Gunderson Teppers Clinical Radiation Oncology 5Th Edition Joel Tepper Full Chapter

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Radiation Oncology 5th Edition Joel

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ASSOCIATE EDITORS
Jeffrey A. Bogart, MD Abram Recht, MD

Professor and Chair Professor
Department of Radiation Oncology Department of Radiation Oncology
SUNY Upstate Medical University Harvard Medical School
Syracuse, New York Vice Chair
Department of Radiation Oncology
Minesh P. Mehta, MBChB, FASTRO Beth Israel Deaconess Medical Center
Professor and Chair Boston, Massachusetts
FIU Herbert Wertheim College of Medicine Christopher L. Tinkle, MD, PhD
Deputy Director and Chief Assistant Member
Miami Cancer Institute Department of Radiation Oncology
Baptist Health South Florida St. Jude Children’s Research Hospital
Miami, Florida Memphis, Tennessee
Andrea K. Ng, MD, MPH Akila N. Viswanathan, MD, MPH

Professor Professor
Department of Radiation Oncology Department of Radiation Oncology and Molecular Radiation Sciences
Harvard Medical School Johns Hopkins University School of Medicine
Dana-Farber Cancer Institute Baltimore, Maryland
Brigham and Women’s Hospital
Boston, Massachusetts
5 th
EDITION
Gunderson & Tepper’s
CLINICAL
RADIATION
ONCOLOGY
SENIOR EDITORS
Joel E. Tepper, MD, FASTRO

Hector MacLean Distinguished Professor of Cancer Research
University of North Carolina Lineberger Comprehensive Cancer Center
University of North Carolina School of Medicine
Chapel Hill, North Carolina
Robert L. Foote, MD, FACR, FASTRO

Hitachi Professor of Radiation Oncology Research
Mayo Clinic College of Medicine and Science, Mayo Clinic
Rochester, Minnesota
Jeff M. Michalski, MD, MBA, FACR, FASTRO

Carlos A. Perez Distinguished Professor
Vice Chair of Radiation Oncology
Washington University School of Medicine in St. Louis
St. Louis, Missouri
Elsevier
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GUNDERSON & TEPPER’S CLINICAL RADIATION ONCOLOGY, ISBN: 978-0-323-67246-7

FIFTH EDITION
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9 8 7 6 5 4 3 2 1
To my wife Laurie, for her support and for teaching me what is important in
life, and who has made me a better person;
and to my family, including Miriam, Adam, Abigail, Agustin,
Zekariah, Zohar, Sammy, Marcelo, Jonah, and Aurelio
for the love and support they have given me for many years.
To my parents, who taught me the importance of education,
learning, and doing that which should be done.
To my many mentors who taught me in the past
and those who continue to teach me.
To my professional colleagues, both at the University
of North Carolina and around the country,
who have made me a better physician.
JET
To Kally, who, during our 40 years of marriage, has

made innumerable, selfless, personal sacrifices
for my patients and for my professional career.
To my father, Leonard, who introduced me to the art,
science, and practice of medicine.
To John Earle, Len Gunderson and John Noseworthy.
Their mentorship has enriched my professional life
with experiences, opportunities, and growth beyond my wildest dreams.
To the Mayo Clinic for providing a patient-centered,
collegial, cooperative, compassionate, respectful,
scholarly, integrated, professional, innovative,
and healing environment in which to work and serve.
RLF
To Sheila, my loving wife of 31 years, for her steadfast

support of my career and academic endeavors
while also encouraging me to enjoy life with our family.
To our children, Basia, Sophie, and Jeffrey, who have
enriched my life with their love and support.
To my parents, Richard and Rita, who both faced
their own challenges of cancer therapy
and taught me perspectives of care that you don’t get in medical school.
To my mentors, especially Drs. Jim Cox and Larry Kun,
who we lost this past year during the development
of this new edition. They inspired me to reach higher.
And finally, to my colleagues at Washington University
in St. Louis who have patiently given me
the time and support to contribute to this work.
JMM
Downloaded for upr@ck7.com upr07 (upr@ck8.com) at Autonomous University of Guadalajara from ClinicalKey.com by Elsevier on April 23, 2020.
For personal use only. No other uses without permission. Copyright ©2020. Elsevier Inc. All rights reserved.
CONTRIBUTORS
Christopher D. Abraham, MD Jonathan B. Ashman, MD, PhD Philippe L. Bedard, MD, FRCPC
Assistant Professor Assistant Professor Medical Oncologist
Department of Radiation Oncology Department of Radiation Oncology Princess Margaret Cancer Centre
Washington University School of Medicine Mayo Clinic College of Medicine and Science Associate Professor
in St. Louis Phoenix, Arizona Department of Medicine
St. Louis, Missouri University of Toronto
Matthew T. Ballo, MD Toronto, Ontario, Canada
Ross A. Abrams, MD Professor
Department of Radiation Oncology Radiation Oncology Jonathan J. Beitler, MD, MBA, FACR,
Rush University Medical Center West Cancer Center and Research Institute FASTRO
Chicago, Illinois Memphis, Tennessee Professor
Departments of Radiation Oncology,
Aydah Al-Awadhi, MBBS Lucia Baratto, MD Otolaryngology, and Hematology and
Department of Cancer Medicine Research Fellow Medical Oncology
University of Texas MD Anderson Cancer Department of Radiology Emory University School of Medicine
Center Division of Nuclear Medicine and Molecular Georgia Research Alliance Clinical Scientist
Houston, Texas Imaging Winship Cancer Institute of Emory
Stanford University University
Kaled M. Alektiar, MD Stanford, California NRG Institutional Principal Investigator
Member Atlanta, Georgia
Department of Radiation Oncology Christopher Andrew Barker, MD
Memorial Sloan Kettering Cancer Center Radiation Oncologist Sushil Beriwal, MD, MBA
New York, New York Director of Clinical Investigation Professor
Jan Alsner, PhD Memorial Sloan Kettering Cancer Center UPMC Hillman Cancer Center
Professor New York, New York University of Pittsburgh School of Medicine
Department of Experimental Clinical Pittsburgh, Pennsylvania
Oncology Adam Bass, MD
Aarhus University Hospital Associate Professor of Medicine Ranjit S. Bindra, MD, PhD
Aarhus, Denmark Dana-Farber Cancer Institute Associate Professor
Harvard Medical School Therapeutic Radiology
K. Kian Ang, MD, PhD† Boston, Massachusetts Yale Medical School
Professor and Gilbert H. Fletcher Endowed New Haven, Connecticut
Chair Brian C. Baumann, MD
Department of Radiation Oncology Assistant Professor Michael W. Bishop, MD
University of Texas MD Anderson Cancer Department of Radiation Oncology Assistant Member
Center Washington University School of Medicine Department of Oncology
Houston, Texas in St. Louis St. Jude Children’s Research Hospital
St. Louis, Missouri Memphis, Tennessee
Lilyana Angelov, MD, FAANS, FRCS(C)
The Kerscher Family Chair for Spine Tumor Beth M. Beadle MD, PhD Rachel Blitzblau, MD, PhD
Excellence Associate Professor Associate Professor
Head, Section of Spine Tumors Department of Radiation Oncology Department of Radiation Oncology
Professor, Department of Neurological Stanford University Duke University Medical Center
Surgery Stanford, California Durham, North Carolina
Cleveland Clinic Lerner College of Medicine
at Case Western Reserve University Staci Beamer, MD Jeffrey A. Bogart, MD
Rose Ella Burkhart Brain Tumor and Neuro- Assistant Professor Professor and Chair
Oncology Center Division of Cardiovascular and Thoracic Department of Radiation Oncology
Department of Neurosurgery Surgery SUNY Upstate Medical University
Neurological Institute and Taussig Cancer Mayo Clinic College of Medicine and Science Syracuse, New York
Institute Phoenix, Arizona
Cleveland Clinic James A. Bonner, MD
Cleveland, Ohio Chairman and Merle M. Salter Professor
The University of Alabama at Birmingham
Birmingham, Alabama
†
Deceased
vii
viii CONTRIBUTORS
J. Daniel Bourland, PhD, MSPH Bruce A. Chabner, MD Stephen G. Chun, MD

Professor Clinical Director, Emeritus Assistant Professor
Radiation Oncology, Biomedical Massachusetts General Hospital Cancer Department of Radiation Oncology
Engineering, and Physics Center The University of Texas MD Anderson
Wake Forest School of Medicine Massachusetts General Hospital Cancer Center
Winston-Salem, North Carolina Professor of Medicine Houston, Texas
Harvard Medical School
Joseph A. Bovi, MD Boston, Massachusetts Christine H. Chung, MD
Department of Radiation Oncology Chair, Department of Head and Neck–
Medical College of Wisconsin Michael D. Chan, MD Endocrine Oncology
Froedtert Memorial Lutheran Hospital Associate Professor and Vice Chairman Moffitt Cancer Center
Milwaukee, Wisconsin Department of Radiation Oncology Tampa, Florida
Wake Forest School of Medicine
Andrew G. Brandmaier, MD, PhD Winston-Salem, North Carolina Peter W. M. Chung, MBChB, MRCP,
Assistant Professor FRCR, FRCPC
Department of Radiation Oncology Samuel T. Chao, MD Radiation Oncologist
Weill Cornell Medical College Associate Professor Princess Margaret Cancer Centre
New York, New York Department of Radiation Oncology Associate Professor
Rose Ella Burkhardt Brain Tumor and Department of Radiation Oncology
John Breneman, MD Neuro-Oncology Center University of Toronto
Professor Cleveland Clinic Toronto, Ontario, Canada
Department of Radiation Oncology Cleveland, Ohio
University of Cincinnati Jeffrey M. Clarke, MD
Cincinnati Children’s Hospital Medical Anne-Marie Charpentier, MD, FRCPC Assistant Professor
Center Radiation Oncologist Department of Medicine
Cincinnati, Ohio Centre Hospitalier de l’Université de Division of Medical Oncology
Montréal Duke University School of Medicine
Juan P. Brito, MD Clinical Assistant Professor Durham, North Carolina
Assistant Professor of Medicine Université de Montréal
Division of Endocrinology, Diabetes, Montréal, Quebec, Canada Louis S. Constine, MD, FASTRO, FACR
Metabolism, and Nutrition The Philip Rubin Professor of Radiation
Mayo Clinic College of Medicine and Aadel A. Chaudhuri, MD Oncology and Pediatrics
Science, Mayo Clinic Assistant Professor Vice Chair, Department of Radiation
Rochester, Minnesota Department of Radiation Oncology Oncology
Washington University School of Medicine James P. Wilmot Cancer Center
Michael D. Brundage, MD, MSc, FRCPC in St. Louis University of Rochester Medical Center
Professor St. Louis, Missouri The Judy DiMarzo Cancer Survivorship
Oncology and Public Health Sciences Program
Queen’s University Nathan I. Cherny, MBBS, FRACP, FRCP James P. Wilmot Cancer Institute
Radiation Oncologist Norman Levan Chair of Humanistic University of Rochester Medical Center
Cancer Centre of Southeastern Ontario Medicine Rochester, New York
Kingston, Ontario, Canada Cancer Pain and Palliative Medicine Service
Shaare Zedek Medical Center Benjamin W. Corn, MD
Matthew R. Callstrom, MD, PhD Jerusalem, Israel Chairman
Professor of Radiology Department of Radiation Medicine
Mayo Clinic College of Medicine and Fumiko Chino, MD Shaare Zedek Medical Center
Science, Mayo Clinic Assistant Professor Jerusalem, Israel;
Rochester, Minnesota Department of Radiation Oncology Professor
Memorial Sloan Kettering Cancer Center Tel Aviv University School of Medicine
Felipe A. Calvo, MD, PhD New York, New York Tel Aviv, Israel
Professor and Chairman
Department of Oncology John P. Christodouleas, MD, MPH Allan Covens, MD, FRCSC
Clinica Universidad de Navarra Attending Physician Professor
Madrid, Spain Department of Radiation Oncology Department of Obstetrics and Gynecology
University of Pennsylvania Division of Gynecologic Oncology
George M. Cannon, MD Philadelphia, Pennsylvania Sunnybrook Health Sciences Centre
Adjunct Assistant Professor University of Toronto
Radiation Oncology Toronto, Ontario, Canada
University of Utah
Salt Lake City, Utah
CONTRIBUTORS ix
Christopher H. Crane, MD Ryan W. Day, MD Hugues Duffau, MD, PhD

Department of Radiation Oncology Instructor of Surgery Professor and Chairman
Memorial Sloan Kettering Cancer Center Mayo Clinic Montpellier University Medical Center
New York, New York Scottsdale, Arizona; Institute for Neurosciences of Montpellier
Senior Fellow Hôpital Gui de Chauliac
Carien L. Creutzberg, MD, PhD Department of Surgical Oncology Montpellier, France
Professor University of Texas MD Anderson Cancer
Department of Radiation Oncology Center Thierry Duprez, MD
Leiden University Medical Center Houston, Texas Medical Imaging and Radiology
Leiden, Netherlands Universite Catholique de Louvain
Amanda J. Deisher, PhD Head of Neurology/Head and Neck Section
Juanita M. Crook, MD, FRCP Instructor Cliniques Universitaires Saint-Luc
Professor Department of Radiation Oncology Brussels, Belgium
Department of Radiation Oncology Mayo Clinic College of Medicine and
University of British Columbia Science, Mayo Clinic Peter T. Dziegielewski, MD, FRCS(C)
Radiation Oncologist Rochester, Minnesota Associate Professor
Center for the Southern Interior Chief, Division of Head and Neck Oncologic
British Columbia Cancer Agency Thomas F. DeLaney, MD Surgery
Kelowna, British Columbia, Canada Andres Soriano Professor of Radiation Microvascular Reconstructive Surgery
Oncology Kenneth W. Grader Professor
Brian G. Czito, MD Harvard Medical School University of Florida College of Medicine
Professor Associate Medical Director Gainesville, Florida
Department of Radiation Oncology Francis H. Burr Proton Therapy Center
Duke Cancer Institute Massachusetts General Hospital Charles Eberhart, MD, PhD
Duke University Boston, Massachusetts Professor
Durham, North Carolina Pathology, Ophthalmology, and Oncology
Phillip M. Devlin, BPhil, MTS, EdM, MD, Johns Hopkins University School of
Bouthaina S. Dabaja, MD FACR, FASTRO, FFRRSCI (Hon) Medicine
Professor Chief, Division of Brachytherapy Baltimore, Maryland
Section Chief, Hematology Dana-Farber/Brigham and Women’s Cancer
Department of Radiation Oncology Center David W. Eisele, MD, FACS
University of Texas MD Anderson Cancer Associate Professor of Radiation Oncology Andelot Professor and Director
Center Harvard Medical School Department of Otolaryngology–Head and
Houston, Texas Institute Physician Neck Surgery
Dana-Farber Cancer Institute Johns Hopkins University School of
Thomas B. Daniels, MD Boston, Massachusetts Medicine
Department of Radiation Oncology Baltimore, Maryland
Mayo Clinic Arizona James J. Dignam, PhD
Assistant Professor of Radiation Oncology Professor Suzanne B. Evans, MD, MPH
Mayo Clinic College of Medicine and Science Department of Public Health Sciences Associate Professor, Therapeutic Radiology
Phoenix, Arizona University of Chicago Associate Director, Residency Program
Chicago, Illinois Yale University School of Medicine
Marc David, MD Statistics and Data Management Center New Haven, Connecticut
Assistant Professor NRG Oncology
Department of Radiation Oncology Michael Farris, MD
McGill University Health Centre Don S. Dizon, MD Assistant Professor
Montreal, Quebec, Canada Associate Professor Department of Radiation Oncology
Warren Alpert Medical School of Brown Wake Forest Baptist Health
Laura A. Dawson, MD University Winston-Salem, North Carolina
Professor Head of Women’s Cancers at Lifespan
Department of Radiation Oncology Cancer Institute Mary Feng, MD
Princess Margaret Cancer Centre Director of Medical Oncology Professor
University of Toronto Rhode Island Hospital Department of Radiation Oncology
Toronto, Ontario, Canada Providence, Rhode Island University of California, San Francisco
San Francisco, California
Jeffrey S. Dome, MD, PhD
Chief Rui P. Fernandes, MD, DMD, FACS,
Hematology and Oncology FRCS(Ed)
Children’s National Health System Associate Professor
Washington, DC OMS, Neurosurgery, Orthopedics, Surgery
University of Florida College of Medicine
Jacksonville, Florida
x CONTRIBUTORS
Gini F. Fleming, MD Adam S. Garden, MD Dennis E. Hallahan, MD

Professor Professor Elizabeth H. and James S. McDonnell
Department of Medicine Department of Radiation Oncology Distinguished Professor of Medicine
University of Chicago Medical Center University of Texas MD Anderson Cancer Chair, Department of Radiation Oncology
Chicago, Illinois Center Washington University School of Medicine
Houston, Texas in St. Louis
John C. Flickinger, MD Barnes Jewish Hospital
Professor Lilian T. Gien, MD St. Louis, Missouri
Department of Radiation Oncology Associate Professor
University of Pittsburgh Division of Gynecologic Oncology Christopher L. Hallemeier, MD
Radiation Oncologist Odette Cancer Center Associate Professor
Department of Radiation Oncology Sunnybrook Health Sciences Centre Department of Radiation Oncology
UPMC Presbyterian-Shadyside Toronto, Ontario, Canada Mayo Clinic College of Medicine and
Pittsburgh, Pennsylvania Science, Mayo Clinic
Mary K. Gospodarowicz, MD, FRCPC, Rochester, Minnesota
Robert L. Foote, MD, FACR, FASTRO FRCR (Hon)
Hitachi Professor of Radiation Oncology Professor and Chair Michele Y. Halyard, MD
Research Department of Radiation Oncology Professor
Department of Radiation Oncology University of Toronto Department of Radiation Oncology
Mayo Clinic College of Medicine and Princess Margaret Hospital Mayo Clinic College of Medicine and
Science, Mayo Clinic Toronto, Ontario, Canada Science, Mayo Clinic
Rochester, Minnesota Phoenix, Arizona
Cai Grau, MD, DMSc
Silvia C. Formenti, MD Professor Marc Hamoir, MD
Sandra and Edward Meyer Professor of Department of Oncology and Danish Centre Head and Neck Surgery
Cancer Research for Particle Therapy Chairman of the Executive Board of the
Chairman, Department of Radiation Aarhus University Hospital Cancer Center
Oncology Aarhus, Denmark Saint-Luc University Hospital Cancer Center
Associate Director, Meyer Cancer Institute Brussels, Belgium
Weill Cornell Medical College Vincent Grégoire, MD, PhD, FRCR
Radiation Oncologist in Chief Radiation Oncology Department Timothy P. Hanna, MD, MSc, PhD,
New York Presbyterian Hospital Centre Léon Bérard FRCPC
New York, New York Lyon, France Clinician Scientist
Cancer Care and Epidemiology
Benedick A. Fraass, PhD, FAAPM, Chul S. Ha, MD Cancer Research Institute at Queen’s
FASTRO, FACR Professor University
Vice Chair for Research, Professor and Department of Radiation Oncology Radiation Oncologist
Director of Medical Physics University of Texas Health Science Center at Cancer Centre of Southeastern Ontario
Department of Radiation Oncology San Antonio Kingston General Hospital
Cedars-Sinai Medical Center San Antonio, Texas Kingston, Ontario, Canada
Health Sciences Professor
Department of Radiation Oncology Michael G. Haddock, MD Paul M. Harari, MD
University of California, Los Angeles Professor of Radiation Oncology Jack Fowler Professor and Chairman
Los Angeles, California; Mayo Clinic College of Medicine and Human Oncology
Professor Emeritus Science, Mayo Clinic University of Wisconsin School of Medicine
Department of Radiation Oncology Rochester, Minnesota and Public Health
University of Michigan Madison, Wisconsin
Ann Arbor, Michigan Ezra Hahn, MD, FRCPC
Radiation Oncologist Joseph M. Herman, MD, MSc, MSHCM
Carolyn R. Freeman, MBBS, FRCPC, Department of Radiation Oncology Professor
FASTRO Princess Margaret Cancer Centre Department of Radiation Oncology
Professor of Oncology and Pediatrics Sunnybrook Health Sciences Centre of the The University of Texas MD Anderson
Mike Rosenbloom Chair of Radiation University of Toronto Cancer Center
Oncology Toronto, Ontario, Canada Houston, Texas
McGill University
Montreal, Quebec, Canada Matthew D. Hall, MD, MBA Michael G. Herman, PhD
Radiation Oncology Professor
Miami Cancer Institute Department of Radiation Oncology
Baptist Health South Florida Mayo Clinic College of Medicine and
Miami, Florida Science, Mayo Clinic
CONTRIBUTORS xi
Caroline L. Holloway, MD, FRCPC Patricia A. Hudgins, MD Brian D. Kavanagh, MD

Clinical Assistant Professor Professor Professor and Chair
Department of Radiation Oncology Department of Radiology and Imaging Department of Radiation Oncology
BC Cancer Agency, Vancouver Island Centre Sciences University of Colorado School of Medicine
Victoria, British Columbia, Canada Emory University School of Medicine University of Colorado Comprehensive
Atlanta, Georgia Cancer Center
Bradford S. Hoppe, MD, MPH Aurora, Colorado
Associate Professor Christine A. Iacobuzio-Donahue, MD,
Department of Radiation Oncology PhD Kara M. Kelly, MD
Mayo Clinic College of Medicine and Director, David M. Rubenstein Center for Waldemar J. Kaminski Endowed Chair of
Science, Mayo Clinic Pancreatic Cancer Research Pediatrics
Jacksonville, Florida Department of Radiation Oncology Department of Pediatric Oncology
Memorial Sloan Kettering Cancer Center Roswell Park Cancer Institute
Michael R. Horsman, PhD, DMSc New York, New York Division Chief, Pediatric Hematology/
Professor Oncology and Research Professor
Department of Experimental Clinical Andrei Iagaru, MD Department of Pediatrics
Oncology Assistant Professor University of Buffalo School of Medicine
Aarhus University Hospital Department of Radiology and Biomedical Sciences
Aarhus, Denmark Division of Nuclear Medicine and Molecular Buffalo, New York
Imaging
Janet K. Horton, MD Stanford University Amir H. Khandani, MD
Adjunct Associate Professor Stanford, California Associate Professor of Radiology
Duke University Medical Center Chief, Division of Nuclear Medicine
Durham, North Carolina Nicole M. Iñiguez-Ariza, MD Department of Radiology
Division of Endocrinology, Diabetes, University of North Carolina at Chapel Hill
Julie Howle, MBBS, MS, FRACS, FACS Metabolism, and Nutrition Chapel Hill, North Carolina
Surgical Oncologist Mayo Clinic College of Medicine and
Westmead Private Hospital Science, Mayo Clinic Deepak Khuntia, MD
Westmead, New South Wales, Australia; Rochester, Minnesota; Vice President, Medical Affairs
Clinical Senior Lecturer Department of Endocrinology and Oncology Systems
Department of Surgery Metabolism Varian Medical Systems, Inc.
The University of Sydney Instituto Nacional de Ciencias Médicas y Palo Alto, California;
Sydney, New South Wales, Australia Nutrición Salvador Zubirán Radiation Oncologist
Mexico City, Mexico Valley Medical Oncology Consultants
Brian A. Hrycushko, PhD Pleasanton, California
Assistant Professor Jedediah E. Johnson, PhD
Department of Radiation Oncology Assistant Professor Ana Ponce Kiess, MD, PhD
UT Southwestern Medical Center Department of Radiation Oncology Assistant Professor
Dallas, Texas Mayo Clinic College of Medicine and Departments of Radiation Oncology and
Science, Mayo Clinic Molecular Radiation Sciences
David Hsu, MD, PhD Rochester, Minnesota Johns Hopkins University School of
William Dalton Family Assistant Professor Medicine
Division of Medical Oncology Joseph G. Jurcic, MD Baltimore, Maryland
Department of Internal Medicine Professor of Medicine
Duke Cancer Institute Director, Hematologic Malignancies Section Joseph K. Kim, MD
Duke University Department of Medicine Resident Physician
Durham, North Carolina Columbia University Irving Medical Center Department of Radiation Oncology
Attending Physician New York University
Chen Hu, PhD New York-Presbyterian Hospital/Columbia New York, New York
Assistant Professor of Oncology University Irving Medical Center
Division of Biostatistics and Bioinformatics New York, New York Susan J. Knox, MD, PhD
Sidney Kimmel Comprehensive Cancer Associate Professor
Center John A. Kalapurakal, MD, FACR, Department of Radiation Oncology
Johns Hopkins University FASTRO Stanford University
Baltimore, Maryland Professor Stanford, California
Statistics and Data Management Center Department of Radiation Oncology
NRG Oncology Northwestern University Wui-Jin Koh, MD
Chicago, Illinois Senior Vice President and Chief Medical
Officer
National Comprehensive Cancer Network
(NCCN)
Philadelphia, Pennsylvania
xii CONTRIBUTORS
Rupesh R. Kotecha, MD Nancy Lee, MD William J. Mackillop, MBChB, FRCR,

Department of Radiation Oncology Radiation Oncologist FRCPC
Miami Cancer Institute Department of Radiation Oncology Professor
Baptist Health South Florida Memorial Sloan Kettering Cancer Center Oncology and Public Health Sciences
Department of Radiation Oncology New York, New York Queen’s University
FIU Herbert Wertheim College of Medicine Kingston, Ontario, Canada
Miami, Florida Percy Lee, MD
Associate Professor Kelly R. Magliocca, DDS, MPH
Matthew W. Krasin, MD Vice Chair of Education Assistant Professor
Member UCLA Department of Radiation Oncology Department of Pathology and Laboratory
Department of Radiation Oncology University of California, Los Angeles Medicine
St. Jude Children’s Research Hospital Los Angeles, California Emory University School of Medicine
Memphis, Tennessee Atlanta, Georgia
Benoît Lengelé, MD, PhD, FRCS, KB
Larry E. Kun, MD† Head of Department Anuj Mahindra, MBBS
Professor and Director of Educational Plastic and Reconstructive Surgery Director, Malignant Hematology
Programs Cliniques Universitaires Saint-Luc Division of Hematology/Oncology
Department of Radiation Oncology Brussels, Belgium Scripps Clinic
Professor, Department of Pediatrics La Jolla, California
UT Southwestern Medical Center William P. Levin, MD
Dallas, Texas Associate Professor Anthony A. Mancuso, MD
Department of Radiation Oncology Professor and Chair
A. Nicholas Kurup, MD Abramson Cancer Center of the University Department of Radiology
Associate Professor of Radiology of Pennsylvania Professor of Otolaryngology
Mayo Clinic College of Medicine and Philadelphia, Pennsylvania University of Florida College of Medicine
Science, Mayo Clinic Gainesville, Florida
Rochester, Minnesota Jeremy H. Lewin, MBBS, FRACP
Medical Oncologist Bindu Manyam, MD
Nadia N. Issa Laack, MD Peter MacCallum Cancer Centre Department of Radiation Oncology
Professor Clinical Senior Lecturer Alleghany Health Network
Chair, Department of Radiation Oncology Sir Peter MacCallum Department of Pittsburgh, Pennsylvania
Mayo Clinic College of Medicine and Oncology
Science, Mayo Clinic University of Melbourne Karen J. Marcus, MD, FACR
Rochester, Minnesota Melbourne, Victoria, Australia Associate Professor and Division Chief
Pediatric Radiation Oncology
Ann S. LaCasce, MD, MMSc Dror Limon, MD Dana-Farber/Boston Children’s Cancer and
Associate Professor of Medicine Head of CNS Radiotherapy Service Blood Disorders Center
Harvard Medical School Radiotherapy Institute Brigham and Women’s Hospital
Dana-Farber Cancer Institute Tel-Aviv Sourasky Medical Center Harvard Medical School
Boston, Massachusetts Tel-Aviv, Israel Boston, Massachusetts
Michael J. LaRiviere, MD Jacob C. Lindegaard, MD, DMSc Stephanie Markovina, MD, PhD
Resident Physician Associate Professor Assistant Professor
Department of Radiation Oncology Department of Oncology Department of Radiation Oncology
University of Pennsylvania Aarhus University Hospital Washington University School of Medicine
Philadelphia, Pennsylvania Aarhus, Denmark in St. Louis
St. Louis, Missouri
Andrew B. Lassman, MD Daniel J. Ma, MD
Chief, Neuro-Oncology Division Associate Professor Lawrence B. Marks, MD, FASTRO
Columbia University Irving Medical Center Department of Radiation Oncology Dr. Sidney K. Simon Distinguished Professor
Medical Director Mayo Clinic College of Medicine and of Oncology Research
Clinical Protocol and Data Management Office Science, Mayo Clinic Chair, Department of Radiation Oncology
Herbert Irving Comprehensive Cancer Center Rochester, Minnesota University of North Carolina School of
New York, New York Medicine
Shannon M. MacDonald, MD Chapel Hill, North Carolina
Colleen A. Lawton, MD Associate Professor
Vice Chair and Professor Department of Radiation Oncology Martha M. Matuszak, PhD
Department of Radiation Oncology Massachusetts General Hospital/Harvard Associate Professor
Medical College of Wisconsin Medical School Department of Radiation Oncology
Milwaukee, Wisconsin Boston, Massachusetts University of Michigan
Ann Arbor, Michigan
†
Deceased
CONTRIBUTORS xiii
Mark W. McDonald, MD Michael Mix, MD Paul Okunieff, MD

Associate Professor Assistant Professor Professor and Chair
Department of Radiation Oncology Department of Radiation Oncology Department of Radiation Oncology
Emory University School of Medicine SUNY Upstate Medical University University of Florida
Atlanta, Georgia Syracuse, New York Gainesville, Florida
Lisa A. McGee, MD Amy C. Moreno, MD Hilary L.P. Orlowski, MD

Assistant Professor Assistant Professor Assistant Professor of Radiology
Department of Radiation Oncology Department of Radiation Oncology Mallinckrodt Institute of Radiology
Mayo Clinic College of Medicine and The University of Texas MD Anderson Washington University School of Medicine
Science, Mayo Clinic Cancer Center in St. Louis
Phoenix, Arizona Houston, Texas St. Louis, Missouri
Paul M. Medin, PhD William H. Morrison, MD Sophie J. Otter, MD(Res), MRCP, FRCR
Professor Professor Consultant Clinical Oncologist
Department of Radiation Oncology Department of Radiation Oncology Department of Oncology
UT Southwestern Medical Center University of Texas MD Anderson Cancer Royal Surrey County Hospital
Dallas, Texas Center Guildford, Surrey, United Kingdom
Houston, Texas
Minesh P. Mehta, MBChB, FASTRO Roger Ove, MD, PhD
Professor and Chair Erin S. Murphy, MD Clinical Associate Professor
FIU Herbert Wertheim College of Medicine Assistant Professor Department of Radiation Oncology
Deputy Director and Chief Department of Radiation Oncology University Hospitals Case Medical Center
Department of Radiation Oncology Rose Ella Burkhardt Brain Tumor and Seidman Cancer Center
Miami Cancer Institute Neuro-Oncology Center Cleveland, Ohio
Baptist Health South Florida Cleveland Clinic
Miami, Florida Cleveland, Ohio Jens Overgaard, MD, DMSc
Professor
William M. Mendenhall, MD, FASTRO Rashmi K. Murthy, MD, MBE Department of Experimental Clinical
Professor Assistant Professor Oncology
Department of Radiation Oncology Department of Breast Medical Oncology Aarhus University Hospital
University of Florida College of Medicine University of Texas MD Anderson Cancer Aarhus, Denmark
Gainesville, Florida Center
Houston, Texas Manisha Palta, MD
Ruby F. Meredith, MD, PhD Associate Professor
Professor Andrea K. Ng, MD, MPH Department of Radiation Oncology
Department of Radiation Oncology Professor of Radiation Oncology Duke Cancer Institute
University of Alabama at Birmingham Dana-Farber Cancer Institute Duke University
Senior Scientist Brigham and Women’s Hospital Durham, North Carolina
UAB Comprehensive Cancer Center Harvard Medical School
University of Alabama at Birmingham Boston, Massachusetts Luke E. Pater, MD
Birmingham, Alabama Associate Professor
Marianne Nordsmark, MD, PhD Department of Radiation Oncology
Jeff M. Michalski, MD, MBA, FACR, Senior Staff Specialist University of Cincinnati
FASTRO Department of Oncology Cincinnati, Ohio
Carlos A. Perez Distinguished Professor Aarhus University Hospital
Vice Chair of Radiation Oncology Aarhus, Denmark Todd Pawlicki, PhD, FAAPM, FASTRO
Washington University School of Medicine Professor and Vice-Chair
in St. Louis Yazmin Odia, MD, MS Department of Radiation Medicine and
St. Louis, Missouri Lead Physician of Medical Neuro-Oncology Applied Sciences
Miami Cancer Institute Director, Division of Medical Physics and
Michael T. Milano, MD, PhD Baptist Health South Florida Technology
Professor Miami, Florida University of California, San Diego
Department of Radiation Oncology La Jolla, California
University of Rochester Desmond A. O’Farrell, MSc, CMD
Rochester, New York Teaching Associate in Radiation Oncology Jennifer L. Peterson, MD
Harvard Medical School Department of Radiation Oncology
Bruce D. Minsky, MD Clinical Physicist Mayo Clinic Florida
Professor of Radiation Oncology Department of Radiation Oncology Associate Professor of Radiation Oncology
Frank T. McGraw Memorial Chair Dana-Farber/Brigham and Women’s Cancer Mayo Clinic College of Medicine and Science
The University of Texas MD Anderson Center Jacksonville, Florida
Cancer Center Boston, Massachusetts
Houston, Texas
xiv CONTRIBUTORS
Thomas M. Pisansky, MD Pablo F. Recinos, MD David P. Ryan, MD

Professor Assistant Professor Clinical Director and Chief of Hematology/
Department of Radiation Oncology Department of Neurological Surgery Oncology
Mayo Clinic College of Medicine and Cleveland Clinic Massachusetts General Hospital Cancer
Science, Mayo Clinic Cleveland, Ohio Center
Rochester, Minnesota Professor of Medicine
Marsha Reyngold, MD, PhD Harvard Medical School
Erqi Pollom, MD, MS Radiation Oncologist Boston, Massachusetts
Stanford University Memorial Sloan Kettering Cancer Center Nabil F. Saba, MD
Stanford, California New York, New York Professor
Departments of Hematology and Medical
Louis Potters, MD, FACR, FASTRO, FABS Nadeem Riaz, MD Oncology and Otolaryngology
Professor and Chairperson Assistant Attending Emory University School of Medicine
Department of Radiation Medicine Department of Radiation Oncology Atlanta, Georgia
Northwell Health and the Zucker School of Memorial Sloan Kettering Cancer Center
Medicine at Hofstra/Northwell New York, New York Joseph K. Salama, MD
Deputy Physician-in-Chief Professor
Northwell Health Cancer Institute Kenneth B. Roberts, MD Department of Radiation Oncology
Lake Success, New York Professor Duke University School of Medicine
Department of Therapeutic Radiology Durham, North Carolina
Harry Quon, MD, MS Yale University School of Medicine
Associate of Radiation Oncology and New Haven, Connecticut John T. Sandlund Jr, MD
Molecular Radiation Sciences Member, Department of Oncology
Johns Hopkins University School of Stephen S. Roberts, MD St. Jude Children’s Research Hospital
Medicine Associate Attending Physician Professor
Baltimore, Maryland Department of Pediatrics Department of Pediatrics
Memorial Sloan Kettering Cancer Center University of Tennessee College of Medicine
David Raben, MD New York, New York Memphis, Tennessee
Professor
Department of Radiation Oncology Claus M. Rödel, MD Michael Heinrich Seegenschmiedt, MD
University of Colorado Professor and Chairman Professor
Aurora, Colorado Radiotherapy and Oncology Strahlentherapie Osnabrück
University Hospital Frankfurt, Goethe Osnabrück, Germany
Ezequiel Ramirez, MS, CMD RT(R)(T) University
Chief Medical Dosimetrist Frankfurt, Germany Amy Sexauer, MD, PhD
University of California, San Francisco Dana-Farber Cancer Institute
San Francisco, California Carlos Rodriguez-Galindo, MD Division of Pediatrics
Member and Chair Hematology/Oncology/Stem Cell Transplant
Demetrios Raptis, MD Department of Global Pediatric Medicine Department of Pediatrics
Assistant Professor of Radiology Member, Department of Oncology Boston Children’s Hospital
Mallinckrodt Institute of Radiology St. Jude Children’s Research Hospital Boston, Massachusetts
Washington University School of Medicine Memphis, Tennessee
in St. Louis Jacob E. Shabason, MD
St. Louis, Missouri C. Leland Rogers, MD Assistant Professor
Professor Department of Radiation Oncology
Michal Raz, MD Department of Radiation Oncology Perelman School of Medicine at the
Neuropathologist Barrow Neurological Institute University of Pennsylvania
Pathology Department Phoenix, Arizona Philadelphia, Pennsylvania
Tel-Aviv Sourasky Medical Center
Tel-Aviv, Israel Todd L. Rosenblat, MD Chirag Shah, MD
Assistant Professor of Medicine Department of Radiation Oncology
Abram Recht, MD Columbia University Irving Medical Center Taussig Cancer Institute
Professor New York, New York Cleveland Clinic
Department of Radiation Oncology Cleveland, Ohio
Harvard Medical School William G. Rule, MD
Vice Chair Assistant Professor Jason P. Sheehan, MD
Department of Radiation Oncology Department of Radiation Oncology Harrison Distinguished Professor
Beth Israel Deaconess Medical Center Mayo Clinic College of Medicine and Neurological Surgery
Boston, Massachusetts Science, Mayo Clinic University of Virginia
Phoenix, Arizona Charlottesville, Virginia
CONTRIBUTORS xv
Arif Sheikh, MD John H. Suh, MD Chiaojung Jillian Tsai, MD, PhD

Mount Sinai Health System Professor and Chairman Radiation Oncologist
New York, New York Department of Radiation Oncology Department of Radiation Oncology
Rose Ella Burkhardt Brain Tumor and Memorial Sloan Kettering Cancer Center
Anup S. Shetty, MD Neuro-Oncology Center New York, New York
Assistant Professor of Radiology Cleveland Clinic
Mallinckrodt Institute of Radiology Cleveland, Ohio Richard W. Tsang, MD, FRCPC
Washington University School of Medicine Professor
in St. Louis Winston W. Tan, MD Department of Radiation Oncology
St. Louis, Missouri Division of Hematology and Oncology University of Toronto
Mayo Clinic Florida Princess Margaret Hospital
Arun D. Singh MD Associate Professor of Medicine Toronto, Ontario, Canada
Professor of Ophthalmology Mayo Clinic College of Medicine and Science
Department of Ophthalmic Oncology Jacksonville, Florida Mark D. Tyson, MD
Cleveland Clinic Department of Urology
Cleveland, Ohio Joel E. Tepper, MD, FASTRO Mayo Clinic Arizona
Hector MacLean Distinguished Professor of Assistant Professor of Urology
William Small Jr, MD, FACRO, FACR, Cancer Research Mayo Clinic College of Medicine and Science
FASTRO Department of Radiation Oncology Phoenix, Arizona
Professor and Chairman University of North Carolina Lineberger
Department of Radiation Oncology Comprehensive Cancer Center Kenneth Y. Usuki, MS, MD
Loyola University Chicago University of North Carolina School of Associate Professor
Stritch School of Medicine Medicine Department of Radiation Oncology
Chicago, Illinois Chapel Hill, North Carolina University of Rochester
Mike Soike, MD Charles R. Thomas Jr, MD
Department of Radiation Oncology Professor and Chair Vincenzo Valentini, MD
Wake Forest Baptist Health Radiation Medicine Professor and Chairman
Winston-Salem, North Carolina Knight Cancer Institute Radiation Oncology
Oregon Health & Science University Policlinico Gemelli-Università Cattolica del
C. Arturo Solares, MD Portland, Oregon Sacro Cuore
Professor Rome, Italy
Department of Otolaryngology Robert D. Timmerman, MD
Emory University School of Medicine Professor and Vice-Chair Julie My Van Nguyen, MD, MSc, FRCSC
Atlanta, Georgia Department of Radiation Oncology Fellow
UT Southwestern Medical Center Division of Gynecologic Oncology
Timothy D. Solberg, PhD Dallas, Texas University of Toronto
Professor and Director, Medical Physics Toronto, Ontario, Canada
Department of Radiation Oncology Christopher L. Tinkle, MD, PhD
University of California, San Francisco Assistant Member Noam VanderWalde, MD, MS
San Francisco, California Department of Radiation Oncology Assistant Professor
St. Jude Children’s Research Hospital Department of Radiation Oncology
Alexandra J. Stewart, DM, MRCP, FRCR Memphis, Tennessee West Cancer Center and Research Institute
Consultant Clinical Oncologist Memphis, Tennessee
St. Luke’s Cancer Centre Betty C. Tong, MD
Royal Surrey County Hospital Associate Professor Ralph Vatner, MD, PhD
Senior Lecturer Department of Surgery Assistant Professor
University of Surrey Division of Cardiovascular and Thoracic Department of Radiation Oncology
Guildford, United Kingdom Surgery University of Cincinnati
Duke University School of Medicine Cincinnati Children’s Hospital Medical
Rebecca L. Stone, MD, MS Durham, North Carolina Center
Assistant Professor Cincinnati, Ohio
Department of Gynecology and Obstetrics Jordan A. Torok, MD
Johns Hopkins Hospital Assistant Professor Michael J. Veness, MD, MMed, FRANZCR
Baltimore, Maryland Department of Radiation Oncology Clinical Professor
Duke University School of Medicine Department of Radiation Oncology
Durham, North Carolina Westmead Hospital
The University of Sydney
Sydney, New South Wales, Australia
xvi CONTRIBUTORS
Vivek Verma, MD Lynn D. Wilson, MD, MPH, FASTRO Joachim Yahalom, MD, FACR
Attending Physician Professor, Executive Vice Chairman, Clinical Professor
Department of Radiation Oncology Director Department of Radiation Oncology
Allegheny General Hospital Department of Therapeutic Radiology Memorial Sloan Kettering Cancer Center
Pittsburgh, Pennsylvania Professor, Department of Dermatology New York, New York
Staff Attending, Yale–New Haven Hospital
Frank A. Vicini, MD Yale University School of Medicine Eddy S. Yang, MD, PhD
Department of Radiation Oncology Smilow Cancer Hospital Professor
21st Century Oncology New Haven, Connecticut Department of Radiation Oncology
Michigan Healthcare Professionals The University of Alabama at Birmingham
Farmington Hills, Michigan Karen M. Winkfield, MD, PhD Birmingham, Alabama
Associate Professor
Akila N. Viswanathan, MD, MPH Department of Radiation Oncology Y. Nancy You, MD, MHSc
Professor Wake Forest Baptist Health Associate Professor
Department of Radiation Oncology and Winston-Salem, North Carolina Department of Surgical Oncology
Molecular Radiation Sciences Associate Medical Director
Johns Hopkins University School of Suzanne L. Wolden, MD Clinical Cancer Genetics Program
Medicine Attending Physician The University of Texas MD Anderson
Baltimore, Maryland Department of Radiation Oncology Cancer Center
Memorial Sloan Kettering Cancer Center Houston, Texas
Daniel R. Wahl, MD, PhD New York, New York
Assistant Professor Ye Yuan, MD, PhD
Department of Radiation Oncology Jeffrey Y.C. Wong, MD, FASTRO Resident Physician
University of Michigan Professor and Chair UCLA Department of Radiation Oncology
Ann Arbor, Michigan Department of Radiation Oncology University of California, Los Angeles
City of Hope National Medical Center Los Angeles, California
Padraig R. Warde, MBBCh, FRCPC Duarte, California
Radiation Oncologist Elaine M. Zeman, PhD
Princess Margaret Cancer Centre Terence Z. Wong, MD, PhD Associate Professor
Professor Professor of Radiology Department of Radiation Oncology
Department of Radiation Oncology Chief, Division of Nuclear Medicine University of North Carolina School of
University of Toronto Department of Radiology Medicine
Toronto, Ontario, Canada Duke Cancer Institute Chapel Hill, North Carolina
Duke University Health System
Christopher G. Willett, MD Durham, North Carolina Peixin Zhang, PhD
Professor and Chair Statistics and Data Management Center
Department of Radiation Oncology William W. Wong, MD NRG Oncology
Duke Cancer Institute Vice Chair, Department of Radiation
Duke University Oncology Tiffany C. Zigras, MD, MSc, MEng,
Durham, North Carolina Mayo Clinic Arizona FRCSC
Professor of Radiation Oncology Fellow
Christopher D. Willey, MD, PhD Mayo Clinic College of Medicine and Science Division of Gynecologic Oncology
Associate Professor Phoenix, Arizona University of Toronto
Department of Radiation Oncology Toronto, Ontario, Canada
The University of Alabama at Birmingham Zhong Wu, MD, PhD
Birmingham, Alabama Research Fellow in Medicine
Dana-Farber Cancer Institute
Grant Williams, MD Harvard Medical School
Assistant Professor Boston, Massachusetts
Division of Hematology and Oncology and
Gerontology, Geriatrics, and Palliative
Care
University of Alabama at Birmingham
Birmingham, Alabama
F O R E WO R D
Joel Tepper and I were approached by the senior medical editor of set of cited references. This allowed a reduction in the length of the
Churchill Livingstone in late 1995 about co-editing a textbook on clinical printed textbook by limiting the number of critical references in
radiation oncology as a counterpart to the multidisciplinary textbook the print version of each chapter to 50. For CRO4 an exciting new
Clinical Oncology, edited by Abeloff, Armitage, Lichter, and Niederhuber. feature was the periodic update of chapters in the online version of
By May 1996, the decision had been made to proceed and a contract the textbook. Periodic changes were made in chapter senior authors
was signed in July. We were interested in producing a new radiation and co-authors and in the associate editors for subsequent editions,
oncology textbook that was easily readable and useful to both residents as appropriate.
and experienced radiation oncologists. As such, we introduced “Key While I was heavily involved in the clinical/content updates for CRO4,
Points” for each disease-site chapter, as well as algorithms for workup I promised my wife, Katheryn, that I would not edit further editions
and treatment for each disease. We thought that, along with careful of CRO. Therefore, when the decision was made to proceed with CRO5,
editing and organization, this would provide a new and valuable resource I conferred with Joel in selecting two new senior editors (Drs. Robert
to the radiation oncology community. While providing a thorough Foote and Jeff Michalski), which resulted in a more diverse group of
coverage of all the topics, we made no attempt to cover all issues but senior editors by virtue of their respective disease-site expertise. At
rather emphasized what was important to the clinician. Joel’s request, I was involved with the three of them in the planning
Since Joel and I had similar disease-site interests, the decision was process for CRO5. As a group we decided to add six new chapters while
made to select associate editors for eight other disease-site sections/ keeping the length of the hardcopy textbook similar to CRO4 by reducing
chapters “to enhance the scientific content and comprehensiveness of the number of critical references in the hardcopy version from 50
the textbook” (breast, central nervous system, childhood, gynecologic, to 25.
genitourinary, head and neck, lymphoma/hematologic, thoracic). The intent of the first edition of CRO was “to be both comprehensive
Associate editors were involved in helping select appropriate senior and authoritative, yet not exhaustive” by virtue of liberal use of tables,
authors for each of the disease-site chapters, in editing the chapters for figures, and treatment algorithms as a supplement to the text. The
scientific content and accuracy, and in writing a section overview for comprehensive/authoritative intent of the print versions of the book
their respective disease-sites. persisted in subsequent editions, but the addition of an online version
The first edition of Clinical Radiation Oncology (CRO), published for CRO3 and subsequent editions has perhaps resulted in some “exhaus-
in 2000 by Churchill Livingstone/Harcourt Science, was a 1300-page, tive” chapters online for those readers who found the additional
black and white textbook containing 63 chapters in three major sec- information useful. It has been both a privilege and a pleasure to be
tions—Scientific Foundations of Radiation, Techniques and Modalities, associated with Clinical Radiation Oncology planning and editing in
Disease Sites. Subsequent editions (CRO2, CRO3, CRO4) were published conjunction with Joel and many other national and international experts
in 2007 (Churchill Livingstone, Elsevier), 2012 (Saunders/Elsevier) and for over 20 years! The contributions of outstanding authors, associate
2016 (Elsevier), with Joel and I as the co-senior editors, plus section editors, and senior editors will allow CRO5 to be a valuable resource
editors for gastrointestinal and sarcoma, while continuing to involve for many readers in the coming years.
associate editors for the other eight disease-site sections. CRO2 was a Leonard L. Gunderson, MD, MS, FASTRO
full-color textbook and expanded to 76 chapters with approximately Professor Emeritus and Consultant
1800 pages. An exciting feature of CRO3 was the availability of an online Department of Radiation Oncology
version of the textbook that contained the entire print component of Mayo Clinic Rochester/Arizona
the textbook along with additional text, figures, tables, and a complete Mayo Clinic College of Medicine and Science
xvii
P R E FA C E
The radiation oncology community has received the four previous tion of chapter authors and in editing the chapters for scientific
editions of Clinical Radiation Oncology very well, and it has become content and accuracy. For most disease sites, the associate editors also
the standard radiation oncology textbook for many physicians. For the wrote an overview in which they discuss issues common to various
fifth edition of Clinical Radiation Oncology, the major change that has disease sites within the section and give their unique perspective on
been made is that Len Gunderson has decided to step down as a senior important issues.
editor, a position he has held since the inception of this textbook. His Features that are retained within Disease Sites section include an
insight and efforts have been essential over the years in making this opening page format summarizing the most important issues, a full-color
book successful. Drs. Gunderson and Tepper thought carefully about format throughout each chapter, liberal use of tables and figures, and
who could replace Dr. Gunderson as a senior editor and decided that a closing section with a discussion of controversies and problems and
two people were needed to fill that role. We have been fortunate to have a treatment algorithm that reflects the treatment approach of the authors.
recruited Robert Foote and Jeff Michalski to be senior editors. As the Chapters have been edited not only for scientific accuracy, but also for
field of radiation oncology has expanded in its scope, having a third organization, format, and adequacy of outcome data (disease control,
senior editor allows us to have broader expertise. survival, and treatment tolerance).
Despite this major change, our intent is to maintain the many excellent We are again indebted to the many national and international experts
features of the previous editions while adding some new features, new who contributed to the fifth edition of Clinical Radiation Oncology as
chapters and chapter authors, and new associate editors. associate editors, senior authors, or co-authors. Their outstanding efforts
The fifth edition has maintained three separate sections—Scientific combined with ours will hopefully make this new edition a valuable
Foundations of Radiation Oncology, Techniques and Modalities, and contribution and resource in the field in the coming years.
Disease Sites. Within Scientific Foundations of Radiation Oncology,
four new chapters have been added: “Radiation Physics: Charged Particle
Therapy,” “Tumor Ablation in Interventional Radiology,” “Radiation
ACKNOWLEDGMENTS
Therapy in the Elderly,” and “Palliative Radiation Medicine,” reflecting We wish to thank our wives, Laurie, Kally, and Sheila, and Dr. Tepper’s
the increasing clinical interest in all of these issues within the oncologic secretary, Betty Bush, for their patience and assistance during the many
community. In the section on Techniques and Modalities, two new months we were involved in the preparation of the fifth edition of
chapters have been added: “Quality and Safety in Radiation Oncology” Clinical Radiation Oncology.
and “Immunotherapy with Radiotherapy.” We also thank the associate editors and the many senior authors
The associate editors for Disease Sites chapters were an important and co-authors for their time, efforts, and outstanding contributions
component of the success of the four previous editions and have been to this edition.
retained. Three associate editor positions have changed—Dr. Michalski We acknowledge the editors and production staff at Elsevier, especially
has taken the lead on genitourinary diseases, Akila Viswanathan has Anne Snyder, Tara Delaney, and Robin Carter, who have done an
become the associate editor for gynecologic tumors, and Abram Recht outstanding job in collating and producing the fifth edition of Clinical
is the associate editor for breast tumors. Larry Kun functioned as the Radiation Oncology.
associate editor for pediatric tumors until his untimely death, and Joel E. Tepper
the remainder of his responsibilities were taken over by Christopher Robert L. Foote
Tinkle and Jeff Michalski. Associate editors are involved in the selec- Jeff M. Michalski
xviii
VIDEO CONTENTS xxi
VIDEO CONTENTS
Video 20.1 Prostate Brachytherapy

Video 22.1 Intraoperative Radiation
Video 36.1 Ocular Melanoma
Video 65.1 Penile Brachytherapy
PART A Radiobiology
1
The Biological Basis of Radiation Oncology
Elaine M. Zeman
WHAT IS RADIATION BIOLOGY? public when it comes to ionizing radiation, who need to be fully
conversant in the potential risks and benefits of medical procedures
In the most general sense, radiation biology is the study of the effects involving radiation.
of electromagnetic radiation on biological systems. Three aspects of this The majority of this chapter will be devoted to so-called “founda-
definition deserve special mention. First, effects may include everything tional” radiobiology, that is, studies that largely predate the revolution
from DNA damage to genetic mutations, chromosome aberrations, in molecular biology and biotechnology during the 1980s and 1990s.
cell killing, disturbances in cell cycle transit and cell proliferation, While the reader might be tempted to view this body of knowledge as
neoplastic transformation, early and late effects in normal tissues, rather primitive by today’s standards, relying too heavily on phenomenol-
teratogenesis, cataractogenesis, and carcinogenesis, to name but a ogy, empiricism, and descriptive models and theories, the real challenge
few. Electromagnetic radiation refers to any type of radiant energy in is to integrate the new biology into the already-existing framework of
motion with wave and/or particulate characteristics that has the capacity foundational radiobiology. Chapter 2 endeavors to do this.
to impart some or all of its energy to the medium through which it
passes. The amount of energy deposited can vary over some 25 orders RADIOTHERAPY-ORIENTED RADIOBIOLOGY:
of magnitude, depending on the type of electromagnetic radiation.
For example, 1 kHz radio waves have energies in the range of 10–11 A CONCEPTUAL FRAMEWORK
to 10–12 eV, whereas x-rays or γ-rays may have energies upwards of Before examining any one aspect of radiobiology in depth, it is important
10 MeV or more. The more energetic forms of electromagnetic radiation, to introduce several general concepts to provide a framework for putting
the ionizing radiations, deposit energy as they traverse the medium the information in its proper perspective.
by setting secondary particles in motion that can go on to produce
further ionizations. Finally, biological systems may be, for example, The Therapeutic Ratio
quite simple cell-free extracts of biomolecules, or increasingly complex, The most fundamental of these concepts is what is termed the therapeutic
from prokaryotes to single-celled eukaryotes, to mammalian cells in ratio—in essence, a risk-versus-benefit approach to planning a radio-
culture, to tissues and tumors in laboratory animals or humans, to therapy treatment regimen. Many of the radiobiological phenomena
entire ecosystems. to be discussed in this chapter are thought to play important roles in
Radiotherapy-oriented radiobiology focuses on that portion of the optimizing, or at least “fine tuning,” the therapeutic ratio. In theory, it
electromagnetic spectrum energetic enough to cause ionization of atoms. should be possible to eradicate any malignant tumor simply by delivering
This ultimately results in the breaking of chemical bonds, which can a sufficiently high dose of radiation. Of course, in practice, the biological
lead to damage to important biomolecules. The most significant effect consequences for normal tissues that are necessarily irradiated along
of ionizing radiation in this context is cell killing, which directly or with the tumor limit the total dose that can be safely administered. As
indirectly is at the root of nearly all of the normal tissue and tumor such, a balance must be struck between what is deemed an acceptable
responses noted in patients. probability of a radiation-induced complication in a normal tissue and
Cytotoxicity is not the only significant biological effect caused by the probability of tumor control. Ideally, one would hope to achieve
radiation exposure, although it will be the main focus of this chapter. the maximum likelihood of tumor control that does not produce
Other important radiation effects—carcinogenesis, for example—will unacceptable normal tissue damage.
also be discussed, although the reader should be aware that radiation The concept of therapeutic ratio is best illustrated graphically, by
carcinogenesis is a large discipline in and of itself, involving investiga- comparing dose-response curves for both tumor control and normal
tors from fields as diverse as biochemistry, toxicology, epidemiology, tissue complication rates plotted as a function of dose. Examples of
environmental sciences, molecular biology, tumor biology, health this approach are shown in Fig. 1.1 for cases in which the therapeutic
and medical physics, as well as radiobiology. Most radiation protec- ratio is either “unfavorable,” “favorable,” or “optimal,” bearing in mind
tion standards are based on minimizing the risks associated with that these are theoretical curves. Actual dose-response curves derived
mutagenic and carcinogenic events. Therefore radiological health from experimental or clinical data are much more variable, particularly
professionals are de facto educators of and advocates for the general for tumors, which tend to show much shallower dose responses.1 This
2
CHAPTER 1 The Biological Basis of Radiation Oncology 3
Desirable probability of tumor control axis (toward lower doses, i.e., radiosensitization) or shifting the normal
95 tissue complication curve to the right (toward higher doses, i.e.,
Unfavorable
radioprotection) or, perhaps, some combination of both. The key,
Probability of effect (%)
therapeutic
ratio however, is to shift these curves differentially, not necessarily an easy
task given that there are not that many exploitable differences in the
radiobiology of cells derived from tumors and those derived from normal
50 Normal tissue tissues.
complication
Tumor control The Radiation Biology Continuum
There is a surprising continuity between the physical events that occur
in the first few femtoseconds after ionizing radiation interacts with
5 Acceptable risk of normal tissue complication
the atoms of a biomolecule and the ultimate consequences of that
interaction on tissues. The consequences themselves may not become
40 60 80 apparent until days, weeks, months, or even years after the radiation
A Total dose (Gy) exposure. Some of the important steps in this radiobiology continuum
are listed in Table 1.1. The orderly progression from one stage of the
Desirable probability of tumor control
continuum to the next—from physical to physicochemical to biochemi-
95 cal to biological—is particularly noteworthy not only because of the
Favorable
vastly different time scales over which the critical events occur but
therapeutic
ratio also because of the increasing biological complexity associated with
each of the endpoints or outcomes. Each stage of the continuum also
offers a unique radiobiological window of opportunity: the potential
50 Normal tissue to intervene in the process and thereby modify all of the events and
complication outcomes that follow.
Tumor control
Levels of Complexity in Radiobiological Systems
Another important consideration in all radiobiological studies is the
5 nature of the experimental system used to study a particular phenomenon,
Acceptable risk of normal tissue complication
the assay(s) used, and the endpoint(s) assessed. For example, one
40 60 80 investigator might be interested in studying DNA damage caused by
B Total dose (Gy) ionizing radiation, in particular, the frequency of DNA double-strand
breaks (DSBs) produced per unit dose. As an experimental system, the
Desirable probability of tumor control
investigator might choose DNA extracted from irradiated mammalian
95 cells and, as an endpoint, use pulsed field gel electrophoresis to measure
Optimal
the distance and rate at which irradiated DNA migrates through the
therapeutic
ratio gel compared with unirradiated DNA. The DNA containing more DSBs
migrates farther than DNA containing fewer breaks, allowing a calibration
curve to be generated that relates migration to the dose received. A
50 Normal tissue second investigator, meanwhile, may be interested in improving the
complication control rate of head and neck cancers with radiation therapy by employing
Tumor control a nonstandard fractionation schedule. In this case, the type of experiment
would be a clinical trial. The experimental system would be a cohort
of patients, some of whom are randomized to receive nonstandard
5 fractionation and the rest receiving standard fractionation. The endpoints
Acceptable risk of normal tissue complication
assessed could be one or more of the following: locoregional control,
6040 80 long-term survival, disease-free survival, normal tissue complication
C Total dose (Gy) frequency, and so forth, evaluated at specific times after completion of
Fig. 1.1 Illustrating the concept of therapeutic ratio under conditions the radiation therapy.
in which the relationship between the normal tissue tolerance and In considering both the strengths and weaknesses of these two
tumor control dose-response curves is unfavorable (A), favorable (B), investigators’ studies, any number of pertinent questions may be asked.
and optimal (C). Which is the more complex or heterogeneous system? Which is the more
easily manipulated and controlled system? Which is more relevant for
the day-to-day practice of radiation oncology? What kinds of results are
serves to underscore how difficult it can be in practice to assign a single gleaned from each and can these results be obtained in a timely manner?
numerical value to the therapeutic ratio in any given situation. In this example, it is clear that human patients with spontaneously arising
Many of the radiobiological properties of cells and tissues can have tumors represent a far more heterogeneous and complex experimental
a favorable or adverse effect on the therapeutic ratio. Therefore, in system than extracted mammalian DNA. However, the DNA system is
planning a course of radiation therapy, the goal should be to optimize much more easily manipulated, possible confounding factors can be
the therapeutic ratio as much as possible; in other words, using our more easily controlled, and the measurement of the desired endpoint
graphical approach, increase the separation between the tumor control (migration distance/rate) plus the data analysis can be completed within
and normal tissue complication curves. This can be accomplished either a day or two. Obviously, this is not the case with the human studies,
by shifting the tumor control curve to the left with respect to the dose in which numerous confounding factors can and do influence results,
4 SECTION I Scientific Foundations of Radiation Oncology
TABLE 1.1 Stages in the Radiobiology Continuum

Time Scale of Events
(“Stage”) Initial Event Final Event Response Modifiers/Possible Interventions
−16 −12
10 to 10 second (“Physical”) Ionization of atoms Free radicals formed in biomolecules Type of ionizing radiation; shielding
10−12 to 10−2 second Free radicals formed DNA damage Presence or absence of free radical scavengers, molecular
(“Physicochemical”) in biomolecules oxygen and/or oxygen-mimetic radiosensitizers
1.0 second to several hours DNA damage Unrepaired or misrejoined DNA damage Presence or absence of functioning DNA damage
(“Biochemical”) recognition and repair systems; repair-inhibiting drugs;
altering the time required to complete repair processes
Hours to years (“Biological”) Unrepaired or Clonogenic cell death, apoptosis, Cell-cell interactions, biological response modifiers,
misrejoined DNA mutagenesis, transformation, adaptive mechanisms, structural and functional
damage carcinogenesis, “early and late effects” organization of tissues, cell kinetics, etc.
normal tissues, whole body radiation
syndromes, tumor control, etc.
manipulation of the system can be difficult, if not impossible, and the can include inherent radiosensitivity, genomic instability, gene expression
experimental results typically take years to obtain. patterns, DNA repair fidelity, mode(s) of cell death, cell cycle regulation,
The issue of relevance is an even thornier one. Arguably, both studies and how the tissue is structurally and functionally arranged. Extrinsic
are relevant to radiation oncology in so far as the killing of cells is at factors, on the other hand, are related to microenvironmental differences
the root of radiation’s normal tissue and tumor toxicity, and that cell between tissues, such as the functionality of the vasculature, availability
killing usually is, directly or indirectly, a consequence of irreparable of oxygen and nutrients, pH, presence or absence of reactive oxygen
damage to DNA. As such, any laboratory findings that contribute to species, cytokines and immune cells, energy charge, and cell-cell and
the knowledge base of radiation-induced DNA damage are relevant. cell-extracellular matrix interactions.
Clearly, however, clinical trials with human patients not only are a more What are the practical implications of normal tissue and tumor
familiar experimental system to radiation oncologists but also, efficacy heterogeneity? First, if one assumes that normal tissues are the more
in conducting trials with cancer patients is ultimately what leads to new uniform and predictable in behavior of the two, then tumor heterogeneity
standards of care in clinical practice and becomes the gold standard is responsible, either directly or indirectly, for most radiotherapy failures.
against which all newer therapeutic strategies are judged. If so, this suggests that a valid clinical strategy might be to identify the
There is a time and place both for relatively simple systems and radioresistant subpopulation(s) of tumor cells and then tailor therapy
more complex ones. The relatively simple, homogeneous, and easily specifically to cope with them—although, admittedly, this approach is
manipulated systems are best suited for the study of the mechanisms much easier said than done. Some clinical studies—both prospective
of radiation action, such as measuring DNA or chromosomal damage, and retrospective—now include one or more determinations of, for
changes in gene expression, activation of cell cycle checkpoints, or the example, extent of tumor hypoxia4,5 or potential doubling time of tumor
survival of irradiated cells in vitro. The more complicated and hetero- clonogens6 or specific tumor molecular/genetic factors. The hope is
geneous systems, with their unique endpoints, are more clinically relevant, that these and other biomarkers can identify subsets of patients bearing
such as assays of tumor control or normal tissue complication rates. tumors with different biological characteristics and that, accordingly,
Both types of assay systems have inherent strengths and weaknesses, patients with particular characteristics can be assigned prospectively
yet both are critically important if we hope to improve the practice of to different treatment groups.
radiation therapy based on sound biological principles. Another consequence of tissue heterogeneity is that any radiobiologi-
cal endpoint measured in an intact tissue necessarily reflects the sum
Heterogeneity total of the individual radiosensitivities of all of the subsets of cells,
Why is radiation therapy successful at controlling one patient’s tumor plus all other intrinsic and extrinsic factors that contribute to the overall
but not another’s when the two tumors in all other clinical respects seem response of the tissue. Since data on normal tissue tolerances and tumor
identical? Why are we generally more successful at controlling certain control probabilities are also averaged across large numbers of patients,
types of cancers than others? The short answer to such questions is heterogeneity is even more pronounced.
that, although the tumors may appear identical “macroscopically,” their
component cells may be quite different genotypically and phenotypically. Powers of Ten
Also, there could be important differences between the two patients’ Tumor control is achieved only when all clonogenic cells are killed or
normal tissues. otherwise rendered unable to sustain tumor growth indefinitely. In
Because normal tissues by definition are composed of more than order to estimate the likelihood of cure, it is necessary to know, or at
one type of cell, they are necessarily heterogeneous. However, tumors, least have an appreciation for, approximately how many clonogenic
owing both to the genomic instability of individual cells and to micro- cells the tumor contains, how radiosensitive these cells are (i.e., some
environmental differences, are much more so. Different subpopulations measure of killing efficiency per unit radiation dose), and what the
of cells isolated from human and experimental cancers can differ with relationship is between the number of clonogenic cells remaining after
respect to differentiation, invasive and metastatic potential, immunogenic- treatment and the probability of recurrence. The latter is perhaps the
ity, and sensitivity to radiation and chemotherapy, to name but a few. easiest to ascertain given our knowledge of both the random and discrete
(For reviews, see Heppner and Miller2 and Suit et al.3) This heterogeneity nature of radiation damage and the general shape of dose-response
is manifest both within a particular patient and, to a much greater curves for mammalian cells and tissues. For a given number of surviving
extent, between patients with otherwise similar tumors. Both intrinsic cells per tumor, the probability of local control can be derived from
and extrinsic factors contribute to this heterogeneity. Intrinsic factors Poisson statistics using the equation P = e−n, where P is the tumor
control probability and n is the average number of surviving clonogenic Finally, it should be noted that while the goal of curative radiation
tumor cells. For example, when an average of one clonogenic cell per therapy is to reduce tumor cell survival by at least nine logs, even for
tumor remains at the end of radiation therapy, the tumor control rate the smallest tumor likely to be encountered, it is much less clear how
will be about 37%. This means that about 6 out of 10 tumors of the many logs of cell killing a particular normal tissue can tolerate before
same size and relative radiosensitivity will recur. Should the treatment it loses its structural and/or functional integrity. This would depend
reduce clonogenic cell numbers to an average of 0.1 per tumor, the on how the tissue is organized structurally, functionally, and prolifera-
tumor control probability would increase to 90%; 0.05 per tumor, 95%; tively, which constituent cells are the most and least radiosensitive, and
and 0.01 per tumor, 99%, respectively. which cells are the most important to the integrity of the tissue. It is
The tumor control probability for a given fraction of surviving cells unlikely, however, that many normal tissues could tolerate a depletion
is not particularly helpful when the total number of cells at risk is of two logs (99%) of their cells, let alone nine or more logs.
unknown; this is where an understanding of logarithmic relationships
and exponential cell killing is useful. For example, estimates are that a RADIATION BIOLOGY AND THERAPY: THE FIRST
1-cm3 (1-g) tumor mass contains approximately 109 cells,7 admittedly
a theoretical (and incorrect) value that assumes that all cells are perfectly 50 YEARS
packed and uniformly sized and that the tumor contains no stroma. A In fewer than 4 years after the discovery of x-rays by Roentgen,8
further assumption, that all such cells are clonogenic (rarely, if ever, radioactivity by Becquerel,9 and radium by the Curies,10 the new modality
the case), suggests that at least 9 logs of cell killing would be necessary of cancer treatment known as radiation therapy claimed its first cure
before any appreciable tumor control (about 37%) would be achieved, of skin cancer.11 Today, more than 120 years later, radiotherapy is most
and 10 logs of cell killing would be required for a high degree of tumor commonly given as a series of small daily dose fractions of approximately
control (i.e., 90%). 1.8 to 2.0 Gy each, 5 days per week, over a period of 5 to 7 weeks to
After the first log or two of cell killing, however, some tumors respond total doses of 50 to 75 Gy. While it is true that the historical development
by shrinking, a so-called partial response. After two to three logs of cell of this conventional radiotherapy schedule was empirically based, there
killing, the tumor may shrink to a size below the current limits of were a number of early radiobiological experiments that suggested this
clinical detection, that is, a complete response. While partial and complete approach.
responses are valid clinical endpoints, a complete response does not In the earliest days of radiotherapy, both x-rays and radium were
necessarily equal a tumor cure. At least six more logs of cell killing used for cancer treatment. Due to the greater availability and convenience
would still be required before any significant probability of cure would of using x-ray tubes and the higher intensities of radiation output achiev-
be expected. This explains why radiation therapy is not halted if the able, it was fairly easy to deliver one or a few large doses in short overall
tumor disappears during the course of treatment; this concept is treatment times. Thus, from about 1900 into the 1920s, this “massive
illustrated graphically in Fig. 1.2. dose technique”12 was a common way of administering radiation therapy.
Normal tissue complications were often quite severe and, to make matters
Tumor worse, the rate of local tumor recurrence was still unacceptably high.
mass Radium therapy was used more extensively in France. Because of
1 kg Number of 2.0-Gy fractions
the low activities available, radium applications necessarily involved
10 20 30
longer overall treatment times in order to reach comparable total doses.
Although extended treatments were less convenient, clinical results were
1g 109
Partial response often superior. Perceiving that the change in overall time was the critical
Number of tumor cells
factor, physicians began to experiment with the use of multiple, smaller

1 mg x-ray doses delivered over extended periods. By that time, there was
106
already a radiobiological precedent for expecting improvement in tumor
control when radiation treatments were protracted.
Complete response
1 µg As early as 1906, Bergonié and Tribondeau observed histologically
103 that the immature, dividing cells of the rat testis showed evidence of
damage at lower radiation doses than the mature, nondividing cells of
1 ng the stroma.13 Based on these observations, they put forth some basic
100 “laws” stating that x-rays were more effective on cells that were (1)
Cure possible
actively dividing, (2) likely to continue to divide indefinitely, and (3)
10 20 30 40 50 60 70 undifferentiated.13 Since tumors were already known to contain cells
Total dose (Gy) that were not only less differentiated but also exhibited greater mitotic
0.9 activity, they reasoned that several radiation exposures might prefer-
Cure
probability 0.37 entially kill these tumor cells but not their slowly proliferating, differenti-
0.1 ated counterparts in the surrounding normal tissues.
(Dose) The end of common usage of the massive dose technique in favor
Fig. 1.2 The relationship between radiation dose and tumor cell survival of fractionated treatment came during the 1920s as a consequence of
during fractionated radiotherapy of a hypothetical 1-g tumor containing the pioneering experiments of Claude Regaud.14 Using the testes of the
109 clonogenic cells. Although a modest decrease in cell-surviving fraction rabbit as a model tumor system (since the rapid and unlimited prolifera-
can cause the tumor to shrink (partial response) or disappear below the tion of spermatogenic cells simulated to some extent the pattern of cell
limits of clinical detection (complete response), few if any cures would
proliferation in malignant tumors), Regaud showed that only through
be expected until at least 9 logs of clonogenic cells have been killed.
In this example, a total dose of at least 60 Gy delivered as daily 2-Gy
the use of multiple, smaller radiation doses could animals be completely
fractions would be required to produce a tumor control probability of sterilized without producing severe injury to the scrotum.15 Regaud
0.37, assuming that each dose reduced the surviving fraction to 0.5. suggested that the superior results afforded the multifraction irradiation
(Modified from Steel G, Adams G, Peckham M, eds. The Biological scheme were related to alternating periods of relative radioresistance
Basis of Radiotherapy. New York: Elsevier; 1983.) and sensitivity in the rapidly proliferating germ cells.16 These principles
were soon tested in the clinic by Henri Coutard, who first used fraction- one, or a few, large doses) for the preferential eradication of tumors
ated radiotherapy for the treatment of head and neck cancers, with while simultaneously sparing normal tissues.25 It was somewhat ironic
spectacularly improved results, comparatively speaking.17,18 Largely as that the Strandqvist curves were so popular in the years that followed,
a result of these and related experiments, fractionated treatment sub- when it was already known that the therapeutic ratio did increase (at
sequently became the standard form of radiation therapy. least to a point) with prolonged, as opposed to very short, overall
Time-dose equivalents for skin erythema published by Reisner,19 treatment times. However, the overarching advantage was that these
Quimby and MacComb,20 and others21,22 formed the basis for the calcula- isoeffect curves were quite reliable at predicting skin reactions, which
tion of equivalents for other tissue and tumor responses. By plotting were the dose-limiting factors at that time.
the total doses required for each of these “equivalents” for a given level
of effect in a particular tissue, as a function of a treatment parameter— THE “GOLDEN AGE” OF RADIATION BIOLOGY AND
such as overall treatment time, number of fractions, dose per fraction,
and so forth—an isoeffect curve could be derived. All time-dose combina- THERAPY: THE SECOND 50 YEARS
tions that fell along such a curve theoretically would produce tissue Perhaps the defining event that ushered in the golden age of radiation
responses of equal magnitude. Isoeffect curves, relating the total dose biology was the publication of the first survival curve for mammalian
to the overall treatment time, derived in later years from some of these cells exposed to graded doses of x-rays. This first report of a quantitative
data,23 are shown in Fig. 1.3. measure of intrinsic radiosensitivity of a human cell line (HeLa, derived
The first published isoeffect curves were produced by Strandqvist from a cervical carcinoma26) was published by Puck and Marcus in
in 194424 and are also shown in Fig. 1.3. When transformed on log-log 1956.27 In order to put this seminal work in the proper perspective, it
coordinates, isoeffect curves for a variety of skin reactions and the cure is first necessary to review the physicochemical basis for why ionizing
of skin cancer were drawn as parallel lines, with common slopes of radiation is toxic to biological materials.
0.33. These results implied that there would be no therapeutic advantage
to using prolonged treatment times (i.e., multiple small fractions versus The Interaction of Ionizing Radiation
With Biological Materials
As mentioned in the introductory section of this chapter, ionizing
80
70 radiation deposits energy as it traverses the absorbing medium through
60 which it passes. The most important feature of the interaction of ionizing
oma
50 carcin radiation with biological materials is the random and discrete nature
f skin
Total dose (Gy)
40 C ure o
of the energy deposition. Energy is deposited in increasingly energetic
30 ” packets referred to as spurs (≤100 eV deposited), blobs (100–500 eV),
nce
“to lera or short tracks (500–5000 eV), each of which can leave from approximately
20 Skin ma
er ythe three to several dozen ionized atoms in its wake. This is illustrated in
Skin Fig. 1.4, along with a segment of (interphase) chromatin shown to scale.
The frequency distribution and density of the different types of energy
10
deposition events along the track of the incident photon or particle are
measures of the radiation’s linear energy transfer (LET; see also the
“Relative Biological Effectiveness” section to come). Because these energy
1 2 3 4 5 10 20 40 60 80100
deposition events are discrete, it follows that while the average energy
A Overall treatment time (days)
deposited in a macroscopic volume of biological material is small, the
distribution of this energy on a microscopic scale may be quite large.
10,000 This explains why ionizing radiation is so efficient at producing biological
sis damage; the total amount of energy deposited in a 70-kg human that
necro oma
Skin carcin
Total dose (R)
5000 k in in
Cure
of s he sk
4000
a t io n of t Incident particle
3000 am
esqu track
Dry d
ema
2000 eryth
Skin
Chromatin
1000 fiber 30 nm
“Spur”: 0–100 eV
1
2 3 4 5 10 20 40 60
B Overall treatment time (days) “Blob”: 100–500 eV
Fig. 1.3 Isoeffect curves relating the log of the total dose to the log of
Short track: 500–5000 eV
the overall treatment time for various levels of skin reaction, and the
cure of skin cancer. (A) Isoeffect curves constructed by Cohen in 1966, Fig. 1.4 Hypothetical α-particle track through an absorbing medium,
based on a survey of earlier published data on radiotherapy “equiva- illustrating the random and discrete energy deposition “events” along
lents.”19–22 See text for details. The slope of the curves for skin complica- the track. Each event can be classified according to the amount of
tions was 0.33 and that for tumor control, 0.22. (B) Strandqvist’s isoeffect energy deposited locally, which, in turn, determines how many ionized
curves, first published in 1944. All lines were drawn parallel and had a atoms will be produced. A segment of chromatin is also shown,
common slope of 0.33. (A, Modified from Cohen L. Radiation response approximately to scale. (Modified from Goodhead DT. Physics of radiation
and recovery: Radiobiological principles and their relation to clinical action: microscopic features that determine biological consequences.
practice. In: Schwartz E, ed. The Biological Basis of Radiation Therapy. In: Hagen U, Harder D, Jung H, et al., eds. Radiation Research 1895-1995,
Philadelphia: J.B. Lippincott; 1966:208; B, modified from Strandqvist M. Proceedings of the 10th International Congress of Radiation Research.
Studien uber die kumulative Wirkung der Roentgenstrahlen bei Frak- Volume 2: Congress Lectures. Wurzburg: Universitatsdruckerei H. Sturtz
tionierung. Acta Radiol Suppl. 1944;55:1.) AG; 1995:43–48.)
will result in a 50% probability of death is only about 70 calories, about compared with the time scale of the initial ionization events but are
as much energy as is absorbed by drinking one sip of hot coffee.28 The still fast relative to normal enzymatic processes in a typical mammalian
key difference is that the energy contained in the sip of coffee is uniformly cell. For all intents and purposes, free radical reactions are complete
distributed, not random and discrete. within milliseconds of irradiation. The OH radical is capable of both
Those biomolecules receiving a direct hit from a spur or blob receive, abstraction of hydrogen atoms from other molecules and addition across
relatively speaking, a huge radiation dose, that is, a large energy deposition carbon-carbon or other double bonds. More complex macromolecules
in a very small volume. For photons and charged particles, this energy that have been converted to free radicals can undergo a series of
deposition results in the ejection of orbital electrons from atoms, causing transmutations in an attempt to rid themselves of unpaired electrons,
the target molecule to be converted first into an ion pair and then into many of which result in the breakage of nearby chemical bonds. In the
a free radical. Further, the ejected electrons—themselves energetic charged case of DNA, these broken bonds may result in the loss of a base or an
particles—can go on to produce additional ionizations. For uncharged entire nucleotide, or a frank scission of the sugar phosphate backbone,
particles such as neutrons, the interaction is between the incident particles involving either one or both DNA strands. In some cases, chemical
and the nuclei of the atoms in the absorbing medium, causing the bonds are broken initially but then rearranged, exchanged, or rejoined
ejection of recoil protons (charged) and lower-energy neutrons. The in inappropriate ways. Bases in DNA may be modified by the addition
cycle of ionization, free radical production, and release of secondary of one or more hydroxyl groups (e.g., the base thymine converted to
charged particles continues until all of the energy of the incident photon thymine glycol), pyrimidines may become dimerized, and/or the DNA
or particle is expended. These interactions are complete within a may become cross-linked to itself or to associated proteins. Again, because
picosecond after the initial energy transfer. After that time, the chemical the initial energy deposition events are discrete, the free radicals produced
reactions of the resulting free radicals predominate the radiation response also are clustered and, therefore, undergo their multiple chemical
(see later discussion). reactions and produce multiple damages in a highly localized area. This
Any and all cellular molecules are potential targets for the localized has been termed the locally multiply damaged site32 or cluster33 hypothesis.
energy deposition events that occur in spurs, blobs, or short tracks. Examples of the types of damage found in irradiated DNA are shown
Whether the ionization of a particular biomolecule results in a measurable in Fig. 1.5.
biological effect depends on a number of factors, including how probable
a target the molecule represents from the point of view of the ionizing Biochemical Repair of DNA Damage
particle, how important the molecule is to the continued health of the DNA is unique insofar as it is the only cellular macromolecule with its
cell, how many copies of the molecule are normally present in the cell own repair system. Until as recently as 35 years ago, little was known
and to what extent the cell can react to the loss of working copies, how about DNA repair processes in mammalian cells, particularly because
important the cell is to the structure or function of its corresponding of the complexities involved and the relative lack of spontaneously
tissue or organ, and so on. DNA, for example, is obviously an important occurring mutants defective in genes involved with DNA repair. As
cellular macromolecule, and one that is present only as a single, double- a consequence, most studies of DNA repair were carried out either
stranded copy. On the other hand, other molecules in the cell may be in bacteria or yeasts and usually employed UV radiation as the tool
less crucial to survival, yet are much more abundant than DNA and, for producing DNA damage. Although these were rather simple
therefore, have a much higher probability of being hit and ionized. By and relatively clean systems in which to study DNA repair, their
far, the most abundant molecule in the cell is water, comprising at least relevance to mammalian repair systems and to the broader spectrum
70% to 80% of the cell on a per weight basis. The highly reactive free of DNA damage produced by ionizing radiation ultimately limited
radicals formed by the radiolysis of water are capable of augmenting their usefulness.
the DNA damage resulting from direct energy absorption by migrating The study of DNA repair in mammalian cells received a significant
to the DNA and damaging it indirectly. This mechanism is referred to boost during the late 1960s with publications by Cleaver34,35 that identified
as indirect radiation action to distinguish it from the aforementioned the molecular defect responsible for the human disease xeroderma
direct radiation action.29 The direct and indirect action pathways for pigmentosum (XP). Patients with XP are exquisitely sensitive to sunlight
ionizing radiation are illustrated below. and highly (skin) cancer prone. Cleaver showed that cells derived from
such patients were likewise sensitive to UV radiation and defective in
Direct Effect the nucleotide excision repair pathway (see later discussion). These cells
DNA → [DNA + + e− ] → DNA • were not especially sensitive to ionizing radiation, however. Several
(irradiate ) (ion pair ) (DNA free radical)
years later, Taylor et al.36 reported that cells derived from patients with
Indirect Effect a second cancer-proneness disorder called ataxia telangiectasia (AT)
H2O → [H2O+ + e− ] → OH + DNA → DNA • + H2O
• were extremely sensitive to ionizing radiation and radiation-mimetic
(irradiate ) (ion pair ) ( other (DNA free radical
radical and water) drugs, but not UV. In the years that followed, cell cultures derived from
reactions )
patients with these two conditions were used to help elucidate the
complicated processes of DNA repair in mammalian cells. Today, dozens
The most highly reactive and damaging species produced by the radiolysis of other clinical syndromes associated with radiosensitivity, cancer
of water is the hydroxyl radical ( OH), although other free radical species proneness, or both have been identified.37,38
are also produced in varying yields.30,31 Cell killing by indirect action Today, many rodent and human genes involved in DNA repair have
constitutes some 70% of the total damage produced in DNA for low been cloned and extensively characterized.39 Some 30 to 40 proteins
LET radiation. participate in excision repair of base damage; about half that many are
How do the free radicals produced by the direct and indirect action involved in the repair of strand breaks.37 Many of these proteins function
of ionizing radiation go on to cause the myriad lesions that have been as component parts of larger repair complexes. Some are interchangeable
identified in irradiated DNA? Since they contain unpaired electrons, and participate in other DNA repair and replication pathways as well.
free radicals are highly reactive chemically and will undergo multiple It is also noteworthy that some are not involved with the repair process
reactions in an attempt to either acquire new electrons or rid themselves per se, but rather link DNA repair to other cellular functions, including
of remaining unpaired ones. These reactions are considered quite slow transcription, cell cycle arrest, chromatin remodeling, and apoptosis.40
with other cellular activities is termed the DNA Damage Response

(DDR).37,41 For example, the defect responsible for the disease AT is
not in a gene that codes for a repair protein but rather in a gene that
acts in part as a damage sensor and signal transducer but also participates
in a related pathway that normally prevents cells from entering S phase
and beginning DNA synthesis while residual DNA damage is present.
This is termed the G1 cell cycle checkpoint response.42 Because of this
genetic defect, AT cells do not experience the normal G1 arrest after
irradiation and enter S phase with residual DNA damage. This accounts
both for the exquisite radiosensitivity of AT cells and the resulting
DNA-DNA genomic instability that can lead to cancer.
Cross-link
The molecular and biochemical intricacies of DNA repair in mam-
malian cells are described in detail in Chapter 2. A brief overview is
Nucleotide loss also presented next.
(with strand break)
Base Excision Repair
Base loss The repair of base damage is initiated by DNA repair enzymes called
glycosylases, which recognize specific types of damaged bases and excise
Modified base them without otherwise disturbing the DNA strand.43 The action of
the glycosylase results in the formation of another type of damage
Single-stranded break observed in irradiated DNA—an apurinic or apyrimidinic (AP) site.
The AP site is then recognized by another repair enzyme, an endonuclease
that nicks the DNA adjacent to the lesion, in effect creating a DNA
single-stranded break. This break then becomes the substrate for an
exonuclease, which removes the abasic site, along with a few additional
Double-stranded break bases. The small gap that results is patched by DNA polymerase using
the opposite, hopefully undamaged, DNA strand as a template. Finally,
A DNA ligase seals the patch in place.
Nucleotide Excision Repair

O O The DNA glycosylases that begin the process of base excision repair do
CH3
not recognize all known forms of base damage, however, particularly
CH3
HN HN
OH bulky or complex lesions.43 In such cases, another group of enzymes,
OH termed structure-specific endonucleases, initiate the excision repair process.
O O N H These repair proteins do not recognize the specific lesion but rather
N
dR dR the structural distortions in DNA that necessarily accompany a complex
base lesion. The structure-specific endonucleases incise the affected
Thymine Thymine glycol
DNA strand on both sides of the lesion, releasing an oligonucleotide
fragment made up of the damage site and several bases on either side
of it. After this step, the remainder of the nucleotide excision repair
process is similar to that of base excision repair. The gap is then filled
O O
by DNA polymerase and sealed by DNA ligase.
N N For both types of excision repair, active genes in the process of
HN HN
OH transcription are repaired preferentially and more quickly. This has
N H2N N been termed transcription-coupled repair.44
H2N N N
dR
dR Single-Strand Break Repair
B Guanine 8-Hydroxyguanine Single-strand breaks (SSBs) in the DNA backbone are common lesions,
produced in the tens of thousands per cell per day as part of normal
Fig. 1.5 Types of DNA damage produced by ionizing radiation. (A) metabolism and respiration45 on top of any additional breaks introduced
Segment of irradiated DNA containing single- and double-stranded breaks,
by radiation exposure. These are repaired using the machinery of excision
cross-links, and base damage. (B) Two types of modified bases observed
repair, that is, gap filling by DNA polymerase and sealing by DNA ligase.
in irradiated DNA include thymine glycol, which results from the addition
of two hydroxyl (OH) groups across the carbon-carbon double bond of
thymine, and 8-hydroxyguanine, produced by OH radical addition to
Double-Strand Break Repair
guanine. Despite the fact that unrepaired or misrejoined double-strand breaks
(DSBs) often have the most catastrophic consequences for the cell in
terms of loss of reproductive integrity,46 how mammalian cells repair
This attests to the fact that the maintenance of genomic integrity results these lesions has been more difficult to elucidate than how they
from a complex interplay between not only the repair proteins themselves repair base damage. Much of what was originally discovered about
but also others that serve as damage sensors, signaling mediators and these repair processes is derived from studies of x-ray-sensitive rodent
transducers, and effectors. Collectively, this complex network of proteins cells that were later discovered to harbor specific defects in strand break
that sense, initiate, and coordinate DNA damage signaling and repair repair.47 Since then, dozens of other rodent and human cells characterized
by DDR defects have been identified and are also used to help probe In normal cells, little or no toxicity caused by PARP inhibition would
these fundamental processes. be expected, as all DDR pathways are intact and salvage repair pathways
With respect to the repair of DSBs, the situation is more complicated to bypass PARP inhibition are active. In tumor cells already harboring
in that the damage on each strand of DNA may be different and, therefore, defects in HR, however, PARP inhibition would be preferentially toxic.
no intact template would be available to guide the repair process. Under One clinical example is the targeting of breast cancers harboring cellular
these circumstances, cells must rely on a somewhat error-prone process defects in the BRCA1/2 proteins—which either orchestrate or are directly
that rejoins the break(s) regardless of the loss of intervening base pairs involved in HR—for PARP inhibition. This overall approach of using
for which there is no template (nonhomologous end joining [NHEJ]) the combined lethal effect of two genetic defects (one inherent HR
or depend on genetic recombination in which a template for presumably defect plus one synthetic one induced by PARP inhibition) that are
error-free repair is obtained from recently replicated DNA of a sister otherwise nonlethal singly is termed synthetic lethality.54,55,58 Synthetic
chromatid (homologous recombination [HR]48) to cope with the damage. lethality approaches targeting DDR proteins (including those other
NHEJ occurs throughout the cell cycle, but predominates in cells that than PARP) likely will play increasingly important roles in the future.
have not yet replicated their DNA, that is, cells in the G1 or G0 phases
of the cell cycle. NHEJ involves a heterodimeric enzyme complex consist- Cytogenetic Effects of Ionizing Radiation
ing of the proteins Ku-70 and Ku-80, the catalytic subunit of DNA When cells divide following radiation exposure, chromosomes frequently
protein kinase (DNA-PKCS), and DNA ligase IV. Cells that have already contain visible structural aberrations that are the result of any unrepaired
replicated most or all of their DNA—in the late S or G2 phases of the or misrejoined DNA damage that persists from the time of irradiation.
cell cycle—depend on HR to repair DSBs. HR involves the assembly of Most chromosome aberrations are lethal to the cell. In some cases,
a nucleoprotein filament that contains, among others, the proteins Rad51 these aberrations physically interfere with the processes of mitosis and
and Rad52. This filament then invades the homologous DNA sequence cytokinesis, resulting in prompt cell death. In other cases, cell division
of a sister chromatid, which becomes the template for repair. The BRCA2 can occur but the loss or uneven distribution of genetic material between
protein is also implicated in HR as it interacts with the Rad51 protein.38 the cell’s progeny is ultimately lethal as well, although the affected cells
Defects in either the BRCA1 (which helps determine which DSB repair may linger for several days before they die, with some even be able to
pathway will be used in a particular situation) or BRCA2 genes are go through a few more cell divisions in the interim.
associated with hereditary breast and ovarian cancer.49 Most chromosome aberrations result from an interaction between
two damage sites; therefore, they can be grouped into three different
Mismatch Repair types of “exchange” categories. A fourth category is reserved for those
The primary role of mismatch repair (MMR) is to eliminate from newly chromosome aberrations that are thought to result from a single damage
synthesized DNA errors such as base/base mismatches and insertion/ site.59 These categories are described here; representative types of aber-
deletion loops caused by DNA polymerase.50 This process consists of rations from each category are shown in Fig. 1.6:
three steps: mismatch recognition and assembly of the repair complex, 1. Intra-arm Exchanges: An interaction between lesions on the same
degradation of the error-containing strand, and repair synthesis. In arm of a single chromosome (example: interstitial deletion).
humans, MMR involves at least five proteins, including hMSH2 and 2. Inter-arm Exchanges: An interaction between lesions on opposite
hMLH1, as well as other members of the DNA repair and replication arms of the same chromosome (example: centric ring).
machinery. 3. Interchanges: An interaction between lesions on different chromo-
Radiation-induced DNA lesions are not targets for mismatch repair somes (example: dicentric).
per se. However, one manifestation of a defect in mismatch repair is 4. “Single Hit” Breaks: The complete severance of part of one arm of
germane to any study of oncogenesis: genomic instability,51 which renders a single chromosome not obviously associated with any more than
affected cells hypermutable. This “mutator phenotype” is associated with a single lesion (example: terminal deletion).
several cancer predisposition syndromes, in particular, hereditary non- These four categories can be further subdivided according to whether
polyposis colon cancer (HNPCC, a.k.a. Lynch syndrome).52,53 Genomic the initial radiation damage occurred before or after the DNA is replicated
instability is considered one of the main enablers of normal cells to (a chromosome- vs. chromatid-type aberration, respectively) and, for
accumulate cancer-causing mutations and also drives tumor progression the three exchange categories, whether the lesion interaction is sym-
to more aggressive and potentially treatment-resistant phenotypes. metrical or asymmetrical. Asymmetrical exchanges always lead to the
formation of acentric fragments that are usually lost in subsequent cell
The DDR as a Clinical Target divisions and, therefore, are nearly always fatal to the cell. These fragments
Historically, attempts to inhibit the repair of radiation-induced DNA may be retained transiently in the cell’s progeny as extranuclear chromatin
damage were of interest to researchers probing these fundamental bodies called micronuclei. Symmetrical exchanges are more insidious
processes. However, clinical translation was typically lacking, mostly in that they do not lead to the formation of acentric fragments and the
out of concern that normal tissues would also be affected in an adverse accompanying loss of genetic material at the next cell division; thus,
way. More recently, it has become clear that the cells of many tumors they do not always kill the cell. As such, they will be transmitted to all
harbor one or more defects in the DDR (as a consequence of genomic progeny of the original cell. Some types of symmetrical exchanges (e.g.,
instability) that are not present in normal cells and that this difference a reciprocal translocation) have been implicated in radiation carcino-
might be exploitable clinically. genesis insofar as they have the net effect of either bringing new combina-
One approach along these lines is the use of inhibitors of the protein tions of genes together or separating preexisting groups of genes.28
poly(ADP-ribose) polymerase (PARP).54,55 As of 2018, dozens of trials Depending on where in the genome the translocation takes place, genes
were underway using PARP inhibitors in combination with chemo- and normally active could be turned off or vice versa, potentially with adverse
immunotherapies.56,57 PARP is a damage sensor involved in both base consequences.
excision and SSB repair that, if inhibited, leads to the persistence of Quantitation of the types and frequencies of chromosome aberrations
SSBs. If left unrejoined, these breaks can cause the collapse of replication in irradiated cells can be used to probe dose-response relationships for
forks in DNA that then impede DNA replication, transcription, and ionizing radiation and, to a first approximation, also can serve as a
HR repair,55 leading to radiosensitization and, ultimately, cell death.58 radiation dosimeter. For example, the dose-response curve for the
Intra-arm exchanges Inter-arm exchanges Interchanges “Single-hit” breaks
X-ray
Terminal
deletion
Interstitial deletion Centric ring and Dicentric and
fragment fragment
Symmetrical
Asymmetrical
Paracentric Paracentric Reciprocal

inversion inversion translocation
Fig. 1.6 Types of radiation-induced chromosome aberrations that are the result of unrepaired or misrejoined
DNA damage. Aberrations are classified according to whether they involve a single or multiple chromosomes,
whether the damage is thought to be caused by the passage of a single charged particle track (“one-hit”
aberration), or by the interaction of damages produced by two different tracks (“two-hit” aberration), and
whether the irradiation occurred prior to or after the chromosomes had replicated (chromosome- vs. chromatid-
type aberrations, respectively; only chromosome-type aberrations are shown). The aberrations can be further
subdivided according to whether broken pieces of the chromosome rearrange themselves symmetrically
(with no net loss of genetic material) or asymmetrically (acentric fragments produced).
induction of exchange-type aberrations after exposure to low-LET preclude the possibility that a cell may remain physically intact, metaboli-
radiation tends to be linear-quadratic in shape, whereas that for single-hit cally active, and continue its tissue-specific functions for some time
aberrations tends to be linear. In mathematical terms, the incidence, I, after irradiation.60
of a particular aberration as a function of radiation dose, D, can be Compared with nearly 65 years ago, when the term clonogenic death
expressed as was first coined and used as an endpoint in assays of cellular radiosensitiv-
ity,27,61 by today’s standards it is clearly an operationally defined term
I = αD + βD2 + c for exchange-type aberrations that encompasses several distinct mechanisms by which cells die, all of
which result in a cell losing its ability to divide indefinitely. These modes
I = αD + c for single-hit aberrations,
of cell death include mitotic catastrophe, apoptosis, necrosis, senescence,
and autophagy. Strictly speaking, differentiation is included as well,
where α and β are proportionality constants related to the yields of the because differentiated cells lose their ability to divide.62,63
particular type of aberration and c is the spontaneous frequency of Mitotic catastrophe is the major mode of radiation-induced death for
that aberration in unirradiated cells. For fractionated doses or continuous most mammalian cells, occurring secondary to chromosome aberrations
low dose rates of low-LET radiation, the yield of exchange-type aber- and/or spindle defects that interfere with the cell division process.64,65
rations decreases relative to that for acute doses, and the dose-response Accordingly, this type of cell death occurs during or soon after an
curve becomes more linear. For high-LET radiations, dose-response attempted cell division postirradiation (although not necessarily during
curves become steeper (higher aberration yields per unit dose) and the very first division attempt), leaving in its wake large, flattened,
more linear compared with those for low-LET radiations. and multinucleated cells that are typically aneuploid. Apoptosis, or
programmed cell death, is a type of nonmitotic or interphase death
Cell Survival Curves and Survival Curve Theory commonly associated with embryonic development and normal tissue
What Is Meant by “Cell Death”? remodeling and homeostasis.66 However, certain normal tissue and tumor
The traditional definition of death as a permanent, irreversible cessation cells also undergo apoptosis following irradiation, including normal cells
of vital functions is not the same as what constitutes “death” to the of hematopoietic or lymphoid origin, crypt cells of the small intestine,
radiation biologist or oncologist. For proliferating cells—including those salivary gland cells, plus a few tumor cell lines of gynecological and
maintained in vitro, the stem cells of normal tissues, and tumor hematological origin.67 Cells undergoing apoptosis exhibit a number of
clonogens—cell death in the radiobiological sense refers to a loss of characteristic morphological (nuclear condensation and fragmentation,
reproductive integrity, that is, an inability to sustain proliferation membrane blebbing, etc.) and biochemical (DNA degradation) changes
indefinitely. This type of “reproductive” or “clonogenic” death does not that culminate in the fragmentation of the cell, typically within 12 to
24 hours of irradiation and prior to the first postirradiation mitosis. activity. A mathematical expression used to fit this type of dose-response
The remains of apoptotic cells are phagocytized by neighboring cells; relationship is
therefore, they do not elicit the type of inflammatory response, tissue
destruction, and disorganization characteristic of necrosis. Apoptosis S = e−D D0
is an active and carefully regulated pathway that involves multiple
proteins and an appropriate stimulus that activates the pathway. The In this equation, S is the fraction of cells that survive a given dose,
molecular biology of apoptosis, the apoptosis-resistant phenotype noted D, and D0 is the dose increment that reduces the cell survival to 37%
for many types of tumor cells, and the role that radiation may play (1/e) of some initial value on the exponential portion of the curve
in the process are discussed in detail in Chapter 2. Senescence refers (i.e., a measure of the reciprocal of the slope). Target theory could
to a type of genetically controlled cellular growth arrest that, while also be applied to survival curves with shoulders at low doses if one
not necessarily eliminating damaged cells, does halt permanently their assumed that either multiple targets or multiple hits in a single target
continued movement through the cell cycle even in the presence of were necessary for radiation inactivation. A mathematical expression
growth factors.68 Radiation can also induce senescence, presumably based on target theory that provided a fairly good fit to survival
due to the permanent triggering of cell cycle checkpoints. However, data was
it might better be termed radiation-induced permanent growth arrest
to distinguish it from the normal process of cell age-related senes- S = 1− (1− e−D D0 )n
cence.69 Autophagy is defined as the controlled lysosomal degradation
of cytoplasmic organelles or other cytoplasmic components70,71 in with n being the back extrapolation of the exponential portion of the
response to cellular stressors, including nutrient deprivation, hypoxia, survival curve to zero dose. Implicit in this multitarget model was that
DNA damage, or an excess of reactive oxygen species. Likewise, damage had to accumulate before the overall effect was registered.
necrosis—characterized by cell swelling followed by membrane rupture It soon became apparent that some features of this model were
and the release of cellular contents into the extracellular space—can inadequate.77 The most obvious problem was that the single-hit,
occur as a somewhat passive response to nutrient deprivation but multitarget equation predicted that survival curves should have initial
also can follow a molecular program initiated by immune cells or slopes of zero, that is, that for vanishingly small doses (e.g., repeated,
various toxins.72,73 small doses per fraction or continuous low dose rate exposure), the
Most assays of radiosensitivity of cells and tissues, including those probability of cell killing would approach zero. This is not what was
described later, use reproductive integrity, either directly or indirectly, observed in practice for either mammalian cell survival curves or as
as an endpoint. While such assays have served the radiation oncology inferred from clinical studies in which highly fractionated or low dose
community well in terms of elucidating dose-response relationships for rate treatment schedules were compared to more conventional frac-
normal tissues and tumors, the interrelationships between the different tionation. There was no fractionation schedule that produced essentially
modes of cell death can be quite complex. For example, Meyn67 has no cell killing, all other radiobiological factors being equal.
suggested that a tumor with a high spontaneous apoptotic index may A somewhat different interpretation of cell survival was proposed
be inherently more radiosensitive because cell death might be triggered by Kellerer and Rossi78 in the late 1960s and early 1970s. The linear-
by lower doses than are usually required to cause mitotic catastrophe. quadratic or “alpha-beta” equation,
Also, tumors that readily undergo apoptosis may have higher rates of cell
2)
loss, the net effect of which would be to partially offset cell production, S = e−(αD+βD
thereby reducing the number of tumor clonogens. On the other hand,
recent studies suggest that the very enzymes that orchestrate the removal was shown to fit many survival data quite well, particularly in the low-
of radiation-damaged cells via apoptosis also may stimulate tumor dose region of the curve, and also provided for the negative initial
cell repopulation during and after radiotherapy.74 Studies also suggest slope that investigators had described.77 In this expression, S is again
that senescent cells can produce inflammatory cytokines that further the fractional cell survival following a dose D, α is the rate of cell
contribute to immunosuppression in the tumor microenvironment.68 kill by a single-hit process, and β is the rate of cell kill by a two-hit
mechanism. The theoretical derivation of the linear-quadratic equa-
Cell Survival and Dose-Response Curve Models tion is based on two sets of observations. Based on microdosimetric
Survival curve theory originated in a consideration of the physics of considerations, Kellerer and Rossi78 proposed that a radiation-induced
energy deposition in matter by ionizing radiation. Early experiments lethal lesion resulted from the interaction of two sublesions. According
with macromolecules and prokaryotes established that dose-response to this interpretation, the αD term is the probability of these two suble-
relationships could be explained by the random and discrete nature of sions being produced by a single event (the “intra-track” component),
energy absorption if it was assumed that the response resulted from whereas βD2 is the probability of the two sublesions being produced
critical “targets” receiving random “hits.”75 With an increasing number by two separate events (the “inter-track” component). Chadwick and
of shouldered survival and dose-response curves being described for Leenhouts79 derived the same equation based on a different set of
cells irradiated both in vitro and in vivo, various equations were developed assumptions, namely, that a DSB in DNA was a lethal lesion and that
to fit these data. Target theory pioneers studied a number of different such a lesion could be produced by either a single energy deposition
endpoints in the context of target theory, including enzyme inactivation involving both strands of DNA or by two separate events, each involving
in cell-free systems,29 cellular lethality, chromosomal damage, and a single strand.
radiation-induced cell cycle perturbations in microorganisms.29,76 Survival A comparison of the features and parameters of the target theory
curves, in which the log of the “survival” of a certain biological activity and linear-quadratic survival curve expressions is shown in Fig. 1.7.
was plotted as a function of the radiation dose, were found to be either
exponential or sigmoid in shape, the latter usually noted for the survival Clonogenic Assays In Vitro
of more complex organisms.29 As mentioned previously, it was not until the mid-1950s that mammalian
Exponential survival curves were thought to result from the single-hit, cell culture techniques were sufficiently refined to allow quantitation
“all or nothing” inactivation of a single target, resulting in the loss of of the radiation responses of single cells.61,80 Puck and Marcus’s acute
n S = 1 – (1 – e–D/D0)n
Dq α Cell kill
Surviving fraction
Surviving fraction β Cell kill
S = e – (αD ! βD2)
0.01
1/e α"β Ratio
0.0037
B Dose (Gy)
D0
A Dose (Gy)
Fig. 1.7 A comparison of two mathematical models commonly used to fit cell survival curve data. (A) The
single-hit, multi-target model and its associated parameters, D0, n, and Dq. Although this model has since
been invalidated, values for its parameters are still used for comparative purposes. (B) The linear-quadratic
model and its associated parameters, α and β. This model provided the conceptual framework for current
isoeffect formulae used in radiation therapy treatment planning.
endpoint for HeLa cells than for prokaryotes or primitive eukaryotes.

100 The value of the extrapolation number, n, was approximately 2.0,
indicating that the survival curve did have a small shoulder but, again,
Surviving fraction
much smaller than typically observed for microorganisms. Puck and

10!1 Marcus suggested that the n value was a reflection of the number of
critical targets in the cell, each requiring a single hit before the cell
would be killed, and further postulated that the targets were, in fact,
the chromosomes themselves.27 However, the potential pitfalls of deducing
10!2 mechanisms of radiation action from parameters of a descriptive survival
curve model were soon realized.81,82
Survival curves for other types of mammalian cells, regardless of
10!3 whether they were derived from humans or laboratory animals, or from
tumors or normal tissues, have been shown to be qualitatively similar
1 3 4 2 5 6 7 to the original HeLa cell survival curve.
Dose (Gy)
Fig. 1.8 Clonogenic survival of HeLa cells in vitro as a function of x-ray Clonogenic Assays In Vivo
dose. Like many mammalian cells of both tumorigenic and nontumorigenic In order to bridge the gap between the radiation responses of cells
origin, the HeLa cell survival curve is characterized by a modest initial grown in culture and in an animal, Hewitt and Wilson developed an
shoulder region (n ≈ 2.0) followed by an approximately exponential final ingenious method to assay single-cell survival in vivo.83 Lymphocytic
slope (D0 ≈ 1.0 Gy). (Modified from Puck TT, Marcus PI. Action of x-rays
leukemia cells obtained from the livers of donor CBA mice were harvested,
on mammalian cells. J Exp Med. 1956;103:653.)
diluted, and inoculated into disease-free recipient mice. By injecting
different numbers of donor cells, a standard curve was constructed that
allowed a determination of the average number of injected cells necessary
dose, x-ray survival curve for the human tumor cell line HeLa is shown to cause leukemia in 50% of the recipient mice. It was determined that
in Fig. 1.8. Following graded x-ray doses, the reproductive integrity of the endpoint of this titration, the 50% take dose (TD50), corresponded
single HeLa cells was measured by their ability to form macroscopic to an inoculum of a mere two leukemia cells. Using this value as a refer-
colonies of at least 50 cells (corresponding to approximately 6 successful ence, Hewitt and Wilson then injected leukemia cells harvested from
postirradiation cell divisions) on petri dishes. Several features of this γ-irradiated donor mice into recipients and again determined the TD50
survival curve were of particular interest. First, qualitatively at least, following different radiation exposures. In this way, the surviving fraction
the curve was similar in shape to those previously determined for many after a given radiation dose could be calculated from the ratio of the
microorganisms, being characterized by a shoulder at low doses and a TD50 for unirradiated cells to that for the irradiated cells. Using this
roughly exponential region at high doses. Of note, however, was the technique, a complete survival curve was constructed that had a D0 of
finding that the D0 for HeLa cells was only 96 R, some 10- to 100-fold 162 R and an n value close to 2.0, values quite similar to those generated
less than D0s determined for microorganisms and 1000- to 10,000-fold for cell lines irradiated in vitro. For the most part, in vivo survival
less than D0s for the inactivation of isolated macromolecules.60 Thus, curves for a variety of cell types were also similar to corresponding in
cellular reproductive integrity was found to be a much more radiosensitive vitro curves.
A similar trend was apparent when in vivo survival curves for Dose-response curves are generated by plotting the amount of growth
nontumorigenic cells were first produced. The first experiments by Till delay as a function of radiation dose.
and McCulloch84,85 using normal bone marrow stem cells were inspired The tumor control assay is a logical extension of the growth delay
by the knowledge that failure of the hematopoietic system was a major assay. The endpoint of this assay is the total radiation dose required to
cause of death following total body irradiation and that lethally irradiated achieve a specified probability of local tumor control—usually 50%
animals could be “rescued” by a bone marrow transplant. The trans- (TCD50)—in a specified period of time after irradiation. The TCD50
planted, viable bone marrow cells were observed to form discrete nodules value is obtained from a plot of the percentage of tumors locally
or colonies in the otherwise sterilized spleens of irradiated animals. controlled as a function of total dose. The slope of the resulting dose-
Subsequently, these authors transplanted known quantities of irradiated response curve may be used for comparative purposes as a measure of
donor bone marrow into lethally irradiated recipient mice. They were the tumor’s inherent “radiosensitivity” and/or its degree of heterogeneity.
able to count the resulting splenic nodules and then calculate the surviving More heterogeneous tumors tend to have shallower dose response curves
fraction of the injected cells in much the same way as was done for in than more homogeneous ones, as do spontaneous tumors relative to
vitro experiments. The D0 for mouse bone marrow was 0. 95 Gy.84 Other experimental ones maintained in inbred strains of mice.
in vivo assay systems based on the counting of colonies or nodules
included the skin epithelium assay of Withers,86 the intestinal crypt Cellular “Repair”: Sublethal and Potentially Lethal
assays of Withers and Elkind,87,88 and the lung colony assay of Hill and Damage Recovery
Bush.89 During the late 1960s and early 1970s, it also became possible Taking the cue from target theory that the shoulder region of the radiation
to do excision assays, in which tumors irradiated in vivo were removed, survival curve indicated that “hits” had to accumulate prior to cell
enzymatically dissociated, and single cells plated for clonogenic survival killing, Elkind and Sutton94,95 sought to better characterize the nature
in vitro. This allowed more quantitative measurement of survival, of the damage caused by these hits and how the cell processed this
avoiding some of the pitfalls of in vivo assays (e.g., Rockwell and damage. Even in the absence of any detailed information about DNA
Kallman90). damage and repair at the time, a few things seemed obvious. First, those
hits or damages that were registered as part of the accumulation process
Nonclonogenic Assays In Vivo yet did not in and of themselves produce cell killing were, by definition,
Some normal tissues and tumors are not amenable to clonogenic assays. sublethal. Second, sublethal damage (SLD) became lethal only when it
Thus, new assays were needed that had clinical relevance yet did not interacted with additional sublethal damage, that is, when the total
rely on reproductive integrity as an endpoint. Use of such assays required amount of damage had accumulated to a sufficient level to cause cell
one leap of faith—namely, that the endpoints assessed would have to killing. But what would be the result of deliberately interfering with
be a consequence of the killing of clonogenic cells, although not neces- the damage accumulation process by, for example, delivering part of
sarily in a direct, one-to-one manner. Because nonclonogenic assays the intended radiation dose, inserting a radiation-free interval, and
do not directly measure cell survival as an endpoint, data derived from then delivering the remainder of the dose? The results of such “split-dose”
them and plotted as a function of radiation dose are properly called experiments turned out to be crucial to the understanding of why and
dose-response curves rather than cell survival curves, although such how fractionated radiation therapy works as it does. The discovery and
data are often analyzed and interpreted similarly. characterization of SLD, as low tech and operational the concept may
Historically, among the first nonclonogenic assays was the mean be by today’s standards, still stands as arguably the single most important
lethal dose or LD50 assay, in which the (whole body) radiation dose to contribution that radiation biology has made to the practice of radiation
produce lethality in approximately 50% of the test subjects is determined, oncology.
usually at a fixed time after irradiation, such as 30 (LD50/30) or 60 days By varying the time interval between two doses of approximately
(LD50/60). Clearly, the LD50 assay is not very specific in that the cause of 5.0 Gy and plotting the log of the surviving fraction of cells after both
death can result from damage to a number of different tissues. doses (i.e., 10 Gy total dose) as a function of the time between the
Another widely used nonclonogenic method to assess normal tissue doses, the resulting split-dose recovery curve was observed to rise to a
radioresponse is the skin reaction assay, originally developed by Fowler maximum after about 2 hours and then level off. In other words, the
et al.91 Pigs were often used because their skin is similar to that of overall surviving fraction of cells following 10 Gy was higher if the dose
humans in several respects. An ordinate scoring system was used to was split into two fractions with a time interval in between than delivered
compare and contrast different radiation schedules, which was derived as a single dose. Elkind interpreted these results as indicating that the
from the average severity of the skin reaction noted during a certain cells that survived the initial dose fraction had “repaired” some of the
time period (specific to the species and whether the endpoint occurs damage during the radiation-free interval and, as such, this damage
early or late) following irradiation. For example, for early skin reactions, was no longer available to interact with the damage inflicted by the
a skin score of 1 might correspond to mild erythema, whereas a score second dose. At the time, Elkind referred to this phenomenon as sublethal
of 4 might correspond to confluent moist desquamation over more damage repair (SLDR). In retrospect, it is perhaps preferable to call it
than half of the irradiated area. sublethal damage recovery, since biochemical DNA repair processes were
Finally, two common nonclonogenic assays for tumor response are not actually measured, only changes in cell survival.
the growth delay/regrowth delay assay92 and the tumor control dose Of additional interest was the observation that the shape of the
assay.93 Both assays are simple and direct, are applicable to most solid split-dose recovery curve varied with the temperature during the
tumors, and are clinically relevant. The growth delay assay involves radiation-free interval (Fig. 1.9). When the cells were maintained at
measurements of a tumor’s dimensions or volume as a function of time room temperature between the split doses, the SLDR curve rose to a
after irradiation. For tumors that regress rapidly during and after maximum after about 2 hours and then leveled off. When the cells were
radiotherapy, the endpoint scored is typically the time in days that it returned to a 37° C incubator for the radiation-free interval, a different
takes for the tumor to regrow to its original volume at the start of pattern emerged. Initially, the split-dose recovery curve rose to a
irradiation. For tumors that regress more slowly, a more appropriate maximum after 2 hours; then, the curve exhibited a series of oscillations,
endpoint might be the time that it takes for the tumor to grow or dropping to a second minimum for a split of about 4 to 5 hours, and
regrow to a specified size, such as three times its original volume. then rising again to a higher maximum for split-dose intervals of 10
hours or more. The interpretation of this pattern of SLDR was that LET) and the oxygenation status of the cells (recovery reduced or
other radiobiological phenomena operated simultaneously with cellular absent at extremely low oxygen tensions).28
recovery. In this case, the fine structure of the split-dose recovery curve 2. The half-time for SLDR in mammalian cells in culture is, on average,
was not caused by an oscillating repair process but rather by a super- about 1 hour, although there is evidence that it may be somewhat
imposed cell cycle effect: the so-called radiation “age response” through longer for late-responding normal tissues in vivo.28
the cell cycle. This is discussed later in the “Ionizing Radiation and the 3. The survival increase between split doses is a manifestation of the
Cell Cycle” section (see also Fig. 1.14). “regeneration” of the shoulder of the radiation survival curve. After
Since Elkind and Sutton’s original work, SLDR kinetics have been an initial radiation dose and an adequate time interval for SLDR,
described for many different types of mammalian cells in culture,60 and the response of surviving cells to graded additional doses is nearly
for most normal and tumor tissues in vivo (e.g., Belli et al.96 and Emery identical to that obtained from cells without previous radiation
et al.97). Pertinent findings include the following: exposure. Thus, the width of the shoulder of the survival curve came
1. The amount of SLD capable of being repaired for a given cell type to be associated with the capacity of the cells for recovery from
varies both with the radiation quality (less for radiations of increasing sublethal damage. This concept is illustrated in Fig. 1.10.
4. Cells are able to undergo repeated cycles of damage and recovery
without a change in recovery capacity. As such, one would predict
an equal effect per dose fraction during the course of fractionated
7.6 Gy
"t
7.9 Gy
radiotherapy. In a more practical sense, this means that a multi-
fraction survival curve can be generated using the formula SFn =
10!1 V79 hamster cells 37° C
(SF1)n, where SF1 is the surviving fraction of cells after a single-dose
Surviving fraction
fraction (determined from a single-dose survival curve), and SFn is

24° C the surviving fraction of cells after n dose fractions. Accordingly,
multifraction survival curves are shoulderless and exponential
(Fig. 1.11).
5. Sublethal damage recovery is largely responsible for the dose rate
10!2 effect for low-LET radiation, which will be discussed in detail later
in this chapter. As the dose per fraction (intermittent radiation)
or dose rate (continuous irradiation) is decreased and the overall
treatment time increased, the biological effectiveness of a given total
4 0 6 2 8 10 12 dose is reduced. (Note that SLDR also occurs during continuous
Time between doses (h)
irradiation, i.e., that a radiation-free interval is not required per se.)
Fig. 1.9 “Split-dose” or sublethal damage recovery demonstrated in A second type of cellular recovery following irradiation is termed
cultured hamster V79 cells that received a first x-ray dose at time = 0,
potentially lethal damage repair or recovery (PLDR), and was first
followed by a second dose after a variable radiation-free interval. Cells
were maintained at either room temperature (24° C) or at 37° C during
described for mammalian cells by Phillips and Tolmach98 in 1966. PLD
the “split” time. (Modified from Elkind M, Sutton-Gilbert H, Moses W, is, by definition, a spectrum of radiation damage that may or may not
et al. Radiation response of mammalian cells grown in culture. V. result in cell killing depending on the cell’s postirradiation environment.
Temperature dependence of the repair of x-ray damage in surviving Environmental conditions that favor PLDR include maintenance of cells
cells (aerobic and hypoxic). Radiat Res. 1965;25:359.) in overcrowded conditions (plateau phase or contact-inhibited99,100) and
Total dose (Gy)

2.5 5.0 7.5 10.0 12.5
100 100
∆t
5 Gy 5 Gy
Surviving fraction
Surviving fraction
24° C
10!1 100 10!1
10!2 10!1 10!2
10!3 10!2 3h 10!3

0h
1h
1 2 0
3
A B Time between doses (h)
Fig. 1.10 Sublethal damage recovery is also manifest as a return of the shoulder on the radiation survival curve
when a total dose is delivered as two fractions separated by a time interval (A). If the interfraction interval is
shorter than the time it takes for this recovery to occur, the shoulder will be only partially regenerated (e.g.,
compare the shoulder regions of the survival curves for intervals of 1 h vs. 3 h). The regeneration of the
shoulder accounts for the observed survival increase in the corresponding split-dose assay (B, and Fig. 1.9).
Total dose (Gy)

Untransformed rodent fibroblasts
2 4 6 8 10 12 (13 Gy)
10!2
Surviving fraction
Fxn 1
100
Transformed rodent fibroblasts
Fxn 2 (10 Gy)
Surviving fraction
10!1
Fxn 3
10!2
10!3
10!3
etc. 4 8 12 24 48 72 96 120
10!4
A Time between irradiation and subculture (h)
Fig. 1.11 Hypothetical multifraction survival curve (dashed line) for

repeated 3.0 Gy fractions under conditions in which sufficient time
Survival relative to tumor explanted

between fractions is allowed for full sublethal damage recovery, and 5 Large rodent tumor
immediately after irradiation

cell cycle and proliferative effects are negligible. The multifraction survival (20 Gy)
4
curve is shallower than its corresponding single dose curve (solid lines)
and has no shoulder, that is, surviving fraction is an exponential function 3
of total dose.
Small rodent tumor
2 (15 Gy)
incubation following irradiation at either reduced temperature101 in the

presence of certain metabolic inhibitors98 or in balanced salt solutions 1
rather than complete culture medium.101 What these treatment conditions
have in common is that they are suboptimal for continued growth of
cells. This gives resting cells more opportunity to repair DNA damage
prior to cell division than cells that continue traversing the cell cycle
immediately after irradiation. Phillips and Tolmach98 were the first to 2 4 6 8
propose this repair-fixation or competition model to explain PLDR. B
Time between irradiation and explant (h)
While, admittedly, some of these postirradiation conditions are not Fig. 1.12 Potentially lethal damage recovery can be demonstrated using
likely to be encountered in vivo, slow growth of cells in general, with a “delayed-plating” assay in which a variable delay time is inserted
or without a large fraction of resting cells, is a common characteristic between exposure to a large single dose of radiation and the harvesting
of many tissues. As might be expected, tumors (and, subsequently, select of the cells for a clonogenic assay. If cells are maintained in overcrowded
normal tissues amenable to clonogenic assay) were shown to repair and/or nutrient-deprived conditions during the delay period, the surviving
PLD.100 Experiments using rodent tumors were modeled after comparable fraction increases relative to that obtained when there is no delay. (A)
studies using plateau phase cells in culture, that is, a delayed-plating Potentially lethal damage recovery in vitro in a nontumorigenic rodent
fibroblast cell line and its transformed tumorigenic counterpart. (B)
assay was used. For such an experiment, irradiated cell cultures or animal
Potentially lethal damage recovery in vivo in both small and large mouse
tumors are left in a confluent state (either in the overcrowded cell
fibrosarcomas. (A, Modified from Zeman E, Bedford J. Dose-rate effects
culture or in the intact tumor in the animal) for varying lengths of in mammalian cells. V. Dose fractionation effects in noncycling C3H
time before removing them, dissociating them into single-cell suspensions 10T1/2 cells. Int J Radiat Oncol Biol Phys. 1984;10:2089; B, modified
and plating the cells for clonogenic survival at a low density. The longer from Little J, Hahn G, Frindel E, et al. Repair of potentially lethal radiation
the delay between irradiation and the clonogenic assay, the higher the damage in vitro and in vivo. Radiology. 1973;106:689.)
resulting surviving fraction of individual cells, even though the radiation
dose is the same. In general, survival rises to a maximum within 4 to
6 hours and levels off thereafter (Fig. 1.12). Repair in Tissues
The kinetics and extent of recovery from both SLD and PLD are When considering the repair phenomenon in intact tissues, it is important
correlated with the molecular repair of DNA and the rejoining of to remember that both the magnitude of the repair (related both to the
chromosome breaks.102,103 For the purposes of radiation therapy, however, shape of the shoulder region of the corresponding dose-response curve
the most important consideration is that both processes have the potential and the dose delivered) and the rate of the repair can influence how
to increase the surviving fraction of cells between subsequent dose the tissue behaves during a course of radiation therapy. For example, a
fractions. Such a survival increase could be manifest clinically as either particular tissue—normal or tumor—may be quite capable of repairing
increased normal tissue tolerance or decreased tumor control. It is also most damage produced by each dose fraction, but if the interfraction
important to appreciate that small differences in recovery capacity interval is so short as to not allow all the damage to be repaired prior
between normal and tumor cells after a single-dose fraction are magnified to the next dose, the tolerance of that tissue will be less than otherwise
into large differences after 30 or more dose fractions. anticipated. Second, while the sparing effect of dose fractionation for
both normal and tumor tissues can be explained largely by SLD recovery a “1X” DNA content would correspond to cells in G1 phase, cells with
between fractions, at sufficiently small doses per fraction, the degree of a “2X” DNA content in G2 or M phase, and cells with DNA contents
sparing will reach a maximum below which no further sparing occurs, between “1X” and “2X” in the S phase of the cell cycle. By performing
all other radiobiological factors being equal. This is a reflection of the a mathematical fit to the DNA histogram, the proportion of cells in
fact that some radiation damage is necessarily lethal and not modifiable each phase of the cell cycle can be determined, the phase durations can
by either further fractionation or changing postirradiation conditions. be derived, and differences in DNA ploidy can be identified. DNA flow
cytometry is quite powerful in that a static measure of cell cycle distribu-
Ionizing Radiation and the Cell Cycle tion can be obtained for a cell population of interest and dynamic
Another basic feature of the cellular response to ionizing radiation is studies of, for example, transit through the various cell cycle phases or
perturbation of the cell cycle. Such effects can modify the radioresponsive- treatment-induced kinetic perturbations can be monitored over time
ness of tissues either directly or indirectly depending on the fraction (Fig. 1.13). Flow cytometers are often outfitted with a cell-sorting feature.
of cycling cells present in the tissue, their proliferation rates, and the In this case, cells analyzed for a property of interest can be collected in
kinetic organization of the tissue or tumor.
Advances in techniques for the study of cell cycle kinetics during
the 1950s and 1960s paved the way for the generation of survival curves A
as a function of cell “age.” Using a technique known as autoradiography,
Howard and Pelc104 were able to identify the S, or DNA synthesis, phase
of the cell cycle. When combined with the other obvious cell cycle
marker, mitosis, they were able to discern the four phases of the cell
cycle for actively growing cells: G1, S, G2, and M.
Methodology
Several techniques were subsequently developed for the collection G1 G2/M
of synchronized cells in vitro. One of the most widely used was the M
mitotic harvest or “shake-off ” technique first described by Terasima B G2 G
1
and Tolmach.105,106 By agitating cultures, mitotic cells, which tend to S
round up and become loosely attached to the culture vessel’s surface,
can be dislodged, collected along with the overlying growth medium,
and inoculated into new culture flasks. By incubating these flasks at
37° C, cells begin to proceed synchronously into G1 phase (and semi-
synchronously thereafter). Thus, by knowing the length of the various
phase durations for the cell type being studied and then delivering a
radiation dose at a time of interest after the initial synchronization, G1 G2/M
the survival response of cells in different phases of the cell cycle can
be determined. C
A second synchronization method involved the use of DNA synthesis
inhibitors such as fluorodeoxyuridine107 and, later, hydroxyurea108 to
selectively kill S phase cells yet allow cells in other phases to continue
cell cycle progression until they become blocked at the border of G1
and S phases. By incubating cells in the presence of these inhibitors for
times sufficient to collect nearly all cells at the block point, large numbers
of cells can be synchronized. The inhibitor technique has two other
advantages: that some degree of synchronization is possible in vivo109 G1 G2/M
as well as in vitro and that, by inducing synchrony at the end of the G1
phase, a higher degree of synchrony can be maintained for longer periods D
than if synchronization had been at the beginning of G1. On the other
hand, the mitotic selection method does not rely on the use of drugs
that could perturb the normal cell cycle kinetics of the population.
Developments in the early 1970s provided what is now considered
among the most valuable tools for the study of cytokinetic effects: the
flow cytometer and its offshoot, the fluorescence-activated cell sorter.110
These have largely replaced the aforementioned longer and more labor-
intensive cell cycle synchronization methods. Using this powerful
G1 G2/M
technique, single cells are stained with a fluorescent probe that binds
stoichiometrically to a specific cellular component, DNA in the case of Fig. 1.13 The analytical technique of flow cytometry has revolutionized
cell cycle distribution analysis. The stained cells are then introduced the study of cell cycle kinetics by allowing rapid determination of DNA
content in cells stained with a fluorescent dye that binds stoichiometrically
into a pressurized flow cell and forced to flow single file and at a high
to cellular DNA. (A) Frequency distribution for a population of exponentially
rate of speed through a focused laser beam that excites the fluorescent growing cells. The large and small peaks correspond to cells with G1
dye. The resulting light emission from each cell is collected by photo- (“1X”) and G2/M (“2X”) phase DNA content, respectively; those cells
multiplier tubes, recorded, and output as a frequency histogram of cell in S phase have an intermediate DNA content. (B–D) DNA histograms
number as a function of relative fluorescence, with the amount of fluo- for a cell population synchronized initially in mitosis and then allowed
rescence directly proportional to DNA content. Accordingly, cells with to progress into G1 (B), S and G2/M (C and D). See text for details.
separate “bins” after they pass through the laser beam and, if possible, Following a single dose of 5 Gy of x-rays, cells were found to be most
used for other experiments. radioresistant in late S phase. Cells in G1 were resistant at the beginning
of the phase, but became sensitive toward the end of the phase, and G2
Age Response Through the Cell Cycle cells were increasingly sensitive as they moved toward the most sensitive
Results of Terasima and Tolmach’s106 age response experiment using M phase. In subsequent experiments by Sinclair,111,112 age-response curves
synchronized HeLa cells are shown in the lower panel of Fig. 1.14. for synchronized Chinese hamster V79 cells showed that the peak in
resistance observed in G1 HeLa cells was largely absent for V79 cells. This
is also illustrated in Fig. 1.14 (upper panel). Otherwise, the shapes of the
0.2
age-response curves for the two cell lines were similar. The overall length
V79 cells (rodent) of the G1 phase determines whether the resistant peak in early G1 will
0.1 7.1 Gy be present; in general, this peak of relative radioresistance is observed
0.05 only for cells with long G1 phases. For cells with short G1 phases, the
entire phase is often of intermediate radiosensitivity. An analysis of
0.02 the complete survival curves for synchronized cells111,113 confirms that
0.01 the most sensitive cells are those in the M and late G2 phases, in which
survival curves are steep and largely shoulderless, and the most resistant
Surviving fraction
0.005 cells are those in late S phase. The resistance of these cells is conferred
by the presence of a broad survival curve shoulder rather than by a
M G1 S G2
significant change in survival curve slope (Fig. 1.15). When high-LET
HeLa cells (human) radiations are used, the age-response variation through the cell cycle
0.1 is significantly reduced or eliminated, since survival curve shoulders
5.0 Gy
0.05
are either decreased or removed altogether by such exposures (see also
“Relative Biological Effectiveness” section to come). Similar age-response
0.02 patterns have been identified for cells synchronized in vivo.109
The existence of a cell cycle age response for ionizing radiation
0.01
provided an explanation for the unusual pattern of SLDR observed for
0.005 cells maintained at 37° C during the recovery interval (see Fig. 1.9). In
Elkind and Sutton’s experiments, exponentially growing cells were used,
G1 S G2 M that is, cells that were asynchronously distributed across the different
phases of the cell cycle. The cells that survived irradiation tended to be
Cell “age” those most radioresistant. Thus, the remaining population became
Fig. 1.14 Cell cycle age response for sensitivity to radiation-induced enriched with the more resistant cells. For low-LET radiation, those
cell killing in a representative rodent cell line (V79, top) having a short cells that were most resistant were in S phase at the time of the first
G1 phase duration, and a representative human cell line (HeLa, bottom), radiation dose. However, at 37° C, cells continued to progress through
having a long G1 phase duration. Both cell lines exhibit a peak of the cell cycle; those surviving cells in S phase at the time of the first
radioresistance in late S phase and maximum radiosensitivity in late
dose may have moved into G2 phase by the time the second dose was
G2/M phase. A second “trough” of radiosensitivity can be discerned
near the G1/S border for cells with long G1 phase durations. (Modified
delivered. Thus, the observed survival nadir in the SLDR curve was not
from Sinclair W. Dependence of radiosensitivity upon cell age. In: Proceed- due to a loss or reversal of repair but rather because the population of
ings of the Carmel Conference on Time and Dose Relationships in cells was now enriched in G2 phase cells, which are inherently more
Radiation Biology as Applied to Radiotherapy. BNL Report 50203. Upton, radiosensitive. For even longer radiation-free intervals, it is possible
NY: Brookhaven National Laboratory; 1969.) that the cells surviving the first dose would transit from G2 to M and
1.0
7.1 Gy
0.2
0.1 0.1
Surviving fraction
0.05
0.02
0.01 0.01
Late S 0.005
0.001 G2/M G1 Early S M G1 S G2
Cell “age”
5.0
10.0 15.0
Dose (Gy)
Fig. 1.15 Cell survival curves for irradiated populations of Chinese hamster cells synchronized in different
phases of the cell cycle (left), illustrating how these radiosensitivity differences translate into the age response
patterns shown at right (and in Fig. 1.14).
back into G1 phase, dividing and doubling their numbers. In this case, the criteria used by these genes to help make the decision whether to
the SLDR curve again shows a surviving fraction increase because the continue traversing the cell cycle or to pause—either temporarily or, in
number of cells has increased. None of these cell cycle-related phenomena some cases, permanently. Cell cycle checkpoint genes are discussed in
occur when the cells are maintained at room temperature during the Chapter 2.
radiation-free interval, because continued movement through the cell
cycle is inhibited under such conditions. In that case, all that is noted Redistribution in Tissues
is the initial survival increase due to SLDR. Because of the age response through the cell cycle, an initially asyn-
chronous population of cells surviving a dose of radiation becomes
Radiation-Induced Cell Cycle Blocks and Delays enriched with S phase cells. Owing to variations in the rate of further
Radiation is also capable of disrupting the normal proliferation kinetics cell cycle progression, however, this partial synchrony decays rapidly.
of cell populations. This was recognized by Canti and Spear in 1927114 Such cells are said to have “redistributed,”118 with the net effect of sensitiz-
and studied in conjunction with radiation’s ability to induce cellular ing the population as a whole to a subsequent dose fraction (relative
lethality. With the advent of mammalian cell culture and synchronization to what would have been expected had the cells remained in their
techniques along with time-lapse cinemicrography, it became possible resistant phases). A second type of redistribution also has a net sensitizing
for investigators to study mitotic and division delay phenomena in effect, in which cells accumulate in G2 phase (in the absence of cell
greater detail. division) during the course of multifraction or continuous irradiation
Mitotic delay, defined as a delay in the entry of cells into mitosis, is because of a buildup of radiation-induced cell cycle blocks and delays.
a consequence of “upstream” blocks or delays in the movement of cells This has been observed during continuous irradiation by several
from one cell cycle phase to the next. Division delay, a delay in the time investigators.119 In some of these cases, a net increase in radiosensitivity
of appearance of new cells at the completion of mitosis, is caused by is seen at certain dose rates. This so-called “inverse dose rate effect,”
the combined effects of mitotic delay and any further lengthening of where certain dose rates are more effective at cell killing than other,
the mitosis process itself. Division delay increases with dose and is, on higher dose rates, was extensively studied by Mitchell, Bedford and
average, about 1 to 2 hours per gray106 depending on the cell line. associates (for a review, see Bedford et al.120). The magnitude of the
The cell cycle blocks and delays primarily responsible for mitotic sensitizing effect of redistribution varies with cell type depending on
and division delay are, respectively, a block in the G2-to-M phase transi- what dose rate is required to stop cell division. For dose rates below
tion, and a block in the G1-to-S phase transition. The duration of the the critical range that causes redistribution, some cells can escape the
G2 delay, like the overall division delay, varies with cell type, but for a G2 block and proceed on to cell division.
given cell type is both dose and cell cycle age dependent. In general,
the length of the G2 delay increases linearly with dose. For a given dose, Densely Ionizing Radiation
the G2 delay is longest for cells irradiated in S or early G2 phase, and Linear Energy Transfer
shortest for cells irradiated in G1 phase.115 Another factor contributing The total amount of energy deposited in biological materials by ionizing
to mitotic and division delay is a block in the flow of cells from G1 into radiation (usually expressed in units of keV, ergs or joules per g or kg)
S phase. For x-ray doses of at least 6 Gy, there is a 50% decrease in the is in and of itself insufficient to describe the net biological consequences
rate of tritiated thymidine uptake (indicative of entry into S phase) in of those energy deposition events. For example, 1 Gy of x-rays, while
exponentially growing cultures of mouse L cells. Little116 reached a physically equivalent in terms of total energy imparted per unit mass
similar conclusion from G1 delay studies using human liver LICH cells to 1 Gy of neutrons or α-particles, does not produce equivalent biological
maintained as confluent cultures. effects. It is the microdosimetric pattern of that energy deposition, that
A possible role for DNA damage and its repair in the etiology of is, the spacing or density of the ionization events, that determines
division delay was bolstered by the finding that certain cell types that biological effectiveness. This quantity—the average energy deposited
either did not exhibit the normal cell cycle delays associated with radiation locally per unit length of the ionizing particle’s track—is termed its
exposure (such as AT cells42) or, conversely, were treated with chemicals linear energy transfer (LET).
that ameliorated the radiation-induced delays117 tended to contain higher LET is a function both of the charge and mass of the ionizing particle.
amounts of residual DNA damage and to show increased radiosensitivity. Photons set in motion fast electrons that have a net negative charge
It is now known that the radiation-induced perturbations in cell but a negligible mass. Neutrons, on the other hand, give rise to recoil
cycle transit are under the control of cell cycle checkpoint genes, protons or α-particles that possess one or two positive charges, respec-
whose products normally govern the orderly (and unidirectional) tively, and are orders of magnitude more massive than electrons.
flow of cells from one phase to the next. The checkpoint genes are Neutrons, therefore, have a higher LET than photons and are considered
responsive to feedback from the cell as to its general condition and densely ionizing, whereas the x-rays or γ-rays are considered sparsely
readiness to transit to the next cell cycle phase. DNA integrity is one of ionizing. The LET concept is illustrated in Fig. 1.16 for both densely
Sensitive
“target”
60 Co γ-rays
250 kVp x-rays
α Particles
Fig. 1.16 Variation in the density of ionizing events along an incident particle’s track for radiations of differing
linear energy transfer. The more closely spaced the ionizing events, the more energy will be deposited in
the target volume and, to a point, the more biologically effective per unit dose the type of radiation will be.
and sparsely ionizing radiations. For a given ionizing particle, the rate
Relative biological effectiveness

of energy deposition in the absorbing medium increases as the particle 10 3
Oxygen enhancement ratio

slows down. Therefore, a beam of radiation can only be described as
8
having an average value for LET.
Representative LET values for types of radiation that have been used 6
for radiation therapy include 0.2 keV/μm for 60Co γ-rays; 2.0 keV/μm 2
for 250 kVp x-rays; approximately 0.5 to 5.0 keV/μm for protons of 4
different energies; approximately 50 to 150 keV/μm for neutrons; 100
to 150 keV/μm for α-particles; and anywhere from 100 to 2500 keV/μm 2
for “heavy ions.”
1
Relative Biological Effectiveness 1 10 100 1000
Insofar as the “quality” (LET) of the type of radiation influences its Linear energy transfer (keV/!m)
biological effectiveness, two questions immediately come to mind. First,
why do seemingly subtle differences in microdosimetric energy deposition X-rays and γ-rays Argon
patterns lead to vastly different biological consequences? Second, how Neon
is this differing biological effectiveness manifest in terms of the commonly
used assays and model systems of foundational radiobiology, and how Carbon
can this difference be expressed in a quantitative way?
Neutrons
Because high-LET radiations are more densely ionizing than their
low-LET counterparts, it follows that energy deposition in a particular Fig. 1.17 Relative biological effectiveness (RBE, left Y-axis) as a function
of linear energy transfer (LET) for a number of biological endpoints,
“micro”-target volume will be greater and therefore, more severe damage
including production of chromosomal aberrations, cell killing, and tissue
to biomolecules would be expected. In this case, the fraction of cell
reactions. The RBE rises to a maximum corresponding to an LET of
killing attributable to irreparable and unmodifiable DNA damage approximately 100 keV/μm and then decreases as the LET continues
increases in relation to that caused by the accumulation of sublethal to rise. Shown below the X-axis are the ranges of LET for photons plus
damage. Because of this, a number of radiobiological phenomena several different types of particulate radiations that have been used
commonly associated with low-LET radiation are decreased or eliminated clinically. Also shown is the dependence of the oxygen enhancement
when high-LET radiation is used. For example, there is little, if any, ratio (OER, right Y-axis) on LET.
sublethal60 or potentially lethal damage recovery.115 This is manifest as
a reduction or loss of the shoulder of the acute dose survival curve,
little or no sparing effect of dose fractionation or dose rate, and a 100
corresponding reduction in the tolerance doses for normal tissue RBE0.5!5.6
complications, particularly for late-responding tissues.121 Variations in

the age response through the cell cycle also are reduced or eliminated 10!1
Surviving fraction
RBE0.05!3.6
for high-LET radiation,109 and the oxygen enhancement ratio (OER),
a measure of the differential radiosensitivity of poorly versus well- 10!2
oxygenated cells (see later discussion), decreases with increasing LET.122 Neutrons X-rays
The dependence of OER on LET is illustrated in Fig. 1.17; at an LET
of approximately 100 keV/μm, the relative radioresistance of hypoxic 10!3
RBE0.0005!2.8
cells is eliminated.
In light of these differences between high- and low-LET radia- 10!4
tions, the term relative biological effectiveness (RBE) has been coined
to compare and contrast two radiation beams of different LET. RBE
is defined as the ratio of doses of a known type of low-LET radiation 10!5
(historically, 250 kVp x-rays were the standard) to that of a higher-LET 6 8 0 10 2 12 4 14
radiation to yield the same biological endpoint. RBE does not increase Dose (Gy)
indefinitely with increasing LET, however, but rather reaches a maximum Fig. 1.18 Theoretical cell survival curves for x-rays and neutrons,
at approximately 100 keV/μm and then decreases again, yielding an illustrating the increase in relative biological effectiveness (RBE) with
approximately bell-shaped curve. decreasing dose. This occurs because higher linear energy transfer radia-
One interpretation as to why the RBE reaches a maximum at an tions preferentially decrease or eliminate the shoulder on cell survival
curves. (Modified from Nias A. Clinical Radiobiology. 2nd ed. New York:
LET of approximately 100 keV/μm is that, at this ionization density,
Churchill Livingstone; 1988.)
the average separation between ionizing events corresponds roughly to
the diameter of the DNA double helix (approximately 2 nm). As such,
radiations of this LET have the highest probability of producing DSBs Factors That Influence Relative Biological Effectiveness
in DNA, the putative lethal lesion, by the passage of a single charged RBE is highly variable and depends on several parameters, including
particle. Lower-LET radiations have a smaller likelihood of producing the type of radiation, total dose, dose rate, dose fractionation pattern,
such “two-hit” lesions from a single particle track and, therefore, are and the biological effect being assayed. Therefore, when quoting an
less biologically effective. Radiation beams of higher LET than the RBE value, the exact experimental conditions used to measure it must
optimum are less efficient because some of the energy is wasted as be stated. Because increasing LET differentially reduces the shoulder
more ionization events than minimally necessary to kill a cell are region of the radiation survival curve compared to its exponential or
deposited in the same local area. This phenomenon has been termed the near-exponential high-dose region, the single-dose RBE increases with
overkill effect. decreasing dose (Fig. 1.18). Second, the RBE determined by comparing
Total dose (Gy)

2 4 6 8 10 12 14
100
Sensitive subpopulation
Surviving fraction
X-rays (multifraction)
Surviving fraction
10!1 Neutrons
(multifraction) X-rays
(single doses)
Neutrons RBE = 4.1

(single doses)
10!2 RBE = 2.7
Fig. 1.19 Increase in the relative biological effectiveness (RBE) of neutrons

relative to x-rays when comparing single doses with fractionated treat-
ment. For a given level of cell killing (or other approximately isoeffective
endpoint), the more highly fractionated the treatment, the higher the
RBE.
two isoeffective acute doses is less than the RBE calculated from two
isoeffective (total) doses given either as multiple fractions or at a low
dose rate. This occurs because the sparing effect of fractionation magnifies
Fig. 1.20 Cell survival curve for a murine lymphosarcoma growing
differences in the initial slope or shoulder region of cell survival or
subcutaneously and irradiated in vivo. The biphasic curve suggests the
tissue dose-response curves (Fig. 1.19). presence of a small but relatively radioresistant subpopulation of cells,
determined in accompanying experiments to represent the tumor’s
The Oxygen Effect clonogenic hypoxic fraction. (Modified from Powers WE, Tolmach LJ.
Perhaps the best-known chemical modifier of radiation action is A multicomponent x-ray survival curve for mouse lymphosarcoma cells
molecular oxygen. As early as 1909, Schwarz recognized that applying irradiated. Nature. 1963;197:710.)
pressure to skin and thereby decreasing blood flow (and oxygen supply)
caused a reduction in radiation-induced skin reactions.123 For many
decades thereafter, radiation oncologists and biologists continued to
suspect that the presence or absence of oxygen was capable of modifying Tolmach125 in 1963. These authors used the dilution assay to generate
radiosensitivity. In 1955, however, Thomlinson and Gray124 brought an in vivo survival curve for mouse lymphosarcoma cells. The survival
this idea to the forefront of radiation biology and therapy by proposing curve for this solid tumor was biphasic, having an initial D0 of about
that tumors contain a fraction of still-clonogenic, hypoxic cells that, if 1.1 Gy and a final D0 of 2.6 Gy (Fig. 1.20). Since the survival curve for
persistent throughout treatment, would adversely affect clinical outcome. lymphoid cells is shoulderless, it was simple to back-extrapolate the
Although commonly considered a negative prognostic indicator for shallower component of the curve to the surviving fraction axis and
radiation therapy, hypoxia nevertheless has one particularly attractive determine that the resistant fraction of cells constituted about 1.5% of
feature: built-in specificity for tumors, to the extent that normal tissues the total population. This was considered compelling evidence (yet did
contain few, if any, hypoxic cells. not unambiguously prove) that this subpopulation of cells was both
By studying histological sections of a human bronchial carcinoma, hypoxic and clonogenic.
Thomlinson and Gray noted that necrosis was always seen in the centers The question then became how to prove that this small fraction
of cylindrical tumor cords having a radius in excess of approximately of tumor cells was radioresistant because of hypoxia as opposed to
200 μm.124 Further, regardless of how large the central necrotic region being radioresistant for other reasons. An elegant, if somewhat macabre,
was, the sheath of apparently viable cells around the periphery of this method was developed to address this dilemma, called the paired survival
central region never had a radius greater than about 180 μm. The authors curve technique.125,126 In this assay, laboratory animals bearing tumors
went on to calculate the expected maximum diffusion distance of oxygen were divided into three treatment groups, one group irradiated while
from blood vessels and found that the value of 150 to 200 μm agreed breathing air, a second group irradiated while breathing 100% oxygen,
quite well with the radius of the sheath of viable tumor cells observed and a third group killed first by cervical dislocation and then irradiated.
histologically. With the advent of more sophisticated and quantitative Within each group, animals received graded radiation doses so that
methods for measuring oxygen utilization in tissues, the average diffusion complete survival curves were generated for each treatment condition.
distance of oxygen has since been revised downward to approximately When completed, the paired survival curve method yielded three differ-
70 μm.28 Thus, the inference was that the oxygenation status of tumor ent tumor cell survival curves: a fully oxic curve (most radiosensitive), a
cells varied from fully oxic to completely anoxic depending on where fully hypoxic curve (most radioresistant), and the survival curve for air-
the cells were located in relation to the nearest blood vessels. Accordingly, breathing animals which, if the tumor contained viable hypoxic cells, was
tumor cells at intermediate distances from the blood supply would be biphasic and positioned between the other two curves. It was then pos-
hypoxic and radioresistant, yet remain clonogenic. sible to mathematically strip the fully aerobic and hypoxic curves from
The first unambiguous demonstration that a solid rodent tumor the curve for air-breathing animals and determine the radiobiologically
did contain clonogenic, radioresistant hypoxic cells was by Powers and hypoxic fraction.
Across a variety of rodent tumors evaluated to date using the paired radioprotector, glutathione, tip the scales in favor of either fixation
survival curve method, the percentage of hypoxic cells was found to (more damage, more cell killing, greater radiosensitivity) or restitution
vary between 0% and 50%, with an average of about 15%.126 (less damage, less cell killing, greater radioresistance), respectively.
Consistent with this free radical–based interpretation of the oxygen
Mechanistic Aspects of the Oxygen Effect effect is the finding that, for all intents and purposes, oxygen need only
A more rigorous analysis of the nature of the oxygen effect is possible be present during the irradiation (or no more than a few milliseconds
with cells or bacteria grown in vitro. Historically, oxygen had been after irradiation) in order to produce an aerobic radioresponse.129,130
termed a dose-modifying agent, that is, that the ratio of doses to achieve The concentration of oxygen necessary to achieve maximum sensiti-
a given survival level under hypoxic and aerobic conditions was constant zation is quite small, evidence for the high efficiency of oxygen as a
regardless of the survival level chosen. This dose ratio to produce the radiosensitizer. A sensitivity midway between a fully hypoxic and fully
same biological endpoint is termed the oxygen enhancement ratio (OER), aerobic radioresponse is achieved at an oxygen tension of about 3 mm
and is used for comparative purposes (Fig. 1.21). The OER typically of mercury, corresponding to about 0.5% oxygen, much lower than
has a value of between 2.5 and 3.0 for large single doses of x-rays or partial pressures of oxygen usually encountered in normal tissues. This
γ-rays, 1.5 to 2.0 for radiations of intermediate LET, and 1.0 (i.e., no value of 0.5% has been termed oxygen’s k-value and is obtained from
oxygen effect) for high-LET radiations. an oxygen k-curve of relative radiosensitivity plotted as a function of
Increasingly, there is evidence that oxygen is not strictly dose modify- oxygen tension131 (Fig. 1.23).
ing. Several studies have shown that the OER for sparsely ionizing
radiation is lower at lower doses than at higher doses. Lower OERs
for doses per fraction in the range commonly used in radiotherapy Endogenous or
DNA Radioprotection
have been inferred indirectly from clinical and experimental tumor X-rays or γ-rays exogenous
thiol compound
data and more directly in experiments with cells in culture.127,128 It has
been suggested that the lower OERs result from an age response for the DNA DNA Oxygen DNA-OO
oxygen effect, not unlike the age responses for inherent radiosensitivity Radiosensitization
and cell cycle delay.28 Assuming that cells in G1 phase of the cell cycle
have a lower OER than those in S phase and since G1 cells are also Oxygen-mimetic
DNA-sensitizer
more radiosensitive, they would tend to dominate the low-dose region radiosensitizer
of the cell survival curve. Fig. 1.22 Schematic representation of the proposed mechanism of action
While the exact mechanism(s) of the oxygen effect are obviously for the oxygen effect. The radical competition model holds that oxygen
acts as a radiosensitizer by forming peroxides in DNA already damaged
complex, a fairly simplistic model can be used to illustrate our current
by radiation, thereby “fixing” the damage. In the absence of oxygen,
understanding of this phenomenon (Fig. 1.22). The radical competition
DNA can be restored to its preirradiated state by hydrogen donation
model holds that oxygen acts as a radiosensitizer by forming peroxides from endogenous reducing species in the cell, such as the free radical
in important biomolecules (including, but not necessarily limited to, scavenger glutathione. An oxygen-mimetic, hypoxic cell radiosensitizer
DNA) already damaged by radiation exposure, thereby “fixing” the may be used to substitute for oxygen in these fast free radical reactions
radiation damage. In the absence of oxygen, DNA can be restored to or an exogenously supplied thiol compound may be used to act as a
its preirradiated condition by hydrogen donation from endogenous radioprotector.
reducing species in the cell, such as the free radical scavenger glutathione,
a thiol compound. In essence, this can be considered a type of very fast
chemical restitution or repair. These two processes, fixation and restitu- 3.0
tion, are considered to be in a dynamic equilibrium, such that changes
Radiosensitivity relative to the
in the relative amounts of either the radiosensitizer, oxygen, or the

fully anoxic response (= 1.0)
Air 100%
100 Oxygen
2.0 O2 “k”-value =
3 mm Hg (0.5%)
Surviving fraction
10"1 OER ! 15.5 Gy/6.25 Gy ! 2.5

“Isoeffect” = 0.05 SF 1.0
Aerobic Hypoxic Range of normal tissue PO2
10"2
10 20 30 40 50 60 70 155 760
(6.25 Gy) (15.5 Gy) Oxygen tension (mm Hg at 37° C)
10"3 Fig. 1.23 An oxygen “k-curve” is used to illustrate the dependence of
0 2
8 4 6
10 12 14 16 18 20 radiosensitivity on oxygen concentration. If a fully anoxic cell culture is
Dose (Gy) assigned a relative radiosensitivity of 1.0, introducing even 0.5% (3 mm
Fig. 1.21 Representative survival curves for cells irradiated with x-rays Hg) oxygen into the system increases the radiosensitivity of cells to
in the presence (aerobic) or virtual absence (hypoxic) of oxygen. The 2.0; by the time the oxygen concentration reaches about 2.0%, cells
oxygen enhancement ratio (OER) is defined as the ratio of doses under respond as if they are fully aerated (i.e., radiosensitivity ≈ 3.0). The
hypoxic:aerobic conditions to yield the same biological effect, in this shaded area represents the approximate range of oxygen concentrations
case, a cell surviving fraction of 0.05. encountered in human normal tissues.
Reoxygenation in Tumors common cause of acute hypoxia) from, for example, vascular shunting,
After the convincing demonstration of hypoxic cells in a mouse tumor,125 longitudinal oxygen gradients, decreased red cell flux or overall blood
it was assumed that human tumors contained a viable hypoxic fraction flow rate, abnormal vascular geometry, and so on.136 Because of this,
as well. However, if human tumors contained even a tiny fraction of the name perfusion-limited hypoxia is perhaps misleading; a better
clonogenic hypoxic cells, simple calculations suggested that tumor moniker might be fluctuant or intermittent hypoxia. While intermittent
control would be nearly impossible with radiation therapy.132 Since hypoxia would explain the rapid reoxygenation observed for some
therapeutic successes obviously do occur, some form of reoxygenation tumors, it does not preclude the simultaneous existence of chronic,
must take place during the course of multifraction irradiation. This was diffusion-limited hypoxia.
not an unreasonable idea since the demand for oxygen by sterilized Intermittent hypoxia has since been demonstrated for rodent tumors
cells would gradually decrease as they were removed from the tumor, by Chaplin et al.137 and human tumors by Lin et al.138 It is still not clear
and a decrease in tumor size, a restructuring of tumor vasculature, or how many human tumors contain regions of hypoxia (although most
intermittent changes in blood flow could make oxygen available to do—see next section), what type(s) of hypoxia is present, whether this
these previously hypoxic cells. varies with tumor type or site, and whether and how rapidly reoxygen-
The reoxygenation process was extensively studied by van Putten ation occurs. However, the knowledge that tumor hypoxia is a diverse
and Kallman,133 who serially determined the fraction of hypoxic cells and dynamic process opens up a number of possibilities for the develop-
in a mouse sarcoma during a course of clinically relevant multifraction ment of novel interventions designed to cope with, or even exploit,
irradiation. The fact that the hypoxic fraction was about the same at hypoxia.
the end of treatment as at the beginning of treatment was strong evidence
for a reoxygenation process because, otherwise, the hypoxic fraction Measurement of Hypoxia in Human Tumors
would be expected to increase over time due to repeated enrichment Despite prodigious effort directed at understanding tumor hypoxia and
with resistant cells after each dose fraction. Reoxygenation of hypoxic, developing strategies to combat the problem, it was not until the late
clonogenic tumor cells during an extended multifraction treatment 1980s that these issues could be addressed for human tumors because
would increase the therapeutic ratio assuming that normal tissues there was no way to measure hypoxia directly. Before that time, the
remained well oxygenated. This is thought to be another major factor only way to infer that a human tumor contained treatment-limiting
in the sparing of normal tissues relative to tumors during fractionated hypoxic cells was by using indirect, nonquantitative methods. Some
radiation therapy. indirect evidence supporting the notion that human tumors contained
What physiological characteristics would lead to tumor reoxygenation clonogenic, radioresistant hypoxic cells includes the following:
during a course of radiotherapy, and at what rate would this be expected 1. An association between anemia and poor local control rates that, in
to occur? One possible cause of tumor hypoxia and, by extension, a some cases, could be mitigated by preirradiation blood transfusions139
possible mechanism for reoxygenation, was suggested by Thomlinson 2. Success of some clinical trials in which hyperbaric oxygen breathing
and Gray’s pioneering work.124 The type of hypoxia that they described was used to better oxygenate tumors140,141
is what is now called chronic or diffusion-limited hypoxia. This results 3. Success of a few clinical trials of oxygen-mimetic hypoxic cell sensitiz-
from the tendency of tumors both to have high oxygen consumption ers combined with radiation therapy139,142
rates and to outgrow their blood supply. It follows that natural gradients In 1988, one of the first studies showing a strong association between
of oxygen tension should develop as a function of distance from blood directly measured oxygenation status in tumors and clinical outcome
vessels. Cells situated beyond the diffusion distance of oxygen would was published by Gatenby et al.143 An oxygen-sensing electrode was
be expected to be dead or dying secondary to anoxia; yet, in regions of inserted into the patient’s tumor, and multiple readings were taken
chronically low oxygen tension, clonogenic and radioresistant hypoxic at different depths along the probe’s track. The electrode was also
cells could persist. Should the tumor shrink as a result of radiation repositioned in different regions of the tumor to assess intertrack
therapy or if the cells killed by radiation cause a decreased demand for variability in oxygen tension. Both the arithmetic mean Po2 value for
oxygen, it is likely that this would allow some of the chronically hypoxic a particular tumor and the tumor volume-weighted Po2 value directly
cells to reoxygenate. However, such a reoxygenation process could be correlated with local control rate. A high tumor oxygen tension was
quite slow—days at minimum—depending on how quickly tumors associated with a high complete response rate, and vice versa. In a
regress during treatment. The patterns of reoxygenation in some similar prospective study, Höckel et al.4,144 concluded that pretreat-
experimental rodent tumors are consistent with this mechanism of ment tumor oxygenation was a strong predictor of outcome among
reoxygenation, but others are not. patients with intermediate and advanced-stage cervical carcinoma
Other rodent tumors reoxygenate very quickly, from minutes to (Fig. 1.24).
hours.134 This occurs in the absence of any measurable tumor shrinkage The use of oxygen electrodes has its limitations. One weakness is
or change in oxygen utilization by tumor cells. In such cases, the model that relative to the size of individual tumor cells, the electrode is large,
of chronic, diffusion-limited hypoxia, and slow reoxygenation does not averaging an outer diameter of 300 μm, a tip recess of 120 μm, and a
fit the experimental data. During the late 1970s, Brown135 proposed sampling volume of about 12 μm in diameter.145 Thus, not only is the
that a second type of hypoxia must exist in tumors, an acute, perfusion- oxygen tension measurement regional, but the insertions and removals
limited hypoxia. Based on the growing understanding of the vascular of the probe no doubt perturb the oxygenation status. Another problem
physiology of tumors, it was clear that tumor vasculature was often is that there is no way to determine whether the tumor cells are clonogenic
abnormal in both structure and function secondary to abnormal or not. If such cells were hypoxic yet not clonogenic, they would not
angiogenesis. If tumor vessels were to close transiently from temporary be expected to impact radiotherapy outcome.
blockage, vascular spasm, or high interstitial fluid pressure in the sur- A second direct technique for measuring oxygenation status takes
rounding tissue, the tumor cells in the vicinity of those vessels would advantage of a serendipitous finding concerning how hypoxic cell radio-
become acutely hypoxic almost immediately. Then, assuming that blood sensitizers are metabolized. Certain classes of radiosensitizers, including
flow resumed in minutes to hours, these cells would reoxygenate. the nitroimidazoles, undergo a bioreductive metabolism in the absence
However, this type of hypoxia also can occur in the absence of frank of oxygen that leads to their becoming covalently bound to cellular
closure or blockage of tumor vessels (which is now considered a less macromolecules.146,147 Assuming that the bioreductively bound drug
Disease-free survival probability the processes of tumor invasion and metastasis169,170), and osteopontin
1.0 (OPN, a glycoprotein that facilitates tumor invasion and metastasis171–173).
Clearly, although aberrant expression of some individual hypoxia
0.8 markers has been associated with poor clinical outcome, no one marker
Median PO2 !10 mm Hg (n = 21)
is likely to be sufficiently robust or reproducible to either be diagnostic
for the presence of a malignancy (or, at least, the presence of hypoxia
0.6
in an already diagnosed tumor) or prognostic of treatment outcome.
Median PO2 "10 mm Hg (n = 23) Thus, there has been increasing interest in the study of patterns of
0.4 hypoxia-associated gene or protein expression for multiple markers
simultaneously, for example, Le et al.,5 Erpolat et al.,173 and Toustrup
0.2 et al.174
Radiosensitizers, Radioprotectors, and

30 40 50 10
60 20
70 80 Bioreductive Drugs
Time (months) The perceived threat that tumor hypoxia posed spawned much research
Fig. 1.24 The disease-free survival probability of a small cohort of cervical into ways of overcoming the hypoxia problem. One of the earliest
cancer patients stratified according to pretreatment tumor oxygenation, proposed solutions was the use of high-LET radiations,175 which were
as measured using an oxygen electrode. (Modified from Höckel M, less dependent on oxygen for their biological effectiveness. Other agents
Knoop C, Schlenger K, et al. Intratumoral pO2 predicts survival in advanced enlisted to deal with the hypoxia problem included hyperbaric oxygen
cancer of the uterine cervix. Radiother Oncol. 1993;26:45.) breathing140; artificial blood substitutes with increased oxygen-carrying
capacity176; oxygen-mimetic hypoxic cell radiosensitizers, such as misoni-
dazole or etanidazole139; hyperthermia177; normal tissue radioprotectors,
could be quantified by radioactive labeling148 or tagged with an isotope such as amifostine178; vasoactive agents that modify tumor blood flow,
amenable to detection using positron emission tomography149 or magnetic such as nicotinamide179; agents that modify the oxygen-hemoglobin
resonance spectroscopy,150 a direct measure of hypoxic fraction can be dissociation curve, such as pentoxifylline180; and bioreductive drugs
obtained. Another approach to detecting those cells containing bound designed to be selectively toxic to hypoxic cells, such as tirapazamine.181,182
drug was developed by the Raleigh group (e.g., Cline et al.151 and Kennedy
et al.152). This immunohistochemical method involved the development Radiosensitizers
of antibodies specific for the bound nitroimidazole metabolites. After Radiosensitizers are loosely defined as chemical or pharmacological
injecting the parent drug, allowing time for the reductive metabolism agents that increase the cytotoxicity of ionizing radiation. “True”
to occur, taking biopsies of the tumor, and preparing histopathology radiosensitizers meet the stricter criterion of being relatively nontoxic
slides, the specific antibody is then applied to the slides and regions in and of themselves, acting only as potentiators of radiation toxicity.
containing the bound drug are visualized directly. This immunostaining “Apparent” radiosensitizers still produce the net effect of making the
method has the distinct advantages that hypoxia can be studied at the tumor more radioresponsive, yet the mechanism is not necessarily
level of individual tumor cells,153 spatial relationships between regions synergistic nor is the agent necessarily nontoxic when given alone. Ideally,
of hypoxia and other tumor physiological parameters can be assessed,154 a radiosensitizer is only as good as it is selective for tumors. Agents that
and the drug does not perturb the tumor microenvironment. However, show little or no differential effect between tumors and normal tissues
the method remains an invasive procedure, is labor intensive, does not do not improve the therapeutic ratio and, therefore, may not be of
address the issue of the clonogenicity of stained cells (although such much clinical utility. Table 1.2 summarizes some of the classes of
cells do have to be metabolically active), and requires that multiple radiosensitizers (and radioprotectors—see later discussion) that have
samples be taken because of tumor heterogeneity. been used in the clinic.
One hypoxia marker based on the immunohistological method Hypoxic cell radiosensitizers. The increased radiosensitivity of cells
detects reductively bound pimonidazole hydrochloride and has been in the presence of oxygen is believed to be due to oxygen’s affinity for
used in experimental and clinical studies around the world (e.g., Bussink the electrons produced by the ionization of biomolecules. Molecules
et al.,155 Nordsmark et al.156). Human tumor specimens stained with other than oxygen also have this chemical property, known as electron
the marker were found to have a wide range of hypoxic fractions (similar affinity,183 including some agents that are not otherwise consumed by
to that for experimental rodent tumors), with a mean value of approxi- the cell. Assuming that such an electron-affinic compound is not used
mately 15%.152,157 This marker can also be used to probe disease states by the cell, it should diffuse further from capillaries and reach hypoxic
other than cancer that may have the induction of tissue hypoxia as part regions of a tumor and, acting in an oxygen-mimetic fashion, sensitize
of their etiology, such as cirrhosis of the liver158,159 and ischemia- hypoxic cells to radiation.
reperfusion injury in the kidney.160 One class of compounds that represented a realistic trade-off between
There is also considerable interest in endogenous markers of sensitizer efficiency and diffusion effectiveness was the nitroimidazoles,
tissue hypoxia161,162 that could reduce to some extent the procedural which include such drugs as metronidazole, misonidazole, etanidazole,
steps involved in, and the invasive aspects of, detecting hypoxia using pimonidazole, and nimorazole. The nitroimidazoles consist of a
exogenous agents. Among the endogenous cellular proteins being nitroaromatic imidazole ring, a hydrocarbon side chain that determines
investigated in this regard are the hypoxia-inducible factor 1-alpha the drug’s lipophilicity, and a nitro group that determines the drug’s
(HIF-1α, which acts as a transcription factor for hypoxia-regulated electron affinity. Misonidazole was extensively characterized in cellular
genes163), the enzyme carbonic anhydrase IX (CA-9 or CAIX, involved and animal model systems, culminating in its use in clinical trials.
in respiration and maintenance of the proper acid–base balance in Clinical experiences with misonidazole and some of its successor
tissues164,165), glucose transporter-1 (GLUT-1, which facilitates glucose compounds are discussed in Chapter 3.
transport into cells and glycolysis166–168), lysyl oxidase (LOX, which The relative efficacy of a particular hypoxic cell radiosensitizer is
oxidizes lysine residues in extracellular matrix proteins that can enhance most often described in terms of its sensitizer enhancement ratio (SER),
TABLE 1.2 Selected Chemical Modifiers of Radiation Therapy

Name (Type of
Chemical Structure Compound) Mechanism of Action Clinic Status
O 5-Bromodeoxyuridine Radiosensitizer of rapidly proliferating No clear evidence of clinical efficacy has
Br (halogenated cells through incorporation into been established to date. The drug
NH
pyrimidine) DNA during S phase. Uptake continues to be used in a research
O
results in decrease or removal of setting.
HO N
O the shoulder of the radiation
survival curve.
OH
OH Misonidazole Radiosensitizer of hypoxic cells. Clinical trial results were disappointing

N (2-nitroimidazole) Principal mechanism of action is overall except in selected sites, most
N O
mimicry of oxygen’s ability to “fix” notably, head and neck cancer. The drug’s
O2N
free radical damage caused by failure largely has been ascribed to a
exposure to radiation and some dose-limiting toxicity, peripheral
toxic chemicals. neuropathy.
O Tirapazamine (organic Bioreductive drug selectively Phase II and Phase III clinical trials to date
N nitroxide) toxic to hypoxic cells. The drug is have been disappointing overall, except in
N
reduced to a toxic intermediate select subsets of patients with head and
N NH2 capable of producing DNA strand neck or lung cancer. Some trials are still
O breaks only in the relative absence ongoing for the drug combined with
of oxygen. radiotherapy and cis-platinum.
HO Amifostine (thiol free Radioprotector capable of FDA-approved indications for amifostine
S
radical scavenger) “restituting” free radical damage include protection against the nephro-
P N NH2
HO H caused by exposure to radiation and ototoxicity of platinum drugs and to
O and some toxic chemicals. reduce the incidence and severity of
xerostomia in patients receiving
radiotherapy for head and neck cancer.
FDA, U.S. Food and Drug Administration.
a parameter similar in concept to the OER. Whereas the OER is the results have prompted a rethinking of the hypoxia problem and novel
ratio of doses to produce the same biological endpoint under hypoxic approaches to dealing with it as well as consideration of other factors
versus aerobic conditions, the SER is the dose ratio for an isoeffect that may have contributed to the lack of success of the nitroimidazole
under hypoxic conditions alone versus hypoxic conditions in the presence radiosensitizers.185 Among the more obvious questions raised are the
of the hypoxic cell sensitizer. If a dose of a sensitizer produces an SER following:
of 2.5 to 3.0 for large single doses of low-LET radiation, it can be 1. Did the patients entered in the various clinical trials actually have
considered to a first approximation “as effective as oxygen.” This statement tumors that contained clonogenic hypoxic cells? At the time of most
can be very misleading, however, in that the dose of the sensitizer required of these studies, hypoxia markers were not yet available; thus, it was
to produce the SER of 3.0 would be higher than the comparable “dose” not possible to triage patients into subgroups in advance of
of oxygen, high enough in some cases to preclude its use clinically. treatment.
Finally, since the primary mechanism of action of the nitroimidazoles 2. Do hypoxic cells really matter to the outcome of radiotherapy? If
is substitution for oxygen in radiation-induced free-radical reactions, reoxygenation is fairly rapid and complete during radiotherapy, the
these drugs need only be present in hypoxic regions of the tumor at presence of hypoxic cells prior to the start of treatment may be of
the time of irradiation. little consequence.
The nitroimidazoles also have characteristics that decrease their 3. Given that the OER is lower for small doses versus large doses, it
clinical usefulness. The hydrocarbon side chain of the molecule deter- follows that the SER would be reduced as well. If so, a benefit in a
mines its lipophilicity; this chemical property affects the drug’s phar- subgroup of patients might not be readily observed, at least not at
macokinetics, which is a primary determinant of drug-induced side a level of statistical significance.
effects.184 The dose-limiting toxicity of the fairly lipophilic agent Bioreductive drugs. In the wake of the failure of most hypoxic cell
misonidazole is peripheral neuropathy, an unanticipated and serious radiosensitizers to live up to their clinical potential, a new approach to
side effect.139,185 Etanidazole was specifically designed to be less lipophilic186 combating hypoxia emerged: the use of bioreductive drugs that are
in hopes of decreasing neurological toxicity. Although this goal was selectively toxic to hypoxic cells. While these agents kill rather than
accomplished as a proof of concept, clinical results with etanidazole sensitize hypoxic cells, the net effect of combining them with radiation
were otherwise disappointing187 (see also Chapter 3). therapy is an apparent sensitization of the tumor due to the elimination
Finally, in considering the prodigious amount of research and clinical of an otherwise radioresistant subpopulation. Such drugs have been
effort that has gone into the investigation of hypoxic cell radiosensitizers shown to outperform the nitroimidazole radiosensitizers in experimental
over the past 50 years, it is difficult not to be discouraged by the pre- studies with clinically relevant fractionated radiotherapy.188 To the extent
dominantly negative results of the clinical trials. However, these negative that hypoxic cells are also resistant to chemotherapy because of tumor
microenvironmental differences in drug delivery, pH, or the cell’s choice of head and neck tumors for this study was far from ideal,
proliferative status, complementary tumor-cell killing might be antici- because the oral mucosa is also a rapidly proliferating tissue and was
pated for combinations of bioreductive agents and anticancer drugs as similarly radiosensitized, causing severe mucositis and a poor therapeutic
well.189 ratio overall. In later years, tumors selected for therapy with halogenated
Most hypoxia-specific cytotoxic drugs fall into three categories: the pyrimidines were chosen in the hopes of better exploiting the differential
nitroheterocyclics, the quinone antibiotics, and the organic nitroxides.190 radiosensitization between tumors and normal tissues.203 Aggressively
All require bioreductive activation by nitroreductase enzymes such as growing tumors surrounded by slowly growing or nonproliferating
cytochrome P450, DT-diaphorase, and nitric oxide synthase to reduce normal tissues, such as high-grade brain tumors or some sarcomas,
the parent compound to its cytotoxic intermediate, typically an oxidizing have been targeted, for example.204,205 Later strategies for further improv-
free radical capable of damaging DNA and other cellular macromolecules. ing radiosensitization by BrdUdR and IdUdR involved changing the
The active species is either not formed or immediately back-oxidized schedule of drug delivery: giving the drug as a long, continuous infusion
to the parent compound in the presence of oxygen, which accounts for both before and during radiotherapy206; and administering the drug as
its preferential toxicity under hypoxic conditions. Examples of nitro- a series of short, repeated exposures.207 Overall, however, the use of
heterocyclic drugs with bioreductive activity include misonidazole and halogenated pyrimidines in the clinic has remained experimental and
etanidazole191,192 and dual-function agents such as RSU 1069.193 The has not become mainstream.
latter drug is termed “dual function” because its bioreduction also Chemotherapy drugs as radiosensitizers. Several chemotherapy
activates a bifunctional alkylating moiety capable of introducing cross- agents have long been known to increase the effectiveness of radiotherapy
links into DNA. Mitomycin C and several of its analogs (including despite not being “true” radiosensitizers, like the nitroimidazoles. This
porfiromycin and EO9) are quinones with bioreductive activity that has driven the clinical practice of treating many more patients today
have been tested in randomized clinical trials in head and neck tumors than in the past with chemotherapy and radiation therapy concurrently.
(e.g., Haffty et al.194). Two drugs in particular used for chemoradiotherapy are 5-fluorouracil
The lead compound for the third class of bioreductive drugs, the (5-FU, effective against gastrointestinal malignancies208) and cisplatinum
organic nitroxides, is tirapazamine (SR 4233, Table 1.2).181,182,188 The (effective against head and neck209 and cervix cancers210).
dose-limiting toxicity for single doses of tirapazamine is a reversible Based on these clinical successes in combining radiation with concur-
hearing loss; other effects observed include nausea and vomiting, and rent chemotherapy, and with ever-increasing numbers of molecularly
muscle cramps.195 targeted drugs and biologics available today, it is naturally of interest
Tirapazamine is particularly attractive because it both retains its whether any of these novel compounds could also act as radiosensitizers.
hypoxia-selective toxicity over a broader range of (low) oxygen concentra- Two classes of such drugs already have entered the clinical mainstream
tions than the quinones and nitroheterocyclic compounds196 and its as sensitizers: the antiepidermal growth factor receptor (EGFR) inhibitors
“hypoxic cytotoxicity ratio,” the ratio of drug doses under hypoxic and the antivascular endothelial growth factor (VEGF) inhibitors.
versus aerobic conditions to yield the same amount of cell killing, averages Cetuximab is a monoclonal antibody raised against the EGFR that has
an order of magnitude higher than for the other classes of bioreductive been shown to improve outcomes in advanced head and neck cancers
drugs.189 Laboratory and clinical data also support a tumor-sensitizing when combined with radiation therapy211 and bevacizumab is a human-
role for tirapazamine in combination with cisplatinum.195 ized monoclonal antibody raised against VEGF that prolongs overall
To date, clinical trials with tirapazamine combined with radiotherapy and progression-free survival in patients with advanced colorectal cancer
and/or chemotherapy have yielded mixed results,195,197,198 although it when combined with standard chemotherapy.212 It is hoped that these
has improved outcomes for some standard treatments. While it is disap- and other targeted agents will play greater roles in cancer therapy in
pointing that tirapazamine has not made a more significant impact on the future.
clinical practice, the search for more effective agents from the same or
similar chemical class continues.199 Normal Tissue Radioprotectors
Proliferating cell radiosensitizers. Another source of apparent Amifostine (WR 2721, see Table 1.2), is a phosphorothioate compound
radioresistance is the presence of rapidly proliferating cells. Such cells developed by the US Army for use as a radiation protector. Modeled
may not be inherently radioresistant but rather have the effect of making after naturally occurring radioprotective sulfhydryl compounds such
the tumor seem refractory to treatment because the production of new as cysteine, cysteamine, and glutathione,213 amifostine’s mechanism of
cells outpaces the cytotoxic action of the therapy. action involves the scavenging of free radicals produced by ionizing
Analogs of the DNA precursor thymidine, such as bromodeoxyuridine radiation, radicals that otherwise could react with oxygen and “fix” the
(BrdUdR) or iododeoxyuridine (IdUdR), can be incorporated into the chemical damage. Amifostine can also detoxify other reactive species
DNA of actively proliferating cells in place of thymidine because of through the formation of thioether conjugates; in part because of this,
close structural similarities between the compounds. Cells containing the drug can also be used as a chemoprotective agent.214 Amifostine is
DNA substituted with these halogenated pyrimidines are more radiosensi- a prodrug that is dephosphorylated by plasma membrane alkaline
tive than normal cells, with the amount of sensitization directly pro- phosphatase to the free thiol WR 1065, the active metabolite. As is the
portional to the fraction of thymidine replaced.200 In general, the case with the hypoxic cell radiosensitizers, amifostine need only be
radiosensitization takes the form of a decrease in or elimination of the present at the time of irradiation to exert its radioprotective effect.
shoulder region of the radiation survival curve. To be maximally effective, In theory, if normal tissues could be made to tolerate higher total
the drug must be present for at least several rounds of DNA replication doses of radiation through the use of radioprotectors, then the relative
prior to irradiation. Although the mechanism by which BrdUdR and radioresistance of hypoxic tumor cells would be less likely to limit
IdUdR exert their radiosensitizing effect remains somewhat unclear, it radiation therapy. However, encouraging preclinical studies demonstrating
is likely that both the formation of more complex radiation-induced radioprotection of a variety of cells and tissues notwithstanding,215,216
lesions in the vicinity of the halogenated pyrimidine molecules and radioprotectors such as amifostine would not be expected to increase
interference with DNA damage sensing or repair are involved.201 the therapeutic ratio unless they could be introduced selectively into
The clinical use of halogenated pyrimidines began in the late 1960s normal tissues but not tumors. The pioneering studies of Yuhas
with a major clinical trial in head and neck cancer.202 In retrospect, the et al.178,217,218 addressed this issue by showing that the drug’s active
metabolite reached a higher concentration in most normal tissues than limited number of cell divisions. Myelocytes of the bone marrow
in tumors and that this mirrored the extent of radio- or chemoprotection. and spermatocytes of the testis are examples of Type II cells.
The selective protection of normal tissues results from slower tumor Type III or reverting postmitotic cells (RPMs) are relatively radioresistant,
uptake of the drug and tumor cells being both less able to convert consisting of those few types of cells that are fully differentiated
amifostine to WR 1065 (owing to lower concentrations of the required and do not divide regularly yet under certain conditions can revert
phosphatases) and less able to transport this active metabolite throughout to a stem cell–like state and divide as needed. Examples of Type
the cell. III cells include hepatocytes and lymphocytes, although the latter
Dose-reduction factors (DRFs; the ratio of radiation doses to produce are unique in that they are a notable exception to the Rubin and
an isoeffect in the presence vs. absence of the radioprotector) in the Casarett classification system—an RPM cell type that is exquisitely
range of 1.5 to 3.5 are achieved for normal tissues, whereas the cor- radiosensitive.
responding DRFs for tumors seldom exceed 1.2. Those normal tissues Type IV or fixed postmitotic cells (FPMs) are the most radioresistant,
exhibiting the highest DRFs include bone marrow, gastrointestinal tract, consisting of the terminally differentiated, irreversibly postmitotic
liver, testes, and salivary glands.178 The brain and spinal cord are not cells characteristic of most normal tissue parenchyma, such as neurons
protected by amifostine, and oral mucosa only marginally so.178 Com- and muscle cells. Should such cells be killed by radiation, they typically
parable protection factors are obtained for some chemotherapy agents, cannot be replaced.
including cyclophosphamide and cisplatin.219,220 The dose-limiting A second, simpler classification system, based on anatomical and
toxicities associated with the use of amifostine include hypotension, histological considerations, has been proposed by Michalowski.229 Using
emesis, and generalized weakness or fatigue.221 this system, tissues are categorized on the basis of whether the tissue
Amifostine is currently indicated for the reduction of renal toxicity stem cells, if any, and the functional cells are compartmentalized (so-called
associated with repeated cycles of cisplatin chemotherapy in patients Type H or hierarchical tissues, such as skin, gut epithelium, testis, etc.)
with advanced ovarian and non–small cell lung cancer. It is also approved or intermixed (Type F or flexible tissues, such as lung, liver, kidney, and
for use in patients receiving radiotherapy for head and neck cancer in spinal cord).
the hopes of reducing xerostomia secondary to exposure of the parotid
glands. Growth Kinetic Parameters and Methodologies
Finally, just as there are apparent radiosensitizers, there are also In order to predict the response of an intact tissue to radiation therapy
apparent radioprotectors that have the net effect of allowing normal in a more quantitative way, a number of kinetic parameters have been
tissues to better tolerate higher doses of radiation and chemotherapy but described that provide a better picture of the proliferative organization
through mechanisms of action not directly related to the scavenging of of tumors and normal tissues (Table 1.3).
free radicals. Various biological response modifiers, including cytokines, Growth fraction. Among the first kinetic characteristics described
prostaglandins (such as misoprostol222,223), anticoagulants (such as was the growth fraction (GF). The presence of a fraction of slowly
pentoxifylline224,225), and protease inhibitors are apparent radioprotectors cycling or noncycling cells in experimental animal tumors was first
because they can interfere with the chain of events that normally follows noted by Mendelsohn et al.230,231 and, subsequently, in human tumors
the killing of cells in tissues by, for example, stimulating compensatory by other investigators. While normal tissues do not grow and therefore
repopulation or preventing the development of fibrosis. Finally, there do not have a GF per se, many are composed of noncycling cells that
is also growing interest in the use of biologics that inhibit apoptosis have differentiated in order to carry out tissue-specific functions. Some
as normal tissue radioprotectors.226,227 Such agents should have little or normal tissues do contain a small fraction of actively proliferating stem
no effect on tumor cells, most of which are already apoptosis resistant. cells. Others contain apparently dormant or “resting” cells that are
temporarily out of the traditional four-phase cell cycle but are capable
CLINICAL RADIOBIOLOGY of renewed proliferation in response to appropriate stimuli. Lajtha gave
these resting but recruitable cells of normal tissues the designation
Growth Kinetics of Normal Tissues and Tumors G0.232 While a tumor counterpart of the G0 cell may or may not exist,
In the simplest sense, normal tissues are normal because the net produc- the majority of slowly cycling or noncycling tumor cells are thought
tion of new cells, if it occurs at all, exactly balances the loss of old cells to be in such a state because of nutrient deprivation, not because of a
from the tissue. In tumors, the production of new cells exceeds cell loss, normal cell cycle regulatory mechanism. Thus, Dethlefsen233 has suggested
even if only by a modest amount. Although the underlying radiobiology that the term Q cell be reserved for quiescent cells in tumors to distinguish
of cells in vitro applies equally to the radiobiology of tissues, the imposi- them from the G0 cell of normal tissues.
tion by growth kinetics of this higher level of organizational behavior Measurement of a tumor’s GF is problematic,234,235 but an estimate
makes the latter far more complex systems. can be obtained with a technique known as continuous thymidine labeling.
Using this method, the tumor receives a continuous infusion of radio-
Descriptive Classification Systems labeled thymidine for a period of time long enough for all proliferating
Two qualitative classification systems based loosely on the prolifera- cells to have gone through at least one round of DNA synthesis and
tion kinetics of normal tissues are in use. Borrowing heavily from the incorporated the radioactive label. Then, a biopsy of the tumor is obtained
pioneering work of Bergonié and Tribondeau,13 Rubin and Casarett’s228 and tissue sections prepared for autoradiography. Once the slides are
classification system for tissue “radiosensitivity” consists of four main processed and scored, the continuous labeling index, that fraction of
categories: the total population of tumor cells containing tritiated thymidine, is
Type I or vegetative intermitotic cells (VIMs) are considered the most calculated. This value is a rough estimate of the tumor’s GF.
radiosensitive, consisting of regularly dividing, undifferentiated stem Cell cycle and volume doubling times. The percent labeled mitosis
cells such as are found in the bone marrow, intestinal crypts, and (PLM) technique of Quastler and Sherman236 was a key development
the basal layer of the epidermis of the skin. in the study of the cell cycle in vivo because it provided a unique window
Type II or differentiating intermitotic cells (DIMs) are somewhat less into the behavior of that fraction of cells within a tissue that was actively
radiosensitive, consisting of progenitor cells that are in the process proliferating. By focusing on cells in mitosis, the assay allowed both
of developing differentiated characteristics yet are still capable of a the overall cell cycle time (Tc) and the durations of the individual cell
TABLE 1.3 Estimated Cell Cycle Kinetic Parameters for Human Tumors
Representative Values for
Parameter Definition How Measured Human Solid Tumors Notes
Tc (Average) Cell Percent labeled mitosis technique; 0.5–6.5 days (median ≈ 2.5) Tc in vivo usually longer than for
cycle time flow cytometry comparable cells cultured in vitro.
GF Growth fraction Estimated from continuous 0.05–0.90 days (median ≈ 0.40?) Difficult to measure directly; not much
labeling technique data available.
Tpot Potential doubling Flow cytometry (relative movement 2–19 days (median ≈ 5) Tpot ≈ Tc as GF approaches 1.0.
time method: Tpot = λTs/LI)
ϕ Cell loss factor 1 − Tpot/Td 0.30–0.95 days (median ≈ 0.90?) Thought to be the major cause of long
Tds for human tumors; particularly
high in carcinomas.
Td Volume doubling Direct measurement of tumor 5–650 days (median ≈ 90) Increases with increasing tumor size,
time dimensions over time often because of increases in Tc and
f, and a decrease in GF.
Teff/Tp Effective clonogen Estimated from clinical data on the 4–8 days Tp approaches Tpot toward the end of a
doubling time loss of local control with course of fractionated radiotherapy.
increasing overall treatment time
Data from Steel GG. Growth Kinetics of Tumours. Oxford: Clarendon Press; 1977, and Joiner M, van der Kogel A. Basic Clinical Radiobiology.
4th ed. London: Hodder Arnold; 2009.
cycle phases to be determined without the uncertainties introduced by cell production. Pathologists and tumor biologists, meanwhile, had
the presence of noncycling cells in the population. Today, flow cytometric ample evidence that tumors routinely lost large numbers of cells, the
methods have largely replaced the arduous and time-consuming PLM result of cell death, maturation, and/or emigration.234,238,239 It is now
assay. clear that the overall rate of tumor growth, as reflected by its Td, is
Briefly, the PLM technique involves tracking over time a cohort of governed by the competing processes of cell production and cell loss.
proliferating cells that initially was in S phase (and exposed briefly to In fact, the cell loss factor, ϕ, the rate of cell loss expressed as a fraction
tritiated thymidine) and then proceeded through subsequent mitoses. of the cell production rate, is surprisingly high for both experimental
Serial biopsy samples from the tissue of interest are obtained at regular and human tumors, as high as 0.9 or more for carcinomas and lower,
intervals following labeling, and the fraction of cells both in mitosis on average, for sarcomas.234 Cell loss is usually the most important
(identified cytologically) and carrying the radioactive label is determined. factor governing the overall volume Td of solid tumors.
A first peak of labeled mitoses is observed within 24 hours after labeling, The clinical implications of tumors having high rates of cell loss
and as cells pass through their second division, a second wave of labeled are obvious. First, any attempts at making long-term predictions of
mitoses is noted. The average Tc for the population of proliferating treatment outcome based on short-term regression rates of tumors
cells corresponds to the peak-to-peak interval of the resulting PLM during treatment are misleading. Second, although regression rate may
curve, a plot of the fraction of labeled mitoses as a function of time not correlate well with eventual outcome, it may be a reasonable indicator
following the radioactive pulse. With sufficiently robust data, the dura- of when best to schedule subsequent therapy on the assumption that
tions of the individual cell cycle phases can be obtained as well. The a smaller tumor will be more radio- and chemosensitive, as well as
PLM technique is illustrated schematically in Fig. 1.25. easier to remove surgically.
Historically, the interpretation of PLM curves was sometimes Potential doubling time and “effective” doubling time. With the
hampered by technical artifacts and by the fact that proliferating cell recognition that cell loss plays a major role in the overall growth rate
populations have distributed cell cycle times.234,235,237 Despite these of tumors and can mask a high cell production rate, a better measure
limitations, it is clear that most cells in vivo proliferate more slowly of the potential repopulation rate of normal tissues and tumors was
than their in vitro counterparts. Although the variation in intermitotic needed.240 One indicator of regenerative capacity is the potential doubling
times is quite large, a median value for Tc of 2 to 3 days is a reasonable time (Tpot).234,241 By definition, Tpot is an estimate of the time that would
estimate.234 be required to double the number of clonogenic cells in a tumor in the
While the cycle times of proliferating cells in vivo are long by cell absence of cell loss. It follows that Td will usually be much longer than
culture standards, they are quite short when compared with the cor- Tpot because of cell loss, and Tc will be shorter than Tpot because of the
responding volume doubling times (Td) for human tumors. Although presence of nonproliferating cells.237
highly variable from tumor type to tumor type and somewhat difficult Tpot can be estimated from a comparison of the S phase pulse labeling
to measure, the Td for human solid tumors averages about 3 to 4 index (LI) and the duration of S phase (TS) by using the following
months.234 In many cases, sample calculations further suggest that the equation:
discrepancy between Tc for proliferating tumor cells and Td for the
tumor as a whole cannot be accounted for solely by the tumor having Tpot = λ TS LI
a low GF.
Cell loss factor. Cell kineticists initially adhered to the notion that where λ is a correction factor related to the nonuniform distribution
the continued growth of tumors over time reflected abnormalities in of cell “ages” in a growing population (usually, λ ≈ 0.8). TS and LI can
G2 M G1
a c e
s
b d
Percent labeled mitoses

80
60
TM TC
Percent labeled mitoses
40
100
TG2 ! TG2
20
TS 0
50 b c e 10 20 30 40
Time after labeling (h)
TG2
0
a d
“Time” after labeling
Fig. 1.25 The technique of labeled mitoses (PLM) for an idealized cell population with identical cell cycle
times (left panels) and for a representative normal tissue or tumor with a dispersion in cell cycle times (right
panel). Top left: Following a brief exposure to tritiated thymidine or equivalent at time = “a,” the labeled
cohort of S phase cells continues (dark shading) around the cell cycle and is sampled at times = “b,” “c,”
“d,” and “e,” respectively. Bottom left panel: For each sample, the percentage of cells both in mitosis and
containing the thymidine label is determined and plotted as a function of time; from such a graph, individual
cell cycle phase durations can be derived. Right panel: In this more realistic example, a mathematical fit to
the PLM data would be needed to calculate the (average) cell cycle phase durations.
be determined by the relative movement method.241,242 This technique advocated.245–247 Estimates of Tp can be obtained from two types of
involves an injection of a thymidine analog, usually BrdUrd, which is experiments. In an experimental setting, Tp can be inferred from the
promptly incorporated into newly synthesized DNA and whose presence additional dose necessary to keep a certain level of tissue reaction constant
can be detected using flow cytometry. The labeled cohort of cells is as the overall treatment time is increased. (When expressed in terms
then allowed to continue movement through the cell cycle and a biopsy of dose rather than time, the proper term would be Deff, although the
of the tumor is taken several hours later, at which point the majority underlying concept is the same.) For example, acute skin reactions
of the BrdUrd-containing cells have progressed into G2 phase or beyond. usually both develop and begin to resolve during the course of radiation
A value for LI is determined from the fraction of the total cell population therapy, suggesting that the production of new cells in response to
that contains BrdUrd, and TS is calculated from the rate of movement injury gradually surpasses the killing of existing cells by each subsequent
of the labeled cohort during the interim between injection of the tracer dose fraction. By intensifying treatment once this repopulation begins,
and biopsy. it theoretically should be possible to reach a steady state wherein the
Values for Tpot for human tumors have been measured and, although tissue reaction remains constant. In a clinical setting, Tp can be estimated
quite variable, typically range between 2 and 20 days.234,240,243 These from a comparison of tumor control rates for treatment schedules in
findings lend support to the important notion that slowly growing which the dose per fraction and total dose used were held approximately
tumors can contain subpopulations of rapidly proliferating cells. To constant but the overall treatment time varied. In some cases, the loss
the extent that these cells retain unlimited reproductive potential, they of local control with increasing overall treatment time provides an
may be considered the tumor’s stem cells (in a generic sense, at least) estimate not only of Tp but also of the delay time before the repopulation
capable of causing recurrences after treatment. These cells represent a begins, sometimes referred to as Tk, the repopulation “kickoff” time.248–250
serious threat to local control of the tumor by conventional therapies, Repopulation in tumors and normal tissues. As discussed earlier,
especially protracted treatments (that provide them additional time to both normal tissues and tumors are capable of increasing their cell
proliferate). production rate in response to radiation-induced cell killing, a process
The use of a cell kinetic parameter such as Tpot as either a predictor known as regeneration or repopulation. The time of onset of the
of a tumor’s response to therapy or as a means of identifying subsets regenerative response varies with the turnover rate of the tissue or
of patients particularly at risk for recurrence has been attempted, with tumor since cell death (and depopulation) following irradiation is usually
some positive, but mostly negative, results.6,141,244 Lest these negative linked to cell division. Generally, tissues that naturally turn over fairly
findings suggest that proliferation in tumors is unimportant, bear in rapidly repopulate earlier and more vigorously than tissues that turn
mind that it is unlikely that a pretreatment estimate of Tpot—or any over slowly. However, it has been shown that the repopulation patterns
other single cell kinetic parameter (e.g., LI) for that matter—would be of normal tissues and tumors following the start of irradiation tend to
relevant once treatment commences and the growth kinetics of the be characterized by a delay (of at least several weeks in many cases; see
tumor are perturbed. Tk discussed earlier) prior to the rapid proliferative response.248–250 Once
One approach to dealing with this problem is to measure proliferative this proliferative response begins, however, it can be quite vigorous.
activity during treatment. Although not without other limitations, the While this is clearly desirable for early-responding normal tissues
use of an “effective clonogen doubling time” (Teff or Tp) has been attempting to recover from radiation injury, rapid proliferation in tumors
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NOTHER December has begun to lower in the dim skies with wintry
wildness, to bind the earth with iron fetters, and to cover its
surface with its snowy mantle, as we enter for the first time
another town, far from that English borough in which we lingered
a year ago. An ancient city is this, within whose time-honoured
walls the flower and pride of whatever was greatest and noblest in
Scotland, has ever been found through long descending ages. Elevated rank,
mighty mental ability, eminent piety, the soundest of all theology, the most
thorough of all philosophy, and the truest patriotism, have ever been
concentrated within its gates. Here men are common, who elsewhere would
be great, and the few who do stand out from amid that mass of intellect
stand out as towers, and above that vast aggregate of genius and goodness
are seen from every mountain in Christendom, from every Pisgah of
intellectual vision, whereon thoughtful men do take their stations, as suns
amid the stars. And alas that we should have to say it, where vice also erects
its head and stalks abroad with an unblushing front, and a fierce hardihood,
lamentable to behold. We cannot, to-night, tread its far-famed halls of
learning, we may not thread our way through the busy, seething multitudes
of its old traditionary streets; but there is one chamber, from whose high
windows a solitary light streams out into the murky air, into which we must
pass.
It is a plain room, not large, and rich in nothing but books; books which
tell the prevalent pursuits, tastes, and studies of its owner, filling the shelves
of the little bookcase, covering the table, and piled in heaps on floor and
chairs: massive old folios, ponderous quartos, and thick, dumpy little
volumes, of the seventeenth century, in faded vellum, seem most to prevail,
but there are others with the fresh glitter of modern times without, and
perhaps with the false polish of modern philosophies within. With each of
its two occupants we have yet to make acquaintance; one is a tall,
handsome man, already beyond the freshness of his youth, well-dressed and
gentleman-like, but having a disagreeable expression on his finely formed
features, and a glittering look in his eye—a look at once exulting and
malicious, such as you could fancy of a demon assured of his prey. The
other, with whom he is engaged in earnest conversation, is at least ten years
his junior; young, sensitive, enthusiastic, he appears to be, with an ample
forehead and a brilliant eye, as different as possible in its expression from
the shining orb of the other. There is no malice to be seen here, no sneer on
those lips, no deceit in that face, open, manly, eloquent and sincere. Famed
in his bygone career, he is covered with academic honours, is full of vigour,
of promise, of hopefulness, with eloquence on his lips, and logic in his
brain, and his mind cultured thoroughly, the favoured of his teachers, the
beloved of his companions, the brother of our gentle Christian, our
acquaintance of last year in his letter to Christian—Halbert Melville.
But what is this we see to-night! How changed does he seem, then so
beautiful, so gallant; there is a fire in his eyes, a wild fire that used not to be
there, and the veins are swollen on his forehead, and stand out like
whipcord. His face is like the sea, beneath the sudden squall that heralds the
coming hurricane, now wild and tossed in its stormy agitation, now lulled
into a desperate and deceitful calmness. His lips are severed one moment
with a laugh of reckless mirth, and the next, are firmly compressed as if in
mortal agony, and he casts a look around as if inquiring who dared to laugh.
His arm rests on the table, and his finger is inserted between the pages of a
book—one of the glittering ones we can see, resplendent in green and gold
—to which he often refers, as the conversation becomes more and more
animated; again and again he searches its pages, and after each reference he
reiterates that terrible laugh, so wild, so desperate, so mad, while his
companion’s glittering serpent eye, and sneering lip, send it back again in
triumph. What, and why is this?
Look at the book, which Halbert’s trembling hand holds open. Look at
this little pile laid by themselves in one corner of the room, the gift every
one of them of the friend who sits sneering beside him, the Apostle of so-
called spiritualism, but in reality, rank materialism, and infidelity, and you
will see good cause for the internal struggle, which chases the boiling blood
through his youthful veins, and moistens his lofty brow with drops of
anguish. The tempter has wrought long and warily; Halbert’s mind has been
besieged in regular form; mines have been sprung, batteries silenced,
bastions destroyed—at least, to Halbert’s apprehension, rendered no longer
tenable; point by point has he surrendered, stone by stone the walls of the
citadel have been undermined, and the overthrowal is complete. Halbert
Melville is an unbeliever, an infidel, for the time. Alas! that fair and
beauteous structure, one short twelvemonth since so grand, so imposing, so
seeming strong and impregnable, lies now a heap of ruins. No worse sight
did ever captured fortress offer, after shot and shell, mine and counter-mine,
storm and rapine had done their worst, than this, that that noble enthusiastic
mind should become so shattered and confused and ruinous.
There is a pause in the conversation. Halbert has shut his book, and is
bending over it in silence. Oh, that some ray of light may penetrate his soul,
transfix these subtle sophisms, and win him back to truth and right again;
for what has he instead of truth and right? only dead negations and
privations; a series of Noes—no God, no Saviour, no Devil even, though
they are his children; no immortality, no hereafter—a perfect wilderness of
Noes. But his tempter sees the danger.
“Come, Melville,” he says rising, “you have been studying too long to-
day; come man, you are not a boy to become melancholy, because you have
found out at last, what I could have told you long ago, that these
nonsensical dreams and figments, that puzzled you a month or two since,
are but bubbles and absurdities after all—marvellously coherent we must
confess in some things, and very poetical and pretty in others—but so very
irrational that they most surely are far beneath the consideration of men in
these days of progress and enlightenment. Come, you must go with me to-
night, I have some friends to sup with me, to whom I would like to
introduce you. See, here is your hat; put away Gregg, and Newman, just
now, the Nemesis can stand till another time—by-the-by, what a struggle
that fellow must have had, before he got to light. Come away.”
Poor Halbert yielded unresistingly, rose mechanically, put away the
books so often opened, and as if in a dream, his mind wandering and
unsettled so that he hardly knew what he was about, he listened to his
companion’s persuasions, placed his arm within his “friend” Forsyth’s, and
suffered himself to be led away, the prey in the hands of the fowler, the
tempted by the tempter. Poor fallen, forsaken Halbert Melville!
The quiet moments of the winter evening steal along, the charmed hour
of midnight has passed over the hoary city, slumbering among its
mountains. Through the thick frosty air of that terrible night no moonbeam
has poured its stream of blessed light; no solitary star stood out on the
clouded firmament to tell of hope which faileth never, and life that endures
for evermore, far and long beyond this narrow circuit of joys and sorrows.
Dark, as was one soul beneath its gloomy covering, lowered the wide wild
sky above, and blinding frost mist, and squalls laden with sleet, which fell
on the face like pointed needles, had driven every passenger who had a
home to go to, or could find a shelter, or a refuge, from the desolate and
quiet streets. In entries, and the mouths of closes, and at the foot of common
stairs, little heaps of miserable unfortunates were to be seen huddled
together, seeking warmth from numbers, and ease of mind from
companionship, even in their vice and wretchedness. Hour after hour has
gone steadily, slowly on, and still that chamber is empty, still it lacks its
nightly tenant, and the faint gleam of the fire smouldering, shining fitfully,
now on the little pile of poison, now on the goodly heaps of what men call
dry books and rubbish, but which a year ago Halbert considered as the very
triumphs of sanctified genius. Hither and thither goes the dull gleam, but
still he comes not.
But hark, there is a step upon the stair, a hurried, feverish, uncertain step,
and Halbert Melville rushes into his deserted room, wan, haggard, weary,
with despair stamped upon his usually firm, but now quivering lip, and
anguish, anguish of the most terrible kind, in his burning eye. He has been
doubting, fearing, questioning, falling away from his pure faith—falling
away from his devout worship, losing himself and his uprightness of
thought, because questioning the soundness of his ancient principles and
laying them aside one by one, like effete and worthless things. He has been
led forward to doubt by the most specious sophistry—not the rigid
unflinching inquiry of a truth-seeker, whose whole mind is directed to use
every aid that learning, philosophy, history, and experience can furnish, to
find, or to establish what is true and of good repute, but the captious search
for seeming flaws and incongruities, the desire to find some link so weak
that the whole chain might be broken and cast off. In such spirit has Halbert
Melville been led to question, to doubt, to mock, at length, and to laugh, at
what before was the very source of his strength and vigour, and the cause of
his academical success. And he has fallen—but to-night—to-night he has
gone with open eyes into the haunts of undisguised wickedness—to-night
he has seen and borne fellowship with men unprincipled, not alone sinning
against God, whose existence they have taught Halbert to deny, whose laws
they have encouraged him, by their practice and example, to despise,
contemn, and set aside, but also against their neighbours in the world and in
society. To-night, while his young heart was beating with generous
impulses,—while he still loathed the very idea of impurity and iniquity, he
has seen the friends of his “friend,” he has seen his favoured companion
and immaculate guide himself, whose professions of purity and uprightness
have often charmed him, who scorned God’s laws, because there was that
innate dignity in man that needed not an extraneous monitor, whose lofty,
pure nature has been to Halbert that long twelvemonth something to
reverence and admire; him has he seen entering with manifest delight into
all the vile foulness of unrestrained and unconcealed sin, into all the
unhallowed orgies of that midnight meeting and debauch. Unhappy Halbert!
The veil has been torn from his eyes, he sees the deep, black fathomless
abyss into which he has been plunged, the hateful character of those who
have dragged him over its perilous brink, who have tempted him to wallow
in the mire of its pollutions and to content himself with its flowing wine, its
hollow heartless laughter, its dire and loathsome pleasures.
The threatenings of the Scriptures, so long forgotten and neglected, ring
now in his terrified ears, like peals of thunder, so loud and stern their dread
denunciations. His conscience adopts so fearfully that awful expression,
“The fool hath said in his heart there is no God,” that the secret tones of
mercy, whispering ever of grace and pardon, are all unheard and unheeded,
and he was in great fear, for the Lord is in the generation of the righteous.
He leans his burning brow upon the table, but starts back as if stung by an
adder, for he has touched one of those fatal books, whose deadly contents,
so cunningly used by his crafty tempter, overthrew and made shipwreck of
his lingering faith, and has become now a very Nemesis to him. With a
shudder of abhorrence and almost fear, he seizes the volume and casts it
from him as an unclean thing, and then starts up and paces the room with
wild and unsteady steps for a time, then throws himself down again and
groans in agony. See! he is trying with his white and quivering lips to
articulate the name of that great Being whom he has denied and
dishonoured, but the accents die on his faltering tongue. He cannot pray, he
fancies that he is guilty of that sin unpardonable of which he has often read
and thought with horror. Is he then lost? Is there no hope for this struggling
and already sore-tired spirit? Is there no succour in Heaven? The gloom of
night gathering thicker and closer round about him, the dying sparkle of the
fire, the last faint fitful gleam of the expiring candle leaping from its socket,
and as it seems to him soaring away to heaven, cannot answer. Surely there
will yet be a morrow.
CHAPTER II.
Alone walkyng. In thought plainyng,
And sore sighying. All desolate
My remembrying Of my livying.
My death wishying Bothe erly and late.
Infortunate, Is so my fate
That wote ye what, Out of mesure
My life I hate; Thus desperate
In such pore estate, Doe I endure.—Chaucer.
few weeks have passed away since that terrible night, and we are
again in Christian Melville’s quiet home. It is on the eve of the
new year, but how different is the appearance of those assembled
within this still cheerful room from the mirth and happiness
which made their faces shine one short twelvemonth since. Our Christian is
here, sitting with her head bent down, and her hands clasped together with
convulsive firmness. Here is little Mary drooping by her side like a stricken
flower, while the only other person in the apartment sits sulkily beside them
with a discontented, ill-humoured look upon her pretty features, which
contrasts strangely, and not at all agreeably, with the pale and anxious faces
of her companions—her sisters—for this unhappy looking, discontented
woman is James Melville’s wife. Strange and terrifying news of Halbert
have reached them, “that he has fallen into errors most fatal and hazardous
to his future prospects, and all unlike as of his proposed vocation so of his
former character, that he had become acquainted and been seen publicly
with most unfit and dangerous companions,” writes a kind and prudent
Professor, who has from the first seen and appreciated the opening promise
of Halbert’s mind. Two or three days ago James, his brother, has set off to
see if these things be true or no, and to bring, if possible and if needful, the
wandering erring spirit—we cannot call him prodigal yet—home. The spirit
of Christian, the guardian sister, had sunk within her at these terrible
tidings; was she not to blame—had she done her part as she ought to have
done—had she not been careless—is she guiltless of this sad catastrophe?
She remembered Halbert’s letter of the past new year—she remembered
how studiously he had kept from home all this weary year—she
remembered how, save for one hurried visit, he had stayed at a distance
from them all, pleading engagements with his friend, that friend that had
now proved so deadly a foe. A thousand things, unheeded at the time,
sprung up in Christian’s memory in lines of fire. The friend of Halbert, free-
thinking at the first, what was he? the unwonted restraint of the young
brother’s correspondence, the studied omission of all reference to sacred
things, or to his own prospective avocations in his letters, which in former
times used to be the chief subjects of his glowing and hopeful anticipations,
the bitterness of tone which had crept into his once playful irony, all these
which had only caused a momentary uneasiness, because of her dependence
on Halbert’s steadfast settled principles, flashed back with almost
intolerable distinctness now. Alas! for Christian’s recollections—“I am to
blame; yes, I ought to have warned him, even gone to him,” she thinks;
“was he not left me as a precious treasure, to be guarded, to be warned, to
be shielded from ill? Oh! that he was home once more.” Alas! for
Christian’s recollections, we say again; the iron fingers of Time measure out
the moments of that last lingering hour; again light hearts wait breathless
for its pealing signal, as they did of old, but these silent watchers here have
no ear for any sounds within their own sorrowful dwelling, though there is
not a passing footstep on the street without that does not ring upon their
anxious ears in echoing agony; there is not a sound of distant wheels
bearing, mayhap, some reveller to and fro, which does not bring an alternate
throb and chill to their painful beating hearts. This stillness is past all
bearing, it is painfully unendurable, and Christian springs to the door and
gazes out upon the cold and cheerless street, and as she does so a
thoughtless passenger wishes her a “happy new year.” Alas! to speak of
happiness, a happy new year to her in such a moment as this!
Mrs. James Melville is astonished at all this grief; she cannot understand
nor fathom it. Suppose Halbert has been foolish, and behaved ill, what
then? Why should her husband have gone off so suddenly, and her sister-in-
law be in such a state? She was sure she could not comprehend it, and
would have been very foolish to have done such foolish things for all the
brothers in the universe. Young men will be young men, and they should be
left to come to themselves, instead of all this to-do being made about them;
it was preposterous and absurd, and put her in a very ridiculous position;
and so Mrs. James pouted and sulked and played with her chains and her
rings, stopping now and then in her agreeable relaxation to cast a glance of
contemptuous scorn at restless, excited, anxious Christian, and drooping,
fragile Mary. A nice way this to bring in the new year, the first anniversary
of her married life, the first return of the day of her wedding; a nice state
James would be in for her party of to-morrow evening; and Mrs. James, by
way of venting her ill-humour, shoved away with her slippered foot, a little
dog which was sleeping before the cheerful fire. How Christian starts as it
cries and creeps to her feet: it is Halbert’s dog, and as her eye falls on it, its
youthful owner seems to stand before her, so young, so frank, so innocent!
now gay as a child, making the walls echo with his overflowing mirth; now
grave and serious, like the dead mother whose latest breath had committed
him as a precious jewel to her, and bidden her watch over him and guard
him with her life. Oh, had she neglected her charge! Was this fault, this
apparent wreck hers?
The passing footsteps grew less and less frequent; what can detain them?
Old Mr. Melville and his son Robert have gone to meet James and—Halbert
—if Halbert be only with him, and Christian trembles as she repeats that
pregnant if. Her heart will break if they come not soon: she cannot bear this
burden of anxiety much longer. Hush! there are footsteps, and they pause at
the door. Sick at heart, Christian rushes to it again with little Mary by her
side; there at the threshold are her father, James, Robert; she counts them
painfully, one by one; but where is Halbert? where is her boy? The long-
cherished expectation is at once put to flight; the artificial strength of
excitement has gone, and Christian would have fallen to the ground, but for
James’s supporting arm.
“Christian,” he whispered, as he led her back to her seat in the parlour
again, “I know you can command yourself, and you must try to do so now,
for you will need all your strength to-night.”
James’s voice was hoarse, and his eyes bloodshot. Where is—what has
become of Halbert? The story is soon told. When James reached Edinburgh,
he had gone straight to Halbert’s lodging, and found when he arrived at it,
that his brother had disappeared, gone away, whither the people knew not;
his fellow-students and professors were equally ignorant; and all that he
could clearly ascertain was, that the reports they had been grieved so much
with were too true; that one night some weeks before the day that James
went to the lodgings, Halbert had gone out, been seen in several places of
the worst character, with men known as profligates, and abandoned, and
had come home very late. That since then he had been like a man in despair
—mad—his simple landlady said; and she pointed to the books he had left,
crowding the shelves and littering the floor of her little room; that two
nights before James had arrived—having been shut up all the day—he had
gone suddenly out, telling her to send a parcel lying on his table as directed,
the next morning. On his mantel-piece was a letter, apparently forgotten, for
Christian. “Here it is,” said James in conclusion, handing it to her, “would
that it could comfort you!”
Christian broke the seal with eager, trembling fingers; perhaps, after all,
there might be some comfort here:
“Christian,
“Do not hate me! do not forsake me!” (thus did it begin; and it seemed
as if the paper was blistered with tears, so that the words were almost
illegible; and thus went on the trembling words of poor Halbert’s almost
incoherent letter). “I am still your brother; but they will tell you how I have
fallen; they will tell you of my guilt—but none—none can tell, can
comprehend my misery. I dare not come near you. I dare not return home to
pollute the air you breathe with my presence. I feel myself a Cain or a
Judas, branded and marked, that all men may shrink from me as from a
pestilence; and I must rush out from their sight afar, and from their contact.
It is enough that I feel the eye of God upon me—of that God whom I have
denied and contemned, whose throne I strove to overturn with my single
arm, feeble and frail as it is—continually upon me, on my secret heart
burning in on the quivering spirit, my sentence of hopeless, helpless
condemnation! They will tell you that I am mad. Oh, that I were, and had
been so for these last months, that now I might lose the sense of my sin and
of the hopeless despair which haunts me night and day! Christian, I am no
infidel, or as the tempters called it, spiritualist now. I shrink and tremble
just the same while alone, and when among the crowd, from that terrible
Spirit that pursues and searches me out everywhere—terrible in holiness;
inexorable in justice, and I cannot pray, ‘Be merciful, O thou holy and
eternal One.’
“Christian, do you remember that fearful word of Scripture, ‘It is
impossible to renew them again unto repentance, seeing they crucify to
themselves the Son of God afresh, and put him to an open shame?’ I have
entered into the unspeakable bitterness of its doom; it rings in my ears
without intermission; ‘it is impossible to be renewed again.’ But you can
pray, Christian; you have not cast all hope behind you; and if it is not sin to
pray for one accursed, pray for me. It may be I shall never see you again; I
know not where I go; I know not what I shall do! There is no peace left for
me on earth; and no peace, no hope, no refuge beyond it, that I can see.
“Your brother,
“Halbert Melville.”
And where is Christian now? She is lying with rigid marble face and
closed eyes, insensible to all the care bestowed upon her, in a dead faint.
They are chafing her cold hands and bathing her temples, and using all the
readiest means at hand for her recovery. Is Christian gone?—can this letter
have killed her?—has she passed away under the pressure of this last great
calamity. No: God has happier days in store for his patient servant yet; and
by-and-by she is raised from her deathlike faint, and sits up once more; but
it seems as if despair had claimed a second prey, so pitiful and mournful is
that face, and its expression so changed, that they are all afraid; and little
Mary clasps her hand in an agony, and lifts up her tear-stained face to her
sister, and whispers—
“Christian! Christian!” in broken, tearful accents.
“We will make every inquiry possible to be made,” said James,
soothingly; “we may yet bring him back, Christian.”
“I don’t know what this frenzy means!” says Mr. Melville. “Depend
upon it Halbert will come back, and he’ll soon see the folly of this outburst
of feeling; and you see, Christian, he says he’s no infidel or atheist now, so
you need not be so put out of the way by his letter.”
Mary says nothing more but “Christian! Christian!” and her arm glides
round her sister, and her graceful head rests on Christian’s bosom. It is
enough: she may not—must not sink down in despair; she has duties to all
those around her; she must not give way, but be up and doing.
And there are words of better comfort spoken in her ear to-night ere
sleep comes near her; the hand that rocked her cradle in infancy, that tended
her so carefully in childhood, draws the curtain gently round her.
“Dinna misdoubt, or lose hope, Miss Christian,” sobs old Ailie, her own
tears falling thick and fast the while she speaks; “the bairn of sae mony
supplications will never be a castaway; he may gang astray for a while, he
may be misled, puir lad, or left to himself and fall, and have a heavy weird
to dree or a’ be done, but he’ll no be a lost ane. No, Miss Christian, no,
dinna think sae, and distress yoursel’ as you’re doing—take my word, ye’ll
baith hear and see guid o’ Mr. Halbert yet.”
Oh, holy and sublime philosophy, what sure consolation flows in your
simple words!
So closes that dawn of the new year on this sorrowing household. Alas,
how strange the contrast! A year ago, all, masters and servants, with fervour
and enthusiasm, and with heartfelt prayers, wished a “Good, a happy New
Year to Halbert” far away; but there is none of that now, Halbert’s first year
has been a year of trial, mental struggle, and failure so far, and though the
same deep love—or even deeper, for these loving hearts cling even more
closely to him now, in his time of distress and despair—animates them still,
they dare not wish each other, far less openly propose for him, the “happy
new year” so usual. Poor household, it may be rich in world’s gear, and
world’s comforts, but the chaplet has lost a rose, and he, so precious to them
all, is lost to their ken, vanished from their sight, as it were, and all the
remembrance of him that remains is that of a “broken man.”
But where is Halbert? Away, in a struggling ship, tossing on the stormy
bosom of the wide Atlantic, alone upon the storm-swept deck, whence
everything, not fastened with wood and iron, has been driven by these
wintry seas; boats, bulwarks, deck load and lumber, are all gone into the
raging deep, and yet he stands on the deck, drenched by every sea, watching
the giant billows, before which all but he are trembling, uncovered, while
the lightning gleams athwart the seething waters, and the thunder peals out
in incessant volleys overhead; unsheltered, while the big raindrops pour
down in torrents from the heavy cloud-laden sky. There is no rest for him;
in vain does he stretch himself in his uneasy cot; in vain forces the hot
eyelid to close upon the tearless eye; since he wrote Christian, all weeping
and tears have been denied him. Sleep, which comes in healing quietness to
all his shipmates, does not visit him; or, if for a moment wrapped in restless
slumbers, dreams of fearful import rise up before him, far surpassing in
their dread imagery the gloomiest and most horrible conceptions of his
waking thoughts or fancy, too horrible to bear; and the wretched dreamer
starts out into the dreary air, thinking himself a veritable Jonah, to whom
this tempest and these stormy seas are sent as plagues, and he stands a fit
spectator of that external elemental warfare, which is but a type and
emblem, fierce though it be, of the raging war within.
See, how he stands, invulnerable in his despair, the strong masts
quivering like wands in the furious tempest, the yards naked, and not a rag
of sail that would stand before it for an instant; the decks swept by the sea
at every moment, and nothing looked for now, by the staunchest seaman on
board, but utter and speedy destruction. “The ship cannot stand this much
longer,” whispers the captain to his chief mate; “she’ll founder in an hour,
or become water-logged, which would be as bad, or worse, at this season
and in this latitude. Stand by for whatever may happen.” And yet, all this
time, there is not an eye in that strained and struggling ship but Halbert’s,
that does not shrink from looking upon the boiling sea; there is not a heart
but his, however hardened or obdurate it be, which does not breathe some
inward prayer, though it be but some half-forgotten infant’s rhyme. But
Halbert Melville stands alone, uncompanioned, and uncomplaining in his
secret grief; no blessed tear of sorrow hangs on the dark lash of his fevered
eye; no syllable of supplication severs his parched lips; the liberal heavens,
which drop grace upon all, are shut, in his agonised belief, to him alone. He
cannot weep; he dare not pray.
CHRISTIAN MELVILLE.
EPOCH III.
There’s joy and mourning wondrously entwined

In all that’s mortal: sometimes the same breeze
That bringeth rest into one weary mind
Heralds another’s sorer agonies;
Sometimes the hour that sees one battle end
Beholds as sad a time of strife begin;
And sometimes, hearts rejoicing as they win
Themselves the victory, tremble for a friend.
Ah me! how vain to think that mortal ken
Can ever, with love-cleared vision, judge aright.
Doth danger dwell alone ’mong stranger men,
Or safety aye ’neath home’s protecting light?
Shield us, our Father! in our every lot
Thou blendest joy and grief that we forget thee not.
CHAPTER I.
Then followed that beautiful season,

Called by the pious Acadian peasants the summer of All Saints!
Filled was the air with a dreamy and magical light; and the landscape
Lay as if new created in all the freshness of childhood.
Peace seemed to reign upon earth, and the restless heart of the ocean
Was for a moment consoled.—Longfellow’s Evangeline.
WO years have worn on their slow course, two tedious, weary years, and
the first days of December have again arrived. We are now under a sunnier
sky than that of England, and on the outskirts of an old, wide-
spreading, perhaps, primeval forest, full of giant pines and
hemlocks, and the monarch oak, beside which the axe of the
back-woodsman has never yet been lifted. It is morning, and the
sun gleams on the brilliant, dewy leaves of trees, in detached and scattered
groups, each clad like the beloved of Jacob in his coat of many colours. The
Indian summer, pleasant and evanescent, is on the wane, and there is a soft
murmur of falling leaves, as the morning breeze steals through the rustling
foliage, and save for that, and the usual sounds of the forest—the diapason
of that natural organ—all is still, and hushed, and silent. We are on the eve
of winter, we witness the russet leaves falling before every breath of wind,
but yet the grass is as green as ever, and wild flowers and creepers,
luxuriant among the tangled under-brush, festoon the branches with their
hanging blossoms. Here is one leafy arcade, where sunshine and shadow
dance in tremulous alternation on the soft velvety turf beneath; and hark!
the silence is broken; there are the sounds of footsteps on the green sward,
the crackling of dried twigs which have fallen, and the sounds of some
approaching creature. The charm is broken, into this natural temple some
one has entered; who can it be, and for what end does he come?
Down this arcade comes the intruder, deeper and deeper into the forest;
he seems to have no settled purpose here, but wanders below the drooping
branches in meditative silence, communing with his heart, and inhaling as it
seems the melancholy tenderness which floats in the shadowy air—
melancholy because anticipating the departure of those bright lingerers in
summer’s lengthened train; and tender, because remembering how Nature,
the universal mother, gathers in beneath her wintry mantle those children of
her care, and nourishes them in her genial bosom till spring robes her anew
with their verdure and their flowers. He seems no stranger to these gentle
sympathies, this solitary wayfarer, but looks upon the gay foliage and
clinging flowers as if they were ancient friends. He is young, but there is a
shadow on his face which tells of mental suffering and grief, though it
seems of grief whose agony and bitterness has past away. His face is thin,
worn, and thoughtful, there are deep furrows on his cheek and brow, the
traces of some great and long-enduring struggle; his eyes are cast down, and
his lips move from time to time, as though they were repeating words of
comfort, with which he was striving to strengthen himself, and ever and
anon he anxiously raises his earnest eyes to heaven, as if he sought for light
to his soul and assurance there; and then again his head is bent down
towards the greensward as if in sudden humility, and a sigh of conscious
guilt or unworthiness breaks from his labouring breast, and he writhes as if
he felt a sudden agonising pang. It is evident that this lonely man seeks for
something which he has not, of the lack of which he is fully conscious, and
which he desires with all the intensity of a soul’s most ardent and earnest
longing to obtain; wealth it cannot be, nor fame, nor honour, for this wild
wilderness, this place so solitary and far from the abodes of men, is not
where these are either sought or found. On the trunk of a giant pine which
lies across the green arcade, a trophy of the last winter’s storms, he seats
himself; the gentle gale breathes through the wood in long low sighings,
which come to the ear like a prolonged moan; the leaves fall softly with a
pleasant plaintive cadence to their mossy grave; the sun looks down from
the heavens, veiling his glory with a cloud, as though he feared to gaze too
keenly on a scene so fair and solemn; the heart of the lonely meditative man
is fairly melted within him; the object he has been searching for, which he
has so longed to possess, which has shone upon him hitherto so distant, so
far off, beyond the reach of his extended hand, and never been seen save in
such transient glimpses of his straining eyes that again and again, and yet
again, his doubts and fears have returned in almost their original force, and
the despair, which almost engulfed him in the old sad time, seems near at
hand to enshroud him once again, is suddenly brought to nearest
neighbourhood. A holy presence fills that quiet air; a voice of love, and
grace, and mercy steals into that long bereaved and mourning heart; he
throws himself down on the dewy grass, and its blades bend beneath
heavier and warmer drops than the soft tears of morn and even. Listen! for
his voice breaks through the stillness with a tone of unspeakable joy,
thrilling in its accents, and its words are “all things;” hark how the wind
echoes them among the trees, as though so worthy of diffusion, so full of
hopeful confidence, that even it loved to linger on and prolong the sound.
“All things are possible—with God.” His trembling form is bent in the
hallowed stillness of unuttered prayer; his frame quivers with an emotion
for years unfelt. Oh! how different from all his past shakings and
tremblings, how different from all that has gone before is this! But who dare
lift the veil which covers the deep humility of that supplicating spirit, or
break in upon the holy confidence with which it approaches, in this its first
communion, its God and Father. It is enough that there is joy in heaven, this
blessed morning, over the returned prodigal, the lost and wandering child,
“he that was dead is alive again, he that was lost is found,” and Halbert
Melville at length is at rest.
Long and fearfully has he struggled since that fearful night in hoary
Edinburgh; been tossed in Atlantic storms, seen the wonders of the Lord on
the great deep, in the thunder, and the lightning, and the tempest, and
experienced His goodness in being brought to land in safety once again. For
years since then, on every wall, his tearless eyes have seen, as though
written by an unseen hand, those terrible words, “It is impossible,” and a
voice heard by no mortal but himself, has rung again and again in sad
reiteration into his despairing ears, “It is impossible,” like an “anathema
maranatha,” ever binding and irreversible. But to-day the whole has
changed, the cloud has been dissipated and the sun shines forth once more;
another voice sweeter than that harp of sweetest sound has brought to him
joy for mourning, and blotted out from his mental horizon his fancied
doom, with that one word of gracious omnipotence, “All things are possible
with God.” It has told him of the might that can save to the uttermost, of the
grace that casts away no contrite heart, and of the love to sinners which
passeth all knowledge; and in the day of recovered hope, and in faith which
has already the highest seal, the spirit’s testimony ennobling its meek
humility, Halbert Melville arises from beneath these witness trees, from that
altar in a cathedral of Nature’s own fashioning for its Maker’s worship,
more grand and noble than the highest conception of man could conceive or
his highest art embellish, with a change wrought upon his enfranchised
spirit, which makes him truly blessed. In his despair and hopelessness he
had pronounced this “impossible,” and he stands now rejoicing in the
glorious words of one of old, “Return unto thy rest, oh, my soul! for the
Lord hath dealt bountifully with thee.” The summer was nearly past, the
winter had nearly come, but Halbert Melville was saved.
But Halbert must go home; alas, he has no home in this wide continent.
In all the multitude of breathing mortals here, there is not one whose eye
grows brighter or heart warmer at his approach. He is, on the contrary,
regarded curiously and with wonder; sometimes indeed with pity, but he is
still the stranger, though nearly two years have passed since first he
received that name. His home, such as it is, is in a great bustling town, at a
distance from this quiet solitude, to which long ago—there are places near it
famed in the catalogue of vulgar wonders,—a sudden impulse drew him, a
yearning to look on nature’s sunny face again, as he had done of old in his
days of peace and happiness, or a desire, it might be, to attain even a deeper
solitude than he, stranger though he was, could find amid the haunts of
men. But now he must return to his distasteful toil, and solitary room. Ah!
Halbert, how different from the solitary room in old Edinburgh, the books,
congenial studies, and pleasant recreations before the tempter came! but it
is with a light step and a contented heart for all that, that Halbert Melville
retraces his steps along the lonely way. Now there is hope in the sparkle of
his brightened eye, and a glow of his own old home affections at his heart,
as he catches the wide sweep of the distant sea, and the white sails swelling
already in the pleasant breeze, that will bear them home. Home! what magic
in the word! it has regained all its gladdening power, that hallowed syllable,
and Halbert is dreaming already of Christian’s tearful welcome, and little
Mary’s joy; when a chill strikes to his heart. Is he sure that the letter of the
prodigal, who has brought such agony and grief upon them, will be received
so warmly? No, he is not, he doubts and fears still, for all the peace that is
in his heart, and Halbert’s first resolution is changed; he dare not write; but
his fare shall be plain, his lodging mean, his apparel scanty, till he has the
means of going home, of seeking pardon with his own lips, of looking on
their reconciled faces with his own rejoicing eye; or of bidding them an
adieu for evermore.
Halbert has reached the noisy town again, and is threading his way
through its busy streets, among as it seems the self-same crowd he traversed
on his departure; but how differently he looks on it now; then he noticed
none but the poor, the aged, the diseased; and thought in the selfishness of
engrossing care, that the burdens which they bore were light in comparison
with that which weighed him down. Now he embraces them all in the wide
arms of his new-born and sympathetic philanthropy, and is as ready in the
fulness of his heart to rejoice with them that do rejoice, as to weep with
those that weep. His was a true, an early love, which flies to make its
master’s presence known to those who are out of the way; who see him not,
or seeing understand not; and laden with its own exceeding joy, yearns to
share it with all who stand in need of such peace and rest as he himself has
found.
But now he has entered his own dwelling, just as the sudden gloom of
the American night, unsoftened by gentle twilight, falls thick and dark
around. It is a place where many like himself in years, station, and
occupations, engaged in the counting-houses of that great commercial city,
have their abode. Young men gay and careless, with little thought among
them for anything beyond the business or amusement of the passing hour. A
knot of such are gathered together in the common sitting-room when
Halbert enters; but they scarcely interrupt their conversation to greet him:
he has kept apart from all of them, and almost eschewed their society or
companionship. The night is cold, it has grown chilly with the lengthening
shadows, and a glowing fire of logs burns brightly and cheerfully upon the
hearth, and Halbert, wearied and cold, seats himself beside it. The
conversation goes on, it flags not because the stranger is an auditor; one
young man there is in this company, a merry scoffer, whose witty sallies are
received with bursts of laughter, the rather because just now, and indeed
usually, they are directed against Scripture and holy things. There is another
who inveighs against the fanaticism and bigotry of some portion of the
Church, which is, according to his foolish notion, righteous over much, and
therefore, in his clear and conclusive logic, the Church universal is only a
piece of humbug; and there is a third whom Halbert has long marked, a cold
argumentative heartless sceptic, who, emboldened by the profane mirth of
the other young men around him, has begun to broach his infidel opinions,
and for them finds a favourable auditory. Look at Halbert’s face now, how it
beams in the fire-light, as he hears the cold-blooded insinuations, and words
of blasphemy, the dead negations, the poison of his own heedless youth,
from which he has suffered so sorely, again propounded in the identical
guise and semblance which bewitched himself of old. See him! how his
dark eyes sparkle with righteous fire; how his bent form grows erect and
stately, and his features expand in unconscious nobility, as though there was
inspiration within his heart, because of which he must interfere, must speak
to these youths, should he perish.
The solitary man bends over the cheerful blaze no longer. See him
among these wondering youths, with the light of earnest truth beaming from
every noble line of his prophet face. Listen to his solemn tone, his words of
weighty import. Hear what he says to them, amazed and confounded that
the stranger has at last found a voice. What does he say? he tells the story of
his own grievous shipwreck; he tells them how he was tempted and how he
fell; tells them of all the wiles and stratagems by which he was overcome,
and how he found out only at the very last, how hollow, false, and vain they
were; bids them remember the miserable man bowed down by secret
sorrow, that they have all along known him, and his voice trembles with
solemn earnestness, as he warns them as they love their lives—as they love
the gladness which God has given them, the heritage of their youth—to
refuse and reject the insinuations of the tempter, and to oppose themselves
to the serpent-cunning of the blasphemer, refusing even to listen to his
specious arguments and hollow one-sided logic, if they wish it to be well
with them. The air of the room has grown suddenly too hot for the
discomfited sceptic, the scoffer has forgotten his gibe, the grumbler his
grievance, and their companions their responsive laughter. Halbert’s words
of sad and stern experience, spoken in solemn warning, sink into their
hearts with much effect, at least for the time. Perhaps the impression will
not last, but at this moment, these thoughtless youths are startled into
seriousness, and whatever the effect may be ultimately, the recollection of
that thrilling appeal will linger, and that for long, in their memories.
Sweet slumber and pleasant dreams has Halbert Melville this night. He
lies in that fair chamber, whose windows open to the rising sun, where
rested after his great fight of afflictions that happy dreamer of old, where
peace is, and no visions of terror can enter, and Halbert Melville, whatever
his future fate may be, whether calm or tempest, fair or foul weather, has
like the pilgrim found rest.
CHAPTER II.
Maiden! with the meek brown eyes,
In whose orb a shadow lies,
Like the dusk in evening skies!
* * * * *
O, thou child of many prayers!
Life hath quicksands—Life hath snares!
Care and age comes unawares!—Longfellow.
T is well that there are swifter ways of mental travel, than even
the very quickest means of transit for the heavier material part, or
we should be too late, even though we crossed the Atlantic in the
speediest steamer of these modern days, and with the fairest
winds and weather, for Mrs. James Melville’s new year’s party. Mrs. James
looks none the worse for these two years that have glided away since we
saw her last; she is dressed in all her holiday smiles to-night, though, as you
pass up the lighted staircase to her drawing-room, you can hear a shrill tone
of complaint coming from some far-off nursery, which shows that James’s
pretty house has got another tenant; and, truly, his paternal honours sit well
on our old friend. The street without is illuminated by the lights which
gleam through the bright windows, and are alive with the mirth and music
that is going on within. There is a large company assembled; and, amid the
crowded faces, all so individual and dissimilar, beaming on each other, here
is one we should know—pale, subdued, and holy, like the Mary of some old
master. It seems out of place, that grave, sweet countenance in this full
room, and among this gay youthful company. It is our old friend Christian,
hardly, if at all, changed since last we saw her, save for her deepened, yet
still not melancholy sadness; it is said that her smiles, since that terrible
time of Halbert’s disappearance, have been more sad than other people’s
tears, but she does smile sweetly and cheerfully still; there is too little of the
gall of humanity about her, too little selfishness in her gentle spirit to permit
the cloud, which hovers over her own mind, to darken with its spectre
presence the enjoyment of others. Christian likes—as may be well believed
—the quietness of her own fireside better than any other place; but James
would have been grieved had she stayed away, and therefore is she here
amid this crowd to-night. But there is a graceful figure near her that we
shall not recognise so easily, though coming from a contemplation of that
thin, worn face, inspired as we saw it last in yonder American city, and

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Gunderson & Tepper’s Clinical

Radiation Oncology 5th Edition Joel

Jeffrey A. Bogart, MD Abram Recht, MD

Andrea K. Ng, MD, MPH Akila N. Viswanathan, MD, MPH

Joel E. Tepper, MD, FASTRO

Robert L. Foote, MD, FACR, FASTRO

Jeff M. Michalski, MD, MBA, FACR, FASTRO

GUNDERSON & TEPPER’S CLINICAL RADIATION ONCOLOGY, ISBN: 978-0-323-67246-7

Library of Congress Control Number: 2019949699

Executive Content Strategist: Robin Carter

To Kally, who, during our 40 years of marriage, has

To Sheila, my loving wife of 31 years, for her steadfast

J. Daniel Bourland, PhD, MSPH Bruce A. Chabner, MD Stephen G. Chun, MD

Christopher H. Crane, MD Ryan W. Day, MD Hugues Duffau, MD, PhD

Gini F. Fleming, MD Adam S. Garden, MD Dennis E. Hallahan, MD

Caroline L. Holloway, MD, FRCPC Patricia A. Hudgins, MD Brian D. Kavanagh, MD

Rupesh R. Kotecha, MD Nancy Lee, MD William J. Mackillop, MBChB, FRCR,

Mark W. McDonald, MD Michael Mix, MD Paul Okunieff, MD

Lisa A. McGee, MD Amy C. Moreno, MD Hilary L.P. Orlowski, MD

Thomas M. Pisansky, MD Pablo F. Recinos, MD David P. Ryan, MD

Arif Sheikh, MD John H. Suh, MD Chiaojung Jillian Tsai, MD, PhD

Video 20.1 Prostate Brachytherapy

TABLE 1.1 Stages in the Radiobiology Continuum

factor, physicians began to experiment with the use of multiple, smaller

with other cellular activities is termed the DNA Damage Response

Nucleotide Excision Repair

Intra-arm exchanges Inter-arm exchanges Interchanges “Single-hit” breaks

Paracentric Paracentric Reciprocal

endpoint for HeLa cells than for prokaryotes or primitive eukaryotes.

much smaller than typically observed for microorganisms. Puck and

fraction (determined from a single-dose survival curve), and SFn is

Total dose (Gy)

10!2 10!1 10!2

10!3 10!2 3h 10!3

Total dose (Gy)

Fig. 1.11 Hypothetical multifraction survival curve (dashed line) for

Survival relative to tumor explanted

immediately after irradiation

incubation following irradiation at either reduced temperature101 in the

0.001 G2/M G1 Early S M G1 S G2

250 kVp x-rays

Relative biological effectiveness

Oxygen enhancement ratio

complications, particularly for late-responding tissues.121 Variations in

Total dose (Gy)

Neutrons RBE = 4.1

Fig. 1.19 Increase in the relative biological effectiveness (RBE) of neutrons

in the relative amounts of either the radiosensitizer, oxygen, or the

10"1 OER ! 15.5 Gy/6.25 Gy ! 2.5

Aerobic Hypoxic Range of normal tissue PO2

Radiosensitizers, Radioprotectors, and

TABLE 1.2 Selected Chemical Modifiers of Radiation Therapy

OH Misonidazole Radiosensitizer of hypoxic cells. Clinical trial results were disappointing

FDA, U.S. Food and Drug Administration.

Percent labeled mitoses

There’s joy and mourning wondrously entwined

Then followed that beautiful season,

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