrome is virtually 100% over a lifetime.340 Although NBCCS
204 Part I: Science and Clinical Oncology
diagnostic criteria are based on the presence of major and/or minor
criteria (two major criteria and one minor criterion, or one major
and two minor criteria),343,344 limited information is available regarding
the sensitivity and specificity of each criterion. As will be described
shortly, a consensus statement from an international colloquium on
basal cell nevus syndrome suggested that a suspected diagnosis should
be considered based on one major criterion and molecular confirmation;
two major criteria; or one major and two minor criteria.345 The
component tumors that are included in these criteria are also a topic
of debate. One study showed that in approximately 5% of patients
with Gorlin syndrome, medulloblastoma develops in the first few
years of life, and that 10% of patients with medulloblastoma diagnosed
at age 2 years or younger have Gorlin syndrome.346 Another study
found that the only histopathologic subtype of medulloblastoma in
patients with NBCCS is the desmoplastic subtype, which occurs in
only 20% of sporadic medulloblastoma cases; it was suggested that
the occurrence of the desmoplastic subtype in a patient younger than
2 years of age is pathognomic.347 The international consensus group
concluded that childhood medulloblastoma (also called primitive
neuroectodermal tumor) should be considered a major criterion. Thus
the major criteria include (1) BCC before 20 years of age, or excessive
numbers of BCCs out of proportion to prior sun exposure and skin
type; (2) odontogenic keratocyst of the jaw before 20 years of age;
(3) palmar or plantar pitting; (4) lamellar calcification of the falx
cerebri; (5) medulloblastoma, typically desmoplastic; and (6) a first-
degree relative with basal cell nevus syndrome. The minor criteria
include (1) rib anomalies; (2) other specific skeletal malformations
and radiologic changes (i.e., vertebral anomalies, kyphoscoliosis, short
fourth metacarpals, and postaxial polydactyly); (3) macrocephaly; (4)
cleft lip or palate; (5) ovarian or cardiac fibroma; (6) lymphomesenteric
cysts; (7) ocular abnormalities (i.e., strabismus, hypertelorism, congenital
cataracts, glaucoma, and coloboma).345
Genetics
In 1996 Johnson and coworkers found mutations in exon 15 of the
human homologue of the Drosophila patched gene (called PTH or
PTCH)348 in 2 of 60 typical NBCCS kindreds. In 86%, the mutations
caused a truncated protein.349 NBCCS is caused by germline loss of
function mutations in the gene PTCH mapped to chromosomal locus
9q22.3. The PTCH gene consists of 23 exons and encodes an integral
membrane protein, PTCH1, with 12 transmembrane regions and
two extracellular loops and a putative sterol-sensing domain. When
unbound by Hedgehog protein, PTCH1 inhibits Smoothened
homologue, which in turn inhibits the Hedgehog signaling pathway,
a pathway upregulated in BCCs.350 Mutations of PTCH are the only
known genetic alterations associated with NBCCS.351 However, several
studies have found no genotype-phenotype correlations,349,352,353 which
suggests that other factors might add to the development of patients’
clinical features. For kindreds with diagnostic clinical findings of
NBCCS that test negative for a mutation by sequence analysis, testing
that identifies deletions and duplications may be performed. About
70% to 80% of probands have inherited the condition from a parent,
and about 20% to 30% of probands have a de novo mutation.
Risk Management Recommendations
Life expectancy in persons with NBCCS is not significantly different
from average. The major clinical issues revolve around the cosmetic
effects of treating multiple skin tumors and jaw keratocysts, which
can recur. The jaw cysts may also undergo malignant transformation.354
Consultation with oral and plastic surgeons and dermatologists is
important. Because of early-onset disease risk, for example, for medul-
loblastomas, genetic testing of children is appropriate for this condition.
Evaluation of members of NBCCS kindreds includes skin examination,
measurement of head circumference, and radiographic examination
of the skull, spine, ribs, and jaws.355 Ophthalmologic and dental
examination and radiographic monitoring of the jaw cysts (oropan-
tomography) may be performed on affected individuals. MRI scans
of at-risk children can be used to diagnose medulloblastomas, although
it is unclear whether this scanning improves outcome. If no physical
or radiographic stigmata are noted by 5 years of age in the child of
an affected patient, the chances that the child is a heterozygote are
small.343 As has been mentioned, radiotherapy for large basal cell
cancers should be avoided, because this treatment can lead to the
development of thousands of BCCs in the radiation field.340,341 Prenatal
testing for NBCCS is possible if the disease-causing mutation has
been identified in an affected family member.
In recent years, the efficacy of an oral small-molecule inhibitor
of Smoothened homologue, GDC-0449 or vismodegib, has been
demonstrated in persons with metastatic or locally advanced BCC.350
In January 2012, vismodegib was the first drug approved by the
FDA for use in the treatment of advanced BCC after a phase II trial
showed a 9.5-month median progression-free survival in patients with
locally advanced disease and metastatic disease who responded (43%
and 30%, respectively).356,357 Other findings have also demonstrated
the usefulness of vismodegib in prevention of BCCs in patients
with NBCCS.358
Hereditary Leiomyomatosis Renal Cell
Cancer Syndrome
Clinical Features
The autosomal dominant hereditary leiomyomatosis and renal cell
cancer syndrome (HLRCC) predisposes to benign cutaneous and
uterine leiomyomas (fibroids), uterine leiomyosarcomas, and papillary
type II RCC or renal collecting duct carcinoma.359–362
Cutaneous leiomyomas arise from the erector pili muscles, existing
as single or multiple lesions. They can be skin-colored or light brown
and are sensitive to touch and cold temperature. Cutaneous lesions
may be diffuse and symmetric or present in segmental bands along
the lines of Blaschko. Of 11 females, 9 had cutaneous leiomyomas
and 9 had uterine leiomyomas.363,364 Leiomyomas of the uterus are
multiple and symptomatic and result in hysterectomy by age 30 years
in more than 44% of women with HLRCC.361 Kidney tumors in
persons with HLRCC are aggressive and often single, small lesions
of high malignant potential. Within an HLRCC kindred, the age at
onset of four cases of kidney cancer ranged from age 33 to 48 years,
and all were diagnosed in females.359,365 A Finnish series of 98 HLRCC
cases found a standardized increased risk of 71-fold for uterine leio-
myosarcoma and 6.5-fold for RCC.366 In a separate series of patients
with HLRCC, renal cancer occurred in 62% of 21 patients, 76% had
cutaneous leiomyomas, and uterine leiomyomas occurred in 90% to
100% of the women.367 Adrenal gland adenoma and kidney cysts, as
well as breast and bladder carcinoma, have also been reported.366
However, a study of 85 patients with breast cancer with a family
history of HLRCC found no increased risk for breast cancer, although
a considerably larger sample size could be more informative.368
Genetics
The gene associated with hereditary leiomyomatosis and RCC on
1q42.3-q43 encodes fumarate hydratase (FH), a tricarboxylic acid/
Krebs cycle, mitochondrial enzyme. Dominant mutations in FH cause
HLRCC in a manner that is consistent with Knudsen’s two-hit tumor
suppressor model. Hypoxia-inducible factor (HIF) levels are increased
in clear cell renal carcinomas. FH is critical for aerobic metabolism
and functions to convert fumarate to malate in Krebs cycle. When
FH is inactivated, fumarate levels build up and competitively inhibit
HIF prolyl hydroxylase (HPH), a regulator of intracellular HIF. When
HPH is inactivated, HIF levels are upregulated, resulting in transcription
of downstream genes.369 Mutations in the FH gene are detected in
about 75% to 100% of patients with multiple cutaneous and uterine
leiomyomatosis.360,362,367 One mutation, R190H, was detected in 11
of 31 unrelated North American HLRCC kindreds, and the mutation
R58X has also been identified in multiple unrelated kindreds.367 In
addition, a mutation, 905-1G>A, was found in four Iranian HLRCC
 Genetic Factors: Hereditary Cancer Predisposition Syndromes  •  CHAPTER 13 205
kindreds, possibly representing a founder mutation in this population.370
Germline biallelic mutations in FH cause the mitochondrial encepha-
lomyopathy FH deficiency, and obligate carriers have developed
HLRCC.371–374 Reduced FH activity in lymphoblastoid and fibroblast
cells of HLRCC patients may facilitate confirmation of clinical diagnosis
via enzymatic testing.375
Clinical Management
Screening at regular intervals for renal lesions with CT with contrast
agent is indicated in patients with FH mutations. MRI is recommended
for uterine leiomyoma screening, and ultrasonography can also be
used.376 Systemic therapy for HLRCC will likely attempt to prevent
the increase in HIF levels or target the transcription products of
VHL-independent HIF accumulation such as VEGF and EGFR. A
phase II trial of erlotinib, an oral EGFR tyrosine kinase inhibitor, in
locally advanced and metastatic papillary renal cell carcinoma reported
an overall Response Evaluation Criteria in Solid Tumors (RECIST)
response rate of 11%, with an additional 24 patients (53%) experiencing
stable disease377; another phase II trial of erlotinib and bevacizumab
(monoclonal antibody against VEGF) is currently underway.378
von Hippel-Lindau Disease
Clinical Features
VHL disease is characterized by hemangioblastomas of the retina,
brain, and spinal cord; pheochromocytomas; renal cysts, and clear
cell renal cell carcinoma; pancreatic endocrine tumor; and endolym-
phatic sac tumors.379,380 Diagnosis depends on the presence of a
VHL-associated tumor and a family history consistent with VHL or
alternatively the presence of at least two VHL-associated tumors.380
Genetics
VHL is an autosomal dominant disease caused by germline mutations
in VHL, the protein product of which degrades hydroxylated HIF.
HIF is a transcription factor for angiogenesis genes, upregulated in
renal cell carcinoma. In normal conditions of oxygenation, HPH
hydroxylyzes HIF and VHL recognizes the hydrolysed HIF and mediates
proteosomal degradation. In hypoxic conditions, HPH does not
hydrolyze HIF, and therefore HIF is not degraded.369
Screening for von Hippel-Lindau Disease
Health screening in persons with VHL disease should include annual
evaluations starting at 1 year of age. It includes screening for neurologic,
vision (retinal angiomas), and hearing symptoms and signs and blood
pressure. Starting at age 5 years, annual blood or urinary fractionated
metanephrines (pheochromocytoma) should be evaluated, audiology
assessment should be performed every 2 to 3 years or annually if the
patient is symptomatic, and MRI should be performed if repeated
ear infections are present (looking for endolymphatic sac tumors).
Starting at age 16 years, an annual abdominal ultrasound examination
and MRI scan of the abdomen are recommended (with scans of the
kidney, pancreas, and adrenal glands every other year), along with an
MRI of the brain and total spine every 2 years.381 No standardized
guidelines exist for the management of other VHL-associated conditions,
and thus an individualized approach is required.380 The literature is
growing regarding management of renal cell carcinoma; early surgery
for lesions greater than 3 cm is recommended, depending on the size
and location of the tumor, along with undertaking nephron-sparing
procedures382 with preservation of the adrenal gland.
Systemic Therapy in von Hippel-Lindau Disease
Understanding the VHL pathway has provided targets for systemic
therapy in patients with advanced renal cell carcinoma. The VHL gene
has been vigorously studied, and its product is vital in targeting HIF-1α
and HIF-2α for ubiquitin-mediated degradation.383 The transcription
products of HIF-1α and HIF-2α are known to regulate a number
of downstream genes that are involved in tumorigenesis, including
VEGF, platelet-derived growth factor, EGFR, and transforming
growth factor–α. Many of the current therapeutic strategies are
based on targeting the receptors of the HIF-regulated genes. Sunitinib,
sorafenib, bevacizumab plus interferon-α, pazopanib, temsirolimus,
and everolimus are drugs that are currently approved for treating
metastatic RCC through targeting the VHL transcription pathway, and
they form the backbone of the treatment of a disease that is otherwise
resistant to other treatment modalities such as radiotherapy and
chemotherapy.384
Birt-Hogg-Dubé Syndrome
Clinical Features
Birt-Hogg-Dubé (BHD) cancer predisposition syndrome was first
described in 1977.385 This rare genodermatosis comprises an unusual
triad of findings: fibrofolliculomas that appear as white or skin-colored
papules on the face and upper torso, spontaneous pneumothorax, and
kidney tumors.362 The cutaneous lesions usually appear in the region
of the head, neck, and upper part of the trunk in the third or fourth
decade of life, and renal tumors develop in about 15% to 30% of
patients with cutaneous BHD syndrome. A recent review of 130 solid
renal tumors resected from 30 patients with BHD in 19 different
families revealed that renal tumors were multiple and bilateral and
were noted at an early age (mean, 50.7 years). The vast majority were
hybrid oncocytic neoplasms with areas reminiscent of chromophobe
renal cell carcinoma and oncocytoma, whereas a significant number
were chromophobe renal cell carcinomas, and a minority were con-
ventional clear cell renal carcinomas,386,387 thus demonstrating the
phenotypic heterogeneity of renal cancer that is unique to this syndrome
compared with other hereditary renal cancer predispositions. Chro-
mophobe and hybrid oncocytic tumors comprise 23% and 67% of
BHD renal tumors, respectively; clear cell tumors comprise 7% and
oncocytomas comprise 3%, whereas papillary renal cell carcinomas
are rarely noted.386 Persons with BHD syndrome are at markedly
increased risk of spontaneous pneumothoraces. About a third, 64 of
198 (32%), of BHD-affected individuals had a history of a spontaneous
pneumothorax.388 The syndrome is also associated with a progressive
flecked chorioretinopathy with constricted visual fields.389 There is an
association with colorectal neoplasia in some families with BHD,390
as well as colorectal polyps in up to half of patients with BHD.390,391
Similarities exist between BHD and tuberous sclerosis (TSC), which
may be explained by the fact that both BHD and TSC genes function
in the same pathway, mammalian target of rapamycin (mTOR). In
this regard, angiomyolipomas have been described in patients with
BHD and fibrofolliculomas were described in patients with TSC in
case reports.392,393
Genetics
BHD is inherited in an autosomal dominant manner and is caused
by germline mutations in the gene FLCN located on chromosome
17p11.2, which encodes folliculin.394,395 FLCN acts as a tumor sup-
pressor protein involved in the regulation of adenosine monophosphate–
activated protein kinase and mTOR signaling pathways.396,397 More
than 50 mutations in the FLCN gene have been reported, located in
all translated exons (4–14) and intronic borders, with exon 11 being
considered a mutation hot spot.397 In a large study, 27 of 52 BHD-
affected families had an insertion/deletion in exon 11 of the gene
(c.1733insC or c.1733delC). Phenotype manifestations among those
with either the insertion or the deletion were similar for fibrofollicu-
lomas and pneumothoraces. However, the incidence of renal tumors
was significantly higher among those with the C-insertion compared
with the C-deletion (33% versus 6%).388
Risk Management
Patients with BHD syndrome and their relatives should undergo
abdominal CT and renal ultrasound screening for renal tumors. Lung
cysts are best seen on CT scans. Dermatologic consultation is advised.
206 Part I: Science and Clinical Oncology
Ophthalmologic examination should be performed in patients with
BHD syndrome because of the high incidence of chorioretinopathy.
Because of the newly discovered association between FLCN and mTOR
activation, there is a potential therapeutic or preventive role of mTOR
inhibitors in patients affected by BHD, which is currently being
investigated in sporadic chromophobe tumors.398
Tuberous Sclerosis and Gastrointestinal
Stromal Tumor
Other syndromes that have shown the promise of targeted therapy
include TSC399 and inherited gastrointestinal stromal tumor (GIST).400
TSC is an autosomal dominant syndrome caused by germline muta-
tions in TSC1 and TSC2. Major features include facial angiofibromas
or forehead plaque, nontraumatic ungual or periungual fibromas,
hypomelanotic macules (three or more), shagreen patch (connective
tissue nevus), multiple retinal nodular hamartomas, cortical tuber,
subependymal nodule, subependymal giant cell astrocytoma (SEGA),
cardiac rhabdomyoma, single or multiple, lymphangiomyomatosis,
and renal angiomyolipoma. Minor features include multiple randomly
distributed pits in dental enamel, hamartomatous rectal polyps, bone
cysts, cerebral white matter radial migration lines, gingival fibromas,
nonrenal hamartoma, retinal achromic patch, “confetti” skin lesions, and
multiple renal cysts. Clinical diagnosis determining the probability of
having TSC is based on a combination of major and minor features.399
Angiomyolipomas are the dominant renal lesions in TSC, whereas
RCC has been reported in 1% to 3% of patients with TSC.401 Clinical
management of angiomyolipomas is by observation, removal, or
embolization, depending on the size and location of the tumor and
its potential for spontaneous bleeding. Recently, medical therapies
targeting the mTOR pathway have been proposed as another treatment
modality. In 2009, the FDA granted everolimus (Afinitor) an orphan
drug designation to treat renal angiomyolipomas and SEGAs in patients
with TSC. (Orphan designations are given to drugs that have shown
promise in the treatment of a disease or condition affecting fewer
than 200,000 patients in the United States.) In April 2012, the FDA
granted approval to specifically treat noncancerous kidney tumors
(renal angiomyolipomas) not requiring immediate surgery in patients
with TSC complex after the results of a phase III randomized controlled
trial (EXIST-2) that enrolled 118 patients at least 18 years of age who
had angiomyolipomas, randomized 2 : 1 to receive oral everolimus
(Afinitor) at 10 mg once daily or placebo until tumor progression or
unacceptable toxicity. With only half of the patients receiving less
than 8 months of treatment, 42% treated with Afinitor showed a
substantial reduction in tumor size lasting, on average, more than 5
months, whereas none of the patients receiving placebo showed any
tumor shrinkage.402 As part of the FDA approval, Afinitor’s manufacturer
(Novartis) will continue to monitor the patients for at least 4 years
to determine the duration of the response and how it affects the need
for surgery and the control of the disease.403
Mesenchymal stromal tumors of the gastrointestinal tract (GISTs)
are associated with germline and sporadic mutations in KIT and
PDGFRA,404–407 leading to constitutive activation. Familial cases are
inherited in an autosomal dominant manner; the mean age of diagnosis
is in the fifth decade, and multicentric disease generally develops in
the small bowel.400 Neurofibromatosis type 1, Carney-Stratakis, and
Carney triad are associated with GIST, although these tumors do not
tend to demonstrate mutations in c-KIT and PDGFR.400 In 2002 the
selective abl, c-kit, and PDGFR tyrosine kinase inhibitor imatinib
was granted approval by the FDA for the treatment of CD117-positive
unresectable or metastatic GIST. In 2006, sunitinib was granted
approval by the FDA for patients whose disease progressed while
taking imatinib or for those who could not tolerate imatinib. Because
of an overlap between germline and sporadic mutations, it is observed
that the use of imatinib stabilized the esophageal GISTs in a patient
harboring a germline mutation in c-KIT, previously reported in the
germline and sporadic setting mutation.408
A common theme relating to the pharmacologic treatment and
prevention of hereditary cancers is the development of drugs targeting
specific pathways. Cancer predisposition syndromes represent human
models of “knockouts” or “knockins” of germline alterations in
oncogenic pathways and as such may be amenable to pathway-specific
approaches. Therefore ongoing advancements in the understanding
of the molecular pathways and the development of targeted agents
will have immediate applicability to the treatment and, ultimately,
chemoprevention of hereditary cancer.
BAP1 INHERITED CANCER SUSCEPTIBILITY
More recently it has been elucidated that germline and sporadic
mutations in BAP1, a nuclear-localized, ubiquitin carboxy-terminal
hydrolase that binds to the RING finger domain of BRCA1,409 are
associated with uveal melanomas and mesothelioma.410–412 Wiesner
and colleagues412 identified germline mutations in BAP1 in two
families, each with a single case of uveal melanoma in addition to
multiple affected individuals with melanocytic lesions inherited in
an autosomal dominant manner. The BAP1-associated melanocytic
neoplasms were described as atypical, with an appearance ranging from
epithelioid nevi to atypical melanocytic proliferations. A follow-up
study suggests that the frequency of uveal melanomas attributable
to germline mutations in BAP1 is low (1 of 53 probands with uveal
melanoma, 5 of whom had a family history of uveal melanoma).413
In this same study, lung adenocarcinoma and meningioma were also
reported in the proband’s carrier family members, the tumors of
which, in addition to the uveal melanoma, demonstrated biallelic
inactivation, suggestive of tumors associated with a new syndrome.413
Testa and colleagues411 found germline mutations in BAP1 in two
families with multiple affected individuals with mesothelioma and
showed an increased frequency of germline mutations in sporadic
mesothelioma samples (2 of 26 cases). Somatic mutations in BAP1 have
also been found to define a new class of renal cell carcinoma, whereby
a germline variant was also reported.414 Epigenetic EZH2 inhibitors
are being explored in targeted treatment of mesothelioma with BAP1
mutations.415
TUMOR-NORMAL SEQUENCING
Inclusion of “normal” or constitutional DNA as a reference for somatic
(tumor) sequencing adds to the sensitivity of the assay416 and poses
opportunities for targeted (“precision”) prevention.417 Anonymized
retrospective analyses of tumor-normal genomic data from several
large centers89,90,416,418–420 Table 13.6) demonstrated 3% to 17.5%
clinically actionable germline findings. These studies will also allow
for targeting treatment as well as prevention, as already evidenced by
the 12% fraction of prostate cases with inherited mutations of DNA
repair genes.421 Included in the DNA repair genes are DNA homologous
repair genes potentially amenable to therapy with PARP inhibitors,
as well as subsets with Lynch-associated mutations, potentially amenable
to immunotherapy.
These findings have led some institutions to collect tumor-normal
DNA sequence in the setting of a consent process that allows com-
munication of these findings. This tumor-normal testing will result
in a “cascade” of genomic information to unaffected relatives for use
in targeted prevention (and even reproductive planning), at the same
time as tumor-derived information from the proband is used to target
therapy. This new model of integrated tumor and inherited (germline)
DNA sequencing poses a challenge for clinical oncology over the next
decade.
CONCLUSION
The relatively high-penetrance cancer susceptibility alleles associated
with the syndromes reviewed in this chapter account for a minority
of human cancers. Nonetheless, the number of molecularly defined
 Genetic Factors: Hereditary Cancer Predisposition Syndromes  •  CHAPTER 13 207

Table 13.6 Tumor-Normal Sequencing Studies and Proportion of Clinically Actionable

Findings89,90,416,418–420
St. Jude
Memorial Sloan Memorial Sloan Children’s
Institutional Johns Hopkins University Kettering Kettering (Clinically Research Baylor College
Series University of Michigan (Anonymized) Annotated) Hospital of Medicine
Number sequenced 815 91 1566 1040 1120 150
Number of germline 27 9 198 182 95 13
actionable findings
Percentage of 3% 10% 12.6% 17.5% 8.5% 8.6%
germline actionable
findings

cancer syndromes continues to grow and will increasingly include a ACKNOWLEDGMENTS

large number of genomic variants associated with a more modest risk
for disease, as well as measures of polygenic interactions of these
pathways and interactions of these alleles with acquired risk factors.
The emergence of next-generation sequencing applied to diagnostic
evaluation of tumor-normal DNA samples offers a major new challenge
to the integration of cancer risk counseling into the “targeted” thera-
peutic management of patients and families affected by inherited
malignancies.
This work was supported by the Robert and Kate Niehaus Center
for Inherited Cancer Genomics at Memorial Sloan Kettering Cancer
Center. We are grateful to Carolyn Stewart and Christina Tran for
their help in preparing the manuscript.
The complete reference list is available online at
ExpertConsult.com.

KEY REFERENCES
2. Offit K. Decade in review—genomics: a decade of
discovery in cancer genomics. Nat Rev Clin Oncol.
2014;11(11):632–634.
3. Offit K. The future of clinical cancer genomics.
Semin Oncol. 2016;43(5):615–622.
4. Robson ME, Bradbury AR, Arun B, et al. American
Society of Clinical Oncology policy statement
update: genetic and genomic testing for cancer
susceptibility. J Clin Oncol. 2015;33(31):3660–3667.
5. Walsh MF, Chang VY, Kohlmann WK, et al.
Recommendations for childhood cancer screening
and surveillance in DNA repair disorders. Clin
Cancer Res. 2017;23(11):e23–e31.
6. Villani A, Greer MC, Kalish JM, et al. Recommen-
dations for cancer surveillance in individuals with
rasopathies and other rare genetic conditions with
increased cancer risk. Clin Cancer Res. 2017;23(12):
e83–e90.
7. Foulkes WD, Kamihara J, Evans DGR, et al. Cancer
surveillance in Gorlin syndrome and rhabdoid
tumor predisposition syndrome. Clin Cancer Res.
2017;23(12):e62–e67.
8. Wasserman JD, Tomlinson GE, Druker H, et al.
Multiple endocrine neoplasia and hyperparathyroid-
jaw tumor syndromes: clinical features, genetics, and
surveillance recommendations in childhood. Clin
Cancer Res. 2017;23(13):e123–e132.
10. Lancaster JM, Powell CB, Chen LM, Richardson
DL. Society of Gynecologic Oncology statement
on risk assessment for inherited gynecologic cancer
predispositions. Gynecol Oncol. 2015;136(1):3–7.
13. Daly MB, Pilarski R, Berry M, et al. NCCN
Guidelines Insights: Genetic/Familial High-Risk
Assessment: Breast and Ovarian, Version 2.2017.
J Natl Compr Canc Netw. 2017;15(1):9–20.
17. Easton DF, Pharoah PD, Antoniou AC, et al. Gene-
panel sequencing and the prediction of breast-cancer
risk. N Engl J Med. 2015;372(23):2243–2257.
18. Domchek SM, Bradbury A, Garber JE, Offit K,
Robson ME. Multiplex genetic testing for cancer
susceptibility: out on the high wire without a net?
J Clin Oncol. 2013;31(10):1267–1270.
19. Stadler ZK, Schrader KA, Vijai J, Robson ME, Offit
K. Cancer genomics and inherited risk. J Clin Oncol.
2014;32(7):687–698.
21. Kuchenbaecker KB, McGuffog L, Barrowdale D,
et al. Evaluation of polygenic risk scores for breast
and ovarian cancer risk prediction in BRCA1 and
BRCA2 mutation carriers. J Natl Cancer Inst. 2017;
109(7).
35. Weitzel JN, Blazer KR, MacDonald DJ, Culver
JO, Offit K. Genetics, genomics, and cancer risk
assessment: state of the art and future directions in
the era of personalized medicine. CA Cancer J Clin.
2011;61(5):327–359.
39. Robson M, Offit K. Clinical practice. Management
of an inherited predisposition to breast cancer. N
Engl J Med. 2007;357(2):154–162.
49. Antoniou AC, Casadei S, Heikkinen T, et al. Breast-
cancer risk in families with mutations in PALB2.
N Engl J Med. 2014;371(6):497–506.
74. Tung N, Battelli C, Allen B, et al. Frequency
of mutations in individuals with breast cancer
referred for BRCA1 and BRCA2 testing using
next-generation sequencing with a 25-gene panel.
Cancer. 2015;121(1):25–33.
84. Tung N, Domchek SM, Stadler Z, et al. Counsel-
ling framework for moderate-penetrance cancer-
susceptibility mutations. Nat Rev Clin Oncol. 2016;
13(9):581–588.
116. National Comprehensive Cancer Centers.
NCCN Clinical Practive Guidelines in Oncol-
ogy, Genetic/Familial High-Risk Assessment:
Breast and Ovarian Version 2.2017; 2016. http://
www.nccn.org/professionals/physician_gls/pdf/
genetics_screening.pdf.
137. Kauff ND, Satagopan JM, Robson ME, et al.
Risk-reducing salpingo-oophorectomy in women
with a BRCA1 or BRCA2 mutation. N Engl J Med.
2002;346(21):1609–1615.
150. Kaufman B, Shapira-Frommer R, Schmutzler
RK, et al. Olaparib monotherapy in patients with
advanced cancer and a germline BRCA1/2 mutation.
J Clin Oncol. 2015;33(3):244–250.
151. Robson M, Im SA, Senkus E, et al. Olaparib for
metastatic breast cancer in patients with a germline
BRCA mutation. N Engl J Med. 2017.
167. Hampel H, Frankel WL, Martin E, et  al.
Screening for the Lynch syndrome (hereditary
nonpolyposis colorectal cancer). N Engl J Med.
2005;352(18):1851–1860.
181. Win AK, Young JP, Lindor NM, et al. Colorectal
and other cancer risks for carriers and noncarriers
from families with a DNA mismatch repair gene
mutation: a prospective cohort study. J Clin Oncol.
2012;30(9):958–964.
185. Barnetson RA, Tenesa A, Farrington SM, et al.
Identification and survival of carriers of mutations
in DNA mismatch-repair genes in colon cancer.
N Engl J Med. 2006;354(26):2751–2763.
189. Recommendations from the EGAPP Working
Group: genetic testing strategies in newly diagnosed
individuals with colorectal cancer aimed at reducing
morbidity and mortality from Lynch syndrome in
relatives. Genet Med. 2009;11(1):35–41.
190. National Comprehensive Cancer Centers. NCCN
Clinical Practice Guidelines in Oncology, Genetic/
Familial High-Risk Assessment: Colorectal Version
1.2017; 2017. https://www.nccn.org/professionals/
physician_gls/pdf/genetics_colon.pdf.
212. Schmeler KM, Lynch HT, Chen L-m, et al. Pro-
phylactic surgery to reduce the risk of gynecologic
cancers in the Lynch syndrome. N Engl J Med. 2006;
354(3):261–269.
220. Le DT, Durham JN, Smith KN, et al. Mismatch-
repair deficiency predicts response of solid tumors
to PD-1 blockade. Science. 2017.
237. Offit K, Kohut K, Clagett B, et al. Cancer genetic
testing and assisted reproduction. J Clin Oncol.
2006;24(29):4775–4782.
208 Part I: Science and Clinical Oncology
242. Guillem JG, Wood WC, Moley JF, et al. ASCO/
SSO review of current role of risk-reducing surgery
in common hereditary cancer syndromes. J Clin
Oncol. 2006;24(28):4642–4660.
249. Nielsen M, Morreau H, Vasen HFA, Hes FJ.
MUTYH-associated polyposis (MAP). Crit Rev
Oncol Hematol. 2011;79(1):1–16.
260. Palles C, Cazier JB, Howarth KM, et al. Germline
mutations affecting the proofreading domains of
POLE and POLD1 predispose to colorectal adenomas
and carcinomas. Nat Genet. 2013;45(2):136–144.
267. Fitzgerald RC, Hardwick R, Huntsman D, et al.
Hereditary diffuse gastric cancer: updated consensus
guidelines for clinical management and directions for
future research. J Med Genet. 2010;47(7):436–444.
306. Boikos SA, Stratakis CA. Carney complex: the first
20 years. Curr Opin Oncol. 2007;19(1):24–29.
314. Neumann HPH, Bausch B, McWhinney SR, et al.
Germ-line mutations in nonsyndromic pheochromo-
cytoma. N Engl J Med. 2002;346(19):1459–1466.
318. Moline J, Eng C. Multiple endocrine neoplasia type
2: an overview. Genet Med. 2011;13(9):755–764.
336. Lam ET, Ringel MD, Kloos RT, et al. Phase II clini-
cal trial of sorafenib in metastatic medullary thyroid
cancer. J Clin Oncol. 2010;28(14):2323–2330.
345. Bree AF, Shah MR, Group BC. Consensus statement
from the first international colloquium on basal
cell nevus syndrome (BCNS). Am J Med Genet A.
2011;155A(9):2091–2097.
350. Von Hoff DD, LoRusso PM, Rudin CM, et al. Inhi-
bition of the hedgehog pathway in advanced basal-cell
carcinoma. N Engl J Med. 2009;361(12):1164–
1172.
361. Linehan WM, Pinto PA, Srinivasan R, et al. Identi-
fication of the genes for kidney cancer: opportunity
for disease-specific targeted therapeutics. Clin Cancer
Res. 2007;13(2 Pt 2):671s–679s.
402. Bissler J, Kingswood JC, Zonnenberg BA, et al.
Everolimus therapy for angiomyolipoma in patients
with tuberous sclerosis complex or sporadic lymphan-
gioleiomyomatosis: results from EXIST-2. J Clin
Oncol. 2012;30(suppl 5):356.
417. Bombard Y, Robson M, Offit K. Revealing the
incidentalome when targeting the tumor genome.
JAMA. 2013;310(8):795–796.
420. Mandelker D, Zhang L, Kemel Y, et al. Mutation
detection in patients with advanced cancer by univer-
sal sequencing of cancer-related genes in tumor and
normal DNA compared to guideline-based germline
testing. JAMA. 2017;In Press.
421. Pritchard CC, Mateo J, Walsh MF, et al. Inherited
DNA-repair gene mutations in men with metastatic
prostate cancer. N Engl J Med. 2016;375(5):443–453.
438. Syngal S, Brand RE, Church JM, Giardiello FM,
Hampel HL, Burt RW. ACG clinical guideline:
genetic testing and management of hereditary
gastrointestinal cancer syndromes. Am J Gastroenterol.
2015;110(2):223–262, quiz 263.
455. Foulkes WD, Bahubeshi A, Hamel N, et al.
Extending the phenotypes associated with DICER1
mutations. Hum Mutat. 2011;32(12):1381–1384.
478. Hasumi H, Baba M, Hasumi Y, Furuya M, Yao M.
Birt-Hogg-Dube syndrome: clinical and molecular
aspects of recently identified kidney cancer syndrome.
Int J Urol. 2016;23(3):204–210.
504. Tabori U, Hansford JR, Achatz MI, et al. Clinical
management and tumor surveillance recommenda-
tions of inherited mismatch repair deficiency in
childhood. Clin Cancer Res. 2017;23(11):e32–
e37.
536. Pilarski R, Burt R, Kohlman W, Pho L, Shannon
KM, Swisher E. Cowden syndrome and the PTEN
hamartoma tumor syndrome: systematic review
and revised diagnostic criteria. J Natl Cancer Inst.
2013;105(21):1607–1616.

REFERENCES
1. Offit K. Clinical Cancer Genetics: Risk Counseling
and Management. New York: Wiley-Liss; 1998.
2. Offit K. Decade in review—genomics: a decade of
discovery in cancer genomics. Nat Rev Clin Oncol.
2014;11(11):632–634.
3. Offit K. The future of clinical cancer genomics.
Semin Oncol. 2016;43(5):615–622.
4. Robson ME, Bradbury AR, Arun B, et al. American
Society of Clinical Oncology Policy statement
update: genetic and genomic testing for cancer
susceptibility. J Clin Oncol. 2015;33(31):3660–3667.
5. Walsh MF, Chang VY, Kohlmann WK, et al. Recom-
mendations for Childhood cancer screening and
surveillance in DNA repair disorders. Clin Cancer
Res. 2017;23(11):e23–e31.
6. Villani A, Greer MC, Kalish JM, et al. Recommen-
dations for cancer surveillance in individuals with
RASopathies and other rare genetic conditions with
increased cancer risk. Clin Cancer Res. 2017;23(12):
e83–e90.
7. Foulkes WD, Kamihara J, Evans DGR, et al. Cancer
surveillance in Gorlin syndrome and rhabdoid tumor
predisposition syndrome. Clin Cancer Res. 2017;
23(12):e62–e67.
8. Wasserman JD, Tomlinson GE, Druker H, et al.
Multiple endocrine neoplasia and hyperparathyroid-
jaw tumor syndromes: clinical features, genetics, and
surveillance recommendations in childhood. Clin
Cancer Res. 2017;23(13):e123–e132.
9. Zon RT, Goss E, Vogel VG, et al. American Society
of Clinical Oncology policy statement: the role of the
oncologist in cancer prevention and risk assessment.
J Clin Oncol. 2009;27(6):986–993.
10. Lancaster JM, Powell CB, Chen LM, Richardson
DL. Society of Gynecologic Oncology statement
on risk assessment for inherited gynecologic cancer
predispositions. Gynecol Oncol. 2015;136(1):3–7.
11. ACOG Practice Bulletin No. 103: Hereditary
breast and ovarian cancer syndrome. Obstet Gynecol.
2009;113(4):957–966.
12. American Gastroenterological Association medical
position statement: hereditary colorectal cancer
and genetic testing. Gastroenterology. 2001;121(1):
195–197.
13. Daly MB, Pilarski R, Berry M, et al. NCCN
Guidelines Insights: Genetic/Familial High-Risk
Assessment: Breast and Ovarian, Version 2.2017.
J Natl Compr Canc Netw. 2017;15(1):9–20.
14. Johnston JJ, Rubinstein WS, Facio FM, et al.
Secondary variants in individuals undergoing exome
sequencing: screening of 572 individuals identifies
high-penetrance mutations in cancer-susceptibility
genes. Am J Hum Genet. 2012;91(1):97–108.
15. Wolf SM, Crock BN, Van Ness B, et al. Managing
incidental findings and research results in genomic
research involving biobanks and archived data sets.
Genet Med. 2012;14(4):361–384.
16. Pon JR, Marra MA. Driver and passenger mutations
in cancer. Annu Rev Pathol. 2015;10:25–50.
17. Easton DF, Pharoah PD, Antoniou AC, et al. Gene-
panel sequencing and the prediction of breast-cancer
risk. N Engl J Med. 2015;372(23):2243–2257.
18. Domchek SM, Bradbury A, Garber JE, Offit K,
Robson ME. Multiplex genetic testing for cancer
susceptibility: out on the high wire without a net?
J Clin Oncol. 2013;31(10):1267–1270.
19. Stadler ZK, Schrader KA, Vijai J, Robson ME, Offit
K. Cancer genomics and inherited risk. J Clin Oncol.
2014;32(7):687–698.
20. Mavaddat N, Pharoah PD, Michailidou K, et al.
Prediction of breast cancer risk based on profiling
with common genetic variants. J Natl Cancer Inst.
2015;107(5).
21. Kuchenbaecker KB, McGuffog L, Barrowdale D,
et al. Evaluation of polygenic risk scores for breast
and ovarian cancer risk prediction in BRCA1 and
Genetic Factors: Hereditary Cancer Predisposition Syndromes  •  CHAPTER 13 208.e1
BRCA2 mutation carriers. J Natl Cancer Inst.
2017;109(7).
22. Lecarpentier J, Silvestri V, Kuchenbaecker KB,
et al. Prediction of breast and prostate cancer risks
in male BRCA1 and BRCA2 mutation carriers
using polygenic risk scores. J Clin Oncol. 2017;
35(20):2240–2250.
23. Stadler ZK, Thom P, Robson ME, et al. Genome-
wide association studies of cancer. J Clin Oncol.
2010;28(27):4255–4267.
24. Malkin D. Li-fraumeni syndrome. Genes Cancer.
2011;2(4):475–484.
25. Lakhani VT, You YN, Wells SA. The multiple
endocrine neoplasia syndromes. Annu Rev Med.
2007;58:253–265.
26. Marx SJ. Molecular genetics of multiple endo-
crine neoplasia types 1 and 2. Nat Rev Cancer.
2005;5(5):367–375.
27. Balmer A, Zografos L, Munier F. Diagnosis and
current management of retinoblastoma. Oncogene.
2006;25(38):5341–5349.
28. Hayward NK. Genetics of melanoma predisposition.
Oncogene. 2003;22(20):3053–3062.
29. Felsani A, Mileo AM, Maresca V, Picardo M, Paggi
MG. New technologies used in the study of human
melanoma. Int Rev Cytol. 2007;261:247–286.
30. Kaelin WG. Molecular basis of the VHL hereditary
cancer syndrome. Nat Rev Cancer. 2002;2(9):
673–682.
31. Woodward ER, Maher ER. Von Hippel-Lindau
disease and endocrine tumour susceptibility. Endocr
Relat Cancer. 2006;13(2):415–425.
32. Sudarshan S, Linehan WM. Genetic basis of cancer
of the kidney. Semin Oncol. 2006;33(5):544–
551.
33. Ferner RE. Neurofibromatosis 1 and neuro-
fibromatosis 2: a twenty first century perspective.
Lancet Neurol. 2007;6(4):340–351.
34. Lee M-J, Stephenson DA. Recent developments
in neurofibromatosis type 1. Curr Opin Neurol.
2007;20(2):135–141.
35. Weitzel JN, Blazer KR, MacDonald DJ, Culver
JO, Offit K. Genetics, genomics, and cancer risk
assessment: state of the art and future directions in
the era of personalized medicine. CA Cancer J Clin.
2011;61(5):327–359.
36. Antoniou A, Pharoah PDP, Narod S, et al. Average
risks of breast and ovarian cancer associated with
BRCA1 or BRCA2 mutations detected in case series
unselected for family history: a combined analysis of
22 studies. Am J Hum Genet. 2003;72(5):1117–1130.
37. Pharoah PD, Guilford P, Caldas C. International
Gastric Cancer Linkage Consortium. Incidence
of gastric cancer and breast cancer in CDH1
(E-cadherin) mutation carriers from hereditary
diffuse gastric cancer families. Gastroenterology.
2001;121(6):1348–1353.
38. Narod SA, Offit K. Prevention and manage-
ment of hereditary breast cancer. J Clin Oncol.
2005;23(8):1656–1663.
39. Robson M, Offit K. Clinical practice. Management
of an inherited predisposition to breast cancer. N
Engl J Med. 2007;357(2):154–162.
40. Claus EB, Risch N, Thompson WD. Genetic analysis
of breast cancer in the cancer and steroid hormone
study. Am J Hum Genet. 1991;48(2):232–242.
41. Whittemore AS, Gong G, Itnyre J. Prevalence
and contribution of BRCA1 mutations in breast
cancer and ovarian cancer: results from three U.S.
population-based case-control studies of ovarian
cancer. Am J Hum Genet. 1997;60(3):496–504.
42. Ford D, Easton DF. The genetics of breast and
ovarian cancer. Br J Cancer. 1995;72(4):805–812.
43. Loveday C, Turnbull C, Ramsay E, et al. Germline
mutations in RAD51D confer susceptibility to
ovarian cancer. Nat Genet. 2011;43(9):879–882.
208.e1
44. Starink TM. Cowden’s disease: analysis of fourteen
new cases. J Am Acad Dermatol. 1984;11(6):
1127–1141.
45. Sidransky D, Tokino T, Helzlsouer K, et al. Inherited
p53 gene mutations in breast cancer. Cancer Res.
1992;52(10):2984–2986.
46. Lim W, Hearle N, Shah B, et al. Further observations
on LKB1/STK11 status and cancer risk in Peutz-
Jeghers syndrome. Br J Cancer. 2003;89(2):308–313.
47. Hearle N, Schumacher V, Menko FH, et al. Fre-
quency and spectrum of cancers in the Peutz-Jeghers
syndrome. Clin Cancer Res. 2006;12(10):3209–3215.
48. Wong MW, Nordfors C, Mossman D, et al. BRIP1,
PALB2, and RAD51C mutation analysis reveals
their relative importance as genetic susceptibility
factors for breast cancer. Breast Cancer Res Treat.
2011;127(3):853–859.
49. Antoniou AC, Casadei S, Heikkinen T, et al. Breast-
cancer risk in families with mutations in PALB2.
N Engl J Med. 2014;371(6):497–506.
50. Slater EP, Langer P, Niemczyk E, et al. PALB2
mutations in European familial pancreatic cancer
families. Clin Genet. 2010;78(5):490–494.
51. Jones S, Hruban RH, Kamiyama M, et al. Exomic
sequencing identifies PALB2 as a pancreatic cancer
susceptibility gene. Science. 2009;324(5924):217.
52. Ford D, Easton DF, Stratton M, et al. Genetic
heterogeneity and penetrance analysis of the BRCA1
and BRCA2 genes in breast cancer families. The
Breast Cancer Linkage Consortium. Am J Hum
Genet. 1998;62(3):676–689.
53. Frank TS, Deffenbaugh AM, Reid JE. Clinical
characteristics of individuals with germline mutations
in BRCA1 and of 10,000 individuals. J Clin Oncol.
2002;20(6):1480–1490.
54. Pathology of familial breast cancer: Differences
between breast cancers in carriers of BRCA1 or
BRCA2 mutations and sporadic cases. Breast Cancer
Linkage Consortium. Lancet. 1997;349(9064):
1505–1510.
55. Johannsson OT, Idvall I, Anderson C, et al. Tumour
biological features of BRCA1-induced breast and
ovarian cancer. Eur J Cancer. 1997;33(3):362–371.
56. Robson M, Rajan P, Rosen PP, et al. BRCA-associated
breast cancer: absence of a characteristic immuno-
phenotype. Cancer Res. 1998;58(9):1839–1842.
57. Foulkes WD, Stefansson IM, Chappuis PO, et al.
Germline BRCA1 mutations and a basal epithelial
phenotype in breast cancer. J Natl Cancer Inst. 2003;
95(19):1482–1485.
58. Bignon YJ, Fonck Y, Chassagne MC. Histo-
prognostic grade in tumours from families with
hereditary predisposition to breast cancer. Lancet.
1995;346(8969):258.
59. Jacquemier J, Eisinger F, Birnbaum D, Sobol H.
Histoprognostic grade in BRCA1-associated breast
cancer. Lancet. 1995;345(8963):1503.
60. Marcus JN, Watson P, Page DL, et al. Hereditary
breast cancer: pathobiology, prognosis, and BRCA1
and BRCA2 gene linkage. Cancer. 1996;77(4):
697–709.
61. Deleted in review.
62. Thompson D, Duedal S, Kirner J, et al. Cancer
risks and mortality in heterozygous ATM mutation
carriers. J Natl Cancer Inst. 2005;97(11):813–822.
63. CHEK2*1100delC and susceptibility to breast
cancer: a collaborative analysis involving 10,860
breast cancer cases and 9,065 controls from 10
studies. Am J Hum Genet. 2004;74(6):1175–1182.
64. Meijers-Heijboer H, van den Ouweland A, Klijn J,
et al. Low-penetrance susceptibility to breast cancer
due to CHEK2(*)1100delC in noncarriers of BRCA1
or BRCA2 mutations. Nat Genet. 2002;31(1):55–59.
65. Weischer M, Nordestgaard BG, Pharoah P, et al.
CHEK2*1100delC heterozygosity in women with
breast cancer associated with early death, breast
208.e2 Part I: Science and Clinical Oncology
cancer-specific death, and increased risk of a second
breast cancer. J Clin Oncol. 2012;30(35):4308–4316.
66. Kilpivaara O, Vahteristo P, Falck J, et al. CHEK2
variant I157T may be associated with increased breast
cancer risk. Int J Cancer. 2004;111(4):543–547.
67. Offit K, Pierce H, Kirchhoff T, et al. Frequency of
CHEK2*1100delC in New York breast cancer cases
and controls. BMC Med Genet. 2003;4:1.
68. Cybulski C, Wokołorczyk D, Jakubowska A, et al.
Risk of breast cancer in women with a CHEK2
mutation with and without a family history of breast
cancer. J Clin Oncol. 2011;29(28):3747–3752.
69. Chenevix-Trench G, Spurdle AB, Gatei M, et al.
Dominant negative ATM mutations in breast cancer
families. J Natl Cancer Inst. 2002;94(3):205–215.
70. Ahmed M, Rahman N. ATM and breast cancer
susceptibility. Oncogene. 2006;25(43):5906–5911.
71. Balmain A, Gray J, Ponder B. The genetics and genom-
ics of cancer. Nat Genet. 2003;33(suppl):238–244.
72. Hunter DJ, Kraft P, Jacobs KB, et al. A genome-
wide association study identifies alleles in FGFR2
associated with risk of sporadic postmenopausal
breast cancer. Nat Genet. 2007;39(7):870–874.
73. Easton DF, Pooley KA, Dunning AM, et al. Genome-
wide association study identifies novel breast
cancer susceptibility loci. Nature. 2007;447(7148):
1087–1093.
74. Tung N, Battelli C, Allen B, et al. Frequency
of mutations in individuals with breast cancer
referred for BRCA1 and BRCA2 testing using
next-generation sequencing with a 25-gene panel.
Cancer. 2015;121(1):25–33.
75. Kuchenbaecker KB, Hopper JL, Barnes DR, et al.
Risks of breast, ovarian, and contralateral breast
cancer for BRCA1 and BRCA2 mutation carriers.
JAMA. 2017;317(23):2402–2416.
76. Köbel M, Kalloger SE, Boyd N, et al. Ovarian
carcinoma subtypes are different diseases: implica-
tions for biomarker studies. PLoS Med. 2008;5(12).
77. Gilks CB, Prat J. Ovarian carcinoma pathology
and genetics: recent advances. Hum Pathol.
2009;40(9):1213–1223.
78. Kalloger SE, Köbel M, Leung S, et al. Calculator for
ovarian carcinoma subtype prediction. Mod Pathol.
2011;24(4):512–521.
79. Alsop K, Fereday S, Meldrum C, et al. BRCA muta-
tion frequency and patterns of treatment response in
BRCA mutation-positive women with ovarian cancer:
a report from the Australian Ovarian Cancer Study
Group. J Clin Oncol. 2012;30(21):2654–2663.
80. Press JZ, De Luca A, Boyd N, et al. Ovarian
carcinomas with genetic and epigenetic BRCA1
loss have distinct molecular abnormalities. BMC
Cancer. 2008;8:17.
81. McAlpine JN, Porter H, Köbel M, et al. BRCA1
and BRCA2 mutations correlate with TP53 abnor-
malities and presence of immune cell infiltrates in
ovarian high-grade serous carcinoma. Mod Pathol.
2012;25(5):740–750.
82. Schrader KA, Hurlburt J, Kalloger SE, et al.
Germline BRCA1 and BRCA2 mutations in ovarian
cancer: utility of a histology-based referral strategy.
Obstet Gynecol. 2012;120(2 Pt 1):235–240.
83. Ryan NA, Evans DG, Green K, Crosbie EJ.
Pathological features and clinical behavior of Lynch
syndrome-associated ovarian cancer. Gynecol Oncol.
2017;144(3):491–495.
84. Tung N, Domchek SM, Stadler Z, et  al.
Counselling framework for moderate-penetrance
cancer-susceptibility mutations. Nat Rev Clin Oncol.
2016;13(9):581–588.
85. Easton DF, Lesueur F, Decker B, et al. No evidence
that protein truncating variants in BRIP1 are
associated with breast cancer risk: implications for
gene panel testing. J Med Genet. 2016;53(5):298–
309.
86. Norquist BM, Harrell MI, Brady MF, et al. Inherited
mutations in women with ovarian carcinoma. JAMA
Oncol. 2016;2(4):482–490.
87. Offit K. BRCA mutation frequency and penetrance:
new data, old debate. J Natl Cancer Inst. 2006;98(23):
1675–1677.
88. Offit K, Levran O, Mullaney B, et al. Shared genetic
susceptibility to breast cancer, brain tumors, and
Fanconi anemia. J Natl Cancer Inst. 2003;95(20):
1548–1551.
89. Zhang J, Walsh MF, Wu G, et al. Germline muta-
tions in predisposition genes in pediatric cancer. N
Engl J Med. 2015;373(24):2336–2346.
90. Parsons DW, Roy A, Yang Y, et al. Diagnostic yield of
clinical tumor and germline whole-exome sequencing
for children with solid tumors. JAMA Oncol. 2016.
91. Walsh MF, Kennedy J, Harlan M, et al. Germ line
BRCA2 mutations detected in pediatric sequencing
studies impact parents’ evaluation and care. Cold
Spring Harb Mol Case Stud. 2017.
92. Casilli F, Tournier I, Sinilnikova OM, et al. The con-
tribution of germline rearrangements to the spectrum
of BRCA2 mutations. J Med Genet. 2006;43(9):
e49.
93. Woodward AM, Davis TA, Silva AGS, Kirk JA,
Leary JA, kConFab I. Large genomic rearrangements
of both BRCA2 and BRCA1 are a feature of the
inherited breast/ovarian cancer phenotype in selected
families. J Med Genet. 2005;42(5):e31.
94. Peshkin BN, DeMarco TA, Brogan BM, Lerman C,
Isaacs C. BRCA1/2 testing: complex themes in result
interpretation. J Clin Oncol. 2001;19(9):2555–2565.
95. Stoppa-Lyonnet D, Laurent-Puig P, Essioux L, et al.
BRCA1 sequence variations in 160 individuals referred
to a breast/ovarian family cancer clinic. Institut Curie
Breast Cancer Group. Am J Hum Genet. 1997;
60(5):1021–1030.
96. Venkitaraman AR. Cancer susceptibility and the
functions of BRCA1 and BRCA2. Cell. 2002;108(2):
171–182.
97. Offit K, Gilewski T, McGuire P, et al. Germline
BRCA1 185delAG mutations in Jewish women with
breast cancer. Lancet. 1996;347(9016):1643–1645.
98. Roa BB, Boyd AA, Volcik K, Richards CS. Ash-
kenazi Jewish population frequencies for common
mutations in BRCA1 and BRCA2. Nat Genet.
1996;14(2):185–187.
99. Neuhausen S, Gilewski T, Norton L, et al. Recurrent
BRCA2 6174delT mutations in Ashkenazi Jewish
women affected by breast cancer. Nat Genet. 1996;
13(1):126–128.
100. Struewing JP, Abeliovich D, Peretz T, et al. The
carrier frequency of the BRCA1 185delAG mutation
is approximately 1 percent in Ashkenazi Jewish
individuals. Nat Genet. 1995;11(2):198–200.
101. Levy-Lahad E, Catane R, Eisenberg S, et al. Founder
BRCA1 and BRCA2 mutations in Ashkenazi Jews
in Israel: frequency and differential penetrance in
ovarian cancer and in breast-ovarian cancer families.
Am J Hum Genet. 1997;60(5):1059–1067.
102. Kauff ND, Perez-Segura P, Robson ME, et al.
Incidence of non-founder BRCA1 and BRCA2
mutations in high risk Ashkenazi breast and ovarian
cancer families. J Med Genet. 2002;39(8):611–
614.
103. Walsh T, Mandell JB, Norquist BM, et al. Genetic
predisposition to breast cancer due to mutations
other than BRCA1 and BRCA2 founder alleles
among Ashkenazi Jewish Women. JAMA Oncol.
2017.
104. Szabo CI, King MC. Population genetics of BRCA1
and BRCA2. Am J Hum Genet. 1997;60(5):
1013–1020.
105. Khoo U-S, Chan KYK, Cheung ANY, et al. Recur-
rent BRCA1 and BRCA2 germline mutations in
ovarian cancer: a founder mutation of BRCA1 identi-
fied in the Chinese population. Hum Mutat. 2002;
19(3):307–308.
106. Thorlacius S, Sigurdsson S, Bjarnadottir H, et al.
Study of a single BRCA2 mutation with high carrier
frequency in a small population. Am J Hum Genet.
1997;60(5):1079–1084.
107. Erkko H, Xia B, Nikkilä J, et al. A recurrent muta-
tion in PALB2 in Finnish cancer families. Nature.
2007;446(7133):316–319.
108. Adank MA, Verhoef S, Oldenburg RA, et al. Excess
breast cancer risk in first degree relatives of CHEK2
*1100delC positive familial breast cancer cases. Eur
J Cancer. 2013;49(8):1993–1999.
109. Han FF, Guo CL, Liu LH. The effect of CHEK2
variant I157T on cancer susceptibility: evidence from
a meta-analysis. DNA Cell Biol. 2013;32(6):329–335.
110. Renwick A, Thompson D, Seal S, et al. ATM
mutations that cause ataxia-telangiectasia are
breast cancer susceptibility alleles. Nat Genet.
2006;38(8):873–875.
111. Paglia LL, Lauge A, Weber J, et al. ATM germline
mutations in women with familial breast cancer and
a relative with haematological malignancy. Breast
Cancer Res Treat. 2010;119(2):443–452.
112. Rafnar T, Gudbjartsson DF, Sulem P, et al. Mutations
in BRIP1 confer high risk of ovarian cancer. Nat
Genet. 2011;43(11):1104–1107.
113. Ramus SJ, Song H, Dicks E, et al. Germline muta-
tions in the BRIP1, BARD1, PALB2, and NBN genes
in women with ovarian cancer. J Natl Cancer Inst.
2015;107(11).
114. Song H, Dicks E, Ramus SJ, et al. Contribution of
germline mutations in the RAD51B, RAD51C, and
RAD51D genes to ovarian cancer in the population.
J Clin Oncol. 2015;33(26):2901–2907.
115. Nelson HD, Huffman LH, Fu R, Harris EL,
Force USPST. Genetic risk assessment and BRCA
mutation testing for breast and ovarian cancer
susceptibility: systematic evidence review for the
U.S. Preventive Services Task Force. Ann Intern Med.
2005;143(5):362–379.
116. National Comprehensive Cancer Centers. NCCN
Clinical Practive Guidelines in Oncology, Genetic/
Familial High-Risk Assessment: Breast and Ovarian
Version 2.2017; 2016. http://www.nccn.org/profes-
sionals/physician_gls/pdf/genetics_screening.pdf.
117. Robson ME, Offit K. Breast MRI for women
with hereditary cancer risk. JAMA. 2004;292(11):
1368–1370.
118. Ziegler LD, Kroll SS. Primary breast cancer
after prophylactic mastectomy. Am J Clin Oncol.
1991;14(5):451–454.
119. Hartmann LC, Sellers TA, Schaid DJ, et al. Efficacy
of bilateral prophylactic mastectomy in BRCA1 and
BRCA2 gene mutation carriers. J Natl Cancer Inst.
2001;93(21):1633–1637.
120. Rebbeck TR, Kauff ND, Domchek SM. Meta-
analysis of risk reduction estimates associated with
risk-reducing salpingo-oophorectomy in BRCA1
or BRCA2 mutation carriers. J Natl Cancer Inst.
2009;101(2):80–87.
121. Rebbeck TR, Friebel T, Lynch HT, et al. Bilateral
prophylactic mastectomy reduces breast cancer risk in
BRCA1 and BRCA2 mutation carriers: the PROSE
Study Group. J Clin Oncol. 2004;22(6):1055–1062.
122. Kauff ND, Domchek SM, Friebel TM, et al.
Risk-reducing salpingo-oophorectomy for the
prevention of BRCA1- and BRCA2-associated breast
and gynecologic cancer: a multicenter, prospective
study. J Clin Oncol. 2008;26(8):1331–1337.
123. Vogel VG, Costantino JP, Wickerham DL,
et al. Effects of tamoxifen vs raloxifene on the
risk of developing invasive breast cancer and
other disease outcomes: the NSABP Study of
Tamoxifen and Raloxifene (STAR) P-2 trial. JAMA.
2006;295(23):2727–2741.
124. Cuzick J, Sestak I, Forbes JF, et al. Anastrozole
for prevention of breast cancer in high-risk post-
menopausal women (IBIS-II): an international,
double-blind, randomised placebo-controlled trial.
Lancet. 2014;383(9922):1041–1048.
125. Goss PE, Ingle JN, Ales-Martinez JE, et al. Exemes-
tane for breast-cancer prevention in postmenopausal
women. N Engl J Med. 2011;364(25):2381–
2391.

126. Narod SA, Brunet JS, Ghadirian P, et al. Tamoxifen
and risk of contralateral breast cancer in BRCA1
and BRCA2 mutation carriers: a case-control study.
Hereditary Breast Cancer Clinical Study Group.
Lancet. 2000;356(9245):1876–1881.
127. King MC, Wieand S, Hale K, et al. Tamoxifen
and breast cancer incidence among women with
inherited mutations in BRCA1 and BRCA2:
National Surgical Adjuvant Breast and Bowel Project
(NSABP-P1) Breast Cancer Prevention Trial. JAMA.
2001;286(18):2251–2256.
128. Gronwald J, Tung N, Foulkes WD, et al. Tamoxifen
and contralateral breast cancer in BRCA1 and BRCA2
carriers: an update. Int J Cancer. 2006;118(9):
2281–2284.
129. Visvanathan K, Hurley P, Bantug E, et al. Use of
pharmacologic interventions for breast cancer risk
reduction: American Society of Clinical Oncology
clinical practice guideline. J Clin Oncol. 2013;31(23):
2942–2962.
130. Bourne TH, Campbell S, Reynolds KM, et al.
Screening for early familial ovarian cancer with
transvaginal ultrasonography and colour blood
flow imaging. BMJ. 1993;306(6884):1025–1029.
131. Scheuer L, Kauff N, Robson M, et al. Outcome
of preventive surgery and screening for breast and
ovarian cancer in BRCA mutation carriers. J Clin
Oncol. 2002;20(5):1260–1268.
132. Menon U, Ryan A, Kalsi J, et al. Risk algorithm
using serial biomarker measurements doubles the
number of screen-detected cancers compared with
a single-threshold rule in the United Kingdom
Collaborative Trial of Ovarian Cancer Screening.
J Clin Oncol. 2015;33(18):2062–2071.
133. U.S. Food and Drug Administration. The FDA
recommends against using screening tests for ovarian
cancer screening: FDA Safety Communication;
2016. https://www.fda.gov/MedicalDevices/Safety/
AlertsandNotices/ucm519413.htm.
134. Medeiros F, Muto MG, Lee Y, et al. The tubal fimbria
is a preferred site for early adenocarcinoma in women
with familial ovarian cancer syndrome. Am J Surg
Pathol. 2006;30(2):230–236.
135. Lee Y, Miron A, Drapkin R, et al. A candidate
precursor to serous carcinoma that originates in the
distal fallopian tube. J Pathol. 2007;211(1):26–35.
136. Finch A, Shaw P, Rosen B, Murphy J, Narod S,
Colgan T. Clinical and pathologic findings of
prophylactic salpingo-oophorectomies in 159
BRCA1 and BRCA2 carriers. Gynecol Oncol.
2006;100(1):58–64.
137. Kauff ND, Satagopan JM, Robson ME, et al.
Risk-reducing salpingo-oophorectomy in women
with a BRCA1 or BRCA2 mutation. N Engl J Med.
2002;346(21):1609–1615.
138. Rebbeck TR, Lynch HT, Neuhausen SL, et al.
Prophylactic oophorectomy in carriers of BRCA1
or BRCA2 mutations. N Engl J Med. 2002;346(21):
1616–1622.
139. Domchek SM, Friebel TM, Neuhausen SL, et al.
Mortality after bilateral salpingo-oophorectomy in
BRCA1 and BRCA2 mutation carriers: a prospec-
tive cohort study. Lancet Oncol. 2006;7(3):223–
229.
140. Struewing JP, Watson P, Easton DF, Ponder BA,
Lynch HT, Tucker MA. Prophylactic oophorectomy
in inherited breast/ovarian cancer families. J Natl
Cancer Inst. 1995;17:33–35.
141. Chen KT, Schooley JL, Flam MS. Peritoneal
carcinomatosis after prophylactic oophorectomy in
familial ovarian cancer syndrome. Obstet Gynecol.
1985;66(suppl 3):93S–94S.
142. Modan B, Hartge P, Hirsh-Yechezkel G, et al.
Parity, oral contraceptives, and the risk of ovarian
cancer among carriers and noncarriers of a BRCA1
or BRCA2 mutation. N Engl J Med. 2001;345(4):
235–240.
143. Narod SA, Risch H, Moslehi R, et al. Oral contra-
ceptives and the risk of hereditary ovarian cancer.
Genetic Factors: Hereditary Cancer Predisposition Syndromes  •  CHAPTER 13 208.e3
Hereditary Ovarian Cancer Clinical Study Group.
N Engl J Med. 1998;339(7):424–428.
144. Narod SA, Sun P, Risch HA. Hereditary Ovarian
Cancer Clinical Study Group. Ovarian cancer, oral
contraceptives, and BRCA mutations. N Engl J Med.
2001;345(23):1706–1707.
145. Narod SA, Dubé M-P, Klijn J, et al. Oral contra-
ceptives and the risk of breast cancer in BRCA1
and BRCA2 mutation carriers. J Natl Cancer Inst.
2002;94(23):1773–1779.
146. Farmer H, McCabe N, Lord CJ, et al. Targeting the
DNA repair defect in BRCA mutant cells as a thera-
peutic strategy. Nature. 2005;434(7035):917–921.
147. Bryant HE, Schultz N, Thomas HD, et al.
Specific killing of BRCA2-deficient tumours with
inhibitors of poly(ADP-ribose) polymerase. Nature.
2005;434(7035):913–917.
148. Swisher EM, Lin KK, Oza AM, et al. Rucaparib in
relapsed, platinum-sensitive high-grade ovarian carci-
noma (ARIEL2 Part 1): an international, multicentre,
open-label, phase 2 trial. Lancet Oncol. 2017;18(1):
75–87.
149. Mirza MR, Monk BJ, Herrstedt J, et  al.
Niraparib maintenance therapy in platinum-
sensitive, recurrent ovarian cancer. N Engl J Med.
2016;375(22):2154–2164.
150. Kaufman B, Shapira-Frommer R, Schmutzler
RK, et al. Olaparib monotherapy in patients with
advanced cancer and a germline BRCA1/2 mutation.
J Clin Oncol. 2015;33(3):244–250.
151. Robson M, Im SA, Senkus E, et al. Olaparib for
metastatic breast cancer in patients with a germline
BRCA mutation. N Engl J Med. 2017.
152. Eng C. Will the real Cowden syndrome please
stand up: revised diagnostic criteria. J Med Genet.
2000;37(11):828–830.
153. Zhou X-P, Marsh DJ, Morrison CD, et al. Germline
inactivation of PTEN and dysregulation of the
phosphoinositol-3-kinase/Akt pathway cause human
Lhermitte-Duclos disease in adults. Am J Hum Genet.
2003;73(5):1191–1198.
154. Eng C, Murday V, Seal S, et al. Cowden syndrome
and Lhermitte-Duclos disease in a family: a single
genetic syndrome with pleiotropy? J Med Genet.
1994;31(6):458–461.
155. Hanssen AM, Fryns JP. Cowden syndrome. J Med
Genet. 1995;32(2):117–119.
156. Fackenthal JD, Marsh DJ, Richardson AL, et al. Male
breast cancer in Cowden syndrome patients with
germline PTEN mutations. J Med Genet. 2001;38(3):
159–164.
157. Riegert-Johnson DL, Gleeson FC, Roberts M, et al.
Cancer and Lhermitte-Duclos disease are common
in Cowden syndrome patients. Hered Cancer Clin
Pract. 2010;8(1):6.
158. Tan M-H, Mester JL, Ngeow J, Rybicki LA, Orloff MS,
Eng C. Lifetime cancer risks in individuals with germ-
line PTEN mutations. Clin Cancer Res. 2012;18(2):
400–407.
159. Li J, Yen C, Liaw D, et al. PTEN, a putative
protein tyrosine phosphatase gene mutated in
human brain, breast, and prostate cancer. Science.
1997;275(5308):1943–1947.
160. Marsh DJ, Kum JB, Lunetta KL, et al. PTEN muta-
tion spectrum and genotype-phenotype correlations
in Bannayan-Riley-Ruvalcaba syndrome suggest a
single entity with Cowden syndrome. Hum Mol
Genet. 1999;8(8):1461–1472.
161. Eng C. Role of PTEN, a lipid phosphatase upstream
effector of protein kinase B, in epithelial thyroid
carcinogenesis. Ann N Y Acad Sci. 2002;968:213–
221.
162. Shiovitz S, Everett J, Huang S-C, Orloff MS, Eng
C, Gruber SB. Head circumference in the clinical
detection of PTEN hamartoma tumor syndrome
in a clinic population at high-risk of breast cancer.
Breast Cancer Res Treat. 2010;124(2):459–465.
163. Carney JA, Gordon H, Carpenter PC, Shenoy
BV, Go VL. The complex of myxomas, spotty
208.e3
pigmentation, and endocrine overactivity. Medicine
(Baltimore). 1985;64(4):270–283.
164. Cannon-Albright LA, Skolnick MH, Bishop DT, Lee
RG, Burt RW. Common inheritance of susceptibil-
ity to colonic adenomatous polyps and associated
colorectal cancers. N Engl J Med. 1988;319(9):
533–537.
165. Houlston RS, Collins A, Slack J, Morton NE.
Dominant genes for colorectal cancer are not rare.
Ann Hum Genet. 1992;56(Pt 2):99–103.
166. Marra G, Boland CR. Hereditary nonpolyposis
colorectal cancer: the syndrome, the genes, and his-
torical perspectives. J Natl Cancer Inst. 1995;87(15):
1114–1125.
167. Hampel H, Frankel WL, Martin E, et  al.
Screening for the Lynch syndrome (hereditary
nonpolyposis colorectal cancer). N Engl J Med.
2005;352(18):1851–1860.
168. Hampel H, Stephens JA, Pukkala E, et al. Cancer
risk in hereditary nonpolyposis colorectal cancer
syndrome: later age of onset. Gastroenterology.
2005;129(2):415–421.
169. Bailey-Wilson JE, Elston RC, Schuelke GS, et al.
Segregation analysis of hereditary nonpolyposis
colorectal cancer. Genet Epidemiol. 1986;3(1):27–38.
170. Scapoli C, Ponz De Leon M, Sassatelli R, et al.
Genetic epidemiology of hereditary non-polyposis
colorectal cancer syndromes in Modena, Italy: results
of a complex segregation analysis. Ann Hum Genet.
1994;58(Pt 3):275–295.
171. Vasen HF, Taal BG, Griffioen G, et al. Clinical
heterogeneity of familial colorectal cancer and its
influence on screening protocols. Gut. 1994;35(9):
1262–1266.
172. Dunlop MG, Farrington SM, Carothers AD,
et al. Cancer risk associated with germline DNA
mismatch repair gene mutations. Hum Mol Genet.
1997;6(1):105–110.
173. Aarnio M, Sankila R, Pukkala E, et al. Cancer risk
in mutation carriers of DNA-mismatch-repair genes.
Int J Cancer. 1999;81(2):214–218.
174. Watson P, Lynch HT. Cancer risk in mismatch repair
gene mutation carriers. Fam Cancer. 2001;1(1):
57–60.
175. Lu KH, Dinh M, Kohlmann W, et al. Gynecologic
cancer as a “sentinel cancer” for women with heredi-
tary nonpolyposis colorectal cancer syndrome. Obstet
Gynecol. 2005;105(3):569–574.
176. Watson P, Vasen HF, Mecklin JP, Järvinen H,
Lynch HT. The risk of endometrial cancer in
hereditary nonpolyposis colorectal cancer. Am J
Med. 1994;96(6):516–520.
177. Lynch HT, Lynch PM, Lanspa SJ, Snyder CL, Lynch
JF, Boland CR. Review of the Lynch syndrome:
history, molecular genetics, screening, differential
diagnosis, and medicolegal ramifications. Clin Genet.
2009;76(1):1–18.
178. Watson P, Lynch HT. Extracolonic cancer in
hereditary nonpolyposis colorectal cancer. Cancer.
1993;71(3):677–685.
179. Raymond VM, Everett JN, Furtado LV, et al. Adre-
nocortical carcinoma is a lynch syndrome-associated
cancer. J Clin Oncol. 2013;31(24):3012–3018.
180. Raymond VM, Mukherjee B, Wang F, et al.
Elevated risk of prostate cancer among men with
Lynch syndrome. J Clin Oncol. 2013;31(14):1713–
1718.
181. Win AK, Young JP, Lindor NM, et al. Colorectal
and other cancer risks for carriers and noncarriers
from families with a DNA mismatch repair gene
mutation: a prospective cohort study. J Clin Oncol.
2012;30(9):958–964.
182. Vasen HF, Watson P, Mecklin JP, Lynch HT. New
clinical criteria for hereditary nonpolyposis colorectal
cancer (HNPCC, Lynch syndrome) proposed by
the International Collaborative group on HNPCC.
Gastroenterology. 1999;116(6):1453–1456.
183. Rodriguez-Bigas MA, Boland CR, Hamilton SR,
et al. A National Cancer Institute Workshop on
208.e4 Part I: Science and Clinical Oncology
Hereditary Nonpolyposis Colorectal Cancer Syn-
drome: meeting highlights and Bethesda Guidelines.
J Natl Cancer Inst. 1997;89(23):1758–1762.
184. Umar A, Risinger JI, Hawk ET, Barrett JC. Testing
guidelines for hereditary non-polyposis colorectal
cancer. Nat Rev Cancer. 2004;4(2):153–158.
185. Barnetson RA, Tenesa A, Farrington SM, et al.
Identification and survival of carriers of mutations
in DNA mismatch-repair genes in colon cancer.
N Engl J Med. 2006;354(26):2751–2763.
186. Chen S, Wang W, Lee S, et al. Prediction of germline
mutations and cancer risk in the Lynch syndrome.
JAMA. 2006;296(12):1479–1487.
187. Balmaña J, Stockwell DH, Steyerberg EW, et al.
Prediction of MLH1 and MSH2 mutations in Lynch
syndrome. JAMA. 2006;296(12):1469–1478.
188. Umar A, Boland CR, Terdiman JP, et al. Revised
Bethesda Guidelines for hereditary nonpolyposis
colorectal cancer (Lynch syndrome) and microsatellite
instability. J Natl Cancer Inst. 2004;96(4):261–268.
189. Recommendations from the EGAPP Working
Group: genetic testing strategies in newly diagnosed
individuals with colorectal cancer aimed at reducing
morbidity and mortality from Lynch syndrome in
relatives. Genet Med. 2009;11(1):35–41.
190. National Comprehensive Cancer Centers. NCCN
Clinical Practice Guidelines in Oncology, Genetic/
Familial High-Risk Assessment: Colorectal Version
1.2017; 2017. https://www.nccn.org/professionals/
physician_gls/pdf/genetics_colon.pdf.
191. Leach FS, Nicolaides NC, Papadopoulos N, et al.
Mutations of a mutS homolog in hereditary nonpol-
yposis colorectal cancer. Cell. 1993;75(6):1215–1225.
192. Bronner CE, Baker SM, Morrison PT, et al. Muta-
tion in the DNA mismatch repair gene homologue
hMLH1 is associated with hereditary non-polyposis
colon cancer. Nature. 1994;368(6468):258–261.
193. Liu B, Parsons R, Papadopoulos N, et al. Analysis of
mismatch repair genes in hereditary non-polyposis
colorectal cancer patients. Nat Med. 1996;2(2):
169–174.
194. Wijnen JT, Vasen HF, Khan PM, et al. Clinical
findings with implications for genetic testing in
families with clustering of colorectal cancer. N Engl
J Med. 1998;339(8):511–518.
195. Liu T, Yan H, Kuismanen S, et al. The role of hPMS1
and hPMS2 in predisposing to colorectal cancer.
Cancer Res. 2001;61(21):7798–7802.
196. Ligtenberg MJL, Kuiper RP, Chan TL, et al.
Heritable somatic methylation and inactivation of
MSH2 in families with Lynch syndrome due to
deletion of the 3′ exons of TACSTD1. Nat Genet.
2009;41(1):112–117.
197. Papadopoulos N, Lindblom A. Molecular basis of
HNPCC: mutations of MMR genes. Hum Mutat.
1997;10(2):89–99.
198. Offit K. MSH6 mutations in hereditary nonpolyposis
colon cancer: another slice of the pie. J Clin Oncol.
2004;22(22):4449–4451.
199. Aaltonen LA, Peltomäki P, Leach FS, et al. Clues
to the pathogenesis of familial colorectal cancer.
Science. 1993;260(5109):812–816.
200. Thibodeau SN, Bren G, Schaid D. Microsatellite
instability in cancer of the proximal colon. Science.
1993;260(5109):816–819.
201. Cunningham JM, Christensen ER, Tester DJ, et al.
Hypermethylation of the hMLH1 promoter in colon
cancer with microsatellite instability. Cancer Res.
1998;58(15):3455–3460.
202. Cunningham JM, Kim CY, Christensen ER, et al.
The frequency of hereditary defective mismatch
repair in a prospective series of unselected colorectal
carcinomas. Am J Hum Genet. 2001;69(4):780–790.
203. Ricciardone MD, Ozçelik T, Cevher B, et al. Human
MLH1 deficiency predisposes to hematological
malignancy and neurofibromatosis type 1. Cancer
Res. 1999;59(2):290–293.
204. Wang Q, Lasset C, Desseigne F, et  al.
Neurofibromatosis and early onset of cancers
in hMLH1-deficient children. Cancer Res.
1999;59(2):294–297.
205. Van Galen P, Perrier R, Bernier FP. Late presentation
of cancer in compound heterozygote PMS2 mutation
carrier. Hered Cancer Clin Pract. 2011;9(suppl 1):
P38.
206. Durno CA, Sherman PM, Aronson M, et al. Phe-
notypic and genotypic characterisation of biallelic
mismatch repair deficiency (BMMR-D) syndrome.
Eur J Cancer. 2015;51(8):977–983.
207. Järvinen HJ, Aarnio M, Mustonen H, et al. Con-
trolled 15-year trial on screening for colorectal cancer
in families with hereditary nonpolyposis colorectal
cancer. Gastroenterology. 2000;118(5):829–834.
208. Renkonen-Sinisalo L, Aarnio M, Mecklin JP, Järvinen
HJ. Surveillance improves survival of colorectal cancer
in patients with hereditary nonpolyposis colorectal
cancer. Cancer Detect Prev. 2000;24(2):137–142.
209. Lindor NM, Petersen GM, Hadley DW, et al.
Recommendations for the care of individuals with
an inherited predisposition to Lynch syndrome: a
systematic review. JAMA. 2006;296(12):1507–1517.
210. Järvinen HJ, Aarnio M. Surveillance on mutation
carriers of DNA mismatch repair genes. Ann Chir
Gynaecol. 2000;89(3):207–210.
211. Syngal S, Weeks JC, Schrag D, Garber JE, Kuntz
KM. Benefits of colonoscopic surveillance and
prophylactic colectomy in patients with hereditary
nonpolyposis colorectal cancer mutations. Ann Intern
Med. 1998;129(10):787–796.
212. Schmeler KM, Lynch HT, Chen L-M, et al. Prophy-
lactic surgery to reduce the risk of gynecologic cancers
in the Lynch syndrome. N Engl J Med. 2006;354(3):
261–269.
213. Offit K, Kauff ND. Reducing the risk of gynecologic
cancer in the Lynch syndrome. N Engl J Med. 2006;
354(3):293–295.
214. Parry S, Win AK, Parry B, et al. Metachronous
colorectal cancer risk for mismatch repair gene
mutation carriers: the advantage of more extensive
colon surgery. Gut. 2011;60(7):950–957.
215. Gryfe R, Kim H, Hsieh ET, et al. Tumor microsatel-
lite instability and clinical outcome in young patients
with colorectal cancer. N Engl J Med. 2000;342(2):
69–77.
216. Popat S, Hubner R, Houlston RS. Systematic review
of microsatellite instability and colorectal cancer
prognosis. J Clin Oncol. 2005;23(3):609–618.
217. Sargent DJ, Marsoni S, Monges G, et al. Defective
mismatch repair as a predictive marker for lack of
efficacy of fluorouracil-based adjuvant therapy in
colon cancer. J Clin Oncol. 2010;28(20):3219–3226.
218. Ribic CM, Sargent DJ, Moore MJ, et al. Tumor
microsatellite-instability status as a predictor of
benefit from fluorouracil-based adjuvant chemo-
therapy for colon cancer. N Engl J Med. 2003;349(3):
247–257.
219. Le DT, Uram JN, Wang H, et al. PD-1 Blockade
in tumors with mismatch-repair deficiency. N Engl
J Med. 2015;372(26):2509–2520.
220. Le DT, Durham JN, Smith KN, et al. Mismatch-
repair deficiency predicts response of solid tumors
to PD-1 blockade. Science. 2017.
221. Bouffet E, Larouche V, Campbell BB, et al.
Immune checkpoint inhibition for hypermutant
glioblastoma multiforme resulting from germline
biallelic mismatch repair deficiency. J Clin Oncol.
2016;34(19):2206–2211.
222. Petersen GM, Slack J, Nakamura Y. Screening
guidelines and premorbid diagnosis of familial
adenomatous polyposis using linkage. Gastroenterol-
ogy. 1991;100(6):1658–1664.
223. Galiatsatos P, Foulkes WD. Familial adenomatous
polyposis. Am J Gastroenterol. 2006;101(2):385–398.
224. Campbell WJ, Spence RA, Parks TG. Familial adeno-
matous polyposis. Br J Surg. 1994;81(12):1722–
1733.
225. Bianchi LK, Burke CA, Bennett AE, Lopez R, Hasson
H, Church JM. Fundic gland polyp dysplasia is
common in familial adenomatous polyposis. Clin
Gastroenterol Hepatol. 2008;6(2):180–185.
226. Lynch HT, Snyder C, Davies JM, et al. FAP, gastric
cancer, and genetic counseling featuring children
and young adults: a family study and review. Fam
Cancer. 2010;9(4):581–588.
227. Groves CJ, Saunders BP, Spigelman AD, Phillips
RKS. Duodenal cancer in patients with familial
adenomatous polyposis (FAP): results of a 10 year
prospective study. Gut. 2002;50(5):636–641.
228. Half E, Bercovich D, Rozen P. Familial adenomatous
polyposis. Orphanet J Rare Dis. 2009;4:22.
229. Kinzler KW, Nilbert MC, Su LK, et al. Identification
of FAP locus genes from chromosome 5q21. Science.
1991;253(5020):661–665.
230. Vasen HFA, Möslein G, Alonso A, et al. Guidelines
for the clinical management of familial adenomatous
polyposis (FAP). Gut. 2008;57(5):704–713.
231. Bisgaard ML, Fenger K, Bülow S, Niebuhr E,
Mohr J. Familial adenomatous polyposis (FAP):
frequency, penetrance, and mutation rate. Hum
Mutat. 1994;3(2):121–125.
232. Aretz S, Stienen D, Friedrichs N, et al. Somatic APC
mosaicism: a frequent cause of familial adenomatous
polyposis (FAP). Hum Mutat. 2007;28(10):985–992.
233. Nieuwenhuis MH, Vasen HFA. Correlations between
mutation site in APC and phenotype of familial
adenomatous polyposis (FAP): a review of the litera-
ture. Crit Rev Oncol Hematol. 2007;61(2):153–161.
234. Bertario L, Russo A, Sala P, et al. Multiple approach
to the exploration of genotype-phenotype correla-
tions in familial adenomatous polyposis. J Clin Oncol.
2003;21(9):1698–1707.
235. Spirio L, Olschwang S, Groden J, et al. Alleles of the
APC gene: an attenuated form of familial polyposis.
Cell. 1993;75(5):951–957.
236. Sieber OM, Lipton L, Crabtree M, et al. Multiple
colorectal adenomas, classic adenomatous polyposis,
and germ-line mutations in MYH. N Engl J Med.
2003;348(9):791–799.
237. Offit K, Kohut K, Clagett B, et al. Cancer genetic
testing and assisted reproduction. J Clin Oncol.
2006;24(29):4775–4782.
238. Giardiello FM, Hamilton SR, Krush AJ, et al. Treat-
ment of colonic and rectal adenomas with sulindac
in familial adenomatous polyposis. N Engl J Med.
1993;328(18):1313–1316.
239. Nugent KP, Farmer KC, Spigelman AD, Williams
CB, Phillips RK. Randomized controlled trial
of the effect of sulindac on duodenal and rectal
polyposis and cell proliferation in patients with
familial adenomatous polyposis. Br J Surg. 1993;
80(12):1618–1619.
240. Steinbach G, Lynch PM, Phillips RK, et al. The
effect of celecoxib, a cyclooxygenase-2 inhibitor,
in familial adenomatous polyposis. N Engl J Med.
2000;342(26):1946–1952.
241. Parc YR, Olschwang S, Desaint B, Schmitt G, Parc
RG, Tiret E. Familial adenomatous polyposis: preva-
lence of adenomas in the ileal pouch after restorative
proctocolectomy. Ann Surg. 2001;233(3):360–364.
242. Guillem JG, Wood WC, Moley JF, et al. ASCO/
SSO review of current role of risk-reducing surgery
in common hereditary cancer syndromes. J Clin
Oncol. 2006;24(28):4642–4660.
243. King JE, Dozois RR, Lindor NM, Ahlquist DA. Care
of patients and their families with familial adenoma-
tous polyposis. Mayo Clin Proc. 2000;75(1):57–67.
244. Knudsen AL, Bisgaard ML, Bülow S. Attenuated
familial adenomatous polyposis (AFAP). A review
of the literature. Fam Cancer. 2003;2(1):43–55.
245. Brensinger JD, Laken SJ, Luce MC, et al. Variable
phenotype of familial adenomatous polyposis in
pedigrees with 3′ mutation in the APC gene. Gut.
1998;43(4):548–552.
246. Bussey HJ. Familial polyposis coli. Pathol Annu.
1979;14(Pt 1):61–81.
247. Al-Tassan N, Chmiel NH, Maynard J, et al.
Inherited variants of MYH associated with somatic

G:C–>T:A mutations in colorectal tumors. Nat
Genet. 2002;30(2):227–232.
248. Goodenberger M, Lindor NM. Lynch syndrome
and MYH-associated polyposis: review and testing
strategy. J Clin Gastroenterol. 2011;45(6):488–500.
249. Nielsen M, Morreau H, Vasen HFA, Hes FJ.
MUTYH-associated polyposis (MAP). Crit Rev
Oncol Hematol. 2011;79(1):1–16.
250. Vogt S, Jones N, Christian D, et al. Expanded
extracolonic tumor spectrum in MUTYH-associated
polyposis. Gastroenterology. 2009;137(6):1976–1985.
e1971.
251. Aretz S, Uhlhaas S, Goergens H, et al. MUTYH-
associated polyposis: 70 of 71 patients with biallelic
mutations present with an attenuated or atypical
phenotype. Int J Cancer. 2006;119(4):807–814.
252. Balaguer F, Castellví-Bel S, Castells A, et al. Identifi-
cation of MYH mutation carriers in colorectal cancer:
a multicenter, case-control, population-based study.
Clin Gastroenterol Hepatol. 2007;5(3):379–387.
253. Jones N, Vogt S, Nielsen M, et al. Increased
colorectal cancer incidence in obligate carriers of
heterozygous mutations in MUTYH. Gastroenterol-
ogy. 2009;137(2):489–494, 494.e481; quiz 725–486.
254. Jones S, Emmerson P, Maynard J, et al. Biallelic
germline mutations in MYH predispose to multiple
colorectal adenoma and somatic G:C–>T:A muta-
tions. Hum Mol Genet. 2002;11(23):2961–2967.
255. Russell AM, Zhang J, Luz J, et al. Prevalence of
MYH germline mutations in Swiss APC mutation-
negative polyposis patients. Int J Cancer. 2006;
118(8):1937–1940.
256. Jo W-S, Bandipalliam P, Shannon KM, et al.
Correlation of polyp number and family history
of colon cancer with germline MYH mutations.
Clin Gastroenterol Hepatol. 2005;3(10):1022–1028.
257. Eliason K, Hendrickson BC, Judkins T, et al. The
potential for increased clinical sensitivity in genetic
testing for polyposis colorectal cancer through the
analysis of MYH mutations in North American
patients. J Med Genet. 2005;42(1):95–96.
258. Fleischmann C, Peto J, Cheadle J, Shah B,
Sampson J, Houlston RS. Comprehensive analysis
of the contribution of germline MYH variation to
early-onset colorectal cancer. Int J Cancer. 2004;
109(4):554–558.
259. Nielsen M, Franken PF, Reinards THCM, et al.
Multiplicity in polyp count and extracolonic
manifestations in 40 Dutch patients with MYH
associated polyposis coli (MAP). J Med Genet. 2005;
42(9):e54.
260. Palles C, Cazier JB, Howarth KM, et al. Germline
mutations affecting the proofreading domains of
POLE and POLD1 predispose to colorectal adenomas
and carcinomas. Nat Genet. 2013;45(2):136–144.
261. Bellido F, Pineda M, Aiza G, et al. POLE and
POLD1 mutations in 529 kindred with familial
colorectal cancer and/or polyposis: review of reported
cases and recommendations for genetic testing and
surveillance. Genet Med. 2016;18(4):325–332.
262. Jaeger E, Leedham S, Lewis A, et al. Hereditary
mixed polyposis syndrome is caused by a 40-kb
upstream duplication that leads to increased and
ectopic expression of the BMP antagonist GREM1.
Nat Genet. 2012;44(6):699–703.
263. Cao X, Eu KW, Kumarasinghe MP, Li HH, Loi C,
Cheah PY. Mapping of hereditary mixed polyposis
syndrome (HMPS) to chromosome 10q23 by
genomewide high-density single nucleotide polymor-
phism (SNP) scan and identification of BMPR1A
loss of function. J Med Genet. 2006;43(3):e13.
264. Cybulski C, Górski B, Huzarski T, et al. CHEK2 is
a multiorgan cancer susceptibility gene. Am J Hum
Genet. 2004;75(6):1131–1135.
265. Laken SJ, Petersen GM, Gruber SB, et al. Familial
colorectal cancer in Ashkenazim due to a hypermu-
table tract in APC. Nat Genet. 1997;17(1):79–83.
266. Rio Frio T, Lavoie J, Hamel N, et al. Homozygous
BUB1B mutation and susceptibility to gastrointestinal
Genetic Factors: Hereditary Cancer Predisposition Syndromes  •  CHAPTER 13 208.e5
neoplasia. N Engl J Med. 2010;363(27):2628–
2637.
267. Fitzgerald RC, Hardwick R, Huntsman D, et al.
Hereditary diffuse gastric cancer: updated consensus
guidelines for clinical management and directions for
future research. J Med Genet. 2010;47(7):436–444.
268. Caldas C, Carneiro F, Lynch HT, et al. Familial
gastric cancer: overview and guidelines for manage-
ment. J Med Genet. 1999;36(12):873–880.
269. Guilford P, Hopkins J, Harraway J, et al. E-cadherin
germline mutations in familial gastric cancer. Nature.
1998;392(6674):402–405.
270. Brooks-Wilson AR, Kaurah P, Suriano G, et al.
Germline E-cadherin mutations in hereditary diffuse
gastric cancer: assessment of 42 new families and
review of genetic screening criteria. J Med Genet.
2004;41(7):508–517.
271. Kaurah P, MacMillan A, Boyd N, et al. Founder and
recurrent CDH1 mutations in families with heredi-
tary diffuse gastric cancer. JAMA. 2007;297(21):
2360–2372.
272. Oliveira C, Senz J, Kaurah P, et al. Germline CDH1
deletions in hereditary diffuse gastric cancer families.
Hum Mol Genet. 2009;18(9):1545–1555.
273. Carneiro F, Oliveira C, Suriano G, Seruca R.
Molecular pathology of familial gastric cancer, with
an emphasis on hereditary diffuse gastric cancer.
J Clin Pathol. 2008;61(1):25–30.
274. Strong VE, Gholami S, Shah MA, et al. Total
gastrectomy for hereditary diffuse gastric cancer at
a single center: postsurgical outcomes in 41 patients.
Ann Surg. 2016.
275. Howlader N, Noone AM, Krapcho M, Miller D,
Bishop K, Kosary CL, et al, eds. SEER Cancer
Statistics Review, 1975-2014. Bethesda, MD:
National Cancer Institute. https://seer.cancer.gov/
csr/1975_2014/, based on November 2016 SEER
data submission, posted to the SEER web site, April
2017.
276. Klein AP. Genetic susceptibility to pancreatic cancer.
Mol Carcinog. 2012;51(1):14–24.
277. Goggins M, Schutte M, Lu J, et al. Germline
BRCA2 gene mutations in patients with appar-
ently sporadic pancreatic carcinomas. Cancer Res.
1996;56(23):5360–5364.
278. Hahn SA, Greenhalf B, Ellis I, et al. BRCA2 germline
mutations in familial pancreatic carcinoma. J Natl
Cancer Inst. 2003;95(3):214–221.
279. Murphy KM, Brune KA, Griffin C, et al. Evaluation
of candidate genes MAP2K4, MADH4, ACVR1B,
and BRCA2 in familial pancreatic cancer: del-
eterious BRCA2 mutations in 17%. Cancer Res.
2002;62(13):3789–3793.
280. Cancer risks in BRCA2 mutation carriers. J Natl
Cancer Inst. 1999;91(15):1310–1316.
281. Risch HA, McLaughlin JR, Cole DEC, et al. Popula-
tion BRCA1 and BRCA2 mutation frequencies and
cancer penetrances: a kin-cohort study in Ontario,
Canada. J Natl Cancer Inst. 2006;98(23):1694–1706.
282. Ferrone CR, Levine DA, Tang LH, et al. BRCA
germline mutations in Jewish patients with pancreatic
adenocarcinoma. J Clin Oncol. 2009;27(3):433–438.
283. Thompson D, Easton DF, Breast Cancer Linkage
C. Cancer incidence in BRCA1 mutation carriers.
J Natl Cancer Inst. 2002;94(18):1358–1365.
284. Stadler ZK, Salo-Mullen E, Patil SM, et al. Prevalence
of BRCA1 and BRCA2 mutations in Ashkenazi
Jewish families with breast and pancreatic cancer.
Cancer. 2012;118(2):493–499.
285. Rogers CD, van der Heijden MS, Brune K, et al. The
genetics of FANCC and FANCG in familial pancre-
atic cancer. Cancer Biol Ther. 2004;3(2):167–169.
286. Tischkowitz MD, Sabbaghian N, Hamel N, et al.
Analysis of the gene coding for the BRCA2-
interacting protein PALB2 in familial and sporadic
pancreatic cancer. Gastroenterology. 2009;137(3):
1183–1186.
287. Schneider R, Slater EP, Sina M, et al. German
national case collection for familial pancreatic cancer
208.e5
(FaPaCa): ten years experience. Fam Cancer. 2011;
10(2):323–330.
288. Kastrinos F, Mukherjee B, Tayob N, et al. Risk of
pancreatic cancer in families with Lynch syndrome.
JAMA. 2009;302(16):1790–1795.
289. Giardiello FM, Brensinger JD, Tersmette AC, et al.
Very high risk of cancer in familial Peutz-Jeghers
syndrome. Gastroenterology. 2000;119(6):1447–1453.
290. Lynch HT, Fusaro RM, Lynch JF, Brand R. Pancreatic
cancer and the FAMMM syndrome. Fam Cancer.
2008;7(1):103–112.
291. Lowenfels AB, Maisonneuve P, DiMagno EP, et al.
Hereditary pancreatitis and the risk of pancreatic
cancer. International Hereditary Pancreatitis Study
Group. J Natl Cancer Inst. 1997;89(6):442–446.
292. Rebours V, Boutron-Ruault M-C, Schnee M, et al.
Risk of pancreatic adenocarcinoma in patients with
hereditary pancreatitis: a national exhaustive series.
Am J Gastroenterol. 2008;103(1):111–119.
293. Vasen H, Ibrahim I, Ponce CG, et al. Benefit of
surveillance for pancreatic cancer in high-risk indi-
viduals: outcome of long-term prospective follow-up
studies from three European expert centers. J Clin
Oncol. 2016;34(17):2010–2019.
294. Atherton DJ, Pitcher DW, Wells RS, MacDonald
DM. A syndrome of various cutaneous pigmented
lesions, myxoid neurofibromata and atrial myxoma:
the NAME syndrome. Br J Dermatol. 1980;103(4):
421–429.
295. Rhodes AR, Silverman RA, Harrist TJ, Perez-Atayde
AR. Mucocutaneous lentigines, cardiomucocutaneous
myxomas, and multiple blue nevi: the “LAMB”
syndrome. J Am Acad Dermatol. 1984;10(1):72–82.
296. Stratakis CA, Carney JA, Lin JP, et al. Carney
complex, a familial multiple neoplasia and lentigi-
nosis syndrome. Analysis of 11 kindreds and linkage
to the short arm of chromosome 2. J Clin Invest.
1996;97(3):699–705.
297. Stratakis CA, Kirschner LS, Carney JA. Clinical and
molecular features of the Carney complex: diagnostic
criteria and recommendations for patient evalua-
tion. J Clin Endocrinol Metab. 2001;86(9):4041–
4046.
298. Bertherat J, Horvath A, Groussin L, et al. Mutations
in regulatory subunit type 1A of cyclic adenosine
5′-monophosphate-dependent protein kinase
(PRKAR1A): phenotype analysis in 353 patients and
80 different genotypes. J Clin Endocrinol Metab. 2009;
94(6):2085–2091.
299. Courcoutsakis NA, Chow CK, Shawker TH,
Carney JA, Stratakis CA. Syndrome of spotty skin
pigmentation, myxomas, endocrine overactivity, and
schwannomas (Carney complex): breast imaging
findings. Radiology. 1997;205(1):221–227.
300. Premkumar A, Stratakis CA, Shawker TH, Papa-
nicolaou DA, Chrousos GP. Testicular ultrasound
in Carney complex: report of three cases. J Clin
Ultrasound. 1997;25(4):211–214.
301. Stratakis CA, Sarlis N, Kirschner LS, et al. Paradoxi-
cal response to dexamethasone in the diagnosis of
primary pigmented nodular adrenocortical disease.
Ann Intern Med. 1999;131(8):585–591.
302. Stratakis CA, Courcoutsakis NA, Abati A, et al.
Thyroid gland abnormalities in patients with the
syndrome of spotty skin pigmentation, myxomas,
endocrine overactivity, and schwannomas (Carney
complex). J Clin Endocrinol Metab. 1997;82(7):
2037–2043.
303. Stratakis CA, Papageorgiou T, Premkumar A, et al.
Ovarian lesions in Carney complex: clinical genetics
and possible predisposition to malignancy. J Clin
Endocrinol Metab. 2000;85(11):4359–4366.
304. Kirschner LS, Sandrini F, Monbo J, Lin JP, Carney
JA, Stratakis CA. Genetic heterogeneity and
spectrum of mutations of the PRKAR1A gene in
patients with the carney complex. Hum Mol Genet.
2000;9(20):3037–3046.
305. Casey M, Vaughan CJ, He J, et al. Mutations in
the protein kinase A R1alpha regulatory subunit
208.e6 Part I: Science and Clinical Oncology
cause familial cardiac myxomas and Carney complex.
J Clin Invest. 2000;106(5):R31–R38.
306. Boikos SA, Stratakis CA. Carney complex: the first
20 years. Curr Opin Oncol. 2007;19(1):24–29.
307. Milanesi U, Mangili F, Milanesi I. Flow-cytometric
study of familial paragangliomas of the carotid body.
Acta Otorhinolaryngol Ital. 1994;14(4):439–447.
308. Netterville JL, Reilly KM, Robertson D, Reiber ME,
Armstrong WB, Childs P. Carotid body tumors: a
review of 30 patients with 46 tumors. Laryngoscope.
1995;105(2):115–126.
309. van der Mey A, Maaswinkel-Mooy PD, Cornelisse
CJ, Schmidt P, van de Kamp J. Genomic imprinting
in hereditary glomus tumours: evidence for new
genetic theory. Lancet. 1989;2:1291–1294.
310. Baysal BE, Ferrell RE, Willett-Brozick JE, et al.
Mutations in SDHD, a mitochondrial complex
II gene, in hereditary paraganglioma. Science.
2000;287(5454):848–851.
311. Hao H-X, Khalimonchuk O, Schraders M, et al.
SDH5, a gene required for flavination of succinate
dehydrogenase, is mutated in paraganglioma. Science.
2009;325(5944):1139–1142.
312. Hensen EF, Jordanova ES, van Minderhout IJHM,
et al. Somatic loss of maternal chromosome 11 causes
parent-of-origin-dependent inheritance in SDHD-
linked paraganglioma and phaeochromocytoma
families. Oncogene. 2004;23(23):4076–4083.
313. Neumann HPH, Pawlu C, Peczkowska M, et al.
Distinct clinical features of paraganglioma syndromes
associated with SDHB and SDHD gene mutations.
JAMA. 2004;292(8):943–951.
314. Neumann HPH, Bausch B, McWhinney SR, et al.
Germ-line mutations in nonsyndromic pheochromo-
cytoma. N Engl J Med. 2002;346(19):1459–1466.
315. Bryant J, Farmer J, Kessler LJ, Townsend RR,
Nathanson KL. Pheochromocytoma: the expanding
genetic differential diagnosis. J Natl Cancer Inst.
2003;95(16):1196–1204.
316. Hensen EF, van Duinen N, Jansen JC, et al. High
prevalence of founder mutations of the succinate
dehydrogenase genes in the Netherlands. Clin Genet.
2012;81(3):284–288.
317. Farndon JR, Leight GS, Dilley WG, et al. Familial
medullary thyroid carcinoma without associated endo-
crinopathies: a distinct clinical entity. Br J Surg. 1986;
73(4):278–281.
318. Moline J, Eng C. Multiple endocrine neoplasia type
2: an overview. Genet Med. 2011;13(9):755–764.
319. Vasen HF, van der Feltz M, Raue F, et al. The
natural course of multiple endocrine neoplasia
type IIb. A study of 18 cases. Arch Intern Med.
1992;152(6):1250–1252.
320. Stoffer SS, Van Dyke DL, Bach JV, Szpunar W,
Weiss L. Familial papillary carcinoma of the thyroid.
Am J Med Genet. 1986;25(4):775–782.
321. Mulligan LM, Eng C, Healey CS, et al. Specific
mutations of the RET proto-oncogene are related
to disease phenotype in MEN 2A and FMTC. Nat
Genet. 1994;6(1):70–74.
322. Eng C, Mulligan LM, Smith DP, et al. Low
frequency of germline mutations in the RET
proto-oncogene in patients with apparently sporadic
medullary thyroid carcinoma. Clin Endocrinol (Oxf).
1995;43(1):123–127.
323. Mulligan LM, Kwok JB, Healey CS, et al. Germ-
line mutations of the RET proto-oncogene in
multiple endocrine neoplasia type 2A. Nature. 1993;
363(6428):458–460.
324. Donis-Keller H, Dou S, Chi D, et al. Mutations in
the RET proto-oncogene are associated with MEN
2A and FMTC. Hum Mol Genet. 1993;2(7):851–
856.
325. Schuffenecker I, Ginet N, Goldgar D, et al.
Prevalence and parental origin of de novo RET
mutations in multiple endocrine neoplasia type
2A and familial medullary thyroid carcinoma. Le
Groupe d’Etude des Tumeurs a Calcitonine. Am J
Hum Genet. 1997;60(1):233–237.
326. Decker RA, Peacock ML, Borst MJ, Sweet JD,
Thompson NW. Progress in genetic screening of
multiple endocrine neoplasia type 2A: is calcitonin
testing obsolete? Surgery. 1995;118(2):257–263,
discussion 263.
327. Lips CJ, Landsvater RM, Höppener JW, et al.
Clinical screening as compared with DNA analysis
in families with multiple endocrine neoplasia type
2A. N Engl J Med. 1994;331(13):828–835.
328. Wells SA, Chi DD, Toshima K, et al. Predictive DNA
testing and prophylactic thyroidectomy in patients
at risk for multiple endocrine neoplasia type 2A.
Ann Surg. 1994;220(3):237–247, discussion 247.
329. Eng C, Clayton D, Schuffenecker I, et al. The rela-
tionship between specific RET proto-oncogene muta-
tions and disease phenotype in multiple endocrine
neoplasia type 2. International RET mutation con-
sortium analysis. JAMA. 1996;276(19):1575–1579.
330. Leboulleux S, Travagli JP, Caillou B, et al. Medullary
thyroid carcinoma as part of a multiple endocrine
neoplasia type 2B syndrome: influence of the stage
on the clinical course. Cancer. 2002;94(1):44–50.
331. Smith DP, Houghton C, Ponder BA. Germline
mutation of RET codon 883 in two cases of de
novo MEN 2B. Oncogene. 1997;15(10):1213–1217.
332. American Thyroid Association Guidelines Task Force,
Kloos RT, Eng C, et al. Medullary thyroid cancer:
management guidelines of the American Thyroid
Association. Thyroid. 2009;19(6):565–612.
333. Lairmore TC, Ball DW, Baylin SB, Wells SA.
Management of pheochromocytomas in patients
with multiple endocrine neoplasia type 2 syndromes.
Ann Surg. 1993;217(6):595–601, discussion 601.
334. Wells SA, Robinson BG, Gagel RF, et al. Vandetanib
in patients with locally advanced or metastatic
medullary thyroid cancer: a randomized, double-
blind phase III trial. J Clin Oncol. 2012;30(2):134–
141.
335. Schlumberger MJ, Elisei R, Bastholt L, et al. Phase II
study of safety and efficacy of motesanib in patients
with progressive or symptomatic, advanced or
metastatic medullary thyroid cancer. J Clin Oncol.
2009;27(23):3794–3801.
336. Lam ET, Ringel MD, Kloos RT, et al. Phase II clini-
cal trial of sorafenib in metastatic medullary thyroid
cancer. J Clin Oncol. 2010;28(14):2323–2330.
337. Mologni L. Development of RET kinase inhibi-
tors for targeted cancer therapy. Curr Med Chem.
2011;18(2):162–175.
338. Phay JE, Shah MH. Targeting RET receptor
tyrosine kinase activation in cancer. Clin Cancer
Res. 2010;16(24):5936–5941.
339. Gorlin RJ, Goltz RW. Multiple nevoid basal-cell
epithelioma, jaw cysts and bifid rib: a syndrome.
N Engl J Med. 1960;262:908–912.
340. Gorlin RJ. Nevoid basal-cell carcinoma syndrome.
Medicine (Baltimore). 1987;66(2):98–113.
341. Strong LC. Genetic and environmental interactions.
Cancer. 1977;40(suppl 4):1861–1866.
342. Evans DG, Farndon PA, Burnell LD, Gattamaneni
HR, Birch JM. The incidence of Gorlin syndrome
in 173 consecutive cases of medulloblastoma. Br J
Cancer. 1991;64(5):959–961.
343. Evans DG, Ladusans EJ, Rimmer S, Burnell
LD, Thakker N, Farndon PA. Complications
of the naevoid basal cell carcinoma syndrome:
results of a population based study. J Med Genet.
1993;30(6):460–464.
344. Kimonis VE, Goldstein AM, Pastakia B, et al.
Clinical manifestations in 105 persons with nevoid
basal cell carcinoma syndrome. Am J Hum Genet.
1997;69(3):299–308.
345. Bree AF, Shah MR, Group BC. Consensus statement
from the first international colloquium on basal
cell nevus syndrome (BCNS). Am J Med Genet A.
2011;155A(9):2091–2097.
346. Cowan R, Hoban P, Kelsey A, Birch JM,
Gattamaneni R, Evans DG. The gene for the
naevoid basal cell carcinoma syndrome acts as a
tumour-suppressor gene in medulloblastoma. Br J
Cancer. 1997;76(2):141–145.
347. Amlashi SFA, Riffaud L, Brassier G, Morandi X.
Nevoid basal cell carcinoma syndrome: relation
with desmoplastic medulloblastoma in infancy. A
population-based study and review of the literature.
Cancer. 2003;98(3):618–624.
348. Johnson RL, Rothman AL, Xie J, et  al.
Human homolog of patched, a candidate gene
for the basal cell nevus syndrome. Science.
1996;272(5268):1668–1671.
349. Wicking C, Shanley S, Smyth I, et al. Most germ-
line mutations in the nevoid basal cell carcinoma
syndrome lead to a premature termination of the
PATCHED protein, and no genotype-phenotype
correlations are evident. Am J Hum Genet. 1997;
60(1):21–26.
350. Von Hoff DD, LoRusso PM, Rudin CM, et al. Inhi-
bition of the hedgehog pathway in advanced basal-cell
carcinoma. N Engl J Med. 2009;361(12):1164–1172.
351. Tate G, Li M, Suzuki T, Mitsuya T. A new germline
mutation of the PTCH gene in a Japanese patient
with nevoid basal cell carcinoma syndrome
associated with meningioma. Jpn J Clin Oncol.
2003;33(1):47–50.
352. Boutet N, Bignon Y-J, Drouin-Garraud V,
et al. Spectrum of PTCH1 mutations in French
patients with Gorlin syndrome. J Invest Dermatol.
2003;121(3):478–481.
353. Lindstrom E, Shimokawa T, Toftgard R, Zaphi-
ropoulos PG. PTCH mutations: distribution and
analyses. Hum Mutat. 2006;27(3):215–219.
354. Anand VK, Arrowood JP, Krolls SO. Malignant
potential of the odontogenic keratocyst. Otolaryngol
Head Neck Surg. 1994;111(1):124–129.
355. Bitar GJ, Herman CK, Dahman MI, Hoard MA.
Basal cell nevus syndrome: guidelines for early detec-
tion. Am Fam Physician. 2002;65(12):2501–2504.
356. National Cancer Institute. FDA approval for vis-
modegib; 2012. http://www.cancer.gov/cancertopics/
druginfo/fda-vismodegib.
357. Food and Drug Administration. Vismodegib; 2012.
http://www.fda.gov/Drugs/InformationOnDrugs/
ApprovedDrugs/ucm289571.htm.
358. Tang JY, Mackay-Wiggan JM, Aszterbaum M,
et al. Inhibiting the hedgehog pathway in patients
with the basal-cell nevus syndrome. N Engl J Med.
2012;366(23):2180–2188.
359. Launonen V, Vierimaa O, Kiuru M, et  al.
Inherited susceptibility to uterine leiomyomas
and renal cell cancer. Proc Natl Acad Sci USA.
2001;98(6):3387–3392.
360. Alam NA, Rowan AJ, Wortham NC, et al.
Genetic and functional analyses of FH mutations
in multiple cutaneous and uterine leiomyomatosis,
hereditary leiomyomatosis and renal cancer, and
fumarate hydratase deficiency. Hum Mol Genet.
2003;12(11):1241–1252.
361. Linehan WM, Pinto PA, Srinivasan R, et al. Identi-
fication of the genes for kidney cancer: opportunity
for disease-specific targeted therapeutics. Clin Cancer
Res. 2007;13(2 Pt 2):671s–679s.
362. Toro JR, Nickerson ML, Wei M-H, et al. Mutations
in the fumarate hydratase gene cause hereditary
leiomyomatosis and renal cell cancer in families in
North America. Am J Hum Genet. 2003;73(1):95–
106.
363. Martinez-Mir A, Glaser B, Chuang GS, et al.
Germline fumarate hydratase mutations in families
with multiple cutaneous and uterine leiomyomata.
J Invest Dermatol. 2003;121(4):741–744.
364. Badeloe S, van Geel M, van Steensel MAM,
et al. Diffuse and segmental variants of cutaneous
leiomyomatosis: novel mutations in the fumarate
hydratase gene and review of the literature. Exp
Dermatol. 2006;15(9):735–741.
365. Sudarshan S, Pinto PA, Neckers L, Linehan WM.
Mechanisms of disease: hereditary leiomyomatosis
and renal cell cancer–a distinct form of hereditary

kidney cancer. Nat Clin Pract Urol. 2007;4(2):
104–110.
366. Lehtonen HJ, Kiuru M, Ylisaukko-Oja SK, et al.
Increased risk of cancer in patients with fumarate
hydratase germline mutation. J Med Genet. 2006;43(6):
523–526.
367. Wei MH, Toure O, Glenn GM, et al. Novel
mutations in FH and expansion of the spectrum
of phenotypes expressed in families with hereditary
leiomyomatosis and renal cell cancer. J Med Genet.
2006;43(1):18–27.
368. Kiuru M, Lehtonen R, Eerola H, et al. No germline
FH mutations in familial breast cancer patients. Eur
J Hum Genet. 2005;13(4):506–509.
369. Isaacs JS, Jung YJ, Mole DR, et al. HIF overexpression
correlates with biallelic loss of fumarate hydratase in
renal cancer: novel role of fumarate in regulation
of HIF stability. Cancer Cell. 2005;8(2):143–
153.
370. Chuang GS, Martinez-Mir A, Geyer A, et al. Germ-
line fumarate hydratase mutations and evidence for
a founder mutation underlying multiple cutaneous
and uterine leiomyomata. J Am Acad Dermatol.
2005;52(3 Pt 1):410–416.
371. Kiuru M, Launonen V, Hietala M, et al. Familial
cutaneous leiomyomatosis is a two-hit condition
associated with renal cell cancer of characteristic
histopathology. Am J Pathol. 2001;159(3):825–
829.
372. Zinn AB, Kerr DS, Hoppel CL. Fumarase deficiency:
a new cause of mitochondrial encephalomyopathy.
N Engl J Med. 1986;315(8):469–475.
373. Alam NA, Bevan S, Churchman M, et  al.
Localization of a gene (MCUL1) for multiple
cutaneous leiomyomata and uterine fibroids to
chromosome 1q42.3-q43. Am J Hum Genet.
2001;68(5):1264–1269.
374. Tomlinson IPM, Alam NA, Rowan AJ, et al. Germline
mutations in FH predispose to dominantly inherited
uterine fibroids, skin leiomyomata and papillary renal
cell cancer. Nat Genet. 2002;30(4):406–410.
375. Pithukpakorn M, Wei MH, Toure O, et al. Fumarate
hydratase enzyme activity in lymphoblastoid cells and
fibroblasts of individuals in families with hereditary
leiomyomatosis and renal cell cancer. J Med Genet.
2006;43(9):755–762.
376. Choyke PL. Imaging of hereditary renal cancer.
Radiol Clin North Am. 2003;41(5):1037–1051.
377. Gordon MS, Hussey M, Nagle RB, et al. Phase II
study of erlotinib in patients with locally advanced
or metastatic papillary histology renal cell cancer:
SWOG S0317. J Clin Oncol. 2009;27(34):
5788–5793.
378. National Institutes of Health Clinical Center
(CC) (National Cancer Institute [NCI]). A phase
II study of bevacizumab and erlotinib in subjects
with advanced hereditary leiomyomatosis and renal
cell cancer (HLRCC) or sporadic papillary renal
cell cancer. Bethesda, MD: National Library of
Medicine (US)2000: http://clinicaltrials.gov/show/
NCT01130519.
379. Lonser RR, Glenn GM, Walther M, et al. von Hippel-
Lindau disease. Lancet. 2003;361(9374):2059–2067.
380. Maher ER, Neumann HP, Richard S. von Hippel-
Lindau disease: a clinical and scientific review. Eur
J Hum Genet. 2011;19(6):617–623.
381. Frantzen C, Links TP, Giles RH. Von Hippel-Lindau
disease. In: GeneReviews at GeneTests Medical
Genetics Information Resource (database online).
Updated June 21, 2012. Copyright University of
Washington, Seattle. 1997-2013. Available at: http://
www.genetests.org. Accessed October 1, 2012.
382. National Comprehensive Cancer Centers. NCCN
Clinical Practice Guidelines in Oncology, Kidney
Cancer Version 2.2017; 2016. http://www.nccn.org/
professionals/physician_gls/pdf/kidney.pdf.
383. Brugarolas J, Kaelin WG. Dysregulation of HIF and
VEGF is a unifying feature of the familial hamartoma
syndromes. Cancer Cell. 2004;6(1):7–10.
Genetic Factors: Hereditary Cancer Predisposition Syndromes  •  CHAPTER 13 208.e7
384. Singer EA, Gupta GN, Srinivasan R. Update on
targeted therapies for clear cell renal cell carcinoma.
Curr Opin Oncol. 2011;23(3):283–289.
385. Birt AR, Hogg GR, Dubé WJ. Hereditary multiple
fibrofolliculomas with trichodiscomas and acrochor-
dons. Arch Dermatol. 1977;113(12):1674–1677.
386. Pavlovich CP, Grubb RL, Hurley K, et al. Evaluation
and management of renal tumors in the Birt-Hogg-
Dubé syndrome. J Urol. 2005;173(5):1482–1486.
387. Durrani OH, Ng L, Bihrle W. Chromophobe renal
cell carcinoma in a patient with the Birt-Hogg-Dube
syndrome. J Urol. 2002;168(4 Pt 1):1484–1485.
388. Schmidt LS, Nickerson ML, Warren MB, et al.
Germline BHD-mutation spectrum and phenotype
analysis of a large cohort of families with Birt-Hogg-
Dubé syndrome. Am J Hum Genet. 2005;76(6):
1023–1033.
389. Walter P, Kirchhof B, Korge B, Heimann K. Flecked
chorioretinopathy associated with Birt-Hogg-Dubé
syndrome. Graefes Arch Clin Exp Ophthalmol. 1997;
235(6):359–361.
390. Khoo SK, Giraud S, Kahnoski K, et al. Clinical
and genetic studies of Birt-Hogg-Dubé syndrome.
J Med Genet. 2002;39(12):906–912.
391. Kluger N, Giraud S, Coupier I, et al. Birt-Hogg-Dubé
syndrome: clinical and genetic studies of 10 French
families. Br J Dermatol. 2010;162(3):527–537.
392. Byrne M, Mallipeddi R, Pichert G, Whittaker S.
Birt-Hogg-Dubé syndrome with a renal angiomyo-
lipoma: further evidence of a relationship between
Birt-Hogg-Dubé syndrome and tuberous sclerosis
complex. Australas J Dermatol. 2012;53(2):151–154.
393. Misago N, Narisawa Y. Fibrofolliculoma in a patient
with tuberous sclerosis complex. Clin Exp Dermatol.
2009;34(8):892–894.
394. Khoo SK, Bradley M, Wong FK, Hedblad MA,
Nordenskjöld M, Teh BT. Birt-Hogg-Dubé syn-
drome: mapping of a novel hereditary neoplasia
gene to chromosome 17p12-q11.2. Oncogene. 2001;
20(37):5239–5242.
395. Nickerson ML, Warren MB, Toro JR, et al. Muta-
tions in a novel gene lead to kidney tumors, lung
wall defects, and benign tumors of the hair follicle
in patients with the Birt-Hogg-Dubé syndrome.
Cancer Cell. 2002;2(2):157–164.
396. Hartman TR, Nicolas E, Klein-Szanto A, et al.
The role of the Birt-Hogg-Dubé protein in mTOR
activation and renal tumorigenesis. Oncogene.
2009;28(13):1594–1604.
397. Toro JR, Wei MH, Glenn GM, et al. BHD muta-
tions, clinical and molecular genetic investigations
of Birt-Hogg-Dube syndrome: a new series of 50
families and a review of published reports. J Med
Genet. 2008;45(6):321–331.
398. Barrisford GW, Singer EA, Rosner IL, Linehan
WM, Bratslavsky G. Familial renal cancer: molecular
genetics and surgical management. Int J Surg Oncol.
2011;(2011):658767.
399. Roach ES, Sparagana SP. Diagnosis of tuberous scle-
rosis complex. J Child Neurol. 2004;19(9):643–649.
400. Agarwal R, Robson M. Inherited predisposition to
gastrointestinal stromal tumor. Hematol Oncol Clin
North Am. 2009;23(1):1–13.
401. Lane BR, Aydin H, Danforth TL, et al. Clinical
correlates of renal angiomyolipoma subtypes in 209
patients: classic, fat poor, tuberous sclerosis associated
and epithelioid. J Urol. 2008;180(3):836–843.
402. Bissler J, Kingswood JC, Zonnenberg BA, et al.
Everolimus therapy for angiomyolipoma in patients
with tuberous sclerosis complex or sporadic lymphan-
gioleiomyomatosis: Results from EXIST-2. J Clin
Oncol. 2012;30(suppl 5):356.
403. FDA. FDA approves Afinitor for non-cancerous
kidney tumors caused by rare genetic disease; 2012.
404. Hirota S, Isozaki K, Moriyama Y, et al. Gain-of-
function mutations of c-kit in human gastrointestinal
stromal tumors. Science. 1998;279(5350):577–580.
405. Hirota S, Ohashi A, Nishida T, et al. Gain-of-
function mutations of platelet-derived growth
208.e7
factor receptor alpha gene in gastrointestinal stromal
tumors. Gastroenterology. 2003;125(3):660–667.
406. Nishida T, Hirota S, Taniguchi M, et al. Familial
gastrointestinal stromal tumours with germline
mutation of the KIT gene. Nat Genet. 1998;
19(4):323–324.
407. Chompret A, Kannengiesser C, Barrois M, et al.
PDGFRA germline mutation in a family with
multiple cases of gastrointestinal stromal tumor.
Gastroenterology. 2004;126(1):318–321.
408. Graham J, Debiec-Rychter M, Corless CL, Reid R,
Davidson R, White JD. Imatinib in the management
of multiple gastrointestinal stromal tumors associated
with a germline KIT K642E mutation. Arch Pathol
Lab Med. 2007;131(9):1393–1396.
409. Jensen DE, Proctor M, Marquis ST, et al. BAP1:
a novel ubiquitin hydrolase which binds to the
BRCA1 RING finger and enhances BRCA1-
mediated cell growth suppression. Oncogene.
1998;16(9):1097–1112.
410. Bott M, Brevet M, Taylor BS, et al. The nuclear
deubiquitinase BAP1 is commonly inactivated by
somatic mutations and 3p21.1 losses in malignant
pleural mesothelioma. Nat Genet. 2011;43(7):
668–672.
411. Testa JR, Cheung M, Pei J, et al. Germline BAP1
mutations predispose to malignant mesothelioma.
Nat Genet. 2011;43(10):1022–1025.
412. Wiesner T, Obenauf AC, Murali R, et al. Germline
mutations in BAP1 predispose to melanocytic
tumors. Nat Genet. 2011;43(10):1018–1021.
413. Abdel-Rahman MH, Pilarski R, Cebulla CM, et al.
Germline BAP1 mutation predisposes to uveal
melanoma, lung adenocarcinoma, meningioma, and
other cancers. J Med Genet. 2011;48(12):856–859.
414. Pena-Llopis S, Vega-Rubin-de-Celis S, Liao A, et al.
BAP1 loss defines a new class of renal cell carcinoma.
Nat Genet. 2012;44(7):751–759.
415. Beguelin W, Popovic R, Teater M, et al. EZH2 is
required for germinal center formation and somatic
EZH2 mutations promote lymphoid transformation.
Cancer Cell. 2013;23(5):677–692.
416. Jones S, Anagnostou V, Lytle K, et al. Personalized
genomic analyses for cancer mutation discovery
and interpretation. Sci Transl Med. 2015;7(283):
283ra253.
417. Bombard Y, Robson M, Offit K. Revealing the
incidentalome when targeting the tumor genome.
JAMA. 2013;310(8):795–796.
418. Schrader KA, Cheng DT, Joseph V, et al. Germline
variants in targeted tumor sequencing using matched
normal DNA. JAMA Oncol. 2016;2(1):104–111.
419. Mody RJ, Wu YM, Lonigro RJ, et al. Integrative
clinical sequencing in the management of refractory
or relapsed cancer in youth. JAMA. 2015;314(9):
913–925.
420. Mandelker D, Zhang L, Kemel Y, et al. Mutation
detection in patients with advanced cancer by univer-
sal sequencing of cancer-related genes in tumor and
normal DNA compared to guideline-based germline
testing. JAMA. 2017;In Press.
421. Pritchard CC, Mateo J, Walsh MF, et al. Inherited
DNA-repair gene mutations in men with metastatic
prostate cancer. N Engl J Med. 2016;375(5):443–453.
422. Lynch HT, Lynch JF, Lynch PM, Attard T. Hereditary
colorectal cancer syndromes: molecular genetics,
genetic counseling, diagnosis and management.
Fam Cancer. 2008;7(1):27–39.
423. Achatz MI, Porter CC, Brugieres L, et al. Cancer
screening recommendations and clinical management
of inherited gastrointestinal cancer syndromes in
childhood. Clin Cancer Res. 2017;23(13):e107–
e114.
424. Locker GY, Kaul K, Weinberg DS, et al. The
I1307K APC polymorphism in Ashkenazi Jews with
colorectal cancer: clinical and pathologic features.
Cancer Genet Cytogenet. 2006;169(1):33–38.
425. Ma X, Zhang B, Zheng W. Genetic variants asso-
ciated with colorectal cancer risk: comprehensive
208.e8 Part I: Science and Clinical Oncology
research synopsis, meta-analysis, and epidemiological
evidence. Gut. 2014;63(2):326–336.
426. Gatti R, Perlman S. Ataxia-telangiectasia. In:
Pagon RA, Adam MP, Ardinger HH, et al, eds.
GeneReviews(R). Seattle (WA): University of
Washington, Seattle University of Washington,
Seattle. GeneReviews is a registered trademark of the
University of Washington, Seattle. All rights reserved;
1993.
427. Suarez F, Mahlaoui N, Canioni D, et al. Incidence,
presentation, and prognosis of malignancies in ataxia-
telangiectasia: a report from the French national
registry of primary immune deficiencies. J Clin
Oncol. 2015;33(2):202–208.
428. Grant RC, Selander I, Connor AA, et al. Prevalence
of germline mutations in cancer predisposition genes
in patients with pancreatic cancer. Gastroenterology.
2015;148(3):556–564.
429. Walsh MF, Nathanson KL, Couch FJ, Offit K.
Genomic biomarkers for breast cancer risk. Adv
Exp Med Biol. 2016;882:1–32.
430. Pilarski R, Rai K, Cebulla C, Abdel-Rahman M.
BAP1 tumor predisposition syndrome. In: Pagon RA,
Adam MP, Ardinger HH, et al, eds. GeneReviews(R).
Seattle (WA): University of Washington, Seattle
University of Washington, Seattle. GeneReviews is a
registered trademark of the University of Washington,
Seattle. All rights reserved; 1993.
431. Sanz MM, German J, Cunniff C. Bloom’s syndrome.
In: Pagon RA, Adam MP, Ardinger HH, et al,
eds. GeneReviews(R). Seattle (WA): University of
Washington, Seattle University of Washington,
Seattle. GeneReviews is a registered trademark of
the University of Washington, Seattle. All rights
reserved; 1993.
432. Bloom D. Congenital telangiectatic erythema
resembling lupus erythematosus in dwarfs; probably
a syndrome entity. Am J Dis Child. 1954;88(6):
754–758.
433. German J, Sanz MM, Ciocci S, Ye TZ, Ellis NA.
Syndrome-causing mutations of the BLM gene in
persons in the Bloom’s Syndrome Registry. Hum
Mutat. 2007;28(8):743–753.
434. Cunniff C, Bassetti JA, Ellis NA. Bloom’s syndrome:
clinical spectrum, molecular pathogenesis, and cancer
predisposition. Mol Syndromol. 2017;8(1):4–23.
435. de Voer RM, Hahn MM, Mensenkamp AR, et al.
Deleterious germline BLM mutations and the risk for
early-onset colorectal cancer. Sci Rep. 2015;5:14060.
436. Prokofyeva D, Bogdanova N, Dubrowinskaja N,
et al. Nonsense mutation p.Q548X in BLM, the
gene mutated in Bloom’s syndrome, is associated with
breast cancer in Slavic populations. Breast Cancer
Res Treat. 2013;137(2):533–539.
437. Larsen Haidle J, Howe JR. Juvenile polyposis
syndrome. In: Pagon RA, Adam MP, Ardinger HH,
et al, eds. GeneReviews(R). Seattle (WA): University
of Washington, Seattle University of Washington,
Seattle. GeneReviews is a registered trademark of
the University of Washington, Seattle. All rights
reserved; 1993.
438. Syngal S, Brand RE, Church JM, Giardiello FM,
Hampel HL, Burt RW. ACG clinical guideline:
genetic testing and management of hereditary
gastrointestinal cancer syndromes. Am J Gastroenterol.
2015;110(2):223–262, quiz 263.
439. Brosens LA, Langeveld D, van Hattem WA, Giardi-
ello FM, Offerhaus GJ. Juvenile polyposis syndrome.
World J Gastroenterol. 2011;17(44):4839–4844.
440. Jass JR. Colorectal polyposes: from phenotype to
diagnosis. Pathol Res Pract. 2008;204(7):431–447.
441. Chow E, Macrae F. A review of juvenile
polyposis syndrome. J Gastroenterol Hepatol.
2005;20(11):1634–1640.
442. Iyer NK, Burke CA, Leach BH, Parambil JG.
SMAD4 mutation and the combined syndrome of
juvenile polyposis syndrome and hereditary haem-
orrhagic telangiectasia. Thorax. 2010;65(8):745–
746.
443. Friedl W, Uhlhaas S, Schulmann K, et al. Juvenile
polyposis: massive gastric polyposis is more common
in MADH4 mutation carriers than in BMPR1A
mutation carriers. Hum Genet. 2002;111(1):108–111.
444. Chen S, Parmigiani G. Meta-analysis of BRCA1
and BRCA2 penetrance. J Clin Oncol. 2007;25(11):
1329–1333.
445. Gonzalez-Angulo AM, Timms KM, Liu S, et al. Inci-
dence and outcome of BRCA mutations in unselected
patients with triple receptor-negative breast cancer.
Clin Cancer Res. 2011;17(5):1082–1089.
446. Gallagher DJ, Gaudet MM, Pal P, et al. Germline
BRCA mutations denote a clinicopathologic subset
of prostate cancer. Clin Cancer Res. 2010;16(7):
2115–2121.
447. Fong PC, Boss DS, Yap TA, et al. Inhibition of
poly(ADP-ribose) polymerase in tumors from BRCA
mutation carriers. N Engl J Med. 2009;361(2):
123–134.
448. Salo-Mullen EE, O’Reilly EM, Kelsen DP, et al. Iden-
tification of germline genetic mutations in patients
with pancreatic cancer. Cancer. 2015;121(24):
4382–4388.
449. Meyer S, Tischkowitz M, Chandler K, Gillespie A,
Birch JM, Evans DG. Fanconi anaemia, BRCA2
mutations and childhood cancer: a developmental
perspective from clinical and epidemiological obser-
vations with implications for genetic counselling.
J Med Genet. 2014;51(2):71–75.
450. Pennington KP, Swisher EM. Hereditary ovarian
cancer: beyond the usual suspects. Gynecol Oncol.
2012;124(2):347–353.
451. van der Post RS, Vogelaar IP, Carneiro F, et al.
Hereditary diffuse gastric cancer: updated clinical
guidelines with an emphasis on germline CDH1
mutation carriers. J Med Genet. 2015;52(6):
361–374.
452. Vasen HF, Gruis NA, Frants RR, van Der Velden
PA, Hille ET, Bergman W. Risk of developing
pancreatic cancer in families with familial atypical
multiple mole melanoma associated with a specific
19 deletion of p16 (p16-Leiden). Int J Cancer.
2000;87(6):809–811.
453. Goldstein AM, Struewing JP, Chidambaram A,
Fraser MC, Tucker MA. Genotype-phenotype
relationships in U.S. melanoma-prone families with
CDKN2A and CDK4 mutations. J Natl Cancer Inst.
2000;92(12):1006–1010.
454. Doros L, Schultz KA, Stewart DR, et al. DICER1-
related disorders. In: Pagon RA, Adam MP, Ardinger
HH, et al, eds. GeneReviews(R). Seattle (WA):
University of Washington, Seattle University of
Washington, Seattle. GeneReviews is a registered
trademark of the University of Washington, Seattle.
All rights reserved; 1993.
455. Foulkes WD, Bahubeshi A, Hamel N, et al.
Extending the phenotypes associated with DICER1
mutations. Hum Mutat. 2011;32(12):1381–1384.
456. Schultz KA, Pacheco MC, Yang J, et al. Ovarian sex
cord-stromal tumors, pleuropulmonary blastoma and
DICER1 mutations: a report from the International
Pleuropulmonary Blastoma Registry. Gynecol Oncol.
2011;122(2):246–250.
457. Slade I, Bacchelli C, Davies H, et al. DICER1
syndrome: clarifying the diagnosis, clinical features
and management implications of a pleiotropic
tumour predisposition syndrome. J Med Genet.
2011;48(4):273–278.
458. Faure A, Atkinson J, Bouty A, et al. DICER1 pleuro-
pulmonary blastoma familial tumour predisposition
syndrome: what the paediatric urologist needs to
know. J Pediatr Urol. 2016;12(1):5–10.
459. Schultz KAP, Rednam SP, Kamihara J, et al.
PTEN, DICER1, FH, and their associated tumor
susceptibility syndromes: clinical features, genetics,
and surveillance recommendations in childhood.
Clin Cancer Res. 2017;23(12):e76–e82.
460. Whitelaw SC, Murday VA, Tomlinson IP, et al. Clini-
cal and molecular features of the hereditary mixed
polyposis syndrome. Gastroenterology. 1997;112(2):
327–334.
461. Plesec T, Brown K, Allen C, et al. Clinicopathological
features of a kindred with SCG5-GREM1-associated
hereditary mixed polyposis syndrome. Hum Pathol.
2017;60:75–81.
462. Porter CC, Druley TE, Erez A, et al. Recom-
mendations for surveillance for children with
leukemia-predisposing conditions. Clin Cancer Res.
2017;23(11):e14–e22.
463. Topka S, Vijai J, Walsh MF, et al. Germline ETV6
mutations confer susceptibility to acute lymphoblas-
tic leukemia and thrombocytopenia. PLoS Genet.
2015;11(6):e1005262.
464. Holmfeldt L, Wei L, Diaz-Flores E, et al. The
genomic landscape of hypodiploid acute lympho-
blastic leukemia. Nat Genet. 2013;45(3):242–252.
465. Dang J, Wei L, de Ridder J, et al. PAX5 is a tumor
suppressor in mouse mutagenesis models of acute
lymphoblastic leukemia. Blood. 2015;125(23):
3609–3617.
466. Mehta PA, Tolar J. Fanconi anemia. In: Pagon RA,
Adam MP, Ardinger HH, et al, eds. GeneReviews(R).
Seattle (WA): University of Washington, Seattle
University of Washington, Seattle. GeneReviews is a
registered trademark of the University of Washington,
Seattle. All rights reserved; 1993.
467. Kutler DI, Singh B, Satagopan J, et al. A 20-year
perspective on the International Fanconi Anemia
Registry (IFAR). Blood. 2003;101(4):1249–1256.
468. Rosenberg PS, Greene MH, Alter BP. Cancer
incidence in persons with Fanconi anemia. Blood.
2003;101(3):822–826.
469. Auerbach AD, Rogatko A, Schroeder-Kurth TM.
International Fanconi Anemia Registry: relation of
clinical symptoms to diepoxybutane sensitivity. Blood.
1989;73(2):391–396.
470. Kutler DI, Auerbach AD, Satagopan J, et al. High
incidence of head and neck squamous cell carcinoma
in patients with Fanconi anemia. Arch Otolaryngol
Head Neck Surg. 2003;129(1):106–112.
471. Faivre L, Guardiola P, Lewis C, et al. Association of
complementation group and mutation type with clin-
ical outcome in Fanconi anemia. European Fanconi
Anemia Research Group. Blood. 2000;96(13):
4064–4070.
472. Brosh RM Jr, Bellani M, Liu Y, Seidman MM.
Fanconi Anemia: A DNA repair disorder character-
ized by accelerated decline of the hematopoietic
stem cell compartment and other features of aging.
Ageing Res Rev. 2017;33:67–75.
473. Pithukpakorn M, Toro JR. Hereditary leiomyoma-
tosis and renal cell cancer. In: Pagon RA, Adam MP,
Ardinger HH, et al, eds. GeneReviews(R). Seattle
(WA): University of Washington, Seattle University
of Washington, Seattle. GeneReviews is a registered
trademark of the University of Washington, Seattle.
All rights reserved; 1993.
474. Stewart L, Glenn GM, Stratton P, et al. Association
of germline mutations in the fumarate hydratase
gene and uterine fibroids in women with hereditary
leiomyomatosis and renal cell cancer. Arch Dermatol.
2008;144(12):1584–1592.
475. Sanz-Ortega J, Vocke C, Stratton P, Linehan
WM, Merino MJ. Morphologic and molecular
characteristics of uterine leiomyomas in hereditary
leiomyomatosis and renal cancer (HLRCC) syn-
drome. Am J Surg Pathol. 2013;37(1):74–80.
476. Schmidt LS, Linehan WM. Hereditary leiomyoma-
tosis and renal cell carcinoma. Int J Nephrol Renovasc
Dis. 2014;7:253–260.
477. Toro JR. Birt-Hogg-Dube syndrome. In: Pagon RA,
Adam MP, Ardinger HH, et al, eds. GeneReviews(R).
Seattle (WA): University of Washington, Seattle
University of Washington, Seattle. GeneReviews is a
registered trademark of the University of Washington,
Seattle. All rights reserved; 1993.
478. Hasumi H, Baba M, Hasumi Y, Furuya M, Yao M.
Birt-Hogg-Dube syndrome: Clinical and molecular

aspects of recently identified kidney cancer syndrome.
Int J Urol. 2016;23(3):204–210.
479. Kuroda N, Furuya M, Nagashima Y, et al. Review
of renal tumors associated with Birt-Hogg-Dube
syndrome with focus on clinical and pathobiological
aspects. Pol J Pathol. 2014;65(2):93–99.
480. Furuya M, Nakatani Y. Birt-Hogg-Dube syndrome:
clinicopathological features of the lung. J Clin Pathol.
2013;66(3):178–186.
481. Forde PM, Cochran RL, Boikos SA, et al. Familial
GI stromal tumor with loss of heterozygosity and
amplification of mutant KIT. J Clin Oncol. 2016;
34(3):e13–e16.
482. Ricci R. Syndromic gastrointestinal stromal tumors.
Hered Cancer Clin Pract. 2016;14:15.
483. Ricci R, Martini M, Cenci T, et al. PDGFRA-mutant
syndrome. Mod Pathol. 2015;28(7):954–964.
484. Lasota J, Miettinen M. KIT and PDGFRA mutations
in gastrointestinal stromal tumors (GISTs). Semin
Diagn Pathol. 2006;23(2):91–102.
485. Giusti F, Marini F, Brandi ML. Multiple endocrine
neoplasia type 1. In: Pagon RA, Adam MP, Ardinger
HH, et al, eds. GeneReviews(R). Seattle (WA):
University of Washington, Seattle University of
Washington, Seattle. GeneReviews is a registered
trademark of the University of Washington, Seattle.
All rights reserved; 1993.
486. Machens A, Schaaf L, Karges W, et al. Age-related
penetrance of endocrine tumours in multiple
endocrine neoplasia type 1 (MEN1): a multicentre
study of 258 gene carriers. Clin Endocrinol (Oxf ).
2007;67(4):613–622.
487. Lemos MC, Thakker RV. Multiple endocrine
neoplasia type 1 (MEN1): analysis of 1336 mutations
reported in the first decade following identification
of the gene. Hum Mutat. 2008;29(1):22–32.
488. Thakker RV, Newey PJ, Walls GV, et al. Clinical
practice guidelines for multiple endocrine neo-
plasia type 1 (MEN1). J Clin Endocrinol Metab.
2012;97(9):2990–3011.
489. Coleman JA, Russo P. Hereditary and familial kidney
cancer. Curr Opin Urol. 2009;19(5):478–485.
490. Rini BI, Campbell SC, Rathmell WK. Renal cell
carcinoma. Curr Opin Oncol. 2006;18(3):289–296.
491. de Vos tot Nederveen Cappel WH, Jarvinen
HJ, Lynch PM, Engel C, Mecklin JP, Vasen HF.
Colorectal surveillance in Lynch syndrome families.
Fam Cancer. 2013;12(2):261–265.
492. Bupathi M, Wu C. Biomarkers for immune therapy
in colorectal cancer: mismatch-repair deficiency and
others. J Gastrointest Oncol. 2016;7(5):713–720.
493. Castro MP, Goldstein N. Mismatch repair deficiency
associated with complete remission to combination
programmed cell death ligand immune therapy
in a patient with sporadic urothelial carcinoma:
immunotheranostic considerations. J Immunother
Cancer. 2015;3:58.
494. Burn J, Gerdes AM, Macrae F, et al. Long-term
effect of aspirin on cancer risk in carriers of
hereditary colorectal cancer: an analysis from the
CAPP2 randomised controlled trial. Lancet. 2011;
378(9809):2081–2087.
495. Shia J. Immunohistochemistry versus microsatellite
instability testing for screening colorectal cancer
patients at risk for hereditary nonpolyposis colorectal
cancer syndrome. Part I. The utility of immunohis-
tochemistry. J Mol Diagn. 2008;10(4):293–300.
496. Zhang L. Immunohistochemistry versus microsatellite
instability testing for screening colorectal cancer
patients at risk for hereditary nonpolyposis colorectal
cancer syndrome. Part II. The utility of microsatellite
instability testing. J Mol Diagn. 2008;10(4):301–307.
497. Bouzourene H, Hutter P, Losi L, Martin P, Benhat-
tar J. Selection of patients with germline MLH1
mutated Lynch syndrome by determination of
MLH1 methylation and BRAF mutation. Fam
Cancer. 2010;9(2):167–172.
498. Vasen HF, Mecklin JP, Khan PM, Lynch HT. The
International Collaborative Group on Hereditary
Genetic Factors: Hereditary Cancer Predisposition Syndromes  •  CHAPTER 13 208.e9
Non-Polyposis Colorectal Cancer (ICG-HNPCC).
Dis Colon Rectum. 1991;34(5):424–425.
499. Perez-Carbonell L, Ruiz-Ponte C, Guarinos C, et al.
Comparison between universal molecular screening
for Lynch syndrome and Revised Bethesda Guidelines
in a large population-based cohort of patients with
colorectal cancer. Gut. 2012;61(6):865–872.
500. South CD, Hampel H, Comeras I, Westman JA,
Frankel WL, de la Chapelle A. The frequency of
Muir-Torre syndrome among Lynch syndrome
families. J Natl Cancer Inst. 2008;100(4):277–281.
501. Hamilton SR, Liu B, Parsons RE, et al. The
molecular basis of Turcot’s syndrome. N Engl J
Med. 1995;332(13):839–847.
502. Bakry D, Aronson M, Durno C, et al. Genetic and
clinical determinants of constitutional mismatch
repair deficiency syndrome: report from the con-
stitutional mismatch repair deficiency consortium.
Eur J Cancer. 2014;50(5):987–996.
503. Walsh MD, Buchanan DD, Cummings MC,
et al. Lynch syndrome-associated breast cancers:
clinicopathologic characteristics of a case series
from the colon cancer family registry. Clin Cancer
Res. 2010;16(7):2214–2224.
504. Tabori U, Hansford JR, Achatz MI, et al. Clinical
management and tumor surveillance recommenda-
tions of inherited mismatch repair deficiency in
childhood. Clin Cancer Res. 2017;23(11):e32–e37.
505. Cleary SP, Cotterchio M, Jenkins MA, et al.
Germline MutY human homologue mutations
and colorectal cancer: a multisite case-control study.
Gastroenterology. 2009;136(4):1251–1260.
506. Lubbe SJ, Di Bernardo MC, Chandler IP, Houlston
RS. Clinical implications of the colorectal cancer risk
associated with MUTYH mutation. J Clin Oncol.
2009;27(24):3975–3980.
507. Aretz S, Tricarico R, Papi L, et al. MUTYH-
associated polyposis (MAP): evidence for the origin
of the common European mutations p.Tyr179Cys
and p.Gly396Asp by founder events. Eur J Hum
Genet. 2014;22(7):923–929.
508. Nielsen M, Poley JW, Verhoef S, et al. Duodenal
carcinoma in MUTYH-associated polyposis. J Clin
Pathol. 2006;59(11):1212–1215.
509. Win AK, Cleary SP, Dowty JG, et al. Cancer risks
for monoallelic MUTYH mutation carriers with
a family history of colorectal cancer. Int J Cancer.
2011;129(9):2256–2262.
510. Jenkins MA, Croitoru ME, Monga N, et al. Risk of
colorectal cancer in monoallelic and biallelic carriers
of MYH mutations: a population-based case-family
study. Cancer Epidemiol Biomarkers Prev. 2006;
15(2):312–314.
511. Zhang G, Zeng Y, Liu Z, Wei W. Significant
association between Nijmegen breakage syndrome
1 657del5 polymorphism and breast cancer risk.
Tumor Biol. 2013;34(5):2753–2757.
512. Dembowska-Baginska B, Perek D, Brozyna A, et al.
Non-Hodgkin lymphoma (NHL) in children with
Nijmegen Breakage syndrome (NBS). Pediatr Blood
Cancer. 2009;52(2):186–190.
513. Friedman JM. Neurofibromatosis 1. In: Pagon RA,
Adam MP, Ardinger HH, et al, eds. GeneReviews(R).
Seattle (WA): University of Washington, Seattle
University of Washington, Seattle. GeneReviews is a
registered trademark of the University of Washington,
Seattle. All rights reserved; 1993.
514. Madanikia SA, Bergner A, Ye X, Blakeley JO.
Increased risk of breast cancer in women with NF1.
Am J Med Genet A. 2012;158a(12):3056–3060.
515. Seminog OO, Goldacre MJ. Risk of benign tumours
of nervous system, and of malignant neoplasms,
in people with neurofibromatosis: population-
based record-linkage study. Br J Cancer. 2013;
108(1):193–198.
516. Walker L, Thompson D, Easton D, et al. A pro-
spective study of neurofibromatosis type 1 cancer
incidence in the UK. Br J Cancer. 2006;95(2):233–
238.
208.e9
517. Evans DGR, Salvador H, Chang VY, et al. Cancer
and central nervous system tumor surveillance in
pediatric neurofibromatosis 1. Clin Cancer Res. 2017;
23(12):e46–e53.
518. Evans DGR, Salvador H, Chang VY, et al. Cancer
and central nervous system tumor surveillance in
pediatric neurofibromatosis 2 and related disorders.
Clin Cancer Res. 2017;23(12):e54–e61.
519. Stadler ZK, Salo-Mullen E, Sabbaghian N, et al.
Germline PALB2 mutation analysis in breast-pancreas
cancer families. J Med Genet. 2011;48(8):523–525.
520. Valle L, Hernandez-Illan E, Bellido F, et al. New
insights into POLE and POLD1 germline mutations
in familial colorectal cancer and polyposis. Hum
Mol Genet. 2014;23(13):3506–3512.
521. Church JM. Polymerase proofreading-associated
polyposis: a new, dominantly inherited syndrome
of hereditary colorectal cancer predisposition. Dis
Colon Rectum. 2014;57(3):396–397.
522. Elsayed FA, Kets CM, Ruano D, et al. Germline
variants in POLE are associated with early onset
mismatch repair deficient colorectal cancer. Eur J
Hum Genet. 2015;23(8):1080–1084.
523. Spier I, Holzapfel S, Altmuller J, et al. Frequency
and phenotypic spectrum of germline mutations
in POLE and seven other polymerase genes in 266
patients with colorectal adenomas and carcinomas.
Int J Cancer. 2015;137(2):320–331.
524. Solomon S, Whitcomb DC, LaRusch J. PRSS1-
related hereditary pancreatitis. In: Pagon RA, Adam
MP, Ardinger HH, et al, eds. GeneReviews(R). Seattle
(WA): University of Washington, Seattle University
of Washington, Seattle. GeneReviews is a registered
trademark of the University of Washington, Seattle.
All rights reserved; 1993.
525. Solomon S, Whitcomb DC. Genetics of pancreatitis:
an update for clinicians and genetic counselors. Curr
Gastroenterol Rep. 2012;14(2):112–117.
526. Rosendahl J, Landt O, Bernadova J, et al. CFTR,
SPINK1, CTRC and PRSS1 variants in chronic
pancreatitis: is the role of mutated CFTR overesti-
mated? Gut. 2013;62(4):582–592.
527. Evans DG, Farndon PA. Nevoid basal cell carcinoma
syndrome. In: Pagon RA, Adam MP, Ardinger HH,
et al, eds. GeneReviews(R). Seattle (WA): University
of Washington, Seattle University of Washington,
Seattle. GeneReviews is a registered trademark of
the University of Washington, Seattle. All rights
reserved; 1993.
528. Soufir N, Gerard B, Portela M, et al. PTCH
mutations and deletions in patients with typical
nevoid basal cell carcinoma syndrome and in patients
with a suspected genetic predisposition to basal cell
carcinoma: a French study. Br J Cancer. 2006;95(4):
548–553.
529. Athar M, Li C, Kim AL, Spiegelman VS, Bickers
DR. Sonic hedgehog signaling in Basal cell
nevus syndrome. Cancer Res. 2014;74(18):4967–
4975.
530. Smith MJ, Beetz C, Williams SG, et al. Germline
mutations in SUFU cause Gorlin syndrome-
associated childhood medulloblastoma and redefine
the risk associated with PTCH1 mutations. J Clin
Oncol. 2014;32(36):4155–4161.
531. Eng C. PTEN Hamartoma tumor syndrome.
In: Pagon RA, Adam MP, Ardinger HH, et al,
eds. GeneReviews(R). Seattle (WA): University of
Washington, Seattle University of Washington,
Seattle. GeneReviews is a registered trademark of the
University of Washington, Seattle. All rights reserved;
1993.
532. Nelen MR, Padberg GW, Peeters EA, et al. Localiza-
tion of the gene for Cowden disease to chromosome
10q22-23. Nat Genet. 1996;13(1):114–116.
533. Tan MH, Mester J, Peterson C, et al. A clinical
scoring system for selection of patients for PTEN
mutation testing is proposed on the basis of a
prospective study of 3042 probands. Am J Hum
Genet. 2011;88(1):42–56.
208.e10 Part I: Science and Clinical Oncology
534. Heald B, Mester J, Rybicki L, Orloff MS, Burke
CA, Eng C. Frequent gastrointestinal polyps and
colorectal adenocarcinomas in a prospective series
of PTEN mutation carriers. Gastroenterology.
2010;139(6):1927–1933.
535. Eng C. PTEN: one gene, many syndromes. Hum
Mutat. 2003;22(3):183–198.
536. Pilarski R, Burt R, Kohlman W, Pho L, Shannon
KM, Swisher E. Cowden syndrome and the PTEN
hamartoma tumor syndrome: systematic review
and revised diagnostic criteria. J Natl Cancer Inst.
2013;105(21):1607–1616.
537. McBride KL, Varga EA, Pastore MT, et al.
Confirmation study of PTEN mutations among
individuals with autism or developmental delays/
mental retardation and macrocephaly. Autism Res.
2010;3(3):137–141.
538. Haibach H, Burns TW, Carlson HE, Burman
KD, Deftos LJ. Multiple hamartoma syndrome
(Cowden’s disease) associated with renal cell car-
cinoma and primary neuroendocrine carcinoma of
the skin (Merkel cell carcinoma). Am J Clin Pathol.
1992;97(5):705–712.
539. Schrager CA, Schneider D, Gruener AC, Tsou HC,
Peacocke M. Clinical and pathological features of
breast disease in Cowden’s syndrome: an under-
recognized syndrome with an increased risk of breast
cancer. Hum Pathol. 1998;29(1):47–53.
540. Pilarski R. Cowden syndrome: a critical review of the
clinical literature. J Genet Couns. 2009;18(1):13–27.
541. Meindl A, Hellebrand H, Wiek C, et al. Germline
mutations in breast and ovarian cancer pedigrees
establish RAD51C as a human cancer susceptibility
gene. Nat Genet. 2010;42(5):410–414.
542. Loveday C, Turnbull C, Ruark E, et al. Germline
RAD51C mutations confer susceptibility to ovarian
cancer. Nat Genet. 2012;44(5):475–476, author reply
476.
543. Lohmann DR, Gallie BL. Retinoblastoma. In:
Pagon RA, Adam MP, Ardinger HH, et al, eds.
GeneReviews(R). Seattle (WA): University of
Washington, Seattle University of Washington,
Seattle. GeneReviews is a registered trademark of
the University of Washington, Seattle. All rights
reserved; 1993.
544. Knudson AG Jr. Mutation and cancer: statistical
study of retinoblastoma. Proc Natl Acad Sci USA.
1971;68(4):820–823.
545. Abramson DH, Beaverson K, Sangani P, et al.
Screening for retinoblastoma: presenting signs
as prognosticators of patient and ocular survival.
Pediatrics. 2003;112(6 Pt 1):1248–1255.
546. Abramson DH, Frank CM. Second nonocular tumors
in survivors of bilateral retinoblastoma: a possible
age effect on radiation-related risk. Ophthalmology.
1998;105(4):573–579, discussion 579–580.
547. Dimaras H, Kimani K, Dimba EA, et al. Retino-
blastoma. Lancet. 2012;379(9824):1436–1446.
548. Kleinerman RA, Yu CL, Little MP, et al. Variation
of second cancer risk by family history of retino-
blastoma among long-term survivors. J Clin Oncol.
2012;30(9):950–957.
549. Klutz M, Brockmann D, Lohmann DR. A parent-
of-origin effect in two families with retinoblastoma
is associated with a distinct splice mutation in the
RB1 gene. Am J Hum Genet. 2002;71(1):174–179.
550. Price EA, Price K, Kolkiewicz K, et al. Spectrum
of RB1 mutations identified in 403 retinoblastoma
patients. J Med Genet. 2014;51(3):208–214.
551. Kamihara J, Bourdeaut F, Foulkes WD, et al. Reti-
noblastoma and neuroblastoma predisposition and
surveillance. Clin Cancer Res. 2017;23(13):e98–e106.
552. Marquard J, Eng C. Multiple endocrine neoplasia
type 2. In: Pagon RA, Adam MP, Ardinger HH,
et al, eds. GeneReviews(R). Seattle (WA): University
of Washington, Seattle University of Washington,
Seattle. GeneReviews is a registered trademark of
the University of Washington, Seattle. All rights
reserved; 1993.
553. Eng C. Seminars in medicine of the Beth Israel
Hospital, Boston. The RET proto-oncogene in mul-
tiple endocrine neoplasia type 2 and Hirschsprung’s
disease. N Engl J Med. 1996;335(13):943–951.
554. Kirmani S, Young WF. Hereditary paraganglioma-
pheochromocytoma syndromes. In: Pagon RA, Adam
MP, Ardinger HH, et al, eds. GeneReviews(R). Seattle
(WA): University of Washington, Seattle University
of Washington, Seattle. GeneReviews is a registered
trademark of the University of Washington, Seattle.
All rights reserved; 1993.
555. Lenders JW, Duh QY, Eisenhofer G, et al. Pheo-
chromocytoma and paraganglioma: an Endocrine
Society clinical practice guideline. J Clin Endocrinol
Metab. 2014;99(6):1915–1942.
556. Gottlieb E, Tomlinson IP. Mitochondrial tumour
suppressors: a genetic and biochemical update. Nat
Rev Cancer. 2005;5(11):857–866.
557. Pai R, Manipadam MT, Singh P, Ebenazer A,
Samuel P, Rajaratnam S. Usefulness of Succinate
dehydrogenase B (SDHB) immunohistochemistry
in guiding mutational screening among patients
with pheochromocytoma-paraganglioma syndromes.
APMIS. 2014;122(11):1130–1135.
558. Rednam SP, Erez A, Druker H, et al. Von
Hippel-Lindau and Hereditary pheochromocytoma/
paraganglioma syndromes: clinical features, genetics,
and surveillance recommendations in childhood.
Clin Cancer Res. 2017;23(12):e68–e75.
559. McGarrity TJ, Amos CI, Baker MJ. Peutz-Jeghers
syndrome. In: Pagon RA, Adam MP, Ardinger HH,
et al, eds. GeneReviews(R). Seattle (WA): University
of Washington, Seattle University of Washington,
Seattle. GeneReviews is a registered trademark of
the University of Washington, Seattle. All rights
reserved; 1993.
560. Beggs AD, Latchford AR, Vasen HF, et  al.
Peutz-Jeghers syndrome: a systematic review and
recommendations for management. Gut. 2010;59(7):
975–986.
561. Tomas C, Soyer P, Dohan A, Dray X, Boudiaf M,
Hoeffel C. Update on imaging of Peutz-Jeghers
syndrome. World J Gastroenterol. 2014;20(31):
10864–10875.
562. Giardiello FM, Trimbath JD. Peutz-Jeghers syn-
drome and management recommendations. Clin
Gastroenterol Hepatol. 2006;4(4):408–415.
563. Jasperson K, Burt RW. The genetics of colorectal
cancer. Surg Oncol Clin N Am. 2015;24(4):683–703.
564. Stoffel EM, Mangu PB, Gruber SB, et al. Hereditary
colorectal cancer syndromes: American Society of
Clinical Oncology Clinical Practice Guideline
endorsement of the familial risk-colorectal cancer:
European Society for Medical Oncology Clinical
Practice Guidelines. J Clin Oncol. 2015;33(2):
209–217.
565. Schneider K, Zelley K, Nichols KE, Garber J.
Li-Fraumeni syndrome. In: Pagon RA, Adam MP,
Ardinger HH, et al, eds. GeneReviews(R). Seattle
(WA): University of Washington, Seattle University
of Washington, Seattle. GeneReviews is a registered
trademark of the University of Washington, Seattle.
All rights reserved; 1993.
566. Li FP, Fraumeni JF Jr. Soft-tissue sarcomas,
breast cancer, and other neoplasms. A familial
syndrome? Ann Intern Med. 1969;71(4):747–752.
567. Li FP, Fraumeni JF Jr, Mulvihill JJ, et al. A cancer
family syndrome in twenty-four kindreds. Cancer
Res. 1988;48(18):5358–5362.
568. Srivastava S, Zou ZQ, Pirollo K, Blattner W, Chang
EH. Germ-line transmission of a mutated p53 gene
in a cancer-prone family with Li-Fraumeni syndrome.
Nature. 1990;348(6303):747–749.
569. Chompret A, Abel A, Stoppa-Lyonnet D, et al.
Sensitivity and predictive value of criteria for
p53 germline mutation screening. J Med Genet.
2001;38(1):43–47.
570. Gonzalez KD, Noltner KA, Buzin CH, et al. Beyond
Li Fraumeni syndrome: clinical characteristics of
families with p53 germline mutations. J Clin Oncol.
2009;27(8):1250–1256.
571. Bougeard G, Renaux-Petel M, Flaman JM, et al.
Revisiting Li-Fraumeni syndrome from TP53 muta-
tion carriers. J Clin Oncol. 2015;33(21):2345–2352.
572. Villani A, Shore A, Wasserman JD, et al. Biochemical
and imaging surveillance in germline TP53 muta-
tion carriers with Li-Fraumeni syndrome: 11 year
follow-up of a prospective observational study. Lancet
Oncol. 2016;17(9):1295–1305.
573. Kratz CP, Achatz MI, Brugieres L, et al. Cancer
screening recommendations for individuals with Li-
Fraumeni syndrome. Clin Cancer Res. 2017;23(11):
e38–e45.
574. Northrup H, Koenig MK, Pearson DA, Au KS.
Tuberous sclerosis complex. In: Pagon RA, Adam
MP, Ardinger HH, et al, eds. GeneReviews(R). Seattle
(WA): University of Washington, Seattle University
of Washington, Seattle. GeneReviews is a registered
trademark of the University of Washington, Seattle.
All rights reserved; 1993.
575. Northrup H, Krueger DA. Tuberous sclerosis
complex diagnostic criteria update: recommenda-
tions of the 2012 International Tuberous Sclerosis
Complex Consensus Conference. Pediatr Neurol.
2013;49(4):243–254.
576. Patel U, Simpson E, Kingswood JC, Saggar-Malik
AK. Tuberose sclerosis complex: analysis of growth
rates aids differentiation of renal cell carcinoma from
atypical or minimal-fat-containing angiomyolipoma.
Clin Radiol. 2005;60(6):665–673, discussion
663–664.
577. Crino PB, Nathanson KL, Henske EP. The tuberous
sclerosis complex. N Engl J Med. 2006;355(13):
1345–1356.
578. Borkowska J, Schwartz RA, Kotulska K, Jozwiak S.
Tuberous sclerosis complex: tumors and tumorigen-
esis. Int J Dermatol. 2011;50(1):13–20.
579. Al-Saleem T, Wessner LL, Scheithauer BW, et al.
Malignant tumors of the kidney, brain, and soft
tissues in children and young adults with the tuberous
sclerosis complex. Cancer. 1998;83(10):2208–2216.
580. Au KS, Williams AT, Roach ES, et al. Genotype/
phenotype correlation in 325 individuals referred
for a diagnosis of tuberous sclerosis complex in the
United States. Genet Med. 2007;9(2):88–100.
581. Qin W, Kozlowski P, Taillon BE, et al. Ultra deep
sequencing detects a low rate of mosaic muta-
tions in tuberous sclerosis complex. Hum Genet.
2010;127(5):573–582.
582. Spurling Jeste S, Wu JY, Senturk D, et al. Early
developmental trajectories associated with ASD in
infants with tuberous sclerosis complex. Neurology.
2014;83(2):160–168.
583. Dworakowska D, Grossman AB. Are neuroendocrine
tumours a feature of tuberous sclerosis? A systematic
review. Endocr Relat Cancer. 2009;16(1):45–
58.
584. Frantzen C, Klasson TD, Links TP, Giles RH, et al.
Von Hippel-Lindau syndrome. In: Pagon RA, Adam
MP, Ardinger HH, eds. GeneReviews(R). Seattle
(WA): University of Washington, Seattle University
of Washington, Seattle. GeneReviews is a registered
trademark of the University of Washington, Seattle.
All rights reserved; 1993.
585. Binderup ML, Bisgaard ML, Harbud V, et al. Von
Hippel-Lindau disease (vHL). National clinical
guideline for diagnosis and surveillance in Denmark.
3rd edition. Dan Med J. 2013;60(12):B4763.
586. Kwon T, Jeong IG, Pak S, et al. Renal tumor size is
an independent prognostic factor for overall survival
in von Hippel-Lindau disease. J Cancer Res Clin
Oncol. 2014;140(7):1171–1177.
587. Grubb RL 3rd, Choyke PL, Pinto PA, Linehan
WM, Walther MM. Management of von Hippel-
Lindau-associated kidney cancer. Nat Clin Pract Urol.
2005;2(5):248–255.
588. Corcos O, Couvelard A, Giraud S, et al. Endocrine
pancreatic tumors in von Hippel-Lindau disease:

clinical, histological, and genetic features. Pancreas.
2008;37(1):85–93.
589. Blansfield JA, Choyke L, Morita SY, et al. Clinical,
genetic and radiographic analysis of 108 patients
with von Hippel-Lindau disease (VHL) manifested
by pancreatic neuroendocrine neoplasms (PNETs).
Surgery. 2007;142(6):814–818, discussion 818.
e811–818.e812.
590. Ford D, Easton DF, Bishop DT, Narod SA,
Goldgar DE. Risks of cancer in BRCA1-mutation
carriers. Breast Cancer Linkage Consortium. Lancet.
1994;343(8899):692–695.
591. Satagopan JM, Offit K, Foulkes W, et al. The lifetime
risks of breast cancer in Ashkenazi Jewish carriers of
BRCA1 and BRCA2 mutations. Cancer Epidemiol
Biomarkers Prev. 2001;10(5):467–473.
592. Lalloo F, Varley J, Ellis D, et al. Prediction of patho-
genic mutations in patients with early-onset breast
cancer by family history. Lancet. 2003;361(9363):
1101–1102.
593. Chen S, Iversen ES, Friebel T, et al. Characterization
of BRCA1 and BRCA2 mutations in a large United
States sample. J Clin Oncol. 2006;24(6):863–871.
594. Birch JM, Alston RD, McNally RJ, et al. Relative
frequency and morphology of cancers in carriers of
germline TP53 mutations. Oncogene. 2001;20(34):
4621–4628.
595. Chompret A, Brugieres L, Ronsin M, et al. P53
germline mutations in childhood cancers and cancer
risk for carrier individuals. Br J Cancer. 2000;82(12):
1932–1937.
Genetic Factors: Hereditary Cancer Predisposition Syndromes  •  CHAPTER 13 208.e11
596. Evans DG, Birch JM, Thorneycroft M, McGown
G, Lalloo F, Varley JM. Low rate of TP53 germline
mutations in breast cancer/sarcoma families not
fulfilling classical criteria for Li-Fraumeni syndrome.
J Med Genet. 2002;39(12):941–944.
597. Masciari S, Dillon DA, Rath M, et al. Breast cancer
phenotype in women with TP53 germline mutations:
a Li-Fraumeni syndrome consortium effort. Breast
Cancer Res Treat. 2012;133(3):1125–1130.
598. Hwang SJ, Lozano G, Amos CI, Strong LC.
Germline p53 mutations in a cohort with childhood
sarcoma: sex differences in cancer risk. Am J Hum
Genet. 2003;72(4):975–983.
599. Brownstein MH, Wolf M, Bikowski JB. Cowden’s
disease: a cutaneous marker of breast cancer. Cancer.
1978;41(6):2393–2398.
600. Starink TM, van der Veen JP, Arwert F, et al. The
Cowden syndrome: a clinical and genetic study in
21 patients. Clin Genet. 1986;29(3):222–233.
601. Zbuk K, Stein JL, Eng C. PTEN Hamartoma tumor
syndrome (PHTS). Gene Reviews at GeneTests:
Medical Genetics Information Resource; 2013.
http://www.genetests.org.
602. Bubien V, Bonnet F, Brouste V, et al. High cumulative
risks of cancer in patients with PTEN hamartoma
tumour syndrome. J Med Genet. 2013;50(4):
255–263.
603. Yoon KA, Ku JL, Yang HK, Kim WH, Park SY,
Park JG. Germline mutations of E-cadherin gene
in Korean familial gastric cancer patients. J Hum
Genet. 1999;44(3):177–180.
208.e11
604. Athma P, Rappaport R, Swift M. Molecular genotyp-
ing shows that ataxia-telangiectasia heterozygotes are
predisposed to breast cancer. Cancer Genet Cytogenet.
1996;92(2):130–134.
605. Berstein JL, Concannon P, Langholz B, et al. Multi-
center screening of mutations in the ATM gene
among women with breast cancer—the WECARE
Study. Radiat Res. 2005;163(6):698–699.
606. Bretsky P, Haiman CA, Gilad S, et al. The relation-
ship between twenty missense ATM variants and
breast cancer risk: the Multiethnic Cohort. Cancer
Epidemiol Biomarkers Prev. 2003;12(8):733–738.
607. Thompson D, Seal S, Schutte M, et al. A multicenter
study of cancer incidence in CHEK2 1100delC
mutation carriers. Cancer Epidemiol Biomarkers Prev.
2006;15(12):2542–2545.
608. Shaag A, Walsh T, Renbaum P, et al. Functional and
genomic approaches reveal an ancient CHEK2 allele
associated with breast cancer in the Ashkenazi Jewish
population. Hum Mol Genet. 2005;14(4):555–563.
609. Rahman N, Seal S, Thompson D, et al. PALB2,
which encodes a BRCA2-interacting protein, is
a breast cancer susceptibility gene. Nat Genet.
2007;39(2):165–167.
610. Bonadona V, Bonaiti B, Olschwang S, et al. Cancer
risks associated with germline mutations in MLH1,
MSH2, and MSH6 genes in Lynch syndrome.
JAMA. 2011;305(22):2304–2310.
611. Watson P, Vasen HF, Mecklin JP, et al. The risk of
extra-colonic, extra-endometrial cancer in the Lynch
syndrome. Int J Cancer. 2008;123(2):444–449.
Genetic and Epigenetic Alterations in Cancer
S UMMARY OF K EY P OI NT S
• A root cause of cancer is the
accumulation of genetic and
epigenetic defects in key cellular
pathways regulating proliferation,
differentiation, and death. The
defects in cancer cells are of two
types: gain-of-function alterations
affecting oncogenes, and loss-of-
function alterations affecting tumor
suppressor genes. Regardless of
whether the defects are genetic or
epigenetic in nature, a common net
consequence is dysregulation of
gene expression in cancer cells. This
intimate relationship between genetic
and epigenetic changes has been
further confirmed by findings of
frequent mutations in chromatin
enzymes.
• More recently, genetic alterations in
noncoding DNA have also been
reported in cancer tissues. They
contribute to tumorigenesis by
affecting the regulatory elements
(e.g., promoters, enhancers) that
influence gene expression of key
cancer-related genes, and some
of them may correspond to
cancer risk genetic variants in
noncoding regions identified by
genome-wide association studies
(GWASs).
• In addition to genetic mutations and
genomic alterations, it is becoming
more evident that clinical and
pathologic studies indicate that
many cancers arise from preexisting
benign lesions, and numerous
cooperating genetic and epigenetic
defects affecting multiple
independent signaling pathways
are likely needed for development
of most clinically recognizable
cancers.
• A process termed clonal selection
has a key role in determining the
particular constellation of genetic
Bin Tean Teh and Eric R. Fearon
and epigenetic defects present in a
cancer cell. Clonal selection is
essentially an evolutionary process
that promotes outgrowth of
precancerous and cancerous cells
carrying those mutations and gene
expression changes that confer the
most potent proliferative and survival
properties on the cancer cells in a
given context.
• Although a sizeable and diverse
array of mutations and gene
expression changes have been
implicated in cancer pathogenesis,
the defects appear to affect a more
limited number of conserved
signaling pathways or networks.
A relatively small collection of
oncogenes and tumor suppressor
genes is recurrently deranged in
cancer cells of various types and
includes the RAS, PIK3CA, EGFR,
RAF, β-catenin, IDH1, and MYC
oncogene proteins and the p53,
p16Ink4a, ARF, RB1, PTEN, APC, NF1,
and ARID1A tumor suppressor
proteins. The proteins that are
recurrently targeted by mutations in
cancer likely represent particularly
critical hubs in the cell’s regulatory
circuitry.
• Although cancer represents a very
heterogeneous collection of
diseases, the development of all
cancers, regardless of type, appears
to be critically dependent on the
acquisition of certain traits that allow
the cancer cells to grow in an
unchecked fashion in their tissue of
origin and to grow as metastatic
lesions in distant sites in the body.
Signature traits that are likely to be
inherent in the majority of, if not all,
cancers include (1) an enhanced
response to proliferative and
growth-promoting signals; (2) a
relative resistance to growth
14 
inhibitory cues; (3) an increased
mutation rate to allow for the rapid
generation of new variant daughter
cells; (4) the ability to attract and
support a new blood supply
(angiogenesis); (5) the capacity to
minimize an immune response and/
or evade destruction by immune
effector cells; (6) the capacity for
essentially limitless cell division;
(7) a failure to respect tissue
boundaries, allowing for invasion into
adjacent tissues and organs with
microenvironments that are markedly
different from the one where the
cancer cells arose; (8) the ability to
escape cell death; (9) altered cell
metabolism to support uncontrolled
proliferation; and (10) the acquisition
of immune cells that promote tumor
progression.1
• Certain gene defects in cancer cells
may contribute to a few or perhaps
even only one of the signature traits.
However, many of the gene defects
and expression changes might have
been selected for in large part
because they exert pleiotropic
effects on the cancer cell
phenotype.
• Despite the fact that some gene
defects may arise early in the
development of certain cancer types,
advanced cancer cells might still be
critically dependent on the “early
gene defects” for continued growth,
survival, and even metastasis.
Studies have shown that metastatic
cancers still harbor the same genetic
alterations as the primary tumors.
Such findings imply that agents that
specifically target key signaling
pathways and proteins could
have use in advanced cancers,
even if the signaling pathway defect
arose very early in cancer
development.
Continued
209
210 Part I: Science and Clinical Oncology

• Genomic and epigenomic
characterization of organ site cancer
has identified a number of subtypes
with different prognoses and
therapeutic relevance.
• Future studies will further clarify the
role of the diverse array of genetic
and epigenetic defects in cancer
phenotypes, even at the level of a
single cell, allowing more definitive
and more specific strategies for
cancer detection, diagnosis, and
therapeutic targeting of cancer
cells.

A genetic basis for human cancer has been recognized for perhaps
more than a century and has been supported by data from familial
and epidemiologic studies and animal studies. However, only during
the past three decades has definitive molecular evidence been accu-
mulated to support the view that all cancer types arise from defects
in the structure and/or regulation of genes. Studies from many different
fields, including tumor virology, chemical carcinogenesis, molecular
biology and biochemistry, somatic cell genetics, developmental biology,
and genetic epidemiology, have all played critical contributing roles
in clarifying the contributions of genetic and epigenetic mechanisms
to cancer development. Although environmental and dietary factors
have substantial roles in cancer development, it is now well established
that the accumulation of multiple genetic and epigenetic alterations
in a single cell plays a fundamental role in cancer initiation and
progression.
The mutations that arise in cancer cells can be divided into two
functionally distinct classes: oncogene and tumor suppressor gene
mutations. In addition to the classic alterations of oncogenes and
tumor suppressor genes, other genetic alterations occur in genes regulat-
ing transcription and translation, as well as in genes responsible for
DNA repair. Although localized mutations are commonly observed,
other genetic variations are also important in oncogenesis, such as
copy number variation, deletion, and translocations. It is becoming
more evident that these genetic changes could occur in both coding
and noncoding DNA and that the latter may involve cis-regulatory
regions affecting gene transcription. All of these genomic and epi­
genomic alterations in the initiation and progression of cancer can
be broadly considered as oncogenic or “gain of function” and suppressor
or “loss of function.” In addition, some of these genetic alterations
may be present in an individual’s germline and may predispose to
particular cancers. Such mutations can also be passed on to future
generations. The nature and role of certain germline mutations in
cancer development are of great interest to cancer biologists because
the mutations provide powerful clues about the identity of genes and
pathways that play particularly critical roles in the malignant conversion
of cells. Nevertheless, germline mutations in oncogenes or tumor
suppressor genes likely have a major contributing role in the develop-
ment of only hereditary cancer, representing a small fraction of all
cancers, and the vast majority of mutations in cancer are somatic (i.e.,
present only in the tumor cells).
A few of the cellular genes that are recurrently affected by inherited
and somatic mutations in human cancer will be discussed in more
detail later. Brief mention will be made here of some general properties
of the mutated genes. Oncogenes act in a positive fashion to promote
tumorigenesis. Their normally functioning cellular counterparts, termed
proto-oncogenes, have been found to be important regulators of many
aspects of cell physiology. The proteins encoded by various proto-
oncogenes can be found in virtually all subcellular compartments.
The term proto-oncogene does not imply that genes of this class lie
dormant in the cell with the purpose of promoting tumorigenesis.
Rather, the terminology reflects the fact that mutations in cancer cells
alter the normal structure and/or expression pattern of the proto-
oncogene, generating oncogenic variants with altered function. In
genetic terms, oncogenic alleles have gain-of-function mutations that
confer enhanced or novel functions.
In contrast to the activating mutations in oncogenes, tumor sup-
pressor genes harbor loss-of-function defects in cancer cells. Historically,
the term antioncogene was sometimes used with respect to the tumor
suppressor gene class. The term suggests that the primary function
of the genes might be to act in direct opposition to activated oncogenes.
Although many of the proteins that are encoded by tumor suppressor
genes do in fact bind to and regulate the function of proto-oncogenes
or function in pathways that regulate proto-oncogene activity, it is
not by necessity a general principle. Hence, genes that contribute to
cancer by virtue of their inactivation or loss-of-function mutations
in human cancers are referred to here as tumor suppressor genes. Similarly
to the proto-oncogenes, the normal functions of tumor suppressor
genes are diverse, and the proteins encoded by these genes are found
in essentially all compartments of the cell.
In addition to the well-established role of oncogene and tumor
suppressor gene mutations in cancer, it is now abundantly clear that
epigenetic mechanisms play critical roles in altering the patterns and
levels of expression of certain proto-oncogenes and tumor suppressor
genes in cancer. For instance, in some cancers, altered transcriptional
regulatory mechanisms can lead to markedly increased levels of proto-
oncogene expression, akin to the level seen in cancer cells with
mutational defects that alter the structure or copy number of the
proto-oncogene. Conversely, gene-silencing mechanisms can exert
dramatic effects on the expression of certain tumor suppressor genes
in cancer cells, essentially rendering the genes functionally inactive
in the absence of any mutations. It is interesting that sequence-based
analyses of many different cancer types have revealed that many of
the mutations in cancer cells directly affect the cancer epigenome.
Specifically, the oncogenes and tumor suppressor gene mutations in
cancer cells often target transcription factors, chromatin modifying
proteins, and other chromatin-associated proteins, leading to potentially
quite dramatic global effects on the structure and activity of chromatin,
DNA methylation, histone modifications, and patterns of gene expres-
sion in cancer cells.
Finally, mutations in genes that regulate the recognition and repair
of DNA damage also play critical roles in tumorigenesis. The DNA
damage recognition and repair genes could be considered to constitute
a distinct class of cancer genes, because at least some of the DNA
repair proteins might have a more passive role in cell proliferation,
differentiation, and cell survival. Their inactivation in tumor cells
might lead predominantly to the acquisition of a “mutator phenotype,”
with a resultant increased rate of mutations in other cellular genes.
More recently, these tumors with higher mutation load, and therefore
presumably generating more tumor-related neoantigens, were found
to be more responsive to checkpoint immunotherapy.2,3
Given the enormous advances during the past two decades, especially
the recent use of next-generation sequencing (NGS) in profiling and
characterizing gene mutations and expression defects in cancer, it will
not be possible in this chapter to review in a comprehensive fashion
the vast collection of genetic and epigenetic defects that have been
catalogued in human cancers. Nor will it be possible to discuss in
detail the possible contributions of the many different gene defects
to alterations in cell signaling and cell physiology. Rather, the primary
aim of this chapter will be to offer a framework for understanding
the relationships between genetic and epigenetic defects in cancer
cells and the impact of the accumulated defects on the cancer cell
phenotype. Although some details on the identity and nature of gene
defects in cancer will be offered here, the emphasis will be on concepts
with biologic and clinical significance.
 Genetic and Epigenetic Alterations in Cancer  •  CHAPTER 14 211

RECURRENT MUTATIONAL TARGETS
IN CANCER
Oncogenic variant alleles that are present in cancer are generated
from the normal counterpart proto-oncogenes by various mutational
mechanisms, including point or localized mutations, gross chromo-
somal rearrangements, or gene amplification. Some representative
oncogene mutations in human cancer are summarized in Table 14.1.
From a brief review of the data in Table 14.1, some generalizations
can be offered. First, the mutations affect proteins functioning in
various compartments of the cell, including growth factor receptors,
cytoplasmic signal transducers, and nuclear proteins, such as transcrip-
tion factors. Second, although some oncogene mutations may be
unique to cancers of a particular type, such as the specific chromosomal
translocations and resultant fusion proteins that are seen in cancers
of hematopoietic origin (e.g., the BCR-ABL translocation that is seen
in chronic myelogenous leukemia and a subset of acute lymphoid
leukemias and the PML-RARα translocation that is seen in acute
promyelocytic leukemia), other mutations, such as those affecting the
KRAS, β-catenin, and c-MYC genes, are found in a broad spectrum
of different cancer types. Third, oncogene mutations in cancer are
nearly always somatic, because only a very limited number of germline

Table 14.1  Selected Oncogene Mutations in Cancer

Gene Activation Mechanism Protein Properties Tumor Types
KRAS Point mutation Signal transducer Pancreatic, colorectal, lung (adeno), endometrial,
other carcinomas
NRAS Point mutation Signal transducer Myeloid leukemia, colorectal cancer
HRAS Point mutation Signal transducer Bladder carcinoma
EGFR (ERBB) Amplification, mutation Growth factor (EGF) receptor Gliomas, lung (non–small cell) carcinoma
NEU (HER2/ERBB2) Amplification Growth factor receptor Breast, ovarian, gastric, other carcinomas
c-MYC Chromosome translocation Transcription factor Burkitt lymphomas
Amplification Small cell lung carcinoma (SCLC); other carcinomas;
glioblastoma
MYCN Amplification Transcription factor Neuroblastoma, SCLC; glioblastoma
MYCL1 Amplification Transcription factor SCLC, ovarian carcinoma
BCL2 Chromosome translocation Antiapoptosis protein B-cell lymphoma (follicular type)
CCND1 Amplification Cyclin D, cell cycle control Breast and other carcinomas
Chromosome translocation B-cell lymphoma, parathyroid adenoma
BCR-ABL Chromosome translocation Chimeric nonreceptor tyrosine kinase CML, ALL (T cell)
RET Chromosome translocation GDNF receptor tyrosine kinase Thyroid cancer (papillary type)
Point mutation Thyroid cancer (medullary type: germline mutations)
CDK4 Amplification
Point mutation Cyclin-dependent kinase Sarcoma, glioblastoma
MET Point mutation Hepatocyte growth factor (HGF) Renal carcinoma (papillary type: germline mutations)
receptor
SMO Point mutations Transmembrane signaling molecule Basal cell skin cancer
in Sonic Hedgehog pathway
CTNNB1 (β-CAT) Point mutation, in-frame deletion Transcriptional coactivator, links Melanoma; colorectal, endometrial, ovarian,
E-cadherin to cytoskeleton hepatocellular, and other carcinomas,
hepatoblastoma, Wilms tumor
FGF4 Amplification Growth factor (FGF-like) Gastric carcinoma
PML-RARA Chromosome translocation Chimeric transcription factor APL
TCF3-PBX1 Chromosome translocation Chimeric transcription factor Pre-B ALL
MDM2 Amplification p53 binding protein Sarcoma
GLI1 Amplification Transcription factor Sarcoma, glioma
TTG2 Chromosome translocation Transcription factor T-cell ALL
AKT2 Amplification Signal transducer (serine/threonine Pancreatic and ovarian carcinoma
kinase; downstream effector of PI3K)
PIK3CA Amplification Catalytic subunit of PI3K Ovarian carcinoma
AURKA Amplification Centrosome-associated kinase Breast, colon, ovarian, and prostate carcinomas
gliomas
TMPRSS2-ERG Chromosome translocation Transcription factor (ETS family) Prostate cancer
TMPRSS2-ETV1
TMPRSS2-ETV4

ALL, Acute lymphocytic leukemia; APL, acute promyelocytic leukemia; CML, chronic myelogenous leukemia; EGF, epidermal growth factor; FGF, fibroblast growth factor; GDNF,
glial-derived neurotrophic factor; HGF, hepatocyte growth factor; PI3K, phosphatidylinositol 3-kinase; SCLC, small cell lung carcinoma.
212 Part I: Science and Clinical Oncology
mutations in proto-oncogenes have been linked to cancer predisposi-
tion thus far. Fourth, some proto-oncogenes, such as KRAS or BCL2,
are somatically altered in cancer by a single mutational mechanism,
namely, point mutations in the KRAS gene and chromosomal transloca-
tions affecting the BCL2 gene. In contrast, other proto-oncogenes,
such as c-MYC, may be activated by more than one mechanism in
cancer, including chromosomal translocation, gene amplification,
and, more recently discovered, association with superenhancer. These
mutational mechanisms lead to increased levels of c-MYC transcripts
and protein. In fact, specific missense mutations at threonine 58
of the c-MYC gene in some lymphomas may further enhance
c-MYC protein levels by abrogating a phosphorylation-ubiquitination
mechanism targeting c-MYC for proteosomal degradation.4 However,
enhanced c-MYC protein levels can also result from alterations in
c-MYC–specific microRNAs, methylation, and other noncoding
regulatory elements functioning to regulate c-MYC transcription and
translation.
In contrast to the activating mutations that generate oncogenic
alleles from proto-oncogenes, inactivation of the normal function of
tumor suppressor genes is critical in tumorigenesis. Akin to the proto-
oncogenes, the functions of tumor suppressor genes are diverse, and
proteins that are encoded by these genes reside in practically all subcel-
lular compartments (Table 14.2). Many tumor suppressor genes were
identified by virtue of the fact that they are mutated in the germline
of persons who are affected by a known mendelian cancer syndrome
or who at the very least display a markedly elevated risk of cancer.
The link between a germline-inactivating mutation in a purported
tumor suppressor gene and increased cancer predisposition provides
very persuasive evidence of the functional significance of the gene in
the cancer process. Nevertheless, for the vast majority of tumor sup-
pressor genes, in terms of their magnitude, somatic inactivating
mutations play a far more significant role in cancer development than
do germline mutations.
Another important point to consider is that much of the attention
for tumor suppressor genes has been focused on demonstrating that
cancer cells carry biallelic inactivating mutations. Clearly, a diverse
array of mechanisms can inactivate gene function, including nonsense,
frameshift, and nonconservative missense mutations, as well as splicing
mutations and gross deletions of the gene or even the chromosome
region that contains the gene. In a number of cases, studies of the
chromosomal mechanisms associated with tumor suppressor gene
inactivation in cancer tissues, such as loss of the parental heterozygosity
(i.e., loss of heterozygosity) that is present in normal tissues, have
even been used to infer the existence of tumor suppressor genes, in
particular, chromosomal regions, before the actual identification of
the tumor suppressor gene of interest. The emphasis on defining
biallelic inactivating mutations in tumor suppressor genes has been
stimulated in large part by the Knudson hypothesis,5,6 which predicted
that recessive genetic determinants played a critical role in retinoblas-
toma and many other cancers and that inactivation of both alleles of
a tumor suppressor gene was needed to abrogate tumor suppressor
gene activity. Nevertheless, as will be discussed in a bit more detail
in the following sections, a variety of observations indicate that epi-
genetic (nonmutational) mechanisms might play a prominent role in
inactivating tumor suppressor gene function in sporadic tumors.
Furthermore, for certain tumor suppressor genes, inactivation of only
one of the two alleles of a tumor suppressor gene might significantly
impair cell growth regulation or programmed cell death. For example,
a number of mutant p53 proteins that carry missense mutations likely
potently interfere via dominant negative mechanisms with the wild-type
p53 protein in the cell because p53 functions as a homotetrameric
protein and all subunits must be wild-type for fully intact p53 function
in transcriptional regulation.7 For other tumor suppressor proteins,
such as the cyclin-dependent kinase inhibitory protein p27, reduction
of protein levels to 50% of the levels present in normal cells might
result in significant detrimental effects on the ability of the cell to
appropriately regulate growth.8
CANCERS ARISE FROM THE
ACCUMULATION OF MULTIPLE GENETIC
AND EPIGENETIC DEFECTS
On the basis of a simple consideration that a cancer involves a large
number of genetic and epigenetic alterations that arise in normal cells
during the many years of life, it would seem quite unlikely that any
adult cancer may arise as the result of single gene defect. Even in
persons who are strongly predisposed to cancer as a result of a germline
mutation in a specific oncogene or tumor suppressor gene, the vast
majority of cells in a person never develop into cancer or even display
definitive morphologic changes akin to those that are seen in benign
tumors. Therefore, any model for cancer must incorporate these data,
which suggest that cancers likely arise as the result of the accumulation
of multiple genetic and epigenetic defects in an affected cell. Another
issue to consider is that clinical and histopathologic data indicate that
the development of nearly all cancers, regardless of the organ site, is
often, if not invariably, preceded by precancerous phases or stages in
which the neoplastic cells manifest increasingly disordered patterns
of differentiation and morphology.
Given this background, it would appear that cancers arise from
accumulated defects affecting multiple genes and pathways, and
precancerous (benign) precursor lesions harbor fewer of the key gene
defects than established cancers. One question, therefore, is how many
rate-limiting defects or “hits” are required for cancer development.
Although a definitive answer to this question cannot be given at this
point, some estimates can be offered. Most common cancers show
dramatically increased incidence with increasing age. On the basis of
analysis of the age-specific incidence of a number of common cancers
and some straightforward assumptions about the rate of mutations
and the size of the target cell population, it was argued as early as the
mid-1950s that most common epithelial cancers arise as the result
of four to seven rate-limiting events.5,9 It was inferred that these
rate-limiting events might represent mutational events. Moreover,
benign lesions harboring fewer gene defects are often shown to have
an age-incidence distribution that was shifted roughly one to two
decades earlier in life than cancers arising in the corresponding organ
or tissue site. Nevertheless, confounding the use of age-incidence
data to model the number of rate-limiting mutations were questions
about the weight of importance of certain key biologic pathways
underlying the multiple hits models. On the other hand, studies of
the genetic alterations of pediatric cancers that occur much earlier
and are perhaps less complex in terms of their genetic signature may
offer insights into key cancer target genes (see Chapter 92 on pediatric
solid tumors).
CLONAL SELECTION AND EVOLUTION
IN CANCER
As previously noted, most if not all cancers arise from preexisting
precancerous populations of cells, and multiple genetic and epigenetic
defects are likely needed for conversion of a normal cell to a cancerous
cell. Molecular studies of various cancer types and their corresponding
associated precancerous lesions have yielded some fundamental insights
into the processes likely to be critical in the emergence of the cancer.
First, although normal tissues and tissues from noncancerous disease
states display polyclonal (balanced) cell populations, the neoplastic
component that is present in benign lesions and cancers displays a
clonally related cell population, consistent with the notion that
neoplastic transformation of one or a number of different cells within
a tissue give rise to all daughter cells or subclones that are present in
the tumor. This is further established by NGS deep sequencing in
studying intratumor heterogeneity.10 Second, in tumors in which it
has been possible to analyze both cancer cells and associated precancer-
ous cell populations, a subset of the somatic gene defects present in
the cancer are clonally represented in the precancerous cell population.
 Genetic and Epigenetic Alterations in Cancer  •  CHAPTER 14 213

Table 14.2 Selected Tumor Suppressor Gene Mutations in Hereditary Cancer Syndromes and
Sporadic Cancers
Associated Inherited Cancer Cancers With Somatic
Gene Syndrome Mutations Presumed Function of Protein
RB1 Familial retinoblastoma Retinoblastoma, osteosarcoma, SCLC, Transcriptional regulator; E2F binding
breast, prostate, bladder, pancreas,
esophageal, others
TP53 Li-Fraumeni syndrome Approximately 50% of all cancers Transcription factor; regulates cell
(rare in some types, such as prostate cycle and apoptosis
carcinoma and neuroblastoma)
CDKN2A (p16INK4a protein) Familial melanoma, familial Approximately 25%–30% of many Cyclin-dependent kinase inhibitor (i.e.,
pancreatic carcinoma different cancer types (e.g., breast, CDK4 and CDK6)
lung, pancreatic, bladder)
CDKN2A (ARF protein) Familial melanoma (?) Approximately 15% of many Regulates MDM2 protein stability and
different cancer types hence p53 stability; alternative reading
frame of CDKN2A gene
APC FAP coli, Gardner syndrome, Colorectal carcinomas, desmoid Regulates levels of β-catenin protein in
Turcot syndrome tumors, hepatocellular carcinoma, the cytosol; binding to EB1 and
breast (rare) microtubules
WT1 WAGR, Denys-Drash syndrome Wilms tumor Transcription factor
NF1 Neurofibromatosis type 1 Melanoma, neuroblastoma p21ras-GTPase
NF2 Neurofibromatosis type 2 Schwannoma, meningioma, Juxtamembrane link to cytoskeleton at
ependymoma adherens junction
VHL von Hippel–Lindau syndrome Renal (clear cell type), Regulator of protein stability
hemangioblastoma
BRCA1 Inherited breast and ovarian Ovarian (~10%), rare in breast cancer DNA repair; complexes with Rad 51
cancer and BRCA2; transcriptional regulation
BRCA2 Inherited breast (both female and Rare mutations in pancreatic, DNA repair; complexes with Rad 51
male), pancreatic cancer others (?) and BRCA1
MEN1 Multiple endocrine neoplasia Parathyroid adenoma, pituitary Nuclear protein; unknown function
type 1 adenoma
Endocrine tumors of the pancreas
PTCH1 Gorlin syndrome, hereditary basal Basal cell skin carcinoma, Transmembrane receptor for Sonic
cell carcinoma syndrome medulloblastoma Hedgehog factor; negative regulator of
Smoothened protein
PTEN Cowden syndrome; sporadic cases Glioma, breast, prostate, follicular Phosphoinositide 3-phosphatase;
of juvenile polyposis syndrome thyroid carcinoma, head and neck protein tyrosine phosphatase
squamous carcinoma; many others
SMAD4 Familial juvenile polyposis Pancreatic (~50%), approximately Transcriptional factor in TGF-β
syndrome 10%–15% of colorectal cancers, rare signaling pathway
in others
BMPR1A Familial juvenile polyposis Not known Receptor for bone morphogenetic
syndrome protein
MSH2, MLH1 PMS1, PMS2, Hereditary nonpolyposis Colorectal, gastric, endometrial, DNA mismatch repair
MSH6 colorectal cancer ovarian
CDH1 Familial diffuse-type gastric Gastric (diffuse type), lobular breast E-cadherin cell-cell adhesion molecule
cancer carcinoma, rare in other types (e.g.,
ovarian)
STK11 (LKB1) Peutz-Jeghers syndrome Lung adenocarcinoma; rare pancreas Serine/threonine protein kinase
cancers; absent in most other
cancers
EXT1 Hereditary multiple exostoses Osteochondroma Glycosyltransferase; heparan sulfate
chain elongation
EXT2 Hereditary multiple exostoses Not known Glycosyltransferase; heparan sulfate
chain elongation
TSC1 Tuberous sclerosis Bladder carcinoma; head and neck Hamartin; binds tuberin (TSC2);
squamous cancer; hepatocellular regulates cell size by inhibiting TOR
carcinoma function and protein synthesis
TSC2 Tuberous sclerosis Head and neck squamous cancer Tuberin (see previous row regarding
TSC1)

FAP, Familial adenomatous polyposis; GTPase, guanosine triphosphatase; SCLC, small cell lung carcinoma; TGF, transforming growth factor; TOR, target of rapamycin; WAGR,
Wilms tumor, aniridia, genitourinary anomalies, and mental retardation.
214 Part I: Science and Clinical Oncology

Other somatic gene defects appear to be acquired during progression
from the precancer subclones to the dominant subclones in the cancer.
These molecular findings in benign and malignant tumors are
essentially consistent with a model initially proposed by Foulds11 and
subsequently advanced by Nowell12 (Fig. 14.1). In brief, the clonal
evolution model predicts that cancers arise as the result of successive
expansions of clonally related cell populations. The successive expansions
are driven by the gradual or punctuated acquisition of mutations and
gene expression changes that endow a particular cell and its progeny
with a selective advantage over cells that do not harbor the gene
defects. In essence, clonal selection is an evolutionary process that
allows the outgrowth of precancerous and cancerous cells that carry
mutations and gene expression changes that confer the most potent
proliferative and survival properties on the cancer cells. However, it
is important to note that the specific constellation of genetic and
epigenetic changes that are present in precancerous and cancerous
cells is context-dependent and certainly varies considerably from one
cancer type to another and even most certainly varies to a significant
degree among patients whose cancers display quite similar clinical
and histopathologic features. The clonal evolution model has biologic
and clinical ramifications, just a few of which will be mentioned here.
First, the clonal gene defects that are present in a cancer can be traced
in precancerous lesions from the same organ site with a goal of
attempting to clarify the preferred order in which gene defects arise
in the natural history of a particular cancer type. The particular order
in which defects accumulate during the initiation and subsequent
progression of one cancer type often differs from that in another
cancer type. As a result, a genetic or epigenetic change that is critical
in tumor initiation in one cancer type might contribute to tumor
progression in another tumor type and vice versa. Second, defects
that arise at “early” stages of tumorigenesis might play a vital role not
only in tumor initiation but also in the aggressive behavior of advanced
stage cancers. Third, the model predicts that the acquisition of further
genetic heterogeneity will be a common and important factor in primary
cancer lesions and metastases. It is becoming more evident that genetic
heterogeneity plays a significant role in resistance to chemotherapy
and targeted therapy and the emergence of aggressive cell populations
in patients with advanced cancer.
Recent comprehensive sequence-based analyses of cancer cell
populations from individual patients in which the tumor cells under
study were distinct from one another in location and/or time has
begun to reveal that quite dramatic intratumoral genetic heterogeneity
may be a “rule” in cancer, rather than an exception.13 Certain, perhaps
key, initiating genetic lesions (e.g., truncal mutations) might be shared
among all neoplastic clones, but geographically distinct regions of
large primary tumors may have very distinct mutation profiles (e.g.,
branched mutations) from those in other portions of the primary
tumor, and the metastatic cell populations may have considerable
mutational divergence from the nonmetastatic cells. Accordingly, it
seems that quite extensively branched evolutionary growth may be
an important feature in both primary tumors and metastatic lesions,
with multiple competing clonal populations evolving through divergent

Normal Competing neoplastic subclones Successful malignant

stem/progenitor subclone
cell population

Time (years to decades)

Mutations and epigenetic defects

Figure 14.1  •  Role of clonal selection in cancer development and progression. Clonal selection is essentially an evolutionary process that promotes outgrowth
of precancerous and cancerous cells that carry the mutations and gene expression changes that confer the most potent proliferative and survival properties on
the cancer cells in a given context. The schematic diagram indicates that the stepwise emergence of benign and malignant cells over time is critically influenced
by mutations and epigenetic defects. Neoplasms most likely arise from a stem cell or progenitor cell population that is capable of additional cell divisions and
acquisition of certain differentiated characteristics. After the accumulation of a particular constellation of mutations and epigenetic defects in oncogenes and
tumor suppressor genes, a successful malignant subclone will outgrow the various competing neoplastic subclones. Further genetic heterogeneity within a
malignant subclone (not depicted) is likely, and the genetic heterogeneity might give rise to new subclones that display increased invasive and metastatic
potential. (Modified from Kern SE. Progressive genetic abnormalities in human neoplasia. In: Mendelsohn J, Howley PM, Israel MA, Liotta LA, eds. The
Molecular Basis of Cancer. 2nd ed. Philadelphia: Saunders; 2001.)

and convergent mutational mechanisms.13 This more recent view of
the potentially quite extensive intratumoral genetic heterogeneity in
any given cancer and the contributions of intratumoral heterogeneity
to tumor progression contrasts with some earlier views. Before the
more recent studies, it was suspected that the cell populations in many
primary cancers might be more homogeneous, wherein somatic
mutations were accumulated in a more stepwise fashion as a result of
multiple sequential clonal sweeps of each variant cell population in
the primary cancer, with metastases perhaps most often arising from
the clonally dominant cell population in the primary tumor.
It is important to note that clonal somatic mutations are often
presumed to have a causal role in promoting further tumor outgrowth
or progression because somatic mutations can become clonal (i.e.,
present in all neoplastic cells) by only a limited number of mechanisms.
For instance, the genetic alteration itself could have been selected for
because it provided the neoplastic cell with a growth advantage, allowing
it to become the predominant cell type in the tumor (clonal expansion).
Genes with critical roles in promoting clonal outgrowth in a given
cancer have been termed drivers.14 Genes that are mutant in a significant
fraction of cancers and for which other lines of evidence link them
to the cancer process can more readily be classified as drivers. Alter-
natively, a somatic mutation, when detected, might have arisen
essentially coincident with another, perhaps undetected, alteration
that was the crucial change underlying clonal outgrowth. Somatic
mutations of this latter type have been termed passengers.14 However,
studies have shown that some of these passengers, which serve as
metabolic and housekeeping genes, can be targeted therapeutically,
leading to tumor suppression.15,16 Therefore, on the basis of extensive
data from large-scale sequencing analyses that reveal large numbers
of distinct genes that are each mutated in only a minority of cancers
of a given type,17–20 sorting out drivers and passengers might not be
entirely straightforward based solely on sequencing data and will likely
require a significant body of functional studies and data. Indeed,
increasingly even noncoding alterations have been shown to play
important roles in causing and maintaining the cancer phenotypes
by introducing new cancer-causing promoters and enhancers that lead
to altered gene expression (see the later discussion of noncoding
mutations). Given this discussion about the potential uncertainties
associated with linking specific somatic mutations to cancer develop-
ment, it is even more challenging to assign causal significance to any
given gene expression change in precancerous and cancerous cells. In
particular, the ambiguities arise when considering whether a gene
expression or epigenetic change merely reflects the relative difference
between cancer and its matched normal tissue or is truly causally
involved in the cancer process. Nonetheless, if a specific gene or
epigenetic alteration can be shown to promote tumorigenesis or
neoplastic transformation in a variety of in vitro and in vivo tumor
models or if the same gene or chromosomal region is recurrently
altered by particular epigenetic changes in tumors, then it might be
reasonable to infer that the particular defect might indeed have a
causal (driver) role in tumorigenesis.
CONTRIBUTION OF GENE DEFECTS TO THE
SIGNATURE TRAITS OF CANCER CELLS
Cancer represents a highly heterogeneous collection of diseases. Each
cancer type has distinct biologic and clinical features and a variable
prognosis. Even cancers that arise at a single organ site, such as the
ovary, kidney, or lung, may represent a hodgepodge of different diseases
at the molecular level. Morphologic features often allow the particular
cancer types to be distinguished to some degree from one another.
Yet even for patients whose cancers have essentially identical gross
and microscopic appearances and very similar clinical manifestations,
there may be vast differences in outcome. In spite of this complexity,
the development of all cancers, regardless of type, is likely to be critically
dependent on the acquisition of certain phenotypic features that allow
the cancer cells not only to grow in an unchecked fashion in their
Genetic and Epigenetic Alterations in Cancer  •  CHAPTER 14 215
tissue of origin but also to gain the ability to disseminate into sur-
rounding tissues and organs, lymphatics, and bloodstream and ultimately
to grow as metastatic lesions in distant sites in the body.1 As indicated
in Fig. 14.2, among the signature traits that are likely to be expressed
in the majority, if not all, of cancers are the following: (1) an increased
tendency to manifest a stem cell or progenitor-like phenotype; (2) an
enhanced response to growth-promoting signals; (3) a relative resistance
to growth-inhibiting cues; (4) an increased mutation rate to allow for
the rapid generation of new variant daughter cells; (5) the ability to
attract and support a new blood supply (angiogenesis); (6) the capacity
to minimize an immune response and/or evade destruction by immune
effector cells; (7) the capacity for essentially limitless cell division; (8)
a failure to respect tissue boundaries, allowing for invasion into adjacent
tissues and organs as well as blood vessels and lymphatics; and (9)
the ability to grow in organ sites with microenvironments markedly
different from the one where the cancer cells arose. The development
of some traits is likely to be associated with certain stages of tumori-
genesis (see Fig. 14.2), but acquisition of signature traits in cancers
is more likely to show a preferred order than an invariant order.
Furthermore, many of the signature traits of cancer cells represent
complex biologic capabilities (e.g., angiogenic activity, immune evasion
and resistance, and metastatic competence). Therefore, it is likely that
substantial changes in many signaling pathways are needed for the
cancer cell to manifest these traits.
Some of the gene defects seen in cancer cells may contribute
predominantly to a few or perhaps even only one of the signature
traits of cancer cells previously listed, but it seems likely that many
of the gene defects and expression changes have been selected for in
large part because they exert pleiotropic effects on the cancer cell
phenotype. Furthermore, defects in certain genes and signaling pathways
might be strongly selected for at a certain point in cancer development
and progression; perhaps in large part the alterations allow the precancer-
ous or cancerous cells to acquire certain critical phenotypic features.
Gene defects that might be nearly uniformly present in early-stage
lesions of one tumor type might preferentially arise in later-stage
tumors in another organ site. A good example will be mutations of
the KRAS oncogene, which is commonly present in early-stage pan-
creatic cancer but appears in late-stage cancer of other types. The data
suggest that cellular and tissue context has critical, albeit poorly
understood, modifying effects on the specific genetic defects that give
rise to neoplastic transformation, clonal outgrowth, and tumor progres-
sion. Despite the elaboration of the so-called hallmarks of cancers,
which involve not just cancer cells but the whole environment and
noncancerous cells such as immune, stromal, and blood vessel cells,
to fully understand the underlying causal mechanisms of each trait
and how to therapeutically target them individually or in combination
remains very challenging.
Alterations in Cancer Target Conserved Signaling
Pathways and Networks
As noted previously and summarized in Tables 14.1 and 14.2, the
protein products of proto-oncogenes and tumor suppressor genes have
been implicated in diverse cellular processes. In light of the potentially
enormous level of complexity suggested by the vast and diverse array
of gene defects in cancer, it is somewhat reassuring that some general
concepts have emerged with respect to the means by which genetic
and epigenetic alterations likely contribute to cancer initiation and
progression. A principal overarching theme is that the protein products
of oncogenes and tumor suppressor genes function in highly conserved
signaling pathways and regulatory networks.
The network in which the retinoblastoma protein (pRb) tumor
suppressor protein (encoded by the RB1 gene) functions is one of the
more intensively studied oncogene–tumor suppressor gene networks.
The pRb protein regulates cell cycle progression, in large part via its
ability to bind to E2F transcription factor proteins.21,22 In addition
to its role as a cell cycle regulator, the pRb protein has been implicated
216 Part I: Science and Clinical Oncology

Normal

Stem/progenitor cell phenotype Benign lesion

Enhanced response
to growth-promoting signals
Carcinoma in situ
Resistance to growth-inhibitory
and apoptosis-inducing cues

Increased mutation rate

Immune evasion/resistance Locally invasive carcinoma

Angiogenic activity

Capacity for limitless cell division

Invasive capacity

Metastatic competence
Distant
metastasis

Figure 14.2  •  Acquisition of signature traits in neoplastic cells during cancer progression. Depicted in the figure are representative stages in the development
of a cancer, perhaps a typical epithelial cancer, such as those that typically arise in the lung, colon, breast, or prostate. The schema suggests that most advanced
cancers arise via clonal selection from subclones present in earlier-stage benign and localized lesions (e.g., carcinoma in situ and locally invasive carcinoma).
Some of the properties of advanced cancer cells are depicted by the ability of the cells to enter the bloodstream and to seed and grow in distant organ sites,
such as the liver (bottom). Nine signature traits of cancer cells are listed. The relative time at which neoplastic cells acquire some of the traits is uncertain,
although it seems likely that some traits, such as the expression of a stem/progenitor cell phenotype or enhanced response to growth-promoting signals, may
be acquired earlier in cancer development. Other traits, such as invasive capacity and/or metastatic competence, may be acquired later. Many signature traits
of cancer cells are in fact complicated biologic capabilities (e.g., angiogenic activity, immune evasion and resistance, and metastatic competence) and likely
depend on defects in a number of different factors and signaling pathways.

in regulation of cellular differentiation, survival, and even angiogenesis
in certain settings.22 The binding of pRb to E2F proteins allows pRb
to silence expression of E2F-regulated or “target” genes, such as those
needed for the DNA synthetic (S) phase of the cell cycle. The ability
of the pRb protein to bind to E2F proteins and to function in
transcriptional repression appears to be tightly linked to its phosphoryla-
tion status, with the hyperphosphorylated forms of pRb being incapable
of binding to and regulating E2F proteins. The cyclin D1 protein
and its associated protein kinase, cyclin-dependent kinase 4 (CDK4),
negatively regulate pRb by phosphorylating it. The p16INK4a tumor
suppressor protein is a critical inhibitor of the CDK4/cyclin D1 complex
(Fig. 14.3) and can therefore prevent the inactivation of pRb. As is
noted in Table 14.2, a subset of sporadic cancers of various types has
inactivating mutations in the RB1 gene. In other cancers, pRb function
appears to be critically compromised as a result of mutations in other
components of the network or pathway. For example, in many cancers
that lack RB1 mutations, inactivating mutations in the CDKN2A
gene have been noted. In other cancers, including some breast cancers,
gene amplification and overexpression of cyclin D1 is found. In yet
others, such as some glioblastomas and sarcomas, amplification and
overexpression of the CDK4 gene have been seen. The net effect of
mutations in the pRb pathway, whether in RB1 itself or in other
genes, such as CDKN2A, CCND1 (cyclin D1), or CDK4, is to inactivate
pRb function and its ability to regulate expression of critical E2F
target genes (see Fig. 14.3).
Studies of other proto-oncogenes and tumor suppressor genes have
also supported the existence of conserved regulatory networks in which
multiple different tumor suppressor gene and proto-oncogene protein
products function. Although the adenomatous polyposis coli (APC)
tumor suppressor protein may have roles in regulating various processes
in the cell,23 a key function of APC is to participate in a multiprotein
complex that regulates the levels of the β-catenin protein in the
cytoplasm and nucleus (see Fig. 14.3). Components of the multiprotein
complex that regulates β-catenin include the APC protein, another
tumor suppressor protein known as AXIN1, and a kinase known as
glycogen synthase kinase 3β (GSK3β). Inactivation of APC or AXIN1
function in cancer cells appears to lead to an inability to phosphorylate
β-catenin and hence target it for recognition and subsequent ubiquitina-
tion by the βTrCP ubiquitin ligase and ultimately its destruction by
the proteasome.23,24 As a result, cancer cells with APC or AXIN1
inactivation display increased levels of β-catenin in the cytoplasm and
nucleus and essentially constitutive complexing of β-catenin with
transcription factors of the T-cell factor (TCF) family, such as TCF4.
When bound to TCF4, β-catenin can function as a transcriptional
coactivator, and in cancers with APC inactivation, such as colorectal
carcinomas, TCF transcriptional activity is clearly deregulated. In a
 Genetic and Epigenetic Alterations in Cancer  •  CHAPTER 14 217

pRb pathway APC/β-catenin pathway p53 pathway

p16 Wnt p19ARF

CYC D1 Cdk4 GSK3β MDM2

APC AXIN1

pRb p53
β-CAT TCF-4

E2F DP

Target genes Target genes Target genes

[e.g., Cyclin E, DHFR, [e.g., c-MYC, CYC D1, MMP-7, [e.g., p21CIP1, GADD45, BAX,
TS, DNA Pol α, RNR] survivin, CD44, EphB2/B3] p53AIP1, TSP1, MDM-2]

Figure 14.3  •  Recurrent gene defects in conserved signaling pathways in cancer. Three signaling pathways that are commonly affected by mutations in
various cancers are shown. Selected interactions between components of the pRb (left), APC/β-catenin (middle), and p53 pathways (right) are shown. Tumor
suppressor proteins are indicated with red symbols, oncogene products are indicated by green, and proteins that are not known to be affected by mutational
or epigenetic defects in human cancer are indicated in yellow. Inhibitory interactions between proteins are indicated by perpendicular lines, and activating
effects are indicated by arrows. Presumptive downstream genes whose expression is affected by the pathways are noted. APC, Adenomatous polyposis coli;
AXIN1, axis inhibition protein 1; β-CAT, β-catenin; Cdk4, cyclin-dependent kinase 4; CYC D1, cyclin D1; DHFR, dihydrofolate reductase; DNA Pol α, DNA
polymerase α; GADD45, growth arrest and DNA damage inducible gene 45; GSK3β, glycogen synthase 3β; MMP-7, matrix metalloproteinase 7; p21, p21
CDK-interacting protein 1; p53AIP1, p53-regulated apoptosis-inducing protein 1; RNR, ribonucleotide reductase; TCF-4, T-cell factor-4; TS, thymidylatesynthase;
TSP1, thrombospondin 1.

subset of the colorectal carcinomas that lack APC inactivation and
in a variety of other cancer types (see Table 14.1), activating (oncogenic)
mutations in the β-catenin protein have been found.24 These missense
and in-frame deletion mutations affect key phosphorylation sites in
the N-terminus of β-catenin, essentially rendering β-catenin resistant
to regulation by the APC/AXIN/GSK3β complex. Hence, the mutant
β-catenin protein accumulates in the cell and deregulates TCF transcrip-
tion. Transcription of selected proto-oncogenes may be activated directly
by the β-catenin/TCF complex, such as c-MYC.25
Other tumor suppressor gene regulatory networks have been defined,
including the p53/MDM2/p19Arf pathway (see Fig. 14.3), the PTCH/
SMO/GLI pathway, and the MSH2/MLH1/PMS2 DNA mismatch
recognition and repair pathway. Similar to the situation for the pRb,
APC/β-catenin, and p53 pathways, mutations in cancer cells not
infrequently target the PTCH/SMO/GLI and MSH2/MLH1/PMS2
pathways, either activating an oncogene within the pathway (e.g.,
SMO or GLI1 for the PTCH/SMO/GLI pathway) or inactivating
one of the key tumor suppressors (e.g., either MLH1 or MSH2 in
the mismatch repair [MMR] pathway).
Although a large collection of genetic and biochemical data support
the proposed protein functions and interactions depicted in Fig. 14.3,
it seems likely that the situation in vivo is far more complex. For
example, on the basis of the regulatory scheme outlined for the pRb
pathway in Fig. 14.3, it might appear that the phenotypic consequences
of RB1 or CDKN2A inactivation are functionally equivalent. However,
patients with germline mutations that inactivate RB1 are predisposed
to retinoblastomas and osteosarcomas, whereas persons with germline
defects in CDKN2A are predisposed predominantly to melanoma and
pancreatic cancer.26,27 Furthermore, whereas persons with germline
mutations that affect RB1 or CDKN2A are predisposed to a rather
limited spectrum of cancers, somatic defects in the pRb pathway (e.g.,
including mutations in RB1, CDKN2A, CCND1 [cyclin D1], and
CDK4) are seen in the majority of a broad array of cancer types.26,27
Unfortunately, at present, although no compelling mechanistic explana-
tion exists for these observations, perhaps a general explanation can
be offered. Specifically, the genetic pathways in which certain oncogenes
and tumor suppressor genes function are not simply linear pathways
as indicated schematically in Fig. 14.3 but more likely represent much
more complex and branched networks. The branches of the network
may even vary considerably, depending on cell type and developmental
context, although it seems reasonable to predict that the genes and
the protein products recurrently affected by mutation in human cancer
represent particularly critical hubs in the pathways and networks.
Epigenetic Mechanisms of Proto-oncogene
Activation and Tumor Suppressor Inactivation
The preceding discussion largely emphasized the role and significance
of somatic and germline mutations in proto-oncogenes and tumor
suppressor genes in cancer. However, changes in the levels and/or
patterns of gene expression, DNA methylation, and chromatin
modifications have also been proven to contribute to tumorigenesis.
Arguably, the most compelling data for assigning a critical and likely
causal role to changes in gene expression in the cancer process are for
the genes that have already been well established in prior mutational
and/or function studies to be tumor suppressor genes, particularly in
the cancer type under study. Hence, the emphasis here is placed on
illustrating how epigenetic mechanisms have been assigned a causal
role in silencing tumor suppressor genes in cancer. The roles of
superenhancer and other epigenetic mechanisms in proto-oncogene
activation, akin to those seen in cancers with high copy amplification
of the respective proto-oncogene, are discussed later.
218 Part I: Science and Clinical Oncology

Mut Me
Mutation Hit #1 Epigenetic
silencing

LOH Epigenetic LOH Epigenetic

Hit #2 silencing Hit #2 silencing
Mut Mut Me Me

Me Me

Mutation Mutation Epigenetic silencing Biallelic epigenetic

+ + + silencing
LOH Epigenetic silencing LOH

Figure 14.4  •  Knudson’s two-hit hypothesis revised. In brief, Knudson’s hypothesis predicted that both alleles of a tumor suppressor gene would need
to be inactivated by germline and/or somatic mutations to elicit critical phenotypic alterations associated with cancer development. The revised version of
Knudson’s two-hit hypothesis considers the possibility that tumor suppressor gene inactivation can result from either genetic (mutation) or epigenetic silencing
events. The two functional alleles of a given tumor suppressor gene are indicated by the two purple boxes (top). The first inactivating event affecting one of
the two tumor suppressor gene alleles could be either a mutation, such as the localized defect indicated by the yellow box (left), or transcriptional silencing
associated with or caused by hypermethylation of CpG-rich sequences in the promoter/regulatory region (right). The inactivating event for the second tumor
suppressor gene allele (i.e., “Hit #2”) could be a nondisjunction event resulting in loss of the chromosome containing the wild-type tumor suppressor gene allele
(loss of heterozygosity [LOH]) or epigenetic silencing. (Modified from Jones PA, Laird PW. Cancer epigenetics comes of age. Nat Genet. 1999;21:163–167.)

As is summarized in Table 14.2, somatic inactivation of selected
tumor suppressor genes has been well established to result from
mutational (genetic) mechanisms in many cancers. However, a
robust and growing body of data supports the view that epigenetic
mechanisms somatically inactivate selected tumor suppressor genes
in certain cancer types28,29 (Fig. 14.4). Although data are still emerg-
ing on the likely diverse transcriptional and chromatin remodeling
mechanisms responsible for epigenetic silencing of particular tumor
suppressor genes, many studies have demonstrated that increases in the
methylation of CpG-rich sequences (CpG islands) in the regulatory
regions (i.e., promoter/enhancer) of tumor suppressor genes are often
linked to loss of tumor suppressor gene expression. For instance,
whereas the VHL gene is inactivated by mutational mechanisms in
roughly 80% of renal carcinomas of clear cell type, in the majority
of the clear cell renal carcinomas in which specific VHL mutations
cannot be detected, loss of VHL gene expression appears to be tightly
linked to hypermethylation of the VHL promoter.28 For some other
tumor suppressor genes, including the CDKN2A, BRCA1, and MLH1
genes, promoter hypermethylation has also been implicated as key
mechanism of inactivation.29 In fact, on the basis of studies of genes
that display extensive CpG island methylation and decreased or absent
gene expression in cancer cells of one type or another, it has been
suggested that aberrant CpG methylation might play a very broad
and important role in silencing tumor suppressor genes in the cancer
process.28,29 Nevertheless, it remains unknown what fraction of the
genes whose promoters show increased methylation in cancers actually
function in vivo as tumor suppressor genes. Promoter hypermethyl-
ation and loss of gene expression coupled with additional data such
as restoration of gene expression by treatment with demethylating
agents and/or other agents that affect chromatin functional state (e.g.,
histone deacetylase inhibitors) might be best to establish their tumor
suppressive roles.
Mutations Affecting DNA Methylation Enzymes
Colorectal cancers (CRCs) were among the cancer types studied initially
in great depth for consistent patterns of epigenetic alterations, and it
was long known that whereas most CRCs display modest to moderate
global loss of 5-methylcytosine (5mC), certain subsets of CRC were
identified that appear to preferentially inactivate many different genes
by promoter hypermethylation—the so-called CpG island methylator
phenotype (CIMP).30 The findings on CIMP-positive CRCs suggested
that unidentified alterations in the methylation/demethylation
machinery might play a role in the development of colorectal and
perhaps other cancer types.
Indeed, during the past few years, intriguing insights into factors
and mechanisms regulating DNA methylation state in cancer have
emerged. Nearly all DNA methylation in somatic mammalian cells
occurs at the cytosine residue of CpG dinucleotides and is catalyzed
by DNA methyltransferases (DNMTs). Whereas DNMT mutations
have been suggested to play a role in several different cancer types, it
is now clear that localized mutations in DNMT3A are present in
roughly 20% to 25% of patients with acute myeloid leukemia (AML).31
Although the DNMT3A mutations are associated with poor prognosis
in persons with AML, it remains unclear exactly if and how the
mutations affect DNA methylation in persons with AML.31 In addition
to mutations in DNMT3A and potentially other DNMT genes, other
gene defects linked to DNA methylation factors and methylation state
have been uncovered. A family of Tet methylcytosine dioxygenase
proteins (TET1, TET2, and TET3) was identified in the past few
years; these proteins modify DNA by hydroxylating 5mC to
5-hydoxymethylcytosine (5hmC) and, as a consequence, result in loss
of 5mC in cellular DNA. More significantly for cancer development,
inactivation of the TET2 gene was found in upward of 30% of all
myeloid malignancies, and the TET2-mutant myeloid neoplastic cells
 Genetic and Epigenetic Alterations in Cancer  •  CHAPTER 14 219

have reduced levels of 5hmC and display a 5mC hypermethylator

phenotype, with resultant changes in chromatin state and gene expres- Table 14.3 Selected Chromatin Regulators and
sion patterns.32 Remarkably, further connections to altered DNA Modifiers Recurrently Mutated in
methylation in cancer have been revealed though studies of the linkages Cancer
between mutations in the isocitrate dehydrogenase genes IDH1 and
IDH2 and the hypermethylator phenotype in myeloid neoplasms and Protein Tumor Types With Mutations
some other tumors.32 Specifically, a critical cofactor for proper TET2 HISTONE ACETYLTRANSFERASES
function is alpha-ketoglutarate, and the IDH proteins normally generate CBP AML, ALL, DLBCL, TCC
alpha-ketoglutarate from isocitrate. The mutant IDH proteins found p300 AML, ALL, DLBCL, TCC, CRC, BrCA, PanCA
in several cancer types, including myeloid cancers and gliomas, generate
2-hydroxyglutarate instead, which renders the TET2 protein in affected MOZ AML, MDS
cells nonfunctional, with a resultant hypermethylator phenotype similar MORF AML, EndometCA
to that seen in TET2-mutant cancers.32,33 It is expected that further HISTONE ACETYLATION READERS
studies will clarify the key genetic and epigenetic mechanisms underlying
CIMP status in colorectal and other cancer types in which the TET BRD1 ALL
and IDH proteins appear to be functionally intact. PBRM1 RenalCA, BrCA
HISTONE METHYLTRANSFERASES
Mutations in Histones, Histone Modifiers, MLL1 AML, ALL, TCC
and Chromatin Remodelers MLL2 Medulloblastoma, RenalCA, DLBCL, FL
Although the progress in defining mutations that can underlie changes MLL3 Medulloblastoma, TCC, BrCA
in 5mC and 5hmC state and DNA methylation in cancer has been SETD2 RenalCA, BrCA
highly encouraging, truly dramatic progress has been achieved during EZH2 DLBCL, MDS, BrCA, ProstateCA, ALL
the past few years in defining a broad array of mutations that are
related to chromatin, including histones, histone modifiers, and HISTONE DEMETHYLASES
chromatin remodelers in different cancer types.34,35 These illustrate JARID1A AML
the importance of chromatin structures and modifications in the JARID1C RenalCA
pathogenesis of cancer. Chromatin is made up of repetitive fundamental
units called nucleosomes, and each nucleosome consists of 147 base UTX AML, TCC, RenalCA, EsophagealCA, MM
pairs of DNA wrapped around an octamer of histones. Accessibility HISTONE METHYLATION READERS
of chromatin to transcription apparatus is integral to gene expression, TRIM33 ThyroidCA
and alterations in structures of chromatin and their modifications will
play an important role in altered gene expression and tumorigenesis. ING1 Melanoma, BrCA
Notably, one of the variant histones, namely histone H3.3, which is ING4 HNSCC
incorporated during mitosis into actively transcribed regions such as MSH6 CRC
promoters, gene bodies, and telomeres, is found to be mutated in
CHROMATIN REMODELERS
upward of one-third of pediatric glioblastomas (Table 14.3).34,36 These
mutations act by inhibiting polycomb repressive complex 2 (PRC2), BRG1 LungCA, medulloblastoma, BrCA, ProstateCA,
a multiprotein complex responsible for the methylation of H3 at PanCA, HNSCC
lysine 27, therefore leading to global reduction in H3K27 trimethyl- BRM HNSCC
ation. These mutations were shown to be therapeutically targetable ARID1A OvCA, EndometCA, BrCA, RenalCA, CRC, GastricCA,
by using EZH2 inhibitors and BET bromodomain inhibitors.37,38 medulloblastoma, TCC
In addition to the DNA and histone mutations, histone modifiers, ARID1B BrCA
which are responsible for reading, writing, and erasing histone modifica-
BAF60A BrCA
tions (e.g., methylation, acetylation) that directly influence gene
expression, are frequently mutated in cancer. For example, EZH2, a ATRX Glioblastoma, PanNET
histone methyltransferase that catalyzes the repressive H3K27me3 DAXX Glioblastoma, PanNET
histone mark and a subunit of the polycomb repressive complex 2 HISTONES
(PRC2), is mutated in diffuse large B-cell lymphoma and follicular
lymphoma. The active mutation causes increased methyltransferase H3.1 Glioblastoma
activity leading to increased H3K27 trimethylation. Its opposite H3.3 Glioblastoma
counterpart, KDM6A or UTX, which is an H3K27 demethylase, is
frequently inactively mutated in different cancer types including ALL, Acute lymphoblastic leukemia; AML, acute myeloid leukemia; BrCA, breast
multiple myeloma and bladder cancer. Interesting to note, both the carcinoma; CRC, colorectal carcinoma; DLBCL, diffuse large B-cell lymphoma;
EndometCA, endometrial carcinoma; EsophagealCA, esophageal carcinoma; FL, follicular
activating mutations of EZH2 and the inactivating mutation of lymphoma; GastricCA, gastric carcinoma; HNSCC, head and neck squamous cell
KDM6A can be targeted therapeutically by EZH2 inhibitors.39,40 To carcinoma; LungCA, lung carcinoma; MDS, myelodysplastic syndrome; MM, multiple
date, many other histone modifiers have been found to be mutated myeloma; OvCA, ovarian carcinoma; PanCA, pancreatic carcinoma; PanNET, pancreatic
in different cancer types including KMT2A (MLL1), KMT2C (MLL3), neuroendocrine tumor; ProstateCA, prostate carcinoma; RenalCA, renal carcinoma;
SETD2, KDM5C (JARID1C), CREBBP, and EP300. TCC, transitional cell carcinoma of the bladder; ThyroidCA, thyroid carcinoma.
Modified from the findings and literature cited in Dawson MA, Kouzarides T.
Chromatin remodelers can cause sliding, ejection, or insertion Cancer epigenetics: from mechanism to therapy. Cell. 2012;150:12–27.
of nucleosomes along the chromatin. Of the four main classes of
chromatin remodelers, SWI/SNF complex is best characterized and
is collectively mutated in approximately 20% of all human cancers.41 are frequently found in clear cell renal cell carcinoma, and BAF250/
It has many subunits subdivided into enzymatic, core, and accessory ARID1A mutations are found in ovarian cell carcinoma and various
subunits. Mutations in these subunits can be organ specific; for gastrointestinal cancers. Exactly how these mutations contribute to
example, the core subunit SMARCB1/SNF5 is invariably mutated tumorigenesis has not been actively studied, but most likely they
in rhaboid tumors, accessory subunit BAF180/PBRM1 mutations affect or alter expression of cancer-related genes. Recent studies have
220 Part I: Science and Clinical Oncology
shown that, for example, SMARCB1, also known as SNF5, INI1,
and BAF47 and a core subunit of the complex invariably inactivated
in pediatric rhabdoid tumors, has been shown to play an important
role in the integrity of SWI/SNF complexes, and its loss affects the
binding of enhancers, particularly those required for differentiation.
Furthermore, it maintains binding to superenhancers that are essential
for rhabdoid tumor survival.42 Animal model studies of ARID1A, the
most frequent subunit of the complex, also show that it targets SWI/
SNF complexes to enhancers, where they work closely with transcription
factors, and its loss impairs enhancer-mediated gene regulation that
drives colon cancer tumorigenesis.43
Alterations in DNA cis-Regulatory Landscape
in Cancer
Although recent large-scale sequencing efforts have focused more
attention on the coding sequence of genes, combined whole-genome
sequencing, DNA chip sequencing, and RNA sequencing technologies
have identified and characterized more and more functionally relevant
alterations in noncoding DNA regions that underlie the transcriptional
programs involved in unannotated transcription start sites.44 Others
have found losses or gains of enhancers in cancer by comparing dif-
ferential H3K4me1Chip-seq levels between tumors and matched
normal tissues, leading to identification of variant enhancer loci predic-
tive of gene expression. More recently, dense clusters of enhancers,
also known as superenhancers or stretch enhancers, have been found to
play an important role not only in cell lineage and identity, but also
in cancer tumorigenesis.45 Cancer cells can acquire superenhancers
that drive expression of key cancer-related genes through different
mechanisms that involve noncoding DNA cis-regulatory regions (Fig.
14.5). One mechanism involves somatic gains of transcription factor
binding sites, either through mutations or microinsertions that can
affect the strength of a promoter or induce a new superenhancer. For
the former, two single nucleotide mutations (C228T and C250T) in
the TERT promoter generate an identical 11 bp nucleotide stretch
(5′-CCCCTTCCGGG-3′) serving as a de novo binding site for the
ETS transcription factor, leading to increased TERT expression.46,47
For the latter, a 12 bp intergenic insertion at the 3′UTR of the TAL1
gene creates a new MYB binding site adjacent to the core members
of the TAL1 complex including RUNX1, GATA3, and ETS1.48 Another
mechanism involves enhancer hijacking, which basically, through
chromosomal deletion or translocation, brings the gene adjacent to
an active superenhancer, as shown by the cases of GF1 genes in
medulloblastoma.49 In other cases, enhancer duplication leading to
gene overexpression has been reported.50 For example, two distinct
focal amplifications of superenhancer 3′ to MYC have resulted in
MYC overexpression in lung adenocarcinoma and endometrial car-
cinoma, respectively. Similarly, focal amplification at the enhancer,
rather than the coding regions, of KLF5 has also been described in
head and neck cancer.50 Finally, removal of regulatory boundaries
marked by the binding sites of insulators (e.g., CTCG and cohesion)
can lead to enhancer spreading and enhanced gene expression.45,51
Genomic deletion of CTCF binding sites has been found to cause
overexpression of TAL1 and LMO2. Equally important, DNA
hypermethylation of the CTCF/cohesin binding sites can disrupt its
binding leading to enhancer spreading.52 These emerging findings of
noncoding variants and their roles in tumorigenesis have led to the
realization that many of the disease risk single-nucleotide polymorphisms
(SNPs) from GWAS findings are indeed located in the noncoding
regions, and they may well contribute to tumorigenesis through the
aforementioned mechanisms.
Noncoding RNAs in Cancer—microRNAs and Long
Noncoding RNAs
It has been recognized during the past decade that the abundance of
messenger RNA (mRNA) transcripts and the translation of mRNA
transcripts into proteins is highly regulated by a novel class of short
noncoding RNAs, the so-called microRNAs (miRNAs). The mature
forms of miRNAs are 20 to 24 nucleotides in length and are generated
by successive cleavages by the Drosha and Dicer nucleases from longer
precursor transcripts that contain characteristic hairpin formations.53
Recognition of target transcripts occurs principally by binding of the
miRNAs in the 3′ untranslated regions (3′UTR). Depending on the
degree of homology to their target sequence, miRNAs induce trans-
lational repression or cleavage of mRNAs. Roughly 1000 human
miRNAs have been described, and each miRNA can target hundreds
to perhaps even a thousand or more mRNAs.53 This ability allows for
substantial combinatorial complexity and functional redundancy,
making the identification of specific functions of miRNAs and their
involvement in oncogenic or tumor suppressive networks difficult.
The fact that miRNAs act via base-pairing of the miRNA with the
3′UTR also implies that alterations of both miRNA sequence and
miRNA target sequence might have functional consequences.
Several lines of evidence make it likely that miRNAs do play a
role in the development of cancers. For instance, global inhibition of
miRNA production seems to facilitate the acquisition of a neoplastic
phenotype in primary mouse cells, suggesting that the net effect of
miRNAs collectively might be a tumor suppressive effect.54 The genomic
location of many miRNAs map close to common chromosomal
breakpoints in cancer. In most cases, however, it is still unclear whether
the miRNAs are actually involved in conferring the selective advantage
that is gained by these translocations or whether miRNAs for other
reasons are localized in regions of high genomic fragility.55 Compre-
hensive analyses of miRNA expression patterns in human cancers
have revealed that different cancer types have distinct miRNA expression
patterns. In fact, in many instances, miRNA expression patterns might
be more precise in determining the tissue of origin than mRNA
expression profiles.56 Similar to the situation with protein-coding genes,
in most cases it is unclear which expression changes are causative and
which are a result of the development of the tumors.57
Several miRNAs seem to play a role as part of classic tumor sup-
pressive or oncogenic signal transduction pathways. The miRNA17-92
locus has been described as a direct transcriptional target of the c-MYC
and E2F oncogenes.58 This polycistron locus encodes for seven miRNAs
and has been shown to be genomically amplified and overexpressed
in some human B-cell lymphomas and lung cancers. When overex-
pressed, it can cooperate with c-MYC to accelerate lymphoma
development in a murine model system. In addition, miRNA372 and
miRNA373 have been implicated as oncogenes in the development
of germ-cell tumors at least in part by inactivating the p53 tumor
suppressor pathway.59 miRNA10a has been suggested to be a driving
force behind the metastatic progression of breast cancer cell lines.60
Members of the let-7 family of miRNAs have been proposed as
tumor suppressor genes in lung cancer, supposedly in part by their
ability to inhibit the translation of the KRAS and HMGA2 oncogenes.61
The members of the miRNA34 family have been shown to be direct
transcriptional targets of the p53 tumor suppressor gene and may play
a significant role in mediating the downstream functional activity of
p53 in some settings.62
Long noncoding RNAs (lncRNAs) represent another important
class of noncoding RNAs in cancer pathogenesis.63 By convention,
lncRNAs are defined as a group of RNAs greater than 200 nucleotides
in length that do not appear to encode a protein. Given the range of
possible variations on this theme, many subclasses of lncRNAs have
been suggested, often based on their location and orientation relative
to other transcribed genes near their chromosome position. There is
much circumstantial evidence, largely based on expression data, to
implicate many different lncRNAs in cancer development and progres-
sion, and space does not permit a full review of these data.63 For the
most part, relatively few functional data are available at this point
that definitively implicate particular lncRNAs in cancer development
or progression. Given the current uncertainties about which lncRNAs
have critical functional roles in cancer development, some general
 Genetic and Epigenetic Alterations in Cancer  •  CHAPTER 14 221

C D
Figure 14.5  •  Various mechanisms by which somatic changes in the noncoding regions can lead to activation of proto-oncogenes. (A) Mutations in the
TERT promoter gene create a de novo binding site for ETS transcription factor. A 12-bp microinsertion in the 3′ untranslated region (UTR) of TAL1 creates
an MYB binding site and activates TAL1 transcriptions. (B) Chromosomal translocation places a superenhancer adjacent to GFI1B or its paralogue, GFI.
(C) Focal amplification of superenhancer by copy number gain induces KLF5 overexpression. (D) Deletion of insulator element at CTCF/cohesion binding
site causes the activation of TAL1 by its neighboring genes. (Modified from Yao X, Xing M, Ooi WF, Tan P, Teh BT. Epigenomic consequences of coding
and noncoding driver mutations. Trends Cancer. 2016;2:585–605.)

concepts are highlighted here instead. lncRNAs may function in a
variety of ways to regulate the expression of various cellular genes,
such as by serving as a scaffold for chromatin regulatory factors that
regulate genes near the locus of the lncRNA and/or genes at other
chromosome locations. Other lncRNAs may contain various miRNA
binding sites and may functionally compete with other coding region
transcripts for binding to particular miRNAs, thus acting in some
settings to quite dramatically modulate the levels and ability of
endogenous miRNAs to regulate transcripts of certain proto-oncogenes
and tumor suppressor genes. Other functions for lncRNAs in cancer
remain to be uncovered and substantiated, but it is possible that given
the many structures that RNA can adopt and the catalytic functions
of certain RNAs, lncRNAs may have a quite broad array of biochemical
functions in cancer.
Genetic and Epigenetic Alterations and
Genomic Integrity
A characteristic feature seen in most cancers is genetic instability,
including high rates of localized point mutations or small insertion/
222 Part I: Science and Clinical Oncology
deletion mutations in a subset of cancers and a chromosomal instability
in a large fraction of many different cancer types.64 The chromosome
instability includes numeric abnormalities (aneuploidy), as well as
simple and complex structural abnormalities. Considerable progress
has been made in defining some of the factors and mechanisms that
contribute to genetic instability in human cancer.
Roughly 2% to 4% of CRCs arise in persons with hereditary
nonpolyposis colorectal cancer (HNPCC), a familial cancer syndrome
attributable to germline mutation in one allele of an MMR gene (see
Table 14.2; e.g., MSH2, MLH1, and MSH6).65 The CRCs arising in
persons with HNPCC arise in large part as a result of the somatic
inactivation of the remaining wild-type MMR allele in an affected
individual with HNPCC. The cancer cells manifest a markedly increased
frequency of point mutations and small insertion and deletion mutations
due to defective MMR function, reflected by the so-called high-
frequency microsatellite instability (MSI-H) phenotype.65 In the other
cancer types arising in individuals and families with HNPCC, such
as ovarian, endometrial, and gastric cancer, similar MSI-H phenotypes
are seen. In the setting of HNPCC, there would appear to be more
rapid tumor progression from an initiated clone to frank malignancy
as a result of an MMR-defective mutator phenotype. In addition to
the HNPCC CRCs, about 10% to 12% of apparently sporadic CRCs
manifest the MSI-H phenotype, and epigenetic silencing of MLH1
by DNA hypermethylation of the promoter region and perhaps other
chromatin modifications plays a critical role in the majority of these
sporadic MSI-H CRC cases.65
Based on the results of recent comprehensive DNA sequence analyses
of human cancers, it seems that hundreds to thousands of localized
mutations can be detected in most primary cancers, yet the specific
factors and mechanisms responsible for the mutator phenotypes in
most of these cancers remain to be defined.20 Possible mechanisms
include mutations and alterations of DNA polymerases, including
aberrant expression of alternative specialized DNA polymerases known
as translesional polymerases.66
Functional studies in cultured cancer cells and in mice provide
quite a broad array of mechanisms that could contribute to numerous
chromosomal abnormalities and the associated chromosomal instability
(CIN) phenotype in cancer. Such chromosomal abnormalities include
mitotic checkpoint defects, chromatin modification or condensation
defects, and chromosome segregation defects due to centrosome
abnormalities and other possibilities, such as altered sister chromatid
cohesion.67 For the most part, although mutations in a few genes that
might contribute to chromosome instability have been reported, the
CIN phenotype in the vast majority of human cancers remains
unexplained. A recent article indicates that the X-chromosome gene
STAG2, a gene encoding a subunit of a sister chromatid separation
complex, is mutated in significant subsets of melanomas, glioblastomas,
and Ewing sarcoma, and that inactivation of STAG2 can promote
aneuploidy in cultured cells.68 The findings highlight the possibility
that further work on STAG2, STAG2-interacting proteins, and the
other proteins that function in regulating proper segregation of
chromosomes at mitosis will uncover more insights into the molecular
basis of the CIN phenotype.
Structural chromosomal alterations are common in many cancer
types and presumably reflect the consequences of DNA double-strand
breaks and nonhomologous end joining to generate chromosomal
deletions, inversions, and translocations, with biologic selection acting
on the variant cells with the structural alterations. If the structural
abnormality activates and/or inactivates a gene or genes that lead to
more robust proliferation and survival properties in a given context,
cells carrying a given structural alteration will be positively selected
for during cancer development. The fact that certain structural altera-
tions are recurrent in cancer offers strong support for this concept.
For the most part, particular structural chromosomal alterations in
cancer were thought to be relatively straightforward, resulting in
recombination of noncontiguous sequences, sometimes accompanied
by deletions. However, recent work indicates that chromotripsis—a
dramatic shattering of a chromosome or chromosomes and recombina-
tion of the fragments—may occur in 2% to 3% of all cancers and
that in certain tumors, such as osteosarcomas, it may occur in up to
25% of cases.69 Chromotripsis may represent another mutator
mechanism, because it would allow multiple genetic abnormalities to
arise and be selected for concurrently rather than sequentially.
Role of Tissue and Context Differences in the
Contributions of Gene Defects to Cancer
Cell Phenotype
The published literature on the potential contributions of gene defects
to the altered phenotype of cancer cells has offered some suggestions
about how to consider the role of the gene defects in cancer patho-
genesis. For instance, terms such as gatekeeper and caretaker have been
used to classify the contributions of genes to cancer development.70
“Gatekeeper” genes have been suggested to be genes that play particu-
larly critical roles in regulating cell proliferation and inhibiting cancer
development in certain tissues, such as the APC gene in CRC; the
tumor suppressive function of the genes must be overcome for cancers
to arise in a given tissue or organ site.70 “Caretakers” have been generally
defined as genes that do not play direct roles in growth control but
rather likely play important roles in a number of tissues in maintaining
the fidelity of the genome via their role in DNA damage recognition
and repair processes. The MLH1 and MSH2 MMR genes have been
proposed to be representative caretaker genes,70 and some researchers
have suggested that perhaps the BRCA1 and BRCA2 genes also represent
caretaker genes.71
The use of terms such as gatekeeper and caretaker might have some
merit. However, as will be illustrated in the following discussion,
given the apparently important role of “gatekeeper” gene defects in
cancers that arise in various organ sites but the quite variable timing
of “gatekeeper” gene defects in the natural history of one cancer type
versus another, the term gatekeeper might be more confusing than
illuminating. In the case of some presumed “caretaker” genes, the
genes might not be playing the passive role in the cancer process that
has been assigned to them. This view is based on three lines of argument:
(1) the apparent tissue specificity of the tumors that arise in persons
who harbor germline mutations in the “caretaker” genes, such as in
persons who are affected by HNPCC and germline MLH1 or MSH2
mutations72; (2) the likely variable time in cancer development at
which “caretaker” mutations may arise from one tumor to the next,
with sporadic colon tumors not usually manifesting MMR gene
inactivation and MSI until the carcinoma stage, despite the fact that
adenomas in persons with HNPCC often show MSI-H73,74; and (3)
the evidence that “caretaker” genes might actually play key roles in
regulating cell proliferation and promoting apoptosis in certain
contexts.56
In general, persons who harbor a germline mutation in a specific
tumor suppressor gene or proto-oncogene are predisposed to a very
limited spectrum of cancer types. This observation is puzzling for a
couple of reasons. The majority of genes that are affected by germline
mutations in specific inherited cancer syndromes are essentially
ubiquitously expressed in adult tissues. Furthermore, for a number
of the tumor suppressor genes, mutations are often found to inactivate
the gene in a much broader collection of sporadic cancer types than
the types that commonly arise in germline mutation carriers. Children
who carry a germline mutation in the RB1 gene have a very elevated
risk of the development of retinoblastoma and a more modest risk of
the development of osteosarcoma but no dramatic increase in the risk
of most common adult cancers. Yet somatic defects in RB1 have been
found and are believed to be critical in the development of many
different cancers, such as small cell lung carcinomas, in which the
vast majority have pRb defects.27,75
Potential explanations exist for these puzzling observations. For
instance, whereas pRb might have an essential role in regulating reti-
noblast cell proliferation and/or differentiation, in other tissues, such

as lung or breast epithelial cells, pRb might have a redundant role in
growth control, perhaps because of the contribution of pRb-related
proteins, such as p107 and p130.21 Under this scenario, RB1 inactivation
in most cell types might not promote neoplastic growth unless other
defects, such as those in pRb-related proteins, are also present. An
alternative and perhaps equally likely possibility is that somatic inactiva-
tion of pRb might trigger apoptosis in many cell types unless other
somatic gene defects have arisen previously and these other defects
interfere with the cell’s ability to undergo apoptosis after disruption
of RB1 function. Evidence that pRb inactivation can act in a context-
dependent fashion to promote apoptosis versus neoplastic transforma-
tion has been offered.76,77 The tissue specificity of cancers that are
seen in persons who carry germline mutations in inherited cancer
genes is not restricted to the case of pRb. Germline p53 mutations
predispose primarily to osteosarcoma, soft tissue sarcoma, leukemia,
brain tumors, and breast cancer in women, and CDKN2A germline
mutations predispose primarily to melanoma and pancreatic cancer.26
In spite of the relatively limited spectrum of cancer types that are
seen in people who carry germline p53 or CDKN2A mutations, the
p53 and p16INK4a genes are very commonly altered in human cancer,
with each of the genes being inactivated in upward of 35% to 50%
of many different sporadic cancer types.
Some genes with prominent roles in the development of a variety
of different cancer types are sometimes presumed to have essentially
singular functions in the cancer process in spite of data that suggest
otherwise. As an example, the E-cadherin protein plays an important
role in cell-cell adhesion via the ability of its extracellular domain to
form adhesive interactions with E-cadherin molecules on opposing
cell surfaces and the ability of the E-cadherin cytoplasmic domain to
link to the actin cortical cytoskeleton via interactions with catenin
proteins at the plasma membrane.78 Early functional studies have
suggested that restoration of E-cadherin in cancer cells that had
endogenous E-cadherin defects interfered with the invasive properties
of cancer cells in selected in vitro assays.79 Perhaps in large part because
of these observations, the loss of E-cadherin expression in cancer has
often been assigned a role in promoting invasive behavior in advanced
cancer cells. Although loss of E-cadherin function might indeed
contribute to invasive behavior in cancers arising in vivo, it is worth
bearing in mind that defects in E-cadherin could in fact play a distinct
role in altering cell growth very early in the neoplastic transformation
process in some tumor types, such as the gastric carcinomas that arise
in patients who carry germline E-cadherin mutations.80
A couple of additional examples of context-dependent effects of
mutations in cancer are offered here, in hopes of highlighting some
of the potential challenges in unambiguously defining certain genes
as oncogene targets or tumor suppressor gene targets in cancer develop-
ment. The p53 gene represents one such example. As previously
indicated, the wild-type p53 protein is a transcriptional regulator that
is mutated in roughly 50% of all cancers. A common theme for the
majority of the mutations is loss of wild-type p53 function as a transcrip-
tion regulator, owing to the mutant p53 protein’s loss of sequence-
specific DNA binding activity and/or misfolding of the protein.7
However, more than 75% of the p53 mutations in human cancer
lead to expression of a mutant p53 protein that may have oncogenic
activity independent of its ability to dominantly interfere with wild-type
p53.7 Although the gain-of-function properties of mutant p53 in
cancer are clear, the mechanisms underlying the oncogenic activity
of mutant p53 are complex and likely vary depending on the particular
mutant p53 protein and cellular context.7 A second example is the
NOTCH1 gene, which is rarely mutationally activated by translocations
in T-cell acute lymphoblastic leukemia (T-ALL) and much more
commonly activated in T-ALL by localized mutations in a juxtamem-
brane domain that allows the mutant NOTCH1 protein to be activated
in a ligand-independent manner or by mutations in a cytoplasmic
domain that lead to increased stability of the cleaved cytoplasmic
domain of NOTCH1.81 In contrast to the gain-of-function mutations
in NOTCH1 in T-ALL, sequence-based studies in head and neck
Genetic and Epigenetic Alterations in Cancer  •  CHAPTER 14 223
squamous cell carcinomas (HNSCCs) indicated that NOTCH1 harbors
various inactivating mutations, including nonsense and frameshift
mutations, in roughly 10% to 17% of primary HNSCCs.81 Based on
the well-known function of NOTCH proteins to regulate cell fate
choices during development and adult tissues, it is perhaps not surprising
that NOTCH1 can function as an oncogene when activated in some
contexts and as a tumor suppressor gene when inactivated in other
contexts.81
CLINICAL IMPLICATIONS
Identification of genetic and epigenetic defects contributing to human
cancer reveals significant clinical implications for the present and the
future, and some current directions and possibilities for the field are
highlighted here. As previously noted, a very great and diverse array
of mutations in oncogenes and tumor suppressor genes and epigenetic
changes underlie different forms of cancer. However, because certain
mutations and epigenetic changes either alone or collectively are
preferentially and sometimes even uniquely associated with certain
cancer types, molecular analyses of cancer specimens and biopsies are
likely to be an important adjunctive approach in the cancer diagnostic
area. For many hematologic malignancies and a subset of solid tumors,
molecular analyses already serve a vital role in the diagnostic arena,
helping, for instance, to distinguish subsets of tumors that manifest
similar or overlapping histologies or to aid in diagnosis when the
primary organ site of a metastatic cancer is uncertain. It seems likely
that cancer diagnostic approaches that incorporate molecular annotation
will continue to expand during the next 5 to 10 years. Moreover,
based on recent reports that cell-free tumor-derived DNA can be
readily detected in the plasma of patients with cancer and that copy
number changes along with specific nucleotide changes can be detected
with comprehensive sequencing approaches,82,83 it seems likely that
molecular interrogation of plasma and/or other body fluids may well
be pursued for improved staging information and to assess for relapse
in patients with a solid tumor after surgery, radiation, and/or chemo-
therapy. Indeed, a recent early study suggests that molecular interroga-
tion of circulating tumor-derived DNA in plasma may be useful for
identifying drug-resistant cancer cells even before the initiation of
certain therapies.84
The genes and the protein products that are recurrently altered by
mutations and/or epigenetic defects in human cancer more than likely
represent particularly critical hubs in the pathways and networks that
regulate cell growth, differentiation, and programmed cell death. Hence
efforts to target the particular proteins and pathways with apparently
central roles in the pathogenesis of certain cancer types would appear
to offer potentially broad impact. Successes with targeted therapy such
as EGFR inhibitors in the setting of EGFR-mutant non–small cell
lung cancer85 and with ALK inhibitors in the setting of lung cancer
harboring translocations activating ALK 86 offer evidence for the notion
of targeting critical driver oncogene defects in cancer, despite the fact
that subsets of these patients may develop resistance.
In spite of the generally optimistic view that is offered as a result
of our rapidly expanding understanding of the nature and contribution
of gene defects in cancer pathogenesis, some significant challenges
remain if novel and specific new anticancer therapies are to be achieved
in the near term. In particular, in a number of the cases with dramatic
initial responses to single agents targeting the specific driver oncogene
lesions, drug resistance and cancer recurrence and progression are
seen. In certain other cancer types carrying some of the same driver
oncogene lesions, such as BRAF-mutant CRCs, the cancers show little
if any evidence for response to the targeted agents.87 Another issue is
that for a fair number of the specific signaling pathways that are
commonly disrupted in cancer, it might prove difficult to define readily
tractable targets for therapeutic intervention. For instance, in the case
of the p53 pathway, it is unclear how effectively p53 function can be
restored for the proteins that carry missense substitutions that inactivate
p53 tumor suppressor activity and perhaps also generate oncogenic
224 Part I: Science and Clinical Oncology

p53 gain-of-function mutants. In the case of cancers with mutations
leading to β-catenin deregulation, a potential goal might be to define
agents that specifically interfere with the nuclear function of β-catenin
in transcriptional activation of β-catenin/TCF-regulated target genes.
Although this is not an unreasonable notion, given the rather limited
successes to date in defining small molecules that specifically affect
transcription factor complexes, it could be challenging to target
β-catenin via conventional pharmacologic approaches. Similar concerns
could perhaps be raised regarding the merits of attempting to specifically
target other nuclear proteins and transcription factors that are deregu-
lated in cancer cells. With increasing understanding of the role of
chromatin biology and its roles in tumorigenesis, we may witness an
increasing number of chromatin-related or epigenetic drugs being
developed for treatment of cancer.
Although the success of approaches to target cancer cells more
selectively remains to be broadly established, it is obvious that ongoing
and future efforts to understand the relationships among gene defects,
altered cell signaling and physiology, and cancer phenotype have already
shaped and will continue to shape our views of how best to proceed
with novel therapeutic interventions. An optimistic view is that
combining various therapeutic approaches including targeted therapy,
immunotherapy or cell therapy, and even conventional chemotherapy
with new molecular diagnostics efforts that inform about various
issues—for example, disease subtypes, occult disease spread beyond
the primary organ site, disease recurrence at earlier time points, and
intratumor heterogeneity and resistance mechanisms—will yield major
improvements in patient survival and quality of life for a range of
cancer types within the next decade.
The complete reference list is available online at
ExpertConsult.com.

KEY REFERENCES
1. Hanahan D, Weinberg RA. Hallmarks of cancer: the
next generation. Cell. 2011;144:646–674.
2. Chan TA, Wolchok JD, Snyder A. Genetic basis for
clinical response to CTLA-4 blockade in melanoma.
N Engl J Med. 2015;373:1984.
3. Le DT, et al. PD-1 Blockade in tumors with
mismatch-repair deficiency. N Engl J Med. 2015;372:
2509–2520.
4. Hemann MT, et al. Evasion of the p53 tumour surveil-
lance network by tumour-derived MYC mutants.
Nature. 2005;436:807–811.
5. Renan MJ. How many mutations are required for
tumorigenesis? Implications from human cancer data.
Mol Carcinog. 1993;7:139–146.
6. Knudson AG. Mutation and cancer: statistical
study of retinoblastoma. Proc Natl Acad Sci USA.
1971;68:820–823.
7. Muller PAJ, Vousden KH. p53 mutations in cancer.
Nat Cell Biol. 2013;15:2–8.
9. Knudson AG. Two genetic hits (more or less) to
cancer. Nat Rev Cancer. 2001;1:157–162.
11. Foulds L. The natural history of cancer. J Chronic
Dis. 1958;8:2–37.
12. Nowell PC. The clonal evolution of tumor cell
populations. Science. 1976;194:23–28.
13. Swanton C. Intratumor heterogeneity: evolution
through space and time. Cancer Res. 2012;72:
4875–4882.
14. Haber DA, Settleman J. Cancer: drivers and pas-
sengers. Nature. 2007;446:145–146.
15. Muller FL, et al. Passenger deletions generate thera-
peutic vulnerabilities in cancer. Nature. 2012;488:
337–342.
16. Nijhawan D, et al. Cancer vulnerabilities unveiled
by genomic loss. Cell. 2012;150:842–854.
22. Sage J. The retinoblastoma tumor suppressor and
stem cell biology. Genes Dev. 2012;26:1409–1420.
23. Aoki K, Taketo MM. Adenomatous polyposis coli
(APC): a multi-functional tumor suppressor gene.
J Cell Sci. 2007;120:3327–3335.
24. Polakis P. The many ways of Wnt in cancer. Curr
Opin Genet Dev. 2007;17:45–51.
25. Myant K, Sansom OJ. Wnt/Myc interactions in intes-
tinal cancer: partners in crime. Exp Cell Res. 2011;317:
2725–2731.
26. Fearon ER. Human cancer syndromes: clues to
the origin and nature of cancer. Science. 1997;278:
1043–1050.
28. Jones PA, Baylin SB. The epigenomics of cancer.
Cell. 2007;128:683–692.
30. Issa JP. CpG island methylator phenotype in cancer.
Nat Rev Cancer. 2004;4:988–993.
32. Cimmino L, Abdel-Wahab O, Levine RL, Aifantis
I. TET family proteins and their role in stem cell
differentiation and transformation. Cell Stem Cell.
2011;9:193–204.
33. Turcan S, et al. IDH1 mutation is sufficient to
establish the glioma hypermethylator phenotype.
Nature. 2012;483:479–483.
35. Yao X, Xing M, Ooi WF, Tan P, Teh BT. Epigenomic
consequences of coding and noncoding driver muta-
tions. Trends in Cancer. 2016;2:585–605.
36. Schwartzentruber J, et al. Driver mutations in histone
H3.3 and chromatin remodelling genes in paediatric
glioblastoma. Nature. 2012;482:226–231.
37. Mohammad F, et al. EZH2 is a potential therapeutic
target for H3K27M-mutant pediatric gliomas. Nat
Med. 2017;23:483–492.
38. Piunti A, et al. Therapeutic targeting of polycomb
and BET bromodomain proteins in diffuse intrinsic
pontine gliomas. Nat Med. 2017;23:493–500.
39. McCabe MT, et al. EZH2 inhibition as a therapeutic
strategy for lymphoma with EZH2-activating muta-
tions. Nature. 2012;492:108–112.
40. Ler LD, et al. Loss of tumor suppressor KDM6A
amplifies PRC2-regulated transcriptional repression in
bladder cancer and can be targeted through inhibition
of EZH2. Sci Transl Med. 2017;9.
42. Wang X, et al. SMARCB1-mediated SWI/SNF
complex function is essential for enhancer regulation.
Nat Genet. 2017;49:289–295.
43. Mathur R, et al. ARID1A loss impairs enhancer-
mediated gene regulation and drives colon cancer
in mice. Nat Genet. 2017;49:296–302.
54. Kumar MS, Lu J, Mercer KL, Golub TR, Jacks
T. Impaired microRNA processing enhances cel-
lular transformation and tumorigenesis. Nat Genet.
2007;39:673–677.
55. Calin GA, Croce CM. Chromosomal rearrangements
and microRNAs: a new cancer link with clinical
implications. J Clin Invest. 2007;117:2059–2066.
58. He L, et al. A microRNA polycistron as a potential
human oncogene. Nature. 2005;435:828–833.
59. Voorhoeve PM, et al. A genetic screen implicates
miRNA-372 and miRNA-373 as oncogenes in tes-
ticular germ cell tumors. Cell. 2006;124:1169–1181.
60. Ma L, Teruya-Feldstein J, Weinberg RA. Tumour
invasion and metastasis initiated by microRNA-10b
in breast cancer. Nature. 2007;449:682–688.
61. Johnson SM, et al. RAS is regulated by the let-7
microRNA family. Cell. 2005;120:635–647.
62. He L, He X, Lowe SW, Hannon GJ. microRNAs
join the p53 network–another piece in the tumour-
suppression puzzle. Nat Rev Cancer. 2007;7:819–822.
65. Boland CR, Goel A. Microsatellite instability
in colorectal cancer. Gastroenterology. 2010;138:
2073–2087.e3.
66. Hoffmann JS, Cazaux C. Aberrant expression of
alternative DNA polymerases: a source of mutator
phenotype as well as replicative stress in cancer. Semin
Cancer Biol. 2010;20:312–319.
67. Janssen A, Medema RH. Genetic instability: tipping
the balance. Oncogene. 2013;32:4459–4470.
68. Solomon DA, et al. Mutational inactivation of
STAG2 causes aneuploidy in human cancer. Science.
2011;333:1039–1043.
69. Stephens PJ, et al. Massive genomic rearrangement
acquired in a single catastrophic event during cancer
development. Cell. 2011;144:27–40.
70. Kinzler KW, Vogelstein B. Cancer-susceptibility genes.
Gatekeepers and caretakers. Nature. 1997;386(761):
763.
72. Lynch HT, de la Chapelle A. Genetic susceptibility to
non-polyposis colorectal cancer. J Med Genet. 1999;36:
801–818.
77. Morgenbesser SD, Williams BO, Jacks T, DePinho
RA. p53-dependent apoptosis produced by Rb-
deficiency in the developing mouse lens. Nature.
1994;371:72–74.
78. Gumbiner BM. Regulation of cadherin-mediated
adhesion in morphogenesis. Nat Rev Mol Cell Biol.
2005;6:622–634.
79. Vleminckx K, Vakaet L, Mareel M, Fiers W, van Roy
F. Genetic manipulation of E-cadherin expression by
epithelial tumor cells reveals an invasion suppressor
role. Cell. 1991;66:107–119.
80. Guilford P, et al. E-cadherin germline mutations in
familial gastric cancer. Nature. 1998;392:402–405.
81. Lobry C, Oh P, Aifantis I. Oncogenic and tumor
suppressor functions of Notch in cancer: it’s NOTCH
what you think. J Exp Med. 2011;208:1931–1935.
82. Forshew T, et al. Noninvasive identification and
monitoring of cancer mutations by targeted deep
sequencing of plasma DNA. Sci Transl Med. 2012;4:
136ra68.
83. Leary RJ, et al. Detection of chromosomal alterations
in the circulation of cancer patients with whole-
genome sequencing. Sci Transl Med. 2012;4:162ra154.
84. Diaz LA, et al. The molecular evolution of acquired
resistance to targeted EGFR blockade in colorectal
cancers. Nature. 2012;486:537–540.
87. Corcoran RB, et al. EGFR-mediated re-activation
of MAPK signaling contributes to insensitivity of
BRAF mutant colorectal cancers to RAF inhibition
with vemurafenib. Cancer Discov. 2012;2:227–
235.

REFERENCES
1. Hanahan D, Weinberg RA. Hallmarks of cancer: the
next generation. Cell. 2011;144:646–674.
2. Chan TA, Wolchok JD, Snyder A. Genetic basis for
clinical response to CTLA-4 blockade in melanoma.
N Engl J Med. 1984;373:2015.
3. Le DT, et al. PD-1 blockade in tumors with mismatch-
repair deficiency. N Engl J Med. 2015;372:2509–2520.
4. Hemann MT, et al. Evasion of the p53 tumour surveil-
lance network by tumour-derived MYC mutants.
Nature. 2005;436:807–811.
5. Renan MJ. How many mutations are required for
tumorigenesis? Implications from human cancer data.
Mol Carcinog. 1993;7:139–146.
6. Knudson AG. Mutation and cancer: statistical study
of retinoblastoma. Proc Natl Acad Sci USA. 1971;68:
820–823.
7. Muller PAJ, Vousden KH. p53 mutations in cancer.
Nat Cell Biol. 2013;15:2–8.
8. Payne SR, Kemp CJ. Tumor suppressor genetics.
Carcinogenesis. 2005;26:2031–2045.
9. Knudson AG. Two genetic hits (more or less) to
cancer. Nat Rev Cancer. 2001;1:157–162.
10. Gerlinger M, et al. Intratumor heterogeneity and
branched evolution revealed by multiregion sequenc-
ing. N Engl J Med. 2012;366:883–892.
11. Foulds L. The natural history of cancer. J Chronic
Dis. 1958;8:2–37.
12. Nowell PC. The clonal evolution of tumor cell
populations. Science. 1976;194:23–28.
13. Swanton C. Intratumor heterogeneity: evolution
through space and time. Cancer Res. 2012;72:
4875–4882.
14. Haber DA, Settleman J. Cancer: drivers and pas-
sengers. Nature. 2007;446:145–146.
15. Muller FL, et al. Passenger deletions generate thera-
peutic vulnerabilities in cancer. Nature. 2012;488:
337–342.
16. Nijhawan D, et al. Cancer vulnerabilities unveiled
by genomic loss. Cell. 2012;150:842–854.
17. Wood LD, et al. The genomic landscapes of human
breast and colorectal cancers. Science. 2007;318:
1108–1113.
18. Greenman C, et al. Patterns of somatic mutation in
human cancer genomes. Nature. 2007;446:153–158.
19. Thomas RK, et al. High-throughput oncogene
mutation profiling in human cancer. Nat Genet.
2007;39:347–351.
20. Loeb LA. Human cancers express mutator phenotypes:
origin, consequences and targeting. Nat Rev Cancer.
2011;11:450–457.
21. Henley SA, Dick FA. The retinoblastoma family
of proteins and their regulatory functions in the
mammalian cell division cycle. Cell Div. 2012;7:10.
22. Sage J. The retinoblastoma tumor suppressor and
stem cell biology. Genes Dev. 2012;26:1409–1420.
23. Aoki K, Taketo MM. Adenomatous polyposis coli
(APC): a multi-functional tumor suppressor gene.
J Cell Sci. 2007;120:3327–3335.
24. Polakis P. The many ways of Wnt in cancer. Curr
Opin Genet Dev. 2007;17:45–51.
25. Myant K, Sansom OJ. Wnt/Myc interactions in intes-
tinal cancer: partners in crime. Exp Cell Res. 2011;317:
2725–2731.
26. Fearon ER. Human cancer syndromes: clues to the
origin and nature of cancer. Science. 1997;278:1043–
1050.
27. Sellers WR, Kaelin WG. Role of the retinoblastoma
protein in the pathogenesis of human cancer. J Clin
Oncol. 1997;15:3301–3312.
28. Jones PA, Baylin SB. The epigenomics of cancer.
Cell. 2007;128:683–692.
29. Esteller M. Epigenetics in cancer. N Engl J Med.
2008;358:1148–1159.
30. Issa J-P. CpG island methylator phenotype in cancer.
Nat Rev Cancer. 2004;4:988–993.
31. You JS, Jones PA. Cancer genetics and epigenetics: two
sides of the same coin? Cancer Cell. 2012;22:9–20.
Genetic and Epigenetic Alterations in Cancer  •  CHAPTER 14 224.e1
32. Cimmino L, Abdel-Wahab O, Levine RL, Aifantis
I. TET family proteins and their role in stem cell
differentiation and transformation. Cell Stem Cell.
2011;9:193–204.
33. Turcan S, et al. IDH1 mutation is sufficient to
establish the glioma hypermethylator phenotype.
Nature. 2012;483:479–483.
34. Dawson MA, Kouzarides T. Cancer epigenetics: from
mechanism to therapy. Cell. 2012;150:12–27.
35. Yao X, Xing M, Ooi WF, Tan P, Teh BT. Epigenomic
consequences of coding and noncoding driver muta-
tions. Trends Cancer. 2016;2:585–605.
36. Schwartzentruber J, et al. Driver mutations in histone
H3.3 and chromatin remodelling genes in paediatric
glioblastoma. Nature. 2012;482:226–231.
37. Mohammad F, et al. EZH2 is a potential therapeutic
target for H3K27M-mutant pediatric gliomas. Nat
Med. 2017;23:483–492.
38. Piunti A, et al. Therapeutic targeting of polycomb
and BET bromodomain proteins in diffuse intrinsic
pontine gliomas. Nat Med. 2017;23:493–500.
39. McCabe MT, et al. EZH2 inhibition as a therapeutic
strategy for lymphoma with EZH2-activating muta-
tions. Nature. 2012;492:108–112.
40. Ler LD, et al. Loss of tumor suppressor KDM6A
amplifies PRC2-regulated transcriptional repression in
bladder cancer and can be targeted through inhibition
of EZH2. Sci Transl Med. 2017;9.
41. Kadoch C, et al. Proteomic and bioinformatic
analysis of mammalian SWI/SNF complexes identifies
extensive roles in human malignancy. Nat Genet.
2013;45:592–601.
42. Wang X, et al. SMARCB1-mediated SWI/SNF
complex function is essential for enhancer regulation.
Nat Genet. 2017;49:289–295.
43. Mathur R, et al. ARID1A loss impairs enhancer-
mediated gene regulation and drives colon cancer
in mice. Nat Genet. 2017;49:296–302.
44. Muratani M, et al. Nanoscale chromatin profiling
of gastric adenocarcinoma reveals cancer-associated
cryptic promoters and somatically acquired regulatory
elements. Nat Commun. 2014;5:4361.
45. Hnisz D, et al. Activation of proto-oncogenes by
disruption of chromosome neighborhoods. Science.
2016;351:1454–1458.
46. Huang FW, et al. Highly recurrent TERT pro-
moter mutations in human melanoma. Science.
2013;339:957–959.
47. Horn SS. TERT promoter mutations in familial
and sporadic melanoma. Science. 2013;339:959–
961.
48. Figueroa ME, et al. Leukemic IDH1 and IDH2
mutations result in a hypermethylation phenotype,
disrupt TET2 function, and impair hematopoietic
differentiation. Cancer Cell. 2010;18:553–567.
49. Northcott PA, et al. Enhancer hijacking activates
GFI1 family oncogenes in medulloblastoma. Nature.
2014;511:428–434.
50. Zhang X, et al. Identification of focally amplified
lineage-specific super-enhancers in human epithelial
cancers. Nat Genet. 2016;48:176–182.
51. Katainen R, et al. CTCF/cohesin-binding sites
are frequently mutated in cancer. Nat Genet.
2015;47:818–821.
52. Flavahan WA, et al. Insulator dysfunction and
oncogene activation in IDH mutant gliomas. Nature.
2016;529:110–114.
53. Valencia-Sanchez MA, Liu J, Hannon GJ, Parker R.
Control of translation and mRNA degradation by
miRNAs and siRNAs. Genes Dev. 2006;20:515–524.
54. Kumar MS, Lu J, Mercer KL, Golub TR, Jacks
T. Impaired microRNA processing enhances cel-
lular transformation and tumorigenesis. Nat Genet.
2007;39:673–677.
55. Calin GA, Croce CM. Chromosomal rearrangements
and microRNAs: a new cancer link with clinical
implications. J Clin Invest. 2007;117:2059–2066.
224.e1
56. Lu J, et al. MicroRNA expression profiles classify
human cancers. Nature. 2005;435:834–838.
57. Garzon R, Fabbri M, Cimmino A, Calin GA, Croce
CM. MicroRNA expression and function in cancer.
Trends Mol Med. 2006;12:580–587.
58. He L, et al. A microRNA polycistron as a potential
human oncogene. Nature. 2005;435:828–833.
59. Voorhoeve PM, et al. A genetic screen implicates
miRNA-372 and miRNA-373 as oncogenes in tes-
ticular germ cell tumors. Cell. 2006;124:1169–1181.
60. Ma L, Teruya-Feldstein J, Weinberg RA. Tumour
invasion and metastasis initiated by microRNA-10b
in breast cancer. Nature. 2007;449:682–688.
61. Johnson SM, et al. RAS is regulated by the let-7
microRNA family. Cell. 2005;120:635–647.
62. He L, He X, Lowe SW, Hannon GJ. microRNAs
join the p53 network—another piece in the tumour-
suppression puzzle. Nat Rev Cancer. 2007;7:819–822.
63. Prensner JR, Chinnaiyan AM. The emergence of
lncRNAs in cancer biology. Cancer Discov. 2011;1:
391–407.
64. Lengauer C, Kinzler KW, Vogelstein B. Genetic insta-
bilities in human cancers. Nature. 1998;396:643–649.
65. Boland CR, Goel A. Microsatellite instability in
colorectal cancer. Gastroenterology. 2010;138:2073–
2087.e3.
66. Hoffmann J-S, Cazaux C. Aberrant expression of
alternative DNA polymerases: a source of mutator
phenotype as well as replicative stress in cancer. Semin
Cancer Biol. 2010;20:312–319.
67. Janssen A, Medema RH. Genetic instability: tipping
the balance. Oncogene. 2013;32:4459–4470.
68. Solomon DA, et al. Mutational inactivation of
STAG2 causes aneuploidy in human cancer. Science.
2011;333:1039–1043.
69. Stephens PJ, et al. Massive genomic rearrangement
acquired in a single catastrophic event during cancer
development. Cell. 2011;144:27–40.
70. Kinzler KW, Vogelstein B. Cancer-susceptibility genes.
Gatekeepers and caretakers. Nature. 1997;386(761):
763.
71. Levitt NC, Hickson ID. Caretaker tumour suppressor
genes that defend genome integrity. Trends Mol Med.
2002;8:179–186.
72. Lynch HT, de la Chapelle A. Genetic susceptibility to
non-polyposis colorectal cancer. J Med Genet. 1999;36:
801–818.
73. Aaltonen LA, et al. Replication errors in benign and
malignant tumors from hereditary nonpolyposis
colorectal cancer patients. Cancer Res. 1994;54:
1645–1648.
74. Young J, et al. Genomic instability occurs in colorectal
carcinomas but not in adenomas. Hum Mutat.
1993;2:351–354.
75. Kaye FJ. RB and cyclin dependent kinase pathways:
defining a distinction between RB and p16 loss in
lung cancer. Oncogene. 2002;21:6908–6914.
76. Khidr L, Chen P-L. RB, the conductor that
orchestrates life, death and differentiation. Oncogene.
2006;25:5210–5219.
77. Morgenbesser SD, Williams BO, Jacks T, DePinho
RA. p53-dependent apoptosis produced by Rb-
deficiency in the developing mouse lens. Nature.
1994;371:72–74.
78. Gumbiner BM. Regulation of cadherin-mediated adhe-
sion in morphogenesis. Nat Rev Mol Cell Biol. 2005;6:
622–634.
79. Vleminckx K, Vakaet L, Mareel M, Fiers W, van Roy
F. Genetic manipulation of E-cadherin expression by
epithelial tumor cells reveals an invasion suppressor
role. Cell. 1991;66:107–119.
80. Guilford P, et al. E-cadherin germline mutations in
familial gastric cancer. Nature. 1998;392:402–405.
81. Lobry C, Oh P, Aifantis I. Oncogenic and tumor
suppressor functions of Notch in cancer: it’s NOTCH
what you think. J Exp Med. 2011;208:1931–
1935.
224.e2 Part I: Science and Clinical Oncology
82. Forshew T, et al. Noninvasive identification and
monitoring of cancer mutations by targeted deep
sequencing of plasma DNA. Sci Transl Med. 2012;4:
136ra68.
83. Leary RJ, et al. Detection of chromosomal alterations
in the circulation of cancer patients with whole-
genome sequencing. Sci Transl Med. 2012;4:162ra154.
84. Diaz LA, et  al. The molecular evolution of
acquired resistance to targeted EGFR blockade
in colorectal cancers. Nature. 2012;486:537–
540.
85. Mok TS, et al. Osimertinib or platinum-pemetrexed
in EGFR T790M-positive lung cancer. N Engl J Med.
2017;376:629–640.
86. Kwak EL, et al. Anaplastic lymphoma kinase inhibi-
tion in non-small-cell lung cancer. N Engl J Med.
2010;363:1693–1703.
87. Corcoran RB, et al. EGFR-mediated re-activation
of MAPK signaling contributes to insensitivity of
BRAF mutant colorectal cancers to RAF inhibition
with vemurafenib. Cancer Discov. 2012;2:227–235.
 C. DIAGNOSIS OF CANCER
Pathology, Biomarkers, and 15 
Molecular Diagnostics
Wilbur A. Franklin, Dara L. Aisner, Kurtis D. Davies, Kristy Crooks,
Miriam D. Post, Bette K. Kleinschmidt-DeMasters, Edward Ashwood,
Paul A. Bunn, and Marileila Varella-Garcia

S UMMARY OF K EY P OI NT S
• Biomarker development has
profoundly affected basic
understanding of carcinogenesis and
expanded means of intervention in
human cancers. Biomarkers have
been applied with variable success
in three broad areas that correspond
to phases of tumor development and
progression: (1) early detection, (2)
diagnosis, and (3) prediction of
clinical outcome (prognosis) and
response to targeted treatment.
• For early detection, biomarkers go
through five stages of development:
(1) identification of promising
directions; (2) validation of a clinical
assay; (3) demonstration that the
biomarker can detect disease before
it is clinically relevant; (4) evaluation
of the biomarker during prospective
screening; and (5) quantification of
the effect of screening on reducing
the burden of disease in a
population.
• Early detection using quite different
approaches has changed the natural
history of cervical, colorectal, and
lung cancer. Progress depends
on improving our understanding
of the biology of site-specific
carcinogenesis and using
intervention strategies informed by
basic biology.
• Cancer diagnosis still relies on an
evaluation of tumor tissue histologic
findings, a complex process
associated with interindividual
variation; however, new approaches
to automated quantitative histologic
evaluation and immunohistochemical
analyses are under active
development.
• New molecular diagnostic
approaches that use assessment of
quantitative messenger RNA
signatures, fluorescence in situ
hybridization, quantitation of
microRNAs, and genetic sequencing
can now be accomplished with
accurate use of formalin-fixed
paraffin-embedded tissues.
• Molecular testing can provide
guidance in prioritizing therapies for
patients most likely to benefit and
can identify patients who have been
proven not to benefit from a targeted
therapy.
• Molecular testing can also provide
clinicians with a rationale to guide
specific patients toward specific
clinical trials.
• As new molecular techniques have
been applied to cancer diagnosis
and treatment, the necessity of
developing standardized processing
approaches for solid tumor
specimens has been appreciated
and has helped to change tissue
collection and specimen-handling
routines.

Since the time of Virchow more than 150 years ago, diagnosis and
treatment of cancer have depended on the appearances of tumor cells
under the microscope and the diverse and often subtle morphologic
differences that distinguish benign from malignant cells. Careful study
of cellular and later of molecular features of both hematologic and
solid tumors led to the idea that cancer results from a series of genetic
and phenotypic changes in progenitor cells that occur over a period
of many years. In epithelial tumors, the final histologic changes that
define transformation of premalignant lesions into malignancy are
the invasion of stromal tissues and the elicitation of a stromal response
including angiogenesis and metastasis to lymph nodes and distant
sites.1 The clinical implications of this multistep process are best
illustrated in the cervix, colon, and lung, where early intervention has
resulted in reduced mortality from cancer. Once invasive carcinoma
has occurred, testing becomes more urgent and intervention more
aggressive. Recently, the great diversity of genetic changes that drive
invasive cancers and the broad range of coverage needed to identify
them have come into focus. This chapter reviews concepts of solid
tumor carcinogenesis and biomarker development for early detection
and distinguishes early detection testing from biomarker applications
in diagnosis, prognosis, and treatment of established solid tumors.
Hematologic biomarkers are described elsewhere in this book.
EARLY DETECTION OF CANCER: THREE
SUCCESSES IN REDUCING CANCER
MORTALITY DISCUSSED AS EARLY
DETECTION MODELS
Remarkable strides have been made in reducing morbidity and mortality
through early intervention in several different kinds of solid tumors.
The triggers for intervention have until now been largely based on

225
226 Part I: Science and Clinical Oncology
Table 15.1 Pepe’s Phases of Biomarker
Development: Early Detection Research
Network
Phase Category Descriptor
1 Preclinical/Exploratory Promising directions identified
2 Clinical Assay and Clinical assay detects established
Validation disease
3 Retrospective/ Biomarker detects disease early,
Longitudinal before it becomes clinically evident
and a screen-positive rule is defined
4 Prospective Screening Extent and characteristics of disease
detected by the test and the false
referral rate are identified
5 Cancer Control Impact of screening on reducing
the burden of disease on the
population quantified
cytologic evaluation of exfoliated cells or advances in imaging technol-
ogy. To date, few molecular markers have been sufficiently sensitive,
specific, or accessible to be useful in population-based studies for early
detection or prevention.
There are several reasons for this. Sensitive biomarkers for risk
assessment and early detection must be inexpensive, noninvasive, and
easily applied to be useful for population-based screening. However,
such biomarkers are rarely sufficiently specific for diagnosis, prognosis,
or response to treatment. A formal process to guide the development
of early detection biomarkers has been defined by Pepe and colleagues.2
In this construct, biomarkers must pass through five phases of develop-
ment that correspond to the strength of evidence that the biomarker
will be a useful population screening tool. These phases of development
are analogous to the structure for therapeutic drug development3 that
has been used for many years. Pepe’s phases are listed in Table 15.1
and are the framework for the discussion of biomarker development
in individual organs later in this chapter.
As will be seen, biomarker development for early detection has
been uneven across the various organs sites, and successes and challenges
faced for three exemplary organs are placed in context below. These
three organs were chosen because they exemplify organ sites where
early detection trials have demonstrated reduction in disease morbidity
and mortality but where success of early detection biomarker develop-
ment has been variable. The organ site for which biomarker development
is most advanced is the cervix, wherein a clear viral cause has been
identified and where the constituents of that virus can be used for
risk assessment, early detection, diagnosis, triggering colposcopic
intervention, and vaccination, achieving Pepe’s stage 5 status of changing
clinical practice as described later.
Cervix and a Validated Biomarker
Invasive cervical carcinoma is one of the few tumors that results from
well-defined premalignant lesions that can be identified and effectively
treated, reducing morbidity and mortality from a tumor that is the
third most common in women worldwide.4 More than 60 years ago,
it was recognized that cellular abnormalities in cervical epithelium
detectable in exfoliated cells could predict the presence of carcinoma.5
By 1988, premalignant cervical cytology had been codified as the
Bethesda system for reporting cervical and vaginal cytologic diagnoses.6
Population-based light microscopic screening for atypical cells in cervical
smears with surgical removal of detected abnormal epithelium in Great
Britain is estimated to have reduced the prevalence of invasive squamous
carcinoma by 80% after 1988.7
Delineation of molecular carcinogenesis in the cervix (Figs. 15.1
and 15.2) has provided alternative methods for detection, risk assess-
ment, and diagnosis. Nearly all cervical carcinomas and their precursor
lesions are caused by human papillomavirus (HPV).8 HPV is a DNA
tumor virus shown by Peyton Rous to produce squamous tumors in
rabbits in 1934.9–11 It was not until 1974 that zur Hausen recognized
the association between HPV and cervical cancer.12–14 Persistent infection
with high-risk strains of HPV, most commonly types 16 and 18, is
an essential step in cervical carcinogenesis (reviewed in zur Hausen15).
The virus encodes three proteins that stimulate cell proliferation and
survival, E5, E6, and E7 (reviewed in zur Hausen16). The viral proteins
E6 and E7 interfere with p53 and Rb, respectively17,18 resulting in
resistance to apoptosis and chromosomal instability. The HPV genome
may be integrated into the host cell genome, accounting for long-term
persistence of the virus. Integration of E6 and E7 genes19,20 is crucial
to progression of the infected cell to a malignant state, whereas other
viral genes may be lost.
Detection of HPV DNA in clinical samples can be accomplished
by a number of methods, with the dominant techniques being based
on PCR or hybridization with signal amplification (reviewed in Leal
and Gulley21). These include the Hybrid Capture 2 method (HC2,
Qiagen), COBAS (Roche Molecular Diagnostics), and Aptima HPV
(HOLOGIC) assays, which have similar sensitivities and specificities22,23;
the latter two systems provide genotyping information in addition to
simple detection information.
Understanding the molecular oncogenesis of cervical squamous
carcinoma has provided a standardized biomarker of risk. More than
95% of HPV infections clear without further consequences. However,
persistent infection for months to years with HPV types 16 or 18
carries a 40% risk of squamous intraepithelial lesions (SIL)24; untreated
high-grade SILs have been estimated to result in invasive carcinoma
in approximately 30% of individuals over 5 to 10 years after diagno-
sis.24,25 Persistent HPV infection thus indicates high risk for eventual
invasive carcinoma and represents an additional biomarker that could
be useful in the prevention of invasive carcinoma.
Testing for HPV DNA has been found to be more sensitive than
cytologic screening for detecting high-grade SIL26,27 but somewhat
less specific. In view of the number of infections that are self-limited,
relying on a single HPV test may result in greater numbers of negative
colposcopy examination results. To address this problem, cotesting
strategies have been evaluated whereby both cytologic evaluation and
HPV testing are performed on initial examination.28 Negative results
have a high negative predictive value and the rescreening interval can
be extended from 1 to 5 years,29 reducing overall screening expense.
Individuals with a positive viral DNA test result but negative cytologic
findings have been rescreened at 1 year and examined with colposcopy
if results are positive. Whether this or other strategies will be universally
adopted for cervical cancer risk management remains to be deter-
mined.30,31 What is clear is that HPV testing provides a means for
improving sensitivity for detection of early cervical lesions, further
reducing mortality from invasive cervical carcinoma while potentially
reducing screening expense.
Finally, HPV not only is a biomarker for risk assessment and
colposcopic intervention, but also is an immunogen that has been
successfully incorporated into bivalent (types 16 and 18; Cervarix),
quadrivalent (types 6, 11, 16, and 18; Gardasil), and nonavalent (types
16, 18, 31, 33, 45, 52, 58, 6; and 11; Gardasil 9) vaccines to prevent
infection and carcinogenesis (reviewed by Stanley32 and Villa33) in both
female and male patients. In-depth discussion of HPV vaccines for
tumor prevention is beyond the scope of this chapter. However, the
many facets of HPV expression in cervical carcinoma and the emerging
role of HPV in other tumors including vulvar and vaginal condylomata,
certain skin cancers, and head and neck carcinoma illustrate how a
valuable biomarker that is integral to oncogenesis can be used to
improve the accuracy of estimating tumor risk and the specificity of
treatment. Unfortunately, such clearly defined and easily accessible
biomarkers are rare. Rather, the more usual case is described here for
 Pathology, Biomarkers, and Molecular Diagnostics  •  CHAPTER 15 227

Cellular Carcinogenesis, Cervix

H&E

p16

Normal
cervix
#1
Low grade
(CIN I)
#2
High grade
(CIN III)
#3
Invasive
carcinoma
#4

Vaccination Colposcopy Resection

Severity of Histological Lesion

Figure 15.1  •  Three potentially premalignant lesions are depicted. 1, Normal cervical mucosa at the squamocolumnar junction (not shown) is composed
of a basilar zone from which squamous cells progressively mature and ultimately give way to flattened cells on the mucosal surface. The p16 immunostain in
the lower frame is negative, reflecting the absence of expression of p16 protein by normal cells. 2, Low-grade squamous dysplasia may occur as a consequence
of HPV infection. In this lesion, the basilar zone is expanded through the lower third of the mucosa, and cells have become more disorderly with irregular
spacing between cells, variable nuclear size and density, and clearing of cytoplasm in some of the cells in the upper mucosa (koilocytosis); p16 immunostains
show strong expression in the lower third of the mucosa. 3, Progression to high-grade squamous dysplasia is indicated by the near complete absence of cellular
maturation toward the surface. Cells contain large, misshapen, and irregularly distributed nuclei. In immunostained sections, p16 is strongly expressed through
the full thickness of the epithelium. 4, Finally, invasive carcinoma occurs when precursor cells acquire the capacity to invade the underlying cervical stroma
as shown in this photomicrograph. Invasive cervical squamous cell carcinoma strongly expresses p16 as shown in the lower frame. Vaccination may prevent
the earliest changes in the progression to invasive carcinoma. Low-grade squamous dysplasia (CIN I) usually regresses and does not require ablation. High-grade
in situ dysplastic changes can be effectively treated by excision of dysplastic epithelium, but if invasive carcinoma occurs, more aggressive surgery and/or radiation
therapy may be required. CIN, Cervical intraepithelial neoplasia.

Molecular Carcinogenesis, Cervix

Mucosal
exposure to HPV
Normal E6 integration, p53 inactivation
cervix E7 integration, pRb inactivation
p16 activation, overexpression
Type 16, 18
HPV infection
Chromosomal instability
Viral persistence other unknown factors
Dysplasia (LSIL)
Dysplasia (HSIL)

Viral persistence
Invasive carcinoma
Emergence from
latency
Clearing of Clearing of
virus virus and dysplasia

Severity of Histological Lesion

Figure 15.2  •  This figure depicts molecular changes that follow infection with high-risk subtypes of human papillomavirus (HPV). In most cases virus is
cleared, but persistent infection may lead to dysplasia. Virus may also be cleared from dysplastic epithelium, but in a subset of cases persistent infection
ultimately leads to carcinoma. HSIL, High-grade squamous intraepithelial lesions; LSIL, low-grade squamous intraepithelial lesions.
228 Part I: Science and Clinical Oncology

colon and lung, wherein potential biomarkers are defined but have not
found a role in early detection, diagnosis, prevention, or treatment.
Colon and Multistep Carcinogenesis With
Hereditary Components
Like cervical carcinoma, colonic adenocarcinoma can be effectively
prevented by extirpation of premalignant epithelium, which in colon
is represented by polyps. Several trials have indicated that removal of
polyps through colonoscopy34–36 reduces the frequency of colorectal
carcinoma (CRC), and consensus opinion is that endoscopic polyp-
ectomy has a significant impact on the incidence of CRC. Data have
also suggested that colonoscopic screening reduces CRC mortality by
more than 50%36 and that the less extensive sigmoidoscopy reduces
mortality from distal CRC by a nearly equal percentage (50%).37 The
latter sigmoidoscopic study was a large randomized population-based
screening trial. Colonoscopic screening for colon polyps beginning at
age 50 has become a standard of care.
Molecular biomarkers to date have not played a direct role in
reduction of morbidity and mortality in CRC. However, molecular
studies have facilitated understanding of colon carcinogenesis (Figs.
15.3 and 15.4) and provided a rationale for colonoscopic surveillance
and polypectomy. In the mid-20th century an association between
between benign polyps and invaive carcinoma was suspected, but at
that time there was no direct evidence that polyps undergo transition
to CRC.38 Suspicions were finally confirmed in the 1980s, when the
clonal nature of both sporadic polyps and CRC was documented.39
Activating mutations in the KRAS oncogene and inactivating mutations
in several tumor suppressor genes (TSGs) were described.40 The same
mutations were demonstrated in both polyps and carcinoma, and the
number of mutations was found to increase with histologic grade of
the adenoma. This suggested a progression to invasive carcinoma
through the accumulation of genetic and epigenetic changes. These
changes provide a proliferative and survival advantage to mutant cells,
which continue to proliferate and mutate until a malignant phenotype
is reached. The combined data from these studies led to the proposal
of a multistep model of colon carcinogenesis,41 which continues as
the standard model.42
Further insight into colon carcinogenesis has been provided by
studies of hereditary forms of colon cancer and the molecular changes
that underlie them. A detailed discussion of hereditary colon cancer
is provided elsewhere in this book and is beyond the scope of this

Cellular Carcinogenesis, Colon

Normal mucosa Low-grade sporadic polyp

#1 #2

Adenomatous polyp High-grade

(FAP, AFAP, MUTYH) sporadic polyp
#5 #3

Serrated adenoma
#6

Invasive carcinoma
#4
Juvenile polyp
(juvenile polyposis)
#7

Peutz-Jeghers polyp
(Peutz-Jeghers syndrome)
#8

Age/years Colonoscopy/excision Resection

Figure 15.3  •  This figure depicts progression from 1, normal mucosa through 2, low-grade and 3, high-grade polyps to 4, invasive carcinoma, the last
represented by poorly formed clusters of cells pushing aside the normal intestinal crypts visible on the right of the frame. Sporadic polyps shown in the upper
row of hematoxylin-eosin (H&E)–stained sections are typically pedunculated and composed of proliferating cells extending to the mucosa surface. As cellular
atypia increases, there is an overlapping of cells along the irregular mucosal surface of the high-grade polyp. 5, Polyps of FAP, AFAP, and MUTYH are mor-
phologically similar to sporadic polyps but occur at an earlier age. In 6, Lynch syndrome, polyps are flat and individual crypts are preserved without the convolutions
present in sporadic polyps. 7, Juvenile polyps are characterized by the presence of mucous cysts (visible on the left side of the frame) and an edematous and
inflamed stroma. The surface of the juvenile polyp may be denuded of epithelium, and there may granulation tissue in the mucosa. Finally, 8, Peutz-Jeghers
polyps have a complex lobulated appearance and contain smooth muscle bundles in the stalk. Removal of sporadic polyps has been found to reduce the frequency
of progression to invasive carcinoma. The presence of invasive carcinoma may require vigorous intervention (discussed elsewhere in this book).
 Pathology, Biomarkers, and Molecular Diagnostics  •  CHAPTER 15 229

Molecular Carcinogenesis, Colon

MGMT, APC, WNT P16, KRAS, BRAF P53, DCC
Mutation/ Mutation/ Mutation/
methylation/ methylation/ methylation/
rearrangement rearrangement rearrangement

Wild type Low-grade High-grade

polyps polyps

MMR mutation MSI Lynch syndrome

CRC
APC mutation FAP
MUTYH mutation MAP
STK11 mutation PJS “Second hit”:
SMAD4 mutation JPS, HHT • Methylation
• Deletion
BMPR1 mutation JPS
• Mutation, etc.
Other/unknown ??

Age/years

Figure 15.4  •  Accumulation of molecular changes that precede colorectal carcinoma (CRC). Sporadic tumors are initiated by a serial accumulation of
somatic mutations that may eventuate in CRC. The initiating event in these polyposes is the mutation present at birth. Second hits may include a variety
molecular changes including tumor suppressor gene promoter methylation, mutations, and copy number changes. Mismatch repair (MMR) and includes
hMLH1, hMLH2, hMSH2, hPMS2, and EpCAMP genes. FAP, Familial adenomatous polyposis; HHT, hereditary hemorrhagic telangiectasia; JPS, juvenile
polyposis syndrome; MAP, MUTYH-associated polyposis; MSI, microsatellite instability; PJS, Peutz-Jeghers syndrome.

Table 15.2  Genetics of Colonic Polyposis

Syndrome (Abbreviation,
Synonym) Gene(s) Molecular Phenotype Noncolonic Organs Affected
Sporadic adenomatous polyp Unknown Chromosomal instability None known
(aneuploidy)
Familial adenomatous polyposis (FAP) APC Chromosomal instability Duodenum, stomach, pancreas, thyroid, liver,
(aneuploidy) central nervous system
Attenuated familial adenomatous APC Chromosomal instability Duodenum, thyroid, liver, central nervous
polyposis (AFAP) (aneuploidy) system
Hereditary nonpolyposis colon cancer hMLH1, hMSH2, hMSH6, MSI Endometrium, stomach, ovary, biliary and
(HNPCC, Lynch syndrome) hPMS2 urinary tracts, small bowel, central nervous
system
MUTYH-linked adenomatous MUTYH Chromosomal instability Duodenum
polyposis (MAP) (aneuploidy)
Peutz-Jeghers syndrome (PJS) STK1 (70%) Unknown Breast, pancreas, stomach, ovary, lung, small
bowel, uterus, testis
Juvenile polyposis syndrome SMAD4, BMPR1A Unknown Blood vessels (hereditary hemorrhagic
telangiectasia)
Hyperplasic polyposis SMAD4, BMPR1A (40%), PTEN Unknown Stomach, pancreas, small bowel

chapter. However, a brief description of the major forms of hereditary
tumors will provide insight into colon carcinogenesis and perspective
on the strengths and limitations of the role of genetic data in early
detection of this prevalent tumor type.
Several forms of hereditary colon cancer are recognized (reviewed
by Jasperson and colleagues43) and are listed in Table 15.2.
Familial Adenomatous Polyposis
The first hereditary colon carcinoma to be described, familial adeno-
matous polyposis (FAP), is characterized in its classic form by the
occurrence of hundreds to thousands of polyps in the colon of affected
individuals with the formation of invasive carcinomas at an early age.
Polyps may also sometimes be found in the upper gastrointestinal
tract. As long ago as 1882, siblings were described with numerous
and recurring polyps,44 and by the mid-20th century FAP had been
established as a hereditary autosomal dominant disorder.45 A gene
mutation associated with FAP was identified on chromosome 5q by
linkage analysis46,47 and positional cloning studies.48–51 The gene,
adenomatous polyposis coli (APC), is now known to encode a tumor
suppressor that modulates Wnt signaling through binding and stabiliza-
tion of a complex of proteins that includes GSK3b, β-catenin, and
axin family members. Mutations in APC truncate the protein at its
230 Part I: Science and Clinical Oncology
carboxyl terminus and disrupt the protein signaling complex. This
results in deregulation of cell cycle control, cell migration, differentia-
tion, and apoptosis (reviewed by Goss and Groden52). In addition to
being responsible for FAP, APC mutation is present in approximately
60% of sporadic colon adenocarcinomas53,54 and an approximately
equal number of sporadic polyps,53,55 suggesting that APC mutation
is an early event in sporadic carcinogenesis as well as in hereditary
disease. An attenuated form of FAP with smaller numbers of tumors
(<100) has been described.54,55 This condition is referred to as attenuated
familial adenomatous polyposis (AFAP)56 and may be driven by the
mutations in APC in the 3′ and 5′ ends rather the central portion of
the gene that is affected in classic FAP.57
Hereditary Nonpolyposis Colorectal Cancer
The most common form of hereditary colon cancer is hereditary
nonpolyposis colorectal cancer (HNPCC). HNPCC is a familial
syndrome that differs from FAP by involving smaller numbers of
polyps in comparison to FAP. The earliest description of this disorder
can again be traced back to the early 20th century,58 but the hereditary
character and syndromic features of the disease were firmly established
by the diligent kindred studies of Lynch and colleagues,59 after whom
the syndrome is named (Lynch syndrome). In brief, the syndrome
consists of CRC occurring in close relatives, often younger than age
50,60,61 without adenomatous polyposis. HNPCC is also associated
with an increased frequency of tumors in the endometrium, stomach,
ovary, hepatobiliary tract, upper urinary tract, small bowel, and central
nervous system (CNS).43 In contrast to FAP polyps, which are frequently
found on the left side of the colon, individuals with HNPCC frequently
have flat lesions on the right side of the colon with a distinct histologic
appearance referred to as serrated adenoma (see Fig. 15.3).
The molecular genetic underpinnings of HNPCC were deciphered
beginning with the linkage to microsatellite markers on chromosome
2p.62 The genetic basis of the disease was established when it was
found that patients with the syndrome harbored mutations in the
mismatch repair (MMR) genes hMLH1,63,64 hMSH2,65,66 hMSH6,67
and hPHS2,68,69 with mutations in hMLH1 and hMSH2 accounting
for over 90% of HNPCC cases. The germline mutations are hetero-
zygous, and colon carcinogenesis results from silencing of the nonmutant
allele by methylation70,71 or by acquired allelic loss.
The phenotypic consequence of gene silencing is loss of MMR
protein and the resulting inability to repair DNA replication errors,
ultimately leading to accumulation of mutations contributing to
carcinogenesis. At a clinical level, protein loss can be demonstrated
by immunochemistry and optimally requires four separate stains. The
inability of affected cells to repair DNA replication errors results
in insertions or deletions in short tandem repeats (microsatellites)
that exist throughout the genome and is referred to as microsatel-
lite instability (MSI).72,73 Currently, MSI is documented by testing
of a standard set of five microsatellite markers recommended by a
consensus panel.74 Results are scored on the basis of the number
of unstable loci and are classified as microsatellite instability–high
(MSI-H), microsatellite instability–low (MSI-L), or microsatellite
instability–stable (MSI-S). More than 90% of HNPCC tumors
are MSI-H, but approximately 15% of all sporadic tumors are
also MSI-H.75
MUTYH-Linked Polyposis
A third hereditary variant of colon carcinoma is MUTYH-linked
polyposis (MAP), the newest polyposis syndrome, first described in
2002.76 This is a disorder similar to AFAP and is characterized by
adenomatous polyps that may number in the hundreds but not in
the thousands, usually ranging from 10 to 1000.77–79 Polyps may also
occur in extracolonic sites, usually the duodenum. Adenomas tend
to occur in the proximal colon.78 MUTYH is a glycosylase and part
of the base excision repair (BER) pathway. Oxidative damage to DNA
frequently results in the modification of guanine (G) to 8-oxoG.
8-oxoG can pair with adenine (A) during DNA replication, a mismatch
that is recognized by MUTYH, which removes the mismatched A
and restores the correct base, cytosine (C). The damaged G (8-oxoG)
is then replaced with an intact G. Mutation of MUTYH at specific
hot spots hinders recognition of the 8-oxoG:A mispairing, resulting
in G:C to A:T transversion at sites of oxidative damage. The mutational
hot spots are found in exon 7, resulting in amino acid substitution
Y179C, and in exon 13, resulting in substitution G496D. Germline
mutation at both hot spots (biallelic mutation) occurs in 0.3% of
patients with CRC. Biallelic mutation confers 28-fold relative risk for
CRC compared with controls. Monoallelic mutation is more common,
with a frequency of approximately 2% in CRC, but also occurs at
approximately the same frequency in control populations. Most studies
suggest that monoallelic mutation confers little or no increased risk
of CRC.77,78 Among control patients without polyposis, biallelic
mutation is rare.78
Hamartomatous Polyposes
The fourth hereditary form of colon cancer are the hamartomatous
polyposes, a category that includes Peutz-Jeghers syndrome (PJS) and
juvenile polyposis syndrome. First described in the 1890s,80,81 PJS was
named for the work of Peutz82 and Jeghers83,84 in 1954 by Bruwer.85
PJS is an autosomal dominant condition characterized by multiple
histologically distinct polyps (see Fig. 15.3) occurring in the large and
small bowel in association with melanin pigmentation of the lips,
mouth, nose, anus, and extremities.86 The condition is associated with
a high risk for cancer in not only the large and small intestine but
also in the esophagus, stomach, pancreas, breast, uterus, ovary, and
lung.87–89 Gene mapping has identified a locus on chromosome
19.3p1390,91 that encodes a serine/threonine kinase, STK11 (LKB1),
a gene that is mutated in more than 90% of affected individuals.92–96
A variety of mutations including deletions, truncations, and missense
point mutations may occur over nearly the full coding region of the
gene.88 STK11 protein is multifunctional, playing a role in cell cycle
arrest, wnt signaling, and the TSC pathway, with effects on mammalian
target of rapamycin (mTOR) signaling.86 Mutation, often associated
with loss of heterozygosity (LOH),97,98 results in loss of function, and
the gene is thus considered to be a TSG. The overall frequency of
PJS is estimated at 0.5 to 2.0 per 100,000 live births.99
Juvenile Polyposis
Juvenile polyps are characteristically solitary peculated lesions that
contain tortuous, often cystically dilated crypts, stromal granulation
tissue, and inflammation and denudation of the surface epithelium.100
These lesions frequently occur in children, and there is no evidence
that solitary juvenile polyps pose an increased risk for carcinoma.100–102
However, multiple juvenile polyps may occur in young individuals
and their family members,103–105 and the presence of more than five
juvenile polyps or a single juvenile polyp in an individual with a
family member with juvenile polyposis now defines the condition
juvenile polyposis. This form of polyposis carries a lifetime risk of
colon carcinoma of 39% and a relative risk of 34.106 Fifty percent to
60% of affected individuals carry germline mutations in SMAD4 or
BMPR1A.107–109 Nearly all patients with SMAD4 mutations also have
hereditary hemorrhagic telangiectasia110 (overlap syndrome). Both
SMAD4 and BMPR1A are involved in the transforming growth
factor–β (TGF-β)/bone morphogenetic protein (BMP) pathway.
Other Polyposes
Finally, a variety of polyposis syndromes with potential genetic linkage
have been described and are best exemplified by hyperplastic polyposis
syndrome (serrated polyposis syndrome).111,112 This condition consists
of the occurrence of multiple sessile polyps variously referred to as
hyperplastic polyps or serrated adenomas, frequently on the right side
of the colon and numbering from 5 to more than 100.113 Although
familial associations of the syndrome114 and linkage to high risk of
colon carcinoma115 have been reported, the genetic basis is complex
and associated with mutations in several driver genes including BRAF
 Pathology, Biomarkers, and Molecular Diagnostics  •  CHAPTER 15 231
and KRAS.112 No single genetic mechanism has been linked to the
syndrome, and the true frequency is not established. To date, the
number of reported cases is small.
Biomarkers for Colon Cancer Screening
Genetic tests
Although the number of highly penetrant mutations that predispose to
colon carcinoma is large, the proportion of colon carcinomas that can
be accounted for by these mutations is approximately 5%43 and has
been overestimated in the past.75 Markers of hereditary predisposition
and molecular progression in sporadic tumors that have been delineated
to date have not been easily converted into an early detection test
suitable for broad population-based applications for several reasons.
First, the types of accessible specimens that can be used for testing for
early detection consist primarily of blood and fecal DNA. Although
DNA from invasive tumors can be detected in the blood by PCR-
based mutation testing, it is not as useful at earlier polyp stages. In
addition, collection and processing of fecal specimens for a biomarker
that is expected to be present at low level and heavily contaminated
by nonneoplastic cells is a logistic challenge. Second, some of the
genetic abnormalities identified in colon carcinogenesis do not readily
lend themselves to screening assays. Mutations in APC, for example,
consist mainly of truncations and deletions that may occur over a
wide region of the gene, so designing a sensitive clinical assay has
been difficult. Finally, wide availability of colonoscopy services that
combine polyp detection and excision reduces the cost and improves the
attractiveness of direct and systematic colonoscopic population-based
surveillance.
Other screening biomarkers
Biomarkers that have been most successful for colon cancer screening
are fecal tests such as the guaiac fecal occult blood test (FOBT), the
fecal immunochemical test (FIT), and the stool DNA (sDNA) test.
The greatest experience has been with the FOBT. This test is based
on a color change whereby α-guaiaconic acid extracted from the wood
of tropical Guaiacum tree is rapidly oxidized by hydrogen peroxide
to a blue quinone in the presence of heme derived from blood. There
is wide variability in results116 as a consequence of the level of compliance
with collection methods, type or brand of kit, frequency of testing,
user interpretation, and other factors. Despite a lack of standardization,
FOBT screening with follow-up colonoscopy is reported to reduce
both incidence117 and mortality118,119 from colon carcinoma. The former
is attributed to polyp removal. FIT is a second test for occult blood
and is reported to be more sensitive and specific than the guaiac assays.
It has been shown to have a cancer detection rate similar to colonoscopy
but has a lower sensitivity for polyps.120
Occult blood tests measure host response (hypervascularity and
bleeding) to tumor. The sDNA test, however, aims to measure a direct
product of tumor cells. A recent version of the sDNA test measures
multiple markers in stool using an array-based platform121 and for
this reason has been referred to as “next generation,” although this
technology does not employ deep sequencing methods and should
not be confused with “next-generation sequencing.” The biomarkers
that are incorporated into the platform include four methylation probes,
as well as mutated KRAS and hemoglobin. Patterns of gene methylation
have been identified in colon tumors that have been characterized as
CpG island methylation (CIMP) high and CIMP low (reviewed by
Kim and colleagues122). When applied to a preliminary cohort of 678
subjects with polyps or CRC, the combination test including methyla-
tion markers, KRAS mutation, and occult blood had a sensitivity of
85% for carcinoma and 54% for polyps.
Despite the availability of these fecal-based tests, the target in early
colon cancer intervention appears to be shifting from detection of
invasive carcinoma to prevention by detection and thorough clearing
of adenomatous polyps from the colon. Defining the biology of colon
carcinogenesis and confirming that polyps are the precursor lesions
along with improvements in colonoscopic methods have led to reduction
in mortality from colon cancer. Colonoscopy enables direct identifica-
tion of potential precursor lesions and at the same time provides a
means for ablating them. This is strikingly evident in the new sig-
moidoscopy findings37 indicating that whereas sigmoidoscopy reduces
mortality from visible distal colon tumors, mortality from proximal
lesions is unaffected. It seems likely that colonoscopy will be the main
vehicle for early detection and intervention in colon cancer for the
foreseeable future.
Lung Carcinogenesis and a Known Carcinogen
For several decades lung carcinoma has had the highest mortality of
all tumors, accounting for more than twice as many deaths as the
next most lethal tumor.123,124 Initial large-scale efforts during the 1970s
and 1980s to reduce mortality by early detection using chest radiography
and/or sputum cytology were not successful. Chemoprevention
using β-carotene and/or α-tocopherol dietary supplementation also
failed.125–128 However, early detection efforts have begun to yield some
success in reducing mortality even in this problematic tumor. Low–
radiation dose computed tomography (LDCT) has a higher clinical
sensitivity for detecting smaller tumors than chest radiography. Early
studies in smokers documented a 2.7% prevalence of malignant disease
detected by LDCT versus 0.7% in the same patients screened by chest
radiography.129 In a multicenter study of more than 30,000 smokers,
LDCT revealed lung cancer in 1.3% at baseline and an additional
0.3% at annual follow-up examinations, for an overall total detection
rate of approximately 1.5%.130 Although this study was not randomized
and had no unscreened control arm, it showed a survival advantage
in comparison to historical controls. Finally, a large randomized trial
of computed tomography (CT) versus radiography conducted annually
in three rounds found that LDCT enabled detection of carcinoma in
2.4% of the screened high-risk population and resulted in a 20%
reduction in lung cancer mortality that was not observed in the chest
radiograph control group.131 The weight of evidence supporting
improved long-term mortality rates in smokers has prompted the
publication of multisociety consensus guidelines recommending annual
LDCT screening for smokers and former smokers aged 55 to 74 years
with access to appropriate follow-up.132 This study has raised awareness
of possible benefits of early detection of lung cancer but has been
criticized for possible overdiagnosis,133 a question that is currently
unresolved.
Premalignant Lesions in the Lung
What is clear is that lung carcinogenesis is a multistep process and
that premalignant lesions exist in both central and peripheral airways
(Figs. 15.5 and 15.6) before invasive carcinoma occurs.
Central airway premalignancy: squamous dysplasia
The predominant premalignant lesion of the central airways is squamous
dysplasia134 (also referred to as atypia). Squamous dysplasia was docu-
mented over 50 years ago in association with carcinoma135 as well as
in smokers without concurrent carcinoma.136 Central squamous lesions
are flat, inconspicuous, and only rarely identified by white light
fiberoptic bronchoscopy. They are somewhat better visualized with
laser-induced fluorescence emission (LIFE) bronchoscopy.137 This
method relies on a loss of autofluorescence and signal from dysplasias;
it may be mimicked by fluorescence loss caused by mucosal hemorrhage
and other reactive changes, so whereas sensitivity of LIFE bronchoscopy
is high, specificity is low.
Prospective studies on the power of dysplasia to prospectively predict
future central airway malignancy are few and face the forbidding
challenges of inaccessibility of the bronchi and the prolonged follow-up
that is required to achieve significant results in a sufficiently powered
cohort. Sputum studies indicate that atypia of cells exfoliated from
the central airways is associated with elevated risk for incidental
carcinoma. Hazard ratio increases with proximity of sputum collection
date to lung cancer diagnosis, but sputum atypia even 3 years before
232 Part I: Science and Clinical Oncology

Cellular Carcinogenesis, Lung

Normal bronchiolar
epithelium
#1 Low-grade
squamous dysplasia
#2 High-grade
squamous dysplasia
#3 Invasive
squamous carcinoma
#4

Normal alveolar septum

#5
Atypical adenomatous
hyperplasia
#6 Adenocarcinoma
in situ
Invasive adenocarcinoma
#7
#8

Progression over time (years) LDCT/resection

Figure 15.5  •  Multistep carcinogenesis in the lung is complicated by the heterogeneity of tumor types that arise in separate locations in the respiratory
tract. Squamous tumors typically form in the central airways, where they are thought to arise from 1, the respiratory mucosa. The earliest premalignant lesion,
2, low-grade squamous dysplasia, exhibits a polarity in which dark basaloid cells mature into large cells that flatten along the surface as seen in the cervix (see
Fig. 15.1). 3, High-grade squamous dysplasia exhibits less distinct maturation and a higher level of cytologic diversity. 4, Invasive squamous cell carcinoma
occurs when tumor cells extend in the underlying stroma, sometimes forming a mass visible on low–radiation dose computed tomography (LDCT). Adeno-
carcinomas typically arise peripherally in 5, the alveoli and terminal bronchioles. The best-defined precursor lesion of adenocarcinoma is 6, atypical adenomatous
hyperplasia (AHH). These are small lesions (<5 mm) in which the alveolar architecture is preserved. 7, Adenocarcinoma in situ (AIS) is a further progression
of peripheral airway epithelium toward malignancy. This category represents a redefinition of the low-grade adenocarcinoma previously known as bronchioloalveolar
carcinoma. These lesions are solitary small (≤3 cm) lesions consisting of a single cell layer that spreads in a nondestructive pattern of growth along the alveolar
septae that is referred to as lepidic and is detectable as a ground-glass nodule by LDCT. 8, Invasive adenocarcinoma spreads beyond the alveolar surface,
forming a mass that appears solid on LDCT. The presence of a mass visible with LDCT suggests tumor. However, by the time this occurs, tumor is often
invasive and must be treated aggressively.

Phases of Molecular Carcinogenesis, Lung

Early premalignant
Tobacco smoke exposure Late premalignant
Inflammatory changes Clonal proliferation
Angiogenesis Lateral spread
Angiogenesis
Diploidy Malignant
Bulky adducts, Vertical spread
Tissue invasion
Mitotic recombination
LOH (allelic loss, UPD)
Methylation myc dysregulation
Telomerase expression TP53, KRAS, APC,
Other TSG mutations
Further mitotic recombination
Methylation
Chromosomal instability
Array signature
Aneuploidy, translocation
Resistance to treatment

Age/years

Figure 15.6  •  The molecular pathology of smoking-related lung cancer begins with the direct interaction between metabolites of carcinogenic polyaromatic
hydrocarbons in cigarette smoke with host DNA. This creates bulky adducts that affect DNA repair and create transcription errors. The result is a wide range
of genomic losses, gains, translocations and point mutations that account for the high degree of molecular heterogeneity in lung tumors with diverse physiologic
effects. LOH, Loss of heterozygosity; UPD, uniparental disomy; TSG, tumor suppressor gene.
 Pathology, Biomarkers, and Molecular Diagnostics  •  CHAPTER 15 233
lung cancer diagnosis is associated with increased risk of lung
carcinoma.138
Bronchial biopsy is an alternative way to evaluate the status of the
airway in high-risk smokers. A recent prospective study of 188 patients
who underwent biopsy at mapped bronchial sites found that the
persistence of high-grade dysplasia at the same bronchial site was
associated with future squamous carcinoma but not adenocarcinoma,
suggesting that persistent dysplasia may be a marker of progressive
field change.139 This study also illustrates the multiyear timeline and
multifocality of premalignant changes.
The corollary question of whether ablation of premalignant central
airway lesions could reduce lung cancer mortality remains an open one.
The findings of a chemoprevention study140 using iloprost, a prosta-
cyclin agonist, have suggested that reversal of premalignant dysplasia
morphological features may be possible, but the study was a small
one and the end point was purely histological. The long-term effect
on mortality of agents that may alter the premalignant morphologic
characteristics of central airway epithelium remains unknown.
Atypical adenomatous hyperplasia, adenocarcinoma in situ,
and lepidic carcinoma
Atypical adenomatous hyperplasia (AAH), adenocarcinoma in situ
(AIS), and pure lepidic carcinomas consist of alveolar epithelial cell
proliferations that do not invade the alveolar wall or elicit a significant
stromal response.141 AAH and AIS are small (<5 mm) solitary lesions
that are usually recognized incidentally in screening trials or in clinical
practice by their ground-glass appearance on CT imaging. AAH and
AIS are characterized by indolent growth rate and 100% survival after
resection. Larger solitary ground-glass lesions of pure lepidic carcinomas
as well as some lesions with areas of consolidation and even multifocal
ground-glass lesions also are characterized by slow growth and excellent
prognosis and may persist for many months to years without changing
size or evincing evidence of alveolar wall invasion.142–145 It appears
that the time to progression of premalignant lesions in both central
and peripheral airways is long and capricious, complicating assessment
of prognosis and intervention strategies.
Molecular Changes During Lung Carcinogenesis
As in other organs, most notably the colon, molecular changes that
accompany the morphologic changes of dysplasia have been investigated
in considerable detail. These studies are informed by the well-established
role of tobacco smoke as a carcinogen in lung cancer. The dominant
carcinogenic compounds found in cigarette smoke are the polyaromatic
hydrocarbons (PAHs) and nicotine-derived nitrosamine ketone
(NNK).146 These compounds are metabolized to highly reactive
intermediates that are inactivated largely by mitochondrial glutathione
transferases (GSTs). However, repair is imperfect and a small proportion
of the intermediates react directly with DNA, forming bulky adducts.
Unless removed by the DNA repair system, adducts can create errors
of replication. Over time, molecular alterations accumulate, providing
a selective advantage to mutant cells for growth and survival.
Components of this pathway have been associated with increased
risk for future lung cancer. Case control studies indicate that current
smokers with tumors have higher levels of adducts in blood or tissue
than non-tumor controls.147 However, bulky adducts appear to reflect
ongoing smoking-related DNA damage rather than biologic changes
specifically associated with malignant transformation. Clinically
applicable adduct levels predictive of lung cancer have not been
established.
GST and DNA repair enzyme mutations and polymorphisms
that affect DNA repair activity could represent a risk factor for lung
cancer because efficient deactivation of reactive carcinogens and
repair of DNA damage caused by bulky adducts are important in
protecting potential precursor cells from critical mutational changes.
However, single-nucleotide polymorphism (SNP) association studies
in many carcinogen-metabolizing and DNA repair enzymes have
demonstrated only weak and inconsistent associations and have not
been sufficiently informative to guide intervention in potentially affected
individuals.148–152
Tumor suppressor gene methylation
DNA damage in lung premalignancy engenders a number of genetic
and epigenetic changes that provide a cellular survival or proliferative
advantage to affected bronchial epithelial cells. Perhaps the earliest
molecular events may be epigenetic and include DNA methylation,
histone modification, and alterations in miRNA expression (reviewed
by Jones and Baylin153 and Berger and colleagues154). The greatest
experience regarding the use of epigenetic changes in the context of
early detection of lung cancer has been obtained with regard to
methylation. Methylation refers to the conversion of cytosine to
5′-methylcytosine by DNA methyltransferase (DNMT). In lung
carcinogenesis, methylation preferentially occurs in CpG-rich regions
(CpG islands) that are present in normally unmethylated promoter
regions of critical genes (hypermethylation) and may silence gene
expression. In lung tumors, interest has focused on genes such as
p16,155 which are silenced in tumors but expressed in normal control
tissue, and are considered to be TSGs. The development of a simple
and sensitive assay based on the conversion of methyl cytosine to
uracil in the presence of metabisulfite followed by PCR with primers
specific for the methylated bases, methylation-specific polymerase chain
reaction (MSP),156 has greatly facilitated the assessment of methylation
status as a risk marker for lung carcinoma.
Methylation of key TSG promoter regions in cells exfoliated from
bronchial surfaces into the sputum is nearly universally associated
with prevalent lung carcinomas157 and frequently also occurs in
sputum of smokers without tumor. TSG methylation is not present
in sputum of never smokers. This finding initially led to optimism
that methylation could be used to screen selected populations at risk
for lung carcinoma. However, the predictive power of single-gene
methylation or methylation of a small number of genes is limited by
the ubiquity of methylation in smokers and the consequent high rate
of false-positive results. This problem led to the strategy of testing
promoter regions in large numbers of genes to estimate overall
methylation burden.158 Identification of panels of genes to optimize
the predictive power of methylation testing is a process that is still
underway. In receiver operating characteristic (ROC) curve analyses, a
seven-gene methylation profile selected from a 31-gene panel achieved
a predictive accuracy of 71% to 77%. However, with a sensitivity
set at 75%, the false-positive rate was 75%.159 Improved results were
obtained in a case-control study of CT-detected nodules by using a
three-gene combination having a sensitivity and specificity of 98% and
71%, respectively.160 Although TSG promoter methylation may prove
problematic as a general screening tool, it may be useful in specific
niche populations or to supplement other methods of screening such
as LDCT, and ongoing studies aim to further evaluate the use of this
approach.
Aneuploidy
A second molecular consequence of tobacco exposure is chromosomal
instability, which is detected as aneuploidy with fluorescence in situ
hybridization (FISH) in bronchial epithelial cells in situ and in
sputum. Aneuploidy is nearly universal in lung carcinoma. It is also
frequently observed in high-grade dysplastic lesions in the central
airways.161 Aneuploidy in sputum has been associated with prevalent
carcinoma162,163 and has recently been shown to have a sensitivity of
76% within a detection window of 18 months before the appearance
of invasive carcinoma.164 However, FISH performed on sputum is
labor-intensive and difficult to perform both in the specimen prepara-
tion and slide interpretation, requiring a high level of expertise at
every step. This has led to the development of automated methods
of FISH screening. However, to date these automated methods have
not been broadly applied. It seems likely that FISH may most suc-
cessfully be applied in restricted settings in conjunction with other
technologies.
234 Part I: Science and Clinical Oncology
TP53 mutation
Mutations in several tumor-associated genes have been documented
in adjacent nonmalignant airways or in subjects at high risk for
carcinoma but without concurrent malignancy. Chief among these is
TP53, which marks clonal populations that may spread laterally across
the bronchial surfaces without invading stroma. Mutation in TP53
appears late in lung carcinogenesis; and although it is one of the most
commonly mutated gene in lung carcinoma, it is mutated in only
half of tumors and in fewer benign airways. It seems unlikely that
single-gene mutation analysis will be sufficiently sensitive for use as
a screening tool.
High-throughput technologies
High-throughput methodologies have been suggested as possible early
detection tools. Genome-wide association studies (GWASs) have
identified an SNP variant at 15q15.2 that is significantly associated
with cancer risk, with an overall odds ratio of 1.15.165 The low odds
ratio suggests that the SNP variant itself will not represent a useful
predictor. However, this region of the gene is still being explored for
more powerful biomarkers.
Gene expression microarrays have identified consistent changes in
the transcriptome of epithelium brushed from nonmalignant bronchial
surfaces that distinguish smokers from nonsmokers.166 An additional
set of changes are reported to identify subjects with cancer from
smokers without cancer with an accuracy of 83%.167 Finally, large-scale
genomic sequencing studies have identified differences in the “genomic
landscape” between smokers and nonsmokers.168 It is expected that
manageable numbers of biomarkers and clinically applicable testing
platforms will emerge from these exploratory studies that will improve
risk assessment and management of high-risk smokers as well as patients
with already invasive carcinomas.
Other Organ Sites
Premalignant cellular and molecular changes are described in several
other tumors including breast, prostate, bladder, and esophagus.
Detailed review of these changes may be found in appropriate chapters
of this text. However, to date, identification of these changes has not
led to mortality reductions.
Conclusion: Cells and Molecules in Early Detection
The examples described above illustrate the uneven progress that has
been made and the challenges that remain in reducing morbidity and
mortality from cancer. What emerges from this review is that progress
depends on improved understanding of site-specific carcinogenesis
and intervention strategies that are informed by basic biology. Relatively
simple technologies such as cytologic evaluation or HPV testing can
profoundly reduce cancer burden. Understanding the biology of
premalignancy provides a lead-time advantage that can be translated
into greatly reduced cancer morbidity and mortality. Appropriate
molecular strategies may supplement and eventually replace morphology
but will require thorough validation.
DIAGNOSIS AND CLASSIFICATION OF SOLID
MALIGNANCIES: HISTOLOGY AND
EXPANDING ROLE OF MOLECULAR
DIAGNOSTICS
Histology of tumor tissue has been the bedrock on which diagnosis
of malignancy is established and is the basis for selecting appropriate
testing to guide treatment. Histologic diagnosis, although simple in
concept, is extremely complex in practice and crucially depends on
the expertise of the examining pathologist. In solid tumors, a nearly
universal feature of malignancy is the capacity of tumor cells to invade
and replace stromal tissue, and nearly all of the following discussion
refers to destructive, invasive malignant tumors that form solid masses.
The primary tool of the pathologist remains the hematoxylin-and-eosin
stained section, but this is now supplemented by an arsenal of
immunohistochemical stains and other tissue testing procedures that
permit much greater specificity of diagnosis than was previously possible.
Diagnostic immunohistochemical tests are relatively simple and
inexpensive and can be assessed in conjunction with in situ cellular
architecture, which ensures a high level of specificity and permits an
assessment of the relevance of results to the overall clinical and anatomic
features. Interpretation of immunohistochemical data is now assisted
by online databases that provide frequencies of immunohistochemical
results in specific tumors and organ sites. Histologic diagnosis was
discussed earlier in the context of early detection and is addressed
elsewhere in this book in relation to individual tumor types.
Although histologic evaluation remains the primary method for
diagnosis of solid tumors, molecular methods are increasingly influential
not only in identification of treatment targets as discussed here, but
also in tumor classification. In CNS tumors, for example, molecular
findings are such powerful predictors of biologic behavior that molecular
features have been formally incorporated into the diagnostic classifica-
tion of this complicated group of neoplasms (see later). Both molecular
and cellular diagnostics continue to depend not only on consistent
and effective fixation and processing of tissue, but also on reliable
accessioning and informatics.
Algorithm for Cellular Molecular Testing
Molecular and cellular tissue testing is a multistep process (Fig. 15.7),
and success requires tight control of collection, accessioning, fixation,
dissection, extraction, and storage of tissue as discussed in the following
sections.
Accessioning and Informatics
Registering patients and biopsy specimens is a demanding exercise
that is often overlooked and underfunded. Although numerous and
variable protocols are available for tracking specimens, virtually all
tracking systems include a secure electronic database with password-
limited access and reliable backup. Appropriate specimen identifiers,
which today are increasingly bar-coded, access to signed consent forms
when associated research studies are underway, and timely data entry
are all essential for any well-run laboratory. A detailed discussion of
laboratory information systems for molecular testing is beyond the
scope of this chapter but must be a major consideration in the manage-
ment and quality assurance programs of all clinical molecular testing
laboratories.
Tissue Collection and Processing
Tissues obtained for solid tumor molecular testing may come from
surgical resection or biopsies (fine-needle aspirates and cores). Most
often, the tissue needed for molecular testing is derived from diagnostic
material, unless special arrangements are made to specifically collect
tissues for molecular studies. Issues regarding specific specimen types
are discussed in the following sections.
Resection Specimens
Resection specimens are specimens obtained as a result of surgical
procedures to remove tumors for therapeutic purposes. These specimens
provide the largest amount of material for molecular testing. Several
aspects of specimen collection must be borne in mind to optimize
resection specimen quality and yield.
Gross dissection. Identification of tumor tissue in resected
specimens requires a high level of morphologic and dissection skill.
For this reason, it is essential that a trained pathologist be involved
in establishing protocols for the collection of specimens for molecular
studies. For DNA-based assays including mutation analysis, timing of
the collection procedure can be somewhat flexible because DNA is a
particularly robust analyte. However, efforts should be made to avoid

Preanalytical
processing
1. Estimate tumor cell content
2. Isolate tumor and/or normal cells
A. Whole sections
B. Coring or
C. Trimming of block
D. Microdissection DNA
a) Scrape extraction
b) Laser capture
1. Deparaffinization
2. Proteinase K digestion
3. Precipitation/capture Library
4. Shear DNA
a) Sonication preparation
b) Enzymatic
1. Target enrichment
a) Hybrid capture
b) Amplicon
2. Quantitation
3. Pooling
Figure 15.7  •  Steps entailed in obtaining optimal results for clinical next-generation sequencing (NGS).
prolonged exposure to formalin fixation, or exposure to harsh acids
such as those that can be encountered during decalcification treatments.
Specimen preservation.  Fresh tissue, tissue preserved in transport
media such as RNAlater, or frozen samples are acceptable starting
materials for molecular testing. However, these samples may be
inconvenient to process in clinical laboratories and have the disadvantage
that tumor cell purification by macrodissection or microdissection
may be more difficult and complicated than with paraffin sections
cut from fixed tissue. Formalin-fixed paraffin-embedded (FFPE)
specimens are now routinely used for molecular testing. The most
cost-effective fixative is 10% buffered neutral (pH 7.4–7.6) formalin.
Buffering is needed to eliminate possible DNA depurination, which
may occur at acidic pH. Phosphate is the preferred buffering salt.
Formalin works by cross-linking amino groups in proteins, introducing
a -CH2- linkage. This linkage is stable and permits long-term storage
of tissues in paraffin with minimal degradation. Fixatives such as
acetone and alcohol work by rapidly dehydrating tissues. These fixatives
may not completely inactivate autolysis and may require modification
of testing platforms. Metal-based (e.g., zinc, mercury) fixatives, which
are favored in some contexts because of the excellent histologic detail
they provide, may inactivate polymerases used in molecular analyses.
For this reason, specimens exposed to metal-based fixatives cannot be
used for molecular biomarker studies. Finally, acid decalcification
causes DNA deterioration and may result in amplification failure.
Decalcification status should be kept in mind in interpretation of
testing results.
Biopsies
Improved imaging and tissue sampling techniques in recent years,
exemplified by advances in lung sampling methods,169,170 have resulted
Pathology, Biomarkers, and Molecular Diagnostics  •  CHAPTER 15 235
Massively parallel
sequencing
1. Ion torrent
2. Illumina Data
analysis
1. Raw data analysis (base calling)
2. Mapping to genome
3. Variant calling Clinical
4. Variant annotation report
1. Adequacy
2. Depth of coverage
3. Frequency of mut
4. Predicted phenotype
5. Drugs/trials available
6. Referral to genetic
counseling (germline)
in increasing reliance on small biopsy specimens for diagnosis and
monitoring of treatment. The primary objective of these tissue acquisi-
tion methods has been to obtain tissue for unequivocal diagnosis,
which can require multiple immunohistochemical stains in addition
to conventionally stained slices. Meeting this objective can leave little
remnant tissue behind for ancillary studies. When tumor is of an
advanced stage and surgery is not appropriate, remnant tissue from
these diagnostic biopsies may be the only tissue available for molecular
testing. As morbidity from biopsy procedures has declined and the
value of information derived from molecular testing has become more
critical, performing biopsies solely for molecular analysis has gained
increasing acceptance by clinicians and patients alike. The major
limitation of biopsy specimens for molecular testing is small tumor
cell yield. Also, testing of DNA extracted from paraffin sections must
take into account shearing of DNA caused by sectioning. Despite
these challenges, isolation of tumor DNA from small specimens of
many types including tissue cores, cell block preparations, smears and
imprinted slides has been well documented.171–174 Individual laboratories
must validate testing protocols for each specimen type.
Enriching for Tumor Cells
Because nonmalignant cells invariably accompany (“contaminate”) all
clinical samples, specimens must be enriched for tumors to a level
that is consistent with the analytic sensitivity of the platform that will
be used for testing. As a rule of thumb, a minimum of 100 viable
tumor cells in a 10-µm section is required for a single PCR-based
assay. Quality of the biopsy specimen may be more important than
quantity for critical molecular studies.175,176
Although tumor enrichment methods may vary from laboratory
to laboratory, it is essential that tumor enrichment methodology be
236 Part I: Science and Clinical Oncology
appropriately matched to the testing platform. At least five different
procedures may be used for tumor cell enrichment, depending on the
amount of contaminating normal tissue that is present in the specimen:
(1) en face sectioning of a trimmed block, (2) coring of paraffin blocks
in regions enriched for tumor cells, (3) macrodissection, (4) manual
microdissection, and (5) laser capture microdissection. Macrodissection
can refer to the practice of scraping of tumor cells from a glass slide,
typically done without direct microscopic visualization. Manual
microdissection, by contrast, involves the use of some form of micro-
scopic assistance to identify tumor cells. Manual microdissection is
typically performed by using a dissecting microscope and usually
involves counterstaining of slides to identify regions of interest, which
can be removed from the slide with a scalpel, needle, or glass micro-
pipette. Laser capture microdissection involves melting of supporting
matrix with a laser beam created with an epifluorescence lens and
removal of the target cells for extraction and quantitative testing of
DNA and other analytes. Although more precise than manual microdis-
section, laser capture microdissection is slower, is costlier, and generates
lower yields of extraction products.
Liquid Biopsy
Obtaining tissue for microscopic examination requires invasive pro-
cedures and a significant commitment of resources but assesses only
a limited portion of tumor. So-called “liquid biopsy” circumvents
these limitations by testing peripheral blood. Testing can use either
isolated circulating tumor cells (CTCs) or extracted cell-free tumor
DNA (ctDNA) from biologic fluids.
CTCs are produced by shedding of tumor cells into the lympho-
vascular system. They are usually found in very low abundance in
blood (on average ~1 cell per milliliter), and therefore require meticulous
methods to isolate them from the large numbers of contaminating
normal blood cells.177 This is typically achieved by using cell-surface
markers specific for cancer cells—for instance, the epithelial cell
adhesion molecule (EpCAM) that is found on most epithelial-derived
tumor cells but not normal blood cells.178 Alternatively, physical
differences between CTCs and normal blood cells, such as size,
deformability, and bioelectric differences, can be used to achieve isola-
tion.177 Regardless of the isolation technique, this approach is hampered
by the low yield of isolation methods. However, CTC analysis, in
particular overall detection of CTCs or quantification of CTCs, has
demonstrated benefits in cancer diagnosis, prognosis assessment, and
monitoring of treatment response.179–182
Circulating ctDNA consists of short (~140–170 bp) fragments of
DNA found in blood or urine. It is derived from both normal and
tumor cells undergoing apoptosis, necrosis, or release from live cells.183
Fragments are typically short because they are derived from DNA
that wraps around the nucleosome and is thus protected from degrada-
tion. The percentage of free DNA derived from tumor varies widely
(0.01%–93%) and is influenced not only by the amount of ctDNA
released by cancer cells but also the total load of cell free DNA, which
itself can vary dramatically from person to person.184 To distinguish
tumor from normal DNA, analysis relies on tumor-specific markers
(e.g., point mutations). The percentage of ctDNA can be lower than
the limits of detection of standard DNA sequencing techniques, and
specialized approaches such as allele-specific PCR, digital PCR, and
presequencing enrichment of mutant alleles are required to achieve
significant sensitivity.185–187 Also, specialized bioinformatics measures
can be undertaken in next-generation tests to reduce background
noise and improve lower limits of detection.188 Despite these efforts,
typical reported sensitivity for detection of ctDNA ranges from 49%
to 78% for localized tumors and 86% to 100% for metastatic tumors,
depending on tumor type,189 the yield of positive results is sufficiently
high that the test may provide diagnostic, prognostic, and disease
burden information in a significant patient fraction without the need
for biopsy.189–191 As ctDNA typically provide sequence data, this
approach can also be used to detect actionable genetic alterations.185
This is particularly useful in the serial monitoring of blood for the
appearance of mutations associated with acquired resistance to targeted
therapies, a setting in which serial biopsy may not be feasible. The
identities and methods of testing for these target sequences are discussed
in the following sections.
TREATMENT TARGETS AND
MOLECULAR ANALYSIS
At the start of the millennium, the sequencing of the human
genome192–194 and the discovery that somatic mutation could activate
transmembrane receptors and tumorigenicity195 led to anticipation
that anticancer drugs affecting this process would be found in abun-
dance.196 The Cancer Genome Atlas (TCGA) program was launched,197
in part, to identify essentially all mutations that might be subject to
external regulation by specific pharmaceuticals. TCGA revealed the
large mutational burden in many solid tumors,198 particularly in those
that have been associated with a high degree of genetic damage caused
by physical or chemical agents such as ultraviolet light in melanoma199,200
and tobacco smoke in lung carcinoma.201,202 TCGA and other deep
sequencing studies have also revealed the complexity, heterogeneity,
and lability of mutations in tumors.203–208
Among the most relevant findings of TCGA has been the confirma-
tion that mutation of proteins in the tyrosine kinase signaling pathway
is common in human tumors and that many are affected by the
increasing number of FDA-approved inhibitors that are now available
for treatment of both solid (Table 15.3) and liquid tumors (Table 15.4;
discussed elsewhere in this book). Tyrosine kinase receptors (TKRs)
are transmembrane immunoglobulin-like molecules209 containing an
extracellular ligand-binding region, a transmembrane region, and an
intracellular tyrosine kinase region that contains tyrosine residues whose
phosphorylation regulates signal transduction. Specific mutations in
tyrosine kinase domains enhance phosphorylation and stimulate
downstream intracellular signaling. The physiologic result is accelerated
cell proliferation, extended cell survival, and increased angiogenesis
and cellular migration, properties that are hallmarks of carcinogenesis.1
Although mutations cluster around hot spots in the receptor, they
may also occur at less common loci. In addition, unexpected tyrosine
kinases and other signaling molecules may drive tumor cells, so it
is increasingly important that sequencing techniques have broad
coverage. For this reason and for economies of scale, next-generation
sequencing (NGS) is now widely used to identify treatment targets in
solid tumors.
DNA: Next-Generation (Massively Parallel)
Sequencing as a Biomarker Predicting Response
to Treatment
A number of small-molecule tyrosine kinase inhibitors (TKIs) specific
for these receptors have been designed to block phosphorylation and
suppress tumor growth and spread. In addition, the extracellular
domains of these molecules have proven to be antigenic, and therefore
the extracellular ligand-binding domain is also a target for therapeutic
antibody development. Several of these drugs have successfully
transferred to the clinic and the number of such drugs available for
cancer treatment is likely to continue to expand, creating an ongoing
demand for more comprehensive molecular testing of malignancies
of virtually all types. The increased demand for sequence coverage is
being filled by NGS.
Definition
NGS refers to techniques in which millions of nucleotide sequences are
deciphered simultaneously (reviewed by Goodwin and colleagues210)
and is often called massively parallel sequencing. The complete sequenc-
ing of the human genome during the early 2000s192–194 engendered
a need for relatively inexpensive, high-throughput sequencing
methods, a need that was met by several NGS platforms that were
quickly commercialized. NGS platforms have proven to be highly
 Pathology, Biomarkers, and Molecular Diagnostics  •  CHAPTER 15 237

Table 15.3  Molecularly Targeted Agents Approved by FDA for Solid Tumors

Tumor(s) Gene(s) or Protein Molecular Lesion(s) Drugs

Breast ER or ER/PR Overexpression Fulvestrant, Tamoxifen, Anastrazole, Exemestane,
Letrazole
Breast CDK4/6 Expression/overexpression Ribociclib, Palbociclib + Fulvestrant or Letrazole
(for ER+, HER2−)
Breast/Stomach HER2/neu Amplification/overexpression Everolimus, Lapatinib, Pertuzumab, Trastuzumab,
Ado-trastuzumab Ematansine, Niratinib
Colon KRAS Absence of mutation Cetuximab, Panitumumab
Colon/NSCLC EGFR Overexpression/increased gene copy number Cetuximab, Panitumumab, Necitmumab
GIST Kit/PDGFRα Activating mutations Imatinib, Midostaurin (PKC412)
Melanoma KIT Activating mutations Imatinib
Melanoma/Colon/Lung BRAF Activating mutations Vemurafenib/cobimetinib, Dabrafinib/trametinib
NSCLC ALK Gene rearrangement (multiple fusion Crizotinib, Ceritinib, Alectinib, Brigatinib
partners)
NSCLC EGFR Activating Mutation (small deletion/point Erlotinib, Gefitinib, Afatinib, Osimertinib
mutation)
NSCLC EGFR T790M Resistance mutation Osimertinib
NSCLC ROS1 Gene rearrangement (multiple fusion Crizotinib
partners)
NSCLC BRAFV600E Activating muation Dabrafinib + Trametinib
Soft tissue sarcoma PDGFR alpha Expression/overexpression Olartumab
Prostate Androgen receptor Expression/overexpression Abiraterone Acetate. Bicalutamide, Cabazitaxel,
Casodex, Degarelix, Enzalutamide, Flutamide.
Ovary BRCA Mutation, PARP inhibition Venatoclax

GIST, Gastrointestinal stromal tumor; NSCLC, non-small cell lung carcinoma.

Table 15.4  FDA Approvals of Targeted Agents for Hematologic Malignancies

Tumor(s) Gene(s) or Protein Molecular Lesion(s) Drugs
ALL B cell CD19, CD3 Expression/overexpression Blinatumomab (bivalent)
ALL B cell CD19-directed genetically Expression/overexpression Tisagenlecleucel
modified autologous T cell
ALL B cell CD22 Expression/overexpression Inotuzumab zogamicin
AML CD33 Expression/overexpression Gemtuzumab ozogamicin
AML FLT3 Activating mutations Midostaurin/lestaurinib
AML IDH2 Activating mutations Enasidenib
B cell lymphoma CD20 antigen Expression/overexpression Rituximab, Tositumomab, Ofatumumab,
Ibritumomab-tiuxetan (radiolabeled)
CLL CDK4/6 Expression/overexpression Idelalisib
CLL/SLL Bruton tyrosine kinase Expression/overexpression Ibrutinib
CLL/SLL CD 52 antigen Expression/overexpression Alemtuzumab
CLL/SLL CD20 antigen Expression/overexpression Obinutuzumab, ofatumumab
CML BCR/ABL Activating mutations Imatinib, Dasatinib, Nilotinib, Bosutinib, Ponatinib
CML, Ph+ ALL c-Kit/PDGFRα Activating mutations Imatinib, Midostaurin, lestaurinib
Follicular lymphoma, relapsed PIK3 Expression/overexpression Copanlisib
Lymphoma CD30 antigen Expression/overexpression Brentuximab-vedotin (antibody-toxin conjugate)
Mantel cell lymphoma Bruton tyrosine kinase Expression/overexpression Ibrutinib
Multiple myeloma CD38 antigen Expression/overexpression Daratumumab
Myelodysplastic syndrome c-Kit/PDGFRα Activating mutations Imatinib
Myelodysplastic syndrome JAK2 Expression/overexpression Ruxolitinib
Peripheral T cell/HTLV Lymphoma CD25 antigen Expression/overexpression Denileukin-difitox (antibody-toxin conjugate)

ALL, Acute lymphoblastic leukemia; AML, acute myeloid leukemia; CLL, chronic lymphocytic leukemia; CML, chronic myeloid leukemia; SLL, small lymphocytic lymphoma.
238 Part I: Science and Clinical Oncology
quantitative211 and adaptable for multiplex marker testing to meet
specific clinical needs.210,212,213 These platforms require a stepwise
approach to tissue processing, nucleotide extraction, and library
preparation for successful biomarker testing; this approach is depicted
in Fig. 15.7.
Platforms
Currently two platforms dominate the field of clinical NGS testing,
Ion Torrent (Thermo Fisher Scientific, Waltham, MA) and Illumina
NGS (Ilumina, San Diego, CA).210 Both methods represent the
so-called “sequence by synthesis” approach to sequencing, and both
require isolation, separation, and amplification of single DNA strands,
creating a vetted library of primary sequences (see later). However,
the platforms differ in their methods of accomplishing these steps.
With Ion Torrent (Fig. 15.8), copies of a single nucleotide template
are linked through an adapter sequence to a single bead. Beads are then
placed in wells engineered into a flow cell, and individual nucleotides
are added one base at a time. Each addition of base into a forming
strand releases a hydrogen ion, resulting a pH change in the well that
is measured in each well, thus registering each base incorporated into
the elongating sequence. The numbers of beads containing identical
strands provides a measure of the frequency of the sequence in the overall
template mixture.
With the Ilumina platform (Fig. 15.9), amplified nucleotide libraries
are created from a sample template using defined probes. Creation of
libraries is a crucial step in NGS that imparts coverage and sensitivity
to testing procedures. Customized reagents214,215 or commercial kits
can be used in this procedure. Regardless of source, libraries must be
continuously monitored for consistency, sensitivity, and specificity.
During Illumina library preparation, end adapters are incorporated
into the DNA template and are used to attach the template to insoluble
matrix in a “flow cell” at a calculated density. The attached single
strand is then amplified in a step referred to as bridge amplification,
in which strands with adapters at both ends can be bent to bond
with matrix at both ends. Replication can proceed from either end of
the strand, thus providing two copies of each strand for each round
of PCR. This process results in the formation of clusters of forward
and reverse strands that can be visualized by incorporating fluorescent
nucleotides. The fluorescent clusters are precisely mapped in the flow cell
and imaged after addition of each nucleotide. Consecutive images are
used to establish the sequence at each cluster location on the flow cell.
Strand length can reach up to 300 bases. Sequences can be assembled
computationally to cover any length required from multiplexed genes
to whole exons to whole genomes.
Informatics pipeline
A series of computational steps is performed to assess the large quantity
of raw data generated by NGS that are collectively known as the
informatics pipeline. Each NGS platform generates base calls from
proprietary algorithms that emerge in the form of tab delimited text
files.216 Ilumina systems typically provide FASTQ files that contain
sequence information in which each base is symbolized by a single
letter and that include a quantitative quality score for each base
(Phred-like or Q score). These files are fed into sequence alignment
software, which, by comparing test sequences with reference sequences,
can detect single nucleotide variants, short indels, large structural
alterations, copy number variations, and gene fusions. These data can
be visualized with several available tools, such as the Integrative
Genomics Viewer217 and SAMTools.218
Quality assurance
NGS testing is a multistep process as diagramed in Fig. 15.7, and
quality must be maintained at each step. The smallest proportion of
tumor cells in a cell mixture (analytic sensitivity) that can be confidently
detected with a molecular test must be determined by each testing
laboratory. Successful testing can be performed with use of as few as
50 intact tumor cells in formalin-fixed material.
Library construction is a crucial step in testing. At this stage, the
number of genes and specific mutations must be specified, ranging
from a few genes to whole exomes. Two techniques are employed in
library creation: PCR amplification and hybrid capture or commonly
a combination of the two. The size of the library determines how
many tests can be performed in a single or multiple flow cells and
thus determines the cost of the test.
Platform-specific sequencing protocols must be thoroughly vetted
by the testing site. Analytic validation or validation of analytic methods
refers to the assessment of sensitivity, specificity, accuracy, precision,
repeatability, reproducibility, detection and quantification limits,
linearity, range, and robustness for each analyte. This can be a major
challenge, and synthetic nucleotides have been introduced to address
this problem. An additional approach to analytic validation consists
of specimen sharing and estimation of concordance among different
institutions.
Genetic alterations detected
Two types of mutation can be detected with conventional NGS
platforms: small variants, including SNVs and small indels, and large
structural abnormalities, including copy number abnormalities and
gene fusions that identify translocations. A remarkable number of
clinically relevant consistent driver mutations have been described in
solid tumors during the past two decades, as shown in Table 15.3.
Several of these specific mutations are described later.
Interpretation and significance of next-generation
sequencing results
Final interpretation of the data must rest on the judgment of experienced
clinical laboratory staff. To accomplish this, filtered NGS data must
be accessible through an online database containing flags for quality
assurance data and for the presence of clinically significant and
insignificant variants. Reporting personnel should assess all of the
available data presented to review and render an opinion on the likely
clinical significance genomics findings. Reporting and interpretation
of NGS data have undergone scrutiny by representatives of several
involved organizations, and consensus guidelines and recommendations
have been published.219 These guidelines recognize the wide range of
biologic effects of mutations that occur in solid tumors, and participat-
ing experts have proposed a four-tiered system to classify mutations,
as shown in Table 15.5.
Table 15.5  Evidence-Based Variant Categorization
Tier Evidence
1. Variants of strong FDA-approved therapy; included in
clinical significance professional guidelines
Well-powered studies with consensus from
experts in the field
2. Variants of FDA-approved for different tumor type or
potential clinical consensus in multiple small published studies
significance Preclinical trials or few case reports without
consensus
3. Variants of Not observed at a significant allele frequency
unknown clinical in the general or specific subpopulation
significance databases, or pan-cancer or tumor-specific
variant databases
No convincing published evidence of cancer
association
4. Benign or likely Observed at significant allele frequency in
benign variants the general or specific subpopulation
databases
No existing published evidence of cancer
association
 Pathology, Biomarkers, and Molecular Diagnostics  •  CHAPTER 15 239

Adapters dNTPs

polymerase

DNA

An emulsion of water and oil is created. The water

The DNA library is produced by ligating adapters to droplets contain beads with covalently-bound
segments of DNA generated by fragmentation or PCR. oligonucleotides, dNTPs, polymerase, primers, and the
DNA library. The oligonucleotides capture the DNA,
and PCR extends the complimentary strand.

The bead-bound DNA is applied to a semiconductor chip

that has thousands of microscopic wells. The beads settle
When cycling is complete, thousands of identical DNA into the wells.
molecules are bound to the bead.

dNTPs
primers

Primers and polymerase are added, then nucleotides are An ion-sensitive layer underneath each well records the
washed over the wells one at a time, in several-second change in Ph associated with hydrogen release and
intervals. Each time a nucleotide is incorporated, records the corresponding nucleotide sequence.
hydrogen is released.

Figure 15.8  •  Diagrammatic representation of Ion Torrent testing platform. dNTP, Deoxynucleotide triphosphate; PCR, polymerase chain reaction.
240 Part I: Science and Clinical Oncology

Adapters

Adapters

DNA

The DNA library is produced by ligating adapters to The DNA is applied to the flow cell and captured by
segments of DNA generated by fragmentation or PCR. adapters that are covalently bound to its surface. A copy
of the original strand is created by PCR.

The original strand is washed away, leaving the copy,

which is covalently bound to the flow cell. The bound
DNA then forms a bridge with a nearby adapter, and the
complementary strand is generated. This process, called
bridge amplification, is repeated to generate thousands of
copies of the original template.
Bridge amplification results in a cluster of DNA
comprising both forward and reverse strands. The
reverse strands are cleaved and washed away, leaving a
cluster of identical DNA strands ready to be sequenced.

ddNTPs

Primers

The sequencing chemistry uses fluorescent ddNTPs,

This process can image thousands of clusters
which cannot be extended. During each cycle, a single
simultaneously and is commonly referred to as
fluorescent ddNTP binds to each strand in the cluster,
massively parallel sequencing
and the flow cell is imaged.

Figure 15.9  •  Diagrammatic representation of Illumina Next Generation Sequencing platform. ddNTP, Dideoxynucleotide triphosphate; PCR, polymerase
chain reaction.
 Pathology, Biomarkers, and Molecular Diagnostics  •  CHAPTER 15 241

Assays for Large-Scale Gene and
Chromosome Rearrangements
Large-scale genomic rearrangements are not easily detected with
conventional gene sequencing, and specialized methods have been
used for decades to demonstrate innumerable complex abnormalities
for which nomenclature is now standardized220 and uniformly applied
in searchable databases (http://cgap.nci.nih.gov/Chromosomes/
Mitelman). Both FISH and molecular methods are validated for
application to clinical samples to identify complex rearrangements.
Each method has its advantages. In FISH, histologic architecture is
maintained and the technique can be accurately applied to small
samples. It is also widely available in clinical laboratories. The large
number of FISH tests currently in active use cannot be fully addressed
here owing to space limitations. However, lung cancer provides an
example of the relevance of discovery and clinical testing for large-scale
gene rearrangements to targeted therapy, as discussed later.
Fluorescence in situ hybridization assays for
chromosome rearrangement
Copy number changes and chromosomal rearrangement are ubiquitous
in lung carcinoma, but to date only a small proportion of those
described are recurrent. An example of a now well-established recurrent
rearrangement is ALK gene inversion and fusion. In this rearrange-
ment, ALK is the oncogenic driver switched on by the rearrangement.
The EML4-ALK fusion is generated by a paracentric chromosomal
inversion in the short arm of chromosome 2, between bands p21
and p23.2 (Fig. 15.10). These two genes originally are separated by
approximately 12.8 MB. The other described ALK fusions in lung
cancer, TFG-ALK,221 KIF5B-ALK,222 KLC1-ALK,223 and PTPN3-ALK,224
are caused by chromosomal translocations t(2;10)(p23.2; p11.22),
t(2;3)(p23.2;q12.1), t(2;9)(p23.2;q31.3), and t(2;14)(p23.2;q32.1),
respectively.
Currently, FISH is the most commonly used assay for ALK
rearrangements in lung cancer. The standard FISH assay uses a
dual-color break-apart (BA) probe set encompassing the 3′ and the
5′ sequences immediately adjacent to the breakpoint area in ALK
(intron 19), labeled in orange and in green fluorophores, respec-
tively.225 Chromogenic in situ hybridization (CISH) BA assay for
ALK rearrangements is also commercially available. Despite interesting
results and a high level of concordance with FISH,226,227 the ALK
CISH assay has not yet been comprehensively explored. The ALK
BA FISH probe was used in the initial clinical studies with crizo-
tinib228,229 and is approved by the US Food and Drug Administration
(FDA) as a companion diagnostic test. The ALK BA FISH assay is
DNA based and is thus robust and resistant to technical artifacts.
It has multiple other advantages, such as being highly suitable for
FFPE tissue sections, and detects all variants of EML4-ALK as well
as any of the reported or uncharacterized partners. On the other
hand, the FISH platform requires specialized resources (fluorescence
microscope, light-protected laboratory space) and highly trained
personnel.
Two other receptor kinase genes have been identified as acti-
vated in lung cancers by fusions, ROS1221 and RET.230 Integrated

ALK
Normal 2

3 5 5 3
ALK EML4

3 5 5 3
EML4 ALK
ALK EML4

Inv(2)(p21p23.2)
A EML4:ALK

B C D
Figure 15.10  •  The EML-ALK fusion is generated by a paracentric inversion in the short arm of chromosome 2. The break-apart fluorescence in situ
hybridization (FISH) probe includes DNA sequences centromeric to the breakpoint (5′ ALK) labeled in SpectrumGreen and sequences telomeric to the
breakpoint (3′ ALK) labeled in SpectrumOrange (A). Normal cells carry only fused 3′ ALK–5′ ALK signals, indicated by yellow arrows (B). Cells with rear-
rangement in the ALK gene exhibit split 3′ (orange) and 5′ (green) signals (C) or additional copies of single 3′ (orange) signals (D). Red arrows indicate 3′
ALK signals; green arrows indicate 5′ ALK signals.
242 Part I: Science and Clinical Oncology
molecular and histopathology-based screening systems have been
successful in detecting a large number of fusion partners in lung
cancer for these two genes, and preclinical studies have confirmed
their tumorigenic potential.222 Clinical trials of targeted agents for
ROS1 are currently underway, and rapid expansion of this field is
expected.
Molecular methods for genomic rearrangement
The past few years have also witnessed rapid advances in molecular
methods for the assessment of large-scale gene rearrangements.
Molecular methods can be readily standardized, automated and
multiplexed, which is now necessary due to the large number of
known actionable gene fusions.
In the majority of cases of pathogenic fusion genes, the genomic
breakpoints occur in intronic regions. Therefore DNA-based detection
of gene rearrangements by molecular methods requires analysis of
introns. Because introns are, in general, far greater in size than exons
(thus requiring far more sequencing), NGS-based assays are typically
used in this setting. This can be achieved either through whole-genome
sequencing231 or by targeted sequencing of selected introns through
hybrid-capture target enrichment (often termed intron “tiling”),232,233
with the targeted approach being much more feasible in the clinical
setting. The detection of rearrangements via DNA-based NGS requires
bioinformatics approaches beyond those typically applied to mutation
calling. In general, these can be divided into two distinct categories. In
one, paired sequencing of DNA molecules of known size is performed.
If the two paired ends map to the reference genome at locations
unexpected based on the size of the input DNA, then it can be deduced
that a rearrangement occurred within that DNA fragment.234,235 In
the other, mapping to the reference genome at a conserved genomic
location of a DNA sequence (termed “soft-clipping”) is used to guide
rearrangement detection.236
To avoid these two complications, many fusion detection assays
query the mRNA (or cDNA derived from the mRNA) instead.
Accordingly, the amount of nucleic acid to be sequenced is far less,
and difficult-to-map repetitive sequences are avoided. The traditional
mRNA-based approach is reverse transcriptase polymerase chain reaction
(RT-PCR), in which primers to both genes involved in the fusion are
used. This technique has been used successfully to detect gene fusions
in clinical samples.237 However, the ability to multiplex traditional
RT-PCR assays is limited, making it a less than ideal approach to query
for multiple gene fusions simultaneously. Furthermore, RT-PCR requires
knowledge of the fusion partner (to design primers for the assay), and
for some clinically actionable gene fusions, dozens of known fusion
partners have been described. Therefore fusion events not in the original
design will not be detected, making this method very biased. NGS-based
assays using mRNA or cDNA input are a less biased approach in this
regard and can be performed on the whole transcriptome238 or in a
targeted fashion by using hybrid capture techniques.239 Amplicon-based
target enrichment for NGS can also be applied. However, similar to
RT-PCR, a priori knowledge of the fusion partner is required.240,241
A clever amplicon-based approach for NGS that does not require
knowledge of the fusion partner is the anchored multiplex PCR
technique.242 In this assay, an adapter sequence is ligated to the end
of cDNA fragments, and this adapter serves as a primer binding site,
thus negating the necessity of having a primer specific to the fusion
partner. Anchored multiplex PCR has been successfully applied to
clinical samples to detect gene fusions in a fusion partner–agnostic
fashion.243
An alternative mRNA-based approach for fusion gene detection
relies on quantification of expression of defined regions of a gene
(generally at least one region on either side of an expected breakpoint).
This quantification can be achieved by several different methods. In
theory, if little to no wild-type expression of a gene is expected in the
tissue type of interest, the region of the gene involved in the fusion
(i.e., 5′ or 3′) should be expressed at a far greater level than the rest
of the gene. Thus when measuring expression levels across the gene,
large discrepancies between 5′ and 3′ regions can indicate the presence
of a fusion.244
Clinical Testing for Predictive Biomarkers by
Immunohistochemistry
Protein biomarkers may be detected in situ by simple and inexpensive
immunohistochemistry (IHC). Cellular morphologic features are
retained in immunohistochemical methods, which adds to specificity
and sensitivity over protein measurement in fluids or homogenates
of disrupted cells. In all immunologic detection methods, binding
characteristics of the primary antibody used are crucially important,
and local familiarity with the idiosyncrasies of each antibody is essential
in assessment of results. Examples of immunohistochemical tests that
are used for clinical decision making are described in the following
sections.
Abnormal Expression of Target Genes
ER, PR, and HER2 in breast cancer
Immunohistochemical staining for estrogen receptors (ERs) and
progesterone receptors (PRs) was one of the earliest applications to
find a place in patient management. Until the early 1980s, antibod-
ies to these proteins were not widely available. However, with the
development of monoclonal antibody technology, antibodies that could
identify hormone receptors were created245,246 and found to reliably label
receptors even in FFPE sections of breast carcinomas.247,248 Unexpected
results of this testing were that these receptors are typically found in
the cell nucleus and that the intratumoral and intertumoral range
of expression is large. Because of this, semiquantitative scoring that
takes into account intensity of staining and percentage of cells stained
has been used to establish expression levels that define positive and
negative results.249,250 These scoring systems continue to prove their
prognostic value.251
A second immunohistochemical test that has been successfully
applied to the management of breast carcinoma is the test for HER2
(HER2/neu, ErbB2) expression. HER2 is a TKR and a member of
the epidermal growth factor receptor (EGFR) family of signaling
molecules. Its role in human carcinogenesis was initially discovered
through DNA screening, where it was found to be amplified in 30%
of the tested breast cancers.252 RNA and protein were shown to be
overexpressed in tumors that are amplified as well as in a subset of
nonamplified tumors.253 HER2 testing became crucial when it was
found that HER2 protein overexpression was a determining factor
for in vitro and xenograft responsiveness to anti-HER2 monoclonal
antibody treatment.254,255 In early clinical trials, anti-HER2 IHC was
used as the method for determination of HER2 status and determined
eligibility for treatment with an anti-HER2 antibody drug, Herceptin.256
A standardized approach to HER2 immunohistochemical testing was
established several years later when expert panels agreed on criteria
for determining HER2 status (Fig. 15.11).257
Tests for multiple immunohistochemical markers have been com-
bined into panels that include ER, PR, HER2, and the proliferation
marker Ki-67,258 in addition to several other markers thought to
be of prognostic importance.259 These panels have been found to
provide prognostic information that is comparable to that of multiplex
mRNA expression assays discussed later and have the added advan-
tage of accessibility in local laboratories and ready correlation with
histologic features.
A substantial subset (~15%) of breast tumors do not express ER,
PR, or HER2 and are now referred to as triple-negative tumors.260
These lesions are notable for their aggressive histologic characteristics
and poor long-term outcome. This category overlaps but is not identical
to the basaloid-like breast cancer category defined by microarray-based
expression profiling261 and includes nonhereditary tumors that frequently
express perturbations of BRCA1 pathway gene expression. The triple-
negative category thus has increased the importance of accuracy
in both positive and negative immunohistochemical testing and
 Pathology, Biomarkers, and Molecular Diagnostics  •  CHAPTER 15 243
HER2 Immunohistochemical Scoring
1+
Figure 15.11  •  Scoring of Her2/neu immunohistochemical predictive marker in breast carcinoma is illustrated in this figure as follows: 1+, faint partial
membrane labeling (brown staining) of more than 10% of tumor cells; 2+, weak to moderate complete membrane labeling of more than 10% of tumor cells;
3+, strong complete membrane labeling of more than 10% of tumor cells.
reemphasizes the need for local validation and controls for accuracy
of immunohistochemical procedures.
CDX2 expression in stage II colon carcinoma
The transcription factor CDX2 is a master regulator of intestinal
development that is expressed in the cell nuclei of intestinal epithelium.
A gene expression study has identified this gene as a prognostic marker
in early-stage (II) colon adenocarcinoma.262 Widely used and well-
described immunohistochemical assays can classify colon tumors as
CDX2 negative or positive. Disease-free survival is worse for CDX2-
negative than for CDX2-positive stage II colon tumors (49% versus
87%), and 5-year disease-free survival is better in patients with
CDX2-negative tumors treated with adjuvant chemotherapy than in
those with untreated tumors (91% versus 56%).262 CDX2 status has
therefore been suggested as an indication for chemotherapeutic treat-
ment in stage II colon tumors.
EGFR expression in lung carcinoma
Finally, EGFR is expressed at high levels in most non–small cell lung
carcinomas (NSCLCs), including both adenocarcinoma and squamous
carcinoma, and expression correlates with gene amplification and copy
number. When a scoring system was applied in a phase III trial testing
the effectiveness of the anti-EGFR chimeric (mouse/human) monoclonal
antibody, cetuximab, in advanced NSCLC, only patients with the
highest levels of EGFR expression (scores >200) experienced a survival
benefit.263 This result suggests that EGFR expression levels determined
with IHC may be a reasonable selection criterion for anti-EGFR
antibody treatment. Properly scored EGFR immunohistochemical
expression may prove to be a valuable predictor of response to anti-
EGFR monoclonal antibody treatment.
Aberrant Expression of Altered Genes
ALK
The availability of monoclonal antibodies to ALK fusion protein has
resulted in the development of highly sensitive and specific immu-
nohistochemical assays for ALK rearrangement, one of which (ALK
[D5F3] CDx Assay; Ventana Medical Systems, Oro Valley, AZ) is now
FDA approved as a stand-alone diagnostic for ALK rearrangement and
selection for anti-ALK therapy. Numerous studies have documented
near 100% sensitivity and specificity of ALK immunohistochemical
tests (reviewed by Lantuejoul and colleagues264). Details of testing
procedures and interpretation of test results have been discussed in
an International Association for the Study of Lung Cancer (IASLC)
monograph.265 Because of the economies and wide availability of
immunohistochemical testing, ALK IHC is now a widely adopted
2+ 3+
screening test for ALK rearrangement in lung cancer. Positive
results should be confirmed by additional testing, including FISH or
molecular testing.
ROS1 and other markers
Preliminary data indicate that immunohistochemical testing for other
markers including ROS1 can possibly substitute for FISH testing. In
a study266 in which lung tumors were tested with both FISH and
IHC, IHC was 100% sensitive and 92% specific, suggesting that this
infrequent rearrangement may be detected as efficiently with IHC as
by FISH. It seems likely that additional genetic lesions will be detectable
by aberrant expression, and this will be a convenient method to screen
for unusual lesions in the future.
Markers of Immune Response
Immunologic responses to tumor have long been observed and quanti-
fied in experimental systems,267 but immunotherapy as a viable clinical
option has only recently emerged with the demonstrated effectiveness
of anti-CTLA4268 and anti-PD-1 therapy.269–273 A large and rapidly
increasing number of immunologic agents are currently approved for
cancer treatment (Table 15.6).
CTLA4 and PD1 are immunologic checkpoints that inhibit the
host immune response. Suppression of inhibitory checkpoints by
monoclonal antibodies to CTLA4 and PD-1 or its ligand PD-L1
promote cytotoxic T-cell responses to antigens expressed by tumor
cells.274 These may consist of nonmutated antigens for which tolerance
is incomplete. More commonly, they are neoantigens that are the
result of tumor cell mutations encoding novel epitopes that can elicit
a T-cell response.275
Because responses to anti-CTLA4 have been weaker and less durable
than responses to anti-PD-1 or anti-PD-L1 and because less than half
of tumors typically respond to anticheckpoint treatment, efforts to
develop biomarkers that predict response have mainly centered on
PD-1 and PD-L1. The markers with greatest clinical potential are
mutational burden, thought to reflect the level of expression of tumor-
associated neoantigens; PD-L1 protein expression by tumor cells; and
quantification of tumor-infiltrating lymphocytes (TILs). Following is
a brief discussion of each of these markers as they relate to clinical
laboratory practice.
Mutational burden
Deep sequencing of solid tumors has shown sharp differences in the
number of mutations occurring in specific tumors with histologic
characteristics and consequently variation in the number of predicted
neoantigens harbored by tumors of various histologic types.275 This
244 Part I: Science and Clinical Oncology

IHC is perhaps the most direct measure of expression but to date has
Table 15.6  FDA Approved Immunotherapeutic been a problematic predictor of response to treatment. Although there
Agents is a general association between immunohistochemical expression of
PD-L1 and response to anti-PD-1/PD-L1 antibody treatment,283–285
Molecular
patients with IHC-negative tumors may respond to treatment, and
Tumor(s) Target Drugs
published data indicate that the negative predictive value of IHC
Melanoma, NSCLC, SCCHN, PD1 Nivolumab for PD-L1 may be as low as 58% for nivolumab in melanoma.283
renal, Hodgkin Moreover, standardization and validation of immunohistochemical
Melanoma, NSCLC, SCCHN, PD1 Pembrolizumab tests has been difficult so that different antibodies with similar
urothelial tumors, reactivities286,287 but different cut points are used with each check-
microsatellite instability-high point inhibitor.288 Finally, the focal distribution of the PD-1 and
(MSI-H) or mismatch repair PD-L1 protein within tumors renders small biopsy and cytologic
deficient (dMMR) specimens of limited use for these immunohistochemical tests. These
NSCLC, urothelial tumors PD-L1 Atezolizumab problems have prevented the widespread adoption of immunohisto-
NSCLC PD-L1 Durvalumab chemical testing for prediction of response to checkpoint inhibition
Urothelial tumors PD-L1 Durvalumab and currently limit the use of these tests to research and clinical
trial settings.
Merkel tumor, urothelial PD-L1 Avelumab
tumors
Tumor-associated lymphocytes
Melanoma CTLA4 Ipilumumab Solid tumors are frequently associated with lymphocytic infiltrates
Melanoma CTLA4 Tremilumumab (TILs); it has been suggested that the level of TILs or TIL subsets
Melanoma CTLA4+ PD1 Ipilumumab+Nivolumab may correlate with outcome.289–291 In addition, attempts have been
made to relate response to immune checkpoint inhibition to response
NSCLC, Non–small cell lung cancer; SCCHN, squamous cell carcinoma of the head to treatment (reviewed by Gibney and colleagues283). At the present
and neck. time, however, no validated studies have produced reproducible cut
points that predict survival or response to immunologic or more
conventional treatment. Evaluation of TILs in solid tumors currently
observation has led to the hypothesis that antitumor responses could remains suitable only for research use.
be strongest in tumors with the greatest number of neoantigens.
Whole-exome sequencing has been found feasible in clinical tumors Examples of Tumor- and Organ-Specific
including lung cancer. In two high-mutation tumors, melanoma and Mutational Profiles
lung cancer, whole-exome sequencing has shown associations between
mutation burden of individual tumors and response and longer survival Although there is considerable overlap in mutational profiles among
with anti-CTLA4276 and anti-PD-1,277 respectively. These findings are tumors of variable tumor type, a consistency of mutations occurs
consistent with the suggestion that neoantigen formation may be a within individual tumor types. A summary of mutational profiles by
crucial determinant of antitumor response. Differences in outcomes, tumor type is provided here.
however, were not sufficient to regard mutational burden as a predictive
marker by itself. At the present time, mutational burden must be Gastrointestinal Stromal Tumors
considered developmental, and continued refinement of mutational One of the earliest successes of the mutation-targeted approach to
cut points will be required before whole-exome sequencing can be cancer treatment was the discovery of mutations in the KIT gene in
considered an actionable predictive test validated for clinical practice. gastrointestinal stromal tumor (GIST). GISTs were until recently a
rare and ill-defined neoplasm of somewhat mysterious origin. In the
Mismatch repair deficiency and microsatellite instability early 1990s these tumors were defined as spindle cell malignancies
A possible source of neoantigen formation is MMR deficiency. As that exhibited no evidence of cell of origin.292 The status of these
described earlier, loss of MMR capacity results in high frequency of tumors began to change as it was recognized in the mid-1990s that
mutation in affected tumors.278–280 The proposed explanation is that GISTs express CD34 but not desmin293 and thus displayed a distinct
mutation may lead to expression of altered protein, possibly stimulating immunophenotype. A watershed discovery was reported by Hirota and
an immune response to the neoantigen. MMR deficiency is reflected colleagues in 1998.294 These investigators described nearly uniform
in a high number of deletions and insertions in tandem repeat sequences, expression of KIT (CD117) in GISTs and suggested that they may arise
a finding referred to as microsatellite instability.72,73 As described earlier, from the immunophenotypically similar paraganglionic interstitial cells
MSI is measured by testing of five standard microsatellite markers. of Cajal. Most important, they identified deletions and point mutations
MMR as reflected by MSI is associated with a high objective response in the juxtamembrane portion of the KIT TKR gene. These mutations
rate (40%) and progression-free survival (PFS; 78%) in patients with were shown to activate KIT signaling, and KIT was thus a potential
colon cancer treated with PD-L1 inhibitor.281 Additional tumor types therapeutic target. KIT is a member of the TKR signaling family and
including carcinomas of endometrium, stomach, liver, ampulla of is encoded at chromosome 4q11-q12. In GISTs, mutations in the KIT
Vater, thyroid, skin (sebaceous tumors and melanoma), ovary, cervix, gene (reviewed by Corless and colleagues295; Lasota and Miettinen296;
esophagus, soft tissue, head and neck, kidney, and bone (Ewing sarcoma) and Martin-Broto and colleagues297) are located predominantly in
also have significant frequency of MMR deficiency and thus are sensitive the juxtamembrane domain (exon 11), with two-thirds of tumors
to PD-1 and PD-L1 therapy.282 MSI testing is an appropriate surrogate harboring mutations in this region of the gene. Exon 11 mutations
for neoantigen expression for a wide variety of tumor types being allow adenosine triphosphate (ATP) to phosphorylate receptor in
considered for PD-1 and PD-L1 therapy until and if direct assays for the absence of ligand (stem cell factor [SCF]). Phosphorylation of
tumor neoantigens are developed. the receptor triggers downstream signaling of RAF, MEK, MAPK,
PIK3CA, and STAT3, transforming mutated cells to malignant status.
PD-1 and PD-l1 expression detected Approximately 10% of mutations are found in exon 9, which encodes
with immunohistochemistry the extracellular domain of the molecule. These mutations induce a
Expression of PD-L1 would seem to be a reasonable biomarker for change in receptor conformation mimicking that seen in the presence
predicting response to inhibition of the PD-1/PD-L1 checkpoint. of ligand and resulting in receptor phosphorylation. Exon 9 mutations
 Pathology, Biomarkers, and Molecular Diagnostics  •  CHAPTER 15 245
are commonly seen in large and small intestinal tumors but rarely
in gastric neoplasms. Finally, mutations in exons 13 (tyrosine kinase
domain) and 17 (activation loop) are occasionally found in GISTs.
The effects of exon 13 and 17 mutations on the receptor are less well
established, but the net result of all these mutations is receptor activation
and oncogenic transformation of mutant cells. KIT mutations occur
in approximately three-fourths of GISTs.295–297
Tumors that do not contain a KIT mutation may contain a mutation
in the closely homologous TKR gene platelet-derived growth factor
receptor alpha polypeptide (PDGFRA).298,299 PDGFR protein has two
isoforms, α and β, and it is the α form that is mutated. Approximately
10% of GISTs contain mutations in PDGFRA.295–297 The mutated
loci in PDGFRA are distributed similarly to those in KIT, but frequencies
differ. The most frequent mutation site (exon 18) encodes a portion
of the tyrosine kinase domain (TK2) and occurs in 6% of all GISTs.
Juxtamembrane mutations in PDGFRA are relatively infrequent,
occurring in less than 1% of GISTs.
Introduction of TKIs that block signaling by mutant receptor has
had a profound effect on the outlook for this tumor. Before 2000,
the outlook for patients with GIST was bleak. In a majority of patients,
disease recurred after surgical resection and the responses to chemo-
therapy were poor. Only 10% of patients were disease free after extended
follow-up.300,301 There was a dramatic reversal of this situation when
it was realized that GIST is exquisitely sensitive to tyrosine kinase
blockade. The drug successfully used to treat GIST was originally
designed to block signaling by the BCR-ABL fusion protein in chronic
myelogenous leukemia. Structural similarities between the intended
target of the drug and KIT led to the discovery that the TKI now
known as imatinib strongly inhibits mutated KIT.302 Subsequent clinical
trials demonstrated high response rates and prolonged PFS,303–305
leading to FDA approval of the drug as discussed elsewhere in this
book. KIT testing is now routinely employed to identify individuals
likely to respond to the drug.
Mutations in PDGFRA are not equivalent to those in KIT. More
than half of mutations in PDGFRA encode a D842V amino acid
substitution that is resistant to imatinib, whereas other mutations in
PDGFRA are sensitive.306 Alternative drugs that show activity against
the D842V resistance mutation, such as midostaurin (PKC412),307
are under investigation.
Colorectal Carcinoma
Molecular markers now guide treatment for CRC, and comprehen-
sive guidelines have been published providing recommendations
on molecular tests that are appropriate for this tumor type.308
The following discussion is intended to provide perspective on
molecular changes that may be important in the management
of CRC.
KRAS as a prognostic marker in colorectal carcinoma
KRAS is a member of a superfamily of guanosine-5-triphosphatase
(GTPase) proteins that also includes NRAS and HRAS and a number
of less well-known proteins. The role of these proteins is to transduce
Table 15.7  KRAS Mutation as a Predictive Biomarker in Clinical Trials
Investigator Treatment
Amado Panitumumab vs best supportive care (third line)
Karapetis Cetuximab versus best supportive care (third line)
Van Cutsem (CYRSTAL) FOLFIRI ± cetuximab (first line)
Bokemeyer (OPUS) FOLFOX ± cetuximab (first line)
a
Progression-free survival.
stimuli from cell surface growth factor receptors. On activation, RAS
undergoes prenylation (addition of a 15-carbon chain) of a CAAX
(C, cysteine; A, aliphatic amino acid; X, serine or methionine) motif
by a farnesyl transferase. This makes RAS more hydrophobic and
adherent to the inner aspect of the cytoplasmic membrane, where it
activates transducers in an intracellular signaling cascade involving
numerous effector molecules including PI3K and MAPK.
Consistent mutations in several components of the RAS signaling
pathway occur in human solid tumors. Mutations in KRAS are par-
ticularly common. They permit stimulus-independent activation of
intracellular signaling. Hydrolysis of GTP bound to RAS must occur
for RAS to return to an inactive state. Typical mutations in codon
12, which normally encodes glycine, result in substitution with an
amino acid that sterically interferes with GTP hydrolysis. This perpetu-
ates independent intracellular signaling and permanently stimulates
proliferation and evasion of apoptosis. Because activation of the KRAS
gene is stimulus independent, targeting of the upstream TKRs has
proven to be ineffective in countering the proliferative and invasive
properties of tumors that are driven by mutation in KRAS.
Mutation in KRAS occurs in approximately 40% of colon carci-
nomas. The independent effect of mutation in KRAS on outcome in
colon cancer is still unclear and remains controversial.309 In a series
of 3439 patients with CRC, it was found that of the 12 possible
mutations on codons 12 and 13, only the glycine-to-valine mutation
on codon 12 (8.6%) had a statistically significant impact on outcome.310
Smaller series have shown similar results, but retrospective data from
other large randomized studies have failed to consistently demonstrate
a meaningful effect of mutation in KRAS on outcome in CRC. In a
study of a large number of untreated patients, there was no difference
in PFS (7.3 weeks) between mutant and nonmutant tumors.311
KRAS as a predictor of treatment response in colorectal carci-
noma.  In 2007 an article published in The New England Journal of
Medicine312 reported that administration of the monoclonal antibody
cetuximab to patients with advanced CRC expressing immunohis-
tochemically detectable EGFR had resulted in improved survival in
comparison to untreated controls. Both treatment and control arms
had been treated with a fluoropyrimidine, irinotecan, and oxaliplatin or
had contraindications to treatment with these drugs. The difference in
survival, although not large, suggested that this specific treatment could
be useful in managing CRC and prompted a closer look at responder
versus nonresponders. Several subsequent studies have indicated that
KRAS mutation status dictates response to anti-EGFR monoclonal
antibody treatment in CRC. In the randomized studies summarized in
Table 15.7,311,313–315 wild-type tumors treated with anti-EGFR antibody
(cetuximab or panitumumab) have a PFS advantage of several months
and a significantly reduced hazard ratio in comparison with mutated
tumors in first-line and third-line settings.
It has been suggested that not all mutations are equally predictive
of poor response to monoclonal anti-EGFR therapy. Patients with
codon 13 mutation (p.G13D) appear to respond to such treatment
with a significantly longer overall survival and a reduced hazard ratio
in comparison with patients with other KRAS mutations.316
KRAS
No. of KRAS Hazard Non-
Patients Mutationa Ratio mutateda HR Reference
427 7.4 wk 0.99 12.3 wk 0.45 311
572 1.8 mo 0.99 3.7 mo 0.40 313
540 7.6 mo 1.07 9.9 mo 0.68 314
233 5.5 mo 1.83 7.7 mo 0.57 315
246 Part I: Science and Clinical Oncology
BRAF mutation and prognosis
A second mutation in colon carcinoma of clinical importance is BRAF.
BRAF mutations were first identified in colon cancer through a survey
of BRAF mutations in all human tumors.317 The reported frequency
of BRAF mutation in clinical samples ranges from approximately
5% to 15%.318–321 BRAF mutation is more frequent in tumors on
the right side of the colon.322–324 Microsatellite instability (MSI-H)
occurs in approximately half of BRAF-mutated tumors.321,324,325
BRAF mutation is associated with aggressive tumor behavior and
shortened survival, but only in tumors that are MSI stable321,324,326,327;
mutation in MSI-H tumors carries no survival disadvantage. MSI in
BRAF-positive tumors is attributed to methylation of MMR rather
than mutation and appears to be unrelated to HNPCC and germline
mutation of MMR.328,329 BRAF mutation in colon tumors confers
resistance to anti-EGFR monoclonal antibody treatments,320,330–332 and
BRAF-mutated tumors respond poorly to BRAF inhibitors. Resistance
to BRAF inhibitors is thought to be due to activation of EGFR in
response to BRAF inhibition.333 It has been suggested that BRAF-
positive tumors may therefore respond to dual inhibition of both EGFR
and BRAF.
Other Mutations
Additional mutations of predictive importance are NRAS and PIK3CA,
which occur at frequencies of less than 5%318,320,334 and 10% to
15%,320,332,334 respectively, in colon carcinoma. PIK3CA mutations
overlap those found in other genes, but NRAS mutations are nonoverlap-
ping with mutations in other genes including KRAS and BRAF.
Mutations in either the PIK3CA or the NRAS gene are associated
with poor response to EGFR monoclonal antibody therapy.320,332 Finally,
initial results of comprehensive molecular analysis by TCGA have
been reported and identify large numbers of mutations that are likely
to become targets of specific molecular inhibitors.334 Comprehensive
molecular testing is expected to contribute greatly to colon carcinoma
management in the near future.
Lung Carcinoma—A Heterogeneous Tumor
Since the 1920s, lung tumors have been divided into small cell (“oat
cell”) lung carcinoma (SCLC) and non-small cell lung carcinoma
(NSCLC), which differ not only morphologically but by molecular
profile as well. SCLCs express a number of markers that are also found
on neural cells and, in current classifications, are categorized as one
of several different types of “neuroendocrine” carcinomas. SCLC is
uniformly aggressive and particularly sensitive to mitotic inhibitors.
Finally, this tumor type is highly genetically unstable and appears to
be driven predominantly by seven transmembrane receptors and has
not been successfully treated with targeted agents.
Most NSCLCs do not express neuroendocrine markers but instead
strongly express cell surface TKR. Further, NSCLC is divided into
(1) adenocarcinomas that are composed of cuboid or columnar cells
that often form acinus-like or papillary structures or grow along
pulmonary alveolar surfaces (as illustrated in the discussion of lung
carcinogenesis earlier), and (2) squamous tumors that form defining
microscopic structures such as keratin pearls and intercellular bridges.
These tumor types are also distinguished by variation in expression
of specific keratin subgroups and other lineage specific markers, most
notably TTF1, which is expressed by adenocarcinoma but not squamous
carcinoma.
The phenotypic heterogeneity of lung carcinoma is reflected in
the recent revision of the anatomical classification of lung carcinoma.335
Genomic changes are also highly heterogeneous. Lung cancer is one
of the most highly mutated of tumors198 containing large numbers
of SNVs, chromosomal arrangements, and copy number abnormalities.
Lung cancers exhibit a high degree of intratumoral heterogeneity and
frequently contain subclones of mutant cells207,208a that may undergo
selection in response to treatment or other microenvironmental changes.
Mutation and clonal selection continue during the life of the tumor
and may affect response to treatment and overall patient survival.208a
Mutational profiles of NSCLC separate according to histologic
type. TKR mutations (discussed later) are far more commonly found
in adenocarcinoma than in squamous carcinoma. Many of the mutations
in adenocarcinoma involve tyrosine kinase receptors and their signal
transducers. The first of these receptors to be defined as a therapeutic
target was epidermal growth factor receptor (EGFR).
Single-Nucleotide Variants, Small Deletions and Insertions
Epidermal growth factor receptor
The original isolation of epidermal growth factor (EGF) from mouse
salivary gland in 1962,336 the demonstration of its binding to cell
membrane receptor,337 and sequencing of the receptor338 established
the role of intercellular signaling in epithelial cell growth and main-
tenance. By the 1990s, overexpression of EGFR had been demonstrated
for squamous carcinoma. Monoclonal antibodies to EGFR that were
effective in localizing EGFR in situ became available in the late 1990s,
documenting the broad expression of EGFR in adenocarcinoma.339
EGFR expression in NSCLC was found to be closely linked to gene
copy number.340
Further studies of EGFR were driven by of the discovery of a
subset of patients who were mostly nonsmokers with adenocarcinoma
who responded dramatically to EGFR TKIs (Fig. 15.12). Simultane-
ously, two groups reported that mutations in the EGFR TK domain
were present in a subset of adenocarcinomas and that their presence
correlated with sensitivity to TK inhibition.341,342 The mutations most
commonly consisted of either single base substitution in exon 21
(c.2573G>C, p.L858R) or in-frame deletion within exon 19 ranging
from 9 to 18 base pairs in the region coding for the ATP-binding
pocket of the tyrosine kinase domain. These resulted in strong activation
of the mutant receptor.
Activating mutations of EGFR result in tumor susceptibility to
TKI therapy.343 However, responses to therapy are temporary, and
hopes of a long-lasting therapy are generally unrealized owing to
resistance to targeted therapy that inevitably arises in treated patients.
Two common mechanisms of resistance to EGFR TKI are MET
amplification344–346 and a point mutation in exon 20 of EGFR (c.2369
C>T, p.T790M),347 the effect of which is depicted in Fig. 15.13. As
a result of the point mutation in EGFR exon 20, a bulky methionine
side chain is thought to sterically hinder binding of TKIs. Tumors
cells harboring this mutation may exist in low numbers in untreated
tumors and emerge through selective pressure exerted by targeted
therapy. In addition, insertions in exon 20 of EGFR are associated
with primary resistance to TKI therapy.348 This also is thought to be
due to steric hindrance of TKIs. Finally, other mechanisms of resistance
including activation of alternative signaling pathways and epithelial-
mesenchymal transition or small cell transformation may also be causes
of resistance. The mechanism of resistance in individual cases can be
ascertained by349,350 cellular or liquid biopsy of recurrent tumor and
is discussed elsewhere in this book.
KRAS
KRAS mutations are more common in lung cancer than EGFR
mutations and, unlike EGFR mutations, occur predominantly in
smokers.351,352 Coexistence of KRAS and EGFR mutation is uncom-
mon but has been reported.353 Because of its position downstream
of EGFR signaling, the hypothesis that an activating mutation in
KRAS would render a tumor nonsusceptible to TKI therapy in
lung tumors has been extensively investigated, with conflicting
results.354
Large-Scale Rearrangements
Large-scale chromosomal rearrangements including translocations,
inversions, duplications, and amplifications are common in solid tumors
and are reflected in global methods for chromosomal imaging such
as chromosome painting or spectral imaging. These rearrangements
may create new fusion genes or activate oncogenes, becoming drivers
of cell proliferation and oncogenesis. Rearrangements that fuse gene
 Pathology, Biomarkers, and Molecular Diagnostics  •  CHAPTER 15 247

Positions of Mutations Detected in EGFR Tyrosine Kinase Domain in NSCLC~

EGF ligand binding Tyrosine kinase Autophosphorylation

TM K DFG Y Y Y Y

718 745 776 835 858 861 869 964

GXGXXG K R H DFG L L Y
Exon: 18 19 20 21 22 23 24

719 747-750 858

Paez:

Lynch:

Pao:

Tumor with point mutation (amino acid substitution)

Tumor with in-frame deletion

EGFR
747-750

G719

L858

Activation
loop

B
Figure 15.12  •  (A) Schematic view of epidermal growth factor receptor (EGFR) and its key domains including the extracellular ligand-binding domain,
the transmembrane domain, the tyrosine kinase domain, and the autophosphorylation domain. The tyrosine kinase domain (exons 18 through 24) is expanded,
showing the sites of described point mutations at G719, S752, R776, H835, L858, and L861, and in-frame deletions at L747-A750. (B) Three-dimensional
ribbon structure of the intracellular domain: the positions of the three most common mutations are shown in relation to the activation loop. EGF, Epidermal
growth factor; NSCLC, non–small cell lung carcinoma; TM, transmembrane.

promoters to the TK domain of growth factors, growth factor receptors,
or transcription factors may result in inappropriate signaling from
the fusion products.
ALK
Examples of activation by gene fusion in lung cancer involve ALK,
ROS1, and RET. In 2007, Soda and colleagues discovered a transcript
derived from a patient with NSCLC that could transform T3T
fibroblasts.355 Further investigation demonstrated the transcript to be
fusion of a gene called echinoderm microtubule-associated protein-like
4 (EML4) with the anaplastic lymphoma kinase (ALK) gene (see
Fig. 15.10), which is also rearranged in large cell lymphomas.
This rearrangement has been detected in 4% to 7% of pulmonary
adenocarcinomas, with an increased proportion seen when cohorts are
selected based on clinicopathologic features including lack of smoking
history.356–359 The driver of tumorigenesis in this rearrangement is
248 Part I: Science and Clinical Oncology

Tyrosine Kinase Scenarios

Ligand
Extracellular

Physiological Mutation Mutation

Inactive activation Mutation TKI inhibition TKI resistance
TM

TKI
ATP PP ATP PP
P P
Tyrosine kinase

ATP PP ATP PP TKI ATP PP

Signal P Signal P Signal P

proteins proteins proteins

Signal Signal
proteins proteins

Proliferation
Proliferation Motility
Motility Cell survival
Cell survival Invasion
Metastasis

Figure 15.13  •  Tyrosine kinase receptors of many types including KIT, EGFR, HER2/neu, HGHR (MET), IGFR1, ROS1, RET, and PDGFRA, have
similar molecular structures comprising extracellular, transmembrane (TM), and tyrosine kinase domains. Physiologic activation of receptor by binding of
ligand induces conformational changes in the intracellular tyrosine kinase domain that permits binding of adenosine triphosphate (ATP) and phosphorylation
of signaling proteins, which ultimately may lead to cell proliferation, increased motility, and cell survival. Activating mutations (zipper line) affect the ATP
binding pocket of the tyrosine kinase domain, causing phosphorylation of signaling proteins in the absence of ligand binding. Receptor mutations may
ultimately lead to uncontrolled cell proliferation and tumor formation. Treatment of mutated tumors with tyrosine kinase small molecular inhibitors (TKI)
blocks ATP binding and phosphorylation of signaling proteins. Resistance to TKI may be caused by additional mutation (double zipper line) affecting the
tyrosine kinase domain. Cells carrying the resistance mutations may exist in low numbers in untreated tumors but selectively proliferate in the presence of
inhibitor.

thought to be ALK, which is not typically expressed in adult lung

tissue. Juxtaposition of a 5′ partner with a promoter results in high-level
expression of the kinase domain of ALK and leads to inappropriate
signaling by this molecule.355,360
Less than 5 years after the initial discovery of this rearrangement,
over 10,000 lung cancers have been tested for its presence. More than 13
molecular variants of EML4-ALK fusion have been detected,357,361 and
three other rare activating gene partners have been identified: TGF,221
KIF5B,222 and KLC1.223 A novel targeted therapy agent—crizotinib—has
been found to be effective against tumors driven by this molecular
marker228 and has received approval for treatment of these specific
tumors by the FDA.
However, as is the case with EGFR, acquired resistance to treatment
inevitably occurs in initially sensitive tumor (reviewed by Lin and
colleagues362). The most important resistance mechanism is mutation
in the ALK tyrosine kinase domain, followed by amplification of the
ALK gene, activation of bypass signaling pathways, change in tumor
phenotype to, for example epithelial-mesenchymal transition or small
cell phenotype, and overexpression for the drug efflux pump protein,
p-glycoprotein. Analysis of specimens from cellular or liquid biopsy
of recurrent tumor can identify which of these mechanisms is responsible
for resistance, and treatment strategies can be adjusted accordingly.
Detailed discussion of strategies for management of ALK resistance
is found elsewhere in this book.
Summary
Mutations in EGFR, KRAS and ALK underscore the diversity of
molecular alterations in NSCLC, and with ongoing extensive efforts,
“driver mutations” have been identified in a significant proportion of
lung cancers. Molecular testing can provide guidance for prioritizing
therapies to those patients who are likely to receive the greatest benefit,
and conversely can identify patients proven to benefit from a targeted
therapy. Finally, molecular testing can provide clinicians with a rationale
to guide certain patients toward clinical trials of targeted therapeutics.
These selected examples of targets are by no means the only ones
of interest in NSCLC, in which a growing number of molecularly
targeted agents with companion molecular alterations are available and
being developed. These include mutations in BRAF,363,364 HER2,365
NTRK,366 and NRAS,367 and rearrangements in RET368 and ROS1,369
among others.370
A graphical summary of the relative frequencies of mutations in
an adenocarcinoma population enrolled in a recent Lung Cancer
Mutation Consortium study is illustrated in Fig. 15.14. Preliminary
data from this study, which tested an expanded set of predictive markers
by NGS, suggest improved outcome for those tumors in which a
targetable driver mutation is found.371 Comprehensive guidelines for
molecular testing in lung cancer and its clinical ramifications have
recently been published.372
 Pathology, Biomarkers, and Molecular Diagnostics  •  CHAPTER 15 249

Mutaon Frequency
Lung Cancer Mutaon Consorum II
Adenocarcinoma veBRAF
MET 2%
oEGFR
2% amp
3%
ALKr
sEGFR 4%
10% oBRAF
1%
RETr
doubletons 2%
2%
KRAS
25% ROS1r ERBB2
2% 1%

PIK3CA [CATEGORY
1% NAME]
<1%

[CATEGORY NAME]
<1%

Unknown
44%

Figure 15.14  •  Pie chart showing relative frequencies (N = 875) of specific mutations in a Lung Cancer Mutation Consortium adenocarcinoma cohort
tested by next-generation sequencing (NGS) and fluorescence in situ hybridization (FISH). Prefix and suffix letters are defined as follows: s, sensitizing; o,
other; ve, V600E; r, rearrangement. (Prepared from data presented at American Society of Clinical Oncology [ASCO] annual meeting 2016.371)

studies reinforce the need for accurate molecular characterization of

Melanoma: Targeted Treatment of an Aggressive Tumor
Melanoma is also a highly mutated tumor and several targetable
mutations have been found in this tumor in recent years involving
the BRAF, NRAS, and KIT genes (see Table 15.3). Of these, the most
common is BRAF.373
BRAF
BRAF protein is a serine/threonine kinase that is encoded on chromo-
some 7q34. It is an important signal transduction molecule mediating
signals from RAS to MEK, ultimately promoting cell proliferation and
mobility. Activating missense mutations in BRAF were first identified
in a screening of over 500 cell lines from multiple organ sites.317
Mutations were found in 60% of melanoma lines and tumors at
lower frequencies in carcinomas of colon (15%), lung (3%), breast
(3%), and ovary (4%) and in glioma (11%). BRAF mutation is now
thought to occur in approximately half of primary and metastatic
melanoma clinical samples,374–377 with some variation according to
melanoma subtype. Activating mutations in BRAF largely occur in
exon 15, which encodes the catalytic domain of BRAF. By far the most
frequent single base change (c.1799T>A) results in an amino acid
change, V600E.317,377 This mutation activates continuous intracellular
signaling, resulting in cell proliferation, increased tumor cell survival,
and enhanced mobility. Two compounds selectively block the kinase
activity of mutated BRAF, vemurafenib and dabrafenib. In clinical trials,
both compounds have produced high response rates and improved
PFS or overall survival in tumors with BRAF mutations.378–381 These
melanoma to allow for selection of effective treatment.
KIT
A second mutation target in melanoma is KIT (reviewed by Woodman
and colleagues373 and Flaherty and Fisher382; discussed earlier with regard
to GIST). KIT mutations differentially affect subsets of melanoma.
Mutation frequencies in sun-damaged cutaneous, acral, and mucosal
melanomas range from 2% to 20%, but KIT mutations are absent
from cutaneous tumors arising in non–sun-damaged skin.373,383 In
addition, a different spectrum of mutations affects melanoma than
GIST.383–385 Whereas KIT mutations in GIST are often short deletions
or insertions in exon 11, the vast majority of mutations in melanoma
reported to date are point mutations. Mutations in exon 9, which
constitute over 10% of GIST mutations, have not been found in
melanoma. Mutations in exon 13, which encoded the kinase domain
of KIT, are more common in melanoma than in GIST. Finally, KIT
gene amplification is frequent in melanoma383–385 but is not described
in GIST.295
Therapeutic trials testing the effectiveness of KIT inhibitors on
KIT-mutated tumors have had limited success. In a phase II trial
enrolling 51 metastatic melanoma patients with genetic alterations of
KIT, durable complete responses to imatinib have been reported in
two patients with mutations in exon 11 and partial durable responses
in four others, for an overall response rate of 16%.385 Another study
has reported similar findings (23% overall partial response rate) in
tumors with exon 11 and 13 mutations.386 Imatinib blockade of KIT
250 Part I: Science and Clinical Oncology
may be a life-extending alternative for those patients with BRAF
wild-type/KIT-mutated metastatic tumors of specific subtype. This
new clinically relevant development will add urgency to the implementa-
tion of multiplex testing at many institutions that are equipped to
perform molecular testing.
NRAS
NRAS, encoded on chromosome 1, is a member of the RAS family
of signaling molecules and was originally identified as an oncogene
in neuroblastoma cells.387,388 Mutation in melanoma was reported
shortly after discovery of the gene.389 NRAS is altered in approximately
20% to 30% of melanomas of all types,373,377 except for uveal mela-
noma. Mutations are restricted mainly to exon 3, codon 61 (>80%),
with a smaller proportion occurring at exon 2, codon 13.377 These
mutations are thought to lock NRAS in its activated state. However,
mutation in NRAS by itself may not be sufficient for malignant
transformation, because both sporadic and congenital nevi of several
types have been shown to harbor NRAS mutation without evidence
of malignant transformation.390,391 To date, no drugs are known to
directly and effectively target NRAS signaling. However, downstream
signal transduction has been the subject of ongoing preclinical
investigation373,382
GNAQ/GNA11/BAP1
Mutations in GNAQ, GNA11, and BAP1 are confined largely to uveal
melanomas, blue nevi, and meningeal melanoma. The GNAQ and
GNA11 genes encode membrane-bound GTPases that regulate signaling
through seven transmembrane receptors. Activating mutations in either
GNAQ or GNA11 are reported in over 80% of uveal melanomas and
blue nevi392,393 at codon Q209 and R183 hot spots. BRCA1-associated
protein 1 (BAP1) is encoded on chromosome 3p21.1 and is mutated
in over 80% of metastatic uveal melanomas.394 BAP1 mutation has
also recently been described in a familial syndrome with develop-
ment of clinically and histologically characteristic tumors with some
features of melanoma. This mutation also occurs in sporadic tumors
with similar features.395 BAP1 mutations cause protein truncation
and therefore inactivation of the protein. For this reason, BAP1 is
thought to be a TSG. BAP1 mutations are of limited prognostic
value and are not yet therapeutic targets but illustrate the highly
specific relationships that may exist between morphology and molecular
pathology.
Central Nervous System: Classification by
Molecular Diagnostics
Tumors of the CNS are a heterogeneous group that range from slow-
growing low-grade gliomas to rapidly growing and often rapidly fatal
glioblastoma (glioblastoma multiforme, GBM). Both low-grade396 and
high-grade397 tumors have been comprehensively tested through the
Cancer Genome Atlas program, and driver mutations are numerous
in these tumors. To a remarkable extent, molecular profiles of tumors
of the brain correlate with or supplement histologic subclassification,
so molecular findings in addition to histologic appearances are used
in the definition of diagnostic categories in the most recent World
Health Organization (WHO) classification of CNS tumors.398 Brain
tumors have moved further toward a molecular classification than
any of the other solid tumors. Mutations of a single dominant gene
are accompanied by consistent associated genotypic changes, so that
a constellation of genomic changes encode consistent histologic and
biologic phenotypes.
IDH1/IDH2
IDH1IDH2 encode two isoforms of isoicitrate hydrogenase. The enzyme
metabolizes isocitrate to α-ketoglutarate (α-KG) in the citric acid
cycle. Mutation of IDH1/IDH2 ablates normal enzymatic function
and shifts the metabolic product to 2-hydroxyglutarate (2-HG). 2-HG
induces widespread hypermethylation (G-CIMP), which inhibits cellular
differentiation and maintains glial cells in stem cell–like state, prone
to self-renewal and tumorigenesis.399
IDH1/IDH2 mutations defines new categories of diffuse low-grade
glioma as listed in Table 15.8. Today, diffuse gliomas in adults are
classified by IDH1/2 mutational status as WHO grade II (diffuse
astrocytoma), III (anaplastic astrocytoma), or IV ([glioblastoma
multiforme (GBM)]). Across all grades, IDH-mutant tumors display
a more favorable prognosis than their like-graded IDH–wild-type
counterparts.400–402 IDH–wild-type grade III tumors are less frequent
than IDH-mutant grade III tumors, but the converse is true in
glioblastoma (6%–13% mutant403–407). In general, histologic and
cytologic features do not distinguish IDH-mutant from IDH–wild-type
gliomas at any grade. Thus assessment of mutational status is key for
classification and prognostication. The large majority (>88%) of IDH
mutations in gliomas are substitutions at codon 132 of histidine for
arginine (R132H). A high-fidelity antibody identifying this mutant
epitope is available,408 obviating need to perform actual mutational
testing if the immunohistochemical test result is positive. Less common
IDH1 mutation or any IDH2 mutation (the latter more often seen
in oligodendroglial lineage tumors) confers the same favorable prognosis
as the canonic mutation.
1p/19q codeletion
Codeletion of recurrent regions in chromosomes 1p and 19q (1p/19q)
was initially described over two decades ago and occurs in 50% to
70% of both low- and high-grade oligodendrogliomas.409–412 Its presence
has been found to strongly correlate with biologic behavior and
prognosis in these tumors.413–415
Histone mutations
Histone mutations occur in many tumor types, but in glioma, specific
histone mutations are closely associated with morphologic features
and biologic behavior and are now incorporated into the WHO glioma
classification. Histones are protein octamers that organize DNA replica-
tion and gene regulation (reviewed by Lee and colleagues416). Mutation
can have the effect of disrupting the many functions of the protein,
including DNA replication, chromosome condensation, mitosis, DNA
repair, telomere and centromere maintenance, and gene expression.
The mutation H3.3 K27M occurs in 70% to 80% of high-grade
midline pediatric tumors417,418 and is now designated as diffuse midline
glioma, H3 K27M mutant in the 2016 edition of the WHO classifica-
tion of gliomas. Other histone mutations along with their phenotypic
expression are listed in Table 15.5.
ATRX
ATRX (α-thalassemia/mental retardation syndrome–X linked) is a
transcriptional regulator that is expressed in the nucleus of normal
cells. Mutation, most often frameshift deletion,396,419,420 results in
loss of expression of the encoded protein that can be demonstrated
by immunohistochemical methods. ATRX mutation occurs in both
adult diffuse gliomas and glioblastoma associated with mutation
with IDH1/IDH2 and TP53 and G-CIMP, suggesting a role in
chromosomal instability. That the combination of mutations occurs
in both diffuse gliomas and glioblastomas suggests that the latter
may be the result of mutational progression of preexisting diffuse
lymphoma.
TERT promoter
TERT (telomerase reverse transcriptase) promoter mutations are frequent
in gliomas of many types and have the predicted effect of upregulating
telomerase expression and thus preventing postmitotic apoptosis.
Mutations of TERT are often associated with a constellation of muta-
tions in other genes that together form a gene profile that is expressed
as a particular glioma type. For example, TERT promoter mutation
may combine with IDH1/IDH2 mutation and 1p19q codeletion to
generate phenotypes oligodendroglioma and anaplastic oligodendro-
glioma, as shown in Table 15.8.
 Pathology, Biomarkers, and Molecular Diagnostics  •  CHAPTER 15 251

Table 15.8 Genetic Mutations, Histology, and World Health Organization (WHO) Classification

Primary Age Histology

Mutation Associated Genotypes Anatomic Location Group (Phenotype) WHO Classification
IDH1/2 mutation TP53, ATRX mutation Cerebral hemispheres Adult Diffuse Diffuse astrocytoma, IDH
astrocytoma mutant
TP53, ATRX mutation Cerebral hemispheres Pediatric Anaplastic Anaplastic astrocytoma, IDH
or adult astrocytoma mutant
1p/19q codeletion, TERT, CIC, Cerebral hemispheres Adult Oligodendroglioma Oligodendroglioma, IDA
FUBP1 mutations mutant and 1p/19q codeleted
1p/19q codeletion, TERT, CIC, Cerebral hemispheres Adult Anaplastic Anaplastic oligodendroglioma,
FUBP1, TCF12 mutation; oligodendroglioma IDH mutant and 1p/19q
CDKN2A deletion codeleted
TP53, ATRX mutation CDKN2A Cerebral hemispheres Adult Glioblastoma Glioblastoma, IDH mutant
homozygous deletion
IDH1/2 wild-type TERT, PTEN, TP53, PIK3CA, Cerebral hemispheres Adult Glioblastoma Glioblastoma, IDH wild-type
PIK3R1, NF1, H3F3A-G34
mutation; CDKN2A, PTEN
homozygous deletion; EGFR,
PDGFRA, MET, CDK4, CDK6,
MDM2, MDM4 amplification;
EGFRvIII rearrangement
H3-K27M mutant TP53, PPMD1, ACVR1, FGFR1 Midline structures, Pediatric Diffuse midline Diffuse midline glioma,
mutations, PDGFRA, MYC, thalamus, brainstem and glioma H3-K62M-mutant
MYCN, CDK4, CDK6, CCND1-3, spinal cord
ID2, MET amplifications
BRAF mutation or RAF1, NTRK2 gene fusions; Cerebral hemispheres Adult Pilocytic Pilocytic astrocytoma
rearrangement NF1, KRAS, FGFR1, PTPN11 astrocytoma
mutation
CDKN2A/p14ARF homozygous Cerebral hemispheres Pediatric Pleomorphic Pleomorphic
deletion xanthoastrocytoma xanthoastrocytoma
MYB or MYBL FGFR1 duplication Cerebral hemispheres Pediatric Well-differentiated Well-differentiated pediatric
rearrangement pediatric diffuse diffuse glioma
glioma

BRAF include TP53, PTEN, PIK3CA, PIK3R1, NF1, and H3F3A-G34

BRAF is a tyrosine kinase pathway gene that is mutated in a limited
number of brain tumor types including pleomorphic xanthoastrocytoma,
ganglioglioma, extracerebellar pilocytic astrocytoma,421 and epithelioid
glioblastoma.422 BRAF mutations may consist of either point muta-
tion, typically V600E, or chromosomal rearrangement resulting in
the fusion gene KIAA1549–BRAF. Both mutations result in activation
of the gene and tumorigenesis, and both have been treated with TK
inhibitors.423–428
C11orf95-RELA fusions
Ependymoma (reviewed by Wu and colleagues429) represents 1.9% of
adult and 5.2% of pediatric brain tumors; most adult tumors are
supratentorial, whereas most pediatric tumors occur in the posterior
fossa and spinal cord.430 C11orf95-RELA fusions in a region of
chromothripsis on chromosome 11 are present in 70% of supratentorial
ependymomas.431 The RELA component of the fusion is the principal
effector of canonic nuclear factor–κB (NF-κB) signaling and is a
potential treatment target, although no targeted drug has yet passed
clinical trials. YAP1-MamlD1 fusions occur in those supratentorial
tumors that lack the C11orf95-RELA fusion431,432 and are associated
with a better prognosis.
Other mutations
Several other mutations shown in Table 15.8 are found in gliomas but
do not uniquely define a tumor type and may be discussed elsewhere
in this book. Although some of these genes may be therapeutic
targets, owing to space limitations they are only mentioned here and
mutations; CDKN2A and PTEN homozygous deletions; FUBP1
and TCF12 mutations; EGFR, PDGFRA, MET, CDK4, CDK6,
MDM2, and MDM4 amplifications; EGFRvIII rearrangement; and
PPMD1, ACVR1, FGFR1, PDGFRA, MYC, MYCN, CDK4, CDK6,
CCND1-3, ID2, and MET amplifications.
REGULATORY CONSIDERATIONS
The wide scope of approaches for biomarker evaluation makes for a
challenging regulatory landscape. Within the United States and around
the world, there have been varying approaches to consideration of
biomarker testing and the types of oversight that should be applied.
For the purposes of this text, the regulatory considerations in the
United States are the focus; however, the framework employed in
the United States sometimes forms the underpinnings of systems
elsewhere.
Testing performed in the United States for the purposes of clinical
decision making is mandated to be compliant with the Clinical Labora-
tory Improvement Amendments (CLIA) of 1988, and accordingly
there is a Clinical Lab Center within the Centers for Medicare and
Medicaid Services (CMS) that maintains the specific regulations (https://
www.cms.gov/Center/Provider-Type/Clinical-Labs-Center.html). CLIA
regulations stipulate “quality standards for proficiency testing (PT),
patient test management, quality control, personnel qualifications and
quality assurance for laboratories performing moderate and/or high
complexity tests” (https://www.cms.gov/Regulations-and-Guidance/
Legislation/CLIA/Program_Descriptions_Projects.html).
252 Part I: Science and Clinical Oncology

Almost all laboratories must obtain a CLIA certificate. Many
laboratories receive accreditation through their state agency or through
one of seven organizations with deeming authority (https://www
.cms.gov/Regulations-and-Guidance/Legislation/CLIA/Accreditation
_Organizations_and_Exempt_States.html). In some circumstances (e.g.,
in New York and Washington states), laboratories are exempt from
CLIA requirements based on equivalent or more stringent requirements
at the state level.
The FDA regulates companies that sell in-home and laboratory
diagnostic tests (in vitro diagnostic devices [IVDs]) to clinical labo-
ratories. The FDA cites the authority of the Medical Device Amend-
ments of 1976 for this oversight. Although IVDs are not specifically
mentioned in the legislation, the FDA considers IVDs to be medical
devices. Per the 1976 law, medical devices are required to be classified
into three categories—low risk (class I), moderate risk (class II), and
high risk (class III)—according to the level of regulatory control needed
to ensure safety and effectiveness. Low-risk IVDs need only be listed
with the FDA. Moderate-risk IVDs must be submitted for premarket
clearance following 510(k) regulations. High-risk IVDs must receive
Premarket Approval (PMA) from the FDA.
In 1992 the FDA stated publicly that its authority extended to
clinical laboratories using laboratory developed tests (LDTs), but that
it chose to exercise “enforcement discretion” by not requiring clinical
laboratories to submit premarket applications. In 2010 the FDA
announced its intent to regulate LDTs, and in 2014 it published a
draft guidance document for comment. As of 2017, no LDT guidance
or regulation had been finalized.
Biomarker assays use a wide variety of techniques, including IHC,
molecular pathologic methods (e.g., PCR, NGS), and proteomics,
among others. The FDA would likely classify most of these tests as
class III. Examples of class III IVDs receiving FDA approval include
BrAF V600, BRCA, and NGS oncological panel (see devices with
codes PQP, NKF, MVC, and PHP at https://www.accessdata.fda.gov/
scripts/cdrh/cfdocs/cfPMA/pma.cfm for examples).
Although high-risk IVDs with FDA approval are available for
purchase, the burdensome regulatory framework limits their availability
to assays that are intended for widespread distribution. In contrast,
a substantial proportion of biomarker assays performed for clinical
decision making are not FDA approved and instead are developed by
individual laboratories as laboratory developed tests (LDTs). The FDA
defines LDTs as in vitro diagnostic test(s) that are manufactured by
and used within a single laboratory. In November 2016, the FDA
announced it would not immediately finalize its proposed regulatory
framework for LDTs and issued a subsequent discussion paper sur-
rounding the issue.
The issues surrounding potential regulatory approaches for LDTs
remain controversial, both with various stakeholders and politically.
Those in favor of increased regulatory requirements for LDTs argue
that lack of oversight makes for potentially inaccurate testing with
the potential for patient harm, whereas those who oppose such oversight
point out that the regulatory framework suggested will significantly
hamper patient access to innovative testing and stifle iterative improve-
ments of existing LDTs. As of the writing of this chapter, no resolution
has been identified, although some legislative solutions are being
considered. As the technology driving biomarker analysis continues
to evolve at a tremendous pace, solutions that account for the pace
of progress will certainly be paramount.
The complete reference list is available online at
ExpertConsult.com.

KEY REFERENCES
15. zur Hausen H. Papillomaviruses causing cancer:
evasion from host-cell control in early events in car-
cinogenesis. J Natl Cancer Inst. 2000;92(9):690–698.
16. zur Hausen H. Papillomaviruses and cancer: from basic
studies to clinical application. Nat Rev Cancer. 2002;
2(5):342–350.
29. Huh WK, Ault KA, Chelmow D, et al. Use of
primary high-risk human papillomavirus testing for
cervical cancer screening: interim clinical guidance.
J Low Genit Tract Dis. 2015;19(2):91–96.
32. Stanley M. Prospects for new human papillomavirus
vaccines. Curr Opin Infect Dis. 2010;23(1):70–75.
33. Villa LL. HPV prophylactic vaccination: The first
years and what to expect from now. Cancer Lett. 2011;
305(2):106–112.
37. Schoen RE, Pinsky PF, Weissfeld JL, et al. Colorectal-
cancer incidence and mortality with screening flexible
sigmoidoscopy. N Engl J Med. 2012;366(25):
2345–2357.
43. Jasperson KW, Tuohy TM, Neklason DW, Burt RW.
Hereditary and familial colon cancer. Gastroenterol-
ogy. 2010;138(6):2044–2058.
59. Lynch HT, Lynch JF. Lynch syndrome: history and
current status. Dis Markers. 2004;20(4–5):181–198.
73. Boland CR, Thibodeau SN, Hamilton SR, et al. A
National Cancer Institute Workshop on Microsat-
ellite Instability for cancer detection and familial
predisposition: development of international criteria
for the determination of microsatellite instability
in colorectal cancer. Cancer Res. 1998;58(22):
5248–5257.
79. Grover S, Kastrinos F, Steyerberg EW, et al.
Prevalence and phenotypes of APC and MUTYH
mutations in patients with multiple colorectal
adenomas. JAMA. 2012;308(5):485–492.
96. Aretz S, Stienen D, Uhlhaas S, et al. High proportion
of large genomic STK11 deletions in Peutz-Jeghers
syndrome. Hum Mutat. 2005;26(6):513–519.
122. Kim MS, Lee J, Sidransky D. DNA methylation
markers in colorectal cancer. Cancer Metastasis Rev.
2010;29(1):181–206.
131. Aberle DR, Adams AM, Berg CD, et al. Reduced
lung-cancer mortality with low-dose computed
tomographic screening. N Engl J Med. 2011;365(5):
395–409.
134. Franklin WA, Muller KM, Wistuba I, et al. Squa-
mous dysplasia and carcinoma in situ. In: Travis WD,
Brambilla E, Muller-Hermelink HK, Harris CC, eds.
Tumours of the Lung, Pleura, Thymus and Heart.
2nd ed. Lyon, France: IARC Press; 2004:68–72.
140. Keith RL, Blatchford PJ, Kittelson J, et al. Oral
iloprost improves endobronchial dysplasia in former
smokers. Cancer Prev Res (Phila). 2011;4(6):793–
802.
146. Hecht SS. Cigarette smoking and lung cancer:
chemical mechanisms and approaches to prevention.
Lancet Oncol. 2002;3(8):461–469.
153. Jones PA, Baylin SB. The epigenomics of cancer.
Cell. 2007;128(4):683–692.
154. Berger SL, Kouzarides T, Shiekhattar R, Shilatifard
A. An operational definition of epigenetics. Genes
Dev. 2009;23(7):781–783.
168. Govindan R, Ding L, Griffith M, et al. Genomic
landscape of non-small cell lung cancer in smokers
and never-smokers. Cell. 2012;150(6):1121–1134.
184. Crowley E, Di Nicolantonio F, Loupakis F, Bardelli
A. Liquid biopsy: monitoring cancer-genetics in the
blood. Nat Rev Clin Oncol. 2013;10(8):472–484.
189. Bettegowda C, Sausen M, Leary RJ, et al. Detection
of circulating tumor DNA in early- and late-stage
human malignancies. Sci Transl Med. 2014;6(224):
224ra224.
195. Hunter T. The Croonian Lecture 1997. The
phosphorylation of proteins on tyrosine: its role
in cell growth and disease. Philos Trans R Soc Lond
B Biol Sci. 1998;353(1368):583–605.
198. Kandoth C, McLellan MD, Vandin F, et al. Muta-
tional landscape and significance across 12 major
cancer types. Nature. 2013;502(7471):333–339.
202. Comprehensive molecular portraits of human breast
tumours. Nature. 2012;490(7418):61–70.
204. Burrell RA, McGranahan N, Bartek J, Swanton C.
The causes and consequences of genetic heterogene-
ity in cancer evolution. Nature. 2013;501(7467):
338–345.
210. Goodwin S, McPherson JD, McCombie WR. Coming
of age: ten years of next-generation sequencing
technologies. Nat Rev Genet. 2016;17(6):333–351.
225. Camidge DR, Kono SA, Flacco A, et al. Optimizing
the detection of lung cancer patients harboring ana-
plastic lymphoma kinase (ALK) gene rearrangements
potentially suitable for ALK inhibitor treatment.
Clin Cancer Res. 2010;16(22):5581–5590.
228. Kwak EL, Bang YJ, Camidge DR, et al. Anaplastic
lymphoma kinase inhibition in non-small-cell
lung cancer. N Engl J Med. 2010;363(18):1693–
1703.
231. Campbell PJ, Stephens PJ, Pleasance ED, et al.
Identification of somatically acquired rearrangements
in cancer using genome-wide massively parallel
paired-end sequencing. Nat Genet. 2008;40(6):722–
729.
241. Beadling C, Wald AI, Warrick A, et al. A multiplexed
amplicon approach for detecting gene fusions by
next-generation sequencing. J Mol Diagn. 2016;18(2):
165–175.
246. Greene GL, Fitch FW, Jensen EV. Monoclonal anti-
bodies to estrophilin: probes for the study of estrogen
receptors. Proc Natl Acad Sci USA. 1980;77(1):
157–161.
258. Cuzick J, Dowsett M, Pineda S, et al. Prognostic
value of a combined estrogen receptor, progesterone
receptor, Ki-67, and human epidermal growth
factor receptor 2 immunohistochemical score

and comparison with the Genomic Health recur-
rence score in early breast cancer. J Clin Oncol.
2011;29(32):4273–4278.
263. Pirker R, Pereira JR, von Pawel J, et al. EGFR
expression as a predictor of survival for first-line
chemotherapy plus cetuximab in patients with
advanced non-small-cell lung cancer: analysis of
data from the phase 3 FLEX study. Lancet Oncol.
2012;13(1):33–42.
264. Lantuejoul S, Varella-Garcia M, Thunnissen E, Yatabe
Y. Comparison of different assay platforms of ALK
testing. In: Tsao MS, Hirsch FR, Yatabe Y, eds.
IASLC Atlas of ALK and ROS1 Testing in Lung
Cancer. 2nd ed. North Fort Myers, FL: Editorial
Rx Press; 2016:73–83.
267. Schreiber RD, Old LJ, Smyth MJ. Cancer
immunoediting: integrating immunity’s roles
in cancer suppression and promotion. Science.
2011;331(6024):1565–1570.
274. Sharma P, Allison JP. Immune checkpoint targeting
in cancer therapy: toward combination strategies
with curative potential. Cell. 2015;161(2):205–214.
283. Gibney GT, Weiner LM, Atkins MB. Predictive
biomarkers for checkpoint inhibitor-based immu-
notherapy. Lancet Oncol. 2016;17(12):e542–e551.
294. Hirota S, Isozaki K, Moriyama Y, et al. Gain-of-
function mutations of c-kit in human gastrointestinal
stromal tumors. Science. 1998;279(5350):577–580.
295. Corless CL, Barnett CM, Heinrich MC. Gastro-
intestinal stromal tumours: origin and molecular
oncology. Nat Rev Cancer. 2011;11(12):865–878.
Pathology, Biomarkers, and Molecular Diagnostics  •  CHAPTER 15 253
296. Lasota J, Miettinen M. Clinical significance of onco-
genic KIT and PDGFRA mutations in gastrointestinal
stromal tumours. Histopathology. 2008;53(3):
245–266.
297. Martin-Broto J, Rubio L, Alemany R, Lopez-
Guerrero JA. Clinical implications of KIT and
PDGFRA genotyping in GIST. Clin Transl Oncol.
2010;12(10):670–676.
308. Sepulveda AR, Hamilton SR, Allegra CJ, et al.
Molecular biomarkers for the evaluation of colorectal
cancer: guideline from the American Society for
Clinical Pathology, College of American Pathologists,
Association for Molecular Pathology, and American
Society of Clinical Oncology. Arch Pathol Lab Med.
2017;141(5):625–657.
333. Prahallad A, Sun C, Huang S, et al. Unresponsive-
ness of colon cancer to BRAF(V600E) inhibition
through feedback activation of EGFR. Nature.
2012;483(7387):100–103.
341. Paez JG, Janne PA, Lee JC, et al. EGFR mutations
in lung cancer: correlation with clinical response to
gefitinib therapy. Science. 2004;304(5676):1497–
1500.
342. Lynch TJ, Bell DW, Sordella R, et al. Activating
mutations in the epidermal growth factor receptor
underlying responsiveness of non-small-cell lung
cancer to gefitinib. N Engl J Med. 2004;350(21):
2129–2139.
343. Mok TS, Wu YL, Thongprasert S, et al. Gefitinib or
carboplatin-paclitaxel in pulmonary adenocarcinoma.
N Engl J Med. 2009;361(10):947–957.
362. Lin JJ, Riely GJ, Shaw AT. Targeting ALK: Precision
medicine takes on drug resistance. Cancer Discov.
2017;7(2):137–155.
372. Ettinger DS, Wood DE, Aisner DL, et al. Non-Small
Cell Lung Cancer, Version 5.2017, NCCN Clinical
Practice Guidelines in Oncology. J Natl Compr Canc
Netw. 2017;15(4):504–535.
373. Woodman SE, Lazar AJ, Aldape KD, Davies MA.
New strategies in melanoma: molecular testing in
advanced disease. Clin Cancer Res. 2012;18(5):
1195–1200.
382. Flaherty KT, Fisher DE. New strategies in metastatic
melanoma: oncogene-defined taxonomy leads to
therapeutic advances. Clin Cancer Res. 2011;17(15):
4922–4928.
396. Cancer Genome Atlas Research Network, Brat
DJ, Verhaak RG, et al. Comprehensive, integrative
genomic analysis of diffuse lower-grade gliomas.
N Engl J Med. 2015;372(26):2481–2498.
416. Lee J, Solomon DA, Tihan T. The role of histone
modifications and telomere alterations in the patho-
genesis of diffuse gliomas in adults and children.
J Neurooncol. 2017;132(1):1–11.
419. Jiao Y, Killela PJ, Reitman ZJ, et al. Frequent
ATRX, CIC, FUBP1 and IDH1 mutations refine
the classification of malignant gliomas. Oncotarget.
2012;3(7):709–722.
429. Wu J, Armstrong TS, Gilbert MR. Biology and
management of ependymomas. Neuro Oncol.
2016;18(7):902–913.

REFERENCES
1. Hanahan D, Weinberg RA. Hallmarks of cancer:
the next generation. Cell. 2011;144(5):646–674.
2. Pepe MS, Etzioni R, Feng Z, et al. Phases of bio-
marker development for early detection of cancer.
J Natl Cancer Inst. 2001;93(14):1054–1061.
3. ICH Harmonised Tripartite Guideline. Statistical
principles for clinical trials. International Conference
on Harmonisation E9 Expert Working Group. Stat
Med. 1999;18(15):1905–1942.
4. Pisani P, Parkin DM, Bray F, Ferlay J. Erratum:
Estimates of the worldwide mortality from 25 cancers
in 1990. Int. J. Cancer, 83, 18-29 (1999). Int J
Cancer. 1999;83(6):870–873.
5. Papanicolaou GN. A survey of the actualities and
potentialities of exfoliative cytology in cancer
diagnosis. Ann Intern Med. 1949;31(4):661–674.
6. The 1988 Bethesda System for reporting cervical/
vaginal cytological diagnoses. National Cancer
Institute Workshop. JAMA. 1989;262(7):931–934.
7. Peto J, Gilham C, Fletcher O, Matthews FE. The
cervical cancer epidemic that screening has prevented
in the UK. Lancet. 2004;364(9430):249–256.
8. Walboomers JM, Jacobs MV, Manos MM, et al.
Human papillomavirus is a necessary cause of inva-
sive cervical cancer worldwide. J Pathol. 1999;189(1):
12–19.
9. Beard JW, Rous P. A Virus-induced mammalian
growth with the characters of a tumor (the Shope
rabbit papilloma): II. Experimental alterations of the
growth on the skin: morphological considerations: the
phenomena of retrogression. J Exp Med. 1934;60(6):
723–740.
10. Rous P, Beard JW. A virus-induced mammalian
growth with the characters of a tumor (the Shope
rabbit papilloma): III. Further characters of the
growth: general discussion. J Exp Med. 1934;60(6):
741–766.
11. Rous P, Beard JW. A virus-induced mammalian
growth with the characters of a tumor (the Shope
rabbit papilloma): I. The growth on implantation
within favorable hosts. J Exp Med. 1934;60(6):
701–722.
12. zur Hausen H, Meinhof W, Scheiber W, Bornkamm
GW. Attempts to detect virus-specific DNA in
human tumors. I. Nucleic acid hybridizations with
complementary RNA of human wart virus. Int J
Cancer. 1974;13(5):650–656.
13. zur Hausen H. Condylomata acuminata and human
genital cancer. Cancer Res. 1976;36(2 Pt 2):794.
14. zur Hausen H. Human papillomaviruses and their
possible role in squamous cell carcinomas. Curr Top
Microbiol Immunol. 1977;78:1–30.
15. zur Hausen H. Papillomaviruses causing cancer:
evasion from host-cell control in early events in car-
cinogenesis. J Natl Cancer Inst. 2000;92(9):690–698.
16. zur Hausen H. Papillomaviruses and cancer: from
basic studies to clinical application. Nat Rev Cancer.
2002;2(5):342–350.
17. Werness BA, Levine AJ, Howley PM. Association of
human papillomavirus types 16 and 18 E6 proteins
with p53. Science. 1990;248(4951):76–79.
18. Dyson N, Howley PM, Munger K, Harlow E. The
human papilloma virus-16 E7 oncoprotein is able
to bind to the retinoblastoma gene product. Science.
1989;243(4893):934–937.
19. Schwarz E, Freese UK, Gissmann L, et al. Struc-
ture and transcription of human papillomavirus
sequences in cervical carcinoma cells. Nature.
1985;314(6006):111–114.
20. Wentzensen N, Vinokurova S, von Knebel Doeberitz
M. Systematic review of genomic integration sites
of human papillomavirus genomes in epithelial
dysplasia and invasive cancer of the female lower
genital tract. Cancer Res. 2004;64(11):3878–
3884.
21. Leal SM Jr, Gulley ML. Current and emerging molec-
ular tests for human papillomavirus-related neoplasia
Pathology, Biomarkers, and Molecular Diagnostics  •  CHAPTER 15 253.e1
in the genomic era. J Mol Diagn. 2017;19(3):
366–377.
22. Arbyn M, Roelens J, Cuschieri K, et al. The
APTIMA HPV assay versus the Hybrid Capture
2 test in triage of women with ASC-US or LSIL
cervical cytology: a meta-analysis of the diagnostic
accuracy. Int J Cancer. 2013;132(1):101–108.
23. Arbyn M, Snijders PJ, Meijer CJ, et al. Which
high-risk HPV assays fulfil criteria for use in
primary cervical cancer screening? Clin Microbiol
Infect. 2015;21(9):817–826.
24. Schlecht NF, Kulaga S, Robitaille J, et al. Persistent
human papillomavirus infection as a predictor of cer-
vical intraepithelial neoplasia. JAMA. 2001;286(24):
3106–3114.
25. Kinlen LJ, Spriggs AI. Women with positive cervical
smears but without surgical intervention. A follow-up
study. Lancet. 1978;2(8087):463–465.
26. Mayrand MH, Duarte-Franco E, Rodrigues I, et al.
Human papillomavirus DNA versus Papanicolaou
screening tests for cervical cancer. N Engl J Med.
2007;357(16):1579–1588.
27. Ronco G, Giorgi-Rossi P, Carozzi F, et al. Efficacy
of human papillomavirus testing for the detection of
invasive cervical cancers and cervical intraepithelial
neoplasia: a randomised controlled trial. Lancet
Oncol. 2010;11(3):249–257.
28. Castle PE, Fetterman B, Thomas Cox J, et al. The
age-specific relationships of abnormal cytology and
human papillomavirus DNA results to the risk of
cervical precancer and cancer. Obstet Gynecol. 2010;
116(1):76–84.
29. Huh WK, Ault KA, Chelmow D, et al. Use of
primary high-risk human papillomavirus testing for
cervical cancer screening: interim clinical guidance.
J Low Genit Tract Dis. 2015;19(2):91–96.
30. Schiffman M, Wentzensen N, Wacholder S, Kinney
W, Gage JC, Castle PE. Human papillomavirus
testing in the prevention of cervical cancer. J Natl
Cancer Inst. 2011;103(5):368–383.
31. Gravitt PE. The known unknowns of HPV natural
history. J Clin Invest. 2011;121(12):4593–4599.
32. Stanley M. Prospects for new human papillomavirus
vaccines. Curr Opin Infect Dis. 2010;23(1):70–
75.
33. Villa LL. HPV prophylactic vaccination: The first
years and what to expect from now. Cancer Lett.
2011;305(2):106–112.
34. Citarda F, Tomaselli G, Capocaccia R, Barcherini
S, Crespi M. Efficacy in standard clinical practice
of colonoscopic polypectomy in reducing colorectal
cancer incidence. Gut. 2001;48(6):812–815.
35. Winawer SJ, Zauber AG, Ho MN, et al. Prevention
of colorectal cancer by colonoscopic polypectomy.
The National Polyp Study Workgroup. N Engl J
Med. 1993;329(27):1977–1981.
36. Zauber AG, Winawer SJ, O’Brien MJ, et al. Colo-
noscopic polypectomy and long-term prevention
of colorectal-cancer deaths. N Engl J Med. 2012;
366(8):687–696.
37. Schoen RE, Pinsky PF, Weissfeld JL, et  al.
Colorectal-cancer incidence and mortality with
screening flexible sigmoidoscopy. N Engl J Med.
2012;366(25):2345–2357.
38. Rider JA, Kirsner JB, Moeller HC, Palmer WL.
Polyps of the colon and rectum; their incidence and
relationship to carcinoma. Am J Med. 1954;16(4):
555–564.
39. Fearon ER, Hamilton SR, Vogelstein B. Clonal
analysis of human colorectal tumors. Science. 1987;
238(4824):193–197.
40. Vogelstein B, Fearon ER, Hamilton SR, et al. Genetic
alterations during colorectal-tumor development. N
Engl J Med. 1988;319(9):525–532.
41. Fearon ER, Vogelstein B. A genetic model for
colorectal tumorigenesis. Cell. 1990;61(5):759–
767.
253.e1
42. Markowitz SD, Bertagnolli MM. Molecular origins
of cancer: molecular basis of colorectal cancer.
N Engl J Med. 2009;361(25):2449–2460.
43. Jasperson KW, Tuohy TM, Neklason DW, Burt RW.
Hereditary and familial colon cancer. Gastroenterol-
ogy. 2010;138(6):2044–2058.
44. Cripps WH. Two cases of disseminated polypus of
the rectum. Trans Pathol Soc London. 1882;33(16):
165–168.
45. Veale AM. Clinical and genetic problems in familial
in-intestinal polyposis. Gut. 1960;1:285–290.
46. Leppert M, Dobbs M, Scambler P, et al. The gene
for familial polyposis coli maps to the long arm of
chromosome 5. Science. 1987;238(4832):1411–1413.
47. Bodmer WF, Bailey CJ, Bodmer J, et al. Localization
of the gene for familial adenomatous polyposis on
chromosome 5. Nature. 1987;328(6131):614–616.
48. Groden J, Thliveris A, Samowitz W, et al. Identifica-
tion and characterization of the familial adenomatous
polyposis coli gene. Cell. 1991;66(3):589–600.
49. Nishisho I, Nakamura Y, Miyoshi Y, et al. Mutations
of chromosome 5q21 genes in FAP and colorectal
cancer patients. Science. 1991;253(5020):665–669.
50. Kinzler KW, Nilbert MC, Su LK, et al. Identification
of FAP locus genes from chromosome 5q21. Science.
1991;253(5020):661–665.
51. Joslyn G, Carlson M, Thliveris A, et al. Identification
of deletion mutations and three new genes at the
familial polyposis locus. Cell. 1991;66(3):601–613.
52. Goss KH, Groden J. Biology of the adenomatous
polyposis coli tumor suppressor. J Clin Oncol. 2000;
18(9):1967–1979.
53. Powell SM, Zilz N, Beazer-Barclay Y, et al. APC
mutations occur early during colorectal tumorigen-
esis. Nature. 1992;359(6392):235–237.
54. Samowitz WS, Slattery ML, Sweeney C, Herrick J,
Wolff RK, Albertsen H. APC mutations and other
genetic and epigenetic changes in colon cancer. Mol
Cancer Res. 2007;5(2):165–170.
55. De Benedetti L, Sciallero S, Gismondi V, et al.
Association of APC gene mutations and histological
characteristics of colorectal adenomas. Cancer Res.
1994;54(13):3553–3556.
56. Lynch HT, Smyrk T, McGinn T, et al. Attenuated
familial adenomatous polyposis (AFAP). A pheno-
typically and genotypically distinctive variant of FAP.
Cancer. 1995;76(12):2427–2433.
57. Knudsen AL, Bisgaard ML, Bulow S. Attenuated
familial adenomatous polyposis (AFAP). A review
of the literature. Fam Cancer. 2003;2(1):43–55.
58. Warthin AS. Heredity with reference to carcinoma.
Arch Intern Med. 1913;12:546–555.
59. Lynch HT, Lynch JF. Lynch syndrome: history and
current status. Dis Markers. 2004;20(4–5):181–198.
60. Vasen HF, Mecklin JP, Khan PM, Lynch HT. The
International Collaborative Group on Hereditary
Non-Polyposis Colorectal Cancer (ICG-HNPCC).
Dis Colon Rectum. 1991;34(5):424–425.
61. Vasen HF, Watson P, Mecklin JP, Lynch HT. New
clinical criteria for hereditary nonpolyposis colorectal
cancer (HNPCC, Lynch syndrome) proposed by
the International Collaborative group on HNPCC.
Gastroenterology. 1999;116(6):1453–1456.
62. Peltomaki P, Aaltonen LA, Sistonen P, et al. Genetic
mapping of a locus predisposing to human colorectal
cancer. Science. 1993;260(5109):810–812.
63. Bronner CE, Baker SM, Morrison PT, et al. Muta-
tion in the DNA mismatch repair gene homologue
hMLH1 is associated with hereditary non-polyposis
colon cancer. Nature. 1994;368(6468):258–261.
64. Papadopoulos N, Nicolaides NC, Wei YF, et al.
Mutation of a mutL homolog in hereditary colon
cancer. Science. 1994;263(5153):1625–1629.
65. Leach FS, Nicolaides NC, Papadopoulos N, et al.
Mutations of a mutS homolog in hereditary non-
polyposis colorectal cancer. Cell. 1993;75(6):1215–
1225.
253.e2 Part I: Science and Clinical Oncology
66. Fishel R, Lescoe MK, Rao MR, et al. The human
mutator gene homolog MSH2 and its association
with hereditary nonpolyposis colon cancer. Cell.
1994;77(1):1 p following 166.
67. Miyaki M, Konishi M, Tanaka K, et  al.
Germline mutation of MSH6 as the cause of
hereditary nonpolyposis colorectal cancer. Nat Genet.
1997;17(3):271–272.
68. Li GM, Modrich P. Restoration of mismatch repair
to nuclear extracts of H6 colorectal tumor cells by a
heterodimer of human MutL homologs. Proc Natl
Acad Sci USA. 1995;92(6):1950–1954.
69. Nakagawa H, Lockman JC, Frankel WL, et al. Mis-
match repair gene PMS2: disease-causing germline
mutations are frequent in patients whose tumors
stain negative for PMS2 protein, but paralogous
genes obscure mutation detection and interpretation.
Cancer Res. 2004;64(14):4721–4727.
70. Kane MF, Loda M, Gaida GM, et al. Methylation
of the hMLH1 promoter correlates with lack of
expression of hMLH1 in sporadic colon tumors
and mismatch repair-defective human tumor cell
lines. Cancer Res. 1997;57(5):808–811.
71. Herman JG, Umar A, Polyak K, et al. Incidence
and functional consequences of hMLH1 promoter
hypermethylation in colorectal carcinoma. Proc Natl
Acad Sci USA. 1998;95(12):6870–6875.
72. Dietmaier W, Wallinger S, Bocker T, Kullmann
F, Fishel R, Ruschoff J. Diagnostic microsatellite
instability: definition and correlation with mis-
match repair protein expression. Cancer Res. 1997;
57(21):4749–4756.
73. Boland CR, Thibodeau SN, Hamilton SR,
et al. A National Cancer Institute Workshop on
Microsatellite Instability for cancer detection and
familial predisposition: development of international
criteria for the determination of microsatellite
instability in colorectal cancer. Cancer Res. 1998;
58(22):5248–5257.
74. Umar A, Boland CR, Terdiman JP, et al. Revised
Bethesda Guidelines for hereditary nonpolyposis
colorectal cancer (Lynch syndrome) and micro-
satellite instability. J Natl Cancer Inst. 2004;96(4):
261–268.
75. Samowitz WS, Curtin K, Lin HH, et al. The colon
cancer burden of genetically defined hereditary non-
polyposis colon cancer. Gastroenterology. 2001;121(4):
830–838.
76. Al-Tassan N, Chmiel NH, Maynard J, et al. Inherited
variants of MYH associated with somatic G:C–
>T:A mutations in colorectal tumors. Nat Genet.
2002;30(2):227–232.
77. Nielsen M, Morreau H, Vasen HF, Hes FJ. MUTYH-
associated polyposis (MAP). Crit Rev Oncol Hematol.
2011;79(1):1–16.
78. Lubbe SJ, Di Bernardo MC, Chandler IP, Houlston
RS. Clinical implications of the colorectal cancer risk
associated with MUTYH mutation. J Clin Oncol.
2009;27(24):3975–3980.
79. Grover S, Kastrinos F, Steyerberg EW, et al.
Prevalence and phenotypes of APC and MUTYH
mutations in patients with multiple colorectal
adenomas. JAMA. 2012;308(5):485–492.
80. Hutchinson J. Pigmentation of lips and mouth.
Arch Surg. 1896;7:290e291.
81. Connor JT. Aesculapian Society of London. Lancet.
1895;2(1):169.
82. Peutz J. Very remarkable case of familial polyposis of
mucous membrane of intestinal tract and nasophar-
ynx accompanied by peculiar pigmentation of skin
and mucous membrane. Nederl Maandschr Geneesk.
1921;10:134e146.
83. Jeghers H, McKusick KV, Katz KH. Generalized
intestinal polyposis and melanin spots of the oral
mucosa, lips and digits; a syndrome of diagnostic
significance. N Engl J Med. 1949;241(25):993, illust;
passim.
84. Jeghers H, McKusick KV, Katz KH. Generalized
intestinal polyposis and melanin spots of the oral
mucosa, lips and digits; a syndrome of diagnostic sig-
nificance. N Engl J Med. 1949;241(26):1031–1036.
85. Bruwer A, Bargen JA, Kierland RR. Surface
pigmentation and generalized intestinal polyposis;
(Peutz-Jeghers syndrome). Proc Staff Meet Mayo Clin.
1954;29(6):168–171.
86. Beggs AD, Latchford AR, Vasen HF, et  al.
Peutz-Jeghers syndrome: a systematic review and
recommendations for management. Gut. 2010;59(7):
975–986.
87. Mehenni H, Resta N, Park JG, Miyaki M, Guanti
G, Costanza MC. Cancer risks in LKB1 germline
mutation carriers. Gut. 2006;55(7):984–990.
88. Hearle N, Schumacher V, Menko FH, et al. Fre-
quency and spectrum of cancers in the Peutz-Jeghers
syndrome. Clin Cancer Res. 2006;12(10):3209–3215.
89. Giardiello FM, Brensinger JD, Tersmette AC, et al.
Very high risk of cancer in familial Peutz-Jeghers
syndrome. Gastroenterology. 2000;119(6):1447–1453.
90. Mehenni H, Blouin JL, Radhakrishna U, et al.
Peutz-Jeghers syndrome: confirmation of linkage
to chromosome 19p13.3 and identification of a
potential second locus, on 19q13.4. Am J Hum
Genet. 1997;61(6):1327–1334.
91. Amos CI, Bali D, Thiel TJ, et al. Fine mapping of a
genetic locus for Peutz-Jeghers syndrome on chromo-
some 19p. Cancer Res. 1997;57(17):3653–3656.
92. de Leng WW, Jansen M, Carvalho R, et al. Genetic
defects underlying Peutz-Jeghers syndrome (PJS)
and exclusion of the polarity-associated MARK/
Par1 gene family as potential PJS candidates. Clin
Genet. 2007;72(6):568–573.
93. Volikos E, Robinson J, Aittomaki K, et al. LKB1
exonic and whole gene deletions are a common cause
of Peutz-Jeghers syndrome. J Med Genet. 2006;43(5):
e18.
94. Jenne DE, Reimann H, Nezu J, et al. Peutz-Jeghers
syndrome is caused by mutations in a novel serine
threonine kinase. Nat Genet. 1998;18(1):38–43.
95. Hemminki A, Markie D, Tomlinson I, et al. A serine/
threonine kinase gene defective in Peutz-Jeghers
syndrome. Nature. 1998;391(6663):184–187.
96. Aretz S, Stienen D, Uhlhaas S, et al. High proportion
of large genomic STK11 deletions in Peutz-Jeghers
syndrome. Hum Mutat. 2005;26(6):513–519.
97. Hemminki A, Tomlinson I, Markie D, et al.
Localization of a susceptibility locus for Peutz-
Jeghers syndrome to 19p using comparative genomic
hybridization and targeted linkage analysis. Nat
Genet. 1997;15(1):87–90.
98. Bignell GR, Barfoot R, Seal S, Collins N, Warren W,
Stratton MR. Low frequency of somatic mutations in
the LKB1/Peutz-Jeghers syndrome gene in sporadic
breast cancer. Cancer Res. 1998;58(7):1384–1386.
99. Giardiello FM, Trimbath JD. Peutz-Jeghers syn-
drome and management recommendations. Clin
Gastroenterol Hepatol. 2006;4(4):408–415.
100. Roth SI, Helwig EB. Juvenile polyps of the colon
and rectum. Cancer. 1963;16:468–479.
101. Giardiello FM, Hamilton SR, Kern SE, et al.
Colorectal neoplasia in juvenile polyposis or juvenile
polyps. Arch Dis Child. 1991;66(8):971–975.
102. Nugent KP, Talbot IC, Hodgson SV, Phillips RK.
Solitary juvenile polyps: not a marker for subsequent
malignancy. Gastroenterology. 1993;105(3):698–700.
103. McColl I, Busxey HJ, Veale AM, Morson BC. Juvenile
polyposis coli. Proc R Soc Med. 1964;57:896–897.
104. Jass JR, Williams CB, Bussey HJ, Morson BC.
Juvenile polyposis—a precancerous condition.
Histopathology. 1988;13(6):619–630.
105. Veale AM, McColl I, Bussey HJ, Morson BC.
Juvenile polyposis coli. J Med Genet. 1966;3(1):5–16.
106. Brosens LA, van Hattem A, Hylind LM, et al.
Risk of colorectal cancer in juvenile polyposis. Gut.
2007;56(7):965–967.
107. Aretz S, Stienen D, Uhlhaas S, et al. High proportion
of large genomic deletions and a genotype phenotype
update in 80 unrelated families with juvenile polypo-
sis syndrome. J Med Genet. 2007;44(11):702–709.
108. Calva-Cerqueira D, Chinnathambi S, Pechman B,
Bair J, Larsen-Haidle J, Howe JR. The rate of germ-
line mutations and large deletions of SMAD4 and
BMPR1A in juvenile polyposis. Clin Genet. 2009;
75(1):79–85.
109. van Hattem WA, Brosens LA, de Leng WW, et al.
Large genomic deletions of SMAD4, BMPR1A
and PTEN in juvenile polyposis. Gut. 2008;57(5):
623–627.
110. O’Malley M, Laguardia L, Kalady MF, et al. The
prevalence of hereditary hemorrhagic telangiectasia
in juvenile polyposis syndrome. Dis Colon Rectum.
2012;55(8):886–892.
111. Hyman NH, Anderson P, Blasyk H. Hyperplastic
polyposis and the risk of colorectal cancer. Dis Colon
Rectum. 2004;47(12):2101–2104.
112. Rosty C, Buchanan DD, Walsh MD, et al. Phenotype
and polyp landscape in serrated polyposis syndrome:
a series of 100 patients from genetics clinics. Am J
Surg Pathol. 2012;36(6):876–882.
113. Young JP, Parry S. Risk factors: Hyperplastic polyposis
syndrome and risk of colorectal cancer. Nat Rev
Gastroenterol Hepatol. 2010;7(11):594–595.
114. Boparai KS, Reitsma JB, Lemmens V, et al. Increased
colorectal cancer risk in first-degree relatives of
patients with hyperplastic polyposis syndrome.
Gut. 2010;59(9):1222–1225.
115. Boparai KS, Mathus-Vliegen EM, Koornstra JJ, et al.
Increased colorectal cancer risk during follow-up in
patients with hyperplastic polyposis syndrome: a mul-
ticentre cohort study. Gut. 2010;59(8):1094–1100.
116. Allison JE, Tekawa IS, Ransom LJ, Adrain AL. A
comparison of fecal occult-blood tests for colorectal-
cancer screening. N Engl J Med. 1996;334(3):
155–159.
117. Mandel JS, Church TR, Bond JH, et al. The effect
of fecal occult-blood screening on the incidence
of colorectal cancer. N Engl J Med. 2000;343(22):
1603–1607.
118. Kronborg O, Fenger C, Olsen J, Jorgensen OD,
Sondergaard O. Randomised study of screening
for colorectal cancer with faecal-occult-blood test.
Lancet. 1996;348(9040):1467–1471.
119. Hardcastle JD, Chamberlain JO, Robinson MH,
et al. Randomised controlled trial of faecal-occult-
blood screening for colorectal cancer. Lancet.
1996;348(9040):1472–1477.
120. Quintero E, Castells A, Bujanda L, et  al.
Colonoscopy versus fecal immunochemical testing
in colorectal-cancer screening. N Engl J Med. 2012;
366(8):697–706.
121. Ahlquist DA, Zou H, Domanico M, et al. Next-
generation stool DNA test accurately detects
colorectal cancer and large adenomas. Gastroenterol-
ogy. 2012;142(2):248–256, quiz e225–e246.
122. Kim MS, Lee J, Sidransky D. DNA methylation
markers in colorectal cancer. Cancer Metastasis Rev.
2010;29(1):181–206.
123. Siegel R, Desantis C, Virgo K, et al. Cancer treatment
and survivorship statistics, 2012. CA Cancer J Clin.
2012.
124. Siegel R, Naishadham D, Jemal A. Cancer statistics,
2012. CA Cancer J Clin. 2012;62(1):10–29.
125. Omenn GS, Goodman GE, Thornquist MD, et al.
Risk factors for lung cancer and for intervention
effects in CARET, the Beta-Carotene and Retinol
Efficacy Trial. J Natl Cancer Inst. 1996;88(21):
1550–1559.
126. Goodman GE, Thornquist MD, Balmes J, et al.
The Beta-Carotene and Retinol Efficacy Trial:
incidence of lung cancer and cardiovascular disease
mortality during 6-year follow-up after stopping
beta-carotene and retinol supplements. J Natl Cancer
Inst. 2004;96(23):1743–1750.
127. Hennekens CH, Buring JE, Manson JE, et al.
Lack of effect of long-term supplementation
with beta carotene on the incidence of malignant
neoplasms and cardiovascular disease. N Engl J Med.
1996;334(18):1145–1149.

128. Heinonen OP, Albanes D. The effect of vitamin E
and beta carotene on the incidence of lung cancer
and other cancers in male smokers. The Alpha-
Tocopherol, Beta Carotene Cancer Prevention Study
Group. N Engl J Med. 1994;330(15):1029–1035.
129. Henschke CI, McCauley DI, Yankelevitz DF, et al.
Early Lung Cancer Action Project: overall design
and findings from baseline screening. Lancet. 1999;
354(9173):99–105.
130. Henschke CI, Yankelevitz DF, Libby DM, Pasmantier
MW, Smith JP, Miettinen OS. Survival of patients
with stage I lung cancer detected on CT screening.
N Engl J Med. 2006;355(17):1763–1771.
131. Aberle DR, Adams AM, Berg CD, et al. Reduced
lung-cancer mortality with low-dose computed
tomographic screening. N Engl J Med. 2011;
365(5):395–409.
132. Bach PB, Mirkin JN, Oliver TK, et al. Benefits and
harms of CT screening for lung cancer: a systematic
review. JAMA. 2012;307(22):2418–2429.
133. Patz EF Jr, Pinsky P, Gatsonis C, et al. Overdiagnosis
in low-dose computed tomography screening for lung
cancer. JAMA Intern Med. 2014;174(2):269–274.
134. Franklin WA, Muller KM, Wistuba I, et al. Squamous
dysplasia and carcinoma in sutu. In: Travis WD,
Brambilla E, Muller-Hermelink HK, Harris CC,
eds. Tumours of the Lung, Pleura, Thymus and Heart.
2nd ed. Lyon, France: IARC Press; 2004:68–72.
135. Auerbach O, Stout AP, Hammond EC, Garfinkel
L. Changes in bronchial epithelium in relation to
cigarette smoking and in relation to lung cancer.
N Engl J Med. 1961;265:253–267.
136. Auerbach O, Stout AP, Hammond EC, Garfinkel L.
Changes in bronchial epithelium in relation to sex,
age, residence, smoking and pneumonia. N Engl J
Med. 1962;267:111–119.
137. Hirsch FR, Prindiville SA, Miller YE, et al. Fluores-
cence versus white-light bronchoscopy for detection
of preneoplastic lesions: a randomized study. J Natl
Cancer Inst. 2001;93(18):1385–1391.
138. Byers T, Wolf HJ, Franklin WA, et al. Sputum
cytologic atypia predicts incident lung cancer:
defining latency and histologic specificity. Cancer
Epidemiol Biomarkers Prev. 2008;17(1):158–162.
139. Merrick DT, Gao D, Miller YE, et al. Persistence of
bronchial dysplasia is associated with development
of invasive squamous cell carcinoma. Cancer Prev
Res (Phila). 2016;9(1):96–104.
140. Keith RL, Blatchford PJ, Kittelson J, et al. Oral
iloprost improves endobronchial dysplasia in former
smokers. Cancer Prev Res (Phila). 2011;4(6):793–802.
141. Travis WD, Naguchi M, Yatabe Y, et al. Adeno-
carcinoma. In: Travis WD, Brambilla E, Burke AP,
Marx A, Nicholson AG, eds. WHO Classification
of Tumours of the Lung, Pleura, Thymus and Heart.
4th ed. Lyon: International Agency for REsearch
on Cancer (IARC); 2015:26–50.
142. Yip R, Yankelevitz DF, Hu M, et al. Lung cancer
deaths in the National Lung Screening Trial attrib-
uted to nonsolid nodules. Radiology. 2016;281(2):
589–596.
143. Henschke CI, Yip R, Smith JP, et al. CT screen-
ing for lung cancer: part-solid nodules in baseline
and annual repeat rounds. AJR Am J Roentgenol.
2016;207(6):1176–1184.
144. Yip R, Wolf A, Tam K, et al. Outcomes of lung
cancers manifesting as nonsolid nodules. Lung
Cancer. 2016;97:35–42.
145. Detterbeck FC, Marom EM, Arenberg DA, et al. The
IASLC Lung Cancer Staging Project: background
data and proposals for the application of TNM
staging rules to lung cancer presenting as multiple
nodules with ground glass or lepidic features or a
pneumonic type of involvement in the forthcoming
eighth edition of the TNM Classification. J Thorac
Oncol. 2016;11(5):666–680.
146. Hecht SS. Cigarette smoking and lung cancer:
chemical mechanisms and approaches to prevention.
Lancet Oncol. 2002;3(8):461–469.
Pathology, Biomarkers, and Molecular Diagnostics  •  CHAPTER 15 253.e3
147. Veglia F, Matullo G, Vineis P. Bulky DNA adducts
and risk of cancer: a meta-analysis. Cancer Epidemiol
Biomarkers Prev. 2003;12(2):157–160.
148. Hung RJ, Christiani DC, Risch A, et al. International
Lung Cancer Consortium: pooled analysis of sequence
variants in DNA repair and cell cycle pathways.
Cancer Epidemiol Biomarkers Prev. 2008;17(11):
3081–3089.
149. Yu H, Zhao H, Wang LE, et al. An analysis of
single nucleotide polymorphisms of 125 DNA repair
genes in the Texas genome-wide association study
of lung cancer with a replication for the XRCC4
SNPs. DNA Repair (Amst). 2011;10(4):398–407.
150. Wei S, Niu J, Zhao H, et al. Association of a novel
functional promoter variant (rs2075533 C>T) in the
apoptosis gene TNFSF8 with risk of lung cancer—a
finding from Texas lung cancer genome-wide associa-
tion study. Carcinogenesis. 2011;32(4):507–515.
151. Dong LM, Potter JD, White E, Ulrich CM, Cardon
LR, Peters U. Genetic susceptibility to cancer: the
role of polymorphisms in candidate genes. JAMA.
2008;299(20):2423–2436.
152. Vineis P, Manuguerra M, Kavvoura FK, et al. A
field synopsis on low-penetrance variants in DNA
repair genes and cancer susceptibility. J Natl Cancer
Inst. 2009;101(1):24–36.
153. Jones PA, Baylin SB. The epigenomics of cancer.
Cell. 2007;128(4):683–692.
154. Berger SL, Kouzarides T, Shiekhattar R, Shilatifard
A. An operational definition of epigenetics. Genes
Dev. 2009;23(7):781–783.
155. Merlo A, Herman JG, Mao L, et al. 5′ CpG island
methylation is associated with transcriptional silenc-
ing of the tumour suppressor p16/CDKN2/MTS1
in human cancers. Nat Med. 1995;1(7):686–692.
156. Herman JG, Graff JR, Myohanen S, Nelkin BD,
Baylin SB. Methylation-specific PCR: a novel PCR
assay for methylation status of CpG islands. Proc
Natl Acad Sci USA. 1996;93(18):9821–9826.
157. Palmisano WA, Divine KK, Saccomanno G, et al.
Predicting lung cancer by detecting aberrant promoter
methylation in sputum. Cancer Res. 2000;60(21):
5954–5958.
158. Belinsky SA, Liechty KC, Gentry FD, et al. Promoter
hypermethylation of multiple genes in sputum
precedes lung cancer incidence in a high-risk cohort.
Cancer Res. 2006;66(6):3338–3344.
159. Leng S, Do K, Yingling CM, et al. Defining a gene
promoter methylation signature in sputum for lung
cancer risk assessment. Clin Cancer Res. 2012;18(12):
3387–3395.
160. Hulbert A, Jusue-Torres I, Stark A, et al. Early
detection of lung cancer using DNA promoter
hypermethylation in plasma and sputum. Clin
Cancer Res. 2017;23(8):1998–2005.
161. Jonsson S, Varella-Garcia M, Miller YE, et al.
Chromosomal aneusomy in bronchial high-grade
lesions is associated with invasive lung cancer. Am
J Respir Crit Care Med. 2008;177(3):342–347.
162. Varella-Garcia M, Kittelson J, Schulte AP, et al.
Multi-target interphase fluorescence in situ hybridiza-
tion assay increases sensitivity of sputum cytology
as a predictor of lung cancer. Cancer Detect Prev.
2004;28(4):244–251.
163. Li R, Todd NW, Qiu Q, et al. Genetic deletions in
sputum as diagnostic markers for early detection of
stage I non-small cell lung cancer. Clin Cancer Res.
2007;13(2 Pt 1):482–487.
164. Varella-Garcia M, Schulte AP, Wolf HJ, et al. The
detection of chromosomal aneusomy by fluorescence
in situ hybridization in sputum predicts lung cancer
incidence. Cancer Prev Res (Phila). 2010;3(4):
447–453.
165. Rafnar T, Sulem P, Besenbacher S, et al. Genome-
wide significant association between a sequence
variant at 15q15.2 and lung cancer risk. Cancer
Res. 2011;71(4):1356–1361.
166. Spira A, Beane J, Shah V, et al. Effects of ciga-
rette smoke on the human airway epithelial cell
253.e3
transcriptome. Proc Natl Acad Sci USA. 2004;101(27):
10143–10148.
167. Spira A, Beane JE, Shah V, et al. Airway epithelial gene
expression in the diagnostic evaluation of smokers
with suspect lung cancer. Nat Med. 2007;13(3):
361–366.
168. Govindan R, Ding L, Griffith M, et al. Genomic
landscape of non-small cell lung cancer in smokers
and never-smokers. Cell. 2012;150(6):1121–1134.
169. Ahrar K, Wallace M, Javadi S, Gupta S. Mediastinal,
hilar, and pleural image-guided biopsy: current
practice and techniques. Semin Respir Crit Care
Med. 2008;29(4):350–360.
170. Cham MD, Lane ME, Henschke CI, Yankelevitz
DF. Lung biopsy: special techniques. Semin Respir
Crit Care Med. 2008;29(4):335–349.
171. Franklin WA, Haney J, Sugita M, Bemis L, Jimeno
A, Messersmith WA. KRAS mutation: comparison
of testing methods and tissue sampling techniques
in colon cancer. J Mol Diagn. 2010;12(1):43–50.
172. Malapelle U, de Rosa N, Rocco D, et al. EGFR
and KRAS mutations detection on lung cancer
liquid-based cytology: a pilot study. J Clin Pathol.
2012;65(1):87–91.
173. Malapelle U, Bellevicine C, Zeppa P, Palombini
L, Troncone G. Cytology-based gene mutation
tests to predict response to anti-epidermal growth
factor receptor therapy: a review. Diagn Cytopathol.
2011;39(9):703–710.
174. Aisner DL, Deshpande C, Baloch Z, et al. Evaluation
of EGFR mutation status in cytology specimens: an
institutional experience. Diagn Cytopathol. 2011.
175. da Cunha Santos G, Saieg MA, Geddie W, Leighl N.
EGFR gene status in cytological samples of nonsmall
cell lung carcinoma: controversies and opportunities.
Cancer Cytopathol. 2011;119(2):80–91.
176. Pirker R, Filipits M. Monoclonal antibodies against
EGFR in non-small cell lung cancer. Crit Rev Oncol
Hematol. 2011;80(1):1–9.
177. Cabel L, Proudhon C, Gortais H, et al. Circulating
tumor cells: clinical validity and utility. Int J Clin
Oncol. 2017.
178. Riethdorf S, Fritsche H, Muller V, et al. Detection of
circulating tumor cells in peripheral blood of patients
with metastatic breast cancer: a validation study of
the CellSearch system. Clin Cancer Res. 2007;13(3):
920–928.
179. Hou JM, Krebs MG, Lancashire L, et al. Clinical
significance and molecular characteristics of circulat-
ing tumor cells and circulating tumor microemboli
in patients with small-cell lung cancer. J Clin Oncol.
2012;30(5):525–532.
180. Cristofanilli M, Budd GT, Ellis MJ, et  al.
Circulating tumor cells, disease progression, and
survival in metastatic breast cancer. N Engl J Med.
2004;351(8):781–791.
181. Bidard FC, Peeters DJ, Fehm T, et al. Clinical validity
of circulating tumour cells in patients with metastatic
breast cancer: a pooled analysis of individual patient
data. Lancet Oncol. 2014;15(4):406–414.
182. Ilie M, Hofman V, Long-Mira E, et al. Sentinel”
circulating tumor cells allow early diagnosis of
lung cancer in patients with chronic obstructive
pulmonary disease. PLoS ONE. 2014;9(10):e111597.
183. Lo YM, Chan KC, Sun H, et al. Maternal plasma
DNA sequencing reveals the genome-wide genetic
and mutational profile of the fetus. Sci Transl Med.
2010;2(61):61ra91.
184. Crowley E, Di Nicolantonio F, Loupakis F, Bardelli
A. Liquid biopsy: monitoring cancer-genetics in
the blood. Nat Rev Clin Oncol. 2013;10(8):472–
484.
185. Kimura H, Kasahara K, Kawaishi M, et al. Detection
of epidermal growth factor receptor mutations in
serum as a predictor of the response to gefitinib
in patients with non-small-cell lung cancer. Clin
Cancer Res. 2006;12(13):3915–3921.
186. Vogelstein B, Kinzler KW. Digital PCR. Proc Natl
Acad Sci USA. 1999;96(16):9236–9241.
253.e4 Part I: Science and Clinical Oncology
187. Thompson JD, Shibahara G, Rajan S, Pel J, Marziali
A. Winnowing DNA for rare sequences: highly
specific sequence and methylation based enrichment.
PLoS ONE. 2012;7(2):e31597.
188. Newman AM, Lovejoy AF, Klass DM, et al.
Integrated digital error suppression for improved
detection of circulating tumor DNA. Nat Biotechnol.
2016;34(5):547–555.
189. Bettegowda C, Sausen M, Leary RJ, et al. Detection
of circulating tumor DNA in early- and late-stage
human malignancies. Sci Transl Med. 2014;6(224):
224ra224.
190. Maheswaran S, Sequist LV, Nagrath S, et al. Detec-
tion of mutations in EGFR in circulating lung-cancer
cells. N Engl J Med. 2008;359(4):366–377.
191. Dawson SJ, Tsui DW, Murtaza M, et al. Analysis of
circulating tumor DNA to monitor metastatic breast
cancer. N Engl J Med. 2013;368(13):1199–1209.
192. Collins FS, Morgan M, Patrinos A. The Human
Genome Project: lessons from large-scale biology.
Science. 2003;300(5617):286–290.
193. Lander ES, Linton LM, Birren B, et  al.
Initial sequencing and analysis of the human
genome. Nature. 2001;409(6822):860–921.
194. Venter JC, Adams MD, Myers EW, et al. The
sequence of the human genome. Science. 2001;
291(5507):1304–1351.
195. Hunter T. The Croonian Lecture 1997. The
phosphorylation of proteins on tyrosine: its role
in cell growth and disease. Philos Trans R Soc Lond
B Biol Sci. 1998;353(1368):583–605.
196. Hunter T. Signaling—2000 and beyond. Cell.
2000;100(1):113–127.
197. Barker A. Q&A: Anna Barker on the cancer genome
atlas. Cancer Discov. 2011;1(3):193.
198. Kandoth C, McLellan MD, Vandin F, et al. Muta-
tional landscape and significance across 12 major
cancer types. Nature. 2013;502(7471):333–339.
199. Cancer Genome Atlas Research Network. Genomic clas-
sification of cutaneous melanoma. Cell. 2015;161(7):
1681–1696.
200. Gupta S, Artomov M, Goggins W, Daly M, Tsao H.
Gender disparity and mutation burden in metastatic
melanoma. J Natl Cancer Inst. 2015;107(11).
201. Cancer Genome Atlas Research Network. Compre-
hensive molecular profiling of lung adenocarcinoma.
Nature. 2014;511(7511):543–550.
202. Comprehensive molecular portraits of human breast
tumours. Nature. 2012;490(7418):61–70.
203. Yates LR, Campbell PJ. Evolution of the cancer
genome. Nat Rev Genet. 2012;13(11):795–806.
204. Burrell RA, McGranahan N, Bartek J, Swanton C.
The causes and consequences of genetic heterogene-
ity in cancer evolution. Nature. 2013;501(7467):
338–345.
205. McGranahan N, Swanton C. Biological and
therapeutic impact of intratumor heterogeneity in
cancer evolution. Cancer Cell. 2015;27(1):15–26.
206. de Bruin EC, McGranahan N, Mitter R, et al.
Spatial and temporal diversity in genomic instability
processes defines lung cancer evolution. Science.
2014;346(6206):251–256.
207. Zhang J, Fujimoto J, Zhang J, et al. Intratumor
heterogeneity in localized lung adenocarcinomas
delineated by multiregion sequencing. Science.
2014;346(6206):256–259.
208. de Bruin EC, McGranahan N, Swanton C. Analysis
of intratumor heterogeneity unravels lung cancer
evolution. Mol Cell Oncol. 2015;2(3):e985549.
208a. Jamal-Hanjani M, Wilson GA, McGranahan
N, et al. Tracking the evolution of non-small-cell
lung cancer. N Engl J Med. 2017;376(22):2109–
2121.
209. Blume-Jensen P, Hunter T. Oncogenic kinase
signalling. Nature. 2001;411(6835):355–365.
210. Goodwin S, McPherson JD, McCombie WR.
Coming of age: ten years of next-generation sequenc-
ing technologies. Nat Rev Genet. 2016;17(6):333–
351.
211. Carter SL, Cibulskis K, Helman E, et al. Absolute
quantification of somatic DNA alterations in human
cancer. Nat Biotechnol. 2012;30(5):413–421.
212. Aziz N, Zhao Q, Bry L, et al. College of American
Pathologists’ laboratory standards for next-generation
sequencing clinical tests. Arch Pathol Lab Med.
2015;139(4):481–493.
213. Horak P, Frohling S, Glimm H. Integrating next-
generation sequencing into clinical oncology: strate-
gies, promises and pitfalls. ESMO Open. 2016;1(5):
e000094.
214. Fisher S, Barry A, Abreu J, et al. A scalable, fully
automated process for construction of sequence-ready
human exome targeted capture libraries. Genome
Biol. 2011;12(1):R1.
215. van Dijk EL, Jaszczyszyn Y, Thermes C. Library
preparation methods for next-generation sequencing:
tone down the bias. Exp Cell Res. 2014;322(1):12–20.
216. Roy S, LaFramboise WA, Nikiforov YE, et al. Next-
generation sequencing informatics: challenges and
strategies for implementation in a clinical environ-
ment. Arch Pathol Lab Med. 2016;140(9):958–975.
217. Robinson JT, Thorvaldsdottir H, Winckler W,
et al. Integrative genomics viewer. Nat Biotechnol.
2011;29(1):24–26.
218. Li H, Handsaker B, Wysoker A, et al. The Sequence
Alignment/Map format and SAMtools. Bioinformat-
ics. 2009;25(16):2078–2079.
219. Li MM, Datto M, Duncavage EJ, et al. Standards
and guidelines for the interpretation and reporting
of sequence variants in cancer: a joint consensus
recommendation of the Association for Molecular
Pathology, American Society of Clinical Oncology,
and College of American Pathologists. J Mol Diagn.
2017;19(1):4–23.
220. Shaffer LG, Slovak ML, Campbell LJ. ISCN 2009:
an international system for human cytogenetic
nomenclature. Karger; 2009.
221. Rikova K, Guo A, Zeng Q, et al. Global survey
of phosphotyrosine signaling identifies oncogenic
kinases in lung cancer. Cell. 2007;131(6):1190–
1203.
222. Takeuchi K, Choi YL, Togashi Y, et al. KIF5B-ALK,
a novel fusion oncokinase identified by an immu-
nohistochemistry-based diagnostic system for ALK-
positive lung cancer. Clin Cancer Res. 2009;15(9):
3143–3149.
223. Togashi Y, Soda M, Sakata S, et al. KLC1-ALK:
a novel fusion in lung cancer identified using a
formalin-fixed paraffin-embedded tissue only. PLoS
ONE. 2012;7(2):e31323.
224. Jung Y, Kim P, Keum J, et al. Discovery of ALK-
PTPN3 gene fusion from human non-small cell
lung carcinoma cell line using next generation RNA
sequencing. Genes Chromosomes Cancer. 2012;51(6):
590–597.
225. Camidge DR, Kono SA, Flacco A, et al. Optimizing
the detection of lung cancer patients harboring ana-
plastic lymphoma kinase (ALK) gene rearrangements
potentially suitable for ALK inhibitor treatment.
Clin Cancer Res. 2010;16(22):5581–5590.
226. Kim H, Yoo SB, Choe JY, et al. Detection of ALK
gene rearrangement in non-small cell lung cancer:
a comparison of fluorescence in situ hybridization
and chromogenic in situ hybridization with cor-
relation of ALK protein expression. J Thorac Oncol.
2011;6(8):1359–1366.
227. Yoshida A, Tsuta K, Nitta H, et al. Bright-field
dual-color chromogenic in situ hybridization for
diagnosing echinoderm microtubule-associated
protein-like 4-anaplastic lymphoma kinase-positive
lung adenocarcinomas. J Thorac Oncol. 2011;6(10):
1677–1686.
228. Kwak EL, Bang YJ, Camidge DR, et al. Anaplastic
lymphoma kinase inhibition in non-small-cell lung
cancer. N Engl J Med. 2010;363(18):1693–1703.
229. Shaw AT, Yeap BY, Solomon BJ, et al. Effect of
crizotinib on overall survival in patients with
advanced non-small-cell lung cancer harbouring
ALK gene rearrangement: a retrospective analysis.
Lancet Oncol. 2011;12(11):1004–1012.
230. Ju YS, Lee WC, Shin JY, et al. A transforming
KIF5B and RET gene fusion in lung adenocarcinoma
revealed from whole-genome and transcriptome
sequencing. Genome Res. 2012.
231. Campbell PJ, Stephens PJ, Pleasance ED, et al. Iden-
tification of somatically acquired rearrangements in
cancer using genome-wide massively parallel paired-
end sequencing. Nat Genet. 2008;40(6):722–729.
232. Lipson D, Capelletti M, Yelensky R, et al. Identifica-
tion of new ALK and RET gene fusions from colorec-
tal and lung cancer biopsies. Nat Med. 2012;18(3):
382–384.
233. Drilon A, Wang L, Arcila ME, et al. Broad, hybrid
capture-based next-generation sequencing identifies
actionable genomic alterations in lung adenocarci-
nomas otherwise negative for such alterations by
other genomic testing approaches. Clin Cancer Res.
2015;21(16):3631–3639.
234. Korbel JO, Urban AE, Affourtit JP, et al. Paired-end
mapping reveals extensive structural variation in the
human genome. Science. 2007;318(5849):420–426.
235. Chen K, Wallis JW, McLellan MD, et al. Break-
Dancer: an algorithm for high-resolution mapping of
genomic structural variation. Nat Methods. 2009;6(9):
677–681.
236. Wang J, Mullighan CG, Easton J, et al. CREST
maps somatic structural variation in cancer genomes
with base-pair resolution. Nat Methods. 2011;8(8):
652–654.
237. Li C, Fang R, Sun Y, et al. Spectrum of oncogenic
driver mutations in lung adenocarcinomas from East
Asian never smokers. PLoS ONE. 2011;6(11):e28204.
238. Stransky N, Cerami E, Schalm S, Kim JL, Lengauer
C. The landscape of kinase fusions in cancer. Nat
Commun. 2014;5:4846.
239. Levin JZ, Berger MF, Adiconis X, et al. Targeted
next-generation sequencing of a cancer transcriptome
enhances detection of sequence variants and novel
fusion transcripts. Genome Biol. 2009;10(10):R115.
240. Pfarr N, Stenzinger A, Penzel R, et al. High-
throughput diagnostic profiling of clinically action-
able gene fusions in lung cancer. Genes Chromosomes
Cancer. 2016;55(1):30–44.
241. Beadling C, Wald AI, Warrick A, et al. A Multiplexed
amplicon approach for detecting gene fusions by
next-generation sequencing. J Mol Diagn. 2016;18(2):
165–175.
242. Zheng Z, Liebers M, Zhelyazkova B, et al. Anchored
multiplex PCR for targeted next-generation sequenc-
ing. Nat Med. 2014;20(12):1479–1484.
243. Shim HS, Kenudson M, Zheng Z, et al. Unique
genetic and survival characteristics of invasive
mucinous adenocarcinoma of the lung. J Thorac
Oncol. 2015;10(8):1156–1162.
244. Suehara Y, Arcila M, Wang L, et al. Identification
of KIF5B-RET and GOPC-ROS1 fusions in lung
adenocarcinomas through a comprehensive mRNA-
based screen for tyrosine kinase fusions. Clin Cancer
Res. 2012;18(24):6599–6608.
245. Greene GL, Nolan C, Engler JP, Jensen EV. Mono-
clonal antibodies to human estrogen receptor. Proc
Natl Acad Sci USA. 1980;77(9):5115–5119.
246. Greene GL, Fitch FW, Jensen EV. Monoclonal anti-
bodies to estrophilin: probes for the study of estrogen
receptors. Proc Natl Acad Sci USA. 1980;77(1):
157–161.
247. Shimada A, Kimura S, Abe K, et al. Immunocyto-
chemical staining of estrogen receptor in paraffin
sections of human breast cancer by use of monoclonal
antibody: comparison with that in frozen sections.
Proc Natl Acad Sci USA. 1985;82(14):4803–
4807.
248. Poulsen HS, Ozzello L, King WJ, Greene GL. The
use of monoclonal antibodies to estrogen receptors
(ER) for immunoperoxidase detection of ER in
paraffin sections of human breast cancer tissue.
J Histochem Cytochem. 1985;33(2):87–92.

249. Harvey JM, Clark GM, Osborne CK, Allred DC.
Estrogen receptor status by immunohistochemistry
is superior to the ligand-binding assay for predicting
response to adjuvant endocrine therapy in breast
cancer. J Clin Oncol. 1999;17(5):1474–1481.
250. McCarty KS Jr, Szabo E, Flowers JL, et al. Use of a
monoclonal anti-estrogen receptor antibody in the
immunohistochemical evaluation of human tumors.
Cancer Res. 1986;46(8 suppl):4244s–4248s.
251. Bartlett JM, Brookes CL, Robson T, et al. Estrogen
receptor and progesterone receptor as predictive
biomarkers of response to endocrine therapy: a
prospectively powered pathology study in the
Tamoxifen and Exemestane Adjuvant Multinational
trial. J Clin Oncol. 2011;29(12):1531–1538.
252. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich
A, McGuire WL. Human breast cancer: correlation of
relapse and survival with amplification of the HER-2/
neu oncogene. Science. 1987;235(4785):177–182.
253. Slamon DJ, Godolphin W, Jones LA, et al. Studies of
the HER-2/neu proto-oncogene in human breast and
ovarian cancer. Science. 1989;244(4905):707–712.
254. Lewis GD, Figari I, Fendly B, et al. Differential
responses of human tumor cell lines to anti-
p185HER2 monoclonal antibodies. Cancer Immunol
Immunother. 1993;37(4):255–263.
255. Shepard HM, Lewis GD, Sarup JC, et al. Monoclo-
nal antibody therapy of human cancer: taking the
HER2 protooncogene to the clinic. J Clin Immunol.
1991;11(3):117–127.
256. Slamon DJ, Leyland-Jones B, Shak S, et al. Use of
chemotherapy plus a monoclonal antibody against
HER2 for metastatic breast cancer that overexpresses
HER2. N Engl J Med. 2001;344(11):783–792.
257. Wolff AC, Hammond ME, Schwartz JN, et al.
American Society of Clinical Oncology/College of
American Pathologists guideline recommendations
for human epidermal growth factor receptor 2 testing
in breast cancer. J Clin Oncol. 2007;25(1):118–145.
258. Cuzick J, Dowsett M, Pineda S, et al. Prognostic
value of a combined estrogen receptor, progesterone
receptor, Ki-67, and human epidermal growth factor
receptor 2 immunohistochemical score and com-
parison with the Genomic Health recurrence score
in early breast cancer. J Clin Oncol. 2011;29(32):
4273–4278.
259. Bartlett JM, Bloom KJ, Piper T, et al. Mammostrat
as an immunohistochemical multigene assay for
prediction of early relapse risk in the tamoxifen
versus exemestane adjuvant multicenter trial pathol-
ogy study. J Clin Oncol. 2012;30(36):4477–4484.
260. Foulkes WD, Smith IE, Reis-Filho JS. Triple-
negative breast cancer. N Engl J Med. 2010;363(20):
1938–1948.
261. Perou CM, Sorlie T, Eisen MB, et al. Molecular portraits
of human breast tumours. Nature. 2000;406(6797):
747–752.
262. Dalerba P, Sahoo D, Paik S, et al. CDX2 as a
prognostic biomarker in stage II and stage III colon
cancer. N Engl J Med. 2016;374(3):211–222.
263. Pirker R, Pereira JR, von Pawel J, et al. EGFR
expression as a predictor of survival for first-line che-
motherapy plus cetuximab in patients with advanced
non-small-cell lung cancer: analysis of data from
the phase 3 FLEX study. Lancet Oncol. 2012;13(1):
33–42.
264. Lantuejoul S, Varella-Garcia M, Thunnissen E,
Yatabe Y. Comparison of different assay platforms
of ALK testing. In: Tsao MS, Hirsch FR, Yatabe Y,
eds. IASLC Atlas of ALK and ROS1 Testing in Lung
Cancer. 2nd ed. North Fort Myers, FL: Editorial
Rx Press; 2016:73–83.
265. Tsao MS, Hirsch FR, Yatabe Y. IASLC Atlas of ALK
and ROS1 Testing in Lung Cancer. 2nd ed. North
Fort Myers, FL: Editorial Rx Press; 2016.
266. Sholl LM, Sun H, Butaney M, et al. ROS1
immunohistochemistry for detection of ROS1-
rearranged lung adenocarcinomas. Am J Surg Pathol.
2013;37(9):1441–1449.
Pathology, Biomarkers, and Molecular Diagnostics  •  CHAPTER 15 253.e5
267. Schreiber RD, Old LJ, Smyth MJ. Cancer immu-
noediting: integrating immunity’s roles in cancer sup-
pression and promotion. Science. 2011;331(6024):
1565–1570.
268. Hodi FS, O’Day SJ, McDermott DF, et al. Improved
survival with ipilimumab in patients with metastatic
melanoma. N Engl J Med. 2010;363(8):711–723.
269. Robert C, Schachter J, Long GV, et al. Pembroli-
zumab versus ipilimumab in advanced melanoma.
N Engl J Med. 2015;372(26):2521–2532.
270. Larkin J, Chiarion-Sileni V, Gonzalez R, et al. Com-
bined nivolumab and ipilimumab or monotherapy
in untreated melanoma. N Engl J Med. 2015;373(1):
23–34.
271. Garon EB, Rizvi NA, Hui R, et al. Pembrolizumab
for the treatment of non-small-cell lung cancer. N
Engl J Med. 2015;372(21):2018–2028.
272. Brahmer J, Reckamp KL, Baas P, et al. Nivolumab
versus docetaxel in advanced squamous-cell non-
small-cell lung cancer. N Engl J Med. 2015;373(2):
123–135.
273. Borghaei H, Paz-Ares L, Horn L, et al. Nivolumab
versus docetaxel in advanced nonsquamous non-
small-cell lung cancer. N Engl J Med. 2015;373(17):
1627–1639.
274. Sharma P, Allison JP. Immune checkpoint targeting
in cancer therapy: toward combination strategies
with curative potential. Cell. 2015;161(2):205–214.
275. Schumacher TN, Schreiber RD. Neoantigens in cancer
immunotherapy. Science. 2015;348(6230):69–74.
276. Snyder A, Makarov V, Merghoub T, et al. Genetic
basis for clinical response to CTLA-4 blockade in
melanoma. N Engl J Med. 2014;371(23):2189–2199.
277. Rizvi NA, Hellmann MD, Snyder A, et al. Cancer
immunology. Mutational landscape determines
sensitivity to PD-1 blockade in non-small cell lung
cancer. Science. 2015;348(6230):124–128.
278. Cancer Genome Atlas N. Comprehensive molecular
characterization of human colon and rectal cancer.
Nature. 2012;487(7407):330–337.
279. Eshleman JR, Lang EZ, Bowerfind GK, et al.
Increased mutation rate at the hprt locus accom-
panies microsatellite instability in colon cancer.
Oncogene. 1995;10(1):33–37.
280. Timmermann B, Kerick M, Roehr C, et al. Somatic
mutation profiles of MSI and MSS colorectal
cancer identified by whole exome next generation
sequencing and bioinformatics analysis. PLoS ONE.
2010;5(12):e15661.
281. Le DT, Uram JN, Wang H, et al. PD-1 blockade
in tumors with mismatch-repair deficiency. N Engl
J Med. 2015;372(26):2509–2520.
282. Dudley JC, Lin MT, Le DT, Eshleman JR. Microsat-
ellite instability as a biomarker for PD-1 blockade.
Clin Cancer Res. 2016;22(4):813–820.
283. Gibney GT, Weiner LM, Atkins MB. Predictive
biomarkers for checkpoint inhibitor-based immu-
notherapy. Lancet Oncol. 2016;17(12):e542–e551.
284. Sacher AG, Gandhi L. Biomarkers for the clinical
use of PD-1/PD-L1 inhibitors in non-small-cell lung
cancer: a review. JAMA Oncol. 2016;2(9):1217–
1222.
285. Gandini S, Massi D, Mandala M. PD-L1 expres-
sion in cancer patients receiving anti PD-1/PD-L1
antibodies: a systematic review and meta-analysis.
Crit Rev Oncol Hematol. 2016;100:88–98.
286. Rimm DL, Han G, Taube JM, et al. A prospective,
multi-institutional, pathologist-based assessment of 4
immunohistochemistry assays for PD-L1 expression
in non-small cell lung cancer. JAMA Oncol. 2017.
287. Hirsch FR, McElhinny A, Stanforth D, et al. PD-L1
Immunohistochemistry assays for lung cancer: results
from phase 1 of the Blueprint PD-L1 IHC Assay
Comparison Project. J Thorac Oncol. 2017;12(2):
208–222.
288. Kerr KM, Tsao MS, Nicholson AG, et al. Pro-
grammed death-ligand 1 immunohistochemistry
in lung cancer: in what state is this art? J Thorac
Oncol. 2015;10(7):985–989.
253.e5
289. Zeng DQ, Yu YF, Ou QY, et al. Prognostic and
predictive value of tumor-infiltrating lymphocytes
for clinical therapeutic research in patients with
non-small cell lung cancer. Oncotarget. 2016;7(12):
13765–13781.
290. Thomas NE, Busam KJ, From L, et al. Tumor-
infiltrating lymphocyte grade in primary melanomas
is independently associated with melanoma-specific
survival in the population-based genes, environment
and melanoma study. J Clin Oncol. 2013;31(33):
4252–4259.
291. Huh JW, Lee JH, Kim HR. Prognostic significance
of tumor-infiltrating lymphocytes for patients with
colorectal cancer. Arch Surg. 2012;147(4):366–372.
292. Lewin KJ, Riddell RH, Weinstein WM. Mesen-
chymal tumors. In: Gastrointestinal Pathology and
Its Clinical Implications. Vol. 1. 1st ed. New York,
Tokyo: Igaku-Shoin; 1992:284–341.
293. van de Rijn M, Hendrickson MR, Rouse RV. CD34
expression by gastrointestinal tract stromal tumors.
Hum Pathol. 1994;25(8):766–771.
294. Hirota S, Isozaki K, Moriyama Y, et al. Gain-of-
function mutations of c-kit in human gastrointestinal
stromal tumors. Science. 1998;279(5350):577–580.
295. Corless CL, Barnett CM, Heinrich MC. Gastro-
intestinal stromal tumours: origin and molecular
oncology. Nat Rev Cancer. 2011;11(12):865–878.
296. Lasota J, Miettinen M. Clinical significance of onco-
genic KIT and PDGFRA mutations in gastrointestinal
stromal tumours. Histopathology. 2008;53(3):
245–266.
297. Martin-Broto J, Rubio L, Alemany R, Lopez-
Guerrero JA. Clinical implications of KIT and
PDGFRA genotyping in GIST. Clin Transl Oncol.
2010;12(10):670–676.
298. van de Rijn M, Lombard CM, Rouse RV. Expression
of CD34 by solitary fibrous tumors of the pleura,
mediastinum, and lung. Am J Surg Pathol. 1994;18(8):
814–820.
299. Hirota S, Ohashi A, Nishida T, et al. Gain-of-
function mutations of platelet-derived growth
factor receptor alpha gene in gastrointestinal stromal
tumors. Gastroenterology. 2003;125(3):660–667.
300. Ng EH, Pollock RE, Munsell MF, Atkinson EN,
Romsdahl MM. Prognostic factors influencing
survival in gastrointestinal leiomyosarcomas. Implica-
tions for surgical management and staging. Ann
Surg. 1992;215(1):68–77.
301. Dematteo RP, Heinrich MC, El-Rifai WM, Demetri
G. Clinical management of gastrointestinal stromal
tumors: before and after STI-571. Hum Pathol. 2002;
33(5):466–477.
302. Heinrich MC, Griffith DJ, Druker BJ, Wait CL, Ott
KA, Zigler AJ. Inhibition of c-kit receptor tyrosine
kinase activity by STI 571, a selective tyrosine kinase
inhibitor. Blood. 2000;96(3):925–932.
303. Verweij J, Casali PG, Zalcberg J, et al. Progression-
free survival in gastrointestinal stromal tumours
with high-dose imatinib: randomised trial. Lancet.
2004;364(9440):1127–1134.
304. Blanke CD, Rankin C, Demetri GD, et al. Phase
III randomized, intergroup trial assessing imatinib
mesylate at two dose levels in patients with unresect-
able or metastatic gastrointestinal stromal tumors
expressing the kit receptor tyrosine kinase: S0033.
J Clin Oncol. 2008;26(4):626–632.
305. Demetri GD, von Mehren M, Blanke CD, et al.
Efficacy and safety of imatinib mesylate in advanced
gastrointestinal stromal tumors. N Engl J Med. 2002;
347(7):472–480.
306. Corless CL, Schroeder A, Griffith D, et al. PDGFRA
mutations in gastrointestinal stromal tumors: fre-
quency, spectrum and in vitro sensitivity to imatinib.
J Clin Oncol. 2005;23(23):5357–5364.
307. Weisberg E, Wright RD, Jiang J, et al. Effects of
PKC412, nilotinib, and imatinib against GIST-
associated PDGFRA mutants with differential
imatinib sensitivity. Gastroenterology. 2006;131(6):
1734–1742.
253.e6 Part I: Science and Clinical Oncology
308. Sepulveda AR, Hamilton SR, Allegra CJ, et al.
Molecular biomarkers for the evaluation of colorectal
cancer: guideline from the American Society for
Clinical Pathology, College of American Pathologists,
Association for Molecular Pathology, and American
Society of Clinical Oncology. Arch Pathol Lab Med.
2017;141(5):625–657.
309. Jimeno A, Messersmith WA, Hirsch FR, Franklin
WA, Eckhardt SG. KRAS mutations and sensitivity
to epidermal growth factor receptor inhibitors in
colorectal cancer: practical application of patient
selection. J Clin Oncol. 2009;27(7):1130–1136.
310. Andreyev HJ, Norman AR, Cunningham D, et al.
Kirsten ras mutations in patients with colorectal cancer:
the ‘RASCAL II’ study. Br J Cancer. 2001;85(5):
692–696.
311. Amado RG, Wolf M, Peeters M, et al. Wild-type
KRAS is required for panitumumab efficacy in
patients with metastatic colorectal cancer. J Clin
Oncol. 2008;26(10):1626–1634.
312. Jonker DJ, O’Callaghan CJ, Karapetis CS, et al.
Cetuximab for the treatment of colorectal cancer.
N Engl J Med. 2007;357(20):2040–2048.
313. Karapetis CS, Khambata-Ford S, Jonker DJ, et al. K-
ras mutations and benefit from cetuximab in advanced
colorectal cancer. N Engl J Med. 2008;359(17):
1757–1765.
314. Van Cutsem E, Kohne CH, Hitre E, et al. Cetuximab
and chemotherapy as initial treatment for metastatic
colorectal cancer. N Engl J Med. 2009;360(14):
1408–1417.
315. Bokemeyer C, Bondarenko I, Makhson A, et al. Fluo-
rouracil, leucovorin, and oxaliplatin with and without
cetuximab in the first-line treatment of metastatic
colorectal cancer. J Clin Oncol. 2009;27(5):663–
671.
316. De Roock W, Jonker DJ, Di Nicolantonio F, et al.
Association of KRAS p.G13D mutation with
outcome in patients with chemotherapy-refractory
metastatic colorectal cancer treated with cetuximab.
JAMA. 2010;304(16):1812–1820.
317. Davies H, Bignell GR, Cox C, et al. Mutations
of the BRAF gene in human cancer. Nature.
2002;417(6892):949–954.
318. Vaughn CP, Zobell SD, Furtado LV, Baker CL,
Samowitz WS. Frequency of KRAS, BRAF, and
NRAS mutations in colorectal cancer. Genes
Chromosomes Cancer. 2011;50(5):307–312.
319. Roth AD, Tejpar S, Delorenzi M, et al. Prognostic
role of KRAS and BRAF in stage II and III resected
colon cancer: results of the translational study on
the PETACC-3, EORTC 40993, SAKK 60-00 trial.
J Clin Oncol. 2010;28(3):466–474.
320. De Roock W, Claes B, Bernasconi D, et al. Effects
of KRAS, BRAF, NRAS, and PIK3CA mutations
on the efficacy of cetuximab plus chemotherapy in
chemotherapy-refractory metastatic colorectal cancer:
a retrospective consortium analysis. Lancet Oncol.
2010;11(8):753–762.
321. Ogino S, Shima K, Meyerhardt JA, et al. Predictive
and prognostic roles of BRAF mutation in stage III
colon cancer: results from intergroup trial CALGB
89803. Clin Cancer Res. 2012;18(3):890–900.
322. Yamauchi M, Lochhead P, Morikawa T, et al.
Colorectal cancer: a tale of two sides or a continuum?
Gut. 2012;61(6):794–797.
323. Yamauchi M, Morikawa T, Kuchiba A, et al. Assess-
ment of colorectal cancer molecular features along
bowel subsites challenges the conception of distinct
dichotomy of proximal versus distal colorectum.
Gut. 2012;61(6):847–854.
324. Samowitz WS, Sweeney C, Herrick J, et al. Poor
survival associated with the BRAF V600E mutation
in microsatellite-stable colon cancers. Cancer Res.
2005;65(14):6063–6069.
325. Ogino S, Nosho K, Kirkner GJ, et al. CpG island
methylator phenotype, microsatellite instability,
BRAF mutation and clinical outcome in colon
cancer. Gut. 2009;58(1):90–96.
326. Saridaki Z, Papadatos-Pastos D, Tzardi M, et al. BRAF
mutations, microsatellite instability status and cyclin
D1 expression predict metastatic colorectal patients’
outcome. Br J Cancer. 2010;102(12):1762–1768.
327. French AJ, Sargent DJ, Burgart LJ, et al. Prognostic
significance of defective mismatch repair and BRAF
V600E in patients with colon cancer. Clin Cancer
Res. 2008;14(11):3408–3415.
328. Weisenberger DJ, Siegmund KD, Campan M, et al.
CpG island methylator phenotype underlies sporadic
microsatellite instability and is tightly associated with
BRAF mutation in colorectal cancer. Nat Genet.
2006;38(7):787–793.
329. Wang L, Cunningham JM, Winters JL, et al. BRAF
mutations in colon cancer are not likely attributable
to defective DNA mismatch repair. Cancer Res. 2003;
63(17):5209–5212.
330. Di Nicolantonio F, Martini M, Molinari F, et al.
Wild-type BRAF is required for response to panitu-
mumab or cetuximab in metastatic colorectal cancer.
J Clin Oncol. 2008;26(35):5705–5712.
331. Laurent-Puig P, Cayre A, Manceau G, et al. Analysis
of PTEN, BRAF, and EGFR status in determining
benefit from cetuximab therapy in wild-type KRAS
metastatic colon cancer. J Clin Oncol. 2009;27(35):
5924–5930.
332. Sartore-Bianchi A, Di Nicolantonio F, Nichelatti
M, et al. Multi-determinants analysis of molecular
alterations for predicting clinical benefit to EGFR-
targeted monoclonal antibodies in colorectal cancer.
PLoS ONE. 2009;4(10):e7287.
333. Prahallad A, Sun C, Huang S, et al. Unresponsive-
ness of colon cancer to BRAF(V600E) inhibition
through feedback activation of EGFR. Nature.
2012;483(7387):100–103.
334. Comprehensive molecular characterization of human
colon and rectal cancer. Nature. 2012;487(7407):
330–337.
335. Travis WD, Brambilla E, Noguchi M, et al.
International Association for the Study of Lung
Cancer/American Thoracic Society/European
Respiratory Society international multidisciplinary
classification of lung adenocarcinoma. J Thorac
Oncol. 2011;6(2):244–285.
336. Cohen S. Isolation of a mouse submaxillary gland
protein accelerating incisor eruption and eyelid
opening in the new-born animal. J Biol Chem.
1962;237:1555–1562.
337. Carpenter G, Lembach KJ, Morrison MM, Cohen
S. Characterization of the binding of 125-I-labeled
epidermal growth factor to human fibroblasts. J Biol
Chem. 1975;250(11):4297–4304.
338. Ullrich A, Coussens L, Hayflick JS, et al. Human epi-
dermal growth factor receptor cDNA sequence and
aberrant expression of the amplified gene in A431
epidermoid carcinoma cells. Nature. 1984;309(5967):
418–425.
339. Franklin WA, Veve R, Hirsch FR, Helfrich BA,
Bunn PA Jr. Epidermal growth factor receptor family
in lung cancer and premalignancy. Semin Oncol.
2002;29(1 suppl 4):3–14.
340. Hirsch FR, Varella-Garcia M, Bunn PA Jr, et al.
Epidermal growth factor receptor in non-small-
cell lung carcinomas: correlation between gene
copy number and protein expression and impact
on prognosis. J Clin Oncol. 2003;21(20):3798–
3807.
341. Paez JG, Janne PA, Lee JC, et al. EGFR mutations
in lung cancer: correlation with clinical response
to gefitinib therapy. Science. 2004;304(5676):
1497–1500.
342. Lynch TJ, Bell DW, Sordella R, et al. Activating
mutations in the epidermal growth factor receptor
underlying responsiveness of non-small-cell lung
cancer to gefitinib. N Engl J Med. 2004;350(21):
2129–2139.
343. Mok TS, Wu YL, Thongprasert S, et al. Gefitinib or
carboplatin-paclitaxel in pulmonary adenocarcinoma.
N Engl J Med. 2009;361(10):947–957.
344. Bean J, Brennan C, Shih JY, et al. MET amplifica-
tion occurs with or without T790M mutations in
EGFR mutant lung tumors with acquired resistance
to gefitinib or erlotinib. Proc Natl Acad Sci USA.
2007;104(52):20932–20937.
345. Nguyen KS, Kobayashi S, Costa DB. Acquired resis-
tance to epidermal growth factor receptor tyrosine
kinase inhibitors in non-small-cell lung cancers
dependent on the epidermal growth factor receptor
pathway. Clin Lung Cancer. 2009;10(4):281–289.
346. Suda K, Murakami I, Katayama T, et al. Reciprocal
and complementary role of MET amplification and
EGFR T790M mutation in acquired resistance to
kinase inhibitors in lung cancer. Clin Cancer Res.
2010;16(22):5489–5498.
347. Pao W, Miller VA, Politi KA, et al. Acquired
resistance of lung adenocarcinomas to gefitinib or
erlotinib is associated with a second mutation in the
EGFR kinase domain. PLoS Med. 2005;2(3):e73.
348. Yasuda H, Kobayashi S, Costa DB. EGFR exon 20
insertion mutations in non-small-cell lung cancer:
preclinical data and clinical implications. Lancet
Oncol. 2012;13(1):e23–e31.
349. Sequist LV, Waltman BA, Dias-Santagata D, et al.
Genotypic and histological evolution of lung cancers
acquiring resistance to EGFR inhibitors. Sci Transl
Med. 2011;3(75):75ra26.
350. Morgillo F, Della Corte CM, Fasano M, Ciardiello
F. Mechanisms of resistance to EGFR-targeted drugs:
lung cancer. ESMO Open. 2016;1(3):e000060.
351. Ahrendt SA, Decker PA, Alawi EA, et al. Cigarette
smoking is strongly associated with mutation of the
K-ras gene in patients with primary adenocarcinoma
of the lung. Cancer. 2001;92(6):1525–1530.
352. Riely GJ, Kris MG, Rosenbaum D, et al. Frequency
and distinctive spectrum of KRAS mutations in never
smokers with lung adenocarcinoma. Clin Cancer
Res. 2008;14(18):5731–5734.
353. Benesova L, Minarik M, Jancarikova D, Belsanova B,
Pesek M. Multiplicity of EGFR and KRAS mutations
in non-small cell lung cancer (NSCLC) patients
treated with tyrosine kinase inhibitors. Anticancer
Res. 2010;30(5):1667–1671.
354. Roberts PJ, Stinchcombe TE, Der CJ, Socinski MA.
Personalized medicine in non-small-cell lung cancer:
is KRAS a useful marker in selecting patients for
epidermal growth factor receptor-targeted therapy?
J Clin Oncol. 2010;28(31):4769–4777.
355. Soda M, Choi YL, Enomoto M, et al. Identification
of the transforming EML4-ALK fusion gene in
non-small-cell lung cancer. Nature. 2007;448(7153):
561–566.
356. Rodig SJ, Mino-Kenudson M, Dacic S, et al. Unique
clinicopathologic features characterize ALK-rear-
ranged lung adenocarcinoma in the western popula-
tion. Clin Cancer Res. 2009;15(16):5216–5223.
357. Sasaki T, Rodig SJ, Chirieac LR, Janne PA. The
biology and treatment of EML4-ALK non-small
cell lung cancer. Eur J Cancer. 2010;46(10):1773–
1780.
358. Takahashi T, Sonobe M, Kobayashi M, et al.
Clinicopathologic features of non-small-cell lung
cancer with EML4-ALK fusion gene. Ann Surg
Oncol. 2010;17(3):889–897.
359. Pillai RN, Ramalingam SS. The biology and clinical
features of non-small cell lung cancers with EML4-
ALK translocation. Curr Oncol Rep. 2012;14(2):
105–110.
360. Soda M, Takada S, Takeuchi K, et al. A mouse model
for EML4-ALK-positive lung cancer. Proc Natl Acad
Sci USA. 2008;105(50):19893–19897.
361. Sanders HR, Li HR, Bruey JM, et al. Exon scanning
by reverse transcriptase-polymerase chain reaction for
detection of known and novel EML4-ALK fusion
variants in non-small cell lung cancer. Cancer Genet.
2011;204(1):45–52.
362. Lin JJ, Riely GJ, Shaw AT. Targeting ALK: precision
medicine takes on drug resistance. Cancer Discov.
2017;7(2):137–155.

363. Villaruz LC, Socinski MA, Abberbock S, et al.
Clinicopathologic features and outcomes of patients
with lung adenocarcinomas harboring BRAF muta-
tions in the Lung Cancer Mutation Consortium.
Cancer. 2015;121(3):448–456.
364. Nguyen-Ngoc T, Bouchaab H, Adjei AA, Peters
S. BRAF alterations as therapeutic targets in non-
small-cell lung cancer. J Thorac Oncol. 2015;10(10):
1396–1403.
365. Ou SI, Schrock AB, Bocharov EV, et al. HER2
transmembrane domain (TMD) mutations (V659/
G660) that stabilize homo- and heterodimerization
are rare oncogenic drivers in lung adenocarcinoma
that respond to afatinib. J Thorac Oncol. 2017;
12(3):446–457.
366. Vaishnavi A, Capelletti M, Le AT, et al. Oncogenic
and drug-sensitive NTRK1 rearrangements in lung
cancer. Nat Med. 2013;19(11):1469–1472.
367. Ohashi K, Sequist LV, Arcila ME, et al. Character-
istics of lung cancers harboring NRAS mutations.
Clin Cancer Res. 2013;19(9):2584–2591.
368. Rosell R, Karachaliou N. RET inhibitors for
patients with RET fusion-positive and RET wild-
type non-small-cell lung cancer. Lancet Oncol.
2016;17(12):1623–1625.
369. Shaw AT, Ou SH, Bang YJ, et al. Crizotinib in
ROS1-rearranged non-small-cell lung cancer. N Engl
J Med. 2014;371(21):1963–1971.
370. Pao W, Girard N. New driver mutations in non-small-
cell lung cancer. Lancet Oncol. 2011;12(2):175–180.
371. Aisner D, Sholl LM, Berry LD, et al. Effect of
expanded genomic testing in lung adenocarcinoma
(LUCA) on survival benefit: the Lung Cancer Muta-
tion Consortium II (LCMC II) experience. J Clin
Oncol. 2016;34(15_suppl):11510.
372. Ettinger DS, Wood DE, Aisner DL, et al. Non-Small
Cell Lung Cancer, Version 5.2017, NCCN Clinical
Practice Guidelines in Oncology. J Natl Compr Canc
Netw. 2017;15(4):504–535.
373. Woodman SE, Lazar AJ, Aldape KD, Davies MA.
New strategies in melanoma: molecular testing in
advanced disease. Clin Cancer Res. 2012;18(5):
1195–1200.
374. Long GV, Menzies AM, Nagrial AM, et al. Prognostic
and clinicopathologic associations of oncogenic
BRAF in metastatic melanoma. J Clin Oncol. 2011;
29(10):1239–1246.
375. Shinozaki M, Fujimoto A, Morton DL, Hoon DS.
Incidence of BRAF oncogene mutation and clinical
relevance for primary cutaneous melanomas. Clin
Cancer Res. 2004;10(5):1753–1757.
376. Edlundh-Rose E, Egyhazi S, Omholt K, et al. NRAS
and BRAF mutations in melanoma tumours in
relation to clinical characteristics: a study based on
mutation screening by pyrosequencing. Melanoma
Res. 2006;16(6):471–478.
377. Hocker T, Tsao H. Ultraviolet radiation and mela-
noma: a systematic review and analysis of reported
sequence variants. Hum Mutat. 2007;28(6):578–588.
378. Hauschild A, Grob JJ, Demidov LV, et al. Dabrafenib
in BRAF-mutated metastatic melanoma: a multi-
centre, open-label, phase 3 randomised controlled
trial. Lancet. 2012;380(9839):358–365.
379. Sosman JA, Kim KB, Schuchter L, et al. Survival in
BRAF V600-mutant advanced melanoma treated with
vemurafenib. N Engl J Med. 2012;366(8):707–714.
380. Long GV, Trefzer U, Davies MA, et al. Dabrafenib in
patients with Val600Glu or Val600Lys BRAF-mutant
melanoma metastatic to the brain (BREAK-MB): a
multicentre, open-label, phase 2 trial. Lancet Oncol.
2012.
381. Flaherty KT, Infante JR, Daud A, et al. Combined
BRAF and MEK inhibition in melanoma with BRAF
V600 mutations. N Engl J Med. 2012;367(18):
1694–1703.
382. Flaherty KT, Fisher DE. New strategies in
metastatic melanoma: oncogene-defined taxonomy
leads to therapeutic advances. Clin Cancer Res.
2011;17(15):4922–4928.
Pathology, Biomarkers, and Molecular Diagnostics  •  CHAPTER 15 253.e7
383. Curtin JA, Busam K, Pinkel D, Bastian BC. Somatic
activation of KIT in distinct subtypes of melanoma.
J Clin Oncol. 2006;24(26):4340–4346.
384. Beadling C, Jacobson-Dunlop E, Hodi FS, et al.
KIT gene mutations and copy number in melanoma
subtypes. Clin Cancer Res. 2008;14(21):6821–
6828.
385. Carvajal RD, Antonescu CR, Wolchok JD, et al.
KIT as a therapeutic target in metastatic melanoma.
JAMA. 2011;305(22):2327–2334.
386. Guo J, Si L, Kong Y, et al. Phase II, open-label,
single-arm trial of imatinib mesylate in patients with
metastatic melanoma harboring c-Kit mutation or
amplification. J Clin Oncol. 2011;29(21):2904–2909.
387. Marshall CJ, Hall A, Weiss RA. A transforming
gene present in human sarcoma cell lines. Nature.
1982;299(5879):171–173.
388. Hall A, Marshall CJ, Spurr NK, Weiss RA.
Identification of transforming gene in two human
sarcoma cell lines as a new member of the ras gene
family located on chromosome 1. Nature. 1983;
303(5916):396–400.
389. Padua RA, Barrass N, Currie GA. A novel transform-
ing gene in a human malignant melanoma cell line.
Nature. 1984;311(5987):671–673.
390. Pollock PM, Harper UL, Hansen KS, et al. High
frequency of BRAF mutations in nevi. Nat Genet.
2003;33(1):19–20.
391. Bauer J, Curtin JA, Pinkel D, Bastian BC. Congenital
melanocytic nevi frequently harbor NRAS muta-
tions but no BRAF mutations. J Invest Dermatol.
2007;127(1):179–182.
392. Van Raamsdonk CD, Bezrookove V, Green G, et al.
Frequent somatic mutations of GNAQ in uveal
melanoma and blue naevi. Nature. 2009;457(7229):
599–602.
393. Van Raamsdonk CD, Griewank KG, Crosby MB,
et al. Mutations in GNA11 in uveal melanoma. N
Engl J Med. 2010;363(23):2191–2199.
394. Harbour JW, Onken MD, Roberson ED, et al.
Frequent mutation of BAP1 in metastasizing uveal
melanomas. Science. 2010;330(6009):1410–1413.
395. Wiesner T, Obenauf AC, Murali R, et al. Germline
mutations in BAP1 predispose to melanocytic
tumors. Nat Genet. 2011;43(10):1018–1021.
396. Cancer Genome Atlas Research Network, Brat
DJ, Verhaak RG, et al. Comprehensive, integrative
genomic analysis of diffuse lower-grade gliomas. N
Engl J Med. 2015;372(26):2481–2498.
397. Brennan CW, Verhaak RG, McKenna A, et al. The
somatic genomic landscape of glioblastoma. Cell.
2013;155(2):462–477.
398. Louis DN, Ohgaki H, Wiestler OD, Cavenee WK,
eds. WHO Classification of Tumours of the Central
Nervous System. Vol 4th ed. Lyon, France: IARC;
2016. Revised 4th edn 10–122 (IARC, 2016).
399. Viswanath P, Chaumeil MM, Ronen SM. Molecular
Imaging of Metabolic Reprograming in Mutant IDH
Cells. Front Oncol. 2016;6:60.
400. Reuss DE, Sahm F, Schrimpf D, et al. ATRX and
IDH1-R132H immunohistochemistry with subse-
quent copy number analysis and IDH sequencing
as a basis for an “integrated” diagnostic approach
for adult astrocytoma, oligodendroglioma and glio-
blastoma. Acta Neuropathol. 2015;129(1):133–146.
401. Reuss DE, Mamatjan Y, Schrimpf D, et al. IDH
mutant diffuse and anaplastic astrocytomas have
similar age at presentation and little difference
in survival: a grading problem for WHO. Acta
Neuropathol. 2015;129(6):867–873.
402. Reuss DE, Kratz A, Sahm F, et al. Adult IDH
wild type astrocytomas biologically and clinically
resolve into other tumor entities. Acta Neuropathol.
2015;130(3):407–417.
403. Yan H, Parsons DW, Jin G, et al. IDH1 and
IDH2 mutations in gliomas. N Engl J Med.
2009;360(8):765–773.
404. Lai A, Kharbanda S, Pope WB, et al. Evidence for
sequenced molecular evolution of IDH1 mutant
253.e7
glioblastoma from a distinct cell of origin. J Clin
Oncol. 2011;29(34):4482–4490.
405. Ichimura K, Pearson DM, Kocialkowski S, et al.
IDH1 mutations are present in the majority
of common adult gliomas but rare in primary
glioblastomas. Neuro Oncol. 2009;11(4):341–347.
406. Balss J, Meyer J, Mueller W, Korshunov A, Hartmann
C, von Deimling A. Analysis of the IDH1 codon
132 mutation in brain tumors. Acta Neuropathol.
2008;116(6):597–602.
407. Nobusawa S, Watanabe T, Kleihues P, Ohgaki
H. IDH1 mutations as molecular signature and
predictive factor of secondary glioblastomas. Clin
Cancer Res. 2009;15(19):6002–6007.
408. Capper D, Weissert S, Balss J, et al. Characteriza-
tion of R132H mutation-specific IDH1 antibody
binding in brain tumors. Brain Pathol. 2010;20(1):
245–254.
409. von Deimling A, Louis DN, von Ammon K, Petersen
I, Wiestler OD, Seizinger BR. Evidence for a tumor
suppressor gene on chromosome 19q associated with
human astrocytomas, oligodendrogliomas, and mixed
gliomas. Cancer Res. 1992;52(15):4277–4279.
410. Rosenberg JE, Lisle DK, Burwick JA, et al. Refined
deletion mapping of the chromosome 19q glioma
tumor suppressor gene to the D19S412-STD
interval. Oncogene. 1996;13(11):2483–2485.
411. Kraus JA, Koopmann J, Kaskel P, et al. Shared
allelic losses on chromosomes 1p and 19q suggest
a common origin of oligodendroglioma and oligoas-
trocytoma. J Neuropathol Exp Neurol. 1995;54(1):
91–95.
412. Reifenberger J, Reifenberger G, Liu L, James
CD, Wechsler W, Collins VP. Molecular genetic
analysis of oligodendroglial tumors shows prefer-
ential allelic deletions on 19q and 1p. Am J Pathol.
1994;145(5):1175–1190.
413. Cairncross JG, Ueki K, Zlatescu MC, et al. Specific
genetic predictors of chemotherapeutic response and
survival in patients with anaplastic oligodendroglio-
mas. J Natl Cancer Inst. 1998;90(19):1473–1479.
414. Fallon KB, Palmer CA, Roth KA, et al. Prognostic
value of 1p, 19q, 9p, 10q, and EGFR-FISH analyses
in recurrent oligodendrogliomas. J Neuropathol Exp
Neurol. 2004;63(4):314–322.
415. van den Bent MJ, Weller M, Wen PY, Kros JM,
Aldape K, Chang S. A clinical perspective on the
2016 WHO brain tumor classification and routine
molecular diagnostics. Neuro Oncol. 2017.
416. Lee J, Solomon DA, Tihan T. The role of histone
modifications and telomere alterations in the patho-
genesis of diffuse gliomas in adults and children.
J Neurooncol. 2017;132(1):1–11.
417. Wu G, Broniscer A, McEachron TA, et al. Somatic
histone H3 alterations in pediatric diffuse intrinsic
pontine gliomas and non-brainstem glioblastomas.
Nat Genet. 2012;44(3):251–253.
418. Khuong-Quang DA, Buczkowicz P, Rakopoulos
P, et al. K27M mutation in histone H3.3 defines
clinically and biologically distinct subgroups of
pediatric diffuse intrinsic pontine gliomas. Acta
Neuropathol. 2012;124(3):439–447.
419. Jiao Y, Killela PJ, Reitman ZJ, et al. Frequent
ATRX, CIC, FUBP1 and IDH1 mutations refine
the classification of malignant gliomas. Oncotarget.
2012;3(7):709–722.
420. Liu XY, Gerges N, Korshunov A, et al. Frequent
ATRX mutations and loss of expression in adult
diffuse astrocytic tumors carrying IDH1/IDH2 and
TP53 mutations. Acta Neuropathol. 2012;124(5):
615–625.
421. Schindler G, Capper D, Meyer J, et al. Analysis
of BRAF V600E mutation in 1,320 nervous
system tumors reveals high mutation frequencies
in pleomorphic xanthoastrocytoma, ganglioglioma
and extra-cerebellar pilocytic astrocytoma. Acta
Neuropathol. 2011;121(3):397–405.
422. Kleinschmidt-DeMasters BK, Aisner DL, Birks
DK, Foreman NK. Epithelioid GBMs show a high
253.e8 Part I: Science and Clinical Oncology
percentage of BRAF V600E mutation. Am J Surg
Pathol. 2013;37(5):685–698.
423. Robinson GW, Orr BA, Gajjar A. Complete clini-
cal regression of a BRAF V600E-mutant pediatric
glioblastoma multiforme after BRAF inhibitor
therapy. BMC Cancer. 2014;14:258.
424. Lee EQ, Ruland S, LeBoeuf NR, Wen PY, Santagata
S. Successful treatment of a progressive BRAF
V600E-mutated anaplastic pleomorphic xanthoas-
trocytoma with vemurafenib monotherapy. J Clin
Oncol. 2016;34(10):e87–e89.
425. Chamberlain MC. Salvage therapy with BRAF
inhibitors for recurrent pleomorphic xanthoastro-
cytoma: a retrospective case series. J Neurooncol.
2013;114(2):237–240.
426. Lassaletta A, Guerreiro Stucklin A, Ramaswamy V,
et al. Profound clinical and radiological response to
BRAF inhibition in a 2-month-old diencephalic
child with hypothalamic/chiasmatic glioma. Pediatr
Blood Cancer. 2016;63(11):2038–2041.
427. Olow A, Mueller S, Yang X, et al. BRAF Status
in personalizing treatment approaches for
pediatric gliomas. Clin Cancer Res. 2016;22(21):
5312–5321.
428. Miller C, Guillaume D, Dusenbery K, Clark HB,
Moertel C. Report of effective trametinib therapy
in 2 children with progressive hypothalamic optic
pathway pilocytic astrocytoma: documentation of
volumetric response. J Neurosurg Pediatr. 2017;19(3):
319–324.
429. Wu J, Armstrong TS, Gilbert MR. Biology and man-
agement of ependymomas. Neuro Oncol. 2016;18(7):
902–913.
430. Ostrom QT, Gittleman H, Liao P, et al. CBTRUS
statistical report: primary brain and central nervous
system tumors diagnosed in the United States in
2007-2011. Neuro Oncol. 2014;16(suppl 4):iv1–iv63.
431. Parker M, Mohankumar KM, Punchihewa C, et al.
C11orf95-RELA fusions drive oncogenic NF-kappaB
signalling in ependymoma. Nature. 2014;506(7489):
451–455.
432. Pajtler KW, Witt H, Sill M, et al. Molecular
classification of ependymal tumors across all CNS
compartments, histopathological grades, and age
groups. Cancer Cell. 2015;27(5):728–743.
 16  Imaging
Richard L. Wahl
S UMMARY OF K EY P OI N T S
• Noninvasive medical imaging often is
essential to cancer management at
multiple times in the course of the
illness.
• Imaging currently is used for
screening to detect cancer,
characterizing lesions, performing
locoregional and systemic staging,
providing prognostic information,
assessing response during and after
therapy, restaging after treatment,
performing follow-up of patients for
recurrence, and precisely guiding
biopsies and therapies such as
external beam or systemic radiation,
brachytherapy, or thermal and other
ablations.
• More invasive interventional
radiologic procedures also can guide
and be used to monitor and deliver
vascular or intraluminal delivery of
treatments such as radioactive
microspheres, embolic materials,
radiofrequency ablation or
cryoablation, and therapeutic drugs.
• Imaging methods range from the
traditional anatomic methods—
radiography, computed tomography
(CT), and ultrasonography—to the
more functional methods of magnetic
resonance imaging (MRI) and nuclear
medicine methods, including
positron emission tomography (PET),
single-photon emission computed
tomography (SPECT), and planar
nuclear imaging. Hybrid methods
combining PET and CT, SPECT and
CT, and PET and MRI are growing in
importance. Optical imaging is
promising but remains limited, by
limited penetration of light through
tissues, to superficial structures in
most cases.
• Plain films and mammography
remain useful techniques, with
mammography (including digital
mammography and tomosynthesis)
254
being the main imaging method and
clearly proven capable of reducing
cancer deaths when applied in the
screening setting. A downside of
screening imaging is the small but
real risk of “overdiagnosis” and
“overtreatment”—detection and
aggressive treatment of cancers that
may not be life-threatening.
• CT remains a cornerstone
technology for most oncologic
imaging, and CT technology allowing
for rapid-sequence angiography is
finding new applications, as is
three-dimensional reconstruction of
CT data sets. Screening data with
CT colonography continues to
improve, and in some studies it has
been found to be comparable to
traditional colonoscopy for colon
cancer screening. CT scanning for
lung cancer screening reduces lung
cancer death rates when applied to
high-risk populations. The radiation
dose from CT is a concern, and
major efforts to reduce the dose
from CT scanning have been
implemented in newer CT systems.
• MRI is the imaging tool of choice for
central nervous system, spinal, and
musculoskeletal neoplasms, as well
as for assessment of vascular and
some hepatobiliary and pelvic
lesions. MRI also can be used frequency in the staging and
to detect breast cancers, follow-up of lung, colorectal, and
especially in women with dense
breasts. Concerns regarding
gadolinium-associated nephrogenic
systemic fibrosis (NSF) have led to
caution in the use of MRI contrast
medium in patients with impaired
renal function. There are also minor
concerns regarding gadolinium
accumulation in the brain, although
these are of no known clinical
significance. Newer MRI techniques
such as diffusion imaging
complement diffusion contrast
enhancement (DCE) MRI and appear
promising in assessing response to
tumor treatment. Multiparametric
MRI sequences are being used more
frequently to characterize the
prostate to determine if and where
biopsies should be performed
and to help in conducting active
surveillance, delaying or reducing
prostatectomies.
• Bone scans using single-photon
methods (technetium-99m [99mTc]
methylene diphosphonate) remain
the dominant procedure for detecting
suspected bone metastases;
however, the PET agent fluorine-18
(18F) sodium fluoride is increasingly
applied since its approval by the US
Food and Drug Administration (FDA).
These techniques may be less
sensitive for marrow involvement
than MRI and other PET techniques
for detecting bone metastases of
many tumors.
• PET and PET-CT technology using
18
F-fluorodeoxyglucose (FDG)
continues to grow in a wide variety
of applications, and its use is
becoming increasingly routine in the
management of patients with cancer
at varying states of the disease
process. PET is used with increasing
head and neck cancers, as well as
lymphomas and other types of
tumors, and is now a routine tool in
lymphoma management at several
points in the disease. PET with
non-FDG tracers is a promising
research area with growing clinical
applications. There has been
particular progress in imaging of
prostate cancer with several imaging
agents including FDA-approved C-11
choline.
 Imaging  •  CHAPTER 16 255

• The fusion of anatomic and
functional images to create hybrid
“anatomolecular images” with
software or dedicated instruments
such as PET-CT, SPECT-CT, or the
newer PET-MRI devices also is
seeing rapid growth in applications
in cancer imaging. Fully diagnostic
CT scans coupled with PET imaging
in the form of PET-CT often provide
valuable composite imaging for
cancer management. PET-MRI is an
evolving technology, but fully
integrated simultaneous systems are
being used much more widely.
• Imaging management for staging
lung cancer and characterizing
solitary pulmonary nodules often
includes FDG-PET in addition to CT
when the technology is available
because PET-CT has higher
accuracy in lung cancer
assessments compared with CT.
• Imaging management of suspected
recurrences of colorectal cancer,
head and neck cancer, lymphoma,
and many other cancers often now
includes the use of PET in addition
to CT. Negative FDG-PET scans of
the neck after chemoradiotherapy
can obviate the need for surgery in
patients with head and neck cancer.
Response criteria for FDG-avid
lymphomas are now mainly PET
based, and PET assessments of
treatment response are increasingly
applied. Use of PET at earlier stages
in the workup is becoming
increasingly common, as is the use
of PET in early assessments of the
efficacy of cancer therapies.
Adapting treatments based on the
response seen on PET-CT is also
increasingly applied with treatment
deintensification approaches for
rapid responders, and treatment
intensification for poor responders
being informed by PET.
Neuroendocrine tumors are now
diagnosed with gallium-68 (68Ga)
radiopeptides binding to the
somatostatin receptors, and these
tumors can be treated with
radiopeptides labeled with
lutetium-177 (177Lu), among other
therapeutic agents.
• In prostate cancer, available imaging
methods remain suboptimal for
detection of primary tumor and early
determination of local or systemic
tumor spread. MRI nodal contrast
agents are promising but not yet
routinely available, and magnetic
resonance spectroscopy has had
only limited success in the prostate.
A variety of MRI sequences including
T2 images, diffusion images, and
DCE MRI appear to improve on
purely anatomic MRI approaches for
lesion detection and detection of
extracapsular involvement. More
standardized reading systems such
as Prostate Imaging–Reporting and
Data System (PI-RADS) may lead to
more consistent use of this
technology to help differentiate
low- and high-risk tumors from each
other and inform potential active
surveillance choices. A variety of
innovative radiotracers for PET show
promise for detecting disease
recurrence, and C-11 choline is now
FDA approved in the United States
for use in prostate cancer, as is the
synthetic amino acid fluciclovine.
68
Ga- or 18F-radiolabeled peptides
with binding to the prostate cancer
prostate-specific membrane antigen
(PSMA) molecule show great
promise diagnostically and can guide
radiopeptide therapies.
• Visceral angiography for diagnostic
purposes has been substantially
supplanted by CT and MRI methods;
however, it remains important as a
tool for intravascular delivery of
therapies such as chemotherapy,
coils, or radioactive microspheres.
• CT, ultrasonography (including with
ultrasonographic contrast media),
fluoroscopy, and innovative MRI
systems can guide interventional
procedures such as thermal and
cryotherapeutic lesion ablations.
Bifunctional methods including a
radionuclide probe and an optical
probe may guide diagnosis and
surgery.
• Highly specific probe-reporter
systems are being developed to
allow for optical and radionuclide
imaging of transfected gene
biodistribution and function. These
approaches face major regulatory
challenges when being translated to
humans.
• Combined anatomic and functional
information is being applied to allow
for more precise planning of external
beam radiation therapy, including
intensity-modulated radiation therapy
(IMRT) and conformal therapy,
methods that potentially allow for
increasing dose escalation and
minimization of toxicity to normal
tissues.
• Emerging imaging methods are
proving increasingly useful in
providing information on the
physiology and molecular
characteristics of lesions. This
means that a multiparametric
biologic imaging phenotype for
tumors can be obtained and makes
it possible to display heterogeneities
in tumors. Given the complexity and
multidimensionality of these imaging
data, artificial intelligence (AI)
approaches are being applied to
augment radiologist performance.
“Radiomics” is the emerging field of
linking detailed analysis of
quantitative images to tumor
genetics and biology. Imaging
phenotypes can more precisely
guide individualized tumor treatment
to yield a higher probability of
success without excessive toxicity
for treatment of the selected
neoplastic process.

Noninvasive imaging is of fundamental and increasing importance in
the daily management of the patient with cancer. Although physical
examination and laboratory diagnosis remain key for planning treat-
ment, for solid tumor management, imaging tests represent a major
objective metric of disease presence or absence and activity and may
be used at different times during the course of the disease to monitor
the efficacy (or lack of efficacy) of treatment. Imaging to determine
tumor size is an objective end point in disease management used to
compare different types of cancer treatment and treatment across
institutions. The use of imaging also is increasing in the drug develop-
ment process and in developing new cancer therapies.
Specific clinical questions addressed by imaging include screening
for the presence of cancer (e.g., breast, lung, or colorectal), characterizing
anatomic lesions as malignant or benign, and staging a neoplasm—that
is, determining the size and local extent of a primary lesion and
determining whether it is localized or locoregionally or systemically
metastatic. Such studies are essential for determining whether the
patient is a candidate for surgical resection, identifying the extent of
the field for radiation therapy, and determining whether systemic
chemotherapy is appropriate. Initial staging of tumor size and extent
also can provide important prognostic data. During the course of
treatment, imaging is used to determine response of the cancer. Imaging
256 Part I: Science and Clinical Oncology
Table 16.1 Imaging in Cancer: Key Current Clinical
Uses in Cancer Management
• Screening
• Lesion characterization: malignant or benign, size, local invasion
• Tumor staging: locoregional, systemic, at initial presentation or on
retreatment
• Size and extent of tumor: to plan radiation or other local therapy
• Prognostic information
• Defining sites for biopsy and subsequent analysis by pathology
• Guidance of interventional therapy
• Assessment of response to treatment
• Restaging tumor after treatment
• Assessment of normal organ function or status before, during, and
after treatment
• Assessment for toxicity or complications of treatment
90% SENSITIVE AND SPECIFIC TEST
• 50% Prevalence of cancer in the population imaged
• 1000 Independent scans
• 50 False-positive findings in healthy patients (10% of 500)
• 450 True-positive findings in patients with disease (90% of 500)
• Positive predictive value (disease positive/test positive): 450/500
(90%)
• Possible conclusion: an excellent test!
90% SENSITIVE AND SPECIFIC TEST
• 10% Prevalence of cancer in the population imaged
• 1000 Independent scans
• 90 False-positive findings in healthy patients (10% of 900)
• 90 True-positive findings in patients with disease (10% of 900)
• Positive predictive value (disease positive/test positive): 50/100
(50%)
• Possible conclusion: a rather lousy test!
But these are the same test!
also is often used to follow patients for recurrence or development of
second malignancies. Imaging is being used more often as a method
to assist in delivery of minimally invasive therapeutic procedures to
ablate cancers, to guide radiation therapy, and to guide the dosage of
therapeutic drugs, including radiopharmaceuticals, more precisely.1
Imaging often is the best means of noninvasively identifying and
assessing tumors. With information gleaned from imaging studies,
the prognosis can be established and treatment decisions made with
greater certainty. Before discussing the varying imaging methods
available for patients with cancer, this chapter considers some general
principles that are applicable to all imaging tests.
Tasks for Imaging
The major roles of imaging in the current and evolving practice of
cancer management are shown in Table 16.1.
GENERAL CONSIDERATIONS
Performance of Imaging Tests
Noninvasive imaging is used to perform a wide variety of important
tasks. Although the best way to determine the medical usefulness of
a diagnostic test can be argued, a few key concepts are required to
understand and compare diagnostic tests. These can be applied to
one of the most basic tasks (i.e., determining whether tumor is present)
and also to the ability of imaging to predict resectability or response
to treatment.
Sensitivity
Sensitivity describes how often the imaging test would give a “positive
result” in a patient with cancer (i.e., true-positive finding). Ideally,
the test precisely detects and locates one or multiple cancers in a given
patient. Thus
% sensitivity = 100 × (test positive disease present )
Sensitivity can be calculated on a per-patient basis or a per–
malignant lesion basis. The per-patient basis most commonly is used
in screening studies for early diagnosis, whereas the per-lesion basis
may be used in patients expected to have multiple sites of tumor.
Per-lesion detection analyses can be misleading because they can be
heavily biased by a single patient’s results if that patient has multiple
tumor foci.
It can sometimes be difficult to judge how “good” a test is by
reading the literature. Sensitivity is supposed to be substantially
independent of study population composition, but as discussed in
the next section, certain imaging tests may be insensitive for some
very early-stage disease, but very, very sensitive for more advanced
disease. Each imaging test has a limit of detection threshold below
which tumors cannot be detected because they are not distinguishable
from the background tissues. Thus the patient population and very
often the tumor burden and average tumor size can make a difference
in the sensitivity of a test for detection of cancer. Virtually all non-
invasive imaging tests are less sensitive for small-volume disease than
for large-volume disease. For example, if an imaging test is used in a
patient population in which patients have advanced disease before
seeking medical attention (e.g., they are symptomatic at presentation),
the imaging test may have far greater sensitivity than if it were used
in patients with earlier-stage, smaller tumors. For example, positron
emission tomography (PET) with Fluorine-18 (18F)–fluorodeoxyglucose
(FDG) has been reported to be more than 90% sensitive for detecting
metastatic melanoma, but it is less than 20% sensitive in detecting
early (low tumor volume) nodal metastases of melanoma at initial
surgical resection. Mammography has higher sensitivity in women
with more radiolucent than radiodense breasts. A test with high sensitiv-
ity has a low number of false-negative results.2 The false-negative
fraction usually is expressed as 1 – sensitivity (in this case, sensitivity
being rated on a 0–1 scale).3
Specificity
Specificity is the frequency with which a test result is negative if
no disease is present, or the true-negative ratio. As a percentage,
specificity is
100 × (test negative disease negative)
Specificity can be calculated on a per-patient, per-lesion, or per-
region basis. The per-patient calculations commonly are performed
in the screening setting. They also can be done per region of the body
(e.g., Is the liver free of tumor? Are the draining lymph nodes free of
tumor?). It is technically difficult and sometimes impossible to know
exactly how many tumor foci are present because this depends on the
reference gold standard. It is not possible to perform “whole-body”
biopsies antemortem, so some very small tumor foci may not be
known to be present when disease is diagnosed. Specificity can be
affected substantially if the imaging test is used in a population that
has a characteristic that can result in false-positive results for the
imaging test. For example, inflammatory and infectious lung disease,
such as active tuberculosis (TB) or sarcoidosis, if present in a patient
population, can result in false-positive findings on PET or computed
tomography (CT) scans or other imaging methods. In this situation,
the specificity of FDG-PET, and likely of CT, for staging the
mediastinum for cancer would vary. Thus the specificity of PET for
assessing mediastinal lymph nodes may be much lower in areas of the

world with endogenous TB than in developed areas without it. Therefore
an imaging test that is very useful in one part of the world may be
far less useful in another part of the world. A highly specific test has
a low frequency of false-positive results (i.e., a low frequency of positive
test results in the patient population that does not have the disease).
The ideal imaging test has both high sensitivity and high specificity,
although none of our current imaging tests have perfect sensitivity
and specificity.2
Accuracy of Imaging
For detection of disease, a binary, yes-or-no answer as to whether
disease is present or absent is desirable. When such binary answers
can be provided, it is simple to mathematically provide an accuracy
value for a diagnostic imaging test. Thus accuracy is
100 × ( TP + TN ) ( TP + FP + TN + FN )
where TP = true positive, TN = true negative, FP = false positive,
and FN = false negative.
In this case, the number of patients with each finding is recorded.
A highly accurate test is one with a low prevalence of false-positive
and false-negative results.
Positive and Negative Predictive Values
Sensitivity and specificity define a test reasonably well, but its perfor-
mance in a specific patient is affected by the characteristics of the
population from which the patient is drawn. A physician normally
wants to know whether an individual patient has cancer and whether
the tumor is localized or metastatic (and where). The correct answer
is binary in most cases, but imaging does not always reveal the true
status of the individual patient. Thus the statistical likelihood of the
accuracy of the result might be conveyed in the clinical imaging test
report. This statistical likelihood will be related to the accuracy of the
test as well as to the patient population characteristics. Therefore the
positive predictive value often is of considerable clinical relevance.
For example, the positive predictive value of a test with 90% sensitivity
and 90% specificity will vary markedly, depending on the frequency
of disease in the population. With these test performance characteristics,
the clinician could reach two different conclusions regarding the
practical value of the same imaging test (Table 16.1).
A test that is effective in a patient population with a high prevalence
of a disease may be far less valuable in a patient population with a
lower prevalence of the same disease because there would be far too
many false-positive results. The most effective use of imaging technology
is in groups of patients in whom the imaging characteristics are expected
to be robust enough to allow for predictions in individual patients.
These challenges are particularly apparent when a test that was developed
and validated in a patient population with disease is used to evaluate
individuals with a lower prevalence of tumor (i.e., screening), or smaller
tumors. In this situation, the number of false-positive findings may
rise dramatically, sometimes nearly completely negating the value of
the test.4
Costs associated with a high false-positive rate can include excessive
biopsies, with both considerable economic and personal costs, as well
as an increased radiation dose in the population tested. Higher radiation
doses in a population may lead to a risk of an increased prevalence
of cancer. A clear balance must be achieved between when imaging
tests are applied, especially in patient groups with a low prevalence
of cancer, to minimize generating risks and maximize disease detection
with the test. Screening programs may have specific challenges discussed
later in this chapter. They may reveal disease that may be clinically
irrelevant, resulting in “overdiagnosis” and “overtreatment.” However,
proving overtreatment is challenging and ultimately requires randomized
trials. That said, high-level evidence exists that screening for breast
cancer and lung cancer in appropriate populations reduces cancer
Imaging  •  CHAPTER 16 257
death rates. In addition, strong data also exist to support virtual
colonoscopy for colorectal cancer screening.
Receiver Operating Characteristic Curves
Cancer imaging tests are interpreted by imaging specialists, often
radiologists. As with all of medicine, there is considerable science
involved in image interpretation, but the human element, or “art,”
as it is referred to in some settings, also is involved. In developed
countries, medical specialty boards have been established to ensure
that practitioners have a basal level of training and knowledge, thereby
providing some level of uniformity to image interpretations. However,
even with board certification and extensive training, not all imaging
specialists interpret a given imaging study in the same manner. Thus
although the goal of an imaging test often is a simple binary “yes,
there is tumor” or “no, there is not tumor” answer, there are varying
degrees of certainty in the interpretation of an image in most instances.
Some readers read with high sensitivity, whereas others read with high
specificity. Unless a test is very robust, it is hard to achieve both high
sensitivity and high specificity.5
An example of a receiver operating characteristic (ROC) curve is
shown in Fig. 16.1. This set of curves reflects the performance of PET
imaging in detecting axillary metastases in patients with newly diagnosed
breast cancer. The axes of the curves are the true-positive fraction
(sensitivity/100), which forms the y-axis, and the false-positive fraction
(1 – specificity/100), which forms the x-axis, on a scale of 0 to 1. A
perfect diagnostic test would yield no false-positive or false-negative
results. The greater the area under an ROC curve, the greater the
accuracy of the test.5
The results shown in Fig. 16.1 are from three readers who graded
PET scans using a five-point certainty scale (i.e., not a simple yes/no,
but a continuum from definitely abnormal to definitely normal). The
three readers had similar ROC curves, indicating that they were of
generally comparable accuracy. For the same test, however, two readers
may be reading at different points on the ROC curve, meaning that
one is more sensitive and one is more specific, but both are of equal
accuracy.
An excellent reader may have a greater area under the ROC curve
(AUC) than a less skilled reader, meaning that the more experienced
(and hopefully more capable) reader is both more sensitive and more
specific than a less experienced (and presumably less capable) reader.
However, virtually none of our imaging tests is perfect, and varying
1.0
0.9
0.8
0.7
0.6
TPF
0.5
0.4
0.3
0.2
Az0.76 Reader 1 Az0.70 Reader 3
0.1 Az0.75 Reader 2 Az0.95 Better test
0.0
0 0.2 0.4 0.6 0.8 1.0
FPF
Figure 16.1  •  Receiver operating characteristic (ROC) curves plotting
the true-positive fraction (TPF; sensitivity) versus the false-positive fraction
(FPF; 1 − specificity) for the three independent readers of the entire analysis
data set. A hypothetical curve for the test if it had 95% accuracy also is shown.
Az, Estimated area under the ROC curve.
258 Part I: Science and Clinical Oncology
“cut points” between disease and normalcy often are made, affecting
the overall performance of the test. In this study, the AUC of 0.7 to
0.76 was not viewed as sufficiently good for the task of nodal detection
of metastatic cancer spread to the axilla.6 Despite this, a very high
sensitivity or a very high specificity can be achieved depending on
which part of the curve one operates in. A higher, hypothetical curve,
with an AUC of 0.9, is shown for a more robust test, such as a
higher-resolution PET system devoted to imaging the axilla.
In practice, sentinel node sampling, often guided by imaging or
a radionuclide-sensitive probe system, is assuming a very important
role in this area of tumor staging.6 Practically, if a rather insensitive
test has a high positive predictive value, then the test may be of value
if the result is positive, but of little value if negative. For example, a
strongly positive PET scan for axillary metastases may obviate the
need for a pretreatment axillary dissection in a patient with newly
diagnosed advanced breast cancer in whom neoadjuvant chemotherapy
could be given.
Other Approaches to Assessing the Value
of Imaging
Although sensitivity, specificity, and accuracy commonly are used
to characterize the tumor detection process, other metrics may be
of greater importance. For example, some studies have focused on
how often imaging substantially changes management. This kind
of study is of great practical interest, but the optimal methods to
assess such changes in treatment decisions are evolving. Ideally,
one would like to show that the use of imaging, especially a new
imaging technology, when applied randomly to half of the study
population provided a reduction in the number of adverse events
in the imaged population, improved survival, or had comparable
outcomes at lower costs than standard treatments. As an example, a
reduction in the number of “futile thoracotomies” has been used as
a metric of success for PET versus CT in planning the treatment of
newly diagnosed lung cancer.7 Ideally, randomization of patients to
imaged versus not imaged groups can be shown to improve survival.
Performance of randomized trials in which a portion of the patients
undergo imaging and the other patients do not (or they obtain differ-
ent kinds of imaging), with an end point of survival, will be of great
interest. Unfortunately, such studies are complex or impossible, because
management of patients after imaging may be altered markedly based
on the imaging results. Therefore it can be difficult to separate the
imaging study effect from the treatment effect. Ultimately, however,
for some imaging studies to be adopted, such evaluations of survival
will be needed. This point is particularly relevant to screening, as
discussed later.
Registry data have been applied with substantial benefit to determine
if planned or actual patient management is altered through the use
of imaging tests. The National Oncologic PET Registry (NOPR) has
provided a great deal of information on the use of FDG-PET imaging
in the management of patients with a variety of cancers. The NOPR
collected questionnaire data from referring physicians on intended
patient management before and after PET. After 1 year, the cohort
included data from 22,975 studies (83.7% PET-CT) from 1178 centers.
Overall, physicians changed their intended management in 36.5%
(95% confidence interval [CI], 35.9–37.2) of cases after PET, supporting
the usefulness of PET for cancer imaging in registry cases, which are
from a wide range of sources.8
Screening Concepts and Challenges
Screening programs for cancer often have taken the form of laboratory
tests such as the Papanicolaou (Pap) smear, or, more recently, blood
tests for tumor markers. The success of the Pap smear in reducing
mortality rates from cervical cancer is incontrovertible. The use of
imaging in screening for cancer is an example of success and is of
considerable interest, but also is a source of considerable controversy.
As discussed in detail in the chapters on breast cancer and lung cancer,
screening mammography programs have been shown to be capable
of saving lives in women older than 50 years. These programs also
may save lives in women 40 to 50 years of age, but the data are less
compelling.9
Studies have been initiated in which CT scanning is used in an
attempt to detect early lung cancer.10 Screening high-risk populations
with CT imaging was proven to reduce lung cancer–specific mortality
in the National Lung Screening Trial (NLST). This followed results
from the Early Lung Cancer Action Project (ELCAP), a large study
screening patients at increased risk of lung cancer with low-dose CT,
which reported promising results in 1999.11 The ELCAP showed that
lung cancers are detected at a smaller size and that patients whose
cancers are detected by screening live longer following diagnosis than
patients whose tumors are not detected by screening. Whether this
translates into longer-term survival for the screened population remains
unclear. The ELCAP study further evaluated 31,567 asymptomatic
persons at risk for lung cancer with use of low-dose CT from 1993
through 2005, and from 1994 through 2005; 27,456 repeated screenings
were performed.12 A diagnosis of lung cancer was made in 484 par-
ticipants based on screening. Of particular note, 412 patients (85%)
had clinical stage I lung cancer. Ten-year survival approached 90%
in this group.11,12 This study demonstrated that annual spiral CT
screening can detect lung cancer that is curable.
The results of another large randomized trial of lung cancer screen-
ing, the NLST, were reported in 2011.13 In this trial, 53,454 persons
at high risk for lung cancer were enrolled from over 30 US sites. Study
participants were randomly assigned to undergo three annual screenings
with either low-dose CT (26,722 participants) or single-view postero-
anterior chest radiography (26,732); 24.2% and 6.9%, respectively,
of the CT-screened and chest radiography–screened groups had positive
screening studies at some point. There was a high false-positive screening
rate: 96.4% of the positive screening results in the low-dose CT group
and 94.5% in the radiography group. The incidence of lung cancer
was significantly higher (1060 versus 941 cancers) in the low-dose
CT group, as compared with the chest radiography group, There were
247 deaths from lung cancer per 100,000 person-years in the low-dose
CT group and 309 deaths per 100,000 person-years in the radiography
group, a relative reduction in mortality from lung cancer with low-dose
CT screening of 20.0% (95% CI, 6.8–26.7; P = .004). The rate of
death from any cause was reduced in the low-dose CT group as
compared with the radiography group by 6.7% (95% CI, 1.2–13.6;
P = .02).
This exciting trial has raised some controversies: the vast majority
of small pulmonary nodules identified are not malignant, the costs
of the screening and of the medical care for incidentally detected
lesions are substantial, and a substantial radiation burden, which in
principle could be carcinogenic, is delivered to patients undergoing
the screening. However, there is considerable hope that screening
programs can achieve a reduction in lung cancer mortality as the
radiation is given to patients later in their lives, when risks of radiation-
induced cancers are reduced. Indeed, the evidence supporting lung
cancer screening with CT was of sufficiently high quality to be included
as a covered benefit in the United States under insurance policies
consistent with the Affordable Care Act (ACA).
Based on the findings of the NLST, and the approval by US Medicare
of screening, many centers have offered lung cancer screening clinics
for high-risk patients. A risk, however, for lower-risk patients is that
screening may have an unacceptably high false-positive rate. It is
important to note that the promising results of NLST are for a higher-
risk population, and extrapolations of benefit to lower-risk populations
may be highly problematic.
As discussed later in this chapter, CT colonography holds excellent
promise as a screening method for colon cancer, as an alternative to
optical colonoscopy, and is increasingly being paid for by health insurers
based on its accuracy.
Other areas in which screening by noninvasive imaging has been
performed include colorectal cancer, where virtual colonoscopy can

be used to look for early colon cancers, and in the pelvis in women
who are at risk for ovarian cancer. More recently, screening CT centers
offering a virtual evaluation of the entire body have become available,
and even more recently, magnetic resonance imaging (MRI) and PET
screening have been offered in some locales. The growth of these
centers has been driven emotionally and economically; these tests
are not yet well founded scientifically in the screening setting. In
some cultures, screening imaging is done more commonly than in
others. The risk of false positives in patients at low cancer risk is
substantial.
Screening carries challenges, risks, and costs that are beyond the
scope of this overview chapter. However, several key points apply to
all screening approaches, including those using noninvasive imaging.
These points include (1) whether a screening program is reasonable
to consider; (2) lead-time bias; (3) length bias; (4) the overall economic
cost implications of screening, especially the costs of investigating
false-positive results, and (5) the risk that radiation used in screening
may include subsequent cancers.
The requirements that a screening program must meet to be
considered “reasonable” may differ substantially based on the specific
society’s values and a specific individual’s perception of risk. However, in
general, the following characteristics are important for cancer screening:
• The cancer must have a considerable public health effect.
• The disease must have an asymptomatic period in which detection
by imaging is possible.
• A therapeutic intervention that should lead to better survival or
quality of life must be available.
• The prevalence of the disease must be sufficient in the population
being screened to justify screening (especially the cost). Low preva-
lence of disease lowers the positive predictive value of positive scans.
• Medical, surgical, or other treatment must be available for the
early-stage cancer identified by screening.
• The screening test itself must not cause disease at a significant rate.
• There must be a high likelihood that the patients in whom early
cancer is identified by image-based screening will go on to undergo
a suitable therapeutic intervention.
Furthermore, the imaging test itself must be acceptable to patients
(in terms of level of discomfort, cost, and radiation burden), and it
must be sufficiently sensitive to identify cancer often and sufficiently
specific to minimize false-positive results. Finally, the costs of the
screening process, and attendant costs related to false-positive results,
must be compatible with the society’s or the individual’s economic
resources, and the screening procedure must pose little or no risk to
the patient.4
Another important consideration in screening programs is lead-time
bias. This concept, simply stated, indicates that if the natural history
of a disease is unchanged but the diagnosis is made earlier in the
course of the illness, the apparent survival will be improved. For
example, let us assume that a tumor has a 6-year natural history from
its beginning until the death of the patient, and that treatment is
ineffective. The disease might become clinically detectable after 4
years and lead to death in 6 years, a 2-year survival after diagnosis.
With screening, if the tumor is detected 3 years after the onset of
disease and no improvement in treatment occurs, then the survival
in the screened population would appear to increase from 2 to 3 years
after diagnosis. This illusion of improved survival in the screened
population is a considerable concern and can lead to inappropriate
enthusiasm for screening programs.4
Another important consideration in screening is the possibility of
length bias. This is a more complex concept, but it may be related
to the types of cancer that can be detected by screening programs. A
possibility is that very rapidly growing and presumably highly lethal
cancers are less likely to be detected by annual screening programs,
whereas more slowly growing cancers, which have an intrinsically
better prognosis, may be detected more frequently by screening. In
fact, some of the early cancers discovered by screening may not be
Imaging  •  CHAPTER 16 259
biologically relevant at all. If so, the patients with cancers identified
in the screened population could appear to have a better survival than
the patients with cancers identified in the unscreened population.
This concern exists for prostate cancer screening by prostate-specific
antigen (PSA), for example, wherein there are major concerns regarding
overdiagnosis of slow-growing, and presumed indolent, cancers that
might not need surgery.14 This concern also exists for breast cancer,
wherein some argue that a small to a more substantial portion of
the cancers diagnosed would not have proven harmful to patients
had they been left undiagnosed and untreated. That said, we do not
fully know which cancers need no treatment, and an issue exist-
ing with “overdiagnosis” is that it really is “overtreatment.” Better
screening tests that identify biologically relevant cancers would
be ideal.
A third factor is the selection bias that is difficult to control for
in retrospective observational studies: how the patients and their
referring physicians determined to have a scan performed. Selection
bias occurs when there are unintended differences between the groups
observed that, although they are associated with the variable used to
sort the groups—for example, exposure in case-control studies and
outcome in cohort studies—affect measurement of the study variable.15
For instance, in a case-control study of the effects on disease-specific
mortality of a particular screening program, investigators would examine
records from patients who have died from the disease in question
versus those who have not, then determine the rates of the screening
intervention in these two populations.
It is possible that those likeliest to have sought screening were the
ones at highest risk for the disease. Results for the screening’s effect
on mortality could be underestimated in such a scenario. Selection
bias also can work in the opposite direction, wherein the high-risk or
poor-prognosis individuals are less likely to seek screening. With the
relative absence of large randomized prospective trials, as is commonly
the case when evaluating imaging as a screening tool, one may be
tempted to base conclusions on the findings of cohort or case-control
studies. However, the most accurate conclusions would be drawn
from a prospective randomized study design.
Collectively, lead-time bias and length bias and, at times, selection
bias can make screening programs appear to improve the survival of
patients with cancer. Because of these major biases intrinsic to screening,
very large, randomized, studies are required to show that overall
cancer-specific mortality (and ideally mortality from all causes) declines
as a result of screening programs.
All-cause mortality is a critical parameter. If the treatment of a
presumed tumor discovered by a screening imaging test carries with
it a risk of death or morbidity, the screening program could lower
cancer-specific mortality but not all-cause mortality. If the screened
population is very young, late adverse effects of screening may be
difficult to detect, such as slightly increased risks of cancer due to
radiation.
The advent of imaging and other screening tools that uncover a
tumor long before it is symptomatic also brings up the need for
biomarker discovery to help physicians to determine whether to treat
what has been discovered through screening. The experience with
PSA screening in prostate cancer serves to illustrate the point that
not all cancers identified by screening eventually lead to death. This
is likely also the case for some early-stage breast cancers of low prolifera-
tive rate and potential. Discovering and validating biomarkers that
can help to distinguish reliably between lethal and nonlethal tumor
types will be of great help in reducing unnecessary treatment in patients
with less active disease. Identifying and phenotyping tumors with the
greatest risk for progression is of great importance because it is clear
that not all cancers carry the same risk of mortality if left untreated.
Screening Costs
The determination of whether a screening program is valuable to
society is often, in part, based on its cost and benefit. The concept
of quality-adjusted life-years (QALYs) often is applied. This concept
260 Part I: Science and Clinical Oncology
is defined as the economic cost to society required to result in 1
additional year of good-quality life for a member of the society. In
many Western countries, a figure of $50,000 has been considered a
useful guide (although many affluent countries are willing to spend
far more per QALY), with QALYs costing less than this amount
considered cost-effective. Such a guideline, however, does not neces-
sarily apply when individuals make their own determinations as to
whether to pay for a screening test. For example, it is reasonable
to expect that those with greater disposable income would be willing
to pay more per QALY than those with less disposable income. Thus
it can be difficult to generalize about the cost efficacy of screening
procedures.16
Even when people choose to undergo a screening test at their own
expense, considerable costs can be transferred to society as a result of
the screening program. For example, if a screening test has a high rate
of false-positive results, a substantial number of follow-up biopsies or
procedures will be performed and can cost a great deal of money.
Such costs can dramatically raise the total cost per QALY. Invasive
procedures also can increase the likelihood of morbidity or death as
a result of additional investigations. To determine the true cost per
QALY associated with screening, these additional costs and risks must
be considered. Thus screening remains an area of great promise, but
also of considerable controversy.
Screening beyond breast imaging must overcome major hurdles
before it is likely to be accepted. There has been considerable
progress for both virtual colonoscopy as an alternative to optical
colonoscopy and for CT scanning of the lungs in patients at high
risk for lung cancer. In both these populations, the Centers for
Medicare and Medicaid Services (CMS) pays for screening, as do
ACA-compatible private insurance policies. It is almost certainly the
case, however, that in high-risk patient groups, such as those with a
family history of cancer or major carcinogen exposure with a high
penetrance or early onset of disease, screening may prove of greater
value than in the general population. As an example, MRI screening
in patients at high risk for breast cancer is increasingly accepted as
appropriate.
Size of Detectable Lesions
Noninvasive imaging methods in humans cannot enable detection
and localization of a single malignant cell, although flow cytometric
methods are very sensitive for finding a very few cancer cells in a patient’s
blood, and there is a great interest in assays for circulating tumor
cells and for tumor DNA and RNA in the blood. Imaging methods
are improving, however, and detection of a much smaller number of
cells is possible in small-animal models. It has been estimated that by
the time a tumor reaches 3 to 5 mm in diameter, which is the lower
limit in size for detection by the best current noninvasive methods
in humans, the tumor has undergone more than 25 doublings and
contains 0.1 to 1 billion cells, depending on their size.1 In contrast,
a cytologist on a very good day or with use of flow cytometric
methods may be able to identify a single cell as malignant by using a
microscope.
Realistically, even for histologic assessment of malignancy, typically
a group of tumor cells must be present before cancer is diagnosed.
However, light microscopy and more sensitive techniques such as
immunohistochemistry and polymerase chain reaction studies mean
that pathologic techniques potentially will be more sensitive than
imaging methods. One very important proviso is that for light
microscopy pathologic methods to be effective, actual examination
of the malignant cells is required; the sample containing the tumor
must be cut appropriately and viewed under a microscope. This task
may be impossible, because the 8-µm sections that are used for
pathologic examination typically are taken only from a small portion
of a tumor or lymph node, whereas most of the tumor or node will
be unexamined (e.g., to assess a 1-cm node by using 8-µm-thick
sections, approximately 125 slices would be required). This large
number of sections is not typically obtained.
Despite the markedly superior sensitivity of histologic methods
over noninvasive imaging, there is a major sampling error issue for
histologic sampling, and, paradoxically, imaging in some diseases
potentially may be more sensitive than histologic examination for
cancer. This situation can arise in mammography, in which small
tumor foci can be seen on the mammogram but can be missed on
cytologic sampling, possibly because of sampling error. When tumors
are imaged with noninvasive methods, the entire tumor is visual-
ized, not just a small portion. So, paradoxically, imaging, despite
limited resolution, can be more sensitive than cytology or pathol-
ogy. However, if exhaustive and thorough sampling is performed,
with microscopic examination of tissue there usually is greater
sensitivity for tumor than there is with current noninvasive imaging
techniques.
With the advent of serum protein tumor markers, circulating tumor
cell assays, and DNA- and RNA-based methods, it is possible that
imaging will not always be a primary tool for cancer detection. These
other methods may be more sensitive than imaging, although imaging
has the distinct advantage of both detection and physical localization
of tumor foci. In addition, noninvasive imaging can detect heterogeneity
in cancers that cannot be identified with circulating cells or DNA.
Such information is potentially more “actionable” medically than
simply detecting whether tumor is present or absent.
Stage Migration
One of the major goals of noninvasive imaging is to stage the tumor
precisely to allow the clinician to best choose treatment and determine
the prognosis. The evolving concepts of tumor staging are discussed
elsewhere in this text, but improvements in detection technology can
change the understanding of the natural history of a given stage of
disease.
Patients with small or microscopic lung cancer metastases to the
mediastinum are likely to do better than patients with bulky metastases,
but both may have the same stage of disease. As the sensitivity for
detecting small lesions improves, it becomes possible to identify more
patients with small primary tumors and small metastases to the lymph
nodes or small systemic foci of metastatic disease.
When primary tumors are detected at ever earlier and less advanced
stages as imaging and other detection methods improve, patients are
assigned to a higher stage than was used historically. Their inclusion
in the advanced-stage group, as opposed to the localized disease group,
appears to improve survival and outcome. The outcome of the lower-
staged group also may improve because a subset of patients with
now-detectable tumors has been removed from that group. The outcome
of the overall group will not have changed because the numbers of
patients in each group will have been altered.
One has to be very cautious in extrapolating historical survival
data in a given advanced cancer stage based on an insensitive
staging method to that observed with a more sensitive method,
which might move some patients to a higher stage, so-called stage
migration.4
MAJOR IMAGING MODALITIES
Broadly stated, cancer imaging can be performed with anatomic or
functional (“molecular”) imaging methods.1 The traditional imaging
of the patient with cancer, and the most established methods, are
based on anatomic imaging. However, interest is increasing in more
functional methods in cancer imaging. Furthermore, several anatomic
imaging methods offer functional components that complement the
anatomic method. Hybrid images, derived from and displaying both
functional and anatomic data, also are becoming more widely available,
often coming from the same hybrid imaging machine, such as in
PET-CT.17,18 Imaging data are almost exclusively digital or digitized
and suitable for postprocessing and image exchange. The major imaging
modalities are discussed in the following section.

Plain-Film Radiographs
The traditional radiograph remains an important part of cancer imaging.
It commonly is used to detect bone tumors and can be used to detect
lung cancers in the thorax. The method displays mainly water, air,
fat, and calcium density and is affected by overlapping tissue in front
of or behind the lesion. Radiographs have become increasingly digitized
in the last few years, with the introduction of film digitization and
solid-state image screen capture devices.
Plain radiographs offer exceptional resolution but provide relatively
little image contrast if there is not much calcium present. The radiation
dose from a plain film radiograph depends on which portion of the
body is being examined. In most centers, plain film radiographs for
cancer management are being used less often, with CT scanning
increasingly replacing radiographs in the abdomen and MRI in the
brain and extremities.19
Mammography
Mammography is a specialized form of plain radiograph. Very
high-resolution images of the breast are obtained using specialized
x-ray sources and devices optimized for breast cancer detection.
Digital mammography is now available and offers greater flexibility
of image display because of the digital image format. A limitation
of the digital format, however, is the field of view of the imaging
phosphor, which may be too small to fully include some breast tissue.
In the last several years, there has been a major shift toward digital
mammography.
The Digital Mammographic Imaging Screening Trial (DMIST),
which included nearly 50,000 women, showed comparable overall
accuracy between film-screen and digital mammography in the overall
study. A higher accuracy for cancer detection in women younger than
50, women with radiodense breasts, and premenopausal and peri-
menopausal women was seen with digital mammography as compared
with film-screen mammography.20 Despite this higher accuracy, the
reported sensitivity for both techniques for cancers was only 41%,
with a positive predictive value of 12%, albeit with 98% specificity.
This is far from ideal performance for a screening test. The move to
digital mammography has occurred in part to increase diagnostic
accuracy but also to allow digital images to be viewed on a picture
archiving and communication system (PACS), as is the case with
virtually all other diagnostic imaging methods in more and more
imaging centers.20,21
A newer technique, tomosynthesis, is under evaluation; in this
technique, a number of “slices” of the breast are generated during a
mammographic image acquisition that involves a moving x-ray source.
This approach offers considerable promise going forward, but is early
in its evolution technically. It has received FDA approval and will
likely improve on the performance of mammography.22 Data suggest
that tomosynthesis may help identify the 15% to 30% of breast cancers
not identified by full-field digital mammography (FFDM). The
specificity of tomosynthesis may be better than that of FFDM, as
well. However, there are challenges with the large volumes of data
and the potential failure to detect micrometastatic disease. The cost
efficacy of such an approach remains in evolution, and larger multicenter
studies will inform medical decisions regarding the precise role of
digital tomosynthesis—for example, whether it can totally replace
mammography.23
Two emerging technologies in breast imaging—contrast-enhanced
breast imaging and molecular breast imaging (MBI)—may ultimately
play an important role. The former involves intravenous administra-
tion of contrast agent to identify areas of altered blood vessel density
and permeability (often seen in cancer). The latter uses a radioactive
tracer to identify areas rich in mitochondrial density and flow, such
as cancer. Both are showing better performance than mammog-
raphy alone in some settings, with performance approaching that
of MRI.
Imaging  •  CHAPTER 16 261
Computed Tomography
CT is now established as the dominant imaging technique for cancer
detection and follow-up. Currently, tumor response and staging criteria
very commonly are based on tumor size as measured on CT. CT
scanners acquire images by using an x-ray source and digital detector
elements. The x-ray source rotates rapidly around the patient, usually
with a single scan level taken in 0.5 seconds or less. Faster and faster
rotation speeds of the scanners, along with multiple simultaneous
detectors capable of imaging multiple slice thicknesses in a rapid spiral
motion, are being used.
Scanners with 16 and 64 simultaneous slices are commonly in use,
with some scanners with 256 or more slices now in use. Large–field-
of-view detectors may allow even more of the body to be evaluated
nearly instantaneously. Current fast scanners can potentially scan the
entire body in a fraction of a minute. Although such evaluations
provide key information about lesion size, some lesions may elude
detection unless contrast medium is given intravenously, orally, or
both. With such devices, it also is possible to capture contrast in
arteries or veins to achieve superior visualization of these structures,
which can then be displayed three dimensionally or in a volume-
rendered fashion.
Although more slices in a CT scanner make for faster CT scans,
it is not clear that having more slices always results in a superior
diagnostic-quality scan. More slices and faster scans do mean that
whole-body scans in a single breath hold can be obtained, which is
advantageous because it can reduce the frequency of breathing
artifacts.
A clear disadvantage of CT is its cost, both technically and in
terms of the radiation dose. In the past several years, increased concern
has developed regarding the total radiation dose being delivered by
CT, particularly in children, because of the potential risks of carci-
nogenesis. Great efforts have been undertaken to reduce the dose from
CT, especially with use of more advanced software methods for
reconstruction.
Although CT is an exceptional technique, it remains a predominantly
anatomic imaging method. Information can be derived based on the
timing of intravenous CT contrast enhancement, such as the ability
to estimate tumor blood flow, but it is not easily extracted without a
substantial radiation dose from repeated images. Thus only a limited
number of post-intravenous contrast images are obtained with CT,
to limit radiation dose. All CT images are digital. Another major
challenge with CT is the large amount of image data generated for
analysis, data that can take the radiologist a long time to interpret
fully. Newer dual-energy or multispectral CT scanners generate
even more data and can provide virtual contrast-enhanced and
non–contrast-enhanced images from a post–contrast administration
acquisition. The large amount of data in a series of CT scans is of
interest for purposes of further characterization beyond a visual one.
Radiomic features can be extracted and analyzed, which may add
information beyond tumor size and location. In addition, artificial
intelligence approaches may be of growing importance to characterize
large data sets.24
Angiography
Historically, angiography has been performed after intravascular inser-
tion of catheters into arteries, followed by rapid injection of iodinated
contrast media, along with rapid-sequence filming of the images. The
improving ability of rapid-sequence CT scanning to show the vascular
anatomy (CT angiography) has caused it to rapidly replace angiography
for diagnostic purposes. Angiography still can be used to produce the
most precise maps of vascular anatomy before organ transplantation
or radical cancer surgery. Most angiography now is performed with
digital image-capture devices, known as digital angiography.
Angiographic delivery of therapy is, however, an important area.
This form of imaging can allow for therapeutic delivery of embolization
262 Part I: Science and Clinical Oncology
materials such as coils, delivery of chemotherapeutic agents regionally,
or delivery of radioactive microspheres, for example.
Angiography has high resolution but typically delivers a high
dose of radiation energy to the patient. The use of angiography
remains essential for studies to evaluate gastrointestinal (GI) bleed-
ing, but with CT angiography continuing to improve in quality, the
use of diagnostic angiography has become less frequent in routine
clinical practice while cancer therapies by catheter have grown in
application.25
Ultrasonography
Ultrasonography uses reflected beams of high-frequency sound rather
than ionizing radiation to generate images. Ultrasonography provides
high resolution and some functional information, specifically about
the presence and direction of blood flow in tissues. It also provides
some information regarding tissue characterization properties and is
very effective in determining whether a tissue is cystic or solid. The
method also is excellent for detecting vascular structures and determin-
ing extent of flow.
Ultrasonography provides real-time imaging capability to guide
biopsies and procedures effectively. It is less effective in the evaluation
of deeper structures and requires access to a sonographic window.
Therefore it has only a modest role in evaluating deep abdominal
structures. Ultrasonography is used commonly in evaluations of the
pelvis, neck (including the thyroid), and gallbladder and liver areas.
Agents that can enhance the visualization of vessels or can be specifically
retained in clots are under evaluation, suggesting that ultrasound
examination can offer some functional information beyond the purely
anatomic.26
Ultrasound contrast agents are being applied to a limited extent.
Currently these agents are mainly for intravascular use. An ultrasound
contrast agent has been approved for use in assessing the liver to help
determine if liver lesions are malignant or benign. High-energy focused
ultrasound remains under development as a tool to ablate, or at least
deliver thermal energy to, tumors with therapeutic intent. Ultraso-
nography coupled with needle aspiration biopsy has been used in
some settings as a minimally invasive procedure to characterize nodal
metastases and primary lesions. Ultrasonography combined with
mammography also is being evaluated as a screening method for breast
cancers, but results in high-risk patients have shown disappointingly
low detection rates relative to MRI and high false-positive rates for
ultrasonography.27
Magnetic Resonance Imaging and Magnetic
Resonance Spectroscopy
In many clinical settings, MRI, as applied in imaging for most cancers,
is used predominantly as an anatomic imaging method that does not
use ionizing radiation. MRI offers superb contrast resolution between
tissues and excellent spatial resolution. MRI also offers a variety of
forms of functional information. However, it does not offer the level
of temporal resolution, in general, seen with ultrasound examination,
fluoroscopy, or recent-generation, multislice CT scanners. However,
MRI technology has moved forward inexorably, and rapid-pulse
sequences allowing gating of images now are available for several types
of units. Magnetic resonance images can be of a variety of pulse
sequences, allowing visualization of several parameters. However,
visualization of hydrogen nuclei is the major approach with conventional
1.5-T and 3-T (tesla) machines. Visualization of blood, especially
with contrast materials such as gadolinium chelates, and of altered
vascular permeability is routinely applied.28
MRI is the preferred procedure in evaluating neoplasms of the brain
and spinal cord regions as well as musculoskeletal tumors. MRI also is
being used with increasing frequency in the evaluation of tumors of
the extremities, such as sarcomas. With gadolinium contrast enhance-
ment, MRI also can be useful in locating tumors of the breast, which
appear as areas of contrast enhancement. Such studies have shown the
higher sensitivity of MRI versus mammography. However, there remain
concerns that MRI may achieve this sensitivity with an accompanying
relatively high false-positive rate, depending on the methods used for
interpretation. Still, MRI probably is our most accurate technique for
detecting and characterizing breast masses.
MRI also can provide valuable information about tumors in close
proximity to vascular structures as well as in the liver and upper
abdomen. Tumors in the upper abdomen and liver can be degraded
in their appearance on MRI because of respiratory motion artifacts.
Therefore this approach is applied less commonly than in other situ-
ations, such as in the lower pelvis, where there often is less respiratory
motion artifact. Research currently is working on developing agents
that can facilitate specific MRI contrast enhancement. More rapid
whole-body MRI acquisition methods also are under evaluation, which
may broaden the use of MRI.
In magnetic resonance spectroscopy (MRS), tissue characterization
can be achieved by sampling its magnetic spectrum at 1.5 T or higher.
More and more scanners now provide a 3-T or higher field strength
signal for evaluation, offering the possibility of more refined tissue
characterization. Opportunities to detect increased content of choline
(often increased in tumor foci) versus other substituents can be helpful
in separating tumor from nonmalignant tissues in the brain and
elsewhere. Spectroscopy also can provide information on lactate
concentration and pH, among other parameters. A limitation of
spectroscopy is resolution, which typically is not nearly as fine as that
of anatomic MRI itself. Thus spectroscopy has only limited application
in most oncologic practices.29
Diffusion MRI has shown promise in tumor response assessment.
This depends on the freer movement of nuclei in areas of necrosis as
compared with the motion of nuclei in fully viable tumors. This
technique is demonstrating considerable potential for response assess-
ment in brain tumors and recently has shown promise in tumors in
the bones, breast, prostate, and other tissues.30
Nephrogenic systemic fibrosis (NSF) has been linked to gadolinium-
containing MRI contrast agents.31 This condition has been described
in patients with substantially impaired renal function who receive
MRI contrast material intravenously. Black box warning labels now
appear on the product inserts for MRI, and we are seeing increased
use of alternative imaging methods to MRI in patients with low
creatinine clearance rates followed by sequential imaging studies.
Gadolinium may be responsible in part for this process as it has been
found in the skin of some of such affected patients.32,33 In 2007, black
box warning labels were added to the package inserts related to
gadolinium-containing MRI contrast (Box 16.1). Gadolinium deposits
in the brain have been identified on MRI scans, with the amount of
gadolinium deposited in rough proportion to the number of gadolinium
doses the patient has received. Although this is an area of active
controversy, no adverse effects have been identified with the gadolinium
deposition. However, the European Medicines Agency (EMA) has
recommended that gadolinium-based contrast agents (GBCAs) be
used with caution and that linear GBCAs not be used, favoring
macrocyclic chelators for gadolinium. A list of approved GBCAs is
shown at the end of this chapter. For practical purposes, it is probably
wisest to use gadolinium contrast only when necessary and in most
cases to use macrocyclic contrast agents, given the EMA’s recom-
mendation. In May 2018, the FDA approved new medication guides
for patients receiving gadolinium contrast.
An exciting area of application of MRI technology is in the field
of whole-body MRI. Such a test may begin to offer whole-body
cancer surveys with no ionizing radiation delivered to the patient.
Whole-body diffusion imaging methods are quite informative
because they can detect a variety of tumors, including in bone. Such
applications to date have been less sensitive than whole-body PET
imaging, but this area is emerging very rapidly34 (Figs. 16.2 and 16.3).
Recently, hyperpolarized C-13 imaging has been investigated. This
field is in its infancy but allows some level of magnetic resonance
 Imaging  •  CHAPTER 16 263

A B C

R L L R

Anterior Posterior

Figure 16.2  •  Representative 64-year-old patient with metastatic disease. (A) Technetium-99m–methylene diphosphonate (MDP) bone scan obtained
after injection of 21 mCi of the radiopharmaceutical. The bone scan demonstrates multiple areas of increased uptake in the calvaria, sternum, ribs, vertebra,
pelvis, and femur. Degenerative and inflammatory changes are noted in the left knee and left ankle (after fracture due to fall). (B) Axial diffusion-weighted
MRI images (b = 300–600 s/mm2) of corresponding areas of metastatic sites. The sternum metastases are clearly seen, as are those in the rib and femur
(arrows). (C) Sagittal image showing whole-body coverage. (Courtesy Dr. Martin Pomper, Johns Hopkins.)

single-photon and positron-emitting isotopes. Single-photon emitters

Box 16.1.  US FOOD AND DRUG
ADMINISTRATION (FDA) BLACK BOX
WARNING REGARDING GADOLINIUM
MAGNETIC RESONANCE IMAGING
(MRI) CONTRAST MEDIUM
Warning: Nephrogenic Systemic Fibrosis (NSF)
Gadolinium-based contrast agents increase the risk for nephrogenic
systemic fibrosis (NSF) in patients with
• Acute or chronic severe renal insufficiency (glomerular filtration rate
<30 mL/min/1.73 m2)
OR
• Acute renal insufficiency of any severity due to the hepatorenal
syndrome or in the perioperative liver transplantation period
With these patients, avoid the use of gadolinium-based contrast
agents unless the diagnostic information is essential and not available
with non–contrast-enhanced MRI. NSF may result in fatal or debilitating
systemic fibrosis affecting the skin, muscle, and internal organs. Screen
all patients for renal dysfunction by obtaining a history and/or
performing laboratory tests. When administering a gadolinium-based
contrast agent, do not exceed the recommended dose, and allow a
sufficient period of time for elimination of the agent from the body
before any readministration.
metabolic imaging in vivo without the need for radioactive tracer
administration.35
Nuclear Medicine and Positron
Emission Tomography
Radionuclide methods can provide a great deal of functional informa-
tion, but the anatomic resolution often is limited. Broadly, there are
typically have longer half-lives than positron emitters, and decay in
a different fashion, emitting gamma rays. Depending on the ligand
attached to the radioactive isotope, a wide variety of processes can be
imaged. For single-photon imaging, the most common isotope is
technetium-99m (99mTc), which can be used to image bone (bone
scan with 99mTc–methylene diphosphonate) or thyroid (technetium
pertechnetate), for example.
With positron emitters, the most commonly used tracer is 18F,
which is used as the radiolabel for FDG, an agent that images glycolysis
in vivo. Because tumors have increased glycolytic metabolism in general,
use of this agent in tumor imaging is increasing very rapidly, especially
in lung and colorectal tumors and lymphomas.36
Both PET and single-photon emission computed tomography
(SPECT) have very high sensitivity for a small number of radioactive
molecules in vivo as well as the ability to quantitate the radioactivity
concentration precisely. Thus these methods are important as both
clinical and research tools. The intrinsic lack of anatomic resolution
for PET and SPECT can be partly addressed by fusing the PET or
SPECT images to CT by using computer software. More recently,
dedicated hybrid imaging devices, including both PET and CT or
SPECT and CT in a single device, have been applied to the imaging
practice of cancer.
In the last several years, combined PET-MRI devices have also
been constructed. These can include a PET scanner housed within
an MRI unit performing both PET and MRI studies at the same
time or adjacent PET and MRI scanners in which the patient table is
precisely aligned to allow for sequential imaging using PET and then
MRI imaging with preservation of patient alignment. PET-MRI is
in evolution, and two current limitations are the cost of the systems
and challenges associated with precise quantitation of the PET
images for radioactivity concentrations in vivo due to the limitations
intrinsic to MRI-based attenuation correction algorithms.37 In clinical
practice, PET-MRI and PET-CT have shown relatively comparable
outcomes.38
264 Part I: Science and Clinical Oncology

A B
Whole Body–DWI ADC map
b=50 b=300 b=600

B
PET CT PET/CT

Figure 16.3  •  This 72-year-old man had colorectal cancer and metastatic disease in the liver. (A) Diffusion-weighted images of different b-values in the
liver. (B) Corresponding positron emission tomography (PET), computed tomography (CT), and PET-CT images.

Table 16.2  Imaging Methods

Modality Resolution Sensitivity Specificity Functional Imaging Ability
Magnetic resonance imaging 1–2 mm Moderate Moderate Moderate with spectroscopy
Computed tomography 1–2 mm Moderate Moderate Low, except angiography
Radiographs 1–2 mm Low Moderate Very little
Single-photon emission computed tomography 1 cm High Moderate Excellent
Positron emission tomography 5 mm High Relatively high Excellent
Ultrasonography 2 mm Low Low Some, especially with contrast agent
Mammography 1–2 mm Moderate Relatively low None
Angiography 1–2 mm Moderate Moderate Low

Adapted from Bragg DG, Rubin P, Hricak H. Imaging strategies for oncologic diagnosis and multidisciplinary treatment. In Bragg D, Rubin P, Hricak H, eds. Oncologic Imaging.
2nd ed. Philadelphia: WB Saunders; 2002:2–20.

Higher doses of radioactive isotopes also can be therapeutic. For
example, iodine-131 (131I) is used for the treatment of thyroid cancer,
as sodium iodide. The same isotope, conjugated to an anti-CD20
monoclonal antibody, was approved by the FDA for the treatment
of low-grade and transformed B-cell non-Hodgkin lymphoma that
is considered refractory to standard treatments. The tracer doses are
used to guide the treatment doses in such instances, or at least to
determine if the radioantibody has any unexpected targeting behavior.39
Optical Imaging Methods
Optical imaging methods are applied in a variety of ways. The external
physical examination of a patient involves visual interrogation of
reflected light. Infrared and transmitted light also are used to a limited
extent in evaluating small parts of the body. Optical imaging has been
limited in clinical deployment by the limited penetration depth of a
variety of forms of light in the body. Therefore this type of approach
may prove of greatest usefulness in evaluation of small animals as part
of experimental studies, or for superficial organs such as the breast.
Similarly, the technique may have greater usefulness in evaluation of
intraoperative procedures.
The possibility of constructing light-emitting contrast media is a
real one, and optical imaging has the potential to provide remarkable
sensitivity and resolution in superficial structures. However, it is not
routinely applied in cancer imaging, with the exception of visualization
of the interior of the eye, visualization of the cervix, and endoscopy,
bronchoscopy from above and below.40 The combination of light-
generating acoustic signals, photoacoustic imaging, also has considerable
promise and potential for areas of the body in which light can be
delivered at sufficient intensity. The strengths and weaknesses of the
major imaging methods are contrasted in Table 16.2.
Radiation Dose and Imaging
Imaging methods that use ionizing radiation are a major source of
population radiation exposure. CT and nuclear medicine procedures

(notably, cardiac nuclear medicine procedures) are the major sources of
the ionizing radiation.41 The most vulnerable population with regard to
radiation exposure is children, in whom the risk of radiation-induced
cancers is higher, with a longer time for manifestation, than seen in
cancers in older individuals.42 Clearly, caution must be applied when
choosing an imaging study using ionizing radiation, because it is quite
likely that there are some increased risks of cancer associated with a low
radiation dose distributed over a very large population of individuals.43
It should be noted that many cancer patients have relatively short
life spans because of their underlying disease, and the risks of the
known cancer greatly exceed the potential risk of cancer from imaging.
It is also important to realize that lifetime risks of cancer may carry
varying significance—for example, a hypothetically “radiation-induced”
cancer developing 40 years after cure of testicular cancer has a less
immediate health care impact than a cancer developing within a few
years of radiation.44 Certainly, concerns about radiation are appropriate,
and every effort should be made to use imaging methods that include
ionizing radiation only when appropriate, and then at the lowest dose
possible consistent with adequate image quality.
ANATOMIC VERSUS FUNCTIONAL IMAGING
Limitations of Anatomic Imaging of Cancer
Anatomic imaging has been the fundamental approach to cancer
imaging for more than 100 years. Anatomic methods are quite robust,
as is supported by their daily use in managing individual patients
with cancer. Anatomic imaging normally enables detection of a
phenotypic alteration that is sometimes, but not invariably, associated
with cancer—a mass. However, with anatomic imaging, we often do
not know whether masses are the result of malignant or benign etiolo-
gies, as in solitary pulmonary nodules or borderline-size lymph nodes.
Similarly, small cancers are undetectable with traditional anatomic
methods, because they have not yet formed a mass.
After surgery, it is even more difficult to assess for the presence of
recurrent tumor with anatomic methods. Posttreatment scans are
complicated by the need for comparisons with normal anatomy to
detect altered morphologic findings as a result of cancer. Anatomic
methods do not predict cancer response to treatment and do not
quickly document tumors that are responding to therapy.45 Despite
these challenges, anatomic images remain routine in cancer manage-
ment. PET, a functional imaging method, helps to address many of
the limitations of anatomic imaging, and when combined with anatomic
images in fusion images, is emerging as a particularly valuable tool,
providing both anatomic precision and functional information in a
single image set.45
Molecular and Functional Alterations in Cancer
The molecular bases of neoplasia are increasingly becoming well defined.
Mutations in genomic DNA precede the development of overt
neoplasia.46 With sufficient alterations in genotype, phenotypic changes
occur. These genotypic and phenotypic changes in cancer antedate
the development of a discrete mass lesion and represent potential
targets for innovative imaging agents. The concepts of altered “genome,”
“proteome,” and “methylome” resulting in alterations in metabolism,
consistent with an altered “metabolome,” are increasingly recognized
as present in cancers along with a variety of typical cancer “hallmarks.”
PET, because of its superb sensitivity to low signal levels, can detect
signals from tracers, targeting such alterations that are preferentially
present in cancer.
PET has led the growing field of molecular imaging to the clinic, in
part because of the quantitative capabilities of PET and the sensitivity
of electronic collimation, but also because of the choice of a proper
radiotracer for cancer imaging. Although a wide variety of molecular,
proteomic, and metabolic alterations occur in cancers, and many of
these can or ultimately may be imaged with PET, the most useful target
Imaging  •  CHAPTER 16 265
in the clinical practice of PET is the increased glucose metabolism
present in most cancers. Other PET tracers, such as those targeting
hypoxia, proliferation, amino acid transport, blood-brain barrier
permeability, and protein synthesis, are discussed in the following
sections. The challenge with all imaging modalities is how best to
integrate them into clinical practice. The next section addresses
these issues.
DISEASE-SPECIFIC IMAGING
RECOMMENDATIONS
Brief discussions of the role and possible role of imaging in managing
several common cancers follow. There is great variation in the imaging
workup of specific types of tumors, depending on the type of therapy
planned.
In general, the more aggressive and radical the planned treatment,
the more critical the need is for accurate determination of the location
of all foci of tumor through imaging. Similarly, the intensity and
frequency of follow-up imaging examinations must be guided by the
potential importance of the information gained. For example, if no
effective salvage therapy is available, intensive surveillance for recurrent
tumor makes little sense, except to provide reassurance to patients
when test results are negative. This may seem self-evident, but it is
surprising how practice patterns vary.
The National Comprehensive Cancer Network continues to update
recommendations on imaging approaches in specific cancers. In
addition, the American College of Radiology and Society of Nuclear
Medicine have provided “appropriateness guidelines” for a variety of
cancer therapies. These guidelines inherently lag behind developments
in technology but are based on careful analyses of the literature and
expert opinion and thus are worth consulting as they continue to be
updated. The CMS in the United States has provided guidance allowing
PET with FDG to be used in the diagnosis and subsequent management
of nearly all cancers based in substantial measure on the results of the
NOPR trial.
Lung Cancer
The initial diagnosis of lung cancer often is based on incidental detection
of an abnormality on an imaging study, although more advanced lung
cancer can cause hemoptysis, cough, weight loss, hoarseness, infection,
or shortness of breath. There has been a great deal of interest in testing
screening programs for lung cancer in high-risk groups such as heavy
smokers. Both chest x-ray screening and CT screening have been
evaluated.
Chest x-ray screening has not been proven to be effective at reducing
mortality from lung cancer. CT-based screening programs are of greater
interest, and it is clear that these programs can detect lung cancers at
an earlier stage, when they are smaller than lung cancers diagnosed
by other methods, and appear to prolong survival. Patients with such
cancers tend to live longer than patients with cancers diagnosed at a
more advanced stage. However, detecting additional cancers is
dependent on detection of small lung nodules and many small lung
nodules, and most of these do not have a malignant etiology. The
costs, both economic and in terms of risks from invasive diagnostic
procedures, are considerable. In addition, concerns exist that the cancers
detected by such an approach may be the more slowly growing cancers.
Results from the NLST support CT screening in a high-risk smoking
population. A summary of the results in a format easily understood
by patients is included.47
The Fleischner Society has offered guidelines for dealing with
small, incidentally detected pulmonary nodules, which involve variable
imaging follow-up for lesions greater than 4 mm in size. Growing
lesions require additional investigation.48
Certainly, if a solitary pulmonary nodule 8 mm or larger in diameter
is identified on a chest radiograph or on screening CT, the workup
to determine whether it represents lung cancer can take a variety of
266 Part I: Science and Clinical Oncology

forms. Comparison with old anatomic images is essential, but if none
are available, a decision must be made whether to perform additional
imaging, perform a biopsy, remove the nodule, or follow the abnormal-
ity. A variety of factors can be considered, including patient age,
smoking history, lesion size, and history of exposure to potential
infectious agents.
The morphology of the nodule is investigated to determine whether
it has characteristics that suggest malignancy or benignity. The margin
and internal density of the lesion are examined. Smooth, well-defined
margins suggest a benign nodule, but these also are seen in 21% of
malignant nodules.49 A lobulated margin suggests cells of different
lines with uneven growth, but can be seen in 25% of benign nodules.
A spiculated margin is highly suggestive of malignancy. Both benign
and malignant nodules can be homogeneous and can cavitate. A cavity
with a wall thickness of 4 mm or less is likely benign in 95% of cases,
a wall thickness of 5 to 15 mm is indeterminate, and a wall thickness
of more than 15 mm is likely malignant in 95% of cases.49 If the
lesion contains fat, it is specific for a hamartoma, and 50% of ham-
artomas have fat on CT.
Benign calcifications are central in the lesion, diffuse and solid,
laminated, or popcornlike. Calcification is seen in 6% of lung cancers
on CT and tends to be eccentric or amorphous.49 Thus calcification
alone does not indicate benignity in a lung nodule. If contrast agent
is administered and the lesion is monitored over 5 minutes, enhance-
ment of less than 15 Hounsfield units (HU) suggests a benign lesion
and enhancement of greater than 20 HU suggests a malignant lesion,
with reported sensitivity of as high as 98%, specificity of 73%, and
accuracy of 85%.50 However, these CT criteria rely on very small
changes in CT attenuation levels. These subtle changes may be insuf-
ficient to allow for reliable stratification of nodules as malignant or
benign, because timing of the CT bolus also is an important consid-
eration. The growth rate of the lesion also can be evaluated, but this
is difficult for subcentimeter lesions. Computer programs for nodule
detection are being developed that also provide lesion volume, and
this may prove useful in follow-up.
However, for a significant number of patients, the risk of cancer
remains intermediate. For such patients, FDG-PET imaging may be
useful. PET has been reported to have sensitivity of approximately
96% for detecting cancer in solitary pulmonary nodules (predominantly
≥1 cm in diameter) in a retrospective meta-analysis,51 with specificity
of approximately 80%. However, some histologic types are less well
detected, such as those of adenocarcinoma in situ (bronchioloalveolar
carcinomas), which have lower FDG uptake.52,53 Nonetheless, the
PET scan can help to determine which patients require immediate
biopsy or excision of a nodule (i.e., a nodule with intense FDG uptake)
versus those who can be observed (some of those with low or no tracer
uptake). The use of PET varies widely, but it can be a valuable tool
for helping to determine which patients need invasive procedures for
pulmonary nodules. Because false-negative findings occur, however,
patients who do not have surgery should be followed up regularly for
up to 2 years to ensure that there has been no lesion growth. Fig.
16.4A shows “hot” and “cold” nodules in the same patient.
Once lung cancer has been diagnosed, an appropriate staging workup
should be undertaken. The workup for non–small cell lung cancer
often is done to determine whether the patient is a candidate for
surgery. Because FDG-PET imaging is at least 20% more accurate
than CT imaging, PET is commonly recommended as a staging
procedure for the mediastinum and for systemic evaluation for
metastases.54 In a prospective randomized trial, PET reduced the
number of futile thoracotomies by half, from 41% to 21%, versus
algorithms in which PET was not performed.55 This occurred, in part,
because remote foci of metastatic disease that were not identified with
standard staging methods were identified with PET.
Consensus is developing on how PET should be used in staging
non–small cell lung cancer, but many centers routinely perform PET-CT
before surgery, with the incremental benefit being most apparent (in
terms of detecting additional remote disease from the primary lesion)
in patients with larger primary lung cancers. It usually is considered
prudent to perform a biopsy on FDG-avid tissues to prove that they
represent cancer. Many would argue that mediastinoscopy is no longer
essential for patients who have negative mediastinal PET and CT
scans, although in some patients, cancer in nodes is detected only
surgically. An example of a positive PET scan with ipsilateral and
contralateral mediastinal tumor involvement is shown in Fig. 16.4B.

A B
Figure 16.4  •  (A) Images obtained with positron emission tomography (PET) and computed tomography (CT). The upper left image is CT, the upper
right image is PET, and the lower left image is a fusion of PET and CT data. The CT scan shows a smaller right apical nodule and a large left upper lung
nodule. PET shows increased uptake of fluorine-18 fluorodeoxyglucose (18F-FDG) in the left apical nodule, consistent with cancer, whereas only minimal
uptake is seen in the right apical lesion, consistent with benign disease or a very low grade malignancy. Fused PET and CT images confirm the location of
increased apical FDG uptake, as in the left lung nodule. (B) Whole-body images from PET scans of the patient shown in (A). The images include coronal,
sagittal, transverse, and “projection” whole-body views, from left to right. They show cancer in the left upper lung, mediastinal involvement, and a right
paratracheal tumor focus. Normal uptake is seen in the heart, brain, and excretory system (bladder).

The role of PET-CT in lung cancer is to provide a thorough whole-body
screening assessment.
For mediastinal nodes, a short-axis diameter of greater than 1 cm
is considered abnormal on CT. Larger nodes can be reactive, and
nodes smaller than 1 cm can contain tumor, leading to sensitivity of
40% to 67% and specificity of 79% to 86% for metastatic disease.56
The hybrid PET-CT technology is significantly superior to PET
alone for the staging of lung cancer.57 The best algorithm for staging
the mediastinum is evolving, but PET and PET-CT are the most
accurate noninvasive methods. Some argue that mediastinoscopy is
necessary in each case, however, because PET may produce false-
negative results in patients with a low tumor burden, although the
negative predictive value of a negative PET scan and a negative
CT scan is approximately 95%. Others, however, would argue that
patients with a low tumor burden, below the level of detectability
with PET and CT, may be suitable candidates for surgery without
mediastinoscopy. Positive PET scans for metastases usually require tissue
confirmation of the most advanced site of tumor to avoid false-positive
imaging findings that indicate a tumor is not resectable. Fig. 16.5
illustrates detection of lung cancer on CT and the use of reformatted
virtual images.
MRI with contrast is recommended for imaging the brain if brain
imaging is indicated. It usually is performed if patients have larger
primary tumors or any symptoms that suggest central nervous system
involvement, although in some centers, MRI with contrast is performed
in every patient with lung cancer in whom resection for cure is planned.
Evaluation for bone metastases currently is performed by bone scan.
Any patient with bone pain or an elevated alkaline phosphatase level
should undergo this study. In practice, where PET is available, bone
scans are increasingly being replaced by PET scans. This area is evolving,
and the bone scan remains the routine procedure in most centers
when bone metastases are suspected, but FDG-PET can reveal lung
cancer metastases to bone that are not detected with CT alone.
In institutions where PET is not available, bone scan, MRI of the
brain, and CT of the chest and abdomen, to include the adrenals,
are recommended. The abdominal CT scan should be performed with
contrast agent to best evaluate the liver. Fig. 16.6 shows extensive
mediastinal disease and liver metastases on CT. In some centers, PET
A B
Figure 16.5  •  Lung cancer. (A) Axial computed tomography (CT) image (lower left) shows a mediastinal mass (arrows) narrowing the airway. Coronal
(top left) and sagittal (top right) reconstructed images show narrowing of the airway by a mass (arrows). The virtual bronchoscopy image (lower right) shows
an endoluminal view of the narrowed airway. (B) Coronal reconstruction of CT data in a different patient shows a cavitating mass in the left upper lobe.
(Courtesy Dr. Leo Lawler, The Johns Hopkins University.)
Imaging  •  CHAPTER 16 267
and diagnostic-quality CT used together provide sufficient diagnostic
information that no additional studies are required.
For small cell lung cancer, it must be determined whether the
disease is extensive or localized before the form of therapy can be
chosen. CT is essential for the chest and upper abdomen. MRI of
the brain typically is performed, and a bone scan is done to search
for bone metastases. PET scans have been shown to upstage about
10% of patients with limited-stage small cell lung cancer to extensive
disease and to find lesions not identified with CT. FDG-PET identifies
essentially all lesions detectable with CT.58 Thus many centers are
using FDG-PET more routinely for this type of staging, and the CMS
has approved FDG-PET for staging of small cell lung cancer.
PET also is useful in the evaluation of mesotheliomas, although
the data are evolving, and CT is the established method for this type
of evaluation. For follow-up of patients with lung cancer, the guidelines
are more challenging because the available therapeutic options often
are more limited. PET has a growing role because it can better separate
residual scarring from viable tumor than can CT.
Evaluation of the Adrenal
Adrenal masses occur in approximately 9% of the population. These
masses can be benign adenomas, metastatic disease from primary
tumors such as lung cancer, or primary adrenal cortical carcinoma.
CT and MRI are used to distinguish adenomas from malignant lesions
in the adrenals. Adrenal adenomas have low attenuation on noncontrast
CT as a result of elevated lipid content. If a threshold value of 0 HU
is used, sensitivity is 47% and specificity is 100% for the diagnosis
of adenoma.59 If a threshold value of 10 HU is used, sensitivity is
71% and specificity is 98% for the diagnosis of adenoma.
Adenomas take up contrast material, but the washout of contrast
material from an adenoma is faster than from a metastatic lesion. On
10-minute delayed images obtained after contrast injection, a decrease
in the density of the lesion greater than 50% is specific for an adenoma.59
If the lesion is atypical on CT, chemical shift imaging on MRI can
be used to determine whether it is an adenoma. An adenoma has
both lipid and water content, and decreases in signal are seen on
out-of-phase T1-weighted images. PET has a growing role in evaluating
268 Part I: Science and Clinical Oncology
A B
Figure 16.6  •  Lung cancer. (A) Axial computed tomography (CT) scan of the chest with intravenous contrast agent shows a mass obstructing the right
upper lobe bronchus. (B) CT scan of the abdomen shows a metastatic hypodense lesion in the left lobe of the liver.
Figure 16.7  •  Positron emission tomography (PET) and computed
tomography (CT) image panel shows a variety of PET, CT, and fused images
that reveal diffusely metastatic lung carcinoma. Notable are adrenal metastases,
best seen on the transverse images, although many metastases are visible in a
variety of locations. This figure also shows the multiple types of images available
from a PET and CT system.
the adrenals, with accuracy of 90% and greater reported in some
series. High FDG uptake typically is seen in metastases to the adrenal.
Caution is in order, however, in that some adrenal adenomas can have
moderately high FDG uptake. Fig. 16.7 illustrates detection of adrenal
and systemic metastases on CT and PET.
Breast Cancer
Mammography is the major imaging tool in breast cancer, allowing
for early detection of tumors. When properly used, mammographic
screening programs have been shown to save lives when compared
with unscreened populations, and these programs are routinely
implemented in many countries.60
Although mammography is a reasonably sensitive and specific
procedure, the relatively low prevalence of cancer means that many
image-directed biopsies show no tumor; thus the results of the mam-
mogram were falsely positive. Stereotactic biopsy devices have greatly
facilitated nonsurgical breast biopsies. Although the sensitivity of
mammography is generally considered “good,” an overall sensitivity
of less than 50% was seen for x-ray mammography in DMIST. Even
though this can be improved on through the use of digital rather than
screen-film mammography, these results indicate clearly that mam-
mography is far from perfect as a screening tool.
Early efforts with tomosynthesis of the breasts, a mammo-
graphic method that provides a number of “slices” of the breast
for analysis, are promising.61 Recently, breast tomosynthesis devices
have received FDA approval, and some studies have shown superior
performance of breast tomosynthesis compared with more standard
mammography. Tomosynthesis helps remove overlying structures
from the breast image, potentially improving lesion detection. It
also is possible that use of intravenous contrast agent may enhance
mammographic results.62
Although other techniques can reveal breast cancer, only ultraso-
nography is used fairly commonly in most imaging centers to help
to separate cystic from solid lesions or to help to locate and evaluate
palpable but mammographically negative lesions. MRI with gadolinium
contrast is used because it is a very sensitive technique and can help
to determine whether disease is unifocal or multicentric. An example
of a positive mammogram is shown in Fig. 16.8A.
The use of mammography in screening has been established as a
technique that saves lives; however, controversies remain regarding
the method. As a tool that delivers ionizing radiation, it is not without
risks. The benefit of mammography in women younger than 50 years
of age is somewhat less clear because they typically have more radiodense
breasts and have a lower frequency of breast cancer than older patients.
The optimal frequency of breast cancer screening has been subject to
varying recommendations as well, with some guidelines recommending
screening every 2 years as opposed to annually. It has also been argued
that mammography may disproportionately detect slower-growing
breast cancers, which may be less medically relevant and possibly lead
to “overdiagnosis” and “overtreatment.”
Another concern is that the rate of detection of advanced breast
cancers has not changed a great deal since the introduction of mam-
mography, and that some of the improvements in outcomes are due
to better overall therapies of breast cancer and not solely to early
detection related to improved diagnosis.63 Although there are some
controversies, available data point to regular mammography in women

A B
Figure 16.8  •  Breast cancer. (A) Mammogram of the right breast shows cancer. (B) Axial computed tomography (CT) image shows an irregular soft tissue
primary mass in the right breast in a different patient. (C) Axial CT image with a lung window shows a metastatic nodule in the right lung. ([A] Courtesy
Dr. Nagi Khouri, The Johns Hopkins University.)
older than 50 and younger than 75 years as a valuable tool for sub-
stantially reducing the risk of death from breast cancer.
Another method of breast imaging using radionuclides is molecular
breast imaging (MBI), in which 99mTc-MIBI is injected intravenously,
and the breasts are imaged with a high-resolution dual-head or single-
head gamma camera, optimized for breast imaging. This approach
has been evaluated in a number of settings. The radiodense breast is
highly problematic for cancer detection with mammography, but it
appears that the MBI approach is more sensitive. In a study in which
MBI was offered as a part of screening for radiodense breasts, of 936
women screened, 11 cancers were identified (one with mammography
only, seven with gamma imaging only, two with both combined, and
one with neither). Diagnostic yield was 3.2 per 1000 (95% CI, 1.1–9.3)
for mammography, 9.6 per 1000 (95% CI, 5.1–18.2) for gamma
imaging, and 10.7 per 1000 (95% CI, 5.8–19.6) for both (P = .016
versus mammography alone).64 The challenge with this method is
radiation dose. Major efforts to reduce radiation dose are ongoing.
With dose reduction, these approaches may find a growing role in
breast cancer screening, but they must compete directly with MRI
approaches, which do not use ionizing radiation.
MRI has been evaluated as a tool for characterizing substantially
abnormal mammograms or ultrasound scans. The prospective mul-
ticenter National Institutes of Health (NIH)–sponsored trial in which
821 patients referred for breast biopsy because of American College
of Radiology category 4 or 5 mammographic assessment or suspicious
clinical or ultrasound findings were studied with MRI found an AUC
of 0.88 in a population with 404 cancers present, with dichotomized
data demonstrating sensitivity of 88% and a specificity of 68%. The
positive predictive value was about 72%. The high sensitivity was still
insufficient to obviate the need for biopsy in these patients, however.65
MRI has been found to reveal breast cancer in the contralateral
breast of women with newly diagnosed primary breast cancers in
approximately 3% to 4% of cases.66 These additional cancers are found
at a rate of about 25% in the lesions identified in this manner (i.e.,
75% of the biopsy results are negative). In addition, MRI has been
used to screen high-risk women (e.g., those with the BRCA1 or BRCA2
mutation) for breast cancer to some advantage. This higher sensitivity
comes at the cost of more false-positive results and challenges in biopsy,
which have limited deployment of the method. However, increased
uniformity of reporting and greater similarities in technique are leading
to more use of this methodology, which has been comprehensively
Imaging  •  CHAPTER 16 269
C
reviewed.67 PET methods occasionally are applied in the breast, but
they are used for diagnosis infrequently owing to the low sensitivity
of whole-body PET scanners to small tumors. PET is used more
commonly in disseminated disease. FDG-PET appears to be superior
to CT for detecting disseminated breast cancer.68
Dedicated PET imaging devices for the breast have been increasingly
applied for diagnosis. Such positron emission mammography (PEM)
devices have relatively good resolution, likely superior to that available
with whole-body PET alone. PEM and MRI performed relatively
comparably. PEM has been directly compared with MRI in a study
of 388 women with newly diagnosed breast cancer. Additional cancers
were found in 21% of women. Thirty-four percent of the 82 cancers
were identified with both PEM and MRI; 26% with MRI only; 17%
with PEM only; and 8.5% with mammography and ultrasonography.
Of 306 breasts without additional cancer, 279 (91.2%) were correctly
assessed with PEM compared with 264 (86.3%) that were correctly
assessed with MRI (P = .03). The positive predictive value of biopsy
prompted by PEM findings (47 [66%] of 71 cases) was higher than
that of biopsy prompted by MRI findings (61 [53%] of 116 cases)
(P = .016).69 MRI was slightly more sensitive, but PEM more specific
in this group. Thus there is promise for the PEM method, especially
in women who cannot undergo MRI. However, it is probable that
tomosynthesis or MBI may provide similar data at a lower radiation
dose, although this area continues to evolve.
Breast cancer often metastasizes first to locoregional lymph nodes.
Current noninvasive imaging methods do not evaluate the axillary
nodes effectively. However, imaging can help to define which lymph
nodes should be resected for histologic sampling. Sentinel node imaging
or detection through use of a radiation-sensitive probe system is
becoming increasingly more important in axillary assessment of patients
with breast cancer.70 This procedure is discussed in more detail elsewhere
in the text, but imaging is used, especially in European centers, to
better localize the axillary nodes for intraoperative assessment and
determine atypical routes of lymphatic drainage.
Studies have shown sentinel node sampling procedures to be at
least 90% sensitive—which is considered useful sensitivity—relative to
axillary dissection, and they are virtually 100% specific.71 FDG-PET is
not sufficiently sensitive to detect small metastases and has an accuracy
of only about 75% in axillary nodal staging.6,72 Data are emerging
that detection of bulky nodal lesions is of greatest importance for
outcomes.
270 Part I: Science and Clinical Oncology
Table 16.3 US Food and Drug Administration
(FDA)–Approved Gadolinium-Based
Contrast Agents
Brand Name Generic Name Structure
Ablavar gadofosveset trisodium Linear
Dotarem gadoterate meglumine Macrocyclic
Eovist gadoxetate disodium Linear
Gadavist gadobutrol Macrocyclic
Magnevist gadopentetate Linear
dimeglumine
MultiHance gadobenate Linear
dimeglumine
Omniscan gadodiamide Linear
OptiMARK gadoversetamide Linear
ProHance gadoteridol Macrocyclic
The extent of systemic imaging required in the initial workup of a
patient with breast cancer depends on the size of the primary tumor
and the status of the axillary nodes at biopsy. It can be argued that
the likelihood of systemic metastatic disease is higher for patients
with positive nodes; therefore more intensive evaluation may be more
appropriate. Some would suggest a baseline bone scan and CT of
the chest and abdomen at this time. The frequency of follow-up is
debated, given the improbability of curing recurrent systemic disease,
and some have advocated only limited biochemical follow-up. However,
this area is controversial. Examples of primary and metastatic breast
cancers imaged on CT are shown in Fig. 16.8B–C. PET is a highly
effective tool for evaluating soft tissue involvement and quickly
assessing treatment response.73 FDG-PET imaging can detect dis-
seminated cancers; however, the optimal role of PET in imaging is
evolving. PET is now approved for monitoring treatment response of
breast cancer and often can predict histological outcomes within 2
weeks of therapy initiation.
Prostate Cancer (Table 16.3)
The role of imaging in the diagnosis and management of prostate
cancer is rapidly evolving. The serum PSA test remains controversial
but when used in the screening setting has allowed the detection of
smaller prostate cancers than generally are detectable clinically, along
with opportunities for earlier interventions. In addition, mortality
from prostate cancer has declined in the past two decades. Many
believe these two observations (more PSA testing and fewer deaths
from prostate cancer) are not random associations, although this is
discussed in more detail elsewhere in this book, and is a topic of
controversy. Although there is some evidence that early surgical resec-
tions are more closely associated with diminished mortality from
prostate cancer than active surveillance, large prospective trials compar-
ing radiation therapy, prostatectomy, and active surveillance (with the
possibility of intervention if there is progression of disease) have shown
similar 10-year survival outcomes. These studies were not image driven
at their outset (i.e., there was not extensive imaging of the prostate
itself ), but they indicate an opportunity to include advanced imaging
in prostate cancer treatment decision making. Imaging in prostate
cancer could play several roles: detecting cancer in the prostate, detecting
the extent of tumor, determining if the tumor is highly likely to be
aggressive, and determining whether the tumor has spread beyond
the prostate locally or systemically at diagnosis or recurrence.
Evaluation of the Prostate
Intraprostate carcinoma is not detected particularly well with imaging
with ultrasound, and there are limitations to imaging with MRI and
other methods, though there has been progress. Although transrectal
ultrasound (TRUS) has been used to guide biopsies, up to 40% of
prostate cancers are isoechoic (and thus undetectable) by this method.
Similarly, the positive predictive value for cancer of hypoechoic lesions
of the prostate typically is well below 50%.
Ultrasound imaging can be used to detect abnormalities and guide
biopsy of the entire prostate because it does an excellent job of defining
the overall shape, size, and location of the prostate for purposes of
systematic biopsy. A sextant approach often is used to sample the
base, middle, and apex of the prostate bilaterally, in addition to taking
biopsy samples of any suspicious areas. Although this technique is
less than perfectly accurate for detecting prostate cancers, on ultrasound
examination, tumors in the peripheral zone are more readily visible
than tumors in the inner gland. The peripheral zone is echogenic,
and tumors that are hypoechoic to it (approximately 60%) can be
detected.74 However, hypoechoic lesions also can be caused by inflam-
matory processes, and the positive predictive value of ultrasound is
approximately 18% to 52%.74
Contrast enhancement of the prostate on ultrasonography has
been used to some advantage to enhance detection rates and positive
yields on biopsies of the prostate.75 Similarly, on MRI, cancers that
are hypointense on T2-weighted images are seen, but the findings are
not specific for tumor. Extension of cancer beyond the prostate capsule
is suggested on ultrasound when the capsule margin is irregular or
the seminal vesicles are abnormal in morphology; however, sensitivity
of as low as 20% has been reported. The sensitivity of MRI for
extracapsular invasion is approximately 50%, and specificity is 95%.74
The range of reported accuracies varies widely.
Previously, with 1.5-T magnets, intrarectal coils were sometimes
required to produce optimal images of the prostate. However, with
newer 1.5-T and 3-T systems, very good images of the prostate can
be obtained, often without the need for a rectal coil This leads to
better patient acceptance and greater dissemination of this methodology.
The typical approach to prostate imaging with MRI has been an
emphasis on T2-weighted images, in which cancers often appear as
hypodense; however, a variety of other pulse sequences are now being
applied, so imaging of the prostate may include T1 imaging (for
detection of neurovascular involvement), T2 imaging for intraprostate
and capsular assessment, and diffusion contrast enhancement (DCE)
and apparent diffusion coefficient (ADC) images. The DCE images
can be kinetically modeled and a variety of parameters extracted and
displayed. Thus a multiparametric MRI approach to prostate cancer
imaging is increasingly being applied, and likely is leading to more
accurate diagnoses and local staging.
A systematic reporting system for prostate cancer, Prostate Imaging–
Reporting and Data System (PI-RADS), has been developed and
continues to be updated.76 At present, although the DCE portion of
the MRI is perhaps the least valuable, it has been reported to have
more value in the peripheral zone of the prostate and is usually included
in multiparametric imaging studies. A prospective multicenter MRI
study from the United Kingdom, PROMIS, compared multiparametric
MRI with grid biopsies of the prostate and TRUS postgrid biopsies
in 740 men, 576 of whom underwent 1.5-T multiparametric MRI
followed by both TRUS biopsy and template prostate mapping (TPM)
biopsy. On TPM biopsy, 408 (71%) of 576 men had cancer, with
230 (40%) of 576 patients having “clinically significant” cancers. For
clinically significant cancer, multiparametric MRI was more sensitive
(93%; 95% CI, 88%–96%) than postgrid biopsy and TRUS biopsy
(48%; 95% CI, 42%–55%; P < .0001) and less specific (41%
[36%–46%] for multiparametric MRI versus 96% CI [94%–98%]
for TRUS biopsy; P < .0001). These authors suggest the potential for
multiparametric MRI to identify patients who do not need biopsies
of the prostate. Of course, some false negatives would occur, which
might be problematic if not subsequently identified at a treatable
time. This study also did not optimally evaluate the TRUS biopsy,
because these were performed after whole-prostate template biopsy
sequences. MRI-guided biopsy techniques are also now being applied

in research and clinical settings, for the purpose of diagnosis and also
as a means of guiding locoregional therapies. Such approaches, when
applied by experts, increase the yield of biopsy results from the prostate
compared with random biopsy approaches.77 This area continues to
evolve, but the precise and optimal method of deploying MRI methods,
and the precise patients who will benefit most from the method,
remain in evolution, and practice patterns vary considerably across
centers, although expert-based guidelines have been developed. It is
clear that biopsy remains essential for findings seen on MRI owing
to a relatively high false-positive rate of lesion identification on MRI.
It is also commonly the case that MRI-informed biopsies can be
performed by importing MRI data into an ultrasound-guided system
to increase the yield of biopsies. These approaches can increase the
yield of “significant” cancers, although there is not full agreement on
what is a significant cancer. Patients at intermediate risk for invasion,
with a PSA level of 10 to 20 ng/mL and a Gleason score of 5 to 7,
may benefit from MRI staging of local extension.74
MRI methods are improving. Diffusion MRI appears particularly
helpful and in some centers is preferred over DCE studies. Some
centers use spectroscopy74,78; however, this is not yet a routine part
of management of prostate cancer. 3-T methods are likely superior
to 1.5-T methods for spectroscopy. Increased field strength for MRI
is now being more systematically assessed and appears promising as
a tool to separate benign from malignant prostate tissue by enabling
detection of tissue with increased ratios of choline to citrate. To date,
it has been hard to prove that spectroscopy greatly improves the
diagnosis of prostate cancer.79 The role of MRI in prostate cancer
imaging continues to evolve. It is quite possible the MRI will in the
future be able to identify patients with small primary tumors who
may be managed with active surveillance. The role of MRI in follow-up
of active surveillance remains in evolution.
CT has no established role in evaluating the prostate itself or
local invasion (25% or less sensitivity for capsular invasion), although
tumors sometimes can be seen on CT (Fig. 16.9A). CT is used to
detect bladder and rectal invasion, adenopathy, and distant metastases,
and MRI is used to assess local extent. Similarly, the sensitivity for
detecting nodal metastases has been reported to be as low as 30%
with CT. Higher sensitivity can be achieved, but with lower specificity.
Pathologic proof is desirable before it is concluded that a patient
has metastatic disease to the lymph nodes, and if metastatic disease
is demonstrated, radical prostatectomy is considered inappropriate.
Large nodal metastases are easily detected with CT imaging, however
(Figs. 16.9B and 16.10).
A B
Figure 16.9  •  Prostate cancer. (A) Axial computed tomography (CT) image shows an enlarged prostate with an enhancing mass and irregular margins.
(B) A more superior image shows enlarged bilateral pelvic nodes from metastatic disease.
Imaging  •  CHAPTER 16 271
It is not clear that MRI is better than CT for nodal staging;
however, data on MRI using a node-specific paramagnetic contrast
agent have shown very high sensitivity and accuracy in the detection
of nodal metastases.78 There is considerable hope for this or related
methods, but MRI- based contrast agents have not yet been approved
by the FDA for this indication, and use of such agents has not yet
been widely accepted.80 Given the traditionally low sensitivity of CT
and MRI to detect nodal metastases, it has been recommended that
the serum PSA level be at least 20 ng/mL before CT is performed.
If the CT findings are positive, then biopsy can be performed of the
enlarged node or nodal sampling can be performed before radical
prostatectomy. CT also is the test of choice for visceral metastases.
Nuclear medicine methods such as C-11 choline and prostate-specific
membrane antigen (PSMA) targeting agents are showing increased
application for nodal staging.
A 99mTc bone scan is a reasonably sensitive technique relative to
radiographs for bone metastases. The results of a bone scan can be
positive when radiographs of bone are essentially normal. The current
recommendation is that a radionuclide bone scan be performed only
if the serum PSA level at presentation is greater than 10 ng/mL. Only
very rarely is a bone scan positive for tumor at lower levels at the time
of diagnosis. Some argue that for large primary tumors or very high
Gleason scores a bone scan still may be appropriate at staging. Bone
scans with 18F-sodium fluoride (18F-NaF) PET imaging or SPECT
are likely more sensitive than standard bone scans. The precise role
for NaF PET in evaluating patients with prostate cancer is uncertain,
but it is increasingly available in the United States with FDA approval
of NaF for bone imaging. Typically, more lesions are seen with NaF
PET than with standard bone scan or bone SPECT imaging. For
example, in a study of 44 men with high-risk prostate cancer, bone
scans (99mTc–methylene diphosphonate [MDP]), SPECT bone scans,
and NaF PET scans were compared. Of the 156 18F-fluoride lesions,
81 lesions (52%), including 34 metastases, were overlooked with
normal appearance on planar bone scan. SPECT revealed 62% of the
lesions overlooked with planar bone scan. 18F-fluoride PET-CT was
more sensitive and more specific than bone scan and more specific
than PET alone (P < .001).
Thus imaging is used selectively in patients with newly diagnosed
prostate cancer. A bone scan commonly is used for recurrent prostate
cancer whenever the PSA level begins to rise.
Much monitoring of prostate cancer now involves sequential
blood tests to measure the PSA level. When the PSA level rises
persistently, the tumor has recurred, but determining the site of
272 Part I: Science and Clinical Oncology

A B C

D E F

G H I
Figure 16.10  •  Prostate cancer of the transition zone in a 52-year-old man with a Gleason score of 3+4 and a prostate-specific antigen level of 19 ng/
mL. Endorectal magnetic resonance imaging was performed at 1.5 T. (A) An axial T2-weighted image (6000/92) shows ill-defined homogeneous dark infiltration
of the central gland (arrows). (B) A sagittal T2-weighted image (3350/92) shows homogeneous dark tissue replacing the central gland (arrows). (C) An axial
color diffusion contrast-enhanced magnetic resonance imaging map shows a large area of high permeability (Ktrans) (red areas) in the transition zone. (D) A
permeability histogram shows a shift toward high permeability values, a finding characteristic of cancer. (E) A kinetic curve (percentage of enhancement over
time) shows a typical washout pattern in the transition zone tumor. (F) The magnetic resonance spectroscopic spectrum from the transition zone tumor shows
a high choline (Ch) peak (arrow) at 3.2 ppm that is above that of citrate (Ci) at 2.64 ppm. Cho + Cr/Ci = 1.31, where Cr = creatine; this value is typical of
prostate cancer. (G) An ex vivo T2-weighted image (4700/42) of the specimen, obtained at 9.4 T, shows highly cellular, compact dark tissue in the central
gland (arrows) surrounding the urethra (U). (H) A photograph of a whole-mount reconstructed histologic section (original magnification ×2; hematoxylin-eosin
[H&E] stain) of the midgland shows a large volume of tumor in the transition zone (outlined in green). Note the excellent correlation with the ex vivo image
in (G) and the in vivo image in (C), which show cancer of high cellular density in the transition zone. (I) A photomicrograph of a histologic section (original
magnification ×40; H&E stain) from the transition zone tumor shows loss of gland units and sheets of cancer cells with randomly scattered lumina. Note the
muscular stroma component between the tumor cells. (From Bonekamp D, Jacobs MA, El-Khouli R, Stoianovici D, Macura KJ. Advancements in MR imaging
of the prostate: from diagnosis to interventions. Radiographics. 2011;31:677–703.)

recurrence often is problematic. Bone scans and CT often are used,
with radio­antibody imaging for anti-PSMA antibodies used only
infrequently because of limited diagnostic accuracy, although some
promising results have been obtained by experienced groups. PET
methods are growing rapidly in prostate cancer; however, FDG-PET
often is falsely negative in prostate cancer, especially for disease in the
earliest stages.62
Several more accurate methods of detecting metastatic prostate
cancer have been developed. C-11 choline PET was approved by the
FDA in September 2012. C-11 choline provides an important imaging
method to help detect the location of prostate cancer in patients
whose blood test results suggest recurrent cancer when other imaging
findings are negative. Per the FDA labeling of the product, the safety
and effectiveness of C-11 choline injection were verified by a systematic
review of published study reports.
Four independent studies examined a total of 98 patients with
elevated blood PSA levels but no sign of recurrent prostate cancer on
conventional imaging. After PET imaging with C-11 choline, the
patients underwent tissue sampling of the abnormalities detected on
the PET scans. In each of the four studies, at least half the patients
who had abnormalities detected on PET scans also had recurrent
prostate cancer confirmed by tissue sampling of the abnormal areas.
PET scan errors also were reported. Depending on the study, falsely
positive PET scans were observed in 15% to 47% of the patients.
These findings underscore the need for confirmatory tissue sampling
of abnormalities detected with C-11 choline injection PET scans.
Other agents are under development for prostate cancer imaging. For
example, 18F anti-FACBC, a synthetic amino acid, shows considerable
promise.81 When compared with FDA-approved ProstaScint for disease
detection in the prostate bed, anti-3-(18)F-FACBC had an accuracy
of 83%. Indium-111 (111In)–capromab pendetide had an accuracy
of 67%. In the detection of extraprostatic recurrence, anti-3-(18)
F-FACBC had an accuracy of 100%. 111In–capromab pendetide had
an accuracy of 47%. 18F-FACBC now known as fluciclovine, has
been FDA approved in the United States for intravenous injection.
It is indicated for PET imaging in men with suspected prostate
cancer recurrence based on elevated blood PSA levels following prior
treatment.

Several radiopeptides that bind to the PSMA target in prostate
cancer are showing great promise in human studies. Gallium-68 (68Ga)
and 18F peptides have been evaluated. Gallium PSMA agents have
better detection capabilities for nodal metastases than anatomic imaging
methods.82 In a study of 130 patients, lymph node metastases were
found in 41 of 130 patients (31.5%). On patient-based analysis, the
sensitivity, specificity, and accuracy of 68Ga-PSMA-PET were 65.9%,
98.9%, and 88.5%, and those of morphologic imaging were 43.9%,
85.4%, and 72.3%, respectively. Direct comparisons of C-11 choline
and 68Ga PSMA binding peptides show higher frequencies of lesion
detection with the PSMA binding ligand.83 In a study of 129 patients
with biochemical recurrence of prostate cancer measured with serum
PSA, and imaged with sequential F choline and, if negative, gallium
PSMA scanning, the overall detection rates were 85.6% (107 of 125)
for the sequential imaging approach and 74.4% (93 of 125) for
18
F-choline-PET-CT alone. 68Ga-PSMA-PET-CT detected sites of
recurrence in 43.8% (14 of 32) of the choline-negative patients.
Detection rates of the sequential imaging approach and 18F-choline-
PET-CT alone increased with higher serum PSA levels. Subgroup
analysis of 68Ga-PSMA-PET-CT in 18F-choline–negative patients
revealed detection rates of 28.6%, 45.5%, and 71.4% for PSA levels
of 0.2 or greater to less than 1 ng/mL, 1 to 2 ng/mL, and greater
than 2 ng/mL, respectively.84 Several 18F-labeled small molecules, such
as 18F DCFBC, which binds to PSMA, and successor molecules such
as 18F DCFPYL, result in exceptionally high-quality images of metastatic
prostate cancer.85–87 Thus there is considerable progress in the molecular
imaging of prostate cancer, with agents approved and under develop-
ment that are likely to alter practice patterns in prostate cancer, especially
metastatic disease assessments.
Colon Cancer
The role of noninvasive imaging in the diagnosis of the primary lesion
in colon cancer has changed over the last several decades. At one time,
barium enema studies were used extensively to search for colorectal
cancer. They have been replaced, in large measure, by fiberoptic
colonoscopy. However, there is a growing level of interest in virtual
colonoscopy, which is performed with use of CT scanning and per-
rectum insufflation after thorough bowel preparation. This procedure
is used to a limited extent for screening. The technique faces challenges
because it requires bowel preparation and the interpretation is quite
time-consuming. Screening for colon cancer is an area of considerable
opportunity for noninvasive imaging. Infrequently, these cancers can
be detected by other methods, such as ultrasonography.
Virtual colonoscopy was directly compared with optical colonoscopy
in a study of over 6000 patients. Patients were randomized to either
an optical or a CT colonography group and were compared for the
detection of advanced neoplasia and the total number of harvested
polyps. Advanced neoplasia was confirmed in 100 of the 3120 patients
in the CT colonography group (3.2%) and in 107 of the 3163 patients
in the optical colonography group (3.4%). There were seven colonic
perforations in the optical colonography group and none in the CT
colonography group. Primary CT colonography and optical colonog-
raphy screening strategies resulted in similar detection rates for advanced
neoplasia, although the numbers of polypectomies and complications
were considerably smaller in the CT colonography group. CT colo-
nography also was much less expensive than optical colonoscopy.88
Interesting to note, the detection rate for polyps requiring biopsy was
statistically identical between the two groups of patients.
In a study of 2531 evaluable participants screened with virtual
colonoscopy for large adenomas and cancers, the mean with standard
error for per-patient estimates of the AUC for CT colonography was
0.89 ± 0.02. The sensitivity of 0.90 (i.e., 90%) indicates that CT
colonography failed to detect a lesion measuring 10 mm or more in
diameter in 10% of patients. The per-polyp sensitivity for large adeno-
mas or cancers was 0.84 ± 0.04. The per-patient sensitivity for detecting
adenomas that were 6 mm or more in diameter was 0.78.89
Imaging  •  CHAPTER 16 273
Evidence supporting the accuracy of CT colonography has continued
to grow. In the United States, a variety of insurance carriers pay for
virtual colonoscopy screening for colon cancer. There is also evidence
that in the 65-year-old and older population, virtual colonoscopy
performs as well as does optical colonoscopy. However, coverage by
the CMS in the United States for screening has been slow in evolution,
and at the time of writing this chapter, virtual colonoscopy has been
approved for diagnostic but not screening purposes by the CMS.
Regardless of the means of detection, the extent of preoperative
imaging performed before resection of primary colon cancer is variable,
based on the institution. In general, the larger the primary tumor,
the more aggressive is the staging procedure required. In most instances,
the primary tumor must be surgically resected for palliation (or cure),
even if metastatic tumor is present. For staging, the most common
studies include CT scan with contrast enhancement of the abdomen
and pelvis, CT scan of the thorax (or chest radiograph), and bowel
imaging to exclude the presence of a second primary colon cancer.
In institutions in which PET imaging is available, PET is used somewhat
more frequently at presentation; however, it is much more commonly
applied in the setting of suspected recurrence.
For colorectal cancer, many advocate regular imaging studies after
surgery for “cure,” because isolated metastases or oligometastases of
colorectal cancer can be resected from the liver or lungs; in some
instances the patient is disease free for a long period. Thus before
such removal of a limited number of metastases is contemplated, a
thorough imaging procedure is performed. This usually includes CT
of the abdomen and pelvis with contrast agent and CT of the thorax
without contrast agent. CT of the thorax may be replaced with a
chest radiograph, but chest radiographs are less sensitive for pulmonary
metastases.
PET is applied very often in this setting and in the setting of a
rising carcinoembryonic antigen level after surgery. The precise timing
of follow-up studies can be variable, but they often are done every 6
to 12 months in the early years after surgery. Considerable evidence
supports the idea that PET can be used to detect more metastatic foci
than CT in the setting of a rising carcinoembryonic antigen level.90
An example of a patient initially believed to have only a limited
number of liver metastases is shown in Fig. 16.11; however, more
extensive disease was identified (Fig. 16.11B). PET-CT has been shown
to offer higher accuracy than PET alone in recurrent colorectal cancer.91
Although immunoscintigraphy has been used, it has been rendered
essentially obsolete because of the availability of FDG-PET. FDG-PET
often does not detect small (<5 mm) tumors, however, and is known
to be less sensitive for tumors of mucinous histology, so opportunities
for improvement remain.
Ultrasonography can reveal many liver lesions and is a very useful
technique for guiding biopsies of the liver.29 For liver metastases, CT
is still the most commonly used procedure, but in a meta-analysis,
PET was a more robust test to identify the presence and location of
hepatic metastases of colorectal cancer compared with CT, MRI, or
ultrasound methods.92
MRI methods have continued to improve, and MRI likely can
reveal more lesions in the liver than CT or PET. Thus for small lesion
detection, MRI may offer advantages over PET, but at the cost of
increased false-positive results.93
Gynecologic Neoplasms
Screening for gynecologic neoplasms usually is not performed with
imaging, except for very limited programs that have evaluated the use
of either transabdominal or, more commonly, transvaginal ultrasound
of the pelvis to detect masses that may represent ovarian cancer. These
programs have been combined with serum biomarkers. Such programs
have not been proven cost-effective and are not widely applied, but
they warrant further study, because this is a very important health
problem. In women with high genetic risk of ovarian cancer, such as
those with the BRCA mutation, screening may have greater value.
274 Part I: Science and Clinical Oncology

A B
Figure 16.11  •  Colon cancer. (A) Positron emission tomography (PET) and computed tomography (CT) image display of a patient with two fluorine-18
(18F)–fluorodeoxyglucose (FDG)–avid lesions in the liver. These are seen on the CT scan (upper left), the attenuation-corrected PET scan (upper right), the
non–attenuation-corrected PET scan (lower right), and fused images (lower left). (B) PET and CT images of the pelvis, oriented as in part (A), show increased
FDG uptake in a left external iliac lymph node metastasis.

In cervical carcinoma, screening with use of the Pap smear has a

large effect in terms of lowering mortality rates by enabling detection
of premalignant changes and early-stage disease. Although much of
the staging of cervical carcinoma is performed through physical
examination, for larger primary tumors, imaging has an important
role. Both CT and MRI are used in the pelvis; however, the use of
PET for tumor staging is increasing. As in other tumors, PET appears
to be more sensitive than anatomic imaging methods. Emerging data
show that PET provides better prognostic value than anatomic imaging
in cervical cancer.94–96
In ovarian cancer, no technique is able to reveal microscopic
metastatic disease. Ultrasound examination is the main method by
which ovarian tumors are identified at their earliest stages; however,
the unfortunate fact is that these tumors are usually diagnosed at an
advanced stage. Imaging can be used in an attempt to determine the
extent of the surgical procedure that will be required. Both CT and
MRI are used to assess the extent of ovarian cancer, with CT the
preferred method (Fig. 16.12). PET is not sensitive to tumor foci
smaller than 5 mm, but it is reasonably reliable in detecting larger
tumor foci. For this reason, some advocate the use of PET to determine
whether tumor debulking should be performed. PET has a role in
the setting of a rising CA125 level in patients with normal CT Figure 16.12  •  Axial computed tomography (CT) image shows a dense,
findings.97 calcified mass in the left pelvis, compatible with a metastatic implant from
The use of serum markers and imaging is recommended for surveil- ovarian cancer.
lance after surgery for ovarian carcinoma. The aggressiveness of imaging
follow-up depends on the treatments available. PET has an emerging
role in this setting, although practice patterns vary widely. PET also
has been shown to provide information related to treatment response 20% more lesions than are seen with CT alone.101 PET can demonstrate
in preliminary studies.98,99 disease in the bone marrow and spleen in some instances. In fact,
marrow involvement seen on FDG-PET often is associated with a
Lymphoma negative CT scan. Because the negative predictive value of a negative
PET scan of the marrow in a patient with Hodgkin lymphoma is
For both Hodgkin and non-Hodgkin lymphomas, accurate staging high, omission of bone marrow biopsy is a reasonable consideration
is important. For both types of lymphoma, accurate definition of the in PET-negative patients.102,103
tumor burden is needed for effective treatment planning, especially CT is traditionally used for follow-up of lymphoma, and there are
treatment with external beam radiation. CT was the historically accepted clear response criteria in place to follow lymphoma therapy. One of
method for noninvasive staging of lymphoma, with PET being used the challenges in lymphoma is that large masses often do not normalize
increasingly because many studies have shown it to be capable of in size after treatment, leading to questions in interpretation of a
locating more tumor foci than CT.100 An example of CT imaging of residual mass lesion. Determining whether these lesions contain a
abdominopelvic lymphoma is shown in Fig. 16.13. In most lymphoma viable tumor is important, because it defines whether more treatment
histologic types, FDG-PET is more sensitive than CT, often showing is needed.
 Imaging  •  CHAPTER 16 275

A B

C
Figure 16.13  •  (A) Axial computed tomography (CT) image with oral and intravenous contrast agent shows paraaortic adenopathy and infiltration of
the left kidney by lymphoma. (B) Image of the lower abdomen in the same patient shows enlarged mesenteric nodes. (C) Positron emission tomography
(PET)–only images (left to right: coronal, sagittal, transverse, and anterior projections) show focal areas of increased fluorine-18 (18F)–fluorodeoxyglucose
(FDG) uptake in multiple lymph node groups, including the axillary, inguinal, and iliac regions, consistent with disseminated lymphoma.

Data indicate that gallium-67 (67Ga) scintigraphy can be
effective for assessing the viability of residual Hodgkin lymphoma
and intermediate- and high-grade non-Hodgkin lymphoma. However,
the results of multiple studies on FDG-PET in this setting now have
been published, and this imaging modality has essentially replaced
67
Ga scintigraphy in assessing the viability of residual masses of
lymphoma. If a residual mass of lymphoma shows increased FDG
uptake, that usually is indicative of residual viable tumor; however,
scans also can be falsely positive because of inflammation and falsely
negative, likely because some tumor foci may be smaller than the
resolution of PET imaging.104,105
Positive midtreatment 67Ga and PET scans predict a poor outcome
from therapy, and positive PET scans at the conclusion of a therapeutic
regimen also indicate a poor prognosis. It is increasingly appreciated
that PET findings soon after treatment is initiated can be highly
predictive of ultimate outcome of the therapy. Indeed, a rapid decline
in FDG uptake to background levels after one cycle of therapy indicates
a highly responsive patient.106–108
Challenges associated with PET include reactive lymph nodes and
nodes involved with inflammatory processes such as sarcoidosis, which
can be very FDG avid. Similarly, uptake of FDG in brown fat in the
neck and thymus can be somewhat confusing.109
Although anatomic imaging has been the key for lymphoma assess-
ment, there is a growing trend toward greater use of PET. PET imaging
and PET-CT imaging, providing additional functional information, are
of growing usefulness. A limitation of PET-only methods had been the
lack of a standardized set of response criteria. Tracer activity in PET
images typically decreases more rapidly than tumor shrinkage occurs (i.e.,
anatomic changes of treatment lag behind metabolic changes detectable
with PET). For these reasons, PET scans may appear normal before
CT scans do. A concern is that there is not strong evidence showing
that a negative PET scan should be used to truncate the duration
of lymphoma therapy. For example, if a PET scan became negative
after two cycles of treatment, this would not justify, based on current
data, discontinuation of treatment. However, if a treatment involved a
standard of four possible courses of treatment and PET findings became
negative after these courses were completed, available data suggest that
this portends a very good prognosis compared with a positive PET
scan. New criteria for response including PET have been developed
for lymphoma (International Workshop Criteria [IWC] + PET). For
FDG-avid lymphomas, a negative PET scan is required to determine
a complete response. The five-point Deauville and Lugano score has
assumed widespread use in lymphoma imaging. Caution is in order
in patients who have received immune checkpoint treatments.110–115
It can be argued that PET is not essential in all patients with
lymphoma. However, the use of PET and PET-CT is increasingly
becoming the norm in centers where this technology is available.
Because PET can find some lymphomatous tumors that cannot be
detected with CT, PET is increasingly finding routine application in
the care of patients with lymphoma (see Fig. 16.13C).
276 Part I: Science and Clinical Oncology
An exciting new opportunity for the use of PET in non-Hodgkin
lymphoma is in the setting of a “response adaptive” approach. In such
treatment approaches, patients who are poorly responding can be
identified with PET performed soon after treatment is initiated. This
approach can allow segregation of responding from nonresponding
patients. In the responding patients, standard treatment is given,
whereas in the poorly responding patients, alternative approaches such
as stem cell transplants are used. This approach currently is investi-
gational, but it is an area of great promise because it allows the potential
for tailoring the therapy to the individual patient’s responsiveness to
the treatment algorithm.116 Recently, use of such an approach has
been shown feasible in a clinical comparative trial on Hodgkin
lymphoma.117
The possibility of increasing dose in patients identified as being
at high risk of progression and decreasing therapy in low-risk groups
is being tested more commonly. In a Hodgkin lymphoma trial involving
602 patients, 571 underwent PET scanning posttreatment. The PET
findings were negative in 426 of these patients (74.6%), 420 of whom
were randomly assigned to a study group (209 to the radiotherapy
group and 211 to no further therapy). The 3-year progression-free
survival rate was 94.6% (95% CI, 91.5–97.7) in the radiotherapy
group and 90.8% (95% CI, 86.9–94.8) in the group that received
no further therapy. Death rates were comparable. This suggests that
PET can be used to deintensify therapy of Hodgkin disease, as long
as patients are managed for recurrence, which is slightly more common
in the nonirradiated group. A concern with FDG-PET in lymphoma
is false-positive results due to inflammation. It is important to note
that both false-positive and false-negative PET study findings can
occur in patients with lymphoma.
PET is changing the therapy of lymphoma, and this field is in
rapid evolution. The ability to measure total tumor volumes and
modify therapies based on PET is leading to a wide range of studies
including dose intensification. Some are discussed in more detail in
the lymphoma therapy chapter elsewhere in this book. PET with
FDG is markedly changing and routinely informing treatment of
patients with lymphoma.
Melanoma
The imaging management of melanoma varies based on the stage of
disease. Radiologic imaging has no significant role in the diagnosis
of primary melanomas. Lymphoscintigraphy—the injection of
radiolabeled colloidal material, typically 99mTc-sulfur colloid, into the
subcutaneous tissues or intradermally to locate lymphatic drainage
routes, and thus lymph nodes with the potential for metastatic
involvement—commonly is performed for primary melanomas of
intermediate thickness. Although practice patterns vary, this method
typically is used for melanomas that are thicker than 1 mm without
other evidence of metastases. If the sentinel node identified at surgery
(often with radionuclide guidance) is involved with tumor, additional
staging procedures often are done.118
Data suggest that SPECT-CT imaging may improve the accuracy
of the sentinel lymph node procedure and reduce the probability of
recurrence of disease in a lymph node basin119 versus more standard
sentinel node identification procedures. These procedures most com-
monly include CT of the chest and abdomen, and of the pelvis if the
melanoma affected the lower extremities. If there are systemic metastases,
brain imaging is performed with MRI with and without gadolinium
contrast enhancement.
PET and PET-CT with FDG also are potent methods for detecting
metastatic melanoma, often revealing more tumor foci than CT. PET
can show nodal metastases but is not as sensitive as sentinel node
imaging and is not a replacement for sentinel node imaging and
removal. PET is particularly good for soft tissue metastases but cannot
reveal microscopic disease. Thus sentinel lymph node biopsy is used
in preference to PET for detection of early metastases. There is some
evidence that ultrasonography can be used to detect small nodal
metastases of melanoma, but it is not widely applied and is also less
sensitive than sentinel node imaging.1 However, PET can show most
tumor foci larger than 6 mm and sometimes can reveal smaller tumor
foci. CT is a more robust technique for small pulmonary nodules
than is PET.120
Melanoma metastases, especially if they are localized, can be resected
surgically. However, identifying whether only one or two versus many
metastases are present is a major challenge. Aggressive surgical pro-
cedures are not appropriate if there are disseminated metastases.
Therefore staging imaging procedures are performed aggressively before
major surgery is undertaken to resect melanoma metastases. PET
commonly is part of such a staging evaluation.
For metastatic melanoma, PET has been reported to have sensitivity
well over 90%. Thus although anatomic imaging dominates, PET
has a growing role in melanoma assessment.121 For bone metastases,
radionuclide bone scanning also is an important diagnostic procedure.
Recently, it has been shown that the performance of FDG-PET in
melanoma is significantly enhanced if the CT scan portion of PET-CT
is analyzed very carefully. This appears to increase both sensitivity and
specificity of the method, Caution is in order for assessing melanomas
undergoing immune checkpoint inhibitor therapies. Increases in FDG
uptake can be seen soon after treatment is started, yet can be associated
with a favorable response. Repeat imaging is often in order122 before
disease is considered nonresponsive.123
Bladder Carcinoma
Bladder carcinoma often manifests at an early stage, and no imaging
evaluation is performed to determine whether there are locoregional
or systemic metastases. However, ultrasound examination has been
used to determine the depth of penetration of primary bladder car-
cinomas. An important consideration in bladder carcinomas is that
uroepithelial tumors are often multicentric. Thus intravenous pyelogram
examinations to evaluate the entire genitourinary system commonly
are performed early in the diagnostic algorithm. Although ultraso-
nography can reveal many bladder cancers 5 mm and larger, trans-
urethral sonography is more sensitive; obviously, however, it is invasive.
MRI is used more commonly than CT for assessing primary bladder
lesions because of its superior soft tissue contrast characterization
abilities.124
For larger primary tumors that are invasive, imaging evaluation
is important to help to determine whether surgery is appropriate.
Metastatic disease to locoregional nodes or systemic metastases indicates
disease with a poorer prognosis, and tumor invading local structures
or metastatic either to nodes or systemically often is considered
unresectable. CT is used most commonly for local nodal staging,
but MRI also can be used. Small nodal metastases usually are not
detected with CT, because they have not enlarged the lymph nodes
sufficiently to allow the tumor-involved nodes to be detectable.
CT has a reported sensitivity of 60% to 70%. Biopsy commonly
is used to determine whether an enlarged node seen on CT truly
contains tumor.
MRI probably is more sensitive than CT in detecting nodal
metastases, and new contrast agents that accumulate in normal nodes
are potentially important for enhancing the diagnostic accuracy of
MRI in detecting nodal metastases.125 Initial data with MRI contrast
agents support the accuracy of this approach, but also point out that
the interpreter’s experience is important.126 Such agents are not yet
routinely available or FDA approved.
FDG-PET has been used and is promising in bladder carcinoma;
however, images of the pelvis can be degraded by intense 18F activity
in the bladder. The use of PET in bladder cancer is enhanced by
PET-CT and iterative reconstruction methods that make for better
assessments of the pelvis with less degradation due to the bladder
radiotracer activity. For bone metastases, radionuclide bone scan remains
the procedure of choice, and MRI also can be sensitive. PET-CT is
quite sensitive for metastatic disease beyond the pelvis and is being
 Imaging  •  CHAPTER 16 277

applied to a greater extent because of the results of the CMS registry
in the United States.127
Follow-up of patients with bladder carcinoma usually involves the
use of CT scans. Follow-up for new or recurrent bladder carcinoma
within the bladder usually requires direct visualization of the bladder
with cystoscopy.
Head and Neck Cancer
Head and neck cancers often are associated with cigarette smoking and
alcohol use or abuse. Human papillomavirus also has been linked to
head and neck cancers, especially in younger women. Most malignancies
in the head and neck (except for lymphomas, as discussed earlier) are
of squamous cell etiology. A key issue in these lesions is determining
whether the disease is localized to the head and neck or whether
metastatic disease or a second primary lesion is present. Thus imaging of
the lungs usually is done to exclude a primary or metastatic lung tumor.
The physical examination is very important in assessing a primary
lesion in the head and neck, but both CT and MRI are very potent
methods. MRI typically is performed before and after gadolinium
contrast is administered; CT scanning usually is performed after contrast
enhancement (Fig. 16.14A). Because MRI is subject to respiratory
and motion artifacts, CT is used somewhat more commonly in the
initial staging of these tumors.128
CT and MRI can characterize the extent of primary tumors; however,
CT is more effective than MRI in assessing the extent of involvement
of cartilage or bone. For nodal metastases, current diagnostic schemes
are based mainly on nodal size. Nodal size is an imperfect indicator
of tumor involvement, however.
Increasingly, FDG-PET is being applied to the assessment of head
and neck cancers. Although several studies have suggested that PET,
MRI, and CT have similar sensitivity, more recent studies have suggested
that PET is more accurate in staging (Fig. 16.14B–C). However, PET
can demonstrate increased glucose uptake in nonmalignantly involved
inflamed nodes (false-positive findings). These can occur in patients
with head and neck or gingival infection or the common cold. Defining
the precise extent of tumor is important to determine whether surgery
or radiation therapy should be performed, because more extensive
tumors are less amenable to surgical resection.
Occasionally, head and neck cancers manifest as isolated nodal
metastases without the location of the primary tumor being evident.
Imaging has a role in such cases. Often MRI is performed in addition

A B

Figure 16.14  •  Head and neck tumors. (A) Axial computed tomography (CT) image of the neck with intravenous contrast agent shows a mass in the
left piriform sinus. (B) Positron emission tomography (PET) and PET-CT images show right vocal cord cancer with intense tracer uptake. (C) PET and
PET-CT images from the lower level of the neck show nodal tracer uptake consistent with metastases. (Courtesy Dr. David Yousem, The Johns Hopkins
University.)
278 Part I: Science and Clinical Oncology
to extensive inspection and biopsies; however, FDG-PET also has
been applied. This method can be used to detect as many as 15% to
30% of primary tumors.
For recurrent tumors, PET appears to be a more robust test than
MRI or CT, especially when timed properly, probably because contrast
enhancement can be seen in both postoperative changes (and postradia-
tion tissue) and tumors. In general, FDG uptake is a more reliable
predictor of tumor than the anatomic methods. PET is useful in
surveillance for the recurrence of these tumors, but it is not yet
considered the standard of care.
An extremely important trial of PET as a tool to inform cancer
management was reported by Mehanna and colleagues.129 They prospec-
tively studied 564 patients with newly diagnosed head and neck cancer
who would typically have been managed by chemoradiation followed
by surgery. In this study, patients were randomized (282 patients to
the planned-surgery group and 282 patients to the surveillance group)
from 37 centers in the United Kingdom. A total of 84% of the patients
had oropharyngeal cancer, and 75% had tumor specimens that stained
positive for the p16 protein, an indicator that human papillomavirus
had a role in the causation of the cancer. The median follow-up was 36
months. The PET-CT–guided surveillance patients (notably those with
a negative PET scan after chemoradiotherapy) underwent fewer neck
dissections than did the patients randomized to the planned dissection
surgery (54 versus 221, respectively); rates of surgical complications
were similar in the two groups (42% and 38%, respectively). The
2-year overall survival rate was 84.9% in the surveillance group and
81.5% in the planned-surgery group. The hazard ratio for death slightly
favored PET-CT–guided surveillance and indicated noninferiority
(upper boundary of the 95% CI for the hazard ratio, <1.50; P =
.004). Quality of life was similar in the two groups. PET-CT–guided
surveillance, as compared with neck dissection, resulted in savings
of £1492 (approximately $2190 in US dollars; as reported in 2016)
per person over the duration of the trial.130 Therefore PET guidance
of treatment in head and neck cancer has strong evidence of “value.”
For head and neck cancers, MRI offers excellent contrast resolution
for soft tissues, but images can be degraded substantially by motion.
For this reason, CT with contrast is much more commonly performed.
FDG-PET is assuming a growing role in cancer management, especially
for recurrence and for assessment of response to treatment. PET with
CT is being used more often to stage and monitor these tumors during
and after treatment and may become the standard of care.131 A recent
comparison of PET-CT with MRI and PET alone showed superior
accuracy of PET-CT versus the other methods.132
Pancreatic Carcinoma
The standard of care for the imaging diagnosis of pancreatic cancers
is CT scanning. Although MRI can be useful, CT, including CT
angiography, is the main method used for staging and assessing tumor
invasion of vessels (Fig. 16.15). Some studies have shown FDG-PET
to be somewhat more sensitive for detection of tumors and to have
moderately high accuracy—approximately 85%—in characterizing
pancreatic lesions as malignant or benign.133–135 Ultrasound examination
is used to assess cystic lesions. After treatment, salvage therapy is
ineffective, and these patients often are less aggressively monitored
than those with other, more treatable cancers.
Neuroendocrine tumors of the pancreas often can be detected with
use of 111In-pentetreotide scanning (OctreoScan), a SPECT procedure.
Positron emitter–labeled analogues of somatostatin have been developed
and show encouraging biologic characteristics compared with single-
photon agents.136 The FDA has approved Ga 68 dotatate (NETSPOT)
for imaging NET.
Liver Cancer
Hepatic malignancies, especially hepatomas, are common worldwide.
Both CT and MRI can be very effective in detection of these lesions.
They sometimes are challenging to assess, because the appearance of
cirrhosis with regenerating nodules and tumors can overlap. Multiphase
CT imaging, CT angiography, and MRI with and without gadolinium
contrast material are commonly used in hepatomas. PET is less reliable,
because approximately half—and sometimes more—of hepatomas are
not FDG avid. There has been some use of alternative PET tracers
such as C-11 acetate to image some hepatocellular cancers, because
some FDG-negative tumors are C-11 acetate avid.137 Ultrasonography
also can be used to assess the liver and guide biopsies.
Metastatic lesions to the liver also are common, especially in the
United States. For most tumors, CT is the initial method used for
assessing whether tumor is present. However, FDG-PET is more
sensitive than CT in detecting liver metastases in common cancers
such as colorectal cancer.90,92 Thus PET is seeing greater application
in assessing suspected liver metastases, although ultrasonography, CT,
and MRI also are important methods and still are more commonly
applied in many centers (Fig. 16.16).
Kidney Cancer
In the past, renal cancers were detected with intravenous pyelograms;
currently, however, the most common method for detection is CT.
Renal cell cancer commonly is detected incidentally because of the
widespread use of cross-sectional imaging. Between 25% and 50% of
surgically treated renal cell cancers are discovered incidentally.138 Renal
lesions are classified as cysts or solid masses, depending on their
characteristics as shown by imaging. Renal cysts are fluid-filled and
appear anechoic with increased through-transmission on ultrasound
examination. They show water density without enhancement on CT,
and appear hyperintense on T2-weighted images, also without enhance-
ment, on MRI.
Renal masses typically are evaluated with CT because of its short
examination times and ease of evaluation, even in patients with a
large body habitus, which can make ultrasonography difficult. MRI
typically is used for problem solving. Multiphasic scanning is performed
with CT to evaluate the density of lesions before administration of
contrast agent and as contrast material filters from the cortex into the
medulla and collecting system. An increase in density of greater than
20 HU corresponds to lesion enhancement and confirms the presence
of a solid mass.
CT is used to stage the tumor by determining the presence of
renal vein invasion, adenopathy, local extension, and distant metastases.
The accuracy of CT for staging is 91%.138 For resectable lesions, CT
can provide information on whether the lesion is amenable to nephron-
sparing surgery or partial nephrectomy. Lesions that are smaller than
4 cm, polar, and cortical and do not involve the renal hilum or collecting
system may be candidates for partial nephrectomy or ablation.
Although CT, MRI, and ultrasonography can all be used to assess
renal lesions, CT with contrast is the dominant method (Fig. 16.17).
PET is useful only when the tumor is FDG-avid. However, the normal
excretion of FDG by the kidneys makes evaluation of the kidneys
more challenging than other tissues, and some renal masses are not
very FDG avid. Thus FDG-PET is not currently recommended for
renal cancers, at least not for reliably characterizing renal masses as
malignant or benign. Metastatic renal cancer is more accurately imaged
on FDG-PET than is primary renal cancer.139,140
Renal cancers can also be imaged by using iodine-124 (124I)–labeled
monoclonal antibodies to carbonic anhydrase IX, which is overexpressed
in clear cell renal cancers. A prospective open-label multicenter study
of 124I-girentuximab PET-CT in patients with renal masses who were
scheduled for resection was performed and PET-CT and contrast-
enhanced CT of the abdomen were compared. In 195 patients, complete
data sets (histopathologic diagnosis and PET-CT and contrast-enhanced
CT results) were available. The average sensitivity was 86.2% for
PET-CT and 75.5% for contrast-enhanced CT (P = .023). The average
specificity was 85.9% for PET-CT and 46.8% for contrast-enhanced
CT (P = .005). These data suggest this investigational agent may be
 Imaging  •  CHAPTER 16 279

A B

C D
Figure 16.15  •  Pancreatic cancer. (A) Axial computed tomography (CT) image shows a mass in the pancreatic body, encasing the splenic artery.
(B) Coronal volume-rendered three-dimensional CT image shows tumor encasing the splenic and left gastric arteries. (C) Coronal volume-rendered maximum-
intensity projection. (D) Sagittal volume-rendered three-dimensional CT images show a mass in the pancreatic head without invasion of the superior mesenteric
artery or vein. A common bile duct stent also is seen.

useful for non-invasive phenotyping of renal masses as malignant
or benign.141
Endocrine Tumors
Imaging is used to study several types of endocrine tumors in a variety
of locations. For adrenal tumors, CT is the procedure of choice, with
metaiodobenzyl-guanidine 123I (MIBG) scanning and MRI scanning
also proving useful for lesion characterization.140 MIBG accumulates
selectively in pheochromocytomas. Adrenal masses with low HU values
(<10 HU) typically are adenomas, which are lipid rich.
For the thyroid gland, radioiodine imaging commonly is used. For
non–radioiodine-avid thyroid cancers, FDG-PET is very useful for
lesion detection and is recommended in the setting of a rising serum
thyroglobulin level with a normal 131I or 23I scan, particularly when
recombinant thyroid-stimulating hormone (TSH) stimulation is used
(Fig. 16.18).142 For neuroendocrine tumors such as carcinoid tumors,
CT and radiolabeled octreotide analogues are very useful, especially
the newer Ga 68 agents.
111
In pentetreotide is FDA approved and used in detecting neu-
roendocrine tumors. 68Ga-labeled DOTA-TOC and related compounds
are typically more effective than 111In-labeled compounds and are
available in a variety of settings. These agents are typically more accurate
than 111In OctreoScan. A meta-analysis of the 68Ga peptide imaging
literature showed the AUC in the task of diagnosing somatostatin
receptor–expressing neuroendocrine tumors in 16 studies comprising
567 patients. The pooled AUC was 0.96. Since FDA approved in the
United States, these Ga 68 dotatate techniques, where available, should
be considered as first-line diagnostic imaging methods in patients
with suspected neuroendocrine tumors.136,143,144
Brain Tumors
The dominant method for the assessment of brain tumors is the MRI
scan, which is the preferred method for initial detection, assessment
of extent of disease, and assessment of efficacy of therapy. The superior
soft tissue contrast provided by MRI makes it superior to CT for
lesion characterization (Fig. 16.19).
280 Part I: Science and Clinical Oncology

A B

C
Figure 16.16  •  (A) Carcinoid metastases to the liver. Axial magnetic resonance image with gadolinium shows multiple enhancing masses in both lobes
of the liver. (B) Hepatoma. Axial magnetic resonance image with gadolinium shows a solid mass in the left lobe of the liver. (C) Intraductal papillary mucinous
tumor of the pancreas. Axial T1-weighted magnetic resonance image of the abdomen shows a low–signal intensity cystic mass in the head of the pancreas.
(Courtesy Dr. Ihab Kamel, The Johns Hopkins University.)

Figure 16.17  •  Axial computed tomography (CT) image of the abdomen

with intravenous contrast agent shows enhancing renal cell carcinoma in the
right kidney. Enhancing tumor invades the right renal vein.
Figure 16.18  •  Positron emission tomography (PET) and computed
tomography (CT) image panel showing an intense fluorine-18 (18F)–fluoro-
deoxyglucose (FDG) uptake focus near clips in the left thyroid bed, consistent
with recurrent thyroid cancer.

Figure 16.19  •  Coronal magnetic resonance image of the brain with
gadolinium shows a mass with peripheral enhancement (arrows), compatible
with tumor, and a more hypointense necrotic area.
Unfortunately, even sophisticated MRI techniques, often relying
on tumor enhancement with gadolinium, cannot be used to detect
microscopic disease. MRI findings, although fairly specific for tumor,
are not completely specific. Thus infarcts, infections, and foci of
demyelination occasionally can mimic tumor foci on MRI. FDG-PET
has a very limited role but can be useful in assessing residual tumor
after radiation therapy and determining whether residual masses are
caused by tumor or tumor necrosis. It is most accurate in highly
aggressive tumors in which brain tumor uptake of FDG is greater
than that of normal brain tissue. Other tracers, such as FDOPA, FET,
and FLT, have shown results more promising than those for FDG.145,146
MRS also can be useful in evaluating this issue because tumors
typically have high levels of choline and low levels of N-acetylaspartate.
Angiography, although a historically useful method, is performed less
frequently for diagnostic purposes in brain tumors. MRI, CT, and
PET can guide biopsies. Furthermore, MRI can be used intraoperatively
to guide therapy.147
Pediatric Tumors
CT usually is the method of choice for evaluation of tumors in children.
For tumors of the central nervous system and for sarcomas, MRI is
preferred. FDG has a growing role, especially for lymphomas and
sarcomas. MIBG scanning often is used in neuroblastomas. CT scanning
should be performed with a reduced tube current and energy to
minimize the radiation dose while preserving the quality of the
diagnostic image.
Multiphase CT should be avoided in children, unless it is clearly
indicated, to minimize the radiation dose to the child.148 PET-CT
has shown broad usefulness in pediatric cancers, and the evolving
literature suggests that PET-CT often offers improvements over CT
alone. Given the radiation dose and long-term risks of carcinogenesis,
it is possible that MRI will have a growing role in pediatric cancer
imaging owing to its non-ionizing nature. PET-MRI, a newer technol-
ogy, may be particularly attractive in children.
Esophageal and Gastric Cancer
CT scanning is the method of choice for initial staging of esophageal
and gastric cancer. Endoscopic ultrasonography is the most sensitive
and accurate method for determining whether tumor has invaded the
Imaging  •  CHAPTER 16 281
wall of the esophagus or involved the periesophageal nodes. PET
scanning is very sensitive in determining whether stage IV disease is
present, and typically is performed as part of the initial staging evalu-
ation, at least for esophageal cancer, to identify patients who are clearly
not candidates for surgery.149 FDG-PET also is being used with
increasing frequency to assess treatment response in patients with
esophageal cancer, because early changes in glycolysis after treatment
appear to be related to treatment efficacy.150
Sarcomas
MRI is the method of choice for detection and assessment of soft
tissue sarcomas. Gadolinium contrast enhancement is also commonly
used. FDG-PET imaging has been useful in assessing primary sarcomas
for aggressiveness and defining possible sites for biopsy in addition
to defining prognosis and grade.151,152 It also has been used to assess
and predict the response of sarcomas to therapy. Early (10 days into
treatment) FDG-PET scans show changes in metabolism predictive
of outcomes, with a rapid decline in FDG uptake associated with a
more favorable survival with some treatments.153,154
Gastrointestinal Stromal Tumors
GI stromal tumors are relatively infrequent, but have been studied
extensively with both CT and PET imaging in the past few years.
These tumors often are responsive to imatinib and other tyrosine
kinase inhibitors, in part because of their constitutive overactivity of
a mutated KIT oncogene. These tumors have been shown to be typically
FDG avid. FDG accumulation declines very rapidly with effective
treatments, earlier than changes in tumor size. Of interest is that
revised CT criteria may allow CT to assess response more quickly to
treatment in such tumors, with changes in tumor Hounsfield units
being one of the characteristic findings of response. Both PET and
CT thus have a role in this somewhat uncommon tumor, especially
for the early assessment of treatment response.155
TREATMENT RESPONSE ASSESSMENT
The use of imaging to assess treatment response is common. The
precise best time to assess response depends on the treatment, its
toxicities, and the alternative therapies available. Typically, the same
methodology used at baseline is used to follow the response of the
cancer to treatment. Early diagnosis of failure of response is a key
goal of treatment response assessments. However, typically response
is assessed after two or more cycles of treatment when anatomic methods
are used.
Functional methods such as PET with FDG or possibly MRI with
diffusion assessments may provide earlier indices of response than
seen with size measures alone. Diffusion and DCE MRI are quite
promising methods, but standardization of diffusion MRI is in its
early phases, although it seems effective in single-center studies,
including in brain tumors.156
There are systematic response criteria available for anatomic imaging
(most recently, Response Evaluation Criteria in Solid Tumors [RECIST]
1.1) and proposed criteria for PET response, the European Organisation
for Research and Treatment of Cancer (EORTC) criteria and the
more recent Positron Emission Tomography Response Criteria In Solid
Tumors (PERCIST) 1.0.157,158 To use quantitation in imaging, close
attention to detail is required in the imaging process. CT or MRI
can provide tumor sizes. PET can provide a standardized uptake value
(SUV), which can be used to phenotype tumors and to quickly assess
treatment response, but such values may vary from device to device
and over time, unless careful standardization is followed. Response
criteria for lymphoma are discussed earlier in this chapter. A challenge
in treatment response is deciphering responses to immunotherapy
with checkpoint inhibitors and other treatments. With these treatments,
transient increases in tumor size, metabolic activity, and lesion number
282 Part I: Science and Clinical Oncology
can be seen before a response or in association with stable disease.
This phenomenon is known as “pseudoprogression.”159 Thus some
caution is in order in assessing mild tumor size increases or increased
metabolism in patients on checkpoint inhibitor therapy.160,161
DEFINING NORMAL ORGAN FUNCTION FOR
CANCER THERAPY
Several tests are used to determine whether a patient is a suitable
candidate for aggressive therapy. These tests include myocardial perfu-
sion imaging at stress to determine whether ischemia is present, because
if present it could increase the risk for a major surgical procedure.
Echocardiography also is used for this purpose. Myocardial function
often is evaluated before chemotherapy is given. This may be done
by using a myocardial blood pool study or, less commonly, an echo-
cardiogram to determine chamber size and ejection fraction.
Pulmonary function usually is determined by pulmonary ventilatory
function tests; however, split lung function and regional function may
be determined with pulmonary perfusion imaging with 99mTc MAA
(macroaggregated albumin; i.e., a quantitative lung scan). Regional
ventilation also can be assessed quantitatively. Such determinations
help to predict the level of pulmonary function expected after surgery.
Split assessment of renal function sometimes is performed before
removal of a renal cancer to ensure that the remaining kidney will be
functional.
Functional imaging can be used to identify the location of eloquent
brain activity and evaluate motor cortex function. It also can help to
guide brain tumor surgery by avoiding key areas of the brain.
GUIDANCE OF RADIATION THERAPY
Radiation therapy can be palliative, or it may be performed with
curative intent. In general, the goal is to deliver maximum radiation
to the tumor while minimizing radiation delivery to normal tissues.
This delicate balance is achieved through increasingly sophisticated
dose delivery systems. The anatomic location of a tumor most com-
monly is defined by treatment-planning CT. The CT data are used
to define tumor and normal tissues with a therapy-planning system.
The planning potentially can be enhanced by better definition of the
gross tumor volume (or biologic tumor volume) versus anatomic tumor
volume, which are not always the same.
FDG-PET is beginning to be used to better define the biologic
tumor volume, often with data from PET-CT. Although this procedure
is not yet fully the norm, it is clear that imaging is key to the optimal
planning of radiation therapy ports. Substantial potential exists to
target areas of tumor that are not identified on CT (expand port size)
or reduce ports to areas that are not involved with tumor, because
the goal is to irradiate tumor while not irradiating normal tissues.162
A substantial number of centers now include PET imaging as part of
their radiation therapy planning in a broad range of diseases.163 Treat-
ment plans for radiation therapy are discussed in a separate chapter
in this book. PET agents identifying areas of tumor hypoxia may be
useful in helping guide escalating doses to areas at high risk of treatment
failure. Another area of opportunity is elimination of radiation therapy
in low-risk patients, in whom the incremental benefit of radiation
may be less than the risks of radiation. This has been studied in
Hodgkin lymphoma, and image-guided elimination of radiation
treatment in low-risk patients is a tenable option.
INTERVENTIONAL PROCEDURES
Increasingly, imaging is being combined with a therapeutic procedure
to provide minimally invasive therapeutics. Examples include the use
of CT to guide thermal or cryoablation of liver or lung tumors and
the use of MRI to guide ablation of brain and prostate tumors (through
heat and cold). The ability to locate tumors and follow, in near real
time, the response to treatment is of tremendous potential.
Catheter-based delivery systems also are important. For example,
angiographic catheters can be used to deliver regional chemotherapy,
emboli, or radioactive or chemical microspheres to treat tumors.
Radioactive microspheres also are assuming a growing role in inter-
ventional oncologic radiology.164
EMERGING OPPORTUNITIES IN IMAGING
Functional imaging methods such as PET and various innovative
MRI techniques are being used increasingly to assess treatment response
early after treatment is begun.30 Beyond this, a variety of imaging
methods are being developed to image key aspects of tumor biology
(Table 16.4). An exciting area for both MRI and PET is in detection
of hypoxia, which is common in a broad range of malignancies and
which represents a possible target for cancer treatments.165 In addition
to hypoxia, tumor perfusion can be imaged with a variety of approaches.
Even though the process is difficult, interest remains in gene
therapeutic approaches, which has led to attempts to determine, through
imaging, whether genes, when delivered, actually reach tumors and,
more importantly, whether they express in vivo the desired levels of
gene product expected to be required to achieve a therapeutic effect.
For example, dopamine receptors have been transfected into cells and
imaged with radioligands capable of binding to the D2 dopamine
receptor. Similarly, genes have been transfected that express varying
thymidine kinase activities. This agent is suitable for gene therapy
and can be imaged with radiolabeled substrates such as FMAU.166,167
Similarly, a great deal of interest has been expressed in stem cell
biology and the potential for stem cells to allow for regeneration of
tissues. Tracking these stem cells in vivo and determining their bio-
distribution and ultimate proliferation are exceptional opportunities
for imaging. These goals have been achieved with both nuclear medicine
and MRI methods.
Small-animal imaging devices of a variety of types are now being
used to help in drug development. Small-animal PET, SPECT, MRI,
and CT scanners have been used to assess treatment response and
aspects of tumor biology. Such methods are of critical importance for
assessing the response of cancers to treatment with newer agents and
understanding therapeutic effects. Even combined human PET-CT
devices have been used for imaging and can provide useful information
for drug development in cancer.
Some methods are of tremendous importance in preclinical studies
but will be more difficult to extend to human studies. One example
is optical imaging. Such methods are capable of tremendous sensitivity
Table 16.4  Emerging Uses of Imaging
INDIVIDUAL PATIENT MANAGEMENT DECISIONS
• Screening
• Phenotyping of the tumor and host
TUMOR
• Viability, proliferation, extent of necrosis or apoptosis, hypoxia,
receptor expression prognosis, type of treatment most likely to be
effective based on receptors or presence of imageable pathways
HOST
• Individualized assessment of pharmacokinetics of the drug and of
organ function and pharmacodynamics
DRUG DEVELOPMENT
• Does the tumor have a relevant target?
• Does the drug reach the target?
• Is the target pathway affected by treatment?
• Is the proper dose of drug being given?
• Does alteration of the target pathway alter the tumor biology?
 Imaging  •  CHAPTER 16 283

and excellent resolution in vivo in small animals. A variety of approaches
can be used, with emitted light, transmitted light, and reflected light.168
Photoacoustic imaging approaches, in which light is transmitted and
sound is received, are also being applied.169
With bioluminescence approaches, a very small number of cancer
cells can be identified in vivo in small animals. Such approaches,
although very potent in vivo in small animals and capable of being
combined with radionuclide and other methods, are not as easily
translated into humans, at least for broad applications, because of the
limited penetration in tissue of light photons. However, in a broad
range of detection issues the light can reach detectors or targets, both
intraoperatively and endoscopically for superficial structures, so this
area is likely to be increasingly translated to practice. Detection of
sentinel lymph nodes and resection margins are both areas of consider-
able promise. Thus small-animal imaging is a key element of progress
allowing for proof of concept and refinement of tumor biologic processes
before translation to human use.170
SUMMARY
Anatomic imaging of cancer using x-rays has been and remains extremely
useful, and in the more modern form of CT is still the dominant
approach to imaging the patient with visceral cancer. Anatomic methods,
although very potent, have clear limitations, but continue to improve,
as evidenced by digital mammography, tomosynthesis, and other
techniques. Screening programs with mammography, CT of the thorax,
and CT colonography are now proven methods to detect disease at
earlier stages and improve outcomes. Anatomic methods can detect
disease early, and such methods reliably show whether a mass is present.
However, they provide limited information regarding the composition
of the mass. In addition, they can be insensitive to the presence of
small tumor foci, may be slow to change in response to therapy, are
not predictive of response, and may be more challenging to apply in
evaluating the postoperative patient.
The functional information provided by MRI, including diffusion
characteristics and, to a substantially lesser extent, MRS, adds to the
anatomic signature of the lesions and continues to grow in application.
Imaging of additional phenotypic alterations of cancers with functional
imaging methods such as PET adds information that often is clinically
valuable for patient management in many common cancers.
Although many molecular alterations are present in cancer, the
one that is by far most exploited in clinical practice and clinical
research is the accelerated glucose metabolism present in most cancers.
This process is well imaged with the radiotracer FDG. The ability to
localize the molecular alterations of cancer spatially, through qualitative
cognitive methods performed by the imaging specialist, computer
fusion of image sets, or fused “anatomolecular” image sets using
dedicated PET-CT, is key to optimal use of the imaging methods.
The newer PET imaging agents for prostate cancer, especially those
targeting PSMA, are expected to have a great impact on prostate
cancer management.
Increasingly, the ability of PET imaging to quickly assess treatment
response is allowing adaptation of treatments depending on the response
of a specific patient’s tumor to treatment. This response adaptive
approach is expected to grow in the coming years. It has had great
impact in lymphoma and in head and neck cancer. Systematic response
criteria for treatment response with PET have been developed
(PERCIST 1.0). Functional imaging also is being revisited in the
approach to radiation therapy treatment planning. Functional
“molecular” imaging methods such as PET, SPECT, and varying
methods of MRI, in addition to optical, photoacoustic, and ultrasound
imaging and technical improvements in CT and interventional
techniques, are expected to enhance the care of the patient with cancer
in the coming years and will likely continue to change the way oncology
is practiced.
The complete reference list is available online at
ExpertConsult.com.

KEY REFERENCES
1. Bragg DG, Rubin P, Hricak H. Imaging strategies for
oncologic diagnosis and multidisciplinary treatment.
In: Bragg D, Rubin P, Hricak H, eds. Oncologic
Imaging. 2nd ed. Philadelphia: WB Saunders; 2002:
3–20.
3. Bossuyt PM, Cohen JF, Gatsonis CA, Korevaar
DA. STARD 2015: updated reporting guidelines
for all diagnostic accuracy studies. Ann Transl Med.
2016;4(4).
5. Hanley JA. Receiver operating characteristic (ROC)
methodology: the state of the art. Crit Rev Diagn
Imaging. 1989;29:307–335.
8. Hillner BE, Siegel BA, Liu D, et al. Impact of
positron emission tomography/computed tomog-
raphy and positron emission tomography (PET)
alone on expected management of patients with
cancer: initial results from the National Oncologic
PET Registry. J Clin Oncol. 2008;2155–2161.
doi:10.1200/JCO.2007.14.5631.
26. DePriest PD, DeSimone CP. Ultrasound screening
for the early detection of ovarian cancer. J Clin
Oncol. 2003;21(suppl 10):194–199.
30. Lee KC, Sud S, Meyer CR, et al. An imaging
biomarker of early treatment response in prostate
cancer that has metastasized to the bone. Cancer
Res. 2007;67:3524–3528.
32. Kuo PH, Kanal E, Abu-Alfa AK, Cowper SE.
Gadolinium-based MR contrast agents and
nephrogenic systemic fibrosis. Radiology. 2007;242:
647–649.
34. Blomqvist L, Torkzad MR. Whole-body imaging
with MRI or PET/CT: the future for single-modality
imaging in oncology? JAMA. 2003;290:3248–
3249.
47. Woloshin S, Schwartz LM, Black WC, et al. Cancer
screening campaigns—getting past uninformative
persuasion. N Engl J Med. 2012;367:1677–
1679.
51. Gould MK, Maclean CC, Kuschner WG, et al.
Accuracy of positron emission tomography for
diagnosis of pulmonary nodules and mass lesions:
a meta-analysis. JAMA. 2001;285:914–924.
55. van Tinteren H, Hoekstra OS, Smit EF, et al.
Effectiveness of positron emission tomography
in the preoperative assessment of patients with
suspected non-small-cell lung cancer: the PLUS mul-
ticentre randomised trial. Lancet. 2002;359:1388–
1393.
57. Lardinois D, Weder W, Hany TF, et al. Staging of
non-small-cell lung cancer with integrated positron-
emission tomography and computed tomography.
N Engl J Med. 2003;348:2500–2507.
89. Johnson CD, Mei-Hsiu Chen MH, Alicia Y,
Toledano AY, et al. Accuracy of CT colonography
for detection of large adenomas and cancers. N Engl
J Med. 2008;359:1207–1217.
104. Spaepen K, Stroobants S, Dupont P, et al. Prognostic
value of positron emission tomography (PET) with
fluorine-18 fluorodeoxyglucose ([18F]FDG) after
first-line chemotherapy in non-Hodgkin’s lymphoma:
is [18F]FDG-PET a valid alternative to conventional
diagnostic methods? J Clin Oncol. 2001;19:414–
419.
105. Schaefer NG, Taverna C, Strobel K, et al. Hodgkin
disease: diagnostic value of FDG PET/CT after
first-line therapy—is biopsy of FDG-avid lesions
still needed? Radiology. 2007;244:257–262.
106. Front D, Bar-Shalom R, Mor M, et al. Hodgkin
disease: prediction of outcome with 67Ga scintig-
raphy after one cycle of chemotherapy. Radiology.
1999;210:487–491.
110. Cheson BD, Pfistner B, Juweid ME, et al. Revised
response criteria for malignant lymphoma. J Clin
Oncol. 2007;25:579–586.
119. Stoffels I, Boy C, Pöppel T, Kuhn J, et al. Associa-
tion between sentinel lymph node excision with or
without preoperative SPECT/CT and metastatic
node detection and disease-free survival in mela-
noma. JAMA. 2012;308(10).
172. Tatsumi M, Miller JH, Wahl RL. 18F-FDG PET/
CT in evaluating non-CNS pediatric malignancies.
J Nucl Med. 2007;48(12):1923–1931.

REFERENCES
1. Bragg DG, Rubin P, Hricak H. Imaging strategies for
oncologic diagnosis and multidisciplinary treatment.
In: Bragg D, Rubin P, Hricak H, eds. Oncologic
Imaging. 2nd ed. Philadelphia: WB Saunders;
2002:3–20.
2. Friedman KP, Wahl RL. Clinical use of positron
emission tomography in the management of cutane-
ous melanoma. Semin Nucl Med. 2004;34:242–253.
3. Bossuyt PM, Cohen JF, Gatsonis CA, Korevaar
DA. STARD 2015: updated reporting guidelines
for all diagnostic accuracy studies. Ann Transl Med.
2016;4(4).
4. Gates TJ. Screening for cancer: evaluating the
evidence. Am Fam Physician. 2001;63:513–522.
5. Hanley JA. Receiver operating characteristic (ROC)
methodology: the state of the art. Crit Rev Diagn
Imaging. 1989;29:307–335.
6. Wahl RL, Siegel BA, Coleman RE, Gatsonis CG.
Prospective multicenter study of axillary nodal
staging by positron emission tomography in breast
cancer. J Clin Oncol. 2004;22:277–285.
7. Verboom P, Van Tinteren H, Hoekstra OS, et al.
Cost-effectiveness of FDG-PET in staging non-small
cell lung cancer: the PLUS study. Eur J Nucl Med
Mol Imaging. 2003;30:1444–1449.
8. Hillner BE, Siegel BA, Liu D, et al. Impact of
positron emission tomography/computed tomog-
raphy and positron emission tomography (PET)
alone on expected management of patients with
cancer: initial results from the National Oncologic
PET Registry. J Clin Oncol. 2008;2155–2161.
doi:10.1200/JCO.2007.14.5631.
9. de Koning HJ. Mammographic screening: evidence
from randomised controlled trials. Ann Oncol.
2003;14:1185–1189.
10. Henschke CI, Yankelevitz DF, McCauley DI,
et al. Guidelines for the use of spiral computed
tomography in screening for lung cancer. Eur Respir
J. 2003;39:45s–51s.
11. Henschke CI, McCauley DI, Yankelevitz DF, et al.
Early Lung Cancer Action Project: overall design and
findings from baseline screening. Lancet. 1999;354:
99–105.
12. Henschke CI, Yankelevitz DF, Libby DM, et al. Sur-
vival of patients with stage I lung cancer detected on
CT screening. N Engl J Med. 2006;355:1763–1771.
13. Aberle DR, Adams AM, Berg CD, et al. National Lung
Screening Trial Research Team: Reduced lung-cancer
mortality with low-dose computed tomographic
screening. N Engl J Med. 2011;365(5):395–
409. doi:10.1056/NEJMoa1102873. [Epub 2011
Jun 29].
14. Carter HB, Partin AW, Walsh PC, et al. Gleason
score 6 adenocarcinoma: should it be labeled as
cancer? J Clin Oncol. 2012;30(35):4294–4296.
15. Jepsen P, Johnsen SP, Gillman MW, et  al.
Interpretation of observational studies. Heart.
2004;90:956–960.
16. Ubel PA, Hirth RA, Chernew ME, Fendrick
AM. What is the price of life and why doesn’t it
increase at the rate of inflation? Arch Intern Med.
2003;163:1637–1641.
17. Wahl RL, Quint LE, Cieslak RD, et al. “Anatometa-
bolic” tumor imaging: fusion of FDG PET with
CT or MRI to localize foci of increased activity.
J Nucl Med. 1993;34:1190–1197.
18. Cohade C, Wahl RL. Applications of positron
emission tomography/computed tomography image
fusion in clinical positron emission tomography:
clinical use, interpretation methods, diagnostic
improvements. Semin Nucl Med. 2003;33:228–237.
19. Gourtsoyiannis N, Grammatikakis J, Prassopoulos P.
Role of conventional radiology in the diagnosis and
staging of gastrointestinal tract neoplasms. Semin
Surg Oncol. 2001;20:91–108.
20. Pisano ED, Gatsonis C, Hendrick E, et al.
Diagnostic performance of digital versus film
mammography for breast-cancer screening. N Engl
J Med. 2005;353:1773–1783.
21. Shah AJ, Wang J, Yamada T, Fajardo LL. Digital
mammography: a review of technical develop-
ment and clinical applications. Clin Breast Cancer.
2003;4:63–70.
22. Svahn TM. Breast tomosynthesis and digital mam-
mography: a comparison of diagnostic accuracy. BJR.
2012;85:1019, e1074–e1082.
23. Gilbert FJ, Tucker L, Young KC. Digital breast
tomosynthesis (DBT): a review of the evidence for
use as a screening tool. Clin Radiol. 2016;71(2):141–
150.
24. Miles KA. Functional computed tomography in
oncology. Eur J Cancer. 2002;38:2079–2084.
25. Singh K. Interventional radiology in the gynaeco-
logical oncology patient. Best Pract Res Clin Obstet
Gynaecol. 2001;15:279–290.
26. DePriest PD, DeSimone CP. Ultrasound screening
for the early detection of ovarian cancer. J Clin
Oncol. 2003;21(suppl 10):194–199.
27. Lehman CD, Isaacs C, Schnall MD, et al. Cancer
yield of mammography, MR, and US in high-risk
women: prospective multi-institution breast cancer
screening study. Radiology. 2007;244:381–388.
28. Laking GR, Price PM, Sculpher MJ. Assessment of
the technology for functional imaging in cancer. Eur
J Cancer. 2002;38:2194–2199.
29. Kwock L, Smith JK, Castillo M, et al. Clinical
applications of proton MR spectroscopy in oncology.
Technol Cancer Res Treat. 2002;1:17–28.
30. Lee KC, Sud S, Meyer CR, et al. An imaging
biomarker of early treatment response in prostate
cancer that has metastasized to the bone. Cancer
Res. 2007;67:3524–3528.
31. Cowper SE, Su LD, Bhawan J, et al. Nephrogenic
fibrosing dermopathy. Am J Dermatopathol. 2001;23:
383–393.
32. Kuo PH, Kanal E, Abu-Alfa AK, Cowper SE. Gad-
olinium-based MR contrast agents and nephrogenic
systemic fibrosis. Radiology. 2007;242:647–649.
33. High WA, Ayers RA, Chandler J, et al. Gadolinium
is detectable within the tissue of patients with
nephrogenic systemic fibrosis. J Am Acad Dermatol.
2007;56:21–26.
34. Blomqvist L, Torkzad MR. Whole-body imaging
with MRI or PET/CT: the future for single-modality
imaging in oncology? JAMA. 2003;290:3248–
3249.
35. Cho A, Lau JY, Geraghty BJ, Cunningham CH,
Keshari KR. Hyperpolarized MRI of cancer
metabolism. J Nucl Med. 2017;jnumed-116.
36. Jerusalem G, Hustinx R, Beguin Y, Fillet G.
PET scan imaging in oncology. Eur J Cancer.
2003;39:1525–1534.
37. Kim JH, Lee JS, Song IC, Lee DS. Comparison of
segmentation-based attenuation correction methods
for PET/MRI: evaluation of bone and liver standard-
ized uptake value with oncologic PET/CT data.
J Nucl Med. 2012;53(12):1878–1882.
38. Fraum TJ, Fowler KJ, McConathy J. PET/MRI:
Emergency applications of oncology. Acad Radiol.
2016;23(2):220–236.
39. Kaminski MS, Zelenetz AD, Press OW, et al.
Pivotal study of iodine I 131 tositumomab for
chemotherapy-refractory low-grade or transformed
low-grade B-cell non-Hodgkin’s lymphomas. J Clin
Oncol. 2001;19:3918–3928.
40. Bremer C, Ntziachristos V, Weissleder R.
Optical-based molecular imaging: contrast agents
and potential medical applications. Eur Radiol.
2003;13:231–243.
41. Fazel R. Exposure to Low-dose ionizing radiation
from medical imaging procedures. N Engl J Med.
2009;361:849–857.
42. Brenner DJ, Elliston CD, Hall EJ, et al. Estimated
risks of radiation-induced fatal cancer from
Imaging  •  CHAPTER 16 283.e1
283.e1
pediatric CT. AJR Am J Roentgenol. 2001;176(2):289–
296.
43. Brenner DJ, Hall EJ. Computed tomography—an
increasing source of radiation exposure. N Engl J
Med. 2007;357:2277–2284.
44. Pandharipande PV, Eisenberg JD, Lee RJ, Gilmore
ME, Turan EA, Singh S, et al. Patients with testicular
cancer undergoing CT surveillance demonstrate a
pitfall of radiation-induced cancer risk estimates: the
timing paradox. Radiology. 2013;266(3):896–904.
doi:10.1148/radiol.12121015. [Epub 2012 Dec 18].
45. Wahl RL. Anatomolecular imaging of cancer. Mol
Imag Biol. 2003;5:49–56.
46. Hanahan D, Weinberg RA. Hallmarks of cancer:
the next generation. Cell. 2011;144(5):646–674.
doi:10.1016/j.cell.2011.02.013.
47. Woloshin S, Schwartz LM, Black WC, et al. Cancer
screening campaigns—getting past uninformative
persuasion. N Engl J Med. 2012;367:1677–1679.
48. Guidelines for management of small pulmonary
nodules detected on CT scans: a statement from
the Fleischner Society. Radiology. 2005;237:395–400.
49. Erasmus JJ, Connolly JE, McAdams HP, Roggli
VL. Solitary pulmonary nodules: Part I. Morpho-
logic evaluation for differentiation of benign and
malignant lesions. Radiographics. 2000;20:43–58.
50. Erasmus JJ, McAdams HP, Connolly JE. Solitary
pulmonary nodules: Part II. Evaluation of the
indeterminate nodule. Radiographics. 2000;20:59–66.
51. Gould MK, Maclean CC, Kuschner WG, et al.
Accuracy of positron emission tomography for
diagnosis of pulmonary nodules and mass lesions:
a meta-analysis. JAMA. 2001;285:914–924.
52. Naidich DP, Bankier AA, MacMahon H. Recommen-
dations for the management of subsolid pulmonary
nodules detected at CT: a statement from the
Fleischner Society. Radiology. 2013;266(1):304–317.
doi:10.1148/radiol.12120628. Published online
October 15, 2012.
53. Goudarzi B, Jacene HA, Wahl RL. Diagnosis and
differentiation of bronchioloalveolar carcinoma from
adenocarcinoma with bronchioloalveolar components
with metabolic and anatomic characteristics using
PET/CT. J Nucl Med. 2008;49:1585–1592.
54. Dwamena BA, Sonnad SS, Angobaldo JO, Wahl RL.
Metastases from non-small cell lung cancer: medias-
tinal staging in the 1990s. Meta-analytic comparison
of PET and CT. Radiology. 1999;213:530–536.
55. van Tinteren H, Hoekstra OS, Smit EF, et al.
Effectiveness of positron emission tomography in the
preoperative assessment of patients with suspected
non-small-cell lung cancer: the PLUS multicentre
randomised trial. Lancet. 2002;359:1388–1393.
56. Beadsmoore CJ, Screaton NJ. Classification,
staging and prognosis of lung cancer. Eur J Radiol.
2003;45:8–17.
57. Lardinois D, Weder W, Hany TF, et al. Staging of
non–small-cell lung cancer with integrated positron-
emission tomography and computed tomography.
N Engl J Med. 2003;348:2500–2507.
58. Bradley JD, Dehdashti F, Mintun MA, et al. Positron
emission tomography in limited-stage small-cell lung
cancer: a prospective study. J Clin Oncol. 2004;22:
3248–3254.
59. Mayo-Smith WW, Boland GW, Noto RB, Lee MJ. State-
of-the-art adrenal imaging. Radiographics. 2001;21:
995–1012.
60. Smith RA, Saslow D, Sawyer KA, et al. American
Cancer Society guidelines for breast cancer screening:
update 2003. CA Cancer J Clin. 2003;53:141–169.
61. Poplack SP, Tosteson TD, Kogel CA, Nagy HM.
Digital breast tomosynthesis: initial experience in
98 women with abnormal digital screening mam-
mography. AJR Am J Roentgenol. 2007;189:616–623.
62. Chen SC, Carton AK, Albert M, et al. Initial clinical
experience with contrast-enhanced digital breast
tomosynthesis. Acad Radiol. 2007;14:229–238.
283.e2 Part I: Science and Clinical Oncology
63. Bleyer A, Welch HG. Effect of three decades of
screening mammography on breast-cancer incidence.
N Engl J Med. 2012;367:1998–2005.
64. Rhodes DJ, Hruska CB, Phillips SW, et al. Dedicated
dual-head gamma imaging for breast cancer screening
in women with mammographically dense breasts.
Radiology. 2011;258(1):106–118. doi:10.1148/
radiol.10100625. [Epub 2010 Nov 2].
65. Bluemke DA, Gatsonis CA, Chen MH, et al.
Magnetic resonance imaging of the breast prior to
biopsy. JAMA. 2004;292:2735–2742.
66. Lehman CD, Gatsonis C, Kuhl CK, et al. MRI
evaluation of the contralateral breast in women with
recently diagnosed breast cancer. N Engl J Med.
2007;356:1295–1303.
67. Kuhl CK. Current status of breast MR imaging.
Part 2. Clinical applications. Radiology. 2007;244:
672–691.
68. Tatsumi M, Cohade C, Mourtzikos KA, et al. Initial
experience with FDG-PET/CT in the evaluation
of breast cancer. Eur J Nucl Med Mol Imaging.
2006;33(3):254–262.
69. Berg WA, Madsen KS, Schilling K, et al. Breast
cancer: comparative effectiveness of positron emission
mammography and MR imaging in presurgi-
cal planning for the ipsilateral breast. Radiology.
2011;258(1):59–72. doi:10.1148/radiol.10100454.
[Epub 2010 Nov 12]; PMID:21076089.
70. Lyman GH, Giuliano AE, Somerfield MR, et al.
American Society of Clinical Oncology guideline
recommendations for sentinel lymph node biopsy
in early-stage breast cancer. J Clin Oncol. 2005;23:
7703–7720.
71. Veronesi U, Paganelli G, Viale G, et al. A randomized
comparison of sentinel-node biopsy with routine
axillary dissection in breast cancer. N Engl J Med.
2003;349:546–553.
72. Galimberti V, Cole BF, Viale G, Veronesi P, Vicini
E, Intra M, et al. Axillary dissection versus no
axillary dissection in patients with breast cancer
and sentinel-node micrometastases (IBCSG 23-01):
10-year follow-up of a randomised, controlled phase
3 trial. Lancet Oncol. 2018.
73. Wu D, Gambhir SS. Positron emission tomography
in diagnosis and management of invasive breast
cancer: current status and future perspectives. Clin
Breast Cancer. 2003;4(suppl 1):55–63.
74. Yu KK, Hricak H. Imaging prostate cancer. Radiol
Clin North Am. 2000;38:59–85.
75. Heijmink SW, Barentsz JO. Contrast-enhanced versus
systematic transrectal ultrasound-guided prostate
cancer detection: an overview of techniques and a
systematic review. Eur J Radiol. 2007;63:310–316.
76. Weinreb JC, Barentsz JO, Choyke PL, Cornud F,
Haider MA, Macura KJ, et al. PI-RADS Prostate
Imaging–Reporting and Data System: 2015, version
2. Eur Urol. 2016;69(1):16–40.
77. Pinto PA, Chung PH, Rastinehad AR, et al.
Magnetic resonance imaging/ultrasound fusion
guided prostate biopsy improves cancer detection
following transrectal ultrasound biopsy and correlates
with multiparametric magnetic resonance imaging.
J Urol. 2011;186(4):1281–1285. doi:10.1016/j.
juro.2011.05.078. [Epub 2011 Aug 17].
78. Harisinghani MG, Barentsz J, Hahn PF, et al.
Noninvasive detection of clinically occult lymph-
node metastases in prostate cancer. N Engl J Med.
2003;348:2491–2499.
79. Scheenen TW, Heijmink SW, Roell SA, et al. Three-
dimensional proton MR spectroscopy of human
prostate at 3 T without endorectal coil: feasibility.
Radiology. 2007;245:507–516.
80. Barentsz JO, Futterer JJ, Takahashi S. Use of
ultrasmall superparamagnetic iron oxide in lymph
node MR imaging in prostate cancer patients. Eur
J Radiol. 2007;63:369–372.
81. Schuster DM, Savir-Baruch B, Nieh PT, et al.
Detection of recurrent prostate carcinoma with
anti-1-amino-3-18F-fluorocyclobutane-1-carboxylic
acid PET/CT and 111In-capromab pendetide
SPECT/CT. Radiology. 2011;259(3):852–861.
doi:10.1148/radiol.11102023. [Epub 2011 Apr 14].
82. Maurer T, Gschwend JE, Rauscher I, Souvatzoglou
M, Haller B, Weirich G, et al. Diagnostic efficacy
of 68 gallium-PSMA positron emission tomography
compared to conventional imaging for lymph node
staging of 130 consecutive patients with intermediate
to high risk prostate cancer. J Urol. 2016;195(5):
1436–1443.
83. Schwenck J, Rempp H, Reischl G, Kruck S, Stenzl
A, Nikolaou K, et al. Comparison of 68Ga-labelled
PSMA-11 and 11C-choline in the detection of
prostate cancer metastases by PET/CT. Eur J Nucl
Med Mol Imaging. 2017;44(1):92–101.
84. Bluemel C, Krebs M, Polat B, Linke F, Eiber M,
Samnick S, et al. 68Ga-PSMA-PET/CT in patients
with biochemical prostate cancer recurrence and
negative 18F-choline-PET/CT. Clin Nucl Med.
2016;41(7):515–521.
85. Cho SY, Gage KL, Mease RC, et al. Biodistribu-
tion, tumor detection, and radiation dosimetry of
18F-DCFBC, a low-molecular-weight inhibitor of
prostate-specific membrane antigen, in patients with
metastatic prostate cancer. J Nucl Med. 2012;53(12):
1883–1891.
86. Rowe SP, Gage KL, Faraj SF, Macura KJ, Cornish
TC, Gonzalez-Roibon N, et al. 18F-DCFBC
PET/CT for PSMA-based detection and charac-
terization of primary prostate cancer. J Nucl Med.
2015;56(7):1003–1010.
87. Szabo Z, Mena E, Rowe SP, Plyku D, Nidal R,
Eisenberger MA, et al. Initial evaluation of [18F]
DCFPyL for prostate-specific membrane antigen
(PSMA)-targeted PET imaging of prostate cancer.
Mol Imaging Biol. 2015;17(4):565–574.
88. Kim DH, Pickhardt PJ, Taylor AJ, et al. CT
colonography versus colonoscopy for the detection
of advanced neoplasia. N Engl J Med. 2007;357:
1403–1412.
89. Johnson CD, Mei-Hsiu Chen M-H, Alicia Y,
Toledano AY, et al. Accuracy of CT colonography
for detection of large adenomas and cancers. N Engl
J Med. 2008;359:1207–1217.
90. Chin BB, Wahl RL. 18F-Fluoro-2-deoxyglucose
positron emission tomography in the evaluation of
gastrointestinal malignancies. Gut. 2003;52(suppl
4):23–29.
91. Cohade C, Osman M, Leal J, Wahl RL. Direct
comparison of 18-FDG PET and PET/CT in
patients with colorectal carcinoma. J Nucl Med.
2003;44:1797–1803.
92. Kinkel K, Lu Y, Both M, et al. Detection of hepatic
metastases from cancers of the gastrointestinal tract
by using noninvasive imaging methods (US, CT,
MR imaging, PET): a meta-analysis. Radiology.
2002;224:748–756.
93. Niekel MC, Bipat S, Stoker J. Diagnostic imaging
of colorectal liver metastases with CT, MR imaging,
FDG PET, and/or FDG PET/CT: a meta-analysis
of prospective studies including patients who have
not previously undergone treatment. Radiology.
2010;257:674–684.
94. Singh AK, Grigsby PW, Dehdashti F, et al. FDG-
PET lymph node staging and survival of patients
with FIGO stage IIIb cervical carcinoma. Int J Radiat
Oncol Biol Phys. 2003;56:489–493.
95. Grigsby PW, Siegel BA, Dehdashti F, et al. Post-
therapy [18F] fluorodeoxyglucose positron emission
tomography in carcinoma of the cervix: response
and outcome. J Clin Oncol. 2004;22:2167–2171.
96. Grigsby PW, Siegel BA, Dehdashti F. Lymph
node staging by positron emission tomography in
patients with carcinoma of the cervix. J Clin Oncol.
2001;19:3745–3749.
97. Zimny M, Siggelkow W, Schroder W, et al.
2-[Fluorine-18]-fluoro-2-deoxy-d-glucose positron
emission tomography in the diagnosis of recurrent
ovarian cancer. Gynecol Oncol. 2001;83:310–315.
98. Avril N, Sassen S, Schmalfeldt B, et al. Prediction
of response to neoadjuvant chemotherapy by
sequential F-18-fluorodeoxyglucose positron emission
tomography in patients with advanced-stage ovarian
cancer. J Clin Oncol. 2005;23:7445–7453.
99. Bristow RE, del Carmen MG, Pannu HK, et al.
Clinically occult recurrent ovarian cancer: patient
selection for secondary cytoreductive surgery using
combined PET/CT. Gynecol Oncol. 2003;90:
519–528.
100. O’Doherty MJ, Macdonald EA, Barrington SF,
et al. Positron emission tomography in the manage-
ment of lymphomas. Clin Oncol (R Coll Radiol).
2002;14:415–426.
101. Nakamoto Y, Tatsumi M, Hammoud D, et al. Normal
FDG distribution patterns in the head and neck:
PET/CT evaluation. Radiology. 2005;234:879–885.
102. Nakamoto Y, Cohade C, Tatsumi M, et al. CT
appearance of bone metastases detected with FDG
PET as part of the same PET/CT examination.
Radiology. 2005;237:627–634.
103. El-Galaly TC, d’Amore F, Mylam KJ, de Nully Brown
P, Bøgsted M, Bukh A, et al. Routine bone marrow
biopsy has little or no therapeutic consequence for
positron emission tomography/computed tomogra-
phy–staged treatment-naive patients with Hodgkin
lymphoma. J Clin Oncol. 2012;30(36):4508–4514.
104. Spaepen K, Stroobants S, Dupont P, et al. Prognostic
value of positron emission tomography (PET) with
fluorine-18 fluorodeoxyglucose ([18F]FDG) after
first-line chemotherapy in non-Hodgkin’s lymphoma:
is [18F]FDG-PET a valid alternative to conventional
diagnostic methods? J Clin Oncol. 2001;19:414–419.
105. Schaefer NG, Taverna C, Strobel K, et al. Hodgkin
disease: diagnostic value of FDG PET/CT after
first-line therapy—is biopsy of FDG-avid lesions
still needed? Radiology. 2007;244:257–262.
106. Front D, Bar-Shalom R, Mor M, et al. Hodgkin
disease: prediction of outcome with 67Ga scintig-
raphy after one cycle of chemotherapy. Radiology.
1999;210:487–491.
107. Kasamon YL, Jones RJ, Wahl RL. Integrating PET
and PET/CT into the risk-adapted therapy of
lymphoma. J Nucl Med. 2007;48(suppl 1):19S–27S.
108. Hutchings M, Kostakoglu L, Zaucha JM, Mal-
kowski B, Biggi A, Danielewicz I, et al. In vivo
treatment sensitivity testing with positron emission
tomography/computed tomography after one cycle
of chemotherapy for Hodgkin lymphoma. J Clin
Oncol. 2014;32(25):2705–2711.
109. Cohade C, Osman M, Pannu HK, Wahl RL.
Uptake in supraclavicular area fat (“USA-Fat”):
description on 18F-FDG PET/CT. J Nucl Med.
2003;44:170–176.
110. Cheson BD, Pfistner B, Juweid ME, et al. Revised
response criteria for malignant lymphoma. J Clin
Oncol. 2007;25:579–586.
111. Juweid ME, Cheson BD. Positron-emission
tomography and assessment of cancer therapy.
N Engl J Med. 2006;354:496–507.
112. Juweid ME, Wiseman GA, Vose JM, et al. Response
assessment of aggressive non-Hodgkin’s lymphoma
by integrated International Workshop Criteria and
fluorine-18-fluorodeoxyglucose positron emission
tomography. J Clin Oncol. 2005;23:4652–4661.
113. Cheson BD, Fisher RI, Barrington SF, Cavalli F,
Schwartz LH, Zucca E, et al. Recommendations for
initial evaluation, staging, and response assessment of
Hodgkin and non-Hodgkin lymphoma: the Lugano
classification. J Clin Oncol. 2014;32(27):3059–
3067.
114. Cheson BD, Ansell S, Schwartz L, Gordon LI,
Advani R, Jacene HA, et al. Refinement of the
Lugano Classification lymphoma response criteria
in the era of immunomodulatory therapy. Blood.
2016;128(21):2489–2496.

115. Gallamini A, Barrington SF, Biggi A, Chauvie S,
Kostakoglu L, Gregianin M, et al. The predictive
role of interim positron emission tomography for
Hodgkin lymphoma treatment outcome is confirmed
using the interpretation criteria of the Deauville five-
point scale. Haematologica. 2014;99(6):1107–1113.
116. Kasamon YL, Wahl RL, Swinnen LJ. FDG PET and
high-dose therapy for aggressive lymphomas: toward
a risk-adapted strategy. Curr Opin Oncol. 2004;16:
100–105.
117. Dann EJ, Bar-Shalom R, Tamir A, et al. Risk-adapted
BEACOPP regimen can reduce the cumulative dose
of chemotherapy for standard and high-risk Hodgkin
lymphoma with no impairment of outcome. Blood.
2007;109:905–909.
118. Alazraki N, Glass EC, Castronovo F, Olmos RA,
Podoloff D. Procedure guideline for lymphos-
cintigraphy and the use of intraoperative gamma
probe for sentinel lymph node localization in
melanoma of intermediate thickness 1.0. J Nucl
Med. 2002;43:1414–1418.
119. Stoffels I, Boy C, Pöppel T, Kuhn J, et al. Associa-
tion between sentinel lymph node excision with or
without preoperative SPECT/CT and metastatic
node detection and disease-free survival in mela-
noma. JAMA. 2012;308(10).
120. Cobben DC, Koopal S, Tiebosch AT, et al. New
diagnostic techniques in staging in the surgical
treatment of cutaneous malignant melanoma. Eur
J Surg Oncol. 2002;28:692–700.
121. Swetter SM, Carroll LA, Johnson DL, Segall GM.
Positron emission tomography is superior to com-
puted tomography for metastatic detection in mela-
noma patients. Ann Surg Oncol. 2002;9:646–653.
122. Cho SY, Lipson EJ, Im HJ, Rowe SP, Mena E,
Blackford A, et al. Prediction of response to immune
checkpoint inhibitor therapy using early time-point
FDG-PET/CT imaging in patients with advanced
melanoma. J Nucl Med. 2017;pp.jnumed-116.
123. Strobel K, Dummer R, Husarik DB, et al. High-risk
melanoma: accuracy of FDG PET/CT with added
CT morphologic information for detection of
metastases. Radiology. 2007;244:566–574.
124. Barentsz JO, Witjes JA, Ruijs JH. What is new
in bladder cancer imaging? Urol Clin North Am.
1997;24:583–602.
125. Lawler LP. MR imaging of the bladder. Radiol Clin
North Am. 2003;41:161–177.
126. Harisinghani MG, Saksena MA, Hahn PF, et al.
Ferumoxtran-10-enhanced MR lymphangiography:
does contrast-enhanced imaging alone suffice for
accurate lymph node characterization? Am J
Roentgenol. 2006;186:144–148.
127. Powles T, Murray I, Brock C, et al. Molecular
positron emission tomography and PET/CT imaging
in urological malignancies. Eur Urol. 2007;51:
1511–1520.
128. Zinreich J. Imaging in laryngeal cancer: computed
tomography, magnetic resonance imaging, positron
emission tomography. Otolaryngol Clin North Am.
2002;35:971–991.
129. Mehanna H, McConkey CC, Rahman JK, et al.
PET-NECK: a multicenter randomized phase III
non-inferiority trial comparing a positron emission
tomography-computerised tomography-guided
watch-and-wait policy with planned neck dissec-
tion in the management of locally advanced (N2/
N3) nodal metastases in patients with squamous
cell head and neck cancer. Health Technol Assess.
2017;21:1–122.
130. Mehanna H, Wong WL, McConkey CC, Rahman
JK, Robinson M, Hartley AG, et al. PET-CT
surveillance versus neck dissection in advanced
head and neck cancer. NEJM. 2016;374(15):1444–
1454.
131. Wong RJ, Lin DT, Schoder H, et al. Diagnostic
and prognostic value of [(18)]fluorodeoxyglucose
positron emission tomography for recurrent head
and neck squamous cell carcinoma. J Clin Oncol.
2002;20:4199–4208.
132. Branstetter BF, Blodgett TM, Zimmer LA, et al. Head
and neck malignancy: is PET/CT more accurate
than PET or CT alone? Radiology. 2005;235:580–
586.
133. Kalra MK, Maher MM, Boland GW, et al. Correla-
tion of positron emission tomography and CT in
evaluating pancreatic tumors: technical and clinical
implications. Am J Roentgenol. 2003;181:387–393.
134. Hanbidge AE. Cancer of the pancreas: the best image
for early detection. CT, MRI, PET or US? Can J
Gastroenterol. 2002;16:101–105.
135. Pakzad F, Groves AM, Ell PJ. The role of positron
emission tomography in the management of pan-
creatic cancer. Semin Nucl Med. 2006;36:248–256.
136. Antunes P, Ginj M, Zhang H, et al. Are radiogallium-
labelled DOTA-conjugated somatostatin analogues
superior to those labelled with other radiometals?
Eur J Nucl Med Mol Imaging. 2007;34:982–993.
137. Ho CL, Chen S, Yeung DW, Cheng TK. Dual-tracer
PET/CT imaging in evaluation of metastatic hepa-
tocellular carcinoma. J Nucl Med. 2007;48:902–909.
138. Sheth S, Scatarige JC, Horton KM, et al. Current
concepts in the diagnosis and management of renal
cell carcinoma: role of multidetector C and three-
dimensional CT. Radiographics. 2001;21:237–254.
139. Aide N, Cappele O, Bottet P, et al. Efficiency of
[(18)]FDG PET in characterising renal cancer and
detecting distant metastases: a comparison with CT.
Eur J Nucl Med Mol Imaging. 2003;30:1236–1245.
140. Pocaro AB, Cavalleri S, Ballista C, et al. Modern
imaging methods and preoperative management of
pheochromocytoma: review of the literature and
case report. Arch Esp Urol. 2000;53:749–753.
141. Divgi CR, Uzzo RG, Gatsonis C, et al. Positron
emission tomography/computed tomography
identification of clear cell renal cell carcinoma: results
from the REDECT Trial. J Clin Oncol. 2013;31(2):
187–194.
142. Chin BB, Patel P, Cohade C, et al. Recombinant
human thyrotropin stimulation of fluoro-d-glucose
positron emission tomography uptake in well-
differentiated thyroid carcinoma. J Clin Endocrinol
Metab. 2004;89:91–95.
143. Treglia G, Castaldi P, Rindi G, et al. Diagnostic
performance of gallium-68 somatostatin receptor
PET and PET/CT in patients with thoracic and
gastroenteropancreatic neuroendocrine tumours:
a meta-analysis. Endocrine. 2012;42(1):80–87.
doi:10.1007/s12020-012-9631-1. [Epub 2012
Feb 20].
144. Kauhanen S, Seppanen M, Minn H, et al.
Fluorine-18-l-dihydroxyphenylalanine (18F-DOPA)
positron emission tomography as a tool to localize an
insulinoma or beta-cell hyperplasia in adult patients.
J Clin Endocrinol Metab. 2007;92:1237–1244.
145. Chen W, Silverman DH, Delaloye S, et al. 18F-
FDOPA PET imaging of brain tumors: comparison
study with 18F-FDG PET and evaluation of
diagnostic accuracy. J Nucl Med. 2006;47:904–
911.
146. van Waarde A, Jager PL, Ishiwata K, et al. Com-
parison of sigma-ligands and metabolic PET tracers
for differentiating tumor from inflammation. J Nucl
Med. 2006;47:150–154.
147. Jacobs AH, Winkler A, Dittmar C, et al. Molecular
and functional imaging technology for the develop-
ment of efficient treatment strategies for gliomas.
Technol Cancer Res Treat. 2002;1:187–204.
148. Frush DP. Pediatric CT: practical approach to
diminish the radiation dose. Pediatr Radiol. 2002;32:
714–717.
149. Reed CE, Eloubeidi MA. New techniques for
staging esophageal cancer. Surg Clin North Am.
2002;82:697–710.
150. Ott K, Weber WA, Lordick F, et al. Metabolic
imaging predicts response, survival, and recurrence
Imaging  •  CHAPTER 16 283.e3
283.e3
in adenocarcinomas of the esophagogastric junction.
J Clin Oncol. 2006;24:4692–4698.
151. Hoffer FA. Primary skeletal neoplasms: osteosarcoma
and Ewing sarcoma. Top Magn Reson Imaging.
2002;13:231–239.
152. Brenner W, Bohuslavizki KH, Eary JF. PET imaging
of osteosarcoma. J Nucl Med. 2003;44:930–
942.
153. Hawkins DS, Schuetze SM, Butrynski JE, et al. [18F]
Fluorodeoxyglucose positron emission tomography
predicts outcome for Ewing sarcoma family of
tumors. J Clin Oncol. 2005;23:8828–8834.
154. Luber BS, Leal JP, Wang H, Bolejack V, Schuetze
SM, Schwartz LH, et al. Response to early treatment
evaluated with 18F-FDG PET and PERCIST 1.0
predicts survival in patients with Ewing sarcoma
family of tumors treated with a monoclonal antibody
to the insulinlike growth factor 1 receptor. J Nucl
Med. 2016;57(5):735–740.
155. Choi H, Charnsangavej C, Faria SC, et al. Cor-
relation of computed tomography and positron
emission tomography in patients with metastatic
gastrointestinal stromal tumor treated at a single
institution with imatinib mesylate: proposal of
new computed tomography response criteria.
J Clin Oncol. 2007;25:1753–1759.
156. Padhani AR, Liu G, Koh DM, et al. Diffusion-
weighted magnetic resonance imaging as a cancer
biomarker: consensus and recommendations.
Neoplasia. 2009;11(2):102–125, 71.
157. Wahl RL, Jacene H, Kasamon Y, et al. From
RECIST to PERCIST: evolving considerations for
PET response criteria in solid tumors. J Nucl Med.
2009;50(suppl 1):122S–150S.
158. Eisenhauer EA, Therasse P, Bogaerts J, et al. New
response evaluation criteria in solid tumours: revised
RECIST guideline (version 1.1). Eur J Cancer. 2009;
45(2):228–247.
159. Hodi FS, Hwu WJ, Kefford R, Weber JS, Daud
A, Hamid O, et al. Evaluation of immune-related
response criteria and RECIST v1. 1 in patients with
advanced melanoma treated with pembrolizumab.
J Clin Oncol. 2016;34(13):1510–1517.
160. Shankar LK, Hoffman JM, Bacharach S, et al. Con-
sensus recommendations for the use of 18F-FDG
PET as an indicator of therapeutic response in
patients in National Cancer Institute Trials. J Nucl
Med. 2006;47(6):1059–1066.
161. Wolchok JD, Hoos A, O’Day S, Weber JS, Hamid
O, Lebbé C, et al. Guidelines for the evaluation
of immune therapy activity in solid tumors:
immune-related response criteria. Clin Cancer Res.
2009;15(23):7412–7420.
162. Ling CC, Humm J, Larson S, et al. Towards mul-
tidimensional radiotherapy (MD-CRT): biological
imaging and biological conformality. Int J Radiat
Oncol Biol Phys. 2000;47:551–560.
163. Brianzoni E, Rossi G, Ancidei S, et al. Radiotherapy
planning: PET/CT scanner performances in the
definition of gross tumour volume and clinical target
volume. Eur J Nucl Med Mol Imaging. 2005;32:
1392–1399.
164. Lin M, Shon IH, Wilson R, et al. Treatment response
in liver metastases following 90Y SIR-spheres:
an evaluation with PET. Hepatogastroenterology.
2007;54:910–912.
165. van Baardwijk A, Dooms C, van Suylen RJ,
et al. The maximum uptake of 18-deoxyglucose
on positron emission tomography scan correlates
with survival, hypoxia inducible factor-1alpha and
GLUT-1 in non–small cell lung cancer. Eur J Cancer.
2007;43:1392–1398.
166. Tjuvajev JG, Avril N, Oku T, et al. Imaging herpes
virus thymidine kinase gene transfer and expres-
sion by positron emission tomography. Cancer Res.
1998;58:4333–4341.
167. Yaghoubi SS, Berger F, Gambhir SS. Study-
ing the biodistribution of positron emission
283.e4 Part I: Science and Clinical Oncology
tomography reporter probes in mice. Nat Protoc.
2007;2:1752–1755.
168. Keereweer S, Hutteman M, Kerrebijn JD, et al.
Translational optical imaging in diagnosis and treat-
ment of cancer. Curr Pharm Biotechnol. 2012;13(4):
498–503.
169. Akers WJ, Edwards WB, Kim C, et al. Multimodal
sentinel lymph node mapping with single-photon
emission computed tomography (SPECT)/computed
tomography (CT) and photoacoustic tomography.
Transl Res. 2012;159(3):175–181.
170. Emerson DK, Limmer KK, Hall DJ, et al. A
receptor-targeted fluorescent radiopharmaceutical
for multireporter sentinel lymph node imaging.
Radiology. 2012;265(1):186–193.
 Imaging  •  CHAPTER 16 283.e5
283.e5

SELF-ASSESSMENT REVIEW QUESTIONS ANSWERS

1. In evaluating CT scanning for lung cancer as reported in the 1. (a, b, d)  Note that to achieve a reduction in lung cancer and all
NLST trial, which of the following statement(s) are true? cancer mortality in this study, a high prevalence of false-positive
a. CT screening reduced mortality from lung cancer by 20%. findings was required.
b. Of the CT findings, 95% were false positives. 2. (b)  Mammography and tomosynthesis with modern systems
c. CT screening and chest x-ray screening were equally have comparable radiation doses. Younger patients are more
ineffective in detecting lung cancer. at risk of carcinogenesis than older patients. MRI scans carry
d. CT screening was associated with decreased all-cause no risk of irradiation but carry risks of adverse reactions
mortality in this trial. to IV contrast, especially in patients with impaired renal
2. Which of the following statement(s) are true regarding radiation function (NSF).
dose in imaging? 3. (c)  Ultrasound may be the least expensive study to perform,
a. Mammography results in a much lower dose than breast but it may not be the most accurate in the abdomen; thus it is
tomosynthesis. not the most cost-effective. MRI has low sensitivity for
b. CT and nuclear medicine procedures represent over 75% of detecting contrast agent molecules versus optical and nuclear
the imaging-related radiation dose in the United States. methods. Thus large amounts of MRI contrast material must be
c. Age at the time of irradiation is not a major factor in given to obtain a suitable signal in vivo. PET can be quantified
potential radiation-induced carcinogenesis. for SUV if performed correctly per RSNA QIBA guidelines.
d. MRI scans with contrast agent carry no risk of side effects.
3. Which of the following statement(s) are true about imaging
methods in cancer?
a. Ultrasound is the most cost-effective method in abdominal
imaging.
b. MRI is the most sensitive method for detecting low numbers
of contrast agent molecules compared with nuclear or optical
methods.
c. Radiographs have higher resolution than ultrasound images
or nuclear imaging scans.
d. FDG-PET studies cannot be quantified.
 D. CLINICAL TRIALS
17  Biostatistics and Bioinformatics in
Clinical Trials
Brian P. Hobbs, Donald A. Berry, and Kevin R. Coombes

S UMMARY OF K EY
• The process of conducting cancer
research must change in the face of
prohibitive costs and limited patient
resources.
• Biostatistics has a tremendous
impact on the level of science in
cancer research, especially in the
design and conducting of clinical
trials.
• The bayesian statistical approach to
clinical trial design and conduct can
be used to develop more efficient
and effective cancer studies.
P OI N T S
• Modern technology and advanced
analytic methods are directing the
focus of medical research to subsets
of disease types and to future trials
across different types of cancer.
• A consequence of the rapidly
changing technology for generating
“omics” data is that biologic assays
are often not stable long enough to
discover and validate a model in a
clinical trial.
• Bioinformaticians must use
technology-specific data
normalization procedures and
rigorous statistical methods to
account for sample collecting, batch
effects, multiple testing, confounding
covariates, and any other potential
biases.
• Best practices in developing
prediction models include public
access to the information, rigorous
validation of the model, and model
lockdown before its use in patient
care management.

BIOSTATISTICS APPLIED TO
CANCER RESEARCH
The modern era of cancer therapy raises major issues regarding statistical
inference and study design. First, as biomarkers become available,
they divide cancers into ever smaller subsets, with unique biomarker-
defined combinations of targeted therapies for each subset. Soon every
patient with cancer will have an orphan disease. A related issue is the
ever-increasing cost of drug development and the consequent cost of
delivering care to persons with cancer. Unless we modify our approaches
to cancer research, we will not be able to afford the therapies we
develop, and innovation will cease.
Biostatistical philosophy is critical in understanding the state of
cancer research today and where it is heading. Biostatistics has had
an immense impact on the level of science in cancer research, especially
in designing and conducting clinical trials. However, as we make
progress in developing better cancer therapeutics, patients’ prognoses
improve, and clinical trials get larger because they are event driven.
The irony is that despite the development of cancer in increasing
numbers of persons in the world, fewer clinical investigations are
possible because of limited resources, and fewer drugs can be developed
despite the burgeoning number of potential anticancer drugs. False-
positive and false-negative conclusions are well known, but the most
frustrating problem in the modern era may be the number of “false
neutrals”—drugs and therapeutic strategies that are never investigated
because of insufficient resources.
The history of biostatistics is dominated by the so-called frequentist
approach. An alternative approach, the bayesian approach, was largely
ignored as biostatistics developed into a discipline. In the frequentist
approach, probabilities are defined as long-run proportions. The
focus is on the final results of a clinical trial, say, with the unit of
inference being the entire trial rather than, for example, a patient within
the trial.
For reasons discussed in this chapter, much interest has recently
been expressed in the bayesian approach to statistics. In the first half
of the 20th century there were few bayesian biostatisticians, and few
biostatisticians knew what it meant to be bayesian.
The bayesian approach predates the frequentist approach. Thomas
Bayes developed his treatise on inverse probabilities in the 1750s, and
it was published posthumously in 1763 as “An Essay Towards Solving
a Problem in the Doctrine of Chances.”
In the 200 years after Bayes, the discipline of statistics was influenced
by probability theory and, in particular, games of chance, dating to
the early 1700s.1–3 This view focused on probability distributions of
outcomes of experiments, assuming a particular value of a parameter.
A simple example is the binomial distribution. This distribution gives
the probabilities of outcomes of a specified number of tosses of a
coin with known probability of heads, which is the distribution’s
parameter. The binomial distribution continues to be important today.
For example, it is used in designing cancer clinical trials in which the
end point is dichotomous (tumor response or not, say) and assuming
a predetermined sample size.
CLINICAL TRIALS
A clinical trial is an experiment involving human subjects with the
goal of evaluating one or more treatments for a disease or condition.
A randomized controlled trial (RCT) compares two or more treatments
in which treatment assignment is determined by chance, such as by
rolling a die or tossing a coin.
The traditional statistical approach is to consider two possible
values of the unknown parameters, such as the tumor response rate

284

0.25
Probability of number of responses
0.20
0.15
0.10
0.05
0.00
0 1 2 3 4 5 6 7
Figure 17.1  •  Probabilities for number of responses when the rate of response is r = 20% (red bars) and when r = 50% (blue bars). Like all probability
distributions, the sums of the heights of the red bars and the blue bars both equal 1.
r. For one value of r the treatment has no useful benefit, the so-called
null hypothesis. The alternative hypothesis is a value of r that is clinically
important in the sense of meriting future development.
Consider a single-arm clinical trial with the objective of evaluating
r. The null value of r is taken to be 20%. The alternative value is r =
50%. The trial consists of treating n = 20 patients. The exact number
of responses is not known in advance, but it is known to be either 0
or 1 or 2 on up to 20. The relevant probability distribution of the
outcome is binomial, with one distribution for r = 20% and a second
distribution for r = 50%. These distributions are shown in Fig. 17.1,
with red bars for r = 20% and blue bars for r = 50%. More generally,
there is a different binomial distribution for each possible value of r.
Both the frequentist and bayesian approaches to clinical trial design
and analysis use distributions such as those shown in Fig. 17.1, but
they use them differently.
Frequentist Approach
The frequentist approach to inference is based on error rates. A type
I error is rejecting a null hypothesis when it is true, and a type II
error is accepting the null hypothesis when it is false, in which case
the alternative hypothesis is assumed to be true. It seems reasonable
to reject the null, r = 20% (red bars in Fig. 17.1), in favor of the
alternative, r = 50% (blue bars in Fig. 17.1), if the number of responses
is sufficiently large. Candidate values of “large” might reasonably be
those in which the red and blue bars in Fig. 17.1 start to overlap,
perhaps 9 or greater, 8 or greater, 7 or greater, or 6 or greater.
The type I error rates for these rejection rules are the respective
sums of the heights of the red bars in Fig. 17.1. For example, when
the cut point is 9, the type I error rate is the sum of the heights of
the red bars for 9, 10, 11, and so on, which is 0.007387 + 0.002031
+ 0.000462 + … = 0.0100. When the cut points are 8, 7, and 6,
the respective type I error rates are 0.0321, 0.0867, and 0.1958. One
convention is to define the cut point so that the type I error rate is no
greater than 0.05. The largest of the candidate type I error rates that
is less than 0.05 is 0.0321, the test that rejects the null hypothesis if
there are eight or more responses. The type II error rate is calculated
from the blue bars in Fig. 17.1, wherein the alternative hypothesis is
assumed to be true. The sum of the heights of the blue bars for 0 up
to 7 responses is 0.1316. Convention is to consider the complementary
quantity and call it “statistical power:” 0.8684, which rounds off
to 87%.
Biostatistics and Bioinformatics in Clinical Trials  •  CHAPTER 17 285
8 9 10 11 12 13 14 15 16 17 18 19 20
Number of responses
The distinction between rate and probability in the aforementioned
description is important, and failing to discriminate these terms has
led to much confusion in medical research.4 The type I error rate is a
probability only when one assumes that the null hypothesis is true.
Probabilities of events requiring the truth of the null hypothesis are
not available in the frequentist approach, and indeed this is a principal
contrast with the bayesian approach (described later).
Suppose a trial is conducted and 9 of 20 patients respond. One
concludes that the null, r = 20%, is rejected because 9 is greater
than the predetermined cut point of 8. However, the results are
more convincing than had the result been exactly 8. Such reasoning
led to the convention of reporting a P value, an “observed type I
error rate.” This is the type I error rate had the predetermined cut
point been set to the observed number. Thus the P value is .0100, as
calculated earlier.
Values of r other than 20% and 50% are possible. The standard
frequentist approach to representing the range of possibilities is to
provide a confidence interval, which is the set of values of r that would
not be rejected as null hypotheses based on the data actually observed:
9 responses out of 20 patients (or 45%). For these data, the values of
r that would be rejected are those less than 26%. Because very large
values of r are also inconsistent with the observed rate of 45%, it is
conventional to also exclude large values from the confidence interval.
Assuming type I error rates of 5% for both small and large values
of r, the resulting confidence interval is from about 26% to 61%,
which is a 90% confidence interval in the sense that 5% is excluded
on both sides. Excluding only 2.5% on each side provides a 95%
confidence interval: 23% to 64%. Ninety-five percent confidence
intervals are commonly used and are always somewhat wider than 90%
confidence intervals.
Bayesian Approach
A major difference between the bayesian and frequentist approaches is
the use of probability distributions to represent unknown values. In the
frequentist approach, probabilities apply only for “data.” Parameters
that index data distributions (such as r in the aforementioned example)
are unknown but are fixed and thus are not subject to probability state-
ments. In the bayesian approach, all unknowns, including parameters,
have probability distributions.
In the example in which 20 patients yielded 9 responses, suppose
that r is assumed to be either 20% or 50%. The bayesian conclusion
286 Part I: Science and Clinical Oncology

is the probability of r = 50%, given the final results. (This is 1 minus of a diagnostic test is the prevalence of the condition or disease in
the probability of r = 20% under the assumption that these are the question. Prior probability depends on other evidence that may be
only two possible values of r.) The calculation uses the Bayes rule. available from previous studies of the same therapy in the same disease,
The method is the same as when finding the positive predictive value or related therapies in the same disease or different diseases. Assessment
of a test as it relates to the condition’s prevalence and the test’s sensitivity may differ depending on the person making the assessment. Subjectivity
and specificity. The Bayes rule is also called the rule of inverse probabilities is present in all of science; the bayesian approach has the advantage
because it relates the probability of some event A, given that event of making subjectivity explicit and open.5
B has occurred, with the probability of event B given that event A When the prior probability equals 0.50, as assumed in Fig. 17.2,
has occurred. the posterior probability of r = 20% is 0.0441. Obviously, this is
The calculation is intuitive when viewed as a tree diagram, as in different from the frequentist P value of 0.0100 calculated earlier.
Fig. 17.2. Fig. 17.2A shows the full set of probabilities. The first Posterior probabilities and P values have very different interpretations.
branching shows the two possible parameters, r = 20% and r = 50%. The P value is the probability of the actual observation, 9 responses,
The probabilities shown in the figure, 0.50 for both, will be discussed plus that of 10 responses, 11 responses, and so on, assuming the null
later. The data are shown in the next branching, with the observed distribution (the red bars in Fig. 17.1). The posterior probability is
data, nine responses (nine resp) on one branch and all other data on computed conditionally with respect to the actual observation of nine
the other. The probability of the data given r, which statisticians call responses and is the probability of the null hypothesis—that the red
the likelihood of r, is the height of the bar in Fig. 17.1 corresponding bars are in fact correct—but assuming that the true response rate is
to nine responses, the red bar for r = 20%, and the blue bar for r = a priori equally likely to be 20% and 50%.
50%. The rightmost column in Fig. 17.2A gives the probability of P values are not intuitive in that they are calculated conditionally
both the data and r along the branch in question. For example, the with respect to a hypothesis that seems unlikely to be true in view of the
probability of r = 20% and “nine resp” is 0.50 multiplied by 0.0074. results (the null hypothesis) and because they depend on probabilities
The probability of r = 20% given the experimental results depends of possible occurrences that were not observed. For example, if there
on the probability of r = 20% without any condition, its so-called are two candidate null distributions with the same probability of
prior probability. The analog in finding the positive predictive value the actual observation, the one with the smaller “tail” of unobserved
values will have a smaller P value. Because they are counterintuitive,
misinterpretations of P values abound. People usually convert a P
value into something they understand but that is wrong, and the
misinterpretation usually has a bayesian ring to it; for example, “The P
Probability Probability
value is the probability that the results could have occurred by chance
Probability Prior of data of data alone.” An objection to bayesian inferences is that they are specific to
of r probability Data given r given r assumed prior probabilities. A sensible type of report that addresses
this concern is the following: “If your prior probability is this, then
9 resp 0.0074 0.0037
your posterior probability is that.”
As an example of such a report, Fig. 17.3 shows the relationship
0.50
r = 20% between the prior and posterior probabilities. The figure indicates
Not 9 0.9926 0.4963 that the posterior probability is moderately sensitive to the prior.
Someone whose prior probability of r = 20% is 0 or 1 will not be
swayed by the data. However, as Fig. 17.3 indicates, the conclusion
9 resp 0.1602 0.0801 that r = 20% has low probability is robust over a broad range of prior
r = 50% probabilities. A conclusion that r = 20% has moderate to high prob-
0.50 ability is possible only for someone who was convinced that r = 20%
in advance of the trial.
A Not 9 0.8398 0.4199 In the example, r was assumed to be either 20% or 50%. It would
be unusual to be certain that r is one of two values and that no other
values are possible. A more realistic example would be to allow r to
9 resp 0.0074 0.0037 have any value between 0 and 1 but to associate weights with different
values depending on the degree to which the available evidence supports
0.50 those values. In such a case, prior probabilities can be represented
r = 20% with a histogram (or density). A common assumption is one that
Not 9 0.9926 0.4963 reflects no prior information that any particular interval of values of
r is more probable than any other interval of the same width. The
9 resp 0.1602 0.0801 corresponding density is flat over the entire interval from 0 to 1 and
r = 50% is shown in red in Fig. 17.4A.
0.50 The probability of the observed results (9 responses and 11
nonresponses) for a given r is proportional to r9(1 − r)11, with the
B Not 9 0.8398 0.4199 likelihood of r based on the observed results. The prior density is
updated by multiplying it by the likelihood. Because the prior density
is constant, this multiplication preserves the shape of the likelihood.
Probability of 9 responses: 0.0037 + 0.0801 = 0.0838 Thus the posterior density equals just the likelihood itself, shown in
Probability that r = 20% given 9 responses: 0.0037/0.0838 = 0.0441 green in Fig. 17.4A.
C Probability that r = 50% given 9 responses: 0.0801/0.0838 = 0.9559
The bayesian analog of the frequentist confidence interval is called
a probability interval or a credibility interval. A 95% credibility interval
Figure 17.2  •  (A) Tree diagram showing probabilities and conditional is shown in Fig. 17.4B: r = 26% to 66%. It is similar to but different
probabilities when there are 20 observations. (B) Modification of (A), restricting from the 95% confidence interval discussed earlier: 23% to 64%.
to the actual number of nine responses (9 resp), with “Not 9” grayed out. Confidence intervals and credibility intervals calculated from flat prior
Possible observations that were not observed are not considered. (C) Calculations densities tend to be similar, and indeed they agree in most circumstances
demonstrating the Bayes rule. when the sample size is large. However, their interpretations differ. A
 Biostatistics and Bioinformatics in Clinical Trials  •  CHAPTER 17 287

credible interval has a particular probability that the parameter lies
in the interval. Statements involving probability or chance or likelihood
cannot be made for confidence intervals.
Any interval is an inadequate summary of a posterior density. For
example, although r = 45% and r = 65% are both in the 95% credibility
interval in the aforementioned example (see Fig. 17.4), the posterior
density shows the former to be five times as probable as the latter.
A characteristic of the bayesian approach is the synthesis of evidence.
The prior density incorporates what is known about the parameter
or parameters in question. For example, suppose another trial is
conducted under the same circumstances as the aforementioned example
trial, and suppose the second trial yields 15 responses among 40
patients. Fig. 17.5 shows the prior density and the likelihoods from
the first and second trials. Multiplying likelihood number 1 by the
prior density gives the posterior density, as shown in Fig. 17.4. This
1
now serves as the prior density for the next trial. Multiplying that
density by likelihood number 2 gives the posterior density based on
the data from both trials, as shown in Fig. 17.5. The order of observation
is not important. Updating first on the basis of the second trial gives
the same result. Indeed, multiplying the prior density, likelihood
number 1, and likelihood number 2 in Fig. 17.5 gives the posterior
density shown in the figure.
The calculations of Fig. 17.5 assume that r is the same in both
trials. This assumption may not be correct. Different trials may well
have different response rates, say r1 and r2. In the bayesian approach,
these two parameters have a joint prior density. One way to incorporate
into the prior distribution the possibility of correlated r1 and r2 is to
use a hierarchical model in which r1 and r2 are regarded to be sampled
from a probability density that is unknown, and therefore this density
itself has a probability distribution. More generally, there may be
multiple sources of information that are correlated and multiple
parameters that have an unknown probability distribution. A hierarchi-
cal model allows for borrowing strength across the various sources
depending in part on the similarity of the results.6–8

0.9 0.82
Posterior probability that r = 20%

0.8
7
0.7
Posterior density
0.6 6 Prior density
0.5 Likelihood 1
5 Likelihood 2
0.4
0.3 0.0441 4

0.2
3
0.1
2
0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
1
Prior probability that r = 20%
0
Figure 17.3  •  Influence of prior probability of rate of response, r = 20%,
on its posterior probability after observing 9 responses among 20 patients. The 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
calculation uses the Bayes rule, a different application of the Bayes rule for r
each possible prior probability. The figure highlights the posterior probability
of 0.0441 when the prior probability is 0.50, as calculated in Fig. 17.2C. If Figure 17.5  •  Two trials, conducted under the same circumstances, with
the prior probability is very high—for example, 0.99—then the posterior data indicated by the likelihoods 1 and 2. The posterior density is that for
probability is about 0.82, still high but much reduced from its prior probability. the data from both trials.

4 4

3 3

2 2

1 1
2.5% 2.5%

0 0
A 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 B 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
r r
Posterior density
Prior density

Figure 17.4  •  Histograms of probability distributions. (A) The horizontal red line shows the prior density, assumed to be flat and hence “noninformative”
in the sense described in the text. The blue curve shows the posterior density on the basis of 9 responses among 20 patients. Because the prior density is flat,
the posterior density is proportional to the probability of the results for a given response rate, r, which is r9(1 − r)11. The proportionality constant makes the
area under the curve equal to 1, the same as the area under the prior density. (B) The area under the posterior density to the left of r = 0.26 equals 2.5%,
and the same is true for that to the right of 0.66. The values between these two limits form a 95% credibility interval for r.
288 Part I: Science and Clinical Oncology
A rather different application of bayesian hierarchical models,
called a tumor agnostic, is destined to become important in cancer
clinical trials. Consider an agent targeted at a particular mutation.
There may be subsets of patients who harbor this mutation across a
broad range of tumor types. The mutation may be rare in some or
all tumor types. Mustering a compelling clinical trial in any given
tumor type may be impossible. Instead, one might conduct a single
trial across 10 tumor types, say, regarding response rates r1, r2, …, r10
as a sample from a population. The population distribution may be
homogeneous, in which case there is substantial “borrowing of strength”
across the tumor types. This may enable a claim of effectiveness in
tumor types that, when left to stand alone, would have wide credibility
intervals because of their small sample sizes. On the other hand, if
the population of response rates is heterogeneous, then the observed
response rates will be highly variable, and borrowing will occur mainly
across neighboring tumor types.
We next apply the bayesian perspective in two important directions
that are the focus of attention in modern cancer research.
Adaptive Designs of Clinical Trials
Randomization was introduced into scientific experimentation by
R.A. Fisher in the 1920s and 1930s and applied to clinical trials
by A.B. Hill in the 1940s.9 Hill’s goal was to minimize treatment
assignment bias, and his approach changed medicine in a funda-
mentally important way. The RCT is now the gold standard of
medical research. A mark of its impact is that the RCT has changed
little during the past 65 years, except that RCTs have gotten bigger.
Traditional RCTs are simple in design and address a small number of
questions, usually only one. However, progress is slow, not because of
randomization but because of limitations of traditional RCTs. Trial
sample sizes are prespecified. Trial results sometimes make clear that
the answer was present well before the results became known. The
only adaptations considered in most modern RCTs are interim analyses
with the goal of stopping the trial on the basis of early evidence of
efficacy or for safety concerns. There are usually few interim analyses,
and stopping rules are conservative. As a consequence, few trials
stop early.
In traditional RCTs, randomization proportions are fixed through-
out. The possibility that the accumulating data in the trial can influence
randomization probabilities or other aspects of a trial’s course may
affect the trial’s performance characteristics, including its type I error
rate and statistical power. Moreover, these effects may be difficult to
analyze with traditional mathematics. However, modern computers
and software make traditional mathematics unnecessary. Any prospective
trial design, however complicated, can be simulated. A prospective
trial design is an automaton. At any time during the trial and based
on the information available from trial participants, the next patient
is assigned a particular therapy, possibly based on an adaptive randomiza-
tion scheme. Any trial that has a prospective design can be simulated.
Virtual patients can be generated with their outcomes depending on
assumed parameter values and treatment assignment according to the
prospective design. Replicating the trial 10,000 times, say, gives a firm
handle on the trial conclusion for the parameter values assumed in
the simulation.
Consider a simple case, a variant of the earlier example in which
the response rate r was assumed to be either 20% or 50%. Set the
maximum number of patients in the trial to 20. Stop the trial with
a claim favoring the alternative hypothesis r = 50% whenever the
probability of r = 50% versus r = 20% is at least 95% (and therefore
the probability of r = 20% is less than 5%).
As a check that the reader is following this description: the binomial
distribution assumed earlier is no longer relevant, even if the final
sample size happens to be 20. Consider an extreme case. The binomial
distribution gives positive probability to 20 responses regardless of
the value of r (unless r = 0). However, the adaptive design would have
stopped long before getting to 20 patients when every patient
represented a response to the treatment—in fact, the trial would have
stopped after four responses in four patients.
It happens that for this simple adaptive design it is possible to find
its operating characteristics algebraically, but the calculations are tedious.
In more complicated circumstances, algebraic calculations may not
be possible and simulations are necessary.
Simulations are easy to describe. To address r = 50%, start with a
fair coin, one with probability of heads equal to 50%, and interpret
“heads” to mean response. Whenever a patient is accrued to the trial,
toss the coin, observe the result, and update the probability that r =
50% on the basis of the tosses thus far. Stop the trial if this probability
is greater than 95% and make a mark so indicating. If the number
of tosses reaches 20 without having made a mark, then stop the trial
because you have hit the maximal sample size. When the trial stops,
make a note of the total number of tosses, say n, in the trial. Repeat
the trial a total of 10,000 times. Count the number of marks and
divide by 10,000. This is the estimated power of the study assuming
r = 50%. Tabulate the 10,000 values of n to give an estimate of the
distribution of the final sample size under the alternative hypothesis.
(You should have a mark for every trial with n <20 and also for some
trials with n = 20.)
To find the type I error rate, repeat the aforementioned process
with another set of 10,000 iterations generated using a device that
has probability of heads equal to 20%. (An example device is a regular
20-sided die with “heads” labeled on four of the sides.) The proportion
of marks is the estimated type I error rate. The tabulated values of n
are the estimated distribution of the final sample size under the null
hypothesis.
Tossing a coin perhaps 100,000 times will take a while, especially
because you will have to keep track of which trial you are in and
whether you have achieved the stopping boundary for that trial. The
good news is that a simple program running on a modern desktop
computer can simulate 10,000 trials in a few seconds. The computer
does all the work. Moreover, an additional few seconds gives you
another 10,000 simulated trials assuming another value of r by simply
changing the value of r in the program.
Fig. 17.6 shows the sample size distribution for 10,000 trials under
the assumption that r = 50%. The estimated type II error rate is
0.1987, which is the proportion of these 10,000 trials that reached
n = 20 without ever concluding that the posterior probability of r =
50% is at least 95%. The sample size distribution when r = 20% is
not shown but is easy to describe: 9702 of the trials (97.02%) went
to the maximum sample size of n = 20 without hitting the posterior
probability boundary and the other 3% stopped early (at various
values of n <20) with an incorrect conclusion that r = 50%. Thus
3% is the estimated type I error rate. The “estimated” aspect of these
statements is because there is some error due to simulation. Based on
10,000 iterations, the standard error of the estimated power is small,
but positive: 0.4%. The standard error of the type I error rate is less
than 0.2%.
Because the bayesian approach allows for updating knowledge
incrementally as data accrue, even after every observation, it is ideally
suited for building efficient and informative adaptive clinical trials.10–12
The bayesian approach serves as a tool. As evinced in the aforementioned
example, simulations enable calculation of traditional frequentist
measures of type I error rate and power. The Institute of Medicine
(IOM) recently advocated for the need to restructure the entire clinical
trial system to advocate for adaptive designs and to address other
deficiencies that limit the effectiveness and efficiency of trials.13 The
initiative was reaffirmed with the 21st Century Cures Act passed by
the United States House of Representatives in July 2015.
Innovations in adaptive design have been proposed to address all
aspects of drug development. Despite the extensive use of traditional
dose-escalation studies, their limitations with monitoring rules based
on algorithmic formulations, such as the 3 + 3 design, have been well
described (see e.g., Wong and colleagues14). Recent innovations have
effectuated designs for phase I trials that use the sequential application

2500
Number of simulated studies
2000
1500
1000
500
0
1 2 3 4
Figure 17.6  •  Sample size distribution when the response rate is r = 50% in the adaptive stopping example in the text. Among the 2353 simulated trials
that finished with maximum sample size of n = 20 were 366 that achieved a posterior probability of r = 50% greater than 0.95 at that point; the other 1987
simulated trials reached n = 20 without such a conclusion, and thus 0.1987 is the estimated type II error rate (equivalently, the estimated power is 0.8013).
The mean sample size in the 10,000 simulations was 12.2, with a standard deviation of 5.7. Some sample sizes never occurred. For example, the stopping
bound for the probability of r = 50% requires at least five responses in n = 6 patients. However, both four and five responses also call for stopping at n = 5,
and thus five responses cannot occur at n = 6.
of decision rules derived from formal probability models15 and have
facilitated designs that are devised to optimize dose and schedule
conjointly.16,17 Several authors have explored the extent to which
platform trials18 (which enable study arms to be dropped or added
during the course of enrollment) may yield improvements in efficiency
as well as improve treatment efficacy for trial participants.
A patient’s experience on receiving a particular therapeutic strategy
is often a complex synthesis of measures that describe both the extent
of induced harm as well as realized clinical benefit achieved. Thus
“patient response” in itself is often difficult to characterize, especially
when designing studies to compare the multimodal treatment strategies
that are actually used as matter of routine in clinical oncology. Clinical
translation has been limited by statistical testing procedures and designs
that rely on reductive characterizations of a patient’s experience based
on binary and univariate endpoints. A few authors have put forth
innovations to address these limitations19–22 and to establish designs
that account for the dynamic nature of cancer care in settings that
require multiple therapies administered in stages over the course of
treatment.23,24
Taking an adaptive approach is fruitless when there is no information
to which to adapt. In particular, for long-term end points there may
be little information available when making an adaptive decision.
However, early indications of therapeutic effect are sometimes available,
including longitudinal biomarkers and measurements of tumor burden,
for example. These indications can be correlated with long-term clinical
outcomes to enable better interim decisions.7,12
A limitation of adaptive approaches is that they require the ability
to update the outcome database and connect it to the patient assignment
algorithm. Another limitation is that an adaptive design, although
fully prospective, can be complicated to convey to investigators, patients,
institutional review boards, and regulators.
BIOINFORMATICS
As a discipline, bioinformatics sits at the interface where biology and
medicine meet a confluence of quantitative sciences, including computer
science, mathematics, and statistics. Its primary application to oncology
is sifting through genome-scale (“omics”) data sets to identify biomarkers
and molecular signatures that can be used to predict clinically relevant
outcomes. Each omics technology focuses on a particular class of
Biostatistics and Bioinformatics in Clinical Trials  •  CHAPTER 17 289
5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
n = Final sample size
biologic molecules: genomics for sequence-based studies of DNA,
epigenomics for DNA modifications, transcriptomics for RNA,
proteomics for proteins, and metabolomics for small molecules. Predic-
tors that can potentially be used for classification, diagnosis, prognosis,
or selection of therapy may be found in any of these classes of molecules.
The role of bioinformatics is to manage and organize the data and
to make possible the statistical analysis needed to discover and validate
predictive models based on omics technologies.
Challenges
Numerous challenges must be overcome to incorporate omics data
into clinical oncology. The technology changes so rapidly that it can
be difficult to develop an assay that will remain stable long enough
to discover and validate a model in a clinical trial. A wide variety of
omics technologies are available to assay different classes of molecules,
which requires researchers and analysts to develop a broad range of
expertise, not only in the individual technologies, but also in statistical
methods to integrate the resulting data. Omics data sets often are
hampered by batch effects, which can be overcome only by careful
attention to experimental design and the development of standard
protocols. Statistically, the analysis of omics data sets is a struggle
against issues of multiple testing and overfitting. The development of
second-generation sequencing technologies poses additional challenges
from the sheer volume of data and the need for substantial computer
power to interpret the data.
Pace of Technologic Change
The first microarrays that could simultaneously measure the messenger
RNA (mRNA) expression levels of thousands of genes were developed
in the mid 1990s.25,26 Within a few years, they were being used to
study cancer.27–31 The technology evolved rapidly. For example, one
of the major manufacturers of microarrays, Affymetrix, began with
HuGeneFL GeneChip arrays that contained 6800 probe sets. They
advanced over the course of little more than a decade through the
U95A (12,000 probe sets), U133A (22,000), U133Plus2.0 (54,000),
Human Gene ST 1.0 and 2.0, and Human Exon ST 1.0 and 2.0
microarrays. The typical time between the introductions of successive
generations of microarray platforms was 2 or 3 years. Nevertheless,
290 Part I: Science and Clinical Oncology
as that decade ended, researchers were increasingly moving away from
microarray technology and toward RNA sequencing technology as
their primary tool to measure mRNA abundance. The technologies
used to measure DNA copy number alterations went through a similar
evolution during this period.
This rapid pace of change poses serious challenges when trying to
apply these technologies in clinical trials, which may take several years
to conduct. Often the technologic platform used to develop a predictive
model differs from the one proposed to test the predictions in a clinical
trial. It is even possible for a manufacturer to discontinue production
of a platform that researchers are using or proposing to use in a clinical
trial. Every time the platform changes, the assay must be recomputed
and revalidated (preferably by biostatisticians and bioinformaticians
working with a laboratory that has been certified according to the
Clinical Laboratory Improvement Amendments [CLIA] of the US
Code of Federal Regulations).
Breadth of Technologies
At the same time that microarray technologies were expanding to
allow the entire transcriptome to be assayed in a single experiment,
new technologies were emerging that focused on other biologically
important molecules. The Cancer Genome Atlas (TCGA) started
assaying approximately 500 tumors per cancer type by using a broad
spectrum of omics technologies.32 Initial plans called for microarray
approaches to measure mRNA expression, microRNA (miRNA)
expression, DNA copy number alteration, and methylation. These
plans were supplemented by direct Sanger sequencing of a predefined
set of cancer-related genes and by proteomic techniques, including
mass spectrometry and reverse-phase protein lysate arrays. More recently,
TCGA began applying second-generation sequencing technology to
study DNA (whole-genome or whole-exome sequencing), RNA
(RNA-Seq), and methylation (chromatin immunoprecipitation on
sequencing [ChIP-Seq]) in these tumor samples.33,34
In at least one case, an entire class of biologically interesting
molecules is itself relatively new. The first miRNA was discovered in
1993 by cloning the lin-4 gene in Caenorhabditis elegans.35 That there
were numerous (highly conserved) miRNAs in different species did
not become known until the simultaneous publication of three papers
in 2001.36–38 A year later, Calin and colleagues39 demonstrated that
miRNAs played an important role in cancer by showing that the
“tumor suppressor genes” contained in the minimal deleted region of
a recurrent deletion of chromosome 13q in chronic lymphocytic
leukemia consisted of miRNA-15a and miRNA-16-1. Version 19 of
the reference database miRBase (http://www.mirbase.org/), maintained
by the Wellcome Trust Sanger Institute, now lists 1600 precursors
and 2042 mature human miRNAs.40–42 In addition, of course, there
are now microarrays that simultaneously measure (almost) all of them.
Each omics technology requires specialized methods for processing
the raw data and converting it into a form that can be analyzed to
discover or validate biomarkers and signatures. Often the raw data
are fluorescence-based images that must be quantified and summarized.
Each assay requires its own form of normalization that is intended to
correct for technologic artifacts that may distort the measurements.
At its simplest, the process of normalization is rescaling the data to
account for differences in starting material between samples; if one
starts an experiment with twice as much total RNA from one sample,
then one would expect all the measurements to be twice as large.
More complicated normalization schemes, based on sophisticated
statistical models, are common. Bioinformaticians must understand
enough about how different biologic assays work to select (or develop)
appropriate methods to process each kind of data.
Historically, when manufacturers introduced new instruments, they
also provided software to quantify and analyze the data that they
produced. Statisticians and bioinformaticians, sometimes with good
reason, tend to be leery of the “black box” software packages that
accompany scientific instruments. For one thing, the structure of the
data after quantification, summarization, and normalization is often
similar even for radically different technologies, in which case similar
statistical methods can be applied. The exact form of the statistical
analysis depends less on the nature of the technology platform and
more on the design of the experiment. Bioinformaticians must consider
several factors, such as the kinds of patient samples that are being
contrasted, whether the outcome measures binary, continuous, or
time-to-event data, and the covariates (e.g., age, stage, grade, and
smoking status) for which they must account in the analysis.
It is unreasonable to expect manufacturers of scientific instruments
to have the expertise required to program the wide variety of sophis-
ticated statistical methods needed to account for differences in
experimental design. It is surprising, however, that manufacturers do
not always know the best ways to process their own raw data. For
example, the first Affymetrix microarrays were designed with use of
multiple pairs of “perfect match” and “mismatch” (MM) probes to
target each gene. The idea was that the MM probes could be used to
estimate nonspecific cross-hybridization, and so their initial algorithm
quantified the expression of a gene as the average difference between
the perfect match and MM probes. Li and Wong43 recognized that
different probes for the same gene have different affinities, and thus
their mean expression will vary even within the same sample. They
replaced the simple average with a statistical model that accounted
for different probe affinities.43 Bolstad and colleagues44 and Irizarry
and associates45 showed that the MM probes increased the noise in
the summary measurements with no compensating gain in signal
clarity; they introduced an improved statistical method known as the
robust multiarray average (RMA). Most current analyses of Affymetrix
gene expression data use RMA.
Many advances in the methods for processing and analyzing
omics data sets have come from academics and are made available as
open source software. Bioconductor (http://www.bioconductor.org)
is the largest repository of such software packages, written for the
R statistical programming environment.46,47 The Bioconductor
repository is the equivalent of a hardware store for statisticians and
bioinformaticians searching for tools with which to analyze their latest
data set. An alternative approach is provided by GenePattern48 (http://
www.broadinstitute.org/cancer/software/genepattern/). GenePattern is
a Web service through which data can be uploaded and analyzed by
biologists as well as statisticians. Modules can be written and shared
in a variety of programming languages, then assembled into reusable
self-documenting pipelines.
Batch Effects and Experimental Design
Batch effects are an unavoidable characteristic of data collected using
cutting-edge technologies on research-grade scientific instruments.49
The instruments are often temperamental, requiring frequent tuning
and calibration. Reagents change, and new printings of the “same”
microarray can be subtly different. As a result, a batch of tumor
samples analyzed in November may differ in many ways (although
not of biologic interest) from a similar batch analyzed in February.
These technologic effects are often large enough to swamp any interest-
ing biology, and they can occur on the timescale of days rather than
months. Unless accounted for in the experimental design, batch effects
can ruin a perfectly good experiment. For example, in 2002, Petricoin
and colleagues50 published an article that claimed they had developed
a tool that could be used to diagnose ovarian cancer based on proteomic
patterns detected in serum samples. Their results were soon questioned;
it eventually transpired that the signals they had detected were
technologic artifacts, caused by running all of the controls on their
mass spectrometer before all the cases.51–53
There are several ways to deal with batch effects. First, pay attention
to experimental design. Apply the basic principles of randomization
and blinding to ensure that the contrasts of interest (tumor versus
normal, or responder versus nonresponder, for example) are not
confounded with any batch effects that may be present. Second, if

batch effects are suspected, there are existing statistical methods to
model them and, to some extent, remove them.54–56
Additional challenges arise in the context of clinical trials. The
standard normalization methods for most omics technologies require
analyzing the entire set of data at once, which is impossible when
patients arrive one by one and a decision about how to randomly
assign them to treatment arms depends on the results of an individual
assay. In the context of Affymetrix microarrays, this issue was addressed
by the introduction of “frozen RMA,” which computes the normaliza-
tion parameters from a training set and then applies them to new
arrays one at a time.57 Not all omics technologies have completely
faced this issue. In many cases, as with the comparative CT (or ΔΔCT)
method for quantitative, real-time, reverse-transcription polymerase
chain reaction (qPCR),58 the best solution may be to run a normalization
or calibration standard alongside every experimental sample.
Multiple Testing and Overfitting
At this point, we can assume that we have settled on the technology
platform to assay a particular class of biologic molecules. We have
collected the patient samples, randomized their run order, and corrected
any batch effects in the processed data. We are now ready to discover
the new biomarkers or molecular signatures that will revolutionize
the practice of clinical oncology. And we run head-on into an enormous
statistical hurdle: multiple testing.
Each omics technology measures thousands of features simultane-
ously. (Feature is the standard term in the machine learning literature
for a potential predictor. The first step in building a complex predictive
model is feature selection—deciding which features to include and which
to leave out of the model. We use the term here for its neutrality; it
covers genes, proteins, miRNAs, single-nucleotide polymorphisms,
enhancers, promoters, or anything else that might help us predict
who will or will not respond to a particular therapy.) Each feature can
be tested separately for its ability to predict the outcome of interest,
and a P value can be associated with each feature in the usual way for
the statistical test being used. However, those P values do not mean
what one might think they mean. Suppose, for example, that 20,000
features are tested, and it is discovered that only 1000 of them have
P values less than the magic number of .05. In that case, a reasonable
conclusion is that nothing that has been measured is actually related
to the outcome the test is trying to predict! The problem is that
1000/20000 = 5%, which is the number of small P values one would
expect to occur by chance when 20,000 statistical tests are performed.
The traditional statistical response to the problem of multiple
testing is to apply a “Bonferroni correction” to the cutoff used to
define significance. If one is testing for N features, then the P value
should be less than 0.05/N in order for one to claim significance at
the 5% level. With 20,000 features, this requirement translates into
a P value below .0000025. One might think that this requirement
is extreme, and perhaps it is. The Bonferroni correction is extremely
conservative. It tries to control the “family-wise error rate”; in other
words, it attempts to make sure there are no (type I) errors in what
one calls significant. Continuing the hypothetical experiment, suppose
20,000 features are tested, and it is found that 50 of them have a P
value below 2.5 × 10−6. In only 5% of such experiments would one
expect to find any errors in the list of 50 features. A less conservative
approach is to control the false discovery rate (FDR), the expected
fraction of false-positive (FP) calls among all positive calls: FP/(FP
+ TP), where TP is a true-positive call.59 Numerous methods have
been developed to estimate the FDR in omics experiments.60–64 A
few of these methods also allow one to estimate the number or rate
of false-negative calls. Sometimes called type II errors, false-negative
calls have an opportunity cost in terms of potential discoveries that
are never made.
Multiple testing becomes much worse when one is evaluating
molecular signatures. First, it is easy to show that random data (created
with a random number generator) containing enough features can be
Biostatistics and Bioinformatics in Clinical Trials  •  CHAPTER 17 291
used to construct a multivariate computational model that will be
able to perfectly predict any desired outcome variable on any given
set of biologic samples. Because the data are random, it is guaranteed
that any such model is bogus: it “overfits” the existing data and will
not generalize to new data. Second, the P value correction methods
that work one feature at a time will not help. For example, the number
of 35-feature signatures that can be selected from a set of 10,000
features is approximately a googol (10100). No molecular signature
will ever survive Bonferroni correction of this magnitude. In addition,
two different 35-feature signatures that share 30 features in common
will be highly correlated, invalidating most of the methods for estimating
the FDR. The solution to this problem, however, is simple: any putative
predictive model based on a complex signature must be validated on
an independent data set, preferably one collected prospectively with
respect to the definition of the predictive model.
Sequencing
The move from hybridization-based microarray assays to second-
generation sequencing technology poses additional bioinformatics
challenges. First, there is the sheer volume of data. Even after discarding
the raw images, sequencing data from one sample takes about 1 terabyte
of disk space. Storing these data, moving them across the network,
and accessing them efficiently requires improvements in computer
hardware and software. Second, raw sequence data require heavy-duty
computer processing before they begin to become useful. Millions of
sequence “reads” must be mapped to the genome before they can be
summarized to provide information about individual genes or other
genomic features. In the hybridization approaches, this computational
burden is front-loaded and performed once when designing probes.
In sequencing, a computational burden exists for every sample processed.
Third, because every individual genome is unique, the mapped reads
must be interpreted to identify single-nucleotide polymorphisms, small
insertions and deletions (indels), structural variations (translocations
or gene fusions), and DNA copy number variation, either present in
germline DNA or acquired somatically. Generating the raw sequence
data currently takes about a week; mapping and interpretation of the
data may take a month to get through the queue on a high-performance
computing cluster.
BEST PRACTICES
In response to problems encountered with the premature use of
gene-expression signatures in clinical trials at Duke University,65 the
IOM convened a committee to clarify the steps needed to move
omics-based signatures from their initial discovery into clinical trials
where they can affect patient management and, ideally, improve patient
outcomes.66 The most important committee recommendation was to
draw a bright line after the discovery and validation of a predictive
model and before its application in a clinical trial. Before crossing that
line, the test should already be validated, preferably in an independent
set of samples or, if that method is not available, by cross-validation. A
clinical assay should be developed in a CLIA-certified laboratory, and
the complete computational procedure must be completely specified
and “locked down.” Any change to the procedure requires revalidation
of the method before it is used to affect patient management. An
example is changing the prespecified cutoffs that distinguish patients
with high-risk disease from those with low-risk disease; this warrants
revalidating the method.
The IOM recommendations build on a long history of related
guidelines. For example, the Early Detection Research Network
proposed a sequence of phases for the development of biomarkers
intended for cancer screening.67 However, because the requirements
for a good screening biomarker are different from those for a prognostic
or predictive biomarker, the Early Detection Research Network
biomarker phases are not universally applicable. The minimum
information about a microarray experiment (MIAME) standard defines
292 Part I: Science and Clinical Oncology
data structures for making microarray data publicly available.68 Many
journals require authors of papers that use microarray data to deposit
them in a public repository (such as the Gene Expression Omnibus
or ArrayExpress) in a format that adheres to the MIAME standard.
The MIAME data structures apply directly to a wide variety of technolo-
gies. One weakness of MIAME and related standards, such as the
minimum information about a proteomics experiment (MIAPE)
standard,69 is that, although they describe the collection of metadata
about the technology used in an experiment, they do not include a
structured way to store clinical or demographic data about the patients
whose samples were used in the experiments. Other important guidelines
include the reporting recommendations for tumor marker prognostic
studies (REMARK),70,71 the CONSORT statement for randomized
trials,72 and the STARD initiative for diagnostic studies.73
Discovery Phase
The discovery phase lasts from the initial experiments through complete
definition of a locked-down computational model to predict an outcome
of interest. During this phase, researchers should:
• Make the data and metadata publicly available
• Make the computer code and analysis protocols publicly available
• Confirm the model by using an independent, preferably blinded,
set of samples
• Lock down the model, including molecular measurements, com-
putational procedures, and intended clinical use
The recommendations to make the data and code available grew
out of the problems encountered at Duke University,74,75 but they
reflect a larger concern with reproducible research in computationally
intensive sciences.76–79 Because it is impossible to fully understand the
biologic rationale behind a complex predictive model involving tens
or hundreds of genes, it is critically important to be able to confirm
that the statistical analysis to discover the model was performed both
sensibly and reproducibly. Checking these results requires access to
both the original data and the computer code used to analyze it.
The requirement to specify the intended clinical use is also critical.
Such information should answer the following questions: In what
group of patients will the signature be tested? Will the result of the
test be used to screen patients for early diagnosis? Will it provide
prognostic information? Will it help select therapy? Will it be used
to monitor minimal residual disease or the possibility of recurrence?
Different applications require different performance characteristics to
demonstrate the usefulness of a biomarker or signature and thus require
different experimental designs and different types of controls. Research
scientists who have the expertise to develop omics signatures but are
not accustomed to designing or running clinical trials can fail to think
carefully about these issues. In one example, researchers reported the
discovery of peptide patterns with nearly 100% sensitivity and specificity
to detect prostate cancer.80 The problem was that the cancer specimens
came from a group of men with a mean age of 67 years, whereas the
control subjects came from a group composed of 58% women, with
a mean age of 35 years.81
Test Validation Phase
The goal of the test validation phase is to take the locked-down predic-
tive model and turn it into a usable clinical assay. During this phase,
researchers should:
• Use a CLIA-certified laboratory
• Design, optimize, validate, and implement the test using current
laboratory standards
• If multiple laboratories will perform the test, ensure that all labo-
ratories meet the analytic validation and CLIA requirements
We have touched briefly on the need to standardize the assay in
our discussion of challenges. The current era of rapidly changing
technology and research-grade scientific instruments does not provide
the stability needed for a clinical assay. One consequence is that the
development of a clinical assay may involve a change of technology.
Instead of using microarrays or RNA-Seq to measure gene expression,
the assay may be converted to use qPCR. Whole-genome sequencing
will be replaced by sequencing targeted at a few relevant genes. Tests
for DNA copy number alterations may be converted to use fluorescence
in situ hybridization. The role of the clinical laboratory is to demonstrate
that the assay is analytically reproducible, and, if necessary, to adjust
the locked-down predictive model to use the measurements made on
the new technology platform.
Evaluation of Clinical Utility
After developing the clinical assay to implement the locked-down
predictive model, researchers can start designing the clinical trial.
At this point, regulatory issues come into play. As with all clinical
trials, researchers must obtain approval from the institutional review
board. However, additional action may also be required. In response
to a proposed test to detect ovarian cancer (OvaCheck) that grew
out of the flawed mass spectrometry experiments on serum, to the
clinical trials at Duke, and to similar issues elsewhere, the US Food
and Drug Administration (FDA) has ruled that complex predictive
models are “medical devices,” the regulation of which falls under
their jurisdiction. If those devices will be used in a clinical trial
to guide patient management in any way, then researchers must
obtain an investigational device exemption (IDE). The IDE is the
medical device equivalent of the better-known investigational new
drug application that is needed for testing a new drug in a clinical
trial. The IOM committee recommends that specific members of the
institutional review board be made responsible for considering the
need for an investigational new drug application or IDE in proposed
clinical trials.
Not every clinical trial evaluating the usefulness of a predictive
model needs an IDE; the fundamental criterion is whether the
molecular signature or assay device is being used to direct patient
management. A prospective-retrospective82 study design in which
the signature is measured on archived specimens to determine
whether it would have been useful should not require an IDE. Simi-
larly, a prospective clinical trial in which the signature is passively
measured but not used to determine patient care would not need
an IDE.
Signatures Are Not Enough
In most of the literature, “signature” is a synonym for “list of genes.”
As should be clear by this point, having a list of genes is not enough
to make predictions about patient outcomes. For that purpose, you
must know exactly how to measure each gene, how to combine those
measurements to produce a numeric score, and how to interpret that
score in a clinical context. Only then do you have a fully specified,
locked-down computational model. The good news, however, is that
for statistical purposes the fully specified model can be considered
to be just one thing. Omics-based predictive signatures are a form
of complex biomarker. As a result, the statistical designs for clinical
trials that will evaluate one such signature are exactly the same as the
designs for evaluating a single biomarker (such as the mutation status
of the EGFR gene). The sample size computations are, in principle,
identical.
Clustering Is Not Prediction
Clustering algorithms, especially in the form of two-way clustered,
colored heat maps, have been ubiquitous in the microarray literature
since its inception.83,84 Just as a list of genes (a signature) is not enough
to make predictions, and neither is clustering. There are certainly
scientific questions that can be appropriately answered by applying a

clustering algorithm; the canonic example involves identifying natural
biologic subtypes within a larger class of cancer samples. It is, however,
possible to identify subtypes with different response profiles. The most
likely way to find subtypes related to outcome is to start by performing
feature-by-feature statistical tests that identify individual predictors
of outcome. After selection of the best predictors, the resulting list of
genes (signature!) can be used to cluster the samples. A statistical test
can then be performed to determine if the resulting “cluster member-
ship” variable is a good predictor of outcome.
The literature distinguishes between supervised and unsupervised
analyses. Supervised methods use the outcome during the analysis;
the simplest example is a t-test to identify genes that are differentially
expressed between responders and nonresponders. By contrast, clustering
is the classic example of an unsupervised approach, because it uses the
gene expression measurements only during clustering. However, the
procedure described in the previous paragraph is not unsupervised;
the process of selecting the best predictive features is a supervised
(and essential) part of the procedure.
Two questions illustrate why this clustering procedure, by itself, is
inadequate for prediction or classification. First, what happens when
you get the data for one new patient? Can you tell to which cluster
he or she belongs? Second, how do you validate the clustering results
on an independent data set? In both cases, clustering is unable to
tell us how to react to new data. Again, this problem has a simple
solution. The clustering results must be supplemented by developing
a completely specified, locked-down predictive model that assigns
new samples to the existing clusters. Not surprisingly, this model
will probably use the same features that were selected to perform
clustering. After this model is constructed, the predictions can be
validated on new data sets and used in prospective clinical trials to
classify patients one at a time.
PRECISION MEDICINE
Precision medicine endeavors to understand the mechanisms of
pharmacogenetics and ultimately to achieve conformality between
therapeutic interventions and the individuals being treated. With
advancements in the understanding of molecular cancer biology, several
treatment strategies that target specific biomarkers, such as crizotinib
for non–small cell lung cancer (NSCLC) characterized by an anaplastic
lymphoma kinase (ALK) gene rearrangement, have been discovered
and translated into clinical practice.85 Early successes of molecularly
targeted interventions have to some extent effectuated precision
medicine. For example, antiestrogen agents, such as tamoxifen, are
recommended for patients with breast tumors that harbor receptors
for estrogen.86 Melanomas with BRAF V600E mutations have been
shown to benefit from vemurafenib.87 In addition, therapy may be
guided by signatures that comprise multiple markers, such as the
Oncotype DX recurrence score (RS). Based on 21 genes, Oncotype
DX is used to predict recurrence and response to adjuvant chemotherapy
for estrogen receptor–positive (ER+) stage I or II invasive breast cancer.88
Implicit to the concept of precision medicine is heterogeneity of
treatment benefit among patients and patient populations. Its successful
implementation relies on our understanding of distinct molecular
profiles and their biomarkers, which can be used as targets to devise
treatment strategies that exploit current understanding of the biologic
mechanisms of the disease. The concept of personalizing clinical care,
although a topic of recent emphasis, is not entirely new. Evaluations
of individual-level characteristics have long been used to inform
treatment selection.89 Advances in informatics technologies, however,
have provided access to enormous databases yielding inputs that facilitate
interrogation of therapeutic options in the presence of diverse types
of molecular and clinical information.
In oncology settings, biomarkers arising from diverse sources of
patient-level informatics are being interrogated for their utility to
inform treatment decisions and treatment monitoring. Biomarkers
used in cancer may derive from clinical characteristics of the patient
Biostatistics and Bioinformatics in Clinical Trials  •  CHAPTER 17 293
and tumor (such as histopathology and cytopathology), genetic variants
and somatic mutations present in malignant cells, or predisposition
to mutation based on identified by simple sequence repeats DNA.89a
Although the evolution of informatics technology has the potential
to generate massive databases for quantitative-based interrogations of
many informatics sources, the success of precision medicine will depend
on the extent to which these rich databases may be used to advance
understanding of the disease profiles and ultimately integrated for
treatment selection, necessitating robust quantitative methodology
for dimension reduction and appropriate trial design. Specifically, the
effectiveness with which a treatment’s usefulness in a given type of
tumor or patient may be estimated from data inherently depends on
the effectiveness with which one may characterize the relative contribu-
tions of determinants that are predictive of treatment effectiveness
versus those features that are prognostic of the disease extent.90 Consider-
ing the oncology context, predictive features could characterize correlates
of the particular mechanisms by which the tumor evades apoptosis,
and prognostic features determine the likelihood of response with an
established standard-of-care strategy.
Although several statistical methods have been proposed to address
challenges arising with the high-dimensionality of omics-type data,
these methods have largely emphasized the manner in which associations
may be identified with clinical outcomes when genomic features have
been acquired from a relatively limited number of patients. These
methods, although useful for identifying associations deriving from
prognostic biomarkers, are often limited for discovering predictive
biomarkers that interact with treatment and fail to elucidate the predic-
tive power of subpopulations. Ma and colleagues91 conducted a survey
of statistical methods for establishing personalized treatment selection
rules that are currently available in the statistical literature. Bayesian
approaches that predict the usefulness of personalized treatment for
a given individual’s tumor on the basis of measures of interpatient
molecular similarity92,93 have been established.
The potential benefits of such biomarker-guided therapy are
seemingly substantial, but discovery of prognostic and predictive
markers from signal in the presence of many noise covariates remains
a challenge.94 Several aspects of the feature inputs, such as process or
interobserver reproducibility, need to be considered carefully before
the proposed methods are used for personalized selection with large-scale
genomic, imaging, and clinical data. A checklist of criteria has been
developed by the US National Cancer Institute to address issues of
specimens, assays, and clinical trial design.95
Biomarker-Driven Adaptive Clinical Trials and
Case Studies
Precision medicine challenges the traditional paradigms of clinical
translation, for which estimates of population-averaged effects from
large randomized trials are used as the basis for demonstrating improve-
ments in comparative effectiveness. Moreover, successes for targeted
therapeutic strategies have been limited. For example, from 2003 to
2011, 71.7% of investigational agents failed in phase II trials, and
only 10.5% attained final market approval from the FDA.96 The low
success rates may be attributable to reliance on inadequate trial designs
for characterizing treatment benefit and on analytic approaches that
are inadequate for characterizing selection rules based on the joint
effects of multiple candidate biomarkers or environmental exposures.97
Despite innovations in trial design for precision medicines, statistical
methods for validating predictive biomarkers remain less established
than for prognostic biomarkers.98 Strategies such as enrichment, all-
comers, and hybrid designs99,100 have been proposed for predictive
biomarker validation. Moreover, current validation designs predomi-
nately consider validation strategies for binary markers arising from
a single source. Most validation approaches rely on linear models that
use interaction terms to characterize the partial effects of receipt of a
type of targeted therapy given observed values of candidate predictive
biomarkers.101,102
294 Part I: Science and Clinical Oncology
Examining interactions in ongoing clinical trials has many advan-
tages.12 One is that requiring biomarker information for randomization
means that information will be available for all patients, thus avoiding
the “data missingness” problem that plagues retrospective biomarker
studies.103 Another advantage is that when patients in a definable
biomarker subset are not benefiting from a particular therapy, that
subset can be dropped from the trial. In a multiarmed trial, biomarker
subtypes that do not respond to a particular treatment can be excluded
from that treatment arm, possibly gradually, through use of adaptive
randomization.11 A consequence of excluding nonresponders is that
focusing on responders means trials can become smaller. And of course,
excluding patients who do not benefit reduces the extent of overtreat-
ment of trial participants.
An adaptive biomarker-driven clinical trial can begin by including
all the patients who meet the enrollment criteria for the trial, but
then can continue by restricting enrollment to individuals with
biomarker profiles that match those of the responding population as
the results accumulate over the course of the trial. Biomarker subsets
can be identified in advance, generated on the basis of the data in the
trial, or some combination of the two can be used by incorporating
historical data via the prior distribution, as previously described. Any
approach is subject to multiplicities.4 Basing an assessment on the
responding subsets is particularly prone to false-positive conclusions
and requires a level of within-trial empiric validation. The extent of
validation is a design characteristic that can be determined prospectively.
False-positive rates and statistical power can be evaluated and controlled,
usually requiring simulations. Comprehensive overviews of recent
advances in designs used in oncology for studying many agent-and-target
combinations in parallel, such as platform trials, basket trials, and
umbrella trials, can be found.104,105
An example of a complicated biomarker-driven, adaptively ran-
domized clinical trial is I-SPY212,106,107 (http://clinicaltrials.gov/show/
NCT01042379) (http://www.ispy2.org/). The setting is neoadjuvant
treatment for breast cancer in which the end point is pathologic complete
response (pathCR), which the FDA has recently determined to be a reg-
istration end point in high-risk early breast cancer (http://www.fda.gov/
downloads/Drugs/GuidanceComplianceRegulatoryInformation/
Guidances/UCM305501.pdf ). Investigational drugs are added to a
taxane in the initial cycles of standard therapy. Similar trials are being
explored in other diseases, including lymphoma.108
The goal of I-SPY2 is to efficiently pair drugs and combinations
with the patients’ biomarker profiles that characterize the disease subset
that responds to that treatment. I-SPY2 is essentially a screening process.
Through use of bayesian updating, assignment to therapy is adaptively
randomized based on the patient’s tumor biomarkers and the drug’s
performance in patients with similar biomarker profiles. A consequence
of adaptive randomization is that better-performing drugs move through
the process faster. Drugs are graduated when they have a sufficiently
high (bayesian) predictive probability of success in a small, focused
phase III trial involving patients who are most likely to benefit from
the drug based on their biomarker profiles (or the molecular signature
of their disease subtype).
The biomarker categories for I-SPY2 are set in advance and apply
to all therapies. The design considers 10 biomarker profiles, or molecular
signatures, that make both marketing and biologic sense. The categories
and signatures are fixed throughout, and any new therapy that is
inserted into the trial conforms to the predefined set of biomarker
signatures. In a trial similar to I-SPY, the tumor categories could be
determined by the drugs’ targets.
Even though the I-SPY2 end point is ascertained relatively
quickly at surgery, waiting 6 months to learn whether a drug is
benefiting a particular patient can be a long time. Therefore, mag-
netic resonance imaging was incorporated to assess tumor volume
after the first cycle of therapy and after the 12 weeks of taxane or
investigational drug cycles. Modeling the longitudinal relationship
between pathCR and tumor volume reduction enables prediction of
whether the patient will have a pathCR. Predictions are not perfect,
but they help improve the design’s performance. Bayesian modeling
appropriately accounts for the uncertainty involved in this prediction
process.
Case Studies
Oncotype DX
The series of studies performed by Genomic Health to establish its
Oncotype DX assay followed most of the procedures recommended
by the IOM committee. First, the researchers asked a well-defined
clinical question about a well-defined patient population. Their goal
was to predict the risk of distant recurrence after tamoxifen treatment
of women with node-negative, ER+ breast cancer. Second, they used
four existing microarray data sets from the published literature to
select 250 candidate genes. Third, they developed a new assay using
qPCR to measure the expression levels of those 250 genes in tumor
samples. Fourth, they validated the assay by obtaining data from tumor
samples that had been collected in three independent clinical trials
of breast cancer, involving a total of 447 women. They performed
univariate analyses and selected 23 genes that were associated with
the risk of recurrence in at least two of three qPCR data sets. They
then constructed multivariate predictive models and further reduced
the set of predictors to a panel of 16 cancer-related genes and five
reference genes. Their approach was simple but elegant, reducing a
multidimensional problem into a single number, an RS, which made
for a straightforward validation process. The algorithm included cutoffs
to separate patients into low-, intermediate-, and high-risk categories
and was completely specified during this step. Fifth, they tested the
prospectively defined qPCR assay and RS algorithm for their ability
to predict recurrence in a retrospective set of 668 samples from the
National Surgical Adjuvant Breast and Bowel Project (NSABP) B14
trial, for which paraffin blocks were available.109 Another study showed
that RS did not predict recurrence in women with node-negative
breast cancer (regardless of ER status) who had received no adjuvant
chemotherapy, which suggests that the signature depends on either
the ER positivity or on the treatment.110 Although the RS was built
to be prognostic, samples from the NSABP B20 trial showed that
it helped predict response to chemotherapy for women with node-
negative, ER+ breast cancer, with no apparent benefit for women with
low RSs.111
BATTLE Trial
The Biomarker-Integrated Approaches of Targeted Therapy for Lung
Cancer Elimination (BATTLE) trial was a prospective phase II,
biomarker-based, adaptively randomized study in 255 patients who
had been pretreated for NSCLC.112,113 The trial consisted of four
treatment arms of targeted therapies, each of which was associated
with a prespecified set of biomarkers that were anticipated to predict
the efficacy of the therapy: erlotinib (EGFR), sorafenib (KRAS/BRAF),
vandetanib (VEGFR2), and bexarotene plus erlotinib (RXR/CCND1).
The primary end point of the trial was 8-week disease control. After
an initial equal randomization period, patients were adaptively random-
ized to one of the treatment arms, based on the molecular biomarkers
analyzed in fresh core needle biopsy specimens. Overall results included
a 46% 8-week disease control rate, median progression-free survival
of 1.9 months (95% confidence interval [CI], 1.8–2.4), and median
overall survival of 8.8 months (95% CI, 6.3–10.6). The results
confirmed that EGFR mutations predicted better 8-week disease control
with sorafenib.
CONCLUSIONS
Innovations in cancer research are developing rapidly. The quantitative
aspects of this research, from assessing individual genes and biomarkers
to evaluating pathways and systems biology, are becoming both more
important and more difficult. Assessing which patients benefit from
which therapies is the holy grail of cancer research, culminating in
 Biostatistics and Bioinformatics in Clinical Trials  •  CHAPTER 17 295

clinical trials to demonstrate benefit, or lack thereof. A single adaptive
clinical trial can be designed to discover responding subsets and validate
the discovery. However, despite the great strides seen in modern cancer
biology, strides of much greater magnitude are yet to come. Advances
in biology lead to ever more and ever smaller subsets. Large clinical
trials will soon be impossible, and traditional statistical approaches
will have to be replaced. We cannot predict what those replacements
will be. Our aim has been to describe some ways in which the scientific
winds are blowing, preparing the reader to understand and appreciate
the path being followed.
The complete reference list is available online at
ExpertConsult.com.

KEY REFERENCES
4. Berry DA. Multiplicities in cancer research: ubiqui-
tous and necessary evils. J Natl Cancer Inst. 2012;104:
1125–1133.
7. Berry DA. Statistical innovations in cancer research.
In: Holland J, Frei T, et al, eds. Cancer Medicine.
8th ed. London: BC Decker; 2009.
8. Berry DA. Bayesian approaches for comparative
effectiveness research. Clin Trials. 2012;9:37–47.
10. Biswas S, Liu DD, Lee JJ, Berry DA. Bayesian clinical
trials at the University of Texas MD Anderson Cancer
Center. Clin Trials. 2009;6:205–216.
12. Berry DA. Adaptive clinical trials in oncology. Nat
Rev Clin Oncol. 2011;9:199–207.
13. Nass SJ, Moses HL, Mendelsohn J. Institute of
Medicine: a national cancer clinical trials system for
the 21st century: reinvigorating the NCI Coopera-
tive Group Program. Washington, DC: National
Academies Press; 2010.
14. Wong KM, Capasso A, Eckhardt SG. The changing
landscape of phase I trials in oncology. Nat Rev Clin
Oncol. 2016;13(2):106–117.
15. Ji Y, Wang SJ. Modified toxicity probability interval
design: a safer and more reliable method than the 3
+ 3 design for practical phase I trials. J Clin Oncol.
2013;31(14):1785–1791.
16. Thall PF, Nguyen HQ, Braun TM, Qazilbash MH.
Using joint utilities of the times to response and
toxicity to adaptively optimize schedule-dose regimes.
Biometrics. 2013;69(3):673–682.
18. Hobbs BP, Chen N, Lee JJ. Controlled multi-arm
platform design using predictive probability. Stat
Methods Med Res. 2018;27(1):65–78.
20. Hobbs BP, Thall PF, Lin SH. Bayesian group
sequential clinical trial design using total toxicity
burden and progression-free survival. J R Stat Soc
Ser C Appl Stat. 2016;65(2):273–297.
24. Kidwell KM. SMART designs in cancer research: past,
present, and future. Clin Trials. 2014;11(4):445–456.
32. Cancer Genome Atlas Research Network. Com-
prehensive genomic characterization defines human
glioblastoma genes and core pathways. Nature.
2008;455:1061–1068.
33. Chin L, Andersen JN, Futreal PA. Cancer genomics:
from discovery science to personalized medicine.
Nat Med. 2011;17:297–303.
39. Calin GA, Dumitru CD, Shimizu M, et al. Frequent
deletions and down-regulation of micro-RNA
genes miR15 and miR16 at 13q14 in chronic
lymphocytic leukemia. Proc Natl Acad Sci USA.
2002;99:15524–15529.
42. Kozomara A, Griffiths-Jones S. miRBase: integrating
microRNA annotation and deep-sequencing data.
Nucleic Acids Res. 2011;39:D152–D157.
48. Reich M, Liefeld T, Gould J, et al. GenePattern
2.0. Nat Genet. 2006;38:500–501.
49. Leek JT, Scharpf RB, Bravo HC, et al. Tackling
the widespread and critical impact of batch effects
in high-throughput data. Nat Rev Genet. 2010;11:
733–739.
50. Petricoin EF, Ardekani AM, Hitt BA, et al. Use
of proteomic patterns in serum to identify ovarian
cancer. Lancet. 2002;359:572–577.
52. Baggerly KA, Edmonson SR, Morris JS, Coombes
KR. High-resolution serum proteomic patterns
for ovarian cancer detection. Endocr Relat Cancer.
2004;11:583–584, author reply 585–7.
53. Baggerly KA, Morris JS, Coombes KR. Reproduc-
ibility of SELDI-TOF protein patterns in serum:
comparing datasets from different experiments.
Bioinformatics. 2004;20:777–785.
54. Luo J, Schumacher M, Scherer A, et al. A comparison
of batch effect removal methods for enhancement of
prediction performance using MAQC-II microar-
ray gene expression data. Pharmacogenomics J.
2010;10:278–291.
55. Chen C, Grennan K, Badner J, et al. Removing batch
effects in analysis of expression microarray data: an
evaluation of six batch adjustment methods. PLoS
ONE. 2011;6(2):e17238.
57. McCall MN, Bolstad BM, Irizarry RA. Frozen
robust multiarray analysis (fRMA). Biostatistics.
2010;11:242–253.
60. Efron B, Tibshirani R. Empirical Bayes methods and
false discovery rates for microarrays. Genet Epidemiol.
2002;23:70–86.
62. Storey JD, Tibshirani R. Statistical significance
for genomewide studies. Proc Natl Acad Sci USA.
2003;100:9440–9445.
63. Pounds S, Cheng C. Improving false discovery rate
estimation. Bioinformatics. 2004;20:1737–1745.
64. Qian HR, Huang S. Comparison of false discovery
rate methods in identifying genes with differential
expression. Genomics. 2005;86:495–503.
65. Baggerly KA, Coombes KR. Deriving chemosen-
sitivity from cell lines: forensic bioinformatics and
reproducible research in high-throughput biology.
Ann Appl Stat. 2009;3:1309–1334.
67. Pepe MS, Etzioni R, Feng Z, et al. Phases of bio-
marker development for early detection of cancer.
J Natl Cancer Inst. 2001;93:1054–1061.
74. Baggerly K. Disclose all data in publications. Nature.
2010;467:401.
75. Baggerly KA, Coombes KR. What information
should be required to support clinical “omics”
publications? Clin Chem. 2011;57:688–690.
79. Peng RD. Reproducible research in computational
science. Science. 2011;334:1226–1227.
82. Simon RM, Paik S, Hayes DF. Use of archived
specimens in evaluation of prognostic and predictive
biomarkers. J Natl Cancer Inst. 2009;101:1446–1452.
86. Simon R. Advances in clinical trial designs for
predictive biomarker discovery and validation.
Current Breast Cancer Reports. 2009;1(4):216–221.
87. Sosman JA, Kim KB, Schuchter L, Gonzalez R,
Pavlick AC, Weber JS, et al. Survival in BRAF
V600-mutant advanced melanoma treated with
vemurafenib. N Engl J Med. 2012;366(8):707–714.
90. Simon R. Clinical trial designs for evaluating the
medical utility of prognostic and predictive bio­
markers in oncology. Per Med. 2010;7(1):33–47.
91. Ma J, Hobbs BP, Stingo FC. Statistical methods for
establishing personalized treatment rules in oncology.
Biomed Res Int. 2015;2015:670691.
92. Ma J, Stingo FC, Hobbs BP. Bayesian predictive
modeling for genomic based personalized treatment
selection. Biometrics. 2016;72(2):575–583.
93. Ma J, Hobbs BP, Stingo FC. Integrating genomic
signatures for treatment selection with Bayesian
predictive failure time models. Stat Methods Med
Res. 2016;[Epub ahead of print].
95. McShane LM, Cavenagh MM, Lively TG, Eberhard
DA, Bigbee WL, Williams PM, et al. Criteria for
the use of omics-based predictors in clinical trials.
Nature. 2013;502(7471):317–320.
96. Hay M, Thomas DW, Craighead JL, Economides C,
Rosenthal J. Clinical development success rates for
investigational drugs. Nat Biotechnol. 2014;32(1):
40–51.
100. Mandrekar SJ, Sargent DJ. Predictive biomarker
validation in practice: lessons from real trials. Clinical
trials. 2010.
101. Mandrekar SJ, Sargent DJ. Design of clinical trials for
biomarker research in oncology. Clinical investigation.
2011;1(12):1627–1636.
107. Berry DA, Herbst RS, Rubin EH. Design strategies for
personalized therapy trials. Clin Cancer Res. 2012;18:
638–644.
108. Younes A, Berry DA. From drug discovery to
biomarker-driven clinical trials in lymphoma. Nat
Rev Clin Oncol. 2012;9:643–653.
109. Paik S, Shak S, Tang G, et al. A multigene assay to
predict recurrence of tamoxifen-treated, node-negative
breast cancer. N Engl J Med. 2004;351:2817–2826.
110. Esteva FJ, Sahin AA, Cristofanilli M, et al. Prognostic
role of a multigene reverse transcriptase-PCR assay
in patients with node-negative breast cancer not
receiving adjuvant systemic therapy. Clin Cancer
Res. 2005;11:3315–3319.
111. Paik S, Tang G, Shak S, et al. Gene expression and
benefit of chemotherapy in women with node-
negative, estrogen receptor-positive breast cancer.
J Clin Oncol. 2006;24:3726–3734.
113. Kim ES, Herbst RS, Wistuba II, et al. The BATTLE
trial: personalizing therapy for lung cancer. Cancer
Discov. 2011;1:44–53.

REFERENCES
1. Bernoulli J. The Art of Conjecturing, Together with
Letter to a Friend on Sets in Court Tennis (English
translation of the 1713 text by Edith Sylla). Baltimore:
Johns Hopkins University Press; 2005.
2. Stigler SM. Gauss and the invention of least squares.
Ann Statist. 1981;9:465–474.
3. Bernoulli D. Exposition of a new theory on
the measurement of risk [1738]. Econometrica.
1954;22:23–36. https://engineering.purdue.edu/
~ipollak/ece302/SPRING12/notes/Bernoulli_1738
.pdf/. Accessed March 15, 2013. 2009.
4. Berry DA. Multiplicities in cancer research:
ubiquitous and necessary evils. J Natl Cancer Inst.
2012;104:1125–1133.
5. Berger JO, Berry DA. Statistical analysis and the
illusion of objectivity. Am Sci. 1988;76:159–165.
6. Berry DA, Stangl DK. Bayesian Biostatistics. New
York: Marcel Dekker; 1996.
7. Berry DA. Statistical innovations in cancer research.
In: Holland J, Frei T, et al, eds. Cancer Medicine.
8th ed. London: BC Decker; 2009.
8. Berry DA. Bayesian approaches for comparative
effectiveness research. Clin Trials. 2012;9:37–47.
9. Yoshioka A. Use of randomisation in the Medical
Research Council’s clinical trial of streptomycin
in pulmonary tuberculosis in the 1940s. BMJ.
1998;317:1220–1223.
10. Biswas S, Liu DD, Lee JJ, Berry DA. Bayesian clinical
trials at the University of Texas MD Anderson Cancer
Center. Clin Trials. 2009;6:205–216.
11. Berry DA. Adaptive clinical trials: the promise and
the caution. J Clin Oncol. 2011;29:606–609.
12. Berry DA. Adaptive clinical trials in oncology. Nat
Rev Clin Oncol. 2011;9:199–207.
13. Nass SJ, Moses HL, Mendelsohn J. Institute of
Medicine: a national cancer clinical trials system for the
21st century: reinvigorating the NCI Cooperative Group
Program. Washington, DC: National Academies
Press; 2010.
14. Wong KM, Capasso A, Eckhardt SG. The changing
landscape of phase I trials in oncology. Nat Rev Clin
Oncol. 2016;13(2):106–117.
15. Ji Y, Wang SJ. Modified toxicity probability interval
design: a safer and more reliable method than the 3
+ 3 design for practical phase I trials. J Clin Oncol.
2013;31(14):1785–1791.
16. Thall PF, Nguyen HQ, Braun TM, Qazilbash MH.
Using joint utilities of the times to response and
toxicity to adaptively optimize schedule-dose regimes.
Biometrics. 2013;69(3):673–682.
17. Zhang J, Braun TM. A phase I Bayesian adaptive
design to simultaneously optimize dose and schedule
assignments both between and within patients.
J American Statistical Assoc. 2013;108:892–901.
18. Hobbs BP, Chen N, Lee JJ. Controlled multi-arm
platform design using predictive probability. Stat
Methods Med Res. 2018;27(1):65–78.
19. Bekele BN, Thall PF. Dose-finding based on multiple
toxicities in a soft tissue sarcoma trial. J. Am.Statist.
Ass. 2004;99:26–34.
20. Hobbs BP, Thall PF, Lin SH. Bayesian group
sequential clinical trial design using total toxicity
burden and progression-free survival. J R Stat Soc
Ser C Appl Stat. 2016;65(2):273–297.
21. Thall PF, Cook JD. Dose-finding based on efficacy-
toxicity trade-offs. Biometrics. 2004;60(3):684–693.
22. Zhang W, Sargent DJ, Mandrekar S. An adaptive
dose-finding design incorporating both toxicity and
efficacy. Stat Med. 2006;25(14):2365–2383.
23. Wang L, Rotnitzky A, Lin X, Millikan RE, Thall
PF. Evaluation of viable dynamic treatment regimes
in a sequentially randomized trial of advanced
prostate cancer. J Am Stat Assoc. 2012;107(498):
493–508.
24. Kidwell KM. SMART designs in cancer research:
past, present, and future. Clin Trials. 2014;11(4):
445–456.
Biostatistics and Bioinformatics in Clinical Trials  •  CHAPTER 17 295.e1
25. Fodor SP, Rava RP, Huang XC, et al. Multiplexed
biochemical assays with biological chips. Nature.
1993;364:555–556.
26. Schena M, Shalon D, Davis RW, Brown PO.
Quantitative monitoring of gene expression patterns
with a complementary DNA microarray. Science.
1995;270:467–470.
27. DeRisi J, Penland L, Brown PO, et al. Use of a
cDNA microarray to analyse gene expression patterns
in human cancer. Nat Genet. 1996;14:457–460.
28. Khan J, Simon R, Bittner M, et al. Gene expression
profiling of alveolar rhabdomyosarcoma with cDNA
microarrays. Cancer Res. 1998;58:5009–5013.
29. Alon U, Barkai N, Notterman DA, et al. Broad
patterns of gene expression revealed by clustering
analysis of tumor and normal colon tissues probed
by oligonucleotide arrays. Proc Natl Acad Sci USA.
1999;96:6745–6750.
30. Golub TR, Slonim DK, Tamayo P, et al. Molecular
classification of cancer: class discovery and class
prediction by gene expression monitoring. Science.
1999;286:531–537.
31. Perou CM, Jeffrey SS, van de Rijn M, et al. Distinc-
tive gene expression patterns in human mammary
epithelial cells and breast cancers. Proc Natl Acad
Sci USA. 1999;96:9212–9217.
32. Cancer Genome Atlas Research Network. Com-
prehensive genomic characterization defines human
glioblastoma genes and core pathways. Nature.
2008;455:1061–1068.
33. Chin L, Andersen JN, Futreal PA. Cancer genomics:
from discovery science to personalized medicine.
Nat Med. 2011;17:297–303.
34. Chin L, Hahn WC, Getz G, Meyerson M. Making
sense of cancer genomic data. Genes Dev. 2011;25:
534–555.
35. Lee RC, Feinbaum RL, Ambros V. The C. elegans
heterochronic gene lin-4 encodes small RNAs
with antisense complementarity to lin-14. Cell.
1993;75:843–854.
36. Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl
T. Identification of novel genes coding for small
expressed RNAs. Science. 2001;294:853–858.
37. Lau NC, Lim LP, Weinstein EG, Bartel DP.
An abundant class of tiny RNAs with probable
regulatory roles in Caenorhabditis elegans. Science.
2001;294:858–862.
38. Lee RC, Ambros V. An extensive class of small
RNAs in Caenorhabditis elegans. Science. 2001;294:
862–864.
39. Calin GA, Dumitru CD, Shimizu M, et al. Frequent
deletions and down-regulation of micro-RNA
genes miR15 and miR16 at 13q14 in chronic
lymphocytic leukemia. Proc Natl Acad Sci USA.
2002;99:15524–15529.
40. Griffiths-Jones S, Grocock RJ, van Dongen S,
et al. miRBase: microRNA sequences, targets and
gene nomenclature. Nucleic Acids Res. 2006;34:
D140–D144.
41. Griffiths-Jones S, Saini HK, van Dongen S, Enright
AJ. miRBase: tools for microRNA genomics. Nucleic
Acids Res. 2008;36:D154–D158.
42. Kozomara A, Griffiths-Jones S. miRBase: integrating
microRNA annotation and deep-sequencing data.
Nucleic Acids Res. 2011;39:D152–D157.
43. Li C, Wong WH. Model-based analysis of oligo-
nucleotide arrays: expression index computation
and outlier detection. Proc Natl Acad Sci U SA.
2001;98:31–36.
44. Bolstad BM, Irizarry RA, Astrand M, Speed TP.
A comparison of normalization methods for high
density oligonucleotide array data based on variance
and bias. Bioinformatics. 2003;19:185–193.
45. Irizarry RA, Hobbs B, Collin F, et al. Exploration,
normalization, and summaries of high density
oligonucleotide array probe level data. Biostatistics.
2003;4:249–264.
295.e1
46. Gentleman RC, Carey VJ, Bates DM, et al.
Bioconductor: open software development for
computational biology and bioinformatics. Genome
Biol. 2004;5:R80.
47. R Core Team. R: A Language and Environment for
Statistical Computing. Vienna: R Foundation for
Statistical Computing; 2012.
48. Reich M, Liefeld T, Gould J, et al. GenePattern
2.0. Nat Genet. 2006;38:500–501.
49. Leek JT, Scharpf RB, Bravo HC, et al. Tackling
the widespread and critical impact of batch effects
in high-throughput data. Nat Rev Genet. 2010;11:
733–739.
50. Petricoin EF, Ardekani AM, Hitt BA, et al. Use
of proteomic patterns in serum to identify ovarian
cancer. Lancet. 2002;359:572–577.
51. Sorace JM, Zhan M. A data review and re-assessment
of ovarian cancer serum proteomic profiling. BMC
Bioinformatics. 2003;4:24.
52. Baggerly KA, Edmonson SR, Morris JS, Coombes
KR. High-resolution serum proteomic patterns
for ovarian cancer detection. Endocr Relat Cancer.
2004;11:583–584, author reply 585–7.
53. Baggerly KA, Morris JS, Coombes KR. Reproduc-
ibility of SELDI-TOF protein patterns in serum:
comparing datasets from different experiments.
Bioinformatics. 2004;20:777–785.
54. Luo J, Schumacher M, Scherer A, et al. A comparison
of batch effect removal methods for enhancement of
prediction performance using MAQC-II microarray
gene expression data. Pharmacogenomics J. 2010;
10:278–291.
55. Chen C, Grennan K, Badner J, et al. Removing batch
effects in analysis of expression microarray data: an
evaluation of six batch adjustment methods. PLoS
ONE. 2011;6(2):e17238.
56. Lazar C, Meganck S, Taminau J, et al. Batch effect
removal methods for microarray gene expression data
integration: a survey. Brief Bioinform. 2012;Jul 31
[Epub ahead of print].
57. McCall MN, Bolstad BM, Irizarry RA. Frozen
robust multiarray analysis (fRMA). Biostatistics.
2010;11:242–253.
58. Livak KJ, Schmittgen TD. Analysis of relative gene
expression data using real-time quantitative PCR
and the 2(-Delta Delta C(T). Method. Methods.
2001;25:402–408.
59. Benjamini Y, Hochberg Y. Controlling the false
discovery rate: a practical and powerful approach
to multiple testing. J R Stat Soc Series B Stat Methodol.
1995;57:289–300.
60. Efron B, Tibshirani R. Empirical Bayes methods and
false discovery rates for microarrays. Genet Epidemiol.
2002;23:70–86.
61. Pounds S, Morris SW. Estimating the occurrence
of false positives and false negatives in microarray
studies by approximating and partitioning the
empirical distribution of p-values. Bioinformatics.
2003;19:1236–1242.
62. Storey JD, Tibshirani R. Statistical significance
for genomewide studies. Proc Natl Acad Sci USA.
2003;100:9440–9445.
63. Pounds S, Cheng C. Improving false discovery rate
estimation. Bioinformatics. 2004;20:1737–1745.
64. Qian HR, Huang S. Comparison of false discovery
rate methods in identifying genes with differential
expression. Genomics. 2005;86:495–503.
65. Baggerly KA, Coombes KR. Deriving chemosen-
sitivity from cell lines: forensic bioinformatics and
reproducible research in high-throughput biology.
Ann Appl Stat. 2009;3:1309–1334.
66. Committee on the Review of Omics-Based Tests for
Predicting Patient Outcomes in Clinical Trials. In:
Christine MM, Sharly JN, Gilbert SO, eds. Evolution
of Translational Omics: Lessons Learned and the Path
Forward. Washington, DC: The National Academies
Press; 2012.
295.e2 Part I: Science and Clinical Oncology
67. Pepe MS, Etzioni R, Feng Z, et al. Phases of bio-
marker development for early detection of cancer.
J Natl Cancer Inst. 2001;93:1054–1061.
68. Brazma A, Hingamp P, Quackenbush J, et al.
Minimum information about a microarray experi-
ment (MIAME)-toward standards for microarray
data. Nat Genet. 2001;29:365–371.
69. Orchard S, Hermjakob H, Taylor CF, et al. Further
steps in standardisation. Report of the second annual
Proteomics Standards Initiative Spring Workshop
(Siena, Italy 17–20th April 2005). Proteomics. 2005;5:
3552–3555.
70. McShane LM, Altman DG, Sauerbrei W, et al.
Statistics Subcommittee of the NCIEWGoCD.
Reporting recommendations for tumor marker
prognostic studies (REMARK). J Natl Cancer Inst.
2005;97:1180–1184.
71. Altman DG, McShane LM, Sauerbrei W, Taube
SE. Reporting Recommendations for Tumor Marker
Prognostic Studies (REMARK): explanation and
elaboration. PLoS Med. 2012;9:e1001216.
72. Moher D, Schulz KF, Altman D, Group C. The
CONSORT statement: revised recommendations
for improving the quality of reports of parallel-group
randomized trials. JAMA. 2001;285:1987–1991.
73. Bossuyt PM, Reitsma JB, Bruns DE, et al. Standards
for Reporting of Diagnostic A. The STARD state-
ment for reporting studies of diagnostic accuracy:
explanation and elaboration. Clin Chem. 2003;49:
7–18.
74. Baggerly K. Disclose all data in publications. Nature.
2010;467:401.
75. Baggerly KA, Coombes KR. What information
should be required to support clinical “omics”
publications? Clin Chem. 2011;57:688–690.
76. Buckheit J, Donoho DL. Wavelab and reproducible
research. In: Antoniadis A, ed. Wavelets and Statistics.
New York: Springer-Verlag; 1995.
77. Leisch F, Rossini AJ. Reproducible statistical research.
Chance. 2003;16:46–50.
78. Gentleman R. Reproducible research: a bioinformat-
ics case study. Stat Appl Genet Mol Biol. 2005;4:
Article2.
79. Peng RD. Reproducible research in computational
science. Science. 2011;334:1226–1227.
80. Villanueva J, Shaffer DR, Philip J, et al. Differential
exoprotease activities confer tumor-specific serum
peptidome patterns. J Clin Invest. 2006;116:271–284.
81. Ransohoff DF, Gourlay ML. Sources of bias in
specimens for research about molecular markers
for cancer. J Clin Oncol. 2010;28:698–704.
82. Simon RM, Paik S, Hayes DF. Use of archived
specimens in evaluation of prognostic and predictive
biomarkers. J Natl Cancer Inst. 2009;101:1446–1452.
83. Weinstein JN, Myers TG, O’Connor PM, et al. An
information-intensive approach to the molecular
pharmacology of cancer. Science. 1997;275:343–
349.
84. Eisen MB, Spellman PT, Brown PO, Botstein D.
Cluster analysis and display of genome-wide expres-
sion patterns. Proc Natl Acad Sci USA. 1998;95:
14863–14868.
85. Kelloff GJ, Sigman CC. Cancer biomarkers: selecting
the right drug for the right patient. Nat Rev Drug
Discov. 2012;11(3):201–214.
86. Simon R. Advances in clinical trial designs for
predictive biomarker discovery and validation.
Current Breast Cancer Reports. 2009;1(4):216–221.
87. Sosman JA, Kim KB, Schuchter L, Gonzalez R,
Pavlick AC, Weber JS, et al. Survival in BRAF
V600-mutant advanced melanoma treated with
vemurafenib. N Engl J Med. 2012;366(8):707–714.
88. Albain KS, Barlow WE, Shak S, Hortobagyi GN,
Livingston RB, Yeh IT, et al. Prognostic and
predictive value of the 21-gene recurrence score
assay in postmenopausal women with node-positive,
oestrogen-receptor-positive breast cancer on chemo-
therapy: a retrospective analysis of a randomised
trial. Lancet Oncol. 2010;11(1):55–65.
89. Byar DP, Corle DK. Selecting optimal treatment in
clinical trials using covariate information. J Chronic
Dis. 1977;30(7):445–459.
89a.  Ellegren H. Microsatellites: simple sequences with
complex evolution. Nat Rev Genet. 2004;5(6):
435–445.
90. Simon R. Clinical trial designs for evaluating the
medical utility of prognostic and predictive biomark-
ers in oncology. Per Med. 2010;7(1):33–47.
91. Ma J, Hobbs BP, Stingo FC. Statistical methods for
establishing personalized treatment rules in oncology.
Biomed Res Int. 2015;2015:670691.
92. Ma J, Stingo FC, Hobbs BP. Bayesian predictive
modeling for genomic based personalized treatment
selection. Biometrics. 2016;72(2):575–583.
93. Ma J, Hobbs BP, Stingo FC. Francesco C Stingo.
Integrating genomic signatures for treatment selec-
tion with bayesian predictive failure time models.
Stat Methods Med Res. 2016;[Epub ahead of print].
94. Food and Drug Administration. Paving the Way
for Personalized Medicine: FDA’s Role in a New
Era of Medical Product Development. http://www.
fda.gov/downloads/ScienceResearch/SpecialTopics/
PersonalizedMedicine, 2013.
95. McShane LM, Cavenagh MM, Lively TG, Eberhard
DA, Bigbee WL, Williams PM, et al. Criteria for
the use of omics-based predictors in clinical trials.
Nature. 2013;502(7471):317–320.
96. Hay M, Thomas DW, Craighead JL, Economides
C, Rosenthal J. Clinical development success
rates for investigational drugs. Nat Biotechnol.
2014;32(1):40–51.
97. Knox SS. From ‘omics’ to complex disease: a systems
biology approach to gene-environment interactions
in cancer. Cancer Cell Int. 2010.
98. Janes H, Pepe MS, Bossuyt PM, Barlow WE.
Measuring the performance of markers for guiding
treatment decisions. Ann Intern Med. 2011;154(4):
253–259.
99. Mandrekar SJ, Sargent DJ. Clinical trial designs for
predictive biomarker validation: one size does not
fit all. J Biopharm Stat. 2009;19(3):530–542.
100. Mandrekar SJ, Sargent DJ. Predictive biomarker
validation in practice: lessons from real trials. Clinical
trials. 2010.
101. Mandrekar SJ, Sargent DJ. Design of clinical trials for
biomarker research in oncology. Clinical investigation.
2011;1(12):1627–1636.
102. Chen JJ, Lu TP, Chen DT, Wang SJ. Biomarker
adaptive designs in clinical trials. Translational Cancer
Research. 2014;3(3):279–292.
103. Rimm DL, Nielsen TO, Jewell SD, et al. Cancer and
Leukemia Group B Pathology Committee guidelines
for tissue microarray construction representing
multicenter prospective clinical trial tissues. J Clin
Oncol. 2011;29:2282–2290.
104. Renfro L, Sargent D. Statistical controversies in
clinical research: basket trials, umbrella trials, and
other master protocols: a review and examples. Ann
Oncol. 2016.
105. Berry DA. The brave new world of clinical cancer
research: adaptive biomarker-driven trials integrating
clinical practice with clinical research. Mol Oncol.
2015;9(5):951–959.
106. Barker AD, Sigman CC, Kelloff GJ, et al. I-SPY2:
an adaptive breast cancer trial design in the setting
of neoadjuvant chemotherapy. Clin Pharmacol Ther.
2009;86:97–100.
107. Berry DA, Herbst RS, Rubin EH. Design strategies
for personalized therapy trials. Clin Cancer Res.
2012;18:638–644.
108. Younes A, Berry DA. From drug discovery to
biomarker-driven clinical trials in lymphoma. Nat
Rev Clin Oncol. 2012;9:643–653.
109. Paik S, Shak S, Tang G, et al. A multigene assay
to predict recurrence of tamoxifen-treated, node-
negative breast cancer. N Engl J Med. 2004;351:
2817–2826.
110. Esteva FJ, Sahin AA, Cristofanilli M, et al. Prognostic
role of a multigene reverse transcriptase-PCR assay
in patients with node-negative breast cancer not
receiving adjuvant systemic therapy. Clin Cancer
Res. 2005;11:3315–3319.
111. Paik S, Tang G, Shak S, et al. Gene expression and
benefit of chemotherapy in women with node-
negative, estrogen receptor-positive breast cancer.
J Clin Oncol. 2006;24:3726–3734.
112. Zhou X, Liu S, Kim ES, et al. Bayesian adaptive
design for targeted therapy development in lung
cancer—a step toward personalized medicine. Clin
Trials. 2008;5:181–193.
113. Kim ES, Herbst RS, Wistuba II, et al. The BATTLE
trial: personalizing therapy for lung cancer. Cancer
Discov. 2011;1:44–53.
 18  Clinical Trial Designs in Oncology
Edward L. Korn and Boris Freidlin

S UMMARY OF K EY
• Phase I trials to determine
recommended doses and schedules
for further testing of new treatments
need to be designed to minimize the
number of patients exposed to
unacceptable toxicity.
• The use of formal interim monitoring
of accruing outcome data in phase II
and phase III trials is important in
order to allow trials to be stopped as
soon as possible because of positive
or negative results, while retaining
the statistical validity of the trial
conclusions.
P OI N T S
• Phase II/III trial designs, multiarm
designs, and the use of master
protocols, which allow treatment
arms to be added to an ongoing
trial, can speed the development of
new treatments but are not without
disadvantages.
• It is important that the primary end
point of a trial be chosen so that the
results of the trial will meet its
clinical objectives.
• Trial designs have been developed
to assess whether a biomarker can
be used to identify a subgroup of
patients who benefit from an agent
that targets tumors that express a
particular molecular abnormality.
• Simultaneous screening of patients
for multiple, possibly rare,
biomarkers in order to assign them
to different biomarker-restricted
subtrials is a highly efficient way to
assess multiple biomarker-driven
treatment hypotheses.

Clinical trials are designed to answer specific clinical questions in the
development of new treatments. In order to address the study question,
it is important for the trial protocol to describe prospectively how
patients will be treated and how the resulting data will be analyzed.
This chapter begins with a review of the traditional classification of
clinical trial designs: phase I trials (to find safe doses and schedules
of new agents), phase II trials (to assess the biologic activity or to
offer a preliminary assessment of clinical efficacy), and phase III trials
(to provide definitive evidence of treatment efficacy for changing
clinical practice). Interim monitoring, which allows trials to be stopped
early based on the accrual of positive or unpromising results, is discussed
next. Phase II/III trials, which combine the phase II and phase III
trials into one trial, can speed the development of new treatments.
For all phases of clinical trials, it is important to choose the primary
end point to meet the objectives of the trial; some commonly used
end points are discussed. The chapter ends with a discussion of trial
designs that use and evaluate biomarkers and treatments together,
considering both situations in which there is a single biomarker and
associated experimental treatment and situations in which there are
multiple biomarkers that can potentially help choose which among
many treatments would be best for the patient. The chapter offers a
brief survey of the important elements of the designs of clinical trials
to address various cancer treatment questions. More detailed expositions
including reviews of statistical methods are given elsewhere.1,2
PHASE I DESIGNS
The purpose of a phase I trial is to find a dose and schedule of a new
therapy to take forward for further evaluation. This is typically done
by sequentially treating small cohorts of patients with advanced disease
(e.g., three patients), starting at a low dose of the agent(s) that is not
expected to cause toxicity, and then increasing dose levels until unac-
ceptable rates of toxicity (dose-limiting toxicity [DLT]) are seen. Phase
I trials are designed to be small and are typically not limited to a
specific histologic type, so that the investigators can quickly move on
to testing the therapy for efficacy.
Although there are many possible phase I dose escalation designs,3
there are two general approaches to choosing the next dose level to
be tested based on the DLTs seen up to that point. One approach
uses only the information on the patients being treated at the current
dose level (and, if available, at the dose levels immediately above and
below it). For example, the commonly used 3 + 3 design4 treats cohorts
of three patients and decides the next dose level (escalate one level,
de-escalate one level, or remain the same level) based on the number
of DLTs seen at the current dose: no DLTs in three patients or one
DLT in six patients, escalate; one DLT in three patients, remain at
the same level; and two or more DLTs in three patients or two or
more DLTs in six patients, de-escalate. The recommended dose is the
highest dose level at which there was one or no DLTs in six patients.
A modification of the 3 + 3 design that is slightly more aggressive,
the “rolling six design,”5 is sometimes used when the accrual is rapid
as compared with the evaluation period. Additional modifications
that accelerate the design with one or two patient cohorts initially
until some (possibly less than DLT) toxicity is seen have also been
considered.4,6,7 These designs implicitly target a 20–25% DLT rate
as maximally acceptable; designs that target different DLT rates are
possible.7a
The other general approach to phase I dose escalation is based on
a statistical model for the probability of DLT as a function of the
dose level. An example of a model-based method is the continual
reassessment method.8 Model-based escalation approaches use the DLT
data from all the current patients treated to determine the dose level
for the next cohort of patients. For example, the decision to escalate
from dose level 3 to level 4 will partially depend on the proportion
of DLTs seen at dose level 1. The benefit of model-based escalations
is that by using all the data, the investigators can better choose the
next dose level at which to treat patients (if the assumed model is
correct). The problem with the methods is that if the assumed model

296

is incorrect, too many patients may be treated at dose levels that are
too high.6 To mitigate this problem, it is useful to specify some
constraints on model-based approaches—for example, not allowing
the treatment of new patients at (or above) a dose level at which 33%
or more of at least four patients have had a DLT. Regardless of whether
one uses a statistical model to guide the dose escalation, one could
use a model to fit the toxicity data (as a function of dose) after the
trial is over to identify the dose to recommend for further testing.
Combinations of Agents
For phase I trials involving a combination of agents, the goal is to
identify the doses of each of the agents to be used in the combination.
There may be many dose combinations that have acceptable toxicity.
For example, for a combination of agents X and Y, both high-dose X
plus low-dose Y and low-dose X plus high-dose Y may have acceptable
toxicity (but not high-dose X plus high-dose Y), so the choice between
the two acceptable combinations (and the dose escalation scheme)
will need to be made based on biologic and clinical considerations.
If it is known that a certain minimal dose of X is necessary for its
effectiveness, then the escalation would be of agent Y, with the dose
of X fixed at its appropriate level. In addition, the toxicity profiles of
the individual agents may suggest dose escalation schemes.9
Late Dose-Limiting Toxicities
For some treatments, some DLTs of concern may occur late—for
example, toxicities occurring up to a year after radiation treatment.
This can lead to a very long phase I trial if one has to wait a lengthy
evaluation period (e.g., 1 year) for each cohort of patients before the
next cohort of patients can be treated. One approach to this problem
is to model the occurrence of DLT times, for example, by assuming
that DLTs are uniformly likely to occur over the evaluation period.
If this were true, then one could extrapolate from the early toxicity
experience of the patients to allow dose escalation before the patients
have been followed for the full evaluation period. An example of this
approach is the time-to-event continual reassessment method10 (although
this particular method has apparently not worked well in practice3).
If one designs a trial assuming the uniform occurrence of DLTs over
the evaluation period and the assumption in reality does not hold,
then one is at risk of exposing too many patients to DLTs. For example,
if all the DLTs typically occur near the end of the evaluation period,
then too many patients may be accrued at that dose level and a higher
dose level based on not seeing any early DLTs. Because of this concern,
additional constraints on these approaches are useful—for example,
not accruing more than three patients at a dose level until the initial
three patients at that dose level have been observed for at least one-half
the evaluation period.
Biologic End Points
Phase I designs that escalate to find the highest dose level with acceptable
toxicity (maximum tolerated dose) are based on the assumptions that
(1) higher doses are more effective than lower doses, (2) higher doses
are associated with more toxicity than lower doses, and (3) there is a
dose with acceptable toxicity that will be effective.11 As opposed to
cytotoxic agents, with targeted agents the first two assumptions may
not hold, and with immunologic agents the first assumption may not
hold. This suggests the use of a biologic (nontoxicity) end point to
guide the dose escalation and to choose the dose for further testing.
Although attractive in theory,12,13 designs using nontoxicity end points
have been infrequent in practice.14 Instead, it may be preferable to
assess the biologic end point at the maximum dose level determined
according to DLT, and possibly at lower dose levels for comparison.15
In the context of a targeted agent, “phase 0 trials” are very small
first-in-human trials that are designed not to find the recommended
dose of the new agent, but instead to assess the agent’s effect on its
Clinical Trial Designs in Oncology  •  CHAPTER 18 297
molecular target through use of low exposures of the agent not expected
to cause any toxicity.16
PHASE II DESIGNS
We first define some statistical parameters used to design clinical trials
to evaluate treatment effects. The three principal parameters are (1)
the target treatment effect—the treatment effect of interest, which
the study should have a reasonable chance to identify (referred to
as the alternative hypothesis, contrasted with the null hypothesis of
no treatment effect); (2) the false-positive error rate (type I error)—the
probability of the study findings being positive when the null hypothesis
is true; and (3) the false-negative error rate—the probability of the
study findings being negative when the alternative hypothesis is true,
that is, the target treatment effect is present. (Note that the study
power, the probability of a positive study result under the alternative
hypothesis, is 1 minus the false-negative error rate.) An appropriate
statistical design is obtained by selecting values for the three parameters
corresponding to the study goal.
Phase II trials are designed to provide preliminary efficacy data to
identify therapies that have sufficient activity to be worthy of further
testing in definitive (phase III) trials. Because they are preliminary,
the designs allow the probability of moving an ineffective therapy
forward to be larger than one would use in a phase III trial (e.g., a
type I error of 10% instead of 2.5%). More important, phase II trials
typically use end points that measure treatment activity (e.g., tumor
shrinkage); the effect of the treatment on these end points is expected
to be larger and available more quickly than the effect of the treatment
on a phase III end point that measures direct patient benefit (e.g.,
survival). Both of these factors allow for a smaller sample size and
shorter time to completion than a phase III trial.
For a phase II trial of a single agent, the historical approach is a
single-arm trial in a specific histologic type designed to assess whether
the experimental agent yields sufficient responses (partial or complete
responses as defined by the Response Evaluation Criteria in Solid
Tumors [RECIST]17); the denominator for calculating the response
rate is typically taken to be all patients who started the agent. For
example, one could consider a trial of 32 patients in which the agent
would be deemed worthy of further study if four or more responses
were seen. This design would have both false-positive and false-negative
error probabilities of less than 10% (a typical value chosen) for the
null and alternative response rates of interest. If the true response rate
was 5% or less (the null hypothesis, considered too low to be interest-
ing), then there would be less than a 10% probability of declaring
the agent worth pursuing, and if the true response rate was 20% or
higher (the alternative hypothesis), then there would be less than a
10% probability of a negative conclusion.
To minimize the number of patients treated with an inactive agent,
various two-stage designs have been developed that allow for early
stopping because of negative results.18,19 For example, in a study with
a Simon minimax two-stage design for targeting 5% versus 20%
response rates (with 10% error rates), 18 patients are treated at the
first stage. If there is at least one response among these 18 patients,
a second stage of 14 patients is accrued. The agent is considered
worthy of further study if there are at least four responses seen among
the 32 patients. The required sample size of a single-arm phase II trial
(either one-stage or two-stage) depends on the hypothesized targeted
response rates of interest (e.g., 20% versus 5%), and the error prob-
abilities (both 10% in the aforementioned examples). The sample size
will be smaller when the difference in target response rates is larger
(e.g., 25% versus 5%) or when the error probabilities are larger (e.g.,
15% instead of 10%).
Randomized Screening Designs
There are two general situations in which a single-arm phase II trial
design will not be appropriate because the results would be difficult
298 Part I: Science and Clinical Oncology

Table 18.1  Examples of Required Total Sample Sizes for Phase II and Phase III Randomized Trials
With a Response Rate End Point to Achieve 90% Power for the Designated Target
Response Rates
Response rates Control 20% 20% 20% 40% 40% 40% 60% 60%
Experimental 30% 40% 50% 50% 60% 70% 80% 90%
PHASE II (ONE-SIDED TYPE I ERROR = 10%)
Sample size 530 156 78 688 182 82 156 66
PHASE III (ONE-SIDED TYPE I ERROR = 2.5%)
Sample size 824 236 116 1076 280 126 236 98

Table 18.2  Examples of Required Total Numbers of Events for Phase II and Phase III Randomized Trials
With a Time-to-Event (Survival) End Point to Achieve 90% Power for the Designated Target
Hazard Ratioa
Hazard ratio 0.90 0.8 0.71 0.67 0.6 0.56 0.50 0.40
1/Hazard ratio 1.11 1.25 1.40 1.50 1.67 1.80 2.00 2.50
PHASE II (ONE-SIDED TYPE I ERROR = 10%)
Number of events 2367 528 232 160 101 76 55 31
PHASE III (ONE-SIDED TYPE I ERROR = 2.5%)
Number of events 3786 844 371 256 161 122 87 50

a
For outcomes that are approximately exponentially distributed, the hazard ratio is approximately equal to the ratio of the median survival in the experimental treatment
arm to the median survival in the control arm.

to interpret. The first is when the agent is thought to be primarily
cytostatic rather than cytotoxic, so an effective agent might not shrink
tumors in a nonnegligible proportion of patients.20 One could, in
theory, use a time-to-event end point such as progression-free survival
(PFS) or overall survival (OS) in this situation. However, because of
trial-to-trial variability in time-to-event outcomes, it can be difficult
to identify a benchmark for an experimental treatment to beat in a
single-arm trial.21 The other situation in which a single-arm phase II
trial design would not be appropriate is when the treatment being
evaluated is a combination of agents (agent X + agent Z), one of
which is known to be active (agent Z). The problem is again identifying
a null benchmark (e.g., response rate), in this case to isolate the
contribution of agent X; there may be some data on the response rate
of agent Z alone, but typically not from enough different trials in
comparable settings that one could feel comfortable moving the
combination therapy forward if its observed response rate was mod-
erately higher than that of the response rate of Z alone.
For situations in which a single-arm design is not appropriate, a
randomized screening design is an alternative.20,22 In this design, patients
are randomized to the experimental treatment versus a control treatment
to demonstrate superior activity with use of a phase II end point.
Some examples of sample sizes for a response rate end point and a
time-to-event end point (e.g., PFS, OS) are shown in Tables 18.1 and
18.2, respectively. Note that for time-to-event end points, in which
the time-to-event (survival) curves are typically compared with a
log-rank statistic, the design can be specified in terms of the hazard
ratio and the required number of events to be observed.23 For example,
a trial to detect a hazard ratio of 0.5 with 90% power and 10% type
I error would require 54 events. If one were targeting an improvement
to a median PFS of 6 months (for the experimental treatment) from
a median PFS of 3 months (for the control treatment), then one could
randomize 62 patients over 1 year; the analysis would be performed
when 54 events were observed, which would be about 9 months after
the last patient was randomized. There is a choice regarding which
patients should be included in the primary analysis of a randomized
screening design: an intent-to-treat analysis, which includes all eligible
randomized patients, or only those eligible randomized patients who
started their assigned treatment. Restricting the analysis to patients
starting treatment will provide a more sensitive assessment of treatment
efficacy but is subject to bias if substantially more patients in one arm
drop out of the trial before starting treatment; in the context of a
phase II trial, this is not an issue if the trial is blinded.
Unequal randomization is possible in a screening design, whereby,
for example, twice as many patients are randomized to the experimental
arm as the control arm. Unequal randomization makes the required
sample size larger—for example, 13%, 33%, or 278% larger for 2 : 1,
3 : 1, or 9 : 1 randomization, respectively. It is sometimes justified
because it is said that the unequal randomization will make the trial
more attractive to patients, thereby speeding accrual (although we
know of no hard evidence on this). A better justification for unequal
randomization is that in situations with limited experience with use
of the experimental agent, it will allow more experience to be obtained
(e.g., 40 patients instead of 30 in a 60-patient trial using 2 : 1 randomiza-
tion instead of 1 : 1 randomization). Other issues concerning randomized
screening designs are discussed later in the section on phase III random-
ized designs.
Randomized Selection Designs
Another type of randomized phase II design is the randomized selection
design. In this design, the two (or more) treatment arms are experi-
mental treatments. The goal of the trial is to select which of the
treatment arms to take forward to further testing.24 At the end of the
trial, the treatment arm that has the better efficacy outcomes is selected
for further development. For example, if response rate was chosen as
the outcome, then the arm with the better response would be selected.
The sample size for this design is chosen so that there is a high
probability that one will not select a worse treatment arm, if in fact
there is one that is worse by a specified indifference margin. Selection
designs require smaller sample sizes than screening designs because

the conclusions from the design are much weaker (demonstrating that
treatment X is not much worse than treatment Y instead of demonstrat-
ing that treatment X is better than treatment Y). When it is possible
to evaluate each arm individually for efficacy—for example, when a
response rate end point is being used for evaluating single agents—it
is possible to embed within the selection design minimum (single-arm)
response rates. For example, one could select the treatment arm with
the better response rate provided that the response rate was at least
15%; otherwise, neither arm would warrant further study. The typical
use of selection designs is for deciding which of several schedules of
a new agent to use for further agent development.
Designs With Biomarkers
Phase II trials offer an opportunity to assess biomarkers for their
ability to enable identification of a patient population for which the
experimental treatment will be effective or especially effective.25 Unless
there is strong evidence that the treatment will work only in biomarker-
positive patients, the phase II trial should not restrict enrollment to
such patients.26 Instead, the trial design should accommodate the
possibility that benefit will be restricted but not assume it. For example,
in a single-arm trial with a response rate end point, two-stage designs
can be used wherein both biomarker-positive and biomarker-negative
patients are accrued at the first stage; depending on what responses
are seen at the first stage, the trial is either stopped or more biomarker-
positive or unselected patients are accrued.27,28
When there is strong evidence that a molecularly targeted agent
will work only in biomarker-positive patients and some evidence that
its efficacy may not depend on tumor histologic type, a single phase
II trial that pools response data across biomarker-positive patients in
different histologic groups may be useful. However, in the absence of
reliable evidence that the activity level is uniform across these histologic
groups, a separate evaluation may have to be conducted in each group
to allow for a reliable evaluation. Note that the use of Bayesian hierarchi-
cal modeling to borrow information across groups does not work in
this setting,29 although simpler pooled futility rules can be useful.30
In settings in which a randomized screening design is used, one
would again generally not restrict enrollment to biomarker-positive
patients. After enrolling all comers, one could consider in an ad hoc
manner the experimental-versus-control treatment effect in the overall
group and the biomarker-positive and biomarker-negative subgroups.
Alternatively, one could use a formal phase II biomarker trial design that
directly addresses the relevant drug development question31: Should a
phase III trial be performed, and if so, how should the biomarker be
incorporated into that design? Phase II designs with multiple biomarkers
are discussed later under Designs With Multiple Biomarkers.
PHASE III DESIGNS
Phase III trials are the definitive randomized comparisons of treatments
and should provide sufficiently compelling evidence to change clinical
practice. Accordingly, they are designed with a small false-positive
rate—for example, a one-sided type I error rate of 2.5%, and high
power for detecting truly positive effects (e.g., 80% to 90%). In
addition to depending on the type I error and power, the required
sample size depends inversely on the magnitude of the targeted treat-
ment effect between the treatment arms and is typically hundreds or
thousands of patients (see Tables 18.1 and 18.2). As noted previously,
the required sample size for time-to-event end points is a function
of the expected number of events, rather than the sample size. Therefore
the required sample size will be larger for a clinical setting with lower
event rates than one with higher event rates. For example, with 3
years of accrual and 3 years of follow-up, a setting with 70% 2-year
event-free survival will have a required sample size roughly 40% larger
than that needed for a setting with 50% 2-year event-free survival.
For time-to-event end points, the definitive analysis and interim analyses
are typically performed when the expected number of events is observed
Clinical Trial Designs in Oncology  •  CHAPTER 18 299
in both treatment arms together, but there are other alternatives32;
the specific timing of the definitive and interim analyses should be
specified in the trial protocol. The trial protocol should also specify
the analyses that will be performed and which patients will be included
the analyses. Typically, the intent-to-treat principle is used wherein
all eligible randomized patients are included in the analyses, and eligibil-
ity is determined before randomization or assessed blindly based on
prerandomization patient samples.
Randomization and Stratification
The randomization in a phase III trial is typically 1 : 1 (to avoid the
inefficiency of an unbalanced randomization), and, after informed
consent has been obtained, is ideally performed right before the
experimental treatment is given (to avoid having to include patients
who drop out of the trial before receiving the treatment under study).
The randomization is typically stratified on a small number of variables
thought to be highly associated with outcome to balance the distribution
of these variables across the treatment arms and thus ensure that
approximately equal proportions of patients in each arm of the trial
will have relatively good and bad prognoses (as determined according
to these stratifying variables). A stratified analysis can be used even if
stratification was not part of the randomization—for example, when
the stratifying variable is defined with a biomarker assay that is per-
formed on a prerandomization sample after the randomization.
Stratification is more important in smaller trials than larger ones
because the chances of a worrying imbalance are larger in a small
trial. When outcomes have a subjective component, blinding of the
treatment assignment with placebos (when feasible) can remove a
concern about potential bias in the trial results.
Multiarm Trials
A trial with multiple experimental arms and a single control arm can be
an efficient way to test multiple treatments in a single disease setting.33
For example, a single randomized clinical trial (RCT) with a control arm
and three experimental arms can be 33% smaller than three separate
RCTs evaluating each experimental treatment separately. Multiarm
trials can add complexity because they may require multiple placebos,
restriction of eligibility to ensure that all the agents can be given safely,
agreement among multiple industry partners to participate and agree-
ment regarding how the data will be analyzed and shared, complex
funding arrangements, and additional regulatory complexities.33,34
A factorial trial design is a multiarm trial in which the arms represent
all possible combinations of the experimental and control treatments.
For example, in a 2 × 2 factorial design evaluating experimental
treatments A and B (relative to no treatment), patients are randomized
to one of the following four arms: (1) no treatment (arm C), (2)
treatment A (arm A), (3) treatment B (arm B), or (4) treatment A
plus treatment B (arm AB). (In some applications, a common backbone
treatment will be given in all the arms.) A factorial analysis of a factorial
design assumes that the effect of treatment A does not depend on
whether the patient received treatment B—that is, the benefit of arm
A over arm C is the same as the benefit of arm AB over arm B. (This
is equivalent to assuming that that effect of treatment B does not
depend on whether the patient received treatment A.) This assumption,
which is referred to as no statistical interaction among the treatments,
is very strong. When the assumption is satisfied, one can assess the
efficacy of A and B in a trial with the same sample size as would be
required to assess a single agent, a remarkable savings. However, if
the assumption is not true, then a factorial analysis can incorrectly
suggest that agents work when they do not, and do not work when
they do.35,36 Even when a factorial analysis is problematic, the factorial
trial design with a nonfactorial analysis is an efficient way to study
two treatments and their possible interaction.36
When a multiarm trial allows new trial arms to be added as it is
ongoing, it is referred to as a “master” protocol or a “platform” trial.
300 Part I: Science and Clinical Oncology
The idea is that when promising new agents become available they
can be added (possibly along with corresponding control arms). In
addition, trial arms are dropped when the questions involving these
arms have been answered. An example of a master protocol is the
STAMPEDE,37 which evaluates various agents in addition to a standard
hormone therapy for advanced prostate cancer. The advantage of a
master protocol is that it allows testing of agents more quickly because
a new protocol does not need to be developed for each new treatment.
This can be especially important when new treatment arms are suggested
by the ongoing discovery of new biomarkers and targeted treatments.
For any master protocol, one must avoid potential pressure to add
new treatment arms or trial questions that may not be of the highest
priority just to keep the trial ongoing.
Noninferiority Trials
In a noninferiority trial, one is interested in showing that the new
treatment is not inferior to the standard treatment. These trials arise
when the new treatment is expected to be less toxic or more convenient
than the standard therapy. The sample size considerations are similar
to those of a superiority trial (see Tables 18.1 and 18.2) with one
important exception: the targeted treatment effect will typically be
smaller, leading to much larger trials. The targeted effect is small because
one does not want to recommend a new treatment that is inferior to
a standard treatment by a nonnegligible amount. In particular, it has
been suggested that the targeted difference should be no greater than
a fraction of the benefit (e.g., 50%) seen for the standard treatment,38
which itself may not be large. In designing a noninferiority trial, the
role of type I error and power are reversed, so that priority is given
to minimizing the probability of declaring noninferiority when the
new treatment is actually inferior; this probability should be kept very
small (e.g., 2.5%), whereas the probability of declaring noninferiority
when the treatments are equally effective could be set at 80% to 90%.
For noninferiority trials, using the intention-to-treat principle can
be problematic if there are a nonnegligible proportion of patients
who do not receive their assigned treatments, because this can lead
to underestimation of any true loss in efficacy38; to accommodate
this, an additional analysis of only those patients who received their
assigned treatments is typically performed.39
In some situations, it is expected that the new treatment will
actually be modestly better than the standard treatment, but because
it is less toxic, it would be sufficient to demonstrate that it is noninferior
to the standard treatment. In these special situations, the required
sample size can be reduced.40
Nonrandomized Trial Designs
If a treatment works dramatically better than the standard treatment,
then one does not need an RCT to obtain the necessary evidence to
change clinical practice. For example, the hematologic and cytogenetic
responses seen for second-line imatinib in single-arm studies of Phila-
delphia chromosome–positive (Ph+) chronic myelogenous leukemia
led the FDA to grant accelerated approval of this agent in that setting.41
Another example is the benefit of imatinib in pediatric Ph+ acute
lymphoblastic leukemia (ALL), in which an 80% 3-year event-free
survival rate was seen in a Children’s Oncology Group trial compared
with 35% seen in three previous trials without imatinib.42 Care must
be taken when drawing conclusions from comparisons with historical
experience; positive-appearing results could be due to selection bias
(the patients treated with the experimental treatment may have better
prognoses than those previously treated with the standard treatment),
or improvements in ancillary care may make an experimental treatment
appear better than a standard treatment. An example of how things
can go wrong was the widespread use of high-dose chemotherapy
with autologous stem cell rescue for metastatic breast cancer. A series
of small single-arm trials had suggested that it was highly beneficial,
but later RCTs demonstrated that it did not work.43 In considering
a relatively rare cancer, it may be impractical to perform a large RCT,
but the challenges in using results from nonrandomized trial designs
to change clinical practice should not be ignored.44
INTERIM MONITORING
Interim monitoring of accruing outcome data from a clinical trial is
motivated by both ethical and resource considerations; it allows one
to stop a trial as soon as the scientific question is answered. It is the
most important adaptive trial element of a clinical trial.
Stopping for Superiority (Efficacy) in a
Randomized Clinical Trial
If it becomes obvious during the course of a trial that the experimental
treatment is much more effective than the standard treatment, then
one would want to stop the trial and release the results to the public.
However, an undisciplined examination of accruing outcome data
can lead to the release of results that do not have acceptable statistical
validity. For example, if one repeatedly examines accruing outcome
data from a trial and stops it as soon as the (one-sided) P value is
below .025, then the chances of incorrectly recommending an ineffective
treatment (a type I error) can be much higher than 2.5% (e.g., 12.5%
with 20 examinations). Therefore the study protocol should include
a formal interim analysis plan that prospectively designates the times
when the interim analyses will be performed, as well as the decision
rules that will be used at each time to decide if the outcome data are
extreme enough to stop.
A large number of formal interim analysis plans are possible.45,46
The analysis times are typically specified in terms of the “information
fraction,” which is the proportion of the information available in the
data at the interim analysis time compared with the information
available at the regularly scheduled end of the trial (100%, by defini-
tion). With a response rate end point, the information fraction is the
proportion of the total sample size that has been evaluated for response,
and with a survival end point it is the proportion of events observed
as compared with the total number of events required at the final
analysis time. Table 18.3 displays the popular Haybittle-Peto47 and
O’Brien-Fleming48 monitoring plans (in terms of P value cutoffs
required for stopping) for interim analyses at 40% and 70% informa-
tion. (Many common interim monitoring designs accommodate flexible
analysis timing using the “spending function approach.”49) Note that
the P value cutoffs for the final analysis are less than the nominal
0.025 type I error of the trial that would be used if no interim analyses
were performed. Because of this, use of interim monitoring necessitates
a small increase in the sample size to maintain the power of the trial
(e.g., 2% increases in sample size for a trial with 90% power for the
O’Brien-Fleming plan described in Table 18.3).
Table 18.3 One-Sided P Value Cutoffs for
Haybittle-Peto and O’Brien-Fleming
Interim Monitoring Plans With Interim
Analyses at 40% and 70% Information
for Overall One-Sided Type I Error
of 2.5%
INFORMATION TIME OF ANALYSIS
Monitoring Plan 40% 70% 100% (Final Analysis)
None — — .0250
Haybittle-Peto .0010 .0010 .0246
O’Brien-Fleming .0004 .0073 .0227

Stopping for Inefficacy (Futility) in a Randomized
Clinical Trial
Most randomized trials are asking one-sided questions: Does the
experimental treatment work better than a standard treatment? Accord-
ingly, there is no need to demonstrate that the experimental arm is
significantly worse that the standard therapy; instead it is sufficient
to show that it is no better.50 Therefore interim monitoring should
stop the trial for inefficacy as soon as tangible benefit over the control
treatment can be ruled out. Formal inefficacy interim analysis plans
allow for such stopping, but they keep a truly effective therapy from
being dismissed.
In a very simple approach to inefficacy monitoring, the outcome
data are examined at 50% information, and the trial is stopped if the
observed treatment effect favors the control arm (by any amount).51
A number of more complex approaches have been suggested (that
allow for more interim looks).52,53 It has been argued that inefficacy
monitoring is so important for protecting patients from ineffective
therapies that if it is not included in the trial protocol, or if the
specified monitoring plan has not been followed during the trial, then
the reasons for this should be stated in the trial report.54 Inefficacy
monitoring is also critical for noninferiority trials, as it allows stopping
a trial early when the new treatment is unlikely to be noninferior to
the standard treatment (and may be non-negligibly inferior).54a
Other Considerations for Efficacy and
Inefficacy Monitoring
With electronic data capture there is the possibility of easily having
many interim analysis looks. Once interim monitoring has commenced,
there is very little cost in having frequent monitoring from that point
onward,55 so one can analyze the data more often and stop earlier
(when appropriate). For example, looking at the data at 40% informa-
tion and then in 10% increments (six instead of two interim looks)
would require increasing the sample size by 3% for the O’Brien-Fleming
monitoring plan.
Potential drawbacks that should be considered when designing an
interim monitoring plan are that if a trial stops early, then there may
be very little or no information about the longer-term effects of the
treatments or the effects of treatments on secondary end points, and
the treatment effects will be estimated less precisely than if the trial
had not been stopped early. Of special concern is when the benefits
of the experimental treatment over the control treatment are expected
to be delayed; in these situations, inefficacy monitoring should not
begin until the benefits of the experimental treatment are expected
to be seen.55a Another concern is that trials that stop early for superiority
overestimate the treatment effect.56 However, it has been shown that
except for stopping at very early interim analyses (≤25% of information),
the overestimation is minor.57 Furthermore, a review of the National
Cancer Institute (NCI) Cooperative Group phase III trials that were
stopped early for positive results confirmed that mature results were
consistent with the early results.58
Interim monitoring is best performed by an independent data
monitoring committee,59,60 who are the only ones who see accruing
outcome data (besides the trial statisticians). In some very special
circumstances, it may be possible to release preliminary data to the
public when a trial is in its follow-up stage and the release would not
compromise the integrity of the trial.61
Outcome-Adaptive Randomization
RCT designs generally keep arm assignment probabilities constant
throughout the trial (e.g., 1 : 1). However, there are designs in which
the accruing outcome data are used to adjust the randomization
ratio so that a higher proportion of patients are randomized to the
treatment arm(s) that appear to be doing better (outcome-adaptive
randomization); this can be thought of as a type of continuous
Clinical Trial Designs in Oncology  •  CHAPTER 18 301
interim monitoring. There are a number of drawbacks as compared
with standard interim monitoring that make outcome-adaptive
randomization not useful or appropriate. The first is that any time
trends in the prognostic characteristics of the patient population
enrolled in the trial will bias the results of the trial, making it
difficult to interpret the results of a completed trial.62–65 A second
problem is statistical inefficiency due to having an unequal number
of patients in the treatment arms.63 This inefficiency means that
trials using outcome-adaptive randomization will necessarily be
larger and will result in delay in getting new effective treatments to
the clinical community, and they also will expose more patients to
bad outcomes. This is the same issue as discussed earlier with fixed
unequal randomization (e.g., 2 : 1 randomization) but can become
more problematic as the randomization ratio becomes more extreme
in outcome-adaptive randomization (e.g., 9 : 1). A third problem with
outcome-adaptive randomization is that even though it will generally
put more patients in the better treatment arm (if there is one), it will
occasionally put a moderately larger proportion of patients on the
worse treatment arm.66 Finally, there has been ongoing discussion
regarding ethical issues (pro and con) involved in outcome-adaptive
randomization.67–74
Monitoring for Rare Serious Toxicities
Most common toxicities of an experimental treatment are identified
and quantified in the early testing of the agent. When the toxicity is
rare, however, one can monitor for it in a large phase II or phase III
trial to assess its magnitude and see whether the treatment should be
stopped or modified. For a rapidly occurring toxicity, one can use a
continuous monitoring plan that identifies the problem as soon as
the number of patients experiencing the toxicity crosses a defined
boundary.75,76 For example, if a 1% (5%) rate of the toxicity is acceptable
(unacceptable), then one could use a Pocock boundary with the first
169 patients treated in the experimental arm that has the following
properties: If the true toxicity rate is 5%, then the problem will be
identified 95% of the time at an average sample size of 54. If the true
toxicity rate is 1%, then the toxicity rate will be deemed acceptable
90% of the time.
PHASE II/III DESIGNS
A phase II/III trial is designed as a phase III trial but with an interim
“phase II” go/no-go rule to decide whether the experimental treatment
is active enough to continue the trial to its phase III sample size.77
The advantages of a phase II/III trial over a phase II trial followed
by a phase III trial are efficiency and speed, in that (1) the phase
II patients can be included in the phase III analysis, and (2) one
does not have to wait for the phase III protocol development after
the phase II results become available. Phase II/III trials are most
useful when the phase II end point (e.g., PFS) can be achieved
earlier than the definitive phase III end point (e.g., OS). What the
phase II end point should be, how high the bar (go versus no-go
threshold) should be set for that end point, and whether there
should be an accrual suspension while waiting for the phase II data
to mature are the key design considerations for phase II/III trials.78
In general, one could choose the phase II end point and could set
the bar at the same level as would be used in a stand-alone phase
II trial. When the same end point needs to be used for both the
phase II and phase III evaluations (e.g., when the treatment is not
expected to affect any end points other than OS), the gains achieved
with a phase II/III design will be modest unless there is an accrual
suspension.78a
There is a close relationship between phase II/III trials and phase
III trials with futility monitoring: in both cases, interim results are
used to decide whether to continue to the full phase III sample size.
However, in a phase II/III trial, the interim results need to be sufficiently
promising, whereas for a typical futility analysis the experimental
302 Part I: Science and Clinical Oncology
treatment just needs to be not worse. This difference is reasonable
because in a phase III trial one has promising evidence from previous
phase II trials. In addition, it is possible to conduct a phase II/III trial
with multiple experimental arms (along with the control arm), with
the most promising (or all of the promising) agents continuing to
phase III.79 Also, if the experimental treatment is a single agent expected
to produce responses (if it is effective), then it is possible for the
interim phase II look to be based solely on the experimental arm
response rate.
A potential disadvantage of a phase II/III trial is that one is eliminat-
ing the flexibility of modifying the phase III trial design based on the
results of the phase II trial—for example, changing the dose or schedule
of the experimental agent, supportive care requirements, or the patient
population.80–82 In addition, committing to the question to be asked
in phase III at an earlier time locks in a sponsoring organization that
may want to keep its options open longer with regard to what phase
III trials to perform.
END POINTS FOR RANDOMIZED TRIALS
The choice of a primary end point in an RCT should reflect the
study goal. In general, a primary end point for a phase III RCT
should measure direct clinical benefit or a surrogate for direct clinical
benefit. On the other hand, the primary end point in a randomized
phase II trial need only be able to suggest that the treatment would
show direct clinical benefit in a larger trial or in a different clinical
setting. By “direct clinical benefit” we mean a “direct measure of how
a patient feels, functions, or survives.”83 This section first consid-
ers some commonly used RCT end points, and whether they are
measuring direct clinical benefit. This is followed by a consideration
of patient-reported outcomes. We end with discussion of surrogate
end points.
Overall Survival
An experimental treatment that improves OS (death from any cause)
benefits patients. However, there are a number of potential complica-
tions when it is used as a primary end point.84 It can lead to very
large and long trials in settings where the standard therapies (including
after relapses) work quite well. This is because the required size of the
trial depends on the number of deaths observed. A related potential
difficulty with OS as the primary end point occurs when the trial
design crosses patients over to the experimental treatment at relapse
or progression. With crossover as part of the trial design, many patients
in the standard arm will eventually receive the experimental treatment,
potentially lessening the OS differences between the treatment arms.
If the experimental treatment is a standard second-line treatment
being tested for first-line treatment (as is frequently the case when
agents are tested and approved in advanced disease settings first), then
this is not a problem; the OS differences observed between the
experimental arm and the control arm (standard treatment followed
by experimental treatment at relapse) exactly captures the clinical
benefit of the experimental treatment.85 In particular, absence of an
improvement in OS means there is no benefit to patients from moving
the standard second-line treatment to the first line. If the experimental
treatment is not a standard second-line treatment, then OS may indeed
underestimate its benefits when a crossover is part of the trial design.
It could be argued that crossover should not be allowed unless there
is definitive evidence that the experimental treatment is beneficial in
the second line.85,86
In evaluating a treatment in a population with high rates of compet-
ing mortality (e.g., an elderly population), noncancer deaths can dilute
the treatment’s effect on OS, especially in an adjuvant setting. However,
the OS effect seen does represent the benefit to that population. If
one was interested in isolating the treatment’s effect on cancer and
was able to accurately separate cancer from noncancer deaths, one
could use cancer-specific survival as the end point.84
Progression-Free Survival
PFS is defined as the time from randomization to progression or
death. (Time to progression is a related, less-preferred end point wherein
deaths without progression are censored observations rather than
counted as events.) Because PFS requires a subjective reading of patient
scans, if there is a concern of bias in a trial in which the treatment
assignment is not blinded, a blinded independent central review is
often considered.87 However, this is not recommended as a general
strategy, because a central review can actually add bias to the trial
results.88 In addition, evaluations comparing trial results using investiga-
tor versus independent assessment of PFS have not shown substantial
differences.88,89 If investigator bias in evaluating PFS is a concern, one
can perform an audit comparing investigator assessments versus
independent assessments of PFS.90 Another potential source of bias
with the use of PFS in an unblinded trial is assessment time bias: if
the assessment times are different between the treatment arms, a
standard PFS time-to-event analysis can give incorrect results.91 If
there is a concern about bias in assessing PFS, it is best to conduct a
double-blind trial when possible.
Because progression is typically measured according to a specified
radiologic increase in tumor size (or a change in blood chemistry
values in hematologic cancers), PFS is a measure of biologic activity
and does not directly measure clinical benefit.17,86 A sufficiently large
increase in PFS in a randomized phase II trial of an experimental
treatment suggests that the treatment should be assessed in a phase
III trial. Similarly, a sufficiently large increase in PFS in an RCT in
a metastatic disease setting may suggest that the experimental treatment
will show direct clinical benefit in a less advanced disease setting.
Related alternatives to PFS that capture later events, such as time
from randomization to second progression or death, have sometimes
been used in trials of maintenance therapies to measure patient benefit
of a therapy in the context of the subsequent therapies.92 Considerations
for PFS as a surrogate end point for clinical benefit are discussed in
a subsequent section.
Disease-Free Survival
Disease-free survival (DFS) is defined as the time from randomization
to recurrence of tumor or death, and it is typically used in the adjuvant
treatment setting. It can be considered a direct measure of clinical
benefit if the magnitude of its prolongation with an experimental
therapy outweighs its toxicity87; a consideration here is the proportion
of patients whose recurrences are symptomatic. For example, if the
recurrence of disease is measured with a very sensitive blood assay
and the cancer is relatively indolent at recurrence, then it could be
years before patients become symptomatic, and it is questionable
whether improving DFS is offering patients a direct clinical benefit.
In some settings DFS may also be a surrogate for OS, because patients
who remain disease free for an extended period of time may be cured.
Tumor Response Rates
A sufficient improvement in tumor response rates in a randomized
phase II trial shows activity that could be tested in a phase III trial.
Durable complete responses might be a surrogate for clinical benefit,
but PFS would be a more efficient end point in a randomized trial.
Patient-Reported Outcomes
Patient-reported outcomes (PROs) related to a specific cancer symptom
are sometimes used to provide primary or supporting evidence that
a treatment is offering direct clinical benefit (e.g., improvement in
cancer-related pain).93 A number of trial design issues should be
addressed when PROs are used.94 A PRO instrument that is appropriate
for the trial population should be chosen to collect the patient evalu-
ations. The design should specify when in patient courses the PROs

100%
75%
Survival
50%
25%
0%
A Time
Figure 18.1  •  Hypothetical example for an individual-level surrogate that is not a trial-level surrogate (green curves, surrogate-positive patients; red curves,
surrogate-negative patients; black curves, overall) demonstrating individual-level but not trial-level surrogacy. (A) Standard treatment for which 30% of patients
have a positive value of the surrogate immediately after treatment. (B) Experimental treatment for which 58% of the patients have a positive value of the
surrogate immediately after treatment. Note that the treatments are equally effective in terms of overall survival (black curves) even though the proportion that
is positive for the (individual-level) surrogate outcome is higher for the experimental treatment.
will be recorded and whether a baseline PRO will be used to adjust
the patient responses before comparisons between treatment arms are
made. Ideally, patients should be blinded as to their treatment assign-
ment, and it may be useful to collect data on what treatment patients
think they received to check for unintentional unblinding. The design
should attempt to minimize missing data. Finally, the analysis strategy
of the PRO data should be specified in the protocol, including how
missing data will be accommodated in the analysis.
In addition to being used to measure treatment efficacy, PROs can
be used to better understand the toxicities of treatments.95 They can
also be used as primary end points to assess agents used to lessen the
toxicities of anticancer treatments.
Functional Outcomes
In some clinical situations the trial end point may be related to how
a patient functions, which is a measure of direct patient benefit. For
example, in a phase III RCT of three different nonsurgical treatment
strategies for locally advanced laryngeal cancer,96 the primary end
point was laryngectomy-free survival (time from randomization to
laryngectomy or death). Another example is a phase II trial of radiation
and chemotherapy for stage T1 bladder cancer (NCT00981656) in
which the primary end point was freedom from radical cystectomy
at 3 years.
Trial-Level Surrogate End Points
A trial-level surrogate end point is one for which the results of the
RCT with this end point can be used to reliably predict what the
results of the trial would be with the definitive end point. A trial-level
surrogate end point may allow the results of the trial to be obtained
earlier and with a smaller sample size than if the definitive end point
was used. It is, however, challenging to demonstrate trial-level surrogacy;
it requires a comparison of the two end points in a series of trials in
a similar disease setting, with the treatments being tested having similar
mechanisms of action.97 For example, Sargent and colleagues98 examined
the results of 18 RCTs of adjuvant fluorouracil-based therapies for
colon cancer and showed that 3-year DFS was a good surrogate for
5-year OS. However, this trial-level surrogacy would not necessarily
carry over to a different clinical setting, different types of treatments
(e.g., antiangiogenic agents or immunotherapies), or later times when
Clinical Trial Designs in Oncology  •  CHAPTER 18 303
100%
75%
Survival
50%
25%
0%
B Time
more effective postrelapse treatments have become available and have
changed the OS outlook.
Trial-level surrogacy is frequently confused with individual-level
surrogacy; an individual-level surrogate end point is measured after
treatment and predicts which patients will do well versus poorly given
a specific treatment. One might think that a good individual-level
surrogate would always be a good trial-level surrogate, but this is not
true.97,99 As a hypothetical example, consider the survival curves in Fig.
18.1. The binary surrogate variable (S) here is a good individual-level
surrogate for survival as seen by the green curves being above the
red curves for both the experimental treatment (Fig. 18.1A) and the
standard treatment (Fig. 18.1B). We assume that 70% of the patients
who receive the experimental treatment (X) have a positive S value,
whereas only 20% of the patients who receive the standard treatment
(Y) have a positive S value. If S were a good trial-level surrogate for
survival, this would mean that treatment X was better than treat-
ment Y. However, as seen in Fig. 18.1, the treatments have the same
survival (black curves in Fig. 18.1). Heuristically, one can imagine a
situation as depicted in Fig. 18.1 when the experimental treatment
moves some of the better prognosis surrogate-negative patients into
being positive on the surrogate, without affecting their survival. A
real example of an individual-level surrogate for which there is no
evidence of trial-level surrogacy is pathologic complete response as
a surrogate end point for event-free survival or OS for early-stage
breast cancer.99 An intermediate end points can be useful for drug
development even though it has not been demonstrated to be a trial-
level surrogate.99a
PHASE III TRIAL DESIGNS WITH A
SINGLE BIOMARKER
Traditional phase III trials focus on an average treatment effect for
an overall population. However, new targeted agents often benefit
only a subset of patients whose tumors express the molecular target.
The use of biomarkers measured before treatment allows one to focus
trials on the patients who are most likely to benefit.
Prognostic and Predictive Biomarkers
Clinical trial biomarkers are generally described as prognostic or
predictive. A prognostic biomarker categorizes patients into subgroups
304 Part I: Science and Clinical Oncology
with distinct prognoses—that is, patients who will do relatively well
versus relatively poorly when treated with no treatment or a standard
treatment. For example, KRAS mutation is a prognostic biomarker
(for OS) for patients with metastatic colorectal cancer treated with
best supportive care, the control arm in an RCT of panitumumab.100
For patients receiving best supportive care, the median OS was 7.6
months for patients who had wild-type KRAS tumors and 4.4 months
for patients who had mutant KRAS tumors. The clinical usefulness
of a prognostic biomarker is measured according to its ability to
improve patient outcome by guiding patient treatment (more treatment
or experimental treatments for high-risk patients and/or less treatment
for low-risk patients).
A predictive biomarker for an experimental treatment identifies
patients for whom that treatment is especially beneficial—that is, the
treatment effect (comparing the experimental treatment and the standard
treatment) is larger for the biomarker-positive patients than for the
biomarker-negative patients. An example of a predictive biomarker (for
PFS) is epidermal growth factor receptor (EGFR) mutation status for
gefitinib for non–small cell lung cancer patients.101 Gefitinib is more
effective than chemotherapy for EGFR mutation–positive patients
but less effective than chemotherapy for EGFR mutation–negative
patients. EGFR mutation status in this case not only is predictive but
also exhibits a “qualitative interaction,” defined by the experimental
treatment working better than the standard treatment for biomarker-
positive patients, but being only equally effective (or worse) for the
biomarker-negative patients.102 Predictive biomarkers—that is, those
exhibiting a qualitative interaction—have clinical usefulness because
they can direct treatment in an obvious manner.103 Biomarkers can
be both predictive and prognostic.
From randomized clinical trial data, one can evaluate a biomarker
to see if it is predictive for the experimental treatment and/or prognostic.
This can be done with a well-defined analysis plan on previously
collected trial data if the biomarker status is available.104 For planning
a phase III trial, the choice of a biomarker design should depend on
the strength of the available evidence that the biomarker can be
successfully used to identify patients who will benefit from the
experimental treatment.105 In all cases, before a biomarker is used in
a phase III trial, its analytic validity should be ensured—that is, the
biomarker assay should consistently capture the biologic characteristics
it is putatively measuring.106
Evaluate biomarker
Biomarker positive Biomarker negative
Randomize
New Standard
treatment treatment Off study
A B
Figure 18.2  •  Two biomarker randomized clinical trial designs. (A) Enrichment design. (B) Biomarker-stratified design. (From Freidlin B, Korn EL.
Biomarker enrichment strategies: matching trial design to biomarker credentials. Nat Rev Clin Oncol. 2014;11:81–90.)
Enrichment Designs
When there is convincing evidence that any treatment benefit of the
experimental treatment would be limited to biomarker-positive patients
(based on clinical and preclinical data), then the trial should include
only patients who are biomarker positive (Fig. 18.2A). By identifying
the subpopulation in which the treatment is expected to work well,
one can frequently target a larger treatment effect than would be seen
in the overall population, thereby reducing the required sample size
of the trial. An example of an enrichment trial is the trial of the BRAF
inhibitor vemurafenib versus dacarbazine in patients with metastatic
melanoma carrying the BRAF V600E mutation107; the hazard ratios
for PFS and OS were 0.26 and 0.37, respectively. However, even if
the sample size required for the randomized trial is small, the number
of patients screened to obtain these biomarker-positive patients may
be large, depending on the prevalence of biomarker positivity and the
ability of the biomarker to identify treatment-responsive patients.108
Use of an enrichment design does not allow assessment of the treatment
effect in the biomarker-negative patients.
Biomarker-Stratified Designs
In a biomarker-stratified design, both biomarker-positive and biomarker-
negative patients are randomized (see Fig. 18.2B). Alternatively, all
patients are randomized and their biomarker status is determined
after randomization. The advantage of the first approach is that one
is guaranteed that all the patients will have their biomarker status
determined. The advantage of the second approach is that one will
not have to wait for the biomarker evaluation to enroll the patient
in the trial.109
There are several different analytic approaches for analyzing a
biomarker-stratified design, which involve various strategies for testing
the treatment effect in the biomarker-positive, biomarker-negative, and/
or overall populations.105 A good strategy should aim at maximizing
the probability that the new treatment is recommended for patients
who benefit and minimizing the probability that the treatment is
recommended for patients who do not benefit. Accordingly, analysis
strategies that recommend the treatment for the biomarker-negative
subpopulation without assessing the treatment effect in that sub-
population should generally be avoided.110,111 Two effective strategies
Evaluate biomarker
Biomarker positive Biomarker negative
Randomize Randomize
New Standard New Standard
treatment treatment treatment treatment
 Clinical Trial Designs in Oncology  •  CHAPTER 18 305

Yes Recommend the treatment

for biomarker-positive and
biomarker-negative patients
Yes Test
biomarker-negative if
significant at level α
Recommend the treatment
Test for biomarker-positive
biomarker-positive if No patients only
significant at level α

STOP Do not recommend treatment

A No

Yes Recommend the treatment

for biomarker-positive and
biomarker-negative patients
Yes Test
biomarker-negative if
significant at level α
Recommend the treatment
for biomarker-positive
No patients only
Test
biomarker-positive if
significant at level α1
Yes Recommend the treatment
for all patients
Test
overall population if
significant at level α−α1
No
Do not recommend treatment
B No

Figure 18.3  •  Two analysis strategies for analyzing a biomarker-stratified design (α is the overall type I error of the design). (A) Sequential subgroup-specific
strategy. The biomarker-positive subgroup is first tested with type I error of α. If this test rejects the null hypothesis of no treatment effect, then the biomarker-
negative subgroup is tested with type I error of α. (B) MaST strategy. The biomarker-positive subgroup is first tested with type I error of α1, a value less than
α. Depending on whether this test rejects the null hypothesis of no treatment effect, the biomarker-negative subgroup or overall randomized population is
tested with type I error of α or α − α1, respectively. (From Freidlin B, Korn EL. Biomarker enrichment strategies: matching trial design to biomarker credentials.
Nat Rev Clin Oncol. 2014;11:81–90.)

for analyzing a biomarker-stratified design are the subgroup-specific
sequential strategy (Fig. 18.3A) and the MaST strategy112 (Fig. 18.3B).
Biomarker-Strategy Designs
In a biomarker-strategy design with a single biomarker, patients are
randomized to a standard therapy versus a “strategy” treatment arm
in which the treatment is chosen based on the biomarker status. If
the biomarker status is positive, then the patients receive the experi-
mental treatment; otherwise they receive the standard treatment.
Although superficially appealing because it directly estimates how well
the biomarker-guided treatment would improve outcomes in practice,
this design is inefficient owing to the dilution of the between-arm
difference in outcomes as a result of a potentially large proportion of
patients being treated with the standard treatment in both arms of
the trial. Therefore use of this design (with a single biomarker) is not
recommended. Instead, one can use a biomarker-stratified design to
estimate the treatment effect in the biomarker-positive and biomarker-
negative subgroups, and then use these subgroup-specific estimates
to estimate more efficiently how well a biomarker strategy would
compare with a standard treatment.109
Designs With a Continuous Biomarker
Ideally, a cut point separating positive and negative values of a biomarker
would be determined before its prospective evaluation in a phase III
trial. For example, cut points for the prognostic Oncotype DX test
were developed from retrospective analyses of tumor specimens from
two completed breast cancer trials before the cut points were applied
prospectively in the TAILORx trial.113,114 If the cut point of a continuous
biomarker is not precisely determined before a planned RCT, sometimes
there is a lower value of it for which it is believed the experimental
treatment will not work at all, and there is also a higher value that is
thought to identify patients who will be most helped by the treatment.
In these situations, one can randomize only patients who have a value
greater than this lower value, but specify the higher value as the
cut point for a biomarker-stratified design. For example, in the
KEYNOTE-010 trial,115 patients with advanced non–small cell lung
cancer patients whose PD-L1 expression was at least 1% were random-
ized to pembrolizumab (an antibody against PD-1) versus docetaxel,
with a biomarker-stratified design stratified on PD-L1 expression
(1%–49% versus ≥50%). In situations in which little pretrial evidence
for a cutoff is available, there are designs116 that allow a rigorous
306 Part I: Science and Clinical Oncology
assessment of different possible cutoffs in the phase III trial (controlling
for the multiple comparisons), but at the cost of lower statistical power
as compared with a design with a predetermined cutoff.
DESIGNS WITH MULTIPLE BIOMARKERS
The promise of precision medicine is the use of multiple biomarkers
to select a treatment that is most likely to work in a given patient.
Development and validation of these biomarkers and treatment strategies
involves clinical trial designs that may or may not use the biomarkers
to direct treatment.
Biomarker-Directed Treatment Designs
Trials using biomarker-directed treatments can have different designs
depending on their objectives.117 In an umbrella trial, patients with
a specific tumor histologic type are directed into different subtri-
als based on their biomarkers. For example, the Adjuvant Lung
Cancer Enrichment Marker Identification and Sequencing Trial
(ALCHEMIST)118 enrolls patients with early-stage nonsquamous
non–small cell lung cancer into one of three phase III RCTs: a trial
of erlotinib versus no treatment for patients with EGFR-mutant
tumors, a trial of crizotinib versus no treatment for patients with
ALK-rearranged tumors, and a trial of nivolumab versus no therapy
for patients without EGFR mutations or ALK rearrangements. The
master protocol FOCUS4 trial119 for patients with advanced or
metastatic colorectal cancer with stable or responding disease after
first-line chemotherapy enrolls patients into separate phase II/III
trials based on their biomarkers, with patients without a biomarker
match currently being enrolled in a phase II/III trial of capecitabine
versus active monitoring. The master protocol LUNG-MAP120 enrolls
patients with previously treated squamous cell lung cancer into
biomarker-directed single-arm phase II trials, with patients not being
positive for any of the specified biomarkers currently being enrolled
in a randomized phase II trial of nivolumab versus nivolumab plus
ipilimumab.
A basket trial is similar to an umbrella trial except the patients
enrolling are not restricted to a specific tumor histologic type. An
example of a basket trial is the master protocol NCI-MATCH,121 in
which patients whose disease has progressed on standard therapies are
assigned to a phase II single-arm trial of a targeted agent when they
have the relevant biomarker target; the trial is expected assess 20 to
30 targeted agents. The raison d’être of a basket trial is the assumption
that response to a targeted treatment will depend on the biomarkers
of the patients and not on (or not much on) the histologic type of
the tumor. If one pools across histologic types, it is feasible to conduct
a subtrial of a targeted agent in which the target may be quite rare.
There is the possibility that the assumption is wrong—for example,
BRAF inhibitors work in BRAF-positive melanoma but not BRAF-
positive colon cancer122—so a negative subtrial does not conclusively
prove that the targeted agent would not be effective in some specific
histologic type with the target. It is also possible to design a basket
trial that assesses the outcome within each histologic type without
pooling. For example, a basket trial was performed to examine the
response rate of the BRAF inhibitor vemurafenib for BRAF-positive
patients in seven histologically specific cohorts.123
Biomarker-strategy designs wherein patients are randomized to an
arm in which the treatment is suggested by the biomarker versus a
standard treatment arm were discussed in the context of a single
biomarker earlier. It was noted that use of biomarker-stratified designs
is preferable because biomarker-strategy designs are inefficient—they
can result in treating a high proportion of patients in both arms with
the same treatment (the standard treatment). In the setting of many
biomarkers, each with a small probability of being positive, a biomarker-
stratified design may not be feasible. In addition, use of many biomark-
ers as targets for experimental treatments increases the probability
that a patient will be positive for some biomarker, reducing the
proportion of patients receiving the standard treatment in the
biomarker-driven treatment arm. A modified biomarker-strategy design
enrolls only patients who are positive for one of the biomarkers, who
are than randomized between a standard treatment arm and the arm
in which the biomarkers are used to determine which experimental
therapy will be recommended; this completely eliminates the problem
of patients receiving a standard therapy in the biomarker-strategy
treatment arm. An example is the histology-agnostic SHIVA trial,124
in which patients with alterations within one of three molecular
pathways were randomized to a matched targeted agent or physician’s
choice of standard cytotoxic therapy. A shortcoming of a biomarker-
strategy design with multiple biomarkers is that it can be hard to
interpret the results of the trial; a negative result may occur because
enough of the biomarker-directed treatments are not working even
though others work quite well, and a positive trial result may occur
because several of the biomarker-directed treatments are working very
well, thus obscuring absence of benefit for the rest of the treatments.
The SHIVA trial results did not show a benefit for the biomarker-driven
strategy, with a possible reason being that the agents did not work
for the subset of patients with hormonal molecular alterations.124
A critical source of the design efficiency of a biomarker-directed
treatment design is the screening component, which allows screening
of patients for multiple biomarkers to direct them into the correspond-
ing biomarker-defined subtrials. Given that many biomarkers have
low prevalence of positivity, it may not be feasible to perform separate
trials for each biomarker-associated treatment.
Nonbiomarker Directed Designs
In nonbiomarker directed designs the biomarkers of the patients are
recorded but the patients are (initially) randomized to the treatment
arms regardless of the status of their biomarkers, with the efficacy
assessments being made within biomarker subsets. The biomarkers
can also be used in the interim monitoring—for example, to stop the
randomization to a certain treatment for a certain biomarker subgroup.
An example is the randomized phase II BATTLE trial,125 in which
chemotherapy-refractory non–small cell lung cancer patients were
randomly assigned to four therapies and were simultaneously categorized
into five biomarker subgroups; the outcome was 8-week disease control
rates compared with historical controls. Another example is the random-
ized phase II BATTLE-2 trial,126 in which advanced non–small cell
lung cancer patients were randomized to a control arm or three
experimental arms; the outcome was 8-week disease control rates
compared with the control arm. Trial designs such as these in which
biomarkers are used for monitoring and assessment are more exploratory
than trials that use biomarkers to direct treatment, and are appropriate
when one has little idea about the relationships between the biomarkers
and the therapies.
CONCLUSIONS
Properly designed clinical trials ensure that patients are treated safely
and that the scientific objectives of the trial can be achieved in an
efficient manner, so that current and future patients can benefit from
the trial results. Judicious use of important trial design elements, such
as interim monitoring and the choice of appropriate end points, can
ensure that trials definitively answer their scientific objectives as quickly
as possible, be that changing clinical practice or screening a new
treatment for further development. The increasing use of biomarkers
in cancer treatment algorithms has great potential for improving patient
care, but does offer some challenges to clinical trial designs. Clinical
trial designs have been developed to assess the clinical benefits of
experimental biomarker-directed agents and to assess the usefulness
of multiple biomarkers for guiding patient treatment.
The complete reference list is available online at
ExpertConsult.com.

KEY REFERENCES
1. Green S, Smith A, Benedetti J, Crowley J. Clinical
Trials in Oncology. 3rd ed. New York: CRC Press;
2012.
3. Tourneau CL, Lee JJ, Siu LL. Dose escalation
methods in phase I cancer clinical trials. J Natl
Cancer Inst. 2009;101:708–720.
8. O’Quigley J, Pepe M, Fisher L. Continual reassess-
ment method: a practical design for phase I clinical
trials in cancer. Biometrics. 1990;46:33–48.
11. Korn EL. Nontoxicity endpoints in phase I trial
designs for targeted, non-cytotoxic agents. J Natl
Cancer Inst. 2004;96:977–978.
14. Parulekar WR, Eisenhauer EA. Phase 1 trial design for
solid tumor studies of targeted, non-cytotoxic agents:
theory and practice. J Natl Cancer Inst. 2004;96:
990–997.
16. Murgo AJ, Kummar S, Rubinstein L, et al.
Designing phase 0 clinical trials. Clin Cancer Res.
2008;14:3675–3682.
17. Eisenhauer EA, Therasse P, Bogaerts J, et al. New
response criteria in solid tumors: revised RECIST
guideline (version 1.1). Eur J Cancer. 2009;45:
228–247.
18. Simon R. Optimal two-stage designs for phase II
clinical trials. Control Clin Trials. 1989;10:1–10.
20. Korn EL, Arbuck SG, Pluda JM, et al. Clinical trial
designs for cytostatic agents: are new approaches
needed? J Clin Oncol. 2001;19:265–272.
21. Korn EL, Liu PY, Lee SJ, et al. Meta-analysis of
phase II cooperative group trials in metastatic stage
IV melanoma to determine progression-free and
overall survival benchmarks for future phase II trials.
J Clin Oncol. 2008;26:527–534.
22. Rubinstein LV, Korn EL, Freidlin B, et al. Design
issues of randomized phase II trials and a proposal
for phase II screening trials. J Clin Oncol. 2005;23:
7199–7206.
23. Rubinstein LV, Gail MH, Santner TJ. Planning the
duration of a comparative clinical trial with loss to
follow-up and a period of continued observation.
J Chronic Dis. 1981;34:469–479.
29. Freidlin B, Korn EL. Borrowing information across
subgroups in phase II trials: is it useful? Clin Cancer
Res. 2012;19:1–9.
31. Freidlin B, McShane LM, Polley MC, Korn EL.
Randomized phase II trial designs with biomarkers.
J Clin Oncol. 2012;30:3304–3309.
33. Freidlin B, Korn EL, Gray R, Martin A. Multi-arm
clinical trials of new agents: some design consider-
ations. Clin Cancer Res. 2008;14:4368–4371.
35. Green S, Liu PY, O’Sullivan J. Factorial design
considerations. J Clin Oncol. 2002;20:3424–3430.
38. U. S. Food and Drug Administration. Guidance for
Industry—Non-Inferiority Clinical Trials to Establish
Effectiveness. Silver Spring, MD. 2016.
40. Freidlin B, Korn EL, George SL, Gray R. Random-
ized clinical trial design for assessing noninferiority
when superiority is expected. J Clin Oncol. 2007;25:
5019–5023.
Clinical Trial Designs in Oncology  •  CHAPTER 18 307
44. Korn EL, McShane LM, Freidlin B. Statistical
challenges in the evaluation of treatments for small
patient populations. Sci Transl Med. 2013;5:178r3.
48. O’Brien PC, Fleming TR. A multiple testing
procedure for clinical trials. Biometrics. 1979;35:549–
556.
49. Lan KKG, DeMets DL. Discrete sequential bound-
aries for clinical trials. Biometrika. 1983;70:659–
663.
51. Wieand S, Schroeder G, O’Fallon JR. Stopping when
the experimental regimen does not appear to help.
Stat Med. 1994;13:1453–1458.
52. Freidlin B, Korn EL, Gray R. A general inefficacy
interim monitoring rule for randomized clinical
trials. Clin Trials. 2010;7:197–208.
54a. Korn EL, Freidlin B. Interim monitoring for
non-inferiority trials: minimizing patient exposure
to inferior therapies. Ann Oncol. 2018;29:573–577.
58. Korn EL, Freidlin B, Mooney M. Stopping or
reporting early for positive results in randomized
clinical trials: the national cancer institute coopera-
tive group experience from 1990 to 2005. J Clin
Oncol. 2009;27:1712–1721.
59. Smith MA, Ungerleider RS, Korn EL, Rubinstein
L, Simon R. Role of independent data-monitoring
committees in randomized clinical trials sponsored
by the national cancer institute. J Clin Oncol.
1997;15:2736–2743.
60. Ellenberg SS, Fleming TR, DeMets DL. Data
Monitoring Committees in Clinical Trials. Chichester,
England.: Wiley; 2002.
63. Korn EL, Freidlin B. Outcome-adaptive random-
ization: is it useful? J Clin Oncol. 2011;29:771–
776.
65. Korn EL, Freidlin B. Adaptive clinical trials:
advantages and disadvantages of various adaptive
design elements. J Natl Cancer Inst. 2017;109(6).
66. Thall P, Fox P, Wathen J. Statistical controversies
in clinical research: scientific and ethical problems
with adaptive randomization in comparative clinical
trials. Ann Oncol. 2015;26:1621–1628.
67. Hey SP, Kimmelman J. Are outcome-adaptive alloca-
tion trials ethical? Clin Trials. 2015;12:102–106.
72. Buyse M. Commentary on Hey and Kimmelman.
Clin Trials. 2015;12:119–121.
78. Korn EL, Freidlin B, Abrams JS, Halabi S. Design
issues in randomized phase II/III trials. J Clin Oncol.
2012;30:667–671.
80. U. S. Food and Drug Administration. Draft Guid-
ance for Industry—Adaptive Design Clinical Trials
for Drugs and Biologics. Rockville, MD. 2010.
85. Korn EL, Freidlin B, Abrams J. Overall survival
as the outcome for randomized clinical trials
with effective subsequent therapies. J Clin Oncol.
2011;29:2439–2442.
87. U. S. Food and Drug Administration. Guidance for
Industry. Clinical Trial Endpoints for the Approval
of Cancer Drugs and Biologics. Rockville, MD.
2007.
88. Dodd LE, Korn EL, Freidlin B, et al. Blinded
independent central review of progression-free
survival in phase III clinical trials: important design
element or unnecessary expense? J Clin Oncol.
2008;26:379–3796.
92. Freidlin B, Little RF, Korn EL. Design issues in
randomized clinical trials of maintenance therapies.
J Natl Cancer Inst. 2015;107(11):djv225.
94. U. S. Food and Drug Administration. Guidance
for Industry. Patient-Reported Outome Measures:
Use in Medical Product Development to Support
Labeling Claims. Rockville, MD. 2009.
97. Korn EL, Albert PS, McShane LM. Assessing sur-
rogates as trial endpoints using mixed models. Stat
Med. 2005;24:163–182.
98. Sargent DJ, Wieand HS, Haller DG, et al. Disease-
free survival versus overall survival as a primary end
point for adjuvant colon cancer studies: individual
patients data from 20,898 patients on 18 randomized
trials. J Clin Oncol. 2005;23:8664–8670.
99. Korn EL, Sachs MC, McShane LM. Statistical
controversies in clinical research: assessing patho-
logic complete response as a trial-level surrogate
end point for early-stage breast cancer. Ann Oncol.
2016;27:10–15.
103. Polley MY, Freidlin B, Korn EL, et al. Statistical
and practical considerations for clinical evalua-
tion of predictive biomarkers. J Natl Cancer Inst.
2013;105:1677–1683.
104. Simon RM, Paik S, Hayes DF. Use of archived
specimens in evaluation of prognostic and predictive
biomarkers. J Natl Cancer Inst. 2009;21:1446–1452.
105. Freidlin B, Korn EL. Biomarker enrichment strate-
gies: matching trial design to biomarker credentials.
Nat Rev Clin Oncol. 2014;11:81–90.
106. Clark GM, McShane LM. Biostatistical consider-
ations in development of biomarkers-based tests to
guide treatment decisions. Stat Biopharm Res. 2011;3:
549–560.
109. Freidlin B, McShane LM, Korn EL. Randomized
clinical trials with biomarkers: design issues. J Natl
Cancer Inst. 2010;102:152–160.
111. Freidlin B, Sun Z, Gray R, Korn EL. Phase III
clinical trials that integrate treatment and biomarker
evaluation. J Clin Oncol. 2013;31:3158–3161.
116. Jiang W, Freidlin B, Simon R. Biomarker-adaptive
threshold design: a procedure for evaluating treat-
ment with possible biomarker-defined subset effect.
J Natl Cancer Inst. 2007;99:1036–1043.
117. Simon R. Genomic alteration-driven clinical trial
designs in oncology. Ann Intern Med. 2016;165:
270–278.
121. Conley BA, Doroshow JH. Molecular analysis for
therapy choice: NCI MATCH. Semin Oncol. 2014;41:
297–299.

REFERENCES
1. Green S, Smith A, Benedetti J, Crowley J. Clinical
Trials in Oncology. 3rd ed. New York: CRC Press;
2012.
2. George SL, Wang X, Pang H. Cancer Clinical Trials.
Boca Raton; 2016.
3. Tourneau CL, Lee JJ, Siu LL. Dose escalation
methods in phase I cancer clinical trials. J Natl
Cancer Inst. 2009;101:708–720.
4. Storer BE. Design and analysis of phase I clinical
trials. Biometrics. 1989;45:925–937.
5. Skolnik JM, Barrett JS, Jayaraman B, Patel D,
Adamson PC. Shortening the timeline of pediatric
phase I trials: the rolling six design. J Clin Oncol.
2008;26:190–195.
6. Korn EL, Midthune D, Chen TT. A comparison
of two phase I trial designs. Stat Med. 1994;13:
1799–1806.
7. Simon R, Freidlin B, Rubinstein L, et al. Accelerated
titration designs for phase I clinical trials in oncology.
J Natl Cancer Inst. 1997;89:1138–1147.
7a. Yuan Y, Hess KR, Hilsenbeck SG, Gilbert MR.
Bayesian optimal interval design: a simple and
well-performing design for phase I oncology trials.
Clin Cancer Res. 2016;22:4291–4301.
8. O’Quigley J, Pepe M, Fisher L. Continual reassess-
ment method: a practical design for phase I clinical
trials in cancer. Biometrics. 1990;46:33–48.
9. Korn EL, Simon R. Using the tolerable-dose diagram
in the design of phase I combination chemotherapy
trials. J Clin Oncol. 1993;11:794–801.
10. Cheung YK, Chappell R. Sequential designs for phase
I clinical trials with late-onset toxicities. Biometrics.
2000;56:1177–1182.
11. Korn EL. Nontoxicity endpoints in phase I trial
designs for targeted, non-cytotoxic agents. J Natl
Cancer Inst. 2004;96:977–978.
12. Hunsberger S, Rubinstein LV, Dancey J, Korn EL.
Dose escalation trial designs based on a molecularly
targeted endpoint. Stat Med. 2005;24:2171–
2181.
13. Ivy SP, Siu LL, Garrett-Mayer E, Rubinstein L.
Approaches to phase 1 clinical trial design focused
on safety, efficiency, and selected patient populations:
a report from the clinical design task force of the
National Cancer Institute Investigational Drug
Steering Committee. Clin Cancer Res. 2010;16:
1726–1736.
14. Parulekar WR, Eisenhauer EA. Phase 1 trial design
for solid tumor studies of targeted, non-cytotoxic
agents: theory and practice. J Natl Cancer Inst.
2004;96:990–997.
15. Booth CM, Calbert AH, Giaccone G, et al. End-
points and other considerations in phase I studies of
targeted anticancer therapy: recommendations from
the task force on Methodology for the Development
of Innovative Cancer Therapies (MDICT). Eur J
Cancer. 2008;44:19–24.
16. Murgo AJ, Kummar S, Rubinstein L, et al.
Designing phase 0 clinical trials. Clin Cancer Res.
2008;14:3675–3682.
17. Eisenhauer EA, Therasse P, Bogaerts J, et al. New
response criteria in solid tumors: revised RECIST
guideline (version 1.1). Eur J Cancer. 2009;45:
228–247.
18. Simon R. Optimal two-stage designs for phase II
clinical trials. Control Clin Trials. 1989;10:1–10.
19. Jung SH, Lee T, Kim KM, George SL. Admissible
two-stage designs for phase II cancer clinical trials.
Stat Med. 2004;23:561–569.
20. Korn EL, Arbuck SG, Pluda JM, et al. Clinical trial
designs for cytostatic agents: are new approaches
needed? J Clin Oncol. 2001;19:265–272.
21. Korn EL, Liu PY, Lee SJ, et al. Meta-analysis of
phase II cooperative group trials in metastatic stage
IV melanoma to determine progression-free and
overall survival benchmarks for future phase II trials.
J Clin Oncol. 2008;26:527–534.
Clinical Trial Designs in Oncology  •  CHAPTER 18 307.e1
22. Rubinstein LV, Korn EL, Freidlin B, et al. Design
issues of randomized phase II trials and a pro-
posal for phase II screening trials. J Clin Oncol.
2005;23:7199–7206.
23. Rubinstein LV, Gail MH, Santner TJ. Planning the
duration of a comparative clinical trial with loss to
follow-up and a period of continued observation.
J Chronic Dis. 1981;34:469–479.
24. Simon R, Thall PF, Ellenberg SS. New designs for
the selection of treatments to be tests in randomized
clinical trials. Stat Med. 1994;13:417–429.
25. McShane LM, Hunsberger S, Adjei AA. Effective
incorporation of biomarkers into phase II trials. Clin
Cancer Res. 2009;15:1898–1905.
26. Seymour L, Ivy SP, Sargent D, et al. The design of
phase II clinical trials testing cancer therapeutics:
consensus recommendations from the clinical trial
design task force of the National Cancer Institute
Investigational Drug Steering Committee. Clin
Cancer Res. 2010;16:1764–1769.
27. Pusztai L, Anderson K, Hess KR. Pharmacogenomic
predictor discovery in phase II clinical trials for breast
cancer. Clin Cancer Res. 2007;13:6080–6086.
28. Jones CL, Holmgren E. An adaptive Simon two-stage
design for phase 2 studies of targeted therapies.
Contemp Clin Trials. 2007;28:654–661.
29. Freidlin B, Korn EL. Borrowing information across
subgroups in phase II trials: is it useful? Clin Cancer
Res. 2012;19:1–9.
30. LeBlanc M, Rankin C, Crowley J. Multiple histology
phase II trials. Clin Cancer Res. 2009;15:4256–4262.
31. Freidlin B, McShane LM, Polley MC, Korn EL.
Randomized phase II trial designs with biomarkers.
J Clin Oncol. 2012;30:3304–3309.
32. Freidlin B, Othis M, Korn EL. Information time
scales for interim analysis of randomized clinical
trials. Clin Trials. 2016;13:391–399.
33. Freidlin B, Korn EL, Gray R, Martin A. Multi-arm
clinical trials of new agents: some design consider-
ations. Clin Cancer Res. 2008;14:4368–4371.
34. Redman MW, Allegra CJ. The master protocol
concept. Semin Oncol. 2015;42:724–730.
35. Green S, Liu PY, O’Sullivan J. Factorial design
considerations. J Clin Oncol. 2002;20:3424–3430.
36. Korn EL, Freidlin B. Non-factorial analyses of two-
by-two factorial trial designs. Clin Trials. 2016;13:
651–659.
37. James ND, Sydes MR, Clarke NW, et al. Addition
of docetaxel, zoledronic acid, or both to first-line
long-term hormone therapy in prostate cancer
(STAMPEDE): survival results from an adap-
tive, multiarm, multistage, platform randomised
controlled trial. Lancet. 2016;387:1163–1177.
38. U. S. Food and Drug Administration. Guidance for
Industry—Non-Inferiority Clinical Trials to Establish
Effectiveness. Silver Spring, MD. 2016.
39. D’Agostino RB, Massaro JM, Sullivan LM. Non-
inferiority trials: design concepts and issues—the
encounters of academic consultants in statistics. Stat
Med. 2003;22:169–186.
40. Freidlin B, Korn EL, George SL, Gray R. Random-
ized clinical trial design for assessing noninferior-
ity when superiority is expected. J Clin Oncol.
2007;25:5019–5023.
41. Johnson JR, Bross P, Cohen M, et al. Approval
summary: imatinib mesylate capsules for treatment
of adult patients with newly diagnosed Philadelphia
chromosome-positive chronic myelogenous leukemia
in chronic phase. Clin Caner Res. 2003;9:1972–1979.
42. Schultz KR, Bowman WP, Aledo A, et al. Improved
early event-free survival with imatinib in Philadelphia
chromosome-positive acute lymphoblastic leukemia:
a Children’s Oncology Group study. J Clin Oncol.
2009;27:5175–5181.
43. Hortobagyi GN. What is the role of high-dose
chemotherapy in the era of targeted therapies?
J Clin Oncol. 2004;22:2263–2266.
307.e1
44. Korn EL, McShane LM, Freidlin B. Statistical
challenges in the evaluation of treatments for small
patient populations. Sci Transl Med. 2013;5:178r3.
45. Jennison C, Turnbull BW. Group Sequential Methods
With Applications to Clinical Trials. Boca Raton:
Chapman & Hall/CRC; 2000.
46. Whitehead J. The Design and Analysis of Sequential
Clinical Trials. Revised. 2nd ed. Chichester: Wiley;
1997.
47. Haybittle JL. Repeated assessment of results in
clinical trials of cancer treatment. Br J Radiol.
1971;44:793–797.
48. O’Brien PC, Fleming TR. A multiple testing proce-
dure for clinical trials. Biometrics. 1979;35:549–556.
49. Lan KKG, DeMets DL. Discrete sequential boundar-
ies for clinical trials. Biometrika. 1983;70:659–663.
50. Freidlin B, Korn EL. Monitoring for lack of benefit:
a critical component of a randomized clinical trial.
J Clin Oncol. 2009;27:629–633.
51. Wieand S, Schroeder G, O’Fallon JR. Stopping when
the experimental regimen does not appear to help.
Stat Med. 1994;13:1453–1458.
52. Freidlin B, Korn EL, Gray R. A general inefficacy
interim monitoring rule for randomized clinical
trials. Clin Trials. 2010;7:197–208.
53. Zhang Q, Freidlin B, Korn EL, et al. Comparison of
futility monitoring guidelines using completed phase
III oncology trials. Clin Trials. 2017;14(1):48–58.
54. Korn EL, Freidlin B. Inefficacy interim monitoring
procedures in randomized clinical trials: the need
to report. Am J Bioeth. 2011;11:2–10.
54a. Korn EL, Freidlin B. Interim monitoring for
non-inferiority trials: minimizing patient exposure
to inferior therapies. Ann Oncol. 2018;29:573–577.
55. Freidlin B, Korn EL, George SL. Data monitoring
committees and interim monitoring guidelines.
Control Clin Trials. 1999;20:395–407.
55a.  Korn EL, Freidlin B. Interim futility monitoring
assessing immune therapies with a potentially delayed
treatment effect. J Clin Oncol. 2018; (published
online before print).
56. Bassler D, Briel M, Montori VM, et al. Stopping
randomized trials early for benefit and estimation
of treatment effects. JAMA. 2010;303:1180–1187.
57. Freidlin B, Korn EL. Stopping clinical trials early
for benefit: impact on estimation. Clin Trials.
2009;6:119–125.
58. Korn EL, Freidlin B, Mooney M. Stopping or
reporting early for positive results in randomized
clinical trials: the national cancer institute coopera-
tive group experience from 1990 to 2005. J Clin
Oncol. 2009;27:1712–1721.
59. Smith MA, Ungerleider RS, Korn EL, Rubinstein
L, Simon R. Role of independent data-monitoring
committees in randomized clinical trials sponsored by
the national cancer institute. J Clin Oncol. 1997;15:
2736–2743.
60. Ellenberg SS, Fleming TR, DeMets DL. Data
Monitoring Committees in Clinical Trials. Chichester,
England.: Wiley; 2002.
61. Korn EL, Hunsberger S, Freidlin B, Smith MA,
Abrams JS. Preliminary data release for randomized
clinical trials of noninferiority: a new proposal.
J Clin Oncol. 2005;23:5831–5836.
62. Byar DP, Simon RM, Friedewald WT, et al. Random-
ized clinical trials—perspectives on some recent ideas.
N Engl J Med. 1976;295:74–80.
63. Korn EL, Freidlin B. Outcome-adaptive random-
ization: is it useful? J Clin Oncol. 2011;29:771–
776.
64. Simon R, Simon NR. Using randomization tests
to preserve type I error with response-adaptive and
covariate-adaptive randomization. Stat Probab Lett.
2011;81:767–772.
65. Korn EL, Freidlin B. Adaptive clinical trials:
advantages and disadvantages of various adaptive
design elements. J Natl Cancer Inst. 2017;109(6).
307.e2 Part I: Science and Clinical Oncology
66. Thall P, Fox P, Wathen J. Statistical controversies
in clinical research: scientific and ethical problems
with adaptive randomization in comparative clinical
trials. Ann Oncol. 2015;26:1621–1628.
67. Hey SP, Kimmelman J. Are outcome-adaptive alloca-
tion trials ethical? Clin Trials. 2015;12:102–106.
68. Berry DA. Commentary on Hey and Kimmelman.
Clin Trials. 2015;12:107–109.
69. Lee JJ. Commentary on Hey and Kimmelman. Clin
Trials. 2015;12:110–112.
70. Saxman SB. Commentary on Hey and Kimmelman.
Clin Trials. 2015;12:113–115.
71. Joffe S, Ellenberg SS. Commentary on Hey and
Kimmelman. Clin Trials. 2015;12:116–118.
72. Buyse M. Commentary on Hey and Kimmelman.
Clin Trials. 2015;12:119–121.
73. Korn EL, Freidlin B. Commentary on Hey and
Kimmelman. Clin Trials. 2015;12:122–124.
74. Hey SP, Kimmelman J. Rejoinder. Clin Trials.
2015;12:125–127.
75. Pocock SJ. Interim analyses for randomized clinical
trials: the group sequential approach. Biometrics.
1982;38:153–162.
76. Ivanova A, Qaqish BF, Schell MJ. Continuous
toxicity monitoring in phase II trials in oncol-
ogy. Biometrics. 2005;61:540–545.
77. Bretz F, Schmidli H, Konig F, Racine A, Maurer W.
Confirmatory seamless phase II/III clinical trials with
hypotheses selection at interim: general concepts.
Biom J. 2006;48:623–634.
78. Korn EL, Freidlin B, Abrams JS, Halabi S. Design
issues in randomized phase II/III trials. J Clin Oncol.
2012;30:667–671.
78a.  Freidlin B, Korn EL, Abrams JS. Bias, operational
bias, and generalizability in phase II/III trials. J Clin
Oncol. 2018;36:1902–1904.
79. Parmar MKB, Barthel FMS, Sydes M, et al. Speeding
the evaluation of new agents in cancer. J Natl Cancer
Inst. 2008;100:1204–1214.
80. U. S. Food and Drug Administration. Draft guidance
for industry—adaptive design clinical trials for drugs
and biologics. Rockville, MD. 2010.
81. Emerson SS, Fleming TR. Adaptive methods:
telling “the rest of the story. J Biopharm Stat.
2010;20:1150–1165.
82. Cuffe RL, Lawrence D, Stone A, Vandemeulebroecke
M. When is a seamless study desirable? Case studies
from different pharmaceutical sponsors. Pharm Stat.
2014;13:229–237.
83. Temple R. Are surrogate markers adequate to assess
cardiovascular disease drugs? JAMA. 1999;282:
290–795.
84. Freidlin B, Abrams J, Korn EL. New challenges
for comparative effectiveness in oncology: choice of
primary end points for randomized clinical trials.
J Comp Eff Res. 2013;2:469–481.
85. Korn EL, Freidlin B, Abrams J. Overall survival
as the outcome for randomized clinical trials
with effective subsequent therapies. J Clin Oncol.
2011;29:2439–2442.
86. Fleming TR, Rothmann MD, Lu HL. Issues in using
progression-free survival when evaluating oncology
products. J Clin Oncol. 2009;27:2874–2880.
87. U. S. Food and Drug Administration. Guidance for
Industry. Clinical Trial Endpoints for the Approval
of Cancer Drugs and Biologics. Rockville, MD.
2007.
88. Dodd LE, Korn EL, Freidlin B, et al. Blinded inde-
pendent central review of progression-free survival
in phase III clinical trials: important design element
or unnecessary expense? J Clin Oncol. 2008;26:
379–3796.
89. Amit O, Mannino F, Stone AM, et al. Blinded
independent central review of progression in cancer
clinical trials: results from a meta-analysis. Eur J
Cancer. 2011;47:1772–1778.
90. Dodd LE, Korn EL, Freidlin B, et al. An audit
strategy for progression-free survival. Biometrics.
2011;67:1092–1099.
91. Freidlin B, Korn EL, Hunsberger S, et al. Proposal
for the use of progression-free survival in unblended
trials. J Clin Oncol. 2007;25:2122–2126.
92. Freidlin B, Little RF, Korn EL. Design issues in
randomized clinical trials of maintenance therapies.
J Natl Cancer Inst. 2015;107(11):djv225.
93. Basch E, Trentacosti AM, Burke LB, et al. Pain
palliation measurement in cancer clinical trials.
Cancer. 2014;120:761–767.
94. U. S. Food and Drug Administration. Guidance
for Industry. Patient-Reported Outome Measures:
Use in Medical Product Development to Support
Labeling Claims. Rockville, MD. 2009.
95. Basch E, Reeve BB, Mitchell SA, et al. Development
of the national cancer institute’s patient-reported
outcomes version of the common terminology
criteria for adverse events (PRO-CTCAE). J Natl
Cancer Inst. 2014;106:dju244.
96. Forastiere AA, Zhang Q, Weber RS, Maor MH,
Goepfert H, et al. Long-term results of RTOG
99-11: a comparison of three nonsurgical treat-
ment strategies to preserve the larynx in patients
with locally advanced larynx cancer. J Clin Oncol.
2013;31:845–852.
97. Korn EL, Albert PS, McShane LM. Assessing sur-
rogates as trial endpoints using mixed models. Stat
Med. 2005;24:163–182.
98. Sargent DJ, Wieand HS, Haller DG, et al. Disease-
free survival versus overall survival as a primary end
point for adjuvant colon cancer studies: individual
patients data from 20,898 patients on 18 randomized
trials. J Clin Oncol. 2005;23:8664–8670.
99. Korn EL, Sachs MC, McShane LM. Statistical
controversies in clinical research: assessing pathologic
complete response as a trial-level surrogate end point
for early-stage breast cancer. Ann Oncol. 2016;27:
10–15.
99a.  Korn EL, Freidlin B. Surrogate and intermediate
endpoints in randomized trials: what’s the goal? Clin
Cancer Res. 2018;24:2239–2240.
100. Amado RG, Wolf M, Peters M, et al. Wild-type
KRAS is required for panitumumab efficacy in
patients with metastatic colorectal cancer. J Clin
Oncol. 2008;26:1626–1634.
101. Mok TS, Wu YL, Thongprasert S, et al. Gefitinib or
carboplatin-paclitaxel in pulmonary adenocarcinoma.
N Engl J Med. 2009;361:947–957.
102. Peto R. Statistical aspects of clinical trials. In: Halnan
KE, ed. Treatment of Cancer. London: Chapman
and Hall; 1982.
103. Polley MY, Freidlin B, Korn EL, et al. Statistical
and practical considerations for clinical evalua-
tion of predictive biomarkers. J Natl Cancer Inst.
2013;105:1677–1683.
104. Simon RM, Paik S, Hayes DF. Use of archived
specimens in evaluation of prognostic and predictive
biomarkers. J Natl Cancer Inst. 2009;21:1446–
1452.
105. Freidlin B, Korn EL. Biomarker enrichment strate-
gies: matching trial design to biomarker credentials.
Nat Rev Clin Oncol. 2014;11:81–90.
106. Clark GM, McShane LM. Biostatistical consider-
ations in development of biomarkers-based tests
to guide treatment decisions. Stat Biopharm Res.
2011;3:549–560.
107. Chapman PB, Hauschild A, Robert C, et al. Improved
survival with vemurafenib in melanoma with
BRAF V600E mutation. N Engl J Med. 2011;364:
2507–2516.
108. Simon R, Maitournam A. Evaluating the efficiency
of targeted designs for randomized clinical trials.
Clin Cancer Res. 2004;10:6759–6763.
109. Freidlin B, McShane LM, Korn EL. Randomized
clinical trials with biomarkers: design issues. J Natl
Cancer Inst. 2010;102:152–160.
110. Rothmann MD, Zhang JJ, Fleming TR. Testing
in a prespecified subgroup and the intent-to-treat
population. Drug Inf J. 2012;46:175–179.
111. Freidlin B, Sun Z, Gray R, Korn EL. Phase III
clinical trials that integrate treatment and biomarker
evaluation. J Clin Oncol. 2013;31:3158–3161.
112. Freidlin B, Korn EL, Gray R. Marker sequential
test (MaST) design. Clin Trials. 2014;11:19–27.
113. Sparano JA, Paik S. Development of the 21-gene
assay and its application in clinical practice and
clinical trials. J Clin Oncol. 2008;26:721–728.
114. Sparano JA, Gray RJ, Makower DF, et al. Prospective
validation of a 21-gene expression assay in breast
cancer. N Engl J Med. 2015;373:2005–2014.
115. Herbst RS, Baas P, Kim DW, et al. Pembrolizumab
versus docetaxel for previously treated, PD-l1-positive,
advanced non-small-cell lung cancer (KEYNOTE-
010): a randomized controlled trial. Lancet. 2016;
387:1540–1550.
116. Jiang W, Freidlin B, Simon R. Biomarker-adaptive
threshold design: a procedure for evaluating treat-
ment with possible biomarker-defined subset effect.
J Natl Cancer Inst. 2007;99:1036–1043.
117. Simon R. Genomic alteration-driven clinical trial
designs in oncology. Ann Intern Med. 2016;165:
270–278.
118. Govindan R, Mandrekar SJ, Gerber DE, et al.
ALCHEMIST trials: a golden opportunity to
transform outcomes in early-stage non-small cell
lung cancer. Clin Cancer Res. 2015;21:5439–5444.
119. Kaplan R, Maughan T, Crook A, et al. Evaluating
many treatments and biomarkers in oncology: a
new design. J Clin Oncol. 2013;31:4562–4568.
120. Herbst RS, Gandara DR, Hirsch FR, et al. Lung
master protocol (Lung-MAP)—a biomarker-driven
protocol for accelerating development of therapies
for squamous cell lung cancer: SWOG s1400. Clin
Cancer Res. 2015;21:1514–1524.
121. Conley BA, Doroshow JH. Molecular analysis
for therapy choice: NCI MATCH. Semin Oncol.
2014;41:297–299.
122. Kopetz S, Desai J, Chan E, et al. PLX4032 in
metastatic colorectal cancer patients with mutant
BRAF tumors. J Clin Oncol. 2010;28:3534. abstract.
123. Hyman DM, Puzanov I, Subbiah V, et al. Vemu-
rafenib in multiple nonmelanoma cancers with BRAF
v600 mutations. N Engl J Med. 2015;373:726–
736.
124. Le Tourneau C, Delord JP, Goncalves A, et al.
Molecularly targeted therapy based on tumour
molecular profiling versus conventional therapy for
advanced cancer (SHIVA): a multicenter, open-label,
proof-of-concept, randomised, controlled phase 2
trial. Lancet Oncol. 2015;16:1324–1334.
125. Kim ES, Herbst RS, Wistuba II, et al. The BATTLE
trial: personalizing therapy for lung cancer. Cancer
Discov. 2011;1:44–53.
126. Papadimitrakopoulou V, Lee JJ, Wistuba II, et al. The
BATTLE-2 study: a biomarker-integrated targeted
therapy study in previously treated patients with
advanced non-small-cell lung cancer. J Clin Oncol.
2016;34:3638–3647.
 19  Structures Supporting Cancer Clinical Trials
Jeffrey S. Abrams, Margaret Mooney, James A. Zwiebel,
Worta McCaskill-Stevens, Michaele C. Christian, and James H. Doroshow

S UMMARY OF K EY
• Cancer clinical trials provide the
evidence on which sound oncology
practice is based.
• Providing greater efficiency in
implementing clinical trials and
achieving enrollment rapidly have
been major goals of the National
Cancer Institute (NCI), cancer centers,
the biopharmaceutical industry, and
patient advocacy groups.
• Oncology specialists in private
practice have many opportunities to
participate in clinical trials sponsored
by the NCI, private foundations, and/
or the biopharmaceutical industry.
• Physicians and patients have online
access to all US-sponsored clinical
trials at ClinicalTrials.gov and have
access to additional information
about cancer trials at cancer.gov.
P OI N T S
• Successful execution of clinical trials
includes the following clinical
components:
○ The attitude and commitment of
the physician leader (medical,
surgical, radiation oncologist)
○ Sufficient preparation and
infrastructure
○ Trained staff, including at least
some of the following: clinical
research nurse, clinical research
associate, pharmacist, pathologist,
radiologist
○ Affiliation with an institution or
network that provides scientific and
administrative support, such as the
following: NCI-supported network
group, cancer center network,
biopharmaceutical industry
network, research institution
○ Access to an authorized
institutional review board
○ Access to adequate laboratory
facilities to process
protocol-required specimens
○ Adherence to good clinical
practices
○ Accurate and timely data reporting
○ Proper maintenance of primary
source documentation
○ Adequate preparation for trial
monitoring and on-site audits
○ Adequate financial support
• Many organizations now provide
access to clinical trials and/or
provide the necessary training and
certification; relevant Web sites are
included in this chapter.

Modern oncology practice is founded on results from thousands of
clinical trials conducted during the past four decades. Thousands
more clinical trials are ongoing at any given time and provide the
evidence base for the rapidly changing therapeutic practices of this
specialty. Motivations for the decision by an oncologist to participate
actively in this extensive system of medical and scientific inquiry range
from the ability to offer patients state-of-the-art treatments, which are
available through well-designed clinical trials, to the personal satisfaction
and educational benefits that can be achieved from participation in
this process. The commitment of time and resources necessary to
participate effectively in clinical research, and sometimes unfamiliarity
with clinical research requirements and procedures, prevent many
oncologists from taking part. It has been estimated that only 3%
to 5% of adults with cancer in the United States are treated while
taking part in clinical trials, with even lower rates of participation
in many other countries. In stark contrast, approximately two-thirds
of children with cancer are enrolled in clinical trials. Although this
discrepancy exists for multiple reasons, a major factor relates to the
relative rarity of cancer in children (approximately 10,500 cases per year
in children younger than 15 years), resulting in substantial centralization
of pediatric care at major academic research centers, a culture in which
participation in trials is supported and fostered. Care for adults with
cancer in the United States is more decentralized, with many adult
oncologists working in private practices that are not tied to academic
institutions. For these practitioners, inadequate understanding of the
clinical trial process plus pressures on time and reimbursement have
made participation very challenging. Fortunately, because of intense
publicity and educational programs by both patient advocacy groups
and clinical trial organizations and widespread access to clinical trial
information on the Internet, a growing number of patients now expect
that clinical trials will be included in the discussion of options for the
treatment of their cancers. The purpose of this chapter is to describe
some of the requirements, resources, and structures that are available
to enable practicing oncologists to participate in clinical trials and to
discuss the responsibilities that come with such participation. Numerous
opportunities now exist for practicing physicians and their patients to
participate in cancer clinical trials, including treatment, prevention,
and cancer control trials, supported by the National Cancer Institute
(NCI), cancer treatment institutions, or the biopharmaceutical industry.
NATIONAL CANCER INSTITUTE–SPONSORED
CLINICAL TRIAL ACTIVITIES
The NCI supports the development of more than 100 agents for
cancer treatment and prevention (often in collaboration with phar-
maceutical and biotechnology companies) and has an extensive clinical
trial system that encompasses treatment, prevention, and cancer control
studies. More than 800 trials are active at any given time, and several
hundred new trials open each year. In the treatment area, the NCI
has programs for early therapeutic development (primarily phase
I and II trials), including many sites with grants to complete these
early trials.

308

Table 19.1  Barriers to Clinical Trial Participation
Physician Related Patient Related
Inadequate funding for data The patient’s doctor never
management personnel discussed or offered the option of
participating in a trial
Burdensome regulatory Unaware of trials as an option
requirements Concerns about insurance coverage
Institutional review board Fear of receiving placebo
Informed consent Many patients have comorbidities
Conflict of interest
Overly strict eligibility criteria
Inadequate reimbursement
Lack of time
Resistance by third-party payers
For several decades the NCI also supported a large program of
clinical trial cooperative groups to provide a standing mechanism for
performing late-phase, definitive, clinical treatment trials. Although
these cooperative group trials provided a significant proportion of the
evidence on which oncology practice is based, public advocacy for
more rapid progress, increasing fiscal pressures on medical practice,
and the accelerated pace of drug discovery, especially in the new era
of precision medicine, caused the NCI to review and restructure aspects
of its late-phase clinical trial program. Surveys of patients and physicians
found that the obstacles to accrual in oncology trials were multifactorial
(Table 19.1).1–5 With these barriers in mind, the NCI undertook a
series of comprehensive analyses of how it conducts and funds clinical
trials by involving a wide range of stakeholders that included leaders
of the Cooperative Group and Cancer Center Programs, patient
advocates, representatives from the US Food and Drug Administration
(FDA) and the pharmaceutical industry, and government staff, including
the analysis described in the 2010 Institute of Medicine (IOM) report.6
Based on these reviews and analyses, the NCI transformed its
long-standing Cooperative Group Clinical Trials program in 2014
into a more highly integrated network. By rebranding the infrastructure
as the National Clinical Trials Network (NCTN), reducing the number
of groups, and changing the review criteria for funding to emphasize
collaboration and coordination, the NCI has refocused its research
effort. Competition among the remaining groups has been reduced
and replaced with more efficient and rapid development of a menu
of Network trials in which all group members are encouraged to
participate in order to facilitate speedy enrollment.
The development of targeted therapies directed against specific
molecular alterations identified in various cancers and the emergence
of successful immunotherapeutic approaches have fundamentally
changed the approach to cancer treatment and have introduced new
challenges to conducting clinical trials. Identification of patient
populations that can potentially benefit from these new therapies
often requires clinical trials that screen large numbers of patients for
specific molecular alterations. With its state-of-the-art clinical trial
infrastructure, the NCTN is well positioned to implement and complete
trials far more rapidly than in the past. The NCTN has streamlined
administrative and regulatory processes related to the conduct of its
clinical trials, including patient enrollment, data management, ethics
review, and tumor banking processes, and has provided a common
menu of trials for all academic and community member sites in the
NCTN program.
Critical to the achievement of a tightly integrated trials network
are the Cancer Trials Support Unit (CTSU) and the Central Institutional
Review Board (CIRB). The CTSU provides an online menu of all
trials conducted by the various NCTN groups. The CIRB provides
a single, expert ethics review of all NCTN trials, avoiding repetitive
costly reviews at individual sites. Although originally conceived to
Structures Supporting Cancer Clinical Trials  •  CHAPTER 19 309
support only the late-phase trials,7 the CTSU and CIRB have been
expanded to now foster efficiency and networking for all of NCI’s
network early- and late-phase trials, as described subsequently.
Cancer Trials Support Unit
The NCI CTSU is designed to facilitate one-stop online access to a
broad menu of clinical trials and selected international collaborative
trials by a national network of investigators who are members of
NCI-supported clinical trial programs. Network investigators can access
the CTSU menu of treatment trials from a public website (www.ctsu.org).
The scientific leadership for each study remains within the organization
that developed the trial, but patient enrollment can come from any
network physician across the country, and all patient enrollment is
handled through CTSU’s central registration system, the Oncology
Patient Enrollment Network (OPEN). By providing more physicians
and their patients with the opportunity to choose from a broader
menu of trials, the CTSU promotes faster accrual to individual trials,
allows increased access and additional treatment options to more
patients nationwide, and renders trials involving uncommon cancers
more feasible. Although the clinical trials menu and centralized patient
enrollment are the most visible aspects of the CTSU, another major
function of the CTSU is its centralized regulatory database. For all
physicians in the network, the CTSU maintains important demographic
information about their sites or practices, including clinical trial program
affiliation and academic and practice affiliation(s), Office for Human
Research Protections assurance numbers for their sites, institutional
review board (IRB) approvals for specific protocols, and conflict of
interest forms for investigators. This process enables physicians, nurses,
and clinical research associates to register once annually instead of
having to register for each clinical trial program or trial in which they
participate. Centralizing regulatory data has reduced the workload
for investigators in the field, consolidated duplicative work, and allowed
clinical trial program staff to partially offload this activity to the CTSU
and focus instead on protocol development and analysis.
Central Institutional Review Board
In NCI-sponsored multicenter trials, the identical protocol is carried
out at many sites, often including as many as 100 different practices;
each site requires its own local institutional review board (LIRB) to
conduct an initial full-board review and subsequent annual reviews,
adverse event reviews, and amendment reviews. These multiple IRB
reviews create a largely redundant, time-consuming workload at these
sites, compounding the ever-mounting pressures on the nation’s IRB
system, which have been well documented.8 To provide an idea of
the scope of the duplicative effort, consider that NCI currently has
more than 19,000 registered investigators participating in all its clinical
trial programs at more than 2700 sites. Under the former NCI-
sponsored Cooperative Group Program, for example, there were about
160 ongoing phase III trials and 30 new trials entering the NCI
program annually, resulting in approximately 16,000 IRB reviews
(3000 initial reviews) conducted each year.8 In addition, investigators
often mention that the amount of time, paperwork, and funding
required for them to obtain IRB approval is a serious barrier to opening
trials. These factors provided the impetus for the NCI to develop the
CIRB and centralize the approach to human subject protection for
its clinical trial programs.
In late 2012, the NCI CIRB initiative began to transition from
its historic “facilitated review” model to an independent model in
which the NCI CIRB is the sole IRB of Record responsible for both
study review and local context considerations for enrolled institutions.
The primary reason for this model change was to further streamline
the IRB review process, increasing efficiency of implementation and
conduct of multisite clinical trials and promoting consistency of ethics
reviews. On December 13, 2012, the Association for the Accreditation
of Human Research Protection Programs (AAHRPP) awarded
310 Part I: Science and Clinical Oncology
accreditation to the NCI CIRB under its independent model, the
first National Institutes of Health (NIH) entity to earn this distinction.
With its adoption of the independent model, the NCI CIRB program
was a forerunner of the new policy of the NIH requiring US centers
participating in NIH-funded nonexempt, multicenter studies to use
a single IRB for initial and ongoing ethics review, a policy that will
be implemented starting in 2018.9
The NCI CIRB program provides a centralized approach to human
subject protection. The program currently consists of four boards:
one primarily for late-phase clinical trials, including all adult oncology
phase III treatment trials; an early-phase clinical trial board; a pediatric
clinical trial board; and a board covering cancer control and prevention.
The NCI CIRBs are composed of distinguished panels of oncology
physicians, nurses, and patient representatives and include a pharmacist,
an ethicist, and a lawyer. Unlike most local IRBs, the NCI CIRBs
are focused exclusively on cancer trials and have sufficient time and
expertise to review each protocol in detail. This model for human
subject protection in multicenter trials is now used by more than
1700 sites, including NCI-designated Cancer Centers, other university
medical centers, and community hospitals.
National Cancer Institute National Clinical
Trials Network
The NCTN Program was conceived and developed so that it could
operate as a true network with incentives for collaboration in the
development and conduct of innovative clinical trials. These trials
prioritize precision medicine approaches, rare cancers, rare subsets of
more common cancers, multimodality therapies, special populations,
and research questions unlikely to be addressed in the private sector.
In the NCTN Program, network groups are expected to collaborate
with one another and with NCI to achieve the overall goal of the
Program. Member institutions or sites of any network group are able
to enroll patients in all adult phase III and phase II/III trials, as well
as most early-phase trials and trials in adolescents and young adults,
irrespective of the specific network group that is leading the trial. The
NCTN Program, including each of its six key components, is illustrated
in Fig. 19.1.
One of the key aims of the NCTN when it was launched in 2014
was to provide for the conduct of precision medicine clinical trials
by harnessing the scientific leadership of the NCTN investigators,
leveraging the Network to screen large numbers of patients to identify
those whose tumors exhibit molecular features that may be responsive
to new, targeted treatments and/or immunotherapy, and fostering
clinical research partnerships with biotechnology and pharmaceutical
companies to collaborate on a series of unique precision medicine
trials in oncology. NCI’s Molecular Analysis for Therapy Choice
(NCI-MATCH) is the archetype of a precision medicine trial. It is
led by ECOG-ACRIN (one of the NCTN groups) and has active
participation by member sites belonging to any group within the
NCTN. NCI-MATCH opened to patient enrollment in 2015 and
includes adult patients with any type of solid tumor or lymphoma
whose disease is no longer responding to standard therapy. By the
end of 2017, over 6000 patients had their tumors screened for molecular
alterations and approximately 1000 were “matched” to one of a series
of small phase II trials that target a specific mutation in their tumor.
Although NCI-MATCH is led by ECOG-ACRIN, investigators from
all the NCTN groups lead the individual phase II trials, illustrating
the collaborative aspect of the NCTN.
OTHER NATIONAL CANCER INSTITUTE–
SPONSORED STRUCTURES SUPPORTING
CLINICAL TRIALS
Although the CTSU, the CIRB, and the restructured NCTN represent
mechanisms to reduce barriers and facilitate broader clinical trial
participation by practicing oncologists, advocates, and translational
scientists, a number of other important mechanisms provide additional
options for support and participation.
National Cancer Institute Community Oncology
Research Program
The NCI Community Oncology Research Program (NCORP) was
established in 2014 to serve as NCI’s single community-based research
program. NCORP was developed to build on the progress made in
NCI’s previously supported community-based programs, the NCI’s
Community Clinical Oncology Program (CCOP) network, the
Minority-Based CCOPs, Research Bases, and the NCI Community
Cancer Centers Program (NCCCP). NCI recognized the need to
respond to the IOM’s report6 to address efficiency in the conduct of
clinical trials and to respond to the dynamic health care environment
and challenges faced by today’s practicing community oncologists.
NCORP is an integrated national network to (1) design and conduct
cancer prevention, control, and screening trials, and quality-of-life
studies embedded in treatment trials; (2) expand its research scope to
design and conduct cancer care delivery research; (3) enhance patient
and provider access to treatment and imaging clinical trials conducted
under the reorganized NCTN; and (4) integrate disparity research
questions into clinical trials and cancer care delivery research. The
overarching goal of NCORP is to provide access to clinical trials and
cancer care delivery research to individuals in their own communities,
generating evidence that contributes to improved patient outcomes
and a reduction in cancer disparities. NCORP preserved a three-
component organizational structure, as a partnership between academic
and community practices. It is composed of 34 community sites, 12
minority/underserved sites, and 7 research bases. Community sites
accrue patients or participants to NCI-approved clinical trials and
cancer care delivery research designed by NCORP research bases and
NCI’s NCTN groups. Minority/underserved sites also accrue patients
and participants from catchment areas in which at least 30% of their
rural resident populations are members of racial or ethnic minorities.
Five of the research bases are funded through the NCORP cooperative
funding mechanism and have the same consolidated configuration as
the NCTN groups. Two additional NCORP research bases are non-
NCTN, academic institutions.
NCORP is a diverse network of practices ranging from single
practices, integrated and nonintegrated health systems, safety-net
hospitals, and academic institutions. Fifteen states with greater than
30% rural populations have NCORP components.10 The NCORP
grants provide up-front funding to enable sites to develop a standing
infrastructure to support patient accrual to network trials. Sites must
document the ability to enroll at least 80 patients or individuals per
year in cancer control, prevention, screening, and treatment. The
program is housed in the NCI Division of Cancer Prevention, but
collaborates closely with both the NCI Division of Cancer Control
and Population Sciences that coordinates the cancer care delivery
research portfolio and the NCI Division of Cancer Treatment and
Diagnosis that oversees the cancer treatment clinical trials. The NCI
Center to Reduce Health Disparities also plays an important role
across all NCORP programs.
In 2017, 4065 physicians and 421 hospitals participated in NCORP.
Forty-six community sites and minority/underserved sites have 860
associated component and subcomponent sites throughout the United
States and Puerto Rico. NCORP sites accrued more than 12,000
patients and participants to cancer treatment, cancer prevention, and
cancer control trials during the first 2 years of the program.
With its diverse populations, NCORP’s network is a resource suitable
as a laboratory for cancer health disparities research. It can serve as
proof-of-principle for the conduct of precision medicine trials and the
infrastructure requirements for success outside of academic institutions;
and it is a resource for the collection of patient-reported symptom
toxicities from cancer and cancer treatments. The incorporation
 Structures Supporting Cancer Clinical Trials  •  CHAPTER 19 311

Figure 19.1  •  National Cancer Institute (NCI) National Clinical Trials Network (NCTN) structure. The key components of the NCTN are completely
integrated into the other initiatives and components of the NCI clinical trial system, including the NCI Cancer Trials Support Unit, NCI Central Institutional
Review Boards, other NCI-funded programs such as the NCI Community Oncology Research Program (NCORP) and the Minority/Underserved NCORP
as well as NCI advisory and review committees (not illustrated in the figure), which help oversee the entire program. The six major components are (1)
Network Group Operations Centers—four adult groups (Alliance, ECOG-ACRIN, NRG Oncology, and SWOG) and one pediatric group (Children’s Oncology
Group [COG])—which provide scientific leadership for developing and implementing multidisciplinary, multiinstitutional trials in a range of diseases and
special populations with specific scientific strategy and goals; (2) Network Group Statistics and Data Management Centers, associated with specific Network
Group Operations Centers, which provide the statistical expertise required to ensure effective scientific design and conduct of clinical trials, in addition to
data management, data analysis, which provide support for leadership and expertise to facilitate incorporation of translational science into network group
clinical trials; (4) Network Lead Academic Participating Sites, which provide scientific leadership in development and conduct of clinical trials in association
with one or more adult network groups, as well as substantial accrual to clinical trials conducted across the entire NCTN; (5) Network Radiotherapy and
Imaging Core Services Center, which provides scientific and technical expertise for incorporation of appropriate, integrated quality assurance and image data
management for applicable clinical trials conducted by the NCTN that require specialized quality assurance or imaging data management and/or assessment;
and the (6) Canadian Collaborating Clinical Trial Network (Canadian Cancer Trials Group), which will be capable of being a full partner with the US Network
in conducting large-scale, multisite clinical trials. In addition, all NCTN Groups use a Common Data Management System to conduct NCTN trials.

of cancer screening and reinvigoration of cancer prevention in the
community setting has enhanced the partnerships with nononcology
specialists and primary care physicians both within and affiliated with
NCORP.11 The introduction of cancer care delivery research into
community-based practices enables investigators to study the provider,
the system, and the patient influences on cancer care and outcomes,
a timely topic in view of the national effort to improve the quality
of routine medical care in the United States.
Details on NCORP can be found at the NCI Division of Cancer
Prevention website at https://ncorp.cancer.gov.
Phase I and II Early Therapeutic Clinical
Trials Networks
The NCI has long supported a network of individual academic
institutions and academic consortia to conduct phase I and early phase
II clinical trials, often performed with pharmacokinetic and correlative
pharmacodynamic studies. These institutions, currently comprising
more than 30 NCI-designated US Comprehensive Cancer Centers
and leading institutions in Canada, have enrolled approximately 2000
patients per year in clinical trials, many of which include novel
combinations of investigational agents. A major feature of this network
is the expertise of the investigators and their institutions in tumor
sample acquisition, pharmacokinetic assay development and monitoring,
and the development of biomarker assays.
In keeping with the many new scientific opportunities afforded by
advances in molecular diagnostics, the NCI Experimental Therapeutics
Clinical Trials Network (ETCTN) has been restructured to facilitate the
exploration of novel targeted agents in appropriately selected patients.
This restructuring involves a number of changes, including:
• Integration of NCI’s funded tumor biology and translational science
programs (e.g., the NCI-designated Cancer Centers and the Special-
ized Programs of Research Excellence) with clinical investigators
managing the ETCTN. This integration has included the formation
of interdisciplinary project teams (see later) for investigational agent
312 Part I: Science and Clinical Oncology
development. These project teams bring together the necessary
expertise to both formulate key questions and execute clinical trials
in the development of specific investigational agents.
• Support for laboratories with validated assays that will carry out
the molecular characterization of patient tumors both before and
after treatment.
• Centralized regulatory, data management, and pharmacologic core
functions for the ETCTN, making multisite studies easier to perform
even in the earliest stages of drug development.
• Extension of the NCI CIRB to these early-phase multicenter trials
by establishment of a new review board with the requisite expertise
to focus on early-phase clinical trials.
• Integration of phase I and phase II clinical trials under a single grant
structure, enabling ETCTN awardees to have the flexibility to expand
phase I studies quickly on detection of early activity signals.
• Inclusion of investigators from NCI-designated Cancer Centers
that are not affiliated with the ETCTN who may now submit
clinical trial proposals, and, if approved, receive full ETCTN support
for the clinical trial.
The overall intent of these changes is the creation of an early
clinical trial network that is integrated both scientifically and operation-
ally to pursue the development of novel therapeutics.
National Cancer Institute Drug Development
Project Teams
The NCI extramural program now has a single pipeline to review
and approve the development of high-priority agents, originating from
industry or academia. Once vetted via this single entry process, called
NExT (NCI Experimental Therapeutics Program), NCI’s Cancer
Therapy Evaluation Program (CTEP) convenes a Drug Development
Project Team. The Project Team members are selected based on their
qualifications and the expertise they provide. The team members will
determine which clinical trials will be conducted across the NCI
CTEP clinical trial network sites, and how best to approach critical
translational studies.
Extramural investigators on the Project Teams may fill one or more
of the following roles:
• Clinician scientists who will lead the clinical trials recommended
by the NCI Drug Development Project Team and who will create
clinical protocols for execution of these studies.
• Translational scientists who will provide guidance on prioritization
of biomarkers for the studies under development, including recom-
mendations for technologies and platforms that meet increasingly
stringent requirements for integral and integrated biomarkers.
• Basic scientists who will provide scientific guidance for the study
design based on the mechanism of action of the investigational
agent and who will help prioritize the clinical study choices based
on published literature and unpublished data. Basic scientists on
the team will have access to the agents in order to conduct additional
laboratory studies in support of the clinical trials.
Once convened, the NCI Drug Development Project Team meets
frequently over a 6- to 8-week period to finalize a comprehensive drug
development plan of biomarker-rich, preclinical and clinical trial
proposals. Once the Project Team’s development plan has been approved,
CTEP will also make the agent available to other qualified investigators,
contingent on the company or academic collaborator’s approval. These
investigators must submit Letters of Intent (LOIs) to conduct clinical
trials, or they can also request agents for preclinical studies.
BIOPHARMACEUTICAL INDUSTRY–
SPONSORED CANCER CLINICAL TRIALS
During the first three decades of the development of the field of
medical oncology, there was relatively little participation by the
pharmaceutical industry. In part, this lack of participation was a
reflection of the complexity of therapeutic development during a
period when an insufficiently detailed understanding of cancer biology
made rational drug development difficult. This relative lack of industry
involvement, coupled with the significant public health problem
presented by cancer, was responsible for the development of the large
NCI-sponsored clinical trial apparatus described in detail in this chapter.
During the past 30 years, however, a remarkable increase in biophar-
maceutical investment in cancer drug discovery research has occurred,
along with a parallel increase in both the extent and sophistication
of industry sponsorship of clinical trials for the development of new
cancer therapeutics. Hundreds of new agents are currently in different
stages of clinical evaluation by industry, either alone or in cooperation
with the NCI or similar noncommercial organizations in the United
States and in other parts of the world.
Purpose and Nature of Industry-Sponsored
Clinical Trials
The primary goal of therapeutic agent investigation by the biophar-
maceutical industry is the evaluation of promising agents for eventual
registration and commercialization. Because registration of a new cancer
drug requires the demonstration of safety and efficacy for the new
agent in the context of currently available therapy for the cancer being
treated, industry tends to focus its efforts on so-called explanatory
trials.12 Explanatory trials attempt to isolate the benefits and risks of
the investigational agent(s) under ideal study circumstances (efficacy
studies), whereas so-called pragmatic trials are more often conducted
by NCI or other non–commercially supported networks. Pragmatic
trials, which can include multimodality treatments, ask which treatment
approach is best in a representative sample of patients (effectiveness
studies). However, the distinction between the types of trials is often
blurred because a long tradition exists of industry providing investi-
gational agents for the conduct of clinical studies through NCI-
sponsored mechanisms, and many new agents or indications have
received FDA approval on the basis of NCI-sponsored clinical trials.
Ethical considerations regarding human investigation and expectations
of adherence to standards for the conduct of clinical trials do not
differ substantially between NCI-sponsored and industry-sponsored
clinical trials; however, NCI-sponsored studies must adhere to the
US Code of Federal Regulations (45 CFR Part 46), whereas trials of
investigational drugs or devices that fall under the FDA’s purview
must comply with the regulations in 21 CFR. When the NCI sponsors
trials with new agents or devices, then both federal codes (along with
their respective guidances and policies) are applicable, and any dif-
ferences between them require the sponsor to adhere to the most
stringent aspects of the regulations.
Particular Characteristics of
Industry-Sponsored Trials
Biopharmaceutical development proceeds in a heavily regulated
environment, with detailed regulations from various government
agencies covering a spectrum of activities ranging from those related
to preparing an agent for first entry into humans, through the years
of clinical investigation in patients, to postapproval restrictions on
public discussion regarding possible uses of the agent for indications
other than those for which the drug was approved. As a result of the
requirements for working in such an environment, corporate clinical
investigations tend to be focused on the “clinical development plan,”
the specific set of clinical trials that will produce an appropriate
evidentiary base to allow for regulatory review of the safety and efficacy
profile of the agent. During the investigational phase of an agent’s
life cycle, therefore, companies might restrict the general availability
of the agent to individual investigators for clinical study. This perceived
need for containment and control can lead to tension between the
investigative community and the industrial sponsor. Other potential

sources of tension in the interaction between companies and clinical
investigators relate to the investigators’ perceptions of the need for
independence and objectivity in the conduct of multicenter trials.
Historically, for example, many companies have generated phase III
protocols internally, although usually with considerable input from
both external advisors and regulatory bodies. These trials would be
conceived in whole or part by company personnel, and conduct of
the trials would occur in a group of institutions, usually academic or
community-based or both (most often selected on the basis of the
sites’ accrual track record), and authorship would be conferred on the
principal investigator of the largest accruing site. Increasingly, a new
model has emerged in which a recognized expert is appointed as the
principal investigator. This individual has a much greater role in the
trial design and the monitoring and eventual analysis and publication
of the trial results than might have been the case historically. Similarly,
in recent years, the almost universal adoption of independent Data
and Safety Monitoring Committees for late-stage clinical trials has
occurred to oversee safety-related information as it emerges from the
ongoing trial and to make recommendations to company staff about
appropriate actions.
Impact of Globalization on
Pharmaceutical Development
Because pharmaceutical products increasingly are marketed globally,
large multinational pharmaceutical companies need to conduct
clinical development from a global perspective. Clinical trials with
investigational anticancer agents involving the FDA are being conducted
around the world. This tendency toward global development has been
greatly accelerated by the International Council for Harmonization
process, which facilitated standardization of many of the activities
that are involved in preparing agents for clinical investigation,
conducting those investigations, and then preparing the informa-
tion for registration. A single set of standards now exists for the
conduct of industry-sponsored trials worldwide (available at http://
www.fda.gov/ScienceResearch/SpecialTopics/RunningClinicalTrials/
GuidancesInformationSheetsandNotices/ucm219488.htm). Attempts
are therefore made to harmonize the development process to produce
a globally accepted drug registration package to the greatest extent
possible.
Models for the Conduct of Industry Clinical Trials
Biopharmaceutical industry sponsors usually produce the investigational
agent in their own facilities because they anticipate eventually being
responsible for the commercial production and distribution of the
agent. They also can conduct the series of required clinical trials
directly using their own clinical trial personnel, which may include
internal company physicians, statisticians, monitors, data managers,
quality assurance auditors, and the rest of the required infrastructure,
such as company standard operating procedures, company information
system support, and drug distribution apparatus. Alternatively, drug
sponsors may use a contract research organization to perform the
clinical trials. In fact, for large development programs, it is not unusual
for large companies to coordinate clinical trial programs using a mixture
of both internal and externally acquired resources. Contract research
organizations are companies in the business of conducting clinical
trials. During the past two decades, after the explosive growth of
biopharmaceutical clinical investigation, a large number of such
companies were created, some capable of conducting global trials.
The actual arrangement—direct or indirect—that a drug sponsor uses
for a particular clinical trial is important to the investigator and staff
at the clinical trial site because it determines the predominant source
of interaction and contact during the conduct of the trial.
Different models also exist for investigator participation in clinical
trials with industry. Traditionally, pharmaceutical sponsors have dealt
either with individual investigators or with individual institutions, as
Structures Supporting Cancer Clinical Trials  •  CHAPTER 19 313
in the case of academic centers. Clinical trial contract budgets have
included direct trial-related costs such as performing additional labora-
tory studies that are not being done as part of usual medical care plus
direct site-related costs associated with the time spent on the trial by
the various participating staff, and indirect costs for institutional
overhead. Recent years have seen the emergence of consortia of
investigators (sometimes under the rubric of a site management
organization) or consortia of institutions presenting themselves to
companies as clinical trial entities, often linked by a centralized IRB,
offering the advantage of working under a single, negotiated contract.
In addition, individual academic centers have sometimes formed
networks of oncologists within their referral area for the purpose of
presenting themselves as more efficient entities for interaction.
CHANGING NATURE OF ONCOLOGY TRIALS:
IMPACT ON INFRASTRUCTURE
The same explosion in our understanding of cancer biology that led
to the increase in the number of new agents under development
brought with it a realization that the most appropriate tests of those
biologically targeted agents are clinical trials in which the patients
who participate have tumors that are biologically appropriate for the
agent. For example, imatinib administered to all newly diagnosed
patients with any form of leukemia would have a response rate much
lower than that observed in newly diagnosed patients with chronic
myelogenous leukemia; selection of patients with chronic myelogenous
leukemia with the BCR-ABL mutation allowed for a focused develop-
ment program for imatinib that led to rapid initial registration. The
same considerations can be extended to matching biologically directed
agents with specific cancer patient populations and argues strongly
for more complete biologic characterization and monitoring of patients
entering cancer clinical trials. Regardless of whether such characteriza-
tion is designed for prospective or retrospective analyses, the increased
need for collection of peripheral blood for immune parameters; germline
and circulating free tumor DNA, tumor tissue for DNA, RNA, and/
or protein; and the need for specialized functional imaging, have
placed increasing demands on the infrastructure required to conduct
trials. These requirements have also introduced the necessity for
enhanced quality control on sample collection and storage that in
turn necessitates increased resources. Nonetheless, these clinical trials
are more informative, and the need for sample collection is likely to
increase in future trials. Numerous examples now exist (e.g., BRAF-
mutated melanoma, EGFR-mutated and ALK-mutated non–small
cell lung cancer, and HER2 amplified breast cancer) to support the
contention that classifying tumors by their molecular characteristics,
as opposed to classic histopathology, will result in more rapid drug
development, and more effective and well-tailored treatments.
EXPECTATIONS OF CLINICAL
RESEARCH SITES
Staffing is foremost among the critical components of an effective
research practice listed in Box 19.1. The number and precise composi-
tion of the necessary staff depend on the number of patients who are
enrolled in clinical trials and the nature of the practice. In some settings
in which the number of patients in studies is small, a single research
nurse can perform many of the required functions. At more active sites,
research nurses, clinical research associates, and research pharmacists
perform separate functions. For budgeting purposes, for example, it
is often estimated that one full-time clinical research associate can
handle 25 new patients and up to 50 patients in follow-up in a year,
although this will undoubtedly vary with the complexity of the trial
menu. Some of the structures that support clinical trial participation,
such as the NCORP program, provide substantial up-front funding
to support salaries for the necessary staff in return for a commitment
to substantial accrual. Many others have a hybrid model, including
the NCTN and some industry-sponsored trials, wherein selected sites
314 Part I: Science and Clinical Oncology
Box 19.1.  COMPONENTS OF AN EFFECTIVE
RESEARCH PRACTICE
• The presence of committed physicians who are willing to devote the
time and energy necessary to conduct clinical research and to
conscientiously accept the significant responsibility inherent in the
conduct of human research
• The availability of suitably trained staff (preferably an experienced
research nurse) with enough time to assist in screening patients for
protocol eligibility and for follow-up of patients receiving protocol
treatment
• The availability of suitable staff to administer the required treatments
in the protocol-prescribed manner; increasingly, this task might
include administration of a wide range of potential therapeutics
including, but not limited to, more conventional intravenous
chemotherapy and, in some cases, radiation
• Adequate and committed pharmacy capabilities to handle and
account for investigational agents if they are part of the protocol
treatment
• Adequate data management staff to handle the data-reporting
requirements for patients who are being treated according to
protocols
• Access to an institutional review board with Office for Human
Research Protections assurances to approve the protocol and
monitor the progress of the research
• Access to suitable laboratory facilities to complete the studies
required by the protocol
• Willingness to comply with certain federal regulatory requirements,
including adequate privacy procedures and training in human
subjects protection (available as an online course through the
National Institutes of Health at http://cme.cancer.gov/c01/)
receive up-front payments but other sites receive funding only on a
“per-case” basis, and thus start-up costs must be borne by the site.
Quality Assurance, Monitoring, and Audits
Because the accuracy of the data collected in clinical trials is critical
to the validity of the conclusions from the trials, all clinical trial
organizations include quality assurance programs that reinforce “good
clinical practice” guidelines by proactively monitoring and auditing
clinical trials. Although these programs are structured somewhat
differently depending on the mechanism of participation (industry
studies involve the most frequent and extensive quality assurance), all
such programs have certain features in common. All send queries to
the site when discrepancies or suspected errors are noted in submitted
information, and all compare data that are submitted from the sites
against the primary medical or research record for verification by
either monitoring or audit visits. The deployment of electronic data
capture systems (EDCs) in clinical trials has enabled the use of built-in
edit checks as an additional safeguard for ensuring data quality. Trial
monitoring is done in real-time primarily to assess a site’s performance
during the conduct of a trial. Protocol deviations and violations at a
site recognized by monitors allow appropriate corrections to be made.
Auditors also review for data accuracy and protocol adherence, but
in addition, informed consents are reviewed, as are adverse event
reporting compliance, pharmacy practices, and timeliness of required
IRB submissions and approvals. Preparation for audits is time-
consuming for the sites, because all relevant records, including laboratory
studies and films (e.g., computed tomography and magnetic resonance
imaging), required to document tumor measurements and response
verification, must be gathered for the audit team. Some participants
consider this work onerous; however, audits fulfill an important
educational role in addition to ensuring the quality of the data and
clinical trial procedures at participating sites. NCI audits are typically
conducted at a site every 3 years and review a sample of patients
enrolled in a variety of protocols, whereas industry audits are typically
focused on a single trial. Data quality initially became a concern when
clinical trial participation moved beyond academic sites to community
practices. A number of evaluations in the 1980s, however, documented
the ability of community sites to perform at a level comparable to
that of academic institutions in terms of data quality, protocol adher-
ence, and patient outcome. Indeed, approximately 50% of the accrual
to adult NCTN trials now comes from community practices in the
NCORP and from other community sites that are affiliated with
academic centers that are NCTN group members.
As noted previously, although regulatory standards regarding the
conduct of clinical trials impose similar overall expectations, for its
own reasons, industry monitoring is both more extensive and frequent
than is usual with public- or nonprofit-funded trial organizations.
The basic tenet of monitoring for both industry-sponsored and NCI-
sponsored clinical trials is similar: the need to verify data accuracy by
comparing case report forms, whether submitted by paper or electroni-
cally, with source data in the patient’s medical record. Increasingly,
industry and public- or nonprofit-funded trial organizations are
implementing risk-based monitoring by applying metrics and trend
analysis to review trial data and assess site performance to detect
potential problems early so that corrective and preventive measures
can be implemented. The sponsor-assigned clinical trial monitor will
visit the site regularly, educate the involved staff about the goals and
particular details of the protocol, and then track the progress of
protocol-related activities throughout the conduct of the trial. Monitor-
ing responsibilities include confirming appropriate CIRB or LIRB
review, investigator registration via completion of the FDA 1572 form,
and the existence of timely informed consent documents for each
patient; inspecting drug accountability records; confirming timely
and complete submission of serious adverse events reports; and ensuring
appropriate and timely handling of amendments. These activities all
fall within the responsibility of the clinical trial monitor, whether the
monitor is provided directly from the company sponsor or through
a contract research organization. Furthermore, regardless of whether
the clinical trial is conducted directly by its own organization or by
a contract research organization, biopharmaceutical companies often
conduct their own quality assurance audits and monitoring associated
with the trial to further ensure the integrity of the data they submit
to the FDA. The intensity of clinical trial monitoring tends to increase
as clinical trials mature and the data management group prepares to
officially “lock” the database before the conduct of prespecified analyses
and reporting activities. The intensity of quality assurance auditing
also increases for a particular clinical trial when it has been identified
as part of a new drug application or biologics license application for
drug registration with the FDA. Monitoring and data management
activities are evolving with the widespread introduction of electronic
data collection and submission systems, which have replaced traditional
paper-based case report form approaches, and in the future it is likely
that less on-site and more central monitoring will occur.
Educational and Training Tools and
New Federal Guidelines
As described previously, participation in clinical trials adds many
complexities to the care of cancer patients and can require that physi-
cians, nurses, and other office staff acquire new and different skills.
Fortunately, because of the widespread interest in clinical trials in the
cancer community, many resources exist for gaining information about
clinical trials and for acquiring the necessary skills. Professional societies
are a good source for educational programs and materials, and some,
such as the Oncology Nursing Society (https://www.ons.org/), the
Society of Clinical Research Associates (http://www.socra.org/), and
the American Society of Health-System Pharmacists (http://
www.ashp.org/), actually offer certification programs that can serve
 Structures Supporting Cancer Clinical Trials  •  CHAPTER 19 315

Box 19.2.  USEFUL WEBSITES FOR CLINICAL Table 19.2  New Requirements for National
TRIAL RESOURCES AND Institutes of Health (NIH)–Supported
INFORMATION and US Food and Drug Administration
(FDA)–Regulated Clinical Trials
• National Cancer Institute (NCI): http://www.cancer.gov
• Cancer Trials Support Unit (including links to NCI National Clinical NIH Supported FDA Regulated
Trials Network [NCTN] and NCI Community Oncology Research Good Clinical Practice (GCP) training
Program [NCORP] network groups and research bases): http://www required every 3 years for
.ctsu.org investigators and NIH staff
• Cancer Therapy Evaluation Program: https://ctep.cancer.gov/ Encourage use of a clinical trial Protocol template was
• Community Clinical Oncology Program: http://www3.cancer.gov/ protocol template to ensure that developed via a collaboration
prevention/ccop protocols contain all the information between NIH and FDA that
• Cancer Centers Program: https://cancercenters.cancer.gov/ needed for institutional review board meets the ICH Good Clinical
• NCI Central Institutional Review Board: http://www.ncicirb.org (IRB) and FDA investigational new Practice Guidance
• NCI Coordinating Center for Clinical Trials: http://ccct.cancer.gov/ drug (IND) applications
• National Institutes of Health: http://ClinicalTrials.gov Requirement for single IRB of record Single IRBs for multisite trials
• US Food and Drug Administration: http://www.fda.gov/ for multisite trials are permitted but not required
All NIH-supported clinical trials must Trials of drugs, devices, and
be registered in the ClinicalTrials.gov biologicals, other than phase I
database within 21 days of investigations, of a product
as important career development incentives to office staff. The American enrollment of the first participant subject to FDA regulation
Society of Clinical Oncology (http://www.asco.org/) also has a very must be registered in the
useful website with links to a variety of sites that provide information ClinicalTrials.gov database
about available clinical trials and detailed information about chemo- within 21 days of enrollment
therapy agents for physicians and nurses. NCI-sponsored NCTN and of first participant
NCORP groups and research bases provide regular educational activities All NIH-funded trials, including phase Trials of drugs, devices, and
for physicians, statisticians, nurses, and data managers who participate I, early device, and behavioral biologicals, other than phase I
in their trials. Furthermore, the biopharmaceutical industry sponsors intervention trials, are required to investigations, of a product
a wide range of educational activities that are conducted by both submit summary results to subject to FDA regulation
academic institutions and professional societies (e.g., the American ClinicalTrials.gov database within 12 must be registered in the
Society of Clinical Oncology and the American Association of Cancer months of the prespecified primary ClinicalTrials.gov database
Research) about the clinical trial process in general and about the outcome measures (primary within 12 months of the
responsibilities of the individual clinical investigator. completion date) prespecified primary outcome
The NCI’s home page (http://www.cancer.gov/) provides a gateway measures (primary completion
to the many websites of the NCI and provides links to many other date)
useful sites (Box 19.2). It contains extensive information about cancer
ICH, International Council for Harmonisation.
in general and cancer statistics, as well as detailed information about
clinical trials. A comprehensive listing of all trials conducted in the
United States, whether they are federally or privately sponsored, can improving cancer treatment and prevention. A growing number of
be found at www.ClinicalTrials.gov/. clinical practices have been able to integrate clinical research into their
Recently, the regulations regarding NIH-sponsored clinical trials activities successfully. Online tool kits can be helpful to sites as they
and FDA-regulated trials underwent important changes.9,13 These are prepare for participation in a clinical trial. For example, the CTSU
outlined in Table 19.2. These new procedures were created to meet the provides IRB submission packets, protocol calendars, and trial sum-
challenge of improving the quality of clinical trials while also enabling maries. The availability of a single IRB for multisite trials, as currently
the sharing of clinical trial data more expeditiously. The changes exists for all NCI-supported network trials, and common electronic
focus on enhancing the review, monitoring, and results reporting of data reporting systems also eliminates critical barriers and has made
NIH-sponsored clinical trials, along with new requirements for Good it easier for community physicians to participate. The biopharmaceutical
Clinical Practice (GCP) training and single IRBs for multisite studies. industry continually seeks interested, conscientious physicians to
Implementation of these comprehensive changes began in 2018. participate in its trials. The shortage of such physicians creates a
potentially serious limitation on the rate of development of new
CONCLUSION treatments for cancer. Surveys1 have suggested that the attitude of the
treating physician is perhaps the most critical factor in patient enroll-
Although involvement by oncologists in clinical trials introduces ment in clinical trials. An increasing number of resources and tools
additional complexities to their daily practice, it also provides substantial are now available to enhance access, with the potential to benefit both
benefits to all participants and ultimately contributes to the goals of current and future patients with cancer.

REFERENCES
1. Comis RL, Aldige CR, Stovall EL, et al. A quantitative
survey of public attitudes towards cancer clinical trials.
Available at: http://www.cancertrialshelp.org/static
_binary/308.pdf.
2. Mills EJ, Seely D, Rachis B, et al. Barriers to participa-
tion in clinical trials of cancer: a meta-analysis and
systematic review of patient-reported factors. Lancet
Oncol. 2006;7(2):141–148.
3. Sung NS, Crowley WF Jr, Genel M, et al. Central
challenges facing the national research enterprise.
JAMA. 2003;289:1278–1287.
4. Unger JM, Barlow WE, Martin DP, et  al.
Comparison of survival outcomes among cancer
patients treated in and out of clinical trials. J Natl
Cancer Inst. 2014;106(3):dju002. doi:10.1093/jnci/
dju002.
5. Dilts DM, Sandler AB. Invisible barriers to clini-
cal trials: the impact of structural, infrastructural,
and procedural barriers to opening oncology
clinical trials. J Clin Oncol. 2008;24:4545–
4552.
6. Institute of Medicine. A National Cancer Clinical
Trials System for the 21st Century: Reinvigorating
the NCI Cooperative Group Program. Washington,
316 Part I: Science and Clinical Oncology
DC: The National Academies Press; 2010. https://
doi.org/10.17226/12879.
7. Christian MC, Goldberg JL, Killen J, et al. Sound-
ing board: a central institutional review board for
multi-institutional trials. N Engl J Med. 2002;346:
1405–1408.
8. Wagner TH, Murray C, Goldberg J, et al. Costs and
benefits of the National Cancer Institute Central
Institutional Review Board. J Clin Oncol. 2009;28:
662–666.
9. Hudson KL, Lauer MS, Collins FS. Toward a new
era of trust and transparency in clinical trials. JAMA.
2016;316(13):1353–1354.
10. NCI Division of Cancer Prevention. NCORP Rural industry compared to publicly sponsored random-
Populations infographic. https://prevention.cancer.gov/ ized controlled trials. PLoS ONE. 2013;8(3):e58711.
news-and-events/infographics/ncorp%E2%80%99s doi:10.1371/journal.pone.0058711.
-first-year-reviewed. Accessed January 12, 2017. 13. Zarin DA, Tse T, Williams RJ, Carr S. Trial reporting
11. McCaskill-Stevens W, Pearson DC, Kramer BS, Ford in ClinicalTrials.gov—the final rule. N Engl J Med.
LG, Lippman SM. Identifying and creating the next 2016;17(375):1998–2004.
generation of community-based cancer prevention
studies: summary of a national cancer institute think
tank. Cancer Prev Res (Phila). 2017;10(2):99–107.
https://www.ncbi.nlm.nih.gov/pubmed/?term=10.11
58%2F1940230.
12. Djulbegovic B, Kumar A, Miladinovic B, Reljic
T, Galeb S, et al. Treatment success in cancer:
 Oncology and Health Care Policy 20 
Steven Kent Stranne, Clifford A. Hudis, and Deborah Y. Kamin

S UMMARY OF K EY P OI NT S
• Policies promulgated by government
at the federal, state, and local levels
have profound effects on virtually
every aspect of the day-to-day
practice of oncology. In the face of
growing financial pressures, policy
makers will continue to make
decisions that may have tremendous
impacts on the cancer community.
• Federal policies play a critical role • Policies adopted by the Medicare
in cancer research. Although the
national resources devoted to cancer
research remain substantial ($5.6
billion in 2017), federal funding has
not kept pace with inflation or
increased scientific opportunities,
and the nation is not making all of
the progress it should. Legislation
enacted in 2015 and 2016 increased
the federal commitment to cancer
research but does not overcome used by Medicare as a starting point
the impact of the combination of
relatively flat funding for the National
Cancer Institute since 2003 and
biomedical inflation. The promise
offered by new, highly effective
cancer therapies has never been
brighter, and as a result the benefits
we are likely to realize from
adequate and consistent research
funding have never been greater.
program regarding the prevention,
diagnosis, and treatment of cancer
have greatly influenced both the
practice of oncology and the
services available to both Medicare
and non-Medicare patients
throughout the United States. Both
public and private insurers often insights effectively. Policy can have a
rely on the coverage policies,
reimbursement levels, and coding
for establishing their own policies. In
the face of growing economic
pressure, oncologists and other
cancer care professionals must
remain engaged in helping to inform
policy makers and to shape these
changes. This effort includes working
to ensure that policies designed to
reduce health care expenditures do
not undermine access to care or the
quality of care received by persons
with cancer and that reimbursement
for cancer care is fair and adequate.
• Oncologists and other cancer care
specialists have unique insights
involving the care of patients with
cancer, and it will be increasingly
important to communicate these
profound impact on practice, making
engagement in the process not only
important but also a professional
responsibility.

Federal and state governments play critical roles in establishing policies
that shape and fund virtually every aspect of cancer care and research
throughout the United States. Although federal health care reform
legislation—including the Affordable Care Act (ACA)1 and subsequent
legislative efforts—has attracted headlines for many years, such high-
profile legislation is just one aspect of cancer policy. Many important
policies arise with much less fanfare. Examples include policies adopted
by the National Cancer Institute (NCI) to guide the funding of research
proposals, national coverage determinations for Medicare promulgated
by the Centers for Medicare and Medicaid Services (CMS), and policies
established by the US Food and Drug Administration (FDA) regarding
products used for the prevention, diagnosis, and treatment of cancer.
For the foreseeable future, cancer policy initiatives must be forged
in a political environment beset by growing financial pressure. Potential
cuts in reimbursement and funding will remain an ongoing threat,
raising the stakes for efforts to advocate for policies that protect and
promote the needs of persons with cancer, the cancer research enterprise,
and oncology professionals.
As the modalities and therapies available to diagnose and treat cancer
have become more successful and more complex, the administrative
burdens and financial constraints placed on all cancer care specialists
have become more extreme. Perhaps more than at any other time,
policy issues confronting the cancer community are most properly
viewed as affecting the oncology community as a whole rather than any
individual segment. The fates of community-based, hospital-based, and
university-based cancer care providers and researchers are increasingly
linked in ways that may not be transparent to the casual observer.
This chapter does not catalogue every policy initiative that affects
the cancer community. Rather, the following discussion provides a
framework for understanding the important roles that federal and
state governments play in regulating, shaping, and funding the oncology
care delivery system and research enterprise.
The roles played by federal and state policy makers in cancer
include:
• Research. Through the NCI and other federal agencies, the federal
government serves a leadership role in and provides significant
funding for cancer research performed throughout the United
States, including basic science research, translational research, and
clinical trials.
• Health care insurance. Through the ACA and subsequent legislative
initiatives, the US Congress is engaged in reforming the health care
system, addressing concerns regarding access to health care insurance
and the affordability of health care insurance. In addition, the
federal government operates and funds the Medicare program, which
covers approximately 60% of all patients with cancer in the United
States.2 The federal government also oversees public health programs
managed by the CMS, the Department of Defense, the Veterans
Health Administration, and other federal agencies. Coverage,
reimbursement, and coding policies established by Medicare have

317
318 Part I: Science and Clinical Oncology
significant influence on the policies adopted by most other public
and private health care insurers. Federal and state laws may also
create patient safeguards that private insurers must follow, such as
the protections established under the ACA to promote patient
access to cancer care through clinical research trials.
• Cost of care. Through its payment policies for providers and
services, the federal government has a major impact on the care of
patients with cancer. Policy makers have placed increasing emphasis
on promoting quality, cost-effectiveness, and value in the health
care system over the past decade. With strong bipartisan support,
Congress enacted the Medicare Access and CHIP Reauthorization
Act (MACRA) in 2015 to create a new framework for promoting
quality and value. The CMS articulated an aggressive timeline for
reforming its long-standing fee-for-service payment framework in
favor of a system driven by value. These are some of the most
significant changes in health care organization and delivery since the
Medicare program was established in 1965.3 They will also motivate
significant transformation on the part of oncology practices in the
coming years.
• Health information technology. Oncology care often involves
complex diagnostic and treatment regimens delivered by multiple
health care providers, and health information technology holds
tremendous promise to promote quality, communication, efficiencies,
and research. Despite a federal investment of over $30 billion to
promote the adoption of health information technology infrastruc-
ture, health care providers continue to face significant challenges
in obtaining health care information stored by other health informa-
tion technology systems. Congress enacted provisions to promote
interoperability as part of the 21st Century CURES Act in 2016.
BACKGROUND
Multiple areas of authority and discretion are relevant to cancer policy
development within federal and state governments. Each of the three
branches of government—the legislature, the executive branch, and
the courts—plays a distinct role in a carefully constructed system of
checks and balances. Although the Supreme Court’s reviews of the
ACA during both 2012 and 2015 provide notable examples of the
interplay among these branches of government, most cancer policies
do not involve litigation.
In 2012 the Supreme Court considered the constitutionality of
two major provisions of the ACA—the individual mandate (a require-
ment that individuals maintain a minimum level of health care insurance
for themselves and their tax dependents or pay a tax penalty) and the
ACA’s requirements for states to expand their Medicaid programs.
The architects of the ACA relied heavily on Medicaid as a vehicle for
expanding coverage to the uninsured. In June 2012 the Supreme
Court upheld the individual mandate but struck down the ACA’s
requirement for states to expand their Medicaid programs.4
In 2015, the Supreme Court again considered the constitutionality
of a major provision in the ACA, this time focusing on the validity
of federal tax credits, which provide financial subsidies for health
insurance, in states without state-operated health insurance exchanges.
With annual federal subsidies to 6.4 million Americans at stake in
the 34 states with insurance marketplaces operated by the federal
government, the Supreme Court upheld the ACA subsidies. In 2017,
Congress eliminated the financial penalty for individuals who fail to
maintain health insurance (effective January 1, 2019). Although
Congress did not repeal the requirement for individuals to maintain
health insurance, the legislation eliminated the ACA’s enforcement
mechanism used to encourage individuals to obtain health insurance
coverage.
Medicaid funding and oversight are a shared responsibility between
states and the federal government, and political leaders in many states
have indicated an initial unwillingness to engage in any significant
expansion of their Medicaid programs. From the perspective of oncology
patients and providers, the issue of Medicaid expansion remains
complicated. The coverage requirements for the expanded population
created under the ACA differ from traditional Medicaid. Although
we do not have much evidence regarding the adequacy of coverage
for cancer care under the ACA expansion population, concerns remain
regarding Medicaid-covered cancer patients because some studies have
indicated that, historically, cancer outcomes are not significantly better
for Medicaid patients in comparison with the uninsured.5–7 The Trump
Administration has refocused federal administration of the Medicaid
program by enhancing opportunities for states to apply for and receive
waivers to redesign the rules for Medicaid program benefits and eligibil-
ity, creating the potential that protections could erode for cancer
patients on a state-by-state basis.
Although some provisions of the ACA will remain highly contro-
versial, a number of relatively noncontroversial provisions are particularly
important for the cancer community. These provisions include protec-
tions for patient access to preventive screening for cancer; protections
to help vulnerable individuals with cancer secure and retain access to
health insurance; safeguards for individuals with cancer and other
preexisting conditions; and protections for patient access to clinical
trials.8,9 These patient safeguards, which have bipartisan support,
warrant the cancer community’s ongoing attention and support.
As with the ACA, cancer-related policies of significance at the
federal level often arise from laws enacted by the US Congress (Table
20.1). However, in virtually every law enacted by Congress, there are
substantial gaps, conflicts, and ambiguities within the legislation that
must be resolved as part of the implementation process. As a result,
the work to influence the final version of a national policy involving
cancer rarely ends with the enactment of legislation. Advocates for
the cancer community must typically devote substantial attention to
educate agency officials and influence policies adopted by agency
officials under both new and preexisting legislative authority. These
dynamics also occur at the state level.
For policies relevant to the cancer community at the federal level,
the discretion to identify and address the gaps, conflicts, and ambiguities
in the statutory language rests with the executive branch, including
the Office of the President and the US Department of Health and
Human Services. In practice, all but the most politically sensitive
issues are typically determined by one of the sister agencies within
the Department of Health and Human Services, including the NCI,
National Institutes of Health (NIH), CMS, FDA, Centers for Disease
Control and Prevention, Health Resources and Services Administration,
and Agency for Healthcare Research and Quality.
These agencies are charged with providing the expertise to make
effective policies in areas involving complex scientific and clinical
issues. As a result, there often are a substantial number of agency
employees who have scientific and clinical backgrounds, although
other agency officials may have limited experience in these areas. In
many instances, agency employees are left with lean staffing to contend
with some of the most challenging issues facing the US health care
system. This situation heightens the importance of efforts by oncologists
to share detailed, practical solutions with federal and state policy
makers regarding the complex issues facing the cancer community
and the health care system.
RESEARCH
The federal government plays an integral role in cancer research in
the United States, including basic science research, translational research,
and clinical trials. Although a number of federal agencies are active
in supporting cancer research, the NCI (one of 27 NIH institutes
and centers) is the primary agency charged with guiding and funding
national research initiatives in oncology. Congress established the NCI
under the National Cancer Institute Act of 1937.10 The NCI is subject
to oversight by Congress and the administration, but it retains significant
discretion to establish overarching policies and make case-by-case
decisions that shape national research efforts.
 Oncology and Health Care Policy  •  CHAPTER 20 319

Table 20.1  Selected Federal Laws Relevant to Cancer Policy

Federal Law Summary of Selected Effects on the Cancer Community

National Cancer Institute Act of
1937
National Cancer Act of 1971
Biomedical Research Training
Amendments (1978)
Veterans Health Care Act of 1992
Omnibus Budget Reconciliation
Act of 1993
Health Insurance Portability and
Accountability Act (1996)
Balanced Budget Act of 1997
Medicare Prescription Drug,
Improvement, and
Modernization Act (2003)
Genetic Information
Nondiscrimination Act (2008)
Affordable Care Act (2010)
Food and Drug Administration
Safety and Innovation Act (2012)
Medicare Access and CHIP
Reauthorization Act (2015)
21st Century Cures Act (2016)
Bipartisan Budget Act of 2018
Established the National Cancer Institute and charged it with conducting, fostering, and coordinating
research and training related to the cause, prevention, diagnosis, and treatment of cancer
Began “War on Cancer,” initiated the National Cancer Program and its various boards, authorized creation of
new cancer centers and training programs, expanded existing research facilities, enhanced collaboration in
cancer research among federal, state, and other entities, and appropriated significant funds for a variety of
research-related purposes
Expanded research on preventing cancer caused by workplace and environmental carcinogens and
emphasized cancer education programs initiated within local communities and hospitals
Amended the Public Health Service Act by adding section 340B, which limits the amount certain safety net
providers must pay to manufacturers for covered outpatient drugs
Required Medicare to cover off-label uses of anticancer drugs recognized in certain medical compendia
and required Medicare to cover orally administered anticancer drugs that have the same indications and
active ingredients as covered infused anticancer drugs
Limited the restrictions that group health plans can place on the coverage of preexisting conditions and
created security and privacy protections
Expanded Medicare coverage for several cancer screening tests, implemented the Sustainable Growth Rate
reimbursement methodology, and provided coverage for antiemetic drugs used as part of anticancer
chemotherapeutic regimens
Created Medicare Part D (prescription drug benefit), changed methodology for calculating relative value of
drug administration services, and instituted new average sales price payment methodology for many drugs
and biological products covered under Medicare Part B
Prohibited employment decisions made on the basis of genetic information and prohibited group health
plans and health insurers from making coverage and premium decisions for healthy persons on the basis
of genetic predispositions for future illnesses
Established safeguards under private health insurance plans to ensure coverage of individuals with
preexisting conditions and for individuals participating in clinical trials, established protections for patient
access to preventive screenings for cancer under Medicare, Medicaid, and private insurance, and
established the Innovation Center within the CMS for evaluating new payment methodologies, including
but not limited to accountable care organizations
Established provisions to address shortages of cancer drugs and other medications, included a notification
requirement for manufacturers 6 months in advance of an anticipated drug shortage, directed the Food
and Drug Administration to include biologicals on drug shortage listings, and included fees for generic
drug applications to support agency resources for more rapid reviews of generic drugs, which also may
help avoid or alleviate drug shortages
Created a new framework for promoting quality and value within the context of Medicare’s traditional
fee-for-service benefits, replacing the previous formula for physician reimbursement
Authorized additional federal funding for cancer research and created mandates to promote
interoperability among health information technology systems.
Repealed the Affordable Care Act’s “individual mandate” tax penalty for failure to maintain health insurance
and made unrelated changes to the Medicare program in a number of ways that benefit the cancer
community, including a correction to avoid reductions to Medicare Part B drug reimbursement.

Most of the NCI’s funds are used to support extramural research
activities in communities located throughout the United States, which
often focus on important scientific questions that industry may have
little incentive to explore (such as comparisons of competing drugs or
drug regimens). The NCI also supports intramural research conducted
at the NCI’s facilities in Maryland. From 1999 to 2003, the NCI and
NIH benefitted from strong patient advocacy and a concerted effort
by Congress to double the NIH budget during this 5-year period.
The NCI budget for fiscal year 2017 exceeded $5.6 billion.
Federal policy makers have recognized the importance of harnessing
the economic benefits of cancer research and supporting the international
leadership position of the United States in this field. For example, as
part of the American Recovery and Reinvestment Act of 2009 (ARRA),
Congress provided nearly $1.3 billion in additional funding to the
NCI for use during fiscal years 2009 and 2010.11 Congress required
that the NCI target the ARRA funding to preserve and create jobs,
promote economic recovery, and increase economic efficiency through
technologic advances in science and health. The cancer community
welcomed ARRA funding, despite the fact that the ARRA funds
did not become part of the permanent baseline for the NCI and
NIH budgets.
In both 2015 and 2016, Congress enacted additional increases in
federal funding for cancer research. When finalizing the annual federal
budget in late 2015 for fiscal year 2016, Congress included an increase
in federal research funding of 6.6%, including $5.2 billion in funding
for the NCI’s cancer research initiatives. In addition, in December
2016, Congress authorized the expenditure of an additional $1.8
billion in funding over multiple years to support cancer research through
the Cancer Moonshot initiative. The initiative, launched by President
Obama and led by Vice President Biden, was intended to bring public
attention and renewed focus on addressing barriers—technologic,
administrative, and clinical—to speedy development of more effective
therapies.
Despite these positive developments and the substantial national
resources devoted to cancer research, the nation has lost opportunities
and efficiencies because of suboptimal funding. Despite the significant
320 Part I: Science and Clinical Oncology
NCI ANNUAL FUNDING
$6,000,000
NCI funding
$5,000,000 Inflation adjusted NCI funding
Dollars (in thousands)
$4,000,000
$3,000,000
$2,000,000
$1,000,000
$0
2003 2004 2005 2006
Figure 20.1  •  National Cancer Institute (NCI) annual funding 2003–2012. Inflation adjustments based on the National Institutes of Health (NIH)
Biomedical Research and Development Price Index. (From the NCI, Office of Budget and Finance. https://www.cancer.gov/about-nci/budget/fact-book/historical-
trends/funding, and NIH, Office of Budget. https://officeofbudget.od.nih.gov/gbipriceindexes.html. 2012.)
successes achieved in oncology research from the long-standing col-
laboration between the federal government and researchers throughout
the country, the uncertainty and inconsistency in federal funding
for the NCI during recent years has taken a substantial toll. For
example, the aggregate funding levels for the NCI and NIH were
relatively flat after the doubling of these budgets concluded in 2003
(Fig. 20.1). This stagnation eroded the effective level of federal support
for cancer research by more than 20% between 2003 and 2015 because
of the rate of biomedical research inflation.
Our research infrastructure faces ongoing uncertainty in large
part because of the annual nature of federal funding levels. The 21st
Century Cures Act, signed into law December 13, 2016, highlights
this concern. Although many advocates urged Congress to guarantee
the level of funding for cancer research over a multiyear timeframe,
the final law authorizes but does not guarantee the level of funding
in future years. Ultimately, the funding levels will be determined
by Congress through the annual appropriations process. The annual
nature of appropriating funds for federally funded research contributes
to the potential threat of cuts in cancer research funding levels in
the future.
The environment of flat budgets and looming cuts is undermining
and delaying realization of the significant scientific progress made
during the past two decades. Without sustained and predictable increases
in annual funding for the NCI, significant efficiencies are being lost
because important studies may be started but not completed, the
awarding of multiyear research grants is compromised, and established
research teams are destabilized. Oscillations in federal funding levels
and uncertainty have the potential to dissuade highly talented young
people from pursuing careers in cancer research.
One issue of particular concern is the need to restructure and
improve funding for the NCI Clinical Trials Cooperative Group
Program. The Institute of Medicine published a report in 2010 recom-
mending changes, and the NCI has worked to implement modifications
designed to both streamline the research process and prioritize clinical
trials with the most promise for improving patient outcomes.12 Even
with these changes, sustained increases in NCI funding are needed
to ensure that clinical trials can be conducted in a timely manner to
translate recent scientific discoveries into benefits for day-to-day patient
care. For example, the Institute of Medicine report recommended
that the NCI increase the payment rate for clinical trials because the
2007 2008 2009 2010 2011 2012 2013 2014 2015 2016
Year
current rate accounts for only one-third to one-half of the actual costs
incurred by providers conducting clinical trials. With current funding
constraints, the NCI has said that implementation of such a recom-
mendation would require the NCI to scale back plans for new trials
or reduce accrual rates of existing studies.
The national commitment to cancer research has continued, even
in challenging economic times, since Congress initially authorized
the NCI in 1937. However, the promise for new, highly effective
cancer therapies has never been brighter, and as a result the need to
amplify our commitment to ensure that adequate research funding
exists has never been greater.
HEALTH CARE INSURANCE
Through the ACA and subsequent legislative initiatives, the US
Congress is engaged in reforming the health care system, including
finding ways to help provide health care for those who are uninsured
and underinsured. Although many noneconomic factors influence
access to cancer care, lack of health insurance poses a major barrier
to receipt of appropriate treatment. Cancer patients without adequate
insurance receive less care, receive it later, and often have worse outcomes
than individuals with insurance. Uninsured and underinsured families
facing a diagnosis of cancer report that they are unable to meet their
out-of-pocket financial responsibilities and often must forgo cancer
care in order to pay other bills. The specific direction that the federal
government will take in the future to address health care reform
remains the subject of debate.
Policies adopted by the Medicare program regarding the prevention,
diagnosis, and treatment of cancer have greatly influenced both the
practice of oncology and the services available to both Medicare and
non-Medicare patients throughout the United States. Medicare is a
federal health insurance program that provides health care coverage
to persons who satisfy eligibility requirements related to age or disability.
The influence of the Medicare program is due in large part to the
significant size of the program, which covers approximately 60% of
all patients with cancer in the United States.2 However, the influence
of Medicare’s policies extends well beyond its patient population.
Both public and private insurers often rely on coverage policies,
reimbursement levels, and coding used by Medicare as a starting point
for establishing their own policies.

Cancer care has changed dramatically since Congress initially created
the Medicare program in 1965, and as Medicare has evolved, the
program has played an important role in defining the modern practice
of oncology. As the number of effective anticancer chemotherapy
regimens grew in the 1980s and 1990s, Medicare’s coverage of
intravenous drug therapies in outpatient settings enabled hospitals
and physician practices to establish sophisticated, full-service facilities
that care for patients with cancer within local communities throughout
the United States.
However, since the late 1990s Congress has enacted changes in
Medicare reimbursement levels for oncology drugs and professional
services that have placed significant financial and administrative burdens
on cancer care providers. For example, Congress enacted provisions
within the Medicare Prescription Drug, Improvement, and Moderniza-
tion Act of 2003 (commonly referred to as the Medicare Modernization
Act) that changed the reimbursement formula for oncology drugs
administered in outpatient settings.13 Despite multiple steps taken by
Congress and the CMS to mitigate adverse effects on oncologists, the
net effect of these changes in reimbursement over time has exacerbated
the financial challenges of providing optimal oncology care.
Policy makers often justify the various forms of cuts in Medicare
payments to oncologists and other physicians on the basis that health
care providers can shift the financial burdens arising from Medicare’s
underreimbursement to private-sector employers and insurers. This
rationale is flawed in multiple ways, especially for oncologists who
must serve increasingly large numbers of elderly (i.e., Medicare) patients.
Congress enacted multiple safeguards over the years to protect
persons with cancer. For example, Congress enacted provisions in 1993
requiring Medicare to cover off-label uses of drugs and biologicals if
such uses were supported by clinical evidence reflected in the peer-
reviewed medical literature or specific compendia.14 This patient
safeguard has been critically important during the past two decades
because pharmaceutical companies historically have been unlikely to
seek FDA approval for multiple supplemental indications that support
the most important therapeutic agents for the treatment of cancer.
Congress enacted a number of changes to Medicare under the
ACA, including the creation of a new center within Medicare called
the Center for Medicare and Medicaid Innovation (the Innovation
Center).1 Congress vested broad authority in the Innovation Center
to test and expand the use of innovative approaches to reimbursement
under Medicare without the need for further action by Congress. In
2016 the Innovation Center enrolled 200 oncology practices in a
reimbursement demonstration program called the Oncology Care
Model (OCM). The OCM operates in concert with Medicare’s tra-
ditional fee-for-service benefit. Central to this initiative, the CMS
recognized the importance of providing additional reimbursement to
medical oncology practices beyond the standard levels available under
the existing codes used for Medicare fee-for-service. The additional
payments are triggered at the initiation of anticancer drug regimens,
providing resources so that medical oncology practices can provide
additional services to help manage complications, avoid hospitalizations,
and otherwise improve cancer care. Participating oncology practices
are evaluated on the basis of clinical quality measures and cost savings.
The ACA also established a safeguard to ensure that persons with
cancer and other life-threatening conditions are covered under private
insurance if they determine with their physicians that enrolling in a
clinical study is in their best interest.1 The legislation requires insurers
to cover the routine costs of care associated with qualifying phase I,
II, III, and IV clinical trials. Ensuring that patients with cancer can
access clinical trials is not solely focused on promoting research, because
it is a long-standing and firmly held belief in oncology that the option
to participate in a clinical trial is a key component of high-quality
cancer care and should be a readily accessible option for any patient
with cancer. In many instances, clinical trials provide persons with
cancer with the best chance for a successful outcome. The ACA
safeguard for patients who need access to clinical trials is similar to a
protection that already exists for Medicare beneficiaries.
Oncology and Health Care Policy  •  CHAPTER 20 321
In the face of extreme political and economic pressure, major
transformations are occurring in both the public and private health
insurance sectors. Policy makers are making piecemeal (but significant)
changes to the traditional reimbursement systems at the same time
that new models of reimbursement are being tested by Medicare’s
Innovation Center and private payers. Cancer patients are a unique
population and require special considerations when evaluating new
methods for health care delivery to protect and promote patient access
to high-quality, high-value cancer care. In many instances, a cancer
diagnosis is both a medical and financial emergency for diagnosed
individuals and their families. Often policy efforts intended to lower
costs or promote affordability have the consequence of eroding the
overall value of health care coverage by providing fewer benefits and
making it more expensive for patients to access their benefits. Oncolo-
gists and other cancer care professionals must remain engaged in
helping to shape these changes to ensure that efforts to reduce health
care expenditures do not undermine the quality of care received by
persons with cancer, as well as to ensure that fair and adequate
reimbursement exists to permit the ongoing delivery of high-quality,
high-value cancer care.
COST OF CARE
Traditional fee-for-service reimbursement rewards the volume of services
provided, but policy makers have raised concerns that fee-for-service
reimbursement alone does not promote the delivery of quality or
cost-effectiveness in health care services. Policy makers have been
working to create incentives and penalties that promote the delivery
of high-quality, high-value health care services for many decades. To
this end, Congress has encouraged the adoption of managed care
under Medicare, Medicaid, and the private insurance sector under
laws such as the Health Maintenance Organization Act of 1973 and
the Balanced Budget Act of 1997.
A large portion of Medicare beneficiaries continue to opt for coverage
under the traditional fee-for-service benefit of Medicare Part B. As a
result, Congress and agency officials have worked to develop initia-
tives that can mesh with traditional Medicare while promoting both
quality and value. The Physician Quality Reporting System (PQRS),
formerly known as the Physician Quality Reporting Initiative (PQRI),
is one such initiative that the CMS implemented between 2006 and
2016. However, one of the long-standing criticisms of PQRS was the
lack of a robust measure set for oncology and other specialties. To
address the need for improved quality measures in oncology and other
medical specialties, Congress enacted a provision under the American
Taxpayer Relief Act of 2012 that permits use of physician-led registries
such as the American Society of Clinical Oncology (ASCO) Quality
Oncology Practice Initiative (QOPI) to satisfy requirements related to
Medicare’s PQRS.
Congress enacted MACRA in 2015 to create a new framework
for promoting quality and value in conjunction with Medicare Part
B’s fee-for-service benefit for physician services. MACRA creates one
pathway called the Merit-based Incentive Payment System (MIPS)
that incorporates and reconfigures Medicare’s system for measuring
and creating financial incentives for quality, resource use, and health
information technology use and a new category for clinical quality
improvement activities. The second MACRA pathway—Advanced
Alternative Payment Models (Advanced APMSs)—enables providers
to share in potential cost savings achieved as part of their participa-
tion in the alternative model. The Advanced APM pathway requires
providers to develop innovative programs for treating patients while
assuming two-sided risk, which refers to accepting the risk of earning
either bonuses or penalties based in large part on overall cost savings
achieved compared with benchmarks established by the Medicare
program.
The reforms established under MACRA hold promise to funda-
mentally transform the Medicare program. The strong bipartisan
support that MACRA received in Congress suggests that the federal
322 Part I: Science and Clinical Oncology
government has identified a general approach to modifying the Medicare
fee-for-service program that may endure in the highly politicized health
policy environment. There remains significant work ahead for oncology
professionals to help shape the implementation of MACRA in ways
that support the needs of individuals with cancer and the oncology
community without adding to the administrative burdens placed on
oncology providers. To this end, ASCO has developed cutting-edge
programs that help support the unique needs of cancer patients under
the MIPS and Advanced APM pathways, including the Patient-Centered
Oncology Payment (PCOP) proposal and the QOPI.
HEALTH INFORMATION TECHNOLOGY
Oncology care often involves complex diagnostic and treatment regi-
mens delivered by multiple health care providers, and health information
technology holds tremendous promise for oncology to promote quality,
efficiencies, and research. Timely and comprehensive access to all
components of an individual’s medical record provides opportunities
for health care providers to select the most appropriate care, avoid
complications, and minimize needless duplication of diagnostic tests
and therapeutic services. Although there has been significant progress
by providers of all types in adopting health information technology
in the United States, we continue to face significant challenges as a
nation with respect to the interoperability of these systems.
The term interoperability refers to the ability of independent health
information technology systems to successfully exchange electronic
health information without special efforts by the users. In practice,
the health information technology systems used by different hospital
systems and by different physician practices often struggle to com-
municate effectively with one another, creating silos of health informa-
tion rather than a seamless source of information about the health
care services provided to each patient.
As part of ARRA, Congress enacted a series of provisions known
as the Health Information Technology for Economic and Clinical
Health (HITECH) Act. This legislation initiated a multiyear subsidy
program to help support health care providers in purchasing and using
health information technology. Before the enactment of the HITECH
Act, only approximately 10% of hospitals and 17% of physician offices
had an electronic health records system in place. Between 2011 and
2016, the federal government awarded over $30 billion in incentive
payments to cover the costs associated with the adoption of health
information technology infrastructure. By 2015, over 96% percent
of hospitals and over 80% of physician offices had established electronic
health records systems that satisfy national certification standards.
The Medicare program established “meaningful use” requirements
intended to determine whether physicians and hospitals are making
use of health information technology.
The failure to achieve interoperability has attracted significant
attention with many Members of Congress and other policy makers
lamenting this barrier to effective use of the substantial federal invest-
ment. In 2015, Congress enacted legislation that required a federal
report on the technical, operational, and financial barriers to interoper-
ability. Subsequently, in 2016 Congress enacted additional provisions
to tackle the issue of interoperability in the 21st Century Cures Act.
Congress mandated the development of an exchange framework and
common agreement to promote interoperability. Although future
adoption of the common standards will be voluntary, the federal
government is empowered to encourage adoption by requiring use of
the common exchange framework for entities contracting with the
federal government.
Our national interoperability challenges are exacerbated by informa-
tion blocking, which refers to situations in which information technol-
ogy vendors, health care providers, or others take proactive steps to
impede the exchange of information between health information
technology systems. Within the 21st Century Cures Act, Congress
established safeguards that are intended to eradicate information
blocking, including the authority to pursue civil monetary penalties
against violators.
In addition to assisting in the treatment of individual patients,
health information technology presents unique opportunities to advance
our knowledge base. Big data initiatives such as CancerLinQ provide
the potential to collect, aggregate, and interpret data on large numbers
of cancer patients, providing observational data that will enable us to
learn information from a high percentage of cancer patients—not just
the small percentage of patients who enroll in traditional randomized
controlled trials. The federal government is adopting policies that
embrace these initiatives. For example, Congress has enacted safeguards
that expressly extend the federal interoperability protections to
physician-led registries such as CancerLinQ and that require the FDA
to evaluate how to incorporate observational data into its drug approval
decisions.
Although issues involving health information technology affect all
medical specialties, there are unique opportunities to harness health
information technology in serving individuals with cancer. The oncology
community has special insights to share with policy makers on how
best to use health information technology as quickly as possible.
CONCLUSION
Policies promulgated by government at the federal, state, and local
levels have profound effects on virtually every aspect of the day-to-day
practice of oncology. In the face of significant financial pressures,
policy makers will continue to make decisions that have tremendous
impact on the cancer community. Oncologists and other cancer care
specialists have unique insights involving the care of patients with
cancer, and it will continue to be increasingly important to communicate
these insights effectively. Policy can have a profound impact on practice,
making engagement in the process not only important but a professional
responsibility.
The complete reference list is available online at
ExpertConsult.com.

REFERENCES
1. The Patient Protection and Affordable Care Act, Pub.
L No. 111-148, as amended by the Health Care and
Education Reconciliation Act of 2010, Pub. L No.
111-52. http://housedocs.house.gov/energycommerce/
ppacacon.pdf.
2. Smith BD, Smith GL, Hurria A, et al. Future of cancer
incidence in the United States: burdens upon an aging,
changing nation. J Clin Oncol. 2009;27:2758–2765.
3. Health Insurance for the Aged Act. Public Law 89-97.
July 30, 1965.
4. National Federation of Independent Business v. Sebel-
ius, No. 11-393, slip op. US June 28, 2012. http://www
.supremecourt.gov/opinions/11pdf/11-393c3a2.pdf.
5. Roetzheim RG, Gonzalez EC, Ferrante JM, et al.
Effects of health insurance and race on breast carci-
noma treatments and outcomes. Cancer. 2000;89:
2202–2213.
Oncology and Health Care Policy  •  CHAPTER 20 322.e1
6. Kelz RR, Gimotty PA, Polsky D, et al. Morbidity
and mortality of colorectal carcinoma surgery differs
by insurance status. Cancer. 2004;101:2187–2194.
7. Roetzheim RG, Pal N, Gonzaez EC, et al. Effects of
health insurance and race on colorectal cancer treat-
ments and outcomes. Am J Public Health. 2000;90:
1746–1754.
8. Moy B, Polite BN, Halpern MT, et al. American
Society of Clinical Oncology Policy Statement:
Opportunities in the Patient Protection and Affordable
Care Act to reduce cancer care disparities. J Clin
Oncol. 2011;29:3816–3824.
9. Stranne SK. An oncology perspective on preventive
services in the context of US healthcare reform.
Oncology. 2012;25:1119–1132.
10. National Cancer Institute Act of 1937. Pub. L No.
75-244. http://legislative.cancer.gov/history/1937.
11. American Recovery and Reinvestment Act of 2009.
Pub. L No. 111-5. http://www.gpo.gov/fdsys/pkg/
PLAW-111publ5/pdf/PLAW-111publ5.pdf.
12. Institute of Medicine of the National Academies.
A National Cancer Clinical Trials System for the
21st Century: Reinvigorating the NCI Cooperative
Group Program, April 15, 2010. http://iom.edu/
Reports/2010/A-National-Cancer-Clinical-Trials-
System-for-the-21st-Century-Reinvigorating-the-
NCI-Cooperative.aspx..
13. Medicare Prescription Drug, Improvement, and
Modernization Act of 2003. Pub. L No. 108-173.
http://www.gpo.gov/fdsys/pkg/PLAW-108publ173/
pdf/PLAW-108publ173.pdf.
14. Omnibus Budget Reconciliation Act of 1993, Pub. L
No. 103-66. http://www.gpo.gov/fdsys/pkg/BILLS-
103 hr2264pp/pdf/BILLS-103 hr2264pp.pdf.
 E. PREVENTION AND EARLY DETECTION
Discovery and Characterization of Cancer 21 
Genetic Susceptibility Alleles
Stephen J. Chanock and Elaine A. Ostrander

S UMMARY OF K EY
• The discovery of cancer
susceptibility regions across the
genome provides opportunities to
understand defining events in tumor
development, specifically identifying
cellular pathways that contribute to
the complex development of cancer.
• Regions of the genome harboring
susceptibility alleles and genes can
be identified in families or unrelated
populations by using association
studies, next-generation sequencing,
and linkage studies.
• Technologic advances in sequence
analysis in concert with
comprehensive annotation of genetic
P OI NT S
variation across the human genome
continue to accelerate the pace of
discovery and characterization of
cancer susceptibility alleles. The
conclusive identification of a gene or
a regulatory region contributes to an
understanding of defining events in
tumor development.
• The spectrum of cancer
susceptibility alleles includes
mutations in genes that are highly
penetrant, which indicates that
individuals born with a mutant allele
have a high probability of developing
cancer; moderately penetrant
mutations, which confer an increase
in probability; and common variants,
which impart a small risk for cancer.
• Both association studies and
family-based studies require
accurate collection of clinical data by
clinicians, and both represent an
important pillar for precision
medicine and precision prevention.
An improved understanding of
molecular aspects of cancer and
specifically the use of biomarkers
such as susceptibility alleles can
inform clinical and public health
decisions.

For generations, investigators have pursued the heritable contribution
to cancer. Seminal studies in families in which members affected with
breast cancer, colorectal cancer, melanoma, or a constellation of cancers
(e.g., Li-Fraumeni syndrome) have provided evidence for rare mutations
with strong effects.1–3 Generations of family-based and twin studies
have repeatedly shown an excess of familial cancer aggregation for
nearly all types of cancers, although the estimates vary greatly across
cancer types. These observations suggested that it would be possible
to map cancer genes and thus estimate the genetic contribution to
each molecular type of cancer, even in unrelated populations. Until
the mid-2000s, progress had been slow. However, the pace at which
new genetic regions harboring cancer susceptibility alleles have been
identified has accelerated substantially because of three converging
factors. First, a high-quality draft sequence of the human genome was
produced.4,5 Second, its subsequent annotation resulted in the apprecia-
tion of a wide spectrum of variation across the genome.6,7 Third, the
development of technical platforms that enable interrogation of genetic
variation across the genome has changed the discovery speed of cancer
susceptibility alleles. In the last decade the scope of studies changed
dramatically, expanding from family-based studies to larger population-
based studies of unrelated individuals. These studies have been fueled
by the precipitous drop in price for interrogating large numbers of
informative single-nucleotide polymorphisms (SNPs), the most common
type of variant in the genome, or massive parallel sequence analysis
of entire or partial genomes, particularly the coding regions of the
genome (known as the exome). To keep pace with new streams of
large data sets, investigators have forged new collaborations and
developed computational tools for analyzing ever-enlarging data sets
in search of new cancer susceptibility alleles. The ability to discover
and validate cancer susceptibility alleles is highly dependent on sharing
of data through accessible databases enabling bona fide and approved
researchers to carry out additional studies.8,9
Cancer susceptibility alleles have been discovered through a spectrum
of approaches, yielding a range of inherited genetic variants from rare
mutations with strong effects (i.e., highly penetrant) to common genetic
polymorphisms, each of which confers a small risk for cancer.2 By
definition, susceptibility alleles increase an individual’s risk of developing
cancer either within families or across populations, but there are
instances of pleiotropy in which a particular variant may confer
susceptibility to one type of cancer yet protect against another type
of cancer. It is notable that not all susceptibility alleles for a given
gene have equal estimated effects. Consequently, the observed spectrum
of established susceptibility alleles reflects an inverse relationship
between the effect size and the frequency of the genetic variation (Fig.
21.1).10–12 So far, approximately 125 hereditary cancer predisposition
genes (CPGs) have been identified, and their presence is most evident
in studies with familial clustering of one or more cancers.13 Moreover,
the majority of the hereditary predisposition genes are known somatic
mutational drivers of cancer, highlighting an important principle first
outlined by Knudson in 1971.14 The first set of CPGs was discovered
in family studies using linkage analysis, but now next-generation
sequencing (NGS) analysis has accelerated the discovery of new genes

323
324 Part I: Science and Clinical Oncology
Effect size
50.0
High Rare alleles
causing
mendelian
disease
3.0
Intermediate
1.5
Modest
1.1
Low
0.001
Very rare Rare
Figure 21.1  •  Feasibility of identifying genetic variants by risk allele frequency and strength of genetic effect (odds ratio). GWA, Genome-wide
association.
and specific mutations in known CPGs. Susceptibility alleles that
have an appreciable frequency in the general population, and smaller
effect sizes, can be discovered through association studies in which
the genomes of a set of affected cases are compared with those of
unaffected cancer-free or population-based controls.11
Genetic mapping of cancer susceptibility genes has shown that
many signals map to nongenic regions, important in the regulation
of genes or pathways of interacting genes. Although the direct public
health impact associated with conclusively establishing a specific
cancer susceptibility allele may not be immediately apparent, its
contribution to understanding tumor development and metastasis
is invaluable, expanding possible pathways and putative targets for
intervention downstream. Moreover, the possible clinical value of known
susceptibility alleles will continue to increase as more comprehensive
maps of susceptibility alleles emerge for specific cancers. In this
regard, sets of variants tested together show great promise for risk
stratification, important for both the individual and at the population
level. Roughly 10% to 15% of cancer susceptibility alleles are shared
among cancer types, and in some circumstances there are distinct
differences by subtype (e.g., specific alleles for estrogen-negative breast
cancer). To define the genetic architecture (see Fig. 21.1), namely
the constellation of susceptibility alleles that contributes to a specific
cancer, continued efforts are required to define comprehensive sets
of variants, which in turn should emerge as vital tools in both public
health and individual (known as precision medicine) assessments of
cancer risk.9,12
FUNDAMENTAL SCIENCE
Genetic Variation in the Human Genome
The annotation of sequence variation in the genome has provided
important clues toward elucidation of the genetic history of distinct
populations, the possible impact of environmental or pathogenic assault
on the genome, and the heterogeneous distribution of human cancers.
The differences in allele frequency spectrum, as well as types of genetic
variation, from SNPs to large copy number variants, have become
Low-frequency
variants with
intermediate effect
Common
variants
implicated in
common disease
by GWA
0.005 0.05
Low frequency Common
Allele frequency
indispensable tools for geneticists in identifying cancer susceptibility
alleles (Fig. 21.2).6,7,15–17
The search for common alleles is predicated on conclusively finding
“markers” that highlight a region of the genome where a disease
susceptibility allele resides; subsequent mapping and laboratory studies
are required to understand the link between the susceptibility allele
and its biologic association with disease.18 Sets of markers to test are
drawn from dense maps of human genetic variation that are publicly
available. Although there has been great value in embracing this indirect
approach (Fig. 21.3), it comes at a price—namely, many additional
steps are needed in order to sort through the correlated variants and
then conduct the functional studies that are necessary to illuminate
the underpinnings of the susceptibility allele.11,19
Until whole human genome sequence became available, the field
of genetics created maps of variant coordinates based on incomplete
assembly of DNA segments spanning the human genome. Sets of
markers can be thought of as molecular street signs, which allowed
scientists to knowingly navigate their way up or down a region of a
chromosome. Early on, “genetic maps” provided a stable reference for
mapping highly penetrant mutations, primarily in families. These
were based on empiric evidence of recombination hot spots. The
long-standing value of functional elements, herein recombination
frequencies, served adequately for the mapping of disease and traits
until genome sequencing emerged as a viable tool. The emergence of
a physical map of the human genome that establishes the true order
of most DNA segments (currently tractable for more than 95% of
the genome) has accelerated the mapping of traits and diseases, as it
produced absolute coordinates, or street signs for variants within the
genome—that is, the nucleotide location of a given marker or gene
in millions of base pairs from the end or terminus of the chromosome
is generally known.
The principle of meiotic recombination is critical to understanding
the relationship between genetic loci, here defined as variants that
map to unique coordinates. The correlation between genetic markers
is fundamental to both association and linkage analysis. In meiosis,
the cell division leading to gamete formation pairs homologous
chromosomes. Each chromosome consists of two identical strands
 Discovery and Characterization of Cancer Genetic Susceptibility Alleles  •  CHAPTER 21 325

Single nucleotide Molecular

Sequence
variation
• Base change – substitution – point mutation genetic
• Insertion-deletions (“indels”) detection
• SNPs – tagSNPs

2 bp to 1,000 bp
• Microsatellites, minisatellites
• Indels
• Inversions
• Di-, tri-, tetranucleotide repeats
• VNTRs

1 kb to submicroscopic
• Copy number variants (CNVs)
• Segmental duplications
• Inversions, translocations
• CNV regions (CNVRs)
Structural variation

• Microdeletions, microduplications

Microscopic to subchromosomal
• Segmental aneusomy
• Chromosomal deletions – losses
• Chromosomal insertions – gains
• Chromosomal inversions
• Intrachromosomal translocations
• Chromosomal abnormality
• Heteromorphisms
• Fragile sites

Whole chromosomal to whole genome

• Interchromosomal translocations
• Ring chromosomes, isochromosomes
• Marker chromosomes
• Aneuploidy
Cytogenetic
• Aneusomy detection

Figure 21.2  •  Spectrum of variation observed in the genome. The figure depicts both the size and scope of variants as a function of their length and
density in the genome. SNPs, Single-nucleotide polymorphisms; VNTRs, variable number tandem repeats.

(chromatids), with each chromosome pairing composed of four strands.
Homologous chromosomes separate from each other during the process
of meiosis except at one or two zones of contact in a process that
leads to genetic recombination (Fig. 21.4). Mendel’s second law,
independent assortment, states that alleles of genes at unlinked loci,
such those on different chromosomes or at the ends of a chromosome,
segregate or assort independently. Deviations from independent
assortment occur when genes are located close to one another, in
which case alleles assort together more than 50% of the time. In this
scenario, the associated loci are “linked.” Distributed throughout the
genome are recombination hot spots, which “divide” the genome
during meiotic formation of egg and sperm. These can vary by popula-
tion genetic history, providing an opportunity to compare groups and
use the differences to pinpoint possible susceptibility alleles, especially
if there are substantive differences between populations with respect
to cancer incidence.
The spectrum of human genetic variation varies by the frequency
of polymorphisms, which is often substantial among populations.
The most common sequence variation is the substitution of a single
base, known as a single-nucleotide polymorphism, which, by definition,
must be observed in at least 1% in one or more populations. The
minor allele frequency (MAF) refers to the lower allele frequency, often
varying by population ancestry. A substantially larger fraction of genetic
variation exists for single base substitutions below 1%—and many of
these are population private, reflecting population genetics history.20,21
The majority of SNPs with an MAF greater than 10% are common
to most human populations, but the actual frequencies can vary.
Reported SNPs are cataloged in db-SNP (http://www.ncbi.nlm.nih.gov/
snp), which is an important reference that is useful for interpreting
variants identified through DNA sequencing.
A small subset of SNPs are located in exons, of which a fraction
change the predicted amino acid. SNPs that can alter the coding
sequence are known as nonsynonymous SNPs, whereas those that are
silent are termed synonymous. Although there has been great interest
in coding SNPs, partly because they appear to be more interpretable,
very few of the known associations between a disease and a common
SNP marker (MAF > 10%) involve coding SNPs. On the other hand,
rare highly penetrant mutations mainly map to coding changes or
preterminal stop codons. Many of the reported disease mutations,
known as cancer predisposition genes, are cataloged in public databases
such as Online Mendelian Inheritance in Man (https://www.omim.org/)
or Catalogue of Somatic Mutations in Cancer (COSMIC; http://
cancer.sanger.ac.uk/cosmic).22
SNP frequencies become fixed in populations over multiple genera-
tions and are generally not inherited independent of the adjacent
variants. Recombination hot spots can separate sets of highly correlated
variants, resulting in haplotype blocks (Fig. 21.5).23 These segments of
a chromosome, usually quite small, are transmitted as a unit from
326 Part I: Science and Clinical Oncology

i
a b c d e f

ii Direct test

a b c d e f

iii Indirect test Indirect test

a b c d e f

iv
1
a c d
Figure 21.4  •  Genetic recombination is the process of exchanging genetic
2 information between two chromatids during meiosis. The recombination events
b f
3 for a single chromosome within a family are illustrated. The father’s homologous
e chromosomes are light and dark purple, and the mother’s are light and dark
green. Recombination events occurring during meiosis create unique parental
chromosomes.
Figure 21.3  •  Direct versus indirect association testing. (i) Six common
single-nucleotide polymorphisms (SNPs) as they would be represented in a
population sample. SNP-c is responsible for conferring a disease phenotype
on carriers. In a direct test (ii), SNP-c would be directly assayed and tested
for association with the disease, perhaps based on prior evidence of structural
or functional consequences of variation at this site. In contrast, the indirect
approach (iii) is agnostic with regard to functional variation. The assayed Unlinked
markers need only be in linkage disequilibrium with the causative variant to
achieve a signal of association. The caveat with this method is that care must
locus 1 AB CC
be taken to type the appropriate markers needed to ensure thorough coverage
of a given region. In the hypothetical example shown, tests of association locus 2 XY ZZ
between disease status and genotype at SNP-b, SNP-e, or SNP-f would prove
nonsignificant. Only SNP-a and SNP-d are indirectly associated with the
disease. The reason (iv) is that SNPs arise on independent haplotypic back-
grounds and that many common haplotypes exist at a given locus (three are
illustrated in the example, but in a true scenario many more are likely to be AC BC AC BC AC BC AC BC
present). If we assume that SNP-c arose on haplotype 1, we can see that XZ YZ YZ XZ XZ XZ YZ YZ
assaying the SNPs that define haplotypes 2 and 3 will not be useful in A NR NR R R NR R R NR
demonstrating an association of this locus with the disease. Instead, to fully
analyze this region, we must assay at least one haplotype “tagging” SNP from
each of the observed haplotypes. Linked

locus 1 AB CC
locus 2 XY ZZ
one generation to the next. The correlation between SNPs is an estimate
of linkage disequilibrium (LD), which is classically defined as the
nonrandom association of alleles at different loci. Individual SNPs
that always track together are said to be in strong LD. This correlation
can be eroded over time by recombination (exchange of genetic material) AC BC AC BC AC BC AC AC
during meiosis, and SNPs can be defined as being in weak LD,24 XZ YZ XZ YZ XZ YZ XZ YZ
signaling a correlation that is not necessarily strong. Measurement of B NR NR NR NR NR NR NR R
LD is with either D′ or r2 statistics.
The concept of LD is important because it enables investigators Figure 21.5  •  Linked and unlinked markers segregating in two families.
to evaluate sets of SNPs and determine proxies for other, untested Below the symbols, the genotypes for both markers are listed. Offspring have
SNPs in a region; this is useful for indirect mapping. If a group of either recombinant (R) or nonrecombinant (NR) haplotypes. The father is
SNPs is in strong LD (e.g., they are inherited together), one can test heterozygous for marker 1 (AB) and marker 2 (XY); and the mother is
for the alleles of just one reference SNP and immediately have informa- homozygous for both markers (CC and ZZ). (A) If the markers were unlinked,
there would be equal numbers of R and NR haplotypes from the father (AX,
tion regarding alleles segregating in a given individual for all adjacent, BY, AY, and BX). (B) There is an excess of NR haplotypes (AX and BY), and
correlated SNPs. By extension, estimates of LD are useful to construct only one R haplotype appears. Therefore these loci are linked.
haplotypes in unrelated subjects. With new reference data sets (e.g.,
1000 Genomes Project), it is possible to reliably determine or “impute”
untested variants using the backbone of stable data sets.25 Computational
efficiencies enable estimation of the correlation between sets of markers
 Discovery and Characterization of Cancer Genetic Susceptibility Alleles  •  CHAPTER 21 327

and the construction of haplotypes.25 Still, the most reliable approach
is to resolve the phase of haplotypes in multigeneration pedigrees, in
which haplotypes can be traced; alternatively, one can infer the relation-
ship of alleles in unrelated subjects with computational tools.26 Phase
refers to the parental (and grandparental) chromosome of origin for
a set of alleles.27 This information can also be useful for determining
where in a family a disease allele originates.27
The annotation of the human genome has revealed a wide spectrum
of structural variations, which may be either cytologically visible or
detected with either microarray chips or actual sequence analysis (see
Fig. 21.2). Historically, short tandem repeats (STRs) are a class of
polymorphisms in which there is a reiteration of a small number of
base pairs, such as CACACA. Polymerase chain reaction (PCR) primers
are used to define the distinct physical location of one STR from the
remaining 50,000 in the genome and to ascertain the number of
repeats for both chromosomes for any given STR. Also known as
microsatellites, they have been used for linkage analysis and forensic
investigation. Structural variants of all sizes can include deletions,
insertions, and duplications collectively known as copy number variations
(CNVs). In addition, there are infrequent inversions and translocations
of pieces of DNA that vary in size. Some of these are quite common;
chromosome 17 harbors an inversion of 3.5 million base pairs in
approximately 20% of the European population.28 CNVs have been
shown to influence gene dosage and therefore can contribute to risk
for cancer, as demonstrated for a chromosome 1 CNV and risk for
childhood neuroblastoma.29 There have been formidable technical
challenges in calling CNVs, but with new resources and new sequencing
technologies termed next-generation sequencing, it is anticipated that
accuracy will continue to improve,30 which in turn should improve
the capacity to associate CNVs with disease outcomes.
Principles of Linkage Mapping
Many epidemiologic studies have indicated the presence of a familial
contribution, such as the observation that family history of a specific
cancer within first-degree relatives is associated with a doubling of
risk or greater among relatives, particularly in twin registries.31,32 In
the case of prostate cancer, for instance, studies of selected hospital-based
patient populations, population-based case-control studies, and cohort
studies all have demonstrated that a family history of disease is correlated
with an increase in an individual’s risk. If the affected family members
are first-degree relatives (i.e., brothers, fathers, sons), the risk increases
from 1.7-fold to 3.7-fold. Younger ages at diagnosis and multiple
affected relatives with the disease tend to be associated with even
higher relative risk (RR).33–35 For example, men with three or more
first-degree relatives with prostate cancer have an almost 11-fold
increased risk of the disease compared with men who have no known
family history of the disease.34 For this reason, families ascertained
for linkage analysis studies tend to be large, have multiple affected
individuals, and feature people who were diagnosed with the disease
at a comparatively young age.
Familial aggregation describes the occurrence of multiple cases of
cancer within a family (Fig. 21.6). Clustering of familial cases can
also be due to shared environment, shared alleles of particular genes,
or simply chance if the tumor is common in the population. In
mapping cancer susceptibility genes for many cancers, particularly for
breast cancer and colon cancer, the most promising pedigrees for
hereditary cancer are families with three or more first-degree relatives
with a given cancer, three successive generations with cancer, or at
least two siblings with the same cancer detected at a relatively young
age. For common cancers, details regarding tumor stage and grade at
diagnosis, histopathology, and response to treatment are key to linkage
analysis study design because individuals who share similar disease
features likely share common susceptibility alleles. For instance, if a
subset of affected individuals in the family in Fig. 21.7 all had tumors
of similar stage and grade, the data from this homogenous subset of
individuals could be considered in isolation from the rest of the affected
cases thus reducing heterogeneity and increasing power. For common
diseases there are many common susceptibility alleles that account
for a substantial fraction of the underlying genetic architecture of
cancer susceptibility,11,36 and this approach generally does not work.
However, it has proven useful in discovery of highly penetrant alleles
for many cancers (e.g., breast and colon cancers).37,38
To identify highly penetrant mutations, success directly correlates
with the identification and collection of high-risk or hereditary families.
To achieve the numbers needed to improve the power to detect a
disease allele, whether using SNP arrays or NGS, large consortia are
often formed, providing an opportunity to increase power through
addition of more families and the chance to define the phenotype—
namely, the required clinical features and family history.39 Large
consortium studies provide an opportunity to conduct segregation
analysis, which determines the most likely genetic model that could
account for the disease (e.g., dominant, codominant, recessive, or
sex-linked). Additional informative analyses include an estimate of

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
00
02 06 82
81 81 03
00 96
63 00
02 00
03 77 02 00 00 61
02 01 00 70 87
21 77 00 77
70 81 97
20 77 70
20 69 97
68 94
26 8 63 81 91
81 83
65 96
32 83
25 83
80

25
25

Figure 21.6  •  Correlation of variants in a linkage disequilibrium plot. A region of the genome is depicted between two recombination hot spots that
shows the relationship between variants based on either D′ or r2 analysis. The red color indicates a high degree of correlation between variants.
328 Part I: Science and Clinical Oncology
76
58 68 70 62
A 65 80 78 45 50 48 45
41
46 47 45
B 46 48 50 42
Figure 21.7  •  Two theoretical breast cancer families. Age at diagnosis is
indicated below the symbol; males are indicated by squares, and females by
circles. (A) The family has many members with breast cancer, but some were
given diagnoses relatively early in life (younger than 50 years), whereas others
were much older at diagnosis (older than 70 years). The usefulness of this
family for genetic mapping studies is therefore limiting because it likely contains
individuals with both sporadic and hereditary breast cancer. (B) All individuals
were affected at an early age, but breast cancer, caused by mutations in either
the same or different genes, is present on both sides of the family. Because
there is no way to distinguish the number of mutant genes, a priori the usefulness
of this family for a genome-wide scan is also somewhat limited.
the frequency and penetrance of the disease allele(s) in the general
population, age-dependent penetrance, and potential number of loci
contributing to the disease.
High-risk or hereditary families must be ascertained through use
of appropriate guidelines for working with human subjects to collect
biospecimens, such as germline DNA from blood or buccal materials,
somatic or tumor tissue for DNA or RNA analyses, and other body
fluids for determination of biomarkers that could be useful in subse-
quent early detection in high-risk settings. Identification of cancer
families and collection of critical medical information including family
history, medical record data, and DNA samples are generally regulated
by institutional review boards (IRBs) and require informed consent.
Families must be identified and approached in a way that is neither
intrusive nor coercive. Although genetic epidemiologists have success-
fully turned to new approaches, such as social media, to ascertain
families, most families are made aware of or referred to studies by
their personal physicians.
To carry out a successful family-based genetic study, medical record
data must be carefully and systematically extracted into well-protected
databases. Family history data must also be obtained redundantly
from multiple family members, with care taken to resolve discrepan-
cies, including nonpaternity events. Consent to contact other family
members regarding the study is needed, as is permission to obtain
medical records and to recontact study participants years after initial
data collection. The protection of individual privacy is paramount,
and personal identifiers such as names and complete addresses are
rigorously protected, even from most of the scientists participating in
the study.
DNA samples from appropriate family members can be screened
by using either a set of highly polymorphic markers that span the
genome at a sufficiently high density or NGS analysis of the whole
genome or the exome.
Theoretically, a given set of affected individuals within a family
would all carry precisely the same underlying mutation in the relevant
susceptibility gene—that is, each member would have inherited not
just a mutated copy of the same gene, but the same deleterious variants
in that gene. Because there are distinct mutations within a gene, each
of which can confer high penetrance, the approach is predicated on
finding a gene and not the specific mutation within a gene. For example,
there are many mutations and variants of unknown significance across
the BRCA1 gene that can confer an increased risk for breast and/or
ovarian cancer, with measurable differences in penetrance now confirmed
by prospective studies.40,41 This latter point suggests that there are
differential effects of disturbances of key biologic pathways. Moreover,
recent genome-wide association studies (GWASs) have begun to identify
secondary, genetic modifiers that further modulate the penetrance of
BRCA1 mutations.42,43
Fig. 21.7 demonstrates two types of seemingly useful families for
linkage mapping studies. Both include a significant number of affected
members. The first family has a large number of affected individuals
(see Fig. 21.5). However, some individuals were affected very early in
life, whereas others were diagnosed at later ages. It is likely that some
individuals have the disease because they inherited mutated copies of
a particular gene, whereas others have the disease for sporadic reasons
unrelated to the disease allele segregating in the family. Age at onset
provides guidance as to which individuals are more likely to have
hereditary versus sporadic forms of the disease; but this is not absolute,
and in the case of a disease with age-dependent penetrance, some
people will be affected late in life, even though they carry a mutant
allele, and others will be affected early in life for sporadic reasons.
The second family shown in Fig. 21.5 appears to be more informative
for linkage mapping studies because there are several affected individuals
in the family and all were affected at a relatively early age. However,
the presence of disease segregating on both sides of the family should
be noted. The affected individuals in the youngest generation could
have cancer because they inherited mutant alleles from one or both
sides of their family, and one or multiple genes could be involved.
This scenario happens frequently in studies of common cancers such
as those of the colon, breast, and prostate. Thus the family is of limited
usefulness for mapping studies.
Challenges in Finding Cancer Susceptibility Genes
Traditional approaches have been successful in identifying highly
penetrant mutations in multiply affected families for both common
and uncommon cancers. A combination of linkage and candidate
gene analyses revealed mutations in CDKN2A or CDK4 in roughly
50% of familial melanomas, although there appears to be heterogeneity
in exposure to a strong carcinogen for melanoma, ultraviolet sunrays.44
Newer approaches involving analysis of smaller family structure together
with functional laboratory data have identified rare, important muta-
tions in melanoma. For a rare familial cancer, chordoma, a gene
duplication of the T (brachyury) gene confers susceptibility.45 Using
NGS, investigators are revisiting families whose disease was not
understood when traditional methods were used, and are now searching
for sets of susceptibility alleles that can explain an oligogenic risk
model—that is, a set of variants with moderate risk that are neither
sufficient nor necessary for development of a cancer (e.g., MITF is a
moderate-risk gene for melanoma).46,47
The early-onset breast cancer susceptibility genes BRCA1 and
BRCA2 were among the first to be mapped because large and well-
characterized families had been meticulously ascertained. In addition,
deleterious alleles were highly penetrant, often at an early age, leaving
little ambiguity as to who in a family should be counted as a “case.”
The presence of ovarian cancer in some families and not others and
 Discovery and Characterization of Cancer Genetic Susceptibility Alleles  •  CHAPTER 21 329
the presence of breast cancer in some male carriers allowed for creation
of data sets enriched for the BRCA1 and BRCA2 genes, respectively.
In turn, the initial identification of the BRCA1 gene and subsequent
removal of BRCA1-linked families from remaining data sets provided
further useful enrichment for BRCA2-linked families.48,49 For the breast
cancer susceptibility genes BRCA1 and BRCA2, founder mutations
have been identified in distinct populations across the globe,50,51 a
phenomenon most notably observed in Jewish Ashkenazi families.52,53
The three common founder mutations in this population, BRCA1-
185delAG, BRCA1-5382insC, and BRCA2-6174delT, have a combined
population prevalence of 2% to 2.5%. With these observations in
mind, investigators have frequently sought families for genetic mapping
studies from regions of the world where marriage between related
individuals is not discouraged and where geographic barriers have
restricted gene flow.
Locus heterogeneity, the presence of deleterious alleles associated
with many genes in a population, can be reduced by studying families
from isolated or inbred populations. Fewer disease alleles are predicted
to segregate with a particular phenotype in a population derived from
a limited number of founders. Studies of colon cancer in Finland and
breast cancer in Iceland as well as Ashkenazi Jewish populations illustrate
this point. In Finland, two variants in the DNA mismatch repair gene
MLH1—mutations 1 and 2—account for 51% of all Finnish families
with verified or putative cases of hereditary nonpolyposis colorectal
cancer.54 Nineteen mutation 1 and six mutation 2 families were further
investigated by haplotype analysis using 15 microsatellite markers
surrounding the MLH1 locus. The presence of two distinct large
conserved disease haplotypes, one in mutation 1 and the other in
mutation 2 families, indicated that these families are likely to descend
from two distinct common ancestors born in the 16th and 18th
centuries, respectively.
Principles of Association Testing
In the past, genetic linkage analysis was the workhorse for discovery
of CPGs, but the new tools of massively parallel genotyping and
sequencing have changed the landscape of discovery, enabling pursuit
of complex diseases, namely those in which multiple distinct genetic
regions plus environmental factors contribute to risk for disease.
Linkage analysis has been of limited success in studies of common
and/or complex diseases, primarily because of insufficient power
to detect association of smaller effect sizes for multiple susceptibil-
ity alleles and complexities in phenotype assignment. Risch and
Merikangas55 pointed out the shortcomings of linkage for complex
disease mapping and made the case for association analyses in populations
of unrelated subjects. Their projections have been borne out in the age
of GWASs.11,56
In the failed candidate gene era, investigators chose specific variants
based on prior hypotheses and analyzed underpowered studies, yielding
a sea of false-positive reports. The most notable examples include
common variants in NAT2 and GSTM1 in bladder cancer and alcohol
dehydrogenase genes (ADH1B and ADH7) in aerodigestive cancers.57–59
As the annotation of the human genome emerged with first the
International HapMap Project and then the 1000 Genomes Project,
the approach shifted toward use of surrogate markers across the genome
designed to capture the majority of common genetic variation using
the GWAS design.60
GWASs have been successfully applied across a spectrum of diseases
and traits, yielding thousands of loci harboring common susceptibility
variants, associated with hundreds diseases and traits.56 In 2017 there
were more than 725 independent cancer susceptibility loci associated
with at least 30 different cancers, most observed in individuals of
European ancestry. The approach is based on an initial scan across
the genome followed by independent replication.61 The results of
scanning hundreds of thousands of SNPs are analyzed with an “agnostic”
statistical approach, using logistic regression analysis that is often, but
not always, adjusted for critical covariates (e.g., age or subtle differences
in underlying population genetics history). GWASs are pursued free
of prior hypotheses, unlike standard linkage analysis.
The GWAS strategy is based on testing indirectly for the actual
genetic variant using surrogate markers in data sets of cases and controls
to highlight regions harboring susceptibility alleles.18 Thus the functional
marker directly responsible for the effect does not have to be actually
tested in the scan. Instead, its surrogate, which is in LD as measured
by a high correlation (r2 > 0.8), can be replicated in subsequent studies,
pointing to the susceptibility allele(s). Hence, substantial effort is
required to fine-map the region—that is, to find all of the correlated
variants before choosing which ones to evaluate in follow-up studies.
In many regions, variants with lower minor allele frequencies are also
highly correlated with the GWAS marker, and on occasion a less
common allele with a stronger effect has been identified, yielding a
so-called synthetic association.62 However, to date, the majority of
susceptibility alleles cannot be explained by less common variants
with stronger effects.63
Patterns of LD vary among populations, both in specific regions
and across the genome.23 This results in major differences in MAF
among populations on a per-SNP basis, as well as the intervals of LD
defined by recombination hot spots. In rare circumstances, the dif-
ferences between incidences in disease, such as the incidence of prostate
cancer between men of European and of African ancestry, can be
explored by using admixture linkage analysis. Notably, one of the first
GWAS signals for prostate cancer on 8q24 was also found by admixture
analysis.64–66
The basic principle behind an association study is that there is a sta-
tistically significant difference in the distribution of one or more alleles
between cases and controls, indicating the location of a susceptibility
allele that contributes to cancer risk. Although association studies can
uncover highly penetrant mutations that are strongly correlated with
cancer risk, much as the family linkage studies discussed earlier, their real
usefulness is in finding common low-penetrant alleles that contribute
to cancer risk. This is reflected in the fact that the estimated odds
ratios for alleles found in GWASs are low, almost always estimated at
less than 1.3, and thus such alleles are not efficiently detected through
linkage.12 These low-effect susceptibility alleles are neither necessary
nor sufficient for development of a cancer. Indeed, for most forms
of cancer we expect that there are many susceptibility alleles, each of
which contributes a small effect to the disease.
Investigators use one of several commercial SNP microarray chips
to scan the genome and compute rank P values for prioritization of
promising genetic markers for replication studies, which are typically
performed using one or more independent data sets of cases and
controls. Because of the daunting challenge of false positives in testing
so many markers, a community-wide standard has emerged, one that
protects against false-positive findings; this is achieved when studies
report markers that surpass the threshold of genome-wide significance,
now generally defined as a trend association test with a P value of 5 ×
10−8. This can be reported in the primary scan, if large enough, or in
a combined analysis of the scan with the replication sets. More recently,
large meta-analyses that combine data from several independently
collected datasets are used for confirmation. Because GWASs discover
loci that are highly associated with specific markers, surpassing this
threshold ensures that there is a very small probability of a false-positive
result. This is particularly important given that extensive follow-up
analyses are required to map and investigate the biologic underpinning
of the susceptibility allele.
Because GWASs can be effectively scaled to accelerate discovery,
the major challenge ahead is to determine how to establish the critical
connection between the genetic markers of susceptibility alleles and
carcinogenesis. The rapid pace of discovery with use of the GWAS
approach has not been matched by research to interpret and understand
the functional significance of different alleles correlated with cancer
phenotypes.67 The gap between the number of new independent
markers and a biologic understanding of the loci continues to widen
at an accelerated pace. This is a result of the differences in scientific
330 Part I: Science and Clinical Oncology

22
21
Mosaic Gain

1
20

10
20
30
30
40

40
20

50
10

60
0

70
80

100
40

90

110
0
30

120
20
10

130
140
150
0
60

160
Mosaic Copy Neutral

50
19

170
40
30

180
20

190
10

200
210
0
60

220
50

230
40

240
0 3
Mosaic Loss

20
10
18

0
10
0

20
70

30
60

40
50

50
40

60
30

70
20

80
10

90

2
11 0
0

10
17

0
70

0
12
60

50 0
13 0
14 0
40

30
20 15 0
16 0
17 0
10 0

18 0
19 0
16 60
70
80
20 0
21 0
50 22 0
40 23 0
30 24
20 0
10 10
0 20
100 30
90 40
80 50

15 60
70 60
70
50
40
80
90 3
30 100
110
20
120
10
130
0 140
100 150
90 160
80 170
180
14 70
60 190
50
40 0
30 10
20 20
10 30
0 40
50
110 60
100 70
90 80
80
70
90
4
13
100
60 110
50 120
40 130
30 140
20 150
10 160
0 170
130 180
190
120
110 0
100 10
90 20
80 30

12
70 40
60 50
50 60
70
40
30 80
20
10 10
90
0
5
0 11
12 0
0 0
13 13
0 0
12 14
0 0
11 15
0 16 0
10
90 17 0
0
80 18
70 0

11
0
60 10
50 20
40 30
30 40
20

50
10
0

60 0
7
12 0
13

80
11 0

90 0
10 0

10 0
0

6
11 0
90

12 0
80

13 0
70

14
60

15 0
50

16
10

0
40

17
30

0
0
20

10
10

20
0

30
140

40
50
130

60
120
110

70
100

80
90
90

100
80

110
70

120
60

130
50

140

7
40

150
30

0
20
10

10
0

20
9

140

30
130

40
110
120

50
100

60
70
90
80
8

Figure 21.8  •  Clonal somatic mosaicism of large structural events, based on genome-wide association study data from over 125,000 patients and control
subjects, the distribution of types of events (loss, gain, and copy-neutral loss of heterozygosity) across the autosomal chromosomes. Autosomal events are
depicted for 1315 events greater than 2 Mb (0.75% overall) (Data from reference 118).

approaches; the GWAS approach is scalable if surrogate markers are alcohol, or weight/height) are collected, the latter to study the degree
used across the genome so that with larger sample sets further discovery to which an exposure is associated with disease incidence. Exposure
is possible. However, despite the fact that SNP chips with over 1 is measured at baseline, when the cohort is initially established, and
million markers are available, rarely is the marker also the functional may be updated over the period of follow-up for those exposures
variant. Thus follow-up analyses are required to characterize each that may change over time. The advantages of cohort studies include
susceptibility allele. The patterns of genetic variation vary greatly across minimized information and selection biases and the ability to directly
regions thus requiring a detailed fine-mapping of each region prior to calculate disease incidence in exposed and unexposed groups, and
choosing individual genetic variants for laboratory study (Fig. 21.8). thus the RR and absolute risk (attributable risk, AR). Disadvantages
include the fact that prospective cohort studies are expensive and
Study Design and Association Studies time-consuming and large numbers of study subjects are typically
required for sufficient numbers of outcomes (i.e., cancer cases) to be
For association studies, two primary types of study design are typi- obtained to have adequate power to determine associations. Loss of
cally used: cohort and case-control studies. Recently, however, the subjects to long-term follow-up is also an issue. Cohort studies can
discovery of GWAS regions have come at the expense of epidemiologic be retrospective (i.e., the exposure and subsequent development of
rigor with respect to control selection. In a cohort study, subjects are the disease occur before the study begins).
selected, individuals with the disease of interest (i.e., prevalent cases) Case-control studies differ from cohort studies in that the selection
are excluded, one or more exposures of interest are measured and of subjects is based on disease status, providing an opportunity to
monitored over time, and biospecimens are archived. Cancer and examine multiple risk factors. They are generally either population-based
intermediate outcomes, such as risk factors for cancer (e.g., smoking, or hospital-based. Selection of patients and control subjects should
 Discovery and Characterization of Cancer Genetic Susceptibility Alleles  •  CHAPTER 21 331
account for confounding factors such as age, sex, race, and ethnic
background. Controls must be selected from the same underlying
population from which the cases were ascertained in order to avoid
stratification, either by differences in genetic background or by risk
or exposure.
Population-based case-control studies draw on a well-defined source
population such as a particular geographic region defined by state,
county, or city for ascertainment of both case patients and unaffected
control subjects. Control subjects should be selected from the same
source population by a method designed to randomly sample individu-
als; historically this has been random digit telephone dialing. Selection
bias, in which selection of case patients, control subjects, or both is
influenced by prior exposures, is a particular concern in case-control
studies. For instance, many studies have shown that nonparticipants
in such studies are more likely to smoke than individuals who agree
to participate. Therefore there is concern that participants may be
more health conscious than nonparticipants. Also, in recent years the
lack of a landline phone in many homes has hindered efforts to enroll
randomly selected controls, potentially compromising the unbiased
nature of control enrollment.
In comparison, hospital-based, case-control studies enlist a sequential
series of patients who are admitted to the hospital or clinic during a
specific period. Case patients are enrolled because they have the cancer
of interest, whereas control subjects are determined to be cancer free,
although they may be patients at the same clinic or hospital for unrelated
reasons. Depending on the clinic or hospital from which patients are
drawn, disease presentation, severity, and treatment outcome may be
nonrandom. Often cases are drawn from so called “high-risk” clinics,
whereas control subjects may come from nonrandom sampling as
well. Controls from clinics can later be determined to be suboptimal
because they have comorbidities or exposures that were initially thought
to be independent of the disease under study but are later found to
be confounders.68–71 Confounders are factors that are associated with
the exposure as well as the disease. Age is a frequent confounder for
cancer studies because the incidence of cancer advances with age, and
often with level of exposures. An important source of bias specific to
case-control studies and retrospective cohorts is recall bias, in which
study subjects inaccurately or incompletely remember information
related to disease susceptibility such as environmental or lifestyle factors
(e.g., diet, smoking, birth control history, exercise). This introduces
error into the calculation of the association between the exposure and
the disease because of the inability to statistically adjust for the effects
of confounders.
Until the age of GWASs, there was a vigorous debate in candidate
gene studies over the impact of possible differences in underlying
population substructure. The testing of thousands of uncorrelated
SNP markers in GWAS have provided investigators with the opportunity
to sift out individuals who differ substantively across the genome.
Several different analytic algorithms can distinguish the degree of
admixture across chromosomes, based on referential continental
population sets (e.g., International HapMap Project or 1000 Genome
Project).72 In fact, this has been used to map a handful of regions that
appear to be specific to one ancestral group compared with another.
Association Studies in Cancer
To date, over 725 genomic regions have been conclusively established
(i.e., achieving the threshold of genome-wide significance, which
protects against the likelihood of a false-positive discovery) for more
than two dozen distinct types of cancer.12 The majority of susceptibility
alleles are specific to one cancer, but roughly 10 to 15 are pleiotropic,
indicating that the effect can be observed for more than one type of
cancer. Nearly all cancer susceptibility alleles discovered by GWASs
have an MAF greater than 10% with a subset in the 5% to 10%
range as reported in large studies. Overall, the per-allele estimated
effect size has been small, with odds ratios between 1.1 and 1.3.
Several of the alleles reported in pediatric cancers have risks of 1.6 to
1.8, such as in Ewing sarcoma, a rare pediatric cancer, yet for the
adult cancers the norm is low.73 Among the most significant is testicular
cancer, known to have a high heritability in families and twin studies;
GWASs identified a susceptibility allele with a per-allele effect estimate
of greater than 2.5 on chromosome 12 (KITLG).74,75
Initially, the commercial SNP microarrays chips were designed to
efficiently capture SNPs with MAFs greater than 5% to 10%. It is
anticipated that new susceptibility alleles in the range of 1% to 10%
will be discovered with use of new SNP microarrays with lower MAF
content. Most of resulting alleles are expected to have small effect
sizes. In retrospect, these findings are understandable in light of the
power estimates for discovery of new alleles because small effect sizes
in less common alleles require larger sample sets. Less than 5% of the
susceptibility alleles conclusively established in cancer GWASs have
led to determination of an actual coding change in a gene. Most
regions map to regulatory regions in and around well-recognized genes,
a few of which have been implicated in cancer biology. Nearly one-
quarter of markers localize to intergenic regions, namely regions between
genes in which all of the SNPs in strong LD (r2 > 0.8) fail to localize
in or near a known gene. These findings suggest that a fraction of the
genetic contribution to cancer may reside in alterations in the regulation
of known or novel pathways.
A small fraction of susceptibility alleles have been associated with
more than one distinct cancer type. These are highly informative and
reveal possible common carcinogenic mechanisms underlying distinct
cancers. Included in these results are sometimes alleles within the
HLA regions, particularly for cancers of the immune system or those
driven by viral infections. Commercial arrays and imputation provide
inadequate discrimination of the region, particularly because of its
complex structure; hence other technologies will be required to
comprehensively explore this region in cancer susceptibility. The region
flanking the MYC oncogene on 8q24 is also extremely complex; it
harbors at least a dozen independent loci associated with prostate
cancer, as well as loci associated with multiple other cancers including
breast, colon, and bladder cancers and chronic lymphocytic leuke-
mia.64,76 The MYC oncogene itself is a plausible candidate gene, and
work has suggested that allelic differences in enhancers could directly
or indirectly interact with MYC, although there is little to suggest
that coding changes are important.77–79
A region on 5p15.33 harbors a range of susceptibility alleles for
many cancers, including rare and common SNP alleles. So far, more
than 10 distinct cancers have been associated with the region by means
of GWASs, including both breast and prostate cancer, and there are
at least five independent alleles in this region, which contains the
telomerase gene (TERT).80,81 Rare mutations in TERT track with
dyskeratosis congenita (an inherited bone marrow failure syndrome),
idiopathic pulmonary fibrosis, acute myelogenous leukemia, and chronic
lymphocytic leukemia.82,83 The pleiotropy in this region hints at complex
gene-gene or gene-environment interactions. For instance, a protective
allele for one cancer appears to be a susceptibility allele for another
cancer; it is remarkable that the same allele has inverse effects for two
skin cancers, basal cell carcinoma and melanoma.84
The discovery of new susceptibility alleles is now being driven by
the use of imputation, a technique based on computational algorithms
that infers untested and highly correlated SNPs based on reference
data sets (e.g., International HapMap Project or 1000 Genome
Project).85 It is predicated on the observed LD between SNPs in refer-
ence populations drawn from distinct regions. A notable exception is
the recent identification of a rare SNP on 8q24 conferring susceptibility
to prostate cancer, which was discovered and characterized in the
isolated population of Iceland.86
The fine-mapping of susceptibility alleles has led to the discovery
of independent signals nearby, indicating that more than one allele
contributes to cancer risk. The first prostate cancer susceptibility allele
on 8q24 has now blossomed to more than a dozen separate alleles,
several of which are distinctive to African Americans. This latter
observation could partially explain the substantial increase in incidence
332 Part I: Science and Clinical Oncology
in prostate cancer in African American men compared with those of
European ancestry.87 An intergenic region of 11q13 harbors several
independent prostate cancer susceptibility alleles88 but also nearby
distinct alleles for renal and breast cancer, each of which works through
separate mechanisms.89,90
The formation of numerous international consortia promises to
maintain the pace of discovery of common susceptibility variants, not
only in populations of European ancestry but also in those of other
ancestry, including studies from Asia and Africa.91 To date, the majority
of reported GWASs have been in subjects of European ancestry, but
progressively more studies have been conducted in subjects of Asian
and African ancestry. In a few instances, alleles have been identified
in specific populations with substantially higher frequency. For instance,
a region of chromosome 17q21 harbors a prostate cancer susceptibility
allele in African Americans, for which the best marker has an MAF
of 5%, whereas in individuals of European background it is below
1%.92 Additional alleles in 8q24 have been reported in men of African
American background, but they do not fully explain the difference
in incidence.
Differences in study design can lead to conflicting conclusions,
including the biologic interpretation of the association. Initially, two
distinct GWASs in prostate cancer reported contrary results for alleles
on chromosome 19q13.33, which harbors the gene responsible for
prostate-specific antigen (PSA).93,94 In a GWAS using cohort studies,
the effect appears to be related to PSA levels, whereas in a study using
advanced cases and control subjects with low PSA levels, the effect points
toward carcinogenesis. Follow-up studies including fine-mapping of the
KLK3 gene (which encodes PSA) and further replication indicate that
the locus could be associated with both prostate cancer susceptibility
and PSA levels.95,96 Recently, large GWASs have identified several dozen
variants specific to the level of PSA, establishing a foundation for
studying the genetics of a complex biomarker for prostate cancer risk.97
Genetic Architecture Underlying
Cancer Susceptibility
The underlying genetic architecture can differ by cancer sites with
respect to the relative contribution of common variants with small
effects through the spectrum to rare variants with strong effects (e.g.,
CPGs). As indicated in Fig. 21.1, the emerging picture suggests that
there are variants of low effect size, commonly seen in the population,
that contribute a fraction of the heritable risk for the cancer. Although
ongoing studies are expected to generate a more comprehensive catalog
of variants in each class (e.g., common and rare), early modeling of
empiric data suggests differences among cancers.98–101 In prostate cancer,
rare, highly penetrant mutations account for a very small component,
whereas the known common variants can account for at least one-third
of familial risk—namely, the estimated risk of other first-degree relatives
of a patient with a newly diagnosed cancer.102 For breast cancer, there
are more than a dozen highly penetrant CPGs, together accounting
for more than 15% of the familial risk, whereas the common variants
found by GWASs account for one-third of the familial risk.103 NGS
studies in families and now population-based studies have identified
breast cancer susceptibility alleles with moderate effects (e.g., estimated
RR between 2 and 4).104 It is interesting to note that the majority of
these susceptibility alleles map to genes in pathways related to BRCA1
activity, including ATM, BRIP1, CHEK2, ERCC2, PALB2, RAD51C,
and RAD51D.105,106 The rate of discovery of new mutated genes for
other cancers, both rare and common, continues to accelerate. Cor-
roborative laboratory work can supplement analyses in families and
population-based studies. For instance, a rare variant in the MITF
gene that has an allele frequency of approximately 1% increases the
risk for melanoma, a dangerous skin cancer.47 After familial testing
showed incomplete penetrance and association studies suggested an
effect in unrelated populations, laboratory investigation revealed that
the mutation resulted in impaired SUMOylation and differentially
regulated MITF targets.
Unraveling the Cancer Biology of Cancer
Susceptibility Alleles
One of the major surprises of cancer GWASs is that only a small
fraction of the more than 725 susceptibility alleles map to well-
characterized genes already implicated in cancer biology. Notably, the
regions defined by GWAS markers do not harbor genes that are
significantly mutated, especially as drivers, in large landscape charac-
terizations of somatic mutations.107 This is in distinct contrast to the
high overlap between CPGs and somatic drivers of cancer.13 Moreover,
the sets of GWAS signals by specific cancers generally do not correlate
with specific patterns or processes fundamental to the cancer. In this
regard, there is no evidence for enrichment for common variants in
DNA repair pathways that are clearly associated with risk for common
or rare cancers. New discoveries point toward possibly new biologic
mechanisms underlying cancer susceptibility. Because the interrogation
of each region is complex and requires extensive bioinformatics,
fine-mapping, and laboratory analysis specific to the region of the
genome, it is understandable why only three dozen susceptibility alleles
have been adequately interrogated. Typically each region harboring
one or more susceptibility alleles has a unique local pattern of LD.
It is notable that the breast cancer susceptibility locus BRCA1 was
first mapped in 1990 and that more than two decades later the biology
of BRCA1 and the consequences of germline mutations are still under
investigation, but specific therapeutic interventions are now clinically
available.
To unravel the biologic underpinnings of cancer susceptibility
regions, investigators are turning to new resources and approaches.67
Consortium guidelines have been developed to accelerate the pace of
characterization of cancer susceptibility alleles, beginning with fine-
mapping using new tools.108 For instance, progress in understanding
regions identified by GWASs has recently received a major boost by
the publication of the ENCODE Project (Encyclopedia of DNA
Elements), a far-reaching project to map the functional genome.109
The ENCODE papers have begun to shed light on the biology of
the regulation of the genome, specifically cataloging signposts and
markers of biologic activity. It has sharpened our vision of the inner
workings of how the genome functions. With ENCODE and its tool
packages, it is possible to trace the errors that contribute to cancer
susceptibility. It is now possible to look at patterns of regulatory
variation in individuals and across populations. With integration of
experimental data and computational tools, several in silico programs
should enable assessment of known susceptibility regions and prioritize
variants for further study, with a higher value assigned to those with
functional significance.110 New insights into breast cancer susceptibility
loci identified by GWAs have been generated through use of ENCODE
insights together with laboratory observations.
The search for functional variants underlying GWAS signals has
uncovered new insights with possible clinical implications. For example,
investigation of the bladder cancer GWAS signal on 8q24.2, followed
by RNA sequencing and genetic and functional analysis, identified a
variant that is strongly associated with increased mRNA expression
of the prostate stem cell antigen (PSCA) gene.111 Furthermore, this
variant creates an alternative translation start site and leads to increased
expression of PSCA on the cell surface, where it can be subjected to
immunotherapy with anti-PSCA antibody, an emerging therapy for
several cancers. Because the genotype of the GWAS variant is predictive
of PSCA protein expression, a genetic test could be used to identify
patients with bladder cancer who could benefit from the anti-PSCA
therapy.
A few of the GWAS susceptibility alleles have been conclusively
shown to interact with environmental exposures. An SNP in NAT2
is important for bladder cancer susceptibility only in people who have
“ever smoked,” whereas in never-smokers there is no effect.112 The
GWASs of lung cancer, strongly driven by tobacco exposure, have
revealed that select alleles are operative only in smokers whereas others
are observed only in nonsmokers. The strongest signal seen in smoking
 Discovery and Characterization of Cancer Genetic Susceptibility Alleles  •  CHAPTER 21 333
lung cancer GWASs on chromosome 15q25 is not evident in nonsmok-
ing women in Asia, suggesting that the contribution of 15q25 is not
associated with lung cancer, independent of smoking.113,114
Previously, many investigators have attempted to elucidate the
functional genetic variant and a mechanism underpinning the genetic
association between clearance of hepatitis C virus (HCV), a risk factor
for liver cancer, and genetic variants upstream of IFNL3, previously
called IL28B on chromosome 19, which were discovered in several
GWASs. However, RNA-sequencing analysis in primary human
hepatocytes has uncovered a new gene, IFNL4, which is generated
by a complex dinucleotide insertion/deletion variant in LD with
associated GWAS markers.115 The deletion allele of this variant causes
a frameshift that leads to a novel protein that induces an interferon-type
response. Genetic analysis showed that the IFNL4 genetic variant has
a strong effect on HCV clearance, especially in individuals of African
ancestry. The strength and the effect size of the genetic association in
IFNL4 leading to spontaneous and treatment-induced clearance of
HCV are large enough to warrant pursuit in clinical studies. Use of
this complex polymorphism is under active study for clinical usefulness
in directing therapy and outcomes in HCV treatment.
During the practice of applying stringent quality control metrics
to GWAS data, a pattern of unexpected deviations emerged that have
led to the investigation of the detection of genetic mosaicism, defined
as two or more distinct karyotypes within an individual.116 It is still
not clear whether the presence of large structural mosaicism is associated
with risk for nonhematologic cancer, as it is for chronic lymphocytic
leukemia.117 With use of SNP data, it is possible to estimate that
approximately 1% of the adult population harbors one or more large
autosomal somatic events (see Fig. 21.8).118,119 Mosaic events for the
sex chromosomes are even more frequent; roughly 15% to 20% of
men older than 50 years have mosaic loss of the Y chromosome, which
is particularly higher in smokers.120 In fact, the propensity to develop
mosaic loss of Y has been mapped by GWAS to nearly 20 distinct
regions in the genome.121,122
X chromosome mosaicism is observed in women and almost always
involves the inactive X chromosome, comparable to the reported higher
frequency of somatic mutations in the inactive X chromosome of
female cancer genomes.119,123 For hematopoietic malignancies, in cohort
Distribution of modifiable risk by deciles of nonmodifiable risk
30
25
20
Absolute risk (%)
15
00 0 0
10
00 0
0000 0
0000 0
5 Never drinker or
0
1 2 3 4
Decile of nonmodifiable risk
Figure 21.9  •  The impact of changes in lifestyle could be targeted at women with higher genetic risk for breast cancer. The distribution of deciles of
women based on the polygenic risk score for known breast cancer susceptibility alleles (n = 77). HRT, Hormone replacement therapy. (Data from reference 98.)
studies it is possible to detect somatic events well in advance of the
diagnosis of chronic lymphocytic leukemia.117 A more refined under-
standing of mosaicism in the aging genome promises new insights
into genomic instability as it relates to carcinogenesis, especially in
the hematopoietic system. Large-scale analyses of exome sequenced
subjects have indicated that point mutations frequently arise as mosaic
events in circulating blood, particularly involving genes critical in
hematopoietic diseases, such as DNMT3A, TET2, and TP53. Ongoing
studies are designed to elucidate the scope and possible risk for one
or more cancers related to mosaic events, from single-nucleotide to
large structural events involving an entire chromosome.124
GWASs in large consortia have also looked at intermediate exposures
strongly implicated in cancer risk, such as body mass and other
quantitative traits (e.g., tobacco use and alcohol consumption), yielding
new insights into biology. The study of height in hundreds of thousands
of subjects with GWAS data has uncovered novel pathways and provided
a more stable assessment of the contribution of common SNPs (MAF
>5%) to the trait. Polygenic models for many SNPs, each with small
effects not yet conclusively discovered by GWAS, define the fraction
of heritability for important traits such as height, weight, smoking
behavior, and, more recently, PSA levels and breast density on mam-
mography, all established to be associated with risk of cancer.97
As the number of individual cancer susceptibility alleles has increased
with larger GWAS studies, it is now possible to conduct polygenic
risk score (PRS) analyses, both in discovery sets and in validation
studies to define the fraction of the genetic architecture explained by
common variants, each contributing only a small fraction.125 Sufficiently
large GWAS efforts have been reported, and models for risk stratification
can be generated by using empiric data to calibrate the model. PRS
analyses can also be applied to large population-based studies to identify
the fraction of the population at highest genetic risk. In turn, this
fraction can be targeted in order to reduce modifiable risk factors and
decrease the burden of the cancer. Ongoing studies are evaluating the
impact of risk reduction programs targeting those at highest risk by
PRS analysis in order to reduce the incidence of breast cancer, a cancer
with a high absolute risk in the population (Fig. 21.9).98,103,126,127 This
approach is not unique to breast cancer and is being pursued for other
cancers, such as prostate, colon, and bladder cancers.
Heavy drinker,
0
smoker, obese/HRT
0 0 0
0000 0 0
0 00
0
00 0
00 00
smoker, no HRT use,
healthy weight
5 6 7 8 9 10
334 Part I: Science and Clinical Oncology
Clinical Implications of Cancer Susceptibility Alleles
It is clear from the association of many known cancer susceptibility
alleles with more than one cancer type that there may be distinct
alleles that influence the etiology of a cancer and a different set that
influence clinical outcomes, such as progression, treatment response,
or metastasis. More productive have been studies that used clinical
indicators such as high Gleason score as a surrogate for aggressive
disease risk. For instance, studies have identified a modest number of
genes associated with metastatic disease or death from prostate cancer
versus indolent disease.128,129 In the childhood cancer neuroblastoma,
regions have been identified that are associated with more aggressive
disease.130
The discipline of pharmacogenomics has been accelerated by the
same trends that have driven the discovery and characterization of
germline genetic susceptibility alleles.131 Pharmacogenomics seeks to
investigate the contribution of germline genetic variation to response
rates or toxicity associated with therapeutic modalities (e.g., medicinal,
surgical, or therapeutic radiation). For instance, susceptibility alleles
have been identified for second cancer risk and include both a
highly penetrant mutation of the retinoblastoma gene (RB) leading
to osteogenic sarcoma and common SNPs on chromosome 1q41
associated with breast cancer risk in pediatric survivors after radiation
therapy.132
The effect of highly penetrant germline mutations on cancer
outcomes and more specifically cancer risk has generated newfound
interest in the contribution of the germline mutations to cancer
outcomes across all cancers. In women with invasive ovarian cancer,
those with germline mutations in BRCA1 or BRCA2 carry an improved
5-year overall survival.133,134 On the other hand, few of the common
susceptibility alleles also map to outcomes, indicating that for the
common variant component the contributions to etiology differ from
those related to outcomes. One exception is BRCA2 mutations and
prostate cancer, in which germline susceptibility alleles have been
shown to be associated with early onset, rapid progression, and poor
outcomes.
Next-Generation Sequencing Analysis
With the availability of NGS platforms, investigators are likely to
be more successful in their search for less common and rare vari-
ants that explain a proportion of heritability of cancer susceptibility.
NGS platforms perform massively parallel sequence analysis of
major fractions of the genome with a high degree of redundancy,
which is needed to minimize the error in calling sequence vari-
ants. These technologies are reshaping the scope of genetic studies,
in both high-risk families and now in population-based studies.
Often, sequencing of tumors is conducted without accompany-
ing germline material, yet new algorithms can effectively identify
many of the germline variants, especially if the sequence coverage is
sufficiently high.
The shift to population-based sequencing, especially in large referral
cancer centers, has already uncovered a surprising observation: roughly
10% of children and adults with cancer harbor one or more CPGs.135
In pediatric cancer, the discovery of a CPG could be a sentinel for
the entire family, particularly as the parents enter the age range of
highest risk for many of the CPG-related syndromes. In osteosarcoma,
the most common pediatric bone tumor, nearly 5% of children without
a family history harbor a TP53 mutation, characteristic of the Li-
Fraumeni syndrome.136 The interpretation of new variants is daunting
and accordingly is stretching the paradigm of genetic counseling,
which will have to evolve to address the ongoing analyses possible in
a patient with the entire genome or exome sequenced. Similarly, clini-
cians will have an increasing role in discussing genetic findings,
determining actionable results, and referring patients to health care
professionals who can assist with further testing, monitoring, or effective
lifestyle changes.
This transition will accelerate the identification of new potential
variants, and the number of both uncommon and rare variants will
increase. In large-scale sequencing of the exome (defined as the targetable
exons of known genes), there are thousands of novel variants per
individual, some reflecting rare and population-private variants.
Consequently, the statistical challenge of parsing rare variants in
unrelated population studies is daunting, especially as the MAF decreases
because the sample sizes required to detect alleles with low to moderate
effect sizes becomes larger. Large databases and studies should be
developed to conduct larger discovery analyses. For the foreseeable
future, the discovery paradigm has shifted to include correlative labora-
tory confirmation of promising variants. At the same time, the value
of large-scale data sharing, including family history, cannot be
understated, because it will more quickly allow investigators to
determine the pathogenicity of most variants. This in turn can provide
evidence for clinicians to provide state-of-the-art surveillance and
interventions, especially in high-risk settings. In the high-risk setting
of Li-Fraumeni syndrome, early and active surveillance has an important
prognostic value.
CLINICAL RELEVANCE AND APPLICATIONS
Genetic Counseling and Testing
In advising patients whether it is appropriate to consider genetic testing,
it is important to remember that many currently available tests have
limitations. Clinical validity is the term used to describe the predictive
value of a test for clinical outcomes. It is affected by both the sensitivity
and the specificity of the test, as well as a host of factors that are
beyond laboratory control, such as penetrance of the mutant allele or
the value of a set of SNPs for a risk profile. Moreover, the degree of
correlation between a variant and cancer risk in any given patients
can be influenced by genetic background, environmental exposures,
or both. Because nearly all mutations associated with cancer susceptibil-
ity genes are not fully penetrant, clinicians will have to continue to
educate themselves and their patients concerning the incorporation
of new science and methods that offer improved sensitivity and/or
specificity as a result of modifiers of risk (either genetic or environmental
exposures). In this regard, it is important to help patients understand
that there may be a small group of individuals who, even if they live
into their 80s, will not get cancer despite carrying protein-truncating
mutations in a gene associated with high risk for a particular cancer.
The exponential increase in available information in the community
and in a patient who undergoes NGS will require new paradigms to
annotate, present, and revisit previously stored sequence data. It will
also be critical for clinicians and researchers to be well educated in
informatics issues related to the privacy of patient sequence data, as
well as the capacity to seek guidance and well-annotated information
that can assist in making informed decisions.
The National Society of Genetic Counselors defines genetic
counseling as “the process of helping people understand and adapt
to the medical, psychological, and familial implications of the genetic
contributions to disease.”136a Therefore the approach should be offered
in consultation with genetic counselors who aim to help patients (1)
comprehend the medical facts and risks associated with their disease;
(2) understand potential alternatives for dealing with both risk of
disease and recurrence; (3) choose a clinical course that best meets
their needs; and (4) when needed, provide support and guidance for
patients experiencing difficulty in dealing with unexpected results.
Patients often approach genetic testing and the likelihood that they
carry a deleterious inherited mutation with strong preconceived notions
based simply on intuition, which a trained genetic counselor must
first overcome.
Patients will frequently approach clinicians with questions about
genetic testing opportunities for specific cancers, or cancer overall,
particularly because the set of targeted genetic tests varies greatly by
center and company. Although formal regulations exist for interpretation
 Discovery and Characterization of Cancer Genetic Susceptibility Alleles  •  CHAPTER 21 335

of results of tests for highly penetrant mutations, new incidental findings
will require adequate education and direction in assisting clinicians
in guiding patients to the optimal decisions, which may sometimes
require additional expertise, especially if the incidental findings point
to nononcologic challenges. Moreover, germline testing can induce
patients at risk to undergo more vigilant screening, such as frequent
colonoscopy examinations for patients at risk for colon cancer. The
impact on one’s quality of life is an individual decision based on many
factors, and the substantial increase in data available through germline
sequencing should be carefully weighted in recommending individual
medical decisions. It is important to note that some patients will
strongly wish to retain their “right not to know,” wishing to postpone
any decision making, such as whether to undergo a prophylactic
oophorectomy in the face of a BRCA1-associated breast cancer, until
a cancer diagnosis appears. One final consideration for clinicians and
genetic counselors is anticipation that the burden that diagnosis of a
deleterious genetic mutation places on parents, who inevitably struggle
with factors related to when and if children should be informed of
their potential risk.
The identification of cancer susceptibility alleles has prompted
rapid transition of common SNPs to individualized disease prevention
and public health policy. With the identification of cancer susceptibility
SNPs, there has been a debate as to when and how to transition them
into clinical care. Because the cumulative set of SNPs for any one
disease now provides a substantial fraction of the population risk for
a cancer, ongoing studies should provide a sound foundation for
developing new paradigms in precision prevention. Eventually it may
be possible to reclassify high-risk versus low-risk individuals in anticipa-
tion of deciding on a preventive or early-detection program. It is also
important to keep in mind that SNPs may not be informative for a
given cancer because differences in the underlying genetic architectures
may influence the putative usefulness of introducing sets of independent
SNPs into clinical practice.
With advent of NGS technologies introduced into the clinical
venue, the clinician may be faced with interpretation of thousands of
variants of unknown significance. For example, which truncating
mutations in the BRCA1 gene confer high risk for breast and ovarian
cancer? There are more than 400 independent missense changes cata-
logued, only a fraction of which (predominately in the RING finger
domain and the C-terminal region of the protein) have been conclusively
linked with disease risk. Many, including some amino acid deletions,
are inconsequential polymorphisms that do not affect protein function,
nor do they likely increase risk for cancer. The data for other genes
is sparser or nonexistent, resulting in the daunting challenge of
interpreting variants in sequence data. As studies progress, a fraction
of data will interpretable as clinically significant, moving from
the indeterminate category to mutations with evidence for clinical
action. Patience will be required as enough data are acquired to
examine the rest.
WHAT THE FUTURE HOLDS
The sequence of the human genome has been referred to as an “instruc-
tion book for human biology,”137 and it has become clear that there
are many dynamic factors that interact in regulating and responding to
the human genome—including environmental, behavioral, and lifestyle
factors. Locked within the sequence of each individual’s DNA is the
genetic code necessary to develop a complete and healthy individual,
but encoded as well is the sequence-level variation that will determine
each person’s susceptibility to a host of diseases and outcomes.
The results of the comprehensive sequence analyses of paired sets
of genome (e.g., germline and somatically altered cancers) have revealed
a large spectrum of mutational events and epigenetic alterations.138
These newfound insights will provide opportunities to carefully unravel
the interrelationship between the germline susceptibility alleles and
cancer etiology plus progression. Already we can see somatic alterations
that can explain exceptional responses to targeted therapies.139 A more
complete understanding of the molecular pathways involved in cancer
susceptibility will suggest avenues for the development of both methods
of diagnosis and treatments. Identification of specific genes offers the
promise of genetic testing to individuals at risk, as well as the hope
for targeted therapeutics.140 Finally, understanding of sets of specific
variations offers great promise for 21st-century precision medicine
and precision prevention141 in which lifestyle, diet, and preventive
therapies come together to allow patients a full spectrum of choices
for maintaining their personal health.98
It is clear that the Human Genome Project has had and will continue
to have a profound effect on human health and biology. What remains
to be seen is the rate at which the successes of the Human Genome
Project will move from bench to bedside. In a sense, that rate will be
determined by practicing physicians. Knowledge of the underlying
principles of genetic analysis is fundamental for today’s practicing
clinician. The ability to accurately record family history and medical
record data affects the integrity of all subsequent studies for which
those data are used. An understanding by physicians of the findings
generated through both association studies and family-based studies
is key to both moving research forward and discovering what in turn
must be tested and carefully integrated in clinical and public health
paradigms. The challenge of clinically translating the information
derived from both the germline and cancer genomes will continue to
be daunting as we try to carefully make individual decisions by using
data generated from increasingly larger studies, of often differing study
design, and databases. The way forward for personal health care choices
in the 21st century will require communicating what genomic medicine
has to offer; doing so in a compassionate and accurate way will be
required of every health care provider.
The complete reference list is available online at
ExpertConsult.com.

KEY REFERENCES
2. Malkin D, Garber JE, Strong LC, Friend SH.
CANCER. The cancer predisposition revolution.
Science. 2016;352:1052–1053.
10. Manolio TA, Collins FS, Cox NJ, et al. Finding
the missing heritability of complex diseases. Nature.
2009;461:747–753.
11. Visscher PM, Wray NR, Zhang Q, et al. 10 Years of
GWAS discovery: biology, function, and translation.
Am J Hum Genet. 2017;101:5–22.
12. Chanock S. Cancer biology: genome-wide associa-
tion studies; 2014. World Cancer Report 2014, ed
Stewart BW WC (International Agency for Research
on Cancer): pp 193–202.
13. Rahman N. Realizing the promise of cancer
predisposition genes. Nature. 2014;505:302–308.
15. Scherer SW, Lee C, Birney E, et al. Challenges
and standards in integrating surveys of structural
variation. Nat Genet. 2007;39:S7–S15.
16. Sebat J, Lakshmi B, Troge J, et al. Large-scale copy
number polymorphism in the human genome.
Science. 2004;305:525–528.
17. Kidd JM, Cooper GM, Donahue WF, et al. Mapping
and sequencing of structural variation from eight
human genomes. Nature. 2008;453:56–64.
18. Orr N, Chanock S. Common genetic variation and
human disease. Adv Genet. 2008;62:1–32.
23. Gabriel SB, Schaffner SF, Nguyen H, et al. The
structure of haplotype blocks in the human genome.
Science. 2002;296:2225–2229.
26. Scheet P, Stephens M. A fast and flexible statistical
model for large-scale population genotype data:
applications to inferring missing genotypes and hap-
lotypic phase. Am J Hum Genet. 2006;78:629–644.
31. Goldgar DE, Easton DF, Cannon-Albright LA,
Skolnick MH. Systematic population-based assess-
ment of cancer risk in first-degree relatives of cancer
probands. J Natl Cancer Inst. 1994;86:1600–1608.
32. Lichtenstein P, Holm NV, Verkasalo PK, et al.
Environmental and heritable factors in the causation
336 Part I: Science and Clinical Oncology
of cancer—analyses of cohorts of twins from
Sweden, Denmark, and Finland. N Engl J Med.
2000;343:78–85.
36. Fletcher O, Houlston RS. Architecture of inherited
susceptibility to common cancer. Nat Rev Cancer.
2010;10:353–361.
40. Rebbeck TR, Mitra N, Wan F, et al. Association of
type and location of BRCA1 and BRCA2 muta-
tions with risk of breast and ovarian cancer. JAMA.
2015;313:1347–1361.
41. Kuchenbaecker KB, Hopper JL, Barnes DR, et al.
Risks of breast, ovarian, and contralateral breast
cancer for BRCA1 and BRCA2 mutation carriers.
JAMA. 2017;317:2402–2416.
43. Kuchenbaecker KB, McGuffog L, Barrowdale D,
et al. Evaluation of polygenic risk scores for breast
and ovarian cancer risk prediction in BRCA1 and
BRCA2 mutation carriers. J Natl Cancer Inst.
2017;109.
48. Miki Y, Swensen J, Shattuck-Eidens D, et al. A
strong candidate for the breast and ovarian cancer
susceptibility gene BRCA1. Science. 1994;266:66–71.
49. Wooster R, Bignell G, Lancaster J, et al. Identifica-
tion of the breast cancer susceptibility gene BRCA2.
Nature. 1995;378:789–792.
55. Risch N, Merikangas K. The future of genetic
studies of complex human diseases. Science.
1996;273:1516–1517.
56. Hindorff LA, Sethupathy P, Junkins HA, et al.
Potential etiologic and functional implications of
genome-wide association loci for human diseases
and traits. Proc Natl Acad Sci USA. 2009;106:9362–
9367.
61. Chanock SJ, Manolio T, Boehnke M, et al. Rep-
licating genotype-phenotype associations. Nature.
2007;447:655–660.
62. Dickson SP, Wang K, Krantz I, Hakonarson H,
Goldstein DB. Rare variants create synthetic genome-
wide associations. PLoS Biol. 2010;8:e1000294.
63. Wray NR, Purcell SM, Visscher PM. Synthetic
associations created by rare variants do not
explain most GWAS results. PLoS Biol. 2011;9:
e1000579.
67. Freedman ML, Monteiro AN, Gayther SA,
et al. Principles for the post-GWAS functional
characterization of cancer risk loci. Nat Genet.
2011;43:513–518.
72. Patterson N, Price AL, Reich D. Population structure
and eigenanalysis. PLoS Genet. 2006;2:e190.
80. Wang Z, Zhu B, Zhang M, et al. Imputation and
subset-based association analysis across different
cancer types identifies multiple independent risk
loci in the TERT-CLPTM1L region on chromosome
5p15.33. Hum Mol Genet. 2014;23:6616–6633.
87. Rebbeck TR, Devesa SS, Chang BL, et al. Global
patterns of prostate cancer incidence, aggressiveness,
and mortality in men of African descent. Prostate
Cancer. 2013;2013:560857.
92. Haiman CA, Chen GK, Blot WJ, et al. Genome-wide
association study of prostate cancer in men of African
ancestry identifies a susceptibility locus at 17q21.
Nat Genet. 2011;43:570–573.
97. Hoffmann TJ, Passarelli MN, Graff RE, et al.
Genome-wide association study of prostate-specific
antigen levels identifies novel loci independent of
prostate cancer. Nat Commun. 2017;8:14248.
98. Maas P, Barrdahl M, Joshi AD, et al. Breast cancer
risk from modifiable and non-modifiable risk factors
among white women in the Untied States. JAMA
Oncol. 2016;2:1295–1302.
101. Chatterjee N, Wheeler B, Sampson J, Hartge P,
Chanock SJ, Park J-H. Hidden heritability and risk
prediction based on genome-wide association studies.
Nat Genet. 2013;45:400–405.
103. Mavaddat N, Pharoah PD, Michailidou K, et al.
Prediction of breast cancer risk based on profiling
with common genetic variants. J Natl Cancer Inst.
2015;107.
104. Easton DF, Pharoah PD, Antoniou AC, et al. Gene-
panel sequencing and the prediction of breast-cancer
risk. N Engl J Med. 2015;372:2243–2257.
105. Southey MC, Goldgar DE, Winqvist R, et al. PALB2,
CHEK2 and ATM rare variants and cancer risk:
data from COGS. J Med Genet. 2016;53:800–811.
107. Machiela MJ, Ho BM, Fisher VA, Hua X, Chanock
SJ. Limited evidence that cancer susceptibility regions
are preferential targets for somatic mutation. Genome
Biol. 2015;16:193.
109. Birney E, Stamatoyannopoulos JA, Dutta A, et al.
Identification and analysis of functional elements in
1% of the human genome by the ENCODE pilot
project. Nature. 2007;447:799–816.
111. Fu YP, Kohaar I, Rothman N, et al. Common genetic
variants in the PSCA gene influence gene expression
and bladder cancer risk. Proc Natl Acad Sci USA.
2012;109:4974–4979.
118. Machiela MJ, Zhou W, Sampson JN, et al.
Characterization of large structural genetic mosa-
icism in human autosomes. Am J Hum Genet.
2015;96:487–497.
120. Dumanski JP, Rasi C, Lonn M, et al. Mutagenesis.
Smoking is associated with mosaic loss of chromo-
some Y. Science. 2015;347:81–83.
122. Wright DJ, Day FR, Kerrison ND, et al. Genetic
variants associated with mosaic Y chromosome loss
highlight cell cycle genes and overlap with cancer
susceptibility. Nat Genet. 2017;49:674–679.
124. Genovese G, Kahler AK, Handsaker RE, et al.
Clonal hematopoiesis and blood-cancer risk
inferred from blood DNA sequence. N Engl J Med.
2014;371:2477–2487.
125. Chatterjee N, Shi J, Garcia-Closas M. Developing
and evaluating polygenic risk prediction models
for stratified disease prevention. Nat Rev Genet.
2016;17:392–406.
127. Michailidou K, Beesley J, Lindstrom S, et al.
Genome-wide association analysis of more than
120,000 individuals identifies 15 new susceptibility
loci for breast cancer. Nat Genet. 2015;47:373–
380.
133. Bolton KL, Chenevix-Trench G, Goh C, et al.
Association between BRCA1 and BRCA2 muta-
tions and survival in women with invasive epithelial
ovarian cancer. JAMA. 2012;307:382–390.
134. Domchek SM, Friebel TM, Singer CF, et al. Associa-
tion of risk-reducing surgery in BRCA1 or BRCA2
mutation carriers with cancer risk and mortality.
JAMA. 2010;304:967–975.
135. Zhang J, Walsh MF, Wu G, et al. Germline mutations
in predisposition genes in pediatric cancer. N Engl
J Med. 2015;373:2336–2346.
136. Mirabello L, Yeager M, Mai PL, et al. Germline
TP53 variants and susceptibility to osteosarcoma.
J Natl Cancer Inst. 2015;107.
138. Garraway LA, Lander ES. Lessons from the cancer
genome. Cell. 2013;153:17–37.
140. Collins FS, Varmus H. A new initiative on precision
medicine. N Engl J Med. 2015;372:793–795.
141. Rebbeck TR. Precision prevention of cancer.
Cancer Epidemiol Biomarkers Prev. 2014;23:2713–
2715.
 Discovery and Characterization of Cancer Genetic Susceptibility Alleles  •  CHAPTER 21 336.e1
REFERENCES
1. Li FP, Fraumeni JF Jr. Soft-tissue sarcomas,
breast cancer, and other neoplasms. A familial
syndrome? Ann Intern Med. 1969;71:747–752.
2. Malkin D, Garber JE, Strong LC, Friend SH.
CANCER. The cancer predisposition revolution.
Science. 2016;352:1052–1053.
3. Malkin D, Li FP, Strong LC, et al. Germ line
p53 mutations in a familial syndrome of breast
cancer, sarcomas, and other neoplasms. Science.
1990;250:1233–1238.
4. Lander ES, Linton LM, Birren B, et al. Initial
sequencing and analysis of the human genome.
Nature. 2001;409:860–921.
5. Venter JC, Adams MD, Myers EW, et  al.
The sequence of the human genome. Science.
2001;291:1304–1351.
6. Frazer KA, Ballinger DG, Cox DR, et al. A second
generation human haplotype map of over 3.1 million
SNPs. Nature. 2007;449:851–861.
7. International HapMap C, Altshuler DM, Gibbs RA,
et al. Integrating common and rare genetic variation
in diverse human populations. Nature. 2010;467:
52–58.
8. Grossman RL, Heath AP, Ferretti V, et al. Toward
a shared vision for cancer genomic data. N Engl J
Med. 2016;375:1109–1112.
9. Clinical Cancer Genome Task Team of the Global
Alliance for Genomics and Health, Lawler M,
et al. Sharing clinical and genomic data on
cancer—the need for global solutions. N Engl J
Med. 2017;376:2006–2009.
10. Manolio TA, Collins FS, Cox NJ, et al. Finding
the missing heritability of complex diseases. Nature.
2009;461:747–753.
11. Visscher PM, Wray NR, Zhang Q, et al. 10 Years of
GWAS discovery: biology, function, and translation.
Am J Hum Genet. 2017;101:5–22.
12. Chanock S. Cancer biology: genome-wide associa-
tion studies; 2014. World Cancer Report 2014, ed
Stewart BW WC (International Agency for Research
on Cancer). pp 193-202.
13. Rahman N. Realizing the promise of cancer
predisposition genes. Nature. 2014;505:302–308.
14. Knudson AG Jr. Mutation and cancer: statistical
study of retinoblastoma. Proc Natl Acad Sci USA.
1971;68:820–823.
15. Scherer SW, Lee C, Birney E, et al. Challenges
and standards in integrating surveys of structural
variation. Nat Genet. 2007;39:S7–S15.
16. Sebat J, Lakshmi B, Troge J, et al. Large-scale copy
number polymorphism in the human genome.
Science. 2004;305:525–528.
17. Kidd JM, Cooper GM, Donahue WF, et al. Mapping
and sequencing of structural variation from eight
human genomes. Nature. 2008;453:56–64.
18. Orr N, Chanock S. Common genetic variation and
human disease. Adv Genet. 2008;62:1–32.
19. Donnelly P. Progress and challenges in genome-
wide association studies in humans. Nature.
2008;456:728–731.
20. 1000 Genomes Project Consortium, Abecasis
GR, Altshuler D, et al. A map of human genome
variation from population-scale sequencing. Nature.
2010;467:1061–1073.
21. Genome of the Netherlands Consortium. Whole-
genome sequence variation, population structure
and demographic history of the Dutch population.
Nat Genet. 2014;46:818–825.
22. Forbes SA, Beare D, Boutselakis H, et al. COSMIC:
somatic cancer genetics at high-resolution. Nucleic
Acids Res. 2017;45:D777–D783.
23. Gabriel SB, Schaffner SF, Nguyen H, et al. The
structure of haplotype blocks in the human genome.
Science. 2002;296:2225–2229.
24. Hinch AG, Tandon A, Patterson N, et al. The land-
scape of recombination in African Americans. Nature.
2011;476:170–175.
25. Li Y, Willer C, Sanna S, Abecasis G. Genotype
imputation. Annu Rev Genomics Hum Genet.
2009;10:387–406.
26. Scheet P, Stephens M. A fast and flexible statistical
model for large-scale population genotype data:
applications to inferring missing genotypes and hap-
lotypic phase. Am J Hum Genet. 2006;78:629–644.
27. Kong A, Frigge ML, Masson G, et al. Rate of de
novo mutations and the importance of father’s age
to disease risk. Nature. 2012;488:471–475.
28. Boettger LM, Handsaker RE, Zody MC,
McCarroll SA. Structural haplotypes and recent
evolution of the human 17q21.31 region. Nat
Genet. 2012;44:881–885.
29. Diskin SJ, Hou C, Glessner JT, et al. Copy number
variation at 1q21.1 associated with neuroblastoma.
Nature. 2009;459:987–991.
30. Li W, Xia Y, Wang C, et al. Identifying human
genome-wide CNV, LOH and UPD by targeted
sequencing of selected regions. PLoS ONE.
2014;10:e0123081.
31. Goldgar DE, Easton DF, Cannon-Albright LA,
Skolnick MH. Systematic population-based assess-
ment of cancer risk in first-degree relatives of cancer
probands. J Natl Cancer Inst. 1994;86:1600–1608.
32. Lichtenstein P, Holm NV, Verkasalo PK, et al.
Environmental and heritable factors in the causa-
tion of cancer—analyses of cohorts of twins from
Sweden, Denmark, and Finland. N Engl J Med.
2000;343:78–85.
33. Spitz MR, Currier RD, Fueger JJ, Babaian RJ,
Newell GR. Familial patterns of prostate cancer: a
case-control analysis. J Urol. 1991;146:1305–1307.
34. Steinberg GD, Carter BS, Beaty TH, Childs B,
Walsh PC. Family history and the risk of prostate
cancer. Prostate. 1990;17:337–347.
35. Hayes RB, Liff JM, Pottern LM, et al. Prostate cancer
risk in US blacks and whites with a family history
of cancer. Int J Cancer. 1995;60:361–364.
36. Fletcher O, Houlston RS. Architecture of inherited
susceptibility to common cancer. Nat Rev Cancer.
2010;10:353–361.
37. Couch FJ, Farid LM, DeShano ML, et al. BRCA2
germline mutations in male breast cancer cases and
breast cancer families. Nat Genet. 1996;13:123–
125.
38. Bodmer WF, Bailey CJ, Bodmer J, et al. Localization
of the gene for familial adenomatous polyposis on
chromosome 5. Nature. 1987;328:614–616.
39. Hunter DJ, Chanock SJ. Genome-wide association
studies and “the art of the soluble.” J Natl Cancer
Inst. 2010;102:836–837.
40. Rebbeck TR, Mitra N, Wan F, et al. Association of
type and location of BRCA1 and BRCA2 muta-
tions with risk of breast and ovarian cancer. JAMA.
2015;313:1347–1361.
41. Kuchenbaecker KB, Hopper JL, Barnes DR, et al.
Risks of breast, ovarian, and contralateral breast
cancer for BRCA1 and BRCA2 mutation carriers.
JAMA. 2017;317:2402–2416.
42. Antoniou AC, Spurdle AB, Sinilnikova OM,
et al. Common breast cancer-predisposition alleles
are associated with breast cancer risk in BRCA1
and BRCA2 mutation carriers. Am J Hum Genet.
2008;82:937–948.
43. Kuchenbaecker KB, McGuffog L, Barrowdale D,
et al. Evaluation of polygenic risk scores for breast
and ovarian cancer risk prediction in BRCA1 and
BRCA2 mutation carriers. J Natl Cancer Inst.
2017;109.
44. Kamb A, Shattuck-Eidens D, Eeles R, et al. Analysis
of the p16 gene (CDKN2) as a candidate for the
chromosome 9p melanoma susceptibility locus. Nat
Genet. 1994;8:23–26.
45. Yang XR, Ng D, Alcorta DA, et al. T (brachyury)
gene duplication confers major susceptibility to
familial chordoma. Nat Genet. 2009;41:1176–1178.
46. Yokoyama S, Woods SL, Boyle GM, et al. A novel
recurrent mutation in MITF predisposes to familial
and sporadic melanoma. Nature. 2011;480:99–103.
47. Bertolotto C, Lesueur F, Giuliano S, et al. A
SUMOylation-defective MITF germline mutation
predisposes to melanoma and renal carcinoma.
Nature. 2011;480:94–98.
48. Miki Y, Swensen J, Shattuck-Eidens D, et al. A
strong candidate for the breast and ovarian cancer
susceptibility gene BRCA1. Science. 1994;266:66–71.
49. Wooster R, Bignell G, Lancaster J, et al. Identifica-
tion of the breast cancer susceptibility gene BRCA2.
Nature. 1995;378:789–792.
50. Kadouri L, Hubert A, Rotenberg Y, et al. Cancer
risks in carriers of the BRCA1/2 Ashkenazi founder
mutations. J Med Genet. 2007;44:467–471.
51. Tonin P, Weber B, Offit K, et al. Frequency of
recurrent BRCA1 and BRCA2 mutations in
Ashkenazi Jewish breast cancer families. Nat Med.
1996;2:1179–1183.
52. Struewing JP, Abeliovich D, Peretz T, et al. The
carrier frequency of the BRCA1 185delAG mutation
is approximately 1 percent in Ashkenazi Jewish
individuals. Nat Genet. 1995;11:198–200.
53. Spurdle AB, Couch FJ, Parsons MT, et al. Refined
histopathological predictors of BRCA1 and BRCA2
mutation status: a large-scale analysis of breast
cancer characteristics from the BCAC, CIMBA,
and ENIGMA consortia. Breast Cancer Res.
2014;16:3419.
54. Moisio AL, Sistonen P, Weissenbach J, de la Chapelle
A, Peltomaki P. Age and origin of two common
MLH1 mutations predisposing to hereditary colon
cancer. Am J Hum Genet. 1996;59:1243–1251.
55. Risch N, Merikangas K. The future of genetic
studies of complex human diseases. Science.
1996;273:1516–1517.
56. Hindorff LA, Sethupathy P, Junkins HA, et al.
Potential etiologic and functional implications of
genome-wide association loci for human diseases and
traits. Proc Natl Acad Sci USA. 2009;106:9362–9367.
57. Garcia-Closas M, Malats N, Silverman D, et al.
NAT2 slow acetylation, GSTM1 null genotype,
and risk of bladder cancer: results from the Spanish
Bladder Cancer Study and meta-analyses. Lancet.
2005;366:649–659.
58. Garcia-Closas M, Hein DW, Silverman D, et al. A
single nucleotide polymorphism tags variation in
the arylamine N-acetyltransferase 2 phenotype in
populations of European background. Pharmacogenet
Genomics. 2011;21:231–236.
59. Hashibe M, McKay JD, Curado MP, et al. Multiple
ADH genes are associated with upper aerodigestive
cancers. Nat Genet. 2008;40:707–709.
60. A map of human genome variation from population-
scale sequencing. Nature. 2010;467:1061–1073.
61. Chanock SJ, Manolio T, Boehnke M, et al. Rep-
licating genotype-phenotype associations. Nature.
2007;447:655–660.
62. Dickson SP, Wang K, Krantz I, Hakonarson H,
Goldstein DB. Rare variants create synthetic genome-
wide associations. PLoS Biol. 2010;8:e1000294.
63. Wray NR, Purcell SM, Visscher PM. Synthetic
associations created by rare variants do not explain
most GWAS results. PLoS Biol. 2011;9:e1000579.
64. Haiman CA, Patterson N, Freedman ML, et al.
Multiple regions within 8q24 independently affect
risk for prostate cancer. Nat Genet. 2007;39:638–
644.
65. Amundadottir LT, Sulem P, Gudmundsson J, et al.
A common variant associated with prostate cancer
in European and African populations. Nat Genet.
2006;38:652–658.
66. Freedman ML, Haiman CA, Patterson N, et al.
Admixture mapping identifies 8q24 as a prostate
cancer risk locus in African-American men. Proc
Natl Acad Sci USA. 2006;103:14068–14073.
336.e2 Part I: Science and Clinical Oncology
67. Freedman ML, Monteiro AN, Gayther SA,
et al. Principles for the post-GWAS functional
characterization of cancer risk loci. Nat Genet.
2011;43:513–518.
68. Wacholder S. Practical considerations in choosing
between the case-cohort and nested case-control
designs. Epidemiology. 1991;2:155–158.
69. Wacholder S, Silverman DT, McLaughlin JK,
Mandel JS. Selection of controls in case-control
studies. III. Design options. Am J Epidemiol.
1992;135:1042–1050.
70. Wacholder S, Silverman DT, McLaughlin JK,
Mandel JS. Selection of controls in case-control
studies. II. Types of controls. Am J Epidemiol.
1992;135:1029–1041.
71. Wacholder S, McLaughlin JK, Silverman DT, Mandel
JS. Selection of controls in case-control studies. I.
Principles. Am J Epidemiol. 1992;135:1019–1028.
72. Patterson N, Price AL, Reich D. Population structure
and eigenanalysis. PLoS Genet. 2006;2:e190.
73. Postel-Vinay S, Veron AS, Tirode F, et al. Common
variants near TARDBP and EGR2 are associated
with susceptibility to Ewing sarcoma. Nat Genet.
2012;44:323–327.
74. Rapley EA, Turnbull C, Al Olama AA, et al. A
genome-wide association study of testicular germ
cell tumor. Nat Genet. 2009;41:807–810.
75. Kanetsky PA, Mitra N, Vardhanabhuti S, et al.
Common variation in KITLG and at 5q31.3
predisposes to testicular germ cell cancer. Nat Genet.
2009;41:811–815.
76. Haiman CA, Le Marchand L, Yamamato J, et al.
A common genetic risk factor for colorectal and
prostate cancer. Nat Genet. 2007;39:954–956.
77. Sur IK, Hallikas O, Vaharautio A, et al. Mice
lacking a Myc enhancer that includes human SNP
rs6983267 are resistant to intestinal tumors. Science.
2012;338:1360–1363.
78. Pomerantz MM, Ahmadiyeh N, Jia L, et al. The
8q24 cancer risk variant rs6983267 shows long-
range interaction with MYC in colorectal cancer.
Nat Genet. 2009;41:882–884.
79. Tuupanen S, Turunen M, Lehtonen R, et al.
The common colorectal cancer predisposition
SNP rs6983267 at chromosome 8q24 confers
potential to enhanced Wnt signaling. Nat Genet.
2009;41:885–890.
80. Wang Z, Zhu B, Zhang M, et al. Imputation and
subset-based association analysis across different
cancer types identifies multiple independent risk
loci in the TERT-CLPTM1L region on chromosome
5p15.33. Hum Mol Genet. 2014;23:6616–6633.
81. Kote-Jarai Z, Saunders EJ, Leongamornlert DA, et al.
Fine-mapping identifies multiple prostate cancer risk
loci at 5p15, one of which associates with TERT
expression. Hum Mol Genet. 2013;22:2520–2528.
82. Calado RT, Regal JA, Hills M, et al. Constitutional
hypomorphic telomerase mutations in patients with
acute myeloid leukemia. Proc Natl Acad Sci USA.
2009;106:1187–1192.
83. Savage SA, Stewart BJ, Weksler BB, et al. Mutations
in the reverse transcriptase component of telomerase
(TERT) in patients with bone marrow failure. Blood
Cells Mol Dis. 2006;37:134–136.
84. Stacey SN, Gudbjartsson DF, Sulem P, et al.
Common variants on 1p36 and 1q42 are associ-
ated with cutaneous basal cell carcinoma but not
with melanoma or pigmentation traits. Nat Genet.
2008;40:1313–1318.
85. Wang Z. Improved imputation of common and
uncommon SNPs with a new reference set. Nat
Genet. 2012;44:6–7.
86. Gudmundsson J, Sulem P, Gudbjartsson DF, et al.
A study based on whole-genome sequencing yields a
rare variant at 8q24 associated with prostate cancer.
Nat Genet. 2012;44:1326–1329.
87. Rebbeck TR, Devesa SS, Chang BL, et al. Global
patterns of prostate cancer incidence, aggressiveness,
and mortality in men of African descent. Prostate
Cancer. 2013;2013:560857.
88. Johanneson B, Deutsch K, McIntosh L, et al. Sugges-
tive genetic linkage to chromosome 11p11.2-q12.2
in hereditary prostate cancer families with primary
kidney cancer. Prostate. 2007;67:732–742.
89. Schodel J, Bardella C, Sciesielski LK, et al. Common
genetic variants at the 11q13.3 renal cancer
susceptibility locus influence binding of HIF to
an enhancer of cyclin D1 expression. Nat Genet.
2012;44:420–425, S421–422.
90. French JD, Ghoussaini M, Edwards SL, et al.
Functional variants at the 11q13 risk locus
for breast cancer regulate cyclin D1 expression
through long-range enhancers. Am J Hum Genet.
2013;92:489–503.
91. Hunter DJ, Thomas G, Hoover RN, Chanock SJ.
Scanning the horizon: what is the future of genome-
wide association studies in accelerating discoveries
in cancer etiology and prevention? Cancer Causes
Control. 2007;18:479–484.
92. Haiman CA, Chen GK, Blot WJ, et al. Genome-wide
association study of prostate cancer in men of African
ancestry identifies a susceptibility locus at 17q21.
Nat Genet. 2011;43:570–573.
93. Eeles RA, Kote-Jarai Z, Giles GG, et al. Multiple
newly identified loci associated with prostate cancer
susceptibility. Nat Genet. 2008;40:316–321.
94. Ahn J, Berndt SI, Wacholder S, et al. Variation in
KLK genes, prostate-specific antigen and risk of
prostate cancer. Nat Genet. 2008;40:1032–1034,
author reply 1035–1036.
95. Kote-Jarai Z, Amin Al Olama A, Leongamornlert
D, et al. Identification of a novel prostate cancer
susceptibility variant in the KLK3 gene transcript.
Hum Genet. 2011;129:687–694.
96. Parikh H, Wang Z, Pettigrew KA, et al. Fine mapping
the KLK3 locus on chromosome 19q13.33 associated
with prostate cancer susceptibility and PSA levels.
Hum Genet. 2011;129:675–685.
97. Hoffmann TJ, Passarelli MN, Graff RE, et al.
Genome-wide association study of prostate-specific
antigen levels identifies novel loci independent of
prostate cancer. Nat Commun. 2017;8:14248.
98. Maas P, Barrdahl M, Joshi AD, et al. Breast cancer
risk from modifiable and non-modifiable risk factors
among white women in the Untied States. JAMA
Oncol. 2016;2:1295–1302.
99. Park JH, Wacholder S, Gail MH, et al. Estimation of
effect size distribution from genome-wide association
studies and implications for future discoveries. Nat
Genet. 2010;42:570–575.
100. Park JH, Gail MH, Weinberg CR, et al. Dis-
tribution of allele frequencies and effect sizes
and their interrelationships for common genetic
susceptibility variants. Proc Natl Acad Sci USA.
2011;108:18026–18031.
101. Chatterjee N, Wheeler B, Sampson J, Hartge P,
Chanock SJ, Park J-H. Hidden heritability and risk
prediction based on genome-wide association studies.
Nat Genet. 2013;45:400–405.
102. Eeles RA, Olama AA, Benlloch S, et al. Identification
of 23 new prostate cancer susceptibility loci using
the iCOGS custom genotyping array. Nat Genet.
2013;45:385–391, 391e381–382.
103. Mavaddat N, Pharoah PD, Michailidou K, et al.
Prediction of breast cancer risk based on profiling
with common genetic variants. J Natl Cancer Inst.
2015;107.
104. Easton DF, Pharoah PD, Antoniou AC, et al. Gene-
panel sequencing and the prediction of breast-cancer
risk. N Engl J Med. 2015;372:2243–2257.
105. Southey MC, Goldgar DE, Winqvist R, et al. PALB2,
CHEK2 and ATM rare variants and cancer risk:
data from COGS. J Med Genet. 2016;53:800–
811.
106. Southey MC, Winship I, Nguyen-Dumont T.
PALB2: research reaching to clinical outcomes for
women with breast cancer. Hered Cancer Clin Pract.
2016;14:9.
107. Machiela MJ, Ho BM, Fisher VA, Hua X, Chanock
SJ. Limited evidence that cancer susceptibility regions
are preferential targets for somatic mutation. Genome
Biol. 2015;16:193.
108. Machiela MJ, Chanock SJ. LDlink: a web-based
application for exploring population-specific
haplotype structure and linking correlated alleles
of possible functional variants. Bioinformatics.
2015;31:3555–3557.
109. Birney E, Stamatoyannopoulos JA, Dutta A, et al.
Identification and analysis of functional elements in
1% of the human genome by the ENCODE pilot
project. Nature. 2007;447:799–816.
110. Boyle AP, Hong EL, Hariharan M, et al. Annota-
tion of functional variation in personal genomes
using RegulomeDB. Genome Res. 2012;22:1790–
1797.
111. Fu YP, Kohaar I, Rothman N, et al. Common genetic
variants in the PSCA gene influence gene expression
and bladder cancer risk. Proc Natl Acad Sci USA.
2012;109:4974–4979.
112. Garcia-Closas M, Rothman N, Figueroa JD, et al.
Common genetic polymorphisms modify the effect
of smoking on absolute risk of bladder cancer. Cancer
Res. 2013;73:2211–2220.
113. McKay JD, Hung RJ, Han Y, et al. Large-scale
association analysis identifies new lung cancer
susceptibility loci and heterogeneity in genetic
susceptibility across histological subtypes. Nat Genet.
2017;49:1126–1132.
114. Lan Q, Hsiung CA, Matsuo K, et al. Genome-wide
association analysis identifies new lung cancer
susceptibility loci in never-smoking women in Asia.
Nat Genet. 2012;44:1330–1335.
115. Prokunina-Olsson L, Muchmore B, Tang W, et al.
A variant upstream of IFNL3 (IL28B) creating
a new interferon gene IFNL4 is associated with
impaired clearance of hepatitis C virus. Nat Genet.
2013;45:164–171.
116. Rodriguez-Santiago B, Malats N, Rothman N, et al.
Mosaic uniparental disomies and aneuploidies as
large structural variants of the human genome.
Am J Hum Genet. 2010;87:129–138.
117. Machiela MJ, Zhou W, Caporaso N, et al. Mosaic
13q14 deletions in peripheral leukocytes of non-
hematologic cancer cases and healthy controls.
J Hum Genet. 2016;61:411–418.
118. Machiela MJ, Zhou W, Sampson JN, et al.
Characterization of large structural genetic mosa-
icism in human autosomes. Am J Hum Genet.
2015;96:487–497.
119. Machiela MJ, Zhou W, Karlins E, et al. Female
chromosome X mosaicism is age-related and
preferentially affects the inactivated X chromosome.
Nat Commun. 2016;7:11843.
120. Dumanski JP, Rasi C, Lonn M, et al. Mutagenesis.
Smoking is associated with mosaic loss of chromo-
some Y. Science. 2015;347:81–83.
121. Zhou W, Machiela MJ, Freedman ND, et al. Mosaic
loss of chromosome Y is associated with common
variation near TCL1A. Nat Genet. 2016;48:563–568.
122. Wright DJ, Day FR, Kerrison ND, et al. Genetic
variants associated with mosaic Y chromosome loss
highlight cell cycle genes and overlap with cancer
susceptibility. Nat Genet. 2017;49:674–679.
123. Jager N, Schlesner M, Jones DT, et al. Hypermuta-
tion of the inactive X chromosome is a frequent
event in cancer. Cell. 2013;155:567–581.
124. Genovese G, Kahler AK, Handsaker RE, et al.
Clonal hematopoiesis and blood-cancer risk
inferred from blood DNA sequence. N Engl J Med.
2014;371:2477–2487.
125. Chatterjee N, Shi J, Garcia-Closas M. Developing
and evaluating polygenic risk prediction models
for stratified disease prevention. Nat Rev Genet.
2016;17:392–406.
 Discovery and Characterization of Cancer Genetic Susceptibility Alleles  •  CHAPTER 21 336.e3
126. Morgan TH. Random segregation versus coupling
in Mendelian inheritance. Science. 1911;34:384.
127. Michailidou K, Beesley J, Lindstrom S, et al.
Genome-wide association analysis of more than
120,000 individuals identifies 15 new susceptibility
loci for breast cancer. Nat Genet. 2015;47:373–380.
128. Helfand BT, Roehl KA, Cooper PR, et al. Associa-
tions of prostate cancer risk variants with disease
aggressiveness: results of the NCI-SPORE Genetics
Working Group analysis of 18,343 cases. Hum Genet.
2015;134:439–450.
129. Berndt SI, Wang Z, Yeager M, et al. Two susceptibil-
ity loci identified for prostate cancer aggressiveness.
Nat Commun. 2015;6:6889.
130. Maris JM, Mosse YP, Bradfield JP, et al. Chromo-
some 6p22 locus associated with clinically aggressive
neuroblastoma. N Engl J Med. 2008;358:2585–2593.
131. Trevino LR, Yang W, French D, et al. Germline
genomic variants associated with childhood acute lym-
phoblastic leukemia. Nat Genet. 2009;41:1001–1005.
132. Morton LM, Sampson JN, Armstrong GT, et al.
Genome-wide association study identifies susceptibil-
ity loci that modify radiation-related risk for breast
cancer after childhood cancer. J Natl Cancer Inst.
2017.
133. Bolton KL, Chenevix-Trench G, Goh C, et al.
Association between BRCA1 and BRCA2 muta-
tions and survival in women with invasive epithelial
ovarian cancer. JAMA. 2012;307:382–390.
134. Domchek SM, Friebel TM, Singer CF, et al. Associa-
tion of risk-reducing surgery in BRCA1 or BRCA2
mutation carriers with cancer risk and mortality.
JAMA. 2010;304:967–975.
135. Zhang J, Walsh MF, Wu G, et al. Germline mutations
in predisposition genes in pediatric cancer. N Engl
J Med. 2015;373:2336–2346.
136. Mirabello L, Yeager M, Mai PL, et al. Germline
TP53 variants and susceptibility to osteosarcoma.
J Natl Cancer Inst. 2015;107.
136a.  National Society of Genetic Counselors’ Definition
Task Force, Resta R, Biesecker BB, et al. A new
definition of Genetic Counseling: National Society
of Genetic Counselors’ Task Force report. J Genet
Couns. 2006;15(2):77–83.
137. Collins FS, McKusick VA. Implications of the
human genome project for medical science. JAMA.
2001;285:540–544.
138. Garraway LA, Lander ES. Lessons from the cancer
genome. Cell. 2013;153:17–37.
139. Iyer G, Hanrahan AJ, Milowsky MI, et al. Genome
sequencing identifies a basis for everolimus sensitivity.
Science. 2012;338:221.
140. Collins FS, Varmus H. A new initiative on precision
medicine. N Engl J Med. 2015;372:793–795.
141. Rebbeck TR. Precision prevention of cancer. Cancer
Epidemiol Biomarkers Prev. 2014;23:2713–2715.
 Lifestyle and Cancer Prevention 22 
Karen Basen-Engquist, Powel Brown, Adriana M. Coletta,
Michelle Savage, Karen Colbert Maresso, and Ernest Hawk

S UMMARY OF K EY
• Approximately one-third to • Cancer interception is a type of
one-half of cancers can be
prevented through adoption of
healthy lifestyles and avoidance of
known risk factors.
• The findings of several large
observational studies from the
United States and Europe developing countries and remains
strongly suggest that following
current cancer prevention
recommendations from either the
American Cancer Society (ACS) or
the American Institute for Cancer
Research (AICR) leads to significant
health benefits.
• Advanced cancers are now
understood to be extremely
genetically heterogeneous on
numerous levels, so intervening at an
earlier time point when there are
fewer derangements to contend with,
at the precancerous lesion stage,
offers a rational approach to cancer
prevention.
P OI NT S
chemoprevention or molecular
prevention that seeks to address
those at high risk because of
established precancers.
• Tobacco use accounts for 30% of all
cancer deaths; it is on the rise in
high among several subpopulations
within the United States.
• In 2015, electronic nicotine delivery
systems, specifically e-cigarettes,
were the most commonly used
tobacco product among both
middle- and high-school students.
• Obesity is now linked to 13 different
cancers, and approximately 17% of
children and 36% of adults are
classified as obese in the United
States.
• Alcohol has been shown to
convincingly increase the risk of at
least six cancers: (1) oral and
pharynx; (2) larynx; (3) esophageal
(squamous cell); (4) colorectal
(in men only); (5) breast; and
(6) liver.
• Both subjective and objective
evidence links regular, structured
physical activity with reduced risk of
chronic disease and certain cancers,
such as colon and breast cancer.
• The link between diet and cancer
risk is complex; not all dietary
components are linked with cancer
risk, and some components exhibit
strong links with cancer risk
individually whereas others exhibit
stronger effects synergistically.
• It is estimated that indoor tanning
results in 400,000 cases of skin
cancer in the United States each
year, and 6000 of these are
melanoma.
• There are 13 agents approved for
the treatment of precancerous
lesions and cancer risk reduction or
prevention.

Cancer prevention is the most cost-effective, long-term strategy for
the control of cancer.1,2 It is now well established that approximately
one-third to one-half of all adult cancers occurring in Western popula-
tions can be prevented through the adoption of “healthy lifestyles.”
A comprehensive view of a healthy lifestyle includes adherence to
published cancer prevention recommendations that seek to eliminate or
minimize exposure to lifestyle risk factors, and the use of screening and
early detection tests as well as molecular preventive agents (e.g., aspirin,
tamoxifen, human papillomavirus [HPV] vaccine) when warranted. This
chapter focuses on the epidemiology of lifestyle risk factors and their
associated interventions, as well as on the use of molecular preventive
agents. Screening and early detection are addressed in another chapter.
RATIONALE FOR PREVENTION
The rationale for an enhanced focus on prevention as the dominant
strategy by which to reduce the cancer burden is supported by at least
three lines of evidence.
First, the preventability of cancer was estimated by Doll and Peto
in their 1981 landmark publication on the relative contributions of
the avoidable causes of cancer.3 They concluded that three-fourths or
more of all cancers occurring in the United States during 1970 could
have been theoretically avoided.3 This estimate as to the “preventability”
of cancer was based on their approximations for the relative contribution
of individual cancer risk factors—30% for tobacco, 2% to 4% for
alcohol, 35% for diet, 1% to 10% for infectious agents, 2% for
pollution, and 4% for occupational exposures.3 Subsequent epide-
miologic data have largely supported these estimates and have identified
obesity and lack of physical activity as additional cancer risk factors,
causing a slight redistribution of the percent of cancer mortality
attributable to the various lifestyle risk factors. A recent review sum-
marizing updated data on this topic found that obesity accounts for
15% to 20% of cancers, physical inactivity accounts for approximately
5%, and the estimate for diet has decreased to 5%, much less than
the initial estimate of 35% by Doll and Peto.4 Although there are
limitations to estimating the relative contributions of individual risk
factors to cancer mortality, and although the estimate of the amount
of cancer that can be prevented may be a best case scenario difficult
to achieve in reality, such studies not only demonstrate a need to
better understand the interplay among cancer-associated lifestyle factors,
but also highlight the potential impact of prevention and underscore
its urgency.
Second, several large observational studies from the United States
and Europe strongly suggest that following current cancer prevention

337
338 Part I: Science and Clinical Oncology
Table 22.1  Current Recommendations for Cancer Prevention From the American Cancer Society (ACS)
and the American Institute for Cancer Research (AICR)
ACS GUIDELINES ON NUTRITION AND PHYSICAL ACTIVITY64a
Achieve and maintain a healthy weight throughout life.
• Be as lean as possible throughout life without being underweight.
• Avoid excess weight gain at all ages.
• Get regular physical activity and limit intake of high-calorie food
and drinks as keys to help maintain a healthy weight.
Be physically active.
• Get at least 150 minutes of moderate intensity or 75 minutes of
vigorous intensity activity each week (or a combination of these),
preferably spread throughout the week.
• Limit sedentary behavior such as sitting, lying down, watching TV,
and other forms of screen-based entertainment.
• Doing some physical activity, above usual activities, no matter what
one’s activity level, can have many health benefits.
Eat a healthy diet, with an emphasis on plant foods.
• Choose foods and drinks in amounts that help you get to and
maintain a healthy weight.
• Limit how much processed meat and red meat you eat.
• Eat at least 2.5 cups of fruits and vegetables each day.
• Choose whole grains instead of refined grain products.
If you drink alcohol, limit your intake.
• Drink no more than 1 drink per day for women or 2 per day for men.
a
Recommendations are in addition to avoiding tobacco, being safe in the sun, and following cancer screening guidelines.
recommendations (Table 22.1) from either the American Cancer Society
(ACS) or the American Institute for Cancer Research (AICR) leads
to significant health benefits.5 Significant reductions in both cancer
risk and cancer mortality, as well as in cardiovascular mortality and
overall mortality, were seen across studies for those individuals who
adhered to a majority of the recommendations compared with those
who adhered to fewer recommendations.
Third, cancer is the last step in a multistep process occurring over
decades. This offers time and opportunity to intervene. Carcinogenesis
is characterized by a progression of genetic changes affecting cellular
identity and growth that culminates in cancer after many years. The
accumulation of these changes at the DNA level is reflected in
cytomorphologic and histopathologic changes termed preinvasive
neoplastic lesions, or simply precancerous lesions. Because advanced
cancers are now understood to be extremely genetically heterogeneous
on numerous levels—within a primary tumor, between two metastases,
within metastatic lesions, and between patients—and because this
heterogeneity negatively affects the response to treatment,6 intervening
at an earlier time point when there are fewer derangements to contend
with, at the precancerous lesion stage, offers a rational approach to
cancer prevention.
This approach to treating precancerous lesions in order to prevent
progression to full-blown cancer is being termed cancer interception
and is rapidly gaining traction as a priority strategy to address the
cancer burden, in part because of recent advances in understanding
preinvasive cancer biology. According to Blackburn, cancer interception
is “the active way of combatting cancer and carcinogenesis at earlier
and earlier stages.”7 It is “active” in the sense that it involves more
than risk avoidance, such as avoidance of smoking or sun exposure,
which is often the domain of primary prevention. Cancer interception
is a type of chemoprevention or molecular prevention that seeks to
address those at high risk because of established precancers.7,8 A timely
example of interception in cancer prevention is the use of aspirin to
reduce the number of colorectal adenomas (i.e., precancers), which
has resulted in significant reductions in colorectal cancer (CRC)
incidence and mortality in randomized trials of use for cardiovascular
event prophylaxis.7 The application of drugs that have been shown
to be effective in advanced cancer to precancerous lesions—that is,
AICR CANCER PREVENTION RECOMMENDATIONS63
Be as lean as possible without becoming underweight.
Be physically active for at least 30 minutes every day. Limit
sedentary habits.
Avoid sugary drinks. Limit consumption of energy-dense foods.
Eat more of a variety of vegetables, fruits, whole grains, and
legumes such as beans.
Limit consumption of red meats (such as beef, pork and lamb)
and avoid processed meats.
If consumed at all, limit alcoholic drinks to 2 per day for men
and 1 per day for women.
Limit consumption of salty foods and foods processed with salts.
Don’t use supplements to protect against cancer.
It is best for mothers to breastfeed exclusively for up to 6
months and then add other liquids and foods.
After treatment, cancer survivors should follow the
recommendations for cancer prevention.
Do not smoke or chew tobacco.
reverse migration—offers a potential pathway to making this strategy
a reality. However, the field is currently challenged by a dearth of data
and understanding around the precancerous genome. Gaining a better
understanding of the type, timing, and sequence of molecular aber-
rations underlying precancerous lesions and their development will
help drive this strategy forward. Establishing a Precancerous Genome
Atlas, analogous to The Cancer Genome Atlas (TCGA), is a research
priority in the field of cancer prevention.9
PREVENTION THROUGH LIFESTYLE
INTERVENTIONS
Tobacco
Tobacco remains the leading preventable cause of death and disability
in the United States. It has been linked to at least 12 different cancers
and accounts for approximately one-third of all cancer deaths, more
than any other risk factor. In those who already have cancer, it leads
to adverse health outcomes, including increasing the risks of second
primary cancers, cancer-related mortality, and all-cause mortality.10
Tobacco-using cancer patients may also have an increased risk of
recurrence, a poorer response to treatment, and increased treatment-
related toxicity.10 Evidence shows that quitting smoking improves the
prognosis of cancer patients.
The prevalence of cigarette smoking among adults in 2015 was
15%, a decline from 21% in 2005.11 Yet this prevalence equates to
nearly 37 million adults who still smoke, and disparities in prevalence
persist. Groups with a prevalence higher than the general population
include males (16.7%); those aged 25 to 44 years (17.7%); American
Indians/Alaska Natives (21.9%); those with only a General Educational
Development (GED) credential (34.1%); those living below the
poverty level (26.1%); residents of the Midwest (18.7%); those who
are uninsured (27.4%) or receiving Medicaid (27.8%); lesbian, gay,
or bisexual individuals (20.6%); and those with serious psychologic
distress (40.6%).11
In addition to these disparities, the growing use of e-cigarettes
among youth is a real concern because it presents a threat to the
last half-century of progress in tobacco control. Although traditional

cigarette use declined among youth in the 2011–2015 period, there
were substantial increases in e-cigarette use among students during this
same time period.12 In 2015, e-cigarettes were the most commonly used
tobacco product among both middle- and high-school students, with
5% and 16% reporting current use, respectively.12 Limited research to
date suggests that e-cigarette users are more likely to take up traditional
tobacco products than are never-users of e-cigarettes.13 However,
regardless of whether e-cigarettes serve as a gateway to traditional
tobacco use, they nevertheless expose youth to nicotine, opening the
possibility for addiction and altered brain development12 and may pose
unique health threats of their own through their fluid and aerosol
composition. E-cigarette fluid is known to contain propylene glycol
(PG), a US Food and Drug Administration (FDA)–approved food
additive. But the effects of PG when heated and inhaled are unclear,
and e-cigarette aerosol has also been shown to contain numerous
carcinogens, including tobacco-specific nitrosamines, volatile organic
compounds, and heavy metals.14 In 2016 the FDA finalized a rule that
extended its authority to the manufacturing, marketing, and distribution
of e-cigarettes. Before this, e-cigarettes were completely unregulated,
making it difficult to know what was in them, to study or generate
data to inform regulations, and to prevent them from being sold to
minors. With the passing of the new rule, e-cigarette manufacturers,
retailers, and consumers must now adhere to a set of regulations
meant to protect the health of all Americans. This includes a ban on
the sale of e-cigarettes to those younger than age 18. It is hoped that
these regulations will improve the ability of the scientific community
to more easily study e-cigarettes and their potential health effects.
Comprehensive tobacco control as established by the Centers for
Disease Control and Prevention (CDC) includes a mix of educational,
clinical, regulatory, economic, and social strategies. The typical goals
of a comprehensive tobacco control program are to (1) prevent initiation
among youth and young adults; (2) promote quitting; (3) eliminate
exposure to second-hand smoke; and (4) identify and eliminate
tobacco-related disparities among population groups.15 The optimal
mix of strategies to achieve these goals for a particular population
requires careful consideration. Implementation of any one type of
strategy is substantially less effective than the coordinated implementa-
tion of some level of all strategies. The combination of these approaches
has been tremendously successful in reducing the number of tobacco
users over the last 50 years.10 According to the CDC, states that have
made larger investments in tobacco control have seen larger declines
in cigarette sales than the United States as a whole, and the prevalence
of smoking among adults and youth has declined faster as spending
for control has risen.15 Consequently, the CDC recommends state-
specific annual funding levels for tobacco control. Unfortunately, most
states are not providing funding at their recommended level, missing
a tremendous opportunity to reduce the burden of tobacco use within
their populations and across the nation.15 Important to note, with
the emergence and rapid adoption of e-cigarette use among youth, it
is critical to ensure that these devices are included among all forms
of tobacco control and prevention strategies.
Internationally, cigarette consumption is increasing in low- and
middle-income countries (LMICs) owing to their population and
economic growth, but also because of increased social acceptability
of smoking and targeted marketing by tobacco companies.16 Of the
world’s smokers, 80% reside in LMICs. China is the largest consumer
of cigarettes in the world, consuming more than one-third of the
world’s cigarettes,16 and with a population of smokers larger than the
entire population of the United States.17 Other countries with high
rates of consumption include Russia and Greece, and many countries
of eastern Europe and in the eastern Mediterranean region.18 Africa
represents the greatest risk for future growth in tobacco use as its
population continues to grow and as it continues to develop economi-
cally.18 In general, the prevalence of smoking among men is much
higher than it is among women, especially in Russia and Indonesia,
where at least half of all men smoke, but just 17% and 4% of women
smoke, respectively.18 In China, 45% of men smoke but just 2% of
Lifestyle and Cancer Prevention  •  CHAPTER 22 339
Table 22.2  The World Health Organization’s
Framework Convention on Tobacco
Control MPOWER Measures
Monitor tobacco use and prevention policies
Protect people from tobacco smoke
Offer help to quit tobacco use
Warn about the dangers of tobacco
Enforce bans on tobacco advertising, promotion, and sponsorship
Raise taxes on tobacco
women smoke. It is feared that the large number of nonsmoking
women in these countries represents a target for tobacco-company
marketing efforts.17 Global tobacco control is tremendously challenging,
given the varying patterns of use within and between countries and
the often less-than-optimal resources available in LMICs. Nevertheless,
it is essential to reduce the tremendous burden of premature death
and disability that many of these countries can expect if current trends
in tobacco use continue. The World Health Organization (WHO)
Framework Convention Alliance for Tobacco Control (FCTC) is a
legally binding treaty that requires its parties to implement particular
evidence-based tobacco control measures. It entered into force in 2005
and is meant to assist LMICs in making tobacco control a reality in
their countries. It provides both supply- and demand-reduction provi-
sions. The WHO’s MPOWER package (Table 22.2) provides technical
measures and resources to help countries implement all of the FCTC
demand-reduction provisions (http://www.who.int/tobacco/mpower/
en/). According to a 2017 world tobacco control report done col-
laboratively by the WHO and the US National Cancer Institute (NCI),
the FCTC has had a galvanizing effect on efforts related to tobacco
control and has resulted in some progress to date, yet the majority of
the world’s population remains uncovered by the most effective tobacco
control interventions.19 The report calls for “continued research and
surveillance of the epidemic and implementation of the evidence-based
strategies set forth in the WHO FCTC, as well as vigilant monitoring
of the tobacco industry’s tactics and strategies to undermine or subvert
tobacco control efforts.”19
For more on tobacco, particularly as it relates to individual risks
and clinical interventions, the reader is referred to Chapter 24.
Alcohol
The acceptability of alcohol in many countries often overshadows the
fact that it is a recreational drug with psychoactive properties and the
potential to induce dependence, in addition to being a component
cause in approximately 200 different injury conditions and diseases,
including cancer.20 There is a complex array of influences on alcohol
use at both the societal and the individual levels. The reader is referred
to the WHO Global Status Report on Alcohol and Health for a more
in-depth review of these factors.20
Based on data from the WHO, more than one-third of the world’s
population aged 15 or older are current drinkers.20 In the United
States alone, almost 90% of those aged 18 or older report use of
alcohol at some point in their lives, and 72% report use within the
last year. Prevalence of alcohol consumption varies widely around the
globe, as do drinking patterns. In general, wealthier nations consume
more alcohol than do poorer nations. High-income countries have a
much higher prevalence of current drinkers and of heavy episodic
drinking, 70% and 22%, respectively, than do low-income countries,
where the equivalent rates are 18% and 12%, respectively.20 There
are also large gender differences in the use of alcohol, with women
more often being lifetime abstainers than men and generally consuming
less alcohol and being less likely to engage in heavy episodic drinking
340 Part I: Science and Clinical Oncology
when they do drink.20 However, alcohol use has been steadily increasing
among women, given globalization and the changing roles of women
in society.21 This trend could have significant public health impacts,
as women may be more vulnerable to alcohol-related harm for a given
level of use,21 but also because of the known health consequences
associated with alcohol use during pregnancy. Age also affects alcohol
consumption, with adolescents having a higher prevalence of monthly
heavy episodic drinking than adults.20 Although the most frequently
consumed type of alcohol varies regionally, spirits constitute half of
the total alcohol consumed globally.20
The Role of Alcohol in Cancer
In 2007, after a week-long review of the evidence by experts, the WHO
International Agency for Research on Cancer (IARC) concluded that
cancers of the mouth, pharynx, larynx, esophagus, liver, colorectum,
and breast were causally related to alcohol consumption.22 Bagnardi
and colleagues provided the most recent relative risk (RR) estimates for
the effects of alcohol consumption on these cancers, and others, based
on 572 studies with over 486,000 cancer cases.23 In this meta-analysis,
light (≤12.5 g of alcohol per day), moderate (≤50 g/day), and heavy
(>50 g/day) consumption of alcohol all increased the risk of oral
cavity–pharyngeal and esophageal squamous cell carcinoma (ESCC) in
a dose-dependent fashion, with heavy drinkers having approximately
five times the risk of these cancers compared with nondrinkers and
occasional drinkers (oral cavity and pharynx: RR, 5.13 [95% CI,
4.31–6.10]; ESCC: RR, 4.95 [95% CI, 3.86–6.34]).23 These were by far
the largest RRs detected in the study. Light drinking was not associated
with cancer of the larynx, although moderate drinking increased the
risk of this cancer by 44% (RR, 1.44 [95% CI, 1.26–1.66]), and
heavy drinking more than doubled the risk (RR, 2.65 [95% CI,
2.19–3.19]).23 Female breast cancer also exhibited a dose-response
relationship with alcohol, with light drinking being associated with a
4% increase in risk (RR, 1.04 [95% CI, 1.01–1.07]), moderate drinking
conferring a 23% increase in risk (RR, 1.23 [95% CI, 1.19–1.28]),
and heavy drinking resulting in a 61% risk increase (RR, 1.61 [95%
CI, 1.33–1.94]).23 As with laryngeal cancer, light drinking was not
associated with CRC in either men or women, but moderate and
heavy drinking significantly increased the risk of this cancer for men by
21% (RR, 1.21 [95% CI, 1.11–1.32]) and 53% (RR, 1.53 [95% CI,
1.30–1.80]), respectively. Cancers for which the risk was elevated only
for heavy drinkers included cancers of the gallbladder (RR, 2.64 [95%
CI, 1.62–4.30]), liver (RR, 2.07 [95% CI, 1.66–2.58]), stomach (RR,
1.21 [95% CI, 1.07–1.36]), pancreas (RR1.19 [95% CI, 1.11–1.28]),
and lung (RR, 1.15 [95% CI, 1.02–1.30]).23 The elevated risk of
lung cancer may be due to residual confounding by smoking, as a
previous meta-analysis did not show an association between alcohol
and lung cancer in never smokers.24 Other findings from the 2014
meta-analysis include a possible positive association between alcohol
use and melanoma and prostate cancer; an inverse linear association
with lymphomas; and no associations between alcohol use and ovarian,
cervical, endometrial, bladder, and brain cancers. With regard to
prostate cancer, a 2016 systematic review and meta-analysis by Zhao
and colleagues demonstrated a statistically significant dose-response
relationship between level of alcohol consumption and increased risk
of prostate cancer, with risks being of similar magnitude to the risks
detected in the Bagnardi and colleagues meta-analysis.25 Nevertheless,
the literature is mixed on this topic, and the World Cancer Research
Fund (WCRF)/AICR currently states that the evidence is “limited—no
conclusion” for alcoholic beverages and prostate cancer.26
An important aspect of alcohol use that has been more difficult to
study, and was not and could not be examined in the comprehensive
meta-analysis by Bagnardi and colleagues, is pattern of use. However,
in a 2015 study by Cao and colleagues, regularity of drinking and
heavy episodic drinking were not associated with cancer risk, after
adjustment for total alcohol intake, in the Nurses’ Health Study and the
Health Professionals Follow-Up Study.27 Analyses of beverage type have
not produced consistent findings; and in general the most-consumed
beverage in each study is the one that demonstrates the greatest risk.28
The amount of alcohol consumed appears to be more important
than the type.
Further supporting the role of alcohol in cancer are studies that
demonstrate the reversal of risk seen after cessation of drinking. At
least three pooled analyses have suggested significant reductions in risk
of head and neck, esophageal, and liver cancers after alcohol cessation,
although significant timeframes were required for former drinkers’ risk
to equal the risk of never-drinkers. In a 2013 meta-analysis of nine
case-control studies examining the effect of cessation on laryngeal
and pharyngeal cancers, a risk reduction of 2% per year was seen, on
average, for quitters.29 Although it took over 35 years for the risks of
laryngeal and pharyngeal cancers among quitters to equal the risks
of never-drinkers, 5 years was enough time to see a 15% reduction
in the alcohol-related elevated risk of these cancers.29 In 2011, Rehm
and colleagues examined the effect of alcohol cessation on risk of
esophageal and head and neck cancers in a pooled analysis of 13
studies.30 This report identified significantly increased risks for both
cancers in the 5- to 10-year period immediately following cessation,
with risks then declining but not reaching levels of never-drinkers
until more than 20 years after quitting.30 Another meta-analysis, by
Heckley and colleagues, found a significantly increased risk of liver
cancer in the 5 to 10 years after cessation, with a 6% to 7% reduction
in risk per year thereafter, with an estimated 23 years required for risk
equivalence between quitters and never-drinkers.31 The results of these
studies are consistent with the long latency period of most cancers
but nevertheless demonstrate the reversibility of the increased risk
conferred by alcohol. Despite long timeframes required for risk among
quitters to equal that of never-drinkers, these findings suggest that
alcohol cessation efforts are likely to result in a public health benefit
because significant and often substantial decreases were seen in the
risks of these alcohol-associated cancers in the short to medium term.
Finally, some studies have documented protective effects of low-
to-moderate alcohol consumption on other health outcomes, notably
those related to coronary heart disease (CHD) and myocardial infarc-
tion.32 However, there is some evidence to suggest that the observed
protective effect of alcohol on cardiovascular disease seen in many
epidemiologic investigations is due to residual or unmeasured confound-
ing and/or misclassification bias.33 Yet, as reported in a review of
findings from the Nurses’ Health Study, moderate (0.1–14.9 g/day)
alcohol intake remained associated with a significantly reduced risk
of CHD in that cohort even after controlling for critical lifestyle risk
factors and incorporating detailed information on amount and frequency
of consumption.34 The review also notes that the same level of consump-
tion was associated with an increased risk of breast cancer and bone
fractures among the Nurses’ Health Study women.34 Such a compre-
hensive assessment of all the potential health effects of alcohol consump-
tion is challenging, and the balance of risks and benefits is likely to
differ by population and/or other factors. At a more global level, a
2015 prospective cohort study of nearly 115,000 adults from 12
countries across the human development index concluded that current
drinking was not associated with a net health benefit.35 However, the
follow-up time of this study was less than 5 years, and more time is
likely needed to observe the full range of risks and benefits associated
with low-to-moderate drinking. More research is needed in this area
to better understand the balance of risks and benefits of alcohol intake
in particular settings.
In sum, according to the latest consensus of evidence, alcohol has
been shown to convincingly increase the risk of at least six cancers:
(1) oral and pharyngeal cancer; (2) laryngeal cancer; (3) ESCC; (4)
CRC (in men only); (5) breast cancer; and (6) liver cancer. The risk
is strongest for oral and pharyngeal cancers, laryngeal cancer, and
ESCC. A strong, positive dose-response relationship is seen for all of
these cancers with the exception of liver cancer, in which only heavy
consumption appears to increase risk. There is evidence for probable
increased risk for stomach cancer and for CRC in women. There is
no safe level of alcohol use for cancer prevention, because even relatively

low levels have been shown to increase risk of particular cancers, and
the amount of alcohol consumed matters more than the type of beverage
consumed. And as with smoking, the increased risk conferred by
alcohol use is reversible, with former drinkers assuming the same level
of risk as never-drinkers within 20 to 35 years of cessation.
Because alcohol use varies across the globe, the attributable frac-
tions of cancer cases and deaths due to alcohol also vary depending
on world region and country. According to recent estimates, alcohol
was responsible for 5.5% of cancer cases and 5.8% of cancer deaths
globally in 2012.36 By gender, men’s alcohol-attributable fractions were
twice those of women’s for both overall incidence and mortality.36 By
geographic region, the attributable fractions for incidence and mortality
were highest in the Western Pacific region, where alcohol accounted
for 7.1% of cancer cases and 7.8% of cancer deaths; but among men
in this region, the fractions rose to 9.1% and 9.6%, respectively.36
This region includes China, Japan, Korea, Australia, New Zealand,
and many other smaller Asian countries and islands of the South
Pacific. Unsurprisingly, the lowest attributable fractions were found
in the Eastern Mediterranean region, which consists largely of Muslim
countries. In this region, alcohol was responsible for less than 1% of
both cancer cases and deaths.36 Among cancer sites, ESCC had the
largest number of cases (44.7%) and deaths (43.7%) attributable to
alcohol, followed by oral cavity and pharynx (36.7% of cases and 34.2%
of deaths due to alcohol) and larynx (26.1% of cases and 24.7% of
deaths due to alcohol). Less than 10% of colorectal and breast cancer
cases and deaths were due to alcohol.36 Among women, breast cancer
was the number one cancer attributable to alcohol, whereas ESCC
was number one among men.36
Proposed Mechanisms Linking Alcohol With Cancer
The mechanisms behind the carcinogenic effects of alcoholic beverages
are not entirely clear, although they are likely to be organ specific.
Although ethanol itself is considered carcinogenic, it is its metabolite,
acetaldehyde (AA), that is likely responsible for much of the carcinogenic
effect of alcohol consumption, at least within the upper and lower
gastrointestinal (GI) tracts, where it could come into direct contact
with tissues and where its concentration would be high enough to
exert an effect.37 Ethanol is metabolized into AA by the alcohol
dehydrogenase (ADH) and cytochrome P450 2E1 enzymes; AA is
subsequently degraded to acetate (noncarcinogenic) by the aldehyde
dehydrogenase (ALDH) enzyme. Salivary AA increases as ethanol
ingestion increases, and increased levels of AA have been documented
in the saliva of those with and without cancer after moderate alcohol
consumption; and experiments in rats and piglets have demonstrated
mutagenic levels of colonic AA after administration of ethanol.37 AA
has been shown to induce DNA damage through the formation of
adducts and the inhibition of DNA repair, as well as being shown to
alter DNA methylation.38 The effect of alcohol and AA has been
shown to be modulated by genetic factors, mainly polymorphisms in
the genes for ADH and ALDH that affect enzymatic activity and the
amount of AA within saliva and other tissues, and the risk of alcohol-
related cancers.28,37 As an example, the ALDH2*2 allele is prevalent
among Asians, and those who are homozygous for this allele are unable
to metabolize AA and therefore cannot tolerate alcohol. Those who
are heterozygous for ALDH2*2 can metabolize only small amounts
of AA and generate threefold higher concentrations of AA in their
serum and saliva when they drink, compared with those who are
homozygous for the normal allele (ALDH2*1).39 A number of studies
carried out in the Japanese population have established the mutant
ALDH2*2 allele as a strong risk factor among drinkers for cancers of
the upper aerodigestive tract—most particularly, esophageal cancer.40,41
The mechanism behind the increased risk of carriers of the mutant
ALDH allele may be the formation of DNA adducts, as studies in
Japanese drinkers have demonstrated that those with the mutant allele
harbor more AA-derived DNA adducts in their peripheral lymphocytes
than do drinkers without the allele,42 in addition to having more sister
chromatid exchanges and micronuclei.43,44
Lifestyle and Cancer Prevention  •  CHAPTER 22 341
Within the liver, it is likely that oxidative stress plays an important
role in alcohol-related carcinogenesis, because hepatic levels of AA
are relatively low after intake of alcohol.45 The CYP2E1 enzyme
involved in the metabolism of ethanol to AA is capable of producing
reactive oxygen species (ROS) that result in the formation of lipid
peroxidation products (e.g., 4-hydroxynonenal) that are capable of
forming highly mutagenic DNA adducts. One such adduct results
in a mutation of TP53 that leads to cells being more resistant to
apoptosis and confers a growth advantage.46 Similar to ADH and
ALDH, CYP2E1 contains polymorphisms that have been associated
with different levels of enzyme activity. However, results of individual
studies examining an association of these variants with liver cancer in
various populations have been inconclusive. A 2017 meta-analysis of
16 studies including 1737 liver cancer cases and 2947 controls found
no association between two CYP2E1 variants and risk of liver cancer.47
In addition to CYP2E1-related mechanisms, chronic consumption of
alcohol may also result in inflammation-driven oxidative stress, hepatic
iron overload, and increased nitric oxide production, all additional
sources of ROS.37,45
Within the breast, alcohol’s effect on estrogen levels is thought
to be an important mechanism of carcinogenesis, in addition to the
aforementioned mechanisms of alcohol metabolism, because breast
tissue has the ability to metabolize ethanol at low levels. Estrogens
have a proliferative effect on breast epithelial cells, and it has been
well documented that prolonged exposure to estrogens increases the
risk for breast cancer.48,49 And alcohol has been shown to increase
estrogen and other sex hormone levels in both premenopausal
and postmenopausal women. A meta-analysis of eight prospective
studies involving postmenopausal women documented significant
increases in all sex hormones in women who consumed 20 g of
alcohol per day compared with nondrinkers.50 A randomized trial of
51 postmenopausal women showed that serum estrone sulfate and
dehydroepiandrosterone sulfate (DHEAS) were significantly elevated
by 8% and 5%, respectively, in those who consumed 15 g of alcohol
per day;51 and in women who used hormone replacement therapy
(HRT), the effect of alcohol was even more potent, resulting in a
threefold increase in plasma estradiol among those who drank 15.6 g
of alcohol per week.52 Among premenopausal women, one of the
earliest studies documented a significant increase in plasma estradiol
levels in healthy women after acute administration of alcohol (0.695 g/
kg), whereas levels did not change significantly after ingestion of a
placebo.53 Controlled-diet studies in this population have found similar
results.54,55 Authors of some of these studies have postulated that the
increase in circulating sex hormones may be due to an increase in the
hepatic redox state that is associated with the catabolism of ethanol.53
Another possible mechanism for the alcohol-mediated increase in
circulating sex hormones is the enhanced conversion of testosterone
to estrogen resulting from increased aromatase activity that is seen
after chronic alcohol exposure.56,57 These effects likely operate through
estrogen receptor (ER)–dependent pathways. Epidemiologic studies
strongly suggest that alcohol use is more strongly associated with ER+
tumors than with ER− tumors.58 In addition, experimental studies
have shown that ethanol can stimulate proliferation of ER+ breast
cancer cells but not ER− ones, and furthermore that ethanol increases
transcriptional activation of the ER.59 However, the ER pathway alone
cannot explain the effects of alcohol on ER+ tumors. Other mechanisms
that are currently being investigated include alcohol’s potential ability
to promote the epithelial-mesenchymal transition in breast cancer
cells, to activate stromal cells and influence tumor-stroma interactions,
and to lead to epigenetic dysregulation by possibly interfering with
folate metabolism.59
Within the colorectum, changes in methylation and folate metabo-
lism are also believed to be responsible for at least some of the carci-
nogenic effects of alcohol.28,37 Numerous epidemiologic studies have
documented a protective effect of folate on risk of CRC and adeno-
matous polyps.60 Alcohol is a known inhibitor of folate metabolism,
leading to reduced methionine synthesis and subsequently resulting
342 Part I: Science and Clinical Oncology
in abnormal DNA methylation, which is frequently observed in
colorectal neoplasia.61 Many studies have documented an interaction
between folate and alcohol, wherein those with low folate intake and
high alcohol consumption have a much greater risk of CRC than do
those exposed to either risk factor alone.61 Heavy use of alcohol can
lead to reduced intake of foods rich in folate and other B vitamins
and may interfere with intestinal absorption and/or the metabolism
of these key nutrients.28 The effect of folate and alcohol on risk of
CRC may be modified by known polymorphisms in the genes involved
in DNA methylation and alcohol and folate metabolism. Perhaps the
most extensively studied polymorphisms are those in the MTHFR
gene, which is central to the formation of methionine. One polymor-
phism in particular, C677T, appears to interact with levels of folate
and alcohol intake to influence risk of CRC.28,62 In addition to alcohol’s
antifolate effects, other alcohol-related carcinogenic mechanisms are
likely at play in the colorectum, including inflammation and the
production of ROS and AA.
Other mechanisms that may be responsible for some of the car-
cinogenic effects of alcohol include the presence of other carcinogens
in alcohol in addition to ethanol and AA, such as N-nitrosamines,
and the ability of alcohol to enhance the absorption or activity of
other carcinogens (e.g., tobacco).30
Evidence-Based Interventions for Cancer Prevention Related
to Alcohol Use
Published cancer prevention recommendations from the ACS and
the AICR/WCRF suggest that if alcohol is consumed, it should be in
moderation, which is defined as up to one drink per day for women
and two per day for men.63,64 Interventions to reduce alcohol consump-
tion are available at both the personal (or clinical) and population
level. At the personal level, the US Preventive Services Task Force
(USPSTF) recommends “that clinicians screen adults aged 18 years
or older for alcohol misuse and provide persons engaged in risky or
hazardous drinking with brief behavioral counseling interventions
to reduce alcohol misuse.”65 This is a Grade B recommendation,
meaning that there is either high certainty that the net benefit of
this intervention is moderate or that there is moderate certainty
that the net benefit is moderate to substantial. As of the writing
of this chapter, this recommendation was in the process of being
updated by the USPSTF. Screening for alcohol can be done in one
of three ways: with (1) the Alcohol Use Disorders Identification Test
(AUDIT); (2) the abbreviated AUDIT-Consumption (AUDIT-C)
screen, or (3) the single-question screen, “How many times in the
past year have you had five (for men) or four (for women and all
adults older than 65 years) or more drinks in a day?”65 Each of these
screening tools has demonstrated good sensitivity and specificity for
detecting alcohol misuse across multiple populations, but the AUDIT
screen has been the most widely studied in the primary care setting.65
After screening, brief interventions that require multiple contacts
between provider and patient, with each contact lasting at least 6
to 15 minutes, demonstrate the strongest evidence for effectiveness;
single-contact interventions lasting 5 minutes or less have limited
effect.65 The USPSTF documents that these types of interventions
delivered in the primary care setting can positively affect unhealthy
drinking behaviors in adults, including reducing the amount of alcohol
consumed weekly, reducing the proportion of persons who engage in
episodes of heavy drinking, and increasing long-term adherence to
drinking recommendations. It is important to note that the USPSTF
recommendation distinguishes between those who “misuse” alcohol and
those who “abuse” alcohol and are dependent on it. There is limited
evidence that these types of brief behavioral interventions are ineffective
in the setting of alcohol abuse or dependence. In such cases the benefits
of specialty treatment are well established, and this type of treatment is
recommended.65
At the population level, a number of actions can be taken by local,
state, and/or federal authorities to reduce the use of alcohol and prevent
its associated harms. Many of these actions mirror actions used in
tobacco control. Taxation, restriction of availability, and regulation
of marketing and advertising are common, effective tools not only
for tobacco control, but also for alcohol control.20 Taxes and pricing
policies are perhaps some of the more impactful interventions that
can be implemented not only to reduce drinking, but also to reduce
alcohol-related mortality. A 2009 meta-analysis of 112 studies providing
over 1000 estimates of the tax/price–consumption relationship found
that a 10% increase in the price of alcohol resulted in a 5% reduction
in drinking; and that price affected drinking of all types of beverages
and across all populations of drinkers, from light to heavy users.66
Authors noted that effects were large compared with what is typically
seen for preventive interventions. A subsequent 2010 systematic review
of 50 studies containing 340 estimates by the same group documented
that tax/price policies could significantly reduce alcohol-related
morbidity and mortality, with a 35% reduction in alcohol-related
mortality expected from a doubling of alcohol taxes.67 Because of their
relatively low cost to implement and their documented large and
significant impacts on drinking and related outcomes, tax and pricing
interventions are strongly recommended by both the US Community
Preventive Services Task Force (https://www.thecommunityguide.org/
topic/excessive-alcohol-consumption) and the WHO for population-
level alcohol control.20
Obesity
Obesity is now considered an epidemic, with approximately
17% of children and 36% of adults classified as obese.68 Accord-
ing to the IARC, there is abundant evidence supporting the link
between obesity and the following 13 different types of cancers,
now considered obesity-related cancers: cancers of the esophagus,
gastric cardia, colon and rectum, liver, gallbladder, and pancreas;
postmenopausal breast cancer; endometrial, ovarian, and renal cell
cancers; meningioma; thyroid cancer; and multiple myeloma.69 In
addition, a systematic review conducted by Birks and colleagues70
demonstrated a link between deliberate weight loss in overweight or
obese individuals and reduced cancer incidence. This section reviews
the role of obesity in cancer and proposed mechanisms linking obesity
with cancer.
Role of Obesity in Cancer
In obesity, adipose tissue typically accumulates in two different regions:
the android region or abdominal area, and the gynoid region, which
consists of the hips and thighs. Android obesity differs from gynoid
obesity such that it contains two different types of adipose tissue
deposits, subcutaneous and visceral. The latter, visceral adipose tissue
(VAT), is more metabolically active compared with subcutaneous
adipose tissue, is considered the impetus for metabolic dysfunction,
and may contribute to increased cancer risk in obese individuals.71,72–76
Therefore, excessive accumulation of VAT is considered to be a major
contributor to obesity’s role in cancer.
Epidemiologic evidence supports the relationship between VAT
and increased cancer risk. In an observational study conducted by
Britton and colleagues,77 adiposity measurements in four different
areas (subcutaneous, visceral, periaortic, and pericardial) were compared
with incident cancer, cardiovascular disease, and all-cause mortality
over a median 5-year follow-up period. In this study, 3086 middle-aged
men and women (average age, 50 years ± 10) were included. VAT,
but not other adipose areas, was significantly associated with cancer
incidence (n = 141 incident cancer cases; hazard ratio [HR], 1.43
[95% CI, 1.12–1.84]) when the model was adjusted for clinical factors
(e.g., age, sex, blood pressure, body mass index).77 When the sample
was stratified by sex, there were no significant differences observed
among adipose areas and cancer incidence; however, HRs were higher
in men compared with women for VAT.77 These findings support the
relationship between VAT and cancer incidence in middle-aged men
and women.

Similarly, Murphy and colleagues78 compared the relationship of
subcutaneous, visceral, intramuscular, and total-body adiposity with
incident cancer over a 13-year follow-up period in a study that included
2519 older men and women (average age, approximately 74 years).
In considering all incident cancers (n = 617), a significant association
between total adiposity, VAT, and cancer incidence was observed in
women (HR per standard deviation [SD] increase, 1.14 [95% CI,
1.01–1.30]; and 1.15 [95% CI, 1.02–1.30], respectively) but not in
men when clinical factors were adjusted for.78 Regarding obesity-related
cancers (n = 224), after adjustment for clinical factors, a significant
association between total adiposity but not other adiposity measures,
and obesity-related cancer incidence was observed in women (HR per
SD increase, 1.23 [95% CI, 1.03–1.46]) and a significant association
between VAT and obesity-related cancer incidence was observed in
men (HR per SD increase, 1.30 [95% CI, 1.06–1.60]).78 Overall,
evidence from these prospective cohort studies support the role of
obesity, and specifically VAT, in cancer.
Proposed Mechanisms Linking Obesity With Cancer
Although the relationship between obesity and cancer is evident from
observational data, the physiologic mechanisms explaining this relation-
ship have recently emerged in the literature.72,79–81 Essentially, the
excessive accumulation of adipose tissue, characterized by adipocyte
hypertrophy and hyperplasia,80 leads to a state of adipocyte hypoxia
and resultant macrophage activity, and altered adipokine profiles; these
factors promote an interplay among systemic inflammation, sex
hormones, and insulin resistance, all of which elicit conditions that
promote tumorigenesis.
According to a review conducted by Teoh and Das,81 compared
with lean counterparts, adipocyte samples from obese humans and
animal models revealed a state of hypoxia within the adipose tissue via
presence of hypoxia-inducible factor (HIF), lower levels of oxygen, and
lactate. This demonstrated state of hypoxia causes macrophage recruit-
ment via signaling pathways such as chemokine ligand 2, interleukin
(IL)-1β/C-X-C motif chemokine ligand 12.81 Macrophages remain
in the adipose tissue because of increased expression of netrin-1, a
protein that has been proposed to block migration of macrophages
and is expressed when fatty acid palmitate is saturated.81 Of note,
fatty acid palmitate is saturated within the obese state secondary to
the increase in lipolysis and consequent increase in circulating free
fatty acids.
Next, the macrophages transition from an M2 prorepair to an M1
proinflammatory state, activating nuclear factor-κB (NF-κB) and signal
transducer activator of transcription 3 (STAT3).79 NF-κB has been
linked with reduction in cellular senescence, facilitation of the epithelial-
mesenchymal transition, and an increase in proinflammatory cytokines
such as tumor necrosis factor–α (TNF-α) and IL-6.81 STAT3 increases
expression of cyclins and antiapoptotic proteins, resulting in an increase
in cellular proliferation and decrease in cellular apoptosis.79 These
cytokines play different roles depending on cancer type. For example,
in breast cancer, macrophage activation of NF-κB leads to increased
expression of cytokines (i.e., IL-1β, TNF-α, prostaglandin E2 [PGE2])
that increase transcription of CYP19, a transcription factor of aromatase,
and result in increased bioavailability of estrogen.80 Overall, macrophage
activity secondary to hypoxic conditions from obesity promotes
tumorigenic conditions.
Along with macrophage activity, adipokine profiles, notably adi-
ponectin and leptin, are altered in the obese state. Normal physiologic
levels of adiponectin are linked with enhancement of insulin sensitivity
and reduction in angiogenesis.72,81 However, in the obese state, adi-
ponectin levels decrease owing to instability of adiponectin mRNA
and subsequent reduction in gene expression.81 In contrast, under
normal physiologic conditions, leptin promotes satiety and feeding
cessation; yet in the obese state, leptin levels increase. The increase
in leptin is associated with increases in cellular proliferation and
inhibition of apoptosis in breast, ovarian, and cervical cancers.81
Furthermore, increased leptin has demonstrated an association with
Lifestyle and Cancer Prevention  •  CHAPTER 22 343
colon cancer in men.80 Currently proposed mechanisms for these
associations vary depending on cancer site. For example, in ovarian,
endometrial, and breast cancers, some studies have shown that cell
growth is facilitated by leptin binding to ER-α.79 Overall, there is
evidence to support obesity-induced alterations in adipokines as an
enabler of tumorigenesis.
Considering that adiponectin is associated with insulin sensitivity,
in the obese state a reduction in adiponectin also contributes to a
reduction in insulin sensitivity and subsequent insulin resistance.81
Insulin resistance facilitates the tumor microenvironment via two
interrelated mechanisms. First, increased insulin levels result in a
reduction of insulin-like growth factor–binding proteins 1 and 2
(IGFBP1, IGFBP2) and subsequent increase in insulin-like growth
factor 1 (IGF1), ultimately resulting in increased cellular proliferation
and decreased apoptosis via the AKT/mTOR pathway.81 Second,
increased insulin, IGF1, and TNF-α reduce the amount of sex
hormone–binding globulin produced, which increases cancer risk
because of the resultant increase in levels of sex hormones.81 Although
the mechanisms related to estrogen and cancer risk vary depending
on cancer type, according to Teoh and Das,81 estrogen may cause
replication errors during DNA synthesis and increase cellular prolifera-
tion; hence the number of mutations accumulates over time, yielding
tumor growth. In addition to insulin serving as a contributing factor
for increased estrogen and protumorigenic effects, it is important to
note that adipose tissue is a major site for estrogen synthesis in
postmenopausal women and men; thus, increased adipose tissue leads
to increased estrogen production, and consequently a greater ratio of
circulating estrogens to androgens may lead to cancer.81 Taken together,
it is evident that excessive accumulation of adipose tissue induces
protumorigenic conditions.
Physical Inactivity
Physical activity consists of two subtypes of activity: exercise or
structured physical activity, which is defined as activity pursued for
the purpose of improving fitness, and lifestyle physical activity (i.e.,
completing house chores, walking to work, taking the stairs). Therefore
physical inactivity is identified when one not only does not engage
in exercise but also has minimal lifestyle activity.
Role of Physical Inactivity in Cancer
Current public health guidelines recommend 150 minutes per week
of moderate-intensity or 75 minutes per week of vigorous-intensity
aerobic activity, and at least 2 days per week of strength training
exercise.82 Compliance with these guidelines may be measured sub-
jectively, through self-report, or objectively, through measurement or
estimation of peak oxygen consumption via a maximal or submaximal
cardiopulmonary exercise test respectively. Overall, both subjective
and objective evidence is available linking regular, structured physical
activity with reduced risk of chronic disease and certain cancers such
as colon and breast cancer.82–88 An inverse relationship has been
demonstrated between increased physical activity levels and reduction
in cancer risk.82,88 Moreover, a retrospective analysis conducted by Lee
and colleagues89 found that individuals who did not engage in regular,
structured activity were 1.32 times more likely to develop colon cancer
and 1.33 times more likely to develop breast cancer than physically
active individuals.
In addition to structured physical activity, when lifestyle activity
decreases, it is typically replaced by increased sedentary behavior. A
meta-analysis by Schmid and Leitzmann90 noted that colon cancer
risk increased by 8% and endometrial cancer risk by 10% for every
2-hour increase in total sitting time per day. In addition, in a prospective
cohort study conducted by Nomura and colleagues,91 a significant
association with sitting time and breast cancer incidence was observed
when individuals spent more than 10 hours sitting per day. Taken
together, sedentary behavior is linked with increased risk of colon,
breast, and endometrial cancer.
344 Part I: Science and Clinical Oncology
Proposed Mechanisms Linking Physical Activity With
Reduced Cancer Risk
Evidence supports the impact of physical activity on cancer risk
reduction through mechanisms related to obesity, skeletal muscle
cytokine production, and immune function. As mentioned previously,
obesity-induced insulin resistance promotes tumorigenesis. However,
physical activity may counteract this by improving insulin sensitivity
through alterations in VAT deposition and training adaptations of
the skeletal muscle.
Exercise without weight loss or changes in diet has demonstrated
effective reduction in VAT,92–99 the primary fat depot connected to
increased metabolic dysfunction and promotion of tumorigenesis.77,78
The proposed mechanism for VAT reduction with exercise alone is
attributed to an increase in β-adrenergic receptor activity in adipo-
cytes,100 which results in an increase in lipolysis from abdominal fat
depots compared with other fat depots during exercise.97,100,101 Moreover,
the reduction in VAT results in an increase in adiponectin.102 As
mentioned previously, adiponectin is linked with enhancement of
insulin sensitivity and reduction of angiogenesis.72,81
In addition, in the obese state, impaired glucose tolerance at the
level of the skeletal muscle exacerbates insulin resistance secondary
to the increase in circulating free fatty acids and consequent increase
in diacylglycerol instead of glucose for substrate utilization.103 However,
exercise-induced skeletal muscle contraction prompts increased
translocation of GLUT4 receptors to the T tubules and plasma
membrane of the working muscles, which improves skeletal muscle
glucose uptake and whole-body insulin sensitivity.104 Therefore, regular
physical activity, even in the obese state, improves insulin sensitivity
and prevents increases in bioavailability of sex hormones, two factors
that help inhibit tumorigenesis.
Along with obesity-related mechanisms, there are also mechanisms
unique to skeletal muscle activity—notably, release of cytokines from
contracting skeletal muscle, commonly referred to as myokines. To date,
identified myokines include IL-6, irisin, IL-15, calprotectin, myonectin,
oncastatin M (OSM), and secreted protein acidic and rich in cysteine
(SPARC).105 According to a review by Goh and colleagues,105 during
exercise myokines are released from the working skeletal muscles, which
increases activity of antiinflammatory cytokines (i.e., IL-1 receptor
agonist and IL-10), decreases activity of proinflammatory cytokines,
and attenuates migration of M1 macrophages and T cells to areas
with high expression of inflammatory cytokines or inflammation (i.e.,
adipose tissue and/or the tumor microenvironment).
Currently, promising evidence is available regarding the roles of
exercise-induced OSM and irisin activity in breast cancer prevention,
and SPARC in colon cancer prevention.106–108 Within the context of
breast cancer prevention, Hojman and colleagues 108 treated MCF-7
cells (malignant, ER+, breast epithelial cells) with mouse serum collected
immediately after exercise and demonstrated a 52% reduction in cell
proliferation and 54% increase in caspase activity. Furthermore, whereas
various myokines were upregulated after exercise, OSM demonstrated
the greatest effects on cellular proliferation, caspase activity, and
apoptosis.108 The efficacy of OSM was further supported in a model
in which OSM signaling was blocked.108
Similarly, Gannon and colleagues107 assessed the influence of irisin
activity specifically on malignant breast epithelial cells. Overall, irisin
reduced cellular proliferation in aggressive breast epithelial cells through
caspase activity and suppression of NF-κB.107 Taken together, current
evidence supports OSM and irisin as two contributing myokines
connected to breast cancer prevention through their impact on caspase
activity.
Colon cancer is also linked with myokine activity. Specifically, the
myokine SPARC has been linked with reduced risk of colon cancer.
Aoi and colleagues 106 assessed SPARC in relation to exercise and colon
cancer among a colon-26 carcinoma cell model, two different animal
models, and a human subject exercise intervention. In humans, SPARC
levels released from working muscles during exercise increased linearly
with training, yet resting levels remained unchanged, supporting the
role of SPARC as a myokine. In the first animal model, exercised mice
with azoxymethane (AOM)-induced colon tumorigenesis demonstrated
a reduction in aberrant crypt foci and aberrant crypts compared with
AOM nonexercised and control groups.106 These findings were also
observed in the cell line model and lend support to the role of exercise
in the reduction of colon tumorigenesis. In addition, the second
animal model compared sedentary and exercised SPARC knockout
mice to exercised wild-type mice and demonstrated an increase in
apoptotic cells via caspase-3 and caspase-8 activity in exercised wild-type
mice, with no change in apoptosis observed in either group of the
SPARC knockout mice, supporting SPARC’s role in attenuation of
tumorigenesis. Similarly, in a review conducted by Sanchis-Gomar and
colleagues,88 evidence supports SPARC activation of caspase-3 and
caspase-8 to promote cellular apoptosis and prevent aberrant crypt foci
formation in colon cancer. Overall, evidence supports SPARC’s role
as a myokine and as a contributing factor in reducing colon cancer.
Interconnected with myokine activity is the impact of exercise-
induced activation of natural killer (NK) cells in relation to physical
activity and cancer prevention. In a review by Idorn and colleagues109
a reduction in tumor incidence of greater than 60% was observed
due to exercise-induced activation of NK cells among various mouse
models. In humans, NK cells seem to function similarly, such that
NK cells recognize and kill cancer cells.110 During exercise the myokine
IL-15 activates NK cells and T cells via transpresentation of IL-15/
IL-15 receptor alpha complex.109 In addition, with exercise, the natural
increase in epinephrine released from β-adrenergic receptors facilitates
the release and mobilization of NK cells.109,111 Moreover, NK cells,
compared with other lymphocytes, have the greatest density of
β-adrenergic receptors.109 Taken together, improvements in fat deposi-
tion and insulin sensitivity, and the activity of myokines and NK
cells, are all viable proposed mechanisms explaining the role of physical
activity in cancer prevention.
Diet
The link between diet and cancer risk is complex, such that not all
dietary components (i.e., types of food, dietary patterns, vitamins,
and minerals) are linked with cancer risk, and some components
exhibit strong links with cancer risk individually whereas others exhibit
stronger effects synergistically. This section presents the dietary
components containing the most evidence in relation to increased or
decreased cancer risk.
Dietary Components Linked With Increased Cancer Risk
Red meat and processed meats
The majority of evidence from systematic reviews and meta-analyses of
epidemiologic evidence, randomized trials, and observational studies
supports the link between high consumption of red or processed
meats and increased risk of CRC.64,112–114 A 15% to 20% increase in
CRC risk has been detected per 100-g and 50-g daily intake of red
and processed meats, respectively.64 A meta-analysis of epidemiologic
evidence revealed a summary RR of 1.11 (95% CI, 1.03–1.19) for
CRC when red and processed meats were consumed regularly.112
Although significant heterogeneity was observed, the relationship was
similar and with statistically insignificant heterogeneity when assessed
by subgroups. For example, when stratified by sex, men exhibited a
significant association with regular intake of red or processed meats and
CRC risk compared with women (RR, 1.16 [95% CI, 1.02–1.32]; and
RR, 1.03 [95% CI, 0.91–1.17], respectively).112 Interesting to note,
when comparing North America and Europe, North Americans were
at greater risk of CRC (RR, 1.11; 95% CI, 1.03–1.19) than Europeans
(RR, 1.07; 95% CI, 0.98–1.16) when red or processed meats were
consumed regularly,112 suggesting that there may be other lifestyle
factors unique to North Americans that may contribute to CRC risk.
Similarly, a systematic review and meta-analysis of case-control
and cohort studies115 revealed an 11% increase in pancreatic cancer

risk per 100-g daily intake of red meat. When consumed regularly,
the summary risk for pancreatic cancer was 1.38 (95% CI, 1.05–1.81)
and 1.62 (95% CI, 1.17–2.26) for red and processed meats, respec-
tively.115 When assessed by sex, red or processed meat consumption
put men at significantly greater risk of pancreatic cancer compared
with women (men: red meat—RR, 1.21 [95% CI, 1.07–1.31];
processed meat—RR, 1.18 [95% CI, 1.06–1.31]; women: red
meat—RR, 1.06 [95% CI, 0.85–1.31]; processed meat—RR, 0.99
[95% CI, 0.84–1.16]).115 In addition, a recent systematic review of
prospective cohort studies revealed a significant increase in gastric
cancer risk with regular intake of processed meats (RR, 1.15 [95%
CI, 1.03–1.29]), notably ham, bacon, and/or sausage (RR, 1.21 [95%
CI, 1.01–1.46]).116 Taken together, evidence supports the link between
regular intake of red or processed meat and risk of colorectal, pancreatic,
and gastric cancer. For colorectal and pancreatic cancer, these effects
seem to be more relevant to men.
High-salt foods and salt intake
Unique to processed meats is the high salt content. Regular consumption
of high-salt foods (i.e., processed and prepackaged foods) has also
been linked with an increased risk of gastric cancer (RR, 1.55 [95%
CI, 1.17–2.05]).116 Similar trends were observed for salted fish (RR,
1.25 [95% CI, 1.07–1.47]) and salt added to foods (RR, 1.11 [95%
CI, 1.05–1.16]).116 Furthermore, a 12% increase in gastric cancer risk
has been observed per 5 g/day increase of total dietary salt intake
(RR, 1.12 [95% CI, 1.02–1.23]).116
Proposed mechanisms of dietary components linked with
increased cancer risk
When red meat is prepared at high temperatures (i.e., frying, charcoal
broiling or grilling, smoking), the high heat elicits production of
heterocyclic amines and polycyclic hydrocarbons,64,117 which are known
carcinogenic compounds secondary to their impact on DNA damage.118
These compounds have been linked with increased colorectal and
pancreatic cancer risk.64,117 In addition, although the high fat content
of certain cuts of red meat is not linked with pancreatic or gastric
cancer, it is associated with CRC.119 This is attributed to excess produc-
tion of bile acids, resulting in increased concentrations of bile acids
in the colon and/or rectum, leading to cellular proliferation and possible
tumorigenesis.64,119
Moreover, the presence of N-nitroso compounds in nitrite-preserved
processed meats may contribute to colorectal, gastric, and pancreatic
cancer risk.64,116,117,119,120 In CRC specifically, the alkaline nature of the
N-nitroso compounds facilitates DNA damage.119 In gastric cancer, the
nitrites in processed red meats interact with gastric acids to produce
N-nitroso compounds, which enables tumorigenesis.116,117,120 Although
processed meats are high in salt, high-salt foods and added salt in
general have exhibited exacerbation of the carcinogenic effects of
Helicobacter pylori, leading to gastric cancer.116,121,122 In addition, high
dietary salt intake alone may damage the lining of the stomach, leading
to inflammation, cellular proliferation, and possible tumorigenesis.116,121
Dietary Components Linked With Decreased Cancer Risk
Fruits and vegetables
Existing evidence has supported fruit and vegetable intake for risk
reduction of the following types of cancer: lung, mouth, pharynx,
larynx, esophagus, stomach, and colorectal.64,113 Dietary patterns
emphasizing increased intake of fruit and vegetables have been associated
with significant reductions in cancer risk. For example, in a systematic
review and meta-analysis of observational studies conducted by Godos
and colleagues,114 healthier dietary patterns, defined as high consump-
tion of fruits, vegetables, and whole grains, compared with unhealthier
dietary patterns, defined as high consumption of red or processed
meat, salty or sweet snacks, and refined grains, resulted in a 24%
reduction in CRC risk with statistically insignificant heterogeneity
(RR, 0.81 [95% CI, 0.71–0.94]).114 The specific aspects of diet that
reduce cancer risk remain unclear.
Lifestyle and Cancer Prevention  •  CHAPTER 22 345
Vegetarian and vegan dietary pattern
In a second meta-analysis of prospective cohort studies conducted by
Godos and colleagues,123 different types of vegetarian dietary patterns
were compared in relation to breast, colorectal, and prostate cancer
risk. The following diets were compared: vegetarian, defined as consump-
tion of meat less than once per month; semivegetarian, defined as
consumption of meat more than once per month but less than once
per week; pescovegetarian, defined as consumption of fish as the only
type of meat, more than once per month; and nonvegetarian, defined
as consumption of meat more than once per week. Compared with
the nonvegetarian dietary pattern, significant findings included a
reduction in CRC risk for semivegetarians (RR, 0.67 [95% CI,
0.53–0.83]) and pescovegetarians (RR, 0.86 [95% CI, 0.79–0.94])
but not vegetarian (RR, 0.88 [95% CI, 0.74–1.05]), with no significant
heterogeneity among studies.123 In a different meta-analysis comparing
vegetarian and vegan dietary patterns with nonvegetarian patterns
among 86 cross-sectional and 10 prospective cohort studies,124 a
significant reduction in cancer incidence was observed in both the
vegetarian and vegan patterns (RR, 0.92 [95% CI, 0.87–0.98]), with
greater risk reduction observed in the vegan pattern (RR, 0.85 [95%
CI, 0.75–0.95]). Taken together, these findings suggest an association
between lower intake of meat and cancer risk reduction.
Mediterranean dietary pattern
Although there is variability in the definition of the Mediterranean
dietary pattern (MDP), it typically consists of the following dietary
components: fruits, vegetables, nuts, legumes, whole grains, fish, high
monounsaturated to saturated fat ratio, and minimal amounts of dairy
and meat or processed meats.125 In a review by Schwingshackl and
Hoffmann125 of randomized controlled trials (RCTs), case-control
studies, and prospective cohort studies, regardless of study design, a
significant reduction in breast, colorectal, prostate, gastric, and liver
cancer was observed among individuals following the MDP compared
with other patterns. When components of MDP were assessed individu-
ally in relation to cancer risk, significant effects were not observed,
suggesting a synergistic effect among the components in relation to
cancer risk.125 In addition, results from a pooled analysis of case-control
studies demonstrated a significant inverse relationship between
components of MDP and endometrial cancer risk, such that as the
number of components consumed increased (i.e., from 0–3 to 7–9),
risk of endometrial cancer decreased.126 The largest number of com-
ponents consumed elicited an RR of 0.43 (95% CI, 0.36–0.68).
Furthermore, a risk reduction of 0.84 (95% CI, 0.80–0.88) was
observed as dietary component consumption increased by one.126
In an RCT comparing a low-fat diet and two different MDPs that
manipulated the primary fat source (i.e., MDP with extra virgin olive
oil [EVOO], high in monounsaturated fatty acids, compared with
MDP with mixed nuts, a mix of saturated, monounsaturated, and
polyunsaturated fatty acids) on cardiovascular-related health outcomes,
breast cancer incidence was assessed as a secondary outcome.127 Overall
MDP with EVOO demonstrated the greatest reduction of breast
cancer risk after a median follow-up of 4.8 years. Incidence rates were
the lowest in the EVOO group (1.1 per 100 person-years) compared
with mixed nuts or low-fat diet (1.8 and 2.9 per 100 person-years,
respectively).127 In addition, the RR of developing breast cancer was
significantly less with EVOO compared with mixed nuts (RR, 0.32
[95% CI, 0.13–0.79]; and RR, 0.59 [95% CI, 0.26–1.35], respec-
tively).127 These findings suggest that a fat source high in polyunsaturated
fatty acids within the MDP may produce the greatest effects in reducing
risk of breast cancer.
Individual micronutrients
In assessing individual micronutrient supplementation in relation to
cancer risk, the majority of evidence has demonstrated null or adverse
health effects.64,113 In a systematic review and meta-analysis conducted
by Fang and colleagues,116 the only micronutrient exhibiting cancer
prevention benefits was vitamin C, which exhibited a significant
346 Part I: Science and Clinical Oncology
reduction in gastric cancer risk with increased consumption (RR, 0.89
[95% CI, 0.85–0.93]).Yet adequate intake of vitamin C can easily be
achieved through consumption of fruits and vegetables. Thus, current
recommendations for nutrition and cancer prevention state that unless
an individual has micronutrient deficiencies, which would require
supplementation beyond dietary intake, micronutrient consumption
from a diet rich in fruits, vegetables, and whole grains will suffice in
terms of health benefits related to cancer prevention.113
Proposed mechanisms of dietary components linked with
decreased cancer risk
Increased fruit, vegetable, and whole grain intake increases dietary
water and fiber content. An increase in dietary water and fiber content
may increase satiety, prevent excessive energy intake, and replace or
limit poor food choices such as refined grains, added sugars, salty
snacks, and processed meats.64 In addition, a high-fiber diet may
decrease transit time in the colon, reducing exposure to potential fecal
carcinogens and reducing CRC risk.
Obesity, Physical Inactivity, and Dietary Recommendations
and Resources for Cancer Prevention
Recommendations for cancer prevention63 are shown in Table 22.1.
As evidenced by results from the systematic review conducted by Birks
and colleagues,70 weight loss in overweight or obese individuals is
linked with reduction in cancer incidence. The underlying theme in
weight loss is energy balance—moving more (increasing energy
expenditure) and eating less (reducing energy intake). Even small
changes can result in large effects over time.
At initiation of an exercise regimen, it is recommended to start
with at least 5 days per week of moderate-intensity aerobic exercise
(40%–60% heart rate reserve or V̇ o2 reserve) for at least 30 minutes
per exercise session, with eventual progression to the goal of at least
60 minutes per day of aerobic exercise at more vigorous intensities
(i.e., >60% heart rate reserve or V̇ o2 reserve).82 If an individual is
unable to complete a continuous exercise session, it is recommended
to break up the exercise time into 10-minute increments.82 Regarding
diet, a caloric reduction of 500 to 1000 kcal/day is typically recom-
mended to elicit a 1- to 2-pound weight loss per week. Dietary changes
that are often suggested for weight loss include reducing portion size
and avoiding high–energy-dense foods, especially those with low
nutrient value (e.g., potato chips, sugar-sweetened beverages).
There are countless resources available to assist individuals with
energy balance to promote weight loss and maintenance of a healthy
weight. The CDC and healthfinder.gov provide a step-by-step guide
to help individuals with weight loss. This guide includes strategies,
such as goal setting and self-monitoring, to establish healthier dietary
habits in efforts to incorporate the behavior change component into
weight loss.
Moreover, the CDC website provides details on the activity
guidelines, tips on how to incorporate structured physical activity
within one’s daily routine and increase lifestyle activity, how to begin
an exercise program, how to measure exercise intensity, and other
worthwhile messages. These resources, in coordination with worksite
and community efforts (e.g., the local gym, YMCA, or community
recreation center; religious organizations; home exercise videos; wearable
devices such as an accelerometer or pedometer), aid in promoting
increases in physical activity. In addition, the AICR’s website is also
a useful resource, providing various aids to assist in meeting cancer
prevention guidelines, including a visual aid to assist with portion
control and consumption of a balanced diet.
Ultraviolet Radiation
Solar radiation, specifically ultraviolet radiation (UVR), and the use
of UVR-emitting tanning devices are classified as carcinogenic to humans
by the IARC.128
The Role of Ultraviolet Radiation in Cancer
Exposure to UVR, whether from the sun or from a tanning device,
is a major risk factor for all forms of skin cancer (basal cell carcinoma
[BCC], squamous cell carcinoma [SCC], and melanoma). Despite
being highly preventable, skin cancer is the most common form of
cancer in the United States, with 5 billion cases treated annually at a
cost of $8.1 billion.129 Melanoma accounts for a small fraction of
these cases, approximately 63,000, but it is the deadliest form of skin
cancer, with nearly 9000 deaths each year.130 Unlike many other
common cancers, data suggest that skin cancer incidence is rising,
underscoring the need for a focus on evidence-based preventive actions.
The risk of skin cancer varies by skin type, race or ethnicity, the
pattern of UVR exposure, and the age at which exposure occurs. In
general, lighter-skinned individuals have a higher risk of all skin cancers
than do darker-skinned individuals. The Fitzpatrick skin type classifica-
tion system classifies individuals based on their likelihood of tanning
or burning, with six categories (I–VI) ranging from “always burns
and never tans” to “never burns, deeply pigmented, least sensitive.”
Those who are classified as types I and II have the highest risk of
burns, damage from UVR, and skin cancer.130
Regarding race, non-Hispanic whites have the highest rates of
invasive melanoma incidence (24.7 per 100,000) and mortality (3.4
per 100,000), whereas African-Americans have the lowest rates (1.0
per 100,000 and 0.4 per 100,000, respectively).130
The type of skin cancer for which one is at risk also varies according
to his or her pattern of UVR exposure. Chronic continuous exposure,
such as that experienced by an outdoor worker, is more strongly
associated with SCC, whereas intermittent exposure, such as that
experienced by someone vacationing at the beach, is more strongly
associated with BCC and melanoma.130 In a meta-analysis of 34 studies,
intermittent sun exposure was associated with a significant 61% excess
risk of melanoma, whereas chronic exposure was slightly inversely
associated with risk, although this was not significant.131 Numerous
studies have documented the increased risk of skin cancer associated
with sunburns.132–134 Sunburns during childhood are strongly associated
with risk of future melanoma,132,133 although data suggest that sunburns
at any age can significantly increase the risk of melanoma.132,133 A
meta-analysis of 26 studies found that the risk of melanoma increases
with increasing number of sunburns during all life periods (i.e.,
childhood, adolescence, and adulthood).132–134 Consequently, prevention
efforts should occur across the lifespan.
Although the sun is the most common source of UVR exposure,
tanning beds are also an important source, particularly for non-Hispanic
white women aged 18 to 25, of whom nearly one-third report tanning
indoors.135 It is estimated that indoor tanning results in 400,000 cases
of skin cancer in the United States each year, and 6000 of these are
melanoma.130 In general, the risk of skin cancer increases with increasing
use, with younger and more frequent users having a steeply elevated
risk.136–141 For “ever” indoor tanning, the excess risks of melanoma,
BCC, and SCC are 20%, 29%, and 67%, respectively.130,139,140 When
“ever” indoor tanning is restricted to those younger than 35 or to those
considered frequent users, the excess risk dramatically increases.139,140
Although the exact magnitude of the increased risk conferred by
indoor tanning varies quite a bit within the literature (e.g., owing to
differences in study settings and populations), studies consistently show
significantly elevated risk. Important to note, there is no evidence to
suggest that indoor tanning is safer than tanning outdoors or that it
protects from the effects of future sun exposure.130
Proposed Mechanism Linking Ultraviolet Radiation to
Skin Cancer
UVR from the sun consists of three different ultraviolet (UV)
wavelengths—UVA (315–400 nm), UVB (280–315 nm) and UVC
(100–280 nm). UVC rays do not reach the Earth’s surface owing to
stratospheric ozone and so are not considered a source of UVR damage

to the skin. The remaining UVR reaching the surface of the earth is
95% UVA and 5% UVB. UVB rays have more energy but are not
absorbed as deeply into the skin as UVA rays, which have less energy
but can penetrate into the dermis. UVB is significantly more carci-
nogenic than UVA, because it can directly and indirectly damage
DNA, whereas UVA is weakly absorbed by DNA and primarily causes
DNA damage through indirect photosensitization reactions.142,143
UVB directly damages DNA through the formation of DNA
“photoproducts” or dimers formed by the covalent bonding of two
adjacent pyrimidine bases as a result of the absorption of UVR
photons.143 A few different types of dimers can form, but the one
most frequently induced by UVB is the cyclobutane pyrimidine dimer
(CPD). If these are not remedied by the nucleotide excision repair
system, they can result in C to T transitions or CC to TT tandem
mutations. These types of mutations were once thought to be a signature
of UVB damage; however, this has been called into question based
on data showing that UVA exposure can also result in CPDs, although
it is likely through an indirect mechanism.142,143 C to T mutations in
the TP53 gene are likely an early event in the development of SCC,
whereas they occur later in BCC and melanoma.143 Genes coding for
proteins of the Hedgehog signaling pathway, mainly the tumor-
suppressor gene PTCH, are frequently mutated in BCC, and many
of these mutations are C to T and CC to TT transitions.143 In mela-
noma, the PTEN and CDKN2A genes have also been shown to harbor
C to T mutations. However, melanomas from intermittently sun-
exposed areas also show a high frequency of T:A to A:T mutations,
such as the well-known V600E mutation in BRAF, suggesting another
type of UVR-induced mutation that is not fully understood.143
UVA rays primarily cause damage to DNA indirectly. Other
chromophores, such as cytochromes and porphyrins, initially absorb
UVA energy and then transfer it to either DNA (a type I photosensitized
reaction), which results in CPD formation, or to molecular oxygen
(a type II photosensitized reaction), generating ROS that ultimately
damage the DNA.143 In addition to DNA, oxidative stress also
damages other cellular components, which can further contribute
to carcinogenesis.
Another mechanism of UVR photocarcinogenesis may relate to
the ability of UVA and UVB to induce persistent genomic instability
through shortening or loss of telomeres as well as through a “bystander
effect,” whereby cells not exposed to UVR are also damaged.142 This
may occur through the generation of increasingly damaged progeny
of UVR-irradiated cells.
Important to note, UVR is a known immunosuppressant. The
ability of UVR to suppress certain parts of the immune system was
first established by Kripke and Fisher in experiments in mice showing
that UVR prevented the rejection of highly immunogenic transplanted
UVR-induced skin tumors.144–146 In addition, UVR is used for its
immunosuppressant effects as local therapy for various skin diseases,
such as psoriasis. Furthermore, it is well known that organ-transplant
recipients who have the immune system suppressed to prevent organ
rejection are susceptible to skin cancer. UVB rays are thought to be
primarily responsible for the immunosuppressive capability of UVR,
but UVA rays may also play a role.143 UVR can induce both systemic
and local immunosuppression, as well as influencing both acquired
and innate immunity. UVB rays suppress the immune system by
inducing immunosuppressive mediators, by damaging and triggering
the premature migration of antigen-presenting cells required to stimulate
antigen-specific immune responses, by inducing the generation of
suppressor cells, and by inhibiting the activation of effector and memory
T cells.142 Immunosuppression via UVA is less well understood but
may relate to its ability to produce ROS and reactive nitrogen species
as well as to its ability to penetrate more deeply into skin tissue,
reaching many more types of cells than UVB, including T cells, mast
cells, and granulocytes.142
In sum, both UVA and UVB are able to induce carcinogenic
changes to DNA and other cellular components, generally through
unique, although at times overlapping, mechanisms. UVR is both a
Lifestyle and Cancer Prevention  •  CHAPTER 22 347
carcinogen and an immunosuppressive agent that has been implicated
in all forms of skin cancer.
Evidence-Based Interventions for Cancer Prevention Related
to Ultraviolet Radiation Exposure
Reducing exposure to UVR can reduce skin cancer risk. There are
many actions that individuals and communities can take to reduce
their UVR exposure. Unfortunately, many individuals are not aware
of the risks posed by UVR exposure. Data indicate that one-third
of US adults have been sunburned in the past year and that most
do not take the recommended preventive actions.147,148 In addition,
indoor tanning rates are high in some groups, and skin cancer rates
are increasing. These factors led the Surgeon General to release a
report in 2014 that serves as a call to action to prevent skin cancer.130
This report outlines the preventive actions that can be taken today
by various constituencies and at various levels of society (Table 22.3).
The report also sets five goals to support skin cancer prevention
across the country and discusses several strategies to achieve each goal
(Table 22.4).
The use of sunscreens is a recommended action for skin cancer
prevention, although there are few studies demonstrating the skin
cancer–protective effect of these products. Data from the Australian
Nambour Skin Cancer Prevention Trial (N = 1621) suggest that the
daily use of sun protective factor (SPF) 15+ sunscreen can reduce
the risk of SCC and melanoma. After 8 years of follow-up, SCC
incidence in those randomized to daily sunscreen application was
35% lower (0.65 [95% CI, 0.43–0.98]) than in those randomized
to discretionary use (including no use).149 Ten years after cessation
of the trial, overall melanoma risk was reduced by 50% (0.50 [95%
CI, 0.24–1.02]) and invasive melanoma risk was reduced by 73%
(0.27 [95% CI, 0.08–0.97]) in the daily sunscreen group compared
with the discretionary use group.150 No clear benefit for BCC was
observed. Numerous cohort and case-control studies have examined
the relationship between sunscreen use and skin cancer, but results are
conflicting and most have serious methodological limitations relating to
measures of sun exposure, sunscreen use, and adjustment for important
confounders.151 For various reasons, the evidence that sunscreens prevent
skin cancer may remain elusive.152 Nevertheless, studies have shown that
sunscreens are capable of preventing or delaying solar keratosis,153–155
a premalignant skin cancer lesion, and the efficacy of sunscreens
to prevent sunburns, an established risk factor for skin cancer, is
undisputed.156
There is much confusion among the public regarding the type,
amount, and frequency of application when using sunscreens. The
American Academy of Dermatology states that sunscreens should
include broad-spectrum protection (protects against UVA and UVB
rays), should have an SPF of at least 30, and should be water resistant.157
It further recommends that sunscreen should be applied 15 minutes
before going outside and then every 2 hours or after swimming or
sweating, and that approximately 1 ounce of sunscreen should be
used—enough to fill a shot glass—to cover all exposed skin.157
Although there is currently a great deal of research and controversy
surrounding the ingredients of sunscreen and their safety, the risk-benefit
ratio indicates that sunscreens are appropriate to include as a skin
cancer preventive intervention.158
Family History
A family history of cancer among first-degree relatives can significantly
increase one’s risk of cancer. Although one’s family history cannot be
modified, the increased risk of cancer transmitted through a positive
family history can, nevertheless, be mitigated through awareness and
adherence to cancer prevention and screening recommendations,
because there are safe and effective interventions for those known to
be at high risk of cancer. Important to note, family history often
determines when an individual begins cancer screenings, how frequently
348 Part I: Science and Clinical Oncology
Table 22.3  Recommended Actions for Skin Cancer
Prevention
FOR THE INDIVIDUAL
• Wear protective clothing.
• Wear a hat and sunglasses.
• Seek shade.
• Avoid times of peak sunlight.
• Use sunscreen.
• Avoid indoor tanning and sunbathing.
FOR THE CLINICIAN (IN PRIMARY CARE SETTING)
• Counsel patients with fair skin aged 10–24 to minimize their
ultraviolet light exposure to reduce their risk of skin cancer.
FOR THE COMMUNITY
IN OUTDOOR OCCUPATIONAL, RECREATIONAL OR TOURISM
SETTINGS
• Implement interventions to promote sun-protective behaviors that
include educational approaches, modeling or demonstrating
behaviors, environmental approaches (e.g., providing sun screen),
and policies to support sun protection strategies (e.g., requiring
hats and/or sun-protective clothing).
IN EDUCATIONAL SETTINGS
• Implement interventions in child care centers and in primary and
middle schools that promote sun-protective behaviors through
educational interventions, supportive behavioral interventions, and
environmental and policy changes.
THROUGHOUT COMMUNITY
• Implement multicomponent community-wide interventions that
include individual-directed strategies, mass media campaigns, and
environmental and policy changes across multiple settings.
FOR THE STATE
SUN PROTECTION POLICIES AND LEGISLATION
• Adopt and support legislation that requires schools to allow
students to wear sun-protective clothing and sunscreen while on
campus.
EDUCATION AND AWARENESS
• Adopt and support legislation to mandate instruction on skin
cancer prevention as part of the health education curriculum in
public schools.
INDOOR TANNING POLICIES AND LEGISLATION
• Adopt and support legislation that prohibits minors from using
tanning beds and requires monitoring of use of indoor tanning
devices; requires warnings to users about health risks associated
with indoor tanning; and enforces such regulations.
Recommendations are based on the 2014 Surgeon General’s Call to Action to
Prevent Skin Cancer report.130
he or she is screened, and how that screening is performed. Genetic
testing may be warranted in patients with a strong positive family
history of cancer. In the case of hereditary breast and ovarian cancer
syndrome, carriers of BRCA1/2 may be offered prophylactic surgery
as a risk-reducing intervention.159 Because family history is likely to
have potential consequences for multiple family members, families
are encouraged to take the time to collect and share a family health
history, including the results of any cancer screening and/or genetic
testing that any family member has undergone. Such results may have
potentially life-saving implications for other members. There are freely
available tools online that enable this, such as the Surgeon General’s
Table 22.4  Goals to Support Skin Cancer
Prevention and Associated Strategies
GOAL 1: INCREASE OPPORTUNITIES FOR SUN PROTECTION
IN OUTDOOR SETTINGS
• Increase shade in outdoor recreational settings
• Support sun-protective behaviors in outdoor settings
• Increase availability of sun protection in educational settings
• Increase availability of sun protection for outdoor workers
GOAL 2: PROVIDE INDIVIDUALS WITH THE INFORMATION
THEY NEED TO MAKE INFORMED, HEALTHY CHOICES
ABOUT EXPOSURE TO ULTRAVIOLET RADIATION (UVR)
• Develop effective messages and interventions for specific
audiences
• Support skin cancer prevention education in schools
• Integrate sun safety into workplace health education and
promotion programs
• Partner with health care systems and providers to implement and
monitor use of recommended preventive services for provider
counseling on skin cancer prevention
• Establish partnerships between public and private sectors to
disseminate effective messages about skin cancer prevention
• Enhance ongoing engagement of federal partners to advance our
nation’s skin cancer prevention efforts
GOAL 3: PROMOTE POLICIES THAT ADVANCE THE NATIONAL
GOAL OF PREVENTING SKIN CANCER
• Support inclusion of sun protection in school policies, construction
of school facilities, and school curricula
• Promote electronic reporting of reportable skin cancers, and
encourage health care systems and providers to use such systems
• Incorporate sun safety into workplace policies and safety trainings
• Support shade planning in land-use development
GOAL 4: REDUCE HARMS FROM INDOOR TANNING
• Monitor indoor tanning attitudes, beliefs, and behaviors in the US
population, especially among indoor tanners, youth, and parents
• Continue to develop, disseminate, and evaluate tailored messages
to reduce indoor tanning among populations at high risk
• Support organizational policies that discourage indoor tanning by
adolescents and young adults
• Enforce existing indoor tanning laws and consider adopting
additional restrictions
• Address the risks of indoor tanning with improved warning labels
and updated performance standards
GOAL 5: STRENGTHEN RESEARCH, SURVEILLANCE,
MONITORING, AND EVALUATION RELATED TO SKIN CANCER
PREVENTION
• Enhance understanding of the burden of skin cancer and its
relationship with UVR
• Evaluate the effect of interventions and policies on behavioral and
health outcomes
• Build on behavioral research and surveillance related to UVR
exposure
• Quantify the prevalence of tanning in unsupervised locations (use
of tanning beds outside of tanning salons)
Goals and strategies are drawn from the 2014 Surgeon General’s Call to Action to
Prevent Skin Cancer report.130
My Family Health Portrait tool (https://familyhistory.hhs.gov/FHH/
html/index.html). These tools are not cancer specific, but are applicable
to all noncommunicable chronic diseases.
For more information related to family history in cancer, the reader
is referred to Chapter 13.

MOLECULAR PREVENTION
Drugs, vaccines, and natural agents represent different strategies for
molecular prevention, with the ultimate goal of inhibiting, reversing,
or delaying the onset of carcinogenesis. Advances in our understanding
of the mechanistic basis for carcinogenesis, with the identification of
molecules and signaling pathway alterations critical for tumorigenesis,
has enabled the development of molecularly targeted therapies to
block tumor development. The novel drugs, signaling inhibitors, and
vaccines that have been developed and tested as cancer preventive
agents are discussed in this section.
Phase II and III clinical trials have demonstrated the effectiveness
of interventions that target oncogenic pathways within the cell for
the prevention of cancer. Several agents have been FDA approved for
treatment of precancerous lesions for cancer risk reduction (Table
22.5), although they are often not used. Major challenges are (1) to
elucidate the critical molecular drivers of early tumorigenesis that can
Table 22.5  Agents Approved for the Treatment of
Precancerous Lesions or to Reduce
Cancer Risk, 2017
Disease Intervention
Anal cancer HPV vaccine (Gardasil-9; for cancer caused by
infection with HPV types 16, 18, 31, 33, 45, 52,
and 58 and for precancerous or dysplastic
lesions caused by infection with HPV types 6, 11,
16, 18, 31, 33, 45, 52, and 58)
Bladder cancer Bacillus Calmette-Guérin
(dysplasia) Valrubicin
Breast cancer Tamoxifen
Raloxifene
Cervical cancer HPV vaccine (Cervarix; for cancer caused by
infection with HPV types 16 and 18)
HPV vaccine (Gardasil; for cancer caused by
infection with HPV types 16 and 18)
HPV vaccine (Gardasil-9; for cancer caused by
infection with HPV types 16, 18, 31, 33, 45, 52,
and 58 and for precancerous or dysplastic
lesions caused by infection with HPV types 6, 11,
16, 18, 31, 33, 45, 52, and 58)
Colonic adenomas Celecoxiba
Esophageal cancer Photofrin plus photodynamic therapy (PTD)
(dysplasia)
Skin cancer (actinic 5-Aminolevulinic acid plus photodynamic
keratosis) therapy (PTD)
Diclofenac sodium
Fluorouracil
Imiquimod
Ingenol mebutate
Masoprocol
Vulvar cancer HPV vaccine (Gardasil-9; for cancer caused by
infection with HPV types 16, 18, 31, 33, 45, 52,
and 58 and for precancerous or dysplastic
lesions caused by infection with HPV types 6, 11,
16, 18, 31, 33, 45, 52, and 58)
Vaginal cancer HPV vaccine (Gardasil-9; for cancer caused by
infection with HPV types 16, 18, 31, 33, 45, 52,
and 58 and for precancerous or dysplastic
lesions caused by infection with HPV types 6, 11,
16, 18, 31, 33, 45, 52, and 58)
a
US Food and Drug Administration (FDA) labeling voluntarily withdrawn by Pfizer,
February 2011.
HPV, Human papillomavirus.
Lifestyle and Cancer Prevention  •  CHAPTER 22 349
be targeted to prevent cancer development; (2) to develop effective,
safe, and tolerable preventive therapies; and (3) to promote the use
of these safe preventive therapies in at-risk individuals. The ultimate
goal of these efforts is to deliver effective, safe, and acceptable interven-
tions to prevent multiple forms of cancer. However, although these
strategies have proven effective for preventing specific tumor subtypes,
they are often ineffective or poorly effective in prevention of other
tumor subtypes (e.g., selective estrogen receptor modulators (SERMs)
and aromatase inhibitors (AIs) for the prevention of ER+ breast cancer).
Thus, there is a clear need for the identification and development of
novel drugs that can more effectively prevent other cancer subtypes
(e.g., ER− breast cancer) (Fig. 22.1). Alternative approaches for
molecular cancer prevention currently under investigation include
cyclooxygenase-2 (COX-2) inhibitors, retinoids, HER2 receptor kinase
inhibitors, IGF inhibitors, kinase inhibitors, metformin, poly (ADP-
ribose) polymerase (PARP) inhibitors, statins, and transcription factor
inhibitors, in addition to novel vaccine and inflammation- and
immunoprevention-based approaches.
Lung Cancer
Many phase II and III behavioral and cancer prevention clinical trials
have been conducted in lung cancer. Although nicotine replacement
therapy has been among the leading behavioral treatment strategies,
preventive therapy using various agents, including vitamins, minerals,
drugs, and signal transduction inhibitors, has been tested. Many agents
have been tested; however, none have been shown to be useful as
cancer prevention drugs in phase III trials
Numerous large-scale observational studies have provided results
of patient use of a variety of agents and have helped support the
development of clinical trials. Among these, the Singapore Chinese
Health Study investigated the effects of the dietary carotenoids and
risk of lung cancer.160 A total of 63,257 men and women aged 47 to
75 years participated from 1993 to 1998, with 8 years of follow-up.
The results of this study suggested that dietary β-cryptoxanthin reduces
lung cancer risk.
Several clinical trials testing the effects of vitamins on lung cancer
were then conducted. The Alpha-Tocopherol, Beta-Carotene Cancer
Prevention Study (ATBC) was a large randomized, placebo-controlled
phase III cancer prevention trial in which 27,111 male smokers were
recruited and randomized to receive vitamin E (alpha-tocopherol),
β-carotene, a combination of these two agents, or placebo.161,162 The
results of this study showed that neither agent alone nor the combination
of vitamin E and β-carotene reduced lung cancer incidence; however,
the study did show that there was a higher incidence of lung cancer
in smokers who took β-carotene.163
β-Carotene
Based on previous findings from observational and epidemiologic
studies that diets rich in β-carotene are associated with reduced cancer
risk, two large phase III clinical trials were conducted to test the
efficacy of β-carotene for the prevention of lung cancer.
Alpha-Tocopherol, Beta-Carotene Cancer Trial (ATBC)
In 1994, the Alpha-Tocopherol, Beta-Carotene (ATBC) Cancer Preven-
tion Study Group published the initial findings of their randomized
double-blind trial of over 29,000 male smokers.161,164 Study participants
received α-tocopherol (20 mg/day), β-carotene (20 mg/day), both
α-tocopherol and β-carotene, or placebo for 5 to 8 years. α-Tocopherol
(vitamin E) was not associated with reduced incidence of lung cancer,
but was associated with decreased incidence of prostate cancer, whereas
β-carotene was associated with an 18% higher incidence of lung cancer
in smokers.163
Beta-Carotene and Retinol Efficacy Trial (CARET)
Soon after the ATBC results were released, findings from a second
phase III lung cancer prevention clinical trial, the Beta-Carotene and
350 Part I: Science and Clinical Oncology

EGFR inhibitors
ErbB1/ErbB2 inhibitors

IGFR IGFR ErbB1 ErbB2 HER2 inhibitors

P P P P
Arachidonic acid
COX-1 EGCG
Cox1/2 & Cox2 inhibitors PI3K, Ras/Raf
COX-2
Prostaglandin G2
B-carotene / Vitamin A PGE2 MAPKs, JNKs, p38, AKT Kinase inhibitors
AMPK Resveratrol
Retinoids / Rexinoids
mTOR mTOR inhibitors
Vitamin D
p70S6K1 & AKT
Growth Statins
Metformin AMP signaling Mevalonate
AMPK
Aromatase
Aromatase inhibitors NR RXR
E2 XXRE
SERMs ER ER

β-catenin
Curcumin
NFκB Tumorigenesis
Growth control genes

PARP inhibitors PARP

Polyamines
Vaccines
ODC Immune response
Immunomodulators
DFMO T-cell
Cell death Checkpoint inhibitors

Figure 22.1  •  Potential strategies for targeting oncogenic pathways in the cell.

Retinol Efficacy Trial (CARET), studying β-carotene for the prevention
of lung cancer, were published.165,166 In this study, 18,314 men and
women at high risk for lung cancer (significant occupational exposure
to asbestos or extensive history of smoking) were randomized to receive
β-carotene (30 mg/day), retinol (vitamin A, 25,000 IU), combined
treatment with both drugs, or placebo. This study was stopped 21
months early because of similar findings to the ATBC trial. CARET
participants who received combination treatment with β-carotene and
retinol developed 28% more lung cancers and had higher all-cause
mortality, cardiovascular mortality, and mortality from lung cancer.167
These studies of vitamin E and β-carotene demonstrated that vitamins
are not harmless, and they should be used carefully.
Selenium
Nutritional Prevention Cancer Trial (NPC)
After results from geographic and environmental studies suggested
that increased selenium is associated with decreased cancer incidence
and mortality for multiple cancer types,168,169 the Nutritional Prevention
Cancer Trial (NPC) was conducted to assess the effects of selenium
versus placebo on risk of skin cancer in patients with a history of
BCC or SCC.170 Secondary end points included diagnosis of other
cancers (not BCC or SCC), cardiovascular disease, and other medical
conditions. A total of 1312 participants with a history of BCC or
SCC were randomized to receive selenium (200 µg) or placebo daily.
Initial results based on 4.5 years of follow-up showed that patients
treated with selenium had significantly lower incidence of lung cancer
(RR, 0.54 [95% CI, 0.30–0.98]; P = .04) than those treated with
placebo. However, after an extended follow-up of 7.9 years, the decrease
in RR of lung cancer was no longer statistically significant (RR, 0.70
[95% CI, 0.40–1.21]; P = .18).171
Selenium and Vitamin E Cancer Prevention Trial (SELECT)
The Selenium and Vitamin E Cancer Prevention Trial (SELECT) was
a large-scale phase III clinical trial testing the efficacy of long-term
treatment with vitamin E, selenium, or both for the prevention of
prostate cancer.172 Incidence of lung, colorectal, and other cancers
served as secondary end points. A total of 35,533 men were randomized
to receive vitamin E (400 IU/day of all-rac-α-tocopheryl acetate),
selenium (200 µg/day from l-selenomethionine), both, or placebo.
After 5 years of treatment, although vitamin E was found to increase
risk of prostate cancer (13%; 99% CI, 0.95–1.35), treatment with
selenium was not associated with decreased incidence of lung, colorectal,
or other cancers. An extended follow-up study through July 2011
showed significantly increased risk of prostate cancer in patients treated
with vitamin E (17%; 99% CI, 1.004–1.36), selenium (9%; 99%
CI, 0.93–1.27), or vitamin E plus selenium (5%; 99% CI, 0.89–1.22).
No significant change was noted from their previously reported findings
for the secondary end points.173
Selenium Supplementation in Patients With Resected Stage I
Non–Small Cell Lung Cancer: ECOG 5597
The Selenium Supplementation in Patients With Resected Stage I
Non–Small Cell Lung Cancer (Eastern Cooperative Oncology Group
[ECOG] 5597) trial accrued and randomized 1561 participants with
non–small cell lung cancer (NSCLC) to receive selenized yeast (200 µg/
day) or placebo for 48 months.174 Although the interim results suggested

increased risk of second primary lung tumors in the selenium arm,
final results from this study confirmed the results of the SELECT
study, finding selenium to be safe, but not beneficial over placebo,
for both disease-free survival and the prevention of second primary
tumors in NSCLC patients.
The results of these trials testing β-carotene and selenium failed
to demonstrate a preventive benefit associated with these compounds.
Moreover, they identified the unexpected potential for significant health
risks following treatment with these compounds.
Budesonide
The effects of inhaled budesonide (800 µg twice daily) compared
with placebo for 1 year was the focus of a phase IIb trial that included
202 current and former smokers with computed tomography (CT)
screen-detected lung nodules.175 This study was nested within the
large phase III Continuous Observation of Smoking Subjects
(COSMOS) lung cancer screening trial, which was composed of 5203
participants. Budesonide was found to be well tolerated but not effective
in reducing lung nodule size compared with placebo in the per-person
analysis, which was the primary end point (reduction rate: budesonide
2%, placebo 1%). However, budesonide was found to produce sig-
nificant regression of existing nonsolid and partially solid nodules
(lung nodules most likely representing adenocarcinoma precursors).
Long-term follow-up of 5 years showed a significant reduction in
nonsolid lung nodule size after treatment with budesonide compared
with placebo.176 The mean maximum diameter of nonsolid nodules
in the budesonide arm dropped from 5.03 mm at baseline to 2.61 mm
after 5 years. Notably, budesonide was not found to reduce the size
of solid nodules. In addition, neither the number of new lesions nor
the number of lung cancers differed in the patients treated with
budesonide or placebo. Despite the lack of effect on lung cancer
incidence in this phase II trial (which was not powered to detect a
difference in lung cancer incidence), budesonide remains a promising
agent for lung cancer prevention.
Nonsteroidal Antiinflammatory Drugs
Sulindac.  Because of the cardiovascular toxicity associated with
selective COX-2 inhibitors, Limburg and colleagues conducted a study
of the nonselective COX-2 inhibitor sulindac for the prevention of
lung cancer.177 They randomized 61 participants to receive sulindac
(150 mg BID) or placebo for 6 months. This study did not find any
statically significant benefit from sulindac treatment on either bronchial
dysplasia (regression: sulindac arm, 58%; placebo arm, 56%) or mucosal
proliferation (measured by Ki67 expression: sulindac arm, 30 versus
10; placebo arm, 30 versus 5; P = .92).
Iloprost
A phase II study of iloprost was conducted to determine its efficacy in
reversing premalignant histologic changes in the bronchial epithelium
in current and former smokers.178 A total of 152 patients at high
risk of lung cancer were treated with iloprost (a 50 to 150 mg/
day dose escalation for 2 months, followed by 150 mg/day for 4
months) or placebo. Former smokers treated with iloprost versus
placebo demonstrated a significant improvement in endobronchial
histologic findings (58.6% versus 28.6%, respectively), whereas current
smokers did not. These results show that iloprost is a promising drug
for lung cancer prevention. A future phase III trial will be required
to conclusively demonstrate the effectiveness of this promising
intervention.
Serine/Threonine Kinase Inhibitors
Based on positive preclinical findings, Gray and colleagues studied
the effects of enzastaurin, which inhibits protein kinase C–β activation,
as a chemopreventive agent for lung cancer.179 In this study, 40 former
smokers were randomized to receive enzastaurin (500 mg/day) or
placebo for 6 months. Although enzastaurin treatment proved tolerable,
no significant reduction in Ki67 labeling index in bronchial biopsy
Lifestyle and Cancer Prevention  •  CHAPTER 22 351
specimens was identified in participants treated with enzastaurin
compared with those treated with placebo.
myo-Inositol
Based on preclinical and early clinical studies that showed a greater
than 40% decrease in preexisting dysplastic lesions in smokers with
bronchial dysplasia following treatment with myo-inositol,180 Lam and
colleagues published results from the first phase IIb trial testing myo-
inositol for chemoprevention of lung cancer in smokers with bronchial
dysplasia.181 A total of 74 participants were randomized to receive
myo-inositol (9 g/day for 2 weeks, followed by 9 g twice daily for 6
months) or placebo. Treatment with myo-inositol was not found to
have a statistically significant effect on bronchial dysplasia, with
complete response rates of 26.3% (myo-inositol) and 13.9% (placebo)
and progressive disease rates of 47.4% (myo-inositol) and 33% (placebo)
(P = .76). However, these results, combined with the findings of PI3K
activation, significant reductions in bronchoalveolar lavage (BAL) fluid,
IL-6 levels, and statistically insignificant reductions in Ki76 levels,
suggest that future studies of myo-inositol for the chemoprevention
of lung cancer in a more defined cohort may identify a preventive
benefit.
Retinoids
Preclinical and epidemiologic studies using retinoids as preventive
and therapeutic agents for head and neck cancer provided the rationale
to conduct clinical trials using retinoids in patients with head and
neck cancer. Results from these studies demonstrated the cancer preven-
tive effects of retinoids in this setting. Subsequently, these findings,
combined with preclinical and early clinical data from use of isotretinoin
(13-cis–retinoic acid) in lung cancer, supported the development of
phase III trials for the prevention of lung cancer.
Lung Intergroup Trial (LIT)
The multicenter Lung Intergroup Trial (LIT) randomized 1166 patients
with stage I NSCLC to receive the retinoid isotretinoin (30 mg/day)
or placebo for 3 years.182 Patients underwent a 3.5-year follow-up
period, at which time the results showed no benefit in the overall rate
of second primary tumors (primary end point) or in recurrence or
mortality (secondary end points). In addition, this study identified a
potential harmful effect of isotretinoin based on smoking status
(non–statistically significant increased recurrence and statistically
significant increased mortality in current smokers compared with never
smokers). Updated results were published in 2010 with an extended
6.2-year follow-up, which confirmed increased overall and cancer-related
mortality, as well as cardiovascular disease in current smokers treated
with isotretinoin.183 Conversely, never and former smokers exhibited
a slight decrease in both overall and cancer-related deaths.
13-cis–retinoic acid with or without α-tocopherol
Kelly and colleagues randomized 75 subjects at high risk for lung
cancer to receive 13-cis–retinoic acid (50 mg/day by mouth [PO])
alone or in combination with α-tocopherol (800 mg/day PO) for 12
months to determine its effect on histologic progression.184 Their results
were consistent with those of other trials examining the preventive
efficacy of 13-cis–retinoic acid in the lung cancer setting, with risk
of treatment failure being 50% in the 13-cis–retinoic acid and the
13-cis–retinoic acid plus α-tocopherol treatment arms and 55.6% in the
observation arm.
Other Agents
Zileuton, a 5-lipoxygenase inhibitor, was studied as a chemopreventive
agent for lung cancer by Kucuk and colleagues in a phase II study of
38 patients with bronchial dysplasia (NCT00056004). The trial, which
is investigating the effects of 6 months of treatment with zileuton,
has been completed, but no results have been released to date. Results
from a second phase I/II study of short-term (6 days) treatment with
zileuton (1200 mg twice per day) with or without celecoxib (200 mg
352 Part I: Science and Clinical Oncology
twice per day) are also anticipated in the near future (NCT01021215).
Preliminary data from this study suggest preventive efficacy associated
with zileuton, which may be increased when combined with celecoxib.
Other novel drugs have been shown to be effective for the treatment
of lung cancer. These include PARP inhibitors185 and immune
checkpoint inhibitors.186 With continued development of effective
and safe inhibitors of these novel targets, it will be possible to test
them in cancer prevention trials. Such agents offer great promise for
prevention of this common cancer. Because lung cancer is the most
common cause of cancer-related death, there are continued efforts to
develop safe and effective strategies for lung cancer prevention.
Decreased incidence of lung cancer in the future will depend on
primary prevention strategies (e.g., smoking cessation or avoidance
and reduced exposure to toxins) and nontoxic preventive therapy.
Head and Neck Cancers
Smoking, alcohol, and HPV are all associated with increased risk for
head and neck cancers. Thus, as with lung cancer, nicotine replacement
therapy with bupropion or varenicline, smokeless tobacco cessation, and
vaccines to induce nicotine binding in the blood have been among the
leading behavioral treatment strategies for the prevention of head and
neck cancer.187,188 In addition, strategies to reduce alcohol use and con-
traction of HPV, including alcohol awareness and intervention programs,
also represent key primary interventions, and HPV and Epstein-Barr
virus (EBV) vaccines will be incorporated as immunologic strategies in
future years.189,190
A variety of agents have been investigated for the prevention of
head and neck cancer. Types of drugs used in this setting include
retinoids, peroxisome proliferator-activated receptor (PPAR) agonists,
and protease inhibitors (Table 22.6).
Retinoids
Clinical trials for the prevention of head and neck cancers have largely
investigated the use of retinoids, but to date toxicity of these drugs
has impeded their translation to the clinical setting for cancer preven-
tion. Consequently, more recent trials have focused on low-dose
administration of retinoids or the testing of novel less toxic synthetic
retinoids for head and neck cancer prevention.
Retinoid Head and Neck Second Primary (HNSP) trial
The first demonstration that retinoids could prevent cancer in humans
was from a clinical trial conducted by Hong and colleagues testing
13-cis–retinoic acid (isotretinoin) for the prevention of second primary
cancer in individuals with previous squamous cell carcinoma of the
head and neck.191 The results of this clinical trial showed that 13-cis–
retinoic acid reduced the incidence of second primary head and neck
cancers by 83%; however, few of the patients were able to complete
Table 22.6  Head and Neck Chemoprevention Trials With Outcomes
Phase Patients Agent (Dose)
IIB 44 Isotretinoin (13-cis–retinoic acid) (1–2 mg/kg/day)
IIB 103 13-cis–Retinoic acid (50-100 mg/m2/day)
IIB 65 Vitamin A (200,000 IU/wk)
IIB 103 β-Carotene (100,000 IU/wk) + retinol (180 mg/wk)
IIB 31 Retinamide (40 mg/day)
IIB 70 Isotretinoin (0.5 mg/day)
IIB 153 Fenretinide (200 mg/day)
IIB 675 β-Carotene (40 mg/day) + retinol (100,000 IU/wk) + vitamin E (8 oz/wk)
III 189 cis-Retinoic acid (10 mg/day)
III 1384 cis-Retinoic acid (30 mg/day)
the planned treatment because of significant toxicities (headache, dry
or cracked skin, and skin rash).
Because toxicity was a major problem with this first trial, sub-
sequent trials tested lower doses of isotretinoin. A phase III trial
testing the efficacy of low-dose isotretinoin (13-cis–retinoic acid;
1–2 mg/kg/day) for prevention of second primary tumor incidence
and survival in individuals previously diagnosed with early-stage
head and neck squamous cell cancer (HNSCC) was conducted by
Hong and colleagues.200 This study showed that isotretinoin did
not reduce second primary cancer incidence (HR, 1.06 [95% CI,
0.83–1.35]) or mortality (HR, 1.03 [95% CI, 0.81–1.32]) as compared
with placebo.
More recently, Hong and colleagues genotyped 9465 single-
nucleotide polymorphisms (SNPs) in 450 of the participants of the
trial to determine any association between these SNPs and second
primary cancers.201 They found that 13 loci correlated with significantly
increased risk of second primary tumor incidence or recurrence
(3.33-fold increased risk [95% CI, 1.67–6.67]). In addition, these
investigators identified several loci that were associated with successful
cancer prevention: an SNP in the retinoid X receptor (rs3118570 in
RXRA), an SNP in the CDC25C gene (s6596428), and an SNP in
the JAK2 gene (rs1887427). These results suggest that it may be
possible to identify host characteristics that predict successful cancer
prevention with isotretinoin.
Eastern Cooperative Oncology Group study
A study conducted by ECOG examined low-dose 13-cis–retinoic acid
for the prevention of second primary tumors in patients with head
and neck cancer.202 A total of 189 participants were randomized to
receive 13-cis–retinoic acid (7.5–10 mg) or placebo. After a 3-year
minimal follow-up, no significant reduction in incidence was observed
between the experimental and control groups.
Peroxisome Proliferator-Activated Receptor Agonists
The PPAR family includes PPAR-α, PPAR-β, and PPAR-γ. PPAR-γ
has been shown to regulate expression of metabolic pathway genes
by heterodimerizing with retinoic acid receptors,203 and to regulate
differentiation and apoptosis.204 PPAR ligands include pioglitazone,
rosiglitazone,and troglitazone, which were developed for patients with
type 2 diabetes mellitus.205 Both rosiglitazone and troglitazone are
not currently in use owing to cardiovascular events and hepatotoxicity,
respectively, but pioglitazone is currently used for some individuals
with type 2 diabetes mellitus.205,206
A multisite phase IIB clinical trial (NCT00951379) conducted
through the NCI chemoprevention consortium investigated the effects
of pioglitazone as a preventive therapy for patients with oral prema-
lignant lesions exhibiting dysplasia or hyperplasia. This trial has
completed accrual, and biomarker data are being analyzed. The 52
Results Reference
Positive Hong et al191
Positive Hong et al192
Positive Stich et al193
Positive Stich et al194
Positive Han et al195
Positive Lippman et al196
Positive Chiesa et al197
Positive Zaridze et al198
Negative Pinto et al199
Negative Khuri et al200

nondiabetic participants were randomized to receive pioglitazone
(45 mg/day) or placebo for 24 weeks. Publication of the results from
this trial are anticipated in the near future. A second phase II trial,
led by Keith at the VA Eastern Colorado Health Care System, has
also completed accrual (NCT00780234). This study is examining
pioglitazone (30 mg/day) versus placebo for 6 months in 391 non-
diabetic patients at risk for lung cancer.
Celecoxib
Celecoxib has been investigated in 2 phase II prevention trials for
head and neck cancers. Of these, Engstrom and colleagues at Fox
Chase are studying the efficacy of celecoxib in preventing head and
neck cancer in individuals with oral leukoplakia (NCT00101335).
A second study of celecoxib in patients with oral leukoplakia and/
or head and neck dysplasia is being conducted at Dana Farber by
Wirth and colleagues (NCT00052611). Although the Fox Chase study
has been completed, results have not yet been released for either of
these studies.
Erlotinib
The epidermal growth factor receptor (EGFR) inhibitor erlotinib was
the focus of the Erlotinib Prevention of Oral Cancer (EPOC) study.
This phase III clinical trial randomized 150 individuals with high-risk
oral premalignant lesions (defined as loss of heterozygosity [LOH])
to receive erlotinib (150 mg/day) or placebo for 12 months.207 The
median follow-up time was 35 months. Erlotinib did not improve
oral cancer-free survival (CFS; HR, 1.27 [95% CI, 0.68–2.38]).
However, this study demonstrated that LOH is a marker of oral cancer
risk, with LOH-positive patients having a significantly lower 3-year
oral CFS compared with LOH-negative patients (HR, 2.19 [95% CI,
1.25–3.83]). LOH positivity was also found to correlate with increased
EGFR copy number.
Bowman-Birk Inhibitor Concentrate
Following positive results from a phase IIa trial, Armstrong and col-
leagues conducted a phase IIb clinical trial of the serine protease
inhibitor Bowman-Birk inhibitor concentrate (BBIC) in patients with
oral leukoplakia.208 A total of 89 subjects received 600 chymotrypsin
inhibitor units of BBIC or placebo for 6 months. The experimental
and control groups did not demonstrate a significant difference in
clinical response.
Other Agents
Other phase II trials are investigating additional strategies for the
prevention of head and neck cancer. The kinase inhibitor vandetanib
is the focus of a study currently recruiting participants that is investigat-
ing its potential as a chemopreventive agent for premalignant lesions
of the head and neck (NCT01414426). Currently underway is a
phase II trial of metformin, originally designed for diabetic patients,
for preventing oral cancer in individuals with oral premalignant lesion(s)
(NCT02581137). Accrual to this trial is ongoing, with an estimated
primary completion date of February 2019. Another study still recruit-
ing participants is investigating the efficacy of GL-0817 vaccination
(10 doses) with cyclophosphamide administered 1 day before the first
three vaccinations. GL-0817 is a vaccine against melanoma-associated
antigen A3 (MAGEA3). This study started in March 2017 and is
expected to be completed by March 2020. Among the dietary strategies
being used in studies to prevent recurrence are lyophilized black
raspberries (NCT01504932), strawberry polyphenols (NCT01514552),
and soy isoflavones (NCT020007200).
Despite decades of investigation of chemopreventive agents for
patients with oral premalignant lesions, there is currently no standard-
of-care drug that has shown consistent and durable benefits in this
setting. One of the greatest challenges of developing a chemopreven-
tion approach for oral premalignant lesions is the identification of
a high-risk group that would be expected to benefit the most from
candidate agents. Toward this end, molecular markers of oral cancer
Lifestyle and Cancer Prevention  •  CHAPTER 22 353
risk have been assessed, and LOH at specific chromosomal sites
(especially 3p14 and 9p21) in oral premalignant lesions has been
identified as one of the most robust prognostic markers.209 Building
on these results, the first molecularly based chemoprevention study
has been completed: the EPOC trial.207 In this study, patients with
oral premalignant lesions were tested for LOH, and only those harbor-
ing high-risk LOH profiles were randomized to receive the EGFR
inhibitor erlotinib versus placebo for 12 months. Although this study
did not demonstrate a reduction in oral CFS in the erlotinib group,
it confirmed, for the first time in a prospective setting within the
context of a clinical trial, the role of LOH in predicting cancer risk,
initiating an era of personalized prevention. The study also allowed
for a rich, clinically annotated biobank to be assembled that could be
interrogated for identification of additional targets for cancer preven-
tion. Subsequently, using samples from the EPOC study, William and
colleagues demonstrated that oral premalignant lesions may express
PD-L1, and that PD-L1 expression is higher in LOH-positive lesions
and independently associated with inferior oral CFS.210 Based on these
findings and approval of PD-1 inhibitors in advanced HNSCC, the
MD Anderson group launched the first PD-1–based Immune Preven-
tion of Oral Cancer (IPOC) randomized clinical study, investigating
the role of pembrolizumab versus observation to improve oral CFS
in patients with high-risk, LOH-positive oral premalignant lesions
(NCT02882282).
Esophageal Cancers
ESCC is the most common type of esophageal carcinoma throughout
the majority of the developing world. Although ESCC was the leading
type of esophageal cancer in the United States in the 1970s, 50% or
more of esophageal cancer diagnoses in the United States, as well as
in a number of other countries, are esophageal adenocarcinoma (EAC).
ESCC and EAC have disparate risk factors, histopathologic charac-
teristics, and patterns of occurrence, but treatment strategies for both
remain fairly similar.
Esophageal Squamous Cell Carcinoma
Although numerous studies of individuals at high risk of ESCC have
investigated the efficacy of dietary interventions (e.g., herbs, minerals,
and vitamins) and have had promising results, these approaches have
failed to translate to the clinic.198,211–216 Because the incidence of ESCC
is particularly high in central China, and individuals in that region
have a chronically low intake of a number of micronutrients, a series
of these trials have been conducted there. One phase III trial recruited
29,584 40- to 69-year-old men and women who were randomized
to receive one of four nutrient combinations over a 5-year period:
retinol plus zinc; riboflavin plus niacin; vitamin C plus molybdenum;
and β-carotene plus vitamin E plus selenium.216 Participants were
administered doses that were equal to or twice the recommended daily
allowance in the United States. Whereas 32% of the 2127 deaths that
occurred during intervention were due to esophageal or stomach cancer,
mortality was significantly lower among participants receiving β-carotene
plus vitamin E plus selenium (RR, 0.91 [95% CI, 0.84–0.99]).
Moreover, this group exhibited lower incidence of both overall cancer
(RR, 0.87 [95% CI, 0.75–1.00]) and stomach cancer (RR, 0.79 [95%
CI, 0.64–0.99]). The other three treatment groups did not show
decreased mortality.
In another study conducted in this region, Limburg and colleagues
examined the effects of selenomethionine (200 µg/day) with or without
celecoxib (200 mg/day) in 238 individuals with mild to moderate
esophageal squamous dysplasia.217 Esophageal squamous carcinogenesis
was not decreased after 10 months of intervention with either sele-
nomethionine or celecoxib. However, the 115 individuals with mild
esophageal squamous dysplasia at baseline exhibited decreased dysplasia
with selenomethionine (P = .02). This protective effect was not observed
with the 123 participants with moderate esophageal squamous dysplasia
(P = 1.00).
354 Part I: Science and Clinical Oncology
Unfortunately, translation of these vitamin and mineral combinations
into clinical use for the prevention of esophageal cancer in high-risk
populations with low micronutrient intake has not occurred, in part
because of the difficulty in acquiring and interpreting the data for
these strategies, and in identifying those individuals best suited to
receive each treatment.
Esophageal Adenocarcinoma
Preventive strategies for EAC have investigated targeting COX-2,
cytosolic phospholipase, MCM2, and NF-κB via antiinflammatory
drugs, antioxidants, and acid suppression agents because of the associa-
tion of this disease with both gastroesophageal reflux disease (GERD)
and chronic inflammation.218
Barrett esophagus is a common complication associated with GERD
and results in the replacement of normal squamous esophageal epi-
thelium with metaplastic intestinal epithelium.219,220 Approximately
10% to 15% of individuals with GERD develop Barrett esophagus,
which then places them at a 30- to 125-fold increased risk of adeno-
carcinoma, which is preceded by dysplasia.221 Although the symptoms
of acid reflux can be relieved by antireflux therapy, including proton
pump inhibitors (e.g., lansoprazole),222 antireflux surgery, and endo-
scopic laser ablation,223,224 the effectiveness of these strategies for
promoting regression of Barrett esophagus and preventing the develop-
ment of adenocarcinoma remains unclear.225,226
Nonsteroidal Antiinflammatory Drugs
Preclinical and early-phase clinical studies suggest that nonsteroidal
antiinflammatory drugs (NSAIDs) may be effective in preventing
EAC by reducing proliferation and/or inducing apoptosis.227,228
Additional findings based on observational studies suggest a potential
30% to 40% reduction in risk of EAC associated with NSAID preven-
tive therapy.229,230
Phase II and one phase III clinical trials have examined the efficacy
of aspirin for prevention of esophageal cancer in patients with Barrett
esophagus. The Chemoprevention for Barrett’s Esophagus Trial (CBET)
accrued 222 patients with low- or high-grade Barrett esophagus with
dysplasia.231 Participants received celecoxib (200 mg) or placebo twice
daily for 48 weeks. Celecoxib did not appear to reduce risk of disease
progression or cancer. However, analysis of the subset of 58 CBET
participants with usable quantitative endoscopy area measurements
demonstrated that patients in the celecoxib group did exhibit a sig-
nificant treatment effect measured by quantitative endoscopy before
and after treatment.232
A second phase II study investigated the preventive effects of aspirin
on disease recurrence in 114 patients with Barrett esophagus with no
or low-grade dysplasia after successful elimination of Barrett esophagus
with radiofrequency ablation. This study, led by Limburg at the Mayo
Clinic published results in 2012.233 Participants received short-term
(28 days) self-administered treatment with esomeprazole (40 mg twice
daily) plus either placebo (once per day), aspirin (81 mg/day), or
aspirin (325 mg/day). The group treated with esomeprazole plus
325 mg of aspirin had significantly lower tissue concentrations of
PGE2, which appear to be inhibited by NSAIDs.229,234–238 This reduction
of PGE2 was not observed in the other two treatment groups.
Another phase II trial, led by Bresalier at the University of Texas
MD Anderson Cancer Center, is currently recruiting patients with
Barrett esophagus with or without dysplasia who have undergone
successful elimination of Barrett esophagus via radiofrequency ablation
(NCT02521285). This multiinstitutional study will investigate the
effect aspirin has on preventing recurrence of Barrett esophagus. A
total of 118 participants will be randomized to receive aspirin (orally
once per day) or placebo for 12 months. This study is expected to be
completed in April 2019.
Jankowski and colleagues at Queen Mary University of London
are conducting the phase III Aspirin Esomeprazole Chemopreven-
tion Trial (AspECT), the largest randomized controlled clinical trial
to study the long-term chemopreventive efficacy of esomeprazole
versus esomeprazole plus aspirin.239,240 This study has recruited 2500
participants to receive esomeprazole (20 mg/day), esomeprazole
(80 mg/day), esomeprazole (20 mg/day) plus aspirin (300 mg/
day), or esomeprazole (80 mg/day) plus aspirin (300 mg/day).241
The results from this trial should provide more information about
the cancer prevention efficacy of both esomeprazole and aspirin in
this setting.
Colorectal Cancer
Among the strategies that have been investigated for the prevention
of CRC and have been found to be of some benefit are calcium,
eicosapentaenoic acid (EPA), HRT, NSAIDs, and selenium.
Nonsteroidal Antiinflammatory Drugs
NSAIDs have been the primary focus of a large number of observational,
preclinical, and clinical CRC prevention studies, which have investigated
the use of aspirin, COX-2 inhibitors, and sulindac.242 These studies
have examined both adenoma recurrence and CRC incidence and/or
mortality.
Selenium
Epidemiologic and clinical studies have been unable to clearly define
a preventive efficacy of selenium for CRC.243–246
Nutritional prevention of cancer trial (NPCT).  The Nutritional
Prevention of Cancer Trial (NPCT) randomized 1312 patients with
a history of BCC or SCC of the skin between 1983 and 1991.170
Randomization included two arms: selenium (200 µg/day) and placebo.
Although selenium was not effective in decreasing incidence of basal or
squamous cell skin cancer, it was associated with decreased incidence of
some secondary end points, including incidence of lung cancer, prostate
cancer, and CRC. Subsequent follow-up results in 2002 demonstrated
decreased incidence only of total and prostate cancer incidence, and
not of CRC incidence (HR, 0.46 [95% CI, 0.21–1.02]).247
Selenium and vitamin E cancer prevention trial (SELECT).  SELECT
was a phase III cancer prevention clinical trial investigating the effects of
selenium and vitamin E (α-tocopherol) on the development of prostate
cancer. A substudy of this clinical trial (the Adenomatous Colorectal
Polyp [APC] study) investigated the effect of these supplements on
the incidence of colorectal adenomas.173 After a 7-year follow-up,
neither drug (nor their combination) was found to significantly affect
colorectal adenoma occurrence.172 The most recent results from an
ancillary study of 6546 participants of the SELECT trial, released
in 2017, confirmed these findings, with a 0.96 RR for adenoma
incidence in participants in the selenium group (95% CI, 0.90–1.02;
P = .194), and 1.03 RR in participants in the vitamin E group (95%
CI, 0.96–1.10; P = .38) compared with placebo.248 In 2014, Vinceti
and colleagues published their findings of an analysis of data from
multiple electronic databases regarding selenium exposure and cancer
risk and the preventive efficacy of selenium for the prevention of
cancer.249 Their analysis of 55 observational studies did not show
a beneficial effect of selenium on cancer risk.250 Results from
four additional ancillary studies of the SELECT trial were con-
ducted, examining the effect of selenium on preventing Alzheimer
disease (PREADVISE; NCT00780689), macular degeneration
(SEE; NCT00784225), loss of lung function (NCT0063453), and
colorectal polyps (ACP; NCT00706121).248,251
Women’s Health Initiative.  The Women’s Health Initiative (WHI)
was an observational study within which a nested study of the effects
of selenium on risk of CRC was conducted.252 No association was
found between treatment with selenium and risk of CRC.
Selenium and Celecoxib (Sel/Cel) Trial. This Selenium and
Celecoxib (Sel/Cel) Trial, a phase III randomized clinical trial, examined
the preventive effects of single and combined treatment with selenized
yeast and celecoxib on colorectal adenoma prevention.253 Following
reports of toxicity associated with celecoxib by Arber, Bertagnolli, and
Solomon,254–256 treatment with celecoxib was prematurely terminated.253

Results from this study were published in 2016 and demonstrated
no overall preventive benefit associated with selenium versus placebo.
The researchers did observe an 18% reduction in adenoma recurrence
following treatment with selenium versus placebo in participants
with advanced adenomas at baseline (P = .01). However, as with
Vinceti249 and Stranges,250 increased risk of type 2 diabetes occurred
in the selenium treatment group (HR, 1.25 [95% CI, 0.75–2.11];
P = .41), and a statistically significant increase was found in older
participants (RR, 2.21 [95% CI, 1.04–4.67]; P = .03).257 Additional
randomized clinical trials testing selenium are currently being con-
ducted by Lance (phase III; NCT00078897) and Benya (phase I;
NCT01211561).
Aspirin
Initial results from both the Women’s Health Study258 and the Physi-
cian’s Health Study, two observational studies, suggested that aspirin
does not reduce risk of CRC. Although long-term follow-up reported
for the Physician’s Health Study supported its earlier findings,259
the most recent results from the Women’s Health Initiative Study
reported a significant 42% posttrial reduction in risk of CRC in
healthy women treated with aspirin versus placebo (HR, 0.58 [95%
CI, 0.42–0.80]; P < .001), but increased risk of both GI bleeding and
peptic ulcers.260
Two Colorectal Adenoma/Carcinoma Prevention Program (CAPP)
studies found preventive efficacy associated with aspirin (600 mg/
day), but not resistant starch (30 g/day), compared with placebo for
the prevention of CRC. CAPP1 examined aspirin and resistant starch
in 206 adolescents (10–21 years of age) with familial adenomatous
polyposis (FAP), and found that aspirin reduced polyp load (and size)
compared with no aspirin, whereas resistant starch did not.261 Subse-
quently, the CAPP2 study investigated aspirin and resistant starch in
861 Lynch syndrome carriers. The initial results were reported in
2008 after 4 years of follow-up and showed no decrease in the number
of advanced adenomas or colorectal incidence with use of either aspirin
or resistant starch.262 However, a subsequent report in 2011 after 55.7
months of follow-up demonstrated that 25 months of treatment with
aspirin was associated with a reduction in CRC incidence (incidence
rate of 0.37 [95% CI, 0.18–0.78]; P = .008) compared with placebo,
with no difference in adverse events between these two treatment
groups.263 The CAPP2 results provide strong rationale to treat patients
with hereditary nonpolyposis colorectal cancer with aspirin for colon
cancer prevention. A subsequent trial, the CAPP3 trial, is now being
conducted to test several doses of aspirin (100 mg, 300 mg, 600 mg
for 2 years, then 75 mg, 300 mg, or 600 mg for 3 more years) to
determine the most effective and safest dose.
Several trials have studied the use of aspirin for preventing sporadic
adenoma recurrence in patients with a history of adenomas or CRC.
Studies by Logan,264 Benamouzig,265 and Baron266 have each demon-
strated a significant preventive benefit of aspirin (300 mg/day, 160 or
300 mg/day, and 81 or 325 mg/day, respectively) versus placebo on
the prevention of colorectal adenoma recurrence in patients with a
history of adenomas. However, the most recent follow-up results from
Benamouzig’s group showed that after 4 years of follow-up the preven-
tive effects of aspirin were no longer apparent.267 Conversely, Sandler
and colleagues reported reduced incidence of colorectal adenomas
after treatment with aspirin in a study of 517 CRC survivors treated
with aspirin (325 mg/day) or placebo.268 In a combined analysis of
the British Doctors Aspirin Trial, which tested 5 years of treatment
with aspirin (500 mg/day) versus placebo, and the United Kingdom
Transient Ischemic Attack (UK-TIA) Aspirin Trial, which tested 1
to 7 years of treatment with aspirin (300 or 1200 mg/day) versus
placebo, 5 years of aspirin treatment (≥300 mg/day) was found to
significantly reduce incidence of CRC, particularly after use for
≥10 years.269
Drew and colleagues are investigating the effects of aspirin (81 or
325 mg/day) versus placebo in patients previously diagnosed with
colorectal adenoma in the Aspirin Intervention for the Reduction of
Lifestyle and Cancer Prevention  •  CHAPTER 22 355
CRC Risk study (ASPIRED; NCT02394769).270 This study will use
liquid chromatography–mass spectrometry to assess changes in urinary
prostaglandin metabolites to determine the mechanisms aspirin exploits
to reduce CRC risk.
Several meta-analyses have been published in recent years, including
a strategic review conducted by the USPSTF, with findings published
in 2015.271 This analysis identified a 10-year reduction in risk of CRC
incidence of 20% to 24% after treatment with aspirin (≥75 mg/day)
versus placebo. In addition, these findings suggest that aspirin may
be associated with a 33% reduction in risk of CRC mortality within
10 years of initiation of treatment versus placebo. A second systematic
review and meta-analysis, by Lotrionte and colleagues, identified a
significant preventive effect of aspirin (100 mg/day) on cancer incidence
and death; this dose was better tolerated than higher doses.272 Finally,
Zhao and colleagues conducted a meta-analysis of randomized trials
that once again confirmed the preventive effects of aspirin on advanced
adenoma recurrence for 1 year (RR, 0.68 [95% CI, 0.49–0.95]; P =
.582), and on recurrence of any adenoma for longer than 1 year (RR,
0.84 [95% CI, 0.72–0.98]; P = .484).273 All these studies provide
very strong evidence that aspirin use, particularly for long durations
(of more than 5 or 10 years) significantly reduces the incidence of
CRC. The major questions in this field currently are (1) what is the
optimal (i.e., effective yet safest) dose of aspirin, and (2) how long a
duration is necessary for effective cancer prevention. These questions
are currently being addressed in the CAPP3 trial in Lynch syndrome
patients.
Cyclooxygenase-2 inhibitors
FDA approval of celecoxib in FAP patients was based on the initial
results of a study by Steinbach and colleagues, which demonstrated
that patients with FAP treated with celecoxib (400 mg twice per day)
exhibited 28% fewer colorectal polyps (P = .003) and a 31% reduced
polyp burden (P = .001) versus patients treated with placebo.274
Subsequent results showed that 6 months of treatment with celecoxib
(400 mg twice per day) was also associated with significantly less
duodenal polyposis compared with placebo.275 Shortly thereafter, both
the Adenoma Prevention With Celecoxib255 and Prevention of Colorectal
Sporadic Adenomatous Polyps (PreSAP)254 trials demonstrated a
significant reduction in colorectal adenoma occurrence after treatment
with celecoxib (400 mg/day) compared with placebo. Unfortunately
both the initial and follow-up results of the Adenoma Prevention
With Celecoxib and PreSAP studies identified a significant increase
in risk of cardiovascular events associated with celecoxib, particularly
for individuals who have a high baseline risk for cardiovascular
disease.256,276 These results have greatly limited further development
of COX-2 inhibitors for CRC prevention. However, recent results
from a phase I trial conducted by Lynch and colleagues show that
treatment with short-term (3 months) treatment with celecoxib (16 mg/
kg/day, a dose that corresponds to 400 mg BID in adults) is well
tolerated and associated with no difference in adverse events from
placebo in children with FAP.277
Calcium
Numerous mechanistic and observational studies have suggested
the potential benefit for calcium in preventing polyp recurrence
and CRC.278–286
The Calcium Polyp Prevention and European Cancer Prevention
Organization Study Groups have both conducted clinical trials
testing the efficacy of calcium in preventing adenoma recurrence
in individuals with a history of adenomas.287,288 Their results show
20% to 30% reduced recurrence after calcium supplementation. In
contrast, the WHI conducted a trial of 36,282 postmenopausal women
from 40 WHI centers and found that supplementation with calcium
(500 mg/day) and vitamin D (200 IU/day) did not decrease colorectal
incidence.289 In 2013, updated results 5 years after active interven-
tion ended revealed still no decrease in CRC (HR, 0.95 [95% CI,
0.80–1.13]).290
356 Part I: Science and Clinical Oncology
Vitamin D/Calcium Polyp Prevention Study
The Vitamin D/Calcium Polyp Prevention Study, a phase II/III clinical
trial, tested calcium and vitamin D for the prevention of colorectal
neoplasia in 2259 participants with recently diagnosed neoplastic
polyp(s) that had been successfully removed. Patients were treated
with vitamin D3 (1000 IU/day) and calcium carbonate (1200 mg/
day), alone or together, or placebo. Although the results of this study,
published in 2015, included few serious adverse events, the study also
did not show a significant decrease in risk of colorectal adenomas
after treatment with vitamin D or calcium or both compared with
placebo over 3 to 5 years.291 However, subsequent findings from this
study, released in 2017, suggested variable preventive benefit from
vitamin D supplementation based on SNPs in common variants of
vitamin D pathway genes.292 Specifically, vitamin D decreased risk of
advanced adenomas (RR, 0.36 [95% CI, 0.19–0.69]; P = .002) in
individuals with the vitamin D receptor SNP re7968585 with the
AA genotype, but significantly increased risk of advanced adenomas
(RR, 1.41 [95% CI, 0.99–2.00]; P = .05) in individuals with 1 or 2
G alleles. The researchers found no correlation between the prevention
of advanced colorectal adenomas treated with calcium supplementation
and SNPs in calcium pathway genes.
In 2016, Bonovas and colleagues published their results from a
meta-analysis of randomized placebo-controlled trials testing calcium
for the prevention of adenomas.293 Four trials met the defined study
criteria; calcium was administered daily at a dose of 1200 to 2000 mg.
Their results show an 11% decrease in adenomas in patients treated
with calcium versus placebo (fixed effects RR, 0.89 [95% CI,
0.82–0.96]). However, no significant decrease was observed in incidence
of advanced adenomas (fixed effects RR, 0.92 [95% CI, 0.75–1.13]).
Collectively, these findings suggest that supplementation with calcium
provides minimal prevention of colorectal adenomas, which may persist
for a year and a half to 5 years.
Hormone Replacement Therapy
HRT has been studied extensively as a possible preventive strategy for
CRC. However, although these studies have suggested a preventive
benefit for HRT in risk of CRC,294–297 neither the WHI study298 nor
the European Prospective Investigation into Cancer and Nutrition
(EPIC)299 study, both of which tested estrogen and estrogen plus
progestin, demonstrated significantly decreased risk of colorectal
associated with HRT. The results from the subsequently published
follow-up results of the WHI study confirm these results.300–303 However,
the most recent results from this study show that treatment with
estrogen alone versus placebo is associated with an increased risk of
CRC incidence in postmenopausal women with prior hysterectomy
and prior colon polyp removal (HR, 13.47 [95% CI, 1.76–103.0],
P < .001), and a statistically insignificant increase in CRC deaths
(HR, 1.46 [95% CI, 0.86–2.46], P = .16) in postmenopausal
women with prior hysterectomy.304 Collectively, these studies do
not support the use of estrogen with or without progestin for the
prevention of CRC.
Other Drugs
Curcumin
Numerous preclinical studies have demonstrated that curcumin
(diferuloylmethane) provides both anticancer and antiinflammatory
effects305 and is capable of targeting cancer stem cells.306 Giardiello at
Johns Hopkins University has completed a phase II trial testing the
effects of 12 months of treatment with curcumin versus placebo in
preventing CRC in 50 patients with FAP (NCT00641147). Results
from this study, which has variations in polyp count and size as a
primary end point, are currently pending.
Eflornithine
Difluoromethylornithine (DFMO, eflornithine) has been used to
control facial hair growth and to treat gambiense sleeping sickness
for well over 20 years,307 and is listed as one of the medicines on the
WHO Model List of Essential Medicines.308 Ornithine decarboxylase
(ODC) is overexpressed in tumor cells and upregulates both cell growth
and division,309 and is overexpressed in the intestinal mucosa of patients
with FAP.310 Eflornithine irreversibly inhibits ODC, lowers colorectal
mucosal polyamine levels, and is associated with minimal toxicity.311–314
Several phase II and III clinical trials have studied eflornithine for the
prevention of CRC. Meyskens and colleagues conducted a trial testing
eflornithine and sulindac for the prevention of CRC.315 This phase
III trial randomized 375 patients with colon polyps to receive DFMO
(500 mg/day) and sulindac (150 mg/day) or placebo for 36 months.
They found that patients treated with DFMO plus sulindac exhibited
significantly lower incidence of both colorectal adenoma recurrence
(RR, 0.30 [95% CI, 0.18–0.49], P < .001) and advanced adenomas
(RR, 0.85 [95% CI, 0.011–0.65], P < .001) with minimal side effects.
Three additional trials, a phase II study testing eflornithine plus aspirin
(NCT00983580) and two phase III studies testing eflornithine with
or without sulindac (NCT01483144; NCT01245816), are currently
ongoing. Moreover, Zell and colleagues have reported that the ODC1
genotype impacts adenoma recurrence in patients treated with eflor-
nithine and sulindac compared with placebo.316
Eicosapentanoic Acid
Eicosapentanoic acid (EPA) is a naturally occurring omega-3 fatty
acid that demonstrated variable results in epidemiologic studies and
minimal efficacy in reducing risk of CRC in a review of 20 cohorts
from seven countries.317 However, EPA has been shown to decrease
polyp count and size by 22% and 30%, respectively, after a treatment
time of 6 months.318 The Seafood Polyp Prevention Trial (AFOP) is
currently testing 12 months of treatment with aspirin (300 mg/day)
with or without EPA free fatty acid (2 g/day) versus placebo in prevent-
ing colorectal adenomas in high-risk patients in the English Bowel
Cancer Screening Programme.319
Mesalamine
Gasche and colleagues throughout Europe are currently conducting
the phase II CRC prevention study Mesalamine for Colorectal Cancer
Prevention Program in Lynch Syndrome (MesaCAPP; NCT03070574).
The aminosalicylate antiinflammatory drug mesalamine is used for
the treatment of patients with inflammatory bowel disease. This
study is testing the efficacy of 2 years of treatment with mesalamine
(1220 or 2400 mg/day) or placebo for the prevention of neoplasia in
540 patients with Lynch syndrome and is estimated to be completed
in 2024.
Metformin
The first trial demonstrating the efficacy of metformin in suppressing
colorectal aberrant crypt foci was obtained by Hosono and colleagues.320
The results of a phase III trial conducted by Higurashi and colleagues
that examined the preventive benefit of metformin for recurrence of
metachronous colorectal adenomas and polyps in 151 nondiabetic
patients enrolled at five hospitals in Japan were published in 2016.321
These researchers found that 1 year of treatment with metformin
(250 mg/day) significantly decreased incidence of total polyps (hyper-
plastic polyps and adenomas; RR, 0.67 [95% CI, 0.47–0.97]) and
adenomas (RR, 0.60 [95% CI, 0.39–0.92]). In addition, minimal
adverse events were reported, all of which were grade 1, demonstrating
the safety of metformin in this setting.
Vaccines
A number of studies are now investigating vaccines for the prevention
of CRC. Schoen is leading a phase II study that is investigating the
effectiveness of a MUC1–poly-ICLC vaccine on preventing polyp
recurrence and the progression of advanced adenomatous polyps into
colon cancer in individuals with a history of advanced colorectal
adenoma(s) (NCT0773097). The MUC1–poly-ICLC vaccine immu-
nizes against the Muc-1 tumor-associated antigen, a mucin expressed
by colonic epithelial cells in the lumen of the large intestine. Initial

results published in 2013 demonstrated an immune response in almost
44% of vaccinated subjects, with no associated toxicity.322 The results
also suggested that lack of immune response in the remaining 66%
of subjects was associated with high levels of circulating myeloid-
derived suppressor cells before vaccination. Future results defining
the impact of this vaccine on adenoma recurrence will determine its
efficacy in preventing CRC. Schoen is testing this vaccine in another
multisite phase II trial in 120 patients with newly diagnosed advanced
colon polyps that is currently ongoing (NCT02134925). Another
ongoing early-phase vaccine study is investigating the prevention of
metastases in CRC patients after vaccination with the adenovirus
5–human guanylyl cyclase (Ad5-hGCC)–PADRE vaccine (phase I,
NCT01972737).
Gastric Cancer
Helicobacter pylori
Risk of both local and distant recurrence of gastric cancer is high,
and both chronic gastritis and most gastric cancers are associated with
chronic mucosal infection with the gram-negative bacterium H. pylori;
in addition, H. pylori accounts for 5.5% of all cancer incidence.323 A
recent meta-analysis of the prevalence of H. pylori infection during
childhood found that approximately one-third of children worldwide
were currently or had previously been infected with H. pylori.324 Con-
sequently, many RCTs investigating the role of H. pylori infection in
the development of noncardia gastric cancer have been conducted, with
conflicting and/or non–statistically significant initial findings.325–330
However, extended follow-up results have demonstrated the association
of H. pylori infection and gastric cancer, as well as the efficacy of H.
pylori eradication in the prevention gastric cancer.331,332
In 2000 Correa and colleagues released results from a randomized
controlled chemoprevention trial of antioxidant supplements (ascorbic
acid and β-carotene) and anti–H. pylori triple therapy (combined
treatment with antibiotics and proton pump inhibitors) in 795
patients with gastric precancerous lesions in a high-risk region of
Colombia.325 After 6 years of follow-up, treatment with anti–H. pylori
triple therapy alone (RR, 4.8 [95% CI, 1.6–14.2]) or ascorbic acid
(RR, 5.0 [95% CI, 1.7–14.4]) or β-carotene (RR, 5.1 [95% CI,
1.7–15.0]) demonstrated significantly increased regression of precur-
sor lesions, suggesting the potential efficacy of these interventions in
preventing gastric cancer. Combined treatment with anti–H. pylori
triple therapy and the antioxidant supplements did not significantly
improve regression. Patients with atrophy (RR, 8.7 [95% CI, 2.7–28.2])
and intestinal metaplasia (RR, 5.4 [95% CI, 1.7–17.6]) demonstrated
significantly increased rates of regression of precancerous lesions after
eradication of the H. pylori infection.333 After an additional 6 years
of follow-up, the benefits associated with both ascorbic acid and
β-carotene were lost; however, eradication of H. pylori continued
to be associated with precursor lesion regression.331 The most recent
results from this study with 16 years of follow-up further defined the
association between long-term exposure to H. pylori and progression
of precancerous lesions, and support the use of anti–H. pylori triple
therapy in high-risk individuals.332 The researchers found that 16
years of continuous H. pylori infection increased risk of progression
of precancerous lesions to more advanced diagnoses versus no change
or regression (P = .0001). In addition, higher risk of progression
occurred in individuals with incomplete-type intestinal metaplasia
versus those with complete-type intestinal metaplasia (odds ratio [OR],
11.3 [95% CI, 1.4–91.4]). Continuous H. pylori infection was also
associated with a 0.20 unit/year increase in histopathology scores
(95% CI, 0.12–0.28), and individuals with atrophy but not intestinal
metaplasia exhibited significantly higher increases in histopathology
scores compared with individuals with intestinal metaplasia (P < .001).
These results demonstrate that risk of progression of precancerous gastric
lesions is significantly increased with long-term H. pylori exposure,
and that individuals with incomplete-type intestinal metaplasia are at
high risk.
Lifestyle and Cancer Prevention  •  CHAPTER 22 357
The Shandong Intervention Trial, conducted in Linqu County,
China, randomized 3365 individuals with advanced precancerous
gastric lesions to receive H. pylori treatment (amoxicillin and omepra-
zole) for 2 weeks with or without 7.3 years of vitamin supplements
(vitamin C, vitamin E, and selenium) and/or garlic supplements.327
Neither the vitamin nor the garlic supplements for 7.3 years of treatment
reduced the prevalence of precancerous gastric lesions or the incidence
of gastric cancer. However, eradication of H. pylori reduced incidence
of precancerous lesions. The 14.7-year follow-up results demonstrated
a 39% significant reduction in gastric cancer incidence (OR, 0.61
[95% CI, 0.38–0.96]; P = .32), as well as a non–statistically significant
comparable reduction (33%; HR, 0.67 [95% CI, 0.36–1.28]) in gastric
cancer mortality.333 In addition, although the vitamin and garlic
supplements demonstrated non–statistically significant decreases in
gastric cancer incidence and death, vitamin supplementation was
associated with a significant fully adjusted (for age, sex, alcohol use,
smoking) reduction in esophageal or gastric cancer mortality, which
was a secondary study end point (HR, 0.51 [95% CI, 0.29–1.03];
P = .014).
The findings of a meta-analysis of the efficacy of adding probiotics
to standard H. pylori triple therapy were published in 2016; data from
30 RCTs with a total of 4302 per-protocol and 4515 intention-to-treat
(ITT) patients were analyzed.334 H. pylori triple therapy plus probiotics
increased eradication of H. pylori by 12.2% in the per-protocol group
(RR, 1.122 [95% CI, 1.091–1.153]; P < .001) and 14.1% in the
ITT group (RR, 1.141 [95% CI, 1.106–1.175]; P < .001) versus H.
pylori triple therapy alone. This increase in eradication was demonstrated
regardless of age or status of being Asian. A reduction in risk of
antibiotic-associated side effects was also associated with the addition
of probiotics.
Population-based H. pylori screening and treatment in populations
at high risk are currently recommended by a growing number of
groups around the world, including the Asian-Pacific Gastric Cancer
Consensus group.335 However, these recommendations are currently
in flux because antibiotic resistance rates of the bacterium vary, and
persistent infection with H. pylori is detrimental for gastric cancer
and mucosa-associated lymphoid tissue (MALT) lymphoma, among
other benign diseases, but is potentially beneficial for individuals at
risk for other diseases, including Barrett esophagus and EAC.336 In
addition, geographic variations in the effects of H. pylori infection on
risk of gastric cancer have been reported, particularly between high-risk
Asian populations that show increased risk337 and low-risk Western
populations that show no or decreased risk.338–340 It has been suggested
that these variations in the level of risk for gastric cardia cancer associated
with H. pylori infection could be a result of etiologically distinct types
of gastric cancer.341 This concept is supported by the results of several
recent meta-analyses, which identified genetic polymorphisms associated
with increased susceptibility to H. pylori infection, and/or increased
risk for H. pylori–mediated gastric cancer. In 2017 alone, study
investigators reported such polymorphisms in (1) DNA repair genes
(TP53, BRIP1, ERCC4, and XRCC3)342; (2) IL-1B-31C/T,
IL-1B-511-C/T, and IL-8-251T/A,343 IL-2,344,345 and IL-6 and IL-6R346;
(3) the glutathione S-transferases (GSTs) GSTM1, GSTP1, and
GSTT1347,348; (4) H. pylori349; (5) long noncoding (Lnc) RNA
rs16901946 (G)350; (6) MUC1351; (7) prostate stem cell antigen (PSCA),
protein kinase AMP-activated catalytic subunit alpha 1 (PRKAA1),
solute carrier family 52, member 3 (SLC52A3), and zinc finger and
BTB domain containing 20 (ZBTB20); and (8) Toll-like receptors
(TLRs) TLR1 and TLR10.352 These studies represent just a small
sample of the results that have been released over the past 5 years but
provide compelling evidence of the existence of etiologically distinct
types of gastric cancer; studies clarifying this issue are currently
underway and will, it is hoped, aid in cancer prevention strategies for
H. pylori–regulated gastric cancer.
To date, H. pylori screening and treatment have not been adopted
by any country.336 However, China is currently recruiting over 180,000
participants to the largest H. pylori screening and treatment trial.353
358 Part I: Science and Clinical Oncology
The results of this and other long-term studies on the relationship
among H. pylori screening and treatment and the risk of gastric cancer,
antibiotic resistance, and other diseases will determine the impact of
this strategy for the prevention of gastric cancer.
Nonsteroidal Antiinflammatory Drugs
The effectiveness of NSAIDs for the prevention of gastric cancer has
been and remains controversial; however, efficacy has been suggested
in numerous observational studies. A meta-analysis published in 2010
across 14 studies with 5640 diagnoses of gastric cancer was unable to
demonstrate a statistically significant difference in risk of gastric cancer
based on aspirin use or nonuse.354 However, on stratification by location
and infection with H. pylori, aspirin users demonstrated reduced risk
of noncardia gastric cancer (OR, 0.62 [95% CI, 0.55–0.69]) and
reduced risk of H. pylori infection (OR, 0.62 [95% CI, 0.42–0.90])
compared with nonusers. Aspirin was also associated with reduced
risk for noncardia gastric cancer (OR, 0.73 [95% CI, 0.62–0.87])
and H. pylori infection (OR, 0.62 [95% CI, 0.55–0.69]) in Caucasians,
suggesting that aspirin may be beneficial in preventing noncardia
gastric cancer in Caucasian and H. pylori–infected patients. Another
meta-analysis of eight trials with a total of 25,570 participants on the
efficacy of aspirin as a preventive strategy for gastric cancer showed
a significant decrease in gastric cancer mortality in patients with more
than 10 years of aspirin use.355 Most recently, Huang and colleagues355a
conducted a meta-analysis of 24 studies and identified reductions in
risk of gastric cancer following use of any NSAIDs (RR, 0.78 [96%
CI, 0.72–0.85]), aspirin (RR, 0.70 [96% CI, 0.62–0.80]), and
nonaspirin NSAIDs (RR, 0.86 [96% CI, 0.80–0.94]), particularly
for noncardia gastric cancer.
Jankowski is leading the currently ongoing aspirin and esomepra-
zole chemoprevention trial AspECT, a national trial in the united
Kingdom that has accrued its goal of 2500 patients (EudraCT
2001-003836-77).240 This study, scheduled to publish results in
2018, is investigating the effects of treatment with the proton pump
inhibitor esomeprazole (20 mg/day or 80 mg/day) with or without
aspirin (300 mg/day) in individuals 18 years of age or older with Barrett
metaplasia in order to determine the effects of aspirin on all-cause
mortality and EAC. Results from this study on all-cause mortality
will, it is hoped, include and demonstrate the effect of individual
and combined treatment with esomeprazole and aspirin on risk of
gastric cancer.
Cyclooxygenase-2 Inhibitors
In 2012 the first results of a randomized trial specifically designed to
assess the efficacy of treatment with a COX-2 inhibitor (celecoxib,
200 mg twice daily for 24 months) with or without prior anti–H.
pylori triple therapy (7 days) and anti–H. pylori triple therapy
alone (7 days) versus placebo for the prevention of gastric cancer
were published.356 This study accrued 1024 participants in Linqu
County, Shandong Provence, China, who were 35 to 64 years of
age and had both H. pylori infection and advanced gastric lesions.
Whereas individual treatment with both celecoxib and anti–H. pylori
therapy versus placebo was associated with significant regression of
both gastric lesions (celecoxib: 52.8% versus 41.2%; anti–H. pylori
therapy: 59.3% versus 42.1%) and overall risk (celecoxib: OR, 1.72
[95% CI, 1.07–2.76]; anti–H. pylori therapy: OR, 2.19 [95% CI,
1.32–3.64]), combined treatment did not improve regression of advanced
gastric lesions.
More recently, Zhang and colleagues published the results of a
trial assessing the relationship between treatment with anti–H. pylori
therapy and/or the COX-2 inhibitor celecoxib and both COX-2
methylation levels and gastric cancer in 809 participants with gastric
lesions.357 Methylation of COX-2 levels were decreased by treatment
with both celecoxib and anti–H. pylori therapy; however, as with the
study by Wong and colleagues, no additive or synergetic effects were
associated with combined treatment. Patients with reductions in COX-2
methylation demonstrated significantly higher regression of gastric
lesions than patients lacking methylation reduction (OR, 1.92 [95%
CI, 1.03–3.60]), suggesting the potential for decreased risk of gastric
cancer due to decreased COX-2 methylation via treatment with
celecoxib or anti–H. pylori therapy.
A 2016 meta-analysis that included 47 studies of antiinflammatory
drugs for gastric cancer prevention showed decreased risk of gastric
cancer with antiinflammatory drug use (RR, 0.78 [95% CI,
0.71–0.85]).358 In addition, this analysis showed that use of COX-2
inhibitors was more effective for the prevention of gastric cancer than
other NSAIDs (RR, 0.45 [95% CI, 0.29–0.70]). Ultimately, additional
long-term studies are need to establish the specific populations most
likely to benefit from preventive treatment with NSAIDs for the
prevention of gastric cancer.
Hepatocellular Cancer
Major risk factors for hepatocellular carcinoma (HCC) include
viruses, particularly hepatitis B virus (HBV) and hepatitis C virus
(HCV),359–361 cirrhosis,362,363 environmental toxins (e.g., ingestion of
aflatoxin in parts of Africa),364 diabetes,365 tobacco use, and alcohol
consumption.366
Consequently, strategies focused on the prevention of HBV and
HCV,367,368 prevention or diminished progression of cirrhosis,369 reduced
exposure to toxins,370 prevention or effective management of diabetes,371
tobacco cessation, and alcohol reduction or elimination constitute
potential techniques for the prevention of liver cancer. In addition,
as with most cancers, behavioral modifications, including diet and
exercise, are beneficial preventive interventions.372 Unfortunately, clinical
data have yet to clearly define a preventive benefit associated with
dietary supplements.373
Hepatitis B Virus and Hepatitis C Virus Vaccine
A large-scale study of HBV vaccination of children in Taiwan dem-
onstrated the effectiveness of this strategy for the prevention of
HCC.368,374–376 This initiative was implemented in July 1984. Although
82,000 HCC diagnoses were listed in the Taiwan National Cancer
Registry from 2003 to 2011, almost all of these were in middle-aged
(50.1%) or elderly (49.1%) individuals. Therefore the launch of this
universal immunization program has resulted in the virtual eradication
of both HBV and HCC in children, who account for only 0.04% of
all HCC diagnoses since 2011.377
The Qidong Hepatitis B Intervention Study was conducted in
Qidong, China from 1985 to 1990.378 This population-based RCT
involved the vaccination of 38,366 newborns who completed an HBV
vaccination series and 34,441 newborns who did not receive the
vaccination series. This neonatal HBV regimen demonstrated a sig-
nificant decrease in HBV surface antigen seroprevalence and risk of
primary liver cancer. The long-term preventive efficacy of HBV
immunization for prevention of chronic hepatitis B, liver fibrosis, and
cirrhosis have since been evaluated, and protection has been demon-
strated to extend to marrying age.379 Results from this study have
shown that the administration of a single HBV vaccine booster at 10
to 14 years of age decreases the incidence of HBV infection in high-risk
individuals.380
An effective prophylactic HCV vaccine has yet to be developed.
However, advances in HCV vaccine formulations and modalities381
and preclinical studies in a variety of animal models have demonstrated
promising candidates for HCV vaccines that may translate to the
clinic in future years.382
Other Strategies
Capecitabine
A phase II/III trial for the prevention of HCC recurrence after curative
resection is using capecitabine, which is an FDA-approved drug for
metastatic colorectal and breast cancers, and gastric and esophageal
cancers (NCT00561522). Anticipated enrollment is 290, and individu-
als will be randomized to receive capecitabine (four to six cycles of

1250 mg twice daily for 14 days, followed by 7 days without drug)
or no intervention.
Atorvastatin
Chen is currently conducting a phase IV trial of the lipid-lowering
drug atorvastatin (10 mg/day) versus placebo for the prevention of
HCC recurrence in individuals with curative treatment of HCC
(NCT03024684). The expected accrual for this study, which opened
in 2017, is 240.
Interferon
Interferon (IFN), a cytokine, has been investigated in a randomized
clinical trial of 101 male patients with chronic HBV infection in
Taiwan.383 Results after long-term follow-up showed a significant
decrease in HCC incidence in the treatment arm (1.5% incidence)
compared with the control arm (12% incidence). Subsequently, a
41% reduction in HCC incidence was reported by Yang and colleagues
in a meta-analysis of RCTs testing IFN therapy in subjects with
HBV-related cirrhosis.384 IFN has also been tested (although not in
a randomized clinical trial) for the prevention of HCC in subjects
with chronic HCV.385–389
Lamivudine
Liaw and colleagues conducted a phase III trial of lamivudine, an
antiretroviral medication for HIV/AIDS and hepatitis B, in the Asian-
Pacific region.390 A total of 651 participants were recruited, of whom
98% were Asian and 85% were male. Subjects received lamivudine
(n = 436) or placebo (n = 215), and the primary end point was time
to disease progression, which was defined as hepatic decompensation,
HCC, spontaneous bacterial peritonitis, bleeding gastroesophageal
varices, or liver disease–related death. Because of the significant protec-
tive effect of lamivudine, the study was terminated prematurely at
32.4 months. Disease progression was reached in 7.8% of lamivudine-
treated participants and 17.7% of placebo-treated participants. In
addition, HCC incidence was reduced in the treatment arm (3.9%)
compared with the control arm (7.4%). Lamivudine was also the
focus of a retrospective study conducted in Japan.391 This study analyzed
2795 patients, of whom 657 had received lamivudine and 2138 had
not. Patients treated with lamivudine (1.1% incidence) had significantly
fewer cases of HCC compared with those who were not (13.3%
incidence).
Glycyrrhizin
Glycyrrhizin, an antiinflammatory agent, was the focus of a retrospective
study that examined the effect of glycyrrhizin therapy on HCC in
individuals with IFN-resistant HCV.392 A total of 244 subjects received
intravenous glycyrrhizin injection, whereas 102 did not; all 346 of
the participants had alanine transaminase values that were at least
twice the upper limit of normal. Glycyrrhizin vaccination significantly
decreased HCC incidence in participants with IFN-resistant HCV
and high alanine transaminase values compared with placebo (HR,
0.49 [95% CI, 0.27–0.86]; P = .014).
Breast Cancer
Several strategies for breast cancer prevention have been studied,
including dietary changes, lifestyle changes with increased exer-
cise, preventive drug therapy, and prophylactic surgery. Of these,
surgery and preventive drug therapy have been shown to be highly
effective preventive strategies, but many women do not want to
experience the side effects of medications or surgical procedures.
Therefore there has been a major emphasis to find more toler-
able, yet effective strategies for breast cancer prevention. Several
large studies of diet and exercise are currently ongoing (reviewed
in Chapter 88). This chapter discusses the progress made with use
of hormones, vitamins, targeted drugs, and vaccines to prevent
breast cancer.
Lifestyle and Cancer Prevention  •  CHAPTER 22 359
Selective Estrogen Receptor Modulators
Over the last 25 years there has been a focused effort to develop drugs
to prevent breast cancer development. The results of clinical trials
testing antiestrogen drugs for breast cancer prevention are shown in
Table 22.7. The rationale for using antiestrogen drugs for cancer
prevention came from therapeutic trials of antiestrogen drugs that
were tested as adjuvant therapy of early-stage breast cancer. These
clinical trials demonstrated that women taking the antiestrogen drug
tamoxifen developed 50% fewer contralateral primary breast cancers
than did women taking placebo.393–395 This observation led to the
development of many phase III breast cancer prevention trials using
tamoxifen as preventive therapy in women without breast cancer (see
Table 22.7). The largest of these trials was the National Surgical
Adjuvant Breast and Bowel Project (NSABP)–P1 trial.396 In this trial,
premenopausal and postmenopausal women at moderately high risk
of breast cancer were treated with tamoxifen or placebo for up to 5
years.396,397 This study was stopped early when initial results showed
that women taking tamoxifen developed 49% fewer breast cancers
than did women taking placebo.396 These results led to FDA approval
of tamoxifen for breast cancer risk reduction in high-risk women.
Several similar trials showed similar results with long-term follow-up
(the UK and Italian studies also showed that tamoxifen reduced the
risk of breast cancer by at least 30%).398–401 Although tamoxifen
prevented the development of ER+ breast cancers, the results of each
of these trials showed that tamoxifen did not prevent ER− breast
cancers.398,399,401,402 Tamoxifen is FDA approved for breast cancer risk
reduction, but fewer than 1% of eligible women take this medication
for breast cancer prevention owing to a concern about the side effects
of tamoxifen (increased risk of hot flashes, vaginal dryness, and blood
clots and a rare increased risk of uterine cancer).
Another SERM, raloxifene, has been shown to also prevent ER+
breast cancers. Raloxifene was initially developed and later approved
as a drug for the treatment of osteoporosis to prevent bone fractures.
However, the MORE (Multiple Outcomes of Raloxifene Evaluation)
clinical trial tested this drug in postmenopausal women with osteo-
porosis for its ability to prevent breast cancer.407 The results of the
MORE study demonstrated that women taking raloxifene had 72%
fewer breast cancers than seen in women taking placebo.415 These
promising results led to the development and conduction of the Study
of Tamoxifen and Raloxifene (STAR) trial, which compared the ability
of tamoxifen or raloxifene to prevent breast cancer in high-risk
postmenopausal women. The initial results of the STAR study showed
that both tamoxifen and raloxifene had similar preventive activity405
(each prevented approximately 50% of the expected breast cancers);
however, with longer follow-up, raloxifene’s ability to prevent breast
cancer eroded over time, with raloxifene being 76% as effective as
tamoxifen at preventing invasive breast cancer after 7 years. Important
to note, the side effects of raloxifene were reduced compared with
tamoxifen. Raloxifene caused fewer side effects, with less frequent hot
flashes, episodes of vaginal dryness, and thrombotic events. Raloxifene
also did not increase the risk of uterine cancer. The promising results
of the STAR trial led to the FDA approval of raloxifene for breast
cancer prevention in high-risk postmenopausal women. However,
despite the improved side effect profile of raloxifene as compared with
tamoxifen, less than 5% of eligible women at high risk of breast cancer
take this medication for breast cancer prevention, because of concerns
about the side effects of this medication.
Two other SERMs, lasofoxifene and arzoxifene, have been tested for
their ability to prevent breast cancer. Like tamoxifen and raloxifene,
these drugs reduced the frequency of ER+ breast cancer without
reducing the frequency of ER− breast cancer. In the PEARL trial,
lasofoxifene (0.25 mg/day and 0.5 mg/day) reduced the incidence of
all invasive breast cancers by 79%, and reduced ER+ breast cancers
by 83%.412 As with raloxifene, lasofoxifene use was associated with
increased thromboembolic events, leg cramps, and hot flushes, but
was not associated with an increased risk of endometrial hyperplasias
360 Part I: Science and Clinical Oncology

Table 22.7  Selective Estrogen Receptor Modulator (SERM) Breast Cancer Prevention Studies
Trial Patient Population Study Design Results Reference
TAMOXIFEN
Royal Marsden 2494 high-risk women Tamoxifen (20 mg/day) or No difference initially; with longer Powles et al, 1998403
30–70 yr of age placebo for 8 yr follow-up, reductions in invasive Powles et al, 2007398
and ER+ breast cancer incidence;
no significant change in ER− or
overall breast cancer incidence
NSABP-P1 (BCPT) 13,388 high-risk women Tamoxifen (20 mg/day) or Reductions in invasive, Fisher et al, 1998396
>35 yr of age placebo for 5 yr noninvasive, and ER+ breast Fisher, 2005397
cancer incidence; no significant Fisher et al, 2005402
change in ER− tumors
Italian 5408 normal-risk women Tamoxifen (20 mg/day) or Reductions in invasive and ER+ Veronesi et al, 1998404
with a hysterectomy placebo for 5 yr breast cancer incidence; no Veronesi et al, 2007399
35–70 yr of age significant change in ER− tumors
IBIS-I 7154 high-risk women Tamoxifen (20 mg/day) or Reductions in invasive, Cuzick et al, 2002400
35–70 yr of age placebo for 5 yr noninvasive (DCIS), ER+, and Cuzick et al, 2007401
overall breast cancer incidence; no
significant change in ER− tumors
NSABP-P2 (STAR) 19,747 high-risk, Tamoxifen (20 mg/day) Reductions in invasive and Vogel et al, 2006405
postmenopausal women or raloxifene (60 mg/day) noninvasive ER+ tumors less Vogel et al, 2010406
>35 yr of age for 5 yr effective (but with decreased
toxicity) with raloxifene than
tamoxifen
RALOXIFENE
MORE 7705 postmenopausal women Raloxifene (60 or 120 mg/ Reductions in invasive, ER+, and Cummings et al, 1999407
with low BMD <80 yr of age day) or placebo for 4 yr overall breast cancer incidence; no Ettinger et al, 1999408
significant change in ER− tumors
CORE 4011 postmenopausal women Raloxifene (60 mg/day) or Reductions in invasive, ER+, and Martino et al, 2004409
with low BMD (reconsented placebo for an additional overall breast cancer incidence; no
from MORE trial) <80 yr of age 4 yr after 4 yr of significant change in noninvasive
raloxifene on MORE trial of ER− tumors
RUTH 10,101 postmenopausal Raloxifene (60 mg/day) or Reduction in invasive ER+ tumors Barrett-Connor et al,
women with CHD placebo for a median of 2006410
>35 yr of age 5.6 yr
LASOFOXIFENE
PEARL 8556 women with low BMD Lasoxifene (0.25 mg or Reduction in invasive ER+ tumors Cummings et al, 2010411
59–80 yr of age 0.5 mg/day) or placebo LaCroix et al, 2010412
ARZOXIFENE
GENERATIONS 9354 women with low BMD Arzoxifene (20 mg/day) Reduction in invasive ER+ tumors Cummings et al, 2010413
60–85 yr of age or placebo for 4 yr Powles et al, 2012414

BMD, Bone mineral density; CHD, coronary heart disease; CORE, Continued Outcomes of Raloxifene Evaluation (CORE); ER, estrogen receptor; IBIS-I, International Breast
Intervention Study; Italian, Italian Randomized Tamoxifen Prevention Trial; MORE, Multiple Outcomes of Raloxifene Evaluation; NSABP-P1, National Surgical Adjuvant Breast and
Bowel Project Breast Cancer Prevention Trial P1; NSABP-P2, National Surgical Adjuvant Breast and Bowel Project Study of Tamoxifen and Raloxifene P2; PEARL, Postmenopausal
Evaluation and Risk Reduction With Lasofoxifene trial; RUTH, Raloxifene Use for the Heart trial; Royal Marsden, Royal Marsden Tamoxifen Prevention Trial.

or endometrial cancers. As with the other SERMs, lasofoxifene had
no effect on the development of ER− breast cancers. In the GEN-
ERATIONS trial, arzoxifene (20 mg/day) was tested for its ability to
prevent bone fractures and breast cancer in postmenopausal women
with osteoporosis.413 After a follow-up period of 36 months (for bone
fracture assessment) or 48 months (for breast cancer assessment), there
were 41% fewer vertebral bone fractures and 56% fewer invasive
breast cancers in women taking arzoxifene than in women taking
placebo (the specific effect on ER+ and ER− breast cancers was not
described). Arzoxifene caused significantly more thromboembolic events,
hot flushes, and vaginal discharge, but did not significantly increase
uterine cancers or endometrial hyperplasias.413 Despite the significant
breast cancer preventive effect and generally tolerable side effect profile
of arzoxifene and lasofoxifene, the drug companies have not sought
FDA approval for a breast cancer risk reduction indication. Therefore
these effective drugs are not generally used for breast cancer prevention.
Cuzick and colleagues published a meta-analysis of studies, showing
that, overall, clinical trials with SERMs have demonstrated that these
antiestrogen drugs can prevent the development of invasive ER+ breast
cancer, but not ER− breast cancer.416 In a consensus statement, Cuzick
and colleagues417 suggested that women at high risk of breast cancer,
such as those with a strong family history of breast cancer, high
mammographic density, and atypical hyperplastic premalignant lesions,
are good candidates for SERM preventive therapy. A study by Reimers
and colleagues suggested that the uptake of these agents may be
increasing in the high-risk groups; they observed in the primary care
setting that 37% of eligible high-risk women took an antiestrogen
SERM for breast cancer prevention.418

Aromatase Inhibitors
AIs (anastrozole, letrozole, and exemestane) have been shown to be
more effective than SERMs for the treatment of breast cancer (reviewed
in Litton and colleagues419); therefore there has been strong interest
in use of these drugs for breast cancer prevention. The first indication
that AI drugs would be effective for breast cancer prevention came
from the ATAC trial, which tested anastrozole, tamoxifen, and the
combination for the treatment of early-stage breast cancer.420 Results
from the ATAC trial showed that anastrozole alone was more effective
than tamoxifen in preventing the development of second primary
breast cancers in women with early-stage breast cancer.421 Women
treated with anastrozole had 58% fewer contralateral primary breast
cancers than those treated with tamoxifen when the ATAC trial
was initially reported (OR, 0.42; P = .007).420 With the report of
the 10-year follow-up data, the results showed that women taking
anastrozole developed 32% fewer contralateral breast cancers than
those taking tamoxifen (HR, 0.68; P = .01 for all breast cancers),
and 38% fewer ER+ contralateral breast cancers (HR, 0.62;
P = .003).421
These provocative results led to the development of several clinical
trials testing AIs for their ability to prevent breast cancer in women
with previous ductal carcinoma in situ (DCIS) breast cancer or in
high-risk women with no prior history of breast cancer (Table 22.8).
Several trials have tested the ability of anastrozole to prevent invasive
breast cancer, either in postmenopausal women with DCIS breast
cancer (the NSABP B35 DCIS trial422 or the IBIS-II DCIS trial423),
or in healthy high-risk postmenopausal women (IBIS-II trial).424 In
each of these trials, anastrozole was found to reduce the incidence of
invasive breast cancer. In the NSABP B35 trial, anastrozole was
compared with tamoxifen for its ability to prevent the development
of invasive breast cancer in postmenopausal women with DCIS breast
cancer. The results from this study showed that anastrozole was slightly
more effective than tamoxifen at reducing the incidence of breast
cancer events (HR, 0.73 [95% CI, 0.56–0.96]; P = .0234) in post-
menopausal women with ER+ DCIS.422 There were also fewer invasive
breast cancers (HR, 0.62 [95% CI, 0.42–0.90]; P = .0123) and fewer
DCIS recurrences (although nonsignificant; HR, 0.88 [95% CI,
0.59–1.30]; P = .52) in women taking anastrozole than in those taking
tamoxifen.422 In addition, in the women taking anastrozole, there was
a significant reduction in contralateral breast cancers (HR, 0.64 [95%
CI, 0.43–0.96]; P = .0322). As with other breast cancer prevention
trials, there was no detectable difference in survival (given the excellent
Table 22.8  Select Aromatase Inhibitor (AI) Breast Cancer Prevention Studies
Trial Patient Population Study Design
DCIS TRIALS
NSABP B-35 3104 women accrued Anastrozole (1 mg/day) vs
Postmenopausal with tamoxifen (20 mg/day) for 5 yr
ER+/PR+ DCIS
IBIS-II 2980 women accrued Anastrozole (1 mg/day) vs
(DCIS) Postmenopausal with DCIS tamoxifen (20 mg/day) for 5 yr
PREVENTION TRIALS
NCIC-MAP.3 4560 women accrued Exemestane (25 mg/day) vs
Postmenopausal, high-risk placebo for 5 yr
≥35 yr of age
IBIS-II 3864 women accrued Anastrozole (1 mg/day) vs
Postmenopausal, high-risk placebo
DCIS, Ductal carcinoma in situ; IBIS-II, Second International Breast Cancer Intervention Study; NSABP B-35, National Surgical Adjuvant Breast and Bowel Project B-35;
NCIC-MAP.3, NCIC Clinical Trials Group Mammary Prevention.3 trial; PR, progesterone receptor.
Lifestyle and Cancer Prevention  •  CHAPTER 22 361
overall outcome of women with DCIS). The IBIS-II DCIS trial had
a similar design as the NSABP B35 trial, and compared anastrozole
and tamoxifen with regard to their ability to prevent invasive and
contralateral breast cancer in postmenopausal women. The results
showed that both anastrozole and tamoxifen were similar in their
ability to prevent DCIS recurrence. There was no statistically significant
difference in their ability to prevent any breast cancer event (HR,
0.89 [95% CI, 0.64–0.123]; P = .49), invasive breast cancers (HR,
0.80 [95% CI, 0.52–1.24]; P = .32), or DCIS recurrences (HR, 0.99
[95% CI, 0.60–1.65]; P = .98).423 As expected, the side effect profiles
for tamoxifen and anastrozole were different, with more muscle spasms,
uterine cancers, hot flushes, and deep vein thromboses with tamoxifen,
and more bone fractures, musculoskeletal events, hypercholesterolemia,
and strokes with anastrozole.423
The first of the AI primary prevention studies to be reported was
the MAP.3 study, which compared the AI exemestane and placebo in
healthy postmenopausal women at high risk of breast cancer.428 The
results showed that women who took exemestane had 65% fewer
invasive breast cancers than those who took placebo (HR, 0.35; P =
.002), and 73% fewer ER+ breast cancers (HR, 0.27; P < .001).
Women taking exemestane had significantly more hot flushes, joint
and muscle pain, and GI symptoms than those taking placebo. However,
there were no significant differences in skeletal fractures, thrombo-
embolic events, cardiovascular events, or other cancers between women
taking exemestane and those taking placebo.428 These results indicate
that exemestane may be particularly useful in preventing ER+ breast
cancers and may be tolerable in high-risk women, particularly those
without osteopenia or osteoporosis.
The IBIS-II prevention trial compared the ability of anastrozole
and placebo to prevent breast cancer in healthy postmenopausal women
at high risk of breast cancer. These women took anastrozole (or placebo)
for 5 years. After a median follow-up of 5 years, the results demonstrated
that women who took anastrozole had 53% fewer breast cancers (HR,
0.47 [95% CI, 0.32–0.68]; P < .0001), 50% fewer invasive breast
cancers (HR, 0.50 [95% CI, 0.32–0.76]; P = .001), 70% fewer
noninvasive breast cancers (HR, 0.30 [95% CI, 0.12–0.74]; P = .009),
and 58% fewer ER+ invasive breast cancers (HR, 0.42 [95% CI,
0.25–0.71]; P = .001), but no difference in ER− invasive breast cancers
(HR, 0.78 [95% CI, 0.35–1.72]; P = .538).424 Anastrozole use was
associated with increased musculoskeletal events, hot flushes, vaginal
dryness, hypertension, and dry eyes, compared with placebo.424 No
difference was seen in the frequency of bone fractures, stroke, other
thrombotic events, or myocardial infarction.424
Primary End Point Reference
Ipsilateral/contralateral breast Margolese et al, 2003465
cancer incidence Richardson et al, 2007425
Margolese et al, 2016422
Ipsilateral/contralateral breast Cuzick, 2003426
cancer incidence Cuzick, 2008427
Forbes et al, 2016423
Invasive breast cancer Goss et al, 2011428
incidence
Invasive breast cancer Cuzick, 2003426
incidence Cuzick, 2008427
Cuzick et al, 2014424
362 Part I: Science and Clinical Oncology
Although the results of these phase III AI clinical trials demonstrate
that AIs are highly effective in preventing ER+ breast cancers in
postmenopausal women, the exact role of AIs in managing breast
cancer risk remains a debatable topic. It is clear that along with SERMs,
the AIs offer a promising strategy to prevent ER+ breast cancer.
However, the decision to use a SERM, an AI, or no drug therapy at
all depends on the physician’s experience and comfort with use of
these drugs for breast cancer prevention, the patient’s desire to use
drug therapy and willingness to accept risk of side effects, and ultimately
the risk-benefit ratio perceived by both the physician and woman at
risk of breast cancer.
Prevention of Estrogen Receptor–Negative Breast Cancer
SERMs and AIs are very effective at suppressing the development of
ER+ breast cancer. However, they do not prevent the 30% to 40%
of breast cancers that are ER−. Thus, a major unmet need is the
identification of drugs or strategies to prevent these particularly
aggressive breast cancers. A variety of drugs are being studied in preclini-
cal models and early clinical trials to determine whether they prevent
ER− breast cancer. These agents include drugs targeting the retinoic
acid X receptor, “rexinoids”; vitamin D; the active agent in green tea,
EGCG; metformin; and drugs targeting the HER2 oncogene, in
addition to drugs being tested for the prevention of “triple-negative”
breast cancers. Preclinical studies of bexarotene429–431 and UAB30432
have demonstrated that these RXR-selective retinoids (rexinoids) prevent
the development of ER− and “triple-negative” breast cancer in mouse
models. These studies supported the early-phase biomarker modulation
trials of these drugs in women with or at risk of breast cancer (Table
22.9). Vitamin D has also been extensively studied as a breast cancer
prevention agent. These studies were based on epidemiologic studies
suggesting that low vitamin D levels are associated with increased risk
of breast cancer.433–436 Multiple vitamin D trials tested the effect of
very high doses of vitamin D on mammographic density (as a surrogate
end point biomarker for breast cancer; see Table 22.9). These studies
have shown that high-dose vitamin D is tolerable and safe, but its
effect on mammographic density has not yet been reported. Another
extensively studied potential breast cancer prevention drug is the
antidiabetic drug metformin. The rationale for these studies came
from epidemiology studies that showed that diabetic individuals taking
metformin had a reduced incidence of breast cancer compared with
those taking insulin. Metformin activates AMP kinase, ultimately
reducing insulin production; in addition, metformin has been shown
to inhibit epithelial cell growth at high doses. Multiple early-phase
cancer prevention trials have been conducted that have demonstrated
the safety of this widely used drug (see Table 22.9). Given the extremely
good safety profile of metformin, a phase III adjuvant breast cancer
clinical trial was proposed and opened by the National Cancer Institute
of Canada to test metformin in women with early-stage breast cancer
after their initial therapy (NCT01101438). This multicenter trial will
assess the effect of metformin on disease-free survival (the primary
end point), as well as on overall survival and, important for cancer
prevention applications, contralateral breast cancer incidence. This
very important trial should clearly demonstrate the activity of metformin
with regard to breast cancer recurrence as well as breast cancer
development.
The natural product EGCG, one of the active ingredients in green
tea, has been studied both in preclinical and early clinical trials for
its ability to prevent breast cancer. The basis for studying green tea
and its active ingredient comes from epidemiologic studies of cancer
incidence in China. Several epidemiologic studies have shown that
green tea consumption is inversely associated with cancer incidence,
including breast cancer.437–439 These findings led to testing of EGCG
in preclinical models, producing results showing that EGCG suppresses
the development of mammary tumors in these animals.440–444 Early-phase
clinical trials have been performed to determine the maximum tolerated
dose (found to be 600 mg twice a day,445 equal to 8 to 10 cups of
green tea per day), with elevated liver enzymes and GI toxicity being
the limiting toxicities.445 A phase II clinical trial testing the ability of
green tea extract to modulate mammographic density has completed
accrual, but results have not yet been reported. Other studies have
tested the anti-HER2 drugs trastuzumab and lapatinib for the ability
to suppress the proliferation of HER2+ DCIS breast cancer cells (see
Table 22.9). These trials demonstrate that anti-HER2 drugs can affect
immune response and signaling pathways in HER2+ DCIS cells.
These studies suggest that ultimately it may be possible to prevent
HER2+ DCIS from progressing to invasive breast cancer.422
Other Strategies to Prevent Breast Cancer
Although traditional targeted drug therapy offers significant potential
to reduce breast cancer incidence, there is strong interest in developing
more effective and safer strategies for breast cancer prevention. To
this end, several investigators have developed vaccines for breast cancer
prevention. Peptide vaccines, pulsed dendritic cell vaccines, and
DNA-based vaccines have been developed and are now being tested
in clinical trials (Table 22.10). Disis and colleagues have studied peptide
vaccines against the HER2 oncoprotein. They have conducted several
studies with an HER2 peptide vaccine, showing that it is safe and
that an anti-HER2 immune response can be generated.453–456 Disis
has also tested a DNA-based anti-HER2 vaccine that also induces
anti-HER2 immunity.457–459 These vaccines are now being tested in
phase II cancer prevention trials. Two other groups have developed
anti-HER2 vaccines. Peoples and Mittendorf have developed several
peptide-based anti-HER2 vaccines, including nelipepimut-S, which
has been tested in phase I, II, and III trials for the treatment of breast
cancer (NCT02636582) and is now being tested in a phase II cancer
prevention trial in patients with DCIS (see Table 22.10). Czerniecki
developed and tested dendritic cell vaccines in which dendritic cells
were pulsed with HER2 peptides and injected either intralesionally
or intranodally. He showed that this dendritic cell vaccine is safe and
can induce anti-HER2 immunity. This anti-HER2 dendritic cell vaccine
is now being tested in a phase II DCIS cancer prevention clinical trial
(see Table 22.10).
Most of these efforts to develop breast cancer vaccines have focused
on targeting HER2. However, Disis has developed a complex DNA-
based vaccine composed of DNA fragments of three different antigens
present in triple-negative breast cancers (i.e., that are negative for ER,
progesterone receptor [PR], and HER2). This STEMVac vaccine is
now being tested in an ongoing phase I clinical trial and is highly
promising for the prevention of triple-negative breast cancer. The
results from these vaccine trials will demonstrate whether breast cancer
prevention using vaccines is feasible and effective. Ultimately, it is
likely that a combination of preventive therapy (behavioral, dietary,
drug, and possibly vaccine interventions) will be required to completely
prevent all forms of breast cancer.
Cervical Cancer
Interest in the association between cervical cancer and sexual contact was
initiated with an observation by Rigoni-Stern in 1842, who observed
that cervical cancer–related deaths were more common in women
who were married, widowed, or prostitutes but rare in virgins and
nuns.466 It was not until the 1970s that research was designed and
conducted to define a causal relationship between HPV infection
and the development of cervical cancer.467 Currently, 12 HPV subtypes
are recognized by the IARC as group 1 carcinogens, including HPV
types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59.468 Of these
high-risk HPV subtypes, cervical adenocarcinoma has been shown to be
caused at particularly high rates by HPV-16 (50.9%), HPV-18 (31.6%),
HPV-45 (11.6%),469 HPV-31 (12.6%), and HPV-33 (8.0%).470
Human Papillomavirus Vaccine
Three vaccines against HPV infection have been developed: a bivalent
vaccine for HPV-16 and HPV-18 (bHPV; Cervarix); a quadrivalent
vaccine for HPV types 6, 11, 16, and 18 (qHPV; Gardasil); and a
 Lifestyle and Cancer Prevention  •  CHAPTER 22 363

Table 22.9  Breast Cancer Prevention Studies Using Other Novel Agents
Phase Patient Population Study Design Results Reference
REXINOID
Bexarotene 87 high-risk women Bexarotene (200 mg/m2) or Reduction in cyclin D1 RNA Brown et al, 2008446
 II Postmenopausal placebo for 28 days (NCT00055991)
9cUAB30 40 women ≥18 yr of age with early- 9cUAB30 (orally daily for 14–28 Primary end point: changes in Ki67 Krontiras et al
  IB stage invasive breast cancer days before surgery) expression. (NCT02876640)
VITAMIN D
II 20 high-risk premenopausal women Vitamin D (20,000 or 30,000 IU/ Increased vitamin D levels and Crew et al, 2015447
≥21 yr of age with LCIS or DCIS wk) for 1 yr decreased IGF1/IGFBP3 ratio (NCT00976339)
II 20 high-risk postmenopausal women Vitamin D (20,000 or 30,000 IU/ Increased vitamin D levels and Crew et al, 2015447
≥21 yr of age wk) for 1 yr decreased IGF1/IGFBP3 ratio (NCT00859651)
IIB 200 premenopausal women 18–74 yr of Vitamin D (weekly and daily), Primary end point: change Crew et al
age at high risk for breast cancer or placebo (1/wk) and vitamin in mammographic density at (NCT01097278)
D (daily) for 1 yr 12 months
Results not reported
III 250 premenopausal women ≤55 yr of Vitamin D (2000 IU/wk) or Primary end point: change Wood et al
age with breast density ≥25% (scattered placebo for 1 yr in mammographic density at (NCT01224678)
fibroglandular densities or greater) 12 months
Results not yet reported
II 30 premenopausal women ≤55 yr of at Vitamin D (10,000 IU/wk) for Primary end point: change Fabian et al
increased risk for breast cancer 6 months in mammographic density at (NCT01166763)
6 months
Results not yet reported
METFORMIN
I 5 women ≥18 yr of age with operable Metformin (850 mg BID) for Primary end points: changes in Storniolo et al
stage I or II breast cancer 7–21 days Ki67 and caspase-3 expression (NCT00984490)
II 200 nondiabetic women with breast Metformin (850 mg BID) or No difference in Ki67, but different Bonanni et al, 2012448
cancer placebo for 28 days insulin resistance–based effects (in
luminal B tumors)
II 150 premenopausal women 21–54 yr Metformin (850 mg/day for 4 Primary end point: change in Chow et al
of age weeks, then 850 mg BID or mammographic density at 12 mo (NCT02028221)
BMI ≥25 kg/m2 placebo for 48 wk) Results not yet reported
III 3649 women 18–74 yr of age with Metformin (850 mg BID in Primary end points: invasive Goodwin et al
invasive breast cancer weeks 1–4, then BID or placebo disease-free survival (not yet (NCT01101438)
for ≤5 yr) reported)
I 40 women ≥21 yr of age with newly Metformin (1500 mg/day) plus Primary end points: changes in Kalinsky et al
diagnosed invasive breast cancer or atorvastatin (80 mg/day) for 2 Ki67 expression (NCT01980823)
DCIS weeks prior to surgery Results not yet reported
EGCG (ACTIVE AGENT IN GREEN TEA)
I 40 women with a history of stage I to III EGCG (200, 600, 800 mg BID) or The maximum tolerated dose for Crew et al, 2012445
ER− breast cancer placebo for 6 months Poly E (EGCG) is 600 mg, BID (NCT00516243)
IB 40 women 21–65 yr of age with prior EGCG (orally BID) or placebo The maximum tolerated dose for Crew et al, 2012445
hormone receptor–negative stage I–III for 6 months EGCG is 600 mg BID (NCT00516243)
breast cancer
II 1075 postmenopausal women with high EGCG (800 mg/day) or placebo Elevated liver enzymes (6.7%) Samavat et al, 2015449
risk of breast cancer due to dense breast for 12 mo Mammographic density not yet Dostal et al, 2015450
tissue with differing COMT genotypes reported (NCT00917735)
TRASTUZUMAB
Pilot 69 women with DCIS Single dose of trastuzumab Augmented antibody-dependent Kuerer et al, 2011451
(8 mg/kg) cell mediated cytotoxicity (NCT00496808)
LAPATINIB
II 20 women with HER2-positive DCIS Lapatinib (1500 mg) vs placebo Decreased expression of pHER2 Estévez et al, 2014452
for 4 weeks and pERK; no change in Ki67 or
p27 expression
II 40 women with EGFR-positive or Lapatinib (1000 mg) vs placebo Primary end points: changes in Brown et al
HER-positive DCIS for 6 weeks Ki67 expression, safety (NCT00555152)

BMI, Body mass index; COMT, catechol-O-methyltransferase; DCIS, ductal carcinoma in situ; EGCG, epigallocatechin gallate; ER, estrogen receptor; LCIS, lobular carcinoma
in situ.
364 Part I: Science and Clinical Oncology
Table 22.10  Breast Cancer Prevention Studies Using Vaccines
Phase Patient Population
I 22 patients with stage IV breast HER2/neu T-helper
cancer who are receiving
trastuzumab
II 56 patients with stage III/IV
breast cancer who completed
chemotherapy, are receiving
trastuzumab, and completed
HER2 ICD plasmid-based
vaccine trial
I/II 23 patients ≥18 yr of age with
HER2-positive stage IV breast
cancer
I 30 HER2-negative stage III–IV
breast cancer patients
I/II 195 high-risk node-negative
patients with HER2-positive
breast cancer
II 300 disease-free node-positive
and ER−/PR− node-negative
HER2 1+/2+ breast cancer
patients
II 108 high-risk women ≥18 yr of
age with DCIS
II 298 disease-free node-positive
and high-risk node-negative
breast cancer patients
II 180 HLA-A2+, disease-free
node-positive and high-risk
node-negative breast cancer
patients with HER2 (IHC1+/3+)-
expressing tumors (estimated
enrollment: 600)
IB 17 disease-free, HLA-A2+/A3+,
HER2-positive breast cancer
patients
I 30 women ≥18 yr of age with
DCIS
I/II 58 women ≥18 yr of age with
HER-2/neu-positive DCIS
I/II 58 women ≥18 yr of age with
HER-2/3-positive DCIS
DCIS, Ductal carcinoma in situ; DFS, disease-free survival; ER, estrogen receptor; FISH, fluorescence in situ hybridization; GM-CSF, granulocyte-macrophage colony-stimulating
factor; HER2, human epidermal growth factor receptor 2; HLA, human leukocyte antigen; IBC, invasive breast cancer; ICD, intracellular domain; IHC, immunohistochemistry; lum,
luminal; pCR, pathologic complete response; PR, progesterone receptor; TNBC, triple-negative breast cancer.
Study Design Results Reference
Minimal toxicity; prolonged, robust, antigen-specific Disis et al, 2009460
peptide-based vaccine immune response (median follow-up of 36 mo from (NCT00194714)
first vaccine)
Intradermal HER2 ICD Primary end point: determine generation of Salazar et al
protein mixture or sterile immunologic memory to the HER2 ICD protein (NCT00363012)
water 6 months after
vaccination with pNGVL3-
hlCD vaccine
HER-2/Neu peptide vaccine Primary end points: toxicity of infusing HER2-specific Disis et al
admixed with GM-CSF plus T cells (NCT00791037)
cyclophosphamide and ex Results not yet reported
vivo expanded HER2-
specific T cells
CD105/Yb-1/SOX2/CDH3/ Primary end points: immunology efficacy (increased Disis et al
MDM2-polyepitope plasmid Th1 cell immunity) and incidence of toxicity per (NCT02157051)
DNA vaccine (“STEMVac”) cancer therapy evaluation
Results not yet reported
HER2 peptide DFS was 94.6% in optimally dosed, 87.1% in Mittendorf et al,
(nelipepimut-S) (E75) suboptimally dosed, versus 80.2% in control patients; 2014461
vaccine with booster vaccine is safe (NCT00841399 and
inoculations versus no NCT00584789)
vaccination
Herceptin (6 mg/kg q3wk Primary end point: DFS at 24 and 36 months Peoples et al
for 1 yr) + NeuVax (E75) (NCT01570036)
vaccine (6 vaccinations, 1
every 3 weeks)
HER2 peptide Primary end point: immune response to the HER2 Mittendorf et al
(nelipepimut-S) plus peptide (NCT02636582)
GM-CSF vaccine, or GM-CSF Results not yet reported
alone (before surgery)
HER2 peptide AE37 vaccine Minimal toxicities; estimated 5-yr DFS in patients Mittendorf et al,
plus GM-CSF vs GM-CSF with IHC 1+/2+ HER2-positive tumors was 77.2% with 2016462
alone vaccine vs 65.7% with control subjects; estimated (NCT00524277)
5-yr DFS in patients with TNBC was 77.7% with
vaccine vs 49.0% with control subjects
HER2 peptide GP2 vaccine Interim report: minimal toxicities; estimated 5-yr DFS Mittendorf et al,
plus GM-CSF versus in IHC3+/FISH+ patients was 94% in vaccinated 2016463
GM-CSF alone patients vs 89% in control patients in the intent-to- (NCT02636582)
treat analysis, and was 100% in vaccinated patients
vs 89% in control patients in the per-treatment
analysis
GP2 vaccine + GM-CSF plus No dose-limiting or grade 3–5 local or systemic Clifton et al, 2017464
standard-of-care toxicities; appropriate dose was 1000 µg of GP2 + (NCT03014076)
trastuzumab 250 µg of GM-CSF
HER-2/Neu pulsed dendritic HER-2/neu was well-tolerated; vaccine induced Czerniecki et al659
cell vaccine by intranodal, decline and/or eradication of HER-2/neu expression; (NCT00107211)
intralesional, or both, no DCIS remained in 40% ER− and 5.9% ER+ patients;
vaccination phenotypes shifted after vaccination: 43.8% ER+
HER2+ lumB to ER+ HER2− lumA and 50% ER− HER2+
to ER− HER2−; depressed Th1 response restored with
HER2-directed Th1 immune interventions
HER-2/Neu pulsed dendritic Vaccination was well tolerated; vaccination route did Czerniecki et al
cell vaccine by intranodal, not affect immune response rate; pCR rate was 28.6% (NCT02061332)
intralesional, or both, in DCIS vs 8.3% in IBC patients; pCR and non-pCR
vaccination DCIS patients had similar peripheral blood anti-HER2
immune responses; all pCR patients had anti-HER2
CD4 immune response in sentinel lymph node
HER-2/Neu pulsed dendritic Primary end point: related adverse events (results not Czerniecki et al
cell vaccine plus yet reported). (NCT02336984)
trastuzumab and
pertuzumab

nonavalent vaccine for HPV types 6, 11, 16, 18, 31, 33, 45, 52, and
58 (9vHPV; Gardasil 9), which was developed from the quadrivalent
vaccine. Although many countries have licensed Cervarix and the
quadrivalent Gardasil, Gardasil 9 has only recently been licensed in
some countries. These HPV vaccines provide maximal benefit when
administered in HPV-uninfected individuals. Evidence of the effective-
ness of these HPV vaccines in both preventing HPV infection and
decreasing incidence of several cancers, including anal, cervical,
oropharyngeal, penile, vaginal, and vulvar cancers, and in decreasing
the risk of genital warts and cervical intraepithelial neoplasia (CIN)
grades 2 and 3 precursor lesions has been provided by several studies.467
However, the effectiveness and immunogenicity of each vaccine vary
based on its unique vaccine composition and which high-risk HPV
subtypes are included.
bHPV (Cervarix)
Cervarix, the HP16/18-AS04-adjuvanted vaccine developed by
GlaxoSmithKline, received FDA approval in 2009 for the prevention
of HPV-16 and HPV-18 infection–related cervical cancer, cervical
adenocarcinoma in situ, and CIN in girls and women 9 to 25 years
of age.471 Cervarix contains 20 µg of HPV-16 capsid protein and
20 µg of HPV-18 capsid protein.467 Numerous randomized phase
II/III trials testing the HPV-16/18 vaccine have been conducted, all
of which involved administration of three doses to female subjects
15 years of age or older at 0, 1, and 6 months. These trials included
(1) the multinational (Brazil, Canada, and the United States) Study
HPV-001 (NCT00689741)472 and two extensions of this study;
(2) Study HPV-007 (NCT00120848)473,474; (3) Study HPV-023
(NCT00518336)475–477; (4) the multinational (14 countries throughout
Asia, Australia, Europe, and North and South America) Study HPV-008,
the Papilloma Trial Against Cancer in Young Adults (PATRICIA;
NCT00122681)478–483; (5) Study HPV-009, the Costa Rica HPV
Vaccine Trial (CVT; NCT00128661)484–491; (6) Study HPV-032 of
Japanese women (NCT00316693)492,493; (7) Study HPV-063, an
extension of Study HPV-032 (NCT00929526)494; (8) Study HPV-039
of Chinese women, currently ongoing (NCT00779766)495; and (9)
Study HPV-015, the multinational (12 countries throughout Australia,
Europe, North and South America, and Southeast Asia) Human Papil-
lomavirus: Vaccine Immunogenicity and Efficacy trial (VIVIANE;
NCT00294047).496,497 Prior HPV infection for the participants in
these trials is unknown, and aluminum or hepatitis A vaccine was
used in the control arm.
Collectively, these studies have demonstrated that in women with
no baseline evidence of HPV-16/18 infection, the final efficacy of the
HPV-16/18 vaccine in reducing (6 or 12 month) persistent HPV-16/18
infection is 90.9% to 100% in women 15 to 25 years of age, and
that the interim efficacy for preventing 6-month persistent HPV-16/18
infection is 77.4% to 93.7% in women older than 25 years (the
ongoing VIVIANE study).497 The prevention of cytologic abnormalities
associated with HPV-16/18 infection ranged from 80.6% to 97.1%,
and the prevention of CIN ranged from 80.0% to 100% in girls and
women 15 to 25 years of age and 75.5% to 100% in women older
than 25 years. The HPV-16/18 vaccine proved to be cross-protective
at varying levels against infection with other HPV subtypes, including
HPV types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 and against
CIN associated with these subtypes. Preventive efficacy against the
HPV subtypes that account for most genital warts was reported to
be moderate for HPV-6 and HPV-11 (34.5%) and HPV-74 (49.5%).483
In addition, the HPV-16/18 vaccine was effective at preventing oral
(93.3% [95% CI, 62.5–99.7]),491 anal (83.6% for women HPV-16/18
negative at baseline who received three doses [95% CI, 66.7–92.8]),490
and vulvar (54.1% [95% CI, 4.9–79.1])489 HPV-16/18 infection.
These preventive effects after administration of the HPV-16/18 vaccine
have been demonstrated in all women, independent of age, geographic
location, or sexual experience.497
Kreimer and colleagues conducted a combined analysis of the
PATRICIA and CVT trials to compare the effectiveness of one, two,
Lifestyle and Cancer Prevention  •  CHAPTER 22 365
and three doses of the HPV-16/18 vaccine.498 Their findings show a
77.0% vaccine efficacy against HPV-16/18 with three doses (95%
CI, 74.7–79.1), 76.0% with two doses (95% CI, 62.0–85.3), and
85.7% with a single dose (95% CI, 70.7–93.7), suggesting that a
similar level of protection is obtained against cervical HPV-16/18
infections with one and two doses of the vaccine. However, cross-
protective efficacy of the vaccine against incident HPV-31, HPV-33,
and HPV-45 infections showed a high level of dose-dependence: 59.7%
with three doses (95% CI, 56.0–63.0), 37.7% with two doses (95%
CI, 12.4–55.9), and 36.6% with one dose (95% CI, −5.4 to 62.2).
In addition, analysis of the CVT trial shows that increased protection
was obtained in women who received two doses when the second
dose was given at 6 months (82.6% [95% CI, 42.3-96.1]) rather than
1 month (75.3% [95% CI, 54.2–87.5]).
qHPV (Gardasil)
Gardasil, developed by Merck, received FDA approval in 2006 and
was the first HPV vaccine to be licensed for the prevention of (1)
vulvar and vaginal cancer; (2) HPV-6, HPV-11, HPV-16, and HPV-18
infection–related cervical cancer, cervical adenocarcinoma in situ, and
cervical, vaginal, and vulvar intraepithelial neoplasia; and (3) HPV-6
and HPV-11 infection–related genital warts.499 This approval is for
girls and women 9 to 26 years of age, and applies to boys and men
9 to 26 years of age only for aforementioned prevention of anal cancer,
anal intraepithelial neoplasia, and genital warts. Gardasil contains
20 µg of both HPV-6 and HPV-18 capsid protein and 40 µg of both
HPV-11 and HPV-16 capsid protein, and is the most widely admin-
istered HPV vaccine around the world.467,500 The initial phase I and
II trials on Gardasil were conducted by Merck501–503 and have been
followed by numerous subsequent phase II/III trials, including the
phase III Females United to Unilaterally Reduce Endo/Ectocervical
Disease (FUTURE) I and II trials, a phase III study by Olsson,504
and postlicensure studies by Brotherton,505 Crowe,506 Kahn,507
Herweijer,508 Giuliano,509 Palefsky,510 and Kash and colleagues.511
Both the phase II and the FUTURE phase III studies mentioned
previously have reported increased rates of localized adverse events at
the site of injection (3% and 10%, respectively), but no increase in
serious adverse events, in patients receiving the quadrivalent vaccine
versus those receiving placebo.512–514 In addition, the phase II studies,
which enrolled 2409 and 277 patients, reported a 90% decrease after
3 years of follow-up in HPV-6, HPV-11, HPV-16, and HPV-18
infection in patients receiving the HPV vaccine versus placebo,502
and a 96% decrease in persistent HPV-6, HPV-11, HPV-16, and
HPV-18 infection with 5 years of follow-up.503 The 5-year follow-up
figures also showed that the HPV vaccine conferred 100% preventive
efficacy for precancerous cervical dysplasia and genital warts. These
two studies (Protocols 005 and 007) tested slightly different dosage
regimens. Protocol 005 administered vaccinations at months 0, 1,
and 6 using 0.5 mL of qHPV vaccine (20 µg of HPV-6, 40 µg of
HPV-11, 40 µg of HPV-16, and 20 µg of HPV-18) or one of two
placebos (225 µg or 450 µg of aluminum adjuvant). Protocol 007
administered vaccinations at months 0, 2, and 6 using low (identical
to the Protocol 005 vaccine: 20 µg each of HPV-6 and HPV-18, and
40 µg each of HPV-11 and HPV-16), medium (40 µg each of HPV-6,
HPV-11, HPV-16, HPV-18), and high (80 µg each of HPV-6, HPV-11,
and HPV-18, and 40 µg of HPV-16) doses of the vaccine versus
one of two placebos. Whereas Protocol 007 demonstrated increased
participants with adverse events in the high-dose group, no serious
adverse events relating to the vaccine were reported.502,512 Although
toxicity did not exclude any of the vaccine doses from moving forward
to phase III testing, the comparable efficacy observed in patients
treated with the lower and higher doses resulted in selection of the
low dose for further development.511
The phase III FUTURE I trial (n = 5455; conducted in Asia,
Australia, Europe, and Latin, North, and South America) and FUTURE
II trial (n = 12,167; conducted in Asia, Europe, and North and South
America) enrolled girls and women 16 to 23 years of age for a three-dose
366 Part I: Science and Clinical Oncology
regimen of vaccine or placebo. These studies demonstrated 100%
efficacy of the vaccine in preventing HPV-16/18 infection–related
CIN grades 2 and 3, adenocarcinoma in situ, and vulvar and vaginal
intraepithelial neoplasia grade 1.515,516 As seen with the bivalent vaccine,
the qHPV vaccine was also shown to be cross-protective by conferring
30%, 48%, and 75% vaccine efficacy against any lesion for cervical,
vaginal, and vulvar intraepithelial neoplasia grade 1, respectively, as
well as 83% (condyloma) preventive efficacy regardless of HPV subtype.
Olsson and colleagues tested three doses of the qHPV vaccine or
placebo in 552 girls and women 16 to 23 years of age, with 3 years
of follow-up.504 In addition, 241 of the subjects were included in an
additional 2 years of follow-up (totaling 5 years of follow-up). This
study did not show increased adverse events in the vaccine and placebo
arms. In addition, a high level of efficacy and a maintenance of anti-
HPV levels were observed for at least 5 years, as was the induction
of a strong immune memory, suggesting long-term efficacy. Additional
post–FDA approval studies have further supported the data provided
in these studies, some of which are still ongoing.
9vHPV (Gardasil 9)
Gardasil 9, developed by Merck through modification of the quadri-
valent Gardasil vaccine, received FDA approval in 2014 for the preven-
tion of (1) HPV type 16, 18, 31, 33, 45, 52, and 58 infection–related
cervical, vulvar, vaginal, and anal cancers; (2) HPV type 6, 11, 16,
18, 31, 33, 45, 52, and 58 infection–related cervical, vaginal, vulvar,
and anal intraepithelial neoplasia and cervical adenocarcinoma in situ;
and (3) HPV-6 and HPV-11 infection–related genital warts.517 This
approval is for girls and women 9 to 26 years of age, and applies to
boys and men 9 to 26 years of age only for the previously described
prevention of anal cancer, anal intraepithelial neoplasia, and genital
warts. Gardasil 9 contains 30 µg of HPV-6 capsid protein, 40 µg of
both HPV-11 and HPV-18 capsid protein, 60 µg of HPV-16 capsid
protein, and 20 µg each of HPV-31, HPV-33, HPV-45, HPV-52,
and HPV-58 capsid protein.467
Eight clinical studies have published results regarding the efficacy,
immunogenicity, and toxicity of Gardasil 9.513,518–525 Collectively, these
studies demonstrate that Gardasil 9 induces a strong immune response
and is associated with an almost 100% seroconversion rate after the
third dose.467 Maximal benefit in terms of immune response for the
five high-risk HPV subtypes uniquely in Gardasil 9 (HPV types 31,
33, 45, 52, and 58) appears to be in naïve populations rather than
individuals previously vaccinated with the quadrivalent Gardasil;
conversely, lower immune response for all four HPV subtypes in the
quadrivalent vaccine (HPV-6 and HPV-11 and high-risk HPV-16
and HPV-18) has been higher in individuals previously vaccinated
with the quadrivalent vaccine.467,513,519 Toxicity is minimal, but inci-
dences of local skin reactions have been higher than those associated
with the quadrivalent Gardasil vaccine.519,523 Skin reactions and
headaches have comprised the majority of reported adverse events.
Nonetheless, although the HPV vaccinations are being integrated into
the national health programs of more and more countries around the
world, Japan withdrew its recommendation for the HPV vaccine
following a fearful national response to highly publicized cases of
adverse events (complex regional pain syndrome).500,526 In addition,
four cases of premature ovarian failure have been reported to the
CDC; these may have been due to an HPV vaccine–triggered auto-
immune or inflammatory response.500,527
Signorelli and colleagues conducted a systematic review of the 16
RCTs on 9vHPV listed on RCT registries, eight of which they included
in their analysis.528 In addition to showing a noninferior immune
response following administration of the 9vHPV vaccine compared
with the quadrivalent HPV vaccine for the four HPV subtypes in
both vaccines (HPV-6, HPV-11, HPV-16, and HPV-18), they found
that the efficacy of the 9vHPV vaccine in preventing high-grade cervical,
vulvar, or vaginal diseases associated with the five additional high-risk
HPV subtypes was 96.7% for girls and women 16 to 26 years of
age (95% CI, 80.9–99.8).528 This analysis also demonstrated an
immunobridging efficacy for boys and men 9 to 26 years of age and
girls and women 9 to 15 years of age.
The potential impact of the Gardasil 9 vaccine on increasing the
prevention of HPV infection–related cervical cancer beyond the
quadrivalent Gardasil and Cervarix vaccines varies globally but is
estimated to be 8% higher in Australia (preventing 86.5% of cases),
13% in North America and Europe (preventing 92% and 90.9% of
cases, respectively), and 18.4% worldwide (preventing 89.9% of
cases).467,529 Thus, whereas the bivalent and quadrivalent vaccines both
confer protection against approximately 70% of the cancerous and
precancerous cervical conditions associated with HPV infection, the
9vHPV vaccine increases this to roughly 90%.530 However, it is possible
that broad-scale vaccination may result in a shift in prevalence of the
HPV genotypes500; in fact, one study has reported increased prevalence
of HPV subtypes 51, 59, 73, 84, and 89 in school-aged girls vaccinated
with the quadrivalent vaccine through the Australian national vaccina-
tion program versus unvaccinated girls.531,532
Future results of epidemiologic data, including the long-term
follow-up results of the studies highlighted earlier, will be critical in
better defining the prevalence of HPV infection and the diseases
associated with HPV infection, and the effective duration of prevention
associated with all three HPV vaccines.497 In addition, the global level
of vaccine uptake, the affordability (these vaccines currently cost
approximately $120 per dose, with three doses recommended500),
global availability, and coverage will all play significant roles in the
ultimate impact of these vaccines on HPV-related diseases and out-
comes.528 However, with the prevalence of HPV infection worldwide,
although educated modifications in sexual behavior could significantly
affect the risk of HPV infection, immunization with the HPV vaccine,
HPV testing, and cervical cancer screening with the Pap test are likely
to remain critical components for the continued prevention of cervical
cancer. Furthermore, despite HPV’s status as the leading sexually
transmitted disease in the United States, only 40% of girls 13 to 17
years of age in the United States completed the three-dose HPV series
in 2014, which was a slight increase from the 37% who completed
the series in 2013.533
The Advisory Committee on Immunization Practices (ACIP)
updated its recommendations for HPV vaccination in December 2016
to include the nonavalent vaccine.534 The current recommendations
are to vaccinate with two doses (0 and 6–12 months) at age 11 to
12, and vaccination can be started at age 9. The ACIP recommends
that girls and women through age 26 and boys and men through age
21, as well as some high-risk boys and men through age 26, be vac-
cinated if they did not previously undergo adequate vaccination (defined
as initiating vaccination [1] before their 15th birthday and receiving
the two-dose, at 0 and 6–12 months, or three-dose, at 0, 1–2, and 6
months, regimens; or [2] on or after their 15th birthday and receiving
three doses at 0, 1–2, and 6 months). The three-dose schedule is
recommended for individuals who are immunocompromised. The
ACIP does not have a recommendation regarding additional vaccination
with the nonavalent HPV vaccine for individuals previously adequately
vaccinated with the bivalent or quadrivalent vaccines. The ACIP also
does not recommend restarting interrupted vaccination schedules.
Prostate Cancer
Both epidemiologic and experimental data have supported the develop-
ment of a broad range of dietary supplements and drug-based interven-
tions for prostate cancer prevention. Although some of these have
now been shown to be associated with increased risk of prostate cancer
(e.g., vitamin D),535 numerous clinical trials testing these agents as
cancer preventive interventions, including studies of calcium, selenium,
vitamin E, and 5α-reductase inhibitors, have been conducted.
5α-Reductase Inhibitors
The conversion of testosterone into 5-dihydrotestosterone, which has
stronger androgenic activity, requires the enzyme 5α-reductase.

Table 22.11  Prostate Cancer Large Scale (Phase III) Chemoprevention Studies With Outcomes
Trial Patients Agent Dose Relative Risk (RR) Absolute Risk (AR) AR: High Grade Reference
PCPT 18,882 Finasteride 5 mg/day 25% decrease
REDUCE 8231 Dutasteride 0.5 mg/day 23% decrease
SELECT 35,533 Selenium 200 µg/day 9% increase
Vitamin E 400 IU/day 17% increase
Selenium + 200 µg/day 5% increase
vitamin E 400 IU/day
400 IU/day
Antiandrogenic agents that block this conversion by inhibiting
5α-reductase, including finasteride and dutasteride, have been the
focus of several large-scale phase III trials (Table 22.11).
Prostate Cancer Prevention Trial (PCPT)
The first large phase III trial for prostate prevention was the PCPT,
which tested 7 years of treatment with finasteride (5 mg/day) versus
placebo in 18,882 men 55 years of age or older with normal prostate-
specific antigen (PSA) levels (≤3.0 ng/mL) and digital rectal examination
findings.536 Incidence of prostate cancer was reduced by 24.8% in
patients treated with finasteride compared with placebo. However,
finasteride was also associated with a 0.6% increase in high-grade
prostate cancer (defined as a Gleason score of 8–10). This increased
risk of high-grade prostate cancer appears to be due to an increase in
detection after finasteride-mediated reduction in prostate size.
These results were further clarified by the 18-year follow-up figures,
which demonstrated a 30% decrease in risk of prostate cancer in
patients treated with finasteride versus placebo (RR, 1.17 [95% CI,
0.65–0.76]; P < .001).537 Increased detection of high-grade prostate
cancer (defined as a Gleason score of 7–10) was also seen with the
extended follow-up (RR, 1.17 [95% CI, 1.00–1.37]; P = .05). Neither
overall survival nor survival after diagnosis of prostate cancer was
significantly different between treatment arms.
Reduction by Dutasteride of Prostate Cancer Events
(REDUCE) trial
The REDUCE trial compared the efficacy of 4 years of treatment
with dutasteride (0.5 mg/day) versus placebo for the prevention
of prostate cancer in 6729 men 50 to 75 years of age with a PSA
of 2.5 to 10.0 ng/mL and a negative prostate biopsy finding.538 A
22.8% reduction in risk of prostate cancer was demonstrated with
dutasteride compared with placebo (95% CI, 15.2–29.8; P < .001).
Dutasteride was also shown to reduce the rate of both acute urinary
retention (by 77.3%) and adverse events related to benign prostatic
hyperplasia. However, as with finasteride, increased detection of high-
grade prostate cancer (Gleason score of 8–10) was associated with
dutasteride in years 3 and 4 of treatment (likely because of a shrinkage
of the prostate gland, facilitating the detection of these high-grade
prostate cancers).
A follow-up study examined incidence of newly diagnosed prostate
cancers in 2715 of the men included in the REDUCE trial during
the 2 years following the 4-year trial described earlier.539 The results
from this study identified no additional high-grade cancers and
demonstrated a similar low rate of new prostate cancer diagnoses
between the two treatment arms.
Another subset of 1617 asymptomatic men with enlarged prostates
in the REDUCE study were included in a post hoc analysis that
investigated the effects of dutasteride on risk of clinical progression
of benign prostatic hyperplasia.540 This study demonstrated a 41%
reduction in RR and a 15% reduction in absolute risk. Furthermore,
a multivariable regression analysis adjusted for covariates demonstrated
a significant reduction in clinical progression of benign prostatic
Lifestyle and Cancer Prevention  •  CHAPTER 22 367
PROSTATE CANCER
6% decrease 0.6% increase Thompson et al 2003536
5% decrease ≤0.5 % increase Andriole et al 2010538
0.8% increase No known effect Klein et al 2011173
1.6% increase No known effect
0.4% increase No known effect
hyperplasia in the dutasteride versus the placebo treatment groups
(OR, 0.47 [95% CI, 0.37–0.59]; P < .001).
Ultimately, the findings from both the PCPT and REDUCE trials,
demonstrating that both finasteride and dutasteride reduce incidence
of low-grade prostate cancer but did not reduce the incidence of
high-grade prostate cancer, prevented the FDA approval of these
5α-reductase inhibitors as preventive agents for prostate cancer.541
Selenium and Vitamin E
Based on positive results of epidemiologic studies and secondary analyses
of randomized trials demonstrating decreased risk of prostate cancer
risk with selenium and vitamin E, SELECT was developed and
conducted.172 A total of 35,533 men aged 50 years (black) or 55 years
(all others) or older with a PSA of 4.0 ng/mL or lower and normal
rectal examination findings in Canada, Puerto Rico, and the United
States were randomized to receive treatment with selenium (200 µg/
day of l-selenomethionine) and/or vitamin E (400 IU/day of all-rac-
α-tocopherol acetate) or placebo with a follow-up of 7 to 12 years.
Interim results after a median of 5.5 years of follow-up revealed increased
risk of prostate cancer in patients treated with selenium (HR, 1.04
[95% CI, 0.87–1.24]), vitamin E (HR, 1.13 [95% CI, 0.95–1.35]),
and selenium plus vitamin E (HR, 1.05 [95% CI, 0.88–1.25]). These
data suggested no preventive efficacy of selenium and/or vitamin E
supplementation for prevention of prostate cancer.
Results after a total of 7 years of follow-up indicated a slightly
increased and statistically significant risk compared with the initial
data, revealing that vitamin E (HR, 1.17 [95% CI, 1.004–1.36]; P
= .008]) increased risk of prostate cancer versus placebo.173 These
findings demonstrate an absolute increased risk of prostate cancer in
healthy men of 1.6 per 1000 person-years for vitamin E.
As with the PCPT, an analysis of a subset of 4934 SELECT
participants demonstrated an association between vitamin D and
prostate cancer risk.542 This study identified increased risk of prostate
cancer with both low and high levels of vitamin D but did show
reduced risk of high-grade prostate cancer (Gleason score of 7–10)
in African American men with higher vitamin D concentrations.
Ultimately, the results of the SELECT trial suggest that neither
vitamin E nor selenium will reduce the risk of prostate cancer and
that exceeding the recommended dietary allowance (RDA) of either
of these supplements could, in fact, increase risk in men older than
55 years.543
Aspirin
Several studies are currently testing aspirin alone or in combination
treatment for the prevention of prostate cancer recurrence. Powles and
Cuzick of Queen Mary University of London and their colleagues
are conducting a multiinstitutional phase II/III study in the United
Kingdom testing aspirin (100 or 300 mg/day) with or without vitamin
D (4,000 IU/day; Vigantol Oil) or placebo for the prevention of
progression of prostate cancer, with prevention of new lesions as one
of the secondary end points (NCT03103152). This study, which
should be complete in October 2019, is currently recruiting, with
368 Part I: Science and Clinical Oncology
a planned accrual of 104 men 16 to 100 years of age with prostate
cancer and an estimated life expectancy of over 3 years. Lu at West
China Hospital is currently recruiting participants for a phase IV
study of aspirin (100 mg QD), the antibiotic levofloxacin (0.5 g QD),
both, or neither for the prevention of prostate cancer occurrence
or disease progression (NCT02757365). A total of 100 men aged
50 to 70 years with benign prostate hyperplasia with lymphocyte
infiltration are to be recruited, and the study should be completed
in April 2019. Finally, the large-scale phase III Add-Aspirin clinical
trial under the direction of Langley at University College, London,
is investigating the efficacy of aspirin in preventing recurrence and
improving survival in individuals with nonmetastatic breast, prostate,
colorectal, or gastroesophageal cancer after standard curative therapy
(NCT2804815). Accrual is currently underway, with a goal of
11,000 participants, who will be randomized in cancer type–specific
parallel cohorts to receive aspirin (100 or 300 mg/day) or placebo
for 5 years. This study is not scheduled to be completed until
October 2026.
A pooled analysis of the National Institutes of Health (NIH)–AARP
Diet and Health Study (composed of 566,398 US AARP members
aged 50–71 years) and the Prostate, Lung, Colorectal and Ovarian
(PLCO) Cancer Screening Trial (composed of 76,685 and 78,216 US
men and women, respectively, aged 55–74 years) examined whether
aspirin and other NSAIDs increase survival in men diagnosed with
prostate cancer.544 Included in this analysis were 221,189 participants
of the NIH-AARP study who responded to a Risk Factor Question-
naire and the male PLCO participants. Although prediagnosis and
postdiagnosis use of aspirin and nonaspirin NSAIDs did not affect
prostate cancer–specific mortality, participants with 5 or more years
of occasional (defined as less than daily) use of aspirin before diag-
nosis of prostate cancer demonstrated an 18% reduction in all-cause
mortality (HR, 0.82 [95% CI, 0.75–0.90]), whereas daily aspirin
users for the 5 years before diagnosis demonstrated a 15% reduction
in all-cause mortality (HR, 0.85 [95% CI, 0.66–0.86]) compared
with participants who did not use aspirin during that timeframe.
Thus, although aspirin did not improve prostate cancer–specific
mortality, long-term or daily or occasional use of aspirin may
improve overall survival by preventing comorbidity in this patient
population.
Other Agents
A cohort study of a subset of 9457 PCPT participants with 7 years
of follow-up showed that statin use is not associated with risk of
total, low-grade, or high-grade prostate cancer.545 However, in a
nested case-control analysis of 3377 PCPT participants, higher
serum 25-hydroxyvitamin D (25[OH]D) levels were associated with
a modest increase in risk of Gleason 2 to 6 disease, but a substantial
reduction in risk of Gleason 8 to 10 prostate cancer, particularly in
men 65 years of age or older.546 Preclinical studies of lower doses
of some agents (e.g., vitamin D3 in combination form with the soy
isoflavone genistein) have been shown to be associated with inhibi-
tion of prostate cancer cells,547 although different expression levels
and polymorphisms in the vitamin D receptor may affect preventive
efficacy.548 The most recent results of the PCPT (2017) further assessed
the association between vitamin D and prostate cancer risk previously
observed by Miles and colleagues in 2014.549 This nested case-control
study of 3387 participants from the PCPT found that above-median
serum 25(OH)D levels in men with higher circulating IGF-2 levels
were associated with increased risk of prostate cancer (OR, 1.33
[95% CI, 1.00–1.65]).
Bladder Cancer
Non–muscle-invasive bladder cancer (NMIBC) accounts for 75% to
85% of all bladder cancer diagnoses at presentation and is confined
to the mucosa or submucosa.550 Bacillus Calmette-Guérin (BCG) was
initially developed as an adjuvant treatment for patients with preinvasive
lesions, and valrubicin has been shown to be effective in patients with
bladder carcinoma in situ that is refractory to BCG.551
Bacillus Calmette-Guérin
Since 1977, post–transurethral resection (TUR) intravesicular treatment
with BCG has been the standard of care for individuals at high risk
for NMIBC. Pinsky and colleagues showed that treatment with standard
therapy (cystoscopy with fulguration) plus 6 weeks of BCG treatment
significantly delayed disease progression compared with standard therapy
alone in high-risk patients (P < .003).552 Long-term follow-up confirmed
these findings by demonstrating disease progression in 42% fewer
BCG-treated patients than control patients.553 The most recent results
showed 62% and 37% progression-free rates in the BCG plus TUR
arm and the control arm (TUR), respectively, and 75% and 55%
10-year disease-specific survival rates between the experimental and
control arms, respectively (P = .03).554
Lamm and colleagues conducted a Southwest Oncology Group
(SWOG) study of intravesical and percutaneous BCG immunotherapy
of BCG induction alone or followed by BCG maintenance for 3
weeks administered at months 3, 6, 12, 18, 24, and 30.555 They found
that BCG maintenance therapy significantly increased the efficacy
(twofold) of BCG induction therapy alone for recurrence-free survival
(log rank P < .0001). Their results also identified significantly longer
worsening-free survival within the patients receiving BCG maintenance.
Based on these findings, a regimen of 3 years of BCG maintenance
was established.
After these studies, the most effective regimen of inductive and
maintenance BCG therapy has been controversial and the focus of a
number of clinical trials.556 A variety of doses (e.g., one-third and full
dose) of intravesical inductive and maintenance BCG therapy regimens
have been investigated for inductive therapy alone or followed by
maintenance therapy for timeframes ranging from 1 to 3 years.
Because of the toxicity-based issues resulting in interruptions
and premature discontinuation of BCG maintenance therapy, the
European Organisation for Research and Treatment of Cancer (EORTC)
conducted a four-arm study of one-third-dose versus full-dose BCG
maintenance therapy for 1 and 3 years.557 A total of 1355 patients
with intermediate- and high-risk NMIBC were randomized after
TUR. Suboptimal efficacy was observed with one-third-dose versus
full-dose treatment for 1 year (HR, 0.75 [95% CI, 0.59–0.94]; P =
.01). There was no additional benefit for intermediate-risk patients
to receive more than 1 year of BCG maintenance therapy. However,
3 years of full-dose (but not one-third-dose) BCG maintenance
therapy did reduce recurrence in high-risk patients (HR, 1.61 [95%
CI, 1.13–2.30]; P = .009). No difference was observed in tumor
recurrence or death in high-risk patients treated for 1 compared
with 3 years.
The French Urological Association Cancer Committee also con-
ducted a phase III multicenter study, titled URO BCG 4, that random-
ized 146 patients with intermediate- or high-risk NMIBC from nine
hospitals, in order to investigate the effects of lower dose and fewer
instillations of BCG maintenance for the treatment for NMIBC.558
Patients were randomized to receive one-third dose of BCG for 3
consecutive weeks every 3 (group 1) or 6 (group 2) months. Tumor
recurrence, muscle invasion, and side effects after 6, 12, and 18 months
of treatment were not significantly different between the two treatment
groups. Updated figures after 3 years of BCG maintenance therapy
were published in 2017 and revealed 18% tumor recurrence in group
1 and 10% recurrence in group 2 (P = .241). Rates of adverse events
were slightly lower in group 1 than in group 2 (P = .037), with group
2 accounting for over three times as many major adverse events, which
suggests that lower toxicity is associated with the 6-month instillation
schedule.559 However, the most recent meta-analysis of nine of these
studies reported that extended BCG maintenance therapy (e.g., 3
years) is not more effective than shorter-term therapy (e.g., 1 year)
in decreasing recurrence, but that 3 years of therapy does not increase
the incidence of side effects.560

Over the last 20 years, several studies have shown BCG to be more
effective than chemotherapy when compared with agents such as
doxorubicin and mitomycin C. Induction and maintenance BCG
therapy has also been shown to be significantly more effective than
treatment with epirubicin plus IFN-α2a in both prevention of recur-
rence (HR, 0.41 [95% CI, 0.28–0.60]; P < .001) and disease-specific
mortality (HR, 0.20 [95% CI, 0.04–0.91]; P = .04). A meta-analysis
of randomized trials by Shelley and colleagues identified significantly
decreased tumor recurrence in high-risk patients treated with BCG
versus mitomycin C (P < .001).561 Similarly, Böhle and Bock reported
significantly higher efficacy with BCG maintenance over mitomycin
C (OR, 0.66 [95% CI, 0.47–0.94]; P = .02).562 However, whereas
intravesicular BCG therapy is more effective than chemotherapy for
both complete response and disease-free survival, the difference in
effect on overall survival remains to be determined.563 More recent
evidence suggests that BCG should be the standard of care for both
intermediate- and high-risk NMIBC.564
Given that recurrence occurs in 30% to 45% of individuals treated
with intravesicular BCG,565 several phase II trials have attempted to
further decrease recurrence by using BCG in combination with other
agents, including chemotherapeutic agents and IFN.566,567 To date,
none of these strategies have proved to significantly improve the preven-
tion of recurrence over treatment with BCG alone.568,569
Two strains of BCG, Connaught and TICE, have been the focus
of several studies to ascertain whether they confer a disparate level of
efficacy in preventing recurrence in patients with NMIBC. A retrospec-
tive comparison of BCG Connaught and BCG TICE demonstrated
that without maintenance therapy Connaught is more effective than
TICE in delaying time to first occurrence (HR, 1.48 [95% CI,
1.20–1.82]; P < .001), but that with maintenance therapy TICE is
more effective than Connaught in delaying time to first occurrence
(HR, 0.66 [95% CI, 0.47–0.93]; P = .019). Conversely, Steinberg
and colleagues conducted a post hoc analysis of their phase II study
testing the BCG Tice and Connaught strains with IFN.570 They found
that in 901 patients with high-grade NMIBC treated with six weekly
intravesicular BCG instillations and IFN (50 million units), the strain
of BCG administered did not significant affect recurrence-free survival.
Currently underway is a phase III study that is investigating different
strains of BCG with or without vaccine in patients with high-grade
NMIBC (NCT03091660). Patients will be randomized to receive
induction and maintenance therapy with TICE BCG (arm 1),
Tokyo-172 strain BCG (arm 2), or Tokyo strain BCG with priming
(vaccine; arm 3). Five-year recurrence and disease-free rates will be
determined for an estimated 969 participants, with a study completion
date of 2025.
The most recent European Association of Urology (EAU) guidelines
for NMIBC were published in 2017.571 They recommend that both
patients with low-risk tumors and patients at intermediate risk receive
treatment with one instillation of chemotherapy immediately, and
that intermediate-risk patients then receive either intravesicular
immunotherapy treatment with BCG at full dose for 1 year (level A)
or a maximum of 1 year of chemotherapy (level B). TUR is recom-
mended for low-, intermediate-, and high-risk patients. In addition,
the EAU recommends that intravesicular BCG be administered at
full dose for 1 to 3 years in individuals with high-risk tumors, and
that re-TUR should be considered for individuals at highest risk of
progression and/or with BCG refractory tumors. The American
Urological Association (AUA) updated its recommendations for NMIBC
in 2016,572 and whereas TUR is similarly recommended for low-,
intermediate-, and high-risk patients, the remaining guidelines vary
somewhat from those of the EUA.573 The AUA guidelines now recom-
mend that one instillation of intravesical chemotherapy (mitomycin
C or epirubicin) be considered for individuals with low- or intermediate-
risk tumors within 24 hours of transurethral resection of bladder
tumor (TURBT). They recommend that intermediate-risk patients
receive 6 weeks of induction chemotherapy or immunotherapy (BCG)
and that maintenance therapy be considered, and that high-risk patients
Lifestyle and Cancer Prevention  •  CHAPTER 22 369
receive a 6-week BCG induction course followed by 3 years of
maintenance BCG.
Valrubicin
Although gemcitabine and mitomycin C (with or without hypothermia)
have been studied for patients with post-BCG recurrence who were
unable to undergo cystectomy,574 valrubicin was approved for this
application by the FDA in 1998 and is currently the only drug other
than BCG that has received FDA approval for NMIBC.575,576 Steinberg
and colleagues of the Valrubicin Study Group conducted an open-label,
noncomparative study that tested valrubicin in patients with BCG-
refractory carcinoma in situ of the bladder.551 Before recruitment, two
or more rounds of BCG were administered to 70% of the study
participants, and one round of BCG followed by one or more rounds
of another agent were administered to 30% of the study participants.
In this multiinstitutional trial, intravesical valrubicin (800 mg/wk)
was administered to 90 patients for 6 weeks. Valrubicin was found
to be well tolerated, and after 30 months of follow-up, 21% of the
patients were disease-free. Only 2 of the 79 patients who demonstrated
recurrence had advanced tumors (stage T2). Reversible local bladder
symptoms accounted for the majority of the reported side effects. In
2008, the number of total responders was updated, lowering slightly
from 21% to 18%.577 In addition, recurrence figures were updated
to show that 10 patients developed metastatic or deeply invasive bladder
cancer, including four deaths and six cases of stage T3 bladder cancer.
Steinberg followed this study with the phase II/III A9303 trial,
another open-label trial, testing 6 versus 9 weeks of valrubicin (800 mg/
wk) in 80 patients with BCG-refractory carcinoma in situ of the
bladder who had previously received BCG treatment or two or more
(39%) or three or more (11%) courses of BCG.576 A total of 78
patients completed treatment and underwent primary disease evaluation
at 3 months. The complete response for this study, as with the previous
study, was 18%. Reversible local bladder adverse events were primarily
mild to moderate.
Chemotherapy
Sternberg and colleagues conducted a study of the deoxycytidine
gemcitabine in high-risk BCG-refractory NMIBC patients who were
unable or unwilling to undergo cystectomy and found it to be active.578
However, a second phase II trial (SWOG S0353) revealed that although
intravesical gemcitabine had activity (2 g in 100 mL of normal saline
weekly for 6 weeks, then monthly to 12 months), only 47% of patients
with NMIBC were disease free at 3 months, and fewer than 30%
and approximately 20% demonstrated a durable response at 12 and
24 months, respectively.579 Shelley and colleagues conducted a systematic
review of gemcitabine for the prevention or delay of rumor recurrence
after resection in NMIBC patients.580 They found that gemcitabine
was more effective than mitomycin C in preventing recurrence, and
showed potential for patients with BCG refractory NMIBC. However,
they did note that additional corroborative evidence is needed to
confirm these findings.
Two additional studies of gemcitabine for the prevention of recur-
rence in patients with NMIBC are currently recruiting subjects. A phase
II trial is examining gemcitabine plus cisplatin versus no intervention
in moderate- to high-risk NMIBC patients who underwent TURBT
and had epirubicin instilled 24 hours afterward (NCT02716961);
this trial is scheduled to be completed in 2020. A phase III trial
comparing the efficacy of intravesicular gemcitabine (2 g in 100 mL
of 0.9% sodium chloride) versus intravesicular mitomycin C (40 mg
in 40 mL of 0.9% sodium chloride) versus no intervention in
NMIBC patients immediately after TURBT is currently ongoing
(NCT02685771).
A phase II study of pirarubicin for prevention of recurrence after
nephroureterectomy for upper tract urothelial carcinoma (UTUC) is
being conducted by Huang at Renji Hospital (NCT03030157). This
trial is comparing long-term versus single intravesical instillation of
pirarubicin (THP; 30 mg in 30 mL of normal saline). The study,
370 Part I: Science and Clinical Oncology
scheduled to be completed in December 2019, has begun accru-
ing, with a goal of 220 participants. Another new phase II study
investigating the effects of prophylactic intravesical chemotherapy of
pharmorubicin versus pirarubicin on the prevention of bladder tumors
in patients who have undergone nephroureterectomy for UTUC is being
conducted by Li at Peking University First Hospital (NCT02547350).
This trial, which has not yet opened for recruitment, is scheduled
to randomize 200 patients to receive a single or multiple intravesical
instillations of pharmorubicin (50 mg) or pirarubicin (30 mg). The
study is to be completed by 2020. Li has a second study currently
accruing participants to receive a single intravesical chemotherapy
instillation of pirarubicin (THP; 40 mg for 30 minutes) immediately
after diagnostic ureteroscopy for UTUC. This study is scheduled to be
completed by 2021.
Celecoxib
An Italian study focused on celecoxib for the prevention of recurrence
in NMIBC patients.581 A total of 58 patients were randomized to
receive celecoxib or intravesical mitomycin C weekly for 4 weeks,
then monthly for 11 months. The results demonstrated improved
efficacy associated with celecoxib versus chemotherapy after 75 months
of follow-up, evidenced by increased disease-free patients (mitomycin
C, 34.85%; celecoxib, 44.8%).
Metformin
With the benefits metformin has been shown to have in reducing
risk of cancer, including breast, colorectal, pancreatic, and prostate
cancers, it has become the focus of studies to prevent recurrence
in NMIBC patients.582 A meta-analysis of 36 studies found that
patients with diabetes mellitus have a 35% increased risk of bladder
cancer (RR, 1 in 35 [95% CI, 1.17–1.56]; P < .001), with highest
risk occurring in patients diagnosed within less than 5 years.583 Seng
investigated the effects of metformin on risk of bladder cancer in
970,708 Taiwanese patients with type 2 diabetes mellitus, including
532,519 never users and 408,189 ever users of metformin.584 He found
decreased bladder cancer risk in patients treated with metformin.
These findings were reinforced by a retrospective cohort study of the
effects of metformin on cancer-specific survival in patients diagnosed
with bladder cancer who were undergoing radical cystectomy.585 Of
the 421 patients, 85 had diabetes; 39 of the 85 (46%) were being
treated with metformin. Metformin improved both recurrence-free
survival (HR, 0.54 [95% CI, 0.33–0.88]; P = .013) and bladder
cancer–specific survival (HR, 0.57 [95% CI, 0.35–0.91]; P = .019).
Another retrospective analysis investigating the association of diabetes
mellitus treated with metformin and oncologic outcomes was conducted
by Rieken and colleagues.586 Of 1117 patients with NMIBC, 125
had diabetes mellitus, 43 of whom (3.8% of 1117 patients) were
being treated with metformin. Once again, these researchers found
increased risk of recurrence (HR, 1.45 [95% CI, 1.09–1.94]; P = .01)
and progression (HR, 2.38 [95% CI, 1.40–4.06]; P = .001), but not
any-cause mortality, in diabetic patients not taking metformin. Finally,
Zhang and colleagues showed in preclinical studies that metformin
suppresses bladder cancer cell proliferation.587 Unfortunately, although
metformin is currently being studied in 33 cancer prevention trials,
none of these are investigating its efficacy in preventing recurrence of
bladder cancer.
Toll-Like Receptor Agonist
Based on positive results observed with the TLR agonist imiquimod for
benign and malignant skin cancer in a phase I clinical trial, Donin and
colleagues studied the efficacy of six doses of intravesical TMX-101,
a liquid version of imiquimod, in a phase II pilot study.588 Their
results demonstrate that TMX-101 0.4% (200 mg/50 mL) is well
tolerated and increases urinary cytokines in patients with NMIBC
in situ. Future results further investigating the efficacy of TMX-101
in preventing recurrence will determine its efficacy for patients
with NMIBC.
Other Drugs
A meta-analysis of randomized controlled clinical trials analyzed the
preventive efficacy of vitamin and antioxidant supplements for bladder
cancer, including vitamins A, B6, C, and E, a multivitamin, β-carotene,
fenretinide, selenium, α-tocopherol, and zinc.589 This study analyzed
14 clinical trials and found no preventive benefit for risk of bladder
cancer (RR, 1.04 [95% CI, 0.92–1.17]) associated with any of the
vitamin and antioxidant supplements, or combinations thereof, and
a marginal increase in risk of bladder cancer with β-carotene (RR,
1.44 [95% CI, 1.00–2.09]).
Molecular targets for immune-based therapies are currently being
investigated, including ILs, IFN, TLRs, and TRAIL.565 In addition,
nanoparticle delivery of BCG cell wall skeleton (BCG-CW) to replace
live BCG and the use of gene and virus therapy are in the early stages
of development.
Skin Cancer
Nonmelanoma Skin Cancers
The progression of cutaneous squamous cell carcinoma (cSCC),
the most prevalent metastatic skin cancer,590–592 is associated with
inflammation and has been well characterized, initially manifesting
as a precancerous lesion known as actinic keratosis (AK), that then
progresses to a cSCC in situ before progressing into an invasive
carcinoma.593 Thus, disruption of the progression of AK to invasive
carcinoma represents a strategy with high potential for the prevention of
skin cancer.
A variety of strategies have been developed to prevent AK and
superficial BCC progression, including cryosurgery,594 photodynamic
therapy,595 electrodissection, curettage, and chemical peels.596 Applica-
tion of the FDA-approved topical agents 5-fluorouracil (5-FU) cream
(a cytotoxic agent), diclofenac gel (an NSAID), imiquimod cream
(an immune response modifier), and ingenol mebutate gel (a cell
death inducer) and phytodynamic therapy with δ-aminolevulinic
acid (ALA-PDT; an endogenous 5-carbon aminoketone that is
the first compound in the porphyrin synthesis pathway) have
been shown to clear AKs and thereby prevent skin cancer, with
comparable efficacy in phase II trials, although the adverse effects
and extent of cosmesis associated with treatment vary by agent.597
The majority of cases of AK and superficial BCC are managed with
cryosurgery because of the low cost, ease of use, acceptable cosmesis,
and low rate of recurrence (potentially 7.5%, depending on the
lesions) versus electrodissection and curettage (7.7%), all non-Mohs
modalities (8.7%), radiation therapy (8.7%), and surgical excision
(10.1%).594
Randomized trials have demonstrated that treatment of AKs,
Bowen disease, and both superficial and thin nodular BCC with
δ-aminolevulinic acid phytodynamic therapy is both effective in reduc-
ing recurrence and providing superior cosmetic outcomes versus standard
therapies.595 Unfortunately, results from long-term follow-up studies
have reported that although recurrence rates in individuals treated with
δ-aminolevulinic acid phytodynamic therapy are comparable to those
of other standard therapies for both Bowen disease and superficial
BCC, sustained efficacy is lower in individuals with nodular BCC
versus surgery.595 5-FU cream (5% twice daily for 2–4 weeks) is the
standard-of-care treatment for AKs, and field therapy with this agent
has been shown to be effective for treating AKs.598 Results published in
2017 from an RCT titled the Veterans Affairs Keratinocyte Carcinoma
Chemoprevention (VAKCC) Trial demonstrated a significant reduction
in the development of new AKs after treatment with 5-FU (5% twice
daily for 2 to 4 weeks) versus control that persisted for 24 to 36 months
after treatment (6 months: 62% reduction [95% CI, 57–67%]; 24
months: 41% reduction [95% CI, 31–49%]; 6 months: 22% reduction
[95% CI, 0.54–0.95]).599 RCTs of topical imiquimod (5% applied
daily 2–3 days per week for 16 weeks) have shown that 45% to 57%
of patients have a complete response, and 59% to 75% of patients

demonstrate a partial response.600–602 Conversely, a study of patients
treated with diclofenac (3%) in hyaluronan gel (twice daily for 90
days) reported a complete response in 50% of participants versus
20% of participants treated with vehicle.603 However, the limited
resolution, prolonged treatment period, and adverse effects associated
with treatment have limited its use.
Ingenol mebutate, the agent most recently approved by the FDA
for the treatment of AKs, induces rapid necrosis of lesions and initiates
an immune-mediated reaction that promotes healing.604 A reduction
of 75% or more in existing AKs has been observed in 60% to 68%
of participants with face and scalp AKs treated with ingenol mebutate
versus those treated with placebo (7%–8% of participants) in multiple
phase III trials.605–606 These studies also show a reduction of 75% or
more of AKs on the trunk and extremities with ingenol mebutate
versus placebo (44%–55% of participants versus 7%), and an induction
of AK necrosis after 2 to 3 days of treatment. A recent randomized
clinical trial compared the efficacy of ingenol mebutate (3-day treatment
cycle) and daylight photodynamic with therapy methyl aminolevulinate
(one treatment), and found similar efficacy for grade I/II AKs, but
decreased adverse events and improved cosmetic outcomes with daylight
photodynamic with therapy methyl aminolevulinate. However, in
2015 the FDA released a Drug Safety Communication warning of
severe adverse events associated with ingenol mebutate, including
severe allergic reactions and herpes zoster (shingles).607
Retinoids
Many RCTs have investigated topical and oral retinoids for the
prevention of AKs and nonmelanoma skin cancers (NMSCs).608 The
retinoid acitretin (30 mg/day) was the focus of a clinical trial of 44
renal transplant recipients.609 This study demonstrated a reduction in
SCCs (11% versus 47%, χ2 = 6.27, P = .01) and a reduction in AKs
(13.4% versus 28.2% [95% CI, 11.5–71.7]) in patients treated for 6
months with acitretin versus placebo. The majority of the associated
side effects were mild mucocutaneous reactions, followed by mild
hair loss; no deterioration in renal function was observed. However,
conflicting results regarding the efficacy and/or safety of retinoids for
the prevention of skin cancer have been also reported. Two years of
treatment with acitretin (25 mg orally 5 days per week) versus placebo
was tested in 70 nontransplant high-risk patients with a recent history
of NMSCs.610 No statistically significant reduction was observed in
patients treated with acitretin versus placebo, but this may have been
a result of low statistical power. Similarly, the Veterans Affairs Topical
Tretinoin Chemoprevention Trial of high-dose topical tretinoin for
keratinocyte carcinoma prevention, which accrued 1191 participants
for 1.5 to 5.5 years of treatment with high-dose tretinoin (0.1%) or
vehicle, demonstrated no preventive benefit associated with this retinoid
in preventing BCC, SCC, or AKs, and increased side effects.611 The
Southwest Skin Cancer Prevention Study (SKICAP-BCC/SCC), one
of a set of SKICAP trials, was a large phase III study of oral retinol
(25,000 units/day) and isotretinoin (5–10 mg/day) versus placebo in 525
individuals with a history of BCC or SCC.612 No difference in the time
to first new occurrence of either BCC or SCC was observed between
treatment arms. However, a reanalysis of this study in a dose-response
format identified an increased risk of SCC development in participants
treated with either retinol or isotretinoin versus placebo in the first
quartile of dose.613 In a second SKICAP study, the SKICAP-AK trial,
2800 high-risk individuals were treated with retinol (25,000 IU) or
placebo for 5 years614; the researchers found that retinol was associated
with a reduction in incidence of first new SCC but not BCC.615 Thus,
before the incorporation of retinoids in standard of care for NMSC
prevention, additional larger-scale clinical trials will be required, with
extended follow-up examining the efficacy and associated toxicity of
these agents in the prevention of AKs and NMSC.608
Nonsteroidal antiinflammatory drugs
Some population-based studies have indicated that NSAIDs are associ-
ated with reduced incidence of AKs and/or SCCs,616,617 but others
Lifestyle and Cancer Prevention  •  CHAPTER 22 371
have found no evidence of preventive efficacy for SCC incidence618
or minimal and inconsistent preventive efficacy for SCC and BCC
incidence.619 Regardless, a meta-analysis published in 2015 reported
a significant reduction in SCC risk in NSAID users compared with
nonusers, which was enhanced in high-risk individuals (reduced risk
of SCC among users of any NSAIDs: 0.82 [95% CI, 0.71–0.94]).620
Clouser and colleagues examined data from the SKICAP-AK trial in
an effort to determine the association of NSAID use and time to first
SCC or BCC in aspirin users and nonusers.621 Their results showed a
significant reduction in SCC (HR, 0.49 [95% CI, 0.28–0.87]) and
BCC (HR, 0.43 [95% CI, 0.25–0.73]) in participants who were
aspirin users for less than the full duration of the study, suggesting
that a shorter duration of NSAID use may be more protective than a
longer duration. Most recently, a meta-analysis by Zhu and colleagues
investigated the association between aspirin use and prevention of
skin cancer in individuals from eight case-control and five prospective
cohort studies.622 Their results demonstrate significantly reduced risk
of skin cancer in individuals taking low-dose (≤150 mg) or higher-
dose (50–400 mg) aspirin daily, particularly for risk of NMSC (OR,
0.97 [95% CI, 0.95–0.99]; P = .22). Additional RCTs are needed to
confirm the potential preventive benefit of aspirin for NMSC. Although
epidemiologic studies and alternative strategies using combined therapy,
lower doses, and variable treatment times are promising, preventive
efficacy has not yet been established for naproxen and sulindac623 for
the prevention of NMSC.
Cyclooxygenase-2 inhibitors
Both COX-1 and COX-2 are known to be present in NMSCs624;
however, studies have shown that the primary COX isoform responsive
to UVR in the human epidermis is COX-2.625 Based on accumulating
evidence from preclinical studies demonstrating the efficacy of COX-2
inhibitors in the prevention of NMSC, clinical trials have been
conducted to investigate COX-2 inhibitors in this setting.626 Elmets
and colleagues conducted a phase III clinical trial of 240 subjects
treated with celecoxib (200 mg twice per day) or placebo for 9
months.627 They found that patients treated with celecoxib versus
placebo demonstrated no reduction in AKs 9 months after randomiza-
tion, but significant reductions in NMSCs (RR, 0.41 [95% CI, =
0.23–0.72]; P = .002), BCCs (RR, 0.40 [95% CI, 0.18–0.93]; P =
.032), and SCCs (RR, 0.42 [95% CI, 0.19–0.93]; P = .032) 11
months after randomization, with similar cardiovascular severe adverse
effects. Regardless, a warning regarding serious or life-threatening
adverse events is now required by the FDA for celecoxib owing to
the potentially fatal side effects associated with the suppression of
prostaglandin synthesis after COX inhibition.628
D,L-α-Difluoromethylornithine
d,l-α-Difluoromethylornithine (DFMO) is an enzyme-activated,
irreversible inhibitor of ODC and is the first enzyme in polyamine
synthesis.629 Based on previous positive clinical results, a phase III
clinical trial tested 4 to 5 years of treatment with DFMO (500 mg/
m2/day) or placebo in 291 participants with a history of NMSC.630
Although DFMO was not associated with reduced incidence of NMSC
or SCC versus placebo, it was associated with a significant reduction
in BCC incidence (DFMO, 0.28 BCC per person per year; placebo,
0.40 BC per person per /year; P = .03), which was characterized by
a significant reduction in ODC activity and minimal adverse events.
Long-term follow-up of this study demonstrated a persistent but
statistically insignificant reduction in NMSC incidence in individuals
treated with DFMO versus placebo after discontinuation from the
study that was not associated with any difference in latent or cumulative
toxicity.631
Vismodegib
In 2012 vismodegib became the first inhibitor of the Hedgehog signaling
pathway to gain approval from the FDA for individuals with advanced
or metastatic BCC and other cancers, which led to a clinical trial
372 Part I: Science and Clinical Oncology
testing its efficacy in preventing BCC recurrence.632,633 Tang and
colleagues treated 41 individuals with basal cell nevus syndrome with
vismodegib (150 mg/day) or placebo for 3 months and found that
vismodegib reduced BCC size (−65% versus −11%) and incidence
of new BCCs (2 versus 29; P < .001) versus placebo; in addition,
83% of patients in the treatment arm had no residual BCC at sites
with previous BCC at 1 month of being on treatment.632 However,
although participants receiving vismodegib commonly demonstrated
grade 1 or 2 adverse events, 54% discontinued the drug because of
adverse events.
Statins
Wang and colleagues analyzed data from the Women’s Health Initiative
Observational Study and the Women’s Health Initiative Clinical Trial
to determine whether use of statins was effective in incidence of
NMSC.634 A total of 118,357 non-Hispanic white women with no
history of NMSC at baseline were included in the analysis; with 10.5
years of follow-up, individuals who used any statins at baseline
demonstrated significantly higher incidence of NMSC, with highest
risk associated with lovastatin (OR, 1.52 [95% CI, 1.08–2.16]),
simvastatin (OR, 1.38 [95% CI, 1.12–1.69]), and lipophilic statins
(OR, 1.39 [95% CI, 1.18–1.64]), suggesting that statins increase risk
of NMSC in Caucasian postmenopausal women. Conversely, other
studies and meta-analyses have suggested no association or increased
risk of NMSC with statin use; additional research is required to
determine whether there is in fact a causal relationship between cancer
risk and use of statins, and to determine the precise biologic mechanisms
involved.635
Vitamins and minerals
A post hoc analysis of the WHI study, in which women were random-
ized to receive calcium (1000 mg/day) plus vitamin D3 (400 IU/day)
or placebo for 7 years, investigated the effects of calcium and vitamin
D supplementation on risk of both NMSC and melanoma.636 No
difference in incidence of NMSC or melanoma compared with placebo
was observed; however, patients with a history of NMSC in the
treatment arm had lower risk of melanoma compared with those with
a history of NMSC in the placebo arm (HR, 0.43 [95% CI, 0.21–0.90]).
These findings suggest that women at high risk of NMSC may benefit
from supplementation with calcium and vitamin D. Conversely, the
Physicians’ Health Study of 12 years of treatment with β-carotene
(50 mg on alternate days) in 22,071 healthy physicians 40 to 84 years
of age found no reduction in risk of NMSC, BCC, or SCC associated
with β-carotene.637
Ferrucci and colleagues investigated the relationship between tea,
coffee, and caffeine and early-onset BCC by using data from 767
non-Hispanic white women younger than 40 years listed in Yale’s
Dermatopathology database.638 Women who regularly consumed caf-
feinated coffee plus hot tea had reduced incidence of early-onset BCC
(OR, 0.60 [95% CI, 0.38–0.96]), which was enhanced in women in
the highest category of caffeine use (OR, 0.57 [95% CI, 0.34–0.95]; P
trend = .037), versus nonconsumers, suggesting that caffeinated coffee
plus tea may provide a base level of protection against early-onset BCC.
More recently, an 11-year prospective study of black tea consumption
reported no difference in risk of BCC or SCC in individuals who
drank four or more cups per day and non–black tea drinkers.639
Another study with positive results was a phase III trial that
investigated vitamin B3 (nicotinamide) for the prevention of mela-
noma.640 A total of 386 participants with a recent history of NMSC
were randomized to receive nicotinamide (500 mg twice per day) or
placebo for 12 months. Reduced incidence of NMSC (23% [95%
CI, 4–38]; P = .02), BCC (20% [95% CI, −6 to 39]; P = .12), SCC
(30% [95% CI, 0–51]; P = .05), and AK (13%; P = .001) was
demonstrated in individuals treated with nicotinamide compared with
placebo. Although no difference in adverse events was demonstrated
between arms, the preventive benefit provided by nicotinamide did
not persist after discontinuation of use.
Melanoma
As with other cancers, melanoma has numerous subtypes (over 30),
and approximately 26% of melanomas are histologically associated
with dysplastic or other nevi.641–642
Nonsteroidal antiinflammatory drugs
The efficacy of NSAIDs as a preventive strategy for melanoma has
been suggested by both preclinical643–646 and epidemiologic647–650 studies.
Based on these findings, Curiel-Lewandrowski and colleagues examined
the effects of 8 weeks of treatment with sulindac (150 twice per day)
versus placebo in 50 individuals with atypical nevi on (1) the bioavail-
ability of sulindac and metabolites, (2) apoptosis, and (3) vascular
endothelial growth factor A (VEGFA) expression in nevi.623 The findings
demonstrated that individuals treated with sulindac had elevated
concentrations of the proapoptotic metabolite sulindac sulfone in
benign nevi, but no difference was observed in cleaved caspase-3 or
VEGFA levels. Although these results demonstrate bioavailability of
sulindac and sulindac metabolites, further studies are needed to
determine its efficacy as a preventive strategy for melanoma.
A UCLA retrospective pilot study of 148 patients with melanoma
examined the association between aspirin use (81 mg or 325 mg)
1 month or more before diagnosis and the aggressiveness of mela-
noma.651 Aspirin use significantly decreased Breslow depth (95% CI,
0.297–0.8127; P = .03517), suggesting the potential benefit of aspirin
for patients with or at high risk of melanoma. In addition, the meta-
analysis by Zhu and colleagues, outlined under NMSC earlier, examined
aspirin for prevention of skin cancer and found significant reductions
associated with aspirin at both 50 to 400 mg/day (OR, 0.94 [95%
CI, 0.90–0.99]; P = .02) and 100 mg/day or less (OR, 0.95 [95%
CI, 0.90–0.99]; P = .15).622 Although these results are promising,
the results from large-scale ongoing trials, such as the Aspirin in
Reducing Events in the Elderly (ASPREE) trial, with an expected
accrual of 19,000 participants to be treated with acetylsalicylic acid
(ASA; 100 mg) or placebo and a secondary end point that includes
melanoma incidence,652 will better define the efficacy of NSAIDs for
the prevention of melanoma.
Statins
Epidemiologic data have suggested that intake of statins and fibrates,
both of which are hypolipidemic (lipid-lowering) agents, is associated
with a 10% and 14%, respectively, decreased risk of melanoma.653
Unfortunately, conflicting results regarding the relationship between
statins and melanoma have been reported. A phase II trial of 80
subjects with multiple atypical nevi examined the impact of 6 months
of treatment with lovastatin versus placebo on biomarkers of melanoma
pathogenesis.654 No significant differences in any of the molecular
biomarkers analyzed were apparent between the two study arms,
suggesting that if lovastatin prevents melanoma, the mechanism though
which it functions is other than reversing precursor atypical nevi.
Vitamins, minerals, and dietary factors
Many studies have examined the effects of vitamins and minerals on
risk of melanoma, most with negative or limited results. Numerous
studies have been conducted to determine the potential of vitamin A
in preventing melanoma. Asgari and colleagues examined dietary and
supplemental vitamin A and carotenoids in 69,635 participants of
the Vitamins And Lifestyle (VITAL) cohort study, and demonstrated
a significant reduction in risk of melanoma in individuals with baseline
use of retinol supplements (HR, 0.60 [95% CI, 0.41–0.89]) and
individuals with high-dose (>1200 µg/day) use of retinol supplements
(HR, 0.74 [95% CI, 0.55–1.00]) versus nonusers.655 Neither dietary
or total intake of vitamin A nor carotenoids were found to be associated
with risk of melanoma. Conversely, an analysis published in 2015 of
nine cohort and intervention studies found no association between
risk of melanoma and consumption of vitamin A (retinol), vitamin
C, vitamin E (tocopherol), carotenoids, or selenium or high fruit and
 Lifestyle and Cancer Prevention  •  CHAPTER 22 373

vegetable intake.639,656 Future studies of different retinoid compounds
alone or in combination with other agents will be required to make
a place for vitamin A in this setting.
As mentioned previously, a post hoc study of the WHI, wherein
36,282 postmenopausal women were treated with calcium (1000 mg/
day) plus vitamin D3 (400 IU/day) or placebo for 7 years, found no
preventive efficacy on melanoma incidence associated with calcium
and vitamin D supplementation versus placebo.636 However, reduced
incidence of melanoma was observed in high-risk patients previously
diagnosed with NMSC (HR, 0.43 [95% CI, 0.21–0.90]). Conversely,
some studies have suggested efficacy of other vitamin, mineral, or
diet-related strategies for reducing risk of melanoma. The large
multicenter European Prospective Investigation into Cancer and
Nutrition (EPIC) cohort study with more than 500,000 participants
aged 25 to 70 years from 10 countries examined the effects of total,
caffeinated, and decaffeinated coffee and tea consumption on melanoma
incidence.657 With 14.9 years of follow-up, this study demonstrated
an inverse association between consumption of caffeinated coffee and
risk of melanoma in men in the highest quartile of consumption
versus those with no consumption (HR, 0.31 [95% CI, 0.14–0.69]).
No statistically significant differences were observed in women, or for
tea or decaffeinated coffee in either sex.
Other agents
Additional agents have been demonstrated to be effective in preventing
melanoma in preclinical studies, but positive results in clinical trials
regarding their efficacy and safety are lacking. Among these are cur-
cumin, EGCG, the naturally occurring flavonoid fisetin, resveratrol,
warfarin, and vitamins C, E, and K.658 Currently, although a number
of agents have been found effective for the chemoprevention of mela-
noma, none have been approved by the FDA.
The complete reference list is available online at
ExpertConsult.com.

KEY REFERENCES
5. Kohler LN, Garcia DO, Harris RB, Oren E, Roe DJ,
Jacobs ET. Adherence to diet and physical activity
cancer prevention guidelines and cancer outcomes:
a systematic review. Cancer Epidemiol Biomarkers
Prev. 2016;25(7):1018–1028.
6. Vogelstein B, Papadopoulos N, Velculescu VE,
Zhou S, Diaz LA Jr, Kinzler KW. Cancer genome
landscapes. Science. 2013;339(6127):1546–1558.
8. Sporn MB, Dunlop NM, Newton DL, Smith JM.
Prevention of chemical carcinogenesis by vitamin
A and its synthetic analogs (retinoids). Fed Proc.
1976;35(6):1332–1338.
15. Centers for Disease Control and Prevention.
Best Practices for Comprehensive Tobacco Control
Programs—2014. Atlanta, GA: Department of
Health and Human Services, Centers for Disease
Control and Prevention, National Center for Chronic
Disease Prevention and Health Promotion, Office
on Smoking and Health; 2014.
18. Eriksen M, Mackay J, Schluger N, Gomeshtapeh
FI, Drope J. The Tobacco Atlas. 5th ed. Atlanta, GA:
The American Cancer Society; 2015.
23. Bagnardi V, Rota M, Botteri E, et al. Alcohol con-
sumption and site-specific cancer risk: a comprehen-
sive dose-response meta-analysis. Br J Cancer. 2015;
112(3):580–593.
26. AICR / WCRF. Continuous Update Project Report:
Diet, Nutrition, Physical Activity, and Prostate
Cancer. World Cancer Research Fund (WCRF) /
American Institute for Cancer Research (AICR);
2014.
34. Mostofsky E, Mukamal KJ, Giovannucci EL,
Stampfer MJ, Rimm EB. Key findings on alcohol
consumption and a variety of health outcomes
from the Nurses’ Health Study. Am J Public Health.
2016;106(9):1586–1591.
58. Scoccianti C, Lauby-Secretan B, Bello PY, Chajes
V, Romieu I. Female breast cancer and alcohol
consumption: a review of the literature. Am J Prev
Med. 2014;46(3 suppl 1):S16–S25.
63. AICR / WCRF. Recommendations for Cancer Preven-
tion; 2016. http://www.aicr.org/reduce-your-cancer
-risk/recommendations-for-cancer-prevention/.
64. Kushi L, Doyle C, McCullough M, et al. American
Cancer Society Guidelines on nutrition and physical
activity for cancer prevention: reducing the risk
of cancer with healthy food choices and physical
activity. CA Cancer J Clin. 2012;62(1):30–67.
69. Lauby-Secretan B, Scoccianti C, Loomis D, et al.
Body fatness and cancer—viewpoint of the IARC
working group. N Engl J Med. 2016;375(8):794–798.
70. Birks S, Peeters A, Backholer K, O’Brien P, Brown
W. A systematic review of the impact of weight
loss on cancer incidence and mortality. Obes Rev.
2012;13(10):868–891.
77. Britton KA, Massaro JM, Murabito JM, Kreger
BE, Hoffmann U, Fox CS. Body fat distribution,
incident cardiovascular disease, cancer, and all-cause
mortality. J Am Coll Cardiol. 2013;62(10):921–
925.
79. Donohoe C, Lysaght J, O’Sullivan J, Reynolds J.
Emerging concepts linking obesity with the hallmarks
of cancer. Trends Endocrinol Metab. 2017;28(1):
46–62.
80. Iyengar NM, Hudis CA, Dannenberg AJ. Obesity
and cancer: local and systemic mechanisms. Annu
Rev Med. 2015;66:297–309.
85. Gonçalves A, Dantas Florencio G, Maisonnette de
Atayde Silva M, Cobucci R, Giraldo P, Cote N. Effect
of physical activity on breast cancer prevention: a
systematic review. J Phys Act Health. 2014;11(2):
445–454.
86. Lakoski S, Willis B, Barlow C, et al. Midlife
cardiorespiratory fitness, incident cancer, and survival
after cancer in men: the Cooper Center Longitudinal
Study. JAMA Oncol. 2015;1(2):231–237.
88. Sanchis-Gomar F, Lucia A, Yvert T, et al. Physical
inactivity and low fitness deserve more attention
to alter cancer risk and prognosis. Cancer Prev Res
(Phila). 2015;8(2):105–110.
89. Lee I, Shiroma E, Lobelo F, et al. Effect of physical inac-
tivity on major non-communicable disease worldwide:
an analysis of burden of disease and life expectancy.
Lancet. 2012;380:219–229.
97. Ross R, Janssen I, Dawson J, et al. Exercise-induced
reduction in obesity and insulin resistance in women:
a randomized controlled trial. Obes Res. 2004;12:
789–798.
130. US Department of Health and Human Services.
The Surgeon General’s Call to Action to Prevent
Skin Cancer. Washington D.C. 2014.
141. Colantonio S, Bracken MB, Beecker J. The associa-
tion of indoor tanning and melanoma in adults:
systematic review and meta-analysis. J Am Acad
Dermatol. 2014;70(5):847–857.e841–818.
151. Lin JS, Eder M, Weinmann S. Behavioral counseling
to prevent skin cancer: a systematic review for the
U.S. Preventive Services Task Force. Ann Intern Med.
2011;154(3):190–201.
159. Domchek SM, Friebel TM, Singer CF, et al. Associa-
tion of risk-reducing surgery in BRCA1 or BRCA2
mutation carriers with cancer risk and mortality.
JAMA. 2010;304(9):967–975.
164. Alpha-Tocopherol, Beta Carotene Cancer Preven-
tion Study Group. The effect of vitamin E and
beta carotene on the incidence of lung cancer and
other cancers in male smokers. N Engl J Med. 1994;
330(15):1029–1035.
165. Omenn GS, Goodman GE, Thornquist MD, et al.
Risk factors for lung cancer and for intervention
effects in CARET, the Beta-Carotene and Retinol
Efficacy Trial. J Natl Cancer Inst. 1996;88(21):
1550–1559.
172. Lippman SM, Klein EA, Goodman PJ, et al. Effect
of selenium and vitamin E on risk of prostate cancer
and other cancers: the Selenium and Vitamin E
Cancer Prevention Trial (SELECT). JAMA. 2009;
301(1):39–51.
173. Klein EA, Thompson IM Jr, Tangen CM, et al.
Vitamin E and the risk of prostate cancer: the
Selenium and Vitamin E Cancer Prevention Trial
(SELECT). JAMA. 2011;306(14):1549–1556.
191. Hong WK, Endicott J, Itri LM, et al. 13-cis-retinoic
acid in the treatment of oral leukoplakia. N Engl J
Med. 1986;315(24):1501–1505.
207. William WN Jr, Papadimitrakopoulou V, Lee JJ, et al.
Erlotinib and the risk of oral cancer: the Erlotinib
Prevention of Oral Cancer (EPOC) Randomized
Clinical Trial. JAMA Oncol. 2016;2(2):209–
216.
315. Meyskens FL Jr, McLaren CE, Pelot D, et al.
Difluoromethylornithine plus sulindac for the
prevention of sporadic colorectal adenomas: a
randomized placebo-controlled, double-blind trial.
Cancer Prev Res (Phila). 2008;1(1):32–38.
322. Kimura T, McKolanis JR, Dzubinski LA, et al.
MUC1 vaccine for individuals with advanced
adenoma of the colon: a cancer immunoprevention
feasibility study. Cancer Prev Res (Phila). 2013;6(1):
18–26.
353. Pan KF, Zhang L, Gerhard M, et al. A large
randomised controlled intervention trial to prevent
gastric cancer by eradication of Helicobacter pylori in
Linqu County, China: baseline results and factors
affecting the eradication. Gut. 2016;65(1):9–18.
355. Rothwell PM, Fowkes FG, Belch JF, Ogawa H,
Warlow CP, Meade TW. Effect of daily aspirin on
long-term risk of death due to cancer: analysis of
individual patient data from randomised trials.
Lancet. 2011;377(9759):31–41.
378. Qu C, Chen T, Fan C, et al. Efficacy of neonatal
HBV vaccination on liver cancer and other liver
diseases over 30-year follow-up of the Qidong
hepatitis B intervention study: a cluster random-
ized controlled trial. PLoS Med. 2014;11(12):
e1001774.
419. Litton JK, Arun BK, Brown PH, Hortobagyi GN.
Aromatase inhibitors and breast cancer prevention.
Expert Opin Pharmacother. 2012;13(3):325–331.
374 Part I: Science and Clinical Oncology
460. Disis ML, Wallace DR, Gooley TA, et al. Concurrent
trastuzumab and HER2/neu-specific vaccination in
patients with metastatic breast cancer. J Clin Oncol.
2009;27(28):4685–4692.
519. Joura EA, Giuliano AR, Iversen OE, et al. A 9-valent
HPV vaccine against infection and intraepithelial
neoplasia in women. N Engl J Med. 2015;372(8):
711–723.
576. Dinney CP, Greenberg RE, Steinberg GD.
Intravesical valrubicin in patients with bladder
carcinoma in situ and contraindication to or
failure after bacillus Calmette-Guérin. Urol Oncol.
2013;31(8):1635–1642.
605. Lebwohl M, Swanson N, Anderson LL, Melgaard A,
Xu Z, Berman B. Ingenol mebutate gel for actinic
keratosis. N Engl J Med. 2012;366(11):1010–1019.
633. Sekulic A, Migden MR, Oro AE, et al. Efficacy
and safety of vismodegib in advanced basal-cell
carcinoma. N Engl J Med. 2012;366(23):2171–
2179.
652. ASPREE Investigator Group. Study design of ASPirin
in Reducing Events in the Elderly (ASPREE): a
randomized, controlled trial. Contemp Clin Trials.
2013;36(2):555–564.

REFERENCES
1. Union of International Cancer Control (UICC).
The Economics of Cancer Prevention and Control:
Data Digest; 2014.
2. World Health Organization (WHO). Cancer
Prevention. www.who.int/cancer/prevention/en/.
Accessed February 8, 2015.
3. Doll R, Peto R. The causes of cancer: quantita-
tive estimates of avoidable risks of cancer in the
United States today. J Natl Cancer Inst. 1981;66(6):
1191–1308.
4. Colditz GA, Wei EK. Preventability of cancer: the
relative contributions of biologic and social and
physical environmental determinants of cancer
mortality. Annu Rev Public Health. 2012;33:137–
156.
5. Kohler LN, Garcia DO, Harris RB, Oren E, Roe DJ,
Jacobs ET. Adherence to diet and physical activity
cancer prevention guidelines and cancer outcomes:
a systematic review. Cancer Epidemiol Biomarkers
Prev. 2016;25(7):1018–1028.
6. Vogelstein B, Papadopoulos N, Velculescu VE,
Zhou S, Diaz LA Jr, Kinzler KW. Cancer genome
landscapes. Science. 2013;339(6127):1546–1558.
7. Blackburn EH. Cancer interception. Cancer Prev
Res (Phila). 2011;4(6):787–792.
8. Sporn MB, Dunlop NM, Newton DL, Smith JM.
Prevention of chemical carcinogenesis by vitamin
A and its synthetic analogs (retinoids). Fed Proc.
1976;35(6):1332–1338.
9. Spira A, Yurgelun MB, Alexandrov L, et al. Precancer
atlas to drive precision prevention trials. Cancer Res.
2017;77(7):1510–1541.
10. U.S. Department of Health and Human Services.
The Health Consequences of Smoking: 50 Years of
Progress. A Report of the Surgeon General. Atlanta,
GA 2014.
11. Jamal A, King BA, Neff LJ, Whitmill J, Babb SD,
Graffunder CM. Current cigarette smoking among
adults—United States, 2005–2015. MMWR Morb
Mortal Wkly Rep. 2016;65(44):1205–1211.
12. Singh T, Arrazola RA, Corey CG, et al. Tobacco use
among middle and high school students—United
States, 2011–2015. MMWR Morb Mortal Wkly Rep.
2016;65(14):361–367.
13. Barrington-Trimis JL, Urman R, Berhane K, et al.
E-cigarettes and future cigarette use. Pediatrics. 2016;
138(1).
14. U.S. Department of Health and Human Services.
E-Cigarettes Use Among Youth and Young Adults: A
Report of the Surgeon General. Atlanta, GA: U.S.
Department of Health and Human Services, Centers
for Disease Control and Prevention, National Center
for Chronic Disease Prevention and Health Promo-
tion, Office on Smoking and Health; 2016.
15. Centers for Disease Control and Prevention.
Best Practices for Comprehensive Tobacco Control
Programs—2014. Atlanta, GA: Department of
Health and Human Services, Centers for Disease
Control and Prevention, National Center for Chronic
Disease Prevention and Health Promotion, Office
on Smoking and Health; 2014.
16. Eriksen M, Mackay J, Ross H. The Tobacco Atlas.
4th ed. Atlanta, GA: The American Cancer Society;
2012.
17. Wipfli H, Samet JM. One hundred years in the
making: the global tobacco epidemic. Annu Rev
Public Health. 2016;37:149–166.
18. Eriksen M, Mackay J, Schluger N, Gomeshtapeh
FI, Drope J. The Tobacco Atlas. 5th ed. Atlanta, GA:
The American Cancer Society; 2015.
19. National Cancer Institute (NCI) and the World
Health Organization (WHO). The Economics of
Tobacco and Tobacco Control, National Cancer
Institute Tobacco Control Monograph 212016.
Located at: NIH Publication No. 16-CA-8029A,
Bethesda, MD.
Lifestyle and Cancer Prevention  •  CHAPTER 22 374.e1
20. World Health Organization (WHO). Global Status
Report on Alcohol and Health, 2014. Geneva,
Switzerland, 2014.
21. Wilsnack SC, Wilsnack RW, Kantor LW. Focus on:
women and the costs of alcohol use. Alcohol Res. 2013;
35(2):219–228.
22. Baan R, Straif K, Grosse Y, et al. Carcinogenicity
of alcoholic beverages. Lancet Oncol. 2007;8(4):
292–293.
23. Bagnardi V, Rota M, Botteri E, et al. Alcohol con-
sumption and site-specific cancer risk: a comprehen-
sive dose-response meta-analysis. Br J Cancer. 2015;
112(3):580–593.
24. Bagnardi V, Rota M, Botteri E, et al. Alcohol
consumption and lung cancer risk in never smokers:
a meta-analysis. Ann Oncol. 2011;22(12):2631–2639.
25. Zhao J, Stockwell T, Roemer A, Chikritzhs T. Is
alcohol consumption a risk factor for prostate cancer?
A systematic review and meta-analysis. BMC Cancer.
2016;16(1):845.
26. AICR/WCRF. Continuous Update Project Report:
Diet, Nutrition, Physical Activity, and Prostate
Cancer. World Cancer Research Fund (WCRF)/
American Institute for Cancer Research (AICR);
2014.
27. Cao Y, Willett WC, Rimm EB, Stampfer MJ,
Giovannucci EL. Light to moderate intake of alcohol,
drinking patterns, and risk of cancer: results from
two prospective US cohort studies. BMJ. 2015;
351:h4238.
28. Boffetta P, Hashibe M. Alcohol and cancer. Lancet
Oncol. 2006;7(2):149–156.
29. Ahmad Kiadaliri A, Jarl J, Gavriilidis G, Gerdtham
UG. Alcohol drinking cessation and the risk of
laryngeal and pharyngeal cancers: a systematic review
and meta-analysis. PLoS ONE. 2013;8(3):e58158.
30. Rehm J, Patra J, Popova S. Alcohol drinking ces-
sation and its effect on esophageal and head and
neck cancers: a pooled analysis. Int J Cancer. 2007;
121(5):1132–1137.
31. Heckley GA, Jarl J, Asamoah BO, G-Gerdtham U.
How the risk of liver cancer changes after alcohol
cessation: a review and meta-analysis of the current
literature. BMC Cancer. 2011;11:446.
32. Ronksley PE, Brien SE, Turner BJ, Mukamal KJ,
Ghali WA. Association of alcohol consumption with
selected cardiovascular disease outcomes: a systematic
review and meta-analysis. BMJ. 2011;342:d671.
33. Connor J. Alcohol consumption as a cause of cancer.
Addiction. 2017;112(2):222–228.
34. Mostofsky E, Mukamal KJ, Giovannucci EL,
Stampfer MJ, Rimm EB. Key findings on alcohol
consumption and a variety of health outcomes
from the Nurses’ Health Study. Am J Public Health.
2016;106(9):1586–1591.
35. Smyth A, Teo KK, Rangarajan S, et al. Alcohol con-
sumption and cardiovascular disease, cancer, injury,
admission to hospital, and mortality: a prospective
cohort study. Lancet. 2015;386(10007):1945–1954.
36. Praud D, Rota M, Rehm J, et al. Cancer incidence
and mortality attributable to alcohol consumption.
Int J Cancer. 2016;138(6):1380–1387.
37. Seitz HK, Stickel F. Molecular mechanisms of
alcohol-mediated carcinogenesis. Nat Rev Cancer.
2007;7(8):599–612.
38. Seitz HK, Mueller S. Alcohol and cancer: an overview
with special emphasis on the role of acetaldehyde
and cytochrome P450 2E1. In: Vasiliou V, Zakhari
S, Seitz HK, Hoek JB, eds. Biological Basis of Alcohol-
Induced Cancer, Advances in Experimental Medicine
and Biology. Switzerland: Springer International
Publishing; 2015.
39. Väkeväinen S, Tillonen J, Agarwal DP, Srivastava
N, Salaspuro M. High salivary acetaldehyde after a
moderate dose of alcohol in ALDH2-deficient sub-
jects: strong evidence for the local carcinogenic action
374.e1
of acetaldehyde. Alcohol Clin Exp Res. 2000;24(6):
873–877.
40. Yokoyama A, Muramatsu T, Ohmori T, et al.
Alcohol-related cancers and aldehyde dehydro-
genase-2 in Japanese alcoholics. Carcinogenesis.
1998;19(8):1383–1387.
41. Matsuo K, Hamajima N, Shinoda M, et al. Gene-
environment interaction between an aldehyde
dehydrogenase-2 (ALDH2) polymorphism and
alcohol consumption for the risk of esophageal
cancer. Carcinogenesis. 2001;22(6):913–916.
42. Matsuda T, Yabushita H, Kanaly RA, Shibutani S,
Yokoyama A. Increased DNA damage in ALDH2-
deficient alcoholics. Chem Res Toxicol. 2006;19(10):
1374–1378.
43. Morimoto K, Takeshita T. Low Km aldehyde
dehydrogenase (ALDH2) polymorphism, alcohol-
drinking behavior, and chromosome alterations in
peripheral lymphocytes. Environ Health Perspect.
1996;104(suppl 3):563–567.
44. Ishikawa H, Yamamoto H, Tian Y, Kawano M,
Yamauchi T, Yokoyama K. Effects of ALDH2 gene
polymorphisms and alcohol-drinking behavior on
micronuclei frequency in non-smokers. Mutat Res.
2003;541(1–2):71–80.
45. Seitz HK, Stickel F. Risk factors and mechanisms
of hepatocarcinogenesis with special emphasis on
alcohol and oxidative stress. Biol Chem. 2006;387(4):
349–360.
46. Hu W, Feng Z, Eveleigh J, et al. The major lipid per-
oxidation product, trans-4-hydroxy-2-nonenal, prefer-
entially forms DNA adducts at codon 249 of human
p53 gene, a unique mutational hotspot in hepa-
tocellular carcinoma. Carcinogenesis. 2002;23(11):
1781–1789.
47. Fu P, Yang F, Li B, et al. Meta-analysis of CYP2E1
polymorphisms in liver carcinogenesis. Dig Liver
Dis. 2017;49(1):77–83.
48. Colditz GA. Relationship between estrogen levels,
use of hormone replacement therapy, and breast
cancer. J Natl Cancer Inst. 1998;90(11):814–
823.
49. Key T, Appleby P, Barnes I, Reeves G, Group
EHaBCC. Endogenous sex hormones and breast
cancer in postmenopausal women: reanalysis
of nine prospective studies. J Natl Cancer Inst.
2002;94(8):606–616.
50. Key TJ, Appleby PN, Reeves GK, et al. Circulat-
ing sex hormones and breast cancer risk factors in
postmenopausal women: reanalysis of 13 studies.
Br J Cancer. 2011;105(5):709–722.
51. Dorgan JF, Baer DJ, Albert PS, et al. Serum hormones
and the alcohol-breast cancer association in post-
menopausal women. J Natl Cancer Inst. 2001;93(9):
710–715.
52. Ginsburg ES, Mello NK, Mendelson JH, et al. Effects
of alcohol ingestion on estrogens in postmenopausal
women. JAMA. 1996;276(21):1747–1751.
53. Mendelson JH, Lukas SE, Mello NK, Amass L,
Ellingboe J, Skupny A. Acute alcohol effects on
plasma estradiol levels in women. Psychopharmacology
(Berl). 1988;94(4):464–467.
54. Reichman ME, Judd JT, Longcope C, et al. Effects
of alcohol consumption on plasma and urinary
hormone concentrations in premenopausal women.
J Natl Cancer Inst. 1993;85(9):722–727.
55. Sarkola T, Mäkisalo H, Fukunaga T, Eriksson
CJ. Acute effect of alcohol on estradiol, estrone,
progesterone, prolactin, cortisol, and luteinizing
hormone in premenopausal women. Alcohol Clin
Exp Res. 1999;23(6):976–982.
56. Gavaler JS, Van Thiel DH. The association between
moderate alcoholic beverage consumption and serum
estradiol and testosterone levels in normal postmeno-
pausal women: relationship to the literature. Alcohol
Clin Exp Res. 1992;16(1):87–92.
374.e2 Part I: Science and Clinical Oncology
57. Sarkola T, Fukunaga T, Mäkisalo H, Peter Eriksson
CJ. Acute effect of alcohol on androgens in
premenopausal women. Alcohol. 2000;35(1):84–90.
58. Scoccianti C, Lauby-Secretan B, Bello PY, Chajes
V, Romieu I. Female breast cancer and alcohol
consumption: a review of the literature. Am J Prev
Med. 2014;46(3 suppl 1):S16–S25.
59. Liu Y, Nguyen N, Colditz GA. Links between
alcohol consumption and breast cancer: a look at the
evidence. Womens Health (Lond). 2015;11(1):65–77.
60. Mason JB, Tang SY. Folate status and colorectal
cancer risk: a 2016 update. Mol Aspects Med. 2017;53:
73–79.
61. Nan H, Lee JE, Rimm EB, Fuchs CS, Giovannucci
EL, Cho E. Prospective study of alcohol consumption
and the risk of colorectal cancer before and after
folic acid fortification in the United States. Ann
Epidemiol. 2013;23(9):558–563.
62. Kantor ED, Giovannucci EL. Gene-diet interactions
and their impact on colorectal cancer risk. Curr Nutr
Rep. 2015;4(1):13–21.
63. AICR / WCRF. Recommendations for Cancer Preven-
tion; 2016. http://www.aicr.org/reduce-your-cancer
-risk/recommendations-for-cancer-prevention/.
64. Kushi L, Doyle C, McCullough M, et al. American
Cancer Society Guidelines on nutrition and physical
activity for cancer prevention: reducing the risk
of cancer with healthy food choices and physical
activity. CA Cancer J Clin. 2012;62(1):30–67.
65. Moyer VA, Force PST. Screening and behavioral
counseling interventions in primary care to reduce
alcohol misuse: U.S. preventive services task force
recommendation statement. Ann Intern Med. 2013;
159(3):210–218.
66. Wagenaar AC, Salois MJ, Komro KA. Effects of
beverage alcohol price and tax levels on drinking:
a meta-analysis of 1003 estimates from 112 studies.
Addiction. 2009;104(2):179–190.
67. Wagenaar AC, Tobler AL, Komro KA. Effects of
alcohol tax and price policies on morbidity and
mortality: a systematic review. Am J Public Health.
2010;100(11):2270–2278.
68. Ogden C, Carroll M, Kit B, Flegal K. Prevalence of
childhood and adult obesity in the United States,
2011-2012. JAMA. 2014;311(8):806–814.
69. Lauby-Secretan B, Scoccianti C, Loomis D, et al.
Body fatness and cancer- viewpoint of the IARC
working group. N Engl J Med. 2016;375(8):794–798.
70. Birks S, Peeters A, Backholer K, O’Brien P, Brown
W. A systematic review of the impact of weight
loss on cancer incidence and mortality. Obes Rev.
2012;13(10):868–891.
71. Esposito K, Chiodini P, Colao A, Lenzi A, Giudliano
D. Metabolic syndrome and risk of cancer: a system-
atic review and meta analysis. Diabetes Care. 2012;
35:2402–2411.
72. Micucci C, Valli D, Matacchione G, Catalano A.
Current perspectives between metabolic syndrome
and cancer. Oncotarget. 2016.
73. Uzunlulu M, Ozge T, Oguz A. Association between
metabolic syndrome and cancer. Ann Nutr Metab.
2016;68:173–179.
74. Despres J, Lemieux I. Abdominal obesity and meta-
bolic syndrome. Nature. 2006;444(14):881–887.
75. Doyle S, Donohoe C, Lysaght J, Reynolds J. Visceral
obesity, metabolic syndrome, insulin resistance and
cancer. Proc Nutr Soc. 2012;71(1):181–189.
76. Donohoe C, Doyle S, Reynolds J. Visceral adiposity,
insulin resistance and cancer risk. Diabetol Metab
Syndr. 2011;3(12).
77. Britton KA, Massaro JM, Murabito JM, Kreger
BE, Hoffmann U, Fox CS. Body fat distribution,
incident cardiovascular disease, cancer, and all-cause
mortality. J Am Coll Cardiol. 2013;62(10):921–925.
78. Murphy RA, Bureyko TF, Miljkovic I, et al. Associa-
tion of total and computed tomographic measures
of regional adiposity with incident cancer risk: a
prospective population-based study of older adults.
Appl Physiol Nutr Metab. 2014;39(6):987–992.
79. Donohoe C, Lysaght J, O’Sullivan J, Reynolds
J. Emerging concepts linking obesity with the
hallmarks of cancer. Trends Endocrinol Metab. 2017;
28(1):46–62.
80. Iyengar NM, Hudis CA, Dannenberg AJ. Obesity
and cancer: local and systemic mechanisms. Annu
Rev Med. 2015;66:297–309.
81. Teoh S, Das S. Tumour biology of obesity-related
cancers: understanding the molecular concept for
better diagnosis and treatment. Tumor Biol. 2015.
82. Pescatello L, Arena R, Riebe D, Thompson P, eds.
ACSM’s Guidelines for Exercise Testing and Prescrip-
tion. 9th ed. Baltimore, MD: Lippincott, Williams
& Wilkins; 2013.
83. Abioye AI, Odesanya MO, Abioye AI, Ibrahim N.
Physical activity and risk of gastric cancer: a meta-
analysis of observational studies. Br J Sports Med.
2015;49:224–229.
84. Friedenreich C. Physical activity and breast cancer:
review of the epidemiologic evidence and biologic
mechanisms. Recent Results Cancer Res. 2011;188:
125–139.
85. Gonçalves A, Dantas Florencio G, Maisonnette de
Atayde Silva M, Cobucci R, Giraldo P, Cote N.
Effect of physical activity on breast cancer preven-
tion: a systematic review. J Phys Act Health. 2014;
11(2):445–454.
86. Lakoski S, Willis B, Barlow C, et al. Midlife
cardiorespiratory fitness, incident cancer, and survival
after cancer in men: the Cooper Center Longitudinal
Study. JAMA Oncol. 2015;1(2):231–237.
87. Wu Y, Zhang D, Kang S. Physical activity and risk of
breast cancer: a meta-analysis of prospective studies.
Cancer Res Treat. 2013;137:869–882.
88. Sanchis-Gomar F, Lucia A, Yvert T, et al. Physical
inactivity and low fitness deserve more attention
to alter cancer risk and prognosis. Cancer Prev Res
(Phila). 2015;8(2):105–110.
89. Lee I, Shiroma E, Lobelo F, et al. Effect of physical
inactivity on major non-communicable disease
worldwide: an analysis of burden of disease and
life expectancy. Lancet. 2012;380:219–229.
90. Schmid D, Leitzmann M. Television viewing and
time spent sedentary in relation to cancer risk: a
meta-analysis. J Natl Cancer Inst. 2014;106(7).
91. Nomura S, Dash C, Rosenberg L, Palmer J, Adams-
Campbell L. Sedentary time and breast cancer
incidence in African American women. Cancer
Causes Control. 2016;27:1239–1252.
92. Giannopoulou I, Ploutz-Snyder L, Carhart R,
et al. Exercise is required for visceral fat loss in
postmenopausal women with type 2 diabetes.
J Clin Endocrinol Metab. 2005;90(3):1511–1518.
93. Hallsworth K, Thoma C, Hollingsworth K, et al.
Modified high-intensity interval training reduces liver
fat and improves cardiac function in non-alcoholic
fatty liver disease: a randomized controlled trial.
Clin Sci. 2015;129(12):1097–1105.
94. Ismail I, Keating S, Baker M, Johnson N. A
systematic review and meta-analysis of the effect
of aerobic vs. resistance exercise training on visceral
fat. Obes Rev. 2012;13:68–91.
95. Keating S, Hackett D, Parker H, et al. Effect of
aerobic exercise training dose on liver fat and visceral
adiposity. J Hepatol. 2015;63(1):174–182.
96. Lee S, Kuk J, Davidson L, et al. Exercise without
weight loss is an effective strategy for obesity reduc-
tion in obese individuals with and without Type 2
diabetes. J Appl Physiol. 2005;99(3):1220–1225.
97. Ross R, Janssen I, Dawson J, et al. Exercise-induced
reduction in obesity and insulin resistance in women:
a randomized controlled trial. Obes Res. 2004;
12:789–798.
98. Thomas E, Brynes A, McCarthy J, et al. Preferen-
tial loss of visceral fat following aerobic exercise,
measured by magnetic resonance imaging. Lipids.
2000;35(7):769–776.
99. Johnson N, Sachinwalla T, Walton D, et al. Aerobic
exercise training reduces hepatic and visceral lipids
in obese individuals without weight loss. Hepatology.
2009;50:1102–1112.
100. Arner P, Kriegholm E, Engfeldt P, Bolinder J.
Adrenergic regulation of lipolysis in situ at rest and
during exercise. J Clin Invest. 1990;85:893–898.
101. Horowitz J, Leone T, Feng W, Kelly D, Klein S.
Effect of endurance training on lipid metabolism
in women: a potential role for PPAR alpha in
the metabolic response to training. Am J Physiol
Endocrinol Metab. 2000;2079:E348–E355.
102. Sturgeon K, Digiovanni L, Good J, et al. Exercise-
induced dose-response alterations in adiponectin and
leptin levels are dependent on body fat changes in
women at risk for breast cancer. Cancer Epidemiol
Biomarkers Prev. 2016;25(8):1195–1200.
103. Attie A, Scherer P. Adipocyte metabolism and obesity.
J Lipid Res. 2009;50:S395–S399.
104. Richter E, Hargreaves M. Exercise, Glut 4, and
skeletal muscle glucose uptake. Physiol Rev. 2013;93:
993–1017.
105. Goh J, Niksirat N, Campbell K. Exercise training and
immune crosstalk in breast cancer microenvironment:
exploring the paradigms of the exercise-induced
immune modulation and exercise-induced myokines.
Am J Transl Res. 2014;6(5):422–438.
106. Aoi W, Naito Y, Takagi T, et al. A novel myokine,
secreted protein acidic and rich in cysteine (SPARC),
suppresses colon tumorigenesis via regular exercise.
Gut. 2013;62(6):882–889.
107. Gannon N, Vaughan R, Garcia-Smith R, Bisoffi
M, Trujillo K. Effects of the exercise-inducible
myokine irisin on malignant and non-malignant
breast epithelial cell behavior in vitro. Int J Cancer.
2015;136(4):E197–E202.
108. Hojman P, Dethlefsen C, Brandt C, Hansen J, Ped-
ersen L, Pedersen B. Exercise-induced muscle-derived
cytokines inhibit mammary cancer cell growth. Am J
Physiol Endocrinol Metab. 2011;301(3):E504–E510.
109. Idorn M, Hojman P. Exercise-dependent regulation
of NK cells in cancer protection. Trends Mol Med.
2016;22(7):565–577.
110. Idorn M, Thor Straten P. Exercise: a new role for
an old tool. Mol Cell Oncol. 2016;3(4):e1163005.
111. Pedersen L, Idorn M, Olofsson G, et al. Voluntary
running suppresses tumor growth through epineph-
rine- and IL-6-dependent NK cell mobilization and
redistribution. Cell Metab. 2016;23(3):554–562.
112. Alexander D, Weed D, Miller P, Mohamed M. Red
meat and colorectal cancer: a quantitative update
on the state of the epidemiologic science. J Am Coll
Nutr. 2015;34(6):521–543.
113. Bail J, Meneses K, Demark-Wahnefried W. Nutri-
tional status and diet in cancer prevention. Semin
Oncol Nurs. 2016;32(3):206–214.
114. Godos J, Bella F, Torrisi A, Sciacca S, Galvano F,
Grosso G. Dietary patterns and risk of colorectal
adenoma: a systematic review and meta-analysis of
observational studies. J Hum Nutr Diet. 2016.
115. Zhao Z, Yin Z, Pu Z, Zhao Q. Association
between consumption of red and processed meat
and pancreatic cancer risk, a systematic review and
meta-analysis. Clin Gastroenterol Hepatol. 2016.
116. Fang X, Wei J, He X, et al. Landscape of dietary
factors associated with risk of gastric cancer: a system-
atic review and dose-response meta-analysis of pro-
spective cohort studies. Eur J Cancer. 2015;51(18):
2820–2832.
117. Nöthlings U, Wilkens L, Murphy S, Hankin J,
Henderson B, Kolonel L. Meat and fat intake as
risk factors for pancreatic cancer: the multiethnic
cohort study. J Natl Cancer Inst. 2005;97(19):1458–
1465.
118. Fahrer J, Kaina B. Impact of DNA repair on
the dose-response of colorectal cancer formation
induced by dietary carcinogens. Food Chem Toxicol.
2016.
119. Cross A, Sinha R. Meat-related mutagens/carcinogens
in the etiology of colorectal cancer. Environ Mol
Mutagen. 2004;44(1):44–55.

120. Song P, Wu L, Guan W. Dietary nitrates, nitrites, and
nitrosamines intake and the risk of gastric cancer: a
meta-analysis. Nutrients. 2015;7(12):9872–9895.
121. Cheng X, Lin J, Tu S. Etiology and prevention of
gastric cancer. Gastrointest Tumors. 2016;3(1):25–
36.
122. Fox J, Dangler C, Taylor N, King A, Koh T, Wang
T. High-salt diet induces gastric epithelial hyperplasia
and parietal cell loss, and enhances Helicobacter pylori
colonization in C57BL/6 mice. Cancer Res. 1999;
59(19):4823–4828.
123. Godos J, Bella F, Sciacca S, Galvano F, Grosso G.
Vegetarianism and breast, colorectal and prostate
cancer risk: an overview and meta-analysis of cohort
studies. J Hum Nutr Diet. 2016.
124. Dinu M, Abbate R, Gensini G, Casini A, Sofi
F. Vegetarian, vegan diets and multiple health
outcomes: a systematic review with meta-analysis
of observational studies. Crit Rev Food Sci Nutr.
2016;6.
125. Schwingshackl L, Hoffmann G. Does a
Mediterranean-type diet reduce cancer risk? Curr
Nutr Rep. 2016;5:9–17.
126. Filomeno M, Bosetti C, Bidoli E, et al. Mediter-
ranean diet and risk of endometrial cancer: a pooled
analysis of three Italian case-control studies. Br J
Cancer. 2015;112(11):1816–1821.
127. Toledo E, Salas-Salvadó J, Donat-Vargas C, et al.
Mediterranean Diet and invasive breast cancer risk
among women at high cardiovascular risk in the
PREDIMED Trial: a randomized clinical trial. JAMA
Intern Med. 2015;175(11):1752–1760.
128. International Agency for Research on Cancer
(IARC). IARC Monographs on the Evaluation of
Carcinogenic Risks to Humans. IARC Monographs.
2009;100(Pt D).
129. Guy GP Jr, Machlin SR, Ekwueme DU, Yabroff
KR. Prevalence and costs of skin cancer treatment
in the U.S., 2002–2006 and 2007–2011. Am J Prev
Med. 2015;48(2):183–187.
130. U.S. Department of Health and Human Services.
The Surgeon General’s Call to Action to Prevent
Skin Cancer. Washington D.C. 2014.
131. Gandini S, Sera F, Cattaruzza MS, et al. Meta-analysis
of risk factors for cutaneous melanoma: II. Sun
exposure. Eur J Cancer. 2005;41(1):45–60.
132. Dennis LK, Vanbeek MJ, Beane Freeman LE,
Smith BJ, Dawson DV, Coughlin JA. Sunburns
and risk of cutaneous melanoma: does age matter?
A comprehensive meta-analysis. Ann Epidemiol.
2008;18(8):614–627.
133. Chang YM, Barrett JH, Bishop DT, et al. Sun
exposure and melanoma risk at different latitudes:
a pooled analysis of 5700 cases and 7216 controls.
Int J Epidemiol. 2009;38(3):814–830.
134. Macbeth AE, Grindlay DJ, Williams HC. What’s
new in skin cancer? An analysis of guidelines and
systematic reviews published in 2008-2009. Clin
Exp Dermatol. 2011;36(5):453–458.
135. Centers for Disease Control and Prevention (CDC).
Use of indoor tanning devices by adults—United
States, 2010. MMWR Morb Mortal Wkly Rep.
2012;61(18):323–326.
136. Ferrucci LM, Cartmel B, Molinaro AM, Leffell DJ,
Bale AE, Mayne ST. Indoor tanning and risk of
early-onset basal cell carcinoma. J Am Acad Dermatol.
2012;67(4):552–562.
137. Lazovich D, Vogel RI, Berwick M, Weinstock MA,
Anderson KE, Warshaw EM. Indoor tanning and
risk of melanoma: a case-control study in a highly
exposed population. Cancer Epidemiol Biomarkers
Prev. 2010;19(6):1557–1568.
138. Gandini S, Stanganelli I, Magi S, et al. Melanoma
attributable to sunbed use and tan seeking behav-
iours: an Italian survey. Eur J Dermatol. 2014;24(1):
35–40.
139. Boniol M, Autier P, Boyle P, Gandini S. Cutaneous
melanoma attributable to sunbed use: systematic
review and meta-analysis. BMJ. 2012;345:e4757.
Lifestyle and Cancer Prevention  •  CHAPTER 22 374.e3
140. Wehner MR, Shive ML, Chren MM, Han J, Qureshi
AA, Linos E. Indoor tanning and non-melanoma
skin cancer: systematic review and meta-analysis.
BMJ. 2012;345:e5909.
141. Colantonio S, Bracken MB, Beecker J. The associa-
tion of indoor tanning and melanoma in adults:
systematic review and meta-analysis. J Am Acad
Dermatol. 2014;70(5):847–857.e841–818.
142. International Agency for Research on Cancer (IARC).
Biological agents. Volume 100 B. A review of human
carcinogens. IARC Monogr Eval Carcinog Risks Hum.
2012;100(Pt B):1–441.
143. Moura Valejo Coelho M, Matos TR, Apetato M.
The dark side of the light: mechanisms of photo-
carcinogenesis. Clin Dermatol. 2016;34(5):563–570.
144. Kripke ML. Antigenicity of murine skin tumors
induced by ultraviolet light. J Natl Cancer Inst. 1974;
53(5):1333–1336.
145. Fisher MS, Kripke ML. Systemic alteration induced
in mice by ultraviolet light irradiation and its
relationship to ultraviolet carcinogenesis. Proc Natl
Acad Sci USA. 1977;74(4):1688–1692.
146. Kripke ML. Immunology and photocarcinogenesis.
New light on an old problem. J Am Acad Dermatol.
1986;14(1):149–155.
147. Centers for Disease Control and Prevention (CDC).
Sunburn and sun protective behaviors among adults
aged 18-29 years—United States, 2000–2010.
MMWR Morb Mortal Wkly Rep. 2012;61(18):
317–322.
148. Buller DB, Cokkinides V, Hall HI, et al. Prevalence
of sunburn, sun protection, and indoor tanning
behaviors among Americans: review from national
surveys and case studies of 3 states. J Am Acad
Dermatol. 2011;65(5 suppl 1):S114–S123.
149. van der Pols JC, Williams GM, Pandeya N, Logan
V, Green AC. Prolonged prevention of squamous
cell carcinoma of the skin by regular sunscreen use.
Cancer Epidemiol Biomarkers Prev. 2006;15(12):
2546–2548.
150. Green AC, Williams GM, Logan V, Strutton
GM. Reduced melanoma after regular sunscreen
use: randomized trial follow-up. J Clin Oncol.
2011;29(3):257–263.
151. Lin JS, Eder M, Weinmann S. Behavioral counseling
to prevent skin cancer: a systematic review for the
U.S. Preventive Services Task Force. Ann Intern Med.
2011;154(3):190–201.
152. Diffey B. New sunscreens and the precautionary
principle. JAMA Dermatol. 2016;152(5):511–512.
153. Thompson SC, Jolley D, Marks R. Reduction of
solar keratoses by regular sunscreen use. N Engl J
Med. 1993;329(16):1147–1151.
154. Naylor MF, Boyd A, Smith DW, Cameron GS,
Hubbard D, Neldner KH. High sun protection factor
sunscreens in the suppression of actinic neoplasia.
Arch Dermatol. 1995;131(2):170–175.
155. Darlington S, Williams G, Neale R, Frost C, Green
A. A randomized controlled trial to assess sunscreen
application and beta carotene supplementation in
the prevention of solar keratoses. Arch Dermatol.
2003;139(4):451–455.
156. Green A, Williams G, Neale R, et al. Daily sunscreen
application and betacarotene supplementation in
prevention of basal-cell and squamous-cell carci-
nomas of the skin: a randomised controlled trial.
Lancet. 1999;354(9180):723–729.
157. American Academy of Dermatology. Sunscreen
FAQs; 2017. https://www.aad.org/media/stats/
prevention-and-care/sunscreen-faqs. Accessed June
22, 2017.
158. Jansen R, Osterwalder U, Wang SQ, Burnett M,
Lim HW. Photoprotection: part II. Sunscreen:
development, efficacy, and controversies. J Am Acad
Dermatol. 2013;69(6):867.e861–814; quiz 881–862.
159. Domchek SM, Friebel TM, Singer CF, et al. Associa-
tion of risk-reducing surgery in BRCA1 or BRCA2
mutation carriers with cancer risk and mortality.
JAMA. 2010;304(9):967–975.
374.e3
160. Yuan JM, Stram DO, Arakawa K, Lee HP, Yu MC.
Dietary cryptoxanthin and reduced risk of lung
cancer: the Singapore Chinese Health Study. Cancer
Epidemiol Biomarkers Prev. 2003;12(9):890–898.
161. Albanes D, Heinonen OP, Taylor PR, et al. Alpha-
Tocopherol and beta-carotene supplements and
lung cancer incidence in the Alpha-Tocopherol,
Beta-Carotene Cancer Prevention Study: effects of
base-line characteristics and study compliance. J Natl
Cancer Inst. 1996;88(21):1560–1570.
162. Wright ME, Mayne ST, Stolzenberg-Solomon RZ,
et al. Development of a comprehensive dietary
antioxidant index and application to lung cancer
risk in a cohort of male smokers. Am J Epidemiol.
2004;160(1):68–76.
163. Albanes D, Heinonen OP, Huttunen JK, et al. Effects
of alpha-tocopherol and beta-carotene supplements
on cancer incidence in the Alpha-Tocopherol Beta-
Carotene Cancer Prevention Study. Am J Clin Nutr.
1995;62(6 suppl):1427S–1430S.
164. Alpha-Tocopherol, Beta Carotene Cancer Preven-
tion Study Group. The effect of vitamin E and
beta carotene on the incidence of lung cancer and
other cancers in male smokers. N Engl J Med. 1994;
330(15):1029–1035.
165. Omenn GS, Goodman GE, Thornquist MD, et al.
Risk factors for lung cancer and for intervention
effects in CARET, the Beta-Carotene and Retinol
Efficacy Trial. J Natl Cancer Inst. 1996;88(21):
1550–1559.
166. Costa A, Formelli F, Chiesa F, Decensi A, De Palo G,
Veronesi U. Prospects of chemoprevention of human
cancers with the synthetic retinoid fenretinide.
Cancer Res. 1994;54(7 suppl):2032s–2037s.
167. Omenn GS, Goodman GE, Thornquist MD, et al.
Effects of a combination of beta carotene and vitamin
A on lung cancer and cardiovascular disease. N Engl
J Med. 1996;334(18):1150–1155.
168. Schrauzer GN, White DA, Schneider CJ. Cancer
mortality correlation studies—III: statistical associa-
tions with dietary selenium intakes. Bioinorg Chem.
1977;7(1):23–31.
169. Clark LC, Cantor KP, Allaway WH. Selenium in
forage crops and cancer mortality in U.S. counties.
Arch Environ Health. 1991;46(1):37–42.
170. Clark LC, Combs GF Jr, Turnbull BW, et al. Effects
of selenium supplementation for cancer prevention
in patients with carcinoma of the skin. A randomized
controlled trial. Nutritional Prevention of Cancer
Study Group. JAMA. 1996;276(24):1957–1963.
171. Reid ME, Duffield-Lillico AJ, Garland L, Turnbull
BW, Clark LC, Marshall JR. Selenium supplementa-
tion and lung cancer incidence: an update of the
Nutritional Prevention of Cancer Trial. Cancer
Epidemiol Biomarkers Prev. 2002;11(11):1285–1291.
172. Lippman SM, Klein EA, Goodman PJ, et al. Effect of
selenium and vitamin E on risk of prostate cancer and
other cancers: the Selenium and Vitamin E Cancer
Prevention Trial (SELECT). JAMA. 2009;301(1):
39–51.
173. Klein EA, Thompson IM Jr, Tangen CM, et al.
Vitamin E and the risk of prostate cancer: the
Selenium and Vitamin E Cancer Prevention Trial
(SELECT). JAMA. 2011;306(14):1549–1556.
174. Karp DD, Lee SJ, Keller SM, et al. Randomized,
double-blind, placebo-controlled, phase III chemo-
prevention trial of selenium supplementation in
patients with resected stage I non–small-cell lung
cancer: ECOG 5597. J Clin Oncol. 2013;31(33):
4179–4187.
175. Veronesi G, Szabo E, Decensi A, et al. Randomized
phase II trial of inhaled budesonide versus placebo in
high-risk individuals with CT screen-detected lung
nodules. Cancer Prev Res (Phila). 2011;4(1):34–42.
176. Veronesi G, Lazzeroni M, Szabo E, et al. Long-term
effects of inhaled budesonide on screening-detected
lung nodules. Ann Oncol. 2015;26(5):1025–1030.
177. Limburg PJ, Mandrekar SJ, Aubry MC, et al.
Randomized phase II trial of sulindac for lung
374.e4 Part I: Science and Clinical Oncology
cancer chemoprevention. Lung Cancer. 2013;79(3):
254–261.
178. Keith RL, Blatchford PJ, Kittelson J, et al. Oral
iloprost improves endobronchial dysplasia in former
smokers. Cancer Prev Res (Phila). 2011;4(6):793–802.
179. Gray JE, Altiok S, Alexandrow MG, et al. Phase 2
randomized study of enzastaurin (LY317615) for
lung cancer prevention in former smokers. Cancer.
2013;119(5):1023–1032.
180. Lam S, McWilliams A, LeRiche J, MacAulay C,
Wattenberg L, Szabo E. A phase I study of myo-
inositol for lung cancer chemoprevention. Cancer
Epidemiol Biomarkers Prev. 2006;15(8):1526–1531.
181. Lam S, Mandrekar SJ, Gesthalter Y, et al. A ran-
domized phase IIb trial of myo-inositol in smokers
with bronchial dysplasia. Cancer Prev Res (Phila).
2016;9(12):906–914.
182. Lippman SM, Lee JJ, Karp DD, et al. Randomized
phase III intergroup trial of isotretinoin to prevent
second primary tumors in stage I non–small-cell lung
cancer. J Natl Cancer Inst. 2001;93(8):605–618.
183. Lee JJ, Feng L, Reshef DS, et al. Mortality in the
randomized, controlled lung intergroup trial of
isotretinoin. Cancer Prev Res (Phila). 2010;3(6):
738–744.
184. Kelly K, Kittelson J, Franklin WA, et al. A ran-
domized phase II chemoprevention trial of 13-CIS
retinoic acid with or without alpha tocopherol or
observation in subjects at high risk for lung cancer.
Cancer Prev Res (Phila). 2009;2(5):440–449.
185. Cardnell RJ, Feng Y, Diao L, et al. Proteomic markers
of DNA repair and PI3K pathway activation predict
response to the PARP inhibitor BMN 673 in small
cell lung cancer. Clin Cancer Res. 2013;19(22):
6322–6328.
186. Blumenthal GM, Zhang L, Zhang H, et al. Milestone
analyses of immune checkpoint inhibitors, targeted
therapy, and conventional therapy in metastatic
non–small cell lung cancer trials: a meta-analysis.
JAMA Oncol. 2017;e171029.
187. Hieda Y, Keyler DE, Vandevoort JT, et al. Active
immunization alters the plasma nicotine concentra-
tion in rats. J Pharmacol Exp Ther. 1997;283(3):
1076–1081.
188. Pentel PR, Malin DH, Ennifar S, et al. A nicotine
conjugate vaccine reduces nicotine distribution
to brain and attenuates its behavioral and cardio-
vascular effects in rats. Pharmacol Biochem Behav.
2000;65(1):191–198.
189. Tyagi A, Singh RP, Ramasamy K, et al. Growth
inhibition and regression of lung tumors by silibinin:
modulation of angiogenesis by macrophage-
associated cytokines and nuclear factor-kappaB and
signal transducers and activators of transcription 3.
Cancer Prev Res (Phila). 2009;2(1):74–83.
190. Yan Y, Wang Y, Tan Q, Lubet RA, You M. Efficacy
of deguelin and silibinin on benzo(a)pyrene-induced
lung tumorigenesis in A/J mice. Neoplasia. 2005;7(12):
1053–1057.
191. Hong WK, Endicott J, Itri LM, et al. 13-cis-retinoic
acid in the treatment of oral leukoplakia. N Engl J
Med. 1986;315(24):1501–1505.
192. Hong WK, Lippman SM, Itri LM, et al. Preven-
tion of second primary tumors with isotretinoin in
squamous-cell carcinoma of the head and neck. N
Engl J Med. 1990;323(12):795–801.
193. Stich HF, Hornby AP, Mathew B, Sankaranaray-
anan R, Nair MK. Response of oral leukoplakias
to the administration of vitamin A. Cancer Lett.
1988;40(1):93–101.
194. Stich HF, Rosin MP, Hornby AP, Mathew B,
Sankaranarayanan R, Nair MK. Remission of
oral leukoplakias and micronuclei in tobacco/
betel quid chewers treated with beta-carotene and
with beta-carotene plus vitamin A. Int J Cancer.
1988;42(2):195–199.
195. Han J, Jiao L, Lu Y, Sun Z, Gu QM, Scanlon
KJ. Evaluation of N-4-(hydroxycarbophenyl)
retinamide as a cancer prevention agent and as a
cancer chemotherapeutic agent. In Vivo. 1990;4(3):
153–160.
196. Lippman SM, Batsakis JG, Toth BB, et al.
Comparison of low-dose isotretinoin with beta
carotene to prevent oral carcinogenesis. N Engl J
Med. 1993;328(1):15–20.
197. Chiesa F, Tradati N, Marazza M, et al. Fenretinide
(4-HPR) in chemoprevention of oral leukoplakia.
J Cell Biochem Suppl. 1993;17F:255–261.
198. Zaridze D, Evstifeeva T, Boyle P. Chemoprevention
of oral leukoplakia and chronic esophagitis in an
area of high incidence of oral and esophageal cancer.
Ann Epidemiol. 1993;3(3):225–234.
199. Pinto H, Li Y, Loprinzi C. Phase 3 trial of low-dose
13-cis-retinoic acid for prevention of second primary
cancers in stage I-II head and neck cancer—an
Eastern Cooperative Oncology Group study. Program
and abstracts of the 37th Annual Meeting of the
American Society of Clinical Oncology; May 12-15,
2001, 2001; San Francisco, California.
200. Khuri FR, Lee JJ, Lippman SM, et al. Randomized
phase III trial of low-dose isotretinoin for preven-
tion of second primary tumors in stage I and II
head and neck cancer patients. J Natl Cancer Inst.
2006;98(7):441–450.
201. Lee JJ, Wu X, Hildebrandt MA, et al. Global assess-
ment of genetic variation influencing response to
retinoid chemoprevention in head and neck cancer
patients. Cancer Prev Res (Phila). 2011;4(2):185–193.
202. Pinto H, Li Y, Loprinzi C, Phase III. Trial of low-dose
13-cis-retinoic acid for prevention of second primary
cancers in stage I-II head and neck cancer—an
Eastern Cooperative Oncology Group Study. Proc
Am Soc Clin Oncol. 2001.
203. Grommes C, Landreth GE, Heneka MT. Antineo-
plastic effects of peroxisome proliferator-activated
receptor gamma agonists. Lancet Oncol. 2004;5(7):
419–429.
204. Ondrey F. Peroxisome proliferator-activated recep-
tor gamma pathway targeting in carcinogenesis:
implications for chemoprevention. Clin Cancer
Res. 2009;15(1):2–8.
205. Yki-Jarvinen H. Thiazolidinediones. N Engl J Med.
2004;351(11):1106–1118.
206. Nissen SE, Nicholls SJ, Wolski K, et al. Comparison
of pioglitazone vs glimepiride on progression of
coronary atherosclerosis in patients with type 2
diabetes: the PERISCOPE randomized controlled
trial. JAMA. 2008;299(13):1561–1573.
207. William WN Jr, Papadimitrakopoulou V, Lee JJ, et al.
Erlotinib and the risk of oral cancer: the Erlotinib
Prevention of Oral Cancer (EPOC) randomized
clinical trial. JAMA Oncol. 2016;2(2):209–216.
208. Armstrong WB, Taylor TH, Kennedy AR, et al.
Bowman Birk inhibitor concentrate and oral
leukoplakia: a randomized phase IIb trial. Cancer
Prev Res (Phila). 2013;6(5):410–418.
209. Monteiro de Oliveira Novaes JA, William WN Jr.
Prognostic factors, predictive markers and cancer
biology: the triad for successful oral cancer chemo-
prevention. Future Oncol. 2016;12(20):2379–2386.
210. William WN, Uraoka N, Peng SA, et al. Immune
profiling of oral pre-malignant lesions (OPLs): an
Erlotinib Prevention of Oral Cancer (EPOC) study
biobank analysis. J Clin Oncol. 2017;2017.
211. Munoz N, Wahrendorf J, Bang LJ, et al. No effect
of riboflavine, retinol, and zinc on prevalence of
precancerous lesions of oesophagus. Randomised
double-blind intervention study in high-risk popula-
tion of China. Lancet. 1985;2(8447):111–114.
212. Munoz N, Hayashi M, Bang LJ, Wahrendorf J,
Crespi M, Bosch FX. Effect of riboflavin, retinol, and
zinc on micronuclei of buccal mucosa and of esopha-
gus: a randomized double-blind intervention study
in China. J Natl Cancer Inst. 1987;79(4):687–691.
213. Wahrendorf J, Munoz N, Lu JB, Thurnham DI,
Crespi M, Bosch FX. Blood, retinol and zinc
riboflavin status in relation to precancerous lesions of
the esophagus: findings from a vitamin intervention
trial in the People’s Republic of China. Cancer Res.
1988;48(8):2280–2283.
214. Lin P, Zhang J, Rong Z, et al. Studies on medi-
camentous inhibitory therapy for esophageal
precancerous lesions—3- and 5-year inhibitory
effects of antitumor-B, retinamide and riboflavin.
Proc Chin Acad Med Sci Peking Union Med Coll.
1990;5(3):121–129.
215. Li JY, Taylor PR, Li B, et al. Nutrition intervention
trials in Linxian, China: multiple vitamin/mineral
supplementation, cancer incidence, and disease-
specific mortality among adults with esophageal dys-
plasia. J Natl Cancer Inst. 1993;85(18):1492–1498.
216. Mark SD, Liu SF, Li JY, et al. The effect of vitamin
and mineral supplementation on esophageal cytology:
results from the Linxian Dysplasia Trial. Int J Cancer.
1994;57(2):162–166.
217. Limburg PJ, Wei W, Ahnen DJ, et al. Randomized,
placebo-controlled, esophageal squamous cell cancer
chemoprevention trial of selenomethionine and
celecoxib. Gastroenterology. 2005;129(3):863–873.
218. Gordon V, Jankowski J. Chemoprevention in Bar-
rett’s oesophagus. Best Pract Res Clin Gastroenterol.
2011;25(4–5):569–579.
219. Barrett NR. The lower esophagus lined by columnar
epithelium. Surgery. 1957;41(6):881–894.
220. Spechler SJ, Goyal RK. The columnar-lined
esophagus, intestinal metaplasia, and Norman
Barrett. Gastroenterology. 1996;110(2):614–621.
221. Haggitt RC. Barrett’s esophagus, dysplasia, and
adenocarcinoma. Hum Pathol. 1994;25(10):982–
993.
222. Sampliner RE. Effect of up to 3 years of high-dose
lansoprazole on Barrett’s esophagus. Am J Gastroen-
terol. 1994;89(10):1844–1848.
223. Lightdale CJ. Photodynamic therapy: a new light
on Barrett’s esophagus. Gastrointest Endosc. 1995;
42(1):96–98.
224. Overholt BF, Panjehpour M. Photodynamic therapy
for Barrett’s esophagus: clinical update. Am J
Gastroenterol. 1996;91(9):1719–1723.
225. Ouatu-Lascar R, Fitzgerald RC, Triadafilopoulos
G. Differentiation and proliferation in Barrett’s
esophagus and the effects of acid suppression.
Gastroenterology. 1999;117(2):327–335.
226. Cooper BT, Chapman W, Neumann CS, Gearty
JC. Continuous treatment of Barrett’s oesophagus
patients with proton pump inhibitors up to 13 years:
observations on regression and cancer incidence.
Aliment Pharmacol Ther. 2006;23(6):727–733.
227. Souza RF, Shewmake K, Beer DG, Cryer B, Spechler
SJ. Selective inhibition of cyclooxygenase-2 suppresses
growth and induces apoptosis in human esophageal
adenocarcinoma cells. Cancer Res. 2000;60(20):
5767–5772.
228. Kaur BS, Khamnehei N, Iravani M, Namburu
SS, Lin O, Triadafilopoulos G. Rofecoxib inhibits
cyclooxygenase 2 expression and activity and reduces
cell proliferation in Barrett’s esophagus. Gastroenterol-
ogy. 2002;123(1):60–67.
229. Corley DA, Kerlikowske K, Verma R, Buffler P.
Protective association of aspirin/NSAIDs and
esophageal cancer: a systematic review and meta-
analysis. Gastroenterology. 2003;124(1):47–56.
230. Abnet CC, Freedman ND, Kamangar F, Leitzmann
MF, Hollenbeck AR, Schatzkin A. Non-steroidal
anti-inflammatory drugs and risk of gastric and
oesophageal adenocarcinomas: results from a cohort
study and a meta-analysis. Br J Cancer. 2009;100(3):
551–557.
231. Heath EI, Canto MI, Piantadosi S, et al. Second-
ary chemoprevention of Barrett’s esophagus with
celecoxib: results of a randomized trial. J Natl Cancer
Inst. 2007;99(7):545–557.
232. Shar AO, Gaudard MA, Heath EI, Forastiere AA,
Yang VW, Sontag SJ. Modeling using baseline
characteristics in a small multicenter clinical trial for
Barrett’s esophagus. Contemp Clin Trials. 2009;30(1):
2–7.

233. Falk GW, Buttar NS, Foster NR, et al. A combination
of esomeprazole and aspirin reduces tissue concentra-
tions of prostaglandin E(2) in patients with Barrett’s
esophagus. Gastroenterology. 2012;143(4):917–926.
e911.
234. Vaughan TL, Dong LM, Blount PL, et al. Non-
steroidal anti-inflammatory drugs and risk of
neoplastic progression in Barrett’s oesophagus: a pro-
spective study. Lancet Oncol. 2005;6(12):945–952.
235. Anderson LA, Johnston BT, Watson RG, et al.
Nonsteroidal anti-inflammatory drugs and the
esophageal inflammation-metaplasia-adenocarcinoma
sequence. Cancer Res. 2006;66(9):4975–4982.
236. Nguyen DM, Richardson P, El-Serag HB. Medica-
tions (NSAIDs, statins, proton pump inhibitors)
and the risk of esophageal adenocarcinoma in
patients with Barrett’s esophagus. Gastroenterology.
2010;138(7):2260–2266.
237. Jayaprakash V, Menezes RJ, Javle MM, et al. Regular
aspirin use and esophageal cancer risk. Int J Cancer.
2006;119(1):202–207.
238. Buttar NS, Wang KK, Anderson MA, et al. The
effect of selective cyclooxygenase-2 inhibition in
Barrett’s esophagus epithelium: an in vitro study.
J Natl Cancer Inst. 2002;94(6):422–429.
239. Jankowski JA, Hooper PA. Chemoprevention in
Barrett’s esophagus: a pill a day? Gastrointest Endosc
Clin N Am. 2011;21(1):155–170.
240. Das D, Chilton AP, Jankowski JA. Chemoprevention
of oesophageal cancer and the AspECT trial. Recent
Results Cancer Res. 2009;181:161–169.
241. Sanders DSA, Grabsch H, Harrison R, et al. Compar-
ing virtual with conventional microscopy for the
consensus diagnosis of Barrett’s neoplasia in the
AspECT Barrett’s chemoprevention trial pathology
audit. Histopathology. 2012;61(5):795–800.
242. Janakiram NB, Rao CV. The role of inflammation
in colon cancer. Adv Exp Med Biol. 2014;816:25–52.
243. Knekt P, Aromaa A, Maatela J, et al. Serum vitamin
E, serum selenium and the risk of gastrointestinal
cancer. Int J Cancer. 1988;42(6):846–850.
244. Knekt P, Aromaa A, Maatela J, et al. Serum
selenium and subsequent risk of cancer among
Finnish men and women. J Natl Cancer Inst. 1990;
82(10):864–868.
245. Clark LC, Hixson LJ, Combs GF Jr, Reid ME,
Turnbull BW, Sampliner RE. Plasma selenium
concentration predicts the prevalence of colorectal
adenomatous polyps. Cancer Epidemiol Biomarkers
Prev. 1993;2(1):41–46.
246. Lee YW, Klein CB, Kargacin B, et al. Carcinogenic
nickel silences gene expression by chromatin con-
densation and DNA methylation: a new model for
epigenetic carcinogens. Mol Cell Biol. 1995;15(5):
2547–2557.
247. Duffield-Lillico AJ, Reid ME, Turnbull BW,
et al. Baseline characteristics and the effect of
selenium supplementation on cancer incidence in
a randomized clinical trial: a summary report of
the Nutritional Prevention of Cancer Trial. Cancer
Epidemiol Biomarkers Prev. 2002;11(7):630–639.
248. Lance P, Alberts DS, Thompson PA, et  al.
Colorectal adenomas in participants of the SELECT
randomized trial of selenium and vitamin E for
prostate cancer prevention. Cancer Prev Res (Phila).
2017;10(1):45–54.
249. Vinceti M, Dennert G, Crespi CM, et al. Selenium
for preventing cancer. Cochrane Database Syst Rev.
2014;(3):CD005195.
250. Stranges S, Marshall JR, Natarajan R, et al. Effects
of long-term selenium supplementation on the
incidence of type 2 diabetes: a randomized trial.
Ann Intern Med. 2007;147(4):217–223.
251. Goodman PJ, Tangen CM, Darke AK, et al. Oppor-
tunities and challenges in incorporating ancillary
studies into a cancer prevention randomized clinical
trial. Trials. 2016;17:400.
252. Takata Y, Kristal AR, King IB, et al. Serum
selenium, genetic variation in selenoenzymes, and
Lifestyle and Cancer Prevention  •  CHAPTER 22 374.e5
risk of colorectal cancer: primary analysis from the
Women’s Health Initiative Observational Study and
meta-analysis. Cancer Epidemiol Biomarkers Prev.
2011;20(9):1822–1830.
253. Thompson P, Roe DJ, Fales L, et al. Design and
baseline characteristics of participants in a phase
III randomized trial of celecoxib and selenium for
colorectal adenoma prevention. Cancer Prev Res
(Phila). 2012;5(12):1381–1393.
254. Arber N, Eagle CJ, Spicak J, et al. Celecoxib for
the prevention of colorectal adenomatous polyps.
N Engl J Med. 2006;355(9):885–895.
255. Bertagnolli MM, Eagle CJ, Zauber AG, et al.
Celecoxib for the prevention of sporadic colorectal
adenomas. N Engl J Med. 2006;355(9):873–884.
256. Solomon SD, Wittes J, Finn PV, et al. Cardiovascular
risk of celecoxib in 6 randomized placebo-controlled
trials: the cross trial safety analysis. Circulation.
2008;117(16):2104–2113.
257. Thompson PA, Ashbeck EL, Roe DJ, et al. Selenium
Supplementation for prevention of colorectal
adenomas and risk of associated type 2 diabetes.
J Natl Cancer Inst. 2016;108(12).
258. Cook NR, Lee IM, Gaziano JM, et al. Low-dose
aspirin in the primary prevention of cancer: the
Women’s Health Study: a randomized controlled
trial. JAMA. 2005;294(1):47–55.
259. Sturmer T, Glynn RJ, Lee IM, Manson JE, Buring JE,
Hennekens CH. Aspirin use and colorectal cancer:
post-trial follow-up data from the Physicians’ Health
Study. Ann Intern Med. 1998;128(9):713–720.
260. Cook NR, Lee IM, Zhang SM, Moorthy MV, Buring
JE. Alternate-day, low-dose aspirin and cancer risk:
long-term observational follow-up of a randomized
trial. Ann Intern Med. 2013;159(2):77–85.
261. Burn J, Bishop DT, Chapman PD, et al. A random-
ized placebo-controlled prevention trial of aspirin
and/or resistant starch in young people with familial
adenomatous polyposis. Cancer Prev Res (Phila).
2011;4(5):655–665.
262. Burn J, Bishop DT, Mecklin JP, et al. Effect of aspirin
or resistant starch on colorectal neoplasia in the Lynch
syndrome. N Engl J Med. 2008;359(24):2567–2578.
263. Burn J, Gerdes AM, Macrae F, et al. Long-term
effect of aspirin on cancer risk in carriers of
hereditary colorectal cancer: an analysis from the
CAPP2 randomised controlled trial. Lancet. 2011;
378(9809):2081–2087.
264. Logan RF, Grainge MJ, Shepherd VC, Armitage
NC, Muir KR. ukCAP Trial Group. Aspirin and
folic acid for the prevention of recurrent colorectal
adenomas. Gastroenterology. 2008;134(1):29–38.
265. Benamouzig R, Deyra J, Martin A, et al. Daily
soluble aspirin and prevention of colorectal adenoma
recurrence: one-year results of the APACC trial.
Gastroenterology. 2003;125(2):328–336.
266. Baron JA, Cole BF, Sandler RS, et al. A random-
ized trial of aspirin to prevent colorectal adenomas.
N Engl J Med. 2003;348(10):891–899.
267. Benamouzig R, Uzzan B, Deyra J, et al. Preven-
tion by daily soluble aspirin of colorectal adenoma
recurrence: 4-year results of the APACC randomised
trial. Gut. 2012;61(2):255–261.
268. Sandler RS, Halabi S, Baron JA, et al. A randomized
trial of aspirin to prevent colorectal adenomas in
patients with previous colorectal cancer. N Engl J
Med. 2003;348(10):883–890.
269. Flossmann E, Rothwell PM. British Doctors Aspirin
Trial and the UK-TIA Aspirin Trial. Effect of aspirin
on long-term risk of colorectal cancer: consistent
evidence from randomised and observational studies.
Lancet. 2007;369(9573):1603–1613.
270. Drew DA, Chin SM, Gilpin KK, et al. ASPirin
Intervention for the REDuction of colorectal cancer
risk (ASPIRED): a study protocol for a randomized
controlled trial. Trials. 2017;18(1):50.
271. Chubak J, Kamineni A, Buist DSM, Anderson
ML, Whitlock EP. Aspirin use for the prevention
of colorectal cancer: an updated systematic evidence
374.e5
review for the U.S. Preventive Services Task Force.
Rockville (MD) 2015.
272. Lotrionte M, Biasucci LM, Peruzzi M, Frati G,
Giordano A, Biondi-Zoccai G. Which aspirin dose
and preparation is best for the long-term prevention
of cardiovascular disease and cancer? Evidence from
a systematic review and network meta-analysis. Prog
Cardiovasc Dis. 2016;58(5):495–504.
273. Zhao TY, Tu J, Wang Y, et al. The efficacy of aspirin
in preventing the recurrence of colorectal adenoma:
a renewed meta-analysis of randomized trials. Asian
Pac J Cancer Prev. 2016;17(5):2711–2717.
274. Steinbach G, Lynch PM, Phillips RK, et al. The
effect of celecoxib, a cyclooxygenase-2 inhibitor,
in familial adenomatous polyposis. N Engl J Med.
2000;342(26):1946–1952.
275. Phillips RK, Wallace MH, Lynch PM, et al. A
randomised, double blind, placebo controlled study
of celecoxib, a selective cyclooxygenase 2 inhibitor,
on duodenal polyposis in familial adenomatous
polyposis. Gut. 2002;50(6):857–860.
276. Bertagnolli MM, Eagle CJ, Zauber AG, et al. Five-
year efficacy and safety analysis of the Adenoma
Prevention with Celecoxib Trial. Cancer Prev Res
(Phila). 2009;2(4):310–321.
277. Lynch PM, Ayers GD, Hawk E, et al. The safety and
efficacy of celecoxib in children with familial adeno-
matous polyposis. Am J Gastroenterol. 2010;105(6):
1437–1443.
278. Giovannucci E, Goldin B. The role of fat, fatty acids,
and total energy intake in the etiology of human
colon cancer. Am J Clin Nutr. 1997;66(6 suppl):
1564S–1571S.
279. Narisawa T, Magadia NE, Weisburger JH, Wynder
EL. Promoting effect of bile acids on colon carci-
nogenesis after intrarectal instillation of N-methyl-
N′-nitro-N-nitrosoguanidine in rats. J Natl Cancer
Inst. 1974;53(4):1093–1097.
280. Bautista D, Obrador A, Moreno V, et al. Ki-ras muta-
tion modifies the protective effect of dietary mono-
unsaturated fat and calcium on sporadic colorectal
cancer. Cancer Epidemiol Biomarkers Prev. 1997;6(1):
57–61.
281. Peters U, Chatterjee N, McGlynn KA, et al. Calcium
intake and colorectal adenoma in a US colorectal cancer
early detection program. Am J Clin Nutr. 2004;80(5):
1358–1365.
282. Kesse E, Boutron-Ruault MC, Norat T, Riboli E,
Clavel-Chapelon F, E3N Group. Dietary calcium,
phosphorus, vitamin D, dairy products and the risk
of colorectal adenoma and cancer among French
women of the E3N-EPIC prospective study. Int J
Cancer. 2005;117(1):137–144.
283. Martinez ME, Willett WC. Calcium, vitamin D,
and colorectal cancer: a review of the epidemio-
logic evidence. Cancer Epidemiol Biomarkers Prev.
1998;7(2):163–168.
284. Flood A, Peters U, Chatterjee N, Lacey JV Jr, Schairer
C, Schatzkin A. Calcium from diet and supplements
is associated with reduced risk of colorectal cancer
in a prospective cohort of women. Cancer Epidemiol
Biomarkers Prev. 2005;14(1):126–132.
285. McCullough ML, Robertson AS, Rodriguez C, et al.
Calcium, vitamin D, dairy products, and risk of
colorectal cancer in the Cancer Prevention Study
II Nutrition Cohort (United States). Cancer Causes
Control. 2003;14(1):1–12.
286. Terry P, Baron JA, Bergkvist L, Holmberg L, Wolk
A. Dietary calcium and vitamin D intake and risk
of colorectal cancer: a prospective cohort study in
women. Nutr Cancer. 2002;43(1):39–46.
287. Baron JA, Beach M, Mandel JS, et al. Calcium
supplements for the prevention of colorectal
adenomas. Calcium Polyp Prevention Study Group.
N Engl J Med. 1999;340(2):101–107.
288. Bonithon-Kopp C, Kronborg O, Giacosa A, Rath
U, Faivre J. Calcium and fibre supplementation
in prevention of colorectal adenoma recurrence: a
randomised intervention trial. European Cancer
374.e6 Part I: Science and Clinical Oncology
Prevention Organisation Study Group. Lancet. 2000;
356(9238):1300–1306.
289. Wactawski-Wende J, Kotchen JM, Anderson GL,
et al. Calcium plus vitamin D supplementation and
the risk of colorectal cancer. N Engl J Med. 2006;
354(7):684–696.
290. Cauley JA, Chlebowski RT, Wactawski-Wende J,
et al. Calcium plus vitamin D supplementation and
health outcomes five years after active intervention
ended: the Women’s Health Initiative. J Womens
Health (Larchmt). 2013;22(11):915–929.
291. Baron JA, Barry EL, Mott LA, et al. A trial of calcium
and vitamin D for the prevention of colorectal
adenomas. N Engl J Med. 2015;373(16):1519–1530.
292. Barry EL, Peacock JL, Rees JR, et al. Vitamin D
receptor genotype, vitamin D3 supplementation, and
risk of colorectal adenomas: a randomized clinical
trial. JAMA Oncol. 2017;3(5):628–635.
293. Bonovas S, Fiorino G, Lytras T, Malesci A, Danese
S. Calcium supplementation for the prevention of
colorectal adenomas: a systematic review and meta-
analysis of randomized controlled trials. World J
Gastroenterol. 2016;22(18):4594–4603.
294. MacLennan SC, MacLennan AH, Ryan P. Colorectal
cancer and oestrogen replacement therapy. A meta-
analysis of epidemiological studies. Med J Aust. 1995;
162(9):491–493.
295. Hebert-Croteau N. A meta-analysis of hormone
replacement therapy and colon cancer in women.
Cancer Epidemiol Biomarkers Prev. 1998;7(8):
653–659.
296. Grodstein F, Newcomb PA, Stampfer MJ. Postmeno-
pausal hormone therapy and the risk of colorectal
cancer: a review and meta-analysis. Am J Med.
1999;106(5):574–582.
297. Nanda K, Bastian LA, Hasselblad V, Simel DL.
Hormone replacement therapy and the risk of
colorectal cancer: a meta-analysis. Obstet Gynecol.
1999;93(5 Pt 2):880–888.
298. Rossouw JE, Anderson GL, Prentice RL, et al. Risks
and benefits of estrogen plus progestin in healthy
postmenopausal women: principal results from the
Women’s Health Initiative randomized controlled
trial. JAMA. 2002;288(3):321–333.
299. Tsilidis KK, Allen NE, Key TJ, et al. Menopausal
hormone therapy and risk of colorectal cancer in
the European Prospective Investigation into Cancer
and Nutrition. Int J Cancer. 2011;128(8):1881–
1889.
300. Chlebowski RT, Wactawski-Wende J, Ritenbaugh
C, et al. Estrogen plus progestin and colorectal
cancer in postmenopausal women. N Engl J Med.
2004;350(10):991–1004.
301. Prentice RL, Pettinger M, Beresford SA, et al.
Colorectal cancer in relation to postmenopausal
estrogen and estrogen plus progestin in the
Women’s Health Initiative clinical trial and obser-
vational study. Cancer Epidemiol Biomarkers Prev.
2009;18(5):1531–1537.
302. Anderson GL, Limacher M, Assaf AR, et al. Effects of
conjugated equine estrogen in postmenopausal women
with hysterectomy: the Women’s Health Initiative
randomized controlled trial. JAMA. 2004;291(14):
1701–1712.
303. Ritenbaugh C, Stanford JL, Wu L, et al. Conjugated
equine estrogens and colorectal cancer incidence and
survival: the Women’s Health Initiative randomized
clinical trial. Cancer Epidemiol Biomarkers Prev.
2008;17(10):2609–2618.
304. Lavasani S, Chlebowski RT, Prentice RL, et al.
Estrogen and colorectal cancer incidence and
mortality. Cancer. 2015;121(18):3261–3271.
305. Samadi AK, Bilsland A, Georgakilas AG, et al. A
multi-targeted approach to suppress tumor-promoting
inflammation. Semin Cancer Biol. 2015;35(suppl):
S151–S184.
306. Li Y, Zhang T. Targeting cancer stem cells by cur-
cumin and clinical applications. Cancer Lett. 2014;
346(2):197–205.
307. Sterverding D. Sleeping sickness and nagana disease
caused by Trypanosoma brucei. In: Marcondes CB,
ed. Arthropod Borne Diseases. Switzerland: Springer
International Publishing; 2017:292.
308. World Health Organization (WHO). WHO Model
List of Essential Medicines; 2015; 19. http://www.
who.int/medicines/publications/essentialmedicines/
EML_2015_FINAL_amended_NOV2015.pdf?ua=1.
309. Raul F. Revival of 2-(difluoromethyl)ornithine
(DFMO), an inhibitor of polyamine biosynthesis,
as a cancer chemopreventive agent. Biochem Soc
Trans. 2007;35(Pt 2):353–355.
310. Giardiello FM, Hamilton SR, Hylind LM, Yang
VW, Tamez P, Casero RA Jr. Ornithine decarboxylase
and polyamines in familial adenomatous polyposis.
Cancer Res. 1997;57(2):199–201.
311. Gerner EW, Meyskens FL Jr. Polyamines and cancer:
old molecules, new understanding. Nat Rev Cancer.
2004;4(10):781–792.
312. Wallace HM, Fraser AV. Inhibitors of poly-
amine metabolism: review article. Amino Acids.
2004;26(4):353–365.
313. Bassiri H, Benavides A, Haber M, Gilmour SK,
Norris MD, Hogarty MD. Translational development
of difluoromethylornithine (DFMO) for the treat-
ment of neuroblastoma. Transl Pediatr. 2015;4(3):
226–238.
314. Ye C, Geng Z, Dominguez D, et al. Targeting orni-
thine decarboxylase by alpha-difluoromethylornithine
inhibits tumor growth by impairing myeloid-derived
suppressor cells. J Immunol. 2016;196(2):915–923.
315. Meyskens FL Jr, McLaren CE, Pelot D, et al.
Difluoromethylornithine plus sulindac for the
prevention of sporadic colorectal adenomas: a
randomized placebo-controlled, double-blind trial.
Cancer Prev Res (Phila). 2008;1(1):32–38.
316. Zell JA, McLaren CE, Chen WP, Thompson
PA, Gerner EW, Meyskens FL. Ornithine
decarboxylase-1 polymorphism, chemoprevention
with eflornithine and sulindac, and outcomes among
colorectal adenoma patients. J Natl Cancer Inst. 2010;
102(19):1513–1516.
317. MacLean CH, Newberry SJ, Mojica WA, et al.
Effects of omega-3 fatty acids on cancer risk: a
systematic review. JAMA. 2006;295(4):403–415.
318. West NJ, Clark SK, Phillips RK, et al. Eicosa-
pentaenoic acid reduces rectal polyp number
and size in familial adenomatous polyposis. Gut.
2010;59(7):918–925.
319. Hull MA, Sandell AC, Montgomery AA, et al. A
randomized controlled trial of eicosapentaenoic acid
and/or aspirin for colorectal adenoma prevention
during colonoscopic surveillance in the NHS Bowel
Cancer Screening Programme (The seAFOod Polyp
Prevention Trial): study protocol for a randomized
controlled trial. Trials. 2013;14:237.
320. Hosono K, Endo H, Takahashi H, et al. Metfor-
min suppresses colorectal aberrant crypt foci in a
short-term clinical trial. Cancer Prev Res (Phila).
2010;3(9):1077–1083.
321. Higurashi T, Hosono K, Takahashi H, et al.
Metformin for chemoprevention of metachronous
colorectal adenoma or polyps in post-polypectomy
patients without diabetes: a multicentre double-
blind, placebo-controlled, randomised phase 3 trial.
Lancet Oncol. 2016;17(4):475–483.
322. Kimura T, McKolanis JR, Dzubinski LA, et al.
MUC1 vaccine for individuals with advanced
adenoma of the colon: a cancer immunoprevention
feasibility study. Cancer Prev Res (Phila). 2013;6(1):
18–26.
323. Parkin DM. The global health burden of infection-
associated cancers in the year 2002. Int J Cancer.
2006;118(12):3030–3044.
324. Zabala Torres B, Lucero Y, Lagomarcino AJ, et al.
Prevalence and dynamics of Helicobacter pylori
infection during childhood. Helicobacter. 2017.
325. Correa P, Fontham ET, Bravo JC, et al. Chemo-
prevention of gastric dysplasia: randomized trial of
antioxidant supplements and anti–Helicobacter pylori
therapy. J Natl Cancer Inst. 2000;92(23):1881–1888.
326. Wong BC, Lam SK, Wong WM, et al. Helicobacter
pylori eradication to prevent gastric cancer in a high-
risk region of China: a randomized controlled trial.
JAMA. 2004;291(2):187–194.
327. You WC, Brown LM, Zhang L, et al. Randomized
double-blind factorial trial of three treatments to
reduce the prevalence of precancerous gastric lesions.
J Natl Cancer Inst. 2006;98(14):974–983.
328. Sung JJ, Lin SR, Ching JY, et al. Atrophy and
intestinal metaplasia one year after cure of H.
pylori infection: a prospective, randomized study.
Gastroenterology. 2000;119(1):7–14.
329. Leung WK, Lin SR, Ching JY, et al. Factors pre-
dicting progression of gastric intestinal metaplasia:
results of a randomised trial on Helicobacter pylori
eradication. Gut. 2004;53(9):1244–1249.
330. Fukase K, Kato M, Kikuchi S, et al. Effect of eradica-
tion of Helicobacter pylori on incidence of metachro-
nous gastric carcinoma after endoscopic resection
of early gastric cancer: an open-label, randomised
controlled trial. Lancet. 2008;372(9636):392–397.
331. Mera R, Fontham ET, Bravo LE, et al. Long term
follow up of patients treated for Helicobacter pylori
infection. Gut. 2005;54(11):1536–1540.
332. Mera RM, Bravo LE, Camargo MC, et al. Dynamics
of Helicobacter pylori infection as a determinant of
progression of gastric precancerous lesions: 16-year
follow-up of an eradication trial. Gut. 2017.
333. Ma JL, Zhang L, Brown LM, et al. Fifteen-year
effects of Helicobacter pylori, garlic, and vitamin
treatments on gastric cancer incidence and mortality.
J Natl Cancer Inst. 2012;104(6):488–492.
334. Lau CS, Ward A, Chamberlain RS. Probiotics
improve the efficacy of standard triple therapy in the
eradication of Helicobacter pylori: a meta-analysis.
Infect Drug Resist. 2016;9:275–289.
335. Fock KM, Talley NJ, Fass R, et al. Asia-Pacific Con-
sensus on the management of gastroesophageal reflux
disease: update. J Gastroenterol Hepatol. 2008;23(1):
8–22.
336. O’Connor A, O’Morain CA, Ford AC. Popula-
tion screening and treatment of Helicobacter pylori
infection. Nat Rev Gastroenterol Hepatol. 2017;14(4):
230–240.
337. Dawsey SM, Mark SD, Taylor PR, Limburg PJ.
Gastric cancer and H. pylori. Gut. 2002;51(3):
457–458.
338. Kamangar F, Dawsey SM, Blaser MJ, et al. Oppos-
ing risks of gastric cardia and noncardia gastric
adenocarcinomas associated with Helicobacter pylori
seropositivity. J Natl Cancer Inst. 2006;98(20):1445–
1452.
339. Hansen S, Melby KK, Aase S, Jellum E, Vollset SE.
Helicobacter pylori infection and risk of cardia cancer
and non-cardia gastric cancer. A nested case-control
study. Scand J Gastroenterol. 1999;34(4):353–360.
340. Helicobacter and Cancer Collaborative Group.
Gastric cancer and Helicobacter pylori: a combined
analysis of 12 case control studies nested within
prospective cohorts. Gut. 2001;49(3):347–353.
341. Cavaleiro-Pinto M, Peleteiro B, Lunet N, Barros
H. Helicobacter pylori infection and gastric cardia
cancer: systematic review and meta-analysis. Cancer
Causes Control. 2011;22(3):375–387.
342. Carrera-Lasfuentes P, Lanas A, Bujanda L, et al.
Relevance of DNA repair gene polymorphisms
to gastric cancer risk and phenotype. Oncotarget.
2017;8(22):35848–35862.
343. Ma J, Wu D, Hu X, Li J, Cao M, Dong W. Associa-
tions between cytokine gene polymorphisms and
susceptibility to Helicobacter pylori infection and
Helicobacter pylori related gastric cancer, peptic ulcer
disease: a meta-analysis. PLoS ONE. 2017;12(4):
e0176463.
344. Melchiades JL, Zabaglia LM, Sallas ML, et al.
Polymorphisms and haplotypes of the interleukin
2 gene are associated with an increased risk of gastric

cancer. The possible involvement of Helicobacter
pylori. Cytokine. 2017;96:203–207.
345. Chang YW, Oh CH, Kim JW, et al. Combination
of Helicobacter pylori infection and the interleukin
8 -251 T > A polymorphism, but not the mannose-
binding lectin 2 codon 54 G > A polymorphism,
might be a risk factor of gastric cancer. BMC Cancer.
2017;17(1):388.
346. Zhang JZ, Liu CM, Peng HP, Zhang Y. Association
of genetic variations in IL-6/IL-6R pathway genes
with gastric cancer risk in a Chinese population.
Gene. 2017;623:1–4.
347. Ribeiro RX, Nascimento C, Silva A. Genotype
association Gstm1 Null and gastric cancer: evidence-
based meta-analysis. Arq Gastroenterol. 2017;
54(2):101–108.
348. Negovan A, Iancu M, Moldovan V, Mocan S, Banescu
C. The Interaction between GSTT1, GSTM1,
and GSTP1 Ile105Val gene polymorphisms and
environmental risk factors in premalignant gastric
lesions risk. Biomed Res Int. 2017;2017:7365080.
349. Cherati MR, Shokri-Shirvani J, Karkhah A, Rajabnia
R, Nouri HR. Helicobacter pylori cagL amino acid
polymorphism D58E59 pave the way toward
peptic ulcer disease while N58E59 is associated
with gastric cancer in north of Iran. Microb Pathog.
2017;107:413–418.
350. He BS, Sun HL, Xu T, et al. Association of genetic
polymorphisms in the LncRNAs with gastric cancer
risk in a Chinese population. J Cancer. 2017;8(4):
531–536.
351. Giraldi L, Michelazzo MB, Arzani D, Persiani R,
Pastorino R, Boccia S. MUC1, MUC5AC, and
MUC6 polymorphisms, Helicobacter pylori infec-
tion, and gastric cancer: a systematic review and
meta-analysis. Eur J Cancer Prev. 2017.
352. Tongtawee T, Bartpho T, Kaewpitoon S, et al.
Genetic polymorphisms in TLR1, TLR2, TLR4,
and TLR10 of Helicobacter pylori–associated gastritis:
a prospective cross-sectional study in Thailand. Eur
J Cancer Prev. 2017.
353. Pan KF, Zhang L, Gerhard M, et al. A large
randomised controlled intervention trial to prevent
gastric cancer by eradication of Helicobacter pylori in
Linqu County, China: baseline results and factors
affecting the eradication. Gut. 2016;65(1):9–18.
354. Yang P, Zhou Y, Chen B, et al. Aspirin use and the
risk of gastric cancer: a meta-analysis. Dig Dis Sci.
2010;55(6):1533–1539.
355. Rothwell PM, Fowkes FG, Belch JF, Ogawa H,
Warlow CP, Meade TW. Effect of daily aspirin on
long-term risk of death due to cancer: analysis of
individual patient data from randomised trials.
Lancet. 2011;377(9759):31–41.
355a. Huang XZ, Chen Y, Wu J, Zhang X, Wu CC,
Zhang CY, Sun SS, Chen WJ. Aspirin and non-
steroidal anti-inflammatory drugs use reduce
gastric cancer risk: A dose-response meta-analysis.
Oncotarget. 2017;8(3):4781–4795.
356. Wong BC, Zhang L, Ma JL, et al. Effects of
selective COX-2 inhibitor and Helicobacter pylori
eradication on precancerous gastric lesions. Gut.
2012;61(6):812–818.
357. Zhang Y, Zeng HM, Nie XR, et al. Alterations
of cyclooxygenase-2 methylation levels before and
after intervention trial to prevent gastric cancer
in a Chinese population. Cancer Prev Res (Phila).
2016;9(6):484–490.
358. Kong P, Wu R, Liu X, et al. The effects of anti-
inflammatory drug treatment in gastric cancer pre-
vention: an update of a meta-analysis. J Cancer. 2016;
7(15):2247–2257.
359. Liu CJ, Jeng YM, Chen CL, et al. Hepatitis B virus
basal core promoter mutation and DNA load cor-
relate with expression of hepatitis B core antigen
in patients with chronic hepatitis B. J Infect Dis.
2009;199(5):742–749.
360. Marcellin P. Hepatitis B and hepatitis C in. Liver
Int. 2009;29(suppl 1):1–8.
Lifestyle and Cancer Prevention  •  CHAPTER 22 374.e7
361. Lavanchy D. The global burden of hepatitis C. Liver
Int. 2009;29(suppl 1):74–81.
362. Benvegnu L, Fattovich G, Noventa F, et al. Concur-
rent hepatitis B and C virus infection and risk of
hepatocellular carcinoma in cirrhosis. A prospective
study. Cancer. 1994;74(9):2442–2448.
363. Chiaramonte M, Stroffolini T, Vian A, et al.
Rate of incidence of hepatocellular carcinoma in
patients with compensated viral cirrhosis. Cancer.
1999;85(10):2132–2137.
364. Ross RK, Yuan JM, Yu MC, et al. Urinary aflatoxin
biomarkers and risk of hepatocellular carcinoma.
Lancet. 1992;339(8799):943–946.
365. Wang P, Kang D, Cao W, Wang Y, Liu Z. Diabetes
mellitus and risk of hepatocellular carcinoma: a
systematic review and meta-analysis. Diabetes Metab
Res Rev. 2012;28(2):109–122.
366. Forner A, Llovet JM, Bruix J. Hepatocellular
carcinoma. Lancet. 2012;379(9822):1245–1255.
367. Leung N. HBV and liver cancer. Med J Malaysia.
2005;60(supplB):63–66.
368. Chang MH, Chen CJ, Lai MS, et al. Universal
hepatitis B vaccination in Taiwan and the incidence
of hepatocellular carcinoma in children. Taiwan
Childhood Hepatoma Study Group. N Engl J Med.
1997;336(26):1855–1859.
369. Schuppan D, Afdhal NH. Liver cirrhosis. Lancet.
2008;371(9615):838–851.
370. Turner PC, Sylla A, Gong YY, et al. Reduction
in exposure to carcinogenic aflatoxins by post-
harvest intervention measures in west Africa: a
community-based intervention study. Lancet. 2005;
365(9475):1950–1956.
371. El-Serag HB, Hampel H, Javadi F. The association
between diabetes and hepatocellular carcinoma: a
systematic review of epidemiologic evidence. Clin
Gastroenterol Hepatol. 2006;4(3):369–380.
372. Dunn BK, Kramer BS. Cancer prevention: lessons
learned and future directions. Trends Cancer. 2016;
2(12):713–722.
373. Glauert HP, Calfee-Mason K, Stemm DN, Tharappel
JC, Spear BT. Dietary antioxidants in the prevention
of hepatocarcinogenesis: a review. Mol Nutr Food
Res. 2010;54(7):875–896.
374. Chen HL, Chang MH, Ni YH, et al. Seroepidemiol-
ogy of hepatitis B virus infection in children: ten
years of mass vaccination in Taiwan. JAMA. 1996;
276(11):906–908.
375. Lee CL, Hsieh KS, Ko YC. Trends in the incidence of
hepatocellular carcinoma in boys and girls in Taiwan
after large-scale hepatitis B vaccination. Cancer
Epidemiol Biomarkers Prev. 2003;12(1):57–59.
376. Chang MH, Shau WY, Chen CJ, et al. Hepatitis B
vaccination and hepatocellular carcinoma rates in
boys and girls. JAMA. 2000;284(23):3040–3042.
377. Hung GY, Horng JL, Yen HJ, Lee CY, Lin LY.
Changing incidence patterns of hepatocellular
carcinoma among age groups in Taiwan. J Hepatol.
2015;63(6):1390–1396.
378. Qu C, Chen T, Fan C, et al. Efficacy of neonatal
HBV vaccination on liver cancer and other liver
diseases over 30-year follow-up of the Qidong
hepatitis B intervention study: a cluster randomized
controlled trial. PLoS Med. 2014;11(12):e1001774.
379. Chen T, Qu C, Yao H, et al. [Long-term efficacy
of neonatal hepatitis B vaccination against chronic
hepatitis B virus infection and chronic liver disease:
a cross-sectional study based on Qidong Hepatitis
B Intervention Study]. Zhonghua Liu Xing Bing Xue
Za Zhi. 2016;37(1):64–67.
380. Wang Y, Chen T, Lu LL, et al. Adolescent booster with
hepatitis B virus vaccines decreases HBV infection
in high-risk adults. Vaccine. 2017;35(7):1064–1070.
381. Roohvand F, Kossari N. Advances in hepatitis C
virus vaccines, part two: advances in hepatitis C
virus vaccine formulations and modalities. Expert
Opin Ther Pat. 2012;22(4):391–415.
382. Baumert TF, Schuster C, Cosset FL, et al. Address-
ing the next challenges: a summary of the 22nd
374.e7
international symposium on hepatitis C virus and
related viruses. J Hepatol. 2016;64(4):968–973.
383. Lau DT, Everhart J, Kleiner DE, et al. Long-term
follow-up of patients with chronic hepatitis B treated
with interferon alfa. Gastroenterology. 1997;113(5):
1660–1667.
384. Yang YF, Zhao W, Zhong YD, Xia HM, Shen L,
Zhang N. Interferon therapy in chronic hepatitis
B reduces progression to cirrhosis and hepatocel-
lular carcinoma: a meta-analysis. J Viral Hepat.
2009;16(4):265–271.
385. Yoshida H, Shiratori Y, Moriyama M, et al. Inter-
feron therapy reduces the risk for hepatocellular
carcinoma: national surveillance program of cirrhotic
and noncirrhotic patients with chronic hepatitis
C in Japan. IHIT Study Group. Inhibition of
Hepatocarcinogenesis by Interferon Therapy. Ann
Intern Med. 1999;131(3):174–181.
386. Imai Y, Kawata S, Tamura S, et al. Relation of
interferon therapy and hepatocellular carcinoma in
patients with chronic hepatitis C. Osaka Hepatocel-
lular Carcinoma Prevention Study Group. Ann Intern
Med. 1998;129(2):94–99.
387. Ikeda K, Saitoh S, Arase Y, et al. Effect of inter-
feron therapy on hepatocellular carcinogenesis in
patients with chronic hepatitis type C: a long-term
observation study of 1,643 patients using statistical
bias correction with proportional hazard analysis.
Hepatology. 1999;29(4):1124–1130.
388. Shindo M, Ken A, Okuno T. Varying incidence of
cirrhosis and hepatocellular carcinoma in patients
with chronic hepatitis C responding differently to
interferon therapy. Cancer. 1999;85(9):1943–1950.
389. Miyake Y, Takaki A, Iwasaki Y, Yamamoto K. Meta-
analysis: interferon-alpha prevents the recurrence
after curative treatment of hepatitis C virus–related
hepatocellular carcinoma. J Viral Hepat. 2010;17(4):
287–292.
390. Liaw YF, Sung JJ, Chow WC, et al. Lamivudine for
patients with chronic hepatitis B and advanced liver
disease. N Engl J Med. 2004;351(15):1521–1531.
391. Matsumoto A, Tanaka E, Rokuhara A, et al. Effi-
cacy of lamivudine for preventing hepatocellular
carcinoma in chronic hepatitis B: a multicenter
retrospective study of 2795 patients. Hepatol Res.
2005;32(3):173–184.
392. Ikeda K, Arase Y, Kobayashi M, et al. A long-term
glycyrrhizin injection therapy reduces hepatocellular
carcinogenesis rate in patients with interferon-
resistant active chronic hepatitis C: a cohort study
of 1249 patients. Dig Dis Sci. 2006;51(3):603–609.
393. Controlled trial of tamoxifen as adjuvant agent in
management of early breast cancer. Interim analysis
at four years by Nolvadex Adjuvant Trial Organisa-
tion. Lancet. 1983;1(8319):257–261.
394. Early Breast Cancer Trialists’ Collaborative Group.
Effects of adjuvant tamoxifen and of cytotoxic
therapy on mortality in early breast cancer. An
overview of 61 randomized trials among 28,896
women. N Engl J Med. 1988;319(26):1681–1692.
395. Cuzick J, Baum M. Tamoxifen and contralateral
breast cancer. Lancet. 1985;2(8449):282.
396. Fisher B, Costantino JP, Wickerham DL, et al.
Tamoxifen for prevention of breast cancer: report
of the National Surgical Adjuvant Breast and Bowel
Project P-1 Study. J Natl Cancer Inst. 1998;90(18):
1371–1388.
397. Fisher B. Tamoxifen for the prevention of breast
cancer: current status of the National Surgical
Adjuvant Breast and Bowel Project P-1 study. J Natl
Cancer Inst. 2005;97:1652–1662.
398. Powles TJ, Ashley S, Tidy A, Smith IE, Dowsett
M. Twenty-year follow-up of the Royal Marsden
randomized, double-blinded tamoxifen breast cancer
prevention trial. J Natl Cancer Inst. 2007;99(4):
283–290.
399. Veronesi U, Maisonneuve P, Rotmensz N, et al.
Tamoxifen for the prevention of breast cancer:
late results of the Italian Randomized Tamoxifen
374.e8 Part I: Science and Clinical Oncology
Prevention Trial among women with hysterectomy.
J Natl Cancer Inst. 2007;99(9):727–737.
400. Cuzick J, Forbes J, Edwards R, et al. First results
from the International Breast Cancer Intervention
Study (IBIS-I): a randomised prevention trial. Lancet.
2002;360(9336):817–824.
401. Cuzick J, Forbes JF, Sestak I, et al. Long-term results
of tamoxifen prophylaxis for breast cancer–96-month
follow-up of the randomized IBIS-I trial. J Natl
Cancer Inst. 2007;99(4):272–282.
402. Fisher B, Costantino JP, Wickerham DL, et al.
Tamoxifen for the prevention of breast cancer:
current status of the National Surgical Adjuvant
Breast and Bowel Project P-1 study. J Natl Cancer
Inst. 2005;97(22):1652–1662.
403. Powles T, Eeles R, Ashley S, et al. Interim analysis of
the incidence of breast cancer in the Royal Marsden
Hospital tamoxifen randomised chemoprevention
trial. Lancet. 1998;352(9122):98–101.
404. Veronesi U, Maisonneuve P, Costa A, et al. Preven-
tion of breast cancer with tamoxifen: preliminary
findings from the Italian randomised trial among
hysterectomised women. Italian Tamoxifen Preven-
tion Study. Lancet. 1998;352(9122):93–97.
405. Vogel VG, Costantino JP, Wickerham DL, et al.
Effects of tamoxifen vs raloxifene on the risk of
developing invasive breast cancer and other disease
outcomes: the NSABP Study of Tamoxifen and
Raloxifene (STAR) P-2 trial. JAMA. 2006;295(23):
2727–2741.
406. Vogel VG, Costantino JP, Wickerham DL, et al.
Update of the National Surgical Adjuvant Breast and
Bowel Project Study of Tamoxifen and Raloxifene
(STAR) P-2 Trial: preventing breast cancer. Cancer
Prev Res (Phila). 2010;3(6):696–706.
407. Cummings SR, Eckert S, Krueger KA, et al. The
effect of raloxifene on risk of breast cancer in
postmenopausal women: results from the MORE
randomized trial. Multiple Outcomes of Raloxifene
Evaluation. JAMA. 1999;281(23):2189–2197.
408. Ettinger B, Black DM, Mitlak BH, et al. Reduction
of vertebral fracture risk in postmenopausal women
with osteoporosis treated with raloxifene: results from
a 3-year randomized clinical trial. Multiple Outcomes
of Raloxifene Evaluation (MORE) Investigators.
JAMA. 1999;282(7):637–645.
409. Martino S, Cauley JA, Barrett-Connor E, et al.
Continuing outcomes relevant to Evista: breast
cancer incidence in postmenopausal osteoporotic
women in a randomized trial of raloxifene. J Natl
Cancer Inst. 2004;96(23):1751–1761.
410. Barrett-Connor E, Mosca L, Collins P, et al. Effects of
raloxifene on cardiovascular events and breast cancer in
postmenopausal women. N Engl J Med. 2006;355(2):
125–137.
411. Cummings SR, Ensrud K, Delmas PD, et al.
Lasofoxifene in postmenopausal women with
osteoporosis. N Engl J Med. 2010;362(8):686–
696.
412. LaCroix AZ, Powles T, Osborne CK, et al. Breast
cancer incidence in the randomized PEARL trial of
lasofoxifene in postmenopausal osteoporotic women.
J Natl Cancer Inst. 2010;102(22):1706–1715.
413. Cummings SR, McClung M, Reginster JY, et al.
Arzoxifene for prevention of fractures and invasive
breast cancer in postmenopausal women. J Bone
Miner Res. 2011;26(2):397–404.
414. Powles TJ, Diem SJ, Fabian CJ, et al. Breast
cancer incidence in postmenopausal women with
osteoporosis or low bone mass using arzoxifene.
Breast Cancer Res Treat. 2012;134(1):299–306.
415. Johnell O, Cauley JA, Kulkarni PM, Wong M, Stock
JL. Raloxifene reduces risk of vertebral fractures
[corrected] in postmenopausal women regardless
of prior hormone therapy. J Fam Pract. 2004;
53(10):789–796.
416. Cuzick J, Powles T, Veronesi U, et al. Overview
of the main outcomes in breast-cancer prevention
trials. Lancet. 2003;361(9354):296–300.
417. Cuzick J, DeCensi A, Arun B, et al. Preventive
therapy for breast cancer: a consensus statement.
Lancet Oncol. 2011;12(5):496–503.
418. Reimers LL, Sivasubramanian PS, Hershman D, et al.
Breast cancer chemoprevention among high-risk
women and those with ductal carcinoma in situ.
Breast J. 2015;21(4):377–386.
419. Litton JK, Arun BK, Brown PH, Hortobagyi GN.
Aromatase inhibitors and breast cancer prevention.
Expert Opin Pharmacother. 2012;13(3):325–331.
420. Baum M, Budzar AU, Cuzick J, et al. Anastrozole
alone or in combination with tamoxifen versus
tamoxifen alone for adjuvant treatment of post-
menopausal women with early breast cancer: first
results of the ATAC randomised trial. Lancet. 2002;
359(9324):2131–2139.
421. Cuzick J, Sestak I, Baum M, et al. Effect of
anastrozole and tamoxifen as adjuvant treatment
for early-stage breast cancer: 10-year analysis of the
ATAC trial. Lancet Oncol. 2010;11(12):1135–1141.
422. Margolese RG, Cecchini RS, Julian TB, et al.
Anastrozole versus tamoxifen in postmenopausal
women with ductal carcinoma in situ undergoing
lumpectomy plus radiotherapy (NSABP B-35): a
randomised, double-blind, phase 3 clinical trial.
Lancet. 2016;387(10021):849–856.
423. Forbes JF, Sestak I, Howell A, et al. Anastrozole
versus tamoxifen for the prevention of locoregional
and contralateral breast cancer in postmenopausal
women with locally excised ductal carcinoma in
situ (IBIS-II DCIS): a double-blind, randomised
controlled trial. Lancet. 2016;387(10021):866–873.
424. Cuzick J, Sestak I, Forbes JF, et al. Anastrozole
for prevention of breast cancer in high-risk post-
menopausal women (IBIS-II): an international,
double-blind, randomised placebo-controlled trial.
Lancet. 2014;383(9922):1041–1048.
425. Richardson H, Johnston D, Pater J, Goss P. The
National Cancer Institute of Canada Clinical Trials
Group MAP.3 trial: an international breast cancer
prevention trial. Curr Oncol. 2007;14(3):89–96.
426. Cuzick J. Aromatase inhibitors in prevention–data
from the ATAC (Arimidex, Tamoxifen Alone or in
Combination) trial and the design of IBIS-II (the
second International Breast Cancer Intervention
Study). Recent Results Cancer Res. 2003;163:96–103;
discussion 264-106.
427. Cuzick J. IBIS II: a breast cancer prevention trial
in postmenopausal women using the aromatase
inhibitor anastrozole. Expert Rev Anticancer Ther.
2008;8(9):1377–1385.
428. Goss PE, Ingle JN, Ales-Martinez JE, et al. Exemes-
tane for breast-cancer prevention in postmenopausal
women. N Engl J Med. 2011;364(25):2381–2391.
429. Brown PH, Subbaramaiah K, Salmon AP, et al. Com-
bination chemoprevention of HER2/neu-induced
breast cancer using a cyclooxygenase-2 inhibitor and
a retinoid X receptor-selective retinoid. Cancer Prev
Res (Phila). 2008;1(3):208–214.
430. Li Y, Zhang Y, Hill J, et al. The rexinoid, bexarotene,
prevents the development of premalignant lesions
in MMTV-erbB2 mice. Br J Cancer. 2008;98(8):
1380–1388.
431. Kim HT, Kong G, Denardo D, et al. Identification
of biomarkers modulated by the rexinoid LGD1069
(bexarotene) in human breast cells using oligo-
nucleotide arrays. Cancer Res. 2006;66(24):12009–
12018.
432. Kapetanovic IM, Horn TL, Johnson WD, Cwik MJ,
Detrisac CJ, McCormick DL. Murine oncogenicity
and pharmacokinetics studies of 9-cis-UAB30, an
RXR agonist, for breast cancer chemoprevention.
Int J Toxicol. 2010;29(2):157–164.
433. Thanasitthichai S, Chaiwerawattana A, Prasit-
thipayong A. Association of vitamin D level with
clinicopathological features in breast cancer. Asian
Pac J Cancer Prev. 2015;16(12):4881–4883.
434. Abulkhair O, Saadeddin A, Makram O, et al.
Vitamin D levels and breast cancer characteristics:
findings in patients from Saudi Arabia. J Steroid
Biochem Mol Biol. 2016;164:106–109.
435. Jorde R, Schirmer H, Wilsgaard T, et al. Polymor-
phisms related to the serum 25-hydroxyvitamin D
level and risk of myocardial infarction, diabetes,
cancer and mortality. The Tromso Study. PLoS ONE.
2012;7(5):e37295.
436. Yao S, Zirpoli G, Bovbjerg DH, et al. Variants in
the vitamin D pathway, serum levels of vitamin D,
and estrogen receptor negative breast cancer among
African-American women: a case-control study. Breast
Cancer Res. 2012;14(2):R58.
437. Clement Y. Can green tea do that? A literature
review of the clinical evidence. Prev Med. 2009;
49(2–3):83–87.
438. Nakachi K, Suemasu K, Suga K, Takeo T, Imai K,
Higashi Y. Influence of drinking green tea on breast
cancer malignancy among Japanese patients. Jpn J
Cancer Res. 1998;89(3):254–261.
439. Park OJ, Surh YJ. Chemopreventive potential of
epigallocatechin gallate and genistein: evidence from
epidemiological and laboratory studies. Toxicol Lett.
2004;150(1):43–56.
440. Sartippour MR, Heber D, Ma J, Lu Q, Go VL,
Nguyen M. Green tea and its catechins inhibit breast
cancer xenografts. Nutr Cancer. 2001;40(2):149–156.
441. Weisburger JH, Chung FL. Mechanisms of chronic
disease causation by nutritional factors and tobacco
products and their prevention by tea polyphenols.
Food Chem Toxicol. 2002;40(8):1145–1154.
442. Baliga MS, Meleth S, Katiyar SK. Growth inhibitory
and antimetastatic effect of green tea polyphenols
on metastasis-specific mouse mammary carcinoma
4T1 cells in vitro and in vivo systems. Clin Cancer
Res. 2005;11(5):1918–1927.
443. Thangapazham RL, Singh AK, Sharma A, Warren J,
Gaddipati JP, Maheshwari RK. Green tea polyphenols
and its constituent epigallocatechin gallate inhibits
proliferation of human breast cancer cells in vitro
and in vivo. Cancer Lett. 2007;245(1–2):232–241.
444. Mineva ND, Paulson KE, Naber SP, Yee AS,
Sonenshein GE. Epigallocatechin-3-gallate inhibits
stem-like inflammatory breast cancer cells. PLoS
ONE. 2013;8(9):e73464.
445. Crew KD, Brown P, Greenlee H, et al. Phase IB
randomized, double-blinded, placebo-controlled,
dose escalation study of polyphenon E in women
with hormone receptor-negative breast cancer. Cancer
Prev Res (Phila). 2012;5(9):1144–1154.
446. Brown P, Arun B, Miller A, et al. Phase II trial of
bexarotene in women at high risk of breast cancer:
comparison of protein and RNA biomarkers. Cancer
Prev Res (Phila Pa). 2008;1(7 suppl):CN4.
447. Crew KD, Xiao T, Thomas PS, et al. Safety,
feasibility, and biomarker effects of high-dose
vitamin D supplementation among women at
high risk for breast cancer. Int J Food Sci Nutr Diet.
2015;2015(suppl 1):1–16.
448. Bonanni B, Puntoni M, Cazzaniga M, et al. Dual
effect of metformin on breast cancer proliferation in
a randomized presurgical trial. J Clin Oncol. 2012;
30(21):2593–2600.
449. Samavat H, Dostal AM, Wang R, et al. The Min-
nesota Green Tea Trial (MGTT), a randomized
controlled trial of the efficacy of green tea extract
on biomarkers of breast cancer risk: study rationale,
design, methods, and participant characteristics.
Cancer Causes Control. 2015;26(10):1405–1419.
450. Dostal AM, Samavat H, Bedell S, et al. The safety
of green tea extract supplementation in postmeno-
pausal women at risk for breast cancer: results of
the Minnesota Green Tea Trial. Food Chem Toxicol.
2015;83:26–35.
451. Kuerer HM, Buzdar AU, Mittendorf EA, et al.
Biologic and immunologic effects of preoperative
trastuzumab for ductal carcinoma in situ of the
breast. Cancer. 2011;117(1):39–47.
452. Estévez LG, Suarez-Gauthier A, Garcia E, et al.
Molecular effects of lapatinib in patients with HER2

positive ductal carcinoma in situ. Breast Cancer Res.
2014;16(4):R76.
453. Knutson KL, Disis ML. Expansion of HER2/
neu-specific T cells ex vivo following immunization
with a HER2/neu peptide-based vaccine. Clin Breast
Cancer. 2001;2(1):73–79.
454. Schiffman K, Rinn K, Disis ML. Delayed type
hypersensitivity response to recall antigens does not
accurately reflect immune competence in advanced
stage breast cancer patients. Breast Cancer Res Treat.
2002;74(1):17–23.
455. Montgomery RB, Makary E, Schiffman K, Goodell
V, Disis ML. Endogenous anti-HER2 antibodies
block HER2 phosphorylation and signaling through
extracellular signal-regulated kinase. Cancer Res.
2005;65(2):650–656.
456. Phan-Lai V, Dang Y, Gad E, Childs J, Disis ML.
The antitumor efficacy of IL2/IL21-cultured
polyfunctional neu-specific T cells is TNFalpha/IL17
dependent. Clin Cancer Res. 2016;22(9):2207–2216.
457. Dols A, Meijer SL, Hu HM, et al. Identification
of tumor-specific antibodies in patients with breast
cancer vaccinated with gene-modified allogeneic
tumor cells. J Immunother. 2003;26(2):163–170.
458. Emens LA, Asquith JM, Leatherman JM, et al. Timed
sequential treatment with cyclophosphamide, doxo-
rubicin, and an allogeneic granulocyte-macrophage
colony-stimulating factor-secreting breast tumor
vaccine: a chemotherapy dose-ranging factorial
study of safety and immune activation. J Clin Oncol.
2009;27(35):5911–5918.
459. Pathangey LB, McCurry DB, Gendler SJ, et al. Surro-
gate in vitro activation of innate immunity synergizes
with interleukin-7 to unleash rapid antigen-driven
outgrowth of CD4+ and CD8+ human peripheral
blood T-cells naturally recognizing MUC1, HER2/
neu and other tumor-associated antigens. Oncotarget.
2017;8(7):10785–10808.
460. Disis ML, Wallace DR, Gooley TA, et al. Concurrent
trastuzumab and HER2/neu-specific vaccination in
patients with metastatic breast cancer. J Clin Oncol.
2009;27(28):4685–4692.
461. Mittendorf EA, Clifton GT, Holmes JP, et al. Final
report of the phase I/II clinical trial of the E75
(nelipepimut-S) vaccine with booster inoculations to
prevent disease recurrence in high-risk breast cancer
patients. Ann Oncol. 2014;25(9):1735–1742.
462. Mittendorf EA, Ardavanis A, Symanowski J, et al.
Primary analysis of a prospective, randomized,
single-blinded phase II trial evaluating the HER2
peptide AE37 vaccine in breast cancer patients to
prevent recurrence. Ann Oncol. 2016;27(7):1241–
1248.
463. Mittendorf EA, Ardavanis A, Litton JK, et al. Primary
analysis of a prospective, randomized, single-blinded
phase II trial evaluating the HER2 peptide GP2
vaccine in breast cancer patients to prevent recur-
rence. Oncotarget. 2016;7(40):66192–66201.
464. Clifton GT, Litton JK, Arrington K, et al. Results
of a phase Ib trial of combination immunotherapy
with a CD8+ T cell eliciting vaccine and trastuzumab
in breast cancer patients. Ann Surg Oncol. 2017;
24(8):2161–2167.
465. Margolese RG, Vallow LA, Albain KS, Whitworth
PW, Cianfrocca M. National Surgical Adjuvant
Breast and Bowel Project (NSABP): Anastrozole
or tamoxifen in treating postmenopausal women
with ductal carcinoma in situ who are undergoing
lumpectomy and radiation therapy; 2003.
466. Rigoni-Stern D. Fatti statistici relative alle mallatie
cancrosi che servirono de base alla poche cose dette
dal dott. [Surgical facts, related to cancer diseases].
G Servire Progr Path Tera. 1842;2:507–517.
467. Lopalco PL. Spotlight on the 9-valent HPV vaccine.
Drug Des Devel Ther. 2017;11:35–44.
468. International Agency for Research on Cancer
WHO. IARC Monographs on the Evaluation of
the Carcinogenic Risks to Human; 2016. http://
monographs.iarc.fr.
Lifestyle and Cancer Prevention  •  CHAPTER 22 374.e9
469. Pirog EC, Lloveras B, Molijn A, et al. HPV preva-
lence and genotypes in different histological subtypes
of cervical adenocarcinoma, a worldwide analysis of
760 cases. Mod Pathol. 2014;27(12):1559–1567.
470. Wheeler CM, Hunt WC, Joste NE, Key CR, Quint
WG, Castle PE. Human papillomavirus genotype
distributions: implications for vaccination and cancer
screening in the United States. J Natl Cancer Inst.
2009;101(7):475–487.
471. U.S. Food and Drug Administration. Cervarix
[Human Papillomavirus Bivalent (Types 16 and 18)
Vaccine, Recombinant]; 2009. https://www.fda.gov/
BiologicsBloodVaccines/Vaccines/ApprovedProducts/
ucm186957.htm.
472. Harper DM, Franco EL, Wheeler C, et al. Efficacy of
a bivalent L1 virus-like particle vaccine in prevention
of infection with human papillomavirus types 16
and 18 in young women: a randomised controlled
trial. Lancet. 2004;364(9447):1757–1765.
473. Harper DM, Franco EL, Wheeler CM, et al.
Sustained efficacy up to 4.5 years of a bivalent L1
virus-like particle vaccine against human papilloma-
virus types 16 and 18: follow-up from a randomised
control trial. Lancet. 2006;367(9518):1247–1255.
474. GlaxoSmithKline Vaccine HPVSG, Romanowski
B, de Borba PC, et al. Sustained efficacy and
immunogenicity of the human papillomavirus
(HPV)-16/18 AS04-adjuvanted vaccine: analysis of a
randomised placebo-controlled trial up to 6.4 years.
Lancet. 2009;374(9706):1975–1985.
475. De Carvalho N, Teixeira J, Roteli-Martins CM,
et al. Sustained efficacy and immunogenicity of
the HPV-16/18 AS04-adjuvanted vaccine up to 7.3
years in young adult women. Vaccine. 2010;28(38):
6247–6255.
476. Roteli-Martins CM, Naud P, De Borba P, et al.
Sustained immunogenicity and efficacy of the HPV-
16/18 AS04-adjuvanted vaccine: up to 8.4 years
of follow-up. Hum Vaccin Immunother. 2012;8(3):
390–397.
477. Naud PS, Roteli-Martins CM, De Carvalho NS,
et al. Sustained efficacy, immunogenicity, and safety
of the HPV-16/18 AS04-adjuvanted vaccine: final
analysis of a long-term follow-up study up to 9.4
years post-vaccination. Hum Vaccin Immunother.
2014;10(8):2147–2162.
478. Paavonen J, Jenkins D, Bosch FX, et al. Efficacy of a
prophylactic adjuvanted bivalent L1 virus-like-particle
vaccine against infection with human papillomavirus
types 16 and 18 in young women: an interim analysis
of a phase III double-blind, randomised controlled
trial. Lancet. 2007;369(9580):2161–2170.
479. Paavonen J, Naud P, Salmeron J, et al. Efficacy
of human papillomavirus (HPV)-16/18 AS04-
adjuvanted vaccine against cervical infection and pre-
cancer caused by oncogenic HPV types (PATRICIA):
final analysis of a double-blind, randomised study in
young women. Lancet. 2009;374(9686):301–314.
480. Lehtinen M, Paavonen J, Wheeler CM, et al.
Overall efficacy of HPV-16/18 AS04-adjuvanted
vaccine against grade 3 or greater cervical intraepi-
thelial neoplasia: 4-year end-of-study analysis of the
randomised, double-blind PATRICIA trial. Lancet
Oncol. 2012;13(1):89–99.
481. Wheeler CM, Castellsague X, Garland SM,
et al. Cross-protective efficacy of HPV-16/18
AS04-adjuvanted vaccine against cervical infection
and precancer caused by non-vaccine oncogenic
HPV types: 4-year end-of-study analysis of the
randomised, double-blind PATRICIA trial. Lancet
Oncol. 2012;13(1):100–110.
482. Szarewski A, Poppe WA, Skinner SR, et al. Efficacy
of the human papillomavirus (HPV)-16/18 AS04-
adjuvanted vaccine in women aged 15-25 years with
and without serological evidence of previous exposure
to HPV-16/18. Int J Cancer. 2012;131(1):106–116.
483. Szarewski A, Skinner SR, Garland SM, et al. Efficacy
of the HPV-16/18 AS04-adjuvanted vaccine against
low-risk HPV types (PATRICIA randomized trial):
374.e9
an unexpected observation. J Infect Dis. 2013;208(9):
1391–1396.
484. Hildesheim A, Wacholder S, Catteau G, et al.
Efficacy of the HPV-16/18 vaccine: final according
to protocol results from the blinded phase of the
randomized Costa Rica HPV-16/18 vaccine trial.
Vaccine. 2014;32(39):5087–5097.
485. Herrero R, Wacholder S, Rodriguez AC, et al.
Prevention of persistent human papillomavirus
infection by an HPV16/18 vaccine: a community-
based randomized clinical trial in Guanacaste, Costa
Rica. Cancer Discov. 2011;1(5):408–419.
486. Rodriguez AC, Solomon D, Herrero R, et al. Impact
of human papillomavirus vaccination on cervical
cytology screening, colposcopy, and treatment.
Am J Epidemiol. 2013;178(5):752–760.
487. Lang Kuhs KA, Porras C, Schiller JT, et al. Effect
of different human papillomavirus serological and
DNA criteria on vaccine efficacy estimates. Am J
Epidemiol. 2014;180(6):599–607.
488. Kreimer AR, Rodriguez AC, Hildesheim A, et al.
Proof-of-principle evaluation of the efficacy of fewer
than three doses of a bivalent HPV16/18 vaccine.
J Natl Cancer Inst. 2011;103(19):1444–1451.
489. Lang Kuhs KA, Gonzalez P, Rodriguez AC, et al.
Reduced prevalence of vulvar HPV16/18 infection
among women who received the HPV16/18 bivalent
vaccine: a nested analysis within the Costa Rica
Vaccine Trial. J Infect Dis. 2014;210(12):1890–1899.
490. Kreimer AR, Gonzalez P, Katki HA, et al. Efficacy
of a bivalent HPV 16/18 vaccine against anal HPV
16/18 infection among young women: a nested
analysis within the Costa Rica Vaccine Trial. Lancet
Oncol. 2011;12(9):862–870.
491. Herrero R, Quint W, Hildesheim A, et al. Reduced
prevalence of oral human papillomavirus (HPV) 4
years after bivalent HPV vaccination in a randomized
clinical trial in Costa Rica. PLoS ONE. 2013;8(7):
e68329.
492. Konno R, Tamura S, Dobbelaere K, Yoshikawa
H. Efficacy of human papillomavirus type 16/18
AS04-adjuvanted vaccine in Japanese women aged 20
to 25 years: final analysis of a phase 2 double-blind,
randomized controlled trial. Int J Gynecol Cancer.
2010;20(5):847–855.
493. Konno R, Tamura S, Dobbelaere K, Yoshikawa H.
Efficacy of human papillomavirus 16/18 AS04-
adjuvanted vaccine in Japanese women aged 20 to
25 years: interim analysis of a phase 2 double-blind,
randomized, controlled trial. Int J Gynecol Cancer.
2010;20(3):404–410.
494. Konno R, Yoshikawa H, Okutani M, et al.
Efficacy of the human papillomavirus (HPV)-
16/18 AS04-adjuvanted vaccine against cervical
intraepithelial neoplasia and cervical infection in
young Japanese women. Hum Vaccin Immunother.
2014;10(7):1781–1794.
495. Zhu FC, Chen W, Hu YM, et al. Efficacy, immu-
nogenicity and safety of the HPV-16/18 AS04-
adjuvanted vaccine in healthy Chinese women aged
18-25 years: results from a randomized controlled
trial. Int J Cancer. 2014;135(11):2612–2622.
496. Skinner SR, Szarewski A, Romanowski B, et al.
Efficacy, safety, and immunogenicity of the human
papillomavirus 16/18 AS04-adjuvanted vaccine
in women older than 25 years: 4-year interim
follow-up of the phase 3, double-blind, randomised
controlled VIVIANE study. Lancet. 2014;384(9961):
2213–2227.
497. Skinner SR, Apter D, De Carvalho N, et al. Human
papillomavirus (HPV)-16/18 AS04-adjuvanted
vaccine for the prevention of cervical cancer and
HPV-related diseases. Expert Rev Vaccines. 2016;15(3):
367–387.
498. Kreimer AR, Struyf F, Del Rosario-Raymundo MR,
et al. Efficacy of fewer than three doses of an HPV-
16/18 AS04-adjuvanted vaccine: combined analysis
of data from the Costa Rica Vaccine and PATRICIA
Trials. Lancet Oncol. 2015;16(7):775–786.
374.e10 Part I: Science and Clinical Oncology
499. U.S. Food and Drug Administration. Gardasil
[Human Papillomavirus Quadrivalent (Types
6, 11, 16, 18) Vaccine, Recombinant]; 2006.
https://www.fda.gov/BiologicsBloodVaccines/
Vaccines/ApprovedProducts/ucm094042.htm.
500. Handler NS, Handler MZ, Majewski S, Schwartz
RA. Human papillomavirus vaccine trials and
tribulations: vaccine efficacy. J Am Acad Dermatol.
2015;73(5):759–767; quiz 767-758.
501. U.S. Food and Drug Administration. VRBPAC
Background Document: Gardasil HPV Quadrivalent
Vaccine; 2006. https://www.fda.gov/ohrms/dockets/
ac/06/briefing/2006-4222B3.pdf.
502. Villa LL, Costa RL, Petta CA, et al. Prophylactic
quadrivalent human papillomavirus (types 6,
11, 16, and 18) L1 virus-like particle vaccine in
young women: a randomised double-blind placebo-
controlled multicentre phase II efficacy trial. Lancet
Oncol. 2005;6(5):271–278.
503. Villa LL, Costa RL, Petta CA, et al. High sustained
efficacy of a prophylactic quadrivalent human papil-
lomavirus types 6/11/16/18 L1 virus-like particle
vaccine through 5 years of follow-up. Br J Cancer.
2006;95(11):1459–1466.
504. Olsson SE, Villa LL, Costa RL, et al. Induction
of immune memory following administration of
a prophylactic quadrivalent human papillomavirus
(HPV) types 6/11/16/18 L1 virus-like particle (VLP)
vaccine. Vaccine. 2007;25(26):4931–4939.
505. Brotherton JM, Fridman M, May CL, Chappell G,
Saville AM, Gertig DM. Early effect of the HPV
vaccination programme on cervical abnormalities
in Victoria, Australia: an ecological study. Lancet.
2011;377(9783):2085–2092.
506. Crowe E, Pandeya N, Brotherton JM, et al.
Effectiveness of quadrivalent human papillomavirus
vaccine for the prevention of cervical abnormali-
ties: case-control study nested within a population
based screening programme in Australia. BMJ.
2014;348:g1458.
507. Kahn JA, Brown DR, Ding L, et al. Vaccine-type
human papillomavirus and evidence of herd protec-
tion after vaccine introduction. Pediatrics. 2012;
130(2):e249–e256.
508. Herweijer E, Leval A, Ploner A, et al. Association
of varying number of doses of quadrivalent human
papillomavirus vaccine with incidence of condyloma.
JAMA. 2014;311(6):597–603.
509. Giuliano AR, Palefsky JM, Goldstone S, et al.
Efficacy of quadrivalent HPV vaccine against
HPV Infection and disease in males. N Engl J Med.
2011;364(5):401–411.
510. Palefsky JM, Giuliano AR, Goldstone S, et al. HPV
vaccine against anal HPV infection and anal intraepi-
thelial neoplasia. N Engl J Med. 2011;365(17):
1576–1585.
511. Kash N, Lee MA, Kollipara R, Downing C, Guidry
J, Tyring SK. Safety and efficacy data on vaccines
and immunization to human papillomavirus. J Clin
Med. 2015;4(4):614–633.
512. Julius JM, Ramondeta L, Tipton KA, Lal LS,
Schneider K, Smith JA. Clinical perspectives on the
role of the human papillomavirus vaccine in the pre-
vention of cancer. Pharmacotherapy. 2011;31(3):280–
297.
513. Garland SM, Cheung TH, McNeill S, et al. Safety
and immunogenicity of a 9-valent HPV vaccine in
females 12-26 years of age who previously received
the quadrivalent HPV vaccine. Vaccine. 2015;33(48):
6855–6864.
514. FUTURE II Study Group. Prophylactic efficacy
of a quadrivalent human papillomavirus (HPV)
vaccine in women with virological evidence of HPV
infection. J Infect Dis. 2007;196(10):1438–1446.
515. FUTURE II Study Group, Dillner J, Kjaer SK,
et al. Four year efficacy of prophylactic human
papillomavirus quadrivalent vaccine against low grade
cervical, vulvar, and vaginal intraepithelial neoplasia
and anogenital warts: randomised controlled trial.
BMJ. 2010;341:c3493.
516. FUTURE II Study Group. Quadrivalent vaccine
against human papillomavirus to prevent high-grade
cervical lesions. N Engl J Med. 2007;356(19):
1915–1927.
517. U.S. Food and Drug Administration. Gardasil 9
[Human Papillomavirus 9-valent Vaccine, Recombi-
nant]; 2014. https://www.fda.gov/BiologicsBloodVac
cines/Vaccines/ApprovedProducts/ucm426445.htm.
518. Castellsague X, Giuliano AR, Goldstone S, et al.
Immunogenicity and safety of the 9-valent HPV
vaccine in men. Vaccine. 2015;33(48):6892–6901.
519. Joura EA, Giuliano AR, Iversen OE, et al. A 9-valent
HPV vaccine against infection and intraepithelial
neoplasia in women. N Engl J Med. 2015;372(8):
711–723.
520. Kosalaraksa P, Mehlsen J, Vesikari T, et al. An
open-label, randomized study of a 9-valent human
papillomavirus vaccine given concomitantly with
diphtheria, tetanus, pertussis and poliomyelitis
vaccines to healthy adolescents 11-15 years of age.
Pediatr Infect Dis J. 2015;34(6):627–634.
521. Schilling S, Samuels-Kalow M, Gerber JS, Scribano
PV, French B, Wood JN. Testing and treatment
after adolescent sexual assault in pediatric emergency
departments. Pediatrics. 2015;136(6):e1495–e1503.
522. Van Damme P, Olsson SE, Block S, et al. Immu-
nogenicity and safety of a 9-valent HPV vaccine.
Pediatrics. 2015;136(1):e28–e39.
523. Vesikari T, Brodszki N, van Damme P, et al. A
randomized, double-blind, phase III study of the
immunogenicity and safety of a 9-valent human
papillomavirus L1 virus-like particle vaccine (V503)
versus Gardasil(R) in 9-15-year-old girls. Pediatr
Infect Dis J. 2015;34(9):992–998.
524. European Medicines Agency. Gardasil 9. Summary
of Product Characteristics; 2016. http://www.ema
.europa.eu/docs/en_GB/document_library/EPAR
_-_Product_Information/human/003852/
WC500189111.pdf.
525. Van Damme P, Meijer CJ, Kieninger D, et al. A phase
III clinical study to compare the immunogenicity
and safety of the 9-valent and quadrivalent HPV
vaccines in men. Vaccine. 2016;34(35):4205–4212.
526. Larson HJ, Wilson R, Hanley S, Parys A, Paterson
P. Tracking the global spread of vaccine sentiments:
the global response to Japan’s suspension of its HPV
vaccine recommendation. Hum Vaccin Immunother.
2014;10(9):2543–2550.
527. Colafrancesco S, Perricone C, Tomljenovic L,
Shoenfeld Y. Human papilloma virus vaccine
and primary ovarian failure: another facet of the
autoimmune/inflammatory syndrome induced by
adjuvants. Am J Reprod Immunol. 2013;70(4):309–
316.
528. Signorelli C, Odone A, Ciorba V, et al. Human
papillomavirus 9-valent vaccine for cancer preven-
tion: a systematic review of the available evidence.
Epidemiol Infect. 2017;1–21.
529. Zhai L, Tumban E. Gardasil-9: a global survey of
projected efficacy. Antiviral Res. 2016;130:101–109.
530. Angioli R, Lopez S, Aloisi A, et al. Ten years of
HPV vaccines: state of art and controversies. Crit
Rev Oncol Hematol. 2016;102:65–72.
531. Gertig DM, Brotherton JM, Saville M. Measuring
human papillomavirus (HPV) vaccination coverage
and the role of the National HPV Vaccination
Program Register, Australia. Sex Health. 2011;8(2):
171–178.
532. Osborne SL, Tabrizi SN, Brotherton JM, et al. Assess-
ing genital human papillomavirus genoprevalence
in young Australian women following the introduc-
tion of a national vaccination program. Vaccine.
2015;33(1):201–208.
533. Reagan-Steiner S, Yankey D, Jeyarajah J, et al.
National, regional, state, and selected local area
vaccination coverage among adolescents aged 13-17
years—United States. MMWR Morb Mortal Wkly
Rep. 2014;64(29):784–792.
534. Meites E, Kempe A, Markowitz LE. Use of
a 2-dose schedule for human papillomavirus
vaccination—updated recommendations of the Advi-
sory Committee on Immunization Practices. MMWR
Morb Mortal Wkly Rep. 2016;65(49):1405–1408.
535. Mondul AM, Weinstein SJ, Layne TM, Albanes D,
Vitamin D. and cancer risk and mortality: state of
the science, gaps, and challenges. Epidemiol Rev.
2017;39(1):28–48.
536. Thompson IM, Goodman PJ, Tangen CM, et al.
The influence of finasteride on the development of
prostate cancer. N Engl J Med. 2003;349(3):215–224.
537. Thompson IM Jr, Goodman PJ, Tangen CM, et al.
Long-term survival of participants in the prostate
cancer prevention trial. N Engl J Med. 2013;369(7):
603–610.
538. Andriole GL, Bostwick DG, Brawley OW, et al.
Effect of dutasteride on the risk of prostate cancer.
N Engl J Med. 2010;362(13):1192–1202.
539. Grubb RL, Andriole GL, Somerville MC, Mahoney
C, Manyak MJ, Castro R. The REDUCE follow-up
study: low rate of new prostate cancer diagnoses
observed during a 2-year, observational, followup
study of men who participated in the REDUCE
trial. J Urol. 2013;189(3):871–877.
540. Toren P, Margel D, Kulkarni G, Finelli A, Zlotta A,
Fleshner N. Effect of dutasteride on clinical progres-
sion of benign prostatic hyperplasia in asymptomatic
men with enlarged prostate: a post hoc analysis of
the REDUCE study. BMJ. 2013;346:f2109.
541. Theoret MR, Ning YM, Zhang JJ, Justice R,
Keegan P, Pazdur R. The risks and benefits of
5alpha-reductase inhibitors for prostate-cancer
prevention. N Engl J Med. 2011;365(2):97–99.
542. Kristal AR, Till C, Song X, et al. Plasma vitamin D
and prostate cancer risk: results from the Selenium
and Vitamin E Cancer Prevention Trial. Cancer
Epidemiol Biomarkers Prev. 2014;23(8):1494–1504.
543. Kristal AR, Darke AK, Morris JS, et al. Baseline
selenium status and effects of selenium and vitamin
e supplementation on prostate cancer risk. J Natl
Cancer Inst. 2014;106(3):djt456.
544. Zhou CK, Daugherty SE, Liao LM, et al. Do aspirin
and other NSAIDs confer a survival benefit in men
diagnosed with prostate cancer? a pooled analysis
of NIH-AARP and PLCO cohorts. Cancer Prev Res
(Phila). 2017.
545. Platz EA, Tangen CM, Goodman PJ, et al. Statin
drug use is not associated with prostate cancer risk in
men who are regularly screened. J Urol. 2014;192(2):
379–384.
546. Schenk JM, Till CA, Tangen CM, et al. Serum
25-hydroxyvitamin D concentrations and risk of
prostate cancer: results from the Prostate Cancer
Prevention Trial. Cancer Epidemiol Biomarkers Prev.
2014;23(8):1484–1493.
547. Aghajanpour M, Nazer MR, Obeidavi Z, Akbari M,
Ezati P, Kor NM. Functional foods and their role in
cancer prevention and health promotion: a compre-
hensive review. Am J Cancer Res. 2017;7(4):740–769.
548. Wan L, Chen M, Cao J, et al. The APC/C E3 ligase
complex activator FZR1 restricts BRAF oncogenic
function. Cancer Discov. 2017;7(4):424–441.
549. Miles FL, Goodman PJ, Tangen C, et al. Interactions
of the insulin-like growth factor axis and vitamin
D in prostate cancer risk in the Prostate Cancer
Prevention Trial. Nutrients. 2017;9(4).
550. Smith A, Balar AV, Milowsky MI, Chen RC. Bladder
cancer. In: Niederhuber JE, Doroshow JH, Kastan
MB, Tepper JE, eds. Abeloff ’s Clinical Oncology. 5th
ed. Philadelphia, PA: Elsevier; 2014:1445–1462.
551. Steinberg G, Bahnson R, Brosman S, Middleton
R, Wajsman Z, Wehle M. Efficacy and safety of
valrubicin for the treatment of bacillus Calmette-
Guérin refractory carcinoma in situ of the bladder.
The Valrubicin Study Group. J Urol. 2000;163(3):
761–767.
552. Pinsky CM, Camacho FJ, Kerr D, et al. Intravesi-
cal administration of bacillus Calmette-Guérin in
patients with recurrent superficial carcinoma of the
urinary bladder: report of a prospective, randomized
trial. Cancer Treat Rep. 1985;69(1):47–53.

553. Herr HW, Laudone VP, Badalament RA, et al.
Bacillus Calmette-Guérin therapy alters the pro-
gression of superficial bladder cancer. J Clin Oncol.
1988;6(9):1450–1455.
554. Herr HW, Schwalb DM, Zhang ZF, et al. Intravesical
bacillus Calmette-Guérin therapy prevents tumor
progression and death from superficial bladder
cancer: ten-year follow-up of a prospective random-
ized trial. J Clin Oncol. 1995;13(6):1404–1408.
555. Lamm DL, Blumenstein BA, Crissman JD, et al.
Maintenance bacillus Calmette-Guérin immuno-
therapy for recurrent TA, T1 and carcinoma in
situ transitional cell carcinoma of the bladder: a
randomized Southwest Oncology Group Study.
J Urol. 2000;163(4):1124–1129.
556. Ehdaie B, Sylvester R, Herr HW. Maintenance
bacillus Calmette-Guérin treatment of non-muscle-
invasive bladder cancer: a critical evaluation of the
evidence. Eur Urol. 2013;64(4):579–585.
557. Oddens J, Brausi M, Sylvester R, et al. Final results
of an EORTC-GU cancers group randomized
study of maintenance bacillus Calmette-Guérin in
intermediate- and high-risk Ta, T1 papillary carci-
noma of the urinary bladder: one-third dose versus
full dose and 1 year versus 3 years of maintenance.
Eur Urol. 2013;63(3):462–472.
558. Pfister C, Kerkeni W, Rigaud J, et al. Efficacy and
tolerance of one-third full dose bacillus Calmette-
Guérin maintenance therapy every 3 months
or 6 months: two-year results of URO-BCG-4
multicenter study. Int J Urol. 2015;22(1):53–60.
559. Nouhaud FX, Rigaud J, Saint F, et al. Final results
of the phase III URO-BCG 4 multicenter study:
efficacy and tolerance of one-third dose BCG
maintenance in nonmuscle invasive bladder cancer.
Anticancer Drugs. 2017;28(3):335–340.
560. Huang Z, Liu H, Wang Y, Zhang C, Xu T. Deter-
mining optimal maintenance schedules for adjuvant
intravesical bacillus Calmette-Guérin immuno-
therapy in non-muscle-invasive bladder cancer:
a systematic review and network meta-analysis.
Curr Med Res Opin. 2017;1–9.
561. Shelley MD, Court JB, Kynaston H, Wilt TJ, Coles
B, Mason M. Intravesical bacillus Calmette-Guérin
versus mitomycin C for Ta and T1 bladder cancer.
Cochrane Database Syst Rev. 2003;(3):CD003231.
562. Böhle A, Bock PR. Intravesical bacille Calmette-
Guérin versus mitomycin C in superficial bladder
cancer: formal meta-analysis of comparative studies
on tumor progression. Urology. 2004;63(4):682–686;
discussion 686–687.
563. Shelley MD, Mason MD, Kynaston H. Intravesical
therapy for superficial bladder cancer: a systematic
review of randomised trials and meta-analyses. Cancer
Treat Rev. 2010;36(3):195–205.
564. Lamm D, Persad R, Colombel M, Brausi M.
Maintenance bacillus Calmette-Guérin: the standard
of care for the prophylaxis and management of
intermediate- and high-risk non-muscle-invasive
bladder cancer. Eur Urol Suppl. 2010;9(9):715–734.
565. Fuge O, Vasdev N, Allchorne P, Green JS. Immu-
notherapy for bladder cancer. Res Rep Urol. 2015;7:
65–79.
566. Bazarbashi S, Raja MA, El Sayed A, et al. Prospec-
tive phase II trial of alternating intravesical Bacillus
Calmette-Guérin (BCG) and interferon alpha IIB
in the treatment and prevention of superficial tran-
sitional cell carcinoma of the urinary bladder: pre-
liminary results. J Surg Oncol. 2000;74(3):181–184.
567. Bazarbashi S, Soudy H, Abdelsalam M, et al.
Co-administration of intravesical bacillus Calmette-
Guérin and interferon alpha-2B as first line in
treating superficial transitional cell carcinoma of the
urinary bladder. BJU Int. 2011;108(7):1115–1118.
568. Joudi FN, Smith BJ, O’Donnell MA, National
BCGIPIG. Final results from a national multicenter
phase II trial of combination bacillus Calmette-
Guérin plus interferon alpha-2B for reducing
recurrence of superficial bladder cancer. Urol Oncol.
2006;24(4):344–348.
Lifestyle and Cancer Prevention  •  CHAPTER 22 374.e11
569. Bazarbashi SN, Azouz HJ, Abu Sabaa AH, Aljubran
AH, Alzahrani AM, Alotaibi MF. Recurrence and
progression in nonmuscle invasive transitional cell
carcinoma of urinary bladder treated with intravesical
bacillus Calmette-Guérin: a single center experi-
ence and analysis of prognostic factors. Urol Ann.
2016;8(3):333–337.
570. Steinberg RL, Brooks NA, Thomas LJ, Mott SL,
O’Donnell MA. Bacillus Calmette-Guérin strain may
not effect recurrence-free survival when used intra-
vesically with interferon-alpha2b for non-muscle-
invasive bladder cancer. Urol Oncol. 2017;35(5):
201–207.
571. Babjuk M, Bohle A, Burger M, et al. EAU
Guidelines on non-muscle-invasive urothelial
carcinoma of the bladder: update 2016. Eur Urol.
2017;71(3):447–461.
572. Chang C, Boorjian SA, Chou R, et al. Diagnosis and
treatment of non-muscle invasive bladder cancer:
AUA/SUO Joint Guideline. American Urological
Association (AUA)/Society of Urologic Oncology
(SUO) Guideline 2016; file:///C:/Users/MISavage/
Downloads/Non-Muscle-Invasive-Bladder-Cancer.
pdf. Accessed June 12, 2017.
573. Reis LO, Moro JC, Ribeiro LF, Voris BR, Sadi MV.
Are we following the guidelines on non-muscle
invasive bladder cancer? Int Braz J Urol. 2016;42(1):
22–28.
574. Eifler JB, Scarpato KR, Clark PE. Management
of noninvasive bladder cancers. Curr Opin Oncol.
2015;27(3):185–190.
575. Lammers RJ, Witjes JA, Inman BA, et al. The role of
a combined regimen with intravesical chemotherapy
and hyperthermia in the management of non-muscle-
invasive bladder cancer: a systematic review. Eur
Urol. 2011;60(1):81–93.
576. Dinney CP, Greenberg RE, Steinberg GD. Intravesi-
cal valrubicin in patients with bladder carcinoma
in situ and contraindication to or failure after
bacillus Calmette-Guérin. Urol Oncol. 2013;31(8):
1635–1642.
577. Steinberg G. Valstar (valrubicin): Sterile Solution
for Intravesical Instillation; 2008. https://www
.accessdata.fda.gov/drugsatfda_docs/label/
2011/020892s013lbl.pdf.
578. Sternberg IA, Dalbagni G, Chen LY, Donat SM,
Bochner BH, Herr HW. Intravesical gemcitabine
for high risk, nonmuscle invasive bladder cancer
after bacillus Calmette-Guérin treatment failure.
J Urol. 2013;190(5):1686–1691.
579. Skinner EC, Goldman B, Sakr WA, et al. SWOG
S0353: phase II trial of intravesical gemcitabine in
patients with nonmuscle invasive bladder cancer and
recurrence after 2 prior courses of intravesical bacillus
Calmette-Guérin. J Urol. 2013;190(4):1200–1204.
580. Shelley MD, Jones G, Cleves A, Wilt TJ, Mason
MD, Kynaston HG. Intravesical gemcitabine therapy
for non-muscle invasive bladder cancer (NMIBC):
a systematic review. BJU Int. 2012;109(4):496–
505.
581. Pagliarulo V, Ancona P, Martines I, et al. Celecoxib
for the prevention of nonmuscle invasive bladder
cancer: results from a matched control study. Ther
Adv Urol. 2015;7(6):303–311.
582. El-Arabey AA. New insight for metformin against
bladder cancer. Genes Environ. 2017;39:13.
583. Zhu Z, Wang X, Shen Z, Lu Y, Zhong S, Xu C. Risk
of bladder cancer in patients with diabetes mellitus:
an updated meta-analysis of 36 observational studies.
BMC Cancer. 2013;13:310.
584. Tseng CH. Metformin may reduce bladder cancer
risk in Taiwanese patients with type 2 diabetes. Acta
Diabetol. 2014;51(2):295–303.
585. Nayan M, Bhindi B, Yu JL, et al. The effect of
metformin on cancer-specific survival outcomes in
diabetic patients undergoing radical cystectomy for
urothelial carcinoma of the bladder. Urol Oncol.
2015;33(9):386.e7–386.e13.
586. Rieken M, Xylinas E, Kluth L, et al. Association of
diabetes mellitus and metformin use with oncological
374.e11
outcomes of patients with non-muscle-invasive
bladder cancer. BJU Int. 2013;112(8):1105–1112.
587. Zhang T, Guo P, Zhang Y, et al. The antidiabetic drug
metformin inhibits the proliferation of bladder cancer
cells in vitro and in vivo. Int J Mol Sci. 2013;14(12):
24603–24618.
588. Donin NM, Chamie K, Lenis AT, et al. A phase
2 study of TMX-101, intravesical imiquimod, for
the treatment of carcinoma in situ bladder cancer.
Urol Oncol. 2017;35(2):39.e31–39.e37.
589. Park SJ, Myung SK, Lee Y, Lee YJ. Effects of vitamin
and antioxidant supplements in prevention of bladder
cancer: a meta-analysis of randomized controlled
trials. J Korean Med Sci. 2017;32(4):628–635.
590. Diepgen TL, Mahler V. The epidemiology of skin
cancer. Br J Dermatol. 2002;146(suppl 61):1–6.
591. Housman TS, Feldman SR, Williford PM, et al.
Skin cancer is among the most costly of all cancers
to treat for the Medicare population. J Am Acad
Dermatol. 2003;48(3):425–429.
592. Rogers HW, Weinstock MA, Harris AR, et al. Inci-
dence estimate of nonmelanoma skin cancer in the
United States, 2006. Arch Dermatol. 2010;146(3):
283–287.
593. Ratushny V, Gober MD, Hick R, Ridky TW, Seykora
JT. From keratinocyte to cancer: the pathogenesis
and modeling of cutaneous squamous cell carcinoma.
J Clin Invest. 2012;122(2):464–472.
594. Kuflik EG, Gage AA. The five-year cure rate achieved
by cryosurgery for skin cancer. J Am Acad Dermatol.
1991;24(6 Pt 1):1002–1004.
595. Morton CA, McKenna KE, Rhodes LE, British
Association of Dermatologists Therapy G, Audit
S, the British Photodermatology G. Guidelines
for topical photodynamic therapy: update. Br J
Dermatol. 2008;159(6):1245–1266.
596. Rigel DS, Stein Gold LF. The importance of early
diagnosis and treatment of actinic keratosis. J Am
Acad Dermatol. 2013;68(1 suppl 1):S20–S27.
597. Gupta AK, Paquet M, Villanueva E, Brintnell W.
Interventions for actinic keratoses. Cochrane Database
Syst Rev. 2012;(12):CD004415.
598. Krawtchenko N, Roewert-Huber J, Ulrich M, Mann
I, Sterry W, Stockfleth E. A randomised study of
topical 5% imiquimod vs. topical 5-fluorouracil vs.
cryosurgery in immunocompetent patients with
actinic keratoses: a comparison of clinical and
histological outcomes including 1-year follow-up.
Br J Dermatol. 2007;157(suppl 2):34–40.
599. Walker JL, Siegel JA, Sachar M, et al. 5-Fluorouracil
for actinic keratosis treatment and chemoprevention:
a randomized controlled trial. J Invest Dermatol.
2017;137(6):1367–1370.
600. Lebwohl M, Dinehart S, Whiting D, et al. Imiqui-
mod 5% cream for the treatment of actinic keratosis:
results from two phase III, randomized, double-blind,
parallel group, vehicle-controlled trials. J Am Acad
Dermatol. 2004;50(5):714–721.
601. Szeimies RM, Gerritsen MJ, Gupta G, et al.
Imiquimod 5% cream for the treatment of actinic
keratosis: results from a phase III, randomized,
double-blind, vehicle-controlled, clinical trial with
histology. J Am Acad Dermatol. 2004;51(4):547–555.
602. Korman N, Moy R, Ling M, et al. Dosing with 5%
imiquimod cream 3 times per week for the treatment
of actinic keratosis: results of two phase 3, random-
ized, double-blind, parallel-group, vehicle-controlled
trials. Arch Dermatol. 2005;141(4):467–473.
603. Wolf JE Jr, Taylor JR, Tschen E, Kang S. Topical 3.0%
diclofenac in 2.5% hyaluronan gel in the treatment
of actinic keratoses. Int J Dermatol. 2001;40(11):
709–713.
604. Rosen RH, Gupta AK, Tyring SK. Dual mechanism
of action of ingenol mebutate gel for topical treat-
ment of actinic keratoses: rapid lesion necrosis
followed by lesion-specific immune response. J Am
Acad Dermatol. 2012;66(3):486–493.
605. Lebwohl M, Swanson N, Anderson LL, Melgaard A,
Xu Z, Berman B. Ingenol mebutate gel for actinic
keratosis. N Engl J Med. 2012;366(11):1010–1019.
374.e12 Part I: Science and Clinical Oncology
606. Inc LP. FDA Approves Picato; 2012. http://www
.prnewswire.com/news-releases/fda-approves-picato
-ingenol-mebutate-gel-the-first-and-only-topical
-actinic-keratosis-ak-therapy-with-2-or-3-
consecutive-days-of-once-daily-dosing-138033448
.html.
607. US Food and Drug Administration. FDA Drug
Safety Communication: FDA warns of severe adverse
events with application of Picato (ingenol mebutate)
gel for skin condition; requires label changes; 2015.
https://www.fda.gov/Drugs/DrugSafety/ucm459142.
htm.
608. Ianhez M, Fleury LF Jr, Miot HA, Bagatin E.
Retinoids for prevention and treatment of actinic
keratosis. An Bras Dermatol. 2013;88(4):585–593.
609. Bavinck JN, Tieben LM, Van der Woude FJ, et al.
Prevention of skin cancer and reduction of keratotic
skin lesions during acitretin therapy in renal trans-
plant recipients: a double-blind, placebo-controlled
study. J Clin Oncol. 1995;13(8):1933–1938.
610. Kadakia KC, Barton DL, Loprinzi CL, et al.
Randomized controlled trial of acitretin versus
placebo in patients at high-risk for basal cell or
squamous cell carcinoma of the skin (North Central
Cancer Treatment Group Study 969251). Cancer.
2012;118(8):2128–2137.
611. Weinstock MA, Bingham SF, Digiovanna JJ,
et al. Tretinoin and the prevention of keratinocyte
carcinoma (basal and squamous cell carcinoma of the
skin): a veterans affairs randomized chemoprevention
trial. J Invest Dermatol. 2012;132(6):1583–1590.
612. Levine N, Moon TE, Cartmel B, et al. Trial of retinol
and isotretinoin in skin cancer prevention: a random-
ized, double-blind, controlled trial. Southwest Skin
Cancer Prevention Study Group. Cancer Epidemiol
Biomarkers Prev. 1997;6(11):957–961.
613. Clouser MC, Roe DJ, Foote JA, Harris RB, Alberts
DS. Dose response of retinol and isotretinoin in the
prevention of nonmelanoma skin cancer recurrence.
Nutr Cancer. 2010;62(8):1058–1066.
614. Moon TE, Levine N, Cartmel B, et al. Design and
recruitment for retinoid skin cancer prevention
(SKICAP) trials. The Southwest Skin Cancer Preven-
tion Study Group. Cancer Epidemiol Biomarkers Prev.
1995;4(6):661–669.
615. Moon TE, Levine N, Cartmel B, Bangert JL.
Retinoids in prevention of skin cancer. Cancer Lett.
1997;114(1–2):203–205.
616. Butler GJ, Neale R, Green AC, Pandeya N,
Whiteman DC. Nonsteroidal anti-inflammatory drugs
and the risk of actinic keratoses and squamous cell
cancers of the skin. J Am Acad Dermatol. 2005;53(6):
966–972.
617. Johannesdottir SA, Chang ET, Mehnert F, Schmidt
M, Olesen AB, Sorensen HT. Nonsteroidal anti-
inflammatory drugs and the risk of skin cancer: a
population-based case-control study. Cancer. 2012;
118(19):4768–4776.
618. Asgari MM, Chren MM, Warton EM, Friedman
GD, White E. Association between nonsteroidal anti-
inflammatory drug use and cutaneous squamous cell
carcinoma. Arch Dermatol. 2010;146(4):388–395.
619. Grau MV, Baron JA, Langholz B, et al. Effect of
NSAIDs on the recurrence of nonmelanoma skin
cancer. Int J Cancer. 2006;119(3):682–686.
620. Muranushi C, Olsen CM, Pandeya N, Green AC.
Aspirin and nonsteroidal anti-inflammatory drugs
can prevent cutaneous squamous cell carcinoma:
a systematic review and meta-analysis. J Invest
Dermatol. 2015;135(4):975–983.
621. Clouser MC, Roe DJ, Foote JA, Harris RB. Effect
of non-steroidal anti-inflammatory drugs on non-
melanoma skin cancer incidence in the SKICAP-
AK trial. Pharmacoepidemiol Drug Saf. 2009;
18(4):276–283.
622. Zhu Y, Cheng Y, Luo RC, Li AM. Aspirin for the
primary prevention of skin cancer: a meta-analysis.
Oncol Lett. 2015;9(3):1073–1080.
623. Curiel-Lewandrowski C, Swetter SM, Einspahr JG,
et al. Randomized, double-blind, placebo-controlled
trial of sulindac in individuals at risk for melanoma:
evaluation of potential chemopreventive activity.
Cancer. 2012;118(23):5848–5856.
624. Thomas RM, Worswick S, Aleshin M. Retinoic acid
for treatment of systemic sclerosis and morphea: a
literature review. Dermatol Ther. 2017;30(2).
625. Muller-Decker K. Cyclooxygenase-dependent
signaling is causally linked to non-melanoma skin
carcinogenesis: pharmacological, genetic, and clini-
cal evidence. Cancer Metastasis Rev. 2011;30(3–4):
343–361.
626. Elmets CA, Ledet JJ, Athar M. Cyclooxygenases: medi-
ators of UV-induced skin cancer and potential targets
for prevention. J Invest Dermatol. 2014;134(10):
2497–2502.
627. Elmets CA, Viner JL, Pentland AP, et al. Chemopre-
vention of nonmelanoma skin cancer with celecoxib:
a randomized, double-blind, placebo-controlled trial.
J Natl Cancer Inst. 2010;102(24):1835–1844.
628. Gurpinar E, Grizzle WE, Piazza GA. NSAIDs
inhibit tumorigenesis, but how? Clin Cancer Res.
2014;20(5):1104–1113.
629. Meyskens FL Jr, Gerner EW. Development of difluo-
romethylornithine (DFMO) as a chemoprevention
agent. Clin Cancer Res. 1999;5(5):945–951.
630. Bailey HH, Kim K, Verma AK, et al. A randomized,
double-blind, placebo-controlled phase 3 skin cancer
prevention study of {alpha}-difluoromethylornithine
in subjects with previous history of skin cancer.
Cancer Prev Res (Phila). 2010;3(1):35–47.
631. Kreul SM, Havighurst T, Kim K, et al. A phase III
skin cancer chemoprevention study of DFMO: long-
term follow-up of skin cancer events and toxicity.
Cancer Prev Res (Phila). 2012;5(12):1368–1374.
632. Tang JY, Mackay-Wiggan JM, Aszterbaum M,
et al. Inhibiting the hedgehog pathway in patients
with the basal-cell nevus syndrome. N Engl J Med.
2012;366(23):2180–2188.
633. Sekulic A, Migden MR, Oro AE, et al. Efficacy
and safety of vismodegib in advanced basal-cell
carcinoma. N Engl J Med. 2012;366(23):2171–2179.
634. Wang A, Stefanick ML, Kapphahn K, et al. Relation
of statin use with non-melanoma skin cancer: pro-
spective results from the Women’s Health Initiative.
Br J Cancer. 2016;114(3):314–320.
635. Wang A, Tang JY, Stefanick ML. Relation of statin
use with non-melanoma skin cancer: prospective
results from the Women’s Health Initiative. Womens
Health (Lond). 2016;12(5):453–455.
636. Tang JY, Fu T, Leblanc E, et al. Calcium plus vitamin
D supplementation and the risk of nonmelanoma
and melanoma skin cancer: post hoc analyses of
the women’s health initiative randomized controlled
trial. J Clin Oncol. 2011;29(22):3078–3084.
637. Frieling UM, Schaumberg DA, Kupper TS,
Muntwyler J, Hennekens CH. A randomized,
12-year primary-prevention trial of beta carotene
supplementation for nonmelanoma skin cancer in the
physician’s health study. Arch Dermatol. 2000;136(2):
179–184.
638. Ferrucci LM, Cartmel B, Molinaro AM, Leffell DJ,
Bale AE, Mayne ST. Tea, coffee, and caffeine and
early-onset basal cell carcinoma in a case-control
study. Eur J Cancer Prev. 2014;23(4):296–302.
639. Miura K, Hughes MC, Arovah NI, van der Pols JC,
Green AC. Black tea consumption and risk of skin
cancer: an 11-year prospective study. Nutr Cancer.
2015;67(7):1049–1055.
640. Chen AC, Martin AJ, Choy B, et al. A Phase 3
Randomized trial of nicotinamide for skin-cancer
chemoprevention. N Engl J Med. 2015;373(17):1618–
1626.
641. Marks R, Dorevitch AP, Mason G. Do all melanomas
come from “moles”? A study of the histological asso-
ciation between melanocytic naevi and melanoma.
Australas J Dermatol. 1990;31(2):77–80.
642. Bevona C, Goggins W, Quinn T, Fullerton J, Tsao
H. Cutaneous melanomas associated with nevi. Arch
Dermatol. 2003;139(12):1620–1624; discussion
1624.
643. Chiu LC, Tong KF, Ooi VE. Cytostatic and cytotoxic
effects of cyclooxygenase inhibitors and their synergy
with docosahexaenoic acid on the growth of human
skin melanoma A-375 cells. Biomed Pharmacother.
2005;59(suppl 2):S293–S297.
644. Ottino P, Duncan JR. Effect of vitamin E and
indomethacin treatment on adenylate cyclase activity,
PGE2 and cAMP levels in murine melanoma cells.
Prostaglandins Leukot Essent Fatty Acids. 1997;56(2):
143–149.
645. Duff M, Stapleton PP, Mestre JR, et  al.
Cyclooxygenase-2 inhibition improves macrophage
function in melanoma and increases the antineoplas-
tic activity of interferon gamma. Ann Surg Oncol.
2003;10(3):305–313.
646. Nakata K, Kashimoto S, Yoshida H, Oku T,
Nakamura S. Augmented antitumor effect of recom-
binant human interleukin-1 alpha by indomethacin.
Cancer Res. 1988;48(3):584–588.
647. Harris RE, Beebe-Donk J, Namboodiri KK. Inverse
association of non-steroidal anti-inflammatory drugs
and malignant melanoma among women. Oncol
Rep. 2001;8(3):655–657.
648. Ramirez CC, Ma F, Federman DG, Kirsner RS. Use
of cyclooxygenase inhibitors and risk of melanoma in
high-risk patients. Dermatol Surg. 2005;31(7 Pt 1):
748–752.
649. Joosse A, Koomen ER, Casparie MK, Herings
RM, Guchelaar HJ, Nijsten T. Non-steroidal
anti-inflammatory drugs and melanoma risk: large
Dutch population-based case-control study. J Invest
Dermatol. 2009;129(11):2620–2627.
650. Curiel-Lewandrowski C, Nijsten T, Gomez ML,
Hollestein LM, Atkins MB, Stern RS. Long-term use
of nonsteroidal anti-inflammatory drugs decreases the
risk of cutaneous melanoma: results of a United States
case-control study. J Invest Dermatol. 2011;131(7):
1460–1468.
651. Famenini S, Duan L, Young L. Aspirin use and
melanoma: a UCLA pilot study. J Am Acad Dermatol.
2015;72(6):1094–1095.
652. ASPREE Investigator Group. Study design of ASPirin
in Reducing Events in the Elderly (ASPREE): a
randomized, controlled trial. Contemp Clin Trials.
2013;36(2):555–564.
653. Uzarska M, Czajkowski R, Schwartz RA, Bajek
A, Zegarska B, Drewa T. Chemoprevention of
skin melanoma: facts and myths. Melanoma Res.
2013;23(6):426–433.
654. Linden KG, Leachman SA, Zager JS, et al. A
randomized, double-blind, placebo-controlled phase
II clinical trial of lovastatin for various endpoints
of melanoma pathobiology. Cancer Prev Res (Phila).
2014;7(5):496–504.
655. Asgari MM, Brasky TM, White E. Association of
vitamin A and carotenoid intake with melanoma
risk in a large prospective cohort. J Invest Dermatol.
2012;132(6):1573–1582.
656. Miura K, Green AC. Dietary antioxidants and
melanoma: evidence from cohort and intervention
studies. Nutr Cancer. 2015;67(6):867–876.
657. Caini S, Masala G, Saieva C, et al. Coffee, tea
and melanoma risk: findings from the European
Prospective Investigation into Cancer and Nutrition.
Int J Cancer. 2017;140(10):2246–2255.
658. Chhabra G, Ndiaye MA, Garcia-Peterson LM,
Ahmad N. Melanoma chemoprevention: current
status and future prospects. Photochem Photobiol.
2017.
659. Sharma A, Koldovsky U, Xu S, et al. HER-2 pulsed
dendritic cell vaccine can eliminate HER-2 expres-
sion and impact ductal carcinoma in situ. Cancer.
2012;118(17):4354–4362.
 Screening and Early Detection 23 
Therese Bevers, Hashem El-Serag, Samir Hanash, Aaron P. Thrift,
Kenneth Tsai, Karen Colbert Maresso, and Ernest Hawk

S UMMARY OF K EY
• Cancer incidence is on the rise in the
United States and worldwide.
Beyond therapeutics, substantial
reduction in cancer mortality
necessitates improved understanding
of cancer risk, implementation of
effective preventive intervention
strategies, and early detection of
cancers that are likely to progress.
• Genomic- and proteomic-based
approaches have the potential to
refine risk assessment and
stratification, allowing for more
tailored screening and early
detection strategies. However, the
use of such approaches is still in its
early stages.
• In the United States, population- as a colorectal cancer screening
based screening tests are currently
recommended for breast, cervical,
colon, and lung cancers.
Individualized decision making after
a discussion with a clinician is
recommended for men in the case of
prostate cancer screening.
• Discordant professional society
recommendations for screening have
accentuated the need to implement
P OI NT S
risk-stratified screening for common
cancers to improve the effectiveness
of screening.
• Overdiagnosis is finding cancers that
would never become clinically
relevant in a person’s lifetime. This
has been an issue mainly in breast
and prostate cancer screening.
• Systematic reviews of randomized
controlled trials (RCTs) of screening
mammography have demonstrated
an approximate 20% mortality
reduction in women ages 40 to identified. The two tests commonly
74 years.
• Guaiac-based fecal occult blood
tests (gFOBTs) have the strongest
direct evidence supporting their use
modality, with a 32% reduction in
colorectal cancer mortality with
annual use.
• Lung cancer screening with
low-dose computed tomography
resulted in an approximate 20%
reduction in lung cancer mortality in
the National Lung Screening Trial.
• Human papillomavirus (HPV)
vaccination does not remove the
need for routine cervical cancer
screening according to age and
clinical history.
• Because most risk factors for liver
cancer in the United States and
other developed countries are
associated with cirrhosis, patients
with cirrhosis are the target of
cancer prevention and surveillance
efforts for hepatocellular carcinoma.
• An effective screening test for
ovarian cancer has not been
investigated for ovarian cancer
screening are CA125 and
transvaginal ultrasound.
• Randomized clinical trials for skin
cancer screening are unlikely to be
practical. The US Preventive
Services Task Force (USPSTF)
statement on skin cancer is
forward-looking because it opens the
door to case-control designs that are
more practical, while acknowledging
that strong evidence of benefits and
harms is lacking, including for
nonmelanoma skin cancer.

Although substantial progress is currently being made in the develop-
ment of novel and more effective therapeutics, cancer remains a largely
unsolved clinical problem with high mortality. Cancer incidence is
on the rise in the United States and worldwide. Between 2005 and
2015, the number of cancer cases increased by 33%, owing in part
to population aging, which contributed 16%, and population growth
and changes in age-specific rates, which contributed the remainder.1
Cancer incidence and the societal cancer burden are expected to increase
further.2 Beyond therapeutics, substantial reduction in cancer mortality
necessitates improved understanding of cancer risk, implementation
of effective preventive intervention strategies, and early detection of
cancers that are likely to progress to avoid the problem of overdiagnosis,
all of which represent substantial challenges that can be overcome.
Identifying individuals at increased risk of developing a particular
cancer type would allow for a range of preventive interventions to be
implemented, from altered lifestyle and health behaviors to the use
of vaccines, and early detection of particular cancer(s) for which these
persons are at risk. A case in point is obesity, which is a risk factor
for at least 13 different cancers. Elucidation of metabolic, immune,
and other molecular profiles that contribute to the increased risk
would allow for more focused screening strategies for persons with
these risk profiles and would allow implementation of targeted preven-
tion strategies aimed at the cancer(s) for which they are at risk.3,4
RISK ASSESSMENT
Guidelines are currently available for risk assessment in the clinical
setting through several organizations. Guidelines allow a determination
of a subject’s risk for the common cancers and the associated options
for screening. Organizations creating guidelines include the National
Comprehensive Cancer Network (NCCN; https://www.nccn.org/),
the American Cancer Society (ACS; https://www.acs.org/), and other
advocacy and professional societies. Family history and other patient
characteristics are often important determinants of risk. In general,
individuals are first defined as being at either average risk or increased
risk for a particular cancer. For example, the NCCN Version 2.2016

375
376 Part I: Science and Clinical Oncology

colorectal cancer (CRC) screening guidelines identify a person as

being at “average risk” based on age, negative family history, and no Table 23.1  Performance Characteristics of
prior personal history of inflammatory bowel disease, adenoma, or Screening Tests
sessile serrated polyp or CRC. Risk is increased with a positive family
Performance Practical
or personal history of cancer or precancer. High-risk syndromes include
Characteristic Definition Formula Application
Lynch syndrome and a number of other syndromes. Screening guidelines
for CRC and the modalities used for screening vary based on the risk Accuracy Proportion of TPs + Have disease and
profile. Other common cancers for which risk assessment and screening correct test results TNs/TPs have positive test
guidelines are available include breast, cervical, lung, and prostate. + TNs + result + do not
Given the widespread use of the prostate-specific antigen (PSA) test FPs + FNs have disease and
for prostate cancer screening, the NCCN guidelines for early detection have negative
test result/all test
of prostate cancer stratify men based on their age and PSA levels.
results
There is currently substantial interest in bolstering traditional
guidelines by adding genomic and other “omic” profiles to other Sensitivity Ability of the test TPs/TPs + Have disease and
personal characteristics and family history to determine the need for to correctly identify FNs have positive test
and frequency of screening.5,6 The field is still in its early stages.7 In someone with the results/true
disease as positive disease
the case of breast cancer, susceptibility genes include highly penetrant
mutations in the BRCA1 and BRCA2 genes, the testing for which is Specificity Ability of the test TNs/TNs Do not have
indicated by a positive family history of breast cancer. Several other to correctly identify + FPs disease and have
moderately penetrant mutations in other genes and more common someone without negative test
genomic variants with a modest increase in risk have been identified.8 the disease as results/true no
negative disease
These may very well influence risk assessments and guide screening
in the near future. Positive Probability that TPs/TPs + Have disease and
predictive someone with a FPs have positive test
value (PPV) positive test result results/all
SCREENING AND EARLY DETECTION actually has the positive test
disease results
Although advances in screening and early detection are essential for
progress in both the prevention and treatment of cancer, the incorpora- Negative Probability that TNs/TNs Do not have
tion of such advances into the clinic is challenging and demands a predictive someone with a + FNs disease and have
careful weighing of all potential risks and benefits. At least three value (NPV) negative test result negative test
does not have the results/all
criteria must be fulfilled for a cancer screening test to be useful9:
disease negative test
results
1. The test must detect the disease earlier than routine methods.
2. Earlier treatment must lead to improved outcomes. FN, False negative; FP, false positive; TN, true negatives; TP, true positive.
3. The benefits of screening must be greater than the risks of any
subsequent diagnostic and therapeutic treatments.

A screening test is assessed by its performance characteristics, which
include its sensitivity and specificity, its accuracy, and its positive
predictive value (PPV) and negative predictive value (NPV) (Table
23.1). Sensitivity can be considered the “true-positive” rate, and
specificity can be considered the “true-negative” rate. If a screening
test were 100% sensitive, every individual with the disease in question
within the population would be identified as such; and if it were
100% specific, it would identify every healthy individual as not having
the disease. In reality, however, there is no screening test that achieves
this ideal. Sensitivity and specificity are inversely related, such that
increasing the sensitivity of a test will result in more false positives,
whereas increasing the specificity will result in more false negatives.9
Striking an acceptable balance between these two performance
characteristics is challenging and depends on the disease in question.
In the case of many types of cancers that are curable only in the earlier
stages of the disease, it may be desirable to minimize false negatives,
because these could result in a patient’s delayed diagnosis and cancer-
specific death, and accept more false positives (i.e., higher sensitivity
than specificity). However, where the balance is struck ultimately
depends on the nature and severity of the specific disease being screened
for and the effectiveness of downstream interventions and treatments
for that disease.
The sensitivity and specificity of a screening test are critical to
consider in the design of any population-based screening program.
However, at least at the clinical level, it is also useful to appreciate
the PPV and NPV of the screening test. The PPV is the proportion
of patients who test positive who actually have the disease, and the
NPV is the proportion of those who test negative who truly do not
have the disease. Thus these values provide a clinician with an estimate
of the probability that his or her patient does or does not have the
disease, given the test result. The PPV is influenced by the prevalence
of the disease in the population screened, such that the PPV will be
higher at higher disease prevalence. In addition, when the disease in
question is relatively rare within a population, as is the case with most
cancers, the specificity of the test being used also matters greatly
because most people who will be screened will not have the disease.
In these patients, increasing the specificity of a screening test will
improve its PPV.
Observational data supporting the use of screening tests do not
rule out the potential for these tests to mislead as a result of at least
two important potential biases: lead-time and length biases (Fig. 23.1).
To minimize the influence of these biases, the development of effective
screening tests should ideally culminate in well-conceived and well-
conducted randomized controlled trials (RCTs) that assess a cancer-
related, if not overall, mortality end point.
Implementation of screening programs for common cancers, notably
cervical, breast, lung, and colon cancers, has been shown to reduce
the mortality associated with these cancers. However, whenever there
is a screenable disease that is prevalent but potentially indolent, there
is the potential for overdiagnosis and overtreatment, resulting in a
range of personal and social costs that may ultimately outweigh the
intended benefits. Overdiagnosis is the identification by screening of
a cancer that would never have caused symptoms or adversely influenced
the health of an individual during his or her lifetime; and overtreatment
is the treatment of such an identified cancer. Breast and prostate
cancer screening are two examples in which these issues have challenged
screening efforts. The use of the PSA test for prostate cancer screening
has been particularly controversial.10 The American Urological Associa-
tion recommended shared decision making for men ages 55 to 69
years considering PSA-based screening, a target age group for whom

Lead-time bias
No screen Sx-Dx
Death
Survival
Screen CT-Dx
Death
Lead-time
With screening, the lead time in diagnosis prolongs
survival even if death is not delayed.
Length bias
Screening
Indolent Symptoms
cancer begins and Dx
Death
Detectable
preclinical phase
Aggressive
Sx and Dx
cancer begins Death Screening tends to
detect more indolent
cancers.
Figure 23.1  •  Schematic depictions of lead-time and length biases. Lead
time refers to the amount of time between screen-detected diagnosis and
symptom-detected diagnosis, and it appears to prolong survival in screened
individuals, although death is not actually delayed. Length bias refers to the
fact that screening is more likely to detect indolent or slow-growing cancers
than it is to detect more aggressive forms. Persons who are screened will
therefore have better survival rates than persons who are not screened; however,
these better rates are not actually a result of the screening itself, but rather of
the more indolent nature of cancers that are detected with screening. (From
National Cancer Institute. https://www.cancer.gov/about-cancer/screening/
research/what-screening-statistics-mean.)
benefits may outweigh harms.11 On the other hand, the US Preventive
Services Task Force (USPSTF) has discouraged (i.e., assigned a D
recommendation to) the use of the PSA for prostate cancer screening,
although the organization is now in the process of updating this recom-
mendation. Nevertheless, it is viewed that the USPSTF decision affected
the number and distribution of prostate cancer diagnoses in the United
States.12 Mammography screening for breast cancer has been widely
adopted in many Western countries. However, there has been growing
debate and concern about the benefits and harms of screening with
mammography.13 Guidelines and recommendations for screening
mammography have varied among countries. A working group
assembled by the International Agency for Research on Cancer (IARC)
assessed the cancer-preventive and adverse effects of different methods
of screening for breast cancer.14 The group recognized that the relevance
of RCTs conducted more than 20 years ago should be questioned.
After a careful evaluation of the balance between the benefits and
adverse effects of mammographic screening, the group concluded that
there is a net benefit from screening women ages 50 to 69 years of
age. However, the merits of mammographic screening have been
questioned by others.15 The ACS updated its 2003 breast cancer
screening guidelines for women at average risk of breast cancer and
issued qualified recommendations. The guidelines of 2015 state that
Screening and Early Detection  •  CHAPTER 23 377
women ages 45 to 54 years should be screened annually, although
women ages 40 to 44 should have the opportunity to begin screening
if they so choose, and women age 55 years and older should transition
to biennial screening or have the opportunity to continue screening
annually. Screening should continue as long as a woman’s overall
health is good and she has a life expectancy of 10 years or longer.
Clearly, guidelines for cancer screening are continually evolving.
Discordant professional society recommendations for screening have
accentuated the need to implement risk-stratified screening for common
cancers to improve the effectiveness of screening.16 Risk stratification
may be based on an individual’s genotype or other types of molecular
markers in addition to subject characteristics. For example, a novel
approach to risk-based breast cancer screening has been proposed that
integrates clinical risk factors, breast density, a polygenic risk score
representing the cumulative effects of genetic variants, and sequencing
for moderate- and high-penetrance germline mutations.17 The added
costs versus benefits of molecular testing to guide screening for common
cancers requires further evaluation.
Table 23.2 presents a summary of the USPSTF classification of the
evidence for various cancer screening tests in average-risk individuals.
BREAST CANCER
Breast cancer is the most commonly occurring cancer in women and
the second leading cause of cancer death. Since 1989, the number of
deaths from breast cancers has decreased owing to improvements in
treatment as well as screening.18 Screening provides an opportunity
for the early detection of breast cancer, which has been shown to
reduce not only breast cancer mortality but also treatment morbidity.
Nonmodifiable risk factors include older age, a personal or
family history of breast or ovarian cancer including women with a
genetic mutation for breast cancer, a history of premalignant breast
lesions such as atypical hyperplasia (AH) or lobular carcinoma in situ
(LCIS), or a history of radiation exposure between the ages of 10
and 30 years.18
Modifiable or potentially modifiable risk factors for breast cancer
include increased breast density, moderate to heavy alcohol use, weight
gain after the age of 18, being overweight or obese (for postmenopausal
breast cancer), physical inactivity, and use of exogenous estrogen plus
progesterone (e.g., in postmenopausal hormone therapy).18
Reproductive factors that increase breast cancer risk include a long
menstrual history (early age of menarche or late age of menopause)
and nulliparity or late age of first pregnancy.18 These endocrine risk
factors demonstrate the responsiveness of breast cancer to antiestrogen
drugs and have led to the use of antiestrogen strategies for the prevention
of breast cancer.
Risk Modeling and Assessment
Breast cancer risk assessment is relatively sophisticated compared with
other cancers. The Gail model, a multivariable model used to assess
risk of breast cancer based on age, family history of breast cancer in
first-degree relatives (FDRs), breast biopsy history, and the presence
of AH and various other endocrine features,19,20 is often used to
determine candidates for preventive therapy with antiestrogen drugs
such as tamoxifen or raloxifene. Although this model is generally
accurate for populations, its ability to predict if an individual woman
will get breast cancer is limited. Newer risk assessment models such
as the Breast Cancer Surveillance Consortium model also consider
mammographic density in determining the risk of development of
breast cancer.21–23
For women with a strong family history of breast cancer (with or
without a family history of ovarian cancer), other models may be
more accurate. The Tyrer-Cuzick model estimates a woman’s risk of
developing breast cancer through use of many of the variables used
in the Gail model but includes a more expansive family history. It
also estimates her risk of a BRCA mutation and may be used to
378 Part I: Science and Clinical Oncology

Table 23.2  US Preventive Services Task Force (USPSTF) Grade Definitions and Associated Cancer
Screening Tests in Average-Risk Individuals as of June 2017a,b
Grade Definition
A The USPSTF recommends the service.
There is high certainty that the net benefit
is substantial.
B The USPSTF recommends the service.
There is high certainty that the net benefit
is moderate or there is moderate certainty
that the net benefit is moderate to
substantial.
C The USPSTF recommends selectively
offering or providing this service to
individual patients based on professional
judgment and patient preferences. There
is at least moderate certainty that the net
benefit is small.
D The USPSTF recommends against the
service. There is moderate or high
certainty that the service has no net
benefit or that the harms outweigh the
benefits.
I The USPSTF concludes that the current
evidence is insufficient to assess the
balance of benefits and harms of the
service. Evidence is lacking, of poor quality,
or conflicting, and the balance of benefits
and harms cannot be determined.
Suggestions for Practice
Offer or provide this service.
Offer or provide this service.
Offer or provide this service for
selected patients depending
on individual circumstances.
Discourage the use of this
service.
Read the clinical considerations
section of USPSTF
Recommendation Statement. If
the service is offered, patients
should understand the
uncertainty about the balance
of benefits and harms.
Current Screening Test Recommendations
Cervical cancer screening
for women ages 21–65 with pap smear
or
pap smear with HPV-testing in women ages 30–65
Colorectal cancer screening in adults ages 50–75
Breast cancer screening (biennial) for women ages 50–74
Lung cancer screening in adults ages 55–80 with a
history of smoking
Breast cancer screening for women ages 40–49
Colorectal cancer screening in adults ages 76–85
Prostate cancer screening in men ages 55–69c
Cervical cancer screening in women younger than age 30
with HPV testing alone or in combination with cytologic
assessment
Cervical cancer screening in women younger than age 21
Cervical cancer screening in women age 65+ who have
had adequate prior screening
Ovarian cancer screening
Pancreatic cancer screening
Prostate cancer screening in men age 70+c
Breast cancer screening in women age 75+
Oral cancer screening
Skin cancer screening

a
Based on grade definitions after July 2012.
b
The USPSTF defines certainty as “likelihood that the USPSTF assessment of the net benefit of a preventive service is correct.” The net benefit is defined as benefit minus
harm of the preventive service as implemented in a general, primary care population. The USPSTF assigns a certainty level based on the nature of the overall evidence available
to assess the net benefit of a preventive service.
c
Draft recommendation as of June 2017.
HPV, Human papillomavirus
Adapted with permission of the U.S Preventive Services Task Force.

determine if genetic testing is recommended.24 Other models developed
for the assessment of a genetic mutation in a specific woman include
the following: Berry-Parmigiani-Aguilar (BRCA-Pro),25–27 Claus,28
Couch,29 and the Breast and Ovarian Analysis of Disease Incidence
and Carrier Estimation Algorithm (BOADICEA).30–32 Geneticists and
physicians specializing in cancer risk assessment commonly use these
models to guide genetic testing decisions as well as recommendations
for breast screening.
Screening
Breast Awareness
Breast self-examination (BSE) was a mainstay for decades in breast
cancer screening recommendations, but this recommendation changed
for most organizations in the early 2000s when findings from a large
RCT of BSE in women in Shanghai, China noted no difference in
breast cancer mortality between women performing BSE versus controls.
This trial randomized 266,064 Chinese women to receive instruction
on BSE or a topic unrelated to breast cancer. During the time this
study was conducted, women in China had access to mammography
only for diagnostic evaluation of a clinical finding. Women in the
BSE arm received intensive instruction in BSE technique. Compliance
was encouraged through feedback and reinforcement sessions as well
as monthly reminders. After 10 to 11 years of follow-up, with 135
breast cancer deaths in the instruction group and 131 in the control
group, there was no statistically significant difference between the two
arms in breast cancer mortality (RR, 1.04; 95% confidence interval
[CI], 0.82–1.33; P = .72). In addition, no statistically significant
difference in breast cancer incidence or stage was seen. However, the
BSE group had a higher rate of false positives.33,34
Nonetheless, it is recognized that women are the most likely person
to find a palpable breast cancer, with most being found during normal
activities of daily living (e.g., showering, dressing). For this reason,
BSE has been replaced with the concept of breast awareness, which
recommends that women be familiar with their breasts and promptly
report any change. Differing from BSE, breast awareness does not
involve formalized instruction in the clinical setting or reminders
(such as shower cards).
Clinical Breast Examination
There are limited data on the effectiveness of clinical breast examination
(CBE). Randomized trials comparing CBE versus no screening have
 Screening and Early Detection  •  CHAPTER 23 379

not been performed. A review of controlled trials and case-control
studies that included CBE as part of the screening modality found
sensitivity of CBE to be 54% and specificity, 94%.35
Whereas the ACS recommends against CBE, the NCCN recom-
mends a clinical encounter noting that it provides the opportunity
to perform a number of important clinical activities that may not
otherwise be done if a woman only obtains a screening mammogram
(Table 23.3). In addition to performing breast cancer risk assessment,
advising on risk reduction strategies, including healthy lifestyle
interventions and risk-based screening recommendations, CBE is a
part of this encounter.36
Screening Mammography
The primary benefit of screening mammography is a reduction in death
from breast cancer.36–41 Early detection with screening mammography
Table 23.3  Clinical Encounter Activities Related to
Breast Cancer Screeninga
1. Assess breast cancer risk and refer to a genetic counselor if a
significant family history is identified.
2. Educate about lifestyles that reduce the risk of breast cancer—
specifically, the importance of exercise, maintaining a healthy body
weight, and limiting alcohol intake.
3. For women at increased risk of breast cancer, counsel regarding
preventive therapies such as tamoxifen, raloxifene, or aromatase
inhibitors.b
4. Provide risk-based breast cancer screening recommendations,
which may include screening breast magnetic resonance imaging
for women at increased risk.
5. Perform clinical breast examination.
a
As recommended by the National Comprehensive Cancer Network (NCCN)
Clinical Practice Guidelines in Oncology: Breast Cancer Screening and Diagnosis
(Version 2.2016).36
b
Not approved by the US Food and Drug Administration for this indication.
Data from NCCN Clinical Practice Guidelines in Oncology: Breast Cancer Screening
and Diagnosis (Version 2.2016)36.
may also result in lower morbidity because surgery is often less extensive
and toxic systemic chemotherapy may be less frequently needed.42–45
Systematic reviews of RCTs of screening mammography have
demonstrated an approximate 20% mortality reduction in women
ages 40 to -74 years.37,38 However, all but the Age Trial46 were initiated
in the early 1980s or earlier. Although RCTs have demonstrated a
mortality reduction, they are limited by the quality of mammography
available at the time the study was conducted, with many cancers
having to be 1 cm or greater to be detected.47 A meta-analysis of
observational case-control studies showed a significant 48% mortality
reduction with modern screening mammography.48 Although obser-
vational data are limited by bias, these data are believed by many to
better reflect contemporary screening practices in which the detection
of subcentimeter breast cancers is commonplace. Modeling studies
have demonstrated a 29% to 54% mortality reduction with annual
mammographic screening, providing further support for a larger
mortality reduction than seen in the RCTs.49
Various guidelines from the ACS, USPSTF, and NCCN and recom-
mendations from professional organizations, including the American
College of Radiology, American College of Obstetrics and Gynecology,
American College of Physicians, and American Academy of Family
Physicians, are currently available to guide breast cancer screening in
women at average risk (Table 23.4). A number of organizations recom-
mend annual mammographic screening beginning in the 40s,36,40,50,51
whereas others recommend biennial screening beginning at age 50.41,52
All recommending organizations agree that screening beginning at
age 40 results in a mortality reduction and that annual screening leads
to fewer breast cancer deaths than does biennial screening. Among
women ages 40 to 49 years, the reduction in mortality ranges from
15% in RCTs to 47% in observational studies.38–40,53 Early detection
and successful treatment results in significantly more life-years gained
for this decade than for any other decade.54 Organizations that recom-
mend against annual mammographic screening and delaying initiation
of mammographic screening to the 50s cite concerns that the false
positives and overdiagnosis of screening may not balance the benefits
of screening for women in their 40s.41 However, the USPSTF analysis
shows that the false-positive rate for the same screening interval (annual
or biennial) is equivalent for women in their 40s and 50s and that the

Table 23.4  Breast Cancer Screening Recommendations by Organization

Organization
American Academy of
Family Physicians (AAFP)
American Cancer Society
(ACS)
American College of
Obstetricians and
Gynecologists (ACOG)
American College of
Physicians (ACP)
American College of
Radiology (ACR)
National Comprehensive
Cancer Network (NCCN)
US Preventive Services
Task Force (USPSTF)
When to Initiate Screening
Follow US Preventive Services Task
Force (USPSTF) recommendations
Opportunity to begin screening at
ages 40–44
Regular screening starting at age 45
Annual screening starting at age 40
Individualized for women ages 40–49
Regular screening starting at age 50
annual screening starting at age 40
Annual screening starting at age 40
Individualized for women ages 40–49
Regular screening beginning at age 50
Frequency of Screening When to Stop Screening
Follow USPSTF recommendations Follow USPSTF recommendations
Annually from age 45–54 Continue screening mammography as
Biennially starting at age 55 with long as overall health is good and life
opportunity to continue annually expectancy is 10 yr or longer
Annually Not specified
Biennially Age 75 yr or older
Women of any age with life expectancy
<10 yr
Annually Should be considered as long as the
patient is in good health and is willing to
undergo additional testing if an
abnormality is detected
Annually Upper age limit is not yet established
Consider comorbid conditions limiting
life expectancy (e.g., ≤10 yr) and whether
therapeutic interventions are planned
Biennially Insufficient evidence to recommend for
or against screening at age 75 or older
380 Part I: Science and Clinical Oncology
biopsy rate is actually higher for women in their 50s.41 In addition, it
is recognized that women undergoing their first screening mammogram
have a higher recall rate owing to a lack of prior mammograms for
comparison. Moving the initiation of screening to age 50 would further
increase the incidence of false-positive mammograms at this age.
Most clinicians recognize overdiagnosis of breast cancer as less an
issue of screening and more of a problem related to overtreatment. It
is clear that we need to understand which “breast cancers” do not
require treatment. Clinical trials are opening to begin to investigate
this question. Having said this, it is important to recognize that the
number of overdiagnosed breast cancers is not necessarily influenced
by the age of initiation of screening mammography or the interval in
which screening occurs. It has been shown that breast cancers that
do not progress (i.e., an overdiagnosed breast cancer) also do not
spontaneously resolve.55 These cancers will be diagnosed at the first
screening after their development, whether that is at age 40 or 50 (or
somewhere in between) and whether the screening occurs annually
or biennially.
Full-field digital mammography (FFDM) is the current standard
of care for breast cancer screening. Early data have shown that
tomosynthesis increases the detection of breast cancer and reduces
the recall rate.56–59 This suggests that this modality has the potential
to provide a more favorable balance of benefits and risks of screening
mammography.
Women who have a high risk of breast cancer because of a known
inherited mutation, a strong family history with a greater than 20%
lifetime risk of breast cancer, or prior exposure to therapeutic radiation
between the ages 10 and 30 years should be more aggressively screened
with supplemental breast magnetic resonance imaging (MRI) in addition
to annual screening mammography.36,60,61 It is suggested that women
with high-risk lesions such as atypical ductal or lobular hyperplasia
or LCIS should also be considered for supplemental screening with
breast MRI based on emerging evidence.36 NCCN outlines screening
strategies for these high-risk women, including age of initiation and
frequency of screening.36 Although the optimal frequency and timing
of breast MRI scans is not well established, it is frequent practice to
perform annual screening mammogram and annual screening breast
MRI scans staggered every 6 months. This paradigm provides the
opportunity for the detection of “interval” cancers (defined as cancers
detected between scheduled screening examinations), which may occur
more frequently in high-risk women. Breast MRI scanning is more
sensitive in detecting breast masses in dense breasts (such as in young
women), whereas mammograms are more sensitive in detecting breast
calcifications. Therefore both are used to obtain optimal breast screening
in high-risk women. Other screening tests, such as breast ultrasonog-
raphy, are not routinely recommended for breast cancer screening.
Over half of US states now mandate that women be informed about
the increased risk of breast cancer associated with dense breasts and
the option of supplemental screening, but specific tests to recommend
in these women remains an area of investigation.
Research is being done to identify even more effective screening
technologies and approaches. In addition to enhanced imaging modali-
ties, these involve the evaluation of biomarkers in serum, breast duct
fluid, saliva, and tears. These strategies offer great hope for improving
breast cancer early detection. It is anticipated that in the future,
blood- or biofluid-based early detection tests will be used in conjunction
with or in place of more standard imaging screening tests.
COLORECTAL CANCER
Despite being largely preventable through lifestyle modifications, an
estimated 140,250 new cases of CRC will be diagnosed in the United
States during 2018, and an estimated 50,630 lives will be lost to
CRC.62 Modifiable risk factors for CRC are obesity, smoking, alcohol,
and red or processed meat consumption. The World Cancer Research
Fund/American Institute for Cancer Research (WCRF/AICR) has
estimated that 47% of CRC cases, or 63,652 cases, could be prevented
each year through a healthy diet, weight management, and physical
activity. Nonmodifiable risk factors include a personal or family history
of CRC or adenomatous polyps, including the inherited conditions
of familial adenomatous polyposis (FAP) and hereditary nonpolyposis
colorectal cancer (HNPCC), and a personal history of chronic inflam-
matory bowel disease. In addition, the presence of adenomas at screening
is a strong predictor of CRC risk.
Globally, the incidence of CRC varies greatly, with as much as a
10-fold variation in both sexes, with the highest rates found in Australia/
New Zealand and the lowest in Western Africa.63
Mortality rates vary somewhat less than do incidence rates, with
mortality being the highest in Central and Eastern Europe, and the
lowest in Western Africa.63 Although more than half of new CRC
cases occur in more developed regions of the world, more than half
of the deaths due to CRC occur in less developed regions.63
Risk Modeling and Assessment
Despite the development of numerous risk prediction models for
different populations, there is no validated tool with high discrimina-
tory power that can be easily applied in various settings to estimate
risk of colorectal neoplasia. Nevertheless, the Freedman risk model/
NCI’s Colorectal Cancer Risk Assessment tool, developed in 2009,
has been validated in an independent cohort (unlike many of the
other proposed models) and is relevant to a large proportion of the
US population that is eligible for CRC screening.64,65 Although the
model was developed and validated based on non-Hispanic white
men and women, and its generalizability to other racial and ethnic
groups is uncertain, it is believed that the variables that contribute to
the model have deep penetration into the US population, regardless
of race and ethnicity, and that it generalizes to nonwhite populations
within the United States better than do non-US models, of which
there are many.66 The Freedman model originally predicted the future
10- and 20-year absolute risk of CRC, with an area under the curve
(AUC) of 0.61 (95% CI, 0.60–0.62) for men and 0.61 (95% CI,
0.59–0.62) for women, comparable to other cancer risk prediction
models.65 In its current form, the model now predicts 5-year, 10-year,
and lifetime absolute CRC risk. Imperiale and colleagues tested the
ability of the model to predict current risk of advanced neoplasia,
reasoning that knowledge of current risk may more directly affect
short-term decisions regarding screening than knowledge of future
risk, and that this could lead to increased adoption of CRC screening
and to tailored screening strategies wherein those at higher risk for
advanced neoplasia would be directed toward colonoscopy as the
preferred method of screening and those with a lower risk of such a
finding toward less invasive screening methods.66 Results of the study
demonstrated that the model’s discriminatory power for predicting
current advanced neoplasia was in the moderate-to-good range, with
an AUC of 0.71 (95% CI, 0.68–0.73) in the 5-year analysis, which
is better than the AUC of the model in its original validation cohort
as well as the Gail model when applied to an independent cohort to
estimate 5-year breast cancer risk. The authors concluded that the tool
can be used to estimate current risk of advanced neoplasia, making it
potentially useful for tailoring and improving CRC screening efficiency
among those at average-risk of CRC.66 The Cleveland Clinic Score
Against Colon Cancer risk model is another tool that is available
online and is similar to the Freedman/NCI model in terms of the
variables it incorporates. However, it is unclear if it has been evaluated
for publication in a peer-reviewed journal.
Screening
The biology of CRC makes it an ideal candidate for early detection
and preventive interventions. The colon is a relatively accessible organ,
and the vast majority of CRCs develop from adenomas, a precancerous
stage that can last as long as 20 to 30 years, providing ample time
for its detection and removal before it evolves into cancer.

The USPSTF currently recommends that average-risk individuals
be screened for CRC beginning at age 50 and continuing until age
75. For average-risk adults between 76 and 85 years of age, the decision
to screen should be an individual one, considering a patient’s past
screening history and overall health. The USPSTF does not recommend
a particular screening test. Multiple CRC screening examinations exist,
although they differ with respect to the level of evidence supporting
their use, their test performance to detect cancer and adenomas, and
their risk of harms. There has been no assessment of the relative
benefits and harms among the tests, because comparative studies are
currently limited in their study design and power to detect cancers,
any mortality benefits, and associated harms.67 No screening test has
been shown to reduce all-cause mortality.
Screening options include stool tests, direct visualization tests, and
a US Food and Drug Administration (FDA)–approved blood test (Epi
proColon). However, because the blood test has very limited data on
its performance, and because the data that are available suggest that
its ability to detect CRC is worse than that of other noninvasive tests,
this section will discuss only the stool and direct visualization tests.
Guaiac-based fecal occult blood tests (gFOBTs), fecal immuno-
histochemical tests (FITs), and a FIT combined wit stool DNA testing
(Cologuard) are the three options for stool-based testing. Because
stool-based tests do not reliably predict adenomas, these tests are early
detection tests and cannot prevent CRC from occurring. The gFOBTs
have the strongest direct evidence supporting their use as a CRC
screening modality, with biennial screening reducing CRC mortality
by 9% to 22% and annual screening reducing CRC mortality by 32%
after 11 to 30 years of follow-up compared with no screening.67 The
gFOBTs are noninvasive and inexpensive, although they can have a
high false-positive rate and patient compliance is often low because
of the test’s dietary restrictions.
FITs are rapidly replacing gFOBTs because they do not have dietary
restrictions and because they offer at least equal and likely better
detection of CRC. Different FITs use different assay methods, so
FIT sensitivity has varied widely across studies. In the largest studies
of the USPSTF evidence review for its 2016 recommendation, the
sensitivity was 73.8% (95% CI, 62.3–83.3) for the quantitative OC
FIT-CHEK and 78.6% (95% CI, 61.0–90.5) for the qualitative OC-
Light, based on single stool samples.67 Sensitivity could be increased
by using three stool samples or by lowering the assay cutoff value;
however, specificity decreased with increasing sensitivity.67 Data are
lacking regarding the impact of FITs on CRC mortality. Finally, FITs
are generally inexpensive, with a Centers for Medicare and Medicaid
Services (CMS) reimbursement of $23 and a mean commercial
reimbursement of $21.68
The newest stool-based screen combines a FIT with DNA marker
analysis. Sensitivity of one-time FIT-DNA testing for CRC was shown
to be 92.3% in one large study, significantly better than the sensitivity
of one-time FIT alone (73.8%); yet, specificity was lower (86.6%
versus 94.9).69 In other studies, the sensitivity of FIT was superior
when multiple samples or lower assay cutoff values were used; and
specificity for the FIT-DNA test is lower than for all FIT assays,
resulting in this test having the highest false-positive rate among FIT
screens.67 Data regarding a possible CRC mortality benefit for the
FIT-DNA test are lacking. Finally, this is the most expensive stool
test, with a CMS reimbursement rate of $493.67
There are few adverse outcomes associated with stool-based tests
directly, aside from the risk of missed cancers. However, serious adverse
events can occur during follow-up colonoscopy after positive stool test
results, and some data suggest that the rate of perforations in these
colonoscopies may be higher than for those performed in average-risk
screening populations. The pooled estimate from the USPSTF evidence
review was 8 (95% CI, 2–32) perforations per 10,000 diagnostic
colonoscopies.67 A modeling study from the Cancer Intervention and
Surveillance Modeling Network (CISNET) Colorectal Cancer Working
Group estimated the number of complications (defined as perforations,
gastrointestinal bleeding, nausea and vomiting, ileus, dehydration,
Screening and Early Detection  •  CHAPTER 23 381
abdominal pain, myocardial infarction, angina, arrhythmias, congestive
heart failure, respiratory arrest, syncope, hypotension, or shock) in a
population of 1000 screened individuals to be 10 to 11 with annual
FIT and 9 to 10 with FIT-DNA at 3-year intervals.70,71
Direct visualization tests offer not only the ability to detect CRC
early, but also to prevent it through detection and subsequent removal
of precancerous lesions. These tests include flexible sigmoidoscopy
(FS), colonoscopy, and computed tomography (CT) colonography
(CTC). In the case of CTC, follow-up endoscopy is required for
removal of polyps detected during the screen.
Meta-analysis of four RCTs of FS in over 450,000 individuals
demonstrated a 27% reduction (incidence rate ratio [IRR], 0.73; 95%
CI, 0.66–0.82) in CRC mortality at 11 to 12 years of follow-up (find-
ings limited to distal CRC).67 A single RCT from Norway demonstrated
a 38% reduction in CRC mortality when FS was combined with
FIT.72 Although use of FS in the United States is uncommon,73 the
2016 USPSTF guideline recommends a 5-year screening interval for
FS alone and a 10-year interval if combined with annual FIT testing.
Despite a lack of RCT evidence supporting the use of colonoscopy,
it remains the gold standard of screening because of its high sensitivity
and specificity for the identification of polyps and CRC and it ability
to concomitantly diagnose and reduce risks associated with biopsied
precancerous lesions. Observational evidence supporting the use of
colonoscopy comes from a large prospective analysis of the Nurses’
Health Study and the Health Professionals Follow-Up Study, which
demonstrated a 68% (hazard ratio [HR], 0.32 [95% CI, 0.24–0.45])
reduction in CRC mortality in those who self-reported screening
colonoscopy compared with those who never underwent screening
endoscopy.74 In this study, statistically significant reductions were seen
for both distal and proximal CRC, although the mortality reduction was
greater in the distal colon. There are currently a number of RCTs under-
way for colonoscopy. At least two of them are comparing colonoscopy
and FIT (NCT01239082 and NCT00906997); the Nordic-European
Initiative on Colorectal Cancer (NordICC) is comparing colonoscopy
and usual care (NCT00883792)75; and one trial in Sweden is comparing
colonoscopy with FIT or no screening (NCT02078804).
Drawbacks of lower endoscopy include its invasiveness, risk of
serious complications, expense, and operator dependence. The estimated
number of complications from the aforementioned CISNET modeling
study is 14 to 15 with colonoscopy at 10-year intervals and 9 to 12
with FS at 5-year intervals.70
As with colonoscopy, there is currently no direct evidence establish-
ing the effectiveness of CTC in reducing CRC incidence and mortality.
However, a number of studies have examined the diagnostic accuracy
of CTCs and are summarized in the 2016 USPSTF evidence review.67
In studies in which bowel preparation was performed before the CTC,
per-person sensitivity and specificity to detect adenomas 10 mm or
larger ranged from 66.7% to 93.5% and 86.0% to 97.9%, respectively.
For detection of adenomas 6 mm or larger, sensitivity and specificity
were 72.7% to 98.0% and 79.6% to 93.1%, respectively. The sensitivity
to detect advanced adenomas ranged from 87.5% to 100%. Limited
data suggest that CTC without bowel preparation results in lower
sensitivity to detect adenomas 10 mm or larger and 6 mm or larger
in addition to advanced adenomas.
Serious harms of CTC in asymptomatic individuals appear to be
uncommon. The risk of perforation is less than 2 per 100,000 examina-
tions. Data are lacking to estimate any serious adverse events from
follow-up colonoscopy after CTC. Exposure to radiation is perhaps
the most important potential harm from CTC; however, the evidence
to date suggests that the benefits of CTC screening every 5 years far
outweigh the potential radiation risks.76 Finally, although this test is
relatively noninvasive, it can result in the identification of extracolonic
findings that may or may not be clinically important. Estimates are
that extracolonic findings occur in 41% to 69% of CTC procedures,
but only 5% to 37% of CTC procedures have extracolonic findings
that require actual diagnostic follow-up, and an even smaller percentage
result in findings that require any definitive treatment.67
382 Part I: Science and Clinical Oncology
CERVICAL CANCER
Cervical cancer provides the “perfect” paradigm for cancer prevention,
having a vaccine to reduce the incidence of infection from the sole
causative agent, human papillomavirus (HPV), and screening tests to
identify women infected with HPV or cytologic findings that indicate
precancerous conditions that can be successfully managed to prevent
the development of invasive disease. The incidence and mortality of
cervical cancer in the United States has decreased by over 75% since
widespread screening with the Papanicolaou (Pap) test and treatment
of precancerous lesions began decades ago.77
Infection with high-risk HPV is a necessary, although not sufficient,
cause of cervical cancer. Most HPV is transient and poses little risk
of persistent infection.78 The natural history of cervical cancer involves
(1) HPV infection; (2) persistent HPV infection; (3) development of
a precancerous cervical lesion; and (4) progression to invasive cervical
cancer.79 Opportunities for interventions exist throughout the con-
tinuum, including education about HPV transmission, HPV vaccina-
tion, screening with HPV and Pap tests, and treatment of precancerous
or cancerous lesions of the cervix. Interventions at steps 1 to 3 provide
the greatest opportunity to reduce the incidence and mortality from
cervical cancer.
HPV is the most common sexually transmitted infection in the
United States, with over 80% of sexually active individuals acquiring
an HPV infection by age 45.80 A number of other cancers have been
associated with HPV infection, including oropharyngeal (largely in
men), vaginal, vulvar, penile, and anal (mostly in men who have sex
with men). Although most HPV is transient in nature, factors that
determine which HPV infections will persist and progress are not
completely understood. The HPV genotype appears to be the most
important determinant. HPV-16 has the highest carcinogenic potential,
causing approximately 55% to 60% of all cases of cervical cancer
worldwide. HPV-18 is the next most carcinogenic genotype, accounting
for 10% to 15% of cervical cancers. Approximately 12 other genotypes
are associated with the remainder of cases of cervical cancer.81,82
The risk of HPV infection is greatest among teens and women in
their early 20s and decreases with increasing age. Most infections with
high-risk HPV do not result in persistent infections or cancer and
are very likely to regress among women with both normal and abnormal
cytology results. However, persistence of HPV infection appears to
increase with age, with HPV infections detected in women older than
30 years being more likely to reflect a persistent infection.83 Identifying
women with a persistent HPV infection is one of the best ways to
distinguish women at increased risk of developing cervical dysplasia
who require closer monitoring.
With the recognition of HPV as the primary etiologic factor in
cervical cancer, variables previously identified as risk factors are now
better classified as cofactors that either increase the risk of HPV infection
or increase the likelihood that an infection will be persistent, thus
increasing the risk of cervical neoplasia. These include age at first
coitus, number of sexual partners for a woman or her partner, history
of sexually transmitted disease including HPV infection, absence of
HPV vaccination, immunosuppression (human immunodeficiency
virus [HIV]–positive individuals, transplant recipients), younger age
at first pregnancy, high parity, long-term oral contraceptive (OC) use,
poor nutritional status, tobacco use, race, or ethnicity [Hispanic,
especially if not English speaking, or African American women),
socioeconomic status, and infrequent Pap smear screening.83
Diethylstilbestrol (DES) exposure in utero is largely a historical
risk factor for the development of cervical cancer. DES is a synthetic
form of the hormone estrogen that was prescribed to pregnant women
between 1940 and 1971 to prevent miscarriage and premature labor.84
It has been well defined that the daughters of women who took DES
while pregnant (so-called “DES daughters”) have a 40-fold increased
risk of developing clear cell adenocarcinoma of the vagina and cervix
compared with unexposed women. However, this type of cancer remains
rare, with only 1 in 1000 DES daughters developing it.85 Risk of
cervical cancer related to DES exposure in utero has been shown to
persist in DES daughters above the age of 40.86
Cervical Cancer Screening
The Pap test is considered to be one of medicine’s most successful
cancer screening tests. Although there are no RCTs that have evalu-
ated the efficacy of the Pap test, there is overwhelming observational
evidence that cervical cancer incidence and mortality have decreased
since Pap testing was integrated into routine clinical practice in the
United States.77
Cervical cancer is characterized by a long sojourn time, with precur-
sor lesions identifiable long before the development of invasive disease.
Cytologic screening for these lesions (e.g., cervical intraepithelial
neoplasia [CIN]) with the Pap test is effective in reducing both the
incidence and mortality from cervical cancer because these premalignant
lesions are generally amenable to interventions that prevent the progres-
sion to invasive disease.77 HPV testing shows whether a person has a
current HPV infection. It does not provide information on previous
HPV infections. Genotype testing is now available for HPV types 16
and 18 and can be used to aid in the clinical management of precursor
lesions, but is not currently used in cervical cancer screening.
Studies of HPV cervical cancer screening have found that HPV
testing alone was more sensitive but less specific than cytologic evalu-
ation alone. For CIN3+ outcomes, sensitivity ranged from 86% to
97% for HPV testing versus 46% to 50% for cytologic evaluation at
a colposcopy referral threshold of “atypical squamous cells of unde-
termined significance” (ASCUS). For CIN2+ outcomes, sensitivity
ranged from 63% to 98% for HPV testing versus 38% to 65% for
cytology. However, specificity for CIN2+ and CIN3+ was consistently
3% to 5% lower for HPV testing than for cytology.87
A 2012 study identified that a negative HPV test result provided
greater reassurance against CIN3+ over an 18-year follow-up than
did a normal Pap result.88 Although baseline Pap and HPV tests
equally predicted who would develop CIN3+ within the first 2 years
of follow-up, only HPV testing predicted who would develop CIN3+
10 to 18 years later. These data support extending the screening interval
in women who have negative cotesting results.
Current Cervical Cancer Screening: Cytologic
Assessment With or Without Human
Papillomavirus Testing
Updated screening guidelines were released in 2012 jointly by the
ACS, the American Society for Colposcopy and Cervical Pathology
(ASCCP) and the American Society for Cervical Pathology (ASCP)
(Table 23.5) and separately by the USPSTF.89,90 These guidelines apply
to women at average risk of cervical cancer who have a cervix.
Increasing evidence and an improved understanding of the natural
history of cervical cancer led to the recognition that previous recom-
mendations for annual screening were excessive and resulted in an
increased rate of false positives. Annual screening leads to a very small
increment in cancers prevented, at the cost of a very large excess of
unnecessary procedures and treatments due to the high prevalence of
transient, benign HPV infections and associated lesions, most of which
will regress within a year or two. Of those that do not regress, on
average, they are many years from causing cancer. Women at average
risk at any age should not be screened annually by any screening
method. Recommended screening intervals for women at average risk
are based on age and clinical history.89
Women ages 21 through 30 should be screened every 3 years with
the Pap test. HPV testing is not recommended in women under age
30 because the risk of HPV infection is high, but the likelihood of a
persistent infection is extremely low. Cotesting every 5 years with
cytologic evaluation and HPV testing provides benefits similar to a
Pap test alone done every 3 years for women 30 to 65 years of age.
Therefore, in women age 30 to 65 years who want to lengthen the

Table 23.5  American Cancer Society, American
Society for Colposcopy and Cervical
Pathology, and American Society for
Clinical Pathology Cervical Cancer
Screening Recommendations for
Women at Average Riska
Age Screening Recommendationb
Aged <21 yr No screeningc
Aged 21–29 yr Cytology alone every 3 yrc
Aged 30–65 yr Human papillomavirus (HPV) and cytology
“cotesting” every 5 yr (preferred)
OR
Cytology alone every 3 yr (acceptable)
Age >65 yr No screening following adequate negative
prior screening
a
The following are not considered to be at average risk: women with a history of
CIN2+ or cervical cancer in the past 20 years, diethylstilbestrol (DES) daughters, and
those with human immunodeficiency virus (HIV) infection or immunosuppression
due to other factors (e.g., transplant patients).
b
c
Average-risk women should not be screened annually at any age by any method.
HPV testing should not be used for screening in this age group.
d
If the hysterectomy was supracervical and the cervix is intact, screening would
be the same as in average-risk women.
CIN, Cervical intraepithelial neoplasia.
Modified from Saslow D, Solomon D, Lawson HW, et al. American Cancer Society,
American Society for Colposcopy and Cervical Pathology, and American Society for
Clinical Pathology screening guidelines for the prevention and early detection of
cervical cancer. CA Cancer J Clin. 2012;62(3):147–172.
screening interval, cotesting every 5 years with Pap and HPV is recom-
mended. ACS, ASCCP, and ASCP preferred cotesting over the Pap
test alone, whereas the USPSTF deemed both cotesting and Pap test
alone as acceptable.89,90
Certain low-risk groups should no longer be screened. These include
women under age 21, low-risk women over age 65, and women who
have undergone a hysterectomy.89,90 Evidence has shown that screening
women age 21 and younger does not reduce cervical cancer incidence
and mortality but is associated with significant harms. Treatment of
CIN in women younger than age 21 has been shown to increase the
risk of adverse pregnancy outcomes, including cervical incompetence
and preterm delivery.89,90 Women age 65 and older who have had the
recommended screening in the past 10 years and are considered to
be at low risk of cervical cancer do not need to continue screening
beyond age 65. Women who are not low risk include those with
immunosuppression (e.g., HIV or transplant patients), DES daughters,
and those with a prior history of a high-grade precancerous lesion of
the cervix or cervical cancer within the past 20 years. It is important
to note that older women who have not had recent screening are a
population at risk for the development of cervical cancer. Although
cervical cancer incidence rates are on the decline, 50% of women
diagnosed with cervical cancer have not been screened 3 to 5 years
prior to diagnosis.83,89,90 Women who have had hysterectomy do not
need to have cervical cancer screening unless the hysterectomy was
done for the management of CIN2+ or cervical cancer, in which case
they should follow the recommended surveillance guidelines. When a
supracervical hysterectomy is performed (i.e., leaving the cervix intact),
screening recommendations should be based on cervical cancer risk.89,90
At this time, women who receive the HPV vaccination should
continue to follow current cervical cancer screening recommendations,
because HPV vaccination does not protect against all types of oncogenic
HPV. In addition, the HPV vaccination rate is far from optimal at
this time, and immunization registries do not identify who has received
the recommended series of vaccinations. Although HPV vaccination
is an important step toward cervical cancer prevention, it does not
remove the need for routine cervical cancer screening.
Screening and Early Detection  •  CHAPTER 23 383
Emerging Tests for Cervical Cancer Screening:
Primary Human Papillomavirus Screening
As previously noted, studies have found that HPV testing alone was
more sensitive than cytologic evaluation alone. In 2014, the FDA
approved a currently marketed HPV test to include the additional
indication of primary cervical cancer screening.
Although HPV testing alone is more sensitive than cytologic
evaluation alone, it is also less specific. Strategies that maximize detection
of CIN3+ by immediate referral to colposcopy with follow-up testing
and triage of women at intermediate risk of CIN maximizes the benefits
of cervical cancer screening while decreasing the potential harm.91
The Addressing the Need for Advanced HPV Diagnostics (ATHENA
HPV) Study evaluated the role of primary HPV screening in women
25 years of age and older and validated an effective triage algorithm.
In this trial, HPV-positive specimens underwent genotyping for HPV-16
and HPV-18. If a specimen was positive for HPV-16 or HPV-18,
colposcopy was performed. If a specimen was negative for HPV-16
or HPV-18, cytologic evaluation was performed on the specimen. If
the cytologic evaluation results were abnormal, colposcopy was per-
formed. If both HPV genotyping and cytologic findings were normal,
repeat cotesting was performed in 1 year. The trial concluded that
primary HPV screening in women 25 years of age or older is as
effective as a screening strategy that uses cytologic evaluation in those
25 to 29 years old and cotesting in those 30 years of age or older. In
addition, this paradigm requires fewer screening tests.92
In 2015, the ASCCP and the Society of Gynecologic Oncology
(SGO) provided interim guidance for the use of the FDA-approved
HPV test for primary cervical cancer screening. They concluded that,
because of its equivalent or superior effectiveness in women 25 years
of age or older, the FDA-approved primary HPV screening test can
be considered an alternative to current cytology-based cervical cancer
screening methods.93 However, primary HPV testing is not currently
considered a standard of care.
Prevention of cervical cancer through education about HPV
transmission, HPV vaccination, and screening with cytologic evaluation
with or without HPV testing has the potential to eliminate invasive
cervical cancer as a disease of concern for women. Primary HPV
screening is emerging as a new screening modality.
LUNG CANCER
Lung cancer is the most common cause of cancer-related death
worldwide and the second most diagnosed cancer in the United States
for both men and women.94 Estimated figures for 2016 in the United
States exceed 224,000 new cases and 158,000 deaths as reported by
the ACS. In China alone, estimated figures for 2015 are 733,000 new
cases and 610,000 deaths.95 Lung cancer incidence and death rates
in the United States have been declining slightly for men and women,
with the magnitude of the decrease varying by race and ethnicity.94
Risk Factors
Smoking continues to represent the major risk factor for lung cancer,
with a dose-response relationship between pack-years of smoking and
risk.96 Exposure to environmental tobacco smoke among nonsmokers
increases lung cancer risk by approximately 20%. Additional risk
factors associated with smoking include age at smoking onset, type
of tobacco product smoked, and depth of inhalation. Risks due to
marijuana, hookah use, and e-cigarettes remain to be determined.97
Other risk factors are related to exposure to other toxins and damaging
agents including asbestos, radon, air pollution, and ionizing radiation.98
Host factors associated with lung cancer risk include history of chronic
obstructive pulmonary disease or infections and a family history of
lung cancer.99 Substantial effort has focused on susceptibility genes
for lung cancer, which have yielded significant associations with
chromosomal regions including 15q25 and 5p15.100
384 Part I: Science and Clinical Oncology
Risk Modeling
At present, there is not a currently recommended risk prediction
model for lung cancer for general clinical use, though several models
have been proposed with varied complexity.101–103 Risk estimates from
predictive models have utility in determining who needs screening
and in counseling individuals at risk, particularly in encouraging
smoking cessation.104 Application of a model to ever-smokers in the
Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO)
cohort intervention arm led to improved sensitivity (80% versus 71%)
and specificity (66% versus 63%) compared with NLST screening
criteria alone.105
The addition of pulmonary function has been shown to improve
risk prediction for lung cancer.106 A recently reported lung cancer
risk-prediction model that incorporated lung function was based on
the UK Biobank prospective cohort study.107 Flexible parametric survival
models were used to estimate the 2-year probability of lung cancer,
accounting for the competing risk of death. Models included sex,
variables related to smoking history and nicotine addiction, medical
history, family history of lung cancer, and lung function measured
with spirometry (forced expiratory volume in 1 second [FEV1]). A
risk prediction model that included lung function had strong predictive
ability and had better discrimination than standard lung cancer
screening eligibility criteria. A potentially important improvement in
risk prediction models will likely result from incorporation of molecular
markers. Cotinine, a circulating metabolite of nicotine, may improve
model discrimination among current smokers. Cotinine has been
found to be associated with lung cancer risk108 and could be used as
a more sensitive assessment of smoking intensity than self-reporting
and thus may improve risk modeling further. A protein, prosurfactant
protein B (pro-SFTPB), which is a target of the oncogene NKX2-1
implicated in lung tumor development, has been shown to be a risk
marker for lung cancer.109 Using pro-SFTPB as an anchor marker,
the addition of the metabolite diacetylspermine resulted in a statistically
significant increase in predictive power of the model.110 Comprehensive
models that incorporate demographic data and smoking information,
lung function, and molecular risk markers thus have great potential
to improve lung cancer risk stratification. Potential improvements in
risk prediction through lab-based tests have to be weighed against
increased costs and practicality.
Screening
A major cause of high mortality in lung cancer is that most cases are
diagnosed at advanced stages of the disease, when current therapeutic
regimens are relatively ineffective.111 There has been long-standing
interest in detecting cancer at an early stage with imaging modalities.
The concept of screening for lung cancer originated in the 1950s with
the Philadelphia Neoplasm Research Project, which performed periodic
screening on some 6000 males with fluoroscopy, but with no effect on
outcome.112 In the following decades, several randomized trials were
conducted to assess the benefits of chest radiography, with similar
conclusions with respect to outcome. These studies reported finding
more early-stage cancers in the screened group, and more surgical
procedures were performed, but with no impact on cancer-related
mortality.113 Interest in screening picked up again with the introduc-
tion of CT scans and with reports of better detection of early-stage
cancers with CT scans compared with chest radiographs.114 The lack
of control groups in the early trials made it difficult to determine
whether screening could decrease lung cancer mortality. The National
Lung Screening Trial (NLST), an RCT comparing CT screening with
chest radiography and with lung cancer mortality as the end point
followed.115 The NLST enrolled 53,000 individuals aged 55 to 74
years with a 30–pack-year smoking history. Participants were randomly
assigned to chest radiography or low-dose computed tomography
(LDCT) and screened at baseline with two annual follow-up scans.
Follow-up lasted up to 7 years. The LDCT group had a 20% reduction
in lung cancer mortality and a 6.7% reduction in all-cause mortality.
A striking finding from the NLST was the high rate of false positives,
which was 27% at baseline and 28% at 1-year follow-up. After the
completion of the NLST, the International Early Lung Cancer Action
Program retrospectively analyzed the outcomes of more than 21,000
prospectively enrolled patients who underwent lung cancer screen-
ing.116 An increase in size threshold for nodule diameter resulted in
an increase in cancer diagnosis rates. An increase in the size threshold
from 5 mm to 6 mm could reduce the workup by 36%, and up to 75%
for 9 mm.
Concerns about the high percentage of false positives and potential
health hazards resulting from exposure to radiation with repeat LDCT
scans provide justification for the application of more informative
risk prediction models to identify individuals at high risk who would
benefit from LDCT screening for early detection, to minimize false
positives. An alternative to screening with LDCT is through further
reduction in radiation dose with ultra-low-dose CT without loss of
image quality.117 A Japanese multicenter study compared performance
of ultra-low-dose CT with LDCT for the detection of lung nodules.118
A total of 161 nodules and 60 ground glass opacities were identified,
with comparable performance between the two modalities.
Guidelines for the management of patients with nodules detected
through CT screening are also evolving. The NCCN provides guidelines
that avoid invasive follow-up for small nodules with low probably of
malignancy. The guidelines incorporate recommendations based on
the NLST findings, the International Early Lung and Cardiac Action
Program protocols, and the Fleischner Society guidelines. A reporting
system has been suggested that classifies nodules observed with screening
CT based on the risk of malignancy, which is linked to suggested
follow-up.119 The goal is to improve communication with patients
and clinicians. Subjects with nodules smaller than 5 mm or with
perifissural or long-term stable nodules would require no further workup
before the next routine screening CT scan. Subjects with larger nodules
would require CT follow-up at an interval dependent on nodule size,
varying from small (5–9 mm) to large (≥10 mm). Other nodules
would be considered to have a high likelihood of malignancy based
on screening CT features.
PROSTATE CANCER
Prostate cancer is the most commonly occurring cancer among men
(excluding nonmelanoma skin cancers [NMSCs]) and was the second
most common cause of cancer death among men in 2014.62 Compared
with non-Hispanic whites, African Americans experience higher
incidence (203.5 versus 121.9 per 100,000 men) and mortality (44.1
versus 19.1 per 100,000 men). The prostate cancer incidence rate has
declined sharply, 10% annually from 2010 to 2013, owing to reduced
use of the PSA test after the 2012 USPSTF recommendation against
routine screening with the test.62 The prostate cancer mortality rate
has dropped 51% from 1993 to 2014.62 Local- or regional-stage
diagnosis is now typical for over 90% of prostate cases, resulting in
5-year survival rates just under 100%, having improved 30% over
the last several decades. However, 5-year survival is just 29% for those
with distant metastases.120 The etiology of prostate cancer is incompletely
understood but involves both modifiable risk factors (diet, tobacco
use, obesity) and nonmodifiable risk factors (age, race or ethnicity,
genetics).121 Prostate cancer progression is typically slow, and clinically
significant progression may not occur during a man’s lifetime.121
Risk Modeling and Assessment
Risk prediction models offer the possibility of tailoring PSA screening
to those who are most likely to benefit from it, thereby reducing
unnecessary biopsies and maximizing the number of clinically significant
prostate cancers detected. Hundreds of risk models have been published,
although just a handful have been validated in various external
populations.122

One such model is the Prostate Cancer Risk Calculator, developed
in 2006 with data from the Prostate Cancer Prevention Trial (PCPT)
and recently updated in 2014 with an expanded set of PCPT biopsy
findings and now reporting on three outcomes (negative biopsy result,
low-grade and high-grade cancer) rather than the initial two (cancer
versus no cancer).123 This 2.0 model was externally validated in 10
international biopsy cohorts and a reference biopsy set from NCI’s
Early Detection Research Network (EDRN). In these cohorts, the
AUC ranged from 0.62 to 0.88, with a median of 0.75, for the
detection of clinically significant prostate cancer.123 In another nine
studies, the AUC ranged from 0.51 to 0.79, with a median AUC of
0.69.121 The AUC of PSA alone in these studies ranged from 0.56 to
0.71 with a median of 0.65.121 The PCPT 2.0 includes age and race,
family history, PSA level, digital rectal examination (DRE) result, and
prior biopsy findings, and also considers percent-free PSA, if available.
A systematic evidence review conducted for the USPSTF’s 2017 update
of its prostate cancer screening recommendation states that although
the calculator improves discrimination between men with and without
high-grade prostate cancer, both its predictive accuracy and calibration
vary substantially across samples.121 The model is freely available online
at http://myprostatecancerrisk.com/.
A second commonly used risk prediction model is derived from
the Rotterdam data of the European Randomized Study of Screen-
ing for Prostate Cancer (ERSPC).122,124 This tool includes PSA level,
DRE result, prior biopsy, and prostate volume. In the development
of the model, prostate volume was based on transrectal ultrasound
(TRUS). But because TRUS is invasive and generally performed during
a biopsy, the model allows for an estimate of prostate volume based
on DRE to be used in place of TRUS. The model with the DRE
substitution was validated in 322 men referred for biopsy during
later screening rounds of the ERSPC, wherein its AUC was 0.78 for
the detection of high-grade cancer.125 The AUC of the PSA test in
these men was 0.68.125 In four external validation studies, the AUC
of the ERSPC model ranged from 0.69 to 0.78, with the AUC of
PSA alone ranging from 0.61 to 0.65.121 This model also allows the
Prostate Health Index (PHI) to be included, if available. The PHI
slightly increases the calculator’s predictive capability.126 This tool is also
freely available online at http://www.prostatecancer-riskcalculator.com/
seven-prostate-cancer-risk-calculators.
In a comparison of the PCPT and ERSPC models, Foley and
colleagues demonstrated that the ERSPC significantly outperforms
PCPT 2.0 in the prediction of prostate cancer, with an AUC of 0.71
(versus 0.64), and an AUC of 0.74 (versus 0.69) for the prediction
of significant prostate cancer in 2001 men from six tertiary referral
centers in Ireland.126 A meta-analysis of six different risk prediction
tools, including the PCPT 2.0 and ERSPC models, found that all
risk models improved the predictive accuracy of PSA testing to detect
prostate cancer, with the exception of the PCPT 2.0 model.122
Important to note, no RCTs have been performed to test the use
of a risk prediction model versus no model on outcomes such as
clinical decision making, biopsy accuracy, or long-term incidence of
advanced prostate cancer.121 Few studies have addressed the implementa-
tion of risk calculators into clinical practice and decision making.
Limitations to current prostate cancer risk models include variable
calibration among populations and/or often poorly reported calibration
statistics.
In addition to risk models, serum and urine tests and multipara-
metric MRI are also being used in an attempt to improve PSA screening,
although none of them have been evaluated in RCTs. For some men
with mildly elevated PSA levels (3–10 ng/dL) at screening, current
NCCN guidelines suggest consideration of free PSA, the 4Kscore, or
the PHI to assist in decision making about whether to proceed to
biopsy or whether to repeat biopsy if the result of the first biopsy is
negative.121,127 The PCA3 (noncoding mRNA overexpressed in prostate
cancer tissue) test is FDA approved for use in men with a prior negative
biopsy result to assist in decision making regarding a repeat biopsy,
and consideration of its use is recommended by the NCCN in such
Screening and Early Detection  •  CHAPTER 23 385
situations.127 More research is needed to determine if these types of
adjunctive tests improve the balance of benefits and harms of PSA-based
prostate cancer screening.
Screening
The ability of the PSA test to reduce prostate cancer–specific mortality
when used in a screening context has been examined in the PLCO
and ERSPC. Results of the US-based PLCO128,129 did not identify
any significant benefit from screening on prostate cancer mortality at
either 7 or 13 years of follow-up.129,130 However, results of this trial
have been called into question, given the extremely high proportion
of the control arm that reported having at least one PSA test during
or before the trial.131 By contrast, the ERSPC,132 a collaborative effort
involving eight European countries (Belgium, Finland, France, Italy,
the Netherlands, Spain, Sweden, and Switzerland), identified a sig-
nificant 20% reduction in prostate cancer mortality as a result of
PSA-based screening. However, these results demonstrated a high risk
of overdiagnosis of prostate cancer in individuals lacking clinical
symptoms. The 12- to 14-year follow-up results from the Swedish
and Netherlands sites of the ERSPC trial found enhanced benefit of
PSA screening, with prostate cancer mortality reduced by almost half
in Sweden and by 20% in the Netherlands, although the reduction
was as great as 32% in those ages 55 to 69.133,134 Conversely, the
12- to 15-year follow-up results from the Finland and Spanish sites
of the ERSPC study failed to demonstrate significant effects of PSA-
based screening on prostate cancer mortality.135,136 Limitations of the
ERSPC trial include substantial variation in methods across sites as
well as unexplained postrandomization treatment differences, which
may partly explain the benefit of screening observed in this trial.121
Neither the PLCO nor the ERSPC documented evidence for a reduction
in overall mortality; and neither trial provides insight into any potential
benefits of screening that may be specific to men with a family history
of prostate cancer or who are African American.
The previous USPSTF prostate cancer screening guideline (2012)
recommended against (grade D) routine use of the PSA test in average-
risk populations because it concluded that the benefits of PSA testing
did not outweigh the potential risks. However, the Task Force is now
in the process of issuing a new prostate cancer screening guideline
and, as of this writing, it is recommending that clinicians inform men
ages 55 to 69 of the potential benefits and harms of PSA testing and
that the decision to screen should be an individual one (grade C).121
This change in recommendation is due, in part, to new evidence
supporting the benefits of screening that comes from longer-term
follow-up, and new reports from subsites, of the ERSPC trial. According
to the latest evidence, PSA-based screening programs in men ages 55
to 69 may prevent up to one to two deaths from prostate cancer and
up to three metastatic prostate cancers per 1000 men screened over
13 years.121 The change in recommendation is also driven by new
data on the use of active surveillance, which may reduce the risk of
overtreatment. The rate of active surveillance in the United States
among men diagnosed with low-risk prostate cancer has increased
sharply, from 14.3% in 2009 to 40.4% in 2013. Results from the
British Prostate Testing for Cancer and Treatment (ProtecT) trial
demonstrate no significant difference in either prostate cancer or overall
mortality among men randomized to active surveillance, radical
prostatectomy, or radiotherapy after 10 years of follow-up.137 However,
men randomized to active surveillance developed more metastases
and experienced higher rates of disease progression than did men in
either the surgery or radiation groups.137 The very high 10-year survival
rate (98%) across all three groups may have limited the trial’s power
to detect differences in mortality among the groups. Longer follow-up
is needed to clarify these results.
The USPSTF also assessed harms related to screening and, because
the benefit of screening can be achieved only through treatment, it
also assessed harms related to the various treatment modalities for
prostate cancer (i.e., radical prostatectomy, radiotherapy, and active
386 Part I: Science and Clinical Oncology

Table 23.6  Benefits and Harms of Prostate Cancer Screening per 1000 Men Screened
Trial
Screening population
Median follow-up, yr
Outcome
Men invited to screening
≥1 positive screen
≥1 biopsy
Additional men
hospitalized for biopsy
complications
Additional prostate
cancer diagnosed
because of screeninga
Additional men with ED
due to treatment
Additional men with UI
after RP
Men treated with radical
prostatectomy or
radiotherapy without
benefits
Metastatic cases averted
Prostate cancer deaths
averted
PLCO ERSPC (Core Age Group) Goteborg, Sweden Notes
Men 55–74 yr; Men 55–69 yr; screened Men 50–64 yr; invited Data for PLCO and Goteborg
screened annually for every 4 yr (biennially in biennially up to 70 yr; PSA derived from reports with 13.0
6 yr; PSA threshold Sweden); PSA threshold threshold 3.0–3.5 ng/mL and 14.0 yr of median follow-up
for biopsy, 4.0 ng/mL most commonly 3.0 ng/mL before 2005 and 2.5 ng/mL as more recent reports with 14.0
from 2005 to 2008 and 18.0 yr median follow-up lack
13.0 13.0 14.0 some required data
Number of men affected
1000 1000 1000
282 274 248 PLCO, ERSPC, Goteborg trial
130 244 231 reports
1.7 3.2 3.0 ProbE cohort study (1.3%
hospitalized due to biopsy
complications)
11.3 34.8 42.0 Absolute cumulative incidence
differences and numbers needed
to invite in trial reports
1.8 6.8 8.3 Men with cancer allocated to
primary treatments as in trial
0.8 2.4 3.3 screening arms. For treatment
harms, number needed to harm is
from EPC pooled meta-analyses
NC 23.9 25.8 Number computed by
subtracting number of men with
prostate cancer death or
metastasis averted from number
of men actively treated
NR 2.9 3.5 ERSPC core age group absolute
risk reduction in metastatic
disease cumulative incidence
(and number need to diagnose)
0 1.3 3.4 Absolute risk reduction in
prostate cancer deaths (and
numbers needed to diagnose)

a
Based on trial data, 99.5, 68.3, and 72.1 would be diagnosed with prostate cancer among 1000 men not invited to screening in the PLCO, ERSPC, and Goteborg, Sweden
settings, respectively.
ED, Erectile dysfunction; EPC, evidence-based practice center; ERSPC, European Randomized Study of Screening for Prostate Cancer; NC, not calculable; NR, not reported;
PLCO, Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial; ProbE, Prostate Biopsy Effects cohort study; PSA, prostate-specific antigen; UI, urinary incontinence.
Adapted with permission of the U.S Agency for Healthcare Research and Quality.

surveillance). Harms from screening include false-positive results and
the potential pain, bleeding, and infection from biopsy. The conclusion
of the Task Force regarding harms of screening is that they are “at
least small.”138 Harms related to treatment include urinary and fecal
incontinence, erectile dysfunction or long-term sexual impotence,
and, in the case of radical prostatectomy, serious surgical complications
and possible death. The conclusion of the Task Force regarding the
harms of overdiagnosis and treatment is that they are “at least moder-
ate.”139 Table 23.6 presents a summary of the benefits and harms of
PSA screening and subsequent treatment, as reported in the USPSTF
evidence review and draft recommendation.
Finally, the USPSTF assessed whether changing the screening
interval or lowering the threshold of PSA testing for diagnostic biopsy
might increase the benefit of screening while simultaneously reducing
harms. An optimal cutoff value for the PSA test that is associated
with both high specificity and sensitivity has not yet been established.140
RCT data suggest that while a greater mortality benefit may be derived
from screening every other year, as opposed to longer intervals, and
from using a lower PSA threshold, harms are also substantially increased,
including overdiagnosis.139 Nevertheless, in a review of modeling studies
an interval of every 2 or 4 years, rather than annually, was found to
offer a reasonable tradeoff between a reduction in overdiagnosis and
a small reduction in mortality benefit.141 Evidence was deemed insuf-
ficient to support the use of alternative PSA screening modalities,
such as single- and adjusted-threshold testing, PSA velocity, and
double-timing.139
Given the new data on the potential benefits of screening (i.e.,
reduced risk of dying from prostate cancer and reduced risk of metastatic
prostate cancer), the USPSTF has stated that the benefits and harms
of PSA screening are more closely balanced than previously estimated.
Therefore it is the intention of the Task Force that, after discussion
of these benefits and harms with a health care provider, each man’s
particular values and preferences tip the balance in favor of either a
net benefit or net harm.
Evidence does not currently support different recommendations
for African American men or men with a family history of the disease,
although many clinicians would choose to screen these men earlier
and/or more often. With regard to PSA screening in men 70 of age
or older, the USPSTF has concluded that there is moderate certainty
that the potential benefits of screening do not outweigh the expected
 Screening and Early Detection  •  CHAPTER 23 387

harms, and so it recommends against screening in this group (grade

D). The 2017 guideline brought the USPSTF into line with the recom- Table 23.7  Nongenetic and Genetic Risk Factors
mendations of most other physician and patient advocacy groups. for Pancreatic Cancer
Variable Risk Increase
PANCREATIC CANCER NONGENETIC RISK FACTORS
Pancreatic cancer is the fourth leading cause of cancer death in Western Chronic pancreatitis 13.3
countries, with an overall 5-year survival rate of around 7% and an New onset type 2 diabetes 7.9
average survival of 6 months.142 The median age at diagnosis is 71
Long-standing diabetes mellitus 2.0
years. The annual incidence and mortality rates closely mirror each
other. Pancreatic cancer is estimated to become the second most Obesity 2.0
common cause of cancer-related deaths by 2030.143 A classification Smoking 1.8
system based on gene expression signatures derived from either tumor Alcohol abuse 1.2
or stromal cells has been proposed that predicts patient outcome.144 Non-O blood group 1.3
A model for progression to pancreatic adenocarcinoma has been
established with occurrence of signature mutations in premalignant GENETIC SYNDROME AND ASSOCIATED GENES
lesions.145 There is a stepwise progression of pancreatic intraepithelial Familial pancreatic cancer (unknown gene)
neoplasia from low grade to high grade in types 1, 2, and 3 with One first-degree relative 9
accumulating genetic alterations and occurrence of KRAS mutations
Three first-degree relatives 32
in the vast majority of pancreatic intraepithelial neoplasia of all grades.146
Low-grade pancreatic intraepithelial lesions are frequently detected Familial adenomatous polyposis (APC) 4.5–6
in autopsies in nondiseased pancreas, whereas advanced lesions are Breast and ovarian cancer syndrome (BRCA1, BRCA2, 2–3.5
usually detected adjacent to adenocarcinoma.147 PALB2)
Intraductal papillary mucinous neoplasm (IPMN) is a heterogeneous Peutz-Jeghers syndrome (STK11/LKB1) 132
entity that represents another type of precursor lesion to pancreatic Hereditary pancreatitis (PRSS1, SPINK1) 69
cancer.148 The prevalence of IPMN lesions can be as high as 20% and Familial atypical multiple mole melanoma 47
increases with age.149,150 Risk stratification with regard to cancer can pancreatic carcinoma syndrome (P16INK4A/CDKN2A)
be accomplished according to high-risk characteristics.148,151 Serous
cystic neoplasm (SCN) represents another type of lesion for which Lynch syndrome (MLH1, MSH2, MSH6, PMS2, EPCAM) 8.6
our understanding of the natural history is rather limited. A retrospective Cystic fibrosis (CFTR) 3.5
follow-up study of patients with SCN over a 3-year period revealed Ataxia-telangiectasia (ATM) Unknown
clinically relevant symptoms in a very small proportion of patients
GENETIC POLYMORPHISMS
and a slow increase in size in fewer than half of the patients.152
ABO 1.3
Risk Factors and Inherited Syndromes NR5A2 1.3
TERT 1.2
Some 10% of all patients with pancreatic cancer have a positive family PDX1 1.2
history for pancreatic cancer or for related genetic syndromes that
justify screening.147 The genetic basis for familial aggregation has not BCAR1, CTRB1, CTRB2 1.5
been identified in a large proportion of cases.153 Genetic syndromes ZNRF3 1.2
include Lynch syndrome, FAP, hereditary breast-ovarian cancer syn- LINC00673 1.3
drome, hereditary pancreatitis, Peutz-Jeghers syndrome, and familial ETAA1 1.1
atypical multiple mole melanoma syndrome. In addition, a multitude TP63 1.1
of polymorphisms have been associated with a slightly increased risk
of pancreatic cancer (Table 23.7). SUGCT 1.1
Risk factors associated with pancreatic cancer development in the
general population include aging, tobacco use, heavy alcohol use,
diabetes, and obesity. Tobacco use is estimated to double the risk of
pancreatic cancer, with population estimates of attributable risk and precursor lesions and likelihood of progression among the latter.
estimated to be as high as 25%.154 Heavy alcohol use is associated As a result, at present, efforts in early detection are largely limited to
with a 22% increased risk of pancreatic cancer.155 There is also a individuals at high risk.
significant association between body mass index (BMI), type 1 dia- Screening recommendations of the International Cancer of the
betes,156 long-standing type 2 diabetes,157 and abnormal glucose toler- Pancreas Screening Consortium concern high-risk subjects—namely,
ance158 and pancreatic cancer risk. individuals from a familial pancreatic cancer kindred with at least two
affected FDRs; patients with Peutz-Jeghers syndrome; and p16 or
Screening BRCA1/2 mutation carriers.159,160 Screening is largely based on imaging,
notably MRI or endoscopic ultrasonography. A prospective observational
Although survival rates are dismal, a diagnosis of pancreatic cancer study was done involving 40 consecutive patients with a genetic risk
at a resectable stage carries a better prognosis, with a 5-year survival for developing pancreatic cancer who were referred to Karolinska
rate of 30%, substantiating the potential merits of early detection. University Hospital for MRI-based surveillance.161 During a median
The relatively long timeline for progression provides an opportunity follow-up of approximately 1 year, pancreatic lesions were detected
for preventive intervention and early detection. However, the low in 40% of the patients, of whom five underwent surgery—three for
prevalence of pancreatic cancer, varying from 9 per 100,000 in the pancreatic ductal adenocarcinoma and two for intraductal papillary
general population to 68 per 100,000 persons older than age 55 years, mucinous neoplasia.
and the common occurrence of early precursor lesions in the elderly Screening of individuals at high risk is recommended starting at
population create challenges for implementing early detection and 40 years of age or 10 years earlier than the age at which the youngest
for development of noninvasive tools to distinguish between benign relative was diagnosed with pancreatic cancer.162 In general, screening
388 Part I: Science and Clinical Oncology
examinations are conducted annually until an abnormality is identified,
at which time, intervals may shorten to every 3 to 6 months. For
IPMNs, an international consensus panel recommended screening
every 3 to 6 months.163 Ideally, prophylactic resection would be recom-
mended in patients with PanIN3, IPMN, mucinous cystic neoplasms
with high-grade atypia, or minimally invasive cancer.164,165
The development of biomarkers to assist in screening or to determine
the malignant potential of pancreatic lesions is currently of great
interest. At present, CA19-9 is the marker primarily being evaluated,
in addition to imaging.166 There is potentially a vast array of circulating
markers that could be explored for such purposes, from nucleic acids
to proteins, metabolites, autoantibodies, and microvesicles. A highly
performing biomarker panel with the prerequisite performance for
screening, demonstrated through prospective blinded validation studies,
has yet to emerge.
LIVER CANCER
Worldwide, primary liver cancer is the sixth most frequent cancer and
the second leading cause of death from cancer. An estimated 782,500
new cases of liver cancer were diagnosed and 745,500 deaths from
liver cancer occurred worldwide during 2012.167 However, incidence
and mortality rates vary greatly around the world and by sex, such
that liver cancer is the second most common cancer among men in
developing countries.167
In the United States, liver cancer rates have more than doubled over
the past two decades, and liver cancer is currently the fastest growing
cause of cancer-related death.168 In 2018, as estimated 42,220 new
cases of liver and intrahepatic bile duct cancer will be diagnosed; and
about 30,200 will die from the disease in the United States.62 Because
of the growing number of patients with advanced hepatitis C virus
(HCV) and/or nonalcoholic steatohepatitis (NASH), it is anticipated
that hepatocellular carcinoma (HCC) incidence will continue to rise
over the next 20 years. At its current rate of increase, HCC is projected
to surpass breast cancer and CRC to become the third leading cause
of cancer-related death in the United States by 2030.143
Of all primary liver and intrahepatic cancers diagnosed in the
United States, more than 70% are classified as HCC.169 HCC incidence
rates among men are twice as high as the incidence rates among
women. HCC is rare among persons aged younger than 40 years.
HCC incidence rates reach a peak at approximately 70 years of age.
Despite improvement in prevention and treatment, currently less than
half of all HCC cases are diagnosed at an early stage, and most patients
do not receive curative therapy.170 Overall 5-year survival rates in
HCC remain less than 20%.169
Risk Modeling and Assessment
Major risk factors for HCC include chronic infection with hepatitis
B virus (HBV) or HCV, alcoholic liver disease, and nonalcoholic fatty
liver disease (NAFLD). Approximately 350 million people worldwide
have HBV infection, and HBV accounts for more than 50% of all
cases of HCC worldwide. Among active HBV carriers, the risk of
developing HCC is approximately 0.02 to 0.2 per 100 person-years.171
HCC risk is substantially higher among HBV patients with compensated
cirrhosis (2.2–3.7 per 100 person-years) compared with those with
chronic HBV infection without cirrhosis (0.3–0.63 per 100 person-
years).171 Furthermore, HCC risk in patients with chronic HBV
infection varies according to sex, age, and race or ethnicity, as well as
by a number of viral factors (higher levels of HBV DNA and hepatitis
B surface antigen; hepatitis B e antigen [HBeAg] positivity; HBV
genotype; duration of infection; and coinfection with HCV, HIV, or
hepatitis D virus [HDV]) and environmental factors (heavy intake
of alcohol and possibly tobacco, exposure to aflatoxin).171
In the United States, most of the burden of HCC is due to chronic
HCV infection (approximately 70% of HCCs in the United States
are attributed to HCV).172 Compared with those not infected, persons
infected with HCV have 15- to 20-fold higher risk of developing
HCC. HCV leads to the formation of cirrhosis (i.e., advanced liver
fibrosis), which is present in about 90% of HCC patients at diagnosis
with cancer.173,174 Once HCV-related cirrhosis is established, HCC
develops at an estimated rate of 3% per year.175 Studies have identified
a number of risk factors for HCC in HCV-infected individuals,
including older age, male sex, Hispanic ethnicity, HCV genotype 3,176
HBV or HIV coinfection, diabetes, obesity, and prolonged, heavy
alcohol drinking.177 HCV viremia of any level is a strong risk factor
for HCC compared with no viremia. Virologic cure of HCV, which
is becoming very common with the advent of directly acting antivirals,
although resulting in a considerable reduction in HCC risk, does not
eliminate this risk, especially in those who have already developed
cirrhosis.
The metabolic syndrome and its components have been associated
with higher risk of HCC. Epidemiologic studies have consistently
reported increased risk of HCC in patients with type 2 diabetes.
Multiple meta-analyses on this topic show a 2.5-fold higher risk of
HCC associated with diabetes. The association did not vary by study
design (in both case-control and cohort studies) and was independent
of viral hepatitis and alcohol use.178,179 Obesity, particularly abdominal
obesity and obesity at an earlier age, is associated with increased risk
for HCC.180–182
Epidemiologic studies show that NAFLD, the hepatic manifestation
of the metabolic syndrome, is associated with a modest increase in
the risk of developing HCC; however, the few population-based cohort
studies of patients with NAFLD have been limited by the low numbers
of HCC cases, and the higher risk is largely limited to those NAFLD
patients who develop cirrhosis.183 Genomic-wide association studies
report an association between variation in the patatin-like phospholipase
domain-containing 3 (PNPLA3) gene and risk of NAFLD.184 PNPLA3
may also determine individual susceptibility to HCC development
and poor prognosis.185
Screening and Surveillance
Because most risk factors for HCC in the United States and other
developed countries are associated with cirrhosis, patients with cirrhosis
are the current target of cancer prevention and surveillance efforts for
HCC.186 However, there are multiple points of failure in this surveillance
process. For example, in the United States, underrecognition of cirrhosis
(e.g., >20% of HCC patients have unrecognized cirrhosis at diagnosis)
is one of the main reasons for the underuse of HCC surveillance.187
This is especially important given that patients with NASH have the
highest rates of unrecognized liver disease,188 and the fraction of HCC
patients missed through surveillance of cirrhosis is likely to increase
as NAFLD becomes the leading cause of HCC in the United States.
Uptake of surveillance as well as the sensitivity of the tools used will
determine the effectiveness of HCC surveillance. A modeling study
found that percentages of surveillance use and sensitivity must exceed
34% and 42%, respectively, for HCC surveillance to be associated
with a survival benefit.189
Results from a large RCT among patients in China with chronic
HBV infection provide the best evidence to date for HCC surveil-
lance.190 The study randomly assigned over 9000 HBV carriers to a
surveillance with ultrasound and α-fetoprotein (AFP) group or a no
surveillance group and showed that the HCC mortality rate was
significantly lower in the screened group (83.2 per 100,000 person-years)
compared with the nonscreened group (131.5 per 100,000 person-years)
(HR, 0.63 [95% CI, 0.41–0.98]).190 The improved outcomes in the
surveillance group included a significantly higher proportion of patients
with HCC who were diagnosed with early-stage tumors (61% versus
none) and received curative therapy (47% versus 8%).
However, in the United States where surveillance targets high-risk
patients with cirrhosis and where methods for cancer detection and
therapy differ from those for HBV patients, these data may be less
applicable.191 Currently, the only evidence around efficacy of HCC

surveillance in patients with cirrhosis comes from observational studies,
with their inherent limitations of lead-time, length-time, and selection
bias. In their meta-analysis of 47 studies, Singal and colleagues reported
that HCC surveillance in patients with cirrhosis was associated with
earlier stage at diagnosis, receipt of curative therapy, and overall
survival.192 Although an RCT among patients in cirrhosis would provide
the best evidence, it may be unethical to withhold surveillance from
some patients, given the data from the observational studies.193,194
Nonetheless, as has been the case in other settings (e.g., colonoscopy
is widely accepted for CRC screening without data from randomized
trials), a lack of RCT data supporting HCC surveillance for patients
with cirrhosis does not mean that surveillance is ineffective.
Currently, liver ultrasound is recommended as the backbone of
HCC surveillance testing. Semiannual surveillance with abdominal
ultrasound has been shown to be efficacious for early tumor detec-
tion,195–197 safe, and cost-effective.198–201 Alternative imaging approaches
(e.g., CT and MRI) have been proposed to increase sensitivity for
each HCC; however, there are limited data on their efficacy in HCC
surveillance. In the only randomized trial to date, ultrasound and
annual CT detected similar proportions of early HCCs (6.0% versus
6.3%), but biannual ultrasound was less costly per HCC detected
($17,000 versus $57,000).202
Finally, although on one hand we should continue to focus on
providing a platform to successfully implement HCC surveillance,203
there is also a clear and urgent need for interventions to address its
shortcomings. For example, interventions could be aimed at the provider
or system level. Although patients are engaging with HCC surveil-
lance,204 providers continue to report difficulty in identifying at-risk
patients with cirrhosis, suboptimal levels of knowledge about HCC
surveillance guidelines, and time constraints, as barriers to effective
HCC surveillance.193 Similarly, focus should be on biomarkers for
improved risk stratification. AFP is one biomarker examined for use
in the early detection of HCC. Studies using assays for AFP in combina-
tion with age, platelet count, and ALT have shown potential for
clinical use.205 New biomarkers, including AFPL3 and DCP (FDA
approved), GP73, and osteopontin, are being evaluated for early HCC
detection in phase III biomarker studies, including the EDRN.206
HCC has become the fastest rising cause of cancer-related deaths
in the United States. Despite screening and surveillance efforts and
improved treatments, overall survival for patients diagnosed with HCC
remains poor. We have identified actionable information that can be
used to reduce the HCC burden; however, multiple challenges at the
patient, provider, and system levels need to be addressed in order
improve on current efforts.
GASTRIC CANCER
Globally, gastric cancer is one of the most common cancers. In 2012,
952,000 new cases of gastric cancer were diagnosed worldwide
(representing 6.8% of all cancers diagnosed in 2012).207 It is the third
most common cause of cancer-related deaths, accounting for 8.8%
of all cancer-related deaths in 2012.207 Gastric cancer is rare among
young persons, with incidence increasing with age and reaching a
plateau between 55 and 80 years, and rates are twofold higher in men
than in women.208 The highest incidence rates of gastric cancer in
men are seen in Eastern Asia (Korea, Mongolia, Japan, and China,
with incidence rates between 40 and 60 per 100,000), Eastern Europe
(35 per 100,000), and Central America and the Andean Region (with
incidence rates between 20 and 30 per 100,000).209,210 The highest
mortality rates for gastric cancer are seen in Eastern Asia (24 per
100,000 in men; 9.8 per 100,000 in women), and the lowest in
Northern America (2.8 and 1.5, respectively). However, high mortality
rates are also seen for men and women in Central and Eastern Europe,
and in Central and South America. Notably, the incidence rates for
gastric cancer also vary among different ethnic groups within a particular
country. For example, in the United States, annual incidence among
Asians is twice as high as in whites (15.6 versus 7.4 per 100,000);211
Screening and Early Detection  •  CHAPTER 23 389
similarly, gastric cancer is more frequent in Hispanics, African
Americans, and Native Indians than whites.209
The epidemiology of gastric cancer has changed over recent
decades.212 Data from the European Cancer Observatory for cancer
registries from 26 European countries confirm a decline in the incidence
of gastric cancer from 1988 to 2008.213 Studies have shown that this
decline is due to decreases in Helicobacter pylori infection, cigarette
smoking, and salt intake, and increases in use of refrigerated foods
and availability of fresh produce.214 However, in the United States
between 1977 and 2006, incidence rates for distal gastric cancer have
increased among whites aged 25 to 39 years; the cause of this increase
remains unknown.212
Risk Modeling and Assessment
Chronic infection with H. pylori is the main cause of gastric cancer,
accounting for approximately 89% of distal gastric cancer cases
worldwide.215–217 In 1994 the IARC classified H. pylori as a class I
carcinogen; it reconfirmed this classification in 2009.218 H. pylori is
a Gram-negative bacterial pathogen that selectively colonizes the human
gastric mucosa and modulates the host’s immune response.219 Most
of H. pylori infection is acquired during childhood; once established,
infection usually persists for life in the absence of treatment. The
prevalence of H. pylori infection in adults exceeds 50% in many
industrialized countries; however, there is substantial regional variation
in prevalence.220 Prevalence of H. pylori infection is higher in Central
and South America and in parts of Asia and Eastern Europe, in
comparison with North America, Australia, and Western Europe.220
Other, less common causes account for up to 10% of gastric cancers
and include infection with the Epstein-Barr virus (EBV), autoimmune
gastritis, and Ménétrier disease. An international pooled analysis of
15 studies and involving 5081 gastric cancer cases reported that
approximately 8% of gastric cancers harbor EBV,221 but there is
insufficient epidemiologic evidence of a clear etiologic role for EBV
in gastric carcinogenesis.218
Whereas most gastric cancers are sporadic, they can be hereditary,
associated with specific mutational profiles. Risk of gastric cancer is up
to 10 times higher in persons with a family history of gastric cancer.215
Gastric cancer may also develop as part of a familial cancer syndrome,
such as hereditary diffuse gastric cancer syndrome, Lynch syndrome
(in particular in patients with an MLH1 or MSH2 mutation222), FAP,
Peutz-Jeghers syndrome, and Li-Fraumeni syndrome.223
Other factors associated with increased risk for gastric cardia and
noncardia cancers include tobacco smoking, low socioeconomic status,
low level of physical activity, and radiation exposure; obesity and
gastroesophageal reflux disease are associated with increased risk of
gastric cardia cancer only.224 A report on dietary factors and cancer
prevention published by the WCRF/AICR stated that nonstarchy
vegetables and fruits probably protect against gastric cancer.225 A
high-salt diet has been associated with a higher risk for gastric cancer,225
and salt may act synergistically with H. pylori infection to confer
especially high risk for gastric cancer.226,227 Diets high in nitroso
compounds may also be associated with increased risk for gastric
cancer.225 Conversely, use of aspirin and nonsteroidal antiinflammatory
drugs and statins may lower risk for gastric cancer.224
Screening and Surveillance
Even in countries where gastric cancer is relatively common, mass
screening for gastric cancer is generally not included in national
strategies for cancer prevention. There are a number of reasons for
this, including high cost, decreasing incidence, and a lack of data on
whom, when, and how to screen. Furthermore, screening services are
complex, with success hinging on the underlying processes and efforts
to ensure its overall quality.228
Globally, various types of tests have been proposed and used for
gastric cancer screening, with the implementation of endoscopic
390 Part I: Science and Clinical Oncology
methods limited to high-risk populations. Several countries including
Venezuela, Chile, and East Asian countries such as China and Japan
have implemented a variety of screening programs. For example, in
Korea, the Korean Gastric Cancer Association and National Cancer
Center established national guidelines for gastric cancer screening in
2001. These guidelines recommend biennial gastric cancer screening
for men and women aged 40 years or older via endoscopy or upper
gastrointestinal series.229 As a result, a nationwide gastric cancer
screening program in Korea was started in 2002. Other screening
methods for gastric cancer include serum pepsinogen, serum gastrin-17,
and H. pylori antibody testing. In 2013 the Japanese government
approved insurance coverage for a gastric cancer prevention program
that includes H. pylori screening and treatment (primary prevention),
as well as posttreatment surveillance (secondary prevention for people
with atrophic gastritis).230,231
Upper gastrointestinal endoscopy studies have been shown to have
the highest detection rates among the screening tests and have been
universally regarded as the gold standard for the diagnosis of gastric
cancer.224 Although the available evidence shows that endoscopic
population screening is cost-effective in areas with high gastric cancer
burden,232 further studies are needed to evaluate the impact of this
technique before its broad use as a primary screening test can be recom-
mended.233 Upper gastrointestinal series has continuously been analyzed
as a rivaling modality for cancer detection but has not shown clear
benefits over endoscopy.234
There is increasing attention given to identifying higher-risk patients
(e.g., patients with advanced precancerous lesions) for gastric cancer
surveillance. In a cohort study conducted among patients in the Dutch
nationwide histopathology registry, the 10-year gastric cancer risk in
patients with mild to moderate dysplasia was 4%, and for severe
gastric dysplasia was 33%.235 In a cohort of high-risk Chinese dysplastic
patients, after 5 years of follow-up, these rates were 3% and 7%,
respectively.236 These patients are the ideal target for gastric cancer
surveillance; however, no evidence-based guidelines for follow-up
strategies have been established, and the few recommendations available
stem mostly from expert opinions.237
However, there are some data among healthy asymptomatic
infected Asians that show that eradicating H. pylori reduces the
incidence of gastric cancer. A systematic review and meta-analysis
of six international RCTs compared risk of incident gastric cancer in
adults who had tested positive for H. pylori and received eradication
therapy or placebo or no therapy.238 The risk of developing incident
gastric cancer was 34% lower among the 3294 individuals who
received eradication therapy compared with the 3203 control subjects
(relative risk [RR], 0.66 [95% CI, 0.46–0.95]). This corresponds
to a number needed to treat to prevent one gastric cancer of 124
(95% CI, 78–843).238
The potential effect of a gastric cancer prevention program that
includes H. pylori screening and treatment is dependent on a patient’s
level of cancer risk at the time that H. pylori is eradicated (e.g., consider-
ing the twofold higher risk of gastric cancer between the most and
the least virulent strains). Under various assumptions about both
effectiveness and costs, performance of population-based screening
for H. pylori and eradication of the infection has also been shown to
be cost-effective.232,239 A phase III RCT in school-aged children reported
the success of a prophylactic oral H. pylori vaccine,240 and a large
community-based intervention trial in China demonstrated that H.
pylori eradication is feasible and acceptable; however, they also identified
several risk factors for the failure of eradication therapy to prevent
gastric cancer.241 Studies have shown that under conditions of low
bacterial burden and acute infection, H. pylori infection may be
prevented, and additional follow-up of these patients should provide
promising data on the preventive effect of eradication on the incidence
of gastric cancer.
Further improvements in screening, treatment, and early diagnosis
are needed for gastric cancer, which remains the third most common
cause of cancer-related death worldwide. In general, there are no
population-based mass screening programs aimed at reducing the
incidence of gastric cancer. Although mass screening strategies could
be beneficial, current modalities are not yet readily implementable in
organized screening settings. Conversely, since the population risk
(based on histology) can change rapidly and H. pylori eradication
eliminates the problem, population-based programs of screening and
treatment for H. pylori may hold the greatest promise for reducing
the burden of gastric cancer.239
ESOPHAGEAL CANCER
When considering screening and early detection of esophageal cancer,
it is essential to take into account the changing epidemiology of the
disease. Esophageal cancer is relatively uncommon; however, it is a
highly fatal malignancy. Worldwide, esophageal cancer is the eighth
most common cancer (456,000 new cases in 2012) and the sixth
most common cause of cancer-related death (400,000 deaths in 2012).207
The highest incidence rates of esophageal cancer are seen along two
geographic belts: one from north central China through the central
Asian republics to northern Iran, and one from eastern to southern
Africa. In the United States, 16,940 new cases of esophageal cancer
and 15,690 deaths from esophageal cancer are expected to occur in
2017.62 Although treatment effectiveness has improved during the
past decade, survival rates for esophageal cancer remain poor. In the
United States, the overall 5-year survival rate for patients diagnosed
with esophageal cancer is less than 20%.242 There are two main histologic
subtypes of esophageal cancer: esophageal adenocarcinoma and
esophageal squamous cell carcinoma. There has been a dramatic shift
in the epidemiology of esophageal cancer in Western populations,
such that esophageal adenocarcinoma, which typically affects the lower
third of the esophagus, has become the predominant subtype of
esophageal cancer in North America, Australia, and Europe.243–245
Among white men in the United States, the annual incidence of
esophageal adenocarcinoma has increased 10-fold (0.6 per 100,000
to 6.0 per 100,000) since the early 1970s.246 In contrast, esophageal
squamous cell carcinoma is now less common than esophageal adeno-
carcinoma in the Unites States. To stem the increasing incidence and
to improve outcomes for patients diagnosed with esophageal cancer,
it is crucial to develop new strategies of screening and surveillance.
Risk Modeling and Assessment
Esophageal Squamous Cell Carcinoma
Unlike the situation in the United States, elsewhere esophageal
squamous cell carcinoma is the most common histologic subtype of
esophageal cancer.207 Although the incidence of esophageal adeno-
carcinoma has increased, the incidence of esophageal squamous cell
carcinoma has decreased in the United States.244 Alcohol consump-
tion and tobacco smoking are the main risk factors for esophageal
squamous cell carcinoma, such that more than 75% of the esophageal
squamous cell carcinoma burden in men could be attributed to smokers
with heavy alcohol consumption.247 The decrease in incidence of
esophageal squamous cell carcinoma in the United States is thought
to be due to the decreasing consumption of alcohol and tobacco.
Squamous cell carcinoma is believed to develop through progres-
sion through a premalignant dysplastic phase before development of
carcinoma.248
Barrett Esophagus and Esophageal Adenocarcinoma
Epidemiologic studies have implicated gastroesophageal reflux
disease, obesity, and cigarette smoking as the main risk factors for
esophageal adenocarcinoma. Together, these three risk factors account
for over 70% of all cases of esophageal adenocarcinoma in Western
populations.249,250 Barrett esophagus, a condition in which the normal
squamous mucosa of the esophagus is replaced by columnar intestinal
epithelium (i.e., metaplasia), is the only known precursor lesion for
esophageal adenocarcinoma, likely because of progression through

dysplasia. Barrett esophagus is present in up to 15% of individuals
with frequent symptoms of gastroesophageal reflux disease and in
1% to 2% of the general adult population.251 Compared with the
general population, patients with Barrett esophagus have at least a
10-fold higher risk for esophageal adenocarcinoma.252 Among those
with nondysplastic Barrett esophagus, 0.33% per year progress to
esophageal adenocarcinoma253; patients with Barrett esophagus who
have low- and high-grade dysplasia progress at rates of 1% and 6%
per year, respectively.254
Screening and Surveillance
Owing to the low incidence of esophageal squamous cell carcinoma
in the United States, screening for this disease in the general US
population is not recommended. Screening may be considered for
specific higher-risk subgroups (e.g., patients with oral and oropharyngeal
cancer, patients with tylosis). Likewise, based on the available evidence,
population-wide mass screening for Barrett esophagus and esophageal
adenocarcinoma is seen as not being cost-effective.255 However, despite
the lack of high-level evidence and remaining questions around whom,
when, and how often to screen, endoscopic screening (and subsequent
surveillance) in high-risk patients is advised, based on expert opinion.256
For example, the 2016 guidelines from the American College of
Gastroenterology recommend endoscopic screening for Barrett
esophagus in men with chronic (>5 years) and/or frequent (weekly
or more) symptoms of gastroesophageal reflux disease and two or
more risk factors for Barrett esophagus and esophageal adenocarcinoma
(e.g., age above 50 years, white race, obesity, history of cigarette
smoking, and history of Barrett esophagus or esophageal adenocarci-
noma in FDRs).257
Multiple professional societies have advocated for endoscopic
surveillance in patients with known Barrett esophagus, with the goal
of detecting early neoplastic changes (e.g., dysplasia) and early-stage
esophageal adenocarcinoma that can be cured with endoscopic or
surgical resection. For patients with nondysplastic Barrett esophagus,
periodic endoscopic surveillance is recommended every 2 to 3 years
and requires obtaining random biopsy specimens from the Barrett
region(s) as well as targeted biopsy specimens from nodules or ulcers.
Although evidence from randomized trials is lacking (and will be very
difficult to obtain, given logistic challenges), there is a growing body
of evidence from observational studies that having a Barrett esophagus
diagnosed is associated with better neoplasia-associated outcomes.258,259
These outcomes include earlier stage and greater chances for receipt
of curative treatment, and importantly reduced cancer-related mortality.
Endoscopic surveillance in patients with known Barrett esophagus
may be cost-effective where curative therapy is available and patients
have limited comorbidities.260,261 Furthermore, continuous endoscopic
screening and surveillance have been fortified by the success associated
with endoscopic radiofrequency ablation in patients with Barrett
esophagus (including resolution of Barrett esophagus and dysplasia,
and durability).262–264 As this technology improves in efficacy and
safety, endoscopic radiofrequency ablation may be an alternative for
nondysplastic Barrett esophagus, at least for those at high risk for
progression (e.g., long segment, severe reflux, family history of Barrett
esophagus or adenocarcinoma).265
Upper endoscopy remains the gold standard for Barrett esophagus
screening, but it has several limitations as a screening tool, including
its cost (i.e., related to endoscopy, histopathology examination, and
sedation), low NPV for short segments of Barrett esophagus (high
NPV is necessary for endoscopy to be a valid screening tool), and
risk to the patient. Consequently, less invasive screening that can be
performed without the need for sedation (unsedated endoscopy) has
been proposed and shown to be tolerable and accurate compared
with conventional sedated endoscopy.266 Transnasal endoscopy is one
particular screening modality that can be performed in an office setting
without sedation. Transnasal esophageal endoscopy has been assessed for
feasibility of Barrett esophagus screening. It has relatively high accuracy
Screening and Early Detection  •  CHAPTER 23 391
and acceptable image quality, and biopsy specimens, although small in
size, are still sufficient for acceptable histologic analysis, demonstrating
its potential.267,268 However, although this modality is generally preferred
by patients and is associated with less patient anxiety than conventional
sedated endoscopy, acceptance and use among primary care providers
have been limited owing to concerns about patient tolerance of an
unsedated procedure and uncertainties regarding the safety, time, train-
ing, and facilities required for this procedure. For gastroenterologists
and other endoscopists, there is an additional financial disincentive
for performing an office-based procedure: lower reimbursement rates
than for standard upper endoscopy with biopsies.269
Molecular screening methods might soon be practical for Barrett
esophagus. Esophageal capsule cytology (Cytosponge; Medical Research
Council, London, UK) is a new technique that has the potential to
provide a noninvasive, nonendoscopic method of screening for Barrett
esophagus.270 The device, which can be used in the office setting
without sedation, is a sponge within a capsule covered in a gelatin
layer, which dissolves after swallowing. After that the sponge is released,
and esophageal epithelial cells are retrieved.271 Immunohistochemistry
is then performed on these cells to analyze for markers such as trefoil
factor 3, which can differentiate the cells of Barrett esophagus from
other columnar cells found in the normal gastric cardia and upper
airway.271 Depending on the length of the Barrett esophagus segment,
prospective cohort studies conducted in the United Kingdom have
shown that this technique has a sensitivity of 80% to 90% with
a specificity of approximately 92%.272 Additional clinical trials to
further validate this technique (and the use of other biomarkers) are
ongoing.
The incidence of esophageal cancer, especially esophageal adeno-
carcinoma, is increasing, and overall survival for patients diagnosed
with esophageal cancer remains poor. The ability to increase survival
in this disease will depend on earlier detection through screening and
surveillance; however, screening for Barrett esophagus has several
limitations. Because patients are often asymptomatic, it is difficult to
identify the entire at-risk group and therefore risk-based screening
may miss a considerable proportion of patients with Barrett esophagus
and esophageal adenocarcinoma. For example, several studies have
shown that about 40% of patients diagnosed with esophageal adeno-
carcinoma report no history of gastroesophageal reflux symptoms.273,274
Furthermore, only 7% of patients with esophageal adenocarcinoma
have a prior diagnosis of Barrett esophagus.258 Therefore, current
approaches to esophageal cancer control are missing 93% of cases
owing to an emphasis on symptoms. If we are to prevent esophageal
adenocarcinoma, much more work needs to be done around screening
strategies for Barrett esophagus.
HEAD AND NECK SQUAMOUS
CELL CARCINOMA
Head and neck squamous cell carcinomas (HNSCCs) can occur on
the lip, in the oral cavity, and in the pharynx (nasopharynx, oropharynx,
and hypopharynx) and larynx. Worldwide, approximately 600,000
cases of HNSCCs occur annually, with oral cancer comprising at least
half of the cases.275 Incidence of HNSCC varies greatly around the
globe because of differences in the prevalence of its primary risk
factors: alcohol, tobacco, betel quid with or without added tobacco
(mainly in Asia), and, more recently identified, HPV. The ACS estimates
that nearly 50,000 cancers of the oral cavity and pharynx will occur
in the United States in 2017, with an additional 13,000 laryngeal
cancers.18 Incidence and mortality are generally higher among men,
with each being nearly three times higher in US men than in US
women. Outcomes are dependent on the anatomic site and stage of
disease at diagnosis. Two-thirds of individuals with oral and pharyngeal
cancers have regional or distant metastases at the time of diagnosis
and therefore have 5-year survival rates of 64.2% and 38.5%, respec-
tively.276 HNSCCs are notable for their high rate of recurrence and
second primary tumors.
392 Part I: Science and Clinical Oncology
Risk Modeling and Assessment
Risk assessment is generally based on an individual’s current or past
history of exposure to one of the primary factors. Numerous epide-
miologic studies have documented the increased risk associated with
each factor.
All forms of tobacco, including smokeless tobacco, are associated
with an increased risk of HNSCC.277 Current cigarette use increases
the risk of oral and oropharyngeal cancers by approximately tenfold
for men and fivefold for women, compared with lifetime nonsmokers.277
Alcohol increases the risk fivefold for those consuming five or more
drinks per day relative to those who do not drink; and an increased
risk has been observed in nonsmokers and in studies that control for
confounding by smoking, demonstrating a direct effect of alcohol.278,279
In combination, these two risk factors confer a risk that is two to
three times greater than the risk expected if one were to assume a
simple multiplicative effect.280 Individuals who smoke two or more
packs per day along with consuming four or more alcoholic drinks
per day have an approximate 35-fold increased risk of oral or oro-
pharyngeal cancers in comparison with those who neither smoke nor
drink. In Asia, the use of betel quid and gutka (betel quid combined
with tobacco) is common. An Indian meta-analysis has suggested that
betel quid doubles the risk of both oral and oropharyngeal cancers
and that gutka confers an eightfold increased risk for oral cancer and
a fourfold increased risk for oropharyngeal cancer.281
Tobacco cessation significantly reduces the risk of both oral and
oropharyngeal cancers, with an approximate 50% risk reduction
observed after 5 to 9 years of quitting and a return to risk comparable
to never-smokers within two decades.282 The data are less strong for
cessation of alcohol. Nevertheless, given the dose-response relationship
of alcohol with oral cancer, it is believed that cessation or a reduction
in consumption would result in reduced risk. A meta-analysis of studies
by the International Head and Neck Caner Epidemiology Consortium
suggests a statistically significant 40% reduction in HNSCC risk
(compared with current drinkers) in those who quit drinking at least
20 years ago.282 By subsite, a 34% risk reduction was seen for oral
cancer after 10 to 19 years of cessation; and a 31% risk reduction
was observed at 20 or more years of cessation for laryngeal cancer.
No statistically significant reduction was observed for oropharyngeal
or hypopharyngeal cancers. Less is known regarding the effect of
cessation of betel quid and gutka on risk of HNSCC. A 2016 study
of tobacco-free betel quid in Taiwan demonstrated that every year of
cessation was associated with a 2.9% risk reduction for HNSCC,
although risk among those who quit never returned to the level of
nonusers, even after 20 years.283 More studies with long-term follow-up
are needed in areas of betel quid and gutka use to more thoroughly
assess the effect of cessation on subsequent risk of HNSCCs.
In the last 15 years, the recognition that HPV is causally linked
to HNSCCs, primarily to oropharyngeal cancer, has led to a paradigm
shift in the prevention, detection, and treatment of these cancers. The
fraction of HNSCCs attributable to HPV varies greatly by geographic
region, gender, and age at diagnosis. Studies have suggested worldwide
attributable fractions in the range of 18.5% to 39.8% of oropharyngeal
cancers, 7.5% to 16.1% of unspecified pharyngeal cancers, 1.1% to
5.9% of nasopharyngeal cancers, 2.2% to 16.3% of oral cancers, 1.5%
to 8.6% of laryngeal cancers, and 2.4% of hypopharyngeal cancers.284–286
However, within the United States, data support an HPV-etiologic
fraction of at least 72% for oropharyngeal cancer.287 The variability
in HPV attributable fractions originates from differences in incidence
of HPV infection and patterns of behavior, such as sexual practices
and tobacco use, around the world and across time. Data show that
oral HPV infection is the major risk factor for HPV-positive oropha-
ryngeal cancer, and that more than 90% of these oral infections are
sexually acquired.287 Indeed, lifetime number of oral sex partners is
strongly, consistently, and specifically associated with oropharyngeal
cancer.287,288 Current studies are examining the risk of oral HPV
infection and cancer in partners of patients with HPV-positive
oropharyngeal cancer. The HPV vaccine has the potential to reduce
oral HPV infections. Herrero and colleagues demonstrated that vac-
cination against HPV strains 16 and 18 prevented more than 90%
of these oral infections within 4 years of vaccination.289 However, it
is still too early to assess whether this will lead to a reduced risk of
HPV-associated oropharyngeal cancer later in life.
Screening
Currently, no organized screening programs for any HNSCC exist
because there is a lack of RCT data to support their use. Screening
for oral cancer can be carried out through visual and tactile inspection
of the oral cavity by a health care provider or by self-examination.
The mouth is easily accessible, and there are known premalignant
lesions associated with oral cancer development. However, to date,
just one RCT has examined the potential benefits of oral cancer
screening.290,291 This was a cluster randomized trial of 13 districts (N
= 191,873) in India, which has some of the highest rates of tobacco-
associated oral cancer in the world, and used advanced health workers
to perform the screening during home visits.292 There was no statistically
significant difference in either oral cancer mortality or incidence between
screened and unscreened groups. However, a stage shift and an increase
in survival were observed in the screened group. Of oral cancer cases
in the screened districts, 41% were stage I or II, whereas just 23% of
cases were stage I or II in the unscreened districts (P = .004); and
5-year survival was 50% in the screened areas versus just 34% in
unscreened areas (P = .009). In a post hoc subgroup analysis of high-risk
individuals (i.e., those using tobacco, alcohol, or both; N = 84,600),
a significant 34% reduction in oral cancer mortality was observed in
the screened group (RR, 0.66 [95% CI, 0.45–0.95]). Although these
results suggest that screening may be beneficial among high-risk groups,
the generalizability of these results to the United States and to other
Western populations is unknown, given differences in disease prevalence,
risk factor distribution, and health care systems. In addition, the trial
suffers from a number of flaws, including possible lead-time and
length-time bias, low compliance with follow-up, and an imbalance
in baseline risk factors.290
In addition to this single RCT, a number of diagnostic accuracy
studies have been conducted for conventional oral cancer screening
(visual and tactile inspection by a health care worker), mouth self-
examination, light-based detection, and vital rinsing (e.g., toluidine
blue). These have been conducted in both apparently healthy individuals
and in those with clinically evident lesions. A review of 10 studies
conducted in apparently healthy populations demonstrated great
variability in the sensitivity of conventional oral examination (COE),
ranging from 0.50 (95% CI, 0.07–0.93) to 0.99 (95% CI, 0.97–1.00)
depending on the prevalence of oral cancer and premalignant lesions,
but found consistently high specificity of around 0.98 (95% CI,
0.97–1.00).293 Two studies of mouth self-examination demonstrated
much lower estimates of sensitivity, 0.18 (95% CI, 0.13–0.24) to
0.33 (95% CI, 0.10–0.65), and variable specificity, 0.54 (95% CI,
0.37–0.69) to 1.00 (95% CI, 1.00–1.00). One RCT found a higher
rate of detection of oral cancer in the arm of conventional oral
examination plus toluidine blue versus conventional oral examination
alone, but results were not significant and the overall risk of bias in
this study was judged as unclear; In addition, there was concern among
the reviewers regarding the overall applicability of the RCT, given its
method of patient selection. A review of 41 studies of individuals
with clinically evident lesions found them to be generally of poor
quality and that none of the adjunctive tests could be recommended
to replace scalpel biopsy and histologic assessment, although cytologic
evaluation showed promise with both high sensitivity (0.91 [95% CI,
0.91–0.96]) and specificity (0.91 995% CI, 0.91–0.950).294 Neither
review found eligible studies of blood or salivary sample (biomarker)
analysis.293,294
Given this limited evidence, the USPSTF concluded in 2013 that
evidence was insufficient to assess the balance of benefits and harms

of oral cancer screening by primary care physicians. The American
The American Dental Association has recently published updated
evidence-based clinical practice guidelines for the evaluation of
potentially malignant disorders in the oral cavity.295
The emergence of HPV as a risk factor for oropharyngeal cancer
suggests the possibility of screening for oral HPV infection as a means
of prevention and/or early detection, but there is currently no FDA-
approved screening test for oral HPV infection.296 The use of cytologic
evaluation and HPV DNA testing, as in the cervix, offers two potential
approaches. However, cytologic assessment may be limited by the
anatomic location and properties of oropharyngeal cancer and the
tonsillar epithelium, and HPV DNA testing faces technical challenges,
including how best to obtain a sample.297 Noninvasive sampling strate-
gies that could be applied in the context of large-scale population-based
screening are oral rinses and gargles; however, these strategies are not
specific to the tonsillar epithelium and may overestimate the actual
rate of tonsillar HPV infection.297 In addition, the lack of knowledge
regarding the natural history of oral HPV infections and the likelihood
of a high-risk HPV infection to persist and progress to cancer challenges
the use of an HPV DNA testing approach to screening. Blood tests
are a third potential screening strategy for HPV-driven HNSCCs,
and recent data suggest that this may be a promising, noninvasive
approach.298 Finally, oropharyngeal examination with narrow band
imaging299 and transcervical and intraoral ultrasonography300–302 hold
potential for diagnostic purposes in individuals with a positive screening
test result.
OVARIAN CANCER
Ovarian cancer is the deadliest of the gynecologic malignancies and
is the fifth leading cause of cancer mortality among American women.18
The high mortality rate is a result of the typically late presentation
of the disease, largely because of vague, nonspecific, or even lack of
early symptoms. For women at high risk of ovarian cancer due to a
BRCA mutation, prevention through risk-reducing surgery and early
detection with screening has the potential to improve the outcomes
of this disease.303
Risk Factors and Etiology
The majority of ovarian cancers are sporadic; only 5% to 10% of
ovarian cancers are related to genetic mutations.304 Absent other relevant
family history, a single FDR with ovarian cancer is not suggestive of
an inherited predisposition. Women having one FDR with the disease
have a threefold to fourfold increased risk of the disease.305 Although
they are at increased risk of ovarian cancer compared with average
risk women, they do not meet the threshold for ovarian cancer screening
or prophylactic surgery. The strongest known risk factor for ovarian
cancer is hereditary breast and ovarian cancer syndrome due to muta-
tions in the BRCA1 and BRCA2 genes.304 In addition, women with
a family history suggestive of a BRCA mutation but who are untested
are considered at high risk until testing definitively proves otherwise.
BRCA1 mutation carriers have a reported 39% to 63% cumulative
risk of ovarian cancer by age 70, whereas the risk for BRCA2 mutation
carriers is 11% to 31%, depending on whether one is using data from
population-based case series or multicase family data (the latter sug-
gesting higher risk).305 Ovarian cancers are less commonly the result
of other inherited genetic syndromes such as Lynch syndrome, Cowden
disease, or Li-Fraumeni syndrome. Genetic testing for inherited
mutations should be discussed with women who have a significant
family history of ovarian and/or breast cancer.
Advancing age is associated with an increased risk of epithelial
ovarian cancer, with half of all cases of ovarian cancer occurring in
women over the age of 63.304 Obese women have a higher risk of
ovarian cancer.304 Some studies have suggested that the effect of BMI
on ovarian cancer risk is greater in premenopausal women than in
postmenopausal women. A pooled analysis of 12 prospective cohort
Screening and Early Detection  •  CHAPTER 23 393
studies showed a 72% increase in risk of ovarian cancer in premeno-
pausal women who were obese (BMI of 30 or higher) compared
with women of a healthy weight (BMI of 18.5–23). The same
comparison in postmenopausal women did not identify a link between
a high BMI and ovarian cancer risk.306 However, results from the
European Prospective Investigation Into Cancer and Nutrition (EPIC)
suggest that being obese after menopause may also increase the risk
of ovarian cancer.307
An interesting paradox exists with regard to hormones and ovarian
cancer risk. Although risk of ovarian cancer appears to be increased
with use of postmenopausal hormones, studies have suggested that
use of OCs decreases the risk of ovarian cancer. A systematic review
of published case-control cohort studies and randomized trials revealed
that use of estrogen-only therapy for 5 years increased the risk of
ovarian cancer by 22%, significantly more than the 10% risk increase
with use of estrogen-progestin hormone therapy (EPT).308 The UK
Million Women Study reported that current users of postmenopausal
hormones had a 20% greater risk of developing ovarian cancer (RR,
1.20 [95% CI, 1.09–1.32]; P = .0002]) and a 23% higher risk of
dying from the disease (RR, 1.23 [95% CI, 1.09–1.38]; P = .0006]).
Although incidence of ovarian cancer was higher with increased duration
of use for current users, it did not appear to differ significantly by
type of hormonal therapy (estrogen-only therapy or EPT). Past users
were not at increased risk of ovarian cancer.309
In contrast, long-term use of OCs has been associated with a
reduced incidence of ovarian cancer in ever-users compared with
never-users (odds ratio [OR], 0.73 [95% CI, 0.66–0.81]). A significant
duration-response relationship was seen, with a reduction in incidence
of more than 50% among women using OCs for 10 or more years.310
In an analysis of 45 epidemiologic studies, this reduction in risk
persisted for more than 30 years after OC use had ceased but became
somewhat attenuated over time. The proportional risk reductions per
5 years of use were 29% (95% CI, 23–34%) for use that had ceased
less than 10 years previously, 19% (95% CI, 14%–24%) for use that
had ceased 10–19 years previously, and 15% (95% CI, 9%–21%) for
use that had ceased 20 to 29 years previously.311 Use of OCs has also
been shown to reduce the risk of ovarian cancer in carriers of BRCA1
mutations (OR, 0.56 [95% CI, 0.45–0.71]; P < .0001) and carriers
of BRCA2 mutations (OR, 0.39 [95% CI, 0.23–0.66]; P = .0004).312
In another twist on hormones and ovarian cancer risk, use of the
fertility drug clomiphene citrate for longer than 1 year may increase
the risk of low malignant potential tumors. However, it should be
noted that infertile women may be at higher risk of ovarian cancer
even if they do not use fertility drugs. Some of this risk may be due
to the fact that they have not carried a pregnancy to term or used
birth control pills long term, both of which are protective.304
With regard to hormonal and reproductive factors, incessant
ovulation has been hypothesized to increase the number of spontaneous
mutations and thus the risk of ovarian cancer.313 This may be the
mechanism by which the risk of ovarian cancer is reduced by OC
use. In line with this hypothesis, ovarian cancer risk is reduced by
factors that interrupt ovulation such as pregnancy and breastfeeding.
Tubal ligation and hysterectomy are associated with a protective effect
of 30% to 40%.313
Genital talcum powder has been suggested to be associated with
an increased risk of ovarian cancer. A 2006 meta-analysis of 21 studies
reported an approximately 35% (95% CI, 1.26–1.46) increase in risk
with genital exposure to talc,314 similar to findings from a 2003 meta-
analysis.315 In a 2006 review by the IARC, talc was classified as possibly
carcinogenic to humans (group 2B) on the basis that most of the 20
epidemiologic studies on talc and ovarian cancer consistently show a
30% to 60% increased risk associated with talc use.316 Before the
mid-1970s, contamination of talc with asbestos fibers was known to
occur, and guidelines were introduced to prevent this in 1975. One
study that examined talc use by year showed that use before 1975
was associated with an increase in risk, whereas use after 1975 was
not.317 However, use after 1975 has been shown in other studies to
394 Part I: Science and Clinical Oncology
be associated with an increased risk of ovarian cancer similar to that
seen before 1975.318,319
Screening
To date, an effective screening test for ovarian cancer has not been
identified. The two tests commonly investigated for ovarian cancer
screening are CA125 and transvaginal ultrasound (TVUS). CA125
is elevated in 90% of advanced ovarian cancers but in only 50% to
60% of early ovarian cancers.320 Any process that disrupts the epithelial
lining of the peritoneum has the potential to raise the CA125 level.
As a result, CA125 has been identified as being elevated in a number
of benign conditions including menstruation, pregnancy, endometrio-
sis, adenomyosis, pelvic inflammatory disease, uterine leiomyoma,
pancreatitis, hepatitis, peritonitis, renal failure, and congestive heart
failure. The low incidence of an elevated CA125 level in early-stage
ovarian cancers and the fact that it is elevated in a number of normal
conditions significantly affects its usefulness in ovarian cancer screen-
ing. TVUS attempts to discriminate between benign and malignant
adnexal masses by using information on morphology, color flow imaging
based on the neovascularity of neoplasms, and measurements of
pulsatility indices.
In 2011, initial results from PLCO were published regarding ovarian
cancer screening in 78,216 women aged 55 to 74 years who were
randomized to receive usual care or annual screening for 6 years with
CA125 and 4 years with TVUS.321 Although screening identified more
ovarian cancers—212 compared with 176 found in women who
received usual care (RR, 1.21 [95% CI, 0.99–1.48])—the majority
of the cancers in both groups were stage III or IV tumors. With a
median of 12.4 years of follow-up, there were 118 deaths caused by
ovarian cancer in the intervention group and 100 deaths in the usual
care group (RR, 1.18 [95% CI, 0.82–1.71]). Although no mortality
reduction was observed, significant harms were identified when
subsequent surgery was considered in screened women with false-positive
results. Of 3285 women with false-positive results, one-third (1080
women) underwent surgical follow-up; 15% (163 women) of these
women experienced at least one serious complication. Of the 10% of
women in the PLCO’s intervention group who had a false-positive
result, one-third had an oophorectomy—33% more oophorectomies
than in the control group—and 15% had a serious complication,
such as infection, surgical complication, or cardiovascular or pulmonary
event. Extended follow-up of the PLCO has continued to demonstrate
a lack of ovarian cancer mortality reduction (RR, 1.06 [95% CI,
0.87–1.30]) after a median of 15 years.322
Retrospective studies have shown that rising levels of CA125 within
the normal range may be indicative of ovarian cancer, whereas stable
CA125 levels, even if somewhat elevated, were not predictive of
malignancy. Hypothesizing that changes in CA125 over time may
predict ovarian cancer, Skates and colleagues developed a risk of ovarian
cancer algorithm (ROCA). They applied linear regression analyses to
longitudinal CA125 measurements from 9233 postmenopausal,
average-risk women who had two or more serial samples and a 6.8-year
median follow-up. Risk was assigned through use of a baseline CA125
and changes in CA125 over time combined with age and other known
risk factors. The ROCA was shown to improve sensitivity for detection
of preclinical disease from 62% to 86%.323 The algorithm is currently
in clinical trials in the United States and the United Kingdom.
The US, single-arm, prospective, multicenter screening study
enrolled postmenopausal women age 50 to 74 with no significant
family history of breast or ovarian cancer. Participants underwent a
CA125 blood test annually. Based on the ROCA result, women were
triaged to the next annual CA125 test (low risk), repeat CA125 test
in 3 months (intermediate risk), or TVUS and referral to a gynecologic
oncologist (high risk). Based on clinical findings and TVUS, the
gynecologic oncologist made the decision regarding whether to proceed
with surgery. The US study enrolled 4051 women over an 11-year
period. The average annual rate of referral for CA125 measurement
in 3 months was 5.8%, and the average annual rate of TVUS and
gynecologic oncologist referral was 0.9%. Ten women subsequently
underwent surgery based on the TVUS findings and referral, revealing
four invasive ovarian cancers (one stage IA, two stage IC, and one
stage IIB), two ovarian tumors of low malignant potential (both stage
IA), one endometrial cancer (stage I), and three benign ovarian tumors,
providing a PPV of 40% (95% CI, 12.2%–73.8%) for detection of
invasive ovarian cancer. The combined specificity of ROCA followed
by TVUS for referral to surgery is 99.9% (95% CI, 99.7%–100%).
All four women with invasive ovarian cancer had been enrolled in
the study for at least 3 years with low-risk annual CA125 levels before
a rising CA125 level.324
The UK Collaborative Trial of Ovarian Cancer Screening
(UKCTOCS) multicenter RCT enrolled 202,638 postmenopausal
women age 50 to 74 women who were not at increased risk of familial
ovarian cancer. Participants were randomly allocated to annual
multimodal screening (MMS) with the ROCA to interpret serial CA125
results and TVUS as a second-line test, annual TVUS only, or no
screening. The ROCA doubled the number of screen-detected cancers
compared with a single-threshold rule, detecting 86.4% of the invasive
epithelial ovarian and tubal cancers, whereas use of annual serum
CA125 fixed cutoffs of more than 35, more than 30, and more than
22 U/mL enabled identification of only 41.3%, 48.4%, and 66.5%,
respectively.325 The overall sensitivity for detection of ovarian cancer,
diagnosed within a year of screening, was 84% (95% CI, 79%–88%)
for MMS and 73% (95% CI, 66%–79%) for TVUS. A higher propor-
tion of invasive epithelial ovarian and peritoneal cancer diagnosed
with low-volume disease (stage I, II, and IIIa) was seen in the MMS
group (40%; P < .0001) than in the no screening group (26%), but
not in the TVUS group (24%; P = .57). With a median of 11.1 years
of follow-up, the relative mortality reduction was 15% in the MMS
group and 11% in the TVUS group, but these reductions were not
significant in the primary prespecified Cox analysis. However, the
prespecified secondary subgroup analysis with exclusion of prevalent
cases in the MMS group was significant, suggesting that the long-term
effect of an MMS screening program is about a 28% mortality reduction
after 7 years of screening.326 Follow-up continues to determine if the
observed stage shift will translate into a significant mortality reduction,
with the next analysis planned to be performed in 2019.
In the United Kingdom Familial Ovarian Cancer Screening Study
(UK FOCSS), all participants had a BRCA1 or BRCA2 mutation or
an estimated lifetime risk of ovarian cancer greater than 10% on the
basis of personal and family history of breast or ovarian cancer. In
the phase I portion of the trial, annual CA125 screening with a single
cut point and TVUS had a sensitivity of greater than 80% but did
not lead to an appreciable stage shift.327 In the phase II portion of
the trial, women not undergoing risk-reducing salpingo-oophorectomy
(RRSO) had CA125, triaged according to the ROCA, every 4 months.
In addition to its use as a second-line test in the ROCA, annual TVUS
was performed if ROCA results were normal. With a median follow-up
of 4.8 years, 19 invasive ovarian or tubal cancers were diagnosed out
of 4348 women who underwent 13,728 woman-years of screening.
Thirteen were screen-detected cancers (1 prevalent, 12 incident); the
other 6 were occult cancers discovered at RRSO. All women were
asymptomatic at cancer detection; only 37% had high-volume/
advanced-stage (IIIB or IIIC) disease at diagnosis. Of the occult cancers
found at RRSO, five of the six were stage I or II. However, four of
the five had normal ROCA findings, as did the occult stage IIIa cancer.
All but one of the screen-detected cancers had intermediate or elevated
ROCA findings; the one incident screen-detected cancer with normal
ROCA findings was found on the annual TVUS. However, only 5
of the 12 incident screen-detected cancers (42%) were stage I or II,
making the main stage shift reported less impressive.328
Currently, there is insufficient evidence to support routine ovarian
cancer screening with CA125 and TVUS in women at average risk
of ovarian cancer. The USPSTF recommends against ovarian cancer
screening in women at average risk (D recommendation).329 The ROCA

screening test was marketed in the United States after publication
of the UKCTOCS findings in December 2015. In 2016, the FDA
issued a Safety Communication, recommending against the use of
ovarian cancer screening tests in the general population of women
and noting that testing higher-risk asymptomatic women for ovarian
cancer has no proven benefit and is not a substitute for preventive
actions that may reduce their risk.330 The ROCA test was volun-
tarily withdrawn in the United States but remains available in the
United Kingdom.
Women with a BRCA mutation or a family history suggestive of
a mutation but as yet untested should be identified and counseled
regarding their ovarian cancer risk, surgical strategies to reduce their
risk, and screening options. Although there are no validated screening
methods for women at high risk of ovarian cancer, mutation carriers
who have not undergone RRSO should be counseled about screening
with pelvic examination, TVUS, and CA125 every 6 months beginning
at age 30 years or 5 to 10 years before the earliest age of diagnosis in
the family.331 Screening is not a substitute for preventive surgery and
should continue only until RRSO is performed. Because these women
are also at increased risk of breast cancer, they should be counseled
regarding breast cancer surgical risk reduction strategies and high-risk
breast cancer screening recommendations (see section on breast
cancer screening).
ENDOMETRIAL CANCER
Endometrial cancer is the most commonly diagnosed gynecologic
malignancy in women in the United States.18 Although most endo-
metrial cancers diagnosed in the United States are localized, suggesting
a limited value to screening, approximately one in four cases are
advanced at the time of diagnosis.
Risk Factors and Etiology
Conditions associated with prolonged progesterone deficiencies promote
endometrial proliferation and an increased risk of endometrial
hyperplasia and cancer. Estrogen drives endometrial epithelial prolifera-
tion. Progesterone inhibits growth and causes cell differentiation.
Progesterone has been described as the ultimate endometrial cancer
tumor suppressor.332 The importance of progesterone as a key inhibitor
of carcinogenesis is reflected by the observation that women who
regularly ovulate and produce progesterone rarely get endometrial
cancer. In addition, studies have demonstrated regression of endometrial
hyperplasia (both usual and atypical) by 80% to 90% after treatment
with progestin.332 However, in premenopausal obese women, lack of
progesterone due to anovulation, such as that observed in polycystic
ovarian syndrome (PCOS), increases a woman’s risk of endometrial
cancer. An Australian case-control study of women younger than 50
years showed that women with PCOS had a fourfold greater risk of
endometrial cancer compared with women without PCOS. When
adjusted for BMI, PCOS remained an independent risk factor and
was associated with a greater than twofold increased risk of endometrial
cancer.333 In addition, the use of unopposed estrogen is a well-established
risk factor for the development of endometrial cancer. Addition of
progesterone to estrogen therapy negates this risk.
A review of 15 case-control studies and four large cohort studies
demonstrated a decrease in the risk of endometrial cancer of about
50% for ever-use of OCs. An increasing protective effect with longer
duration of OC use has been found in most studies. This protective
effect persisted for as long as 20 years after cessation of use.334 Increased
parity has been shown to decrease risk of endometrial cancer. In
EPIC, a 35% decreased risk of endometrial cancer was observed in
parous women (HR, 0.65 [95% CI, 0.54–0.77]) compared with
nulliparous women, with a trend of decreasing risk with increasing
number of full-term pregnancies. A shorter menstrual lifespan (late
menarche, early menopause) has been associated with a reduced risk of
endometrial cancer.335
Screening and Early Detection  •  CHAPTER 23 395
Among all cancers, increasing BMI and obesity are most strongly
associated with endometrial cancer incidence and death.336 In a meta-
analysis, Renehan and colleagues found that each increase in BMI of
5 kg/m2 significantly increased a woman’s risk of endometrial cancer by
59% (95% CI, 1.50–1.68).337 A large study of patients who underwent
gastric bypass surgery revealed a 78% reduced risk of endometrial
cancer (HR, 0.22 [95% CI, 0.13–0.40]), underscoring the profound
role of obesity in the development of this disease.338 Obesity has been
associated with several factors known to increase endometrial cancer risk,
including upper body or central adiposity, PCOS, physical inactivity,
and a diet high in saturated fat.339 It has long been presumed that
the peripheral conversion of androstenedione to estrone in adipose
tissue resulted in excess circulating estrogen and subsequent risk of
endometrial cancer in obese women. However, more recently, other
mechanisms have been suggested, including elevated serum insulin
levels and low adiponectin levels, a marker of insulin resistance that
has also been linked to endometrial cancer risk.336
In a meta-analysis of 33 studies, the average endometrial cancer
risk reduction associated with high versus low physical activity was
20%.340 There is some evidence that the association between physical
activity and endometrial cancer risk may reflect the effect of physical
activity on obesity, a known risk factor for endometrial cancer.340–342
The aforementioned findings suggest an opportunity to reduce the
incidence of endometrial cancer by obesity management and increasing
physical activity.
In the National Surgical Adjuvant Breast and Bowel Project Breast
Cancer Prevention Trial, endometrial cancer risk was increased with
use of tamoxifen.343 The annual rate was 2.3 per 1000 for women on
tamoxifen versus 0.91 for those on placebo. Women older than 50
years experienced the greatest effect. Raloxifene, a second-generation
selective estrogen receptor modulator, does not have this association.344
Although Lynch syndrome (also called hereditary nonpolyposis
colorectal cancer), typically manifests as familial clustering of early-onset
colon cancer, there is also an increased incidence of endometrial cancer
in women with Lynch syndrome. About 3% to 5% of endometrial
cancers are attributable to the inherited mutations in the DNA
mismatch repair (MMR) genes that cause Lynch syndrome.345 Lifetime
risk of endometrial cancer in women with MLH1 or MSH2 mutations
is approximately 40%, with diagnosis at a median age of 49. Women
with MSH6 mutations have a similar risk of endometrial cancer but
a later age of diagnosis.346 In a large series of women with Lynch
syndrome who developed both colorectal and gynecologic cancers,
endometrial and/or ovarian cancer was, in half of the cases, the “sentinel
cancer” preceding the development of colon cancer.347
Screening
There are no RCTs that have demonstrated that screening with
endometrial biopsy (EMB) and/or TVUS is superior to simply
performing a biopsy if abnormal uterine bleeding occurs. Given that
most endometrial cancers are found at an early stage based on the
development of symptoms (e.g., abnormal uterine bleeding) and that
survival rates are high, the efficacy of endometrial cancer screening is
questionable. Studies of screening for endometrial cancer with EMB
and/or TVUS in asymptomatic women receiving tamoxifen or with
Lynch syndrome348–351 reported disappointing results and associated
harms including unnecessary EMBs in women with suspicious findings
on TVUS, uterine perforations, and an increase in operative procedures.
At this time, no organization recommends routine screening for
endometrial cancer in average-risk women. In addition, screening is
not recommended for obese women, women with PCOS, women
taking tamoxifen or unopposed estrogen, or other women at increased
risk. However, they should be informed about the risks and symptoms
of endometrial cancer and strongly encouraged to report any unexpected
bleeding or spotting to their physicians.
Although there are no validated screening methods for patients at
high risk of endometrial cancer, women with known or suspected
396 Part I: Science and Clinical Oncology
Lynch syndrome may be offered annual EMB beginning at age 30 to
35, pending definitive management with total hysterectomy with
BSO.352,353 It has been shown that EMB performed at the time of
colonoscopy is a patient-centered option that decreases pain associated
with the biopsy and increases patient satisfaction.354
SKIN CANCER
Skin cancer screening has traditionally been done by dermatologists
and is considered a major distinguishing professional responsibility
within the specialty. The accessibility of skin and the substantial burden
of skin disease make it an ideal clinical setting for cancer screening
and prevention. That said, there is significant controversy regarding
how effective total body skin examination (TBSE) is in terms of
improving survival rates in skin cancer.355,356 This is largely a result
of the difficulty with which evidence to support its widespread use
in the general population can be gathered and is reflected in the recent
USPSTF recommendation of I, or insufficient evidence, a reiteration
of its previous rating from 2009.357 It is very important to recognize
that this rating pertains to evidence for the benefit of screening
asymptomatic adults with no elevated risk of skin cancer and does
not address individuals who have had a prior history of skin cancer
or precancerous lesions.357,358
The evidence that TBSE is effective in reducing skin cancer mortality
is largely derived from two ecologic studies: one focused on employees
at Lawrence Livermore National Laboratory (LLNL),359 and one
population-based study in Northern Germany.360,361 Understandably,
the focus has been on melanoma, which accounts for over 75% of
deaths attributed to skin cancer, and the consensus is that melanoma
survival is enhanced when lesions are diagnosed earlier. The end point
of skin cancer screening is often the selection of appropriate lesions
for biopsy, and measures of efficacy other than survival or mortality
include the number of lesions that require biopsy per melanoma
diagnosis, changes in cancer incidence, earlier stage of diagnoses, and
cost-effectiveness of avoiding management of more advanced disease.
However, these end points are not used in formulating USPSTF
recommendations.
German SCREEN Study
The results of the landmark Skin Cancer Research to Provide Evidence
for Effectiveness of Screening in Northern Germany (SCREEN) study
have garnered significant attention, and this study is perhaps the
one cited most frequently in the assessment of TBSE.360–362 In 2003,
98% of dermatologists and 64% of general practitioners and other
specialists in the state of Schleswig-Holstein, Germany participated in an
8-hour training course on TBSE; from July 1, 2003 to June 30, 2004,
360,288 adults aged 20 years or older (19% of the population) were
screened for melanoma by TBSE in the state of Schleswig-Holstein.360,361
Initially, 84.5% were screened by nondermatologists, and 12.9% of
total participants were ultimately triaged for further evaluation by
dermatologists. A total of 9.3% of screened individuals had skin lesions
suspicious for skin cancers, and ultimately 585 melanomas, 1961 basal
cell carcinomas, and 293 squamous cell carcinomas were identified
through biopsy.360,361
In the period from 1980 to 2002, melanoma mortality in Schleswig-
Holstein was significantly higher than the German average by over 24%,
but in 2002, this decreased significantly. In the 5-year period following
SCREEN, from 2003 to 2008, a 50% reduction in mortality was
observed, corresponding to a decrease of 0.8 death per 100,000.360,361
Additional analyses demonstrated that melanoma incidence increased
shortly after SCREEN, as did incidences of NMSCs.363 Analysis of
interval melanomas between a negative examination result at SCREEN
and a subsequent regular screening in over 350,000 individuals
showed a moderate 29% reduction in invasive melanomas, but a
61% increase in in situ melanomas. Possible reasons for this increase
are difficult to identify, given that overall incidences rose and the
unquantifiable impact of self-selection both into SCREEN and for
subsequent examinations, which were not mandated or tracked in
the study.364
The trend of increased survival was short-lived and based on routine
cause-of-death statistics in Schleswig-Holstein, rather than individual-
level data of screened versus unscreened individuals. This has been
questioned as a potential source of poor-quality data,365,366 and the
inability to link outcomes to individual-level data precludes firm
establishment of causal relationships between increased screening and
increased survival. In addition, the lack of TNM-based stage-specific
data or subgroup data (e.g., in older men) makes it difficult to hone
in on the high-risk groups mostly likely to benefit quickly from
screening, although it presents opportunities for guiding future
studies.365,366 Finally, it is difficult, if not impossible, to deconvolute
the potential for increased public awareness during the pre-SCREEN
advertising campaign to have increased self-selection into the ranks
of those screened during the study.367 Ultimately the absolute magnitude
of reduced mortality was modest, only 19% of the general population
was screened, and a substantial number of patients were lost to follow-up
(37%).361 Nevertheless, SCREEN was performed only for 1 year and
mortality tracked for a 10-year period before and after the intervention.
It is possible that extending this effort over time would have yielded
a more significant, larger, or sustained effect.367
Nevertheless, as a result of the SCREEN study, Germany adopted
the Screen Cancer Screening program for all adults older than age
35, who since 2008 may undergo TBSE every 2 years.361,367 This
nationwide screening effort has not shown a sustained decrease in
melanoma mortality, and although direct comparisons have been made
to the initial SCREEN effort, there are important differences, including
older age of eligibility, lack of tracking of referral patterns, and lack
of a public information campaign.367,368 The SCREEN study was
monumental in its scope, longevity, and cost and generated considerable
discussion in how such efforts should be studied in the future, especially
with regard to who should be screened, evidentiary standards of risk
and benefits, and economic impact.
Patient Populations
Even within the SCREEN study, it is clear that women and younger
individuals participated the most. This reduced men as a major source
of higher-risk disease, because there is good evidence that survival of
men with melanoma, even when controlled for stage, is significantly
inferior to that of women. This is thought to be partially due to less
favorable clinicopathologic factors and older age at diagnosis, suggesting
that there may be a particularly positive impact of screening on men.62,369
A comparison between TBSE and lesion-directed skin (LDS)
examination conducted in two areas of Belgium suggested that detection
rates for skin cancer were similar (2.3% for TBSE versus 3.2% for
LDS examination), although TBSE identified more lesions overall.370
The authors concluded that LDS examination might be a more practical
way to increase skin cancer surveillance without burdening a system
with a population-based effort.
Another model for this might be patterned on what has been
recently reported in Australia, in which a high-risk melanoma clinic
was formed for biannual surveillance of patients with a personal or
strong family history of melanoma, including those with high-penetrance
germline mutations. This study identified significant economic and
quality-adjusted life-year (QALY) benefit as compared with similar
patients receiving standard care in the community, but did not assess
survival.371
Evidentiary Standards and End Points Other
Than Survival
There are several reasons why cancer screening may not translate into
a meaningful increase in overall survival, although there is evidence
that diagnoses of melanoma at earlier stages confers improved cancer-free
 Screening and Early Detection  •  CHAPTER 23 397

survival.372 It has also been argued that improved survival is not the
only meaningful end point of screening. Screening may decrease
morbidity by virtue of permitting less extensive management (e.g.,
avoiding sentinel lymph node biopsy). Screening also may have collateral
benefits of increased education and awareness about avoidable risk
factors among patients and their family members.372
The costs of screening for melanoma and economic benefits of
treating earlier disease are not often studied. Although the Belgian
experience with LDS examination had calculated a cost of $8000 per
melanoma diagnosis,370 analysis of QALYs and years of life saved have
been conducted for melanoma screening.372 Many of these studies
have suggested that focusing on high-risk populations is more
cost-effective.372
Although major therapeutic gains have been made for advanced
disease, the economic burden of targeted and immune-therapies is
likely to increase enormously, making it impractical to ignore potential
savings of screening versus therapy, especially since these burdens can
be devastating for patients.
Overdiagnosis and Harms of Screening
The major harms cited by the panel were overdiagnosis and the risk
of poor cosmetic outcomes, although the latter issue is poorly studied.
The trend of increasing melanoma incidence has been reported to be
matched by the increase in biopsy rates. This trend was interpreted
as due to overdiagnosis by the Task Force, which then raised the
concern that a screening effort would exacerbate this problem.357 Factors
such as pressure to detect lesions earlier and diagnostic drift have been
suggested and may well represent nonmedical (e.g., medicolegal)
concerns that do not necessarily reflect an improved understanding
of the underlying biology of cancer. Conversely, the expected increase
in cancer incidence as a result of screening may only be temporary
because most individuals will be of average (low) risk and are unlikely
to be continuously screened at close intervals.
An additional harm that was suggested in the 2009 statement but
not in the 2016 one is the diagnosis of often nonlethal NMSCs.367
Given that cutaneous SCCs have an appreciable metastatic rate and
NMSCs account for over 3.5 million cases of cancer in 2.2 million
individuals with an annual treatment cost of over $4.8 billion in the
United States alone,373,374 the promise of ameliorating the overall burden
of disease would seem to be of benefit. Viewing the increased diagnosis
and presumably earlier treatment of these diseases as a potential harm
precludes a more nuanced perspective on what could be regarded as
a collateral benefit.
The Way Forward
The general consensus appears to be that a randomized clinical trial
for skin cancer screening is unlikely to be practical. In this regard,
the USPTF statement on skin cancer is forward-looking because
it opens the door to case-control designs that are more practical,
while acknowledging that strong evidence of benefit and harms are
lacking, including for NMSC. The people most likely to benefit from
skin cancer screening are those at high risk by virtue of familial
syndromes or, more commonly, a history of skin cancer or precancerous
lesions, and the evidence review panel for the USPSTF cited this as
an appropriate area of focus in the future.375 Although the panel
recognized, for example, that individuals with high numbers of nevi
are at elevated risk for melanoma, the way to detect these individuals
is through TBSE.
The SCREEN study, among others, demonstrated the importance
of a standardized curriculum to boost the sensitivity and specificity
of screeners, particularly nondermatologists, who perform TBSE,376
and the Belgian experience suggests that a combination of self-directed
initial screenings370 may be an effective way of balancing the high cost
of population-based screening with access to specialists. Ultimately,
screening must fit into a comprehensive cost-effective medical system,
and benefits that include decreased morbidity, increased QALYs, and
treatment savings should be considered alongside survival.
The complete reference list is available online at
ExpertConsult.com.

KEY REFERENCES
4. Iyengar NM, Gucalp A, Dannenberg AJ, Hudis
CA. Obesity and cancer mechanisms: tumor
microenvironment and inflammation. J Clin Oncol.
2016;34(35):4270–4276.
19. Gail MH, Brinton LA, Byar DP, et al. Projecting indi-
vidualized probabilities of developing breast cancer
for white females who are being examined annually.
J Natl Cancer Inst. 1989;81(24):1879–1886.
69. Imperiale TF, Ransohoff DF, Itzkowitz SH, et al.
Multitarget stool DNA testing for colorectal-cancer
screening. N Engl J Med. 2014;370(14):1287–1297.
89. Saslow D, Solomon D, Lawson HW, et al. American
Cancer Society, American Society for Colposcopy
and Cervical Pathology, and American Society for
Clinical Pathology screening guidelines for the
prevention and early detection of cervical cancer.
CA Cancer J Clin. 2012;62(3):147–172.
121. Fenton J, Weyrich M, Durbin S, Liu Y, Bang
H, Melnikow J. Prostate-Specific Antigen-Based
Screening for Prostate Cancer: A Systematic Evidence
Review for the US Preventive Services Task Force.
Rockville, MD: Agency for Healthcare Research and
Quality; 2017.
130. Andriole GL, Crawford ED, Grubb RL, et al.
Prostate cancer screening in the randomized Prostate,
Lung, Colorectal, and Ovarian Cancer Screening
Trial: mortality results after 13 years of follow-up.
J Natl Cancer Inst. 2012;104(2):125–132.
148. Tanaka M, Fernandez-del Castillo C, Adsay V, et al.
International consensus guidelines 2012 for the
management of IPMN and MCN of the pancreas.
Pancreatology. 2012;12(3):183–197.
175. El-Serag HB, Kanwal F, Davila JA, Kramer J,
Richardson P. A new laboratory-based algorithm to
predict development of hepatocellular carcinoma in
patients with hepatitis C and cirrhosis. Gastroenterol-
ogy. 2014;146(5):1249–1255.
188. Blais P, Husain N, Kramer JR, Kowalkowski M, El-
Serag H, Kanwal F. Nonalcoholic fatty liver disease
is underrecognized in the primary care setting. Am
J Gastroenterol. 2015;110(1):10–14.
217. Plummer M, Franceschi S, Vignat J, Forman D,
de Martel C. Global burden of gastric cancer
attributable to Helicobacter pylori. Int J Cancer. 2015;
136(2):487–490.
225. World Cancer Research Fund/American Institute for
Cancer Research (WCRF/AICR). Food, Nutrition,
Physical Activity, and the Prevention of Cancer: a
Global Perspective. Washington DC: AICR 2007.
231. Asaka M, Kato M, Sakamoto N. Roadmap to
eliminate gastric cancer with Helicobacter pylori
eradication and consecutive surveillance in Japan.
J Gastroenterol. 2014;49(1):1–8.
238. Ford AC, Forman D, Hunt RH, Yuan Y, Moayyedi
P. Helicobacter pylori eradication therapy to prevent
gastric cancer in healthy asymptomatic infected
individuals: systematic review and meta-analysis of
randomised controlled trials. BMJ. 2014;348:g3174.
240. Zeng M, Mao XH, Li JX, et al. Efficacy, safety, and
immunogenicity of an oral recombinant Helicobacter
pylori vaccine in children in China: a randomised,
double-blind, placebo-controlled, phase 3 trial.
Lancet. 2015;386(10002):1457–1464.
246. Vaughan TL, Fitzgerald RC. Precision prevention of
oesophageal adenocarcinoma. Nat Rev Gastroenterol
Hepatol. 2015;12(4):243–248.
248. Taylor PR, Abnet CC, Dawsey SM. Squamous
dysplasia—the precursor lesion for esophageal squa-
mous cell carcinoma. Cancer Epidemiol Biomarkers
Prev. 2013;22(4):540–552.
259. El-Serag HB, Naik AD, Duan Z, et al. Surveillance
endoscopy is associated with improved outcomes
of oesophageal adenocarcinoma detected in patients
with Barrett’s oesophagus. Gut. 2016;65(8):1252–
1260.
275. Ferlay J, Soerjomataram I, Dikshit R, et al. Cancer
incidence and mortality worldwide: sources, methods
and major patterns in GLOBOCAN 2012. Int J
Cancer. 2015;136(5):E359–E386.
280. Blot WJ, McLaughlin JK, Winn DM, et al. Smoking
and drinking in relation to oral and pharyngeal
cancer. Cancer Res. 1988;48(11):3282–3287.
284. Castellsagué X, Alemany L, Quer M, et al. HPV
involvement in head and neck cancers: compre-
hensive assessment of biomarkers in 3680 patients.
J Natl Cancer Inst. 2016;108(6):djv403.
303. Domchek SM, Friebel TM, Singer CF, et al. Associa-
tion of risk-reducing surgery in BRCA1 or BRCA2
mutation carriers with cancer risk and mortality.
JAMA. 2010;304(9):967–975.
398 Part I: Science and Clinical Oncology
322. Pinsky PF, Yu K, Kramer BS, et al. Extended mortal-
ity results for ovarian cancer screening in the PLCO
trial with median 15 years follow-up. Gynecol Oncol.
2016;143(2):270–275.
337. Renehan AG, Tyson M, Egger M, Heller RF, Zwahlen
M. Body-mass index and incidence of cancer: a
systematic review and meta-analysis of prospective
observational studies. Lancet. 2008;371(9612):
569–578.
338. Adams TD, Stroup AM, Gress RE, et al. Cancer
incidence and mortality after gastric bypass surgery.
Obesity (Silver Spring). 2009;17(4):796–802.
343. Fisher B, Costantino JP, Wickerham DL, et al.
Tamoxifen for prevention of breast cancer: report
of the National Surgical Adjuvant Breast and Bowel
Project P-1 Study. J Natl Cancer Inst. 1998;90(18):
1371–1388.
344. Vogel VG, Costantino JP, Wickerham DL, et al.
Effects of tamoxifen vs raloxifene on the risk of
developing invasive breast cancer and other disease
outcomes: the NSABP Study of Tamoxifen and
Raloxifene (STAR) P-2 trial. JAMA. 2006;295(23):
2727–2741.
361. Breitbart EW, Waldmann A, Nolte S, et al. Systematic
skin cancer screening in Northern Germany. J Am
Acad Dermatol. 2012;66(2):201–211.
366. Stang A, Jockel KH. Does skin cancer screening
save lives? A detailed analysis of mortality time
trends in Schleswig-Holstein and Germany. Cancer.
2016;122(3):432–437.
370. Hoorens I, Vossaert K, Pil L, et al. Total-body
examination vs lesion-directed skin cancer screening.
JAMA Dermatol. 2016;152(1):27–34.

REFERENCES
1. Global Burden of Disease Cancer Collaboration,
Fitzmaurice C, Allen C, et al. Global, regional, and
national cancer incidence, mortality, years of life lost,
years lived with disability, and disability-adjusted
life-years for 32 cancer groups, 1990 to 2015: a
systematic analysis for the Global Burden of Disease
Study. JAMA Oncol. 2016.
2. Levin PB, B. International Agency for Research on
Cancer. World Cancer Report, 2008. Lyon, France:
International Agency for Research on Cancer; 2008.
ISBN 978 92 832 04237.
3. Nimptsch K, Pischon T. Obesity Biomarkers, metab-
olism and risk of cancer: an epidemiological perspec-
tive. Recent Results Cancer Res. 2016;208:199–217.
4. Iyengar NM, Gucalp A, Dannenberg AJ, Hudis
CA. Obesity and cancer mechanisms: tumor
microenvironment and inflammation. J Clin Oncol.
2016;34(35):4270–4276.
5. Meerzaman D, Dunn BK, Lee M, Chen Q, Yan C,
Ross S. The promise of omics-based approaches to
cancer prevention. Semin Oncol. 2016;43(1):36–48.
6. Foulkes WD, Knoppers BM, Turnbull C. Population
genetic testing for cancer susceptibility: founder muta-
tions to genomes. Nat Rev Clin Oncol. 2016;13(1):
41–54.
7. Bombard Y, Bach PB, Offit K. Translating genomics
in cancer care. J Natl Compr Canc Netw. 2013;
11(11):1343–1353.
8. Walsh MF, Nathanson KL, Couch FJ, Offit K.
Genomic biomarkers for breast cancer risk. Adv
Exp Med Biol. 2016;882:1–32.
9. Wilson J, Jungner G. Principles and Practice of
Screening for Disease. In. Geneva, Switzerland:
World Health Organization; 1968.
10. Gill H, Wu J. Prostate specific antigen: the past,
present and future. Curr Urol. 2013;6(4):175–178.
11. Carter HB, Albertsen PC, Barry MJ, et al. Early
detection of prostate cancer: AUA Guideline. J Urol.
2013;190(2):419–426.
12. Barocas DA, Mallin K, Graves AJ, et al. Effect of
the USPSTF Grade D Recommendation against
screening for prostate cancer on incident prostate
cancer diagnoses in the United States. J Urol. 2015;
194(6):1587–1593.
13. Kearney AJ. Viewpoint: it is time to reconsider policy
for population-based mammography screening.
J Public Health Policy. 2015;36(3):259–269.
14. Lauby-Secretan B, Scoccianti C, Loomis D, et al.
Breast-cancer screening—viewpoint of the IARC
Working Group. N Engl J Med. 2015;372(24):
2353–2358.
15. Wise J. Breast screening benefits have been overstated,
Danish study finds. BMJ. 2017;356:j94.
16. Hall AE, Chowdhury S, Hallowell N, et al. Imple-
menting risk-stratified screening for common cancers:
a review of potential ethical, legal and social issues.
J Public Health (Oxf ). 2014;36(2):285–291.
17. Shieh Y, Eklund M, Madlensky L, et al. Breast cancer
screening in the precision medicine era: risk-based
screening in a population-based trial. J Natl Cancer
Inst. 2017;109(5).
18. American Cancer Society (ACS). Cancer Facts &
Figures; 2017. Atlanta, GA2017.
19. Gail MH, Brinton LA, Byar DP, et al. Projecting
individualized probabilities of developing breast
cancer for white females who are being examined
annually. J Natl Cancer Inst. 1989;81(24):1879–1886.
20. Costantino JP, Gail MH, Pee D, et al. Validation
studies for models projecting the risk of invasive
and total breast cancer incidence. J Natl Cancer
Inst. 1999;91(18):1541–1548.
21. Tice JA, Cummings SR, Smith-Bindman R,
Ichikawa L, Barlow WE, Kerlikowske K. Using
clinical factors and mammographic breast density
to estimate breast cancer risk: development and
validation of a new predictive model. Ann Intern
Med. 2008;148(5):337–347.
Screening and Early Detection  •  CHAPTER 23 398.e1
22. Tice JA, O’Meara ES, Weaver DL, Vachon C, Ballard-
Barbash R, Kerlikowske K. Benign breast disease,
mammographic breast density, and the risk of breast
cancer. J Natl Cancer Inst. 2013;105(14):1043–1049.
23. Tice JA, Miglioretti DL, Li CS, Vachon CM,
Gard CC, Kerlikowske K. Breast density and
benign breast disease: risk assessment to identify
women at high risk of breast cancer. J Clin Oncol.
2015;33(28):3137–3143.
24. Tyrer J, Duffy SW, Cuzick J. A breast cancer predic-
tion model incorporating familial and personal risk
factors. Stat Med. 2004;23(7):1111–1130.
25. Berry DA, Parmigiani G, Sanchez J, Schildkraut
J, Winer E. Probability of carrying a mutation of
breast-ovarian cancer gene BRCA1 based on family
history. J Natl Cancer Inst. 1997;89(3):227–238.
26. Parmigiani G, Berry D, Aguilar O. Determining
carrier probabilities for breast cancer-susceptibility
genes BRCA1 and BRCA2. Am J Hum Genet. 1998;
62(1):145–158.
27. Berry DA, Iversen ES, Gudbjartsson DF, et al.
BRCAPRO validation, sensitivity of genetic
testing of BRCA1/BRCA2, and prevalence of other
breast cancer susceptibility genes. J Clin Oncol.
2002;20(11):2701–2712.
28. Claus EB, Risch N, Thompson WD. Autosomal
dominant inheritance of early-onset breast cancer.
Implications for risk prediction. Cancer. 1994;73(3):
643–651.
29. Couch FJ, DeShano ML, Blackwood MA, et al.
BRCA1 mutations in women attending clinics that
evaluate the risk of breast cancer. N Engl J Med.
1997;336(20):1409–1415.
30. Antoniou AC, Pharoah PD, McMullan G, et al.
A comprehensive model for familial breast cancer
incorporating BRCA1, BRCA2 and other genes. Br
J Cancer. 2002;86(1):76–83.
31. Antoniou AC, Pharoah PP, Smith P, Easton DF.
The BOADICEA model of genetic susceptibility to
breast and ovarian cancer. Br J Cancer. 2004;91(8):
1580–1590.
32. Antoniou AC, Cunningham AP, Peto J, et al. The
BOADICEA model of genetic susceptibility to breast
and ovarian cancers: updates and extensions. Br J
Cancer. 2008;98(8):1457–1466.
33. Thomas DB, Gao DL, Self SG, et al. Random-
ized trial of breast self-examination in Shanghai:
methodology and preliminary results. J Natl Cancer
Inst. 1997;89(5):355–365.
34. Thomas DB, Gao DL, Ray RM, et al. Randomized
trial of breast self-examination in Shanghai: final
results. J Natl Cancer Inst. 2002;94(19):1445–1457.
35. Barton MB, Harris R, Fletcher SW. The rational
clinical examination. Does this patient have breast
cancer? The screening clinical breast examination:
should it be done? How? JAMA. 1999;282(13):
1270–1280.
36. National Comprehensive Cancer Network (NCCN).
The NCCN Clinical Practice Guidelines in Oncol-
ogy: Breast Cancer Screening and Diagnosis (Version
2.2016). In: National Comprehensive Cancer
Network; 2016.
37. Marmot MG, Altman DG, Cameron DA, Dewar
JA, Thompson SG, Wilcox M. The benefits and
harms of breast cancer screening: an independent
review. Br J Cancer. 2013;108(11):2205–2240.
38. Myers ER, Moorman P, Gierisch JM, et al. Benefits
and harms of breast cancer screening: a systematic
review. JAMA. 2015;314(15):1615–1634.
39. Nelson H, Cantor A, Humphrey L, et al. Screening
for Breast Cancer: A Systematic Review to Update
the 2009 U.S. Preventive Services Task Force
Recommendation. Evidence Synthesis No. 124.
Rockville, MD: Agency for Healthcare Research
and Quality;2016.
40. Oeffinger KC, Fontham ET, Etzioni R, et al. Breast
cancer screening for women at average risk: 2015
398.e1
guideline update from the American Cancer Society.
JAMA. 2015;314(15):1599–1614.
41. Siu AL, Force USPST. Screening for breast cancer:
U.S. Preventive Services Task Force recommendation
statement. Ann Intern Med. 2016;164(4):279–296.
42. Barth RJ, Gibson GR, Carney PA, Mott LA,
Becher RD, Poplack SP. Detection of breast cancer
on screening mammography allows patients to be
treated with less-toxic therapy. AJR Am J Roentgenol.
2005;184(1):324–329.
43. Zorzi M, Puliti D, Vettorazzi M, et al. Mastectomy
rates are decreasing in the era of service screening:
a population-based study in Italy (1997-2001). Br
J Cancer. 2006;95(9):1265–1268.
44. Malmgren JA, Parikh J, Atwood MK, Kaplan HG.
Impact of mammography detection on the course of
breast cancer in women aged 40-49 years. Radiology.
2012;262(3):797–806.
45. Malmgren JA, Parikh J, Atwood MK, Kaplan
HG. Improved prognosis of women aged 75 and
older with mammography-detected breast cancer.
Radiology. 2014;273(3):686–694.
46. Moss SM, Wale C, Smith R, Evans A, Cuckle H,
Duffy SW. Effect of mammographic screening
from age 40 years on breast cancer mortality in the
UK Age trial at 17 years’ follow-up: a randomised
controlled trial. Lancet Oncol. 2015;16(9):1123–
1132.
47. Miller AB, Wall C, Baines CJ, Sun P, To T, Narod
SA. Twenty five year follow-up for breast cancer
incidence and mortality of the Canadian National
Breast Screening Study: randomised screening trial.
BMJ. 2014;348:g366.
48. Broeders M, Moss S, Nyström L, et al. The impact of
mammographic screening on breast cancer mortality
in Europe: a review of observational studies. J Med
Screen. 2012;19(suppl 1):14–25.
49. Mandelblatt JS, Cronin KA, Bailey S, et al. Effects
of mammography screening under different screening
schedules: model estimates of potential benefits and
harms. Ann Intern Med. 2009;151(10):738–747.
50. The American Congress of Obstetricians and
Gynecologists (ACOG). ACOG Statement on Breast
Cancer Screening Guidelines; 2016. www.acog.org/
About-ACOG/News-Room/Statements/2016/
ACOG-Statement-on-Breast-Cancer-Screening
-Guidelines.
51. American College of Radiology. American College
of Radiology Position Statement on Screening
Mammography and Health Care Coverage. In: 2016.
52. Wilt TJ, Harris RP, Qaseem A, Physicians HVCT-
FotACo. Screening for cancer: advice for high-value
care from the American College of Physicians. Ann
Intern Med. 2015;162(10):718–725.
53. Roder D, Houssami N, Farshid G, et al. Population
screening and intensity of screening are associated
with reduced breast cancer mortality: evidence of
efficacy of mammography screening in Australia.
Breast Cancer Res Treat. 2008;108(3):409–416.
54. Mandelblatt JS, Stout NK, Schechter CB, et al.
Collaborative modeling of the benefits and harms
associated with different U.S. breast cancer screening
strategies. Ann Intern Med. 2016;164(4):215–225.
55. Arleo EK, Monticciolo DL, Monsees B, McGinty G,
Sickles EA. Persistent untreated screening-detected
breast cancer: an argument against delaying screening
or increasing the interval between screenings. J Am
Coll Radiol. 2017.
56. Ciatto S, Houssami N, Bernardi D, et al. Integration
of 3D digital mammography with tomosynthesis
for population breast-cancer screening (STORM):
a prospective comparison study. Lancet Oncol.
2013;14(7):583–589.
57. Skaane P, Bandos AI, Gullien R, et al. Comparison
of digital mammography alone and digital mam-
mography plus tomosynthesis in a population-based
screening program. Radiology. 2013;267(1):47–56.
398.e2 Part I: Science and Clinical Oncology
58. Friedewald SM, Rafferty EA, Rose SL, et al. Breast
cancer screening using tomosynthesis in combina-
tion with digital mammography. JAMA. 2014;
311(24):2499–2507.
59. Destounis S, Arieno A, Morgan R. Initial experience
with combination digital breast tomosynthesis plus
full field digital mammography or full field digital
mammography alone in the screening environment.
J Clin Imaging Sci. 2014;4:9.
60. Saslow D, Boetes C, Burke W, et al. American Cancer
Society guidelines for breast screening with MRI as
an adjunct to mammography. CA Cancer J Clin.
2007;57(2):75–89.
61. National Comprehensive Cancer Network (NCCN).
The NCCN Clinical Practice Guidelines in Oncol-
ogy: Genetic/Familial High-Risk Assessment:
Breast and Ovarian (Version 2.2017). In: National
Comprehensive Cancer Network; 2017.
62. American Cancer Society. Cancer Facts & Figures
2018. Atlanta: American Cancer Society; 2018.
63. World Health Organization, International Agency
for Research on Cancer(IARC). Globocan; 2012.
64. Freedman AN, Slattery ML, Ballard-Barbash R, et al.
Colorectal cancer risk prediction tool for white men
and women without known susceptibility. J Clin
Oncol. 2009;27(5):686–693.
65. Park Y, Freedman AN, Gail MH, et al. Validation
of a colorectal cancer risk prediction model among
white patients age 50 years and older. J Clin Oncol.
2009;27(5):694–698.
66. Imperiale TF, Yu M, Monahan PO, et al. Risk
of advanced neoplasia using the National Cancer
Institute’s Colorectal Cancer Risk Assessment Tool.
J Natl Cancer Inst. 2017;109(1).
67. Lin J, Piper M, Perdue L, et al. Screening for
Colorectal Cancer: A Systematic Review for the U.S.
Preventive Services Task Force. Evidence Synthesis
No. 135. Rockville, MD: Agency for Healthcare
Research and Quality;2016.
68. Ladabaum U, Levin Z, Mannalithara A, Brill JV,
Bundorf MK. Colorectal testing utilization and
payments in a large cohort of commercially insured
US adults. Am J Gastroenterol. 2014;109(10):
1513–1525.
69. Imperiale TF, Ransohoff DF, Itzkowitz SH, et al.
Multitarget stool DNA testing for colorectal-cancer
screening. N Engl J Med. 2014;370(14):1287–1297.
70. Zauber A, Knudsen A, Rutter CM, Lansdorp-
Vogelaar I, Kuntz KM. Evaluating the Benefits and
Harms of Colorectal Cancer Screening Strategies: A
Collaborative Modeling Approach. Rockville, MD:
Agency for Healthcare Research and Quality; 2015.
71. Inadomi JM. Screening for colorectal neoplasia. N
Engl J Med. 2017;376(2):149–156.
72. Holme Ø, Løberg M, Kalager M, et al. Effect of
flexible sigmoidoscopy screening on colorectal cancer
incidence and mortality: a randomized clinical trial.
JAMA. 2014;312(6):606–615.
73. Centers for Disease Control and Prevention(CDC).
Vital signs: colorectal cancer screening test use—
United States, 2012. MMWR Morb Mortal Wkly
Rep. 2013;62(44):881–888.
74. Nishihara R, Wu K, Lochhead P, et al. Long-term
colorectal-cancer incidence and mortality after lower
endoscopy. N Engl J Med. 2013;369(12):1095–1105.
75. Kaminski MF, Bretthauer M, Zauber AG, et al.
The NordICC Study: rationale and design of a
randomized trial on colonoscopy screening for
colorectal cancer. Endoscopy. 2012;44(7):695–702.
76. Berrington de González A, Kim KP, Knudsen
AB, et al. Radiation-related cancer risks from CT
colonography screening: a risk-benefit analysis. AJR
Am J Roentgenol. 2011;196(4):816–823.
77. Scarinci IC, Garcia FAR, Kobetz E, et al. Cervical
cancer prevention: new tools and old barriers. Cancer.
2010;116(11):2531–2542.
78. Trottier H, Franco EL. The epidemiology of
genital human papillomavirus infection. Vaccine.
2006;24(suppl 1):S1–S15.
79. Schiffman M, Castle PE, Jeronimo J, Rodriguez AC,
Wacholder S. Human papillomavirus and cervical
cancer. Lancet. 2007;370(9590):890–907.
80. Chesson HW, Dunne EF, Hariri S, Markowitz LE.
The estimated lifetime probability of acquiring
human papillomavirus in the United States. Sex
Transm Dis. 2014;41(11):660–664.
81. Wheeler CM, Hunt WC, Joste NE, Key CR, Quint
WG, Castle PE. Human papillomavirus genotype
distributions: implications for vaccination and cancer
screening in the United States. J Natl Cancer Inst.
2009;101(7):475–487.
82. Bosch FX, de Sanjosé S. Chapter 1: human
papillomavirus and cervical cancer—burden and
assessment of causality. J Natl Cancer Inst Monogr.
2003;31:3–13.
83. Vesco KK, Whitlock EP, Eder M, Burda BU, Senger
CA, Lutz K. Risk factors and other epidemiologic
considerations for cervical cancer screening: a nar-
rative review for the U.S. Preventive Services Task
Force. Ann Intern Med. 2011;155(10):698–705,
W216.
84. Exposure in utero to diethylstilbestrol and related
synthetic hormones. Association with vaginal and
cervical cancers and other abnormalities. JAMA.
1976;236(10):1107–1109.
85. National Cancer Institute(NCI). Diethylstilbes-
trol (DES) and Cancer; 2011. www.cancer.gov/
about-cancer/causes-prevention/risk/hormones/
des-fact-sheet. Accessed March, 2017.
86. Verloop J, van Leeuwen FE, Helmerhorst TJM, van
Boven HH, Rookus MA. Cancer risk in DES daugh-
ters. Cancer Causes Control. 2010;21(7):999–991007.
87. Whitlock EP, Vesco KK, Eder M, Lin JS, Senger
CA, Burda BU. Liquid-based cytology and human
papillomavirus testing to screen for cervical cancer:
a systematic review for the U.S. Preventive Services
Task Force. Ann Intern Med. 2011;155(10):687–697,
W214–W685.
88. Castle PE, Glass AG, Rush BB, et al. Clinical human
papillomavirus detection forecasts cervical cancer
risk in women over 18 years of follow-up. J Clin
Oncol. 2012;30(25):3044–3050.
89. Saslow D, Solomon D, Lawson HW, et al. American
Cancer Society, American Society for Colposcopy
and Cervical Pathology, and American Society for
Clinical Pathology screening guidelines for the
prevention and early detection of cervical cancer.
CA Cancer J Clin. 2012;62(3):147–172.
90. Moyer VA. Screening for cervical cancer: U.S.
Preventive Services Task Force recommendation
statement. Ann Intern Med. 2012;156(12):880–891.
91. Cox JT, Castle PE, Behrens CM, et al. Comparison
of cervical cancer screening strategies incorporating
different combinations of cytology, HPV testing,
and genotyping for HPV 16/18: results from
the ATHENA HPV study. Am J Obstet Gynecol.
2013;208(3):184.e1–184.e11.
92. Wright TC, Stoler MH, Behrens CM, Sharma A,
Zhang G, Wright TL. Primary cervical cancer screen-
ing with human papillomavirus: end of study results
from the ATHENA study using HPV as the first-line
screening test. Gynecol Oncol. 2015;136(2):189–197.
93. Huh WK, Ault KA, Chelmow D, et al. Use of
primary high-risk human papillomavirus testing for
cervical cancer screening: interim clinical guidance.
Obstet Gynecol. 2015;125(2):330–337.
94. Torre LA, Siegel RL, Jemal A. Lung cancer statistics.
Adv Exp Med Biol. 2016;893:1–19.
95. Chen W, Zheng R, Baade PD, et al. Cancer statistics
in China, 2015. CA Cancer J Clin. 2016;66(2):
115–132.
96. Malhotra J, Malvezzi M, Negri E, La Vecchia C,
Boffetta P. Risk factors for lung cancer worldwide.
Eur Respir J. 2016;48(3):889–902.
97. Schwartz AG, Cote ML. Epidemiology of lung
cancer. Adv Exp Med Biol. 2016;893:21–41.
98. Guan WJ, Zheng XY, Chung KF, Zhong NS. Impact
of air pollution on the burden of chronic respiratory
diseases in China: time for urgent action. Lancet.
2016;388(10054):1939–1951.
99. Dai J, Yang P, Cox A, Jiang G. Lung cancer and
chronic obstructive pulmonary disease: from a
clinical perspective. Oncotarget. 2017.
100. Marshall AL, Christiani DC. Genetic susceptibility
to lung cancer—light at the end of the tunnel?
Carcinogenesis. 2013;34(3):487–502.
101. Tammemagi MC. Application of risk prediction
models to lung cancer screening: a review. J Thorac
Imaging. 2015;30(2):88–100.
102. Spitz MR, Etzel CJ, Dong Q, et al. An expanded
risk prediction model for lung cancer. Cancer Prev
Res (Phila). 2008;1(4):250–254.
103. Marcus MW, Chen Y, Raji OY, Duffy SW, Field
JK. LLPi: Liverpool Lung Project Risk Prediction
Model for Lung Cancer Incidence. Cancer Prev Res
(Phila). 2015;8(6):570–575.
104. Sherratt FC, Marcus MW, Robinson J, Field
JK. Utilizing lung cancer risk prediction models
to promote smoking cessation: two randomized
controlled trials. Am J Health Promot. 2016.
105. Tammemagi MC, Church TR, Hocking WG,
et al. Evaluation of the lung cancer risks at which
to screen ever- and never-smokers: screening rules
applied to the PLCO and NLST cohorts. PLoS
Med. 2014;11(12):e1001764.
106. Tammemagi CM, Pinsky PF, Caporaso NE, et al.
Lung cancer risk prediction: prostate, lung, colorectal
and ovarian cancer screening trial models and valida-
tion. J Natl Cancer Inst. 2011;103(13):1058–1068.
107. Muller DC, Johansson M, Brennan P. Lung
cancer risk prediction model incorporating lung
function: development and validation in the UK
Biobank Prospective Cohort Study. J Clin Oncol.
2017;JCO2016692467.
108. Timofeeva MN, McKay JD, Smith GD, et al. Genetic
polymorphisms in 15q25 and 19q13 loci, cotinine
levels, and risk of lung cancer in EPIC. Cancer
Epidemiol Biomarkers Prev. 2011;20(10):2250–2261.
109. Sin DD, Tammemagi CM, Lam S, et al. Pro-
surfactant protein B as a biomarker for lung cancer
prediction. J Clin Oncol. 2013;31(36):4536–4543.
110. Wikoff WR, Hanash S, DeFelice B, et al. Diacetyl-
spermine is a novel prediagnostic serum biomarker
for non-small-cell lung cancer and has additive
performance with pro-surfactant protein B. J Clin
Oncol. 2015;33(33):3880–3886.
111. Facts ACSC, Figures ACS, Atlanta. American Cancer
Society, Atlanta (2015). American Cancer Society;
2015.
112. Boucot KR, Weiss W. Is curable lung cancer detected
by semiannual screening? JAMA. 1973;224(10):
1361–1365.
113. Marcus PM, Bergstralh EJ, Fagerstrom RM, et al.
Lung cancer mortality in the Mayo Lung Project:
impact of extended follow-up. J Natl Cancer Inst.
2000;92(16):1308–1316.
114. Henschke CI, Yankelevitz DF, Libby DM, et al.
Early lung cancer action project: annual screening
using single-slice helical CT. Ann N Y Acad Sci.
2001;952:124–134.
115. Hillman BJ, ACRIN. Economic, legal, and ethical
rationales for the ACRIN national lung screening
trial of CT screening for lung cancer. Acad Radiol.
2003;10(3):349–350.
116. Henschke CI, Yip R, Yankelevitz DF, Smith JP.
International Early Lung Cancer Action Program
I. Definition of a positive test result in computed
tomography screening for lung cancer: a cohort
study. Ann Intern Med. 2013;158(4):246–252.
117. Sui X, Meinel FG, Song W, et al. Detection and size
measurements of pulmonary nodules in ultra-low-
dose CT with iterative reconstruction compared to
low dose CT. Eur J Radiol. 2016;85(3):564–570.
118. Nagatani Y, Takahashi M, Murata K, et al. Lung
nodule detection performance in five observers on
computed tomography (CT) with adaptive iterative
dose reduction using three-dimensional processing

(AIDR 3D) in a Japanese multicenter study: com-
parison between ultra-low-dose CT and low-dose
CT by receiver-operating characteristic analysis. Eur
J Radiol. 2015;84(7):1401–1412.
119. Manos D, Seely JM, Taylor J, Borgaonkar J, Roberts
HC, Mayo JR. The Lung Reporting and Data System
(LU-RADS): a proposal for computed tomography
screening. Can Assoc Radiol J. 2014;65(2):121–134.
120. National Cancer Institute (NCI). SEER Cancer
Statistics Fact Sheet: Prostate Cancer. Accessed
April 25, 2017.
121. Fenton J, Weyrich M, Durbin S, Liu Y, Bang
H, Melnikow J. Prostate-Specific Antigen-Based
Screening for Prostate Cancer: A Systematic Evidence
Review for the U.S. Preventive Services Task Force.
Rockville, MD: Agency for Healthcare Research and
Quality; 2017.
122. Louie KS, Seigneurin A, Cathcart P, Sasieni P. Do
prostate cancer risk models improve the predictive
accuracy of PSA screening? A meta-analysis. Ann
Oncol. 2015;26(5):848–864.
123. Ankerst DP, Hoefler J, Bock S, et al. Prostate
Cancer Prevention Trial risk calculator 2.0 for the
prediction of low- vs high-grade prostate cancer.
Urology. 2014;83(6):1362–1367.
124. Roobol MJ, Steyerberg EW, Kranse R, et al. A
risk-based strategy improves prostate-specific
antigen-driven detection of prostate cancer. Eur
Urol. 2010;57(1):79–85.
125. Roobol MJ, van Vugt HA, Loeb S, et al. Prediction
of prostate cancer risk: the role of prostate volume
and digital rectal examination in the ERSPC risk
calculators. Eur Urol. 2012;61(3):577–583.
126. Foley RW, Maweni RM, Gorman L, et al. European
Randomised Study of Screening for Prostate Cancer
(ERSPC) risk calculators significantly outperform the
Prostate Cancer Prevention Trial (PCPT) 2.0 in the
prediction of prostate cancer: a multi-institutional
study. BJU Int. 2016;118(5):706–713.
127. Carroll PR, Parsons JK, Andriole G, et al. NCCN
guidelines insights: prostate cancer early detec-
tion, version 2.2016. J Natl Compr Canc Netw.
2016;14(5):509–519.
128. Prorok PC, Andriole GL, Bresalier RS, et al. Design
of the Prostate, Lung, Colorectal and Ovarian
(PLCO) Cancer Screening Trial. Control Clin Trials.
2000;21(suppl 6):273S–309S.
129. Andriole GL, Crawford ED, Grubb RL, et al. Mortal-
ity results from a randomized prostate-cancer screen-
ing trial. N Engl J Med. 2009;360(13):1310–1319.
130. Andriole GL, Crawford ED, Grubb RL, et al.
Prostate cancer screening in the randomized Prostate,
Lung, Colorectal, and Ovarian Cancer Screening
Trial: mortality results after 13 years of follow-up.
J Natl Cancer Inst. 2012;104(2):125–132.
131. Shoag JE, Mittal S, Hu JC. Reevaluating PSA
testing rates in the PLCO Trial. N Engl J Med.
2016;374(18):1795–1796.
132. Schröder FH, Hugosson J, Roobol MJ, et al.
Screening and prostate-cancer mortality in a
randomized European study. N Engl J Med. 2009;
360(13):1320–1328.
133. Hugosson J, Carlsson S, Aus G, et al. Mortality
results from the Göteborg randomised population-
based prostate-cancer screening trial. Lancet Oncol.
2010;11(8):725–732.
134. Roobol MJ, Kranse R, Bangma CH, et al. Screening
for prostate cancer: results of the Rotterdam section
of the European Randomized Study of Screening
for Prostate Cancer. Eur Urol. 2013;64(4):530–539.
135. Luján M, Páez A, Angulo JC, et al. Prostate cancer
incidence and mortality in the Spanish section of
the European Randomized Study of Screening for
Prostate Cancer (ERSPC). Prostate Cancer Prostatic
Dis. 2014;17(2):187–191.
136. Kilpeläinen TP, Tammela TL, Malila N, et al.
Prostate cancer mortality in the Finnish Randomized
Screening Trial. J Natl Cancer Inst. 2013;105(10):
719–725.
Screening and Early Detection  •  CHAPTER 23 398.e3
137. Hamdy FC, Donovan JL, Lane JA, et al. 10-Year
outcomes after monitoring, surgery, or radio-
therapy for localized prostate cancer. N Engl J Med.
2016;375(15):1415–1424.
138. Routine aspirin or nonsteroidal anti-inflammatory
drugs for the primary prevention of colorectal cancer:
U.S. Preventive Services Task Force recommendation
statement. Ann Intern Med. 2007;146(5):361–364.
139. U.S. Preventive Services Task Force (USPSTF).
Draft Recommendation Statement: Prostate Cancer:
Screening. 2017; 2017. www.uspreventiveservices
taskforce.org/Page/Document/Recommendations
StatementDraft/prostate-cancer-screening1. Accessed
April 12, 2017.
140. Thompson IM, Ankerst DP, Chi C, et al. Operating
characteristics of prostate-specific antigen in men
with an initial PSA level of 3.0 ng/ml or lower.
JAMA. 2005;294(1):66–70.
141. Petitti D, Lin J, Burda B. Overdiagnosis in Prostate
Cancer Screening Decision Models: A Contextual
Review for the U.S. Preventive Services Task Force.
Rockville, MD: Agency for Healthcare Research and
Quality; 2017.
142. Siegel RL, Miller KD, Jemal A. Cancer statistics,
2016. CA Cancer J Clin. 2016;66(1):7–30.
143. Rahib L, Smith BD, Aizenberg R, Rosenzweig AB,
Fleshman JM, Matrisian LM. Projecting cancer
incidence and deaths to 2030: the unexpected burden
of thyroid, liver, and pancreas cancers in the United
States. Cancer Res. 2014;74(11):2913–2921.
144. Moffitt RA, Marayati R, Flate EL, et al. Virtual
microdissection identifies distinct tumor- and
stroma-specific subtypes of pancreatic ductal adeno-
carcinoma. Nat Genet. 2015;47(10):1168–1178.
145. Hruban RH, Maitra A, Schulick R, et al. Emerging
molecular biology of pancreatic cancer. Gastrointest
Cancer Res. 2008;2(suppl 4):10–15.
146. Ryan DP, Hong TS, Bardeesy N. Pancreatic adeno-
carcinoma. N Engl J Med. 2014;371(11):1039–1049.
147. Canto MI, Hruban RH, Fishman EK, et al.
Frequent detection of pancreatic lesions in
asymptomatic high-risk individuals. Gastroenterology.
2012;142(4):796–804, quiz e714–e795.
148. Tanaka M, Fernandez-del Castillo C, Adsay V, et al.
International consensus guidelines 2012 for the
management of IPMN and MCN of the pancreas.
Pancreatology. 2012;12(3):183–197.
149. Zhang XM, Mitchell DG, Dohke M, Holland
GA, Parker L. Pancreatic cysts: depiction on
single-shot fast spin-echo MR images. Radiology.
2002;223(2):547–553.
150. de Jong K, Nio CY, Hermans JJ, et al. High
prevalence of pancreatic cysts detected by screen-
ing magnetic resonance imaging examinations. Clin
Gastroenterol Hepatol. 2010;8(9):806–811.
151. Del Chiaro M, Ateeb Z, Hansson MR, et al. Survival
analysis and risk for progression of intraductal papil-
lary mucinous neoplasia of the pancreas (IPMN)
under surveillance: a single-institution experience.
Ann Surg Oncol. 2016.
152. Jais B, Rebours V, Malleo G, et al. Serous cystic
neoplasm of the pancreas: a multinational study of
2622 patients under the auspices of the International
Association of Pancreatology and European Pancre-
atic Club (European Study Group on Cystic Tumors
of the Pancreas). Gut. 2016;65(2):305–312.
153. Klein AP, Brune KA, Petersen GM, et al. Prospective
risk of pancreatic cancer in familial pancreatic cancer
kindreds. Cancer Res. 2004;64(7):2634–2638.
154. Vrieling A, Bueno-de-Mesquita HB, Boshuizen HC,
et al. Cigarette smoking, environmental tobacco
smoke exposure and pancreatic cancer risk in the
European Prospective Investigation into Cancer
and Nutrition. Int J Cancer. 2010;126(10):2394–
2403.
155. Tramacere I, Scotti L, Jenab M, et al. Alcohol drink-
ing and pancreatic cancer risk: a meta-analysis of
the dose-risk relation. Int J Cancer. 2010;126(6):
1474–1486.
398.e3
156. Stevens RJ, Roddam AW, Beral V. Pancreatic cancer
in type 1 and young-onset diabetes: systematic review
and meta-analysis. Br J Cancer. 2007;96(3):507–509.
157. Huxley R, Ansary-Moghaddam A, Berrington de
Gonzalez A, Barzi F, Woodward M. Type-II diabetes
and pancreatic cancer: a meta-analysis of 36 studies.
Br J Cancer. 2005;92(11):2076–2083.
158. Gapstur SM, Gann PH, Lowe W, Liu K, Colangelo L,
Dyer A. Abnormal glucose metabolism and pancreatic
cancer mortality. JAMA. 2000;283(19):2552–2558.
159. Talamonti MS. Screening strategies for pancreatic
cancer in high-risk patients: opportunities to make
a real impact but many questions and challenges
still ahead. JAMA Surg. 2015;150(6):518–519.
160. Poruk KE, Firpo MA, Adler DG, Mulvihill SJ.
Screening for pancreatic cancer: why, how, and
who? Ann Surg. 2013;257(1):17–26.
161. Del Chiaro M, Verbeke CS, Kartalis N, et al.
Short-term results of a magnetic resonance imaging-
based Swedish screening program for individuals
at risk for pancreatic cancer. JAMA Surg. 2015;
150(6):512–518.
162. Canto MI, Goggins M, Hruban RH, et al. Screening
for early pancreatic neoplasia in high-risk individuals:
a prospective controlled study. Clin Gastroenterol
Hepatol. 2006;4(6):766–781, quiz 665.
163. Tanaka M, Chari S, Adsay V, et al. International
consensus guidelines for management of intraductal
papillary mucinous neoplasms and mucinous cystic
neoplasms of the pancreas. Pancreatology. 2006;
6(1–2):17–32.
164. Davis B, Lowy AM. Surgical management of
hereditary pancreatic cancer. Med Clin North Am.
2000;84(3):749–759.
165. Goggins M, Canto M, Hruban R. Can we screen
high-risk individuals to detect early pancreatic
carcinoma? J Surg Oncol. 2000;74(4):243–248.
166. Langer P, Kann PH, Fendrich V, et al. Five years
of prospective screening of high-risk individuals
from families with familial pancreatic cancer. Gut.
2009;58(10):1410–1418.
167. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent
J, Jemal A. Global cancer statistics, 2012. CA Cancer
J Clin. 2015;65(2):87–108.
168. Ryerson AB, Eheman CR, Altekruse SF, et al.
Annual report to the nation on the status of cancer,
1975–2012, featuring the increasing incidence of
liver cancer. Cancer. 2016;122(9):1312–1337.
169. Altekruse SF, Henley SJ, Cucinelli JE, McGlynn
KA. Changing hepatocellular carcinoma incidence
and liver cancer mortality rates in the United States.
Am J Gastroenterol. 2014;109(4):542–553.
170. Njei B, Rotman Y, Ditah I, Lim JK. Emerging trends
in hepatocellular carcinoma incidence and mortality.
Hepatology. 2015;61(1):191–199.
171. El-Serag HB. Epidemiology of viral hepatitis
and hepatocellular carcinoma. Gastroenterology.
2012;142(6):1264–1273.e1261.
172. Beste LA, Leipertz SL, Green PK, Dominitz JA,
Ross D, Ioannou GN. Trends in burden of cirrhosis
and hepatocellular carcinoma by underlying liver
disease in US veterans, 2001-2013. Gastroenterology.
2015;149(6):1471–1482.e1475, quiz e1417–e1478.
173. Lok AS, Seeff LB, Morgan TR, et al. Incidence
of hepatocellular carcinoma and associated risk
factors in hepatitis C–related advanced liver disease.
Gastroenterology. 2009;136(1):138–148.
174. Yang JD, Kim WR, Coelho R, et al. Cirrhosis
is present in most patients with hepatitis B and
hepatocellular carcinoma. Clin Gastroenterol Hepatol.
2011;9(1):64–70.
175. El-Serag HB, Kanwal F, Davila JA, Kramer J,
Richardson P. A new laboratory-based algorithm to
predict development of hepatocellular carcinoma in
patients with hepatitis C and cirrhosis. Gastroenterol-
ogy. 2014;146(5):1249–1255.
176. Kanwal F, Kramer JR, Ilyas J, Duan Z, El-Serag HB.
HCV genotype 3 is associated with an increased risk
of cirrhosis and hepatocellular cancer in a national
398.e4 Part I: Science and Clinical Oncology
sample of U.S. Veterans with HCV. Hepatology.
2014;60(1):98–105.
177. Chang KC, Wu YY, Hung CH, et al. Clinical-guide
risk prediction of hepatocellular carcinoma develop-
ment in chronic hepatitis C patients after interferon-
based therapy. Br J Cancer. 2013;109(9):2481–2488.
178. El-Serag HB, Hampel H, Javadi F. The association
between diabetes and hepatocellular carcinoma: a
systematic review of epidemiologic evidence. Clin
Gastroenterol Hepatol. 2006;4(3):369–380.
179. Wang P, Kang D, Cao W, Wang Y, Liu Z. Diabetes
mellitus and risk of hepatocellular carcinoma: a
systematic review and meta-analysis. Diabetes Metab
Res Rev. 2012;28(2):109–122.
180. Saunders D, Seidel D, Allison M, Lyratzopoulos G.
Systematic review: the association between obesity
and hepatocellular carcinoma—epidemiological
evidence. Aliment Pharmacol Ther. 2010;31(10):
1051–1063.
181. Schlesinger S, Aleksandrova K, Pischon T, et al.
Abdominal obesity, weight gain during adulthood
and risk of liver and biliary tract cancer in a European
cohort. Int J Cancer. 2013;132(3):645–657.
182. Hassan MM, Abdel-Wahab R, Kaseb A, et al.
Obesity early in adulthood increases risk but does
not affect outcomes of hepatocellular carcinoma.
Gastroenterology. 2015;149(1):119–129.
183. White DL, Kanwal F, El-Serag HB. Association
between nonalcoholic fatty liver disease and risk
for hepatocellular cancer, based on systematic review.
Clin Gastroenterol Hepatol. 2012;10(12):1342–1359.
e1342.
184. Romeo S, Kozlitina J, Xing C, et al. Genetic variation
in PNPLA3 confers susceptibility to nonalcoholic
fatty liver disease. Nat Genet. 2008;40(12):1461–
1465.
185. Hassan MM, Kaseb A, Etzel CJ, et al. Genetic
variation in the PNPLA3 gene and hepatocellular
carcinoma in USA: risk and prognosis prediction.
Mol Carcinog. 2013;52(suppl 1):E139–E147.
186. Bruix J, Sherman M. Management of hepatocellular
carcinoma: an update. Hepatology. 2011;53(3):1020–
1022.
187. Singal AG, Yopp AC, Gupta S, et al. Failure rates
in the hepatocellular carcinoma surveillance process.
Cancer Prev Res (Phila). 2012;5(9):1124–1130.
188. Blais P, Husain N, Kramer JR, Kowalkowski M, El-
Serag H, Kanwal F. Nonalcoholic fatty liver disease
is underrecognized in the primary care setting. Am
J Gastroenterol. 2015;110(1):10–14.
189. Mourad A, Deuffic-Burban S, Ganne-Carrie
N, et al. Hepatocellular carcinoma screening in
patients with compensated hepatitis C virus (HCV)-
related cirrhosis aware of their HCV status improves
survival: a modeling approach. Hepatology. 2014;
59(4):1471–1481.
190. Zhang BH, Yang BH, Tang ZY. Randomized con-
trolled trial of screening for hepatocellular carcinoma.
J Cancer Res Clin Oncol. 2004;130(7):417–422.
191. Finberg HJ. Whither (wither?) the ultrasound special-
ist? J Ultrasound Med. 2004;23(12):1543–1547.
192. Singal AG, Pillai A, Tiro J. Early detection, cura-
tive treatment, and survival rates for hepatocellular
carcinoma surveillance in patients with cirrhosis: a
meta-analysis. PLoS Med. 2014;11(4):e1001624.
193. Dalton-Fitzgerald E, Tiro J, Kandunoori P, Halm EA,
Yopp A, Singal AG. Practice patterns and attitudes
of primary care providers and barriers to surveillance
of hepatocellular carcinoma in patients with cir-
rhosis. Clin Gastroenterol Hepatol. 2015;13(4):791–
798.e791.
194. Poustchi H, Farrell GC, Strasser SI, Lee AU,
McCaughan GW, George J. Feasibility of con-
ducting a randomized control trial for liver cancer
screening: is a randomized controlled trial for liver
cancer screening feasible or still needed? Hepatology.
2011;54(6):1998–2004.
195. Singal A, Volk ML, Waljee A, et al. Meta-analysis:
surveillance with ultrasound for early-stage
hepatocellular carcinoma in patients with cirrhosis.
Aliment Pharmacol Ther. 2009;30(1):37–47.
196. Santi V, Trevisani F, Gramenzi A, et al. Semiannual
surveillance is superior to annual surveillance for
the detection of early hepatocellular carcinoma and
patient survival. J Hepatol. 2010;53(2):291–297.
197. Trinchet JC, Chaffaut C, Bourcier V, et al. Ultraso-
nographic surveillance of hepatocellular carcinoma
in cirrhosis: a randomized trial comparing 3- and
6-month periodicities. Hepatology. 2011;54(6):
1987–1997.
198. Andersson KL, Salomon JA, Goldie SJ, Chung RT.
Cost effectiveness of alternative surveillance strate-
gies for hepatocellular carcinoma in patients with
cirrhosis. Clin Gastroenterol Hepatol. 2008;6(12):
1418–1424.
199. Arguedas MR, Chen VK, Eloubeidi MA, Fallon MB.
Screening for hepatocellular carcinoma in patients
with hepatitis C cirrhosis: a cost-utility analysis. Am
J Gastroenterol. 2003;98(3):679–690.
200. Patel D, Terrault NA, Yao FY, Bass NM, Ladabaum
U. Cost-effectiveness of hepatocellular carcinoma
surveillance in patients with hepatitis C virus–related
cirrhosis. Clin Gastroenterol Hepatol. 2005;3(1):
75–84.
201. Thompson Coon J, Rogers G, Hewson P, et al.
Surveillance of cirrhosis for hepatocellular carcinoma:
a cost-utility analysis. Br J Cancer. 2008;98(7):
1166–1175.
202. Pocha C, Dieperink E, McMaken KA, Knott A,
Thuras P, Ho SB. Surveillance for hepatocellular
cancer with ultrasonography vs. computed tomogra-
phy—a randomised study. Aliment Pharmacol Ther.
2013;38(3):303–312.
203. Kanwal F, El-Serag HB, Ross D. Surveillance for hepa-
tocellular carcinoma: can we focus on the mission?
Clin Gastroenterol Hepatol. 2015;13(4):805–807.
204. Singal AG, Volk ML, Rakoski MO, et al. Patient
involvement in healthcare is associated with higher
rates of surveillance for hepatocellular carcinoma.
J Clin Gastroenterol. 2011;45(8):727–732.
205. El-Serag HB, Kanwal F. Epidemiology of hepato-
cellular carcinoma in the United States: where are
we? Where do we go? Hepatology. 2014;60(5):
1767–1775.
206. Marrero JA, Feng Z, Wang Y, et al. Alpha-fetoprotein,
des-gamma carboxyprothrombin, and lectin-bound
alpha-fetoprotein in early hepatocellular carcinoma.
Gastroenterology. 2009;137(1):110–118.
207. Ferlay J, Soerjomataram I, Ervik M, et al. GLOBO-
CAN 2012 v1.1, Cancer Incidence and Mortality
Worldwide: IARC CancerBase No. 11. Lyon, France:
International Agency for Research on Cancer; 2014.
208. Bosman FT, World Health Organization, International
Agency for Research on Cancer. WHO Classification
of Tumours of the Digestive System. Lyon: IARC Press;
2010.
209. Correa P. Gastric cancer: overview. Gastroenterol Clin
North Am. 2013;42(2):211–217.
210. Tsukanov VV, Butorin NN, Maady AS, et al. Heli-
cobacter pylori infection, intestinal metaplasia, and
gastric cancer risk in Eastern Siberia. Helicobacter.
2011;16(2):107–112.
211. Lui FH, Tuan B, Swenson SL, Wong RJ. Ethnic
disparities in gastric cancer incidence and survival in
the USA: an updated analysis of 1992–2009 SEER
data. Dig Dis Sci. 2014;59(12):3027–3034.
212. Anderson WF, Camargo MC, Fraumeni JF Jr, Correa
P, Rosenberg PS, Rabkin CS. Age-specific trends in
incidence of noncardia gastric cancer in US adults.
JAMA. 2010;303(17):1723–1728.
213. Arnold M, Karim-Kos HE, Coebergh JW, et al.
Recent trends in incidence of five common cancers
in 26 European countries since 1988: analysis of
the European Cancer Observatory. Eur J Cancer.
2015;51(9):1164–1187.
214. Bertuccio P, Chatenoud L, Levi F, et al. Recent
patterns in gastric cancer: a global overview. Int J
Cancer. 2009;125(3):666–673.
215. Gonzalez CA, Agudo A. Carcinogenesis, prevention
and early detection of gastric cancer: where we
are and where we should go. Int J Cancer. 2012;
130(4):745–753.
216. Lochhead P, El-Omar EM. Helicobacter pylori
infection and gastric cancer. Best Pract Res Clin
Gastroenterol. 2007;21(2):281–297.
217. Plummer M, Franceschi S, Vignat J, Forman D,
de Martel C. Global burden of gastric cancer
attributable to Helicobacter pylori. Int J Cancer. 2015;
136(2):487–490.
218. International Agency for Research on Cancer (IARC).
Biological agents. Volume 100 B. A review of human
carcinogens. IARC Monogr Eval Carcinog Risks Hum.
2012;100(Pt B):1–441.
219. Cover TL, Blaser MJ. Helicobacter pylori in health and
disease. Gastroenterology. 2009;136(6):1863–1873.
220. Peleteiro B, Bastos A, Ferro A, Lunet N. Prevalence of
Helicobacter pylori infection worldwide: a systematic
review of studies with national coverage. Dig Dis
Sci. 2014;59(8):1698–1709.
221. Camargo MC, Murphy G, Koriyama C, et al.
Determinants of Epstein-Barr virus–positive gastric
cancer: an international pooled analysis. Br J Cancer.
2011;105(1):38–43.
222. Capelle LG, Van Grieken NC, Lingsma HF, et al.
Risk and epidemiological time trends of gastric
cancer in Lynch syndrome carriers in the Nether-
lands. Gastroenterology. 2010;138(2):487–492.
223. Oliveira C, Seruca R, Carneiro F. Genetics, pathology,
and clinics of familial gastric cancer. Int J Surg Pathol.
2006;14(1):21–33.
224. Karimi P, Islami F, Anandasabapathy S, Freedman
ND, Kamangar F. Gastric cancer: descriptive epi-
demiology, risk factors, screening, and prevention.
Cancer Epidemiol Biomarkers Prev. 2014;23(5):
700–713.
225. World Cancer Research Fund/American Institute for
Cancer Research (WCRF/AICR). Food, Nutrition,
Physical Activity, and the Prevention of Cancer: a
Global Perspective. Washington DC: AICR 2007.
226. Toyoda T, Tsukamoto T, Hirano N, et al. Synergistic
upregulation of inducible nitric oxide synthase and
cyclooxygenase-2 in gastric mucosa of Mongolian
gerbils by a high-salt diet and Helicobacter pylori
infection. Histol Histopathol. 2008;23(5):593–599.
227. Loh JT, Torres VJ, Cover TL. Regulation of Heli-
cobacter pylori cagA expression in response to salt.
Cancer Res. 2007;67(10):4709–4715.
228. Leung WK, Wu MS, Kakugawa Y, et al. Screening for
gastric cancer in Asia: current evidence and practice.
Lancet Oncol. 2008;9(3):279–287.
229. Choi KS, Suh M. Screening for gastric cancer: the
usefulness of endoscopy. Clin Endosc. 2014;47(6):
490–496.
230. Asaka M. A new approach for elimination of gastric
cancer deaths in Japan. Int J Cancer. 2013;132(6):
1272–1276.
231. Asaka M, Kato M, Sakamoto N. Roadmap to
eliminate gastric cancer with Helicobacter pylori
eradication and consecutive surveillance in Japan.
J Gastroenterol. 2014;49(1):1–8.
232. Areia M, Carvalho R, Cadime AT, Rocha Goncalves
F, Dinis-Ribeiro M. Screening for gastric cancer and
surveillance of premalignant lesions: a systematic
review of cost-effectiveness studies. Helicobacter.
2013;18(5):325–337.
233. Choi KS, Jun JK, Park EC, et al. Performance
of different gastric cancer screening methods in
Korea: a population-based study. PLoS ONE. 2012;
7(11):e50041.
234. Tashiro A, Sano M, Kinameri K, Fujita K, Takeuchi
Y. Comparing mass screening techniques for gastric
cancer in Japan. World J Gastroenterol. 2006;12(30):
4873–4874.
235. de Vries AC, van Grieken NC, Looman CW, et al.
Gastric cancer risk in patients with premalignant
gastric lesions: a nationwide cohort study in the
Netherlands. Gastroenterology. 2008;134(4):945–952.

236. You WC, Li JY, Blot WJ, et al. Evolution of
precancerous lesions in a rural Chinese popula-
tion at high risk of gastric cancer. Int J Cancer.
1999;83(5):615–619.
237. Choi IJ. Endoscopic gastric cancer screening and sur-
veillance in high-risk groups. Clin Endosc. 2014;47(6):
497–503.
238. Ford AC, Forman D, Hunt RH, Yuan Y, Moayyedi
P. Helicobacter pylori eradication therapy to prevent
gastric cancer in healthy asymptomatic infected
individuals: systematic review and meta-analysis of
randomised controlled trials. BMJ. 2014;348:g3174.
239. Helicobacter pylori Eradication as a Strategy for
Preventing Gastric Cancer. Lyon, France: International
Agency for Research on Cancer (IARC Working
Group Reports, No. 8). 2014.
240. Zeng M, Mao XH, Li JX, et al. Efficacy, safety, and
immunogenicity of an oral recombinant Helicobacter
pylori vaccine in children in China: a randomised,
double-blind, placebo-controlled, phase 3 trial.
Lancet. 2015;386(10002):1457–1464.
241. Pan KF, Zhang L, Gerhard M, et al. A large
randomised controlled intervention trial to prevent
gastric cancer by eradication of Helicobacter pylori in
Linqu County, China: baseline results and factors
affecting the eradication. Gut. 2016;65(1):9–18.
242. Thrift AP. The epidemic of oesophageal carcinoma:
where are we now? Cancer Epidemiol. 2016;41:88–95.
243. Pohl H, Welch HG. The role of overdiagnosis and
reclassification in the marked increase of esophageal
adenocarcinoma incidence. J Natl Cancer Inst. 2005;
97(2):142–146.
244. Thrift AP, Whiteman DC. The incidence of esopha-
geal adenocarcinoma continues to rise: analysis of
period and birth cohort effects on recent trends.
Ann Oncol. 2012;23(12):3155–3162.
245. Edgren G, Adami HO, Weiderpass E, Nyren O. A
global assessment of the oesophageal adenocarcinoma
epidemic. Gut. 2013;62(10):1406–1414.
246. Vaughan TL, Fitzgerald RC. Precision prevention of
oesophageal adenocarcinoma. Nat Rev Gastroenterol
Hepatol. 2015;12(4):243–248.
247. Pandeya N, Olsen CM, Whiteman DC. Sex dif-
ferences in the proportion of esophageal squamous
cell carcinoma cases attributable to tobacco smoking
and alcohol consumption. Cancer Epidemiol. 2013;
37(5):579–584.
248. Taylor PR, Abnet CC, Dawsey SM. Squamous dys-
plasia—the precursor lesion for esophageal squamous
cell carcinoma. Cancer Epidemiol Biomarkers Prev.
2013;22(4):540–552.
249. Engel LS, Chow WH, Vaughan TL, et al. Population
attributable risks of esophageal and gastric cancers.
J Natl Cancer Inst. 2003;95(18):1404–1413.
250. Olsen CM, Pandeya N, Green AC, Webb PM,
Whiteman DC. Population attributable fractions
of adenocarcinoma of the esophagus and gastro-
esophageal junction. Am J Epidemiol. 2011;174(5):
582–590.
251. Ronkainen J, Aro P, Storskrubb T, et al. Prevalence
of Barrett’s esophagus in the general population:
an endoscopic study. Gastroenterology. 2005;129(6):
1825–1831.
252. Hvid-Jensen F, Pedersen L, Drewes AM, Sorensen
HT, Funch-Jensen P. Incidence of adenocarcinoma
among patients with Barrett’s esophagus. N Engl J
Med. 2011;365(15):1375–1383.
253. Desai TK, Krishnan K, Samala N, et al. The
incidence of oesophageal adenocarcinoma in non-
dysplastic Barrett’s oesophagus: a meta-analysis. Gut.
2012;61(7):970–976.
254. Rastogi A, Puli S, El-Serag HB, Bansal A, Wani S,
Sharma P. Incidence of esophageal adenocarcinoma
in patients with Barrett’s esophagus and high-grade
dysplasia: a meta-analysis. Gastrointest Endosc. 2008;
67(3):394–398.
255. di Pietro M, Chan D, Fitzgerald RC, Wang KK.
Screening for Barrett’s esophagus. Gastroenterology.
2015;148(5):912–923.
Screening and Early Detection  •  CHAPTER 23 398.e5
256. Spechler SJ, Sharma P, Souza RF, Inadomi JM,
Shaheen NJ. American Gastroenterological Associa-
tion medical position statement on the management
of Barrett’s esophagus. Gastroenterology. 2011;140(3):
1084–1091.
257. Shaheen NJ, Falk GW, Iyer PG, Gerson LB.
ACG Clinical Guideline: Diagnosis and manage-
ment of Barrett’s esophagus. Am J Gastroenterol.
2016;111(1):30–50.
258. Bhat SK, McManus DT, Coleman HG, et al.
Oesophageal adenocarcinoma and prior diagnosis
of Barrett’s oesophagus: a population-based study.
Gut. 2015;64:20–25.
259. El-Serag HB, Naik AD, Duan Z, et al. Surveillance
endoscopy is associated with improved outcomes of
oesophageal adenocarcinoma detected in patients with
Barrett’s oesophagus. Gut. 2016;65(8):1252–1260.
260. Hur C, Choi SE, Rubenstein JH, et al. The cost
effectiveness of radiofrequency ablation for Barrett’s
esophagus. Gastroenterology. 2012;143(3):567–575.
261. Gordon LG, Mayne GC, Hirst NG, Bright T,
Whiteman DC, Watson DI. Cost-effectiveness of
endoscopic surveillance of non-dysplastic Barrett’s
esophagus. Gastrointest Endosc. 2014;79(2):242–
256.e246.
262. Rees JR, Lao-Sirieix P, Wong A, Fitzgerald RC. Treat-
ment for Barrett’s oesophagus. Cochrane Database
Syst Rev. 2010;(1):Cd004060.
263. Gupta M, Iyer PG, Lutzke L, et al. Recurrence of
esophageal intestinal metaplasia after endoscopic
mucosal resection and radiofrequency ablation
of Barrett’s esophagus: results from a US Multicenter
Consortium. Gastroenterology. 2013;145(1):79–
86.e71.
264. Orman ES, Li N, Shaheen NJ. Efficacy and
durability of radiofrequency ablation for Barrett’s
Esophagus: systematic review and meta-analysis. Clin
Gastroenterol Hepatol. 2013;11(10):1245–1255.
265. El-Serag HB, Graham DY. Routine polypectomy for
colorectal polyps and ablation for Barrett’s esopha-
gus are intellectually the same. Gastroenterology.
2011;140(2):386–388.
266. Sorbi D, Gostout CJ, Henry J, Lindor KD. Unsedated
small-caliber esophagogastroduodenoscopy (EGD)
versus conventional EGD: a comparative study.
Gastroenterology. 1999;117(6):1301–1307.
267. Saeian K, Staff DM, Vasilopoulos S, et al. Unsedated
transnasal endoscopy accurately detects Barrett’s
metaplasia and dysplasia. Gastrointest Endosc. 2002;
56(4):472–478.
268. Shariff MK, Bird-Lieberman EL, O’Donovan
M, et al. Randomized crossover study comparing
efficacy of transnasal endoscopy with that of standard
endoscopy to detect Barrett’s esophagus. Gastrointest
Endosc. 2012;75(5):954–961.
269. Peery AF, Hoppo T, Garman KS, et al. Feasibility,
safety, acceptability, and yield of office-based,
screening transnasal esophagoscopy (with video).
Gastrointest Endosc. 2012;75(5):945–953.e942.
270. Lao-Sirieix P, Rous B, O’Donovan M, Hardwick
RH, Debiram I, Fitzgerald RC. Non-endoscopic
immunocytological screening test for Barrett’s
oesophagus. Gut. 2007;56(7):1033–1034.
271. Kadri SR, Lao-Sirieix P, O’Donovan M, et al.
Acceptability and accuracy of a non-endoscopic
screening test for Barrett’s oesophagus in primary
care: cohort study. BMJ. 2010;341:c4372.
272. Ross-Innes CS, Debiram-Beecham I, O’Donovan
M, et al. Evaluation of a minimally invasive
cell sampling device coupled with assessment of
trefoil factor 3 expression for diagnosing Barrett’s
esophagus: a multi-center case-control study. PLoS
Med. 2015;12(1):e1001780.
273. Lagergren J, Bergstrom R, Lindgren A, Nyren O.
Symptomatic gastroesophageal reflux as a risk factor
for esophageal adenocarcinoma. N Engl J Med.
1999;340(11):825–831.
274. Rubenstein JH, Taylor JB. Meta-analysis: the
association of oesophageal adenocarcinoma with
398.e5
symptoms of gastro-oesophageal reflux. Aliment
Pharmacol Ther. 2010;32(10):1222–1227.
275. Ferlay J, Soerjomataram I, Dikshit R, et al. Cancer
incidence and mortality worldwide: sources, methods
and major patterns in GLOBOCAN 2012. Int J
Cancer. 2015;136(5):E359–E386.
276. National Cancer Institute (NCI). SEER Stat Fact
Sheets: Oral Cavity and Pharynx Cancer. Bethesda,
MD SEER 18 2007-2013.
277. U.S. Department of Health and Human Services
(DHHS). The Health Consequences of Smoking:
A Report of the Surgeon General. Atlanta, GA:
Department of Health and Human Services, Centers
for Disease Control and Prevention, National Center
for Chronic Disease Prevention and Health Promo-
tion, Office on Smoking and Health; 2004.
278. Goldstein BY, Chang SC, Hashibe M, La Vecchia
C, Zhang ZF. Alcohol consumption and cancers of
the oral cavity and pharynx from 1988 to 2009: an
update. Eur J Cancer Prev. 2010;19(6):431–465.
279. Huber MA, Tantiwongkosi B. Oral and oropharyngeal
cancer. Med Clin North Am. 2014;98(6):1299–1321.
280. Blot WJ, McLaughlin JK, Winn DM, et al. Smoking
and drinking in relation to oral and pharyngeal
cancer. Cancer Res. 1988;48(11):3282–3287.
281. Guha N, Warnakulasuriya S, Vlaanderen J, Straif
K. Betel quid chewing and the risk of oral and
oropharyngeal cancers: a meta-analysis with implica-
tions for cancer control. Int J Cancer. 2014;135(6):
1433–1443.
282. Marron M, Boffetta P, Zhang ZF, et al. Cessation of
alcohol drinking, tobacco smoking and the reversal
of head and neck cancer risk. Int J Epidemiol.
2010;39(1):182–196.
283. Wu YH, Yen CJ, Hsiao JR, et al. A Comprehensive
analysis on the association between tobacco-free betel
quid and risk of head and neck cancer in Taiwanese
men. PLoS ONE. 2016;11(10):e0164937.
284. Castellsagué X, Alemany L, Quer M, et al. HPV
involvement in head and neck cancers: compre-
hensive assessment of biomarkers in 3680 patients.
J Natl Cancer Inst. 2016;108(6):djv403.
285. de Martel C, Plummer M, Vignat J, Franceschi S.
Worldwide burden of cancer attributable to HPV
by site, country and HPV type. Int J Cancer. 2017.
286. Ndiaye C, Mena M, Alemany L, et al. HPV DNA,
E6/E7 mRNA, and p16INK4a detection in head and
neck cancers: a systematic review and meta-analysis.
Lancet Oncol. 2014;15(12):1319–1331.
287. Gillison ML, Chaturvedi AK, Anderson WF, Fakhry
C. Epidemiology of human papillomavirus–positive
head and neck squamous cell carcinoma. J Clin
Oncol. 2015;33(29):3235–3242.
288. Heck JE, Berthiller J, Vaccarella S, et al. Sexual
behaviours and the risk of head and neck cancers:
a pooled analysis in the International Head and Neck
Cancer Epidemiology (INHANCE) consortium. Int
J Epidemiol. 2010;39(1):166–181.
289. Herrero R, Quint W, Hildesheim A, et al. Reduced
prevalence of oral human papillomavirus (HPV)
4 years after bivalent HPV vaccination in a ran-
domized clinical trial in Costa Rica. PLoS ONE.
2013;8(7):e68329.
290. Olson C, Burda B, Beil T, Whitlock E. Screening for
Oral Cancer: A Targeted Evidence Update for the
U.S. Preventive Services Task Force. In. Rockville,
MD: Agency for Healthcare Research and Quality;
2013.
291. Brocklehurst P, Kujan O, O’Malley L, Ogden G,
Shepherd S, Glenny A. Screening programmes for
the early detection and prevention of oral cancer
(Review). Cochrane Database Syst Rev. 2013;(11).
292. Sankaranarayanan R, Ramadas K, Thomas G, et al.
Effect of screening on oral cancer mortality in Kerala,
India: a cluster-randomised controlled trial. Lancet.
2005;365(9475):1927–1933.
293. Walsh T, Liu J, Brocklehurst P, et al. Clinical assess-
ment to screen for the detection of oral cavity cancer
and potentially malignant disorders in apparently
398.e6 Part I: Science and Clinical Oncology
healthy adults. In. Cochrane Database of Systematic
Reviews, The Cochrane Collaboration: John Wiley
& Sons, Ltd.; 2013.
294. Macey R, Walsh T, Brocklehurst P, et al. Diagnostic
tests for oral cancer and potentially malignant
disorders in patients presenting with clinically
evident lesions. In. Cochrane Database of Systematic
Reviews, The Cochrane Collaboration: John Wiley
& Sons, Ltd.; 2015.
295. Lingen MW, et al. Evidence-based clinical practice
guideline for the evaluation of potentially malignant
disorders in the oral cavity: a report of the American
Dental Association. J Am Dent Assoc. 2017;148:
712–727.
296. Moyer VA, Force USPST. Screening for oral cancer:
U.S. Preventive Services Task Force recommendation
statement. Ann Intern Med. 2014;160(1):55–60.
297. Mirghani H, Jung AC, Fakhry C. Primary, secondary
and tertiary prevention of human papillomavirus-
driven head and neck cancers. Eur J Cancer. 2017;
78:105–115.
298. Kreimer AR, Johansson M, Waterboer T, et al.
Evaluation of human papillomavirus antibodies
and risk of subsequent head and neck cancer.
J Clin Oncol. 2013;31(21):2708–2715.
299. Ebisumoto K, Okami K, Sakai A, Sugimoto R,
Iida M. Successful detection of a minute tonsillar
cancer lesion on transoral examination with narrow
band imaging: a report of 2 cases. Head Neck.
2016;38(suppl 1):E2421–E2424.
300. Coquia SF, Hamper UM, Holman ME, et al.
Visualization of the oropharynx with transcervical
ultrasound. AJR Am J Roentgenol. 2015;205(6):
1288–1294.
301. Fakhry C, Agrawal N, Califano J, et al. The use
of ultrasound in the search for the primary site of
unknown primary head and neck squamous cell
cancers. Oral Oncol. 2014;50(7):640–645.
302. Blanco RG, Califano J, Messing B, et al. Transcervical
ultrasonography is feasible to visualize and evaluate
base of tongue cancers. PLoS ONE. 2014;9(1):
e87565.
303. Domchek SM, Friebel TM, Singer CF, et al. Associa-
tion of risk-reducing surgery in BRCA1 or BRCA2
mutation carriers with cancer risk and mortality.
JAMA. 2010;304(9):967–975.
304. American Cancer Society (ACS). Ovarian Cancer:
Causes, Risk Factors and Prevention; 2017. https://
www.cancer.org/cancer/ovarian-cancer/causes-risks
-prevention/risk-factors.html. Accessed May 12, 2017.
305. Gayther SA, Pharoah PD. The inherited genetics of
ovarian and endometrial cancer. Curr Opin Genet
Dev. 2010;20(3):231–238.
306. Schouten LJ, Rivera C, Hunter DJ, et al. Height,
body mass index, and ovarian cancer: a pooled
analysis of 12 cohort studies. Cancer Epidemiol
Biomarkers Prev. 2008;17(4):902–912.
307. Lahmann PH, Cust AE, Friedenreich CM, et al.
Anthropometric measures and epithelial ovarian
cancer risk in the European Prospective Investiga-
tion into Cancer and Nutrition. Int J Cancer.
2010;126(10):2404–2415.
308. Pearce CL, Chung K, Pike MC, Wu AH. Increased
ovarian cancer risk associated with menopausal
estrogen therapy is reduced by adding a progestin.
Cancer. 2009;115(3):531–539.
309. Beral V, Bull D, Green J, Reeves G. Million Women
Study Collaborators. Ovarian cancer and hormone
replacement therapy in the Million Women Study.
Lancet. 2007;369(9574):1703–1710.
310. Havrilesky LJ, Moorman PG, Lowery WJ, et al.
Oral contraceptive pills as primary prevention for
ovarian cancer: a systematic review and meta-analysis.
Obstet Gynecol. 2013;122(1):139–147.
311. Beral V, Doll R, Hermon C, Peto R, Reeves G.
Ovarian cancer and oral contraceptives: collaborative
reanalysis of data from 45 epidemiological studies
including 23,257 women with ovarian cancer and
87,303 controls. Lancet. 2008;371(9609):303–314.
312. McLaughlin JR, Risch HA, Lubinski J, et al. Repro-
ductive risk factors for ovarian cancer in carriers of
BRCA1 or BRCA2 mutations: a case-control study.
Lancet Oncol. 2007;8(1):26–34.
313. Reid BM, Permuth JB, Sellers TA. Epidemiology
of ovarian cancer: a review. Cancer Biol Med. 2017;
14(1):9–32.
314. Langseth H, Hankinson SE, Siemiatycki J, Weider-
pass E. Perineal use of talc and risk of ovarian cancer.
J Epidemiol Community Health. 2008;62(4):358–360.
315. Huncharek M, Geschwind JF, Kupelnick B. Perineal
application of cosmetic talc and risk of invasive
epithelial ovarian cancer: a meta-analysis of 11,933
subjects from sixteen observational studies. Anticancer
Res. 2003;23(2C):1955–1960.
316. Baan R, Straif K, Grosse Y, et al. Carcinogenicity
of carbon black, titanium dioxide, and talc. Lancet
Oncol. 2006;7(4):295–296.
317. Wu AH, Pearce CL, Tseng CC, Templeman C,
Pike MC. Markers of inflammation and risk of
ovarian cancer in Los Angeles County. Int J Cancer.
2009;124(6):1409–1415.
318. Ness RB, Grisso JA, Cottreau C, et al. Factors related
to inflammation of the ovarian epithelium and risk of
ovarian cancer. Epidemiology. 2000;11(2):111–117.
319. Mills PK, Riordan DG, Cress RD, Young HA.
Perineal talc exposure and epithelial ovarian cancer
risk in the Central Valley of California. Int J Cancer.
2004;112(3):458–464.
320. Bast RC, Badgwell D, Lu Z, et al. New tumor
markers: CA125 and beyond. Int J Gynecol Cancer.
2005;15(suppl 3):274–281.
321. Buys SS, Partridge E, Black A, et al. Effect of
screening on ovarian cancer mortality: the Prostate,
Lung, Colorectal and Ovarian (PLCO) Cancer
Screening Randomized Controlled Trial. JAMA.
2011;305(22):2295–2303.
322. Pinsky PF, Yu K, Kramer BS, et al. Extended mortal-
ity results for ovarian cancer screening in the PLCO
trial with median 15 years follow-up. Gynecol Oncol.
2016;143(2):270–275.
323. Skates SJ, Menon U, MacDonald N, et al. Calcula-
tion of the risk of ovarian cancer from serial CA-125
values for preclinical detection in postmenopausal
women. J Clin Oncol. 2003;21(suppl 10):206s–210s.
324. Lu KH, Skates S, Hernandez MA, et al. A 2-stage
ovarian cancer screening strategy using the Risk of
Ovarian Cancer Algorithm (ROCA) identifies early-
stage incident cancers and demonstrates high positive
predictive value. Cancer. 2013;119(19):3454–3461.
325. Menon U, Ryan A, Kalsi J, et al. Risk algorithm
using serial biomarker measurements doubles the
number of screen-detected cancers compared with
a single-threshold rule in the United Kingdom
Collaborative Trial of Ovarian Cancer Screening.
J Clin Oncol. 2015;33(18):2062–2071.
326. Jacobs IJ, Menon U, Ryan A, et al. Ovarian cancer
screening and mortality in the UK Collaborative
Trial of Ovarian Cancer Screening (UKCTOCS):
a randomised controlled trial. Lancet. 2016;387
(10022):945–956.
327. Rosenthal AN, Fraser L, Manchanda R, et al.
Results of annual screening in phase I of the United
Kingdom familial ovarian cancer screening study
highlight the need for strict adherence to screening
schedule. J Clin Oncol. 2013;31(1):49–57.
328. Rosenthal AN, Fraser LSM, Philpott S, et al. Evi-
dence of stage shift in women diagnosed with ovarian
cancer during phase ii of the United Kingdom
Familial Ovarian Cancer Screening Study. J Clin
Oncol. 2017;35(13):1411–1420.
329. Moyer VA, Force USPST. Screening for ovarian
cancer: U.S. Preventive Services Task Force reaf-
firmation recommendation statement. Ann Intern
Med. 2012;157(12):900–904.
330. U.S. Food and Drug Administration (FDA). The
FDA Recommends Against Using Screening Tests for
Ovarian Cancer Screening: FDA Safety Communica-
tion; 2016. http://www.fda.gov/MedicalDevices/
Safety/AltersandNotices/ucm519413.htm. Accessed
June 6, 2017.
331. Isaacs C, Peshkin B. Management of Patients at
High-Risk for Breast and Ovarian Cancer. UpTo-
Date. 2016. https://www.uptodate.com/contents/
management-of-patients-at-high-risk-for-breast-and
-ovarian-cancer. Accessed June 9, 2017.
332. Yang S, Thiel KW, Leslie KK. Progesterone: the
ultimate endometrial tumor suppressor. Trends
Endocrinol Metab. 2011;22(4):145–152.
333. Fearnley EJ, Marquart L, Spurdle AB, Weinstein
P, Webb PM, Group AOCSGaANECS. Polycystic
ovary syndrome increases the risk of endometrial
cancer in women aged less than 50 years: an Aus-
tralian case-control study. Cancer Causes Control.
2010;21(12):2303–2308.
334. Mueck AO, Seeger H, Rabe T. Hormonal contracep-
tion and risk of endometrial cancer: a systematic
review. Endocr Relat Cancer. 2010;17(4):263–271.
335. Dossus L, Allen N, Kaaks R, et al. Reproductive
risk factors and endometrial cancer: the European
Prospective Investigation into Cancer and Nutrition.
Int J Cancer. 2010;127(2):442–451.
336. Schmandt RE, Iglesias DA, Co NN, Lu KH.
Understanding obesity and endometrial cancer risk:
opportunities for prevention. Am J Obstet Gynecol.
2011;205(6):518–525.
337. Renehan AG, Tyson M, Egger M, Heller RF, Zwahlen
M. Body-mass index and incidence of cancer: a
systematic review and meta-analysis of prospective
observational studies. Lancet. 2008;371(9612):
569–578.
338. Adams TD, Stroup AM, Gress RE, et al. Cancer
incidence and mortality after gastric bypass surgery.
Obesity (Silver Spring). 2009;17(4):796–802.
339. Troisi R, Potischman N, Hoover RN, Siiteri P,
Brinton LA. Insulin and endometrial cancer. Am
J Epidemiol. 1997;146(6):476–482.
340. Schmid D, Behrens G, Keimling M, Jochem C,
Ricci C, Leitzmann M. A systematic review and
meta-analysis of physical activity and endometrial
cancer risk. Eur J Epidemiol. 2015;30(5):397–412.
341. Du M, Kraft P, Eliassen AH, Giovannucci E,
Hankinson SE, De Vivo I. Physical activity and risk
of endometrial adenocarcinoma in the Nurses’ Health
Study. Int J Cancer. 2014;134(11):2707–2716.
342. Friedenreich C, Cust A, Lahmann PH, et al. Physical
activity and risk of endometrial cancer: the European
prospective investigation into cancer and nutrition.
Int J Cancer. 2007;121(2):347–355.
343. Fisher B, Costantino JP, Wickerham DL, et al.
Tamoxifen for prevention of breast cancer: report
of the National Surgical Adjuvant Breast and Bowel
Project P-1 Study. J Natl Cancer Inst. 1998;90(18):
1371–1388.
344. Vogel VG, Costantino JP, Wickerham DL, et al.
Effects of tamoxifen vs raloxifene on the risk of
developing invasive breast cancer and other disease
outcomes: the NSABP Study of Tamoxifen and
Raloxifene (STAR) P-2 trial. JAMA. 2006;295(23):
2727–2741.
345. Watson P, Vasen HF, Mecklin JP, Järvinen H, Lynch
HT. The risk of endometrial cancer in hereditary non-
polyposis colorectal cancer. Am J Med. 1994;96(6):
516–520.
346. Lu KH, Daniels M. Endometrial and ovarian cancer
in women with Lynch syndrome: update in screening
and prevention. Fam Cancer. 2013;12(2):273–277.
347. Lu KH, Dinh M, Kohlmann W, et al. Gynecologic
cancer as a “sentinel cancer” for women with heredi-
tary nonpolyposis colorectal cancer syndrome. Obstet
Gynecol. 2005;105(3):569–574.
348. Barakat RR, Gilewski TA, Almadrones L, et al.
Effect of adjuvant tamoxifen on the endometrium
in women with breast cancer: a prospective study
using office endometrial biopsy. J Clin Oncol. 2000;
18(20):3459–3463.
349. Gerber B, Krause A, Muller H, et al. Effects
of adjuvant tamoxifen on the endometrium in

postmenopausal women with breast cancer: a
prospective long-term study using transvaginal
ultrasound. J Clin Oncol. 2000;18(20):3464–3470.
350. Rijcken FE, Mourits MJ, Kleibeuker JH, Hollema
H, van der Zee AG. Gynecologic screening in
hereditary nonpolyposis colorectal cancer. Gynecol
Oncol. 2003;91(1):74–80.
351. Dove-Edwin I, Boks D, Goff S, et al. The outcome
of endometrial carcinoma surveillance by ultrasound
scan in women at risk of hereditary nonpolyposis
colorectal carcinoma and familial colorectal carci-
noma. Cancer. 2002;94(6):1708–1712.
352. Lindor NM, Petersen GM, Hadley DW, et al.
Recommendations for the care of individuals
with an inherited predisposition to Lynch syndrome:
a systematic review. JAMA. 2006;296(12):
1507–1517.
353. American Cancer Society (ACS). Endometrial
Cancer: Early Detection, Diagnosis, and Staging;
2017. https://www.cancer.org/cancer/endometrial-
cancer/detection-diagnosis-staging.html. Accessed
June 16, 2017.
354. Huang M, Sun C, Boyd-Rogers S, et al. Prospective
study of combined colon and endometrial cancer
screening in women with lynch syndrome: a patient-
centered approach. J Oncol Pract. 2011;7(1):43–47.
355. Tripp MK, Gershenwald JE, Davies MA, et al.
Assessment of compliance with Texas legislation
banning indoor UV tanning by minors. JAMA
Dermatol. 2016.
356. Brunssen A, Waldmann A, Eisemann N, Katalinic
A. Impact of skin cancer screening and secondary
prevention campaigns on skin cancer incidence and
mortality: a systematic review. J Am Acad Dermatol.
2016.
357. US Preventive Services Task Force (USPSTF),
Bibbins-Domingo K, Grossman DC, et al. Screen-
ing for skin cancer: US Preventive Services Task
Screening and Early Detection  •  CHAPTER 23 398.e7
Force recommendation statement. JAMA. 2016;
316(4):429–435.
358. Tsao H, Weinstock MA. Visual inspection and the US
Preventive Services Task Force recommendation on
skin cancer screening. JAMA. 2016;316(4):398–400.
359. Schneider JS, Moore DH, Mendelsohn ML. Screen-
ing program reduced melanoma mortality at the
Lawrence Livermore National Laboratory, 1984 to
1996. J Am Acad Dermatol. 2008;58(5):741–749.
360. Katalinic A, Waldmann A, Weinstock MA, et al.
Does skin cancer screening save lives?: an obser-
vational study comparing trends in melanoma
mortality in regions with and without screening.
Cancer. 2012;118(21):5395–5402.
361. Breitbart EW, Waldmann A, Nolte S, et al. Systematic
skin cancer screening in Northern Germany. J Am
Acad Dermatol. 2012;66(2):201–211.
362. Breitbart EW, Choudhury K, Anders MP, et al.
Benefits and risks of skin cancer screening. Oncol
Res Treat. 2014;37(suppl 3):38–47.
363. Eisemann N, Waldmann A, Geller AC, et al. Non-
melanoma skin cancer incidence and impact of skin
cancer screening on incidence. J Invest Dermatol.
2014;134(1):43–50.
364. Hubner J, Waldmann A, Geller AC, et al. Interval
cancers after skin cancer screening: incidence,
tumour characteristics and risk factors for cutaneous
melanoma. Br J Cancer. 2017;116(2):253–259.
365. Stang A, Garbe C, Autier P, Jockel KH. The many
unanswered questions related to the German skin
cancer screening programme. Eur J Cancer. 2016;
64:83–88.
366. Stang A, Jockel KH. Does skin cancer screening
save lives? A detailed analysis of mortality time
trends in Schleswig-Holstein and Germany. Cancer.
2016;122(3):432–437.
367. Tripp MK, Watson M, Balk SJ, Swetter SM,
Gershenwald JE. State of the science on prevention
398.e7
and screening to reduce melanoma incidence and
mortality: The time is now. CA Cancer J Clin. 2016.
368. Boniol M, Autier P, Gandini S. Melanoma mortality
following skin cancer screening in Germany. BMJ
Open. 2015;5(9):e008158.
369. Scoggins CR, Ross MI, Reintgen DS, et al. Gender-
related differences in outcome for melanoma
patients. Ann Surg. 2006;243(5):693–698, discussion
698–700.
370. Hoorens I, Vossaert K, Pil L, et al. Total-body
examination vs lesion-directed skin cancer screening.
JAMA Dermatol. 2016;152(1):27–34.
371. Watts CG, Cust AE, Menzies SW, Mann GJ,
Morton RL. Cost-effectiveness of skin surveillance
through a specialized clinic for patients at high risk
of melanoma. J Clin Oncol. 2017;35(1):63–71.
372. Curiel-Lewandrowski C, Kim CC, Swetter SM,
et al. Survival is not the only valuable end point in
melanoma screening. J Invest Dermatol. 2012;132(5):
1332–1337.
373. Guy GP Jr, Machlin SR, Ekwueme DU, Yabroff
KR. Prevalence and costs of skin cancer treatment
in the U.S., 2002–2006 and 2007–2011. Am J Prev
Med. 2015;48(2):183–187.
374. Rogers HW, Weinstock MA, Feldman SR, Coldiron
BM. Incidence estimate of nonmelanoma skin cancer
(keratinocyte carcinomas) in the U.S. population,
2012. JAMA Dermatol. 2015;151(10):1081–
1086.
375. Wernli KJ, Henrikson NB, Morrison CC, Nguyen
M, Pocobelli G, Blasi PR. Screening for skin cancer
in adults: updated evidence report and systematic
review for the US Preventive Services Task Force.
JAMA. 2016;316(4):436–447.
376. Shaikh WR, Geller A, Alexander G, et al. Developing
an interactive web-based learning program on skin
cancer: the learning experiences of clinical educators.
J Cancer Educ. 2012;27(4):709–716.
 Nicotine Dependence: Current Treatments 24 
and Future Directions
Jeffrey M. Engelmann, Maher Karam-Hage, Vance A. Rabius,
Jason D. Robinson, and Paul M. Cinciripini

S UMMARY OF K EY P OI NT S
Prevalence of Tobacco Use and (e.g., identifying triggers and number of randomized controlled
Nicotine Dependence in Patients managing withdrawal), quitlines, and trials of smoking cessation
With Cancer self-help material (e.g., booklets and treatments have been conducted
• Approximately one in six American videos). among patients with cancer. These
adults is a current smoker. • Nonpharmacologic approaches are reports suggest that a combination
• Smoking accounts for one-third of all popular but yield relatively low quit of medications and a behavioral
cancer deaths. rates if used alone. approach are needed to make a
• In general, patients with cancer have • Pharmacologic treatments approved difference.
a higher dependence on nicotine and by the US Food and Drug • Patients with cancer who use
are more likely to be smokers or Administration, including nicotine tobacco should be treated according
ex-smokers. replacement therapies (e.g., to evidence-based treatment
• Approximately 15.1% of adult cancer transdermal patch, gum, nasal spray, guidelines, with particular attention
survivors are current smokers. inhaler, and lozenge) and bupropion, paid to tailoring education about
Biologic Characteristics and have been shown to double smoking their disease-tobacco link,
Genetics cessation rates compared with pharmacotherapy, comorbid
• The reward pathway and dopamine placebo. Varenicline has efficacy medical and psychiatric disorders,
are involved in the use and abuse superior to that of nicotine and family and household tobacco
of and dependence on all such replacement therapies and use.
substances, including nicotine. bupropion. Barriers to Cessation Treatment
• Pharmacogenetics research has the Smoking Among Cancer Patients in the Oncology Setting
premise to tailor treatment to • About half of patients with cancer • Health care providers have limited
enhance efficacy and reduce continue to smoke after diagnosis, time and expertise to address
toxicity. even though tobacco use smoking among patients with
Diagnosis and Evaluation complicates cancer treatment, cancer, and patients may have
• Several instruments measure reduces survivorship rates, comorbid substance use or
nicotine dependence among increases the risk for a second dependence or other emotional and
cigarette smokers; among them are primary tumor, and diminishes mental disorders that undermine
the Fagerström Test for Nicotine quality of life. their ability to quit smoking.
Dependence (FTND) and Heaviness • Few studies have examined • Systems-level changes and tailored
of Smoking Index (HSI). However, predictors of continued smoking treatment approaches are needed to
the evaluation and final among patients with cancer, but identify all tobacco users, lower the
recommendation are clinical, and some studies have reported on such rate of persistent tobacco use, and
the treatment plan must be factors as nonsmoking-related reduce recidivism among patients
individualized. cancers, comorbid depression, and with cancer.
poor prognosis.
Current Treatment • Several retrospective studies have
Recommendations shown the detrimental effect of
• Nonpharmacologic treatments continuing to smoke on cancer
include behavioral counseling treatment outcomes. A limited

399
400 Part I: Science and Clinical Oncology
Tobacco use has become a worldwide epidemic, and cigarettes are the
most common form of tobacco consumed. Smoking results in some
of the deadliest yet most preventable diseases, such as respiratory
diseases, cardiovascular diseases, and cancer.1 Tobacco use accounts
for at least 30% of all cancer deaths in the United States, and smoking
has been causally linked to 18 different cancers, including lung, head
and neck, esophageal, pancreatic, bladder, kidney, cervical, endometrial,
and gastric cancers and acute myeloid leukemia.2,3 The scare of a
diagnosis of cancer often impels smokers to quit, and although some
people resume use of tobacco once their cancer is in remission, others
manage to avoid a relapse.
The high recidivism rates could be attributed to comorbid conditions
and to the neurobiology of tobacco addiction. Tobacco addiction is
a complex phenomenon. Nicotine receptors are spread throughout
most areas of the brain, and nicotine consumption activates the reward
pathway, as do all other substances of addiction.4 In addition, several
other neurotransmitter systems are implicated in the process of
establishing an addiction to nicotine.5 After years of tobacco consump-
tion, complex neuronal involvements and adaptations develop, rendering
the extinguishing of tobacco addiction a challenging task. However,
pharmacologic and behavioral treatments have proven to be essential
and highly effective in comparison with other medical treatments of
chronic diseases.6 In recent years, individualized treatment has become
the gold standard, with hopes that advancement in genetics will allow
us to eventually match the best treatment to each individual profile.7
Unfortunately, multiple hurdles and obstacles remain in extending
basic treatment to all smokers and tobacco users, including those
diagnosed with cancer and cancer survivors.8 This chapter summarizes
these aspects of tobacco use disorder and their implication for patients
with cancer. For the purpose of this chapter, the newer term tobacco
use disorder is used interchangeably with older ones such as nicotine
dependence (ND) and tobacco addiction.
EPIDEMIOLOGY AND TOBACCO USE IN
PATIENTS WITH CANCER
Tobacco use, in particular smoking cigarettes, began an upward trend
around the beginning of the 20th century. This trend has been mostly
attributed to James Buchanan “Buck” Duke, who introduced the
mechanized manufacturing of cigarettes and used aggressive marketing
techniques,9 in addition to other societal forces and circumstances
such as the free cigarettes provided with daily rations to all enlisted
American soldiers during World Wars I and II.10 That upward trend
continued until a pivotal moment: the release of the landmark first
Surgeon General’s report on smoking and health in 1964,11 at which
point the evidence from more than 7000 scientific publications
on harm from tobacco smoke was put together and received the
endorsement of Surgeon General Luther Terry, the highest officer
for public health within the US government. That report had an
enduring effect on public beliefs and attitudes about tobacco. As an
example, a Gallup survey conducted in 1958 found that only 44%
of Americans believed smoking caused cancer, whereas 70% believed
so by 1969, 5 years after the report was published. The belief that
smoking causes cancer continued to rise, and the rate of belief reached
94% by 1990.12 Furthermore, since 1964 a gradual and almost annual
decrease in smoking rates has occurred in the United States, with the
rate reaching 15.2% in 2015.13 This lower rate nonetheless encompasses
45 million Americans who, more than 50 years after the landmark
report, continue to use tobacco despite the clear knowledge of harm
from its consumption. Other Surgeon General reports subsequently
solidified the case against tobacco consumption, and to date 37 of
those reports have been dedicated to tobacco (most recently the 50th
anniversary report14), focusing on specific topics such as nicotine addic-
tion, secondhand smoke, minorities, women, and youth.15 Although
these efforts have improved public health, disparities in health care
unfortunately extend to tobacco consumption, as evidenced by higher
smoking rates among American Indians/Alaskan Natives, persons with
a diagnosis of mental health or substance use disorders, those who
are under the poverty level of income, and the unemployed.16 It is
of importance to note that level of education is inversely related to
smoking rates.17
Although the trend for smoking among patients older than 45 years
who are recovering from cancer has decreased to about the same as for
the general population, the prevalence among survivors in the younger
age group is still high. Around 40.4% of persons 18 to 44 years of
age in the cancer survivors cohort still smoke cigarettes,18 compared
with 24.4% of persons 18 to 24 and 24.1% of persons 25 to 44
years of age in the general population.13 Nevertheless, one potential
confounding factor contributing to these statistics is the high mortal-
ity rate in patients with smoking-related tumors. Therefore smokers
who would survive less lethal cancers are counted within the younger
age group.19
Smoking cessation rates in the cancer population seem highly
dependent on the disease (tumor) site and its relationship to smoking.20
As an example, around 80% of patients with lung cancer and 65%
of patients with head and neck cancer were reported to have stopped
smoking in the year after their treatment21,22 compared with 31% of
patients with bladder cancer.23 Other factors increase the likelihood
of smoking cessation among patients with cancer, including having
a good prognosis, having spent a long time in the hospital (a smoke-free
environment), and having treatment-related adverse effects. In particular,
adverse effects of treatment such as nausea, difficulty breathing, facial
surgery, or difficulty swallowing may make it harder or more difficult
to smoke. Consequently, physicians should pay attention to the
particular smoking-cessation pattern in patients with cancer. Typically,
cancer survivors have an increased chance of a delayed relapse to
smoking 1 to 6 months after treatment,24 in contrast to persons in
the general population, who generally relapse within the first week
or so of cessation. Unfortunately, it has been reported that some
patients with lung cancer who have already abstained from smoking
before their diagnosis tend to return to smoking once they are informed
of their cancer.25 Many studies have been published on the benefits
of smoking cessation in patients with cancer, with many focusing on
cancer treatment–related outcomes (Table 24.1). Unfortunately, almost
all published studies to that effect have methodological limitations,
such as use of retrospective analyses, nonstandardized tobacco assess-
ment, no biochemical confirmation, and no tracking of behavior after
diagnosis. Addressing smoking behavior at the time of diagnosis and
throughout treatment and recovery is thus essential for persons with
cancer, constituting a continuum of potential teachable moments (i.e.,
health events that might motivate individuals to adopt risk-reducing
behaviors).26
Another opportunity for teachable moments arises during lung
cancer screening using low-dose computed tomography (LDCT). The
US Preventive Services Task Force recommends LDCT screening in
individuals 55 to 80 years old with a 30 pack-year smoking history
who currently smoke or who have quit within the past 15 years.27
A systematic review of two randomized controlled trials (RCTs)
and three observational studies found that although LDCT itself
does not influence smoking behavior, positive results (i.e., finding
a nodule or a tumor) were associated with increased abstinence.28
A more recent analysis of 18,840 current and former smokers from
the National Lung Screening Trial found that false-positive screening
results were associated with modest increases in abstinence among
smokers over the course of a 5-year follow-up period.29 These studies
have similar limitations to other studies about smoking cessation in
cancer patients, including nonstandardized assessment of smoking
behavior and no biochemical verification of abstinence. Therefore more
research is needed on the effectiveness of lung cancer screening as a
teachable moment for smoking cessation. It will also be important to
study the effectiveness of offering evidence-based smoking cessation
interventions at the time of lung cancer screening, which have the
potential to further motivate smokers to quit beyond lung cancer
screening itself.30
 Nicotine Dependence: Current Treatments and Future Directions  •  CHAPTER 24 401

Table 24.1 Continued Smoking Versus Cessation as It Relates to Cancer Treatment Outcomes in

Retrospective Studies
Sample
Authors (Journal) Year Cancer Outcome if Continued Smoking Size Tumor Site
Fortin et al242 (Int J Radiat Oncol Biol Phys) 2009 Smoking and drinking at baseline were associated with poor 1871 Head and neck
outcomes
Rades et al243 (Int J Radiat Oncol Biol Phys) 2008 Smoking during radiation had a significant effect on 181 Lung
locoregional control
Marin et al244 (Plast Reconstr Surg) 2008 Serum cotinine concentration greater than 10 ng/mL may 89 Head and neck
predict an increased risk of wound complication
Chen et al245 (BJU Int) 2007 Higher recurrence rate for patients with non–muscle- 297 Bladder
invasive bladder cancer
Tammemagi et al246 (Chest) 2004 Worse survival in smokers 1155 Lung
Garces et al228 (Chest) 2004 Smoking after a lung cancer diagnosis negatively affects 1506 Lung
quality-of-life scores
Pytynia et al247 (J Clin Oncol) 2004 Threefold increases in risk for overall death, death as a result 100 Head and neck
of disease, and recurrence of disease
Fox et al248 (Lung Cancer) 2004 Poorer prognosis for survival after radiation therapy of non– 237 Lung
small cell cancer
Nordquist et al249 (Chest) 2004 Worse survival 654 Lung
Videtic et al250 (J Clin Oncol) 2003 Poor survival rates if patients continued to smoke during 215 Lung
radiation and chemotherapy
Chelghoum et al251 (Ann Oncol) 2002 Lower survival in patients with acute myeloid leukemia by 643 Leukemia
shortening complete remission duration and subsequent
survival; severe infections during aplasia
Kreuzer et al252 (Br J Cancer) 2000 Higher risk of developing lung cancer among men than 9792 Lung
among women
Marshak et al253 (Int J Radiat Oncol Biol Phys) 1999 Smoking correlated with decreased local control 207 Glottis
Fleshner et al254 (Cancer) 1999 Diminished recurrence-free survival 286 Bladder
Daniell et al255 (J Urol) 1995 Greater 5-yr tumor-specific mortality rate with stage D2 359 Prostate
disease
Daniell et al256 (Am J Clin Pathol) 1993 More often advanced-stage disease (stages II–IV) 157 Endometrium
Risch et al257 (Am J Epidemiol) 1993 In both sexes, a greatly elevated risk of developing lung 845 Lung
cancer, and an appreciably stronger risk for females than for
males
Rugg et al258 (Br J Radiol) 1990 A highly significant correlation of cancer during and/or after 41 Head and neck
treatment
Archimbaud et al259 (Cancer) 1989 Associated with early blast crisis and short survival 173 Leukemia
Daniell260 (Cancer) 1988 Tobacco and obesity potentiate the early spread of 623 Breast
malignant disease
Daniell261 (Cancer) 1986 More advanced stage of tumors 392 Colon
Phillips et al262 (Cancer) 1985 Marked depression in natural killer activity that was 68 Breast
comparable with that of patients with carcinoma of the lung
Stevens et al263 (Arch Otolaryngol) 1983 Smoking after diagnosis had a fourfold increase in the 200 Head and neck
recurrence rate
Daniell264 (N Engl J Med) 1980 Fared less well than nonsmokers with breast cancer 78 Breast
Hinds et al265 (J Natl Cancer Inst) 1982 Greater probability of dying during the 12 mo after 223 Lung
diagnosis
Johnston-Early et al266 (JAMA) 1980 Decreased survival 112 Lung

REWARD PATHWAY AND BIOLOGIC
CHARACTERISTICS OF ADDICTION
Reward Pathway
Like most drugs associated with abuse and dependence, nicotine
stimulates a variety of receptors in the brain, in particular nicotinic
acetylcholine receptors (nAChRs). These nAChRs are spread throughout
most areas of the brain. Nicotine use leads to a rapid increase in
dopamine release in the nucleus accumbens and the ventral tegmental
area. This stimulation typically occurs within 10 seconds of smoking
a cigarette.31,32 It has been established that natural rewards such as
food consumption, social affiliation, and sexual activity, which are
linked to survival of the individual or species, normally activate these
two central areas of the reward pathway within the brain. Furthermore,
the reward pathway projects from the nucleus accumbens and ventral
tegmental area to the prefrontal cortex, the amygdala, and the olfactory
tubercle (Fig. 24.1), leading to a complex interaction among the
402 Part I: Science and Clinical Oncology
VTA
Prefontal
cortex
Nucleus
accumbens
Figure 24.1  •  The reward pathway, consisting of the nucleus accumbens
and ventral tegmental area (VTA) with projections to the prefrontal cortex.
(Modified from National Institute on Drug Abuse. The Neurobiology of Drug
Addiction. Available at http://www.nida.nih.gov/pubs/teaching/Teaching2/
Teaching.html.)
reward, its saliency, and the ability to inhibit the intake of a drug. In
addition to dopamine, several other brain systems (neurotransmitters,
receptors, and pathways) have been implicated in addiction, including
the γ-aminobutyric acid, glutamate, serotonin, norepinephrine, can-
nabinoid, opioid, and cholinergic systems.33–35 It is important to note
that these major neurotransmitter systems in the brain are in constant
and dynamic interplay, possibly resulting in different types of neuro-
adaptations that lead to and maintain the addiction to a substance in
a given individual.5
Neuronal Adaptation
A pleasurable sensation from the activation of the reward pathway
is associated with the acute use of an addictive substance such as
nicotine. However, repeated administration of a substance, in this
case nicotine, can lead to increased tolerance, which in turn pro-
duces a state of withdrawal in the absence of nicotine. Tolerance
and withdrawal are the physiologic hallmarks of dependence and are
thought to be the result of neuroadaptive effects occurring within
the brain.36 It is interesting to note that the chronic use of drugs
of abuse and dependence, including nicotine, seems to result in a
generalized decrease in dopaminergic neurotransmission. Although
this decrease can be a prodrome for vulnerability to addiction, it is
likely to be a homeostatic response to the intermittent yet repetitive
increases in dopamine induced by the frequent and sustained use of
such drugs.37
Sensitization and counteradaptation are two of the major neu-
roadaptive models that have been proposed to explain how changes
that start in the reward pathway may result in the development of
substance dependence. Sensitization can be thought of as the gradual
increase in “wanting” a drug after intermittent but repeated use, which
then can facilitate the transition from occasional use to chronic use
and tolerance.37,38 The counteradaptation model postulates that the
initial positive feelings of reward (resulting from the use of a drug)
are followed by an opposing rather than synchronous development
of tolerance, as the brain’s homeostatic (counterbalance) response
to the exposure. Other models of nicotine addiction are based on
mechanisms associated with capacity for or the lack of cognitive control
and the development of reinforcement learning,39 in particular the
negative reinforcement associated with withdrawal from nicotine,
which builds up with the reduction in negative effect from smoking
a cigarette.40
GENETICS
Genes Associated With Smoking, Nicotine
Dependence, and Lung Cancer
Several recent genome-wide association (GWA) and candidate gene
studies have revealed significant associations among smoking, ND,
and lung cancer in a region of interest on the long arm of chromosome
15 (15q24/15q25.1), encompassing the nAChR CHRNA3-A5-B4
subunit cluster. Neuronal nAChRs are pentameric ligand-gated channels
composed of alpha and beta subunits,41 several of which contribute
to nicotine-stimulated dopamine release in the striatum, a region
associated with reward learning and vital to the development of
substance dependence.42 The CHRNA3-A5-B4 subunit cluster dem-
onstrates extensive linkage disequilibrium, and the single nucleotide
polymorphisms on that cluster are frequently correlated. Some of the
statistically strongest associations with ND in persons of European
or African ancestry have been obtained for the nonsynonymous
CHRNA5 single-nucleotide polymorphism rs16969968.43–51 Other
gene variants in this region (CHRNA3-A5-B4) identified by GWA
and candidate gene studies as being significant contributors to the
risk of smoking, ND, and lung cancer include CHRNA3 rs1051730,48,52–55
CHRNA3 rs578776,43,45,50 and AGPHD1 rs8034191.43,52,54 Finally,
candidate gene studies have identified associations between ND and
genetic markers associated with dopamine expression (ANKK1,54,56–58
DRD2,58,59 and SLC6A359–62), serotonin expression (SLC6A4),63–65 and
nicotine metabolism (CYP2A6).55,66
Genes Predicting Treatment Outcome
A growing body of genome-wide association studies (GWASs) and
candidate gene studies have examined genetic predictors of responsive-
ness to smoking-cessation therapies (see references 51 and 67–69 for
reviews). Cessation outcome for nicotine replacement therapy (NRT)
has been found to be associated with several polymorphisms in the
D2 receptor gene (DRD2), including C957T rs6277,70 −141C ins/del
rs1799732,70 and ANKK1 (rs1800497).71–73 Other markers that influence
dopamine reuptake and expression, including COMT rs468074,75 and
a variable number tandem repeat (VNTR) in DRD4 C-521T,76 have
been shown to predict cessation outcome for NRT. Genes associated
with the opioid (OPRM1),77–79 serotonergic (SLC6A4),77,80,81 canna-
binoid,82,83 and CHRNA3-A5-B484 pathways have also been associated
with NRT outcome.
Similarly, variants of many of these same markers have been associ-
ated with successful smoking cessation treatment using bupropion,
including ANKK1,85–87 DRD2 −141C ins/del,70 COMT,88 DRD4,89
SLC6A3,90 CYP2B6,91 and the CHRNA3-A5-B4 subunit cluster.92–94
Varenicline, the most recently approved smoking cessation
pharmacotherapy, is the next target for pharmacogenetic inquiry. An
analysis of 1175 smokers who received varenicline, bupropion, or
placebo found that abstinence was associated with multiple CHRNB2,
CHRNA7, and CHRNA4 subunit genes for persons given varenicline
and with CYP2B6 for persons given bupropion.7 However, a more
recent analysis of 1026 smokers found that CHRNA5-A3-B4 genetic
variants were not associated with smoking cessation outcome regard-
less of treatment (placebo, NRT, or varenicline).50 Thus additional
research is needed to identify reliable genetic predictors of varenicline
treatment.
Recently, a genetically informed biomarker has been shown to
be predictive of smoking cessation.95 The nicotine metabolite ratio
(NMR) is the ratio of 3′-hydroxycotinine to cotinine, which reflects
the activity of CYP2A6, the primary enzyme responsible for nicotine
metabolism. Retrospective studies have shown that slow metabolizers
of nicotine (i.e., those with NMRs that fall in the lowest quartile)
have higher smoking cessation rates than normal metabolizers,96–98 but
that normal metabolizers are more likely to benefit from bupropion
treatment than slow metabolizers.99 In a large prospective study,100
 Nicotine Dependence: Current Treatments and Future Directions  •  CHAPTER 24 403

1246 smokers were randomly assigned to receive varenicline, NRT, or PATIENT ASSESSMENT
placebo, stratified by NMR. Among normal metabolizers, varenicline
resulted in significantly higher abstinence rates than NRT, but among In the absence of specific biological markers, the diagnosis of nicotine
slow metabolizers, varenicline was not more efficacious than NRT. addiction remains a clinical one. The Diagnostic and Statistical Manual
Also, slow metabolizers reported significantly more side effects while on of Mental Disorders, fifth edition (DSM-5)119 uses universal criteria
varenicline versus placebo. This suggests that treating slow metabolizers for all substance use disorders (traditionally referred to as addiction,
with NRT and normal metabolizers with varenicline may maximize and referred to as dependence in previous editions of the DSM120),
the likelihood of smoking cessation while minimizing the risk of including nicotine. Because of their universality, the DSM-5 criteria
side effects. are not particularly specific to nicotine and therefore do not capture
In summary, GWASs and candidate gene studies point to a variety many of the peculiar aspects of tobacco use and ND (addiction). This
of genes that may be related to one or more smoking phenotypes, lack of specificity has led to the development of specific scales to
including ND, initiation, cessation, and possibly nicotine withdrawal. quantify ND. Traditionally, the six-item Fagerström Test for Nicotine
In all but a few studies, these relations have been studied in smokers Dependence (FTND)121 has been used, and more recently the two-item
of European decent. The areas of interest include nicotine cholinergic Heaviness of Smoking Index (HSI),122 which was derived from the
receptor genes (CHRNA3-A5-B4; CHRNA2, CHRNA4, CHRNA6, first two items of the FTND.
CHRNB1, CHRNB2, and CHRNB3), nicotine and drug metabolism Despite their limitations, the DSM-5 criteria offer ease of use for
genes (CYP2A6 and CYP2B6) and associated biomarkers (NMR), clinicians because of their universality for all substances of dependence.
and dopaminergic signaling genes (DRD2-ANKK1, DRD4, COMT, According to the DSM-5, the diagnosis is made when someone meets
SLC6A3, and DBH). two or more of the 11 criteria for at least a year (the more criteria
that are met, the more severe is the disorder). As previously mentioned,
COMORBID CONDITIONS some criteria are more pertinent than others to nicotine. For instance,
after prolonged smoking the user develops nicotine tolerance and
The smoking rates among persons with no mental illness, past-month begins to exhibit withdrawal symptoms when nicotine is absent, often
mental illness, and lifetime mental illness have been reported to be overnight, largely because nicotine’s half-life in blood is 1 to 2 hours.123
22%, 34%, and 41%, respectively, which means that having a current Having tolerance and/or withdrawal used to be referred to as physiologic
or past mental disorder effectively doubles the likelihood of being a dependence in the DSM-IV-TR. In addition to tolerance and with-
smoker. Furthermore, in a nationally representative sample, it was drawal, nicotine may also be responsible for four other criteria for
reported that smokers with a mental disorder in the past month consume dependence in DSM-5: craving for cigarettes, loss of control over
44% of all cigarettes smoked.101 The evidence that smoking is more smoking (e.g., not being able to reduce or stop smoking, or smoking
prevalent in and closely linked with several psychiatric comorbidities, more than intended), compulsive smoking (e.g., smoking all day long
including abuse of other substances, suggests a shared biologic pathway and giving up important events or activities if there is no place to
between ND and these psychiatric and substance use conditions. For smoke), and continued smoking despite adverse consequences (e.g.,
example, several studies have demonstrated a positive correlation heart attack, emphysema, or cancer).
between smoking and alcohol use, abuse, or dependence, substance The FTND (Table 24.2) is one of the most widely recognized and
abuse or dependence, and other psychiatric disorders.101–108 Conversely, used scales for nicotine dependence. A person with three points or
the prevalence of lifetime alcohol dependence and/or drug abuse with
the adult smoker population is estimated to be 23% to 30%.109,110
Similarly, among current smokers, the lifetime rates of mood and
anxiety disorders have been reported to range from 33% to 46% and
from 12% to 26%, respectively, regardless of nicotine dependence.109 Table 24.2 Items and Scoring for Fagerström Test
Furthermore, among current smokers who are tobacco dependent, for Nicotine Dependence
even the 12-month prevalence of any mood or anxiety disorder is
reported to be 22%, a relatively high proportion.111 Adding support Question Answer Points
to the idea of a possible causal link between smoking and mental 1. How soon after you wake up do Within 5 min 3
health disorders is the fact that smokers also have an elevated risk of you smoke your first cigarette? 6–30 min 2
a first onset of major depression, panic disorder, or generalized anxiety 31–60 min 1
disorder.112–116 Cognitive dysfunction has also been highly linked to After 60 min 0
smoking, as evidenced by the odds ratio comparing persons who have 2. Do you find it difficult to refrain Yes 1
ever smoked with persons who have never smoked, and smoking from smoking in places where it is No 0
is positively related to the number of attention-deficit/hyperactivity forbidden (e.g., in church, at the
disorder (ADHD) symptoms. There seems to be an effect of early library, in a cinema)?
onset on the severity of ADHD or vice versa, because lifetime regular 3. Which cigarette would you hate The first one in 1
smoking has an inverse relationship between the number of ADHD most to give up? the morning
symptoms and the age at onset. In addition, a positive relationship All others 0
between the number of symptoms and the number of cigarettes smoked 4. How many cigarettes per day do ≤10 0
has been observed.117 The aforementioned evidence of co-occurrence you smoke? 11–20 1
supports the importance of screening for and treating mental health 21–30 2
disorders among smokers, whether the relationship is causal or a ≥31 3
simple correlation. Treating these disorders may increase the ability 5. Do you smoke more frequently Yes 1
to focus on quitting smoking, or at least reduce the impact of nega- during the first hours after waking No 0
tive affect or comorbid mental disorders on a person’s ability to quit than during the rest of the day?
using tobacco, and may have an impact on his or her resilience with 6. Do you smoke if you are so ill that Yes 1
regard to maintaining tobacco abstinence. Treating these disorders you are in bed most of the day? No 0
is of particular importance among patients with cancer and cancer
survivors, because higher levels of confidence about the ability to quit From Heatherton TF, Kozlowski LT, Frecker RC, Fagerström KO. The Fagerström Test
smoking were found to be related to lower depression scores and lower for Nicotine Dependence: a revision of the Fagerström Tolerance Questionnaire. Br J
tumor stage.118 Addiction. 1991;86(9):1119-1127.
404 Part I: Science and Clinical Oncology
more out of a possible maximum of 10 points on the FTND is
customarily considered to be nicotine dependent.124,125 The HSI consists
of two items from the FTND (“How soon after you wake up do you
smoke your first cigarette?” and “How many cigarettes do you smoke
per day?”).122 These tools measure physiologic dependence (i.e., tolerance
and withdrawal) reliably well. However, they do not capture the
psychosocial and behavioral dimensions of ND.126
TREATMENT OF TOBACCO USE
Tobacco treatment among patients with cancer needs to be as intensive
and tailored as possible. Hence, if treatment programs are to be
comprehensive and to make a difference, they ought to provide at a
minimum both behavioral- and medication-based interventions.127
Comprehensive treatments, by definition, would need to address
the biologic, psychologic, and social aspects of ND. Moreover, these
approaches ought to be on a long-term basis, because tobacco addiction
is a chronic disease that is often characterized by multiple relapses before
full remission is reached.128 The main components of a biopsychosocial
treatment are behavioral therapies and social support in conjunc-
tion with medications. Among the various therapies, motivational
interviewing, skill-building, problem-solving, and cognitive behavioral
therapies have been empirically supported.6 Although the main first-line
medications that have been approved by the US Food and Drug
Administration (FDA) are NRTs, bupropion (Zyban), and varenicline
(Chantix), the second-line medications clonidine and nortriptyline have
been used off-label and tested in clinical trials with similar positive
results, although with increased side effect profiles.
Among several other medications that have been or are being
researched, three have shown promising preliminary results but have
faced different challenges: topiramate, rimonabant, and nicotine
vaccines. Although a care provider may prescribe any available medica-
tions off-label, to date, none of the aforementioned three medications
is FDA approved for smoking cessation purposes, and only topiramate
is available in the United States. However, their importance lies in
their mechanisms of action, which may lead to improved or similar
agents in the future. Topiramate, a voltage-dependent sodium channel
blocker, augments γ-aminobutyric acid, antagonizes glutamate, and
inhibits carbonic anhydrase activity, and has FDA label indication as
an adjunct treatment for seizure disorders and migraine headaches.129
Topiramate is reported to have a beneficial effect in the treatment of
alcohol dependence and ND treated independently130,131 but does not
seem to have an effect on nicotine dependence among abstinent
alcohol-dependent patients during their first 6 months of abstinence
from alcohol132; furthermore, it has several adverse effects that limit
its use.133 Rimonabant is a selective CB1 endocannabinoid receptor
antagonist reported to have beneficial effects for the treatment of
obesity134 and ND.135 It was shown to be relatively efficacious for
smoking cessation compared with placebo (odds ratio 1.6)136 in large
multinational trials. It was available in Europe from mid-2006 until
January 2009; then the European Medicines Agency recommended
its withdrawal because of neuropsychiatric adverse effects (i.e., depres-
sion and several reports of suicides).137 Although rimonabant is currently
available in other countries, it did not receive FDA approval in the
United States because of similar concerns about neuropsychiatric adverse
effects. Several vaccines against nicotine have been under study for
some years and have shown initial promising results in animal and
early human trials. The most advanced stage III clinical multisite
human trial on one of the nicotine vaccines did not find it to be
different from placebo as a treatment for smoking cessation. However,
several newer methods to develop better vaccines are still being explored.
The vaccine concept is important and merits further investigation
because it may have a role in relapse prevention and as part of mul-
tifaceted approach to smoking cessation that includes behavioral and
pharmacologic intervention.138
The Clinical Practice Guideline for Treating Tobacco Use and
Dependence (CPG-TTUD), which provides a comprehensive
review of all tobacco treatment–related literature, was last updated
in 2008.6 The key points are presented in an executive summary
in Box 24.1. These approaches are summarized and discussed in
the following section, specifically their applicability to the cancer
population.
Pharmacologic Interventions
Some persons are not interested in or lack the motivation to quit
smoking, and some persons are not ready for change. Even in these
instances, pharmacologic treatments have been shown to increase the
odds of reducing the number of cigarettes per day by half.6 This
decrease in cigarettes smoked can in return positively influence patients
by increasing their self-efficacy and motivation to quit.139
The FDA has approved the use of several NRTs, among them the
patch, nasal spray, buccal inhaler, gum, and lozenge. The abstinence
results for NRTs are compelling and largely encouraging based on
hundreds of studies.139 Despite that, recent debate over their long-term
efficacy is taking place, in particular now that they are available over
the counter.140 However, data on using a combination of a patch with
other intermittent NRTs show that combining NRTs is more effective
than use of single NRT.141 Longer-term NRT use can be effective; a
recent study demonstrated the efficacy of NRTs in sustaining abstinence
when used continuously and up to 24 weeks.142
Nicotine is the key ingredient in NRTs, with the premise that
replacing the addictive substance in cigarettes helps the user to quit.
Nicotine in NRTs is at a much lower dose and is delivered through
the venous system, compared with a higher dose from smoking
a cigarette that has an arterial (direct) delivery to the brain. The
intent with NRTs is to reduce cravings and the withdrawal effects of
tobacco cessation. Interesting to note, and despite some controversies,
NRTs have been found to be effective with and without the aid
of a therapeutic support.143 However, one should not forget that
management of expectations and patient education before initiation
of pharmacologic treatment are key to the success of smoking cessation
interventions. In addition, NRTs are relatively safe to use regardless of
the individual’s smoking status and may be used while the individual
is still smoking.144 Most NRT studies did not identify any noteworthy
nicotine toxicity or adverse events; however, the results of other studies
showed that NRT used before quitting helped reduce the number of
cigarettes per day by half even in persons who were not interested
in quitting.145–148 Some smoker characteristics (e.g., the metabolism
rate of nicotine) can affect response to NRTs, suggesting that the
tailored use of NRT for subgroups of smokers could improve NRT
efficacy.149 For example, the level of nicotine dependence affects the
response to NRTs, with more dependent smokers showing higher rates
of quitting when they receive higher doses of NRTs.150,151 NRTs are
an important treatment option for patients with cancer and have been
reported to have similar efficacy to that among other populations of
smokers.127 However, some animal and laboratory cell culture studies
in normal and cancer tissue have shown that nicotine promotes tumor
growth, whereas others report it to also have a tumor-promoting
effect.152,153 Thus far, human studies have not found a significant causal
relationship between NRTs and cancer genesis,154 even after 5 years of
NRT use.155,156
Bupropion
Bupropion was first introduced on the market as an antidepressant
(Wellbutrin) before being found to be effective in the treatment of
tobacco dependence. This medication was consequently approved for
and is currently used by smokers with or without depression (Zyban).157
To date, bupropion is the only antidepressant approved for smoking
cessation on the market; however, no controlled studies have been
published that examine the role of depression as a moderator of the
response to bupropion in depressed smokers. Bupropion is a relatively
well-tolerated medication, with dry mouth, insomnia, and fine tremor
being the most common adverse effects. A meta-analysis of 44 controlled
 Nicotine Dependence: Current Treatments and Future Directions  •  CHAPTER 24 405
Box 24.1.  TEN KEY GUIDELINE RECOMMENDATIONS FROM THE CLINICAL PRACTICE GUIDELINE
ON TREATING TOBACCO USE AND DEPENDENCE
The overarching goal of these recommendations is that clinicians
strongly recommend the use of effective tobacco dependence
counseling and medication treatments to their patients who use
tobacco and that health systems, insurers, and purchasers assist
clinicians in making such effective treatment available.
1. Tobacco dependence is a chronic disease that often requires
repeated intervention and multiple attempts to quit. However,
effective treatments exist that can significantly increase rates of
long-term abstinence.
2. It is essential that clinicians and health care delivery systems
consistently identify and document tobacco use status and treat
every tobacco user seen in a health care setting.
3. Tobacco dependence treatments are effective across a broad range
of populations. Clinicians should encourage every patient willing
to make a quit attempt to use the counseling treatments and
medications recommended in this guideline.
4. Brief tobacco dependence treatment is effective. Clinicians should
offer every patient who uses tobacco at least the brief treatments
shown to be effective in this guideline.
5. Individual, group, and telephone counseling are effective, and their
effectiveness increases with treatment intensity. Two components
of counseling are especially effective, and clinicians should use
these when counseling patients making a quit attempt:
• Practical counseling (problem solving, skills training)
• Social support delivered as part of treatment
6. Numerous effective medications are available for tobacco
dependence, and clinicians should encourage their use by all
patients attempting to quit smoking—except when medically
Reprinted from Fiore MC, Jaen CR, Baker TB, et al. Treating tobacco use and dependence: 2008 Update U.S. Public Health Service Clinical Practice Guideline executive
summary. 2008;53(9):1217-22. Updated March 4, 2015.
clinical trials showed bupropion to be twice as effective as placebo in
reaching end-of-treatment abstinence (8 weeks), as well as in long-term
cessation (pooled risk ratio = 1.62).158
Unfortunately, certain patients with cancer may be unable to use
bupropion because it is known to lower the seizure threshold and is
contraindicated in persons with active seizures or a history of seizures,
head trauma, elevated liver enzymes, bulimia, or anorexia nervosa. It
would be considered risky to give bupropion to patients with cancer
who have a lower threshold for seizures, such as those with metastases
to the brain or with low appetite or body weight as a result of cancer
treatment. However, bupropion has been reported to be effective for
smoking cessation including in populations with mental disorders
such as schizophrenia,159 depression,160 and posttraumatic stress dis-
order.161 Moreover, research has shown that bupropion limits the weight
gain commonly associated with smoking cessation162 and restores sexual
functioning.161 These added advantages can increase its usefulness
among cancer survivors with fatigue from chemotherapy or radiation
or those with comorbid disorders, in addition to persons who are
overweight or have sexual dysfunction.
Varenicline
Varenicline, marketed as Chantix in the United States and Champix
in other countries, is a non–nicotine-based medication for smoking
cessation that was introduced in 2006. Varenicline’s unique properties
as a partial nAChR agonist (traditionally referred to as a mixed agonist-
antagonist) provide some relief from withdrawal symptoms (agonist
property) usually associated with tobacco cessation, while diminishing
the pleasurable sensation from smoking (antagonist property). Initial
clinical trials of varenicline showed that when it was used at 1 mg
contraindicated or with specific populations for which there is
insufficient evidence of effectiveness (i.e., pregnant women,
smokeless tobacco users, light smokers, and adolescents).
• Seven first-line medications (five nicotine and two non–nicotine-
based medications) reliably increase long-term smoking
abstinence rates: bupropion sustained-release (SR), nicotine gum,
nicotine inhaler, nicotine lozenge, nicotine nasal spray, nicotine
patch, and varenicline.
• Clinicians also should consider the use of certain combinations
of medications identified as effective in this guideline.
7. Counseling and medication are effective when used by themselves
for treating tobacco dependence. The combination of counseling
and medication, however, is more effective than either alone. Thus
clinicians should encourage all individuals making a quit attempt
to use both counseling and medication.
8. Telephone quitline counseling is effective with diverse populations
and has broad reach. Therefore both clinicians and health care
delivery systems should both ensure patient access to quitlines
and promote quitline use.
9. If a tobacco user currently is unwilling to make a quit attempt,
clinicians should use the motivational treatments shown in this
guideline to be effective in increasing future quit attempts.
10. Tobacco dependence treatments are both clinically effective and
highly cost-effective relative to interventions for other clinical
disorders. Providing coverage for these treatments increases quit
rates. Insurers and purchasers should ensure that all insurance
plans include the counseling and medication identified as effective
in this guideline as covered benefits.
twice per day, it was twice as effective as bupropion and three times
more effective than placebo at the end of treatment (12 weeks) and
at 1-year follow-up.163–165 An additional 12 weeks of randomized
varenicline or placebo therapy (for a total of 24 weeks) was provided
in a double-blind design to patients who had abstained from smoking
during the first 3 months of open-label varenicline treatment; 70%
of those randomly assigned to receive varenicline stayed abstinent at
the end of the continuation phase of 3 months (a total of 6 months
of treatment with varenicline) compared with 50% of persons randomly
assigned to placebo. Furthermore, the 1-year follow-up abstinence
rate (after medication treatment) was double that of patients who had
received only 3 months of varenicline (25% and 12%, respectively).166
Subsequent clinical trials have confirmed that varenicline is more
efficacious for smoking cessation than bupropion or placebo,167–170
but a recent open-label study found no differences in efficacy among
varenicline, nicotine patch, or combined NRT (nicotine patch plus
nicotine lozenge), although adherence to treatment was very low,
which may be the reason for the lack of difference in this particular
study.171 A recent open-label study of varenicline for smoking cessation
in 132 cancer patients found similar abstinence rates at end of treatment
(40.2%) and a similar side effect profile to what is seen in the general
population.172 Although further study is needed, the findings of this
study support the use of varenicline for smoking cessation in cancer
patients.
The most common adverse event reported was nausea, which is
reported by almost 30% of the persons using varenicline.165,163 Although
the nausea was mild or moderate in the majority of cases, this adverse
effect limits the use of this drug by patients with cancer undergoing
treatment because nausea is a common side effect of cancer treatments.
406 Part I: Science and Clinical Oncology
Other commonly reported adverse effects were vivid dreams and
flatulence. Less common were the neuropsychiatric events among the
general population reported in the postmarketing phase to the FDA
via the voluntary reporting tool MedWatch, with these events consisting
mainly of difficulty with coordination, depressive symptoms, aggression,
irritability, and (more rarely) suicidal ideations.173 In light of these
reports, the FDA issued a black box warning for varenicline regarding
neuropsychiatric events and commissioned the manufacturer to deepen
its data analyses and conduct postmarketing studies to directly compare
psychiatric versus nonpsychiatric populations to determine the
magnitude of these adverse events and their potential link to varenicline.
The findings of three of those studies were recently published. The
first study reported on varenicline’s efficacy and adverse effect profile
among stable patients with schizophrenia. Although the quit rates
were somewhat lower than expected (19% with varenicline versus 5%
with placebo), no exacerbation of the schizophrenia symptoms occurred,
and varenicline was well tolerated.174 The second study was a placebo-
controlled study of the safety and efficacy of varenicline in those who
had a history of or currently stable depression; no difference was
found between the groups in terms of new occurrence of neuropsy-
chiatric adverse events.175 The third and largest study to date was a
placebo-controlled study of the safety and efficacy of varenicline,
bupropion, or nicotine patch in 4116 smokers with psychiatric disorders
and 4028 smokers without psychiatric disorders. The study did not
show a significant increase in neuropsychiatric adverse events attributable
to varenicline, bupropion, or nicotine patch compared with placebo
but did find, as prior studies have shown, that varenicline was more
effective for smoking cessation than bupropion, nicotine patch, or
placebo.170 Before these studies, two meta-analyses found no evidence
of increased neuropsychiatric side effects in smokers taking vareni-
cline.176,177 As a result of these studies, in December 2016 the FDA
removed the black box warning regarding neuropsychiatric adverse
effects. Furthermore, varenicline has been reported to decrease alcohol
consumption among quitting smokers,178 which is of particular
importance given that alcohol and tobacco use disorders are common
comorbidities among patients with head and neck cancer, with decreased
risk after cessation.179,180
A Cochrane review of partial agonists on nicotine receptors con-
sidered a number of studies. Two studies found that cytisine (a product
based on natural ingredients) was effective for smoking cessation
(pooled relative risk [RR], 3.98), whereas another trial found that
dianicline (a synthetic product similar to varenicline) was not effective
(RR, 1.2). Fifteen trials found that varenicline at the standard dose
(2 mg/day) increased the chances of quitting more than twofold
compared with placebo (RR, 2.27). However, low-dose varenicline
(1 mg/day) roughly doubled the chances of quitting (RR, 2.09) and
reduced the number and severity of adverse effects. In the same review,
varenicline was reported to be better than bupropion in three com-
parison trials (pooled RR, 1.52), but two other trials did not show a
clear benefit of varenicline over the nicotine patch.181
Combinations of First-Line Medications
Combining medications is becoming a trend, in particular for such
traditionally hard-to-treat diseases as tuberculosis, gastric ulcers,
human immunodeficiency virus, and others. Tobacco dependence is
not an exception, as combination therapy is reported to be better
than monotherapy for smoking cessation (Table 24.3).182 It has been
found that all smokers (except light smokers and those who live with
a smoker) may respond better to combination pharmacotherapy than
to monotherapy.183 Before varenicline was available, the options for
combination were to add NRTs to bupropion or to use a patch with
episodic NRTs (e.g., lozenges and gums).184 Studies designed to compare
these smoking cessation medications alone or in combination reported
that certain combinations were superior: specifically, bupropion plus
lozenges and the patch plus lozenges resulted in higher cessation
rates than did monotherapy bupropion, patch, or lozenges.144,184,185
Now that varenicline is available, combining this medication with
bupropion has gained attention as another option. This combination
is of particular interest because it combines two effective medications
that have different mechanisms of action, with the possibility of
potentiating each other. A particular advantage of this combination
might be the beneficial effect of bupropion as an antidepressant in
the mitigation of negative affect and neuropsychiatric effects resulting
from smoking cessation. A single-arm, open-label pilot study tested
the effectiveness of varenicline-bupropion combination in 38 smokers.
The point prevalence smoking abstinence rates were as high as 70% at
3-month follow-up and almost 60% at 6-month follow-up.186 Three
randomized clinical trials followed, and their results seem to support
the efficacy of combination therapy among heavy smokers (highly
addicted to nicotine), and in particular among male heavy smokers.
The first study of 506 smokers randomized to varenicline-bupropion
combination versus varenicline monotherapy found that smokers given
combination therapy had significantly higher prolonged abstinence
rates at the end of treatment (53.0% for combination versus 43.2%
for monotherapy) and at 6-month follow-up (36.6% for combination
versus 27.6% for monotherapy), but not at 1-year follow-up (30.9%
for combination versus 24.5% for monotherapy).187 The second RCT,
in which pretreatment nicotine patch nonresponders were randomized
to receive the varenicline-bupropion combination versus varenicline
monotherapy, reported increased abstinence rates in the combination
group of up to 6 months among male and highly dependent smokers.188
The third study consisted of 122 male heavy smokers and consolidated
further the findings of the second study, showing a significance of
efficacy for the combination in this group.189 Other controlled trials are
underway to further test the efficacy and safety of this combination.
Second-Line Medications
Smokers who cannot use or tolerate first-line medications are usually
referred to second-line medications such as clonidine or nortripty-
line.158,190,191 Although several clinical trials have already proven their
efficacy, these agents do not have FDA approval for treatment of
tobacco use because they are older medications already available in
generic forms. Specifically, clonidine has shown superiority to placebo
in two meta-analyses, with odds ratios ranging between 2.0 and 2.4.190
Nevertheless, it can be administered three times per day and has a
considerable adverse effect profile (dry mouth, dizziness, somnolence,
and orthostatic hypotension); therefore the use of clonidine remains
narrow even as a second-line treatment. Clinical trials of nortriptyline
found it to be effective, with an odds ratio of 2.1 compared with
placebo.192 However, the potential lethality in an overdose, as well as
anticholinergic and cardiac adverse effects similar to those of clonidine,
limit its use in the general and cancer populations.
Psychosocial Interventions
The Centers for Disease Control and Prevention emphasized the need
to screen all patients at all visits in all health care settings for tobacco
use, and to advise smokers to quit, and refer them to or offer them
treatment.193 Gritz and colleagues194 postulated that the cancer popula-
tion is at the heart of the concern. Smoking rates in certain patients
with cancer are higher than average, and mounting evidence links
smoking to outcomes related to cancer treatment success, survival,
and quality of life.195–200 In patients receiving advanced treatments
such as stem cell transplantation, those who never smoked or recent
quitters were reported to have almost double survivorship rates and
about half as many hospitalization days in comparison with smokers.201
Therefore particular attention must be given to the oncology
setting.22,202–204 In general, patients with cancer were found to be more
motivated to quit smoking than were persons in the general popula-
tion.22,205 This phenomenon can be seen as an advantage and an
opportunity for care providers to intervene and promote smoking
cessation while the tobacco user is motivated by the immediacy of
the consequences of continued use on his or her disease treatment
and recovery.
 Nicotine Dependence: Current Treatments and Future Directions  •  CHAPTER 24 407

Table 24.3 Meta-analysis (in CPG-TTUD 2008)a: Effectiveness and Abstinence Rates for Various
Medications and Medication Combinations Compared With Placebo 6 Months After Quitting
(N = 83 Studies)
No. of Estimated Odds Ratio Estimated Abstinence
Medication Arms (95% CI) Rate (95% CI)
Placebo 80 1.0 13.8
MONOTHERAPIES
Varenicline (2 mg/day) 5 3.1 (2.5–3.8) 33.2 (28.9–37.8)
Nicotine nasal spray 4 2.3 (1.7–3.0) 26.7 (21.5–32.7)
High-dose nicotine patch (>25 mg) (these patches included both 4 2.3 (1.7–3.0) 26.5 (21.3–32.5)
standard and long-term duration)
Long-term nicotine gum (>14 wk) 6 2.2 (1.5–3.2) 26.1 (19.7–33.6)
Varenicline (1 mg/day) 3 2.1 (1.5–3.0) 25.4 (19.6–32.2)
Nicotine inhaler 6 2.1 (1.5–2.9) 24.8 (19.1–31.6)
Clonidine 3 2.1 (1.2–3.7) 25.0 (15.7–37.3)
Bupropion sustained release (SR) 26 2.0 (1.8–2.2) 24.2 (22.2–26.4)
Nicotine patch (6–14 wk) 32 1.9 (1.7–2.2) 23.4 (21.3–25.8)
Long-term nicotine patch (>14 wk) 10 1.9 (1.7–2.3) 23.7 (21.0–26.6)
Nortriptyline 5 1.8 (1.3–2.6) 22.5 (16.8–29.4)
Nicotine gum (6–14 wk) 15 1.5 (1.2–1.7) 19.0 (16.5–21.9)
COMBINATION THERAPIES
Patch (long-term; >14 wk) + ad lib NRT (gum or spray) 3 3.6 (2.5–5.2) 36.5 (28.6–45.3)
Patch + bupropion SR 3 2.5 (1.9–3.4) 28.9 (23.5–35.1)
Patch + nortriptyline 2 2.3 (1.3–4.2) 27.3 (17.2–40.4)
Patch + inhaler 2 2.2 (1.3–3.6) 25.8 (17.4–36.5)
Patch + second generation antidepressants (e.g., paroxetine, venlafaxine) 3 2.0 (1.2–3.4) 24.3 (16.1–35.0)
MEDICATIONS NOT SHOWN TO BE EFFECTIVE
Selective serotonin reuptake inhibitors 3 1.0 (0.7–1.4) 13.7 (10.2–18.0)
Naltrexone 2 0.5 (0.2–1.2) 7.3 (3.1–16.2)

a
The articles in this meta-analysis can be found at ww.surgeongeneral.gov/tobacco/gdlnrefs.htm.
CCP-TTUD, Clinical Practice Guideline for Treating Tobacco Use and Dependence; CI, confidence interval; NRT, nicotine replacement therapy.
From Fiore MC, Jaen CR, Baker TB, et al. Treating Tobacco Use and Dependence: 2008 Update. U.S. Public Health Service Clinical Practice Guideline. 2008.

Certain issues are particular to the application of smoking cessation Quitlines

programs among patients with cancer. On the one hand, patients
with cancer may be susceptible to guilt and blame attributions206; on
the other hand, the clinician must be knowledgeable and alert to
comorbid disorders, oncologic treatment adverse effects, and medical
contraindications for medications when treating patients undergoing
treatment for cancer. The CPG-TTUD, in 2000 and again in 2008,
recommended that all physicians and health care providers advise
their patients to quit.207 This guideline included the five necessary
steps in tobacco-dependence treatment, also known as the five As:
ask, advise, assess, assist, and arrange. Fiore and colleagues208 suggested
adding smoking to the vital signs checkup of every patient to ensure
proper documentation, systematic screening, and ongoing monitoring
of tobacco use status. Furthermore, electronic health records can be
used to increase the identification of smokers and facilitate referrals
to treatment.209,210 Interesting to note, research has shown that even
short interventions (3 minutes or less) would have an effect on
abstinence. However, as the number of sessions increases, in particular
for face-to-face interventions, abstinence rates rise as well.6 Therefore
the CPG-TTUD recommends at least a minimal intervention of brief
support in addition to medications, in particular if the patient does
not have access to or will not ultimately receive a full tobacco treatment
intervention.
Quitlines provide physicians with an alternative easy-access treatment,
which can be especially helpful for physicians who are not affiliated
with hospitals or work in hospitals that do not have a tobacco treatment
program (TTP). Quitlines are a good option for patients who are
unable or unwilling to travel to have an in-person appointment. In
addition, quitlines provide telephone-based, individual counseling for
smoking cessation and are recommended as effective in the CPG-
TTUD6 and a Cochrane review.211 Quitlines have been referred to as
an underrecognized success story212 and a tool that has been effective
for both young adult smokers and older smokers213 and for African
Americans as well as white smokers.214
In the United States, each state offers telephone-based quit-
line counseling for smoking cessation. Many quitlines also offer
Internet-based assistance. Tobacco users can access telephone quitline
counseling in all 50 states, the District of Columbia, Guam, and
Puerto Rico by calling the national quitline portal at 1-800-QUIT-
NOW. Callers are routed to their appropriate state quitline based
on the area code of the landline they are calling from or the loca-
tion of the cell phone tower that routes the call. For more general
information about quitlines, see http://www.naquitline.org/?page
=whatisquitline.
408 Part I: Science and Clinical Oncology
Most state quitlines also have health care provider referral programs,
wherein patients can be referred to quitline services by their physician
or other health care provider via fax or e-mail, or the referral can be
integrated into an electronic health record.215 Quitlines then make
proactive outbound calls to referred tobacco users to enroll them in
quitline services and provide feedback to the referring clinic. Research
indicates that patients referred to a quitline by their health care provider
are more likely to quit smoking successfully than are those who
self-refer.216
Once enrolled in quitline services, tobacco users also have access to
free NRTs, when a state program’s budget allows that expenditure, in
70% of states and in a few cases prescription medications (bupropion
or varenicline). Information about each state quitline, including specific
services offered, hours of operations, and contact information for fax
and e-referral programs, can be found on the North American Quitline
Association website at http://map.naquitline.org. The National Cancer
Institute also provides cessation assistance that includes online and
telephone-based support. Telephone counseling and texted support are
available at https://www.smokefree.gov or by calling 1-877-44U-QUIT.
Pharmacotherapy is encouraged but not provided with those innovative
programs.
Self-Help Materials
Self-help smoking cessation interventions are significantly better than
no intervention at all; they are reported to produce quit rates in the
range of 4% to 11%.217 An important new vehicle is the Internet,
through which self-help smoking cessation interventions can be delivered
effectively.218,219 Although few studies have evaluated the efficacy of
Internet-based cessation approaches, in one study a tailored Internet-
based cessation intervention resulted in a quit rate of 24% compared
with 5% in the control condition at 3-month follow-up.220,221 This
finding is consistent with other preliminary work showing the efficacy
of Internet-based cessation interventions.222 More controlled trials of
Internet-based approaches are needed for this medium to be recom-
mended for the delivery of tobacco and ND treatment.
CESSATION TREATMENTS FOR PATIENTS
WITH CANCER: AVAILABILITY
AND CHALLENGES
Cancer treatments and their contraindications impose certain limitations
for clinicians who treat smokers who wish to quit. Particular attention
should be given to the type of pharmacologic or adjuvant treatment
given to smokers and any interaction with the current cancer type
and cancer treatment type, in addition to psychiatric comorbidity
(e.g., alcohol dependence and depression).197,223 More research is needed
to identify proper and tailored smoking cessation treatments for the
cancer population.128
Gritz and colleagues194 have identified several intervention studies
that addressed smoking cessation among patients with cancer. Two
studies have assessed the role and efficacy of physician-patient interven-
tions.224,225 In the first study, the authors compared patients with
cancer who were randomly assigned to full physician advice, tailored
booklets, and booster advice sessions versus patients who were offered
minimal advice sessions, as a control group. In the second study, the
authors compared intensive cognitive behavioral interventions and
patient education and advice. Both studies found no significant dif-
ference between the two conditions; however, in the first study up to
70% of the participants remained abstinent at 1-year follow-up, and
in the latter study about 40% remained abstinent at 3-month follow-up.
Even though no single treatment was found to be superior in helping
patients with cancer to quit smoking, assistance and advice in general
generated good abstinence rates. The findings thus support incorpora-
tion of some level of advice that could be tailored to the patient and
type of cancer diagnosis.
Other studies examined nurse-delivered interventions in smoking
cessation across various cancer diagnoses. A single-session intervention
had lower cessation rates and multisession interventions had the highest
rates.226,227 Although the samples differed greatly in size and composi-
tion, these authors believed that these results were promising and that
nurse-based interventions should be explored further. Moreover, a
retrospective study by Garces and colleagues228 emphasized the need
for early interventions. The analysis showed higher abstinence rates
among patients with cancer who were treated within 3 months of
diagnosis versus those treated more than 3 months after diagnosis.
Finally, a trial assessed peer-based counseling versus self-help interven-
tion in young adults who had survived childhood cancer.229 Higher
rates of smoking cessation were found in the counseling group at
12-month follow-up. Another study comparing a mixed group of
patients with cancer who received motivational interviewing–based
intervention versus those who received usual care consisting of brief
smoking cessation advice did not find the results to be significantly
different.230
In general, the combination of psychosocial therapies and medication
doubles the odds that patients will quit smoking, despite the multiple
types and levels of intensity for the psychosocial therapies (see Table
24.3). A review of controlled trials among patients with cancer suggested
that the combination of medication and therapy is needed to make
a significant difference in smoking cessation in the oncology setting.127
Furthermore, the American Society of Addiction Medicine has described
several possible levels of care and different types of treatment programs
for all addictions, with those levels of care and types of programs
based on whether concurrent psychiatric disorders are diagnosed and
treated.231
Tobacco Treatment Program at MD Anderson
The TTP at MD Anderson Cancer Center was conceived and built
as a center of excellence program with a multidisciplinary team. The
TTP goal is to tailor tobacco dependence treatment to each person,
because each individual is thought to be unique in terms of facing
addiction. The treatment is adapted to each patient’s own history,
current level of ND, intensity of distress due to the cancer diagnosis,
and any concurrent psychiatric disorder. The counseling staff includes
both masters- and doctoral-level counselors who address the psychosocial
aspect of the addiction, and the patient further benefits from the
involvement of a physician assistant, a nurse, and an addiction psy-
chiatrist, who focus on the medical and psychiatric aspect of the
patient’s treatment. The TTP’s mission is to evaluate and treat all
patients with cancer and employees at MD Anderson Cancer Center,
as well as their household members, when their smoking is an obstacle
to the success of the patient with cancer in quitting. Every patient
who indicates tobacco use in the past 12 months is automatically
referred to the TTP via the electronic health record. Referred patients
are proactively contacted by TTP staff, who provide a motivational
interaction and program description. Patients may elect to receive
self-help materials or assistance from a counselor by phone or in
person. Those who choose in-person assistance also receive pharma-
cotherapy and psychologic and psychiatric treatment. The TTP has
been expanded to all three Anderson regional care centers currently
in operation. Videoconferencing is used to optimize centralized expertise
across a diverse infrastructure and to expand treatment as needed
(with a fast Intranet connection and a new generation of video cameras
and software). In addition, the TTP is currently piloting delivery of
follow-up sessions by Web camera to the patient’s home, using special
software and a high security standard to safeguard patients’ confidential-
ity. This approach is in lieu of the usual telephone counseling that
currently takes place for follow-up when patients are outside the
metropolitan area or are simply not able to come to the medical center
regularly.
Currently, over 5000 patients are referred to the TTP each year.
Over 80% receive a motivational interaction, and approximately 20%
 Nicotine Dependence: Current Treatments and Future Directions  •  CHAPTER 24 409

to 25% choose assistance from a counselor. Since the program’s opening
in 2006, TTP counselors have served over 5000 unique patients, with
36% to 47% reporting abstinence at 9-month follow-up.232
CHALLENGES AND FUTURE DIRECTIONS
Many challenges remain to be overcome in treating tobacco dependence,
in particular in the oncology setting. For example, geographic distance
has often been a major obstacle for highly specialized and high-quality
interventions. However, with advances in videoconferencing and
telemedicine and their availability in remote locations, this problem
can be solved.233
As mentioned in the genetics section, a major challenge and yet
an opportunity to advance the current pharmacologic treatments for
smoking cessation is to match a person’s genetic profile7 both to a
successful pharmacologic intervention and to a lower side effect profile
within clinical practice and clinical trial guidelines.
Single-medication trials have led to around 40% cessation rates
by the end of treatment. However, another 40% of participants have
a partial response (they reduce their smoking but do not achieve total
abstinence). Therefore combining medications is being suggested as
a strategy for all hard-core smokers.183 This approach can be imple-
mented by augmentation (i.e., adding another medication or agent
to the one already in place for partial responders), which is a natural
and logical approach for a patient who is engaged in treatment and
is motivated to quit. Another strategy is prescribing a combination
of medications to all or certain smokers from the beginning of cessation
efforts. However, further studies are needed in this area to determine
whether one strategy is superior.
The traditional approach and the vast majority of pharmacotherapy
clinical trials ask participants to choose a quit date within 1 to 2
weeks of starting medications. However, that practice was born out
of the practical need to test a hypothesis in a study within a short
8- to 12-week period. Allowing patients to choose their own interval
for a quit date or not to set a quit date but rather to reduce gradually
and then quit has been suggested in some studies167; however, these
options have not been systematically tested against the strategy of
setting a fixed quit date. Therefore more work is needed to clarify
this important area.
Another area for future study involves the impact that increased
use of electronic nicotine delivery systems (ENDS) such as electronic
cigarettes (e-cigarettes) have on nicotine dependence and smoking
cessation. ENDS were introduced in the United States in 2007. Public
awareness among adults had grown to over 75% by 2012, and 88%
of current smokers were aware of them.234 The technology is rapidly
changing, and many ENDS are available.235 Most e-cigarette users
also consume combustible tobacco products.234 Although first-generation
e-cigarettes were ineffective at delivering nicotine levels comparable
to those obtained from smoking cigarettes,236 e-cigarette technology
is becoming more sophisticated.237 In 2016, the US Surgeon General
issued his first report on e-cigarettes, which indicates that the rate of
e-cigarette use is increasing among youth and young adults and that
this population is at risk of transitioning from the use of e-cigarettes
to combustible tobacco, although 80% appear to be using nonnicotine
e-cigarettes.238 Although the US Surgeon General does not recommend
the use of e-cigarettes for smoking cessation, public health authorities
in the United Kingdom have stated that the use of e-cigarettes is
preferable to combustible tobacco because of reduced exposure to
carcinogens, especially in patients who would otherwise not quit
smoking.239 The use of e-cigarettes among cancer patients has not
been systemically studied, except in one retrospective study that did
not find them to be helpful for cancer patients to quit smoking.240
Thus more work is needed to assess the current prevalence of e-cigarette
use among cancer patients and any impact that e-cigarettes may have
on smoking cessation in this population.
In summary, making tobacco use and dependence a major focus
of patient-centered behavioral change during cancer treatment and
throughout survivorship is a key issue for oncology providers and
cancer care health systems. The field of tobacco dependence treat-
ment is evolving rapidly, with significant increases in cessation rates
and the promise of more powerful pharmacotherapy combined
with greater reach and intensity of treatment. The goal will be to
integrate state-of-the-art and emerging treatments with ever-advancing
oncology care.
The complete reference list is available online at
ExpertConsult.com.

KEY REFERENCES
1. World Health Organization. The Top 10 Causes of
Death. www.who.int/mediacentre/factsheets/fs310/
en/index.html. Updated June 28, 2012.
2. IARC. IARC Monographs on the Evaluation
of Carcinogenic Risks to Humans. http://
monographs.iarc.fr/ENG/Monographs/vol97/
index.php. Updated July 19, 2012.
4. Pontieri FE, Tanda G, Orzi F, Di CG. Effects
of nicotine on the nucleus accumbens and
similarity to those of addictive drugs. Nature.
1996;382(6588):255–257.
6. Fiore MC, Jaen CR, Baker TB, et al. Treating
Tobacco Use and Dependence: 2008 Update,
Clinical Practice Guideline. PM:18807274.
7. King DP, Paciga S, Pickering E, et al. Smoking
cessation pharmacogenetics: analysis of varenicline
and bupropion in placebo-controlled clinical
trials. Neuropsychopharmacology. 2012;37(3):641–
650.
8. McBride CM, Ostroff JS. Teachable moments
for promoting smoking cessation: the context
of cancer care and survivorship. Cancer Control.
2003;10(4):325–333.
13. Jamal A, King BA, Neff LJ, Whitmill J, Babb
SD, Graffunder CM. Current Cigarette Smoking
Among Adults—United States, 2005–2015.
MMWR Morb Mortal Wkly Rep. 2016;65(44):1205–
1211.
14. US Department of Health and Human Services.
The Health Consequences of Smoking—50 Years of
Progress: A Report of the Surgeon General. Atlanta,
GA; 2014. Surgeon General’s Report.
17. US Centers for Disease Control and Prevention.
The CDC Health Disparities and Inequalities
Report—United States. 2011. http://www.cdc.gov/
minorityhealth/reports/CHDIR11/FactSheets/
Smoking.pdf. Updated June 29, 2012.
24. Gritz ER, Schacherer C, Koehly L, Nielsen IR,
Abemayor E. Smoking withdrawal and relapse
in head and neck cancer patients. Head Neck.
1999;21(5).
25. Cooley ME, Sarna L, Kotlerman J, et al. Smoking
cessation is challenging even for patients recovering
from lung cancer surgery with curative intent. Lung
Cancer. 2009;66(2):218–225.
26. McBride CM, Emmons KM, Lipkus IM. Under-
standing the potential of teachable moments:
the case of smoking cessation. Health Educ Res.
2003;18(2):156–170.
27. Moyer VA. Screening for lung cancer: U.S. Preventive
Services Task Force recommendation statement. Ann
Intern Med. 2014;160(5):330–338.
31. Nisell M, Marcus M, Nomikos GG, Svensson TH.
Differential effects of acute and chronic nicotine on
dopamine output in the core and shell of the rat
nucleus accumbens. J Neural Transm. 1997;104:1–10.
32. Pidoplichko VL, DeBiasi M, Williams JT, Dani JA.
Nicotine activates and desensitizes midbrain dopa-
mine neurons. Nature. 1997;390(6658):401–404.
36. Benowitz NL. Neurobiology of nicotine addiction:
implications for smoking cessation treatment. Am
J Med. 2008;121(4 suppl 1):S3–S10.
37. Volkow ND, Fowler J, Wang G-J. Role of dopamine
in drug reinforcement and addiction in humans:
Results from imaging studies. Behav Pharmacol.
2002;13:355–366.
38. Robinson TE, Berridge KC. Incentive-sensitization
and addiction. Addiction. 2001;96(1):103–114.
48. Tobacco and Genetics Consortium. Genome-wide
meta-analyses identify multiple loci associated with
smoking behavior. Nat Genet. 2010;42(5):441–447.
52. Amos CI, Wu X, Broderick P, et al. Genome-wide
association scan of tag SNPs identifies a susceptibil-
ity locus for lung cancer at 15q25.1. Nat Genet.
2008;40(5):616–622.
69. Schnoll RA, Leone FT. Biomarkers to optimize
the treatment of nicotine dependence. Biomarkers.
2011;5(6):745–761.
100. Lerman C, Schnoll RA, Hawk LW Jr, et al. Use
of the nicotine metabolite ratio as a genetically
informed biomarker of response to nicotine patch
or varenicline for smoking cessation: a randomised,
double-blind placebo-controlled trial. Lancet Respir
Med. 2015;3(2):131–138.
410 Part I: Science and Clinical Oncology
101. Lasser K, Boyd JW, Woolhandler S, Himmelstein
DU, McCormick D, Bor DH. Smoking and mental
illness: A population-based prevalence study. JAMA.
2000;284(20):2606–2610.
102. Breslau N, Johnson EO, Hiripi E, Kessler R. Nicotine
dependence in the United States: prevalence, trends,
and smoking persistence. Arch Gen Psychiatry.
2001;58(9):810–816.
103. Covey LS, Hughes DC, Glassman AH, Blazer
DG, George LK. Ever-smoking, quitting, and
psychiatric disorders: Evidence from the Durham,
North Carolina, epidemiologic catchment area. Tob
Control. 1994;3:222–227.
104. Glassman AH, Helzer JE, Covey LS, et al. Smoking,
smoking cessation, and major depression. JAMA.
1990;264(12):1546–1549.
105. Williams JM, Ziedonis D. Addressing tobacco among
individuals with a mental illness or an addiction.
Addict Behav. 2004;29(6):1067–1083.
111. Grant BF, Hasin DS, Chou SP, Stinson FS, Dawson
DA. Nicotine dependence and psychiatric disorders
in the United States: results from the national epide-
miologic survey on alcohol and related conditions.
Arch Gen Psychiatry. 2004;61(11):1107–1115.
115. Johnson RA, Hoffmann JP. Adolescent cigarette
smoking in U.S. racial/ethnic subgroups: findings
from the National Education Longitudinal Study.
J Health Soc Behav. 2000;41(4):392–407.
116. Kendler KS, Neale MC, MacLean CJ, Heath
AC, Eaves LJ, Kessler RC. Smoking and major
depression: a casual analysis. Arch Gen Psychiatry.
1993;50(1):36–43.
128. US National Cancer Institute. Smoking Cessation
and Continued Risk in Cancer Patients. Bethesda,
MD 2012. http://www.cancer.gov/cancertopics/pdq/
supportivecare/smokingcessation/HealthProfessional.
Updated July 2, 2012.
143. Silagy C, Lancaster T, Stead L, Mant D,
Fowler G. Nicotine replacement therapy for
smoking cessation. Cochrane Database Syst Rev.
2004;(3):CD000146-CD000146.
147. Karam-Hage M, Cinciripini PM. Pharmaco-
therapy for tobacco cessation: nicotine agonists,
antagonists, and partial agonists. Curr Oncol Rep.
2007;9(6):509–516.
163. Gonzales D, Rennard SI, Nides M, et al. Var-
enicline, an alpha4beta2 nicotinic acetylcholine
receptor partial agonist, vs sustained-release
bupropion and placebo for smoking cessation: a
randomized controlled trial. JAMA. 2006;296(1):47–
55.
164. Gonzales DH, Rennard SI, Billing CB, Reeves K,
Watsky E, Gong J. A pooled analysis of varenicline,
an alpha 4 beta 2 nicotinic receptor partial agonist
vs bupropion, and placebo for smoking cessation.
February; Orlando, FL.
165. Jorenby DE, Hays JT, Rigotti NA, et al. Efficacy of
varenicline, an alpha4beta2 nicotinic acetylcholine
receptor partial agonist, vs placebo or sustained-
release bupropion for smoking cessation: a
randomized controlled trial. JAMA. 2006;296(1):
56–63.
170. Anthenelli RM, Benowitz NL, West R, et al.
Neuropsychiatric safety and efficacy of varenicline,
bupropion, and nicotine patch in smokers with and
without psychiatric disorders (EAGLES): a double-
blind, randomised, placebo-controlled clinical trial.
Lancet. 2016;387(10037):2507–2520.
184. Piper ME, Smith SS, Schlam TR, et al. A random-
ized placebo-controlled clinical trial of 5 smoking
cessation pharmacotherapies. Arch Gen Psychiatry.
2009;66(11):1253–1262.
185. Smith SS, McCarthy DE, Japuntich SJ, et al.
Comparative effectiveness of 5 smoking cessation
pharmacotherapies in primary care clinics. Arch
Intern Med. 2009;169(22):2148–2155.
194. Gritz ER, Lam CY, Vidrine DJ, Fingeret MC.
Tobacco dependence and its treatment. In: DeVita V,
Lawrence T, Rosenberg S, eds. Cancer: Principles and
Practice of Oncology. 9th ed. Philadelphia: Lippincott
Williams & Wilkins; 2012:529–542.
196. Gritz ER, Dresler C, Sarna L. Smoking, the
missing drug interaction in clinical trials: ignoring
the obvious. Cancer Epidemiol Biomarkers Prev.
2005;14(10):2287–2293.
197. Gritz ER, Fingeret MC, Vidrine DJ, Lazev AB,
Mehta NV, Reece GP. Successes and failures of the
teachable moment: Smoking cessation in cancer
patients. Cancer. 2006;106(1):17–27.
198. Jensen K, Jensen AB, Grau C. Smoking has a nega-
tive impact upon health related quality of life after
treatment for head and neck cancer. Oral Oncol.
2007;43(2):187–192.
206. Gritz ER, Fingeret MC, Vidrine DJ. Tobacco control
in the oncology setting. 2007. Cancer Prevention:
An ASCO Curriculum.
212. Lichtenstein E, Zhu SH, Tedeschi GJ. Smoking
cessation quitlines: an underrecognized intervention
success story. Am Psychol. 2010;65(4):252–261.
222. Walters ST, Wright JA, Shegog R. A review of com-
puter and Internet-based interventions for smoking
behavior. Addict Behav. 2006;31(2):264–277.

REFERENCES
1. World Health Organization. The Top 10 Causes of
Death. www.who.int/mediacentre/factsheets/fs310/
en/index.html. Updated June 28, 2012.
2. IARC. IARC Monographs on the Evaluation of
Carcinogenic Risks to Humans. http://monographs.
iarc.fr/ENG/Monographs/vol97/index.php. Updated
July 19, 2012.
3. Jacobs EJ, Newton CC, Carter BD, et al. What
proportion of cancer deaths in the contemporary
United States is attributable to cigarette smoking?
Ann Epidemiol. 2015;25(3):179–182.
4. Pontieri FE, Tanda G, Orzi F, Di CG. Effects
of nicotine on the nucleus accumbens and
similarity to those of addictive drugs. Nature.
1996;382(6588):255–257.
5. Volkow ND, Fowler JS, Wang GJ. The addicted
human brain viewed in the light of imaging studies:
Brain circuits and treatment strategies. Neurophar-
macology. 2004;47(suppl 1):3–13.
6. Fiore MC, Jaen CR, Baker TB, et al Treating
tobacco use and dependence: 2008 Update U.S.
Public Health Service Clinical Practice Guideline
executive summary. Respir Care. 2008;53(9):1217–
1222.
7. King DP, Paciga S, Pickering E, et al. Smoking
cessation pharmacogenetics: analysis of varenicline
and bupropion in placebo-controlled clinical trials.
Neuropsychopharmacology. 2012;37(3):641–650.
8. McBride CM, Ostroff JS. Teachable moments
for promoting smoking cessation: the context
of cancer care and survivorship. Cancer Control.
2003;10(4):325–333.
9. Carson T, Bonk M. James Buchanan Duke. Gale
Encyclopedia of U.S. Economic History. Detroit: Gale
Group; 1999.
10. James R. Cigarette Advertising. http://www.time.
com/time/magazine/article/0,9171,1905530,00.
html. Updated June 29, 2012.
11. US Department of Health and Human Services.
The health consequences of smoking: a report of
the surgeon general. http://profiles.nlm.nih.gov/
NN/B/B/M/Q/.
12. Newport F, Moore DW, Saad L. Long-term Gallup
poll trends: a portrait of American public opinion
through the century. http://www.gallup.com/
poll/3400/longterm-gallup-poll-trends-portrait-
american-public-opinion.aspx. Updated June 29,
2012.
13. Jamal A, King BA, Neff LJ, Whitmill J, Babb SD,
Graffunder CM. Current Cigarette Smoking Among
Adults—United States, 2005-2015. MMWR Morb
Mortal Wkly Rep. 2016;65(44):1205–1211.
14. US Department of Health and Human Services.
The Health Consequences of Smoking—50 Years of
Progress: A Report of the Surgeon General. Atlanta,
GA; 2014. Surgeon General’s Report.
15. US Centers for Disease Control and Prevention.
Tobacco data statistics Pre-1994. http://www.cdc.
gov/tobacco/data_statistics/sgr/pre_1994/index.htm.
Updated June 29, 2012.
16. Martell BN, Garrett BE, Caraballo RS. Disparities in
Adult Cigarette Smoking—United States, 2002-2005
and 2010-2013. MMWR Morb Mortal Wkly Rep.
2016;65(30):753–758.
17. US Centers for Disease Control and Prevention.
The CDC Health Disparities and Inequalities
Report—United States, 2011. http://www.cdc.
gov/minorityhealth/reports/CHDIR11/FactSheets/
Smoking.pdf. Updated June 29, 2012.
18. NCI. Cancer Trends Progress Report—2009/2010
Update. http://progressreport.cancer.gov/. Updated
June 29, 2012.
19. Bellizzi KM, Rowland JH, Jeffery DD, McNeel T.
Health behaviors of cancer survivors: examining
opportunities for cancer control intervention.
J Clin Oncol. 2005;23(34):8884–8893.
Nicotine Dependence: Current Treatments and Future Directions  •  CHAPTER 24 410.e1
20. Schnoll RA, Zhang B, Rue M, et al. Brief
physician-initiated quit-smoking strategies for
clinical oncology settings: a trial coordinated by
the Eastern Cooperative Oncology Group. J Clin
Oncol. 2003;21(2):355–365.
21. Gritz ER, Nisenbaum R, Elashoff R, Holmes EC.
Smoking behavior following diagnosis in patients
with stage I non-small cell lung cancer. Cancer Causes
Control. 1991;2:105–112.
22. Ostroff JS, Jacobsen PB, Moadel AB, et al. Prevalence
and predictors of continued tobacco use after treat-
ment of patients with head and neck cancer. Cancer.
1995;75(2):569–576.
23. Ostroff J, Garland J, Moadel A, et al. Cigarette
smoking patterns in patients after treatment of
bladder cancer. J Cancer Educ. 2000;15(2):86–90.
24. Gritz ER, Schacherer C, Koehly L, Nielsen IR,
Abemayor E. Smoking withdrawal and relapse in
head and neck cancer patients. Head Neck. 1999;
21(5).
25. Cooley ME, Sarna L, Kotlerman J, et al. Smoking
cessation is challenging even for patients recovering
from lung cancer surgery with curative intent. Lung
Cancer. 2009;66(2):218–225.
26. McBride CM, Emmons KM, Lipkus IM. Under-
standing the potential of teachable moments:
the case of smoking cessation. Health Educ Res.
2003;18(2):156–170.
27. Moyer VA. Screening for lung cancer: U.S. Preventive
Services Task Force recommendation statement. Ann
Intern Med. 2014;160(5):330–338.
28. Slatore CG, Baumann C, Pappas M, Humphrey
LL. Smoking behaviors among patients receiving
computed tomography for lung cancer screen-
ing. Systematic review in support of the U.S.
preventive services task force. Ann Am Thorac Soc.
2014;11(4):619–627.
29. Clark MA, Gorelick JJ, Sicks JD, et al. The rela-
tions between false positive and negative screens
and smoking cessation and relapse in the National
Lung Screening Trial: implications for public health.
Nicotine Tob Res. 2016;18(1):17–24.
30. Fucito LM, Czabafy S, Hendricks PS, Kotsen C,
Richardson D, Toll BA. Pairing smoking-cessation
services with lung cancer screening: a clinical
guideline from the Association for the Treatment
of Tobacco Use and Dependence and the Society
for Research on Nicotine and Tobacco. Cancer.
2016;122(8):1150–1159.
31. Nisell M, Marcus M, Nomikos GG, Svensson TH.
Differential effects of acute and chronic nicotine on
dopamine output in the core and shell of the rat
nucleus accumbens. J Neural Transm. 1997;104:1–10.
32. Pidoplichko VL, DeBiasi M, Williams JT, Dani JA.
Nicotine activates and desensitizes midbrain dopa-
mine neurons. Nature. 1997;390(6658):401–404.
33. Carr LA, Basham JK, York BK, Rowell PP. Inhibition
of uptake of 1-methyl-4-phenylpyridinium ion and
dopamine in striatal synaptosomes by tobacco smoke
components. Eur J Pharmacol. 1992;215:285–287.
34. Le Foll B, Forget B, Aubin H-J, Goldberg SR. Block-
ing cannabinoid CB1 receptors for the treatment of
nicotine dependence: insights from pre-clinical and
clinical studies. Addict Biol. 2008;13(2):239–252.
35. Lowinson J, Ruiz P, Millman R, Langrod J, eds.
Substance Abuse A Comprehensive Textbook. Maryland:
Williams & Wilkins; 1992.
36. Benowitz NL. Neurobiology of nicotine addiction:
implications for smoking cessation treatment. Am
J Med. 2008;121(4 suppl 1):S3–S10.
37. Volkow ND, Fowler J, Wang G-J. Role of dopamine
in drug reinforcement and addiction in humans:
Results from imaging studies. Behav Pharmacol.
2002;13:355–366.
38. Robinson TE, Berridge KC. Incentive-sensitization
and addiction. Addiction. 2001;96(1):103–114.
410.e1
39. Curtin JJ, McCarthy DE, Piper ME, Baker TB.
Implicit and explicit drug motivational processes:
A model of boundary conditions. In: Reinout R,
Stacy A, eds. Handbook of Implicit Cognition and
Addiction. Thousand Oaks, CA.: SAGE Publications;
2006:233–250.
40. Baker TB, Piper ME, McCarthy DE, Majeskie MR,
Fiore MC. Addiction motivation reformulated: An
affective processing model of negative reinforcement.
Psychol Rev. 2004;111(1):33–51.
41. Gotti C, Zoli M, Clementi F. Brain nicotinic acetyl-
choline receptors: native subtypes and their relevance.
Trends Pharmacol Sci. 2006;27(9):482–491.
42. De Biasi M, Dani JA. Reward, addiction, withdrawal
to nicotine. Annu Rev Neurosci. 2011;34:105–
130.
43. Conlon MS, Bewick MA. Single nucleotide poly-
morphisms in CHRNA5 rs16969968, CHRNA3
rs578776, and LOC123688 rs8034191 are
associated with heaviness of smoking in women
in northeastern Ontario, Canada. Nicotine Tob Res.
2011;13(6):498–503.
44. Saccone NL, Culverhouse RC, Schwantes-An TH,
et al. Multiple independent loci at chromosome
15q25.1 affect smoking quantity: a meta-analysis
and comparison with lung cancer and COPD. PLoS
Genet. 2010;6(8):1–16.
45. Bierut LJ, Stitzel JA, Wang JC, et al. Variants in
nicotinic receptors and risk for nicotine dependence.
Am J Psychiatry. 2008;165(9):1163–1171.
46. Lips EH, Gaborieau V, McKay JD, et al. Association
between a 15q25 gene variant, smoking quantity and
tobacco-related cancers among 17 000 individuals.
Int J Epidemiol. 2010;39(2):563–577.
47. Liu JZ, Tozzi F, Waterworth DM, et al. Meta-analysis
and imputation refines the association of 15q25 with
smoking quantity. Nat Genet. 2010;42(5):436–440.
48. Tobacco and Genetics Consortium. Genome-wide
meta-analyses identify multiple loci associated
with smoking behavior. Nature Genet. 2010;
42(5):441–447.
49. Olfson E, Saccone NL, Johnson EO, et al. Rare, low
frequency and common coding variants in CHRNA5
and their contribution to nicotine dependence in
European and African Americans. Mol Psychiatry.
2016;21(5):601–607.
50. Tyndale RF, Zhu AZ, George TP, et al. Lack of
associations of CHRNA5-A3-B4 Genetic variants
with smoking cessation treatment outcomes in
Caucasian smokers despite associations with baseline
smoking. PLoS ONE. 2015;10(5):e0128109.
51. Chen L-S, Horton A, Bierut L. Pathways to precision
medicine in smoking cessation treatments. Neurosci
Lett. 2016.
52. Amos CI, Wu X, Broderick P, et al. Genome-wide
association scan of tag SNPs identifies a susceptibil-
ity locus for lung cancer at 15q25.1. Nat Genet.
2008;40(5):616–622.
53. Thorgeirsson TE, Geller F, Sulem P, et al. A
variant associated with nicotine dependence, lung
cancer and peripheral arterial disease. Nature.
2008;452(7187):638–642.
54. Huang W, Payne TJ, Ma JZ, et al. Significant
association of ANKK1 and detection of a functional
polymorphism with nicotine dependence in an
African-American sample. Neuropsychopharmacology.
2009;34(2):319–330.
55. Thorgeirsson TE, Gudbjartsson DF, Surakka I,
et al. Sequence variants at CHRNB3-CHRNA6
and CYP2A6 affect smoking behavior. Nat Genet.
2010;42(5):448–453.
56. Gelernter J, Yu Y, Weiss R, et al. Haplotype spanning
TTC12 and ANKK1, flanked by the DRD2 and
NCAM1 loci, is strongly associated to nicotine
dependence in two distinct American populations.
Human Mol Genet. 2006;15(24):3498–3507.
410.e2 Part I: Science and Clinical Oncology
57. Ruyck K, de Nackaerts K, Beels L, et al. Genetic
variation in three candidate genes and nicotine
dependence, withdrawal and smoking cessation in
hospitalized patients. Pharmacogenomics. 2010;11(8):
1053–1063.
58. Voisey J, Swagell CD, Hughes IP, et al. A DRD2
and ANKK1 haplotype is associated with nicotine
dependence. Psychiatry Res. 2012.
59. Bergen AW, Conti DV, van Den BD, et al. Dopamine
genes and nicotine dependence in treatment-seeking
and community smokers. Neuropsychopharmacology.
2009;34(10):2252–2264.
60. Wang J, Li MD. Common and unique biological
pathways associated with smoking initiation/progres-
sion, nicotine dependence, and smoking cessation.
Neuropsychopharmacology. 2010;35(3):702–719.
61. Ling D, Niu T, Feng Y, Xing H, Xu X. Association
between polymorphism of the dopamine transporter
gene and early smoking onset: an interaction
risk on nicotine dependence. J Human Genet.
2004;49:35–39.
62. Segman RH, Kanyas K, Karni O, et al. Why do
young women smoke? IV. Role of genetic variation
in the dopamine transporter and lifetime traumatic
experience. Am J Med Genet B Neuropsychiatr Genet.
2007;144B(4):533–540.
63. Chu SL, Xiao D, Wang C, Jing H. Association
between 5-hydroxytryptamine transporter gene-linked
polymorphic region and smoking behavior in Chinese
males. Chin Med J. 2009;122(12):1365–1368.
64. Ishii T, Wakabayashi R, Kurosaki H, Gemma
A, Kida K. Association of serotonin transporter
gene variation with smoking, chronic obstructive
pulmonary disease, and its depressive symptoms.
J Human Genet. 2010;56(1):41–46.
65. Lerman C, Caporaso NE, Audrain J, Main D,
Boyd NR, Shields PG. Interacting effects of the
serotonin transporter gene and neuroticism in
smoking practices and nicotine dependence. Mol
Psychiatry. 2000;5(2):189–192.
66. Bergen AW, Michel M, Nishita D, et al. Drug
metabolizing enzyme and transporter gene variation,
nicotine metabolism, prospective abstinence, and
cigarette consumption. PLoS ONE. 2015;10(7):
e0126113.
67. Kortmann GL, Dobler CJ, Bizarro L, Bau CH. Phar-
macogenetics of smoking cessation therapy. Am J Med
Genet B Neuropsychiatr Genet. 2010;153B(1):17–28.
68. Lessov-Schlaggar CN, Pergadia ML, Khroyan TV,
Swan GE. Genetics of nicotine dependence and
pharmacotherapy. Biochem Pharmacol. 2008;75(1):
178–195.
69. Schnoll RA, Leone FT. Biomarkers to optimize
the treatment of nicotine dependence. Biomarkers.
2011;5(6):745–761.
70. Lerman C, Jepson C, Wileyto EP, et al. Role of
functional genetic variation in the dopamine D2
receptor (DRD2) in response to bupropion and
nicotine replacement therapy for tobacco depen-
dence: results of two randomized clinical trials.
Neuropsychopharmacology. 2006;31(1):231–242.
71. Breitling LP, Twardella D, Hoffmann MM, Witt
SH, Treutlein J, Brenner H. Prospective association
of dopamine-related polymorphisms with smoking
cessation in general care. Pharmacogenomics.
2010;11(4):527–536.
72. Johnstone EC, Yudkin PL, Hey K, et al. Genetic
variation in dopaminergic pathways and short-term
effectiveness of the nicotine patch. Pharmacogenetics.
2004;14(2):83–90.
73. Yudkin P, Munafo M, Hey K, et al. Effectiveness of
nicotine patches in relation to genotype in women
versus men: Randomised controlled trial. BMJ.
2004;328(7446):989–990.
74. David SP, Johnstone EC, Churchman M, Aveyard
P, Murphy MF, Munafo MR. Pharmacogenetics of
smoking cessation in general practice: results from
the patch II and patch in practice trials. Nicotine
Tob Res. 2011;13(3):157–167.
75. Munafò MR, Johnstone EC, Guo B, Murphy MFG,
Aveyard P. Association of COMT Val108/158Met
genotype with smoking cessation. Pharmacogenet
Genomics. 2008;18(2):121–128.
76. David SP, Munafo MR, Murphy MF, Proctor M,
Walton RT, Johnstone EC. Genetic variation in the
dopamine D4 receptor (DRD4) gene and smoking
cessation: follow-up of a randomised clinical trial
of transdermal nicotine patch. Pharmacogenomics J.
2008;8(2):122–128.
77. Lerman C, Wileyto EP, Patterson F. The functional
mu opioid receptor (OPRM1) Asn40Asp variant
predicts short-term response to nicotine replace-
ment therapy in a clinical trial. Pharmacogenomics
J. 2004;4:184–192.
78. Munafò MR, Elliot KM, Murphy MF, Walton RT,
Johnstone EC. Association of the mu-opioid receptor
gene with smoking cessation. Pharmacogenomics J.
2007;7(5):353–361.
79. Ray R, Jepson C, Patterson F, et al. Association of
OPRM1 A118G variant with the relative reinforc-
ing value of nicotine. Psychopharmacology (Berl).
2006;188(3):355–363.
80. David SP, Munafo MR, Murphy MF, Walton RT,
Johnstone EC. The serotonin transporter 5-HTTLPR
polymorphism and treatment response to nicotine
patch: follow-up of a randomized controlled trial.
Nicotine Tob Res. 2007;9(2):225–231.
81. Gilbert DG, Zuo Y, Rabinovich NE, Riise H,
Needham R, Huggenvik JI. Neurotransmission-
related genetic polymorphisms, negative affectiv-
ity traits, and gender predict tobacco abstinence
symptoms across 44 days with and without nicotine
patch. J Abnorm Psychol. 2009;118(2):322–334.
82. Bergen AW, Javitz HS, Krasnow R, Nishita D, Michel
M, Conti DV, et al. Nicotinic acetylcholine receptor
variation and response to smoking cessation thera-
pies. Pharmacogenetic Genomics. 2013;32(2):94–103.
83. Lee W, Bergen AW, Swan GE, et al. Gender-stratified
gene and gene-treatment interactions in smoking
cessation. Pharmacogenomics J. 2012;12(6):521–532.
84. Munafo M, Johnstone EC, Walther D, Uhl GR,
Murphy MF, Aveyard P. CHRNA3 rs1051730
genotype and short-term smoking cessation. Nicotine
Tob Res. 2011;13(10):982–988.
85. David SP, Brown RA, Papandonatos GD, et al.
Pharmacogenetic clinical trial of sustained-release
bupropion for smoking cessation. Nicotine Tob Res.
2007;9(8):821–833.
86. David SP, Strong DR, Munafo MR, et al. Bupropion
efficacy for smoking cessation is influenced by the
DRD2 Taq1A polymorphism: analysis of pooled
data from two clinical trials. Nicotine Tob Res.
2007;9(12):1251–1257.
87. Swan GE, Valdes AM, Ring HZ, et al. Dopamine
receptor DRD2 genotype and smoking cessation
outcome following treatment with bupropion SR.
Pharmacogenomics J. 2005;5:21–29.
88. Berrettini WH, Wileyto EP, Epstein L, et al.
Catechol-O-methyltransferase (COMT) gene variants
predict response to bupropion therapy for tobacco
dependence. Biol Psychiatry. 2007;61(1):111–118.
89. Leventhal AM, David SP, Brightman M, et al.
Dopamine D4 receptor gene variation moderates
the efficacy of bupropion for smoking cessation.
Pharmacogenomics J. 2012;12(1):86–92.
90. Lerman C, Shields PG, Wileyto EP, et al. Effects of
dopamine transporter and receptor polymorphisms
on smoking cessation in a bupropion clinical trial.
Health Psychol. 2003;22(5):541–548.
91. Lee AM, Jepson C, Hoffmann E, et al. CYP2B6 gen-
otype alters abstinence rates in a bupropion smoking
cessation trial. Biol Psychiatry. 2007;62(6):635–641.
92. Conti DV, Lee W, Li D, et al. Nicotinic acetylcholine
receptor beta2 subunit gene implicated in a systems-
based candidate gene study of smoking cessation.
Human Mol Genet. 2008;17(18):2834–2848.
93. Heitjan DF, Guo M, Ray R, Wileyto EP, Epstein LH,
Lerman C. Identification of pharmacogenetic markers
in smoking cessation therapy. Am J Med Genet B
Neuropsychiatr Genet. 2008;147B(6):712–719.
94. Sarginson JE, Killen JD, Lazzeroni LC, et al.
Markers in the 15q24 nicotinic receptor subunit
gene cluster (CHRNA5-A3-B4) predict severity of
nicotine addiction and response to smoking cessation
therapy. Am J Med Genet. 2011;156(3):275–284.
95. Allenby CE, Boylan KA, Lerman C, Falcone M.
Precision medicine for tobacco dependence: devel-
opment and validation of the nicotine metabolite
ratio. J Neuroimmune Pharmacol. 2016;11(3):
471–483.
96. Lerman C, Tyndale R, Patterson F, et al. Nicotine
metabolite ratio predicts efficacy of transdermal
nicotine for smoking cessation. Clin Pharmacol
Ther. 2006;79(6):600–608.
97. Schnoll RA, Patterson F, Wileyto EP, Tyndale
RF, Benowitz N, Lerman C. Nicotine metabolic
rate predicts successful smoking cessation with
transdermal nicotine: A validation study. Pharmacol
Biochem Behav. 2009;92(1):6–11.
98. Ho MK, Mwenifumbo JC, Al Koudsi N, et al.
Association of nicotine metabolite ratio and CYP2A6
genotype with smoking cessation treatment in
African-American light smokers. Clin Pharmacol
Ther. 2009;85(6):635–643.
99. Patterson F, Schnoll RA, Wileyto EP, et al. Toward
personalized therapy for smoking cessation: a
randomized placebo-controlled trial of bupropion.
Clin Pharmacol Ther. 2008;84(3):320–325.
100. Lerman C, Schnoll RA, Hawk LW Jr, et al. Use
of the nicotine metabolite ratio as a genetically
informed biomarker of response to nicotine patch
or varenicline for smoking cessation: a randomised,
double-blind placebo-controlled trial. Lancet Respir
Med. 2015;3(2):131–138.
101. Lasser K, Boyd JW, Woolhandler S, Himmelstein
DU, McCormick D, Bor DH. Smoking and mental
illness: a population-based prevalence study. JAMA.
2000;284(20):2606–2610.
102. Breslau N, Johnson EO, Hiripi E, Kessler R. Nicotine
dependence in the United States: prevalence, trends,
and smoking persistence. Arch Gen Psychiatry.
2001;58(9):810–816.
103. Covey LS, Hughes DC, Glassman AH, Blazer
DG, George LK. Ever-smoking, quitting, and
psychiatric disorders: evidence from the Durham,
North Carolina, epidemiologic catchment area. Tob
Control. 1994;3:222–227.
104. Glassman AH, Helzer JE, Covey LS, et al. Smoking,
smoking cessation, and major depression. JAMA.
1990;264(12):1546–1549.
105. Williams JM, Ziedonis D. Addressing tobacco among
individuals with a mental illness or an addiction.
Addict Behav. 2004;29(6):1067–1083.
106. Barkley RA, Fischer M, Edelbrock CS, Smallish
L. The adolescent outcome of hyperactive children
diagnosed by research criteria: I. An 8-year pro-
spective follow-up study. J Am Acad Child Adolesc
Psychiatry. 1990;29(4):546–557.
107. Borland BL, Heckman HK. Hyperactive boys and
their brothers. A 25-year follow-up study. Arch Gen
Psychiatry. 1976;33(6):669–675.
108. Hartsough CS, Lambert NM. Pattern and progres-
sion of drug use among hyperactives and controls:
a prospective short-term longitudinal study. J Child
Psychol Psychiatry. 1987;28(4):543–553.
109. Breslau N. Psychiatric comorbidity of smoking and
nicotine dependence. Behav Genet. 1995;25(2):
95–101.
110. Batel P, Pessione F, Maitre C, Rueff B. Relationship
between alcohol and tobacco dependencies among
alcoholics who smoke. Addiction. 1995;90(7):
977–980.
111. Grant BF, Hasin DS, Chou SP, Stinson FS, Dawson
DA. Nicotine dependence and psychiatric disorders
in the United States: results from the national epide-
miologic survey on alcohol and related conditions.
Arch Gen Psychiatry. 2004;61(11):1107–1115.

112. Breslau N, Klein DF. Smoking and panic attacks:
an epidemiologic investigation. Arch Gen Psychiatry.
1999;56(12):1141–1147.
113. Breslau N, Peterson EL, Schultz LR, Chilcoat HD,
Andreski P. Major depression and stages of smoking:
a longitudinal investigation. Arch Gen Psychiatry.
1998;55:161–166.
114. Brown DC. Smoking cessation in pregnancy. Can
Fam Physician. 1996;42:102–105.
115. Johnson RA, Hoffmann JP. Adolescent cigarette
smoking in U.S. racial/ethnic subgroups: findings
from the National Education Longitudinal Study.
J Health Soc Behav. 2000;41(4):392–407.
116. Kendler KS, Neale MC, MacLean CJ, Heath
AC, Eaves LJ, Kessler RC. Smoking and major
depression: a casual analysis. Arch Gen Psychiatry.
1993;50(1):36–43.
117. Kollins SH, McClernon FJ, Fuemmeler BF.
Association between smoking and attention-deficit/
hyperactivity disorder symptoms in a population-
based sample of young adults. Arch Gen Psychiatry.
2005;62(10):1142–1147.
118. Martinez E, Tatum KL, Weber DM, et al. Issues
related to implementing a smoking cessation clini-
cal trial for cancer patients. Cancer Causes Control.
2009;20(1):97–104.
119. American Psychiatric Association. Diagnostic and
Statistical Manual of Mental Disorders. 5th ed.
Arlington, VA: American Psychiatric Association;
2013.
120. American Psychiatric Association. Diagnostic and
Statistical Manual of Mental Disorders. 4th revised.
Washington, D.C.: American Psychiatric Association;
2000.
121. Heatherton TF, Kozlowski LT, Frecker RC,
Fagerström KO. The Fagerström Test for Nicotine
Dependence: a revision of the Fagerström Tolerance
Questionnaire. Br J Addict. 1991;86(9):1119–
1127.
122. Heatherton TF, Kozlowski LT, Frecker RC, Rickert
W, Robinson J. Measuring the heaviness of smoking:
using self-reported time to the first cigarette of the
day and number of cigarettes smoked per day.
Br J Addict. 1989;84(7):791–799.
123. Benowitz NL, Kuyt F, Jacob PI. Circadian blood
nicotine concentrations during cigarette smoking.
Clin Pharmacol Ther. 1982;32:758–764.
124. Payne TJ, Smith PO, McCracken LM, McSherry
WC, Antony MM. Assessing nicotine dependence: a
comparison of the Fagerström Tolerance Question-
naire (FTQ) with the Fagerström Test for Nicotine
Dependence (FTND) in a clinical sample. Addict
Behav. 1994;19(3):307–317.
125. Pomerleau CS, Pomerleau OF, Majchrzak MJ, Kloska
DD, Malakuti R. Relationship between Fagerström
Tolerance Questionnaire scores and plasma cotinine.
Addict Behav. 1990;15:73–80.
126. Dijkstra A, Tromp D. Is the FTND a measure of
physical as well as psychological tobacco dependence?
J Subst Abuse Treat. 2002;23(4):367–374.
127. Nayan S, Gupta MK, Sommer DD. Evaluating
smoking cessation interventions and cessation
rates in cancer patients: a systematic review and
meta-analysis. ISRN Oncol. 2011;2011:849023.
128. US National Cancer Institute. Smoking Cessation
and Continued Risk in Cancer Patients. Bethesda,
MD 2012. http://www.cancer.gov/cancertopics/pdq/
supportivecare/smokingcessation/HealthProfessional.
Updated July 2, 2012.
129. PDR Staff. Physician Desk Reference 2012. Montvale:
Thompson Publications; 2012.
130. Anthenelli RM, Blom TJ, McElroy SL, Keck PEJ.
Preliminary evidence for gender-specific effects of
topiramate as a potential aid to smoking cessation.
Addiction. 2008;103(4):687–694.
131. Blodgett JC, Del Re AC, Maisel NC, Finney JW. A
meta-analysis of topiramate’s effects for individuals
with alcohol use disorders. Alcohol Clin Exp Res.
2014;38(6):1481–1488.
Nicotine Dependence: Current Treatments and Future Directions  •  CHAPTER 24 410.e3
132. Anthenelli RM, Heffner JL, Wong E, et al. A
randomized trial evaluating whether topiramate aids
smoking cessation and prevents alcohol relapse in
recovering alcohol-dependent men. Alcohol Clin Exp
Res. 2017;41(1):197–206.
133. Johnson BA, Rosenthal N, Capece JA, et al.
Improvement of physical health and quality of life
of alcohol-dependent individuals with topiramate
treatment: US multisite randomized controlled trial.
Arch Intern Med. 2008;168(11):1188.
134. Gadde KM, Allison DB. Cannabinoid-1 receptor
antagonist, rimonabant, for management of obesity
and related risks. Circulation. 2006;114(9):974–
984.
135. Tonstad S. Rimonabant: a cannabinoid receptor
blocker for the treatment of metabolic and cardio-
vascular risk factors. Nutr Metab Cardiovasc Dis.
2006;16(2):156–162.
136. US Food and Drug Administration. Briefing
Information for FDA Advisory Committee Meeting
on ZIMULTI (Rimonabant). May 10, 2007. http://
www.fda.gov/ohrms/dockets/ac/07/briefing/2007-
4306b1-01-sponsor-backgrounder.pdf. Updated
April 13, 2016. Accessed April 13, 2016.
137. European Medicines Agency. Public statement on
Acomplia (rimonabant): withdrawal of the marketing
authorisation in the European Union. http://www.
ema.europa.eu/docs/en_GB/document_library/
Public_statement/2009/11/WC500012189.pdf.
Updated April 20, 2016.
138. Raupach T, Hoogsteder PHJ, Onno CP. Nicotine
vaccines to assist with smoking cessation: current
status of research. Drugs. 2012;72(4):e1–e16.
139. Moore D, Aveyard P, Connock M, Wang D,
Fry-Smith A, Barton P. Effectiveness and safety of
nicotine replacement therapy assisted reduction to
stop smoking: systematic review and meta-analysis.
BMJ. 2009;338:b1024.
140. Alpert HR, Connolly GN, Biener L. A prospec-
tive cohort study challenging the effectiveness of
population-based medical intervention for smoking
cessation. Tob Control. 2013;22(1):32–37.
141. Fagerström KO, Tejding LR, Ake W, Lunell E. Aiding
reduction of smoking with nicotine replacement
medications. A strategy for the hopeless? Tob Control.
1997;6(4):311–316.
142. Schnoll RA, Goelz PM, Veluz-Wilkins A, et al.
Long-term nicotine replacement therapy: a random-
ized clinical trial. JAMA Intern Med. 2015;175(4):
504–511.
143. Silagy C, Lancaster T, Stead L, Mant D,
Fowler G. Nicotine replacement therapy for
smoking cessation. Cochrane Database Syst Rev.
2004;(3):CD000146-CD000146.
144. Kozlowski LT, Giovino GA, Edwards B, et al. Advice
on using over-the-counter nicotine replacement
therapy-patch, gum or lozenge-to quit smoking.
Addict Behav. 2007;32(10):2140–2150.
145. Bolliger CT, Zellweger JP, Danielsson T, et al.
Smoking reduction with oral nicotine inhalers:
double blind, randomised clinical trial of efficacy
and safety. BMJ. 2000;321(7257):329–333.
146. Etter JF, Laszlo E, Zellweger JP, Perrot C, Perneger
TV. Nicotine replacement to reduce cigarette
consumption in smokers who are unwilling to
quit: a randomized trial. J Clin Psychopharmacol.
2002;22(5):487–495.
147. Karam-Hage M, Cinciripini PM. Pharmaco-
therapy for tobacco cessation: nicotine agonists,
antagonists, and partial agonists. Curr Oncol Rep.
2007;9(6):509–516.
148. Wennike P, Danielsson T, Landfeldt B, Westin A,
Tonnesen P. Smoking reduction promotes smoking
cessation: results from a double blind, randomized,
placebo-controlled trial of nicotine gum with 2-year
follow-up. Addiction. 2003;98(10):1395–1402.
149. McClure JB, Swan GE. Tailoring nicotine replace-
ment therapy: rationale and potential approaches.
CNS Drugs. 2006;20(4):281–291.
410.e3
150. Garvey AJ, Kinnunen T, Doherty K, Vokonas PS.
Effects of nicotine gum on smokers differing in
level of dependence. Paper presented at The Society
for Research on Nicotine and Tobacco; January 1,
1996; Washington, D.C.
151. Shiffman S, Dresler CM, Hajek P, Gilburt SJ,
Targett DA, Strahs KR. Efficacy of a nicotine
lozenge for smoking cessation. Arch Intern Med.
2002;162(11):1267–1276.
152. Warren GW, Rangnekar V, McGarry R, Arnold
S, Valentino J, Kudrimoti M. Pathways of resis-
tance: potential effects of nicotine on cancer and
treatment response. Int J Radiat Oncol Biol Phys.
2008;72(1):S715.
153. Warren GW, Randall M, McGarry R, Kudrimoti
M, Rangnekar V. Abstract 1408: Nicotine decreases
the therapeutic efficacy of radiotherapy and chemo-
radiotherapy in vivo in Proceedings: AACR 101st
Annual Meeting 2010, April 17–21, 2010. Cancer
Res. 2010;70(suppl):8.
154. Shields PG. Long-term nicotine replacement therapy:
cancer risk in context. Cancer Prev Res (Phila).
2011;4(11):1719–1723.
155. Murray RP, Connett JE, Zapawa LM. Does nico-
tine replacement therapy cause cancer? Evidence
from the Lung Health Study. Nicotine Tob Res.
2009;11(9):1076–1082.
156. Murray RP, Bailey WC, Daniels K, et al. Safety of
nicotine polacrilex gum used by 3,094 participants in
the Lung Health Study. Chest. 1996;109(2):438–445.
157. Ascher JA, Cole JO, Colin JN, et al. Bupropion: a
review of its mechanism of antidepressant activity.
J Clin Psychiatry. 1995;56:395–401.
158. Hughes JR, Stead LF, Hartmann-Boyce J, Lancaster
T. Antidepressants for smoking cessation. Cochrane
Database Syst Rev. 2014;(1):CD000031.
159. Evins AE, Cather C, Deckersbach T, et al. A
double-blind placebo-controlled trial of bupropion
sustained-release for smoking cessation in schizophre-
nia. J Clin Psychopharmacol. 2005;25(3):218–225.
160. Beckham JC. Smoking and anxiety in combat
veterans with chronic posttraumatic stress disorder:
a review. J Psychoactive Drugs. 1999;31(2):103–
110.
161. Hertzberg MA, Moore SD, Feldman ME, Beckham
JC. A preliminary study of bupropion sustained-
release for smoking cessation in patients with chronic
posttraumatic stress disorder. J Clin Psychopharmacol.
2001;21(1):94–98.
162. Mooney ME, Sofuoglu M. Bupropion for the treat-
ment of nicotine withdrawal and craving. Expert Rev
Neurother. 2006;6(7):965–981.
163. Gonzales D, Rennard SI, Nides M, et al. Varenicline,
an alpha4beta2 nicotinic acetylcholine receptor
partial agonist, vs sustained-release bupropion
and placebo for smoking cessation: a randomized
controlled trial. JAMA. 2006;296(1):47–55.
164. Gonzales DH, Rennard SI, Billing CB, Reeves K,
Watsky E, Gong J. A pooled analysis of varenicline,
an alpha 4 beta 2 nicotinic receptor partial agonist
vs bupropion, and placebo for smoking cessation.
February; Orlando, FL.
165. Jorenby DE, Hays JT, Rigotti NA, et al. Efficacy of
varenicline, an alpha4beta2 nicotinic acetylcholine
receptor partial agonist, vs placebo or sustained-
release bupropion for smoking cessation: a random-
ized controlled trial. JAMA. 2006;296(1):56–63.
166. Tonstad S, Tonnesen P, Hajek P, Williams KE, Billing
CB, Reeves KR. Effect of maintenance therapy with
varenicline on smoking cessation: a randomized
controlled trial. JAMA. 2006;296(1):64–71.
167. Rennard S, Hughes J, Cinciripini PM, et al. A
randomized placebo-controlled trial of varenicline
for smoking cessation allowing flexible quit dates.
Nicotine Tob Res. 2012;14(3):343–350.
168. Cinciripini PM, Robinson JD, Karam-Hage
M, et al. Effects of varenicline and bupropion
sustained-release use plus intensive smoking ces-
sation counseling on prolonged abstinence from
410.e4 Part I: Science and Clinical Oncology
smoking and on depression, negative affect, and
other symptoms of nicotine withdrawal. JAMA
Psychiatry. 2013;70(5):522–533.
169. Ebbert JO, Croghan IT, Hurt RT, Schroeder DR,
Hays JT. Varenicline for smoking cessation in light
smokers. Nicotine Tob Res. 2016;18(10):2031–2035.
170. Anthenelli RM, Benowitz NL, West R, et al.
Neuropsychiatric safety and efficacy of varenicline,
bupropion, and nicotine patch in smokers with and
without psychiatric disorders (EAGLES): a double-
blind, randomised, placebo-controlled clinical trial.
Lancet. 2016;387(10037):2507–2520.
171. Baker TB, Piper ME, Stein JH, et al. Effects of
nicotine patch vs varenicline vs combination
nicotine replacement therapy on smoking cessation
at 26 weeks: a randomized clinical trial. JAMA.
2016;315(4):371–379.
172. Price S, Hitsman B, Veluz-Wilkins A, et al. The use
of varenicline to treat nicotine dependence among
patients with cancer. Psychooncology. 2016.
173. US Food and Drug Administration. Public Health
Advisory, Important Information on Chantix
(varenicline). Rockville, MD; 2008.
174. Williams JM, Anthenelli RM, Morris CD, et al. A
randomized, double-blind, placebo-controlled study
evaluating the safety and efficacy of varenicline for
smoking cessation in patients with schizophrenia
or schizoaffective disorder. J Clin Psychiatry.
2012;73(5):654–660.
175. Anthenelli RM, Morris C, Ramey TS, et al. Effects
of varenicline on smoking cessation in adults with
stably treated current or past major depression: a
randomized trial. Ann Intern Med. 2013;159(6):390–
400.
176. Gibbons RD, Mann JJ. Varenicline, smoking ces-
sation, and neuropsychiatric adverse events. Am J
Psychiatry. 2013;170(12):1460–1467.
177. Thomas KH, Martin RM, Knipe DW, Higgins JP,
Gunnell D. Risk of neuropsychiatric adverse events
associated with varenicline: systematic review and
meta-analysis. BMJ. 2015;350:h1109.
178. Mitchell JM, Teague CH, Kayser AS, Bartlett SE,
Fields HL. Varenicline decreases alcohol consump-
tion in heavy-drinking smokers. Psychopharmacology
(Berl). 2012;223:299–306.
179. Lubin JH, Purdue M, Kelsey K, et al. Total
exposure and exposure rate effects for alcohol and
smoking and risk of head and neck cancer: a pooled
analysis of case-control studies. Am J Epidemiol.
2009;170(8):937–947.
180. Marron M, Boffetta P, Zhang Z-F, et al. Cessation of
alcohol drinking, tobacco smoking and the reversal
of head and neck cancer risk. Int J Epidemiol.
2010;39(1):182–196.
181. Cahill K, Stead LF, Lancaster T. Nicotine receptor
partial agonists for smoking cessation. Cochrane
Database Syst Rev. 2011;(2):CD006103-CD006103.
182. Shah SD, Wilken LA, Winkler SR, Lin SJ. Sys-
tematic review and meta-analysis of combination
therapy for smoking cessation. J Am Pharm Assoc.
2008;48(5):659–665.
183. Loh WY, Piper ME, Schlam TR, et al. Should
all smokers use combination smoking cessation
pharmacotherapy? Using novel analytic methods
to detect differential treatment effects over 8 weeks
of pharmacotherapy. Nicotine Tob Res. 2012;14(2):
131–141.
184. Piper ME, Smith SS, Schlam TR, et al. A random-
ized placebo-controlled clinical trial of 5 smoking
cessation pharmacotherapies. Arch Gen Psychiatry.
2009;66(11):1253–1262.
185. Smith SS, McCarthy DE, Japuntich SJ, et al.
Comparative effectiveness of 5 smoking cessation
pharmacotherapies in primary care clinics. Arch
Intern Med. 2009;169(22):2148–2155.
186. Ebbert JO, Croghan IT, Sood A, Schroeder DR, Hays
JT, Hurt RD. Vareniclin and bupropion sustained
release combination therapy for smoking cessation.
Nicotine Tob Res. 2009;11(3):234–239.
187. Ebbert JO, Hatsukami DK, Croghan IT, et al.
Combination varenicline and bupropion SR for
tobacco-dependence treatment in cigarette smokers:
a randomized trial. JAMA. 2014;311(2):155–
163.
188. Rose JE, Behm FM. Combination treatment with
varenicline and bupropion in an adaptive smoking
cessation paradigm. Am J Psychiatry. 2014;171(11):
1199–1205.
189. Rose JE, Behm FM. Combination varenicline/
bupropion treatment benefits highly dependent
smokers in an adaptive smoking cessation paradigm.
Nicotine Tob Res. 2017;19(8):999–1002.
190. Gourlay SG, Stead LF, Benowitz NL. Clonidine
for smoking cessation. Cochrane Database Syst Rev.
2004;(3):CD000058.
191. Hughes JR, Stead LF, Lancaster T. Antidepressants
for smoking cessation. Cochrane Database Syst Rev.
2003;(2):CD000031.
192. Hughes JR, Stead LF, Lancaster T. Nortriptyline
for smoking cessation: A review. Nicotine Tob Res.
2005;7(4):491–499.
193. US Centers for Disease Control and Prevention.
Cigarette smoking among adults and trends in
smoking—United States 2008. MMWR Morb Mortal
Wkly Rep. 2012;Updated July 3.
194. Gritz ER, Lam CY, Vidrine DJ, Fingeret MC.
Tobacco dependence and its treatment. In: DeVita V,
Lawrence T, Rosenberg S, eds. Cancer: Principles and
Practice of Oncology. 9th ed. Philadelphia: Lippincott
Williams & Wilkins; 2012:529–542.
195. Gritz ER, Carmack CL, de Moor C, et al. First
year after head and neck cancer: quality of life.
J Clin Oncol. 1999;17(1):352–360.
196. Gritz ER, Dresler C, Sarna L. Smoking, the
missing drug interaction in clinical trials:
ignoring the obvious. Cancer Epidem Biomar.
2005;14(10):2287–2293.
197. Gritz ER, Fingeret MC, Vidrine DJ, Lazev AB,
Mehta NV, Reece GP. Successes and failures of the
teachable moment: smoking cessation in cancer
patients. Cancer. 2006;106(1):17–27.
198. Jensen K, Jensen AB, Grau C. Smoking has a nega-
tive impact upon health related quality of life after
treatment for head and neck cancer. Oral Oncol.
2007;43(2):187–192.
199. Park SM, Lim MK, Shin SA, et al. Impact of
prediagnosis smoking, alcohol, obesity, and insulin
resistance on survival in male cancer patients:
National Health Insurance Corporation Study.
J Clin Oncol. 2006;24(31):5017–5024.
200. Yu GP, Ostroff JS, Zhang ZF, Tang J, Schantz SP.
Smoking history and cancer patient survival: a
hospital cancer registry study. Cancer Detect Prev.
1997;21(6):497–509.
201. Ehlers SL, Gastineau DA, Patten CA, et al. The
impact of smoking on outcomes among patients
undergoing hematopoietic SCT for the treatment
of acute leukemia. Bone Marrow Transplant.
2011;46(2):285–290.
202. Gritz ER. Smoking and smoking cessation in cancer
patients. Br J Addict. 1991;86:549–554.
203. Lippman SM, Lee JJ, Karp DD, et al. Random-
ized phase III intergroup trial of isotretinoin to
prevent second primary tumors in stage I non-
small-cell lung cancer. National Cancer Institute.
2001;93(8):605–618.
204. Walker MS, Vidrine DJ, Gritz ER, et al. Smoking
relapse during the first year after treatment for
early-stage non-small-cell lung cancer. Cancer Epidem
Biomar. 2006;15(12):2370–2377.
205. US Centers for Disease Control and Prevention.
Tobacco use among adults—United States, 2005.
MMWR Morb Mortal Wkly Rep. 2006;55:1145.
206. Gritz ER, Fingeret MC, Vidrine DJ. Tobacco Control
in the oncology setting. 2007. Cancer Prevention:
An ASCO Curriculum.
207. Fiore MC, Bailey WC, Cohen SJ, et  al.
Treating tobacco use and dependence.
Clinical practice guideline. http://rc.rcjournal.com/
content/53/9/1217.short. Updated March 4, 2015.
208. Fiore MC, Jorenby DE, Schensky AE, Smith SS,
Bauer RR, Baker TB. Smoking status as the new
vital sign: effect on assessment and intervention
in patients who smoke. Mayo Clinic Proceedings.
1995;70:209–213.
209. Lindholm C, Adsit R, Bain P, et al. A demonstra-
tion project for using the electronic health record
to identify and treat tobacco users. Wisc Med J.
2010;109(6):335–340.
210. Rigotti NA. Integrating comprehensive tobacco
treatment into the evolving US health care system:
it’s time to act: comment on “A randomized trial
of Internet and telephone treatment for smoking
cessation”. Arch Intern Med. 2011;171(1):53–55.
211. Stead LF, Hartmann-Boyce J, Perera R, Lancaster
T. Telephone counseling for smoking cessation.
Cochrane Database Syst Rev. 2013;(8):CD002850.
212. Lichtenstein E, Zhu SH, Tedeschi GJ. Smoking
cessation quitlines: an underrecognized intervention
success story. Am Psychol. 2010;65(4):252–261.
213. Rabius V, McAlister AL, Geiger A, Huang P,
Todd R. Telephone counseling increases cessation
rates among young adult smokers. Health Psychol.
2004;23(5):539–541.
214. Rabius V, Wiatrek D, McAlister AL. African
American participation and success in telephone
counseling for smoking cessation. Nicotine Tob Res.
2012;14(2):240–242.
215. Karn S, Fernandez A, Grossberg LA, et al. Systemati-
cally improving tobacco cessation patient services
through electronic medical record integration. Health
Promot Pract. 2016;17(4):482–489.
216. Guy MC, Seltzer RG, Cameron M, Pugmire J,
Michael S, Leischow SJ. Relationship between
smokers’ modes of entry into quitlines and treatment
outcomes. Am J Health Behav. 2012;36(1):3–11.
217. Lancaster T, Stead LF. Self-help interventions for
smoking cessation. Cochrane Database Syst Rev.
2005;(3):CD001118.
218. Graham AL, Cobb NK, Papandonatos GD, et al.
A randomized trial of Internet and telephone
treatment for smoking cessation. Arch Intern Med.
2011;171(1):46.
219. Rabius V, Pike KJ, Wiatrek D, McAlister AL.
Comparing internet assistance for smoking cessa-
tion: 13-month follow-up of a six-arm randomized
controlled trial. J Med Internet Res. 2008;10(5):e45.
220. Civljak M, Sheikh A, Stead LF, Car J. Internet-based
interventions for smoking cessation—Updated 2010.
Cochrane Database Syst Rev. 2010;(9):CD007078,
Updated July 5, 2012.
221. Swartz LH, Noell JW, Schroeder SW, Ary DV.
A randomised control study of a fully automated
internet based smoking cessation programme. Tob
Control. 2006;15(1):7–12.
222. Walters ST, Wright JA, Shegog R. A review of com-
puter and Internet-based interventions for smoking
behavior. Addict Behav. 2006;31(2):264–277.
223. Duffy SA, Ronis DL, Valenstein M, et al. A tailored
smoking, alcohol, and depression intervention for
head and neck cancer patients. Cancer Epidemiol
Biomarkers Prev. 2006;15(11).
224. Gritz ER, Carr CR, Rapkin D, et al. Predictors of
long-term smoking cessation in head and neck cancer
patients. Cancer Epidem Biomar. 1993;2:261–270.
225. Schnoll RA, Rothman RL, Wielt DB, et al. A
randomized pilot study of cognitive-behavioral
therapy versus basic health education for smoking
cessation among cancer patients. Ann Behav Med.
2005;30(1):1–11.
226. Griebel B, Wewers ME, Baker CA. The effectiveness
of a nurse-managed minimal smoking-cessation
intervention among hospitalized patients with cancer.
Oncology Nurses Forum. 1998;25:897–902.
227. Wewers ME, Bowen JM, Stanislaw AE, Desimone
VB. A nurse-delivered smoking cessation intervention
among hospitalized postoperative patients—influence

of a smoking-related diagnosis: a pilot study. Heart
Lung. 1994;23(2):151.
228. Garces YI, Yang P, Parkinson J, et al. The
relationship between cigarette smoking and
quality of life after lung cancer diagnosis. Chest.
2004;126(6):1733–1741.
229. Emmons KM, Puleo E, Park E, et al. Peer-delivered
smoking counseling for childhood cancer survivors
increases rate of cessation: the partnership for health
study. J Clin Oncol. 2005;23(27):6516–6523.
230. Wakefield M, Olver I, Whitford H, Rosenfeld E.
Motivational interviewing as a smoking cessation
intervention for patients with cancer: randomized
controlled trial. Nurs Res. 2004;53(6):396.
231. Mee-Lee D, Shulman GD, Fishman M, Gastfriend
DR, Griffith JH, eds. ASAM Patient placement criteria
for the treatment of substance-related disorders, Second
Edition-Revised (ASAM PPC-2R). Chevy Chase:
American Society of Addiction Medicine, Inc; 2001.
232. Karam-Hage M, Oughli HA, Rabius V, et al.
Tobacco cessation treatment pathways for patients
with cancer: 10 years in the making. J Natl Compr
Canc Netw. 2016;14(11):1469–1477. http://www.
jnccn.org/content/14/11/1469.full.
233. American Telemedicine Association. Telemedicine
Standards and Guidelines. http://www.american-
telemed.org/i4a/pages/index.cfm?pageID=3311.
Updated July 3, 2012.
234. Zhu SH, Gamst A, Lee M, Cummins S, Yin L, Zoref
L. The use and perception of electronic cigarettes
and snus among the US population. PLoS ONE.
2013;8(10):e79332.
235. Brown CJ, Cheng JM. Electronic cigarettes: product
characterisation and design considerations. Tob
Control. 2014;23(suppl 2):ii4–ii10. http://tobac-
cocontrol.bmj.com/content/23/suppl_2/ii1.full.
pdf+html.
236. Eissenberg T. Electronic nicotine delivery devices:
Ineffective nicotine delivery and craving sup-
pression after acute administration. Tob Control.
2010;19(1):87–88.
237. Vansickel AR, Eissenberg T. Electronic cigarettes:
effective nicotine delivery after acute administration.
Nicotine Tob Res. 2013;15(1):267–270.
238. US Department of Health and Human Services.
E-cigarette use among youth and young adults: A
report of the Surgeon General. Rockville MD; 2016.
239. Dautzenberg B, Garelik D. Patients with lung
cancer: are electronic cigarettes harmful or useful?
Lung Cancer. 2016.
240. Borderud SP, Li Y, Burkhalter JE, Sheffer CE, Ostroff
JS. Electronic cigarette use among patients with
Nicotine Dependence: Current Treatments and Future Directions  •  CHAPTER 24 410.e5
cancer: characteristics of electronic cigarette users
and their smoking cessation outcomes. Cancer.
2014;120(22):3527–3535.
241. NIDA. The Neurobiology of Drug Addiction.
http://www.nida.nih.gov/pubs/teaching/Teaching2/
Teaching.html.
242. Fortin A, Wang CS, Vigneault E. Influence of
smoking and alcohol drinking behaviors on treatment
outcomes of patients with squamous cell carcinomas
of the head and neck. Int J Radiat Oncol Biol Phys.
2009;74(4):1062–1069.
243. Rades D, Setter C, Schild SE, Dunst J. Effect of
smoking during radiotherapy, respiratory insuf-
ficiency, and hemoglobin levels on outcome in
patients irradiated for non-small-cell lung cancer.
Int J Radiat Oncol Biol Phys. 2008;71(4):1134–
1142.
244. Marin VP, Pytynia KB, Langstein HN, Dahl-
strom KR, Wei Q, Sturgis EM. Serum cotinine
concentration and wound complications in head
and neck reconstruction. Plast Reconstr Surg.
2008;121(2):451–457.
245. Chen CH, Shun CT, Huang KH, et al. Stop-
ping smoking might reduce tumour recurrence
in nonmuscle-invasive bladder cancer. BJU Int.
2007;100(2):281–286.
246. Tammemagi CM, Neslund-Dudas C, Simoff
M, Kvale P. Smoking and lung cancer survival:
the role of comorbidity and treatment. Chest.
2004;125(1):27–37.
247. Pytynia KB, Grant JR, Etzel CJ, Roberts DB, Wei
Q, Sturgis EM. Matched-pair analysis of survival
of never smokers and ever smokers with squamous
cell carcinoma of the head and neck. J Clin Oncol.
2004;22(19):3981–3988.
248. Fox JL, Rosenzweig KE, Ostroff JS. The effect
of smoking status on survival following radiation
therapy for non-small cell lung cancer. Lung Cancer.
2004;44(3):287–293.
249. Nordquist LT, Simon GR, Cantor A, Alberts WM,
Bepler G. Improved survival in never-smokers vs
current smokers with primary adenocarcinoma of
the lung. Chest. 2004;126(2):347–351.
250. Videtic GM, Stitt LW, Dar AR, et al. Continued
cigarette smoking by patients receiving concurrent
chemoradiotherapy for limited-stage small-cell lung
cancer is associated with decreased survival. J Clin
Oncol. 2003;21(8):1544–1549.
251. Chelghoum Y, Danaila C, Belhabri A, et al.
Influence of cigarette smoking on the presentation
and course of acute myeloid leukemia. Ann Oncol.
2002;13(10):1621–1627.
410.e5
252. Kreuzer M, Boffetta P, Whitley E, et al. Gender
differences in lung cancer risk by smoking: a
multicentre case-control study in Germany and
Italy. Br J Cancer. 2000;82(1):227–233.
253. Marshak G, Brenner B, Shvero J, et al. Prognostic
factors for local control of early glottic cancer:
the Rabin Medical Center retrospective study
on 207 patients. Int J Radiat Oncol Biol Phys.
1999;43(5):1009–1013.
254. Fleshner N, Garland J, Moadel A, et al. Influence
of smoking status on the disease-related outcomes
of patients with tobacco-associated superficial
transitional cell carcinoma of the bladder. Cancer.
1999;86(11):2337–2345.
255. Daniell HW. A worse prognosis for smokers with
prostate cancer. J Urol. 1995;154(1):153–157.
256. Daniell HW. More advanced-stage tumors among
smokers with endometrial cancer. Am J Clin Pathol.
1993;100(4):439–443.
257. Risch HA, Howe GR, Jain M, Burch JD, Holowaty
EJ, Miller AB. Are female smokers at higher risk for
lung cancer than male smokers? Am J Epidemiol.
1993;138:281–293.
258. Rugg T, Saunders MI, Dische S. Smoking and
mucosal reactions to radiotherapy. Br J Radiol.
1990;63:554–556.
259. Archimbaud E, Maupas J, Lecluze-Palazzolo C, Fiere
D, Viala JJ. Influence of cigarette smoking on the
presentation and course of chronic myelogenous
leukemia. Cancer. 1989;63(10):2060–2065.
260. Daniell HW. Increased lymph node metastases at
mastectomy for breast cancer associated with host
obesity, cigarette smoking, age, and large tumor
size. Cancer. 1988;62(2):429–435.
261. Daniell HW. More advanced colonic cancer among
smokers. Cancer. 1986;58(3):784–787.
262. Phillips B, Marshall E, Brown S, Thompson JS. Effect
of smoking on human natural killer cell activity.
Cancer. 1985;56:2789–2792.
263. Stevens MH, Gardner JW, Parkin JL, Johnson LP.
Head and neck cancer survival and lifestyle change.
Arch Otolaryngol. 1983;109:746–749.
264. Daniell HW. Estrogen receptors, breast cancer,
and smoking. N Engl J Med. 1980;302(26):
1478.
265. Hinds MW, Yang HY, Stemmermann G, Lee
J, Kolonel LN. Smoking history and lung
cancer survival in women. J Natl Cancer Inst.
1982;68(3):395–399.
266. Johnston-Early A, Cohen MH, Minna JD, et al.
Smoking abstinence and small cell lung cancer
survival. JAMA. 1980;244:2175–2179.
 F. TREATMENT
Cancer Pharmacology 25 
Jerry M. Collins

S UMMARY OF K EY
• Cancer pharmacology encompasses
the spectrum of therapeutic issues
from everyday clinical treatment to
the earliest stages of new drug
discovery.
• Translational research has led to
more sophisticated identification of
targets that are relevant for therapy,
P OI NT S
and clinical success has
attracted wide corporate
involvement.
• Regulatory flexibility has made new
therapies available to patients at
an unprecedented rate and has
provided a variety of options for
developmental strategies.
• The era of oral drugs for cancer
treatment is well underway.
• Once the drug is selected to match
the tumor, drug delivery must
provide adequate systemic exposure
for tumors while minimizing toxicity
and adjusting for concomitant
therapy.

Cancer pharmacology offers an integrated body of practical knowledge
that is valuable for the full range of activities related to cancer thera-
peutics, from everyday clinical treatment decisions, to assisting clinical
trial design in drug development, to discovery and evaluation of
compounds with potential for advancing to the clinic.
One of the most obvious metrics for the importance of cancer
pharmacology is the rapid and continuing expansion in the number
of drugs available to treat cancer. More than 200 agents have been
approved for marketing in oncology, including 51 that were approved
by the US Food and Drug Administration (FDA) from 2011 to 2015.
Notably, 15 were approved in 2015 (Table 25.1). As a point of reference,
at the first meeting of the American Society of Clinical Oncology
(ASCO) in 1964, there were only 12 drugs that had been previously
approved for marketing in oncology.
When ASCO was founded, the National Cancer Institute (NCI)
was the primary source for new cancer drugs, and the NCI continues
to conduct an extensive program of cancer drug development. NCI
also collaborates extensively with industry and other organizations.
Over the past 25 years, the pharmaceutical industry has dramatically
increased the resources invested in cancer pharmacology. Various
nonprofit organizations also are engaged in the development of cancer
drugs, but the private sector is the largest contributor to the effort to
bring new treatments to patients with cancer.
Beyond the impressive increase in the number of drugs available
to treat cancer, other factors are fundamentally changing the way that
oncologists and drug developers discover, develop, and prescribe drugs
for treatment of patients with cancer. In most cases, there is a strong
trend toward narrow indications, as the broad histopathologic categories
of cancer have become fractionated into many subtypes. There is also
a countertrend: seeking drug approvals based on the target for a drug,
rather than the tissue site. In addition, because effective therapy is
more widely available for first-line treatment of many cancers, initial
approvals are further stratified by the extent of prior treatment.
The approval of imatinib in 2001 heralded several of the most
dramatic changes in cancer therapy, foreshadowing entirely new para-
digms that became commonplace during the next 15 years: molecular
targeting, diagnostic tests associated with selection of therapy, and
the beginning of the shift from intravenous to oral drug delivery in
patients with cancer.
Although this chapter focuses on small, synthetic molecules, one
of the most exciting developments in cancer pharmacology is the
successful transition of antibodies from the research stage to full
integration with small molecules in the everyday treatment of patients.
There is also a role for toxins that are linked to antibodies, which can
help to guide the toxin to the tumor (see Chapter 30). Although very
different in terms of their size and manufacturing techniques, the
principles of development for these new therapeutic classes have the
same goals as for small molecules—for example, modulation of specific
molecular targets and linkage to a diagnostic test for that target.
Trastuzumab embodies all of these elements, including its extension
into the realm of antibody-drug conjugates with the 2013 FDA approval
of ado-trastuzumab emtansine.
The rapid increase in availability of new drugs and new data on
how to optimally use drugs, including the ability to avoid drug-drug
and drug-food interactions, is an enormously valuable situation for
patients with cancer. How will oncologists keep up with the avalanche
of information for all of these issues? Everything an oncologist needs
to know can no longer be squeezed into a small book that fits in the
pocket of the physician’s white coat. Electronic versions of textbooks
and centralized electronic databases help provide the intricate pieces
of data. In this chapter the focus is on the principles of cancer
pharmacology and its ability to help categorize data and organize it
into useful information.
FUNDAMENTAL SCIENCE
The two major disciplines within cancer pharmacology are drug actions
(pharmacodynamics [PD]) and drug delivery (pharmacokinetics [PK]).
Both of these areas are highly dependent on knowledge derived from
pharmacogenetics and pharmacogenomics.
In cancer pharmacology the primary basis for success or failure of
drug therapy is the ability to find a treatment that is matched to the
intrinsic sensitivity of the tumor. No therapeutic benefit will be achieved
if the target for drug action is not present in the tumor. However,

411
412 Part I: Science and Clinical Oncology

Table 25.1 Cancer Drugs Approved by the Food Box 25.1. STRATEGIC FACTORS FOR DRUG
and Drug Administration in 2015a COMBINATIONS
Drug Route Type • Rationale for target-based combinations
Daratumumab Intravenous Monoclonal antibody • Addition of new drug to established regimen
• Systematic evaluation of all combinations
Dinutuximab Intravenous Monoclonal antibody
• Translation of findings into clinical trials
Elotuzumab Intravenous Monoclonal antibody
Necitumumab Intravenous Monoclonal antibody
Alectinib Oral Small synthetic molecule
Cobimetinib Oral Small synthetic molecule (CML) that is Philadelphia chromosome positive. These early successes
Ixazomib Oral Small synthetic molecule fueled emphasis on other targets. ALK and BRAF are among the
Lenvatinib Oral Small synthetic molecule many descendants of the approach. In 2017, the target became more
Osimertinib Oral Small synthetic molecule
pivotal with the approval for pembrolizumab for treatment of tumors
with tumors that demonstrate mismatch repair or microsatellite
Panobinostat Oral Small synthetic molecule instability, regardless of tumor’s site of origin.
Palbociclib Oral Small synthetic molecule In addition to the obvious attraction of matching drugs to the
Sonidegib Oral Small synthetic molecule molecular characteristics of the tumor, the therapeutic index can also
Trabectedin Intravenous Small synthetic molecule be improved by examining host tissues. In his last major research
Trifluridine/tipiracil Oral Small synthetic molecule
publication,1 Dr. Abeloff was a leader in a large multicenter study of
the pharmacogenetics of cyclophosphamide. The goal was to determine
Uridine triacetate Oral Small synthetic molecule whether the germline DNA of patients altered the toxicity profile of
a
cyclophosphamide. The researchers found a subgroup of women who
Of the 15 new drugs, 4 are monoclonal antibodies delivered intravenously. Of the
11 small molecules, 10 are delivered orally.
had variant GSTP1 alleles that were associated with less susceptibility
to adverse hematologic toxicity in regimens containing cyclophospha-
mide. Subsequent work by large cooperative groups in the United
Kingdom for paclitaxel in breast cancer2 and in the United States for
the perfect match between target and therapy can still fail if adequate docetaxel in prostate cancer3 found other links between germline DNA
systemic exposure of drug is not delivered to the tumor. and sensory neuropathy.
Systemic exposure is pivotal. For a responsive tumor, higher systemic Although these studies were only hypothesis generating, the examples
exposure can be associated with greater benefit but also more adverse illustrate the possible ways that the therapeutic index for a drug can
effects in normal tissue. Lower systemic exposure can have lower be improved with examination of host tissues. Later in this chapter,
toxicity in normal tissues, but at the risk of reduced effect on the in the discussion of drug delivery, the pharmacogenetics of metabolism
tumor. The therapeutic index for a cancer drug is the ratio of its and cellular membrane transporter cell membranes in host tissues are
antitumor activity versus the adverse effects on normal tissues. discussed.
The principles that connect dose, systemic exposure, and toxicity
have been demonstrated regularly for normal tissues in the body, Combinations of Drugs
but the relationships of systemic exposure to antitumor effect are far
more difficult to discern. Understanding the linkage between drug As described in the various chapters relating to specific malignancies
delivery and drug actions continues to be a major challenge in cancer in Part III of this book, it is rare for a drug to be used as a single
pharmacology. agent. The development of combinations would be an extensive topic
in itself. Box 25.1 provides a set of points to consider for combinations.
Principles of Cancer Drug Action Within a combination chemotherapy regimen, the drugs are intended
to interact at the pharmacodynamic level. Originally, a major strategy
Genetics is a fundamental part of the study of cancer research (see was to combine cancer drugs with nonoverlapping toxicities. Increas-
Chapter 1). The range of applications for genetics in cancer includes ingly, more detailed knowledge regarding pathways for cancer drugs
hereditary predisposition and interactions of heredity with nonhereditary permits the design and evaluation of combination strategies that target
factors (see Chapter 13). parallel and/or sequential pathways.
In cancer pharmacology, molecular targets that could be relevant Most combinations are based on a preexisting scientific rationale
to therapy have received the most intense emphasis of pharmacogenom- or by addition of a new agent to an established regimen. However,
ics. This focus includes unique proteins coded by translocations, it would be presumptuous to assume that all possibilities are already
differences in gene expression between tumors and host tissues, and understood. One tool to expand the scope of hypothesis generation
mutations in sequences. The identification of the most attractive is the systematic study of all cancer drugs in combination with one
molecular targets and the ability to monitor target engagement during another or in combination with approved agents outside oncology,
therapy are cornerstones for customized treatment. or the testing of every investigational agent versus all approved cancer
As described in Chapter 26 and elsewhere (e.g., Chapter 8), our drugs.4 Although this screening approach begins as an empiric exercise,
understanding of the physiology and molecular biology of tumors has the successful combinations generate challenges for explaining the
provided a wealth of potential targets for anticancer therapy. Although underlying mechanisms of action. The focus on drugs that are already
the relative intensity has magnified, the concept of molecular targeting approved provides a potentially fast route to clinical implementation.5
and linkage to diagnostics for selection and monitoring of individual
patients has a long history. The selection of hormonal therapies for Molecular Imaging of Cancer Drug Action
patients whose tumors express the estrogen receptor was among the
first successful uses of molecular targeting. With the widespread availability of fluorine-18 fluorodeoxyglucose
The current era of targeting began with approval of trastuzumab (18F-FDG), a consensus was building in the late 1990s that positron
(1998) only in patients with Her2-positive tumors and approval of emission tomography (PET) would emerge as a tool for functional
imatinib (2001) only in patients with chronic myelogenous leukemia assessment, which would complement anatomic imaging modalities.6

Box 25.2.  DRUG DELIVERY CONCEPTS
Clearance (dose adjustment)
• Impaired organ function
• Drug-drug interactions
• Metabolic
• Transport
• Pharmacogenetic factors
Bioavailability
• How much drug reaches systemic circulation
• Includes food effects
Half-life (dose interval)
Volume of distribution, protein binding
Since then, FDG has been highly successful as a general probe in
many tumor types, providing additional information to help separate
malignant from benign lesions and to monitor response to therapy.
FDG is now an approved agent for imaging. Its success has spurred
interest in the development of other probes for PET imaging that are
currently in the investigational stage of clinical evaluation, including
fluorine-18 fluorothymidine (18F-fluorothymidine; FLT). Liu et al.
demonstrated the potential for FLT in a patient with uterine cancer.7
At baseline, the FLT image shows major uptake by the tumor, indicative
of active proliferation. During treatment with sunitinib, uptake of
FLT in the tumor decreased, which was interpreted as reduced prolifera-
tion. The image following the withdrawal of treatment exhibited a
flare—that is, intensity that rebounded above the baseline value.
Anatomic imaging by computed tomography (CT) readily identified
the tumor and could monitor tumor size over the subsequent weeks
or months, but FLT provided the ability to follow molecular pathways
in real time.
Cancer Drug Delivery: Systemic Exposure
Once the appropriate drug has been chosen, the most important
everyday question in clinical cancer pharmacology is the selection of
an appropriate dose. The standard dose determined previously in
population studies is a starting point for consideration, but what
factors could make this dose too high or too low for individual patients?
For many cancer drugs, evidence-based advice is not available. In
some cases, guidance for dose adjustment is available based on prior
courses of treatment or age.
Increasingly, cancer drugs are approved with information about
dosage adjustment based on renal function testing, food intake, and
the concurrent use of other drugs. All of these factors can produce
variability in systemic exposure. Thus the delivery of drugs to both
the tumor and normal tissues is affected. In addition to empiric data
from clinical investigations for specific drugs, what are the principles
that underlie these decisions based on drug delivery?
Clearance is at the top of the list for drug delivery parameters in
Box 25.2 because it is the underlying factor with the greatest impact
on dose adjustment in cancer drug delivery. As stated in Box 25.3,
clearance is the summation of all mechanisms for a drug to be removed
from the systemic circulation, sometimes called total body clearance.
Several specific applications of clearance for dosage adjustment are
covered in the next few sections.
Bioavailability, which is the second concept listed in Box 25.2, is
a measure of how much drug enters the systemic circulation. Because
of the shift toward oral therapy, bioavailability has risen sharply in
importance. This concept is described further in the section Oral
Cancer Drugs.
Half-life was previously the most discussed parameter for cancer
drug delivery. However, its role is primarily in the development stage,
to help explore frequency of administration. Once a drug has been
Cancer Pharmacology  •  CHAPTER 25 413
Box 25.3. ROLE OF CLEARANCE IN DOSAGE
ADJUSTMENT
Clearance: summation of all routes of drug elimination from the
body―renal, hepatic, other
Primary application: guide dose adjustment
• Low clearance → high exposure (consider reduced dose)
• High clearance → low exposure (consider increased dose)
approved for clinical practice, oncologists do not have a frequent need
to know the specific value for half-life of a cancer drug. Other drug
delivery parameters such as volume of distribution or protein binding
can have value in specific situations, but only on rare occasions.
Dosage Adjustment Based on Clearance to Achieve
Consistent Systemic Exposure
Compared with most other medical areas in which fixed doses are
prevalent, dosage in cancer pharmacology has long been titrated on
the basis of each patient’s body weight or body surface area. These
measurements are intended to serve as first approximations for clearance
in the absence of data, but their incremental value compared with
simple use of a standard dose is unfortunately small, and most current
trials have now adopted fixed doses—that is, “flat dosing.”8
The premise for the comments about dose adjustment in Box 25.3
is that the standard dose was selected on the basis of the needs of
patients with average drug clearance. If a patient has lower clearance
than the average, then using the standard dose will lead to higher
than average systemic exposure, with the probability of higher than
average toxicity. Using a dose reduction to match the lower clearance
will normalize the exposure of this patient relative to the average in
the population. Similarly, a patient with higher than average clearance
will have lower systemic drug exposure than will patients with the
average exposure for the population. Although this lower systemic
drug exposure will probably reduce the frequency or severity of toxicity,
it has the potentially lethal consequence of reducing the response of
the tumor.
Information on drug clearance for individual patients is rarely
available. However, the validation of this overall strategy for dosage
adjustment has been elegantly demonstrated for the first scenario in
Box 25.2, namely, patients with impaired renal function. The seminal
clinical study of carboplatin by Egorin and colleagues9 in patients
with variable renal function remains the prototype for dosage adjustment
based on impaired organ function. For many drugs, renal clearance
is not the primary mode of clearance. But for those drugs for which
renal clearance does dominate total body clearance, most recent drug
approvals have included advice in the product label for dosage adjust-
ment based on renal clearance.
For the other two areas in Box 25.2 (drug-drug interactions and
pharmacogenetic factors), many practical cases of dosage adjustment
exist. Specific examples are discussed in the following section and
Table 25.2.
Drug-Drug Interactions
Many categories of drug-drug interactions exist. The anticancer drugs
in a chemotherapy regimen, which are generally intended to work at
the level of mechanisms of drug action, are only the tip of the iceberg
for drug-drug interactions. Patients with cancer are simultaneously
treated with drugs for various other conditions, including diseases of
the heart, gastrointestinal (GI) tract, and lungs; infections; depression;
and the relief of pain. All of these permutations in polypharmacy can
potentially affect the systemic exposure and thus the delivery of cancer
drugs to the tumor and normal tissues. As a consequence of these
414 Part I: Science and Clinical Oncology

In addition to the interactions of cancer drugs with transporters

Table 25.2 Interaction of Drugs With Enzymes and in the tumor, interactions occur among cancer drugs and cellular
Transporters for Recently Approved transport systems in host tissues that control uptake and elimination
Oral Cancer Drugs of drugs from the GI tract, the liver, and the kidneys. The expression
of transporter function can be modulated by other drugs in a manner
Is This Drug Affected Does This Drug similar to drug-drug metabolic interactions. Indeed, some of the same
by Inducers or Cause Change for inducers of metabolism (phenytoin or rifampin) are also major inducers
Drug Inhibitors? Other Drugs?a of transporters. In contrast, ketoconazole, itraconazole, and certain
Abiraterone No data 2D6 psychopharmacologic agents are inhibitors. This focus on drug transport
Bosutinib 3A No data is important regardless of the route of drug delivery, but similar to
Crizotinib 3A 3A metabolic interactions, additional factors exist that are related to control
of absorption of cancer drugs from the GI tract. Transporters are also
Enzalutamide 2C8, 3A 3A, 2C9, 2C19
the underpinning of the blood-brain barrier and control the entry of
Imatinib 3A 3A, 2D6 cancer drugs into the brain to treat metastases or primary brain tumors.11
Lapatinib 3A 3A, 2D6, ABCB1
Palbociclib 3A 3A Interactions Via Self-Medication
Pazopanib 3A 3A, 2D6, 2C8
The source of drugs for patients with cancer is not restricted to those
Regorafenib 3A No data
prescribed by oncologists, or even medicines prescribed by other medical
Ruxolitinib 3A Not reported practitioners. Self-medication with over-the-counter (OTC) drugs and
Vemurafenib No data 3A, 1A2, 2D6 alternative therapies is prevalent in patients with cancer and includes
Venetoclax 3A, ABCB1 3A, ABCB1 substances that interact with cancer drugs (see Chapter 31).
Far less is known about the interactions of cancer drugs with these
a
The drug metabolism pathways are: 1A2 = CYP1A2; 3A = CYP3A; 2D6 = CYP2D6; substances than is known for prescription drugs. St. John’s wort is a
and various members of the CYP2C family, 2C8, 2C9, 2C19. For transport, the ABCB1 particularly troublesome case. It is marketed OTC for relief of depres-
(MDR1) pathway is cited. sion, which is a major issue for patients with cancer. However, this
Data retrieved from US Food and Drug Administration–approved labeling, found
at http://www.accessdata.fda.gov/scripts/cder/drugsatfda/.
product is a strong inducer of CYP3A enzymes, which inactivate
many newer oral cancer drugs. This situation once again reminds us
of the potentially serious consequences of reduced systemic exposure
and inadequate delivery of cancer drugs to the tumor.

interactions, the standard starting doses of drugs may need to be
either reduced or increased.
Most drug-drug interactions are assessed as safety issues because
of increased systemic exposure subsequent to reduced clearance.
However, induction (increasing) of clearance can decrease toxicity.
Unfortunately, although this type of interaction is “silent” in terms
of enhanced toxicity, reduced exposure to drug can also generate the
worst toxicity of all: decreases in efficacy.
Drug-drug metabolic interactions are possible for parenteral routes
of drug delivery, but they are far more common for the oral route.
Because metabolism is the primary determinant of clearance for most
drugs, it is the dominant controller for changes in plasma concentra-
tions. Following the paradigm in Box 25.3, inhibition of drug
metabolism via a drug-drug interaction will produce lower clearance
and thus higher systemic exposure. A reduction in dose may be necessary
to avoid increased toxicity. Conversely, drugs such as phenytoin can
induce the expression of certain metabolizing enzymes, which would
decrease systemic concentrations for the drugs cleared via those enzymes,
because the rate of their metabolism would be faster. Decreased exposure
increases the probability of inadequate delivery of the cancer drug to
the tumor. If safety information is available at higher doses, one option
is to increase the dose. Otherwise, it might be necessary to change to
a different therapeutic regimen. In the section Oral Cancer Drugs,
Table 25.2 provides some examples of the enzymatic pathways that
metabolize cancer drugs.
Drug-Drug Transporter Interactions
In cancer pharmacology, the primary focus for transport of drugs has
been the flux across the cell membranes of tumors. A major mechanism
of drug resistance has been described for tumors with high expression
of efflux pumps that reduce the availability of cancer drugs to their
targets. Investigations into the influx of cancer drugs into tumors have
also been conducted. White and colleagues10 demonstrated that patients
with CML had more long-term benefit from imatinib if expression
of its influx pump, OCT-1, was higher.
ORAL CANCER DRUGS
Approval of imatinib in 2001 marked a major change in emphasis for
cancer drugs. Oral drugs are overwhelmingly used in essentially all
other therapeutic areas. In contrast, the intravenous route of administra-
tion was the dominant form for cancer drugs. Exceptions included
mercaptopurine, some uses of methotrexate, and a few alkylating
agents. In the decade before the approval of imatinib, some attempts
were made to convert approved cancer drugs from the intravenous
route to oral delivery. Although those efforts were mostly unsuccessful,
capecitabine was approved in 1998 as a prodrug for 5-fluorouracil
(5-FU), which had been used intravenously for four decades.
Parenteral administration will continue to be important, both for
the many legacy drugs that remain part of regimens with established
therapeutic value and for occasional cases in which an agent cannot
be successfully delivered via the oral route. However, based on the
up-front goal to achieve oral delivery for most new cancer drugs under
development, especially for chronic daily administration, it is easy to
project that the ratio of oral to intravenous cancer drugs will continue
to rise. As shown in Table 25.1, the FDA approved 15 cancer drugs
in 2015. Overall, 10 of these drugs are administered orally. Only one
synthetic compound is administered intravenously.
Contemporary excitement regarding immuno-oncology has been
driven by early success with antibodies and therapy with cells that
have been reengineered ex vivo. Potential applications for small
molecules in this area are currently in earlier stages of development.
In general, the advantages of oral cancer drugs are compelling, and
the shift from the prior dominance of parenteral routes is irreversible.
As a consequence, issues regarding oral drugs have now emerged as
the aspect of cancer drug delivery that requires the most attention.
As indicated in Box 25.4, adherence to the prescribed regimen is
one of the first questions to explore. Although patients with cancer are
highly motivated to carefully follow their administration instructions,
it is naïve to expect complete compliance and especially to ignore the
problems of persistence over a long period. Marin and colleagues12
concluded that poor adherence may be the predominant reason for

Box 25.4. ISSUES UNIQUE TO ORAL
DRUG DELIVERY
Adherence: Does the patient take the drug?
Variability in absorption due to:
• Degradation in gastrointestinal tract
• Food effect on drug absorption
• Metabolism and transport in liver and intestines
• Drug-drug interactions prior to systemic exposure
• Emesis before complete absorption
• Comorbid difficulties in delivering oral drugs
failure to achieve adequate molecular responses with oral imatinib.
Partridge and colleagues13 examined the complex scenario of elderly
patients with cancer (older than 65 years), a population that has a high
frequency of polypharmacy and perhaps a fading memory. For adjuvant
therapy of early-stage breast cancer with tamoxifen, 25% of patients
took fewer than 80% of the expected doses, which is problematic.
The concept known as bioavailability was introduced in Box 25.2
as a measure of the drug delivery to the systemic circulation. Most
discussion in the literature compares the ratios of equal oral and
intravenous doses:
Bioavailability
Systemic drug exposure ratio, at equal doses:
Systemic drug exposure for oral delivery
Systemic drug exposure foor intravenous delivery
Among cancer drugs, imatinib has one of the highest values for
oral bioavailability relative to intravenous administration: 98%. By
definition, the bioavailability of an intravenous drug is 100%. Although
it seems preferable to have high values for relative bioavailability, the
most important test for oral administration is whether adequate systemic
exposure can be obtained with undue variability, irrespective of its
relative value. For many recently approved oral drugs, there are no
data for systemic exposure for an intravenous dose in humans.
Although flexibility of oral cancer drug administration is generally
a major advantage, it can become a liability when comorbid conditions
make swallowing drugs a major problem (see Chapter 40). Cancer
chemotherapy can produce emesis (see Chapter 39), which thwarts
the reliability of retaining swallowed doses. In addition, the treatment
of these complications of cancer therapy can include the use of
additional drugs to complicate the drug-drug interaction scenarios.
Some drugs are impractical for oral administration because they
degrade at the low pH of the stomach. At the other end of the pH
spectrum, large segments of the population are chronic users of a
variety of OTC agents that reduce acid in the stomach and raise pH.
For some drugs, high pH creates a solubility or degradation problem.
Unless the drug substance is stable and soluble over a wide range of
pH, the extensive use of acid reducers widens the variability among
patients.
Capecitabine is a drug for which systemic exposure is reduced at
high pH, which raises a concern about drug efficacy. Indeed, recent
clinical studies have demonstrated a negative association between intake
of proton pump inhibitors and efficacy in colorectal cancer14 and
gastroesophageal cancer.15
Effect of Food on Systemic Exposure for Oral
Drug Delivery
The most pragmatic question about oral therapy is whether the drug
should be taken with food. When patients take medicines for a variety
Cancer Pharmacology  •  CHAPTER 25 415
Table 25.3 Impact of Food on Drug Absorption for
Recently Approved Oral Oncology
Drugsa
Drug Take Drug With Food? Effect of Food
Venetoclax Yes Fourfold increase
Palbociclib Yes ?? (reduces variability)
Imatinib Yes ??
Lapatinib No Fourfold increase
Pazopanib No ??
Ruxolitinib ± NS
Vemurafenib ± NS
Crizotinib ± NS
Abiraterone No Tenfold increase
Bosutinib Yes ??
Regorafenib Yes ??
Enzalutamide ± NS
a
If no preference is provided for taking with food or fasting, ± is indicated, and the
effect size is not significant (NS). If no advice is provided, ?? is indicated.
Data retrieved from Food and Drug Administration approved labeling, found at
http://www.accessdata.fda.gov/scripts/cder/drugsatfda/.
of disorders, it is difficult to keep track of the instructions relating to
food, which are often in conflict. Patients cannot simultaneously be
fasting and eating a full meal. Fortunately, as each new set of cancer
drugs is approved, the quality and quantity of advice regarding the
interaction of drugs with food have improved.
For the representative set of 12 recently approved cancer drugs
described in Table 25.3, the advice for four drugs (ruxolitinib,
vemurafenib, crizotinib, and enzalutamide) is that the timing of food
intake is not important. For three drugs (abiraterone, pazopanib,
lapatinib), the advice is to take while fasting. For five drugs (venetoclax,
palbociclib, imatinib, bosutinib, regorafenib), the advice is to take
the drug with food.
If these recommendations are unintentionally reversed, potentially
serious issues of reduced efficacy or increased toxicity occur. On the
other hand, if clinical studies are conducted, there might be potential
economic benefit for creating new regimens that exploit increased
absorption of some drugs with food. The fourfold increase in absorption
of lapatinib with food has been the source of substantial public discus-
sion,16 which has been extended to cover other oral cancer drugs.17
For abiraterone, the tenfold difference in absorption is sufficiently
large to capture everyone’s attention. Perhaps abiraterone has a wider
therapeutic index than most cancer drugs, but a tenfold difference in
exposure seems unlikely to be without consequences.
In addition to the general effects of food on solubility of drugs
and passage into the body, a few food substances have highly specific
effects on cancer drug metabolism and cancer drug transporters. The
most notable example is the interaction of grapefruit juice with
metabolism via the CYP3A family and the ABC family of transporters.18
The specificity of effects with grapefruit and drugs closely resembles
the drug-drug interactions described later. More generally, the whole
conceptual framework for interactions of drugs with food parallels
the approach for drug-drug interactions.
When cancer drugs are administered parenterally, metabolism by
the luminal portion of the intestine is bypassed, and metabolism by
the liver is diluted because most of the drug is distributed systemically
before reaching the liver. In contrast, the entire dose of an oral cancer
drug is exposed to both intestinal and hepatic metabolism and
transporters before any systemic exposure.
For the same set of 12 representative oral cancer drugs described
in Table 25.3 for food effects, Table 25.2 provides information from
416 Part I: Science and Clinical Oncology
the FDA website for drug interactions with enzymes and transporters.
The entries in Table 25.2 indicate that all of these drugs have at least
some information about enzymatic pathways. The largest category of
potential interactions is with the CYP3A family of enzymes, which
are present in both the intestines and the liver and have potential
interactions with 10 of the 12 drugs. Scattered information is presented
for other members of the CYP superfamily: 1A2, 2C8, 2C9, 2C19,
and 2D6. Interest is expanding rapidly in the role of transporters19
interacting with cancer drugs, but only two drugs in this set, lapatinib
and venetoclax, have information about the transporter pathways.
CLINICAL RELEVANCE AND APPLICATIONS
The role of cancer pharmacology for clinical applications is to inform
or guide oncologists in therapeutic decision making. Cancer pharmacol-
ogy is also a major factor in moving drugs from the laboratory to
clinical use and includes studies of formulation, animal model valida-
tion, toxicology, PK, and PD.
A major path forward for advancing our understanding of cancer
drug actions includes the search for biomarkers and evaluation of treat-
ment effects. In the long term, the identification and implementation of
potential predictive biomarkers for selection and monitoring of therapy
is perhaps the most clinically relevant area of cancer pharmacology.
Currently, testing is underway to develop strategies to select treat-
ment for patients based on molecular profiling of their tumors, including
DNA (especially mutations), RNA (expression arrays and real-time
polymerase chain reaction), target proteins and their modified states
(e.g., phosphorylation, enzyme-linked immunosorbent assays, and
Western blots), and functional assays such as enzymatic activity (less
frequently). It is too early to draw conclusions about the ultimate
roles for such studies. It has become more widely appreciated that
each of these end points has various requirements for sample handling
that are crucial to interpretation of the results. Furthermore, the issue
of interlaboratory validation has moved to the top of the list of
requirements for definitive progress in applying these tools.
Cancer drug delivery assists in the selection of feasible doses,
schedules, and routes of administration. The most appealing approach
to customizing doses for patients would provide advice prior to the
first dose to avoid untoward effects and maximize therapeutic delivery.
Earlier sections on impaired organ function and drug-drug interactions
discussed some modest advances toward this goal.
Once therapy is ongoing, adjustments are frequently required.
Dosage adjustment based on adverse effects is always the most urgent
need. If life-threatening or unbearable toxicity occurs, then the
antitumor effects will be moot. An adjustment in dosage based on
lack of antitumor activity has its attraction, particularly if the toxicity
is less than expected, a potential signal of inadequate exposure. However,
recognition of the narrow therapeutic index for cancer drugs and
many other cautionary clinical factors or concerns about liability tend
to discourage escalating a dose higher than the standard, unless adequate
safety data are available for higher doses. In addition, there is always
a sense that if the current treatment is not working, switching to an
alternate therapy is urgent before the tumor becomes even more
resistant.
Subcutaneous Route of Cancer Drug Delivery
The subcutaneous route of delivery has emerged as an occasional
solution to difficulties in drug delivery. The two issues that drive
consideration of the subcutaneous route are (1) inability of an oral
dose to reliably traverse the GI tract and avoid presystemic metabolism,
and (2) impracticality of the intravenous route for relatively frequent
administration.
The approval of 5-azacytidine in 2004 for myelodysplastic syndromes
is an ideal example to demonstrate the advantages of subcutaneous
drug delivery. In addition to incompatibility with oral delivery,
5-azacytidine requires daily parenteral administration for 7 consecutive
days. Subcutaneous and intravenous delivery were both approved.
The patient population for myelodysplastic syndromes has a substantial
fraction of elderly patients, for whom there could be additional chal-
lenges in obtaining daily intravenous treatment.
In 2012 the approval of bortezomib was extended from intravenous
delivery to include the subcutaneous route.
The primary limitation for subcutaneous delivery is the low volume
of drug solution that can be delivered. The success of chronic subcutane-
ous insulin delivery is driven by injection of small volumes, which
eases patient acceptance of injection site issues.
In 2017 the FDA approved rituximab in combination with
hyaluronidase for subcutaneous administration.20 Use of hyaluronidase
to facilitate absorption of complex compounds from the subcutaneous
space could widen use of this route of delivery.
Regional Cancer Drug Delivery
When cancer is localized between barriers to cancer drug delivery,
administering the drug directly to the region of disease is an attractive
concept. Nonetheless, many investigations of regional drug delivery
have failed to demonstrate increased benefit compared with systemic
treatment. The choice of drug is the first consideration. Cyclophos-
phamide and other prodrugs that must be metabolized to their active
species are not suitable for regional delivery. The potential advantage
of local delivery is lost because the drug must enter the body and be
metabolized and then circulate throughout the body as if it had been
systemically delivered.
Intraarterial catheterization has been technically feasible for delivery
of cancer drugs to a variety of places in the body for decades. The
use of an implanted pump for the intraarterial delivery of fluorode-
oxyuridine is approved for the treatment of liver metastases from
colorectal cancer and is the most common application of this approach.20
In 2017 a large international study demonstrated a survival advantage
for this approach.21 Intraperitoneal delivery of drugs for treatment of
localized ovarian cancer and other intraperitoneal malignancies has
undergone many evaluations. Three randomized phase III studies
have shown a survival benefit for intraperitoneal delivery compared
with the intravenous route.22 Despite this success, the lack of familiarity
with the catheters, uncertainty about uniform distribution within the
peritoneal cavity, and other factors have slowed the penetration of
this modality into routine practice.
Intrathecal delivery remains a mainstay for pediatric leukemia. As
previously mentioned, the transporters that constitute the blood-brain
barrier tightly control the passage of drugs into the brain. Direct
injection of drug into the cerebral spinal fluid produces very high
local concentrations for the areas close to the tissue–cerebrospinal
fluid interfaces. The major limitation of intrathecal delivery to treat
solid tumors is the inability of cancer drugs to penetrate deeply into
tissue to reach all of the disease.
The intravesicular route is standard care for some stages of bladder
cancer. Bacillus Calmette-Guérin (BCG) is an approved drug for this
indication, and several other drugs are less commonly used.
Brand Names, Generics, Biosimilars
When a small synthetic drug is initially approved, oncologists and
patients become accustomed to its brand name. After patents and
exclusivity rights expire, everyone needs to readjust to the generic
name and to additional suppliers. For small synthetic compounds,
generic drugs contain the same active ingredients as the initial drug.
The manufacturing and quality control standards are identical. For
oral cancer drugs, there is the additional requirement for bioequivalence,
a term that means that the original drug and the generic drug produce
the same range of plasma concentrations. The fundamental assumption
is that patients should obtain similar benefits (and toxicity profiles)
if the same exposure of the active ingredient is delivered. In addition
to its use for generics, this principle is also applied for changes in the

Box 25.5.  RECENT TRENDS FOR NEW CANCER
DRUGS
1. Rapid pace of approvals
2. Predominantly oral drugs
3. Narrow indications
4. Links to molecular targeting and diagnostic tests
5. Involvement of industry, nonprofit organizations, and government
production of brand name drugs, including changes in suppliers of
bulk ingredients, manufacturing processes, or locations for producing
pills. All of these events occur throughout the marketing history of
the drug but are not highly visible to oncologists and their patients.
The principles for ensuring similarity or equivalence are the same for
all marketed small synthetic drugs.
A parallel set of procedures has evolved for biologicals and other
complex drug substances. The expectations for therapeutic and toxic
effects of biologicals are somewhat different than for small synthetic
compounds, and the approval paths reflect these differences. Most
important, biologicals are manufactured by living organisms, and the
precise characterization of the active ingredient is more difficult to
replicate. The overall program creates a category of drugs known as
biosimilars. Europe already has 20 biosimilars.23
The FDA approved the first biosimilar for the United States in
2015. The important feature for the FDA approach is the designa-
tion of two categories: biosimilars and interchangeable biological
products. Both categories require extensive preclinical data, and
sufficient clinical data to ensure no meaningful differences from the
reference product. Interchangeable products have additional informa-
tion that demonstrates that patients can switch between the reference
product and its interchangeable counterpart, with similar tolerance
and activity.
In terms of dispensing, the name of the biosimilar product is
specifically written by the prescriber. For products that are interchange-
able, pharmacists can substitute the interchangeable product when
the prescription is written for the reference product.
New Indications and Repurposing (Repositioning)
Terms such as repurposing and repositioning have become commonplace
as researchers and companies seek new uses for approved drugs. Because
the safety of the drug has already been established and the supply of
clinical-grade drug is readily available, the road to evaluation in patients
is accelerated (Box 25.5). Initial approval for a drug is not the end
of its clinical development.
The simplest version of this concept is to search within the same
therapeutic area. Additional uses for cancer drugs are often actively
sought. Once again, imatinib sets the standard. In this case, the approved
indications were extended from CML to GI stromal tumor, and again
to various rare cancers.
When therapeutic boundaries are crossed, potential therapies could
move from oncology to other diseases or from other diseases into
oncology. Methotrexate is used in juvenile and rheumatoid arthritis.
Cyclophosphamide is used in both children and adults for nephrotic
syndrome. Paclitaxel is used as a coating for cardiac stents. In the
other direction, cancer researchers have a recurring interest in evaluation
of potential anticancer effects of marketed drugs from outside oncology.
Recurring examples include metformin and simvastatin.
Clinical Phases of Drug Development
As indicated in Box 25.6, the clinical stages of drug development
have been divided historically into phases that are related to specific
goals. These stages also have regulatory significance, but the boundaries
Cancer Pharmacology  •  CHAPTER 25 417
Box 25.6.  “HISTORICAL” PHASES OF HUMAN
EVALUATION FOR CANCER DRUGS
• Phase 0: Target-related study and feasibility of delivery
• Phase I: Safety and early signs of activity
• Phase II: Is anticancer activity promising?
• Phase III: Improvement versus current therapy?
• Phase IV: Postmarketing studies
across phases are always being pushed. It is not possible to assign
every compound into the same bins. For patients with cancer, this
view of clinical development is continually challenged by the compelling
need to provide therapeutic options when no treatments have been
established. The boundaries that are calibrated for less serious diseases
may be a bit more flexible in oncology, just as maximum tolerated dose
is a relative term that depends on the medical scenario. Phase I, phase
II, and phase III are long-established terms. Phase IV was created by
a formal change in FDA regulations. Phase 0 is a less formal term
used by some investigators to describe early explorations to seek
information before deciding to launch full-scale development.
Working backward, phase III clinical studies are the largest and
most expensive periods of clinical development and have the highest
profile in the oncology community. The results of these studies are
carefully followed and are prominently featured at large national
meetings. These trials are designated as potentially “practice changing”
because of their potential for an immediate improvement in the standard
of care for the specific stage and type of cancer. The pivotal hallmark
of phase III trials is a randomized comparison of two or more treatment
groups. These trials are undertaken based on preliminary evidence of
anticancer activity, and the key question is simply stated: How does
the new therapy compare with established therapy?
When phase III trials include an investigational agent, the data
from these trials are the ordinary basis for the regulatory decision to
permit an indication for marketing in the population studied in the
trial. Thus these trials are sponsored by the commercial organizations
that will be seeking marketing approval. If the phase III trial is a
comparison of groups for which all of the drugs are off-patent, then
the NCI is usually the only organization with the resources and the
mission to support such studies. Nonprofit organizations could also
serve in this role, but they tend to support earlier stages of clinical
studies because of the cost of phase III trials.
Phase II clinical trials are the setting for determining if an agent
or combination of agents has sufficient activity in well-defined groups
of patients who have no established treatments.24 In the majority of
phase II studies, there is only a single treatment group, but randomized
designs of two or more groups have been increasingly used. The goal
is to determine if the activity of the agent or combination is sufficient
to move into much larger phase III trials with comparison to standard
treatments. Unfortunately, areas within oncology remain for which
no standard treatment has been established. In such cases, phase II
studies can be sufficient for regulatory approval.
Phase I clinical studies have traditionally been the first-in-human
studies for new agents or for combinations of investigational or approved
drugs. In diseases other than cancer, these safety studies are generally
conducted with healthy volunteers. Until recently, phase I clinical
studies of cancer drugs were conducted only with patients with cancer
because of the potential for acute, severe toxicity, as well as longer-term
issues such as carcinogenicity or mutagenicity for many classes of
older cancer drugs. As the biologic properties of cancer drugs have
evolved, some investigational agents have adverse effect profiles that
are similar to those of agents outside oncology. For compounds
sponsored by the pharmaceutical industry, there has been a trend
toward use of healthy volunteers in phase I clinical studies for certain
classes of cancer drugs. It is likely that both strategies will coexist,
418 Part I: Science and Clinical Oncology
and the approach will be customized for each investigational agent.
Phase I studies in healthy volunteers have a track record of lower
costs, as well as faster and more reliable recruitment of subjects.
On the other hand, the unique aspect for inclusion of patients
with cancer in phase I is the enormous value for determination of
molecular effects in tumors via biopsies or imaging as well as signals
of therapeutic benefit. Although expectations are modest for anticancer
effects during initial studies of new agents, favorable responses can
accelerate development programs.
Phase 0 clinical studies are an approach to first-in-human studies
that extends the preclinical search for specific types of information into
the clinic. Frequently, the question is whether systemic exposure can
be achieved that is considered essential to activity, or whether a specific
degree of target engagement, can be reached.25,26 A small number of
analogs might be compared in a phase 0 study before final selection of
the lead candidate. These and other questions can often be explored
with single doses, or short courses. Some of these same questions can
also be answered during phase I studies. There is a smaller package of
preclinical studies required for phase 0, so the choice may be driven
by degree of commitment to full-scale clinical evaluation.
Phase IV was added to the process for clinical development to
permit approval when sufficient demonstration of safety and efficacy
has been established, but before all studies are completed. For example,
during the review of the marketing application it could be determined
that certain subpopulations were not included in the pivotal studies.
In return for faster approval, the sponsor of the drug makes a com-
mitment to conduct “postmarketing” studies to fill any gaps defined
by the FDA. Situations include clinical studies of patients with impaired
organ function, drug-drug interactions, or expanded studies of elderly
patients.
The numeric order of the clinical phases provides a logical progres-
sion for development but should not be considered as universally
linear. As previously noted, a new drug is sometimes approved based
on phase II (or even phase I) studies, and phase III trials are not
conducted. At the other extreme, an unanticipated problem with
administration during a phase II investigation might prompt the return
to phase I to improve characterization of doses and schedules. Precedents
exist for skipping phase II studies and moving directly from phase I
to phase III. Evidence of activity in phase I may seem so compelling
that it warrants immediate comparison with standard of care. This
strategy has a high risk in terms of the resources invested in a compound
without much understanding of the probable activity. Of course, the
potential exists for a high reward in terms of bringing new treatments
to patients, as well as financially. Of course, this strategy is feasible
only if the phase I study is conducted in patients with cancer.
CANCER PHARMACOLOGY ACROSS
THE DRUG DISCOVERY AND
DEVELOPMENT PROCESS
Applications of the principles of cancer pharmacology are embedded
in the continuum from early drug discovery through preclinical
development, and then bridging to the preapproval stages of clinical
development.
During the development phases for a cancer drug, it is essential
to monitor the relative drug delivery, as assessed by systemic concentra-
tion. When comparing activity studies in murine models, safety studies
in various mammals, or early clinical studies, plasma concentrations
can be monitored. Variation across animal models might signal
important interspecies differences in factors related to either PK (e.g.,
metabolism) or PD (e.g., inherent host cell sensitivity).
In the same manner, when making comparisons between observa-
tions of activity in cell culture versus concentrations that can be safely
achieved in vivo, it is prudent to consider reasons for any discrepancies.
Skepticism is particularly appropriate if “interesting” activity is observed
in vitro at concentrations much higher than are achievable in vivo.
The fruits of cancer pharmacology are found in the paths that
guide drugs into clinical use and in the adjustment of therapy in
patients to balance therapeutic and toxic effects. Improving the con-
nections between cancer drug delivery and the quantitative aspects
of the activity and toxicity of cancer drugs is essential for further
progress. Cancer drug delivery can only provide therapeutic concentra-
tions and avoid toxic concentrations if exploitable differences between
tumors and host tissues have been identified and reliable assays are
available at the laboratory-clinic interface.
WHAT THE FUTURE HOLDS
Three new regulatory actions related to the marketing of pembrolizumab
have stimulated further reexamination of the drug approval process:
1. FDA approval of pembrolizumab for second-line treatment of
metastatic melanoma was based on objective and durable responses
in a phase I trial.27 Surely, this action validates the recent efforts
to learn as much as possible, as early as possible.
2. For the initial approval of pembrolizumab, FDA and the sponsor
worked together to rely on amendments to the initial clinical
protocol from phase I throughout the subsequent clinical develop-
ment.28 FDA staff indicated that this “seamless” approach to
oncology drug development has some disadvantages, and is not
appropriate for all development programs. Nonetheless, FDA
convened several public meetings to create dialog on the potential
advantages and disadvantages. Every development plan will not
fit into this box, but the concept of an integrated approach to
clinical development is surely boosted by this action.
3. The FDA issued its first “tissue-agnostic” approval, for the use of
pembrolizumab based on its target, DNA mismatch repair and
microsatellite instability, rather than the tissue site.29 The molecular
interrogation of tumor samples to help determine appropriate
therapy can be viewed as a direct extension of the historical applica-
tion of histopathologic classification to guide recommended therapy.
These developments were not foreseen as recently as 5 years ago,
and indicate that the “future” is closer than we imagined. Much work
remains to be done in the area of acquisition and handling of biopsy
specimens, as well as systematic evaluation using validated methods.
Fortunately, many complementary options are also evolving, such as
the collection and analysis of circulating tumor cells, as well as molecular
imaging probes that can provide real-time confirmation of target
engagement in situ.
All models have limitations, but we certainly need improvement
in preclinical models of cancer to improve the clinical success rate for
new drugs. It is likely that the continued evolution of patient-derived
xenografts and genetically engineered mice will provide part of the
progress.
Another potential model is emerging from investigation and
treatment of spontaneous tumors in dogs.30 Some tumor types have
very similar pathologic appearance in dogs and humans. The ability
to use a larger mammal with an intact immune system may have some
advantages in the current era of immune-oncology. There is already
a well-organized set of veterinary researchers and clinicians, and the
connections to human oncology are growing.
Finally, with the large and increasing number of drugs available
for use (see Appendix),31 we must recognize that it is not feasible to
remember all the details about each one. The widespread availability
and use of online resources, including FDA databases, supplements
traditional textbooks. Assembling the key pieces of data is only the
first step. Professional interpretation and judgment remain as central
features.
The complete reference list is available online at
ExpertConsult.com.

REFERENCES
1. Yao S, Barlow WE, Albain KS, et al. Gene polymor-
phisms in cyclophosphamide metabolism pathway,
treatment-related toxicity, and disease-free survival
in SWOG 8897 clinical trial for breast cancer. Clin
Cancer Res. 2010;16:6169–6176.
2. Abraham JE, Guo Q, Dorling L, et al. Replication
of genetic polymorphisms reported to be associated
with taxane-related sensory neuropathy in patients
with early breast cancer treated with paclitaxel. Clin
Cancer Res. 2014;20:2466–2475.
3. Hertz DL, Owzar K, Lessans S, et al. Pharmaco-
genetic discovery in CALGB (Alliance) 90401 and
mechanistic validation of a VAC14 polymorphism
that increases risk of docetaxel-induced neuropathy.
Clin Cancer Res. 2016;22:4890–4900.
4. Kummar S, Chen HX, Wright J, et al. Utilizing
targeted cancer therapeutic agents in combination:
novel approaches and urgent requirements. Nat Rev
Drug Discov. 2010;9:843–856.
5. Holbeck SL, Camalier R, Crowell JA, et al. The
National Cancer Institute ALMANAC: A comprehen-
sive screening resource for the detection of anticancer
drug pairs with enhanced therapeutic activity. Cancer
Res. 2017;77:3564–3576.
6. Niederhuber JE. Future of positron-emission
tomography in oncology. Ann Surg. 1998;227:324–
325.
7. Liu G, Jeraj R, Vanderhoek M, et al. Pharmaco-
dynamic study using FLT PET/CT in patients
with renal cell cancer and other solid malignancies
treated with sunitinib malate. Clin Cancer Res.
2011;17:7634–7644.
8. Hartford CM, Desai AA, Janisch L, et al. A phase
I trial to determine the safety, tolerability, and
maximum tolerated dose of deforolimus in patients
with advanced malignancies. Clin Cancer Res.
2009;15:1428–1434.
9. Egorin MJ, Van Echo DA, Tipping SJ, et al.
Pharmacokinetics and dosage reduction of cis-
diammine(1,1-cyclobutanedicarboxylato) platinum
in patients with impaired renal function. Cancer
Res. 1984;44:5432–5438.
10. White DL, Dang P, Engler J, et al. Functional activ-
ity of the OCT-1 protein is predictive of long-term
outcome in patients with chronic-phase chronic
myeloid leukemia treated with imatinib. J Clin Oncol.
2010;28:2761–2767.
11. Umans RA, Taylor MR. Zerbrafish as a model to
study drug transporters at the blood-brain barrier.
Clin Pharmacol Ther. 2012;92:567–570.
12. Marin D, Bazeos A, Mahon FX, et al. Adherence is
the critical factor for achieving molecular responses in
patients with chronic myeloid leukemia who achieve
complete cytogenetic responses on imatinib. J Clin
Oncol. 2010;28:2381–2388.
13. Partridge AH, Archer L, Kornblith AB, et al. Adher-
ence and persistence with oral adjuvant chemotherapy
in older women with early-stage breast cancer in
CALGB 49907: adherence companion study 60104.
J Clin Oncol. 2010;28:2418–2422.
14. Sun J, Ilich AI, Kim CA, et al. Concomitant admin-
istration of proton pump inhibitors and capecitabine
is associated with increased recurrence risk in early
stage colorectal cancer patients. Clin Colorectal Cancer.
2016;15:257–263.
15. Chu MP, Hecht JR, Slamon D, et al. Association of
proton pump inhibitors and capecitabine efficacy in
advanced gastroesophageal cancer: secondary analysis
of the TRIO-013/LOGiC randomized clinical trial.
JAMA Oncol. 2017;3:767–773.
16. Ratain MJ, Cohen EE. The value meal: how to save
$1,700 per month or more on lapatinib. J Clin Oncol.
2007;25:3397–3398.
17. Ratain MJ. Flushing oral oncology drugs down the
toilet. J Clin Oncol. 2011;29:3958–3959.
18. Dolton MJ, Roufogalis BD, McLachlan AJ. Fruit
juices as perpetrators of drug interactions: the role of
polypeptides. Clin Pharmacol Ther. 2012;92:622–630.
19. Morrissey KM, Wen CC, Johns SJ, et al. The UCSF-
FDA TransPortal: a public drug transporter database.
Clin Pharmacol Ther. 2012;92:545–546.
20. Niederhuber JE, Ensminger W, Gyves J, et al. Regional
chemotherapy of colorectal cancer metastatic to the
liver. Cancer. 1984;53:1336–1343.
Cancer Pharmacology  •  CHAPTER 25 419
21. Groot Koerkamp B, Sadot E, Kemeny NE, et al.
Perioperative hepatic arterial infusion pump che-
motherapy is associated with longer survival after
resection of colorectal liver metastases: a propensity
score analysis. J Clin Oncol. 2017;35:1938–1944.
22. Alberts DS, Markman M, Armstrong D, et al.
Intraperitoneal therapy for stage III ovarian cancer:
a therapy whose time has come! J Clin Oncol.
2002;20:3944–3946.
23. Camacho LH. Current status of biosimilars in
oncology. Drugs. 2017;77:985–997.
24. Sharma MR, Stadler WM, Ratain MJ. Randomized
phase II trials: a long-term investment with promising
returns. J Natl Cancer Inst. 2011;103:1093–1100.
25. Murgo AJ, Kummar S, Rubinstein L, et al. Design-
ing phase 0 cancer clinical trials. Clin Cancer Res.
2008;14:3675–3682.
26. Kummar S, Kinders R, Gutierrez ME, et al. Phase
0 clinical trial of the poly (ADP-ribose) polymerase
inhibitor ABT-888 in patients with advanced
malignancies. J Clin Oncol. 2009;27:2705–2711.
27. Chuk MK, Chang JT, Theoret MR, et al. FDA
approval summary: accelerated approval of pem-
brolizumab for second-line treatment of metastatic
melanoma. Clin Cancer Res. 2017 Feb 24. pii:
clincanres.0663.2016. E-Pub ahead of print (as of
11-July-2017).
28. Prowell TM, Theoret MR, Pazdur R. Seamless
oncology-drug development. N Engl J Med.
2016;374:2001–2003.
29. Poh A. First tissue-agnostic drug approval issued.
Cancer Discov. 2017;7:656.
30. LeBlanc AK, Breen M, Choyke P, et al. Perspec-
tives from man’s best friend: National Academy of
Medicine’s Workshop on Comparative Oncology. Sci
Transl Med. 2016;8:324ps5.
31. Freter C, Perry W. Systemic therapy. In: Abeloff MD,
Armitage JO, Niederhuber JE, et al, eds. Abeloff ’s
Clinical Oncology. 4th ed. Philadelphia: Churchill
Livingston; 2008.

Appendixa
Drug Name
Abiraterone acetate (Zytiga)
Ado-trastuzumab emtansine
(Kadcyla)
Afatinib (Gilotrif )
Aflibercept, ziv-aflibercept
(Zaltrap)
Alectinib (Alecensa)
Alemtuzumab (Campath)
Altretamine (Hexalen),
hexamethylmelamine, HMM
Amifostine (Ethyol), WR-2721,
ethiofos
Anastrozole (Arimidex)
Cancer Pharmacology  •  CHAPTER 25 419.e1
419.e1
Drug Class and/or Pharmacokinetics Dosage and
Mechanism and Metabolism Toxicity Indications Administration
Inhibits androgen Do not take with food; Joint swelling or Metastatic prostate 1000 mg QD PO with
biosynthesis inhibits CYP2D6 discomfort, edema; cancer; castration 5 mg prednisone BID
monitor liver enzymes resistant with prior
use of docetaxel
Antibody-drug Avoid strong CYP3A Hepatotoxic, cardiotoxic, Her2+ BC metastasis, 3.6 mg/kg IV every
conjugate inhibitor fetal harm prior trastuzumab and 3 wk
a taxane
Kinase inhibitor Reduce dose if PgP Diarrhea, severe First-line NSLC 40 mg QD PO
inhibitor used cutaneous reaction metastasis with EGFR
exon 19 del or exon
21 sub per FDA test
Fusion protein No studies for renal, Hemorrhage, GI Metastatic colorectal 4 mg/kg via 1-h
binding VEGF and hepatic, or drug-drug perforation, wound cancer resistant or intravenous infusion
related ligands interactions healing problems, fistula, progressed while (not bolus) every
hypertension, arterial taking oxaliplatin 2 wk
thrombosis proteinuria
Kinase inhibitor With food Hepatotoxic—monitor; ALK+ metastasis, 600 mg BID PO
pneumonitis, bradycardia, NSCLC, failed or
myalgia, fetal harm, intolerant to crizotinib
fatigue, constipation,
edema
Monoclonal antibody Long half-life; no dose Infusion reactions; B-cell CLL Intravenous infusion
against CD52 present adjustments based on prolonged with gradual dose
on B cells and other age myelosuppression, escalation up to
immune cells; hemolytic anemia, 30 mg/kg 3 times
produces ADCC and immune per week with
following binding thrombocytopenia; appropriate
immunosuppression and premedication
infection; cardiac
dysrhythmias
Alkylating agent Well absorbed by Myelosuppression is dose Refractory ovarian 4–12 mg/kg/day in
mouth, metabolized limiting; leukopenia, carcinoma divided doses for
in the liver; half-life thrombocytopenia, 3–6 wk or 50 mg/m2/
4–13 h; metabolism nausea, vomiting, day for 14 days each
may be slowed by neurologic toxicity are cycle; higher doses
cimetidine or common, including have been used
enhanced by confusion, lethargy,
phenobarbital weakness, and sensory
changes
Cytoprotectant; After intravenous Transient hypotension is Pretreatment with 740 mg/m2
free-radical infusion, the drug is dose limiting; nausea, cisplatin; useful as intravenous infusion
scavenger metabolized to a thiol vomiting, and somnolence a bone marrow, over 15 min given
metabolite, which is are common; sneezing, kidney, and nerve 15–30 min before
responsible for its hypocalcemia, and cytoprotectant; useful the cytotoxic agent
beneficial activity flushing can be seen with other alkylators or radiation; lower
as well; also FDA doses and
approved as a subcutaneous
radiation protectant administration have
to reduce xerostomia also been used
Nonsteroidal Well absorbed from Very well tolerated; Indications: As 1 mg PO every day;
aromatase inhibitor; GI tract, maximum asthenia, headache, and adjuvant therapy of higher doses are no
blocks estrogen plasma levels within hot flashes occur in fewer BC and for treatment more effective
production 2 h; half-life is 50 h; no than 15% of women; of postmenopausal
selectively adjustments needed diarrhea, abdominal pain, women with breast
for abnormal function anorexia, nausea, and carcinoma that has
of these organs vomiting occur in 10% or progressed during
because of the wide fewer; thrombophlebitis treatment with
therapeutic index has been reported tamoxifen
Continued
419.e2 Part I: Science and Clinical Oncology
Appendixa—cont’d
Drug Name
Arsenic trioxide (Trisenox)
L-Asparaginase (Elspar),
colaspase
Pegaspargase (Oncaspar)
Asparaginase Erwinia
chrysanthemi (Erwinaze)
Atezolizumab (Tecentriq)
Avelumab (Bavencio)
Axitinib (Inlyta)
Drug Class and/or Pharmacokinetics Dosage and
Mechanism and Metabolism Toxicity Indications Administration
Novel arsenical Half-life of this Differentiation syndrome is Relapsed acute 0.15 mg/kg/day in
differentiating agent compound is dose limiting—includes promyelocytic 100–250 mL of D5W
unknown; it is leukocytosis, fever, leukemia until remission, not
methylated in the liver dyspnea, chest pain, to exceed 60 doses,
and eliminated in the tachycardia, hypoxia, then up to 25 doses
urine sometimes death; over 5 wk for
corticosteroids seem to consolidation
benefit this syndrome; QT starting 3–6 wk after
prolongation is common; achievement of
also rash, pruritus, remission
headache, arthralgias,
anxiety, bleeding, nausea,
vomiting; liver and renal
toxicity uncommon
Naturally occurring After intravenous or Hypersensitivity can be ALL; also used in AML, After a 2-unit
enzyme derived from intramuscular life-threatening, late-stage CML, CLL, intradermal test
Escherichia coli or injection, the drug is necessitating anaphylaxis and non-Hodgkin dose, an
Erwinia carotovora metabolized precautions and 2-unit lymphomas intramuscular dose
that cleaves intravascularly by test dose; coagulopathy is of 6000–10,000 IU/
asparagine, an proteolysis; common and requires m2 every 3 days for 9
essential amino acid elimination half-life of monitoring; nausea, doses, or 1000 IU/kg/
required by rapidly 8–30 h vomiting, abdominal day IV over 30 min
proliferating cells cramps, anorexia, elevated for 10 days, has been
liver function test results, used
transient renal
insufficiency are common;
lethargy, somnolence,
fatigue, depression,
confusion, pancreatitis,
and fever are seen
Naturally occurring When given by Less immunogenic that ALL, and like 2,500 IU/m2 IM every
enzyme, covalently intramuscular non-PEGylated form, but asparaginase, it is also 14 days with other
linked to injection, it has an hypersensitivity and used for other chemotherapy
polyethylene glycol elimination half-life of anaphylaxis can still occur; leukemias and agents for induction
to reduce approximately 5 days; toxicities similar to those non-Hodgkin or maintenance
immunogenicity, clearance is not of non-PEGylated forms lymphomas
slow metabolism, dependent on renal or including elevated liver
and prolong half-life; hepatic function enzymes, coagulopathy,
the enzyme cleaves hypercholesterolemia,
asparagine, an pancreatitis,
essential amino acid hyperglycemia, fever,
required by rapidly chills, anorexia, lethargy,
proliferating cells confusion, headache,
seizures, and azotemia
Substitute for the Not characterized Severe hypersensitivity ALL 25,000 IU IM 3 times
naturally occurring reactions, including per wk for 6 doses
enzyme anaphylaxis per cycle
PD-L1 blocking Potential effect of Pneumonitis, hepatitis, Locally advanced or 1200 mg IV over
antibody renal or hepatic colitis, endocrinopathies, metastatic urothelial 24 h; every 3 wk
impairment unknown myasthenia gravis, ocular cancer following
toxicity, pancreatitis, platinum
infusion reaction, fetal chemotherapy
harm
PD-L1 blocking Premedicate with Fatigue, musculoskeletal Merkel cell carcinoma 10 mg/kg IV over
antibody acetaminophen, effects, diarrhea, infusion metastasis, in adults or 60 min; every 3 wk
antihistamine reaction, rash, nausea, pediatric patients
peripheral edema 12 yr and older
Avoid strong CYP3A Decrease dose 50% Diarrhea, hypertension, Advanced RCC after 5 mg BID PO
inhibitors or reduce for CYP3A inhibitors, fatigue, nausea one treatment failure
dose renal or hepatic
impairment

Appendixa—cont’d
Drug Name
Azacitidine (Vidaza)
Bacillus Calmette-Guérin (TICE
BCG, TheraCys), BCG
Belinostat (Beleodaq)
Bendamustine (Treanda)
Bevacizumab (Avastin)
Bexarotene (Targretin)
Cancer Pharmacology  •  CHAPTER 25 419.e3
419.e3
Drug Class and/or Pharmacokinetics Dosage and
Mechanism and Metabolism Toxicity Indications Administration
Antimetabolite; Not orally bioavailable; Myelosuppression is dose MDS 75–100 mg/m2 for 7
induces metabolized by the limiting; leukopenia, days; repeat every
hypomethylation of liver and excreted in thrombocytopenia, and 4 wk for 4–6 cycles
DNA, either inducing urine; elimination transient elevation of liver
apoptosis or half-life of 4 h function test results are
restoring normal common; nausea and
function; at higher vomiting and abdominal
doses, acts as a pain are common
cytidine analogue
Immunostimulant, BCG is a live, Urinary symptoms Intravesical instillation 81 mg per treatment,
vaccine; induces a attenuated bacterial predominate, including for noninvasive in 53 mL total
cellular immune culture; it does not dysuria, hematuria, bladder cancer after volume, given once
response at the site enter the body in hesitancy, urgency, removal of papillary weekly for 6 doses
of instillation viable form; it has frequency, and secondary tumors; also used for and then at 3, 6, 12,
no detectable infection; other toxicities some experimental 18, and 24 mo after
pharmacokinetic fate; include fever, chills, vaccine programs as the induction
in rare cases a clinical malaise, myalgias and an adjuvant to the
infection can result arthralgias, anorexia, vaccine
from treatment, nausea, vomiting, and
indicating invasion of anemia; clinical
the body at site of mycobacterial infection is
administration into rare, usually seen only in
systemic circulation immunocompromised
patients
Inhibition of HDAC Avoid strong UGT1A1 Nausea, fatigue, pyrexia, Relapsed or refractory 1000 mg/m2 IV over
inhibitors anemia, vomiting, T-cell lymphoma 30 min, days 1–5
decreased platelets and every 21 days
WBCs, infection,
hepatotoxicity, tumor lysis
syndrome, fetal harm
Alkylating agent Reduce dose for Nausea, pyrexia, vomiting, CLL and B-cell CLL: 100 mg/m2 over
hematologic toxicity hematologic non-Hodgkin 60 min, day 1, 2 of
abnormalities lymphoma 28-day cycle; B-cell:
120 mg/m2 over
30 min on day 1, 2 of
21-day cycle
Recombinant Administered by Asthenia, pain, nausea, Metastatic colorectal 5–15 mg/kg/day IV
humanized intravenous infusion;vomiting, diarrhea, cancer and NSCLC; every 3 wk
monoclonal antibody half-life is 20 days; the
anorexia, stomatitis, RCC
that binds to all fate of parent drug dermatitis, hypertension,
forms of VEGF, and metabolites is proteinuria; infusion-
preventing binding unknown related reactions rare;
to its receptors hemoptysis, hemorrhage,
delayed wound healing, GI
perforations; increased
risk of thromboembolic
events can be severe or
fatal
Synthetic retinoid, Good oral Hyperlipidemia is dose CTCL (mycosis 300 mg/kg/day PO,
differentiating agent bioavailability limiting and should be fungoides) refractory dose adjusted for
increased by a monitored during therapy to at least one prior toxicity
high-fat meal; and treated as therapy
metabolized in the appropriate; pruritus,
liver to oxidative leukopenia, diarrhea,
metabolites by fatigue, headache, and
cytochrome P450 3A4, liver function test result
glucuronidated, elevation can also be dose
eliminated in bile limiting; rash, edema,
fever, chills, and nausea
are uncommon; excessive
bleeding and back or
abdominal pain are rare
Continued
419.e4 Part I: Science and Clinical Oncology
Appendixa—cont’d
Drug Name
Bicalutamide (Casodex)
Bleomycin (Blenoxane)
Blinatumomab (Blincyto)
Bortezomib (Velcade)
Bosutinib (Bosulif )
Brentuximab vedotin
(Adcetris)
Brigatinib (Alunbrig)
Buserelin (Suprefact), HOE 766
Drug Class and/or Pharmacokinetics Dosage and
Mechanism and Metabolism Toxicity Indications Administration
Nonsteroidal Well absorbed orally; Constitutional symptoms Stage D2 prostate 50 mg PO daily, in
antiandrogen highly protein bound; dominate: hot flashes, cancer, in combination combination with an
converted to inactive decreased libido, with an LHRH agonist LHRH agonist agent
metabolites in liver depression, weight gain, agent
via oxidation and edema, gynecomastia,
glucuronidation; early disease-site pain
half-life of several (flare reaction);
days constipation, nausea,
vomiting, anorexia,
diarrhea, dizziness
uncommon; dyspnea,
anemia, fever, and rashes
rare
Antitumor antibiotic; Elimination half-life of Pulmonary toxicity, Germ cell tumors, Observe 1–6 h; after
causes DNA strand 3–5 h; incompletely including reversible and Hodgkin disease, and 2-unit intravenous
breaks directly in metabolized by irreversible fibrosis, is dose squamous cell test dose over
normal and intracellular limiting; other common cancers; used off-label15 min, the full dose
neoplastic cells aminopeptidases; toxicities include fever, for melanoma, ovarian can be given; usual
excreted in the kidney chills, rash, exfoliation, cancer, and Kaposi dose is 10–20 units/
as unchanged drug anorexia; nausea, sarcoma; also used as m2 IV, IM, or SC 1–2
and metabolites vomiting, a sclerosing agent for times per wk, or
myelosuppression, malignant pleural or 15–20 units/m2/day
anaphylaxis, mucositis rare pericardial effusions as a continuous
infusion over 3–7
days; as a sclerosing
agent, 60 units
generally used
CD19-directed Premedicate with Severe cytokine release Relapsed or refractory Induction: 28 days
antibody to target T dexamethasone syndrome, severe B-cell ALL in adults via continuous
cells neurotoxicity and children intravenous infusion;
variable dosage rates,
see product labeling
Proteasome inhibitor Metabolized in the Cardiovascular; peripheral Multiple myeloma; 1.3 mg/m2 IV or SC
liver by CYP2C19 and neuropathy; skin rash; mantle cell lymphoma days 1, 4, 8, 11, 22, 25,
CYP3A4; reduce dose nausea and diarrhea 29, and 32 of a
for hepatic 42-day treatment
dysfunction cycle for 4 initial
cycles
Kinase inhibitor Take with food; reduce GI toxicity should Ph+ CML, resistant or 500 mg/day PO
to 200 mg if hepatic be monitored; intolerant to prior
impaired; CYP3A myelosuppressive therapy
substrate
CD30-directed Monitor patients Progressive multifocal Hodgkin lymphoma 1.8 mg/kg
antibody conjugate taking CYP3A inducers leukoencephalopathy; and anaplastic large intravenous infusion
or inhibitors peripheral neuropathy; cell lymphoma over 30 min every
neutropenia 3 wk
Kinase inhibitor With or without food Pneumonitis, ALK+ metastasis, 90 mg QD × 7 days
hypertension, bradycardia, NSCLC, failed or PO; increase to
visual effects, enzyme intolerant to crizotinib 180 mg/day if
elevations, hyperglycemia, tolerated
nausea, diarrhea, fatigue,
cough, headache
LHRH agonist; shuts Intravascular and Flare reactions, which can Prostatic cancer 500 µg SC TID for the
off LH and FSH extravascular be prevented; castration first wk, then 200 µg/
secretion, producing proteolysis symptoms such as hot day, or intranasally
chemical castration flashes and decreased 800 µg TID followed
libido common; other by 400 µg TID
nonspecific symptoms
include headache, nausea,
vomiting, diarrhea,
constipation, weakness

Appendixa—cont’d
Drug Name
Busulfan (Myleran Oral),
(Busulfex Injection), BSF
Cabazitaxel (Jevtana)
Cabozantinib
Capecitabine (Xeloda)
Carboplatin (Paraplatin), carbo, Atypical alkylator;
CBDCA
Carfilzomib (Kyprolis)
Cancer Pharmacology  •  CHAPTER 25 419.e5
419.e5
Drug Class and/or Pharmacokinetics Dosage and
Mechanism and Metabolism Toxicity Indications Administration
Alkylating agent Excellent oral Myelosuppression partly PO: palliative 4–8 mg/day PO
bioavailability, with chronic and cumulative, is treatment of chronic initially, decreasing
peak levels in serum dose limiting; other myelogenous based on blood
occurring at about common toxicities: (myeloid, myelocytic, counts, with 1–3 mg
1 h; elimination nausea, vomiting, granulocytic) daily used for
half-life of 2.5 h; anorexia, mucositis, leukemia; IV: in maintenance;
metabolized partially hyperpigmentation, combination with intravenous
in liver; parent drug elevated liver function cyclophosphamide as conditioning dose
and metabolites tests (or venoocclusive a conditioning 0.8 mg/kg every 6 h
excreted in the urine; disease of the liver at regimen before for 4 days
also available IV transplant doses); allogeneic
neurologic toxicity, hematopoietic
including blurred vision, progenitor cell
dizziness, and confusion, transplantation for
and ILD are less common CML
Microtubule inhibitor Caution for patients Neutropenia and Metastatic hormone 25 mg/m2 every 3 wk
taking strong CYP3A hypersensitivity refractory prostate 1-h intravenous
inducers or inhibitors cancer when infusion with oral
docetaxel fails prednisone 10 mg
daily
Inhibits many 55-h half-life; take Perforations, fistulas, and Metastatic medullary 140 mg daily
receptor tyrosine without food hemorrhage thyroid cancer
kinases
Oral antimetabolite Readily absorbed, Myelosuppression and Metastatic BC and 1250 mg/m2 every 12 
prodrug metabolized in vivo to palmar-plantar metastatic colorectal
h for 14 days every
fluorouracil by erythrodysesthesia are cancer; used also in
21 days; dose
carboxylesterase, dose limiting; diarrhea, head and neck reductions are often
cytidine deaminase, fatigue, stomatitis, and squamous cell cancer
required; treatment
thymidine hyperbilirubinemia are delays are sometimes
phosphorylase uncommon; nausea, required; other doses
vomiting, and rash are and schedules have
rare been used
Rapidly cleared from Hemorrhage; Ovarian cancer and Dosage based on
produces intrastrand the bloodstream after thrombocytopenia used extensively in square meters or
and interstrand intravenous infusion, testicular cancer, via formulas that
cross-links in DNA via with a terminal squamous cell cancers take into account
association bonds half-life of 2.5 h; it is of the head and neck renal function and
with the platinum cleared largely as and cervix, and lung desired level of
molecule, leading to unchanged drug by cancer thrombocytopenia
DNA strand breakage the kidneys (such as Calvert’s
during replication formula); typical
doses with normal
renal function are in
300–500 mg/m2
range as intravenous
infusion
Proteosome inhibitor Drug-drug Fatigue, anemia, nausea, Multiple myeloma 20 mg/m2/day IV
interactions unlikely; thrombocytopenia, over 2–10 min, days
no adjustment for dyspnea, diarrhea, and 1, 2, 8, 9, 15, 16; rest
renal impairment; no pyrexia; monitor for period (days 17–28);
experience with cardiac, pulmonary, tumor 27 mg/m2/day other
hepatic impairment lysis, infusion reactions, cycles if tolerated
thrombocytopenia
Continued
419.e6 Part I: Science and Clinical Oncology
Appendixa—cont’d
Drug Name
Carmustine (BiCNU), BCNU,
bischloronitrosourea
Carmustine impregnated
wafer (Gliadel), polifeprosan 20 mechanism for
with carmustine implant
Ceritinib (Zykadia)
Cetuximab (Erbitux)
Chlorambucil (Leukeran)
Drug Class and/or Pharmacokinetics Dosage and
Mechanism and Metabolism Toxicity Indications Administration
Alkylator agent in the After an intravenous Myelosuppression slow in Melanoma, stomach Single-agent dose is
nitrosourea class; cell infusion, the drug is onset, cumulative and cancer, colon cancer, 150–200 mg/m2
cycle independent rapidly taken up by dose limiting; nausea, and liver cancer; FDA every 6 wk; for
mechanism tissues, including the vomiting common, approved for brain transplant, the dose
CNS; extensively can be severe; tumors, multiple is as high as 600 mg/
metabolized in the hyperpigmentation, renal myeloma, Hodgkin m2, along with other
liver; the serum toxicity can occur; ILD disease, lymphoma; drugs
half-life is only with fibrosis rare but can also used for BC
15–20 min occur with any dose;
transplant doses can
cause severe liver toxicity
and more frequent lung
toxicity
Novel delivery More than 70% of the None Patients with newly Up to 8 wafers are
copolymer degrades diagnosed high-grade placed in the
classic nitrosourea by 3 wk; minimal malignant glioma as resection cavity at
alkylating agent systemic exposure to an adjunct to surgery the time of
carmustine and radiation; also, craniotomy and
patients with operative resection
recurrent
glioblastoma
multiforme as an
adjunct to surgery
Kinase inhibitor No food within 2 h Severe GI toxicity: ALK+ metastasis, 750 mg daily PO
diarrhea, nausea, hepatic NSCLC, failed or
enzymes, abdominal pain, intolerant to crizotinib
fatigue, lesser appetite,
constipation
Recombinant Metabolism poorly Infusion reaction with Metastatic colorectal 400 mg/m2 IV,
humanized understood; half-life rapid-onset dyspnea, fever, cancer, head and neck followed by 250 mg/
monoclonal antibody, 5–7 days with minimal chills, urticaria, flushing, cancer in combination m2 weekly
targeting EGFR; clearance by kidneys angioedema, hypotension with radiation
competitively inhibits or liver is seen in 40%–50%
growth factor of patients; acneiform
binding; inhibits rash is common;
autophosphorylation constitutional symptoms;
and cell signaling hypomagnesemia; ILD
rare
Alkylating agent; cell Excellent oral Myelosuppression dose CLL and low-grade 16 mg/m2/day for 5
cycle–independent bioavailability; limiting, universal, lymphomas; also used days every 4 wk, or
maximum plasma can be cumulative; for Waldenström 0.4 mg/kg every
level at 1 h; half-life of nausea, vomiting, diarrhea macroglobulinemia, 2–4 wk, or
1–2 h; extensively mild and uncommon; multiple myeloma, 0.1–0.2 mg/kg/day
metabolized in liver to sterility, alopecia minor hairy cell leukemia, for 3–6 wk
active and inactive occurrence; pulmonary and rarely in some
metabolites fibrosis and neurologic solid tumors
adverse effects quite rare

Appendixa—cont’d
Drug Name
Cisplatin (Platinol)—cDDP,
DDP, cisplatinum, cis-
diamminedichloroplatinum (II) and interstrand
Cladribine (Leustatin)
Clofarabine (Clofarex, Clolar)
Cobimetinib (Cotellic)
Crizotinib (Xalkori)
Cyclophosphamide
Cancer Pharmacology  •  CHAPTER 25 419.e7
419.e7
Drug Class and/or Pharmacokinetics Dosage and
Mechanism and Metabolism Toxicity Indications Administration
Atypical alkylator; Adequate renal Nephrotoxicity dose Used for almost every Cisplatin can be
produces intrastrand perfusion and urine limiting for single dose class of solid tumor given all in one
output are critical for Neurotoxicity, especially and lymphoma; FDA intravenous infusion
cross-links in DNA via minimizing renal painful peripheral approved for testicular or daily as an
association bonds toxicity; therefore neuropathy, dose limiting and ovarian cancer intravenous infusion
with the platinum prehydration and for cumulative doses and transitional cell for several days, (the
molecule, leading to adequate Mild myelosuppression carcinoma latter method is
DNA strand breakage posttreatment Nausea, vomiting somewhat better
during replication hydration are used, common but manageable; tolerated); total dose
usually with normal Anorexia, diarrhea per cycle ranges
saline solution with or common; cumulative from 80–160 mg/m2;
without mannitol, ototoxicity also common; continuous infusion
potassium, and chronic renal magnesium can also be used;
magnesium and potassium wasting dose should be
common, sometimes reduced for
not reversible creatinine clearance
Elevated liver below 60 mL/min
transaminases seen, but along with the
alopecia or cardiac cisplatin; cisplatin
conduction abnormalities 100–200 mg/m2 is
rare also used
intraperitoneally for
ovarian cancer
Purine Dose adjusted for Fatigue, headache; skin AML; CLL; mantle cell IV, dosage regimens
antimetabolite; renal dysfunction rash; nausea; bone lymphoma are disease specific
activated by marrow suppression;
deoxycytidine kinase infection; fever
and incorporated
into DNA, resulting in
DNA strand breaks
Purine Eliminated by the Blood count suppression; ALL in adults at 52 mg/m2, 2-hour IV
antimetabolite; kidney; clearance rash; capillary leak relapse; refractory infusion; QDx5;
inhibits decreased as syndrome; tachycardia adult AML in patients 28-day cycle
ribonucleotide creatinine clearance is and hypotension; nausea younger than 70 yr
reductase affecting reduced or vomiting; diarrhea; Relapsed/refractory
DNA synthesis; also headache; altered liver ALL in patients 1-21
alters mitochondrial function tests; infection years old after at least
membranes and fever 2 prior regiments
enhancing apoptosis
Inhibits MEK1 and With or without food; Diarrhea, photosensitivity, Unresectable or 60 mg QD PO for 21
MEK2 in the MAPK avoid strong or new primary malignancy, metastatic melanoma days on 28-day cycle
cascade moderate CYP3A hemorrhage, with BRAF mutation,
inducers or inhibitors cardiomyopathy, retinal, combined with
hepatic, rhabdomyolysis vemurafenib
ALK kinase inhibitor Avoid strong CYP3A Hepatotoxicity; ALK-positive NSCLC 250 mg BID; reduce
inhibitors, inducers, or pneumonitis; QT interval or interrupt based on
substrates prolonged toxicity
Alkylating agent Metabolized to its Myelosuppression; Hodgkin and 500 mg to several
active alkylating immunosuppression; non-Hodgkin grams IV on a variety
species by mixed hemorrhagic cystitis; lymphoma; leukemias; of schedules;
function oxidases in myocarditis; pneumonitis; ovarian cancer; BC; 1–5 mg/kg PO on an
hepatic microsomes secondary malignancies; retinoblastoma; intermittent
including CYP2A6 and infertility; hyponatremia; neuroblastoma schedule
CYP3A4; decreased venoocclusive disease;
activation to nausea and vomiting;
therapeutic species in mucositis
the face of severe
hepatic dysfunction
Continued
419.e8 Part I: Science and Clinical Oncology

Appendixa—cont’d
Drug Class and/or Pharmacokinetics Dosage and
Drug Name Mechanism and Metabolism Toxicity Indications Administration
Cytarabine Antimetabolite that Metabolized in the Myelosuppression; AML in adults and Intravenous; dose
inhibits DNA liver and excreted by immunosuppression; CNS, children and schedule vary by
polymerase after the kidney cardiac, and pulmonary treatment regimen
activation to its toxicity; infection; fever,
nucleotide bone and chest pain, and
triphosphate, is rash within hours of
incorporated into administration (ara-C
DNA and RNA, and syndrome); nausea,
blocks cell cycle diarrhea; mucositis;
progression from G1 hepatic dysfunction
to S phase
Dabrafenib (Tafinlar) Kinase inhibitor Take 1 h before or 2 h Hyperkeratosis, headache, Unresectable or 150 mg PO BID ±
after food; avoid use hemorrhage, pyrexia, metastatic melanoma 2 mg of trametinib
with strong CYP3A arthralgia, papilloma, with BRAF mutation,
or CYP2C8 inhibitors chills, abdominal pain, single-agent or
or /inducers fatigue, rash, peripheral combined with
Sensitive substrates of edema, constipation, trametinib
CYP3A, CYP2C8, myalgia
CYP2C9, CYP2C19 may
lose efficacy
Daratumumab (Darzalex) CD38-directed Premedicate with Infusion reaction, fatigue,
Multiple myeloma 16 mg/kg IV weekly
antibody corticosteroids, nausea, back pain, pyrexia,