JC TH

OWNED BY THE SECOND AFFILIATED HOSPITAL

Associate Editors

Dr. Timothy Billiar Prof. Lungen Lu

Editorial Board

Dr.Subrat Kumar Acharya Prof. Ying Han Dr. Qingfeng Sun

OF CHONGQING MEDICAL UNIVERSITY F-1281 Presbyterian University Hospital Shanghai Jiaotong University School of New Delhi, India Xi'an, China Wenzhou, China
Pittsburgh, USA Medicine
Shanghai, China
Dr. Piero Luigi Almasio Prof. Saeed Hamid Prof. Gloria Taliani
Dr. John Birk Palermo, Italy Karachi, Parkistan Roma, Italy
Editors-in-Chief University of Connecticut Dr. John Luk
Dr. Costica Aloman Dr. Kazuhiko Hayashi Prof. Hong Tang
Farmington, USA Johnson & Johnson Medical Ltd.
Shanghai, China New York, USA Nagoya, Japan Chengdu, China
Prof. Hong Ren Prof. Limin Chen
Peking Union Medical College Prof. Yen-Hsuan Ni Prof. Gianfranco D. Alpini Dr. Wasim Jafri Dr. Claudio Tiribelli
General Editor-in-Chief
Chengdu, China National Taiwan University Bryan, USA Aga Khan, Parkistan Trieste, Italy
The Second Affiliated Hospital of Chongqing
Medical University, China Prof. Chengwei Chen Taipei, Taiwan Dr. Masahiro Arai Dr. Di Jia Prof. Zhengkun Tu
Dr. Harry Hua-Xiang Xia Nanjing Military Command Prof. Kannika Phornphutkul Tokyo, Japan Boston, USA Changchun, China
Shanghai, China Chiang Mai University
Editor-in-Chief Dr. Gyorgy Baffy Dr. Wei Jia Dr. Adriana Vince
Prof. Aziz A. Chentoufi Chiangmai, Thailand
Novartis Pharmaceuticals Corporation, USA Boston, USA Kunming, China Zagreb, Croatia
Medicine King Fahad Medical College Dr. Kittichai Promrat
Prof. George Y. Wu Riyadh, Saudi Arabia Dr. Savino Bruno Dr. Jiaji Jiang Dr. Farzin Roohvand
Alpert Medical School of Brown University
Comprehensive Editor-in-Chief Prof. Xiaoguang Dou Providence, USA Milan, Italy Fuzhou, China Paris, France/Iran
University of Connecticut Heath Center, USA
Shengjing Hospital of China Medical Prof. Cheng Qian Dr. Chalermrat Bunchorntavakul Dr. Jianning Jiang Dr. Fusheng Wang
University Third Military Medical University Southwest Bangkok, Thailand Nanning, China Beijing, China
Shengyang, China Hospital
Managing Editors Prof. Zhongping Duan Chongqing, China
Dr. Wendy Cao Dr. Anastasios Koulaouzidis Prof. Genshu Wang
New York, USA Edinburgh, United Kingdom Guangzhou, China
Affiliated Beijing You'an Hospital of Capital Dr. Arielle Rosenberg
Medical University Prof. Mark J. Czaja Dr. Ashish Kumar Dr. Leyi Wang
University Paris Descartes
Dr. Huaidong Hu Beijing, China Paris, France New York, USA New Delhi, India Columbus, USA
The Second Affiliated Hospital of Chongqing
Dr. Jean-François Dufour Prof. Naoya Sakamoto Dr. Andres Cardenas Prof. Laurentius A. Lesmana Prof. Benjamin Wong
Medical University, China
University of Bern Hokkaido University Barcelona, Spain Jakarta, Indonesia Hong Kong, China
Dr. Zhi Peng Bern, Switzerland Sapporo, Japan
Prof. Flair José Carrilho Dr. Bing Liu Prof. Catherine Y. Wu
The Second Affiliated Hospital of Chongqing Prof. Marko Duvnjak Dr. Michael Schilsky São Paulo, Brazil Guangzhou, China Farmington, USA
Medical University, China Yale University
Clinical Hospital Centre “Sestre milosrdnice”
New Haven, USA Dr. Phunchai Charatcharoenwitthaya Dr. Rohit Loomba Dr. Yongning Xin
Zagreb, Croatia
Dr. Tawesak Tanwandee Bangkok, Thailand San Diego, USA Qingdao, China
Prof. Faripour Forouhar
Siriraj Hospital, Mahidol University Prof. Zhi Chen Prof. Eduardo Martinez Prof. Huiping Yan
University of Connecticut
Contact information
Bangkok, Thailand Hangzhou, China Hidalgo, Mexico Beijing, China
Farmington, USA
Prof. Lai Wei Dr. Silvia Degli-Esposti Prof. Jane McKeating Prof. Ming Yan
Dr. Johannes Haybaeck
Peking University People’s Hospital
Telephone +86 23 63727251 Medical University Graz Providence, USA Birmingham, United Kingdom Shangdong, China
Beijing, China
Graz, Austria Prof. Lei Dong Prof. Albert D. Min Prof. Eric M. Yoshida
Fax +86 23 63701383 Prof. Jinlin Hou Dr. Xuefeng Xia Xi’an, China San Diego, USA Vancouver, Canada
Nanfang Hospital of Southern Medical The Methodist Hospital Research Institute
E-mail jcthcq@gmail.com Houston, USA
Dr. Yu-Chen Fan Prof. Ming Kuang Dr. Hong You
University
Guangzhou, China
Jinan, China Guangzhou, China Beijing, China
Address 74 Linjiang Road, Dr. Kecheng Xu
Dr. Peter Ferenci Dr. Mohamed Othman Prof. Cihan Yurdaydin
Yuzhong District, Prof. Keqin Hu Fuda Cancer Hospital
University of California Guangzhou, China Heidelberg, Austria El Paso, USA Ankara, Turkey
Chongqing, P. R. China,
400010 Orange, USA Prof. Eduardo Fernández-Martínez Prof. Piero Portincasa Prof. Mikio Zeniya
Prof. Man-Fung Yuen
Prof. Ailong Huang Hidalgo, Mexico Bari, Italy Tokyo, Japan
The University of Hong Kong
Chongqing Medical University Queen Mary Hospital Dr. Heather L. Francis Dr. Farzin Roohvand Prof. Xinxin Zhang
Chongqing, China Hong Kong, China Bryan, USA Tehran, Iran Shanghai, China
Dr. Hitoshi Ikeda Dr. Jinxiang Zhang Dr. James Y.Y. Fung Dr. Regina Santella Dr. Yuanyuan Zhang
The University of Tokyo Tongji Medical College Hong Kong, China New York, USA Chengdu, China
Tokyo, Japan Huazhong University of Science and
Prof. Paul J. Gaglio Dr. Ke-Qing Shi Prof. Yuexin Zhang
Prof. Jidong Jia Technology
Capital Medical University Wuhan, China New York, USA Wenzhou, China Xinjiang, China
Beijing, China Prof. Dazhi Zhang Ping Gu Prof. Gamal Shiha Prof. Jingmin Zhao
Prof. Yong Liao The Second Affiliated Hospital of Chongqing New York, USA Mansoura, Egypt Beijing, China
The Second Hospital Affiliated Chongqing Medical University
Prof. Steven-Huy Bui Han Dr. Robert Smolic Dr. Minghua Zheng
Medical University Chongqing, China
Los Angeles, USA Osijek, Croatia Wenzhou, China
Chongqing, China Dr. Lanjing Zhang
Prof. Tao Han Dr. Martina Smolic Dr. Senlin Zhu
Dr. Joseph Lim University Medical Center of Princeton
Plainsboro, USA Tianjin, China Osijek, Croatia Guangzhou, China
Yale University
New Haven, USA
JC TH
OWNED BY THE SECOND AFFILIATED HOSPITAL
Associate Editors

Dr. Timothy Billiar Prof. Lungen Lu

Editorial Board

Dr.Subrat Kumar Acharya Prof. Ying Han Dr. Qingfeng Sun

OF CHONGQING MEDICAL UNIVERSITY F-1281 Presbyterian University Hospital Shanghai Jiaotong University School of New Delhi, India Xi'an, China Wenzhou, China
Pittsburgh, USA Medicine
Shanghai, China
Dr. Piero Luigi Almasio Prof. Saeed Hamid Prof. Gloria Taliani
Dr. John Birk Palermo, Italy Karachi, Parkistan Roma, Italy
Editors-in-Chief University of Connecticut Dr. John Luk
Dr. Costica Aloman Dr. Kazuhiko Hayashi Prof. Hong Tang
Farmington, USA Johnson & Johnson Medical Ltd.
Shanghai, China New York, USA Nagoya, Japan Chengdu, China
Prof. Hong Ren Prof. Limin Chen
Peking Union Medical College Prof. Yen-Hsuan Ni Prof. Gianfranco D. Alpini Dr. Wasim Jafri Dr. Claudio Tiribelli
General Editor-in-Chief
Chengdu, China National Taiwan University Bryan, USA Aga Khan, Parkistan Trieste, Italy
The Second Affiliated Hospital of Chongqing
Medical University, China Prof. Chengwei Chen Taipei, Taiwan Dr. Masahiro Arai Dr. Di Jia Prof. Zhengkun Tu
Dr. Harry Hua-Xiang Xia Nanjing Military Command Prof. Kannika Phornphutkul Tokyo, Japan Boston, USA Changchun, China
Shanghai, China Chiang Mai University
Editor-in-Chief Dr. Gyorgy Baffy Dr. Wei Jia Dr. Adriana Vince
Prof. Aziz A. Chentoufi Chiangmai, Thailand
Novartis Pharmaceuticals Corporation, USA Boston, USA Kunming, China Zagreb, Croatia
Medicine King Fahad Medical College Dr. Kittichai Promrat
Prof. George Y. Wu Riyadh, Saudi Arabia Dr. Savino Bruno Dr. Jiaji Jiang Dr. Farzin Roohvand
Alpert Medical School of Brown University
Comprehensive Editor-in-Chief Prof. Xiaoguang Dou Providence, USA Milan, Italy Fuzhou, China Paris, France/Iran
University of Connecticut Heath Center, USA
Shengjing Hospital of China Medical Prof. Cheng Qian Dr. Chalermrat Bunchorntavakul Dr. Jianning Jiang Dr. Fusheng Wang
University Third Military Medical University Southwest Bangkok, Thailand Nanning, China Beijing, China
Shengyang, China Hospital
Managing Editors Prof. Zhongping Duan Chongqing, China
Dr. Wendy Cao Dr. Anastasios Koulaouzidis Prof. Genshu Wang
New York, USA Edinburgh, United Kingdom Guangzhou, China
Affiliated Beijing You'an Hospital of Capital Dr. Arielle Rosenberg
Medical University Prof. Mark J. Czaja Dr. Ashish Kumar Dr. Leyi Wang
University Paris Descartes
Dr. Huaidong Hu Beijing, China Paris, France New York, USA New Delhi, India Columbus, USA
The Second Affiliated Hospital of Chongqing
Dr. Jean-François Dufour Prof. Naoya Sakamoto Dr. Andres Cardenas Prof. Laurentius A. Lesmana Prof. Benjamin Wong
Medical University, China
University of Bern Hokkaido University Barcelona, Spain Jakarta, Indonesia Hong Kong, China
Dr. Zhi Peng Bern, Switzerland Sapporo, Japan
Prof. Flair José Carrilho Dr. Bing Liu Prof. Catherine Y. Wu
The Second Affiliated Hospital of Chongqing Prof. Marko Duvnjak Dr. Michael Schilsky São Paulo, Brazil Guangzhou, China Farmington, USA
Medical University, China Yale University
Clinical Hospital Centre “Sestre milosrdnice”
New Haven, USA Dr. Phunchai Charatcharoenwitthaya Dr. Rohit Loomba Dr. Yongning Xin
Zagreb, Croatia
Dr. Tawesak Tanwandee Bangkok, Thailand San Diego, USA Qingdao, China
Prof. Faripour Forouhar
Siriraj Hospital, Mahidol University Prof. Zhi Chen Prof. Eduardo Martinez Prof. Huiping Yan
University of Connecticut
Contact information
Bangkok, Thailand Hangzhou, China Hidalgo, Mexico Beijing, China
Farmington, USA
Prof. Lai Wei Dr. Silvia Degli-Esposti Prof. Jane McKeating Prof. Ming Yan
Dr. Johannes Haybaeck
Peking University People’s Hospital
Telephone +86 23 63727251 Medical University Graz Providence, USA Birmingham, United Kingdom Shangdong, China
Beijing, China
Graz, Austria Prof. Lei Dong Prof. Albert D. Min Prof. Eric M. Yoshida
Fax +86 23 63701383 Prof. Jinlin Hou Dr. Xuefeng Xia Xi’an, China San Diego, USA Vancouver, Canada
Nanfang Hospital of Southern Medical The Methodist Hospital Research Institute
E-mail jcthcq@gmail.com Houston, USA
Dr. Yu-Chen Fan Prof. Ming Kuang Dr. Hong You
University
Guangzhou, China
Jinan, China Guangzhou, China Beijing, China
Address 74 Linjiang Road, Dr. Kecheng Xu
Dr. Peter Ferenci Dr. Mohamed Othman Prof. Cihan Yurdaydin
Yuzhong District, Prof. Keqin Hu Fuda Cancer Hospital
University of California Guangzhou, China Heidelberg, Austria El Paso, USA Ankara, Turkey
Chongqing, P. R. China,
400010 Orange, USA Prof. Eduardo Fernández-Martínez Prof. Piero Portincasa Prof. Mikio Zeniya
Prof. Man-Fung Yuen
Prof. Ailong Huang Hidalgo, Mexico Bari, Italy Tokyo, Japan
The University of Hong Kong
Chongqing Medical University Queen Mary Hospital Dr. Heather L. Francis Dr. Farzin Roohvand Prof. Xinxin Zhang
Chongqing, China Hong Kong, China Bryan, USA Tehran, Iran Shanghai, China
Dr. Hitoshi Ikeda Dr. Jinxiang Zhang Dr. James Y.Y. Fung Dr. Regina Santella Dr. Yuanyuan Zhang
The University of Tokyo Tongji Medical College Hong Kong, China New York, USA Chengdu, China
Tokyo, Japan Huazhong University of Science and
Prof. Paul J. Gaglio Dr. Ke-Qing Shi Prof. Yuexin Zhang
Prof. Jidong Jia Technology
Capital Medical University Wuhan, China New York, USA Wenzhou, China Xinjiang, China
Beijing, China Prof. Dazhi Zhang Ping Gu Prof. Gamal Shiha Prof. Jingmin Zhao
Prof. Yong Liao The Second Affiliated Hospital of Chongqing New York, USA Mansoura, Egypt Beijing, China
The Second Hospital Affiliated Chongqing Medical University
Prof. Steven-Huy Bui Han Dr. Robert Smolic Dr. Minghua Zheng
Medical University Chongqing, China
Los Angeles, USA Osijek, Croatia Wenzhou, China
Chongqing, China Dr. Lanjing Zhang
Prof. Tao Han Dr. Martina Smolic Dr. Senlin Zhu
Dr. Joseph Lim University Medical Center of Princeton
Plainsboro, USA Tianjin, China Osijek, Croatia Guangzhou, China
Yale University
New Haven, USA
 JOURNAL OF CLINICAL AND
TRANSLATIONAL HEPATOLOGY
Call for papers
JCTH is a new, comprehensive specialist journal focusing on the recent progress in clinical and basic
research with direct applications to clinical management of liver diseases. The studies published in JCTH
will represent the most current trends in the field of hepatology, highlighting the topically relevant
subjects of nations worldwide. Publications in JCTH will be presented in formats that emphasize clarity
of the study’s objectives and implications of its findings, using high quality visual aids to enhance the
manuscript’s esthetic appeal as well as its impact. For our upcoming issue, we encourage you and your
group to submit original articles that showcase your work in hepatology and topically relevant reviews to
promote our readers’ understanding of the field.

CONTENTS 2014 2(3):143–216

Review Articles

Stem Cell Strategies to Evaluate Idiosyncratic Drug-induced Liver Injury

Winfried Krueger, Urs A. Boelsterli and Theodore P. Rasmussen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

Acetaminophen-induced Liver Injury: from Animal Models to Humans

Hartmut Jaeschke, Yuchao Xie and Mitchell R. McGill . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153

MicroRNAs in Drug-induced Liver Injury

Li-Min Li, Dong Wang and Ke Zen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162

Chinese Herbal Medicine-induced Liver Injury

Xin Ma, Jing-Hua Peng and Yi-Yang Hu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170

Hepatocellular Carcinoma and Liver Transplantation: State of the Art

Andrea Mancuso and Giovanni Perricone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176

Percutaneous Cryoablation for Liver Cancer

Li-Zhi Niu, Jia-Liang Li and Ke-Cheng Xu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182

Hepatitis C Recurrence after Orthotopic Liver Transplantation: Mechanisms and Management

Bobby Kakati and Anil Seetharam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189

Antiviral Therapy of Liver Cirrhosis Related to Hepatitis B Virus Infection

Lun-Gen Lu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197

The Molecular and Structural Basis of HBV-resistance to Nucleos(t)ide Analogs

Nidhi Gupta, Milky Goyal, Catherine H. Wu and George Y. Wu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202

Hepatobiliary Schistosomiasis
Yehia Shaker, Nervana Samy and Esmat Ashour . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
Review Article
Stem Cell Strategies to Evaluate Idiosyncratic Drug-induced
Liver Injury
Winfried Krueger1, Urs A. Boelsterli1 and Theodore P. Rasmussen*1,2,3
1
Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT, USA; 2Department of Molecular and Cell Biology,
University of Connecticut, Storrs, CT, USA; 3University of Connecticut Stem Cell Institute, Storrs/Farmington, CT, USA
Abstract
The host-dependent nature of idiosyncratic drug-induced
liver injury (iDILI) suggests that rare genetic polymorphisms
may contribute to the disease. Indeed, a few mutations in key
genes have already been identified using conventional human
genetics approaches. Over 50 commonly used drugs can
precipitate iDILI, making this a substantial medical problem.
Only recently have human induced pluripotent stem cells
been used as a research tool to discover novel iDILI genes
and to study the mechanisms of iDILI in vitro. Here we review
the current state of stem cell use in the investigation of iDILI,
with a special focus on genetics. In addition, the concerns and
difficulties associated with genetics and animal model
research are discussed. We then present the features of
patient-specific pluripotent stem cells (which may be derived
from iDILI patients themselves), and explain why these cells
may be of great utility. A variety of recent approaches to
produce hepatocyte-like cells from pluripotent cells and the
associated advantages and limitations of such cells are
discussed. Future directions for the use of stem cell science
to investigate iDILI include novel ways to identify new iDILI
genes, a consideration of epigenetic impacts on iDILI, and the
development of new and improved strategies for the produc-
tion of hepatocytes from human pluripotent cells.
E 2014 The Second Affiliated Hospital of Chongqing Medical
University. Published by XIA & HE Publishing Ltd. All rights
reserved.
Keywords: DILI; Embryonic stem cell; iPS; Toxicity; Genetics.
Abbreviations: AFP, a-fetoprotein; ALB, albumin; BMP4, bone morphogenic
protein 4; ChIP, chromatin-immunoprecipitation; CRISPR, clustered regularly
interspaced short palindromic repeats; c-Myc, proto-oncogene myc; CYP,
cytochrome P450; FGF2, fibroblast growth factor 2; GWAS, genome-wide
association study; GST, glutathione-S-transferase; hESC, human embryonic stem
cell; HGF, hepatocyte growth factor; hiPSC, human induced pluripotent stem cell;
HLA, human leukocyte antigen; HNF4a, hepatocyte-specific nuclear receptor/
transcription factor a; ICM, inner cell mass; iDILI, idiosyncratic drug-induced liver
injury; iHLC, induced hepatocyte-like cell; Indels, insertion/deletions; iPSCs,
induced pluripotent stem cells; Klf4, kruppel-like factor; LEM, liver extracellular
matrix; LIF, leukemia inhibitory factor; LPS, lipopolysaccharide; mESCs, mouse
embryonic stem cells; MHC, major histocompatibility complex; Oct4, octamer-
binding transcription 4; POLG, mitochondrial polymerase c; SHH, sonic hedgehog;
SNP, single nucleotide polymorphisms; Sox2, sry-related HMG box 2; TALENS,
transcription activator-like effector nucleases.
Received: 04 April 2014; Revised: 13 May 2014; Accepted: 07 June 2014
q
DOI: 10.14218/JCTH.2014.00012.
*Correspondence to: Theodore P. Rasmussen, Department of Pharmaceutical
Sciences, University of Connecticut, 69 North Eagleville Road, U-3092, Storrs,
CT 06269, USA. Tel: +86-486-8339, Fax: +86-486-5792, Email: theodore.
rasmussen@uconn.edu
Journal of Clinical Translational Hepatology 2014 vol. 2 | 143–152
Introduction
Idiosyncratic drug-induced liver injury (iDILI) is a complex
disorder that is typified by the onset of severe (even fatal)
liver damage after exposure to drugs that are otherwise well
tolerated in the human patient population. The host-depen-
dent (idiosyncratic) nature of iDILI suggests that genetic
polymorphisms that cause a predisposition to DILI events
likely exist in the population. In these cases, iDILI occurs only
when an individual with a predisposing genotype is exposed
to the precipitating drug. Put another way, iDILI is a type of
drug-genotype interaction, and most individuals with a
potential underlying iDILI genotype will never know of their
underlying genetic susceptibility because they will fail to
encounter the interacting drug.
At present, the only way to ‘‘find’’ an iDILI genotype is for
an individual with a predisposing genotype to be treated
(unintentionally) with an iDIL-precipitating drug. Current
medical practice dictates that all patients are assumed to be
normal, and they are treated routinely with potentially DILI-
causing drugs. Importantly, these patients are monitored for
the onset of liver damage only after the onset of drug
treatment, usually by monitoring levels of liver enzymes,
such as ALT and others in the blood. Currently, there is no
effective way to detect potential iDILI responders prior to
drug treatment. As more iDILI polymorphisms in the genome
become known, it may be possible to devise genetic tests that
identify at-risk individuals. Such a goal will require the
comprehensive identification of iDILI risk alleles for each of
the 50 or more highly problematic drugs, and an expedient
way to gather this genetic information is needed. One way
that such information could be gathered is via genome-wide
association studies (GWAS). However, about 50 commonly
used drugs can cause iDILI, often by completely independent
mechanisms, and it is likely that 50 complete GWAS studies
would be required. Although GWAS, a recently developed and
powerful approach, would work in theory, practicalities of
scale and relatively low numbers of iDILI patients for specific
drugs prohibit the widespread use of GWAS to comprehen-
sively identify iDILI genes and polymorphisms.
Recently, an entirely different and new approach uses
human induced pluripotent stem cells (hiPSCs) that are exact
genetic matches to iDILI patients (Fig. 1). These are obtained
by a cellular reprogramming approach that takes relatively
non-invasive biopsies (either blood or a small skin sample)
and reprograms them to an early developmental state that is
functionally equivalent to embryonic stem cells. This is
achieved by expressing key embryonic transcription factors
that induce the state of pluripotency (i.e. the potential to

Fig. 1. Strategy for using iPS reprogramming to investigate iDILI. Somatic
cell samples such as skin or blood are collected from iDILI and control patients.
These are used to derive hiPSCs that are exact genetic matches to the donor
patients. Such hiPSCs can be indefinitely expanded and passaged, so long as they
are maintained under pluripotent conditions. hiPSCs can then be differentiated to
iHLCs (hepatocyte-like cells). iHLCs from iDILI and control patients can then be
compared using functional tests (A., such as exposure to the iDILI-precipitating
drug) or using transcriptome comparisons (B.), which may lead to identification of
iDILI genes. Subsequently, causative mutations (C.) can be identified, using
transcriptome alterations as a guide.
differentiate into any desired cell type, including hepatocytes,
present in the adult body). To do this, a patient who has
suffered iDILI donates blood or skin that can be derived into
hiPSCs and then hepatocytes. These induced hepatocyte-like
cells (iHLCs) are subjected to functional toxicity studies, and
more importantly, iDILI-causing mutations can be identified
in such cells using strategies that are described below.
Therefore, it may be possible to identify the majority of
144 Journal of Clinical Translational Hepatology 2014 vol. 2 | 143–152
Krueger W. et al: Using stem cells for iDILI research
iDIL-predisposing genes using at most a few hundred patients
rather than several hundred thousand, as would be required
for GWAS. In addition, disease mechanisms can never be
addressed with GWAS, but iHLCs can be easily studied in
vitro, thus providing insight into cellular and molecular
mechanisms that govern iDILI.
Drug-induced liver injury
Idiosyncratic drug-induced liver injury
iDILI is a rare form of iatrogenic liver injury that manifests
itself as a wide spectrum of symptoms, ranging from mild and
transient increases in plasma markers of hepatocellular injury
to more severe forms that present with abdominal symp-
toms, cholestasis, and, in rare cases, the development of liver
necrosis and fulminant hepatic failure. Despite its relatively
low frequency (for individual drugs ranging from 1:100,000,
e.g., for amoxicillin/clavulanic acid, to 1:100, e.g., for
isoniazid),1 iDILI has become a major clinical concern for a
number of reasons. First, an increasing number of drugs
(.600), belonging to a variety of therapeutic classes, have
been implicated in iDILI, with approximately 50 drugs
featuring a strong causality. Therefore, the absolute number
of all iDILI cases has become impressive when iDILI cases
from over 600 drugs are considered in aggregate. Second,
the occurrence of iDILI is unpredictable, both for a new drug
on the market and for an individual patient. Third, iDILI has
been associated with high morbidity and mortality, particu-
larly when combined with jaundice, that can lead to acute
liver failure and the need for liver transplants in the most
severe cases.2 Finally, iDILI is one of the major reasons for
the withdrawal of successfully launched drugs from the
market, which may result in the loss of an otherwise
important drug.3 Numerous reviews have addressed caus-
ality criteria, clinical hallmarks, underreported incidence,
potential mechanisms, and prediction of iDILI.2,4–12
Determinants of susceptibility to iDILI
By definition, iDILI is ‘‘idiosyncratic’’, i.e., dependent on the
specific characteristics of the recipient (patient). Thus,
besides the toxicodynamic properties of the drug itself
(chemotype) and its potential to cause toxicity to hepatocytes
by a number of mechanisms, host-specific factors also
greatly contribute to the precipitation of toxicity (Fig. 2).
There are two major types of host-specific factors: acquired/
environmental factors and genotype. Regarding acquired/
environmental factors, the underlying disease (indication)
against which the patient is being treated,13 underlying
infections, and episodes of inflammatory reactions14 can
influence the risk. Coadministration with other potentially
hepatotoxic drugs or alcohol is another important factor.
Taken together, acquired/environmental and genetic
determinants of susceptibility to iDILI are responsible for
increasing the penetrance and for modulating the expres-
sivity of drug toxicity.10 For these reasons, it is under-
standable that, despite many attempts with limited success,
it is extremely difficult to develop an animal model for iDILI,
using, e.g., inbred, virtually genetically identical mice, that
are expected to recapitulate the clinical situation in individual
patients.
Krueger W. et al: Using stem cells for iDILI research
Fig. 2. Determinants of susceptibility to drug-induced liver injury (DILI).
A number of patient-specific (idiosyncratic) factors greatly modulate the
sensitivity to the potential hepatotoxic effects of certain drugs.
Role of the genotype
Mutations and/or rare single nucleotide polymorphisms
(SNPs) in critical genes have been implicated in modulating
the risk for iDILI.15,16 These specific haplotypes or mutations
could be responsible for modulating the degree of toxicity at
different levels. For example, they could enhance the pro-
toxicant pathways in the liver, they could interfere with
mechanisms involved in adaptation to the insult or impair
defense mechanisms, or they could weaken immune toler-
ance. While traditional studies have focused on polymorph-
isms in cytochrome P450 forms that modulate metabolic
bioactivation/hepatic clearance of drugs, more recent
approaches have addressed the role of genetic variation in
genes involved in the adaptive immune system, in detoxifica-
tion pathways, or in mitochondrial function. For example, the
human leukocyte antigen (HLA)-B*5701 haplotype of the
major histocompatibility complex (MHC) has been associated
with a .80-fold higher risk for developing liver injury from
flucloxacillin treatment than individuals with other MHC
haplotypes.17 Similarly, carriers of the HLA-A*3303 haplo-
type have a 36-fold higher risk for iDILI from ticlopidine
treatment than other patients.18 These risk factors are
statistically robust and point to a key role of these genes in
the susceptibility to liver injury. Another example consists of
mutations in certain forms of glutathione-S-transferase
(GST), which have been implicated in the conjugation of
reactive intermediates of many drugs including isoniazid or
troglitazone. Phenotypes from null mutations are also quite
frequent; specifically, homozygous null mutations encoding
the GSTT1 form and the GSTM1 form of GST are present in
50% and 10–25% of Caucasians, respectively. In a study
involving 154 patients with a diagnosis of iDILI, 18.2% of
patients had the GSTT1/GSTM1 double-null genotype, while
only 7.6% of control patients carried this genotype.19 Finally,
Journal of Clinical Translational Hepatology 2014 vol. 2 | 143–152
other studies have analyzed the role of polymorphisms in the
mitochondrial polymerase c (POLG) gene that is involved in
mtDNA replication. It was found that the risk for valproic acid-
induced DILI was .20-fold increased in individuals carrying
the pQ1236H and pE1143G mutations in the POLG gene as
compared to wild-type.20
One important point is that these polymorphic risk alleles
are quite common in the general population, which raises the
question why iDILI is not more common. Most likely, these
haplotypes become dangerous only in specific contexts, e.g.,
exposure to a particular drug. Furthermore, these analyses
showed, at best, only a strong correlation with iDILI episodes,
and there must undoubtedly be other risk factors. While the
identification of such ‘‘risky’’ mutations does not necessarily
explain the underlying mechanism of toxicity, it can be used
to identify certain individuals or patient subsets who are
predisposed to higher risk of iDILI. For the vast majority of
drugs, however, the genetic determinants of susceptibility are
not known. More recent approaches to establish a link
between iDILI and underlying genetic risk factors have aimed
at identifying entire functional pathways rather than single
genes.
Lessons from current models and the need for novel
patient-specific models
Currently there are no fully validated animal models that
recapitulate the clinical features of iDILI. It makes sense that
normal healthy inbred animals cannot model a disease that is
driven by a variety of underlying patient-specific geno-
types.21 While certain environmental factors can be modeled
in vitro or in vivo, genetic variations are more difficult to
adapt in animal models because in most cases, the most
relevant ‘‘risk genes’’ are not known. For example, the
lipopolysaccharide (LPS) rat or mouse model22 can mimic
the underlying presence of sporadic inflammatory episodes,
and the heterozygous Sod2 mouse model23 can emulate
increased mitochondrial oxidant stress as it occurs with
certain forms of underlying mitochondrial disease. However,
applications useful for wide-scale screening platforms have
not been possible to date. Hepatic cells can be harvested and
cultured from patients who had developed iDILI from a
particular drug and compared with cells from unaffected
patients. At present, however, it is extremely difficult to
obtain viable liver tissue from such patients, and primary
hepatocytes cannot be easily cultured for extended periods of
time. One promising approach is the use and study of stem
cells derived from both diseased and healthy patients.
Stem cell approaches
Pluripotent cells and induced pluripotent stem cells
Pluripotent cells can in theory give rise to any cell type
present in the adult mammalian body plan. The first widely-
used pluripotent stem cells were mouse embryonic stem cells
(mESCs).24,25 These cells were derived from day 3.5 pre-
implantation embryos at the blastocyst stage. At this stage of
development, there are only a small set of cell types in the
embryo. These include trophectoderm cells, which are
destined to form the embryonic portion of the placenta, and
the inner cell mass (ICM), which subsequently gives rise to all
the cells of the embryo proper, and eventually, the adult
mouse. It is the ICM cells that can be explanted and used to
145

derive mESCs. Although the ICM exists in the embryo for only
a few hours at most, mESCs can be cultured indefinitely in
vitro, under conditions that maintain their pluripotency.26
Leukemia inhibitory factor (LIF) is a key cell signaling factor
necessary for the maintenance of mESCs in a state of
protracted pluripotency.27,28 The LIF receptor signals via
Stat3 to the nucleus where key pluripotency transcription
factors, including octamer-binding transcription factor 4
(Oct4), sry-related HMG box 2 (Sox2), and Nanog, collude
to regulate a large set of genes, whose expression has been
aptly described as a pluripotency network.29–32 Human ESCs
(hESCs) were first derived from human blastocysts that
would otherwise have been discarded from human fertility
clinics.33 Unlike mESCs, hESCs depend upon the fibroblast
growth factor 2 (FGF2) for maintenance of pluripotency,34 but
like mESCs, hESCs contain a pluripotency transcriptional
network that is mediated by the master transcription factors
OCT4, SOX2, and NANOG. Collectively mESCs and hESCs
have been used to model many developmental processes in
vitro via directed differentiation experiments. Several dozens
of differentiated cell types have been produced in this way,
and they have been derived from all three of the principle
germ layers, ectoderm, endoderm, and mesoderm. In many
cases, these differentiated cells are rather similar to cells
from primary organ culture as they become post-mitotic and
exhibit gene and protein expression patterns similar to
analogous cells in vivo. Such differentiated cells derived from
pluripotent cells are far more similar to endogenous cells than
traditional immortalized cell lines, which are transformed and
usually without a normal complement of chromosomes.
The first example of reprogramming a differentiated
vertebrate cell to an early embryonic state was achieved
with Xenopus frogs, which were successfully cloned in the
early 1960s from intestinal epithelial cells transferred into
frog oocytes.35,36 The first time differentiated mammalian
cells were successfully reprogrammed was over 40 years
later with the advent of Dolly the Sheep.37 In this case, the
resulting reprogrammed totipotent cell, achieved by nuclear
transfer into an enucleated recipient sheep oocyte, was a one
cell embryo that was cultured briefly in vitro to the blastocyst
stage and then implanted in a surrogate pseudopregnant
female to yield the live-born cloned sheep named Dolly.
These successes with animal cloning showed that terminally
differentiated vertebrate cells could be reversed to a state of
pluripotency, albeit with reprogramming activities only found
in the oocyte. In 2006, Shinya Yamanaka succeeded in
directly reprogramming cultured adult cells to a state of
pluripotency by introducing a set of genes encoding key
transcription factors (Oct4, Sox2, kruppel-like factor 4 (Klf4),
and proto-oncogene myc (c-Myc)) into mouse fibroblasts
using retroviruses.38 The resulting pluripotent cells were
deemed induced pluripotent stem cells (iPSCs). After initial
viral transduction, the somatic cells were shifted to mESC
culture conditions. The mechanism of iPS reprogramming
depends upon the forced expression of a defined set of
transcription factors, which includes members of the master
regulatory transcription factors of the pluripotency network.
IPSCs are remarkably similar to ESCs, and most importantly,
exhibit a full range of pluripotency. Soon after this achieve-
ment, human iPSCs (hiPSCs) were developed.38,39 These,
like their mouse counterparts, exhibited nearly complete
resemblance to their hESC equivalents. It is crucial to point
out that hiPSCs contain exactly the same genetic composition
as the human fibroblast donor, thus opening the door to their
146 Journal of Clinical Translational Hepatology 2014 vol. 2 | 143–152
Krueger W. et al: Using stem cells for iDILI research
use as a stem cell platform for personalized medicine. Early
mouse iPS cells (miPSC) and hiPSC cell lines were made with
integrating retroviruses and lentiviruses, and thus had subtly
altered genomes. Since then it has become possible to make
iPS cell lines that avoid the use of integrating retrovirus or
lentivirus, including the use of messenger RNA encoding the
reprogramming factors,40 transposon systems that excise
with barely a trace,41,42 and episomes that transiently (but
efficiently) express reprogramming factors prior to episomal
loss by cell division.43 All of these strategies leave the
genome nearly or completely untouched by the iPS repro-
gramming process.
Disease in a dish: Differentiation of iPS cells to
hepatocytes
Most strategies to produce hepatocytes from hESCs or hiPSCs
attempt to recapitulate liver development in vivo. In addition,
strategies to differentiate hESCs and hiPSCs to human
hepatocyte-like cells (hereafter termed ‘‘iHLCs’’ if derived
from hiPSCs) have been greatly informed by similar (and
earlier) successes with mESCs. Because liver is an endo-
derm-derived organ, all strategies for production of iHLCs
begin with a preculture of hiPSCs. In one set of strategies,
hiPSCs are shifted from hiPSC culture medium to an
endoderm induction medium containing the signaling factor
Activin A with insulin, transferrin, and selenium in trace
concentration.44,45 Activin A is a potent inducer of endoderm
and rapidly leads to cellular expression of the early endoderm
marker SOX17. Cells are then shifted to a medium containing
FGF2 and bone morphogenic protein 4 (BMP4), both of which
are known to play a role in liver organogenesis in vivo.46–48
These cells are then shifted to a medium containing
hepatocyte growth factor (HGF), and then finally to a medium
containing oncostatin M and dexamethasone. This typical four
stage approach yields hepatocyte-like cells that express both
albumin (ALB) and a-fetoprotein (AFP), indicating that these
iHLCs are similar to hepatocytes of late embryonic or
perinatal origin.44 This same protocol yielded hepatocytes
that can also secrete cholesterol and respond to statins, as
normal hepatocytes should.44 Human ES and iPS cells seem
to form hepatocytes with similar efficiency.49 Fully mature
iHLCs are quite difficult to produce with entirely in vitro
approaches, but subsequent transplantation into immuno-
compromised mouse liver seems to affect final stages of
maturation.50
Similarly, in one seminal study using analogous
approaches to that described above, produced hepatocyte-
like cells that expressed a spectrum of mature hepatocyte
markers with residual AFP expression.45 Alternate designs for
the cell culture have also been tested. For example, one
approach yielded cells with at least some level of CYP3A4
expression, the ability to take up indocyanine green, and
store glycogen.51 In a different strategy, a cDNA encoding the
hepatocyte-specific nuclear receptor/transcription factor
HNF4a was ectopically expressed in hiPSCs that were
otherwise induced to form hepatocyte-like cells.26 These cells
exhibited increased cytochrome P450 (CYP) expression, a
finding that is consistent with the fact that many CYP genes
contain HNF4a response elements near their promoters.
Finally, direct reprogramming of fibroblasts to induced
hepatocyte-like cells (iHLC) has been achieved by induced
transdifferentiation, in most cases using mouse tail tip
fibroblasts as a starting material.52–54
Krueger W. et al: Using stem cells for iDILI research
Limitations of stem cell models
iHLCs are a tool not only for the discovery of new iDILI genes
but also for the elucidation of the cellular and molecular
mechanisms of iDILI. However, iHLCs are not without
potential problems. First and foremost, it has been exceed-
ingly difficult to produce iHLCs that completely exhibit normal
hepatocellular function. Most notably, iHLCs do not exhibit an
adult-like range of CYP expression. Improvements in the
maturation of iHLCs will be needed in order to fully realize
their potential. Another difficulty (though soluble) is rooted in
human genetic variation. Hypothetically, one might repro-
gram somatic cells from three iDILI patients and three
control patients and then compare the two sets of iHLCs.
Although the iDILI patients may contain polymorphisms
relevant to iDILI, there exists a very large amount of
extraneous genetic variation between the two groups. Thus,
the confounding factor of genetic background may dilute
iDILI phenotypes in vitro. A new tool that partially addresses
this problem involves the use of new site-specific recombi-
nase systems such as clustered regularly interspaced
short palindromic repeats (CRISPR) or transcription activa-
tor-like effector nucleases (TALENS) to correct putative
SNPs or other polymorphisms suspected of causing iDILI,
thus yielding an isogenic pair of cell lines. This is discussed
further below.
Finally, a major limitation of stem cell models should be
recognized. Any cells derived from stem cells, even sophis-
ticated co-culture models, cannot fully recapitulate the
situation in vivo. For instance, it is likely that immune
reactions are important for some (though probably not all)
cases of iDILI. It is currently not feasible to combine iHLCs
with reconstituted immune systems in vitro. Placing iHLCs
into mice is also not a reasonable option to assess the
contribution of immune functions since the mouse immune
system differs in fundamental ways from the human immune
system.
In the future, improvements will need to occur if stem cell
approaches are to more faithfully model iDILI. Perhaps the
area most in need of improvement is the differentiation of
stem cells into fully functional hepatocytes that express a
normal range of CYP genes.55 Progress is being made in this
area through the use of three-dimensional differentiation
methods that more closely model cellular positions within
organs. Furthermore, new approaches that yield ‘‘organoids’’
(small three-dimensional multicellular structures with orga-
nized cells) may lead to better expression of CYP genes.56
Current applications and future directions
iDILI is a disorder that can arise from mutations in at least
several key genes, and it likely involves multiple cell types,
including hepatocytes and immune cells. It is also possible
(though little investigated) that epigenetic alterations in the
hepatocyte may lead to changes in gene expression and
contribution to the onset of iDILI. The advent of iHLCs
constitutes a novel step toward identifying genetic and
epigenetic mechanisms leading to predisposition to iDILI
and mechanisms involved in the acute phase of iDILI. A
number of useful attributes of these cells make them useful
for toxicological applications. Foremost, iHLCs are genetically
identical to the donor subject who donated somatic cells to
produce hiPSCs. In addition, the iPS reprogramming process
erases epigenetic modifications that accumulate in primary
Journal of Clinical Translational Hepatology 2014 vol. 2 | 143–152
cells in response to life exposure to toxicants.57–60 Finally,
iPSCs and iHLCs, can be produced in unlimited quantities,
whereas primary hepatocytes cannot be readily expanded.
Hence, the need for repeated liver biopsies from single
donors, necessary for conventional experimentation, can be
avoided. 61–65
Identification and study of genetic variants causing
iDILI
An obvious way to use iPS strategies to investigate iDILI is to
collect somatic cell samples (such as skin or nucleated blood
cells) and subject them to iPS-mediated reprogramming. The
use of iPSCs to investigate iDILI is only now coming to
fruition, but here is a reasonable workflow: Cells from at least
several subjects who have suffered from iDILI and normal
subjects are reprogrammed to yield a set of wild-type and
affected (iDILI) iPSCs. Since episodes of DILI affect the liver
by definition, it is quite possible that liver biopsies from
subjects who have survived (or succumbed) to DILI might be
abnormal in terms of their mutational load, gene expression,
or even epigenetic content. Since iPSCs are derived from non-
liver tissues, these cells are less likely to have undergone
drug-induced mutations or other alterations. Sets of iHLCs
derived from multiple normal and iDILI patients can then be
prepared and investigated to identify changes in gene
expression pathways that are potentially involved in iDILI.
The iPS approach can yield cells that can be useful to actually
test specific mutations in putative iDILI genes such as GSTT1,
GSTM1, POLG, and others, as described above.
A notable advantage of the iHLC-based iDILI cell culture
model stems from the ability to rescue causative mutations
through genome editing.66,67 Using TALENs 68,69 or CRISPR70
homologous recombination genome editing strategies, puta-
tive iDILI associated mutations can be repaired in iPSCs. The
resulting cells are isogenic with respect to the unedited iPSC
cells except for the mutation site. Subsequent differentiation
of the repaired and unrepaired iPSCs (which can differ by only
a single SNP of interest) to iHLCs yields a powerful pair of
experiment and control cell cultures, which may then be
interrogated for their response to iDILI-causing drugs. In
analogous fashion, using the same recombinase strategies,
iDILI associated mutations can also be introduced into the
genome of wild-type iPSCs to yield an equivalent pair of wild
type and mutant cell lines that are isogenic except for the
mutation under investigation. In addition to drug assays,
transcriptome and epigenome analyses can be utilized in such
paired cell culture approaches to learn more about the role of
specific iDILI-promoting mutations and the genes that host
such mutations.
Assessment of the contribution of epigenetic
regulation to iDILI
Exposure to iDILI-causing drugs may lead to the onset of
iDILI in response to epigenetic alterations in hepatocytes.
Such alterations could, in theory, be a direct result of
exposure to the drug. Alternatively, epigenetic changes might
occur over time due to indirect (but consequential)
responses71–73 that result in some patients in the deregula-
tion of the expression of proteins that are protective for iDILI.
Such genes may be epigenetically silenced more permanently
through trimethylation of lysine 27 of histone 3 and methyla-
tion of cytosine residues in CpG dinucleotides in gene
147

regulatory regions.74 Similar to perturbed histone acetylation
homeostasis in cancer,75 further histone deacetylation may
then compact the associated chromatin domains and render
the respective genes difficult to transcribe upon exposure to
subsequent treatment with an iDILI causing drug. It is likely
that candidate genes include not only those encoding proteins
but also microRNAs that are protective for iDILI. Certain
haplotypes may favor the acquisition of epigenetic marks
leading to silencing of iDILI protective genes, adding an
additional genetic component that may only be identifiable
through whole genome sequencing. In addition, insertion/
deletions (indels) and copy number variation for iDILI
protective genes may also have a role in the pathogenesis
of iDILI by affecting transcription levels and dosage of iDILI
protective genes. At present, little is known about an
epigenetic influence on iDILI and future research will need
to address this issue.
Transcriptome profiling provides a readout that integrates
both epigenetic and genetic contributions to gene regula-
tion.76,77 During diagnosis of iDILI in the clinic, it is common-
place to collect a small number of liver cells by needle
biopsy.78 From these, it is possible to isolate hepatocytes by
laser capture or magnetic bead purification approaches. RNA
can be extracted from the iDILI patient hepatocytes and
iHLCs are derived from the same individual. Since iPS
reprogramming erases only epigenetic information, differ-
ences in transcriptome profiles between the patient hepato-
cytes and iHLCs could be due to either iDILI-caused
epigenetic changes in the patient hepatocytes, or incomplete
accumulation of proper epigenetic chromatin modification
patterns due to infidelities that arise during iHLC differentia-
tion. Future research will be needed to determine if iDILI
epigenetic changes can be detected by such strategies.
However, such experiments may provide candidate genomic
targets for further evaluation using targeted chromatin-
immunoprecipitation (ChIP). The alternate approach using
ChIP-Seq instead of ChIP based comparisons may currently
be quite challenging due to the limited availability of primary
cells. In addition, iHLCs are exquisitely well suited for ‘‘omics’’
approaches since their nearly unlimited numbers allow for
sufficient transcriptional profiling experiments to achieve a
level of experimental replication that is sufficient for the
minimization of type one and type two errors.79 Such
replication cannot easily be carried out with primary cells.
Certain limitations, however, are inherent to this approach.
For instance, cell type heterogeneity among collected biop-
sies confounds the interpretation of large datasets. Laser-
capture microscopy based approaches may provide a viable
alternative to mixed cell populations normally retrieved via
biopsies. However, the limitations in the sample amounts
inherent to this technology require amplification based
strategies for expression profiling that can introduce a bias
sufficient to obscure the contribution of low abundance
transcripts to the pathogenesis of iDILI.80–82 Magnetic bead
capture of hepatocytes may supply a better alternative as
cells could be collected en masse. Alternatively, hepatocytes
could be expanded from the initial biopsy but primary
hepatocytes have only a limited life span in culture, are
difficult to expand, and suffer from drifting gene expression
that again can introduce bias sufficient to prohibit the
identification of transcripts relevant to iDILI. Clearly, iHLC
based transcription surveys, though currently not free from
drawbacks, are less prone to these limitations.
148 Journal of Clinical Translational Hepatology 2014 vol. 2 | 143–152
Krueger W. et al: Using stem cells for iDILI research
Possible xenobiotic-induced predisposition to iDILI
It is possible that prior or concomitant exposure to drugs, other
xenobiotics, or even high levels of endogenous metabolites
might predispose the onset of iDILI upon treatment with iDILI-
causing drugs. Little is known, however, about chemical
sensitization to iDILI, so this idea must be considered a
working hypothesis. Conventional medical studies typically do
not control for prior exposure to compounds that might induce
a predisposition to iDILI. Likewise, cell culture models using cell
lines such as HepG283 or HepaRG84 are limited since these cell
lines cannot capture genotype-based predispositions to the
disease. In contrast, iHLCs provide an excellent option to study
how prior treatment with predisposing compounds can make
cells susceptible to iDILI, since these cells are derived from
patients that have already been known to have developed
iDILI. For instance, two-staged high throughput screens can be
developed where iDILI-iHLCs are treated first with a compound
known to elevate oxidative stress in liver cells without causing a
disease phenotype. Subsequently, either in the presence or
absence of the predisposing compound, the iDILI-inducing
drug can be administered and toxicity can be monitored by
assays for increased cell death, reduced energy metabolism, or
induction of proinflammatory cytokine expression.
Certain types of toxicity, e.g., functional hepatic changes,
are difficult to assess in cell culture models. For example,
cholestasis, which is a frequent hallmark of DILI, has only
recently been modeled in vitro.85,86 Such functional assays
have not yet been applied to or validated for iHLCs. Because
polymorphisms and rare variants in bile salt transporters
have been implicated in the development of drug-induced
cholestasis, a genetic analysis of ABCB11 (BSEP), ABCC2
(MRP2), and other genes encoding for canalicular transpor-
ters could be analyzed.
Another possibility for xenobiotic-induced predisposition
to iDILI is immunological in nature. Since major histocompat-
ibility complex (MHC) genes have already been implicated in
iDILI, it follows that dysregulation of the immune system is
one route to the onset of iDILI. It may be that iDILI has much
in common with an allergic response, where the immune
system is first sensitized to a cellular antigen, perhaps by
an initial exposure to a DILI drug or a chemically-similar
compound. This initial exposure could induce the formation
of self-directed (hepatocyte-directed) immune cells, which
then become activated and amplified upon later exposure
to the iDILI-causing drug. Of note, iHLC approaches can
readily be used to study cell-autonomous mechanisms of
chemical sensitization but not immunological sensitization
mechanisms.
Future goals and directions with iHLC cell culture
systems
In order to fully leverage the power of the iHLCs as a cell culture
model for iDILI, iHLCs must be produced that more accurately
resemble the hepatocytes in vivo. In all current differentiation
protocols, iHLCs exhibit a phenotype resembling that of late
embryonic or early postnatal primary hepatocytes.87 For
instance, current state-of-the-art iHLCs express high levels of
a-fetoprotein (AFP) and CYP3A7,44,45,88–90 both markers for
embryonic hepatocytes.91,92 Consistent with fairly robust AFP
expression, ALB, a marker for adult primary hepatocytes, is
expressed at significantly lower levels. Similarly, current iHLCs
express low CYP3A4 and CYP2E1.44,45,88–90 It therefore
Krueger W. et al: Using stem cells for iDILI research
appears that, despite demonstrating other typical liver cellular
activities such as indocyanin green uptake, urea production,
glycogen storage, LDL uptake,93 and cholesterol secretion,44
current iHLCs may express only low levels of drug metabolizing
enzymes that have a key role in the pathogenesis in iDILI. The
low expression level of CYPs in particular may lead to a
significantly attenuated iDILI phenotype in vitro. Future
characterization of iHLCs may require the use of global
transcriptome comparisons between fresh primary hepato-
cytes, but this in itself may not guide the design of improved
methods to differentiate detoxification-competent iHLCs from
pluripotent cells.
A related technical problem is that primary hepatocytes
can be maintained in culture for only a short time, prior to loss
of detoxification activities typical of the hepatocyte in vivo.
For instance, cultured primary hepatocytes rapidly down-
regulate many CYPs, including CYP3A4, to nearly undetect-
able levels.94,95 It is possible that cell culture effects lead to
transcriptional deregulation of genes essential for the patho-
genesis of iDILI. Although significant improvements in
primary hepatocyte culture have been made over the last
few years64,96,97 permitting the maintenance of some key
hepatocyte enzyme expression for up to several weeks, the
length of the drug treatment required for eliciting an iDILI
response in culture is unknown and drug dependent. Perhaps
of even greater concern is that hepatocytes obtained from
iDILI patients by needle biopsy may be abnormal after an
episode of iDILI, and thus not suitable for experiments
designed to uncover the mechanisms whereby naı̈ve hepa-
tocytes are degraded due to a DILI event.
Based on the above, it is clear that there is much room for
improvement in the methodologies leading to the production
of iHLCs from human pluripotent cells. Three major strategies
to improve iHLCs are described here:
1. Use of decellularized animal liver matrices for iHLC
cultures. Promising recent advances have been made
using decellularized liver tissue to produce a native liver
extracellular matrix (LEM) as substrate for the growth of
primary hepatocytes in subsequent two-dimensional and
three-dimensional applications.98 Using LEM, the authors
reported significantly improved hepatic functions includ-
ing elevated albumin secretion and urea synthesis.
Moreover, long-term viability was also increased, but a
comparison of genome-wide expression profiles between
nascent hepatocytes and cells cultured long term was not
carried out. Nevertheless, the use of LEM represents an
exciting route that may lead to improved iHLC matura-
tion. Likewise, inclusion of sonic hedgehog (SHH) into the
stage two (hepatic induction) differentiation cocktail may
result in increased transcription of FOXA2,51,99 a tran-
scription factor that is critical at this stage for hepatic
induction. Further optimization of growth factor concen-
trations and timing of addition to the culture medium may
be another way to optimize iHLCs.
2. Three-dimensional and organoid cultures. Three-dimen-
sional cultures represent a family of approaches to produce
high quality iHLCs with more adult characteristics. Three-
dimensional approaches include the use of bioreactors,100
tethered spheroids, 96 three-dimensional-capable
substrates such as matrigel sandwich cultures,101–103
caprolactones,104 and alginates.105,106 In addition, three-
dimensional co-cultures of mouse embryonic fibroblasts
with hepatocytes61 as well as spontaneous organoid
Journal of Clinical Translational Hepatology 2014 vol. 2 | 143–152
formation in co-cultures of hepatocytes, immuneprogeni-
tors, and human umbilical cord vascular endothelial cells107
have given rise to greatly improved primary hepatocyte
cultures and are becoming more commonplace in the
pharmaceutical industry.
3. Humanized mouse livers. Transplantation of human
hepatocytes into mice constitutes a system that recapi-
tulates many features of the human liver. A major
advantage is the ability to produce engrafted human liver
tissue that is appropriately vascularized and contains the
major cell types present in the human liver. 50,108
Moreover, pharmacokinetic studies can be carried out
with a dose-response regimen that mimics the treatment
protocol in patients and apparent hepatic injury can be
monitored over several months, though interim tissue
harvesting would be necessary in most cases to assess
the state of the graft. Both the immune-compromised
FAH and the ALB-HSV-TK mouse models permit the
ablation of the host liver in a timed and controlled fashion
during which the host liver can be replaced by the human
implant.50,108 While in the FAH mouse dietary constraints
are required for maintenance of the host liver, in the ALB-
HSV-TK mouse, the hepatocytes are ablated in a tissue
specific fashion through uptake of dietary gancyclovir
where the albumin promoter ensures tissue specific
expression of HSV-TK. In addition, these liver replace-
ment models have the advantage that the iDILI-causing
drug is not metabolized primarily by the mouse liver,
which might result in a greatly attenuated response in the
human graft. A particularly attractive variation may be
the combination of liver organoid grafts and either the
FAH or the ALB-HSV-TK mouse as hosts.
Conclusions
In this review article, we have discussed existing knowledge
about iDILI, stem cell approaches to find iDILI genes and to
study iDILI mechanisms, and also provided a discussion of
current applications and future directions for this field of
research. Conventional human genetic approaches are unli-
kely to yield a comprehensive set of iDILI-associated poly-
morphisms. New approaches using pluripotent stem cells
(especially hiPSCs) promise to provide an expedient way to
find new iDILI mutations and to offer a way to study iDILI
mechanisms in vitro. Finally, future uses of hiPSCs from iDILI
patients were discussed, including the use of iHLCs to study
epigenetic contributions to iDILI, and xenobiotic-induced
sensitivity for iDILI.
Acknowledgements
This review article was supported by Connecticut Stem Cell
Research Grant 11SCDIS02.
Conflict of interest
None
Author contributions
Writing the review (WK, UAB, TPR).
149

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Review Article
Acetaminophen-induced Liver Injury: from Animal Models to
Humans
Hartmut Jaeschke*, Yuchao Xie and Mitchell R. McGill
Department of Pharmacology, Toxicology & Therapeutics, University of Kansas Medical Center, Kansas City, KS, USA
Abstract
Drug-induced liver injury is an important clinical problem and
a challenge for drug development. Whereas progress in
understanding rare and unpredictable (idiosyncratic) drug
hepatotoxicity is severely hampered by the lack of relevant
animal models, enormous insight has been gained in the area
of predictable hepatotoxins, in particular acetaminophen-
induced liver injury, from a broad range of experimental
models. Importantly, mechanisms of toxicity obtained with
certain experimental systems, such as in vivo mouse models,
primary mouse hepatocytes, and metabolically competent
cell lines, are being confirmed in translational studies in
patients and in primary human hepatocytes. Despite this
progress, suboptimal models are still being used and experi-
mental data can be confusing, leading to controversial
conclusions. Therefore, this review attempts to discuss
mechanisms of drug hepatotoxicity using the most studied
drug acetaminophen as an example. We compare the various
experimental models that are used to investigate mechan-
isms of acetaminophen hepatotoxicity, discuss controversial
topics in the mechanisms, and assess how these experi-
mental findings can be translated to the clinic. The success
with acetaminophen in demonstrating the clinical relevance
of experimental findings could serve as an example for the
study of other drug toxicities.
E 2014 The Second Affiliated Hospital of Chongqing Medical
University. Published by XIA & HE Publishing Ltd. All rights
reserved.
Keywords: Drug hepatotoxicity; Translational studies; Necrosis, Sterile inflam-
mation.
Abbreviations: AIF, apoptosis inducing factor; ALT, alanine aminotransferase;
APAP, acetaminophen; ASK1, apoptosis signal-regulating kinase 1; AST, aspartate
aminotransferase; Bax, Bcl2-associated X protein; CAD, caspase-activated
DNase; DAMPs, damage-associated molecular patterns; DILI, drug-induced liver
injury; EGF, epidermal growth factor; DMSO, dimethyl sulfoxide; GSH, glu-
tathione; GSK-3b, glycogen synthase kinase-3beta; HGF, hepatocyte growth
factor; HMGB1, high mobility group box 1 protein; IL, interleukins; JNK, c-jun N-
terminal kinase; MAP, mitogen-activated protein; Mkp-1, MAP kinase phosphatase
1; MPT, mitochondrial membrane permeability transition; mtDNA, mitochondrial
DNA; NAC, N-acetylcysteine; NAD, nicotinamide-adenine dinucleotide; NAPQI, N-
acetyl-p-benzoquinone imine; Nrf-2, nuclear factor-like 2; PARP, poly (ADP-
ribose) polymerase; PGC, proliferator-activated receptor gamma coactivator;
PHH, primary human hepatocytes; RIP, receptor interacting protein kinase; SECs,
sinusoidal endothelial cells; TLR, toll like receptors; TNF, tumor necrosis factor;
TUNEL, terminal-deoxynucleoitidyl transferased UTP nick end labeling; VEGF,
vascular endothelial growth factors.
Received: 08 May 2014; Revised: 06 June 2014; Accepted: 12 June 2014
q
DOI: 10.14218/JCTH.2014.00014.
*Correspondence to: Hartmut Jaeschke, Department of Pharmacology,
Toxicology & Therapeutics, University of Kansas Medical Center, Kansas City, KS,
USA. Tel: +1-913-588-7969, Fax: +1-913-588-7501, Email: hjaeschke@kumc.
edu
Journal of Clinical Translational Hepatology 2014 vol. 2 | 153–161
Introduction
Drug-induced liver injury (DILI) is a significant clinical problem
worldwide. Fundamentally, DILI can be divided into two
categories: idiosyncratic and predictive DILI. Idiosyncratic
DILI is mainly caused by therapeutic doses of drugs in
susceptible patients after days or months of treatment.1 It
generally affects only ,1 in 10,000 patients taking the drug
and the symptoms can range from mild, transient liver injury
and dysfunction to acute liver failure.2 The epidemiology and
disease patterns of many idiosyncratic hepatotoxic drugs have
been described; although the involvement of innate and
adaptive immune mechanisms is thought to be critical in most
cases, the actual mechanisms of liver injury remain largely
unclear.3 The limited progress in the understanding of the
pathogenesis of idiosyncratic DILI comes from the lack of
relevant animal models and the rarity of the disease, which
makes it difficult to study it in the first place. In addition,
genome-wide association studies in a large cohort of DILI
cases have failed to identify general risk factors for idiosyn-
cratic DILI.4 These findings suggest that genetic determinants
of DILI risk may be drug specific.4
In contrast, predictive DILI occurs mainly after intentional
or accidental overdose of a drug. The most clinically relevant
drug in this category is acetaminophen (APAP, paracetamol).
In the US, APAP overdose is responsible for 78,000 emer-
gency room visits and about 500 deaths per year.5 In
addition, APAP hepatotoxicity is the most frequent cause of
acute liver failure of any etiology, accounting for approxi-
mately 50% of all cases.6 Unlike idiosyncratic DILI, APAP
hepatotoxicity can be modeled in rodents, primary hepato-
cytes and in certain cell lines. An increasing number of
translational studies demonstrate that these animal models
are valuable tools to investigate the mechanisms of toxicity
and to identify potential therapeutic targets.7 However,
despite the unique situation of having relevant in vivo and
in vitro experimental systems available, there are still many
controversies that hamper progress in understanding the
mechanisms of APAP hepatotoxicity and, consequently, the
reliable identification of clinically relevant therapeutic targets.
This review will address these controversial topics, including
intracellular signaling mechanisms of toxicity, mode of cell
death, and the role of sterile inflammation from animal
models to the most recent clinical findings.
Acetaminophen: Intracellular mechanisms of toxicity
Many drugs cause hepatotoxicity by forming reactive meta-
bolites, which either initiate cell toxicity mechanisms directly
or, through formation of protein adducts (haptens), can
 Jaeschke H. et al: Acetaminophen hepatotoxicity

trigger immune-mediated toxicity.8,9 For APAP, it is well
established that the cell death mechanisms are initiated by
the formation of the presumed reactive metabolite N-acetyl-
p-benzoquinone imine (NAPQI), which is generated mainly by
the cytochrome P450 enzymes Cyp2e1 and 1a2 in mice and
humans.10,11 Although NAPQI can be readily detoxified by
conjugation with glutathione (GSH), the availability of GSH is
limited in case of an overdose.12 The resulting depletion of
GSH enables reactions of NAPQI with protein sulfhydryl
groups of cysteine, causing the binding of APAP to cellular
proteins.12 Originally, it was thought that the protein adducts
formed could directly trigger cell death, but no critical protein
target could be identified.13 In contrast, it was recognized
that the binding to mitochondrial proteins after APAP over-
dose correlated with toxicity,14 suggesting that mitochondria
could be a critical target. This observation was in agreement
with the impaired mitochondrial respiration and increased
mitochondrial oxidant stress observed after APAP overdose in
mice (Fig. 1).15,16 The enhanced superoxide formation leads
to generation of the potent oxidant peroxynitrite in mitochon-
dria.17 This oxidant stress, together with the uptake of
lysosomal iron,18 causes the formation of the mitochondrial
membrane permeability transition (MPT) pore, which is
responsible for the collapse of the membrane potential and
cessation of ATP synthesis.19–21 The critical role of this
oxidant stress for mitochondrial dysfunction and cell necrosis
has been shown by the protective effects of delayed treat-
ment with GSH or N-acetylcysteine (NAC)22–24 and the
aggravation of injury in mice with reduced MnSod (Sod2)
activity in mitochondria.25,26
Although the importance of the mitochondrial oxidant
stress is well established,27 there is a discrepancy between
early GSH depletion and mitochondrial dysfunction with some
oxidant stress and the delayed necrosis.28 This led to the
hypothesis that the initial oxidant stress is insufficient to
trigger the MPT and a ‘‘second hit’’ is needed to amplify this
oxidant stress. This second hit appears to be the mitogen-
activated protein (MAP) kinase c-jun-N-terminal kinase (JNK)
(Fig. 1), which is activated (phosphorylated) in the cytosol
very early during APAP toxicity in mice.29 P-JNK then
translocates to the mitochondria and triggers the MPT by
amplifying the mitochondrial oxidant stress.29,30 The effect of
P-JNK on the mitochondria is mediated by interaction with the
mitochondrial protein Sab (SH3 domain-binding protein that
preferentially associates with Btk), which is located in the
outer membrane.31 Knock-down of Sab attenuated JNK
activation and prevented APAP-induced liver injury, suggest-
ing that Sab is a critical link between JNK activation and
mitochondrial dysfunction.31 The critical role of JNK in the
pathophysiology of APAP-induced liver injury has been
documented by the protective effect of a JNK inhibitor and
by gene knock-down experiments.32 However, JNK does not
seem to be directly activated by the early events of APAP
toxicity. Instead, a number of upstream kinases have been
identified, all of which can be activated directly or indirectly
by the early oxidant stress generated during APAP-induced
mitochondrial dysfunction. These kinases include apoptosis
signal-regulating kinase 1 (ASK1),33 glycogen synthase
kinase-3beta (GSK-3b),34 mixed-lineage kinase 3 (MLK
3),35 receptor interacting protein kinase (RIP) 136 and
RIP3.37 However, several phosphatases that counteract JNK
activation have been shown to protect against APAP toxicity,
including MAP kinase phosphatase 1 (Mkp-1) and protein
tyrosine phosphatase 1B.38,39 Thus, the amplification of the

Fig. 1. Experimental models to study acetaminophen (APAP) hepatotoxicity. The most common models used to study APAP hepatotoxicity are mice, rats, primary
mouse and human hepatocytes (PMH and PHH, respectively), and hepatoma cell lines. However, the mechanisms of injury and mode of cell death differ. In mouse models
and in humans, APAP-induced liver injury involves mitochondrial damage, oxidative stress, c-jun N-terminal kinase (JNK) activation, and nuclear DNA fragmentation. The
mode of cell death in these models is oncotic necrosis. However, rats develop little or no oxidative stress and thus no injury, while hepatoma cells may develop injury but
through different mechanisms than mouse or human hepatocytes. In the latter case, the mode of cell death is almost always apoptosis. The results suggest that mice and
PHH are the best available models for the study of APAP toxicity.

154 Journal of Clinical Translational Hepatology 2014 vol. 2 | 153–161

Jaeschke H. et al: Acetaminophen hepatotoxicity
mitochondrial oxidant stress involves a complex and redun-
dant network of kinases and phosphatases. However, the
JNK-dependent and independent kinase network may not
only be involved in pro-death signaling pathways but can also
promote survival mechanisms such as promoting autophagy,
as discussed later.40
A consequence of the mitochondrial dysfunction is nuclear
DNA damage (Fig. 1). Genomic DNA fragmentation as a
hallmark of APAP toxicity in mice and mouse hepatocytes has
been recognized for some time.41,42 DNA fragments can be
internucleosomal fragments of approximately 180 base pairs
and multiples thereof, as detected by DNA ladder.41 This
implicates a role for endonucleases in this process rather than
oxidant stress.17 The nuclear translocation of endonuclease G
and apoptosis inducing factor (AIF) from the mitochondria
correlates with the nuclear DNA fragmentation. 43,44
Endonuclease G and AIF, which are mitochondrial intermem-
brane proteins, are released initially by mitochondrial outer
membrane permeabilization through a Bcl2-associated X
protein (bax) pore and later, after the MPT and mitochondrial
matrix swelling, by rupture of the outer membrane.45 The
DNA damage can activate DNA repair processes including
poly(ADP-ribose) polymerase (PARP), which may accelerate
cell death by excessive consumption of nicotinamide-adenine
dinucleotide (NAD). Although APAP-induced DNA damage
activates PARP and depletes NAD, the absence of the enzyme
actually enhanced the injury, indicating that activation of DNA
repair is beneficial.46 Thus, DNA damage caused by mito-
chondria-derived endonuclease and others contribute to cell
necrosis, and attempts to repair the damage limit the injury
and support recovery.
Damaged mitochondria are critical for the pathophysiology
of APAP-induced cell death. Endogenous defense systems
(autophagy) can remove damaged cell organelles and mod-
ified protein by enveloping them with membranes (autopha-
gosomes) and fusing these structures with lysosomes.47
Autophagy is activated after APAP overdose.48 Inhibition of
autophagy attenuates liver injury, suggesting that autophagy
is an adaptive mechanism to stress and limits injury.48 In
addition to classical autophagy mechanisms, newly recognized
mitochondrial spheroids can also contribute to the removal of
damaged mitochondria.49 However, these processes are only
effective in the outer area of risk.47 Additional effects, such as
drp1 translocation to mitochondria that promotes mitochon-
drial fission, appear to enhance cell death.37 The role of
mitochondria biogenesis remains unclear because the protec-
tion against APAP-induced liver damage by peroxisome
proliferator-activated receptor gamma coactivator (PGC)-
1alpha,was caused by the activation of nuclear factor-like 2
(Nrf-2)-dependent antioxidant genes.50 Thus, adaptive
mechanisms to drug-induced cellular stress are clearly affect-
ing liver injury and could be potential drug targets.
The vast majority of APAP-induced liver injury studies focus
on hepatocytes. However, non-parenchymal cells may also
play a role. In addition to resident and newly recruited
inflammatory cells, which will be discussed later, sinusoidal
endothelial cells (SECs) can be affected by an APAP over-
dose.51 Direct morphological evidence of SEC damage and
indirect evidence of SEC dysfunction (hemorrhage) have been
reported.52–54 Nitrotyrosine staining in SECs precedes staining
of hepatocytes, suggesting that SEC damage may be inde-
pendent of parenchymal cell injury.55 Isolated SECs exposed
to APAP from specific mouse strains show substantial GSH
depletion56 and lactoferrin protects SECs from APAP-induced
Journal of Clinical Translational Hepatology 2014 vol. 2 | 153–161
injury in specific mouse strains.57 The strain-dependent SEC
injury correlates with Cyp2E1 levels in these SECs.57 In
addition, severe SEC injury in susceptible strains leads to
extensive congestion and microvascular dysfunction and also
impacts parenchymal cell injury.52,56,57
All of the discussed mechanisms of APAP-induced liver
injury were investigated using a number of different in vivo
and in vitro models. Selecting the most appropriate experi-
mental model, which closely resembles the human patho-
physiology, is critical for the clinical relevance of the
mechanisms derived from these models.
In vivo models of drug hepatotoxicity
The most frequently used preclinical species for drug
hepatotoxicity are rats and mice. Testing new drugs in rats
is still required for standard safety evaluations by regula-
tory agencies. In the case of APAP-induced DILI, the rat is
clearly a poor model.12 Rats can metabolize APAP to form a
reactive metabolite and cause GSH depletion and protein
adducts formation, even in mitochondria.58 However, they
do not develop mitochondrial oxidant stress or JNK activa-
tion and consequently develop very little if any liver injury
(Fig. 1).58 It remains unclear at this point if the lack of
progression in rat liver is due to the delay in protein
adducts formation, or if as of yet unidentified specific
protein targets are not hit by NAPQI. Although there is
the potential to learn more about the initiating events, the
mechanisms of injury in rats and the severity of injury do
not reflect the human pathophysiology. Nevertheless, large
numbers of studies are still being published that test the
hepatoprotective potential of natural products using the rat
model (reviewed59). In addition to using an insensitive
animal model for their drug hepatotoxicity studies, another
caveat of most of these investigations is that the plant
extracts are largely uncharacterized and the potential effect
as a P450 inhibitor of one or more of the chemicals in the
extract is rarely tested.
The mouse model of APAP hepatotoxicity was established
in the 1970s.12 Based on the early mechanistic understand-
ing, NAC was developed as an antidote against APAP over-
dose. 60 More recently, the use of gene knock-out or
transgenic mice has helped to further popularize the mouse
model for investigation of DILI. Most of the mechanistic
insight into the pathophysiology has been gained from
experiments with the murine system. Fortunately, many
fundamental mechanisms of APAP hepatotoxicity in the
mouse have been confirmed in patients with APAP overdose61
and in human hepatocytes.62,63 In addition, the severity of
the overall liver injury is very similar between mice and
humans.61 However, the injury process progresses much
faster in mice than in humans, with peak alanine amino-
transferase (ALT) values, as indicator of liver cell death,
between 12 and 24 h in the mouse64 and 36–48 h in humans
after overdose.65 The reason for this delay in humans does
not appear to be reduced drug metabolism and reactive
metabolite formation but may be related to delayed mito-
chondrial protein binding and delayed JNK activation.63 Thus,
the mouse model of APAP hepatotoxicity is superior to other
animal models and most closely resembles the human
pathophysiology in terms of liver injury and recovery. In the
future, large animal models may be needed to better mimic
the acute liver failure observed in humans.66
155

In vitro models of APAP-induced hepatocyte cell death
In vitro models are indispensable tools to identify drug
toxicity and assess molecular mechanisms. Many of the
mechanistic details described in previous paragraphs were
investigated in freshly isolated mouse hepatocytes. However,
in addition to the obvious advantages of an isolated cell
preparation, there are limitations that need to be con-
sidered.67 Among others, primary hepatocytes lack non-
parenchymal cells, they are exposed to artificial media during
isolation and culture, and they are generally cultured under
hyperoxic conditions (room air oxygen levels).67 This may
lead to numerous gene expression changes, enhanced
oxidant stress, and many other potential modifications.67,68
Loss of cytochrome P450 enzyme activity over time in
primary cells in culture is one of the most critical gene
expression changes in studies of drug toxicity.69 This will
affect the sensitivity to drugs such as APAP. Recent advances
in sandwich culture techniques and 3D culture ameliorate
some of these problems,67 but this improvement comes with
a price. These additional manipulations are more cumber-
some and time consuming and likely increase the cost of the
experiments. Nevertheless, studies with primary cultured
hepatocytes can provide new mechanistic understanding
if the limitations are considered and the data are verified
in vivo.
In contrast to primary hepatocytes, many established
hepatocyte cell lines are readily available. An advantage of
these cells is that they proliferate easily, can be cryopre-
served and thawed out without much loss of functionality, are
easy to work with, and are available in large quantities.
However, most of these immortalized cell lines, including the
most popular hepatoma cell lines (e.g. HepG2, Hep3B, Huh7)
have a drastically modified gene expression profile and lack,
among other critical proteins, essential drug metabolizing
enzymes and transporters (Fig. 1).70,71 The caveat of work-
ing with these types of cells is that they still respond to
cellular stress such as APAP exposure with changes in gene or
protein expression.72 However, the relevance to human
pathophysiology is questionable because the nature of the
stress is different than a cell that has the capacity to generate
a reactive metabolite. This discrepancy is most clearly
demonstrated by the fact that APAP causes apoptotic cell
death in hepatoma cell lines73 but necrosis in primary
hepatocytes.19,28,42,63 If these hepatoma cells are trans-
fected with a specific cytochrome P450, e.g. CYP2E1, some of
the sensitivity can be restored.74
HepaRG cells are hepatoma cells isolated and differen-
tiated from a patient with hepatocellular carcinoma that are
metabolically competent, i.e. express a large number of drug
metabolizing enzymes, including P450 enzymes, and trans-
porter similar to primary hepatocytes.70 Although there are
some limitations of these cells, such as being derived from a
single donor and differentiating into both hepatocytes and
biliary epithelial cells, the fact that they are metabolically
competent hepatoma cells with unlimited availability is a
major advantage over most other hepatoma cell lines.70
Consequently, upon exposure to APAP, HepaRG cells develop
cell necrosis with many mechanistic characteristics similar to
mouse hepatocytes but with a time line closely resembling
that of human overdose patients.62
Primary human hepatocytes (PHH) are the most relevant
in vitro model for studying human pathophysiology. However,
availability is limited and it can be prohibitively expensive.
156 Journal of Clinical Translational Hepatology 2014 vol. 2 | 153–161
Jaeschke H. et al: Acetaminophen hepatotoxicity
Cryopreservation of cells can make them more easily avail-
able, but in general, cryopreserved cells are of lower
quality.While these cryopreserved cells may be suitable for
drug metabolism studies, they are not useful for many other
types of experiments. This is especially important for drug
toxicity studies. Freshly isolated cultured human cells are
clearly superior as they reflect closely the gene expression
pattern of the liver in vivo. Recent studies with APAP
hepatotoxicity documented the very close correlation
between the time line of toxicity (onset and peak of cell
death) in PHH and onset and peak of ALT elevation as an
indicator of liver injury in overdose patients.63,65 However,
the overall sequence of events leading to cell death after
APAP in PHH is very similar to mouse hepatocytes, including
GSH depletion, cytosolic and mitochondrial protein adducts
formation, JNK activation and translocation to mitochondria,
mitochondrial dysfunction, and collapse of the mitochondrial
membrane potential preceding cell death (Fig. 1). 63
Importantly, APAP induces necrotic cell death in PHH similar
to mouse hepatocytes.63 Overall, like the translational work
with overdose patient, these studies with PHH confirm that
the mouse model of APAP hepatotoxicity is the most relevant
animal model for studying these mechanisms.
Despite using clinically relevant experimental models,
some topics remain controversial. This is almost always an
issue of experimental design and interpretation of experi-
mental data.
Mechanisms of drug-induced cell death: Apoptosis
versus necrosis
The mode of cell death during APAP hepatotoxicity is con-
troversial. Although the vast majority of animal studies have
concluded that the injury is caused by necrosis, an increasing
number of reports suggest that apoptotic cell death plays a
significant role. The key problem is that there are very few
specific parameters for apoptosis, and even those can be
misinterpreted. For example, frequently used parameters
such as mitochondrial cytochrome c release, increased bax
protein expression and bax translocation to the mitochondria,
BH3 interacting domain death agonist (bid) cleavage, DNA
strand breaks (terminal deoxynucleotidyltransferase dUTP
nick end labeling (TUNEL) assay), and DNA laddering (inter-
nucleosomal DNA cleavage) are all observed during apoptosis
and necrosis, not allowing any distinction between these forms
of cell death.75
One of the more specific features of apoptosis is the
activation of caspases, including the initiator caspase-8 and
the executioner caspase-3.76 An extensive increase in
caspase activity in combination with processing of procas-
pases to the active fragments can be easily detected during
hepatic apoptosis.77 Consequently, caspase inhibitors are
highly effective in preventing apoptosis in the liver.77 In
contrast, during APAP-induced liver injury, no increase in
caspase-3 activity is detectable, and caspase inhibitors offer
no protection.78 A caveat of the use of pancaspase inhibitors
is the fact that they generally require the solvent dimethyl
sulfoxide (DMSO), a potent inhibitor of P450 enzymes,79 and
higher doses of caspase inhibitors may inhibit other proteases
through off-target effects.80
DNA fragmentation is observed during apoptotic and
necrotic cell death. Predominantly small fragments are
formed during apoptosis, and larger fragments are generated
during APAP-induced necrosis.81 The characteristic smaller
Jaeschke H. et al: Acetaminophen hepatotoxicity
DNA fragments during apoptosis (multiples of 180–185 base-
pairs) are formed by caspase-activated DNase (CAD).76 In
contrast, DNA fragmentation during APAP-induced cell death
is caused by mitochondria-derived endonuclease G and AIF,
as discussed above.43,44 One of the most frequently used
assays to visualize DNA strand breaks is the TUNEL assay.
Because the assay detects DNA strand breaks, apoptotic and
necrotic cells will stain positive. However, the staining
patterns are different, with mainly nuclear staining in
apoptotic cells and both nuclear and cytosolic staining in
necrotic cells.75 The cytosolic staining is likely caused by the
release of large DNA fragments due to karyorrhexis and
karyolysis, which are still recognized by the terminal deoxy-
nucleotidyltransferase.
In patients, nuclear DNA fragments were detectable in
plasma after APAP overdose.61 However, similar to the mouse
model, extensive procaspase-3 protein levels were observed
in blood of patients with APAP-induced liver injury but not in
patients without liver injury.61 In support of these findings, no
caspase-3 enzyme activity was measured in any APAP over-
dose patient.61 In addition, the caspase-cleaved cytokeratin-
18 fragment was either not detectable or represented only a
minor fraction (,15%) of the total full-length cytokeratin-18
released into the blood in these patients.82 Together these
results in patients and, as discussed in the previous para-
graph, in PHH support the hypothesis that, similar to mice
and mouse hepatocytes, the primary mode of cell death in
APAP hepatotoxicity in humans is necrosis.
As the example of APAP-induced liver injury demon-
strates, a clear distinction between apoptotic and necrotic
cell death cannot be achieved by assessing a single para-
meter. Use of a combination of parameters, e.g., caspase
activation, cell morphology, and DNA fragmentation, is
mandatory for solid conclusions. In addition, the use of
positive controls will give insight into how much parameters
change with a certain percentage of apoptosis and allow more
confident conclusions regarding the mode of cell death.
Knowledge of the type of cell death is not only important
because of the intracellular signaling pathways involved, but
also because it determines the degree of inflammation that
occurs in response to the tissue damage.
Pathophysiological implications of sterile
inflammation
Extensive cell necrosis causes the release of cell contents, as
indicated by the massive increase of liver enzymes in serum.
In recent years, it has been recognized that many of these
cellular components can activate various pattern recognition
receptors, such as toll like receptors (TLRs).83,84 These
damage-associated molecular patterns (DAMPs) released by
cells include high mobility group box 1 (HMGB1) protein,
nuclear DNA fragments, mitochondrial DNA (mtDNA), heat
shock proteins, hyaluronic acid, and many more.83,84 DAMPs
can activate TLRs on macrophages (e.g. DNA binds to TLR9,
HMGB1and heat shock proteins work through TLR4) to induce
the transcription of pro-inflammatory cytokines and activate
the inflammasome, which can trigger the cleavage of pro-
forms of certain interleukins (IL) and other cytokines (e.g.
pro-IL-1b, and pro-IL-18). Extensive cytokine formation and
release recruits neutrophils and monocytes into the liver with
the potential to aggravate the initial injury. This general
scheme of a sterile inflammatory response has been inves-
tigated in the mouse model of APAP-induced liver injury in
Journal of Clinical Translational Hepatology 2014 vol. 2 | 153–161
vivo. During APAP hepatotoxicity, HMGB1, DNA fragments,
heat shock proteins, and others are detectable in plas-
ma,61,85,86 as are the formation of cytokines53 and the
recruitment of first neutrophils53,87 and later monocytes.88
Thus, there is no doubt that the severe cell necrosis induces
an extensive sterile inflammatory response in mice. However,
it is controversial whether neutrophils and macrophages
actually enhance the injury or contribute to the repair and
recovery of the damaged liver by removing cell debris.
Although a few studies have suggested direct involvement
of neutrophils, most studies do not find evidence for
neutrophil cytotoxicity aggravating APAP-induced liver injury
(reviewed84). Importantly, liver neutrophils are not activated
during the injury phase, and deficiency of key neutrophil
adhesion molecules has no effect on APAP toxicity.89 In
addition, release of acetylated HMGB1 as indicator of macro-
phage activation is delayed in mice and occurs at the end of
the injury phase.85 Taken together, these findings suggest
that neutrophils, in addition to monocytes, are recruited into
the liver in preparation for regeneration.84,88 Cytokines
generated during the sterile inflammatory response may
modulate intracellular events and promote injury by inducing
inducible nitric oxide synthase and enhancing peroxynitrite
formation.90
In APAP overdose patients, DAMPs such as nuclear DNA
fragments, mtDNA, and HMGB1 are extensively released into
the plasma with a time course similar to ALT and aspartate
aminotransferase (AST).61,82 Interestingly, serum levels of
these DAMPs are better predictors of poor outcome (liver
failure and death) than ALT or AST.82,91 Certain cytokines,
including IL-6, IL-8, and monocyte chemoattractant protein
1, are substantially elevated during APAP toxicity.92 However,
neutrophils are not activated in patients during the peak of
liver injury.93 Neutrophil activation correlates with declining
ALT levels, i.e. with the recovery phase during APAP overdose
in patients.93 These findings suggest that no direct neutrophil
cytotoxicity is involved in the human pathophysiology.
Similarly, hepatic macrophages derived from resident
Kupffer cells and recruited monocytes contribute to the tissue
repair process during APAP-induced acute liver failure rather
than aggravate the injury.94 In addition to removing necrotic
tissue, the lost liver cells need to be replaced by proliferation
of surviving cells.
Regeneration
Liver regeneration is an intricate and well-orchestrated
process. The remnant liver will proliferate to reestablish the
original architecture and function after surgical or toxicologi-
cal insult.95,96 In some cases, the remaining hepatocytes will
proliferate in a hyperplastic manner to restore the functional
liver mass, while in others, liver progenitor cells will
participate to generate new hepatocytes.96–98 Generally, the
production of tumor necrosis factor (TNF) a and IL-6 by non-
parenchymal cells will prime the hepatocytes for proliferation,
and cooperative signals from vascular endothelial growth
factors (VEGF), epidermal growth factors (EGF), hepatocyte
growth factors (HGF), and cytokines will stimulate hepato-
cytes to overcome cell-cycle checkpoints to proliferate and
regain the normal liver size. This is followed by termination of
regeneration, which is primarily mediated by TGF-b and
renewed quiescence.95,97
Considerable progress has been made in our understand-
ing of the central mediators of liver regeneration. TNF
157

receptor knockout mice exhibit exaggerated injury and
impaired regenerative responses after APAP, which can be
explained by delayed mitogenic cytokine signaling.99 In mice,
APAP time-dependently induces IL-6 levels in serum and
liver.23,100,101 IL-6 knockout mice are more susceptible to
APAP challenge101 and display a delayed injury resolution due
to impaired liver regeneration.102 In addition, APAP triggers a
significant upregulation of VEGF in mice,103 while pharmaco-
logical inhibition of VEGF resulted in similar injury but
decreased recovery.103 Administering human recombinant
VEGF mitigates the injury after APAP and facilitates regen-
eration in mice.104 Indirectly, scavenging peroxynitrite by
glutathione treatment also enhances regeneration in mice
after APAP.100
More recent data have provided insights about additional
players in liver regeneration after APAP. The Wnt/b-catenin
pathway is emerging as a central player in the regulation of
liver development, growth, and regeneration;105 and its
importance in APAP-induced hepatotoxicity has been
explored. Biphasic increases of b-catenin expression are
observed in mice, and b-catenin knockout mice have reduced
liver regeneration.106 Interestingly, the evaluation of human
liver biopsy samples has indicated that b-catenin activation is
strongly associated with patients who undergo spontaneous
regeneration without liver transplantation.106 Similarly,
increased serum alpha-fetoprotein, a sign of hepatic regen-
eration,107 correlates with a favorable outcome in APAP
overdose patients.108,109 From studies like these, a better
understanding of liver regeneration may lead to improved
prognostic indicators that can supplement existing criteria to
determine candidates for liver transplantation among APAP
overdose patients.
Conclusions
APAP-induced liver injury is the most frequently encountered
drug hepatotoxicity and cause of acute liver failure in the
United States. In addition, APAP is the most used experi-
mental model to generate DILI. During the last decade, there
has been substantial progress in understanding the intracel-
lular mechanisms of cell death, sterile inflammation, and
recovery after APAP overdose. The most relevant models that
have fueled this new knowledge are in vivo mouse models,
primary mouse and human hepatocytes, and certain meta-
bolically competent hepatoma cell lines (e.g. HepaRG)
(Fig. 1). Additional models, such as humanized mice, are in
development and could expand the experimental tool box.
Importantly, translational studies with patients provide sup-
port for the clinical relevance of many experimental models.
It can be expected that this increased insight into toxicity
mechanisms in experimental models and in humans will lead
to improved therapeutic interventions and better strategies
to detect potential relevant hepatotoxic drugs in development.
Furthermore, the success with APAP from understanding
experimental systems to the human pathophysiology could
serve as an example for making relevant progress in other
areas of drug hepatotoxicity.
Acknowledgements
Work in the authors’ laboratory was supported in part by
grants from the National Institutes of Health (R01 DK070195
and R01 AA12916), the National Center for Research
158 Journal of Clinical Translational Hepatology 2014 vol. 2 | 153–161
Jaeschke H. et al: Acetaminophen hepatotoxicity
Resources (5P20RR021940-07), and the National Institute
of General Medical Sciences (8 P20 GM103549-07) of the
National Institutes of Health. Additional support came from
the ‘‘Training Program in Environmental Toxicology’’ T32
ES007079-26A2 (to M.R.M.) from the National Institute of
Environmental Health Sciences.
Conflict of interest
None
Author contributions
Writing the review and approval of the final version (HJ, YX,
MRM).
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161
Review Article
MicroRNAs in Drug-induced Liver Injury
Li-Min Li, Dong Wang and Ke Zen*
State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu, China
Abstract
Drug-induced liver injury (DILI) is a leading cause of acute
liver failure, and a major reason for the recall of marketed
drugs. Detection of potential liver injury is a challenge for
clinical management and preclinical drug safety studies, as
well as a great obstacle to the development of new, effective
and safe drugs. Currently, serum levels of alanine and
aspartate aminotransferases are the gold standard for
evaluating liver injury. However, these levels are assessed
by nonspecific, insensitive, and non-predictive tests, and
often result in false-positive results. Therefore, there is an
urgent need for better DILI biomarkers to guide risk assess-
ment and patient management. The discovery of microRNAs
(miRNAs) as a new class of gene expression regulators has
triggered an explosion of research, particularly on the
measurement of miRNAs in various body fluids as biomarkers
for many human diseases. The properties of miRNA-based
biomarkers, such as tissue specificity and high stability and
sensitivity, suggest they could be used as novel, minimally
invasive and stable DILI biomarkers. In the current review,
we summarize recent progress concerning the role of miRNAs
in diagnosing and monitoring both clinical and preclinical
DILI, and discuss the main advantages and challenges of
miRNAs as novel DILI biomarkers.
E 2014 The Second Affiliated Hospital of Chongqing Medical
University. Published by XIA & HE Publishing Ltd. All rights
reserved.
Introduction
Drug-induced liver injury (DILI) is a growing challenge
because of the increasing number of drugs used in medical
care, and the increasing number of individuals who take
them.1,2 A database (www.livertox.nih.gov) has been estab-
lished to provide up-to-date, comprehensive clinical informa-
tion on DILI for both the general physician and the specialist.
A recent epidemiologic study by Bjornsson and co-workers
suggested that approximately 20 new cases of DILI per
100,000 persons occur each year.2 DILI is also one of the
most frequent causes for the termination of drug develop-
ment or withdrawal of approved drugs, and therefore, has an
enormous economic impact on health care expenditures.3–6
Moreover, it is the leading cause of acute liver failure.7 Of the
Keywords: Biomarker; Diagnosis; Drug-induced liver injury (DILI); MicroRNA.
Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransfer-
ase; DILI, Drug-induced liver injury; miRNAs, microRNAs; ULN, upper limit of
normal.
Received: 15 May 2014; Revised: 2 August 2014; Accepted: 4 August 2014
q injury from specific drugs.38
DOI: 10.14218/JCTH.2014.00015.
* Correspondence to: Ke Zen, Nanjing University School of Life Sciences, 22
Hankou Road, Nanjing, Jiangsu 210093, China. E-mail: kzen@nju.edu.cn
162 Journal of Clinical and Translational Hepatology 2014 vol. 2 | 162–169
estimated 10000 documented drugs developed for humans,
more than 1000 have been associated with DILI.8 The main
causes of DILI in the United States are antibiotics, agents for
the central nervous system, health foods and dietary supple-
ments.4,9,10 In China, traditional Chinese medicine and anti-
tuberculosis drugs are the major causes of DILI.4,11,12
Children, women, and the elderly are more vulnerable to
DILI, and the susceptibility is related to genetic and environ-
mental factors.13–15 Although there are many consistent
features of DILI, early diagnosis is still challenging due to
the lack of specific and sensitive clinical features.8,16
Classification of DILI
DILI is usually categorized as non-idiosyncratic (predictable)
and idiosyncratic (unpredictable).17–19 The most common
example of non-idiosyncratic DILI is from acetaminophen,
which is one of the most commonly used medicines with a
very high safety profile when used properly.20,21 However, if
misused, either intentionally or accidentally, significant liver
injury can occur,22 which has a short latency period, and is
dose-related. This hepatotoxicity can also be studied in
animal models.23–26 Although idiosyncratic DILI has a
longer/variable latency and is less common, it comprises
the majority of clinical DILI cases.27–29 Idiosyncratic DILI
generally cannot be recapitulated in traditional animal models
or in clinical trials.28,30–32 Examples of this kind of DILI
include those related to amoxicillin/clavulanate, non-steroi-
dal anti-inflammatory drugs, and isoniazid.13,33,34
Pathogenesis and treatment of DILI
The bulk of drug metabolism occurs in the liver, which is also
vulnerable to damage from drug metabolites. The more
common idiosyncratic type of DILI has no significant relation-
ship with drug or dose,27–29 and the mechanism can be
divided into allergic35 and metabolic idiosyncratic.36 The
allergic form is closely related to the high variability of the
human leukocyte antigen system on chromosome 6,37–39
whereas the metabolic form is closely associated with the
genetic polymorphisms of individual drug-metabolizing
enzymes, such as cytochrome P450 enzymes,40 uridine
diphosphate glucuronosyltransferase,41 and N-acetyltrans-
ferase.42 In addition, oxidative stress and the host inflam-
matory response also play important roles in the
development of idiosyncratic DILI. There is mixed evidence
to support the role of host factors such as age, sex,
obesity,43,44 and chronic liver disease in the development of
DILI, and genetic predisposition appears to be a risk factor for
Treatment for most forms of DILI is focused on supportive
care and requires longitudinal monitoring of the patient and
Li M.L. et al: MicroRNAs as DILI biomarkers
laboratory work. Suspected cases of idiosyncratic DILI can be
categorized as hepatitic, cholestatic, or mixed on the basis of
the degree/ratio of abnormalities in the alanine aminotrans-
ferase (ALT) and alkaline phosphatase.45 However, a careful
evaluation for other causes of liver disease should be
performed, and sometimes, a liver biopsy is needed. The
mainstay of treatment for DILI is the immediate discontinua-
tion of the offending drugs, and avoidance of drugs with
similar chemical structures or pharmacologic effects. The
timing of discontinuation of hepatotoxic drugs is controver-
sial. Some investigators believe that the elevation of liver
enzymes, including aspartate aminotransferase (AST) or ALT
. 36 upper limit of normal (ULN) or alkaline phosphatase .
1.56 ULN, accompanied by elevation of bilirubin levels (. 36
ULN), indicate serious liver damage, and should trigger
immediate discontinuation of suspected causative drugs.46–49
The Federal Drug Administration has proposed the following
criteria for immediate cessation of the drug in question: ALT .
86 ULN, ALT . 56 ULN for two weeks, ALT . 36 ULN
accompanied by serum bilirubin . 26 ULN, pro-thrombin time-
international normalized ratio . 1.56 ULN, or the appearance
of liver damage symptoms. For oral agents, the severity of
many cases of DILI can be decreased by reducing the residual
drug in the gastrointestinal tract by means of gastric lavage,
catharsis, and adsorption, or the use of diuresis, hemodialysis
and other methods, within six hours of ingestion.50,51
Diagnosis of DILI
It is very important to have a reliable and predictable
biomarker for DILI, and considerable effort has been made
to identify markers specifically for early detection. The
Federal Drug Administration recently endorsed four standard
serum biomarkers, including ALT, AST, total bilirubin and
alkaline phosphatase, to help identify severe DILI events in
clinical trials.21,49,51 However, limitations of these current
blood-based biomarkers include an unacceptable frequency
of false positives/negatives, poor sensitivity, and lack of
tissue specificity. For example, serum ALT activity is also
associated with kidney damage and muscle necrosis.52–54
Liver damage is only considered when serum ALT levels reach
two to four times that of the control group, thus the best
treatment period can easily be missed.55 Fenofibrate can
induce elevated serum transaminases, but does not cause
significant liver damage, resulting in false positives.56,57 In
addition, the markers may become elevated only after
substantial and sometimes irreversible tissue damage.58 All
of these characteristics decrease confidence in the utility of
aminotransferases as biomarkers for DILI. Ideally, more
sensitive and predictive biomarkers that respond very early
before irreversible injury has occurred would offer improved
outcomes.
MicroRNAs
MicroRNAs (miRNAs) are a recently discovered class of small,
endogenous, non-coding, single-stranded RNAs that are 19–
24 nucleotides in length.59 They are highly conserved, and
regulate approximately 30% of all gene expression at a post-
transcriptional level.59 miRNAs were first reported in 1993 in
a study by Lee et al. who showed that Lin-4 controlled the
timing of sexual development in Caenorhabditis elegans.60 To
date, there are 24521 entries representing hairpin precursor
miRNAs, which correspond to 30432 mature miRNAs
Journal of Clinical and Translational Hepatology 2014 vol. 2 | 162–169
expressed in 206 species (miRBase Release 20; http://
www.mirbase.org). The major function of miRNA is to
modulate gene expression either by translational repression
or mRNA degradation. Binding of a miRNA to the 39-
untranslated region of mRNA with partial complementarity,
as occurs in animals,59,60 will inhibit translation, whereas
perfect complementarity, such as occurs in plants,59,61 will
specifically direct mRNA cleavage resulting in target gene
degradation.
Similar to other molecules involved in regulating gene
expression, the expression levels of miRNAs differ signifi-
cantly in various tissues and at distinct developmental
stages.59,61–63 The diversity of miRNA sequences, structures,
abundance and expression make them powerful regulators of
mRNA that are involved in development, proliferation,
differentiation, apoptosis, energy metabolism and other
physiologic processes.63 Therefore, alterations in miRNA
expression may reflect a change in the physiologic and
pathologic states. Fig. 1 shows the number of retrievable
manuscripts over the past 12 years in PubMed using
‘‘microRNA’’ and ‘‘biomarkers’’ as the keywords. An impress-
ive and increasing number of related studies were observed,
showing that the use of miRNA as a biomarker is currently
one of the most important topics in the field.
Recent studies have shown that there are a large number
of circulating miRNAs, and that altered miRNA expression
profiles are closely related to disease. The discovery of
miRNAs in circulating blood was first reported by Lawrie and
co-workers in 2008.64 Subsequently, the presence of stable
miRNAs in plasma and serum was reported by other
researchers.65,66 Gilad and co-workers found that miRNA
can be detected in serum, urine, saliva, and amniotic and
other fluids, and placenta-associated serum miRNAs are
significantly elevated during pregnancy.67 These studies
demonstrated that miRNAs are present in bodily fluid of
humans and other animals, such as mice, rats, bovine
fetuses, calves and horses, which laid the foundation for the
use of miRNAs as noninvasive biomarkers, and their applica-
tion in cancer prevention and disease diagnosis.68–71
MicroRNA and pathogenesis of DILI
Responses to xenobiotics can be regulated or modulated by
miRNAs in liver. For example, the exposure of rats to liver
hepatotoxins, such as acetaminophen or carbon tetrachloride,
results in altered expression of various miRNAs, including a
decrease in miR-298 and miR-370, which are thought to
regulate an oxidative stress-related gene.72 An increase in
levels of several oncogenic miRNAs, such as the 17–92 cluster,
miR-106a, and miR-34, was detected in rat livers following
exposure to tamoxifen, a potent hepatocarcinogen.73 Another
research group demonstrated that dioxins, which are ubiqui-
tously present in the environment and tend to accumulate in
humans and wildlife, alter the expression of miR-101a and its
target, cyclooxygenase-2, which plays a significant role in liver
damage.74 Using the same method, Endo et al. found that in
patients with suppressed expression of miR-106b, the hepa-
totoxic drug halothane caused upregulation of signal transdu-
cer and activator of transcription 3, which was involved with
the resultant severe liver injury.75 Furthermore, there are
many studies reporting that drug-metabolizing enzymes, such
as cytochrome P450 1B1, which is highly expressed in human
liver, are targeted by certain miRNAs.76–78 Taken together,
these studies demonstrate that miRNAs play a significant role
163

Fig. 1. The number of articles listed on Pubmed concerning miRNAs as biomarkers published from 2002 to 2013
in the pharmacologic and toxicologic progression of hepato-
toxicity. As shown in Fig. 2, miRNAs may be involved in
multiple DILI developmental processes via regulation of their
target genes.
Circulating miRNAs as potential biomarkers of DILI
Although studies of miRNAs as biomarkers are primarily
concentrated in cancer research,69,70 their potential as
toxicologic biomarkers have also been recently explored.79
Over the past several years, many animal and clinical studies
have been published showing that miRNAs have an advan-
tage over the conventional biomarkers for DILI. They are
exceptionally stable, can be highly liver-specific and remark-
ably altered in pathologic states, are readily detectable in
easily accessible bodily fluids, and are strictly conserved
among species. Table 1 summarizes the published reports
using circulating miRNAs in the blood and urine as new DILI
biomarkers.
Serum or plasma miRNAs as biomarkers in
experimental DILI
A pioneer study by Wang et al. examined miRNAs as novel
biomarkers in a well-established mouse model of acetamino-
phen-induced liver injury.80 The authors demonstrated that
the liver-enriched miR-122 and miR-192 were the top two
miRNAs elevated in blood in a dose-and exposure time-
dependent manner. The levels of these miRNAs preceded and
paralleled serum ALT and AST levels and corresponding liver
histopathology.
Several independent groups have provided additional
data supporting the use of miRNAs as DILI biomarkers.
In 2010, one group confirmed the time-dependent increase
of plasma miR-122 levels in mice that correlated with liver
164 Journal of Clinical and Translational Hepatology 2014 vol. 2 | 162–169
Li M.L. et al: MicroRNAs as DILI biomarkers
histopathology induced by D-galactosamine and alcohol.81
They also confirmed that the changes of miR-122 were larger
than and preceded the changes in ALT. Importantly, signifi-
cant increases in miR-122 were detected before obvious
histopathologic changes in the liver, suggesting that miR-122
could be used for diagnosing and monitoring disease at early
stages. Bala and co-workers discovered that serum plasma
miR-122 and miR-155 were predominantly associated with
the exosome-rich fraction in alcoholic and inflammatory liver
injuries, whereas in acetaminophen-induced liver injury,
these miRNAs were present mainly in the soluble protein-
rich fraction.82 These results suggest that circulating miRNAs
may serve as biomarkers to differentiate between hepatocyte
injury and inflammation, and the exosome- or protein-
associated miRNAs may provide further specific mechanisms
of liver pathology. The same group also compared 40 plasma
miRNAs that were dysregulated with lethal or sub-lethal
doses of acetaminophen, and found that miR-574-5p, miR-
135a, miR-466g, miR-1196, miR-466f-3p and miR-877 were
upregulated in the setting of lethal hepatotoxicity, while miR-
342-3p, miR-195, miR-375, miR-29c, miR-148a and miR-652
were markedly downregulated.83 Thus, differential expres-
sion of miRNAs in human plasma may be useful to distinguish
lethal and sub-lethal hepatotoxicity.
Compared with mice, the susceptibility to acetaminophen-
induced liver injury is lower in rats.84 Using rat models
created by the administration of chemical or special diets,
Yamaura et al. examined the levels of the plasma miRNAs in
acute liver injury (hepatocellular injury or cholestasis) and
chronic liver injury (steatosis, steatohepatitis, and fibrosis).85
Their results showed that miR-122 levels increased more
quickly and dramatically than levels of aminotransferases,
reflecting the extent of hepatocellular injury. Importantly,
their study also demonstrated that the expression profiles of
plasma miRNAs differed according to the type of liver injury,
Li M.L. et al: MicroRNAs as DILI biomarkers

Fig. 2. Pathways by which miRNAs may be involved in the development of drug-induced liver injury (DILI). Drugs induce hepatocyte injury through their toxic
metabolites generated via CYP450. The toxic metabolites can induce the consumption of ATP, production of reactive oxygen species and formation of drug-protein/DNA
complexes, which result in mitochondrial uncoupling and hepatocellular apoptosis. When there is no ATP consumption, the reactive metabolites increase the mitochondrial
permeability, causing an increase in cytosolic Ca2+ and cell necrosis. All of these processes can affect the expression of miRNAs, and the deregulated miRNAs can, in turn,
regulate the development of DILI through their target genes.

suggesting that these miRNAs could be specific and sensitive
biomarkers for various types of liver injury. Su et al. also
reported that miR-122, miR-192 and miR-193 have the
potential to serve as sensitive, specific and noninvasive
biomarkers for the diagnosis of herb-induced liver damage.86
Furthermore, Starckx and co-workers demonstrated that the
levels of miR-122 in rat plasma were significantly increased
following administration of four well-characterized com-
pounds associated with different types and mechanisms of
liver toxicity: acetaminophen, allyl alcohol, alpha-naphthyl
isothiocyanate, and phenobarbital.87 The response of miR-
122 secretion by mouse liver cells paralleled that of other
markers, and was consistent with liver injury as indicated by
ALT/AST and histopathologic evaluation. The changes of
plasma miR-122 were also detected significantly earlier than
other conventional biomarkers, and exhibited a wide dynamic
range. Taken together, all the above discoveries demonstrate
that miR-122 has great potential to be used as a biomarker of
liver injury and may provide added value for assessing liver
toxicity in both preclinical studies and the development of
new drugs.
Urinary miRNAs as noninvasive biomarkers in
experimental DILI
The discovery of circulating miRNAs in the urine and other
bodily fluids has created a new approach for the discovery of
noninvasive biomarkers of organ injury.88 For example,
urine-derived miRNAs could become useful biomarkers for
kidney89 and bladder diseases,90 or even for DILI. In a rat
DILI model, urinary levels of some miRNAs were elevated,
likely released from the liver after injury.91 High-dose
acetaminophen-treated rats showed elevations of serum
ALT/AST, histologic signs of liver injury, and significant
increases in urinary miRNAs levels. Although low-dose
acetaminophen-treated rats did not show histologic signs of
liver injury or changes in serum ALT/AST, urinary levels of
nine miRNAs were substantially increased. Similarly, carbon

Journal of Clinical and Translational Hepatology 2014 vol. 2 | 162–169 165

166
Table 1. Circulating miRNAs in drug-induced liver injury (DILI)

Sample Quantification
Drug or chemical Species type method miRNA expression in DILI Clinical relevance Reference
72
Acetaminophen, CCl4 Rat Liver miRNA array, miR-153q, miR-302bq, miR-337q, Early diagnosis
qPCR miR-363q, miR-409-5pq,
miR-542-3pq, miR-29cQ, miR-298Q,
miR-327Q, miR-342Q, miR-370Q,
miR-376cQ,
miR-494Q, miR-503Q
80
Acetaminophen Mouse Plasma miRNA array, miR-122q, miR-192q, miR-193q, Early diagnosis
qPCR miR-710Q, miR-711Q, miR-483Q
81
D-GalN/LPS, alcohol Mouse Plasma qPCR miR-122q Diagnosis and monitoring
92
Acetaminophen, unidentified drugs Human Plasma qPCR miR-122q, miR-192q Diagnosis and
pharmaceutical evaluation
82
Alcohol, CpG/LPS, acetaminophen Mouse Plasma, qPCR miR-122q, miR-155q, miR-146aq Differentiate hepatocyte
Serum injury and inflammation
87
Acetaminophen, ANIT, allyl alcohol, Rat Plasma qPCR miR-122q Diagnosis and monitoring
phenobarbital, doxorubicin
96
Paraquat Human Serum qPCR miR-122q, miR-192Q, miR-483Q, Diagnosis
miR-711Q
93
Acetaminophen Human Whole miRNA array, miR-29cq, miR-19aq, miR-19bq, Diagnosis
Blood qPCR miR-802q, miR-374aQ, miR-505Q
86
Acetaminophen, herb drug Rat Serum miRNA array, miR-122q, miR-192q, miR-193q, Diagnosis
qPCR miR-200aq, miR-101aq, miR-323Q,
miR-322Q, miR-327Q, miR-380Q,
miR-214Q, miR-342-3pQ
83
Acetaminophen Mouse Plasma miRNA array, miR-574-5pq, miR-135a*q, Medication guidance
qPCR miR-466gq, miR-1196q,
miR-466f-3pq, miR-877q,
miR-342-3pQ, miR-195Q, miR-375Q,
miR-29cQ, miR-148aQ, miR-652Q
85
Acetaminophen, ANIT, methapyrilene, Rat Plasma miRNA array, miR-200a*q, let-7c-1*q, miR-503q, Diagnosis of different
CCl4 qPCR miR-337-3pq, miR-10bq, miR-190Q, types of liver injury

Journal of Clinical and Translational Hepatology 2014 vol. 2 | 162–169

miR-743bQ, miR-449cQ, miR-410Q,
miR-10b*Q
91
Acetaminophen, CCl4, penicillin Rat Urine miRNA array, miR-185q, miR-296q, miR-484q, Diagnosis of different
qPCR miR-434q, miR-20b-3pq, miR-330*q, types of liver injury
miR-433q, miR-664q, miR-291a-5pq,
miR34c*q

Abbreviations: ANIT, alpha-naphthyl isothiocyanate; CpG, cytidine-phosphate-guanosine; CCl4, carbon tetrachloride; D-GalN, d-galactosamine; LPS, lipopolysaccharide; qPCR, quantitative real-time polymerase chain
reaction.
Li M.L. et al: MicroRNAs as DILI biomarkers
Li M.L. et al: MicroRNAs as DILI biomarkers
tetrachloride also led to an increase in urinary levels of 28
miRNAs, among which ten overlapped with the 44 identified
in the acetaminophen-induced model. Therefore, urinary
miRNAs appear to be another form of noninvasive DILI
biomarker and may be useful for the classification of
hepatotoxins. However, further studies are needed to exam-
ine their organ-specificity in detecting DILI.
Circulating miRNAs as biomarkers in human DILI
A study examining human subjects who suffered DILI showed
that the plasma levels of both miR-122 and miR-192 were
substantially higher in patients who suffered acetaminophen-
induced liver injury than in those who did not.92 Moreover,
levels of the liver-enriched miR-122, but not miR-192,
correlated with serum ALT levels, consistent with results
from a previous study in a mouse model.80 Their findings also
showed that the level of circulating miR-122 decreased to
baseline much earlier than serum ALT did, suggesting that
miR-122 has a shorter circulatory half-life. This work provided
the first convincing evidence that circulating miRNAs could be
used as a human DILI biomarker. Jetten et al. also demon-
strated that miR-19b and miR-29c were upregulated in
human blood cells after treatment with low-dose acetamino-
phen.93 However, the level of ALT was not altered. These
results suggest that the expression profile of circulating
miRNAs may be altered at a very early stage when liver
damage is undetectable using conventional markers. Indeed,
another study showed that miR-122, along with high mobility
group box-1 and full-length and caspase-cleaved keratin-18,
were more sensitive than ALT at identifying acetaminophen-
induced acute liver injury.94 Thulin and colleagues also
showed that serum keratin-18 and miR-122 levels were
significantly increased at an earlier time point and to a
greater extent than ALT in patients with DILI.95
Paraquat is one of the most common toxic herbicides, and
is widely used around the world. By comparing paraquat-
exposed human subjects with healthy donors, Dingand and
co-workers found that the serum levels of miR-122 were
strongly increased and correlated well with the status of liver
function.96 This pattern was similar to that was seen in the
mouse acetaminophen-induced liver injury model. 80
However, miR-192, also identified in the mouse study, was
unexpectedly decreased two-to-eight-fold in human hepatic
samples.
Conclusions
The prediction, diagnosis and management of DILI are very
complicated issues. The current biomarkers or approaches to
assess DILI include biochemical markers such as amino-
transferases, total bile acids, histopathology or ultrastructural
pathology, active metabolites and immune-related markers.
However, due to the poor sensitivity, stability or specificity,
these conventional toxicologic biomarkers cannot provide
accurate information for early evaluation of liver hepatotoxi-
city. The development of novel, reliable and informative
biomarkers remains an important issue in clinical and
preclinical settings. Liver-specific miR-122 has been shown
to be involved in various processes of liver development,
differentiation, metabolism and stress responses.97–99
Compared with the conventional hepatotoxic markers, circu-
lating miR-122 can effectively and consistently distinguish
intrahepatic from extrahepatic damage with higher sensitivity
Journal of Clinical and Translational Hepatology 2014 vol. 2 | 162–169
and specificity.92–96 Thus, miR-122 is expected to be a
preclinical and clinical biomarker of DILI.
Although the use of circulating miRNA levels for early
diagnosis of DILI shows promise, further study is required
before it is used in clinical applications. The major issues to be
addressed include: 1) lack of association between miRNAs
and mechanistic information on DILI; 2) lack of drug-specific
miRNAs to diagnose various types of DILI; 3) lack of larger
sample sizes validating the assay; and 4) lack of conventional
detection methods that can be carried out in routine clinical
laboratories, including standardized detection systems, stan-
dard reference materials, and quality control materials.100,101
More studies are required to demonstrate the mechanisms
underlying the phenotypes of DILI, including research and
investigation on the alterations of miRNAs in liver tissues
after treatments with various drugs representing a broad
range of DILI types, combining miRNAs with other biomar-
kers, and multicenter collaborations.
In conclusion, the use of miRNAs, especially miR-122, as
noninvasive biomarkers for DILI shows great potential, but is
still in its infancy. More research on circulating miRNAs is
needed to validate the use of miRNAs as novel biomarkers of
DILI.
Acknowledgments
This work was supported by grants from the National Basic
Research Program of China (973 Program, 2012CB517603
and 2011CB504803), the National Natural Science
Foundation of China (No.31301061), the Natural Science
Foundation of Jiangsu Province (No. BK2011013,
BK20130564), and the Specialized Research Fund for the
Doctoral Program of Higher Education (20130091120037).
Conflict of interest
None
Author contributions
Analyzing the data (LML, DW), drafting the manuscript (LML,
KZ).
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169
Review Article
Chinese Herbal Medicine-induced Liver Injury
Xin Ma1, Jing-Hua Peng*1,2 and Yi-Yang Hu1,2,3,4
1
Institute of Liver Diseases, Shuguang Hospital affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai,
China; 2Key Laboratory of Liver and Kidney Diseases, Shanghai University of Traditional Chinese Medicine, Ministry of Education,
Shanghai, China; 3E-institute of Shanghai Municipal Education Committee, Shanghai University of Traditional Chinese Medicine,
Shanghai, China; 4Shanghai Key Laboratory of Traditional Chinese Clinical Medicine, Shanghai, China
Abstract
The widespread use of Chinese herbal medicine (CHM) and the
associated adverse reactions has attracted the attention of
researchers and physicians. Reports have shown that several
types of CHM can cause liver injury, with increasing numbers of
cases reported every year. The difficulty in characterizing CHM-
induced liver injury stems from clinical manifestations, diag-
nosis and pathogenesis. The clinical manifestations are varied,
but gastrointestinal symptoms are the majority. The Council for
International Organizations of Medical Sciences scale is cur-
rently the most commonly used method for assessing causality
in cases of medicine-induced liver injury with excellent sensi-
tivity, specificity and predictive validity. However, the pathogen-
esis of CHM-induced liver injury is not well understood. The
classic view encompasses a contribution from ‘‘toxic metabo-
lites’’ that either elicit an immune response or directly affect
cellular biochemical processes or functions. In addition, poor
quality and inappropriate clinical use of CHMs contribute to
safety concerns. To ensure the safe use of CHMs and decrease
the number of hepatotoxic cases, clinicians, researchers and
pharmaceutical companies should share responsibility by
regulating clinical use, strengthening basic toxicology research
and establishing a strict quality control system.
E 2014 The Second Affiliated Hospital of Chongqing Medical
University. Published by XIA & HE Publishing Ltd. All rights
reserved.
Introduction
For thousands of years, herbs have been used to treat disease
in China. Based on the prolonged experience and the original
treatment principles, traditional Chinese medicines, which
include pharmacy components, are being used in clinics today
as an alternative and complementary option to modern
Western medicine. Many herbal medicines are commonly
used to treat the elderly, pregnant women, and children.
Nearly 1.5 million people in the United States have tried
herbal treatments, with an annual cost as much as $1.5
Keywords: Chinese herbal medicine; Liver injury.
Abbreviations: CHM, Chinese herbal medicine; CIOMS, Council for International
Organizations of Medical Sciences; DILI, drug-induced liver injury; HILI, herbal
drug-induced liver injury.
Received: 27 February 2014; Revised: 13 August 2014; Accepted: 15 August
2014
q
DOI: 10.14218/JCTH.2014.00009.
* Correspondence to: Jinghua Peng, No. 528 Zhangheng Road, Pudong District,
Shanghai 201203, China. Telephone: +86-021-20256526; Fax: +86-021-
20256521, Email address: pengjinghua2004@163.com
170 Journal of Clinical Translational Hepatology 2014 vol. 2 | 170–175
billion.1 The use of traditional Chinese medicine has been
popular in Germany since the 1980s, and more than 58% of
the German population use herbal remedies,2 which are
accessible in pharmacies.
Reports involving adverse reactions caused by Chinese
herbal medicine (CHM) have gradually emerged. In recent
years, clinical cases and laboratory data have shown that
CHMs and their preparations may cause varying degrees of
liver damage. Therefore, an understanding of herbal hepato-
toxicity and rational use of CHM is of great importance. The
aims of this review were to discuss clinical cases of liver injury
caused by CHM, describe the diagnosis, clinical patterns and
mechanisms of hepatotoxicity, explore the common causes of
adverse reactions, detail current shortcomings and make
suggestions for future prospects.
Clinical overview
Reported cases
A comprehensive analysis of Chinese academic journals
indicated that of the 9355 total cases of drug-induced liver
injury (DILI) reported between 2001 and 2010, approximately
20% of these (1979 cases) were associated with herbs.3
Another report by Jie et al. examining 337 cases of DILI from
2009 to 2012 found that 44.2% of the cases of liver injury were
caused by CHM.4 Although the incidence rates of herbal drug-
induced liver injury (HILI) are different among the various
regions of China, the percent of CHM-induced liver injury from
literature published in 2013 ranged from 26.4 to 49%. These
data indicate that CHM-induced liver injury in China is an issue
that cannot be ignored. Although there are fewer relevant
reviews and case reports of liver injury caused by herbal
medicine from other countries, the annual average number of
herbal hepatotoxicity-related publications in PubMed has
increased from 4.2 in 1990–1999 to 9.9 in 2000–2009. There
are currently 185 articles, and nearly 60 kinds of herbs
implicated in possible liver damage.5 Based on available data
of DILI cohorts from the US and Europe, herbal products may
be a cause of hepatotoxicity in 2–11% of patients with DILI,
and in 5–10% of patients with drug-induced acute liver failure.6
Clinical characteristics
In general, compared with Western medicine, liver damage
caused by CHM develops slowly with clinical symptoms
appearing within one week to one month.7 The clinical
manifestations can be classified as acute hepatitis, sub-acute
hepatitis, and cirrhosis. Clinical symptoms such as fatigue
Ma X. et al: Chinese herbal medicine-induced liver injury
(67.3%), jaundice (60.3%), anorexia (58.0%), nausea
(35.9%), and fever (19.3%) are more common, but signs
such as rash, pruritus and clay-colored stools have also been
reported.8 CHM-induced liver injury can also develop into
liver failure. A study of 418 patients with DILI by Teng et al.
reported 81 cases of liver failure, 19.4% of which had clinical
manifestations similar to those caused by viral or alcoholic
hepatitis.8 Investigations regarding HILI also suggest that
women are more susceptible,4,9 especially for those 60–65
years of age.10 The susceptibility may be due to age- and sex-
related differences in hepatic microsomal enzyme activity.
Diagnosis
Because of the variety of clinical features and time of onset,
there is no gold standard for diagnosing DILI or CHM-induced
liver injury. The Council for International Organizations of
Medical Sciences (CIOMS) scale is the most commonly used
method for assessing causality in cases of DILI11 and HILI,12
and is currently considered as the best causality assessment
method. For physicians treating a patient with suspected DILI
or HILI, the CIOMS scale can be used to prospectively collect
all necessary parameters without requiring an expert panel.13
Compared to other assessment methods, including the
Maria–Victorino scale,14 Aithal and Day method,15 Aithal,
Rawlins, and Day method,16 and Takikawa, Takamori, and
Kumagi scale,17 the CIOMS scale offers excellent sensitivity,
specificity and predictive validity (based on cases with
positive re-exposures).18 Due to the complexity of Chinese
medicinal ingredients, it is difficult to determine the reference
data on herbal hepatotoxicity, and thus misdiagnoses and
missed diagnoses are more likely to occur. Therefore, the
clinical diagnosis of liver injury caused by herbal medicine
focuses more on medical and medication histories. Physicians
should first make a comprehensive comparison of clinical
manifestations and related enzymology indices before, during
and after treatment, followed by a diagnosis of exclusion. The
important conditions to exclude are hepatitis A, B, and C,
alcoholic liver disease, biliary obstruction, and liver injury due
to ischemia or other medications.19 Although causality
assessment methods, such as the CIOMS scale, are better
approaches for reaching the correct diagnosis of DILI and
HILI than liver histology,20 liver biopsy should still be
considered as a final diagnostic approach in rare instances
of diagnostic uncertainty.
CHM that have been linked to hepatotoxicity
The following CHMs reportedly can cause liver injury:
Dioscorea bulbifera, Gynura segetum, Fructus xanthii,
Polygonum multiflorum, Tripterygium wilfordii Hook F,
Folium artemisiae argyi, Occidentalis, Atractylodes lancea,
trichosanthin, Loranthaceae, Cyrtomium fortunei, cattail
pollen, ephedra, Bupleurum, Folium senna, centipede,
Albizia julibrissin, clove, Fructus toosendan, Brucea javanica,
Ilex pubescens, castor beans, Veratrum nigrum, Salvia
miltiorrhiza, Papaver somniferum, Rhizoma Pinelliae, Alisma
orientalis, rhubarb, Polygonum cuspidatum, groundsel,
Menispermaceae, Dysphania ambrosioides, nutmeg,
Phytolacca acinosa, antipyretic Dichroa, chaulmoogra, cinna-
bar, Rafetus swinhoei, pangolin, Scutellaria, valerian, aconite,
and ginkgo.21 The known hepatotoxic components in CHM
can be divided into five categories according to their chemical
structures: alkaloids, terpenoids, glycosides, toxic proteins,
Journal of Clinical Translational Hepatology 2014 vol. 2 | 170–175
and tannins.22 Some of the most common CHMs that cause
hepatotoxicity are shown in Table 1.
The pathogenesis of liver injury caused by CHM
Although little is known about the pathogenesis of CHM-
induced liver injury, there are various theories on the
pathogenesis of DILI, including the participation of ‘‘toxic
metabolites’’ that either elicit an immune response or directly
affect cellular biochemical processes or functions.23
Direct toxicity
Drugs are generally safe when given at recommended doses.
However, the intrinsic toxicity of a drug at higher doses is a
major cause of acute liver failure and transplantation.24 DILI
is initiated by direct hepatotoxic effects of a drug or its
reactive metabolites.25 Hepatocyte death, which can be
either apoptotic or necrotic,26 is the primary outcome,
although bile duct epithelium or sinusoidal endothelial cells
may also be involved in liver injury.27 Dioscorea bulbifera L.
presents a classical example of direct liver injury.28 It has
been shown that diosbulbin from Dioscorea bulbifera L.
causes excessive exhaustion of liver glutathione amounts,
and reduces levels of normal liver antioxidants, resulting in
oxidative stress injury.29 The reactive oxygen species pro-
duced during oxidative stress can modify and inactivate
lipids, proteins, DNA, and RNA, and thus lead to apoptotic
or necrotic cell death.30
Idiosyncratic reactions
The majority of adverse liver reactions are idiosyncratic,
occurring in most instances 5–90 days after the causative
medication was last taken. Such idiosyncratic reactions can
be divided into metabolic and immune idiosyncrasy.
Metabolic idiosyncrasy is often associated with genetic
polymorphisms of cytochrome P450. Electrophilic and free
radical metabolites are produced by the activation of
cytochrome enzymes, and levels of intermediate metabo-
lites, protein-DNA complexes and other metabolites are
increased, which can form toxic metabolites that cause liver
damage.31 In this mechanism, the alkaloids mentioned
above produce toxic metabolites resulting in liver toxicity.
Pyrrolizidine alkaloids in Sedum aizoon and Senecio are
transformed by cytochrome P450 into unstable toxic meta-
bolites, which damage sinusoidal endothelial cells and cause
blood disorders.32
Immune-mediated idiosyncratic liver injury can be seen as
an immune response. Drugs or their metabolites act as
haptens and are processed by immunocompetent cells and
converted to new antigens. These antigens then stimulate the
body to produce antibodies that can induce an immune
response and cause liver damage. It has been shown that
Tripterygium wilfordii can cause unpredictable liver injury,
which has a certain relationship with the activation of Kupffer
cells in the liver and the release of large amounts of tumor
necrosis factor and carbon monoxide.33 Because liver injuries
caused by idiosyncratic reactions frequently have a long
incubation period and are unpredictable, timely detection of
liver damage and treatment is difficult, which results in
adverse prognoses.
171
172
Table 1. Most common Chinese herbal medicines with hepatotoxicity

Most common form

Chinese name Scientific name Family Hepatotoxic components Intended use of hepatotoxicity
Ai Ye43 Artemisia argyi Asteraceae Volatile oil44,45 Malaria, hepatitis, cancer46 Hepatocellular45
Bi Ma Zi47 Ricinus communis Euphorbiaceae Toxic proteins (ricin)48 Ulcers, cancers, tumors, warts49 Hepatocellular50
51 53
Cang Er Zi Xanthium Asteraceae Glycosides (kaurene), diterpenoids, Asthma, allergies, nasal diseases Hepatocellular54
toxic proteins52
Chang Shan Dichroa febrifuga Lour Hydrangeaceae Alkaloids (dichroine)55 Malaria, cancer, fibrosis, Hepatocellular57
inflammatory diseases56
He Huan Pi Albizia julibrissin Fabaceae Glycosides (saponin)58 Insomnia, anxiety, depression59 Hepatocellular54
60 61 62
He Shou Wu Polygonum multiflorum Polygonaceae Anthraquinones Baldness, hair loss Hepatocellular63
64 65 67
Huang Yao Zi Dioscorea bulbifera L. Dioscoreaceae Glycosides(steroid, diosgenin), Thyroid diseases, tumors Hepatpcellular68
diterpenoid-lactones
(bafoudiosbulbins A and B)66
Ku Lian Zi69 Melia azedarach Meliaceae Glycosides (tetranortriterpenoids)69 Skin diseases69 Hepatocellular69
70 73
Lei Gong Teng Tripterygium wilfordii Celastraceae Glycosides (tripterygium), Immune inflammatory diseases Hepatocellular74,75
hook F diterpenoid-lactones,71
alkaloids (pyrrolizidine)71,72
Qian Li Guang76 Senecio scandens Asteraceae Alkaloids (pyrrolizidine)77 Inflammatory diseases78 Hepatocellular75
79
Shang Lu Phytolacca acinosa Phytolaccaceae Alkaloids (phytolaccine), Human immunodeficiency, Hepatocellular81
Roxb. glycosides (phytolaccatoxin, viral diseases82
esculentoside A)80,81

Journal of Clinical Translational Hepatology 2014 vol. 2 | 170–175

Xiang Si Zi Abrus Precatorius Fabaceae Toxic proteins (abrin)83 Cancers84 Hepatocellular85
Ma X. et al: Chinese herbal medicine-induced liver injury
Ma X. et al: Chinese herbal medicine-induced liver injury
Factors contributing to CHM-induced liver injury
Misunderstanding of CHM
The most frequent factor involved in CHM-induced liver injury
is a misunderstanding of CHM. The public generally regards
CHM as ‘‘natural’’ and ‘‘safe’’. However, the earliest Chinese
medical classic ‘‘The Inner Canon of Huangdi’’ classifies CHMs
as having high, moderate, or low toxicity, or as being
nontoxic. Furthermore, recent toxicologic studies found that
some chemical compositions of the herbs classified as
nontoxic can cause liver toxicity.34
Misuse, abuse and overdose
Liver injury caused by the misuse or abuse of CHM and some
folk remedies is common. Clinical data showed that of the 427
cases of liver injury at the Beijing Ditan Hospital that were
caused by CHM, two cases were due to accidental ingestion,
and in 62 cases, the medications were taken without a
prescription.35 In another 25 cases of HILI caused by Fallopia
multiflora, excessive dosage was the suspected cause in ten
patients.36
Poor quality
The quality of the drug is one of the most important factors
affecting CHM safety. CHMs are likely contaminated during
the process of growth, collection, storage, transportation and
manufacture.37 Uncontrolled processing can also result in the
production of toxic components. For example, cocklebur is
usually stir-fried before use in the clinic. Both reduced frying
time and uneven heating can lead to toxicity by incomplete
degeneration of the toxic protein.38
Improper compatibility of medicines
Compatibility means that two or more kinds of herbs can be
used in any combination to achieve the most effective and
safe treatment through the interactions among them. On the
other hand, incompatibility can not only reduce the effective-
ness, but also increase toxicity. The compatibility can
significantly reduce the extent of liver cell damage via the
antagonism of Angelica against Dioscorea bulbifera L. on
mRNA expression levels of CYP2E1 and CYP1A2.39
These situations can be improved by increasing public
awareness, precise diagnosis and treatment, and strict
quality supervision. However, the adverse reactions due to
differences in sensitivity, reactivity, and tolerance of indivi-
duals still may not effectively be controlled.
Future improvements and prospects
The growing number of clinical case reports and laboratory
research results clearly shows that some Chinese herbs have
a certain liver toxicity. Studies on the diagnosis, treatment
and mechanism have not kept pace. Not only doctors and
researchers, but also the media and pharmaceutical compa-
nies, should share the responsibility to avoid CHM-induced
liver injury and to use herbs safely. These issues deserve
attention in future studies.
Journal of Clinical Translational Hepatology 2014 vol. 2 | 170–175
Limitations of diagnosis
Due to the lack of specific diagnostic criteria, there exists a
certain degree of difficulty in diagnosing liver injury caused by
CHM, resulting in misdiagnoses or missed diagnoses, as well
as delays in treatment.40 On the other hand, because of the
limitation in the diagnosis, in some clinical reports, a past
medical history of CHM is sometimes the only clue to the
diagnosis.41 The European Medicines Agency assessed 31
spontaneous cases of assumed HILI by black cohosh reported
from countries within the European Union. Following the
application of the CIOMS scale, only one case of suspected
HILI from black cohosh remained, equivalent to 3% of the
reported cases.42 Therefore, the establishment of diagnostic
criteria is a top priority, and for doctors, the knowledge and
understanding of the pathogenesis, clinical manifestations
and treatment will also assist in detecting liver injury and
administering timely and effective treatment.
Development of toxicology studies
Toxicology studies on CHM are still limited, and further
research is needed. The focus of future studies should be on
determination of toxic doses, and limiting of doses, providing
important reference for rational herbal medicine use.
Dissemination of the standardized application of CHM
The public awareness of CHM also needs to be improved. This
is the responsibility of the public media and the government.
Public media needs to educate the public on the rational use
of medicinal herbs, and national regulatory authorities should
monitor the quality of the herbs, strictly regulate the doses of
medicine, and make regulations to forbid non-prescription
remedies.
Conclusions
With the popularity of traditional Chinese medicine in the
global market, adverse reactions caused by CHM are increas-
ing. CHM-induced liver injury presents many clinical patterns
and clinical manifestations. There is still no gold standard for
diagnostic criteria. Advances in the understanding of the
frequency, the clinical manifestations and the pathogenesis
can improve the accuracy of diagnosis and improve the safety
of CHM.
Conflict of interest
None
Author contributions
Writing the article (XM), polishing the manuscript (YYH),
polishing and organizing the manuscript, revising the manu-
script and replying the reviewer’s advice (JHP).
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175
Review Article
Hepatocellular Carcinoma and Liver Transplantation:
State of the Art
Andrea Mancuso*1,2 and Giovanni Perricone1
1
Epatologia e Gastroenterologia, Ospedale Niguarda Ca’ Granda, Milano, Italy; 2Medicina Interna 1, Azienda di Rilievo Nazionale
ad Alta Specializzazione Civico - Di Cristina – Benfratelli, Palermo, Italy
Abstract
Hepatocellular carcinoma (HCC) is an aggressive tumor that
often occurs in chronic liver disease and cirrhosis. The
incidence of HCC is growing worldwide.
With respect to any other available treatment for liver
cancer, liver transplantation (LT) has the highest potential to
cure. LT allows for removal at once of both the tumor (‘‘seed’’)
and the damaged-hepatic tissue (‘‘soil’’) where cancerogen-
esis and chronic liver disorders have progressed together. The
Milan criteria (MC) have been applied worldwide to select
patients with HCC for LT, yielding a 4-year survival rate of
75%. These criteria represent the benchmark for patient
selection and are the basis for comparison with any other
suggested criteria.
However, MC are often considered to be too restrictive,
and recent data show that between 25% and 50% of patients
with HCC are currently transplanted beyond conventional
indications. Consequently, any unrestricted expansion of
selection criteria will increase the need for donor organs,
lengthen waiting periods, increase drop-out rates, and impair
outcomes on intention-to-treat analysis. Management of HCC
recurrence after LT is challenging. There are a few reports
available regarding the safety and efficacy of sorafenib for
HCC recurrence after LT, but the data are heterogeneous. A
multi-center prospective randomized controlled trial compar-
ing placebo with sorafenib is advised. Alternatively, a meta-
analysis of patient survival with sorafenib for HCC recurrence
after LT could be helpful to characterize the therapeutic
benefit and safety of sorafenib.
Here, we review the use of LT for HCC, with particular
emphasis on the selection criteria for transplantation in
patients with HCC and management of HCC recurrence
after LT.
Keywords: Hepatocellular carcinoma; Liver transplantation; Selection criteria;
Milan criteria; Recurrence; Sorafenib.
Abbreviations: AAIR, age-adjusted incidence rate; EASL-EORTC, European
Association for the Study of the Liver – European Organisation for Research and
Treatment of Cancer; ELTR, European Liver Transplant Registry; HAART, highly
active anti-retroviral therapy; HBV, hepatitis B virus; HCC, hepatocellular
carcinoma; HCV, hepatitis C virus; HIV, human immunodeficiency virus; ITT,
intention-to-treat; LDLT, living donor liver transplantation; LT, liver transplanta-
tion; MC, Milan criteria; NASH, non-alcoholic steatohepatitis; OPTN, organ
procurement and transplantation network; RFA, radio-frequency ablation; RFTA,
radio-frequency thermal ablation; TACE, trans-arterial chemoembolization;
UNOS, United Network for Organ Sharing; UCSF, University of San Francisco.
Received: 23 April 2014; Revised: 30 June 2014; Accepted: 01 July 2014
q
DOI: 10.14218/JCTH.2014.00013.
*Correspondence to: Andrea Mancuso, Medicina Interna 1, A.R.N.A.S. Civico,
Piazzale Liotti 4, Palermo 90100, Italy. Tel: +39-329-899-7893, Fax:
+39-091-609-0252, E-mail: mancandrea@libero.it
176 Journal of Clinical and Translational Hepatology 2014 vol. 2 | 176–181
E 2014 The Second Affiliated Hospital of Chongqing Medical
University. Published by XIA & HE Publishing Ltd. All rights
reserved.
Introduction
Liver cancer is the second most common cause of death from
cancer worldwide, estimated to be responsible for nearly
746,000 deaths in 2012 (9.1% of the total). The prognosis for
liver cancer is very poor, with an overall ratio of mortality to
incidence of 0.95, and the geographical patterns in incidence
and mortality are similar (see Table 1).1
HCC represents more than 90% of primary liver cancers
and is a major global health problem. The incidence of HCC
increases progressively with advancing age in all populations,
reaching a peak at 70 years.2 HCC has a strong male
preponderance, with a male to female ratio estimated to be
2.4.1 The pattern of HCC occurrence has a clear geographical
distribution.1,3 In 2012, 83% of the estimated 782,000 new
cancer cases worldwide occurred in less developed regions.1
The incidence of HCC is growing worldwide.
A variety of important risk factors for the development of
HCC have been identified. These include chronic hepatitis B
virus (HBV) infection, chronic hepatitis C virus (HCV) infec-
tion, alcohol intake, hereditary hemochromatosis, aflatoxin
exposure, and cirrhosis of almost any cause. However, HCC
can also occur in patients without known risk factors.
Approximately 90% of HCC are associated with a known
underlying risk factor (see Table 2). Cirrhosis may be caused
by chronic viral hepatitis, alcohol, inherited metabolic dis-
eases such as hemochromatosis or alpha-1-antitrypsin
deficiency or Wilson’s disease, non-alcoholic fatty liver
disease, autoimmune hepatitis, and Budd-Chiari syndrome.
All etiologic forms of cirrhosis may be complicated by tumor
formation, but the risk is higher in patients with hepatitis
infection. Overall, one-third of cirrhotic patients will develop
HCC during their lifetime.3–7 Rarely, HCC can develop in the
absence of cirrhosis.
Overall, the incidence of HCC is increasing not only in the
general population of patients with cirrhosis, but also in some
subgroups of patients, like those with human immunodefi-
ciency virus (HIV) infection or thalassemia. In fact, significant
improvement in outcomes due to iron chelating drugs in
thalassaemic patients and to highly active anti-retroviral
therapy (HAART) in HIV patients has recently revealed HCC
as a main complication of the underlying hepatic disease.8–13
Liver transplantation (LT) is an established treatment for
HCC.14,15 If donor graft supply was unlimited, every cirrhotic
patient with HCC would be offered LT as the optimal treatment
Mancuso A. et al: Hepatocellular carcinoma and liver transplantation: State of the art

Table 1. Liver cancer. Estimated incidence, mortality, and 5-year prevalence worldwide in 2012

Estimated Men Women Both sexes

numbers
(thousands) Cases Deaths 5-yr prev Cases Deaths 5-yr prev Cases Deaths 5-yr prev
World 554 521 453 228 224 180 782 746 633
More developed 92 80 112 42 43 51 134 123 164
regions
Less developed 462 441 341 186 182 129 648 622 469
regions
USA 23 17 21 8 7 7 30 24 27
China 293 282 220 101 101 71 395 383 291
India 17 17 8 10 10 5 27 27 13
European Union 36 32 33 16 17 14 52 48 47

Source: Globoscan 2012

for their disease. Relative to any other available treatment for
liver cancer, LT has the highest potential to cure4,6 because of
its ability to remove at once both the seeded-HCC and the
damaged-hepatic tissue where cancerogenesis and chronic
liver disorders have together progressed.16
The criteria for determining whether a HCC patient is
eligible, upfront, for LT are very heterogeneous and relatively
ill-defined. Another controversy is how to manage HCC
recurrence after LT because there is little evidence available
regarding improvement of survival with any treatment
after LT.
Here, we present a state-of-the-art review on LT for HCC.
In particular, this review concentrates on two topics, respec-
tively, selection criteria for LT in patients with HCC and
management of HCC recurrence after LT.
Selection criteria for liver transplantation in patients
with hepatocellular carcinoma: Milan criteria or
expanded criteria?
The number of LTs for HCC has increased worldwide; and
currently in Europe, about 27% of all LT patients have HCC.18
More specifically, in some Mediterranean areas of France,
Italy, and Spain, LT for HCC represents more than 40% of
transplants. Over the last decade in Western countries, HCC
has had the highest growth increment of all indications for
LT.17,18 This trend is even more pronounced in Eastern
countries, where HCC, in some instances, has equalized or
even overtaken cirrhosis as the leading indication for LT.19
The broad selection criteria applied two decades ago led to
poor results in terms of recurrence (32–54% at 5 years) and
survival (5-year survival ,40%), but allowed for the identi-
fication of the best candidates for this procedure.6,20,21 Since
the initial results of LT for HCC were negative, likely because
LT was initially reserved for advanced HCC not suitable for
resection, the reliability of the procedure was questioned. In
1996, the publication of a pivotal prospective study on 48
patients transplanted for HCC under predefined criteria
(single HCC f5 cm or 3 HCC f3 cm each), the so called
‘‘Milan criteria’’ (MC), showed a 4 year survival of 75%.22
Successively, some pioneering groups selecting ‘‘optimal
candidates’’ reported 70% 5-year survival with a recurrence
rate below 15%.23–26 Due to these data, LT is now considered
to be the first-line treatment for patients with single HCC

Table 2. Geographical distribution of main risk factors for HCC worldwide

Geographic area AAIR M/F Risk factors HCV (%) HBV (%) Alcohol (%) Others (%)

Europe 6.7/2.3 60–70 10–15 20 10

Southern 10.5/3.3
Northern 4.1/1.8
North America 6.8/2.3 50–60 20 20 10
(NASH)
Asia and Africa 20 70 10 10
(Aflatoxin)
Asia 21.6/8.2
China 23/9.6
Japan 20.5/7.8 70 10–20 10 10
Africa 1.6/5.3
World 16/6 31 54 15

AAIR, Age-adjusted incidence rate; HBV, Hepatitis B virus; HCV, Hepatitis C virus.
Updated from Llovet et. al. according to IARC1

Journal of Clinical and Translational Hepatology 2014 vol. 2 | 176–181 177

 Mancuso A. et al: Hepatocellular carcinoma and liver transplantation: State of the art
Table 3. Recommendations on liver transplantation for hepatocellular
carcinoma based on the level of evidence and the strength of the data
(classification of evidence adapted from National Cancer Institute) and
the strength of recommendations following previously reported systems
(GRADE systems), according to EASL-EORTC clinical practice guidelines
on the management of HCC6
Levels of Grade of
evidence* recommendation˚
LT Milano IN 2A 1A
LDLT 2A 2B
LT extended 2B 2B
Down-staging 2D 2C
*
Adapted from National Cancer Institute
˚GRADE system
LT, Liver transplantation; LDLT, Living donor liver transplantation.
f5 cm or 3 HCC f3 cm each (see Table 3).4–6 All publication
to date use the strict guidelines set by the MC when
considering LT for treatment of HCC.4–6 A meta-analysis has
confirmed the strong association of MC with a survival
advantage (HR 1.7) and with a low risk of selecting an
aggressive biologic behavior with respect to patients exceed-
ing them. In fact, G3 tumors and microvascular invasion are
less frequent when MC are met, with a HR of 4.8 and 2.5,
respectively.27
According to European (ELTR) and American (OPTN)
registries, the overall 5-year survival of patients transplanted
within the MC (65–78%) is similar to non-HCC indications
(65–87%).27–29 As a consequence of their success, the MC
have been integrated in the BCLC staging system30,31 and in
the United Network for Organ Sharing (UNOS) pre-transplant
staging for organ allocation in the US,32 and remain the
benchmark for any other prognostic criteria proposed for
expanding the indication for LT in cirrhotic patients with
HCC.33
The need to obtain the optimal benefit from the limited
number of organs that are available has prompted the
maintenance of strict selection criteria so as to list only those
patients with early HCC who have the highest likelihood of
survival after transplant. However, this means that some
patients with slightly more advanced HCC in whom transplant
would offer an acceptable, but not excellent outcome, are
excluded from the procedure.34–36
Nevertheless, MC are often considered to be too restrictive
and a plethora of ‘‘expanded criteria’’ have been suggested.
In summary, in order to establish a new policy allowing for
expansion of criteria for transplantation, it is essential to
develop robust data for the specific category of patients
included in the proposed expansion. Novel criteria might have
a major impact on all transplant programs and the data
needed to support any change should be impeccable. In
addition, the impact of the expansion on non-HCC patients
waiting for LT should be taken into account.3
None of these expanded criteria have been prospectively
and independently validated. The partial exceptions are the
University of San Francisco (UCSF) criteria (single tumor
f6.5 cm or multiple HCC f3 nodules f4.5 cm, with the total
tumor diameter f8 cm). Notably, there is a significant
overlap between patients meeting the Milan and the UCSF
criteria, resulting in a modest expansion of the exact number
of HCC patients eligible for LT (estimated to be 5–10%).37 In
fact, UCSF criteria on explant identified retrospectively a
178 Journal of Clinical and Translational Hepatology 2014 vol. 2 | 176–181
cohort of patients whose survival was not significantly
different from those of patients transplanted for HCC inside
the MC.38 The same results have been demonstrated by other
retrospective experiences from other centers using UCSF
criteria. Moreover, a recent prospective study showed a 5
year survival not significantly different in patients trans-
planted for HCC inside Milan and UCSF criteria.39
Today, expansion to UCSF criteria has already been
challenged from the pathological point of view by the up-to-
seven criteria (i.e. those HCC having the number 7 as the sum
of the size of the largest tumor and the number of tumors).36
In a multicenter retrospective study on over 1,700 explants,
the authors demonstrated that HCC inside the ‘‘up-to-seven’’
criteria at explant and without microvascular invasion had a 5
year survival not significantly different from those inside the
MC, while survival was significantly worst in cases of HCC
inside the ‘‘up-to-seven’’ criteria and with microvascular
invasion.36 This pathology-based proposal has been recently
validated in an independent series,40 but the findings must be
further validated with prospective studies and is not suitable
clinical practice.
The major concerns about the expansion proposals are the
lack of specific data on overall survival and drop-out rate on
the waiting list for the patients outside the current criteria but
fulfilling the expanded criteria. Other recent studies challen-
ging the Milan criteria have proposed different algorithms to
optimize patient selection. Nonetheless, 5-year outcome
prediction could vary from 70% to 40% depending on the
presence of microvascular invasion. Thus, preoperative
markers of vascular invasion would be required prior to
adopting these criteria.6
Presently, it is likely that a modest expansion of the
number of potential candidates may be considered for
validated criteria (such as the up-to-seven criteria that
largely include the UCSF criteria), which demonstrate com-
parable survival for patients outside the MC (see Table 3).6,16
As repeatedly noted in scientific and regulatory contexts, any
unrestricted expansion of selection criteria will increase the
need for donor organs, lengthen waiting periods, increase
drop-out rates, and impair outcomes on intention-to-treat
(ITT) analyses.
In the present context, the term ‘‘down-staging’’ defines
the reduction of the intra-hepatic HCC burden to meet
acceptable criteria for LT,41 ‘‘acceptable’’ criteria being driven
by good expected survival after LT.42 This equates to a 5-year
survival comparable to that of HCC patients who meet
transplant criteria without requiring down-staging. 33
Namely, the principle of down-staging is to select a more
favorable tumor biology, as determined by response to
treatment in the perspective of positive post-LT outcome.41
From the seminal experience of the UCSF group, most of the
published reports have used the MC as the endpoint for down-
staging. In doing so, for a minimal observation period of 3
months (suggested to confirm a sustained response to
treatment), a comparable or only slightly reduced survival
than that achieved with HCC meeting MC before LT has been
reported.41
According to recommendations, either MC or a modest
expansion can be targeted in elective LT for HCC, while for
down-staging only conversions to conventional MC are
acceptable.33 Trans-arterial chemoembolization (TACE) is
the single treatment modality most often applied to HCC
down-staging, followed by radiofrequency ablation (RFA),
radioembolization, and surgical resection. 43–48 In the
Mancuso A. et al: Hepatocellular carcinoma and liver transplantation: State of the art
large majority of centers, the choice of treatment – either
alone or in combination – is made within multidisciplinary
transplant-tumor boards.49 Although a treatment strategy
based on the individual components of patients/tumors is
largely justified, it raises concerns of selection bias when a
comparison among treatment strategies is attempted. The
same applies to the criteria of exclusion from down-staging
procedures, which remain largely undescribed.16
Considering the current data, down-staging of patients
beyond MC cannot be adopted as a tool to refine patient
selection and further research is required (see Table 3).6 This
research should be based on the principle that 5-year survival
outcomes of patients undergoing transplantation after suc-
cessful down-staging should be similar to those of patients
transplanted following MC. The EASL-EORTC panel considers,
though, that a special policy should be adopted for patients
already on the waiting list for LT with tumors progressing
beyond MC and liver only disease. In this special circum-
stance, it is recommended to place the candidate on hold until
down-staging by local ablation or chemoembolization is
achieved and maintained for at least 3 months.6
In summary, guidelines still indicate LT only to HCC inside
MC.4–6 However, as published experiences show, many
centers perform LT outside the MC, using criteria that are
different from center to center. Recent data show that even in
large areas, between 25% and 50% of patients with HCC
are currently transplanted beyond conventional indica-
tions.33,36,38,39,50–54 As a results, any unrestricted expansion
of selection criteria will increase the need for donor organs,
resulting in lengthened waiting periods, increased drop-out
rates, and impaired outcomes in ITT analyses. Extreme
deviations from efficiency and equity endpoints should be
avoided in criteria expansion.
Whatever the criteria adopted, a significant problem of
HCC candidates for LT is drop-out, patients who do not reach
the goal of LT because of progression of HCC or causes
unrelated to HCC. Many studies have investigated the risks
for drop-out, despite its difficulty to define, and these factors
include tumor multinodularity, neoadjuvant treatment
failures, and elevated AFP or MELD. From an opposing point
of view, given the organ shortage, some patients with
single HCC ,2 cm may benefit from alternative treatments
and should avoid LT unless recurrence occurs, highlighting
the possibility of salvage transplantation in the low risk
population.
Living donor liver transplantation (LDLT), where the right
hepatic lobe of a healthy donor is used, has emerged as an
alternative to deceased LT.55–56 In 2000, there was great
enthusiasm for LDLT, and it was estimated that it would
represent a significant proportion of the patients transplanted
with HCC.57 Unfortunately, the associated risks of death
(estimated in 0.3%) and life-threatening complications (2%)
for the healthy donor have diminished the interest of the
transplant community.58,59,60 Due to the complexity of the
procedure, LDLT must be restricted to centers of excellence in
hepatic surgery and transplantation.
Outcome results with LDLT compared with deceased LT
have been controversial. Although some studies suggested
that LDLT was associated with higher risk of recurrence, these
data have not been confirmed.61,62 Cost–effectiveness stu-
dies suggested that LDLT can be offered to patients with HCC
if the waiting list exceeds 7 months,63 a policy adopted by the
panel of EASL-EORTC.6
Journal of Clinical and Translational Hepatology 2014 vol. 2 | 176–181
Hepatocellular carcinoma recurrence after liver
transplantation: the need of treatment despite no
evidence of survival benefit
Management of HCC recurrence after LT is complex and
challenging. Despite the metastatic nature of such a recur-
rence, treatments resemble those used for HCC in cirrhosis,
although there is no evidence that such treatments could
improve survival of patients with HCC recurrence after LT.
Approachable localized HCC recurrence should undergo
surgery, with indication resembling those of HCC in cirrhosis.
Recurrences not suitable for surgery should be treated with
RFTA or TACE. However, both surgery and other treatments
are empirical, as no evidence of survival benefit exists.
Sorafenib is the treatment of choice for advanced HCC,
since survival in sorafenib patients with underlying liver
cirrhosis is longer than placebo control.64,65 Theoretically,
such a systemic therapy could be the best approach for HCC
recurrence after LT. However, different observational studies
of patients treated with sorafenib for HCC recurrence after LT
have reported contradictory results regarding safety and
efficacy. In fact, while some authors emphasize the effec-
tiveness and safety of sorafenib, pushing for the general
acceptance of the treatment for HCC recurrence after LT,
others had safety concerns.66–84 In particular, one group
reported grade 3–4 adverse events in 92% of 13 patients,
resulting in sorafenib discontinuation in 77%.72 Another
study of 11 consecutive patients described a high rate of
intolerance or side-effects, causing drug discontinuation in
36%.70 Moreover, some patients in another study died
because of massive gastrointestinal bleeding, possibly due
to an interaction between everolimus and sorafenib that could
facilitate gastrointestinal bleeding.69,75,85 In fact, a concern
for the concomitant use of sorafenib and HAARTs in HIV has
also been reported.86
Taken all together, the experiences on treatment with
sorafenib for HCC recurrence after LT are too heterogeneous
to draw a definite conclusion and further studies are needed.
It is advisable that the effort of a multi-center prospective
randomized controlled sorafenib versus placebo trial should
be made to address this controversial topic. Alternatively, a
meta-analysis of survival of patients treated with sorafenib
for HCC recurrence after LT could help to identify possible
solutions.
Conclusions
LT has the highest potential cure rate for HCC relative to other
options, but the proportion of individuals with HCC on the
waiting list for transplant is growing. The MC are the bench-
mark for patient selection and the basis for comparison with
any other suggested criteria. Expansion of criteria is not a
widely recommended strategy, since there is a lack of
relevant evidence. Any expansion should avoid the saturation
of the listing system and poor long-term outcomes after LT.
Any unrestricted expansion of selection criteria will increase
the need for donor organs, resulting in lengthened waiting
periods, increased drop-out rates, and impaired outcomes in
ITT analyses. Extreme deviations from efficiency and equity
endpoints should be avoided in criteria expansion. ‘‘Down-
staging’’ is the reduction of the intra-hepatic HCC burden to
meet acceptable criteria for LT. The principle of the strategy is
to select individuals whose tumors have a more favorable
biology, as determined by treatment response in the
179
 Mancuso A. et al: Hepatocellular carcinoma and liver transplantation: State of the art
perspective of positive post-LT outcome. However, treatment
may not necessarily change the outcome of LT. Overall, down-
staging is weakly recommended as a LT selection tool, unless
it is applied in prospective studies with survival and disease-
progression endpoints.
Conflict of interest
None
Author contributions
Writing the review (AM, GP).
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181
Review Article
Percutaneous Cryoablation for Liver Cancer
Li-Zhi Niu1,2, Jia-Liang Li2 and Ke-Cheng Xu*1,2
1
Fuda Cancer Hospital, Jinan University School of Medicine, Tianhe District, Guangzhou, China; 2Guangzhou Fuda Cancer
Institute, Tianhe District, Guangzhou, China
Abstract
Based on the primary tumor site, liver cancer can be divided
into two categories: (1) primary liver cancer and (2)
metastatic cancer to the liver from a distant primary site.
Guided cryoablation via many imaging methods induces
iceball formation and tumor necrosisand is an attractive
option for treating unresectable hepatocellular carcinoma
(HCC) and metastatic liver cancer. There are several advan-
tages to using cryoablation for the treatment of liver cancer:
it can be performed percutaneously, intraoperatively, and
laparoscopically; iceball formation can be monitored; it has
little impact on nearby large blood vessels; and it induces a
cryo-immunological response in situ. Clinically, primary
research has shown that percutaneous cryoablation of liver
cancer is relatively safe and efficient, and it can be combined
with other methods, such as radiation therapy, chemother-
apy, and immunology, to control disease. Although research
is preliminary, cryosurgery is fast becoming an alternative
treatment method for HCC or liver tumors. Here, we review
the mechanisms of liver tumor cryoablation, cryoablation
program selection, clinical efficiency, and complications
following treatment.
E 2014 The Second Affiliated Hospital of Chongqing Medical
University. Published by XIA & HE Publishing Ltd. All rights
reserved.
Introduction
Liver cancer can be divided into two groups based on the
primary tumor site: (1) primary liver cancer (including
hepatocellular carcinoma [HCC] and cholangiocarcinoma)
and (2) metastatic cancer to the liver from a distant primary
site. The most common type of adult primary liver cancer is
HCC, which is usually discovered late in the disease course
and generally has poor prognosis.1 Although surgical resec-
tion (SR) is the best option for patients with HCC, only about
20–30% of patients are eligible for tumor resection at the
time of diagnosis.2,3 Systemic chemotherapy does not
significantly increase survival in patients with advanced
Keywords: Cryosurgery; Hepatocellular carcinoma; Metastatic disease; Efficacy.
Abbreviations: AFP, alpha-fetoprotein; CIK, cytokine-induced killer; CT, com-
puted tomography; DCH, dendritic cells; HCC, hepatocellular carcinoma; MRI,
magnetic resonance imaging; OS, overall survival; PC, percutaneous cryosur-
gery; SR, surgical resection; TACE, transarterial chemoembolization; US, ultra-
sound.
Received: 22 May 2014; Revised: 15 June 2014; Accepted: 27 June 2014
q
DOI: 10.14218/JCTH.2014.00017.
*Correspondence to: Kecheng Xu, No.2 of Tangde West Rd, Tianhe District,
Guangzhou, China. Tel: +86-020-38993994-8700, Fax: +86-020-38993994-
8700, Email: fudaclab@gmail.com
182 Journal of Clinical Translational Hepatology 2014 vol. 2 | 182–188
HCC.4,5 Some cancers; such as colorectal, breast, carcinoid,
and pancreatic cancer, melanoma, and renal cell carcinoma,
that can metastasize to the liver are generally associated with
poor prognosis as well. Given the lack of treatment options
for these patients, new locoregional and systemic therapies
are needed. Cryoablation under the guidance of many
imaging methods can induce iceball formation and tumor
necrosis and is an attractive option for treating unresectable
HCC and metastatic liver cancer. Cryoablation has many
advantages, including ability to visualize the iceball, 6,7
activation of cryo-immunology in cancer,8 lack of severe
damage to large blood vessels,9 and no causation of severe
pain.10 With the emergence of argon–helium cryoablation
units, percutaneous cryosurgery (PC) is becoming a main
treatment method in the field of cancer cryosurgery. The
present review describes the mechanisms of liver tumor
cryoablation, percutaneous cryoablation program selection,
clinical efficiency, and the side effects associated with
treatment.
Mechanisms of tissue injury
Currently, two different argon-based cryoablation units are
used in the clinical setting: the CRYO care System (EndoCare,
Irvine, CA, USA) and Cryohit (Galil Medical, Yokneam, Israel).
When high-pressure argon gas is circulated through the
lumen of a cryoprobe, the temperature drops to 2160 ˚C
and an iceball forms at the tip of the probe. The iceball is
thawed when helium gas is circulated through the probe
lumen. This freezing and thawing process induces direct and
indirect cell damage, causing tumor cell death. When the
temperature drops during the freezing process, ice crystals
form inside and outside the cell, osmotic pressure increases
inside the cell, cell dehydration occurs, and the cell mem-
brane is damaged.11 Organelles, such as the mitochondria
and endoplasmic reticulum, are irreversibly damaged, even-
tually leading to cell death. During the thawing process,
recrystallization or the mutual confluence of tiny ice crystals
leads to the formation of large ice crystals, which induce more
damage to the cell. Extracellular water re-enters the cell at
this stage, causing the cell to swell, eventually leading to
more cell membrane damage. If the temperature of the
frozen tissue is not sufficiently decreased, not all tumor cells
will be killed despite apoptosis as the main form of cell
death.12 Since apoptosis does not guarantee the elimination
of all target cells, incomplete tumor cell death will likely lead
to tumor recurrence.
Cryosurgery is indirectly linked to tumor cell death by
promoting arteriovenous injury via the formation of micro-
and small arteriovenous ice crystals that cause vascular
endothelial damage, increased vascular wall permeability,
Niu L.Z. et al: Cryoablation for liver cancer

intravascular fluid extravasation, and interstitial edema.13
Platelet aggregation, thrombosis, and eventual occlusion of
blood flow also cause tumor tissue ischemia and hypoxia,
which induces tumor cell death. The flow of blood in micro-
and small arteries is three times faster than that of
intravenous blood, and blood flow influences ice crystal
formation. In the first freezing cycle, circulation of micro-
and small arteries of the frozen area recovers 10,20 min
after thawing. In the second freezing cycle, micro- and
small artery blood flow does not recover, whereas the
circulation in large arteries recovers within 24 hours after
cryosurgery.14–17 After repeated freezing and thawing, cell
rupture, cell membrane dissolution, and cell antigen release,
the cryo-immunological response is triggered8,18 and the
anti-tumor immune response that targets additional tumor
cells is induced.19
Cryoablation program selection
Several factors influence the effect of freezing, including
freezing temperature, freezing time, thawing, and freezing
time cycle number.20 First, the freezing time controls the size
of the iceball, which increases as freezing time increases until
a dynamic balance of cold/heat exchange is achieved. As
iceball formation can be visualized with ultrasound (US),
computed tomography (CT), and magnetic resonance ima-
ging (MRI),20–22 it can be monitored accurately to ensure
complete ablation. Second, the freezing temperature influ-
ences the effect of the cryosurgery. The temperature at the
margin of the iceball is 0 ˚C, a temperature at which tumor
cells can survive. Five mm inside the edge of the iceball, the
temperature is approximately 220 ˚C to 250 ˚C, and tumor
cells can be killed. Therefore, in order to achieve complete
ablation of tumor tissue, the edge of the iceball must exceed
that of the tumor by 1 cm.23 Third, thawing and cycle times
will affect the degree of necrosis,12 where a longer thawing
time leads to more tissue necrosis.11 Whittaker24 noted that
thawing time will affect iceball formation, where large-scale
intracellular ice crystals will form after the second freeze
cycle. Repeated freezing cycles will increase the size of the
iceball,14,25,26 and the degree and area of permanent tissue
necrosis caused by the iceball spreads outward as freezing
cycles increase, thereby increasing the area of frozen
necrosis.27,28 Since their freezing times are the same
(20 min), the triple-freeze protocol may be a more powerful
liver ablation method than the dual-freeze protocol demon-
strated in a Tibetan pig model.28
As a single probe causes a smaller range of damage, more
than two freezing probes are used for tumors exceeding 3 cm
to increase the effective freezing range.29 Hinshaw and Lee23
reported that if there are no great vessels around the tumor,
the best cryoprobe placement is two needles not greater than
2 cm apart within 1 cm of the edge of the tumor. If there
is a large vessel adjacent to one of the tumor borders,
the cryoprobes should be positioned closer to the vessel
and more closely apposed, as ‘‘heat sink’’ has a critically
important effect on isotherm size and shape. If there are
multiple large blood vessels around the tumor, it is best to
place the two cryoprobes less than 1 cm apart, 5 mm from
the edge of the tumor.23
Altogether, the efficacy of cryosurgery is affected by argon
flow to specific probes, the cryoablation program, and multi-
ple probe selection. For liver cryosurgery, different cryoabla-
tion programs are carried out to ablate different lesions or
lesions in different locations.
Clinical research (summarized in Table 1)
In this decade, PC is the main therapeutic intervention for the
treatment of unresectable HCC. In 2003, Xu et al.44 reported
the use of PC in 105 masses from 65 patients with HCC.
Among 41 patients who were followed for more than 1 year;
32 (78%) were alive despite tumor recurrence, seven
patients (10.8%) died due to disease recurrence, and three
patients (5%) died of non-cancer–related diseases (Fig. 1).
In a study of four patients with small HCC who underwent one
PC with multiple 17-G cryoprobes, it was determined that this
percutaneous technique, and not the laparotomic approach,
should be applied for unresectable HCC.30 In 300 cases of
HCC, the therapeutic effect, safety, and complications of PC
were evaluated, and the mean survival (months) of early-,
intermediate-, and advanced-stage patients was 38.7 ± 3.8,

Table 1. Survival rates of patients after cryosurgery of hepatic tumors

1 year 2 year 3 year 4 year 5 year

survival rate survival survival survival survival
Tumor type Therapy type Patients (%) rate (%) rate (%) rate (%) rate (%)
32–33
Zhou et al. HCC CRY 48 81.3 62.1 47.6 44.4 -
Chen et al.20 HCC CRY - 81.4 - 60.3 - -
Li et al.36 SHCC CRY - 100.0 - 77.6 70.7 -
Xu et al.37 Colorectal liver CRY 326 78.0 62.0 41.0 34.0 23.0
metastases
Qian et al.38 Metastatic liver CRY 22 81.8 22.7 - - -
malignancy
Chen et al.39 Secondary Liver cancer CRY - 80.2 - - - -
Recurrent liver cancer CRY - 46.2 - - - -
Han et al.40 HCC CRY 116 85.7 64.3 32.1 - -
Xu et al.42 HCC CRY 420 73.0 54.0 - 49.0 39.0
HCC TACE+CRY - 71.0 61.0 - 29.0 23.0

Note: SHCC, small HCC; CRY, cryotherapy

Journal of Clinical Translational Hepatology 2014 vol. 2 | 182–188 183


Fig. 1. CT images of two patients (A and B) with HCC who underwent PC. (A1) Space-occupying lesion in the upper right part of the liver. (A2) Cryoprobe placement
and resultant iceball formation. (A3) No lesion five years after treatment. (B1) Space-occupying lesion measuring 73 6 62 mm before treatment. (B2) Ultrasonography
showing shrinkage of the tumor to 42 6 40 mm. Arrows indicate the tumor site.
26.5 ± 4.2, and 16.9 ± 1.4, respectively.31 There were
serious complications in 17 patients (6%), including six with
cold shock (2%), five with bleeding due to tumor rupture
(2%), four with bleeding from stress gastric ulcer (1%), and
two with liver failure (1%). In another study, the clinical
effectiveness of percutaneous cryosurgical ablation for treat-
ing 48 patients with early-stage HCC was evaluated, and it
was found that the 1, 2, 3, and 4 year survival rates were
81%, 62%, 48%, and 44%, respectively.32,33 In a series of
124 unresectable HCC cases, serum alpha-fetoprotein (AFP)
levels were reduced in 76 patients (83%), and 205 of the 222
tumors (92%) diminished or were unchanged after cryoabla-
tion. The median survival time was 31.3, 17.4, and 6.8
months in the early-, intermediate-, and advanced-stage
groups, respectively. In eight patients with primary or
secondary small HCC (f5 cm) after PC, six patients became
AFP negative with a coagulation necrosis rate of 63%, and the
6 month and 1 year local recurrence–free survival rates were
100% and 88%, respectively.34 Zhang investigated 227
patients with different stages of HCC treated with PC and
found that advanced HCC was most sensitive to cryosurgery
and that survival was significantly prolonged.35 Using PC to
treat patients with unresectable HCC, Chen et al.20 found that
the 1 and 3 year overall survival (OS) rates were 81%, and
60%, respectively, while the 1 and 3 year disease-free
survival rates were 68% and 21%, respectively. The 1 and
3 year OS rates of patients with recurrent HCC were 70% and
29%, respectively, while the 1 and 3 year disease-free
survival rates were 54% and 8%, respectively. When the
therapeutic effect of PC and SR of small HCC were compared,
it was found that the respective 1, 3, and 5 year OS rates of
the PC and SR groups were 100%, 75%, and 67%, and
100%, 78%, and 71%.36 The respective 1, 3, and 5 year
recurrence-free survival rates of the PC and SR group were
83%, 46%, and 29%, and 84%, 48%, and 33%. These
results suggested that PC is as effective as SR for treating
solitary small HCC even though it is less invasive, requires a
184 Journal of Clinical Translational Hepatology 2014 vol. 2 | 182–188
Niu L.Z. et al: Cryoablation for liver cancer
shorter hospitalization time, and less expensive than SR.
Overall, these data demonstrate that PC is a viable treatment
option for HCC.
Similar to its use in primary liver cancer, cryosurgery also
is advantageous for the treatment of metastatic liver cancer.
In 326 patients with non-resectable hepatic colorectal
metastases, 526 procedures of cryosurgery under US or CT
guidance were performed.37 Three months after cryosurgery,
the elevated carcinoembryonic antigen levels in 78% of
patients were restored to normal levels. Postoperative CT
scan of the cryotreated lesions showed complete response in
15% of patients, partial response in 41% of patients, and
stable disease in 24% of patients. The 1, 2, 3, 4, and 5 year
OS rates after the treatment were 78%, 62%, 41%, 34%,
and 23%, respectively (Fig. 2). In 2005, Qian and collea-
gues38 reported survival rates of 22 patients with histologi-
cally or clinically confirmed metastatic liver malignancy
treated with PC. The 12, 18, and 24 month survival rates
were 82%, 64%, and 23%, respectively. Chen et al.39
reported that the 1 year survival rates of secondary and
recurrent liver cancer patients treated with cryosurgery were
80% and 46%, respectively. In another study, 116 patients
with unresectable HCC were divided into three groups: (A)
interventional therapy, (B) 3-dimensional conformal radio-
therapy, and (C) cryoablation therapy. The respective 1, 2,
and 3 year survival rates in the A, B, and C groups were 15%,
64%, and 86%; 11%, 32%, and 64%; and 0%, 0%, and
32%. Taken together, these data suggest that PC is an
acceptable new locoregional therapy for metastatic liver
cancer.
In order to optimize treatment strategies for liver cancer,
cryoablation is often combined with other therapies, such as
transarterial chemoembolization (TACE). Between July 2001
and June 2002, Qian et al.41 assessed the indications,
efficacy, and clinical significance of PC following TACE in 34
patients with histologically or clinically confirmed primary or
metastatic carcinoma. During the 3–15 month follow-up
Niu L.Z. et al: Cryoablation for liver cancer
Fig. 2. Liver imaging in two patients (A and B) with hepatic colorectal metastases. Complete ablation of histologically proven tumor was achieved after PC. (A1)
MRI before cryosurgery. (A2) MRI during CT-guided PC. (A3) MRI at 12 months after cryosurgery. In the second patient, the massive lesion regressed completely in
comparison to its size before cryosurgery (B1). (B2) Eight months after cryosurgery and TACE. Arrows indicate the tumor site.
period, 41% of patients were classified as clinically cured or
effectively treated because their serum tumor markers were
normal, or CT and MRI images revealed that the lesion had
become completely necrotic. In a study on the use of
sequential TACE and PC for treatment of unresectable HCC,
the 1 and 2 year survival rates in the sequential group (71%
and 61%, respectively) were similar to that of the cryo-alone
group (73% and 54%, respectively). The 4 and 5 year
survival rates of the sequential group were 49% and 39%,
respectively, which were higher than that of the cryo-alone
group (29% and 23%, respectively). While 18 patients with
large HCC (.5 cm diameter) survived for more than 5 years
after sequential TACE and cryosurgery, no patient in this
category survived more than five years with cryosurgery
alone. The authors suggested that pre-cryosurgical TACE
may increase the efficacy of cryoablation and decrease its
adverse effects, especially bleeding. Therefore, sequential
TACE and cryosurgery may be a better therapeutic option for
unresectable HCC, especially large HCC. In another study
comparing PC followed by TACE with TACE alone for advanced
liver cancer, the rate of effectiveness of the combined group
was 100% and only 47% in the TACE alone group.43 When
determining the effect of order for combination cryosurgery
and TACE in 40 patients with advanced primary hepatic
carcinoma, about half of patients exhibited complete tumor
necrosis, and there was no significant difference between the
TACE/sequential cryosurgery and cryosurgery/sequential
TACE groups.44 However, progression-free survival in the
TACE/sequential cryosurgery group was significantly longer
than that of the cryosurgery/sequential TACE group.
Regarding combining cryoablation with gemcitabine + oxali-
platin for the treatment of advanced liver cancer, combination
therapy was found to be more efficacious, reliable and safe
than monotherapy.45
In a study with combination hepatic PC and percutaneous
ethanol injection for unresectable HCC, 33 patients (51%)
were tumor-free and 22 patients (34%) were alive with tumor
Journal of Clinical Translational Hepatology 2014 vol. 2 | 182–188
recurrence during a mean follow-up period of 14 months
(range 5–21 months). As cryoablation of tumors in multiple
organs (i.e., liver, lung, kidney, breast, pancreas, and
prostate47) has been demonstrated to be safe and effective,
comprehensive cryoablation (simultaneous cryoablation of
intrahepatic and extrahepatic tumors) significantly improves
the survival of metastatic hepatocellular cancer, and timely or
multiple treatments may both contribute more towards the
therapeutic effects than delayed or single treatment.48
Although cryosurgery can induce a cryo-immunological
response,8,19 the anti-tumor immune response is insuffi-
ciently strong to prolong patient life. At the same time,
immunotherapy mediated by autologous dendritic cells (DCs)
is a promising treatment option for the long-lasting control of
unresectable HCC.49–51 Increased knowledge of vaccination
using DCs co-cultured with cytokine-induced killer (CIK) cells
has led to the formulation of better strategies in the clinical
treatment of hepatic cancer.52 Combining these approaches,
Niu et al.53 evaluated whether combination PC and DC-CIK
immunotherapy can improve the therapeutic effect of cryo-
surgery for patients with metastatic hepatocellular cancer. To
stimulate DC recognition of tumor antigens in vivo, harvested
DC-CIKs were transfused into patients one week after
cryosurgery. The median OS of the cryo–DC-CIK immu-
notherapy group (32 months) was higher than that of the
cryotherapy group (18 months) and the untreated group (3
months), and multiple cryo-immunotherapy was associated
with better prognosis than single cryo-immunotherapy.53
Perhaps cryosurgery induces continuous antigen release in
vivo, and the adoptive transfer of CIK cells along with DCs can
present tumor antigens and destroy tumor cells in the body.
Another advantage of cryosurgery is the destruction of the
organ-like structural environment of a tumor, which is
considered hostile toward immune cells. Altogether, it
appears that PC and DC-CIK immunotherapy may be an
alternative therapeutic option for improving survival in
metastatic hepatocellular cancer.
185
 Niu L.Z. et al: Cryoablation for liver cancer

Table 2. Serious complications after hepatic cryoablation

Yang et al.56 Xu et al.37 Mu et al.48

Colorectal liver Metastatic

Tumor type HCC metastases hepatocellular cancer
Number of patients 300 326 33
Cryoshock syndrome 6 (2.0%) 1 (0.3%) -
Bleeding 5 (1.6%) 5 (1.5%) 6 (18.0%)
Liver abscess 2 (0.6%) 3 (0.9%) 2 (6.0%)
Liver failure 2 (0.6%) 1 (0.3%) -
Liver dysfunction 7 (2.3%) - -
Biliary fistulas - 3 (0.9%) -
Intestinal fistulas 1 (0.3%) - -
Renal insufficiency - 5 (1.5%) -
Acute myocardial infarction and severe arrhythmia 6 (2%) 2 (0.6%) -
Pain - 103 (31.6%) 13 (76.0%)
Fever 6 (2%) 108 (33.1%) -
Increased liver enzymes - 124 (38.0%) -
Thrombocytopenia - 58 (17.8%) 7 (21.0%)
Pleural effusion - 20 (6.1%) 2 (6.0%)
Liver capsular cracking 1 (0.3%) - 1 (3.0%)
Gastric mucosal lesion–induced hemorrhage 4 (1.3%) - -

Adverse effects (summarized in Table 2)
Adverse effects of varying degrees of severity during and
after hepatic cryoablation have been documented. In liquid
nitrogen ablation, the frequency of adverse effects may be as
high as 40% and include liver parenchyma fracture,
cryoshock, acute myocardial infarction, biliary fistula, hemor-
rhage, cold-induced lesions in nearby structures, thrombo-
cytopenia, and coagulopathy.54,55 Hepatic cryoablation–
related mortality is 1.5%,56 almost the same as that for liver
resection. There are relatively fewer and milder adverse
effects associated with percutaneous cryoablation than liquid
nitrogen cryoablation. Serious complications, including hepa-
tic failure, rupture, and cold shock, are occasionally observed
after the procedure.37,48
In one study of 300 HCC patients who underwent US-
guided PC, 6% developed serious complications, including
cryoshock syndrome in six patients, hepatic bleeding in five,
stress-induced gastric bleeding in four, liver abscess in one,
intestinal fistulas in one, and death due to liver failure in
two.57 Cold shock, a rare and serious complication of hepatic
cryoablation, has garnered much attention from clinical
researchers. The clinical features of cold shock include
varying degrees of renal failure, disseminated intravascular
coagulation, and adult respiratory distress syndrome. Its
occurrence is proportional to the size of the frozen tissue, and
often happens when the diameter of the area frozen exceeds
6 cm.58
Alternatively, freezing time rather than frozen area may be
much more important for the occurrence of cold shock.59
Another potentially serious complication that can lead to
death is an argon–helium gas embolism.60 Minor complica-
tions due to PC are chills, fever, hepatic region pain, and
elevated serum enzymes, which can be efficiently controlled
with symptomatic treatment.37,48 Coagulopathy, another
complication of hepatic cryoablation, occurs due to the
trapping and destruction of local platelets within the margin
of the cryolesion.61–63 The development of thrombocytopenia
is associated with increased length of stay and mortality.64–66
Although thrombocytopenia is considered relative to the
volume of frozen tissue,63,67 the number of freeze–thaw
cycles, and the number of cryoprobes,68 Shi et al.69 showed
that the diameter of the ablation does not limit the tendency
of platelets to change. Slight coagulopathy (platelet count
[70–100] 6 109/L) can recover naturally within a week, and
recombinant human interleukin-11 can assist recovery from
severe coagulopathy (platelet count , 70 6 109/L).69 At the
same time, PC of hepatic tumors may cause short-term pain
in some patients, which disappears within 15 days, and the
effect of alleviating cancer pain is sustained for .8 weeks
without severe side effects.70 Tumor seeding can be detected
in 0.8% of patients at 1–24 months (median, 6 months) after
cryoablation.71,72
Therefore, monitoring liver function and detecting blood
coagulation and renal function are very important before or
after hepatic cryoablation. Cryosurgery program selection,
accuracy of preoperative localization, and the frozen range all
can affect the occurrence of complications. To avoid serious
complications, all patients should be given diuretics and
mannitol, and their urine alkalized to prevent myoglobinuria
and renal injury.
Conclusions
Experimental and clinical applications have shown that hepatic
cryosurgery is safe and efficacious. Iceball formation can be
monitored visually, and cryoablation has little impact on
nearby large blood vessels. Cryosurgery can be performed
on its own or in combination with other methods, such as
radiation therapy, chemotherapy, immunology, or surgery, to

186 Journal of Clinical Translational Hepatology 2014 vol. 2 | 182–188

Niu L.Z. et al: Cryoablation for liver cancer
control disease. In the future, the safety and efficacy of hepatic
cryosurgery in specific areas of the liver lobe close to the
diaphragm and gall bladder should be examined. Presently,
there are several ablation techniques available for hepatic
cancer, such as radiofrequency, microwave, irreversible
electroporation, and cryosurgery. The ability to compare these
techniques and to know which patients are the best candidates
for each technique is of great importance. Simultaneously,
optimizing therapeutic conditions to enhance the efficacy and
safety of combination cryosurgery with other therapies
requires further investigation.
Conflict of interest
None
Author contributions
Performing all the cryosurgeries (LZN), Writing the paper
(JLL), Conducting and reviewing the article (KCX).
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Review Article
Hepatitis C Recurrence after Orthotopic Liver
Transplantation: Mechanisms and Management
Bobby Kakati1 and Anil Seetharam*1,2
1
Banner Transplant and Advanced Liver Disease Center, Phoenix, AZ, USA; 2University of Arizona College of Medicine-Phoenix,
Phoenix, AZ, USA
Abstract
Chronic Hepatitis C (HCV) infection is the leading indication
for orthotopic liver transplantation and recurrence is nearly
universal. Chronic HCV infection is frequently established
through evasion of the innate immune system. Priming of
adaptive immune responses modulate the severity and rate
of fibrosis progression. Those with demonstrable viremia
entering the transplant period uniformly suffer recurrence
post-transplant. Progression to cirrhosis is accelerated post-
transplant secondary to systemic immunosuppression. In
addition, a number of factors, including donor, host, and viral
characteristics, influence severity and rate of fibrosis pro-
gression. Interferon-based therapy, the previous standard of
care, in those with advanced cirrhosis or post-transplant has
been limited by a number of issues. These include a relative
lack of efficacy and poor tolerability with higher incidence of
infection and anemia. Recently, approval of direct acting
antivirals have ushered in a new era in HCV therapeutics and
have applicability in these special populations. Their use
immediately prior to or post-transplant is expected to
improve both morbidity and mortality.
E 2014 The Second Affiliated Hospital of Chongqing Medical
University. Published by XIA & HE Publishing Ltd. All rights
reserved.
Introduction
Chronic hepatitis C (HCV) infection is a major public health
problem; with an estimated 180 million people infected
worldwide and approximately 4 million infected in the
Keywords: Innate immunity; Fibrogenesis; Sofosbuvir; Simeprevir.
Abbreviations: AASLD, American Association for Liver Disease; ALT, alanine
aminotransferase; CMV, cytomegalovirus; CsA, cyclosporine; CTP, Child Turcotte
Pugh; CUPIC, compassionate use of protease inhibitors in viral C cirrhosis; DAA,
direct acting antiviral; DCD, donation after cardiac death; DDLT, deceased donor
liver transplantation; FCH, fibrosing cholestatic hepatitis; FDA, Food and Drug
Administration; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HHV,
human herpes virus; IDSA, Infectious Disease Society of America; IGD, immune-
mediated graft dysfunction; IL, interleukin; ISG, interferon stimulated gene;
LADR, low accelerated dose regimen; LDLT, liver donor liver transplantation;
MELD, model for end-stage liver disease; NS, non-structural; Peg-IFN, pegylated
interferon; PTVR, post-transplant virologic response; RBV, ribavirin; RNA,
ribonucleic acid; SNP, single nucleotide polymorphism; SVR, sustained virologic
response; Tac, tacrolimus; Th, T-helper; Treg, t-regulatory; US, United States.
Received: 14 May 2014; Revised: 06 June 2014; Accepted: 07 July 2014
q
DOI: 10.14218/JCTH.2014.00016.
*Correspondence to: Anil Seetharam, University of Arizona College of Medicine-
Phoenix, Banner Transplant and Advanced Liver Disease Center, Phoenix, AZ
85006, USA. Tel: +1-602-839-7000, Fax: +1-602-839-7050, E-mail: Anil.
Seetharam@bannerhealth.com
Journal of Clinical and Translational Hepatology 2014 vol. 2 | 189–196
United States (US).1 For approximately two decades, therapy
has been relatively expensive and difficult to complete. More
recently, direct acting antivirals (DAAs) have been developed
and are revolutionizing treatment paradigms.2 Their timely
application prior to the development of cirrhosis is projected
to lessen the incidence of end stage liver disease (and
associated complications) attributable to chronic HCV infec-
tion. In addition, their use is expected to improve outcomes
after orthotopic liver transplantation.
Transplantation for hepatitis C virus
HCV infection is currently the leading indication for orthotopic
liver transplantation, and it is estimated that approximately
one-third of patients on the waiting list for transplant are
infected.3 Although the incidence of new infection with
progression to cirrhosis may be declining, there has been a
concomitant rise in transplant listing for hepatocellular
carcinoma (HCC) related to HCV cirrhosis.4
While graft and patient survival rates have steadily
improved for non-HCV related indications, this is not true for
HCV related transplants.5 For over a decade, relatively poorer
results have been noted for those with HCV indications.6 In a
recent review of the Organ Procurement Transplant Network/
United Network of Organ sharing (OPTN/UNOS), 3 year
survival was at 78% for 7,459 anti-HCV positive recipients
and 82% for 20,734 anti-HCV negative recipients.7 Similar
results were reported in Europe: 73% survival in non-HCV
recipients compared with only 66% in HCV positive recipi-
ents.8 Significant interest has focused on reasons for this
discrepancy in outcomes with HCV. In this context, systemic
immunosuppression and advancing donor age appear to be
important.9,10 The phenomenon of HCV recurrence after
transplant is the driving force for these poorer outcomes.11
HCV reinfection
Reinfection of liver allografts is considered universal and
occurs at the time of allograft reperfusion.12,13 During the
anhepatic phase of transplant surgery, HCV ribonucleic acid
(RNA) levels decline to undetectable levels, but after only a
few hours, increase rapidly to peak by the fourth post-
operative month.14 At 1 year, HCV RNA levels are generally 1
to 2 logs higher than prior to liver transplant.15 The diagnosis
of recurrent HCV infection requires detection of HCV RNA in
serum; and the diagnosis of recurrent disease requires
compatible histology as well. Histologic features of liver injury
will typically develop after 3 months and resemble those seen
in the native liver.16 Once re-infection is established, the
 Kakati B. et al: HCV recurrence after orthotopic liver transplantation
disease progresses; and 20% to 54% of liver transplant
recipients develop bridging fibrosis-cirrhosis within 5 years
post-transplantation.17 Persistent low level inflammation and
loss of viral control mechanisms from systemic immunosup-
pression account for this accelerated progression of cirrhosis.
In addition, co-factors, such as diabetes mellitus (preexisting
or promoted by use of calcineurin based immunosuppression),
may further promote disease progression. Accelerated pro-
gression post-transplant is variable; dependent upon the
degree of immunosuppression and a number of patient co-
factors and baseline demographics. Variability in the afore-
mentioned manifests through multiple clinical variations of
recurrence: ranging from standard ‘‘run of the mill’’ HCV
recurrence to the highly lethal, e.g. fibrosing cholestatic
hepatitis (FCH).
Clinical presentations of HCV recurrence
‘‘Standard’’ HCV recurrence
Transition from acute hepatitis to chronic hepatitis usually
occurs between 3 and 9 months after transplantation.
Transplant recipients may develop histologic features of acute
hepatitis C accompanied by a rapid rise in HCV viral load. The
most common pattern of recurrence is the evolution over
time to chronic hepatitis, as it occurs in immunocompetent
patients (albeit at higher levels of viremia and faster
progression of fibrosis). Fibrosis can progress linearly, but
its course is variable and often unpredictable.18 Interestingly,
a non-Markov analysis based on 901 fibrosis measurements
in 401 patients showed that risk of progression decreases as
time in a given stage increases.19 However, a longer time to
reach a stage does not predict a lower risk of progressing to a
higher stage. Serial biopsies in patients with recurrent
hepatitis C have demonstrated annual rates of fibrosis
progression between 0.3 and 0.6 stages/year (score F0-F4)
versus 0.1 to 0.2 stages/year in immunocompetent patients
with chronic hepatitis C.20–22 The median interval from
transplantation to cirrhosis is 9.5 years versus 30 years from
infection until cirrhosis in immunocompetent patients. The
best predictor of cirrhosis risk at 5 years is severity of
necroinflammatory activity in the allograft at 12 months post-
transplant.23 Often, protocol biopsies are performed by
transplant programs to monitor fibrosis progression.
Fibrosing cholestatic hepatitis
FCH is a presentation of HCV largely unique to liver allograft
recipients and often occurs within the first year after
transplantation. FCH can be present in over-immunosup-
pressed recipients, and studies typically show homogeneous
viral quasispecies and massive HCV RNA levels in the
peripheral circulation (usually .30–50 million IU/mL).24
Liver damage in FCH is due to a direct viral cytopathic effect
on hepatocytes from massive HCV replication.25 The typical
case of FCH HCV is characterized by extensive hepatocyte
swelling necrosis, cholestasis, and Kupffer cell hypertrophy in
combination with portal expansion due to prominent ductular
reaction (often with absent or rare Cytokeratin-7+ intermediate
cells) and fibrotic-type interface activity with mild mixed or
even neutrophil-predominant portal inflammation.26 The intra-
hepatic immune response in FCH HCV is typically T-helper (Th)-
2–like; whereas it is Th-1–predominant in conventional
recurrent HCV, and the few infiltrating lymphocytes often lack
190 Journal of Clinical and Translational Hepatology 2014 vol. 2 | 189–196
HCV specificity.27 Apart from immunosuppression, clear risk
factors with regard to genotype and patient demographics have
not been well established. FCH usually leads to liver failure/
graft loss within 1 to 2 years after transplantation.
Autoimmune HCV recurrence
Recurrent chronic HCV can present with plasma cell–rich,
interface necroinflammatory activity resembling autoimmune
hepatitis.28 Determining whether this presentation repre-
sents an ‘‘autoimmune’’ variant of HCV, acute cellular
rejection, de novo autoimmune hepatitis, or a combination
is often very difficult and requires extensive history and
evaluation. This recurrence variant is often recognized during
the transition from acute hepatitis to chronic hepatitis or after
the onset of chronic hepatitis. Typical histologic findings
include ‘‘sheets of plasma cells’’ at the sites of severe
interface and/or perivenular necroinflammatory activity.29
In clinical scenarios where histology is highly consistent with
autoimmune hepatitis, immunosuppressive therapy can be
used to abrogate the severity of liver damage. However, such
therapies may enhance HCV replication and promote HCV
specific induced inflammation and fibrosis progression.
Factors affecting recurrence
Multiple factors are thought to affect HCV recurrence after
liver transplant. These can be organized as traits of the host
(recipient of new liver) or donor (transplanted liver) and viral
factors.23
Host factors
Gender
An epidemiologic study in the non-transplant setting demon-
strated that females exhibit a slower rate of fibrosis progres-
sion per year and a lower overall incidence of end-stage liver
disease than men.30 Studies suggested a possible anti-
fibrogenic effect of estrogen on hepatic stellate cells and less
rapid progression pre-transplant.31,32 The situation appears
to be different post-transplant; however, as one multicenter
study of more than 500 HCV-positive recipients found the risk
of severe hepatitis C recurrence following transplantation
from a donor older than 60 years was doubled in female
recipients compared to males.33 Higher rates of graft loss and
advanced fibrosis in women have been corroborated by a
more recent study.34
Host immune responses
Strong multispecific CD4+ and CD8+ T-cell responses are
associated with spontaneous clearance and successful
antiviral therapy during the course of HCV infection in the
native liver–signifying their importance in abrogation of
inflammation/fibrosis progression. The blunting of these
seemingly protective adaptive immune responses by immu-
nosuppression may actually contribute to the universal
reinfection and accelerated disease progression observed
after transplantation.35 Detection of vigorous multispecific
CD4+ T-cell responses in the early post-transplant period may
predict mild graft injury and a greater response to antiviral
therapy. The presence of strong innate immune responses
(natural killer T cells) prior to transplantation may also
Kakati B. et al: HCV recurrence after orthotopic liver transplantation
provide protection from severe graft injury following liver
transplantation.36
In addition to the aforementioned risks, older age, African
American ethnicity, presence of metabolic syndrome, and
coinfection with HIV have been associated with worse
outcomes.37,38 It is speculated that these co-factors likely
modulate the natural history of recurrence and are well
established negative predictors of response to interferon
based antiviral therapy.39
Donor issues
Donor/transplant surgery-related issues
Multiple donor-related issues affect the outcome of hepatitis
C infection in the post-transplant setting. Worldwide, the
shortage of organs has led to an increasing use of ‘‘extended
criteria donors.’’ Such donor grafts of reduced quality may be
more sensitive to damaging events such as ischemia/
reperfusion injury and recurrent hepatitis C.40
Age and donation after cardiac death
In liver recipients with HCV, older donor age has emerged as
an important factor influencing disease recurrence and
progression.41 The mean age of donors has increased over
the last several years.42 Donation after cardiac death
(DCD) liver transplantation is associated with worse patient
and graft survival than donation after brain death liver
transplantation, with increased incidence of biliary and
vascular complications in HCV recipients, especially with
older donors.43–45
Steatosis
The role of donor steatosis and recurrence in HCV patients is
controversial. Fibrosis evolution appears to be higher when
graft steatosis is over 30%,46 and steatosis over 30–45% in
the donor liver is often avoided when HCV is the transplant
indication.47 Apart from sensitivity to ischemia/reperfusion
injury and HCV induced inflammation, there may be an
influence of steatosis on adaptive immune responses.48
Cold ischemia time
Recently implemented ‘‘Share 35’’ protocols have increased
regional sharing of liver allografts that in turn may increase
cold ischemia times. Recipients of livers from donors aged 45
years or older and cold ischemia times more than 12 hours
showed increased risk of graft failure compared with recipi-
ents of livers from donors younger than 45 years and cold
ischemia less than 12 hours.49
Live donor transplantation
Living donor liver transplantation (LDLT) is a rapidly evolving
field with expanding utilization in the face of significant organ
shortage. The Adult-to-Adult Live Donor Liver Transplant
Cohort Study found that graft survival in HCV-positive LDLT
recipients (once there was sufficient LDLT experience at a
given center) was similar to that of deceased donor liver
transplant (DDLT) recipients.50 Additionally, studies using
protocol liver biopsies to assess disease severity found no
significant difference in the rate of fibrosis progression
Journal of Clinical and Translational Hepatology 2014 vol. 2 | 189–196
between recipients of LDLT and DDLT within a 5 year follow-
up periods.51
Donor/host IL28B
Genome-wide studies have demonstrated a strong associa-
tion between allelic variations in the Interleukin-28 (IL28B)
gene and response to HCV therapy with interferon. The
precise mechanism through which the IL28B single nucleo-
tide polymorphism (SNP) genotype influences response to
antiviral treatment has not been fully characterized. IFN-
lambda 3, the product of the IL28B gene, belongs to the type
III interferon family and induces interferon-stimulated genes
(ISGs), differentiation of dendritic cells, modulation of Th1
and Th2 immune responses, and inhibition of T-regulatory
(Treg) cells that serve as a critical link between innate and
adaptive immune responses to viral infection.52 In a recent
study, recipients with a CC genotype (favorable response to
interferon based antiviral therapy) have relatively slower
histologic recurrence, with decreased alanine aminotransfer-
ase (ALT) levels and viral load when compared with non-CC
genotypes. The opposite association is seen with donor CC
genotype.53
Viral factors
Viral load and genotype
Higher HCV RNA levels in both serum and liver at the time of
transplantation are linked with increased risk of progression
to cirrhosis, graft loss, and death.35 There is some contro-
versy regarding the relationship between viral genotype and
severity of recurrence, as genotype 1b has been associated
with more severe recurrent hepatitis C in some patient series
but not in others.54,55
Viral genomic heterogeneity (Quasispecies)
Quasispecies diversity increases with the duration of chronic
HCV infection and may be most robust in end-stage liver
disease. Following liver transplantation, diversity falls during
the early period of intense immunosuppression and remains
low in those cases with rapidly progressive cholestatic
hepatitis C.56 Diversity frequently increases in those who
develop mild chronic hepatitis, reflecting increased selective
pressure during maintenance immunosuppression.57
Concomitant virus post transplant
Both cytomegalovirus (CMV) and human herpesvirus 6 (HHV
6) infection have been associated with increased progression
of fibrosis during HCV reinfection after liver transplantation.58
These relationships highlight the importance of CMV prophy-
laxis in transplant populations.59
Peri-transplant management of HCV
Historical perspective
The goal of any effective antiviral therapy is eradication with
minimal side effects. Historically, previous standard of care
treatment with pegylated interferon (Peg-IFN) and ribavirin
(RBV) contributed to a number of side effects and was largely
ineffective with sustained virologic response (SVR) rates
191
 Kakati B. et al: HCV recurrence after orthotopic liver transplantation
varying from 7–30% in HCV genotype 1 patients and 44–50%
in non 1 genotypes.60 Major predictors of achieving SVR
included: low pretreatment viral load, Child Turcotte Pugh
(CTP) score class A (genotype 1 only), and completion of
treatment.61 These early studies highlighted the risk of
decompensation with interferon based therapy in those with
advanced fibrosis and a particularly high risk for infection.62
Despite the significant incidence of adverse effects and
relative lack of efficacy, attempts at treatment were made
in an effort to ‘‘deliver the patient aviremic’’ to transplant
to lessen the chance of recurrence post-operatively.
Investigators further evaluated the strategy of a low accel-
erating dose regimen (LADR) in populations with advanced
fibrosis prior to transplant to prevent recurrence.63 Treatment
using a LADR was initiated with Peg-IFN-alpha-2b (0.75 mg/
kg/week) and RBV (600 mg/day). Dose escalations were
then performed at weeks one (Peg-IFN 1.5 microg/kg/week
and RBV 800 mg/day), two (RBV 1.0 g/day), and three (RBV
1.2 g/day for patients weighing greater than 75 kg) based on
tolerance, side effects, and weekly serum labs. Pre-transplant
treatment prevented post-transplant recurrence of HCV
infection in 25% of transplanted cases—22% in HCV geno-
types 1/4/6 and 29% in HCV genotypes 2/3. The strongest
predictor of post-transplant virologic response (pTVR) was
duration of treatment prior to transplant; however, Peg-IFN
and RBV were poorly tolerated with an increase in serious
adverse events (cytopenias, infection, and hepatic decom-
pensation) in the LADR treated cohort.
Directing acting antivirals pre-transplant
With the introduction of first generation direct acting anti-
virals telaprevir and boceprevir (NS3/4A protease inhibitors)
in 2011, much interest focused upon their applicability in
treatment experienced populations with advanced fibrosis.
However, phase III studies did not include a large number of
cirrhotics.64,65 Recently, results from the Compassionate Use
of Protease Inhibitors in Viral C Cirrhosis (CUPIC) cohort
evaluating the effectiveness of first generation protease
inhibitor with Peg-IFN and RBV in treatment experienced
HCV genotype 1 patients with cirrhosis were reported.66 In
511 patients who did not respond to a prior course of Peg-IFN
and RBV, telaprevir (n 5 299) or boceprevir (n 5 212) was
used for 48 weeks. Among the telaprevir treated cohort,
74.2% of previous relapsers, 40.0% of partial responders,
and 19.4% of null responders achieved SVR12. Among the
boceprevir cohort, 53.9% of relapsers, 38.3% of partial
responders, and none of the null responders achieved
SVR12. While efficacy was certainly improved with addition
of a first generation protease inhibitor, issues of tolerability
remained. Severe adverse events occurred in 49.9% of
cases, including liver decompensation and death in 2.2%.
On multivariate analysis, baseline parameters, including prior
null response and serum albumin level , 35 g/L, and platelet
count f 100,000), predicted serious adverse events and use
was cautioned in patients with these factors.
Towards the end of 2013, two additional agents were
added to the HCV armamentarium: simeprevir, a second
generation once daily NS3/4A inhibitor, and sofusbuvir, an
NS5B polymerase inhibitor of HCV.67,68 Clinical studies that
led to Food and Drug Administration (FDA) approval for
sofosbuvir demonstrated relatively high efficacy and safety in
patients with cirrhosis.69 Both agents are approved as agents
in combination with either Peg-IFN or RBV. As previously
192 Journal of Clinical and Translational Hepatology 2014 vol. 2 | 189–196
mentioned, there has been a rise in transplant listings for HCC
related to HCV cirrhosis, and HCC may develop in those with
well compensated cirrhosis (i.e., low native Model for End-
Stage Liver Disease (MELD). Such a patient population with
relatively well compensated cirrhosis listed for orthotopic
liver transplant by virtue of concomitant HCC represented an
excellent opportunity to evaluate second generation DAA
efficacy prior to transplant in preventing post-transplant
recurrence. Curry et al. recently reported results from a
Phase 2, open-label study investigating use of sofosbuvir plus
RBV for up to 48 weeks in patients with HCV listed for liver
transplant with HCC.70 Patients with chronic HCV infection of
any genotype listed for liver transplantation for HCC received
up to 48 weeks of sofosbuvir (400 mg/day) and RBV (1000–
1200 mg/day) before transplantation. Overall, sofosbuvir
and RBV therapy was safe with well compensated cirrhosis
and prevented post-transplant HCV recurrence in 64% of
patients who had HCV RNA , 25 IU/mL prior to transplant.
The number of consecutive days with HCV RNA target not
detected prior to transplant appeared to be the strongest
predictor of post-transplant HCV recurrence. Certainly, as
new agents are developed, their applicability will be tested in
cirrhotic populations to further optimize efficacy and mini-
mize serious adverse events (Table 1).
Treatment of recurrence post-transplant
Given the difficulty in treating cirrhotic populations prior to
transplant, much attention has focused on treatment post-
transplant. In this context, two approaches have been
examined: 1) a pre-emptive approach where antiviral ther-
apy is used in the first weeks following transplantation and 2)
a histologic, recurrence-based approach for patients with
established hepatitis. Early post transplantation therapy
administered during the first 2–7 weeks post-transplantation
before there is clinical evidence of liver damage has been
evaluated.71 The results have been disappointing overall in
terms of antiviral efficacy and tolerability. With PegIFN based
therapy, SVR rates of about 20% have been documented,
ranging from 18 to 39% (5–33% in genotype 1 and 14–100%
in genotypes 2/3).71 About 30% of patients discontinue
treatment, and dose reductions are required in 70% second-
ary to side effects, such as bacterial infections, hematological
toxicity, and rejections (0–26%).72
The most widely used strategy involves the initiation of
antiviral therapy once histologic consequences of HCV
recurrence are detected on allograft biopsy. With PEG-IFN
and RBV, the previous standard of care, studies estimate
SVR at a rate of 30%.73,74 Transplanted patients were
particularly predisposed to hematologic toxicities, especially
anemia (60–80%), necessitating careful and frequent RBV
dose adjustments.
In addition, patients with recurrent HCV infection treated
with Peg-IFN (PEG) after liver transplantation can develop
severe immune-mediated graft dysfunction (IGD) character-
ized by plasma cell hepatitis or rejection. A recent multicenter
case-control study of 52 liver transplant recipients with
hepatitis C assessed the incidence of, risk factors for, and
outcomes of PEG-IGD.75 Overall incidence of PEG-IGD during
a 10-year study period was 7.2%. Variables associated with
increased mortality included acute rejection as the PEG-IGD
sub-type and lack of a SVR. Variables associated with graft
failure included a high level of alkaline phosphatase at PEG
initiation and lack of a SVR.
Kakati B. et al: HCV recurrence after orthotopic liver transplantation

Table 1. Summary of Treatment Trials Pre-Transplant: Recent studies have evaluated the effectiveness of pegylated interferon (Peg-IFN) and ribavirin
(RBV) in conjunction with newer direct acting antiviral agents including: sofosbuvir (SOF), simeprevir (SMV) and ledipasvir (LED). Identification of
increasingly tolerable regimens prior to transplant able to achieve high SVR12 or reliable post transplant virologic response (pTVR) are in development.

Patients Virologic response (%) Adverse Influential

Study Agents (n) (SVR 12/pTVR) events (%) predictors
Everson et al.,63 Peg-IFN alfa2b 59 22% GT 1 68-Serious Duration of treat-
2013 & RBV (with 29% GT 2/3 ment .16 weeks
dose escalation)
Hezode et al.,66 Peg-IFN alfa 2b 511 SVR12/Agent/Previous history 49.9-Serious Response to prior
2014 & RBV with 299-Telaprevir Telaprevir therapy
Telaprevir or 212-Boceprevir 74.2/relapsers Albumin ,35 g/L
Boceprevir 40/partial responders Plts,100,000
19.4/null responders
Boceprevir
53.9/relapsers
38.3/partial responders
0/null responders
Curry et al.,70 Sofosbuvir & 61 64% 18-Serious Number of days
2013 RBV with HCV RNA
below lower limit
of detection
Lawitz et al.,84 Simeprevir & 87 SVR12/Duration/Agent 4.6-Serious ? Q80K poly-
2014 Sofosbuvir +/2 93/12 weeks/SOF + SMV morphism
RBV 93/12 weeks/SOF + SMV + RBV
100/24 weeks SOF + SMV
93/24 weeks/SOF +SMV + RBV
Afdahl et al.,85 Ledipasvir & 440 total trea- SVR12/Duration/Agent 0-Serious ? NS5A resis-
2014 Sofosbuvir ted 86/12 weeks/SOF + LED 67-90% mild to tance at baseline
+/2 RBV (88 Cirrhotic) 82/12 weeks/SOF + LED + RBV moderate
99/24 weeks SOF + LED depending on
99/24 weeks/SOF + LED + RBV regimen/duration

Directing antivirals post-transplant
Interferon based therapy post-transplantation is fraught with
a number of issues, including: comparative lack of efficacy,
infection, anemia, and immune mediated graft dysfunction.
However, similar to the case of their applicability immediately
prior to transplant, much interest has focused on their
efficacy in treatment of recurrence. A major limitation with
first generation DAAs (telaprevir and boceprevir) are inter-
actions with calcineurin inhibitors cyclosporine (CsA) and
tacrolimus (Tac), the cornerstones of immunosuppression
post liver transplant. Boceprevir and telaprevir are potent
inhibitors of cytochrome P450 3A4. In a study of healthy
volunteers, boceprevir was shown to increase the area under
the curve of CsA and Tac by 2.7 and of 17, respectively.76 In
another study, the concomitant administration of a calci-
nuerin inhibitor with telaprevir in healthy volunteers
increased CsA and Tac exposures approximately 4.6-fold
and 70-fold, respectively.77
Recently, interim results from Europe evaluating the
efficacy of telaprevir in conjunction with Peg-IFN and RBV
post-transplant have been presented.78 Patients receiving
calcineurin based immunosuppression (6 months to 10 years
after LT), with fibrosis stage F0-F3, received telaprevir, Peg-
IFN, and RBV for 12 weeks and Peg-IFN and RBV for an
additional 36 weeks. The study included 74 post-transplant
patients, and at week 12, 40 (80%) patients on tacrolimus
and 21 (87.5%) on cyclosporine achieved undetectable HCV
RNA. Common adverse events included anemia, pruritus, and
rash. These results show improved efficacy with first genera-
tion DAAs and, importantly, manageable levels of immuno-
suppression with close monitoring.
A US based retrospective cohort study of 81 patients with
genotype 1 HCV treated with boceprevir (10%) or telaprevir
(90%) plus Peg-IFN and RBV was performed with SVR12 as
the primary endpoint.79 The intent-to-treat SVR12 rate was
63% in this post-transplant cohort. Patients with extended
rapid virologic response (undetectable HCV RNA at 4 and 12
weeks after starting boceprevir or telaprevir) had higher rates
of SVR12 compared to all other patients (85% vs. 15%).
Similar to the European study, adverse effects were common;
21% of patients experienced hemoglobin ,8 g/dL, and 57%
required blood transfusions during the first 16weeks. Twenty
seven percent were hospitalized, and 9% died.
In another investigation of 37 liver recipients with
advanced HCV recurrence (either o F2 fibrosis [n531] or
fibrosing cholestatic hepatitis [n56]) treated with Peg-IFN,
RBV, and first generation DAA, SVR12 was observed in 20%
(1/5) and 71% (5/7) of patients in telaprevir and boceprevir
groups, respectively (p50.24).80 Treatment was discontinued
in 16 patients (treatment failures (n511), serious adverse
events (n55)).
Emerging data with second generation DAAs shows
promise in the post-transplant population regarding efficacy
and tolerability. Preliminary results presented at AASLD 2013
showed that in a population of transplant recipients with HCV
recurrence (predominately genotype 1) treated with sofos-
buvir (400 mg daily) along with RBV (400 mg daily) with dose

Journal of Clinical and Translational Hepatology 2014 vol. 2 | 189–196 193

 Kakati B. et al: HCV recurrence after orthotopic liver transplantation

escalation based on hemoglobin, 77% of patients achieved
SVR 4 and all patients demonstrated HCV RNA below the
lower limit of detection 4 weeks after treatment.81 Treatment
was well tolerated and no interactions with immunosuppres-
sive agents were observed. In addition, recent data from the
sofosbuvir compassionate use program for patients with
severe recurrent hepatitis C, including fibrosing cholestatic
hepatitis following liver transplantation, showed higher rates
of SVR12 (62%) than standard therapies in this context,
improved liver function tests, and improved clinical out-
comes82 (Table 2).
Recently, the use of sofosbuvir and simeprevir in combina-
tion has been examined.83 At EASL 2014: Cohort 2 of the
COSMOS trial consisting of those with advanced fibrosis (F3-
F4) showed overall SVR rates of over 90% with a 12 week
regimen not containing RBV.84 Phase III clinical studies
evaluating the combination of sofosbuvir and simeprevir are
currently underway.
In the upcoming months, further regimens will be added
to the HCV armamentarium. It is expected that newer
approved regimens with agents such as ledipasvir (NS5A
inhibitor) will have sufficient data to make recommendations
for usage in those with cirrhosis and possibly on the road
to liver transplant.85 In addition, studies for the afore-
mentioned agents are being conducted in post-transplant
populations. In another recent study of a regimen containing
ABT-450/r/ABT-267 along with ABT-333 and RBV in liver
transplant recipients with genotype 1 recurrence, 96% of
patients achieved an SVR12 with 24 weeks of therapy.86 An
unprecedented number of treatment regimens are on the
horizon.
The American Association for Liver Diseases (AASLD) and
Infectious Disease Society of America (IDSA) have published
recommendations regarding treatment of HCV recurrence
in the allograft.87 Here is a new recommended treatment
option for treatment naı̈ve patients with genotype 1 recur-
rence in the allograft includes: sofosbuvir (400 mg) plus
simeprevir (150 mg), with or without RBV (initial dose
600 mg/day, increased monthly by 200 mg/day as tolerated
to weight-based dose of 1,000 mg [,75 kg] to 1200 mg
[.75 kg]), for 12 weeks to 24 weeks. Another alternate
regimen for treatment naı̈ve patients with genotype 1 HCV in
the allograft liver, including those with compensated cirrho-
sis, is as follows: sofosbuvir (400 mg) and RBV (initial dose
600 mg/day, increased monthly by 200 mg/day as tolerated
to weight-based dose of 1000 mg [,75 kg] to 1200 mg
[.75 kg]) with or without Peg-IFN (in the absence of contra-
indication to its use), for 24 weeks in patients with compen-
sated allograft HCV genotype 1 infection. A recommended
regimen for treatment-naı̈ve patients with HCV genotype 2 or
3 in the allograft liver, including those with compensated
cirrhosis, is as follows: sofosbuvir (400 mg) and RBV (initial
dose 600 mg/day, increased monthly by 200 mg/day as
tolerated to weight-based dosages) with consideration of
the patient’s creatinine clearance value and hemoglobin level
for 24 weeks. These guidelines are dynamic and are expected
to be revised as new agents become approved and clinical
experience grows.
Conclusions
Chronic Hepatitis C remains the leading indication for liver
transplantation, and recurrence post-transplantation is uni-
versal for those who enter the transplant period with viremia.
Recurrence is characterized by an accelerated progression to
fibrosis that is thought to be related to loss of viral control in
the context of systemic immunosuppression. A number of
donor, host, and viral characteristics influence the clinical
manifestations of recurrence. Previous standard of care
therapy with Peg-IFN in end-stage liver disease was limited
by decompensation, infection, and post-transplant by ane-
mia. Newer additions to the direct acting antiviral armamen-
tarium have been introduced with increasing applicability in
cirrhotic and post-transplant populations. Their timely use
prior to transplant is expected to improve outcomes post-
transplant; and their use post-transplant is expected to
improve morbidity and mortality.

Table 2. Summary of Treatment Trials Post-Transplant: Recent studies have evaluated the effectiveness of pegylated interferon (Peg-IFN) and ribavirin
(RBV) in conjunction with newer direct acting antiviral agents including post-transplant

SVR 12
Patients (%) (n where
Study Agents (n) applicable) Adverse events
78
Forns et al., 2014 Peg-IFN alfa 2b & 74 59.6% (19/32) 11%-serious
(interim analysis) RBV & Telaprevir 60%-anemia
Burton et al.,79 2014 Peg-IFN alfa 2b & 81 63% Common
RBV & Telaprevir or (10% boceprevir/ 21% Hgb,8g/dL
Boceprevir 90%telaprevir) 57% requiring transfusions
9% liver related death
Coilly et al.,80 2014 Peg-IFN alfa 2b & 37 71% (5/7) Common
RBV & Telaprevir or Boceprevir (n518) Boceprevir 16 discontinuations
Boceprevir Telaprevir (n519 20% (1/5) Telaprevir 92%-anemia
Charlton et al.,81 2013 Sofosbuvir & RBV 40 SVR 4–77% 15%-serious
(abstract)
Forns et al.,82 2014 Sofosbuvir & RBV 104 62% 48%-serious
(abstract)
Kwo et al.,86 2014 ABT-450/r/ABT-267 + 34 96.2% 5.8%-serious
(abstract) ABT-333 + Ribavirin 17.6%-anemia

194 Journal of Clinical and Translational Hepatology 2014 vol. 2 | 189–196

Kakati B. et al: HCV recurrence after orthotopic liver transplantation
Conflict of interest
Dr. Seetharam reports the following: speaker’s bureau:
Merck, Gilead; advisory boards: Janssen, Gilead.
Author contributions
Writing the review (BK, AS).
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[69] Jacobson IM, Gordon SC, Kowdley KV, Yoshida EM, Rodriguez-Torres M,
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[73] Xirouchakis E, Triantos C, Manousou P, Sigalas A, Calvaruso V, Corbani A,
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Review article
Antiviral Therapy of Liver Cirrhosis Related to Hepatitis B
Virus Infection
Lun-Gen Lu*
Department of Gastroenterology, Shanghai First People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
Abstract
Hepatitis B virus (HBV) infection is a leading cause of liver
disease worldwide, with 75% of those affected distributed in
the Asia-Pacific region. Approximately one million HBV-
infected patients die of liver cirrhosis and hepatocellular
carcinoma (HCC) each year. If left untreated, 6–20% of
chronic hepatitis B (CHB) patients will develop cirrhosis over
five years. The cumulative incidence of HBV-related cirrhosis,
disease progression, and prognosis are closely associated
with serum HBV DNA levels. Antiviral therapy in HBV-related
cirrhosis has been documented by several long-term cohort
studies to decrease disease progression to hepatic decom-
pensation and HCC. The approval and availability of oral
antiviral agents with better safety profiles has greatly
improved the prognosis for HBV-related cirrhosis. Here, we
discuss the significance of antiviral therapy for HBV-related
cirrhosis and the management of HBV-related diseases in the
future.
E 2014 The Second Affiliated Hospital of Chongqing Medical
University. Published by XIA & HE Publishing Ltd. All rights
reserved.
Introduction
Hepatitis B virus (HBV) infection, the major cause of liver
cirrhosis and hepatocellulcar carcinoma (HCC), is a serious
global health concern. The World Health Organization (WHO)
reported that approximately 45% of the population lives in an
area with a high prevalence of chronic hepatitis B (CHB).1
CHB is particularly prevalent in the Asia-Pacific region. Of
approximately 350–400 million chronically infected carriers,
approximately 75% are found in Asia.2,3 HBV is the tenth
leading cause of death worldwide, and each year more than
one million chronic HBV carriers worldwide die of liver
cirrhosis and HCC, the most severe adverse sequelae of
CHB.4 Approximately 6–20% of CHB subjects will develop
cirrhosis within five years if left untreated. The incidence of
Keywords: Hepatitis B virus; Chronic HBV infection; Liver cirrhosis; Antiviral
therapy.
Abbreviations: AASLD, American Association for the Study of Liver Disease;
ADV, adefovir dipivoxil; ALT, alanine transaminase; CHB, chronic hepatitis B; CI,
confidence interval; ETV, entecavir; HBeAg, hepatitis B envelope antigen; HBV,
hepatitis B virus; HCC, hepatocellular carcinoma; IFNa, interferon alpha; LAM,
lamivudine; Ldt, telbivudine; TDF, tenofovir; WHO, World Health Organization.
Received: 15 June 2014; Revised: 26 July 2014; Accepted: 27 July 2014
q
DOI: 10.14218/JCTH.2014.00022.
*Correspondence to: Lun-Gen Lu, No 100, Haining Road, Department of
Gastroenterology, Shanghai First People’s Hospital, Shanghai Jiaotong University
School of Medicine, Shanghai 200080, China. Tel: +86-21-63240090, Fax: +86-21-
63241357, Email: lungenlu1965@163.com
Journal of Clinical and Translational Hepatology 2014 vol. 2 | 197–201
HBV-related cirrhosis, disease progression, and outcomes are
closely associated with serum HBV DNA levels. Despite the
availability since 1982 of a highly effective immunization
program against HBV that decreased the incidence of HBV,5,6
the mortality from HBV-related cirrhosis and HCC has actually
increased because of the high rate of HBV infection.7 Given
the natural course of HBV infection and disease progression,
timely management of CHB is of great importance for
effective HBV therapy. The goal of antiviral treatment for
HBV cirrhotic patients is to prevent disease progression to
decompensated cirrhosis, end-stage liver disease, and HCC
and to improve survival.8,9 Here, we review the significance
of antiviral therapy for HBV-related cirrhosis and discuss
concerns regarding the future management of HBV-related
liver diseases.
Progression of CHB from fibrosis to cirrhosis
HBV primarily interferes with liver function by replicating in
hepatocytes, and it is not directly cytopathic. However,
infection with HBV does cause irritation and swelling (inflam-
mation) of the liver. Acute hepatitis B infection does not
usually require treatment, and most adults clear the infection
spontaneously.10,11 If the virus remains in the blood for
more than six months, this is chronic HBV infection. CHB
is a dynamic state of interactions among HBV, hepatocytes,
and the host immune system. CHB elicits varying degrees of
predominantly lymphocytic infiltrates in the portal tracts
associated with portal inflammation, interface hepatitis, and
spotty lobular inflammation.12 Inflammation can lead to liver
damage, including fibrosis (light to medium scarring of the
liver), cirrhosis (extensive scarring of the liver), and liver
cancer. Liver fibrosis refers to the hyperplasia of extracellular
matrix, including collagen, in liver tissues. It is a scarring
process that represents the liver’s response to injury.
Advanced liver fibrosis results in cirrhosis, liver failure, and
HCC.
The replication of HBV in hepatocytes plays a key role in
driving the progression of liver disease. Following develop-
ment of fibrosis or compensated cirrhosis in patients with
CHB, liver disease may continue to progress and decom-
pensation or HCC may occur, especially in those with active
viral replication. If left untreated, approximately 20% will
decompensate over five years and develop complications of
end-stage liver disease.9 Once decompensation occurs, the
cost for CHB therapy increases and the prognosis is poor.
Therefore, the primary aim of therapy is to eliminate or
permanently suppress HBV to reduce disease activity and
thereby reduce the risk or slow the progression of liver
disease. The ultimate long-term goal is to achieve a sustained
 Lu L.G.: Antiviral therapy liver cirrhosis hepatitis B virus infection
response to prevent or reduce the development of hepatic
decompensation, cirrhosis, or HCC and to prolong survival.13
Although none of the available drugs can clear the
infection, they can stop the virus from replicating, thus
minimizing liver damage and decreasing the progression to
cirrhosis, hepatic decompensation, and HCC. According to the
guideline on prevention and treatment of chronic hepatitis B
in China (2010),14 the goal of antiviral therapy for CHB is to
maximize the suppression or elimination of HBV, decrease
liver inflammation and fibrosis, decrease the liver disease
progression to cirrhosis, hepatic decompensation, HCC, and
complication of end-stage liver disease, and prolong survival.
The American Association for the Study of Liver Disease
(AASLD) Practice Guidelines on Chronic Hepatitis B (2009)
stated that to gain the goal of preventing cirrhosis, liver
failure, and HCC, antiviral therapies are needed to restore
liver function and improve survival in patients with CHB.15 A
recent meta-analysis by Mommeja-Marin et al. showed that
antiviral therapy in hepatitis B envelope antigen (HBeAg)
positive patients was associated with continuing improve-
ment in liver histology.16 The 2012 update of the Asian-Pacific
consensus statement on the management of CHB also stated
that active HBV replication is the key driver of liver injury and
disease progression, and thus, sustained viral suppression is
of paramount importance.17 Oral antiviral therapy for CHB
should be instituted regardless of HBV DNA level as early as
possible to prevent the occurrence of hepatic decompensa-
tion and HCC. Therefore, the key to treating HBV-related
cirrhosis is to decrease infectivity and pathogenicity of the
virus by using antiviral agents.
Serum HBV DNA level and risk of cirrhosis and HCC
Cirrhosis develops during CHB disease progression as a result
of hepatic inflammation and subsequent fibrosis. Risk factors
associated with cirrhosis progression include viral replication,
age, alcohol intake, co-infection with other viruses, and
possibly HBV genotype.18 Several cohort studies have
revealed the link between HBV replication and the develop-
ment of cirrhosis and HCC. Multivariate analysis of a
prospective study with 93 CHB patients who developed
cirrhosis during regular follow-up (mean 102 months)
showed that persistent HBeAg seropositivity was significantly
(p50.035) associated with the development of decompensa-
tion. The risk of hepatic decompensation was six-fold higher
in persistently HBeAg positive patients than in patients who
were seronegative for HBeAg at entry.19 Another population-
based prospective cohort study of 3,582 untreated hepatitis
B-infected patients in Taiwan found that having a baseline
HBV DNA level .104 copies/ml was the strongest indepen-
dent predictor of cirrhosis.13 During a mean follow-up time
of 11 years, it was found that when the baseline HBV DNA
level increased from ,300 copies/ml to 16106 copies/ml,
the cumulative incidence of cirrhosis significantly
increased. For those with HBV DNA levels ,300 copies/ml,
300-9.9610 3 copies/ml, 1610 4 –9.9610 4 copies/ml,
16105–9.96105 copies/ml, and o16106 copies/ml, the
incidence of cirrhosis was 4.5%, 5.9%, 9.8%, 23.5%, and
36.2%, respectively (p , 0.001), and the relative risk of
cirrhosis was 1.0, 1.4, 2.5, 5.9, and 9.8, respectively
(p,0.001). After adjusting for age, sex, smoking, alcohol,
HBeAg status, and serum alanine transaminase (ALT) level,
the multivariate-adjusted relative risk (95% confidence
interval, CI) of cirrhosis in all patients was 1.0, 1.4, 2.5,
198 Journal of Clinical and Translational Hepatology 2014 vol. 2 | 197–201
5.6, and 6.5, respectively (p,0.001). In the 2,923 HBeAg-
negative patients with a normal ALT level, the adjusted
relative risk (95% CI) of cirrhosis was 1.0, 1.4, 2.5, 5.6, and
6.6, respectively (p,0.001). These data suggested that
active HBV replication following the onset of cirrhosis is an
important prognostic factor for disease progression and that
the progression to HBV-related cirrhosis is strongly correlated
with the level of circulating virus. The risk for cirrhosis
increases significantly with increasing HBV DNA levels and
is independent of HBeAg status and serum ALT level.
Intervention of HBV-related cirrhosis disease
progression by antiviral therapy
The immediate goal of antiviral therapy of CHB is to improve
hepatic dysfunction and rescue patients from mortality.9,14
Antiviral agents in use clinically are interferon alpha (IFNa)
and nucleos(t)ide analogues, such as lamivudine (LAM),
adefovir dipivoxil (ADV), telbivudine (LdT), entecavir (ETV)
and tenofovir (TDF). Both IFNa and nucleos(t)ide analogues
can be effective, but the duration of treatment, side effects,
treatment costs, and drug resistance must be taken into
consideration when determining an optimal choice based on
individual patient characteristics.20
An effective antiviral agent should be able to achieve
potent and durable viral suppression to low or undetectable
levels and possess a high genetic barrier to prevent the
occurrence of resistance.21
IFN
Long-term follow-up studies of IFN therapy show inconsistent
results.22–29 An early pilot study by Ikeda et al. demonstrated
that IFNa decreased hepatocellular carcinogenesis in patients
with cirrhosis caused by HBV infection.23 Of the 313 patients
evaluated, the cumulative occurrence of HCC was signifi-
cantly lowered by the intermittent administration of IFNa
(n594) compared with those receiving no therapy (n5219) at
three (4.5% vs. 13.3%), five (7.0% vs. 19.6%), and 10 years
(17.0% vs. 30.8%), respectively. The rate of HCC develop-
ment in the treated group was significantly lower than that of
the untreated group (P50.0124). In another study of 233
HBeAg-positive IFN-treated patients (8.1% with cirrhosis)
and 233 matched controls (10.7% with cirrhosis),24 the
incidence of HCC appeared to be reduced by therapy (2.7%
vs. 12% in controls, p, 0.01) but was limited to patients with
pre-existing cirrhosis. However, other studies have shown no
benefit of IFNa in the prevention of HCC in HBV infected
patients because HBeAg seroconversion was used as the
endpoint of treatment and detectable serum HBV DNA, a
relevant risk factor for HCC, remained in most of the
patients.13 It was also reported that pegylated (PEG)-IFNa
did not influence the development of HCC in patients with
mild or moderate fibrosis.25–27,30 Thus, the efficacy of IFNa in
preventing HBV-related HCC is controversial, and there are
several limitations to most studies with positive results. A
recent meta-analysis13 evaluated the efficacy and safety of
adjuvant IFN therapy in patients with HCC who have under-
gone hepatic resection, transplantation, or locoregional
ablation therapy. It showed that adjuvant IFN therapy can
improve both recurrence-free survival and overall survival.
However, the benefits of using this agent should be weighed
against its side effects.
Lu L.G.: Antiviral therapy liver cirrhosis hepatitis B virus infection
IFNa is not recommended for patients with decompen-
sated cirrhosis. No significant benefit was found in two
studies evaluating the use of IFN treatment in HBV patients
with Child-Pugh B or C cirrhosis.31,32 Virologic and biochem-
ical responses were observed in only 33% and 0% of Child-
Pugh B and C patients, respectively.32 In addition, even at low
doses of IFNa, severe side effects due to bacterial infections
and worsening liver failure occurred.32 At present, IFN
therapy is now contraindicated for the treatment of decom-
pensated cirrhotic patients, even with the availability of safer
alternative agents and modalities.
Nucleos(t)ide analogues
Currently, the use of nucleos(t)ide analogues has been widely
reported for antiviral therapy in active and decompensated
HBV-related cirrhosis. Relative to placebo therapy, treatment
with nucleos(t)ide analogues offered improved liver histology
and survival benefits.
A recent study investigated long-term outcomes and
prognostic factors in patients with HBV-related cirrhosis
in the era of oral nucleos(t)ide analog antiviral agents.33 It
was found that in 78% of patients who received antiviral
treatment, sustained viral suppression (serum HBV DNA ,
105 copies/mL) was achieved during a mean follow-up period
of 46 months. The five year cumulative incidences of death,
hepatic decompensation, and HCC were 19.4% vs 43.9%
(log-rank P , 0.001), 15.4% vs 45.4% (p 5 0.001), and
13.8% vs 23.4% (p 5 0.074), respectively. Therefore, oral
antiviral agents have improved the prognosis of patients with
HBV-related cirrhosis.
In a study with 651 CHB patients who had confirmed
cirrhosis or advanced fibrosis, LAM therapy was shown to
delay clinical progression by significantly reducing the
incidence of hepatic decompensation and the risk of HCC.34
The patients were randomly assigned in a 2:1 ratio to receive
LAM (100 mg per day, n5436) or placebo (n5215) for a
maximum of five years. An increase of two points or more in
the Child-Pugh score was pre-set as the primary endpoint for
disease progression. Due to a significant difference between
treatment groups in the number of end points reached, the
study was terminated after a median treatment duration of
32.4 months (range, 0 to 42). At the time of data analysis,
the Child-Pugh score increased in 3.4% of patients receiving
LAM and 8.8% of those receiving placebo (hazard ratio, 0.45;
P50.02). LAM treatment significantly reduced the rate of
disease progression relative to placebo controls (7.4% vs.
18.0%, p50.001) and HCC development (3.9% vs. 7.4%
placebo controls, hazard ratio, 0.49; p50.047). Overall, the
magnitude of protection conferred by LAM is substantial, with
an approximately 50% reduction in disease progression. This
study suggested that long-term LAM treatment can prevent
complications of CHB and reduce the incidence of hepatic
decompensation and HCC.
However, LAM-resistant HBV mutations with amino acid
substitutions in the tyrosine-methionine-aspartate-aspartate
(YMDD) motif of the RNA-dependent DNA polymerase
(rtM204 I/V) developed in 49% of the patients treated with
LAM, and the Child-Pugh score was more likely to increase in
patients with these mutations than in patients without the
mutations (7% vs. ,1%). The emergence of rtM204 I/V is
followed by viral breakthrough and ALT elevation (. 5 times
the upper limit of normal) in over 90% of the patients.35 A
study by Liaw et al.36 compared the efficacy and safety profile
Journal of Clinical and Translational Hepatology 2014 vol. 2 | 197–201
of ADV monotherapy in 18 cirrhotic patients and ADV add-on
LAM therapy in 10 comparable cirrhotic patients with LAM-
resistant rtM204 I/V. After switching to ADV monotherapy or
ADV add-on, Child-Pugh’s score, serum ALT, bilirubin, albu-
min, and HBV DNA response (defined as HBV DNA decreased
to below 105 copies/mL or o2 log10 reduction form baseline)
improved significantly (p,0.01). A transient ALT flare without
concurrent changes in serum bilirubin or prothrombin time
was observed in only two patients (11%).
Another three large, randomized, multicenter, phase III
studies37 evaluated the efficacy and safety of entecavir in
CHB patients and advanced liver fibrosis/cirrhosis. Histologic
improvement was observed in 57%, 59%, and 43% of
nucleos(t)ide-naı̈ve HBeAg(+) patients, nucleos(t)ide-naive
HBeAg(2) patients, and LAM-refractory HBeAg(+) patients
with ETV treatment. The overall performance in histologic,
virologic, biochemical, and serologic outcomes in ETV-
treated groups were better than those observed in LAM-
treated patients with advanced liver fibrosis/cirrhosis, which
were comparable with the overall study populations in each
trial.
A recent randomized, open-label comparative study38 with
a mean duration of 280 weeks comparing ETV and ADV
therapy was performed in CHB subjects with hepatic decom-
pensation (Child-Pugh score o7). It was found that 96% of
patients achieved liver histological improvement by reducing
necroinflammation (a decrease of Ishak score o1). The ETV
group exhibited a greater change from baseline in HBV DNA
levels for the primary and key secondary virologic endpoints
and had a higher proportion of subjects achieve HBV DNA ,
300 copies/mL at weeks 24 (ETV 49%; ADV 16%; P,0.0001)
and 48 (ETV 57%; ADV 20%; P,0.0001). Eight-six percent of
patients achieved ALT,1-fold of the upper limit of normal.
Approximately two-thirds of subjects in both groups showed
improvement/stabilization in Child-Pugh status. Therefore,
both ETV and ADV treatments provided clinical improvements
with suppression of serum HBV DNA level. Given the
favorable safety profile, potency, and high barrier to resis-
tance, ETV appears to be a favorable choice for naı̈ve patients
who have decompensated liver disease.
Marcellin et al.39 recently reported the results of a five year
open-label follow-up study of a double-blind phase III trial of
the nucleotide analogues tenofovir disoproxil fumarate and
adefovir dipivoxil for chronic hepatitis B. Of the 348 patients
who had paired biopsies both at baseline and at year five, 304
(87%) had histological improvement, defined as a o2-point
decrease in Knodell necroinflammatory score (0–18) and no
worsening in Knodell fibrosis score (0–4). Moreover, 176
(51%) had regression of fibrosis, defined as a o1-point
decrease in Ishak fibrosis score (0–6) at year five. Of note, 71
of 96 (74%) patients who had cirrhosis at baseline no longer
had cirrhosis at year five. In the on-treatment analyses, 98%
of the patients had HBV DNA ,169 copies/ml (,35 IU/ml).
These results are remarkable and demonstrate that long-
term therapy with a potent antiviral agent that has a high
genetic barrier to resistance can maintain viral suppression in
nearly 100% of patients.
Treatment selection for HBV-related cirrhosis
In the management of HBV related cirrhosis, viral suppres-
sion with safe and effective antiviral agents is essential as
patients with HBV-related cirrhosis have compromised
liver function and may manifest with endocrine dyscrasia,
199
 Lu L.G.: Antiviral therapy liver cirrhosis hepatitis B virus infection
jaundice, portal hypertension, bleeding from varices, hepatic
encephalopathy, and bacterial peritonitis. In addition, side
effects from antiviral treatments should also be weighed.
Once drug-resistance occurs, the treatment regimen should
be adjusted or a second agent should be added as an
alternative. Recent studies showed that the one to five year
occurrences of drug resistance were 23%, 46%, 55%, 71%,
and 65% in LAM-naı̈ve HBeAg-positive patients,40 0%, 3%,
11%, 18%, and 29% in ADV-naı̈ve HBeAg-negative
patients,41 and 0.2%, 0.5%, 1.2%, 1.2%, 1.2% in ETV-
naı̈ve HBeAg-positive and ETV- naı̈ve HBeAg-negative
patients. 42 The one to two year occurrences of drug
resistance were 5% and 22% in LdT-naı̈ve HBeAg-positive
patients, whereas they were 2% and 9%, respectively, in LdT-
naı̈ve HBeAg-negative patients.43
Conclusions
Taking both efficacy and drug resistance profiles into account,
an ideal antiviral treatment for HBV-related cirrhosis should
be safe and affordable in order for long-term use to achieve a
high rate of sustained HBV suppression with a low risk of drug
resistance. With the ultimate goal to confer beneficial effects
on liver functions and survival, the optimal choice for HBV-
related cirrhosis in each case depends on specific patient
characteristics.
Acknowledgement
This work was supported by the National Key Technologies
Research and Development Program of China during the 11th/
12th Five-year Plan Period (no. 2008ZX1002-006,
2012ZX10002007-001-040 and 2013ZX10002004-002-003).
Conflict of interest
None
Author contributions
Writing the paper (LGL).
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201
Review Article
The Molecular and Structural Basis of HBV-resistance to
Nucleos(t)ide Analogs
Nidhi Gupta1, Milky Goyal2, Catherine H. Wu1 and George Y. Wu*1
1
Department of Medicine, Division of Gastroenterology-Hepatology, University of Connecticut Heath Center, Farmington, USA;
2
Department of Microbiology, College of Basic Sciences and Humanities, Punjab Agriculture University, Ludhiana, Punjab, India
Abstract
Infection with hepatitis B virus (HBV) is a worldwide health
problem. Chronic hepatitis B can lead to fibrosis, liver cirrhosis,
and hepatocellular carcinoma (HCC). Management of the
latter two conditions often requires liver transplantation.
Treatment with conventional interferon or pegylated interferon
alpha can clear the virus, but the rates are very low. The
likelihood, however, of viral resistance to interferon is minimal.
The main problems with this therapy are the frequency and
severity of side effects. In contrast, nucleos(t)ide analogs
(NAs) have significantly lower side effects, but require long
term treatment as sustained virological response rates are
extremely low. However, long term treatment with NAs
increases the risk for the development of anti-viral drug
resistance. Only by understanding the molecular basis of
resistance and using agents with multiple sites of action can
drugs be designed to optimally prevent the occurrence of HBV
antiviral resistance.
E 2014 The Second Affiliated Hospital of Chongqing Medical
University. Published by XIA & HE Publishing Ltd. All rights
reserved.
Introduction
The hepatitis B virus (HBV) is estimated to affect more than
400 million individuals worldwide,1 and approximately two
million people in United States. Chronic hepatitis B can lead to
fibrosis, liver cirrhosis, and hepatocellular carcinoma (HCC).
The geographical distribution of HBV infection varies widely
(Fig. 1). High genomic variability of HBV has resulted in the
evolution of eight distinct genotypes (A-H), some of which are
further divided into subtypes (1, 2 etc.).2
HBV was discovered in 1967,3 but treatment was not
available until the development of interferon, which was first
Keywords: HBV; HCC; Nucleos(t)ide analogs.
Abbreviations: 3TC, lamivudine; ADV, adefovir; cccDNA, covalently closed
circular DNA; dTTP, 29-deoxythymidine 59-triphosphate; HCC, hepatocellular
carcinoma; HBcAg, hepatitis B core antigen; HBeAg, hepatitis B e antigen;
HBsAg, hepatitis B surface antigens; HBV, hepatitis B virus, HIV, human
immunodeficiency virus; LdT, telbivudine; NA, nucleos(t)ide analog; ORF, open
reading frame; pgRNA, pre-genomic RNA; PMPA, 9-((R)-2-((Bis (((isopropox-
ycarbonyl)oxy)methoxy)phosphinyl)methoxy)propyl)adenine fumarate; TDF,
tenofovir disoproxil fumarate.
Received: 13 June 2014; Revised: 26 July 2014; Accepted: 27 July 2014
q
DOI: 10.14218/JCTH.2014.00021.
* Correspondence to: George Y. Wu, Department of Medicine, Division of
Gastroenterology-Hepatology, University of Connecticut Health Center,
Farmington, CT 06030, USA. Tel: +1-800-535-6232, E-mail: wu@nso.uchc.edu
202 Journal of Clinical and Translational Hepatology 2014 vol. 2 | 202–211
used in 1976.4 This was the mainstay therapy until 1998
when the mechanistic similarity between human immunode-
ficiency virus (HIV) reverse transcriptase and HBV polymer-
ase led to the study and approval of lamivudine, the first oral
nucleos(t)ide analog (NA) approved for HBV treatment.
Subsequently, several NAs were developed for HBV treat-
ment, including adefovir in 2002, entecavir in 2005, telbivu-
dine in 2006, and tenofovir in 2008.5
Current treatment for HBV infection consists of two major
classes:6 immuno-modulatory agents (interferon-based
therapies) and oral NAs. Advantages of treatment with
conventional interferon or pegylated (long-acting) interferon
alpha include low risk of developing viral resistance and
slightly increased clearance of HBV with time. However,
severe side effects such as anxiety, depression, and anemia
have limited its benefits. Although NAs have significantly
lower side effects, long term treatment with NAs is generally
required because the rate of sustained virological response is
extremely low.5 Since the replication rate of HBV is high and a
proof reading mechanism of its polymerase is absent, the
mutation rate is very high, 1.4–3.6 6 1025 nucleotide
substitutions per site per cycle. This results in a high risk for
the development of drug resistance7 against NA therapy.
Genome organization and genotypes
HBV is one of smallest known animal viruses, 32–42 nm in
diameter. The viral particle consists of an outer envelope and
an inner nucleocapsid that enclose a viral DNA genome and
other proteins (Fig. 2a). The outer lipoprotein envelope has
hepatitis B surface antigens (HBsAg) embedded in it. These
antigens are found in blood either free or bound to viral
particles. Free HBsAg is non-infectious, but is involved in
induction of long-term immunity against HBV.9–11 The viral
core consists of hepatitis B core antigen (HBcAg), hepatitis B
e antigen (HBeAg), a partially double stranded DNA molecule,
and DNA polymerase. HBcAg is a non-secretory marker of
infectious viral material found inside HBV-infected cells.
HBeAg is a form of HBcAg secreted in blood, and is usually
an accurate index of viral load.12–14 The polymerase, a DNA-
dependent DNA polymerase, is involved in reverse transcrip-
tion for viral replication15–17 and is research target for the
development of drugs against HBV.5,18–20
The compact organization and multiple overlapping open
reading frames (ORFs) has required the HBV to utilize its
genome efficiently (Fig. 2b).16,21 The viral genome consists
of a 3.2 kb partially double-stranded DNA molecule with one
end bound to DNA polymerase. There are four ORFs in the
HBV genome that result in the transcription and translation of
Gupta N. et al: The basis of HBV-resistance to nucleos(t)ide analogs

Fig. 1. Global prevalence of chronic hepatitis B virus (HBV) infection in adults. Source: Centers for Diseases Control and Prevention, CDC 2010

different types of proteins.22 The core (C) gene encodes
capsid proteins HBcAg and HBeAg; the P gene encode viral
DNA polymerase; the S gene has three in-frame start codons
and codes for three envelope proteins that differ in lengths of
the N-termini (Fig. 2c). They are designated as large (L),
major (M), and small (S) surface protein. L and M are surface
proteins and are involved in hepatocyte receptor binding. The
S surface protein is the protein detected in commercial assays
as HBsAg. Finally, an X gene codes for a 16 kDa protein HBx,
whose function is still not completely understood. It has been
reported to modulate cellular signals, promoters, and cell-
cycle pathways, some of which have been implicated in the
development of hepatocellular carcinoma.23–26
The eight genotypes of HBV (A-H) have a distinct
geographical distribution (Table 1) and are classified based
on .8% divergence in complete genome sequence.27–29 The

Fig. 2. Structural and genetic organization of HBV. a, structure of HBV; b, compact organization of the HBV genome showing multiple overlapping open reading
frames; c, expression of three envelope proteins from three in-frame start codons from the S-gene of the HBV genome.

Journal of Clinical and Translational Hepatology 2014 vol. 2 | 202–211 203

 Gupta N. et al: The basis of HBV-resistance to nucleos(t)ide analogs
prevalence of HBV genotypes varies highly, with A and C
being most common (.30%), and E and F being least
common (,0.6%) in the US. Four of these (A, B, C, and F)
are further divided into sub-genotypes based on genomic
variability of .4%.29,30 Disease progression and response to
anti-viral therapies differs in patients infected with different
genotypes.31
Life cycle and replication
To understand current therapeutic approaches to HBV infec-
tion, knowledge of the HBV life cycle and replication is very
important. HBV is a DNA virus, but it replicates like a
retrovirus using reverse transcription. The initial steps of
HBV entry into cells have been difficult to characterize. They
are thought to involve cell membrane binding and endocy-
tosis of viral particles.32 Several proteins have been proposed
to assist in viral attachment to the cell surface and entry into
the cell. For example, apolipoprotein H,33,34 poly-human
serum albumin,35 fibronectin,36 interleukin-637,38 and car-
boxypeptidase D (gp 180)39,40 have all been proposed to play
a role as HBV receptors. Recently, another 80 kDa protein has
been identified that binds HBV surface proteins.41 To date, the
exact mechanism of HBV entry into cells is still not well
elucidated.
Once viral particles enter the cell, the viral nucleocapsid is
uncoated and delivered to the cell nucleus.42–44 In the
nucleus, partially double-stranded HBV DNA is repaired to
form covalently closed circular DNA (cccDNA), which serves
as a template for transcription by cellular enzymes.45–47 HBV
DNA polymerase does not have repair mechanism activity.48
Also, unlike retroviruses, HBV DNA integration into host cell
genome is not required for HBV replication. After re-
circularization, HBV DNA starts to express transcripts
required for HBV protein synthesis and pre-genomic RNA
(pgRNA) required for viral replication. Two types of tran-
scripts are expressed: sub-genomic and genomic. Sub-
genomic, smaller transcripts, serve as templates for surface
proteins and HBx expression. Genomic transcripts longer
than one genome in length serve as templates for e, core, and
polymerase expression. The HBV DNA polymerase selects
genomic transcripts lacking the start ‘ATG’ codon at the 59
terminus as pgRNA for packaging into virions.49 Sub-genomic
and genomic transcripts are then transported and translated
in the cytoplasm. Binding of DNA polymerase protein to
pgRNA results in encapsidation,50,51 and formation of RNA-
Table 1. Geographic Distribution of HBV Genotypes
HBV
genotypes Geographic distribution
A Northwest Europe, North America, Africa
B Asia
C Asia
D Worldwide distribution
E Africa
F South America, Central America (Hispanic
origin)
G France, UK, Italy, Germany, USA
H Central America (Amerindian populations)
Source: Ref [28] [145–153]
204 Journal of Clinical and Translational Hepatology 2014 vol. 2 | 202–211
containing nucleocapsids. After encapsidation, reverse tran-
scription is then initiated upon interaction of the C-terminus
of the DNA polymerase with core protein. Polymerase bound
to pgRNA starts to reverse transcribe pgRNA into (-) DNA
strand using a bulge in the stem loop of pgRNA.52–55 The
RNase H activity of the polymerase degrades pgRNA after (-)
DNA strand synthesis,56 leaving 13–20 nt on the 59-end that
serve as primers for (+) DNA strand synthesis.16,57 Following
the synthesis of viral DNA and nucleo-capsid packaging, HBV
surface proteins aggregate in cellular Golgi bodies, forming
vesicles around viral nucleocapsids that result in enveloped
HBV particles. These particles are then secreted as mature
virions by exocytosis. This occurs along with secretion of non-
infectious particles, viral surface protein aggregates without
enclosed nucleo-capsid. Although several steps of the HBV
life cycle have been well characterized,17,46,58 many aspects
remain unclear and need to be explored in order to fully
understand the life cycle of HBV.
In vitro screening assays for HBV resistance
Currently, there are no practical animal models for testing
antiviral drug efficacy. Drugs are tested pre-clinically with in
vitro assays to determine both their efficiency and resistance
profiles.59,60 In these assays, liver cells are infected with wild
type or mutated HBV, and the effects of NAs are determined
by measuring HBV replication or cytotoxicity.61 In vitro
reverse transcription assays are performed to determine the
effects of various mutations on resistance to NAs. Wild type or
mutant HBV DNA polymerase is expressed from plasmids and
tested for priming and elongation of viral DNA.62
Clinical drug resistance rates
Determining the rate of the development of resistance from
clinical experience varies considerably depending on the
agents. For lamivudine, the one year resistance rate is
30%, and for telbivudine it is 15%. When used individually,
entecavir and adefovir have higher barriers of resistance,
with rates less than 2% after two years. No resistance to
tenofovir has yet been reported. After five years of treatment,
the rates of resistance were about 70% for lamivudine, 28%
for adefovir, less than 1% for entecavir,63–65 and 0% for
tenofovir and interferon.
Current treatment and mechanisms of HBV resistance
Interferon therapy
Conventional interferon and pegylated (long-acting) inter-
feron alpha are two drugs currently available and approved
for HBV therapy.66–69 Interferon is an immunomodulator,70
acting indirectly to eliminate HBV-infected host cells by
activating various host genes and innate immune response
pathways.71–73 Its use has a number of advantages including
a finite treatment course,74 lack of drug resistance,75 and a
decreased risk of cirrhosis and HCC with long term use.76
However, the sustained virological response rate is generally
poor (6%) for PEG-interferon. However, there are substantial
side effects, including fatigue, diarrhea, alopecia,
flu-like symptoms, insomnia, and psychiatric complications,
that often require additional medications to correct.
Gupta N. et al: The basis of HBV-resistance to nucleos(t)ide analogs
NAs
The presence of extremely stable cccDNA HBV in infected
hepatocytes is the major obstacle to permanent elimination
of HBV.77,78 The cccDNA acts as template for viral RNA
genome production. The DNA-dependent DNA polymerase of
HBV catalyzes synthesis of RNA, RNase H, and protein
priming activities along with replication of HBV genome.
Recently, NAs have been developed to directly inhibit the
function of HBV DNA polymerase by either competitive
binding to the enzyme or by terminating viral replication
prematurely, which inhibits viral genome production. NAs
currently available for HBV treatment are shown in Fig. 3.
Lamivudine
Lamivudine [(-)-b-L-29-39 dideoxythiacytidine)] or 3TC was
the first oral NA approved for the treatment of HBV (Fig. 3).
3TC is a cytidine analogue, and is phosphorylated intracellu-
larly to 59-triphosphate active metabolites. It inhibits DNA
polymerase function by competing with a natural substrate
(dCTP), resulting in DNA chain termination. Treatment with
3TC has been shown to not only suppress viral replication and
HBV serum levels, but also to reverse fibrosis to varying
degrees with long term use.79,80 Furthermore, it has been
shown to decrease the risk of HCC.81,82 Lamivudine is a
potent drug, but it unfortunately also has a high rate of drug
resistance, which increases with duration of treatment.82–86
According to molecular model studies, residues F88, L180,
and M204 play indirect, but crucial roles in the formation of a
hydrophobic pocket and other non-covalent interactions in
the DNA polymerase of HBV.87,88 These have been shown to
be involved in the binding of NAs to the polymerase. Although
slightly different structurally from natural cytosine, 3TC
Fig. 3. Molecular structures of approved NAs
Journal of Clinical and Translational Hepatology 2014 vol. 2 | 202–211
interacts with the HBV DNA polymerase active site perfectly,
inducing a fitting movement of the M204 residue that results
in significant inhibition of viral replication (Fig. 4). Long term
use of 3TC for HBV treatment frequently leads to mutation of
the M204 residue in the HBV DNA polymerase gene and gives
rise to 3TC-resistant HBV. Three major mutations (M204I,
M204V, and L180M) individually or in combinations in HBV
DNA polymerase result in significant resistance against 3TC
anti-viral characteristics (Table 2). Mutation of L180 to M180
orients the residue away from the hydrophobic pocket,
resulting in reduced interaction of 3TC with HBV DNA
polymerase. Mutations of M204 to I204 or V204 add a bulky
group of branched residues to the hydrophobic pocket,
creating steric hindrance that prevents accommodation of
the 3TC ring (Fig. 4). This ultimately causes resistance by
increasing the thermodynamic stability of binding of the
natural substrate to HBV DNA polymerase compared to
3TC.89 Dual mutations (L180M + M204I or L180M + M204V)
can further reduce the binding affinity of 3TC with HBV DNA
polymerase by combining effects of reduced interaction
(mutated L180) and steric effects (mutated M204) (Fig. 5).
Adefovir dipivoxil (Adefovir, ADV)
Adefovir (ADV), an acyclic adenosine analogue (Fig. 3), is
phosphorylated to yield an active metabolite that inhibits HBV
DNA polymerase activity by competing with the natural
substrate (dATP). In doing so, it causes chain termination.
Decreased potency of ADV is due to lower effective intracel-
lular drug levels compared to other NAs. In addition, ADV has
been associated with a dose-dependent nephrotoxicity and
can lead to reversible renal impairment.90–92
Molecular modeling studies have shown that ADV interacts
with HBV DNA polymerase in a way similar to dATP.89 Major
205
 Gupta N. et al: The basis of HBV-resistance to nucleos(t)ide analogs

Fig. 4. Increased resistance to NAs caused by inhibition of induced fitting movement by a mutation near the active site. a, HBV DNA polymerase can bind to
either natural substrate for HBV DNA synthesis or NAs by induced fitting movement at the active site; b, mutated HBV DNA polymerase can bind to natural substrate for HBV
DNA synthesis. A mutation near the active site inhibits induced fitting movement of polymerase resulting in steric inhibition of binding of NA to polymerase.

mutations in the HBV DNA polymerase gene that cause ADV
resistance are A181V, A181T, N236T, and I233V.93–95 Other
mutations (P237H, N238T/D, V84M, S85A, V214A, and
Q215S) have also been reported to decrease sensitivity to
ADV, although the exact mechanism remains unclear96
(Fig. 5). ADV binding to HBV DNA polymerase is not affected
by mutations (M204I, M204V and L180M) that result in
resistance to 3TC. As such, ADV is effective against lamivu-
dine-resistant HBV polymerase.97,98
Entecavir
Entecavir (ETV) is a guanosine analogue (Fig. 3) that is
phosphorylated intracellularly to an active metabolite. It
inhibits multiple functions of HBV DNA polymerase, including
polymerase priming, reverse transcription, and DNA synth-
esis.99–101 Unlike 3TC and ADV, ETV does not cause chain
termination because it possess a 39-hydroxyl group,102 so
addition of nucleotides to the nascent chain continues.
Molecular modeling studies have demonstrated that ETV
forms most interactions with the active site of HBV DNA
polymerase in a manner similar to the natural substrate
(dGTP). It does not, however, require induced fit movements
of any residue in the polymerase.89,100 This allows ETV to be
more effective and less susceptible to the development of drug
resistance.103–105 ETV inhibits HBV DNA elongation after
addition of a few nucleotides by increasing steric hindrance
and thereby preventing addition of more bases to the DNA.100
ETV is more potent than other NAs and has a higher rate of viral
clearance.106–110 It also has a high barrier to the development
of resistance because a single mutation is not sufficient to
cause resistance. Rather, multiple mutations are required:
I169T, T184G, S202I, or M250V in addition to L180M and
M204V/I, the latter two result in resistance to 3TC111–113
(Fig. 5). Therefore, the risk of resistance is higher with HBV
already resistant to 3TC.111

206 Journal of Clinical and Translational Hepatology 2014 vol. 2 | 202–211

Gupta N. et al: The basis of HBV-resistance to nucleos(t)ide analogs

Table 2. Anti-HBV Agents and Reported Resistance Mutations

Analog Type HBV polymerase mutation

Lamivudine Cytosine analog M204I/V, L180M, L180M-M204V,
[(-)-b-L-29-39 dideoxythiacytidine] L180M-M204I
Adefovir Adenosine analog A181V/T, N236T, I233V
9-[2-[[bis[(pivaloyloxy)methoxy]-phosphinyl]methoxy]
ethyl]adenine
Entecavir Guanosine analog I169T, T184G, S202I, M250V, M204I/V
2-amino-1,9-dihydro-9-[(1S,3R,4S)-4hydroxy-3-(hydroxymethyl)-
2-methylenecyclopentyl]-6H-purin-6-one, monohydrate
Telbivudine Thymidine analog M204I, L80I/V, L180M, A181 T/V, L229
1-(2-deoxy-b-L-ribofuranosyl)-5methyluracil W/V
Tenofovir Adenosine analog A181V, N236T
9-((R)-2-((Bis(((isopropoxycarbonyl)oxy)methoxy) phosphinyl)-
methoxy)propyl) adenine fumarate (PMPA)

Table 3. Reported Mutations and Associated Resistance Determined by in Vitro Assays

Mutation Lamivudine Adefovir* Entecavir Telbivudine Tenofovir*

I169T .1000 1.0 .1000 .100 No data
A181T 2–6 1–5 No data No data 1–1.5
A181V 2–6 1–5 No data No data 2.9–10
T184G .1000 2.0 .1000 .1000 0.6–6.9
S202I .1000 2.0 .1000 .1000 0.6–6.9
M204I (YMDD) .1000 0.7 30 .1000 0.7–3.4
M204V (YMDD) .1000 0.7 No data 1.2 0.7–3.4
V214A 10–20 7–10 No data 7–10 .10
Q214S 10–20 7–10 No data 7–10 .10
N236T 3–8 7–10 0.67 2.4 2.9–10
M250V .1000 1.0 .1000 1.0 0.6–6.9
L80I + M204I .1000 .10 No data .1000 .10
L180M + M204V .1000 0.2 30 133 3.4

Source: Ref [59]

general weakness, have also been reported in various clinical

Telbivudine
Telbivudine (LdT) is an analog of thymidine (Fig. 3) with an L-
configuration rather than the D-configuration of natural
nucleosides. It competes with natural dTTP (29-deoxythymi-
dine 59-triphosphate) for the HBV DNA polymerase active site
after phosphorylation intracellularly, leading to premature
chain termination.114 Although LdT is more potent and less
prone to drug resistance than 3TC,115–118 the drug resistance
rate is still relatively high.119,120 An M204I mutation greatly
decreases LdT inhibition of HBV replication by altering the
orientation and position of key methyl groups89 (Fig. 5). The
L180M mutation results in the loss of another methyl group
required for LdT affinity to the polymerase. Hence, dual
mutations (M204I + L180M) make HBV DNA polymerase
highly resistant to LdT. Other mutations L80I, A181T/V, and
L229W/V have been linked to LdT resistance.5,121 Besides the
high resistance rate, adverse reactions of LdT, such as
myopathy,122 myalgia,123 creatine kinase elevations,124 and
trials. Mechanisms of these adverse reactions are not clearly
understood, and further studies are required to further
characterize them.
Tenofovir
Tenofovir disoproxil fumarate (TDF), 9-((R)-2-((Bis (((iso-
propoxycarbonyl)oxy)methoxy)phosphinyl)methoxy)propy-
l)adenine fumarate (PMPA) is an adenine analog (Fig. 3) that
competes with dATP after intracellular phosphorylation,
resulting in chain termination. TDF is more potent than ADV
because of a high tolerance to increased doses of TDF
compared to ADV.125–127 Despite long-term treatment, no
evident resistance has been reported with TDF.128 However,
some mutations, A181V and N236T, have been found to
decrease susceptibility to TDF,129–131 although the exact
mechanism by which this occurs is unknown. Mild renal
toxicity132–134 and osteomalacia135,136 has been observed
with long term TDF treatment.

Journal of Clinical and Translational Hepatology 2014 vol. 2 | 202–211 207

 Gupta N. et al: The basis of HBV-resistance to nucleos(t)ide analogs
Fig. 5. DNA synthesis occurs at the active site of DNA polymerase.
Induced fitting and mutations alter the structure of the active site.
Combination therapy and HBV resistance
Recently, the effects of combination therapy were studied and
shown to result in reduced drug resistance. Several studies
have demonstrated that addition of interferon therapy to NAs,
such as lamivudine and adefovir, is beneficial by decreasing
viral load. There have been few studies, however, examining
the efficacy of combination therapy with regard to the
development of drug resistance. One study showed that the
rate of resistance in a lamivudine monotherapy group was
25% compared to 0% in a combination treatment group.137
Another study reported a decrease in HBV replication in
patients with lamivudine resistance after adding adefovir to
therapy without developing resistance against adefovir over a
period of three years.138 A tenofovir study with lamivudine-
experienced and -naive individuals showed that combination
therapy with tenofovir and lamivudine was more effective in
preventing resistance mutations compared to a group treated
with lamivudine alone.139,140 Tenofovir was also found to be
more effective in viral suppression in combination with
emtricitabine in patients with adefovir resistance.141 In one
study, the rate of adefovir resistant mutations was 15% in
patients treated with combination therapy of lamivudine and
adefovir compared to 0% in patients treated with entecavir
and adefovir. Entecavir resistant mutations were found in 4%
patients in the same group.142 In contrast, studies have
shown an increased rate of HBV resistance in patients
sequentially treated with monotherapies. 143,144 Taken
together, the data suggest that while efficacy, in terms of
the extent of viral suppression, might not be affected, the
rates of resistance appear to be lower with combination
therapy. However, more studies are required in larger
populations to demonstrate this and whether combination
therapy to prevent HBV drug resistance is a cost effective
strategy.
Conclusions
Despite the availability of an effective vaccine for the
prevention of HBV infection, thousands of people acquire
208 Journal of Clinical and Translational Hepatology 2014 vol. 2 | 202–211
HBV infections every year, and many go on to develop chronic
hepatitis. Introduction of NAs have significantly impacted
HBV treatment. Although these agents are highly potent,
there is potential for the selection of viral resistance to NA
therapy because of the necessity for long-term treatment,
possible exposure to sequential monotherapy, and possible
non-compliance. Molecular modeling, in vitro, and clinical
studies examining, at the molecular level, the interactions
between NAs and other agents with HBV DNA polymerase will
assist in the development of new therapeutic agents. These
drugs will hopefully lead to complete clearance of HBV from
hepatocytes without the risk of developing viral resistance.
Overall, the data on drug resistance suggests that the rates of
resistance are lower with combination therapy than conven-
tional monotherapy.
Acknowledgements
We thank the Herman Lopata Chair in Hepatitis Research for
supporting our academic activities.
Conflict of interest
One of the authors (GYW) is a member of medical advisory
boards for Gilead, and Bristol-Myers-Squibb.
Author contributions
Writing the paper (NG, MG), reviewing and editing the paper
(CHW, GYW).
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211
Review Article
Hepatobiliary Schistosomiasis
Yehia Shaker*, Nervana Samy and Esmat Ashour
Biochemistry Department, National Research Center, Cairo, Egypt
Abstract
Schistosomiasis is an ancient parasitic disease that has
afflicted Egyptians since the time of the pharaohs. The
disease is caused by lodged schistosome eggs in the host
liver, evoking an immune response and leading in some
patients to the development of hepatic granuloma and
fibrosis. Here, we review the epidemiology, immunopatho-
genesis, and clinical profile of schistosomiasis. This informa-
tion may aid in the development of more efficacious
treatments and improved disease prognosis.
E 2014 The Second Affiliated Hospital of Chongqing Medical
University. Published by XIA & HE Publishing Ltd. All rights
reserved.
Introduction
Of the more than 207 million people infected with schistoso-
miasis, 85% live in Africa. An estimated 700 million additional
people are at risk of infection in 76 countries where the
disease is considered endemic due to exposure to infested
water agriculturally, domestically, or recreationally. 1
Worldwide, 200,000 deaths are attributed to schistosomiasis
annually.2 There is a long history in Egypt of diseases caused
by schistosomiasis, notably hematuria. This symptom classi-
cally appeared in young boys and was at one time deemed a
sign of puberty. Schistosomiasis, sometimes referred to as
bilharzia, bilharziasis, or snail fever, was discovered in 1851
by Theodore Bilharz, a German surgeon working in Cairo. He
identified the etiological agent as Schistosoma hematobium.4
Most schistosomiasis is caused by S. haematobium, S.
mansoni, and S. japonicum. Less prevalent species include
S. mekongi and S. intercalatum, which may also cause
systemic human disease.
When schistosoma eggs are trapped in tissues, they
activate an immunologic reaction and induce schistosomiasis.
Antigens released from the egg stimulate a granulomatous
reaction involving T cells, macrophages, and eosinophil that
cause clinical disease. Symptoms and signs depend on the
number and location of eggs. The initial inflammatory
Keywords: Schistosomiasis; Immunopathogenesis; Hepatosplenic schistoso-
miasis; Co-infection and complications; Anti-schistosomal vaccine.
Abbreviations: APC, antigen-presenting cells; CCA, circulating cathodic antigen;
DSRS, distal splenorenal shunts; EGDS, esophagogastric devascularization with
splenectomy; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HCV,
hepatitis C virus; HIV, human immunodeficiency virus; HSS, hepatosplenic
schistosomiasis; IL, interleukin; MHC, major histocompatibility complex; SRS,
splenorenal shunt.
Received: 9 June 2014; Revised: 23 July 2014; Accepted: 24 July 2014
q
DOI: 10.14218/JCTH.2014.00018.
*Correspondence to: Yehia Shaker, Biochemistry Dept, National Research
Centre, Dokki, Giza 12622, Egypt. Email: ymshaker@gmail.com
212 Journal of Clinical Translational Hepatology 2014 vol. 2 | 212–216
reaction is readily reversible. However, as the disease
progresses, there is collagen deposition and fibrosis, resulting
in organ damage that may be only partially reversible.5
Life cycle of parasite
The three species of schistosomes that commonly affect
humans (S. hematobium, S. mansoni and S. japonicium)
have similar life cycles and develop by a succession of stages:
the egg, miracidium, first stage sporocyst, second stage
sporocyst, cercariae, schistosomule, and adult. All three
species of schistosomes infect in the same manner: direct
contact with surface water containing free-living form known
as cercariae, which can penetrate the skin. Schistosome
ceracariae consist of a tail (used for motility in the water), a
head region (used for attachment to host skin), and glands
that contain proteolytic enzymes to facilitate penetration of
the skin.
In addition to several other major changes, the tail of the
cercariae is shed during penetration of the skin to yield a new
form called the schistosomulum. After penetrating the wall of
a nearby vein, schistosomula are carried in the host blood
flow, eventually reaching the portal venous system. It is here
where they grow and reach sexual maturity, the mature male
and female worms pair, and then, depending on species,
migrate to the vessels of the bowel or bladder for egg
production. Many eggs pass through the intestinal or bladder
wall and are excreted in the feces or urine. The schistosome
lifecycle is completed when the eggs hatch, releasing free-
swimming miracidia that can in turn re-infect freshwater snails.
However, it is the eggs, not the worms, that cause the
pathophysiology of schistosomiasis, and some eggs remain
lodged in the tissues of the host. In intestinal schistosomiasis,
eggs lodged in the mucosa or submucosa of the gut cause
granulomatous reactions, which may then extend into the gut
lumen as pseudopapillomas. These pseudopapillomas can
cause colonic obstruction and blood loss. Eggs residing in the
liver may cause portal fibrosis, leading to portal hypertension,
splenomegaly, and esophageal and gastric varices.
Exsanguination by esophageal variceal bleeding is the major
cause of death by schistosomes.7
Immunopathogenesis
Hepatosplenic schistosomiasis (HSS) is due to inflammation
and fibrosis in the presinusoidal portal areas of the liver.
Investigations in an experimental murine model have
unequivocally shown that the egg stage of the parasite s
responsible for these pathological changes.8
Shortly after developing into schistosomula, the parasites
shed surface antigens and acquire blood group glycolipid and
major histocompatibility complex (MHC) antigens from the
Shaker Y. et al: Hepatobiliary schistosomiasis
host. Since they express host antigens on their tegument, the
schistosomes resemble the host tissue and can thereby avoid
attack by the immune system. Although the host suffers little
damage directly from the adult worms, the eggs elicit an
intense granulomatous inflammatory response. The egg
granulomas are well-circumscribed aggregates of inflamma-
tory cells composed of eosinophils, lymphocytes, macro-
phages, and neutrophils that are embedded in a collagenized
extracellular matrix. Hepatic granuloma formation in S.
mansoni infections is mediated by MHC class 11-restricted
CD4+ Th cells.9
During the early stages of granuloma formation, the
immune system initiates a Th-1 type response to schistoso-
mal egg antigens.10 The proinflammatory cytokines produced
by the activated antigen-presenting cells (APC) stimulate the
Th-1 lymphocytes to secrete interferon gamma and inter-
leukin (IL)-2, which in turn further stimulate the APC.
Subsequently, with the aid of B7 (mostly B7-2) co-stimula-
tory signaling and the cytokines IL-4 and IL-10, there is a
switch from a Th-1 to a Th-2 response. The Th-2 response is
characterized by increased levels of the anti-inflammatory
cytokines IL-4, IL-5, IL-10, and IL-13, antibody production by
B cells, and recruitment of eosinophils. Eosinophils generate
IL-4, which serves to secure the Th-2 immune response and
to induce APCs with alternative activation and secretion
profiles. The hepatic egg granulomas that form during the
Th-2 immune response appear to be beneficial by limiting the
spread of egg secretions and causing little or no parenchymal
inflammation of the liver. In contrast, granulomas formed in
Th-1 environment are less efficient and can be accompanied
by severe hepatocellular microvesicular change, inflamma-
tion, and necrosis.11
A well-known phenomenon, originally described in the
mouse, is the progressive downsizing of granuloma formation
in response to eggs arriving in the liver after the period of
acute disease. An important mechanism in the immunomo-
dulatory process may involve the anti-inflammatory cytokine
K-10, which can inhibit accessory cell function and inflam-
matory cytokine production.12
Clinical manifestation
Disease syndromes associated with schistosomiasis include
cercarial dermatitis, larval pneumonitis, acute schistosomia-
sis (Katayama fever), and HSS.
Acute schistosomiasis
In a previously uninfected person, Katayama fever will
develop in about 4 to 6 weeks. The onset of symptoms
coincides with the initiation of egg deposition. Katayama
fever has been described in people with initial heavy
infections caused by S. mansoni, S. haematobium, and
especially S. japonicum. It is believed to represent a serum
sickness-like reaction that results from circulating immune
complexes. Symptoms include fever, chills, arthralgias,
myalgias, dry cough, wheezing, abdominal pain, and diar-
rhea. Physical findings include rash, tender hepatomegaly
splenomegaly, and lymph- adenopathy. Peripheral eosinophi-
lia and high levels of immunoglobulin (Ig)G and IgE are
usually present. Symptoms will usually resolve sponta-
neously after several weeks in most patients.13
Journal of Clinical Translational Hepatology 2014 vol. 2 | 212–216
Hepatosplenic schistosomiasis
The pathology of hepatic schistosomiasis was first described
in Egypt by the great British pathologist Symmers (1904)
before either its life cycle or clinical aspects were understood.
He found that HSS with portal hypertension and esophageal
varices was due to a presinusoidal block of portal blood flow
and increased flow from the spleen.14
While all schistosome species may cause some degree of
hepatobiliary disease, S. hematobium is less likely to do so,
and affects primarily the urinary tract. The main species that
cause hepatobiliary disease are S. mansoni and S. japoni-
cum. Intestinal and hepatosplenic syndromes may overlap to
some degree, but in most cases one or the other prevails.
Severe HSS is likely due to failure of immunomodulation
processes. Predictably, severely affected patients exhibit a
Th-1 types cytokine profile with little if any detectable IL-10.
Conversely, the mild intestinal form of schistosomiasis is
associated with a Th-2 cytokine profile.15 Symptoms of HSS
include hepatomegaly, hepatic fibrosis, portal hypertension,
and, with chronic infection, portosystemic collateral blood
flow. As the infection progresses, splenomegaly, esophago-
gastric varices, and hemorrhoids develop. Bleeding from
esophageal varices leads to hematemesis and melena.
Patients with HSS generally tolerate variceal bleeding epi-
sodes better than patients with portal hypertension asso-
ciated with viral or alcoholic hepatitis. A feature characteristic
of HSS is the maintenance of liver perfusion by an increase in
hepatic arterial flow, allowing for the preservation of hepato-
cyte function in the absence of hepatotropic viral co-
infections. Neither the eggs nor the adult form of S. mansoni
cause damage to hepatocytes.16
In the compensated stage of HSS, patients present with
predominantly left hepatic lobe enlargement and moderate
splenomegaly. Laboratory findings include normal transami-
nases, mildly elevated alkaline phosphatase, increased IgG,
leukopenia, mild hemolytic anemia, and thrombocytopenia.
The decompensated phase of HSS is characterized by a
shrunken liver, splenomegaly, esophageal varices, hepatic
encephalopathy, massive ascites, hypoalbuminemia, and
muscle wasting. There is a remarkable lack of other stigmata
of chronic liver disease such as jaundice, spider nevi, palmar
erythema, testicular atrophy, and gynecomastia. Patients
generally do not develop cirrhosis unless they are co-infected
with another hepatotropic organism.16,17 HSS should be
considered in any patient without stigmata of cirrhosis, from
an endemic area, who presents with splenomegaly, portal
hypertension, and variceal bleeding.18
Co-infections and complications
Co-infection of hepatitis, HIV, and malaria with schistoso-
miasis can increase the risk for hepatocellular carcinoma and
mortality. Patients with schistosomiasis have higher rates of
hepatitis B surface antigen (HBsAg) carriage and hepatitis C
virus (HCV) seropositivity than non-infected controls. This
association may be due, at least in part, to transmission of
hepatitis viruses via contaminated needles during blood
transfusion and parenteral therapy for schistosomiasis.
Schistosomiasis potentiates hepatitis B virus (HBV) and
HCV infections by prolonging hepatic inflammation after an
episode of acute viral hepatitis and by increasing the risk of
chronic infection.19
213

Fig 1. Global distribution of schistosomiasis. Adapted from Gryseels et al.3
Increased levels of aminotransferases and bilirubin asso-
ciated with histological and periportal inflammation on liver
biopsy have been reported in patients with HSS and HBV or
HCV co-infections. In addition, liver function deteriorates
more rapidly, often with the development of severe, irrever-
sible periportal fibrosis and a more rapid progression toward
end-stage liver disease. Development of hepatocellular
carcinoma has also been reported in schistosomiasis.20
However, some studies have failed to demonstrate a defini-
tive relationship between chronic hepatitis B or C infection
and positive ultrasonography or histologic evidence of
schistosomal hepatic fibrosis. Potential explanations for these
discrepancies include false-positive anti-HCV tests secondary
to anti-schistosomal antibodies, small sample size, study
design, short duration of follow-up, and bias in recruitment,
selection, or observation of patients.21
One way to prevent exacerbation of liver disease in
schistomiasis with co-infection of hepatitis B is immunization
against HBV. Schistosome-infected school children appear to
respond well to the hepatitis B vaccine, but hepatomegaly or
splenomegaly can significantly hamper sero-conversion.
Therefore, vaccination in early childhood should be universal
in countries endemic for the hepatotropic schistosomes.22
Because of the increasing prevalence of immunodeficiency
virus (HIV) infection in many countries that are highly
endemic for schistosomiasis, the interaction between these
two infectious diseases is being monitored closely. A recent
study from Kenya found significantly lower fecal egg counts in
subjects with HIV infection, and a direct correlation between
the CD4+ T-cell count and the level of egg excretion. No
214 Journal of Clinical Translational Hepatology 2014 vol. 2 | 212–216
Shaker Y. et al: Hepatobiliary schistosomiasis
difference, however, was seen in plasma levels of circulating
cathodic antigen (CCA), a direct measure of the worm
burden, between HIV-infected and non-HIV patients with S.
mansoni infection. The therapeutic response, as measured by
a reduction of fecal egg excretion and schitosomal CAA levels,
to standard doses of praziquantel in HIV-seropositive sub-
jects was found to be equal to non-infected controls. Clearly,
given the large overlap in their geographic distribution,
further studies are needed regarding the interactions
between HIV and schistosomiasis.23
Although no clear link has been made between S. mansoni
and cancer, epidemiologic studies in China and Japan have
implicated S. japonicum infection as a risk factor for both
primary hepatocellular carcinoma and colorectal cancer. Risk
factors for the development of liver cancer, including viral co-
infection with HBV or HCV and alcohol abuse, are usually
present as well.2
Prognosis
HSS carries a relatively good prognosis because hepatic
function is preserved until the end of the disease (unless
variceal bleeding occurs). Although treatment is indicated for
patients with end-stage complications of portal hypertension
and severe pulmonary hypertension, these patients are not
likely to benefit from it. Indeed, cor pulmonale usually does
not improve significantly with treatment. Three operations
have been used for the successful treatment of portal
hypertension due to schistosomiasis: esophagogastric devas-
cularization with splenectomy (EGDS), distal splenorenal
Shaker Y. et al: Hepatobiliary schistosomiasis
Fig 2. Life cycle of S. mansoni, S. japonicum, and S. haematobium. Adapted from McManus DP et al.6
shunts (DSRS), and classic splenorenal shunt (SRS). SRS is
associated with higher rates of death and hepatic encephalo-
pathy than both EGDS and DSRS. However, high rates of
variceal rebleeding after EGDS (27%) and incidences of
encephalopathy after DSRS (19%) have been reported.25
Fig 3. Liver fibrosis in hepatosplenic schistosomiasis. Adapted from
Lambertucci JR.32
Journal of Clinical Translational Hepatology 2014 vol. 2 | 212–216
Following diagnosis of schistomiasis, praziquantel should
be administered as soon as possible. Praziquantel can safely
be given to pregnant and lactating women, and it decreases
the disease burden and improves pregnancy and fetal
outcomes. The implementation of programs targeting women
during reproductive years will improve their reproductive life
and overall well-being.26
Morbidity and mortality
Acute schistosomiasis is associated with a mortality rate as
high as 25% in some studies. Although most individuals with
chronic schistosomiasis have few or no symptoms, significant
morbidity can develop. Complaints are difficult to quantitate
because of the geographic distribution of schistosome infec-
tion in developing nations and the frequency of co-morbidity
with other conditions, such as viral hepatitis. Hepatosplenic
disease with portal hypertension is the most common long-
term serious complication, followed by cardiopulmonary
involvement, obstructive nephropathy, bacteremia, and
malignancy. Female genital infection can contribute to
pregnancy complications, including reports of related ectopic
pregnancy. Urogenital schistosomiasis is considered to be a
risk factor for HIV infection, especially in women.27 End-stage
hepatosplenic disease with variceal bleeding, pulmonary
hypertension with cor pulmonale, and central nervous system
215

disease are associated with high mortality rates; and
carcinoma of the liver and gallbladder may cause death.
Although effective anti-helminthic treatment exists, it may
not be readily available in endemic areas and may not reverse
existing fibrosis. Re-infection is extremely common in people
who live in or return to endemic areas. Therefore, repetitive
treatment is necessary to prevent disease progression in this
situation.27
Anti-schistosomal vaccination
Researchers are optimistic in the successful development and
implementation of an anti-schistosomal vaccine. The greatest
advances in schistosomiasis vaccine technology have been in
the integrated genomic and proteomic study on antigen
selection. The apical membrane proteins expressed on the
surface of the schistosomulum and the adult worm are logical
vaccine targets, and there is some data available in support of
this hypothesis.28,29 Another putative target, although not
yet explored, is the mRNAs encoding novel, putatively
secreted proteins with no known homologues that are lodged
in the tegument membrane.There are very few schistoso-
miasis vaccine trials that target proteins completely unique to
schistosomes and do not share sequence identity with any
other proteins.30,31
Anti-schistosome vaccines should not be considered as a
panacea. It is just one component of integrated schistoso-
miasis control programs that include chemotherapy and
health education. Although controversial, praziquantal resis-
tance is a major concern, and the necessity for vaccines is
now more important than ever.6
Conclusions
HSS is a highly prevalent and preventable parasitic disease in
which damage to the hepatobiliary system is mediated by the
host immune response. Knowledge of the immunopathogen-
esis and clinical aspects of the infestation may lead to the
development of new treatment strategies and improved
disease prognosis.
Conflict of interest
None
Author contributions
Writing the review (YS, NS, EA).
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