Liver Cancer

Consolato M. Sergi, MD, PhD, MPH, FRCPC, FCAP

Editor
Liver Cancer
 Liver Cancer
Edited by
CONSOLATO M. SERGI, MD, PHD, MPH, FRCPC, FCAP
Departments of Pediatrics, Laboratory Medicine and Pathology,
Stollery Children’s Hospital, University of Alberta,
Edmonton, AB, Canada
Liver Cancer
ISBN: 978-0-6450017-2-3
DOI: https://doi.org/10.36255/exonpublications.livercancer.2021

Edited by
Consolato M. Sergi, MD, PhD, MPH, FRCPC, FCAP, Departments of Pediatrics,
Laboratory Medicine and Pathology, Stollery Children’s Hospital, University of
Alberta, Edmonton, AB, Canada

Published by
Exon Publications, Brisbane, Australia

Copyright© 2021 Exon Publications

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First Published in April 2021

Printed in Australia
 Contents v

CONTENTS

Foreword vii
Preface ix
List of Contributors xi

1 Carcinoma of the Liver in Children and Adolescents 1

Consolato M. Sergi

2 Hepatocellular Carcinoma in Adults 39

Aducio L. Thiesen

3 Non-Alcoholic Fatty Liver Disease Progression to Non-

Alcoholic Steatohepatitis-Related Primary Liver Cancer 55
Utibe-Abasi Udoh, Juan D Sanabria, Pradeep K Rajan,
Moumita Banerjee, Mathew Schade, Jacqueline A Sanabria,
Gary Smith, Gideon Udoh, Komal Sodhi, Sandrine Pierre,
Joseph I Shapiro, Juan R Sanabria

4 Gross Dissection of Liver for Hepatocellular Carcinoma
Using AJCC Cancer Staging Manual 8th Edition:
Anatomical and Practical Considerations 77
Marla Beach, Laura Henao Caviedes, Consolato M. Sergi

5 Radiologic-Pathologic Correlation of Liver Tumors 87

Eric Lachance, Jake Mandziuk, Consolato M. Sergi, Justin Bateman,
Gavin Low

6
Achieving a Cure: The Next Frontier in Hepatitis B
Treatment 109
Tina Boortalary, Brianna Shinn, Dina Halegoua-DeMarzio,
Hie-Won Hann
vi Contents

7 Locoregional Therapies for Bridging and Downstaging
Hepatocellular Carcinoma Prior to Liver Transplant 127
Sandeep A. Ponniah, Andreas G. Zori, Roniel Cabrera

8 Hepatoblastoma 145
Josef Hager, Consolato M. Sergi

9 Undifferentiated Embryonal Sarcoma of the Liver

in Adults 165
Jingyang Huang

Index 179

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 Foreword vii

FOREWORD

Investigating liver tumors in children and adults is a task for the intrepid,
undaunted, and naturally curious scientist. As I count myself among those who
wade in these treacherous and thoroughly stimulating waters, I understand the
value and rare nature of a book that successfully explores both widely established
general concepts and new discoveries with ease. Liver Cancer achieves these goals,
and I am delighted to be part of the effort to bring it to all of you.
Liver tumors are a heterogeneous and complex mix of benign and malignant
neoplasms that may arise in the setting of chronic liver injury or due to no prior
insult. In children, hepatoblastoma is the most common malignant primary liver
tumor and hepatocellular carcinoma is rare. In adults, however, hepatocellular
carcinoma is most common and undifferentiated embryonal sarcoma is vanish-
ingly rare. Liver Cancer explores these, and the myriad of other entities in between,
with a depth and precision that is highly informative and practical to the modern
physician scientist. Descriptions of grossing techniques, histopathologic features,
ancillary testing modalities, molecular/genetic abnormalities, imaging characteris-
tics, treatment options, clinical signs/symptoms and surgical approaches are con-
temporary contributions to this exciting field.
Liver Cancer represents the latest knowledge of primary liver tumors.
Refreshingly, it focuses on tumors and underlying processes that affect both chil-
dren and adults. This is uncommon in recent books and, therefore, places Liver
Cancer among the best in its field. Written by and for practicing Pathologists,
Oncologists, Surgeons, Gastroenterologists and Radiologists, its comprehensive
content makes it THE go to reference for primary liver tumors. It is easy to read
format also makes it a valuable tool for teaching clinical trainees, medical students
and graduate students.
It has been my pleasure to preview this important book and help bring it forth
to readers. The authors and editors should be commended for their tremendous
work. It is my hope readers will include this book as an important reference on
their bookshelves and consult it often. I know I will.
Florette K. Hazard, MD
Medical Director and Chief of Pathology,
Lucile Packard Children’s Hospital Stanford
Director of Pediatric Pathology,
Stanford University School of Medicine
Stanford, CA, USA
March 2021
Doi: https://doi.org/10.36255/exonpublications.livercancer.2021.fr
 Preface ix

PREFACE

Liver cancers are complex neoplastic diseases with varied etiologies. Despite
numerous research programs and clinical trials targeting several molecules in the
last 50 years, hepatocellular carcinoma, the most common form of liver cancer, is
still difficult to cure, and has become the second most common cause of cancer-
related deaths in adults. This, at least in part, is due to the rising incidence of
obesity, non-alcoholic fatty liver diseases, and excessive alcohol consumption. An
adequate knowledge of the disease, including its etiology and pathology, is essen-
tial to develop effective therapies. Contributed by some of the leading hepatolo-
gists and pathologists in the field, this book is an effort to provide students, basic
scientists, clinicians, and pathologists with a comprehensive understanding of
the  pathology, diagnosis, treatment, and management of various types of liver
cancers. There are nine chapters in the book.
Chapter 1 provides a comprehensive review of malignant epithelial tumors of
the liver in children and adolescents. Hepatocellular carcinoma, lipid-rich hepa-
tocellular carcinoma, fibrolamellar carcinoma, and cholangiocellular carcinoma
are discussed. This is followed by an in-depth discussion of hepatocellular carci-
noma in adults in Chapter 2, covering the etiology and pathogenesis of hepatocel-
lular carcinoma, various diagnostics tests, and treatment options.
While the etiology of liver cancers is multifactorial, obesity and non-alcoholic
fatty liver disease are emerging as significant risk factors for liver cancer. Chapter 3
highlights our current knowledge on the progression of non-alcoholic fatty liver
disease to malignancy. It gives insight into the diagnosis, treatment options, and
future directions for non-alcoholic steatohepatitis-related tumorigenesis. A com-
prehensive understanding of the functional liver segments and anatomy of resec-
tion samples is essential to generate clinically relevant pathology reports. Based on
the College of American Pathologists Cancer Protocols, Chapter 4 guides the gross
dissection procedure for producing a valuable pathology report. Radiology plays
a vital role in the screening, diagnosis, and treatment of liver lesions. Pathology
provides further diagnostic insight into material obtained through biopsy or surgi-
cal resection. Gross appearances guide sampling approaches, and microscopic
examination can lead to a final diagnosis. Chapter 5 introduces the reader to the
radiologic and histopathologic features of common tumor or tumor-like lesions
within the liver, emphasizing hepatocellular lesions, biliary lesions, vascular
­mesenchymal lesions, and metastatic malignancies.
Chapters 6 and 7 focus on therapy and management. Although antiviral thera-
pies have reduced the incidence of hepatitis B virus-related hepatocellular carci-
noma, patients are still at risk of developing hepatocellular carcinoma. Chapter 6
discusses the immunopathogenesis of the virus, the effectiveness of nucleos(t)ide
analogues, and recent therapeutic developments. Hepatocellular carcinoma is a
common indication for liver transplantation. While awaiting liver transplantation,
locoregional therapy can be used as bridging therapy to maintain the tumor
­burden. Chapter 7 provides a comprehensive review of various locoregional
therapies, including trans-arterial chemoembolization, transcatheter arterial
­
radioembolization, thermal ablation, and stereotactic body radiation therapy.
x Preface

Chapter 8 and 9 deal with two relatively rare entities: hepatoblastoma and
undifferentiated embryonal sarcoma of the liver. Hepatoblastoma is the most
common liver cancer in children aged three years and younger. Chapter 8 pro-
vides an overview of the etiology, pathogenesis, epidemiology, incidence, symp-
toms, and therapeutic considerations of hepatoblastoma. The diagnostic measures
necessary from a surgical point of view and the essential operational and technical
considerations for the various hepatoblastoma stages are discussed. Undifferentiated
embryonal sarcoma of the liver is an aggressive malignancy that most commonly
affects the pediatric age group. This tumor very rarely occurs in adults. Chapter 9
outlines the clinical, radiological, and pathological features of hepatic undifferen-
tiated embryonal sarcoma in adults. An in-depth discussion is undertaken to
describe the diagnostic approach and the differential diagnosis for this rare and
challenging tumor.
I thank the authors for their dedication and professionalism in contributing to
this book. I believe this book will encourage readers to delve deeper into this field
and take up the critical challenge of working toward effective treatments for liver
cancers.
Consolato M. Sergi, MD, PhD, MPH, FRCPC, FCAP
University of Alberta
Edmonton, Canada
Doi: https://doi.org/10.36255/exonpublications.livercancer.2021.pr
 List of Contributors xi

LIST OF CONTRIBUTORS

ADUCIO L. THIESEN, MD, PHD, MSC, FRCP

Department of Laboratory Medicine and Pathology, University of Alberta,
Edmonton, AB, Canada

ANDREAS G. ZORI, MD
Division of Gastroenterology, Hepatology, and Nutrition, University of Florida,
Gainesville, FL, USA

BRIANNA SHINN, MD
Division of Gastroenterology and Hepatology, Department of Medicine,
Thomas Jefferson University Hospital, Philadelphia, PA, USA

CONSOLATO M. SERGI, MD, PHD, MPH, FRCPC, FCAP

Departments of Pediatrics, Laboratory Medicine and Pathology, Stollery
Children’s Hospital, University of Alberta, Edmonton, AB, Canada

DINA HALEGOUA-DEMARZIO, MD
Division of Gastroenterology and Hepatology, Department of Medicine,
Thomas Jefferson University Hospital, Philadelphia, PA, USA

ERIC LACHANCE, MD
Department of Radiology and Diagnostic Imaging, University of Alberta,
Edmonton, Alberta, Canada

GARY SMITH, BSC

Department of Surgery, Marshall University Joan Edwards School of Medicine,
Huntington WV, USA

GAVIN LOW, MBCHB, MPHIL, MRCS, FRCR

Department of Radiology and Diagnostic Imaging, University of Alberta,
Edmonton, Alberta, Canada

GIDEON UDOH, BSC

Marshall Institute for Interdisciplinary Research, Marshall University
Joan Edwards School of Medicine, Huntington WV, USA
xii List of Contributors

HIE-WON HANN, MD, FAASLD

Division of Gastroenterology and Hepatology, Department of Medicine,
Thomas Jefferson University Hospital, Philadelphia, PA, USA

JACQUELINE A. SANABRIA, BSC

Department of Surgery, Marshall University Joan Edwards School of Medicine,
Huntington WV, USA

JAKE MANDZIUK, MD
Department of Laboratory Medicine and Pathology, University of Alberta,
Edmonton, Alberta, Canada

JINGYANG HUANG, MD
Department of Pathology and Laboratory Medicine, University of Alberta,
Edmonton, Canada

JOSEF HAGER, MD, MSC

Department of Pediatric Surgery University Hospital of Surgery,
Medical University of Innsbruck, Innsbruck, Austria

JOSEPH I. SHAPIRO, MD
Marshall University Joan Edwards School of Medicine,
Huntington WV, USA

JUAN D. SANABRIA, BSC

Department of Surgery, Marshall University Joan Edwards School of Medicine,
Huntington WV, USA

JUAN R. SANABRIA, MD, MSC, FRCSC, FACS

Department of Surgery, Marshall University Joan Edwards School of Medicine,
Huntington WV, USA

JUSTIN BATEMAN, MD, FRCPC

Department of Laboratory Medicine and Pathology, University of Alberta,
Edmonton, Alberta, Canada

KOMAL SODHI, MD
Department of Surgery, Marshall University Joan Edwards School of Medicine,
Huntington WV, USA
 List of Contributors xiii

LAURA HENAO CAVIEDES, MSC

Department of Laboratory Medicine and Pathology, University of Alberta
Hospital and Stollery Children’s Hospital, Edmonton, AB, Canada

MARLA BEACH, BSC

Department of Laboratory Medicine and Pathology, University of Alberta
Hospital and Stollery Children’s Hospital, Edmonton, AB, Canada

MATHEW SCHADE, MSC

Department of Surgery, Marshall University Joan Edwards School of Medicine,
Huntington WV, USA

MOUMITA BANERJEE, PHD

Marshall Institute for Interdisciplinary Research, Marshall University
Joan Edwards School of Medicine, Huntington WV, USA

PRADEEP K. RAJAN, MSC, MPHIL, PHD

Department of Surgery, Marshall University Joan Edwards School of Medicine,
Huntington WV, USA

RONIEL CABRERA, MD, MS

Division of Gastroenterology, Hepatology, and Nutrition, University of Florida,
Gainesville, FL, USA

SANDEEP A. PONNIAH, MD
Division of Gastroenterology, Hepatology, and Nutrition, University of Florida,
Gainesville, FL, USA

SANDRINE PIERRE, PHD

Marshall Institute for Interdisciplinary Research, Marshall University
Joan Edwards School of Medicine, Huntington WV, USA

TINA BOORTALARY, MD
Department of Medicine, Thomas Jefferson University Hospital,
Philadelphia, PA, USA

UTIBE-ABASI UDOH, BSC, MSC, PHD

Department of Surgery, Marshall University Joan Edwards School of Medicine,
Huntington WV, USA

Doi: https://doi.org/10.36255/exonpublications.livercancer.2021.cont
 1
Carcinoma of the Liver in Children
and Adolescents
Consolato M. Sergi
Departments of Pediatrics, Laboratory Medicine and Pathology, Stollery Children’s
Hospital, University of Alberta, Edmonton, AB, Canada.
Author for Correspondence: Consolato M. Sergi, Departments of Pediatrics,
Laboratory Medicine and Pathology, Stollery Children’s Hospital, University of Alberta,
Edmonton, AB, Canada. Email: [email protected]
Doi: https://doi.org/10.36255/exonpublications.livercancer.2021.ch1

Abstract: Liver cancer, predominantly hepatocellular carcinoma, is the second

most common cause of cancer-related death in adults. Although infrequent in
children, hepatocellular carcinoma is a terrifying diagnosis. Rising levels of
­obesity and obesity-associated lipid metabolic reprogramming of hepatocytes
are increasing the prevalence of lipid-rich hepatocellular carcinoma in young
adults. Most pediatric liver cancers occur in otherwise healthy liver, with some
exceptions such as progressive familial intrahepatic cholestasis, hereditary
­tyrosinemia, alpha-1-antitrypsin deficiency, and genetic hemochromatosis. In
the last decade, although aggressive multidisciplinary treatments including
surgical resection and chemotherapy have remarkably improved patient
­
­outcomes in terms of decreased recurrence rate and increased overall survival
rate, in children with unresectable liver cancer, the 5-year survival rate is still
less than 20%. This chapter provides an overview of malignant epithelial tumors
of the liver in children and adolescents. Hepatocellular carcinoma, lipid-rich
hepatocellular carcinoma, fibrolamellar carcinoma, and cholangiocellular
­carcinoma are discussed.

Keywords: cholangiocarcinoma; fibrolamellar carcinoma; hepatocellular ­carcinoma;

lipid-rich hepatocellular carcinoma; liver tumor in children

In: Liver Cancer. Sergi CM. (Editor). Exon Publications, Brisbane, Australia.
ISBN: 978-0-6450017-2-3; Doi: https://doi.org/10.36255/exonpublications.livercancer.2021
Copyright: The Authors.
License: This open access article is licenced under Creative Commons Attribution-NonCommercial
4.0 International (CC BY-NC 4.0) https://creativecommons.org/licenses/by-nc/4.0/

1
2 Sergi C M

INTRODUCTION

Liver tumors in children and adolescents are catastrophic events. While most
occur in otherwise healthy liver, they may be associated with congenital ­anomalies,
genetic syndromes or metabolic conditions. Two significant entities are hepato-
blastoma (HBL) and liver carcinoma/hepatocellular carcinoma (HCC). Both
­neoplasms account for 0.5–1.5% of all childhood neoplasms (1) and 4% of all
pediatric orthotopic liver transplantations (OLT). HBL accounts for 67–80% of
all pediatric liver cancers worldwide, while 20–33% are HCC. Prevalence of HCC
is geographically and demographically dependent and is associated with the
prevalence of Hepatitis B in the population (1). Differential diagnosis can be
genuinely challenging considering the number of variants and subtypes that have
been delineated over the last decade. Known predisposing factors for HCC in
­childhood and youth are alpha-1-antitrypsin deficiency, hereditary tyrosinemia,
genetic hemochromatosis, Wilson’s disease, acute intermittent porphyria,
progressive familial intrahepatic cholestasis type 2, mitochondrial ETC disorders,
glycogen storages diseases, and transaldolase deficiency. Several neoplastic
conditions must also be considered in the differential diagnosis of HCC, including
­epithelioid hemangioendothelioma of the liver, hemangiosarcoma, malignant
rhabdoid tumor of the liver, undifferentiated embryonal sarcoma of the liver,
biliary tract rhabdomyosarcoma, and the unique variant of neuroblastoma IVS.
Some note should be given about fibrolamellar carcinomas of the liver, which are
tumors of adolescence with a predilection for the left liver and have a tendency to
be ­challenging to resect (2–6).

HEPATOCELLULAR CARCINOMA

HCC is the second most common primary liver malignancy in a pediatric setting
(27%) following HBL. Vascular tumors and sarcomas constitute the remaining
(1, 7). HCC is becoming a common indication for liver transplantation in child-
hood with an incidence rate of 0.3 to 0.45 individuals per million per year (23%).
HCC is diagnosed more commonly in adolescents (10–14 years) but has been
described in children age 5 and younger. Male children have a greater predisposi-
tion to HCC (3:1). HCC typically presents at a more advanced stage in children
than in adults. Overall, only 0.5–1% of all HCCs occur in childhood. HCC has a
broad etiologic spectrum including alcoholic cirrhosis, hepatitis B virus (HBV)
infection, hepatitis C virus (HCV) infection and ingestion of aflatoxins from
Aspergillus flavum in contaminated food, but these factors show a geographic vari-
ation. HCC is most common in central Africa (sub-Saharan regions) and southeast
Asia, with an incidence rate of up to 1 per 1,000, most commonly as a result of
HBV infection. HCC remains a common diagnosis in areas with high endemic
HBV infection rates. There are rates of up to 100% positivity for HBV infection in
Taiwan, and 64% positivity for HBV infection in Hong Kong SAR (Special
Administrative Region of the People’s Republic of China) (8, 9). The integration of
the HBV genome into the neoplasm genome can be detected by immunohisto-
chemistry and demonstrated at the molecular level, but this event is not necessar-
ily oncogenic; alone it does not result in liver carcinogenesis. A secondary
 Liver Cancer in Children and Adolescents 3

promoter (for example, environmental influences or genetic variations) is required

for the development of HCC. In several countries, vaccination of neonates has led
to a drop in HCC rate in childhood and adulthood.
Numerous genetic abnormalities are associated with HBV-HCC in adults, such
as mutations in the cytotoxic T-lymphocyte antigen 4 (CTL A-4) gene, deletion or
chromosomal loss of DLC1 (Deleted in Liver Cancer 1) or loss of heterozygosity
on the KIF1b promoter region of the Mini-Chromosome Maintenance protein-7
(MCM7) gene and enhancer II (EnhII) (1, 10, 11). Moreover, alterations of the
basal core promoter (BCP) and precore regions of HBV have been found. It has
been shown that Pre-S mutations (C1653T, T1753V, and A1762T/G1764A) are
explicitly associated with an amplified risk of HCC (11, 12). HCV infection is
rarer than HBV infection in children and so is less significant etiologically (13).
Other non-viral conditions associated with HCC include glycogen storage ­diseases
type 1, 3, and 4 (poor metabolic control, size and number of adenoma, malignant
transformation in adenoma), congenital portosystemic shunts (complete absence
of portal vein or type 1a, presence of nodular regenerative hyperplasia, adenoma,
and dysplasia), hepatic venous outflow tract obstruction/Budd-Chiari syndrome
(long-standing obstruction, presence of nodular regenerative hyperplasia, inferior
caval vein (IVC) obstruction, Factor V Leiden mutation (14), acute intermittent
porphyria, porphyria cutanea tarda, hyper-citrullinemia, tyrosinemia (non-usage
of [2-(2-nitro-4-trifluoromethyl-benzoyl)-1,3-cyclohexanedione (Nitisinone,
NTBC) or late introduction of NTBC), familial intrahepatic cholestasis (BSEP
deficiency, TJP2 mutations, MDR3 deficiency), alpha-1-antitrypsin deficiency
(AATD), biliary atresia, Alagille syndrome, autoimmune hepatitis, Wilson’s
­disease, and genetic hemochromatosis (1). Genetic syndromes predisposing to
HCC include ataxia telangiectasia, Fanconi anemia, familial adenomatous
­polyposis, and neurofibromatosis. Non-metabolic conditions are presented in the
following section. Metabolic diseases are discussed separately.

Biliary atresia
Biliary atresia (BA) is characterized by a necro-inflammatory process with destruc-
tive cholangitis (15, 16). The prevalence rate of HCC in BA is 1.3% (five out of
387 patients) with all but one patient below five years of age. This figure is par-
ticularly alarming in the West where rates of BA are up to 30%, although it has
been argued that the said figure may be confounded by surveillance bias (1).

Congenital portosystemic shunts

Congenital portosystemic shunts (CPSS) are another predisposing factor for liver
oncogenesis. CPSS causes nodular transformation in the pediatric liver. Of all
types of CPSS, patent ductus venosus, portal vein to IVC, or left portal venous
system to IVC are the most common (17–23). Liver tumors have been observed
in both extra- (35%) and intra-hepatic (13%) types at a median age of eight years;
however, the malignant tumors (HBL, HCC, and sarcoma) are exclusively
seen  alongside the extrahepatic variety. HCC occurs in 2.5% of patients with
CPSS  (24). In the case of benign tumors like focal nodular hyperplasia (FNH)
and  hepatic adenoma, shunt occlusion leads to a decrease in the size of the
4 Sergi C M

tumor masses, but resection and shunt occlusion, as well as OLT, are necessary for
malignant tumors (20).

Budd-Chiari syndrome
Budd-Chiari syndrome (BCS) is characterized by abdominal pain, ascites, and
liver enlargement caused by blockage of the hepatic veins that drain the liver. BCS
can be primary (75%) (polycythemia rubra vera, pregnancy, postpartum state, use
of oral contraceptives, paroxysmal nocturnal hemoglobinuria, and lupus antico-
agulants) or secondary (25%) (compression of the hepatic vein by an outside
structure [for example, a neoplasm]). HCC is a known sequela of BCS in adults,
with a 5-year cumulative incidence of 4% (1). Nodular regenerative hyperplasia is
more commonly associated with the onset of BCS, and is more often associated
with male gender, FVL mutation, and inferior vena cava (IVC) obstruction,
but can also be seen in tuberculosis, congenital venous webs, and IVC stenosis
(25, 26).

Alagille syndrome
Alagille is a multisystem genetic disorder characterized by interlobular bile ductal
paucity and chronic hepatocellular cholestasis (27). Alagille syndrome is a result
of mutations on one of the two genes: JAG-1 in 90% of cases, and NOTCH2 in
about 1% of cases. Liver carcinogenesis in Alagille syndrome has been occasion-
ally reported (28–32). In the first two decades of life, patients can develop HCC
in conjunction with Alagille syndrome and cirrhosis (32). Upregulation of Notch2
in hepatocytes results in proliferation of hepatocyte and biliary epithelial cells; it
also triggers diethylnitrosamine induced HCC formation in mice (33). Neoplastic
cells commonly lose contact inhibition to undergo anchorage-independent prolif-
eration. They become resistant to programmed cell death by inactivating the
Hippo signaling pathway. It results in activation of the transcriptional co-activator
yes-associated protein (YAP). The transcriptional regulator YAP upregulates
JAGGED-1 and activates NOTCH pathways in mouse models and humans with
HCC and colorectal and pancreatic carcinomas (34). YAP is a transcriptional co-
activator and is responsible for the rapid proliferation of neoplastic cells becoming
apoptosis-resistant.

Autoimmune hepatitis
Autoimmune hepatitis (AIH) is a non-metabolic disorder that predisposes to
HCC (35). The risk of HCC in adults with AIH is 3.06 per 1,000. Absolute risk of
HCC as a sequela of AIH is slightly lower than the risk of HCC attributable to
other causes of cirrhosis in adults like hepatitis B, C, or primary biliary cholangitis
(PBC) (36). The risk of HCC is related to the extent of cirrhosis at the time of clini-
cal diagnosis of AIH (OR = 4.08). Abnormal liver enzymes (transaminases) are
seen on final observation (OR = 3.66) (36, 37). Although the incidence of HCC in
children is rare, AIH remains a common pediatric liver condition. Non-alcoholic
fatty liver disease (NAFLD), type 2 diabetes mellitus, genetic hemochromatosis,
and porphyria (acute intermittent and porphyria cutanea tarda) carry a high risk
 Liver Cancer in Children and Adolescents 5

of development of HCC in adults, but not in children (38). About 33–50% of all
HCC cases in childhood are sporadic (1).

Symptoms
Symptoms of HCC in noncirrhotic individuals are similar to those seen in patients
harboring other liver tumors. The “tumor quia tumet” or mass-effect includes an
abdominal mass, pain, and jaundice. Abnormal liver biochemistry alongside spe-
cific clinical signs and symptoms may be present if the tumor originates as a result
of liver disease. Clinical signs may include splenomegaly due to portal hyperten-
sion and spider nevi, among others. There are no specific findings on full blood
count associated with HCC. Liver function tests frequently give abnormal results
particularly in children in whom HCC has occurred with frank cirrhosis. Alpha-
fetoprotein (AFP) is increased in 50–70% of patients with HCC, although it
should be kept in mind that persistent elevations of AFP are found in hepatic
regeneration. On the other hand, AFP levels greater than 400 to 500 ng/mL in a
patient with liver cirrhosis is strongly suggestive of HCC. HCC should be sus-
pected when AFP levels fall between 200 and 300 ng/mL. AFP has prognostic
value and has been used for years as a prognostic (relapse) indicator after chemo-
therapy. Elevations of the vitamin-B12-binding protein transcobalamin I is a useful
indicator for the fibrolamellar variant of HCC or fibrolamellar carcinoma (FLC)
(vide infra). Finally, the exclusion of known risk factors, such as HBV/HCV
­serology, plasma and urine amino acid, urinary succinylacetone for hereditary
tyrosinemia, biochemical serum level, and isoelectricfocusing-based phenotype
for AAT should be routinely performed. If there is a suspicion of a genetic disease,
a genetic confirmation of PFIC 2, hereditary tyrosinemia, and Alagille syndrome
should be carried out as soon as possible.

Imaging
Imaging is useful to determine the site and characteristics of the tumor, to estab-
lish the presence of tumor metastases, and to help the surgeon to assess the suit-
ability for prompt resection. HCC is difficult to distinguish from HBL on imagery.
Both HCC and HBL are typically large and multifocal. In both tumors, there may
be evidence of venous invasion, tissue calcification, and lung metastases.
Ultrasound of HCC shows a large, heterogeneous, generally hyperechoic vascular
mass. Computed tomography (CT) provides detailed information on the anatomic
boundaries of liver tumors, which is essential for the pediatric surgeon. MRI scan-
ning gives information about tumor infiltration into surrounding tissue, enabling
precise evaluation of segmental involvement. MRI findings of HCC show a hetero-
geneous, mostly hypointense neoplasm on T1-weighted images. MRI findings on
T2-weighted images of HCC is mildly hyperintense in comparison with healthy
liver. Contrast-enhanced T1-weighted images identify a pattern like CT. There is
early arterial enhancement and reduced signal intensity in the portal venous
phase. Imaging data is essential for clinical staging and surgical intervention. PET
scans show areas of high metabolism, which is useful in localizing early metasta-
ses or recurrence of the neoplastic disease. In all children without cirrhosis, unless
primary surgery is feasible, a biopsy is necessary. Image-guided needle biopsy is
6 Sergi C M

usually favored to open biopsy due to the multifocal nature of the hepatocellular
neoplasm. To avoid tumor seeding in children, the tumor should be approached
along a short route through unaffected liver, avoiding Coinaud liver segments that
will not be removed at subsequent surgery. Moreover, a coaxial biopsy should be
used to obtain several cores of tissue with a single shot. The needle track should
be plugged, either with gelatin foam or with a slurry of collagen. HCC in adults
has been studied in greater detail than pediatric HCC in terms of cytogenetic and
molecular characteristics. Future studies aim to outline molecular pathology of
HCC in children (39).

Morphology
Morphology of adult and pediatric HCC are similar. There are several growth
­patterns including trabecular (plate-like), solid/compact, pseudoglandular/acinar,
and scirrhous (scleroting architectural pattern). Cytologic variants include
­pleomorphic cells, clear cells, and sarcomatous cells (spindle cells or bizarre giant
cells). The cytoplasm of the tumor cells is stained with Hep Par 1, a monoclonal
antibody (clone OCH1E5.2.10) in a granular manner, without canalicular or
zonal accentuation. The antigen detected by Hep Par 1 appears specifically associ-
ated with the mitochondrial membrane of hepatocytes. The use of antigen retrieval
(high pH buffer) and a detection kit which eliminates non-specific biotin reactiv-
ity allows the pathologist to use Hep Par 1, which is highly effective on formalin-
fixed, paraffin embedded tissues. Cautious use of Hep Par 1 antibody in a panel
with other positive (AFP, carcinoembryonic antigen with a polyclonal antibody,
CD10) as well as negative (epithelial membrane antigen, carcinoembryonic anti-
gen with a monoclonal antibody, CD15) markers of hepatocellular differentiation
may assist in accurate diagnosis of hepatocellular carcinoma (Figure 1). Epithelial
cell adhesion molecule (EPCAM) expression seems to be far more robust in
­pediatric than adult HCC (7, 40). In the future, transcriptomics studies may reveal
additional data comparing adult and pediatric HCC.

Differential diagnosis
Distinguishing HBL from HCC may be straightforward in a few cases. HBL may
be easily recognized if both epithelial and mesenchymal components are present,
but when it shows a macrotrabecular architecture with well-differentiated fetal
epithelial cells and no mesenchymal elements, distinction may be practically
impossible histologically (7, 41). In this scenario, liaison with the clinician is
critical. The pathologist should consider that HBL occurs more frequently than
HCC in patients aged 5 or younger, very-low-birth-weight infants and premature
infants, and with a multiple congenital anomaly syndrome, such as Beckwith-
Wiedemann syndrome. On the other hand, the presence of underlying hepatic
disease, especially with a known risk factor, such as hereditary tyrosinemia,
favors a diagnosis of HCC over HBL. In the most recent pediatric liver tumor
consensus classification, the category of “hepatocellular neoplasm not otherwise
specified” has been introduced to acknowledge the challenge of differentiating
sporadic cases which show cells that are “transitional” between hepatoblasts and
­hepatocytes (39, 42).
 Liver Cancer in Children and Adolescents 7

Figure 1.  Microphotographs of hepatocellular carcinoma. A. Solid pattern showing large

hepatocytes organized in a solid architecture (top of the figure) contrasting with the lobular
arrangement of non-tumoral hepatocytes (bottom of the figure) (Hematoxylin-Eosin staining,
scale bar: 50 µm). B. Macrotrabecular pattern of growth showing trabeculae of hepatocytes,
which are more than two cells thick. The cytoplasm of the tumor cells is stained with Hep
Par 1, which is a monoclonal antibody (clone OCH1E5.2.10) in a granular manner, without
canalicular or zonal accentuation. The antigen detected by Hep Par 1 appears to be quite
specifically associated with the mitochondrial membrane of hepatocytes. The use of an
antigen retrieval (high pH buffer) and a detection kit which eliminates non-specific biotin
reactivity allows the pathologist to use Hep Par 1, which is very effective on formalin-fixed,
paraffin embedded tissues. It seems that cautious use of Hep Par 1 antibody in a panel with
other positive (alpha fetoprotein, carcinoembryonic antigen with a polyclonal antibody,
CD10) as well as negative (epithelial membrane antigen, carcinoembryonic antigen with a
monoclonal antibody, CD15) markers of hepatocellular differentiation may assist in the
accurate diagnosis of hepatocellular carcinoma (Hep Par 1 immunostaining, Avidin-Biotin
Complex, scale bar: 50 µm). C. Macrotrabecular pattern of growth showing trabeculae of
hepatocytes, which are more than two cells thick. The cytoplasm of the tumor cells is not
stained with FP2 (FP2 immunostaining, Avidin-Biotin-Complex, 50 µm). Mutations in the
PKHD1 (polycystic kidney and hepatic disease 1) gene are responsible for all typical forms of
autosomal recessive polycystic kidney disease (ARPKD). The PKHD1 gene is associated with a
complex splicing pattern and its longest open reading frame product, fibrocystin, also
known as polyductin, is a single transmembrane domain glycoprotein. FP2, a fusion protein
containing the polyductin intracellular carboxy-terminus has been used to generate a
polyclonal antibody that was applied to study the polyductin expression profile in human
tissues. The anti-FP2 purified antiserum stains the renal collecting ducts, the thick ascending
limbs of Henle in humans and the branching ureteric bud in mouse, the murine intrahepatic
and extrahepatic biliary system, and cholangiocarcinoma (inset on the top of the
microphotograph), but not hepatocellular carcinoma making an important negative marker
of hepatocellular differentiation. Immunohistochemical detection of polyductin and
co-localization with liver progenitor cell markers during normal and abnormal development
of the intrahepatic biliary system and in adult hepatobiliary carcinomas. J Cell Mol Med. 2009
Jul;13(7):1279-90).
8 Sergi C M

Staging
Staging pediatric liver neoplasms is crucial, but the reality is that there are
no  ­uniformly and broadly accepted staging systems for the pediatric HCC (7).
In adulthood, the Barcelona Clinic Liver Cancer score is probably the most widely
accepted system, because it assesses the state of the tumor, the state of the patient,
and the condition of the liver. Use of the tumor node metastasis (TNM) staging
system is controversial. TNM does not seem to be well-suited for predicting prog-
nosis in either pediatric HCC or adult HCC (43). Other staging systems include
the Cancer of the Liver Italian Program (CLIP) scale and the Okuda system, among
others (43).
Once a diagnosis is reached, in the United States of America and Canada, the
Children’s Oncology Group (COG) provides vital information to children and
their families. This support continues throughout treatment and carries on
after cure. The COG’s adoption of a system based mostly on surgical findings in
the late 1980s has been particularly important for the treatment of HCC. In the
late  80’s, the PRETEXT (PRETreatment EXTent) system was developed by the
International Society of Pediatric Oncology (SIOP) Liver Tumor Study (SIOPEL)
Group. The PRETEXT was revised in 2005, and again in 2017 (7, 44). This sys-
tem divides children with liver neoplasms into four categories consistent with the
amount of liver segments not involved by the tumor (7, 45). A separate classifica-
tion includes the involvement of the portal and hepatic veins and IVC as well as
metastatic disease. The interobserver reliability of the system remains neverthe-
less quite high. The significant advantage of PRETEXT staging is that it allows
stratifying of patients to determine the most effective treatment. Involvement of
hepatoduodenal lymph nodes has a role in staging. It is important therefore to
take samples from lymph nodes associated with the hepatoduodenal ligament at
the time of surgical resection. After resection of the tumor, the 5-year survival rate
(5-YSR) ranges between 35and 50%, while the 5-YRR (5-years recurrence rate) is
20–30% (7).

Treatment and management

Traditionally, treatment of malignant liver neoplasm consists of a combination of
chemotherapy and surgical resection. The highest cure rates are associated with
complete surgical resection with tumor-free resection margins (R1 and R2 equal
to zero using the TNM staging). In the case of a child with a liver neoplasm, when
a predisposing condition is present, and HCC is likely, primary resection is indi-
cated without biopsy. PRETEXT grade I and II tumors (that is, having 3 or 2 adja-
cent sectors, respectively, tumor-free) are quite easily removed by surgery, but
PRETEXT III tumors (in which only one sector or two nonadjacent sectors are
tumor-free) necessitate a surgeon that specializes in liver surgery, and may also
require intensive care facilities and liver transplantation teams. PRETEXT IV
tumors (in which there are no tumor-free liver segments) are considered unresect-
able due to extensive liver involvement. Although complete tumor clearance with
margins of at least 1 cm have been considered safe, recent discussion suggests that
any clear margin (that is, less than 1 cm) may be acceptable. This suggests that all
options should be investigated before declaring a tumor nonresectable. Segmental
hepatic removal and resection require the application of an intraoperative
 Liver Cancer in Children and Adolescents 9

ultrasound examination, which must correlate with the volume of the liver that
needs to be removed using CT- or MRI-based calculations (46). In pediatric HCC,
the usual limit of resection is calculated by dividing the remnant liver volume
(milliliters) by the patient’s body weight (kilograms). This value can safely exceed
the usual 0.6–0.8 mL/kg. Orthotopic liver transplantations (OLT) as a treatment
for HCC is still controversial, although the criteria for OLT in adults has been
modified over time from unrestricted tumor limits to precise tumor burden which
meets the conventional Milan criteria (CMC). The CMC-based safe limits for OLT
are either a single tumor smaller than 5 cm in size, or in case of multiple tumors,
no more than 3, and each not exceeding 3 cm in diameter. Satisfying this criterion
allows a 5-YSR of about 70%. Unfortunately, there are no randomized prospective
studies comparing liver resection with OLT as treatments for childhood HCC,
although we must acknowledge that experience with OLT in pediatric HCC is
limited (47). A low 5-YSR has been observed in earlier reports (48, 49) with val-
ues ranging between 29–35% in children with unresectable neoplasms. More
recent reports from Pittsburgh and other centers show an improvement in 5-YSR
rates, with values of up to 60% (50). Contraindications for OLT include extrahe-
patic disease and FLC. It has been proposed that decisions on which treatment is
most appropriate should be made on a case-by-case basis.
Ablative therapies include radiofrequency ablation and percutaneous ethanol
injection (51), although these techniques have been explored mostly for adult
HCC. Ablative therapy is comparable to surgery for tumor size equal to 3 cm or
below, but its effect on HCC up to 5 cm or more is not confirmed to be better than
tumor removal. Usefulness of ablative therapy for treatment of HCC of a size
greater than 5 cm seems to be uncertain.
Transfemoral hepatic artery chemoembolization (TACE) is an option for the
management of adult HCC with improved 5-YSR in randomized controlled trials
(52). TACE indications for pediatric HCC is similar to adult HCC; it may be con-
sidered a bridge to OLT or resection with attempted conversion of tumors from
nonresectable to resectable. Advantages of TACE include the delivery of high con-
centrations of cytotoxic drugs to the tumor center, prolonged intratumoral dwell
time of drug, and reduced systemic toxicity. TACE appears to be a safe and effec-
tive method in children with unresectable hepatocellular neoplasm and is useful
for handling hepatic tumor burden, downstaging, and bridging to OLT (53–55).
Occasionally, pulmonary embolism and thrombosis of some branches of the
hepatic artery have been recorded as severe complications. Despite widespread
use of neoadjuvant and adjuvant chemotherapy, there is little evidence to suggest
that this translates to a benefit in survival as shown in the North American
Intergroup Hepatoma study (INT-0098) and the International and in the
Childhood Liver Tumor Study Group (SIOPEL) study (7, 44). Nevertheless, adju-
vant chemotherapy may be of benefit for children with completely resected HCC.
The outcome was uniformly poor for patients with advanced-stage disease. These
patients showed a 5-year event-free survival (5-YEFS) with stage III and IV HCC
of 23% and 10% respectively. The resection of the neoplasm after neoadjuvant
chemotherapy was practicable in two patients only. Similarly, the SIOPEL identi-
fied a 5-YEFS of 17% (1). In the three cooperative studies of the German Society
for Pediatric Oncology and Hematology (GPOH), HCC has been targeted with the
same regimens as HBL, with surgical resection highly recommended as the pri-
mary treatment in all patients (7, 56).
10 Sergi C M

Ifosfamide, a chemotherapy drug used to treat testicular cancer, soft tissue

sarcoma, osteogenic sarcoma, urinary bladder cancer, small cell lung cancer, cer-
vical cancer, and ovarian cancer, is given in combination with cisplatin and doxo-
rubicin in a combination termed “IPA”. Cisplatin is a chemotherapy drug that
interferes with DNA replication using chloride ions to displace water to give the
aquo-complex cis-[PtCl(NH3)2(H2O)]+, while doxorubicin acts to stop growth of
cancer cells by blocking topoisomerase 2. IPA was initially present as a chemo-
therapeutic combination in HB89, but in HB94 carboplatin and etoposide are
added to the IPA combination (57). However, the results were not satisfactory,
with disease-free survival of only 32% in HB94. Currently, high-dose carboplatin
and etoposide are given for nonresectable HCC. The rationale is to achieve tumor
regression to allow for surgical resection. A microscopic residual tumor (termed
an R1 resection in relation to TNM classification) may be observed and requires
careful histological examination. New targeted therapies for pediatric HCC are
being explored and are proving to be promising.
Sorafenib, a multikinase inhibitor that targets vascular endothelial growth
­factor, in combination with doxorubicin has been demonstrated to be advanta-
geous in advanced adult HCC (58). This study showed improved progression-free
survival time and tumor shrinkage. Moreover, GPOH showed that in 50% (6 of 12)
of pediatric patients receiving PLADO (cisplatin/doxorubicin) and sorafenib as
first-line therapy for HCC, there was complete remission at a median follow-up of
20 months. Of these six children, four received PLADO/sorafenib and had a liver
resection, whereas two children underwent OLT after a localized relapse. Alternate
approaches with first- and second-line chemotherapy regimens include gem-
citabine/oxaliplatin (GemOx), 5-fluoruracyl (FU)/cisplatin, capecitabine/­cisplatin,
5-FU/mitomycin, 5-FU/oxaliplatin, gemcitabine/cisplatin, 5-FU/­interferon, and
monotherapy with sorafenib (59, 60). These approaches are derived from studies
on adult patients because pediatric experience is limited. Some chemotherapy
protocols have been investigated in pediatrics in small, single-­institution trials.
They include: (i) the combination of irinotecan, v­ incristine, temozolomide, and
bevacizumab; (ii) the combination of oxaliplatin, irinotecan, and gemcitabine;
and (iii) the combination of gemcitabine and oxaliplatin (7). Experience in adult
HCC suggests that GemOX is both efficient and well-tolerated in relapsed/refrac-
tory neoplastic disease (59).
Knowledge of the biological pathways of liver carcinogenesis has increased
in the last decade, generating potential for exploration of new targeted ­therapies.
Erlotinib, which targets the epidermal growth factor (EGF) pathway, may be
useful for pediatric HCC (61–64). The mTOR or mammalian target of r­ apamycin
pathway inhibitor everolimus has been demonstrated to have antitumor a­ ctivity
in clinical trials of adult HCC (61, 65, 66). cMET, a tyrosine kinase receptor for
the hepatocyte growth factor, is implicated in neoplastic formation and prolif-
eration in both HBL and HCC. In a randomized phase 2 study, Tivantinib, a
cMET inhibitor, has shown activity in a subset of patients with advanced HCC
who had progressed on sorafenib (66–73). Cabozantinib, an inhibitor of c-Met,
VEGFR2, AXL and RET, is used to treat medullary thyroid cancer and as a sec-
ond line treatment for renal cell carcinoma among others. It offers the potential
of deactivating multiple mechanisms by which neoplastic cells proliferate (66,
67, 74–78).
 Liver Cancer in Children and Adolescents 11

Treatment-response follow-up
Repeat CT scans or ultrasonography are useful to observe response to treatment.
Imaging can document shrinkage of the liver cancer and chest radiography or CT
scans can monitor potential progress of pulmonary metastases. Also, the presence
of AFP levels alongside the lack of radiological disease is highly suspicious of
minimal residual disease and more sophisticated investigation is mandatory. An
increase in AFP levels after initiation of chemotherapy is usually a sensitive marker
of treatment failure or relapse. Transcobalamin I has been used as a biomarker of
fibrolamellar carcinoma of the liver or fibrolamellar variant of HCC.
Long-term outcomes are dependent on demographics and treatment type.
Survival of children aged 0–4 years is better than that of children aged five years
and older, with a 5-YSR of 53% (unadjusted) compared with 32% for children
aged 5–19 years. Prognosis is better in male children when compared with female
children. Some ethnic evaluation studies identified that Asian children have rela-
tively low survival (13%), compared with white and black children (survival of
33% and 46% respectively) (7). On the other hand, results of Surveillance,
Epidemiology and End Results (SEER) database are puzzling. A large series of 218
pediatric patients with HCC seem to show no significant difference in the overall
survival by age at diagnosis, gender, race/ethnicity, or use of radiation (79–81).
Pediatric excisable HCC seems to bear a better prognosis irrespective of histologic
subtype. These patients seem to do worse than those with HBL (48). Analysis of
post-OLT showed that 1-, 5-, and 10-YSR were 86%, 63%, and 58%, respectively.
The primary cause of death of these patients was metastatic or recurrent disease,
responsible for 86% of deaths in children with HCC (49).

HEPATOCELLULAR ADENOMA AND FOCAL NODULAR

HYPERPLASIA

In the differential diagnosis, we should mention hepatocellular adenoma (HCA)

and focal nodular hyperplasia (FNH). A large multicenter study resulted in
­genotype-phenotype classifications of four subtypes of adenomas, including
biallelic loss of TCF-1 gene/HNF1α HCA (42%), WNT pathway mutation/​
β-catenin-activated HCA (12.5%), inflammatory HCA, and nonmutated/­non-
inflammatory HCA (82, 83). The genotype-phenotype HCA classification is
­paramount because certain genotype-phenotypes are associated with HCC: 7%
in HNF1α mutated adenoma, 13% in noninflammatory/nonmutated adenomas,
and 46% in β-catenin-activated HA (83). An inflammatory HCA has the
distinctive features of sinusoidal dilatation and perivascular lymphocytic
­
­inflammation with immunohistochemical evidence of serum amyloid associated
(SAA) protein. Immunohistochemical staining of FNH with glutamine synthe-
tase shows characteristic map-like labeling. FNH is a hyperplastic liver lesion
and should be considered a response to a pre-existing arterial malformation
(84). Although FNH can occur as multiple lesions in one-fifth of cases, it is
­usually a solitary hepatic lesion discovered incidentally. FNH is a common
tumor lesion, second only to hemangioma and at least ten times more common
12 Sergi C M

than HCA. Imaging is crucial; ultrasound is highly distinctive in detecting FNH,

and both CT scan and MRI are highly reliable techniques in diagnosing FNH
(85). Although FNH does not require a liver biopsy for the diagnosis due to
good imaging criteria, biopsy may be important if the patient is outside of the
typical age range. FNH is a benign lesion with very low risk of rupture and
hemorrhage, and is treated with conservative management unless it becomes
symptomatic (86).
FNH presents grossly as a well-demarcated non-encapsulated lesion of 4–5 cm
in diameter and harboring a central or slightly eccentric scar. Histologically, FNH
shows a nodular-shaped architecture with normal hepatic plate architecture and
no atypia, fibrous bands radiating from the central or slightly eccentric scar which
contain “portal tract-like areas” with bile ductular proliferation, lack of interlobu-
lar bile ducts, and thickening of eccentrically located blood vessels. Reticulin stain
demonstrates intact reticulin framework which is characteristically lost in malig-
nant hepatic lesions (87). On macroslides, the central scar contains large vessels
with fibromuscular hyperplasia, intense lymphocytic infiltrate, and bile duct pro-
liferation, as well as normal hepatocytes with regeneration. Polyclonal carcinoem-
bryonic antigen is useful to reveal a hepatic origin but does not distinguish
between benign and malignant tumors. Reticulin histochemical stain plays a
major role in pinpointing hepatic adenomas.
Although hepatic adenomas are more common in women of reproductive age,
they are not extremely rare in children and adolescents. Hepatic adenomas are
usually rare in males and have been associated with oral contraception and ana-
bolic steroid use, but most importantly various metabolic disorders, such as gly-
cogen storage disorder and galactosemia, can exhibit hepatic adenomas, even in
childhood. It is rare to see marked elevations in liver enzymes. Genetic analysis
can prove neoplastic etiology. Although multiple adenomas can occur, they usu-
ally are solitary. There is no liver cirrhosis as background, and differential diagno-
sis from macro-regenerative nodules is essential. Grossly, hepatic adenomas do
not have a capsule, and there is no surrounding fibrosis. Microscopically, hepato-
cytes are arranged 1 to 3 cell plates thick with a normal appearance. Cytologically,
the nucleus to cytoplasmic ratio is normal, but some mild variation in cell size
may occur. Atypia is minimal or absent. Mitotic figures are nil or occasionally
present, but steatosis, lipofuscin pigment deposition, and Mallory’s hyaline can be
seen. There are no portal tracts, which are seen in both adenomas and HCCs.
A useful feature in distinguishing an adenoma from a well-differentiated HCC is
the presence of an intact reticulin framework. The protein FPA can be detected in
the serum of children with malignant hepatocellular tumors, but it may not be
specific. AFP is absent in adenomas. Similarly, glypican-3, which is a protein
encoded by the GPC3 gene that is absent in adenomas and positive in HCCs or
dysplastic nodules, although HCCs with negative glypican-3 immunostaining,
can be seen. The differential diagnosis with FNH relies on the presence of fibrotic
areas with bile ductules and abnormal blood vessels. Liver cirrhosis can help dif-
ferentiate an adenoma from the large regenerative nodule. Therapy consists of
surgical excision. Because of the risk of life-threatening hemorrhage, excisional
procedures are strongly indicated for lesions that could not be differentiated on
needle biopsy. In the presence of adenomatosis, liver transplantation is essential.
In young patients with a gene mutation predisposing to colorectal carcinoma
(for example, TP53 and FAP), colorectal malignancy with metastatic disease can
 Liver Cancer in Children and Adolescents 13

occur at an early stage. Metastatic colon cancer needs to be excluded in these

patients, and immunohistochemistry may help in this direction. Two antibodies
may be beneficial: CDX2 and CK20. CDX2 (Caudal Type Homeobox 2), a gene
that directs early embryogenesis in mice, is a marker for gastrointestinal differen-
tiation, especially colorectal differentiation. CK20 or K20, is an antibody for anti-
gen 20 of the keratin intermediate filaments of the cytoskeleton (88). If the tumor
is metastatic in origin and arises from the colorectal portion of the gut, both stains
should be positive. Also, in the differential diagnosis, a primary cholangiocellular
carcinoma must be excluded. The antibodies CK7 and CK19 are appropriate
markers for differentiation. Both antibodies are positive in the tissue of a primary
cholangiocellular carcinoma (89–91).
As indicated above, three antibodies are useful in highlighting the diagnosis of
a primary hepatocellular carcinoma: HepPar1, glypican-3, and AFP. The combina-
tion of three positive antibodies aids in the diagnosis of HCC. Three antibodies
may help in the differential diagnosis with malignant lymphoma: CD45 or com-
mon leukocyte antigen, CD3 (T-lymphocyte marker), and CD20 (B-lymphocyte
marker). However, immunohistochemistry may not be necessary because cell
cytology, tumor growth pattern, and desmoplasia help to exclude a malignant
hematological neoplasm. Malignant lymphomas have little cytoplasm and no
nested and trabecular growth pattern with a fibrotic stroma. Appropriately, the
presence of synaptophysin and chromogranin are useful in identifying a neuroen-
docrine tumor, considering the nested and trabecular growth pattern can be a
classic pattern for neuroendocrine neoplasms.

LIPID-RICH HEPATOCELLULAR CARCINOMA

The most common histological patterns of HCC are solid, trabecular, pseudoglan-
dular, and scirrhous, with three recognized cytologic variants: pleomorphic, clear
cell, and sarcomatoid. In the clear cell variant, cytoplasmic changes are related to
glycogen and/or lipid accumulation (lipid-rich HCC, LR-HCC) within the tumor
cells. In cytologic samples of low-grade lipid-containing hepatic neoplasms, dif-
ferentiation of neoplastic cells from benign steatotic cells can be extremely chal-
lenging (92–94). Fine-needle aspirates of benign lesions contain rigid cores and
large tissue fragments, while LR-HCC shows a breakdown of the reticulin frame-
work (visualized with a routine special stain) with scattered cells and a fine gran-
ular-appearing background. LR-HCC is an uncommon HCC variant and needs to
be taken into consideration, particularly now that non-alcoholic steatohepatitis
and fatty cell change are more common in adolescence and youth (95–98).

FIBROLAMELLAR CARCINOMA

FLC or fibrolamellar variant of HCC is a rare variant of HCC, and recent investiga-
tions have identified a specific molecular signature (99–103). This neoplasm
affects adolescents and youth with no significant gender prevalence primarily.
FLC chiefly affects Caucasians. Unlike HCC, FLC is never associated with a
14 Sergi C M

history of parenchymal liver disease. Liver cirrhosis and underlying metabolic

disorders are not associated with this tumor. Although broadly considered a vari-
ant of HCC, it has been suggested that it ought to be reported as a distinct clinical
and histopathologic entity (41, 100). FLC accounts for up to almost one-tenth of
all HCCs. Moreover, FLC accounts for up to one-third of all pediatric HCCs.
The characteristic histological features of this neoplasm provide the grounds
for the singularity of this neoplasm. The Fibrolamellar Carcinoma Consortium is
an institutional collaboration involving Memorial Sloan-Kettering Cancer Center
(MSKCC), the University of California at San Francisco (UCSF), and Johns
Hopkins Hospital. The Consortium was developed to address the challenges asso-
ciated with investigating FLC, such as small study samples and the lack of aware-
ness of the disease within the oncology community, and with identifying new
treatment options. The Consortium presented the pooled demographic, clinical,
pathologic, treatment, and survival data of 95 patients with FLC seen at the three
institutions from 1986–2011 (104).

Presentation
Clinically, abdominal pain, hepatomegaly, obstructive jaundice, ascites, systemic
manifestations (for example, fever or sense of “fullness”), and rarely gynecomastia
and BCS have been observed in patients harboring an FLC (100). Laboratory val-
ues that need to be taken into consideration are neurotensin, vitamin B12 binding
capacity (transcobalamin), and des-gamma-carboxyprothrombin (104). Serum
AFP is increased in just 10% of FLC cases, unlike with HCC where AFP is elevated
in 60% of cases. X-ray imaging shows a central scar which resembles the central
scar observed in FNH, a benign lesion with no correlation with FLC. The central
scar of FLC is often calcified, which is unusual with FNH.
Grossly, FLC is typically larger than conventional HCC. FLC has an odd
propensity to metastasize to regional lymph nodes. Thus, the pediatric surgeon
should attempt to sample the regional nodes. FLC generally appears as well-
circumscribed, single white-brown hard mass that shows fibrous bands, similar to
FNH. It usually involves the left lobe of the liver, but it has been reported in both
lobes.

Pathology
Microscopically, the tumor is made of large polygonal cells with copious eosino-
philic cytoplasm (about 50 microns, that is, approximately double the size of a
normal human hepatocyte, which is 20–30 microns) with granular character and
large nuclei containing prominent nucleoli. At low magnification, thick fibrous
collagen bands are seen transecting the tumor (Figure 2A). They may partially
encircle or surround the tumor cells. On histology, pale cytoplasmic bodies (pale
bodies) or ground glass cells and PAS-positive hyaline globules and copper depo-
sitions are also seen (Figure 2B). Bile is observed using negative Prussian Perl’s
blue iron stain. In the differential diagnosis of FLC, sclerotic variant of HCC
should be mentioned, which rarely harbors the parallel (or almost parallel) run-
ning collagen bands. Vascular invasion and necrosis may be seen. Radiological
calcification may also be confirmed on histology.
 Liver Cancer in Children and Adolescents 15

Figure 2.  Fibrolamellar carcinoma. A. Tumor tissue showing extensive intratumoral lamellar
bands of collagen (blue) separating large polygonal cells (Masson’s trichromic stain, scale bar:
50 µm). B. Tumor tissue showing large polygonal cells with vesiculated nuclei, large nucleoli
and intracytoplasmic pale bodies (Hematoxylin and Eosin staining, scale bar: 100 µm).
16 Sergi C M

A controversial issue is the “mixed” FLC and HCC, but molecular pathology
may resolve this dilemma in the future. Additional features that can be encoun-
tered are trabecular, adenoid or pelioid patterns that need to be taken into
­consideration when evaluating needle or open biopsy. Metastases to the regional
lymph nodes, peritoneum, and lungs are commonly seen. Immunohistochemistry
is an important ancillary tool. FLC shows HepPar-1, keratin 7, epithelial mem-
brane antigen (EMA), CD68 and carcinoembryonic antigen (polyclonal anti-
body, p-CEA).
Immunohistochemically, there is usually a lack of expression of AFP, synapto-
physin, and chromogranin. The p-CEA is shown in a canalicular pattern due to
cross-reactivity to biliary glycoprotein I existing in bile canaliculi of healthy liver
and HCC. Of note, the monoclonal CEA is usually negative. These findings have
suggested to some authors that FLC may be a hybrid neoplasm of hepatobiliary
type. FLC may also be positive for antibodies against CAM5.2 (a low molecular
weight cytokeratin panel against keratin 7 and keratin 8 present on secretory epi-
thelia but not on stratified squamous epithelia), AE1–3 (a pan-cytokeratin mark-
ers grouping low and high molecular weight antibodies), and neurotensin. AAT is
usually inconsistently expressed. CK19 is usually negative (105). There may be an
expression of epithelial growth factor receptor (EGFR), and human epidermal
growth factor receptor (Her-2) (106). Electron microscopy reveals copious mito-
chondria (107). Pale bodies contain fibrinogen. There is an abundance of rough
endoplasmic reticulum (ER) in the neoplastic cells, which may also show dense
core neuroendocrine-like granules. However, the neoplastic cells are not positive
for neuroendocrine markers by immunohistochemistry (vide supra) or immuno-
gold-marked electron microscopy. Differential diagnoses include sclerosis-
accompanied adenosquamous carcinoma, cholangiocellular carcinoma, which is
markedly glandular and mucin-positive (108), and FNH. FNH is smaller in size
than FLC. FNH contains bile ductular proliferating associated stroma (fibrous)
and inflammatory cells. Microscopically, FNH has neither gross bile staining nor
hepatocyte atypia. Also, FLC must be differentiated from the sclerosing variant of
HCC, which shows no oncocytic change of the neoplastic cells. This tumor is
smaller in size than the tumor cells of FLC and shows a typical pseudoglandular
pattern. Other differential diagnoses include metastatic carcinoma with sclerotic
stroma (a full history is invaluable), neuroendocrine tumors (neuroendocrine
markers proof by immunohistochemistry is useful), and paraganglioma, which
harbors a Zellballen growth pattern, round/ovoid nuclei without atypia, vascular
stroma but no dense fibrosis and is positive for neuroendocrine markers (109).
A clear cell variant of FLC has also been described. (110). In addition to classic
FLC, the neoplastic clear cells with empty cytoplasm have ballooning and rarefac-
tive changes to their mitochondria (100).
A fusion gene between DNAJB1 and PRKACA characteristically distinguishes
FLC from conventional HCC (103). This genetic abnormality is found with loss
of genetic material on chromosome 19. It leads to the unusual combination of
two genes, DNAJB1 and PRKACA. DNAJB1 is a DnaJ (Hsp40) homolog, subfam-
ily B, member 1 (DNAJB1) or a transcriptional target of forkhead box protein E3
(FOXE3) in a pathway apparently crucial for the development of the eye.
PRKACA encodes one of the catalytic subunits of protein kinase A. The ­“chimeric”
gene, which in turn produces an abnormal protein product, is an unusual com-
bination of the two normal proteins. Common pathways that are altered in HCC,
 Liver Cancer in Children and Adolescents 17

such as β-catenin and p53, are not differentially controlled in FLC. Occasionally,
there is also an upregulation of other pathways including EGFR, MAPK, PI3K,
and RAS (111). In 2015, Riehle et al. highlighted some of these pathways, show-
ing that mTORC1 is activated in FLC and has been found in association with
fibroblast growth factor receptor 1 (FGFR1) overexpression. This data supports
the use of FGFR1 inhibitors in patients with FLC (112). Recently, data from the
Fibrolamellar Carcinoma Consortium has identified and updated overall survival
of FLC (104). About 3/4 of patients with FLC undergo surgical resection and/or
OLT and 1/3 receive perioperative therapy. Preoperative chemotherapy, includ-
ing external beam radiation, and TACE with doxorubicin are the milestones of
the perioperative therapy. Chemotherapy agents used in FLC include cisplatin,
doxorubicin, fluoropyrimidines, gemcitabine, irinotecan, and oxaliplatin.
Postoperative adjuvant chemotherapy and/or radiation should also be consid-
ered as relevant options (104).

PEDIATRIC METABOLIC CONDITIONS PREDISPOSING TO

HEPATOCELLULAR CARCINOMA

This section lists metabolic conditions that may predispose to the development of
HCC. Liver cirrhosis has been considered a precursor of HCC irrespective of etiol-
ogy because dysplasia can occur in the setting of a regenerating nodule, but this
argument is mainly relevant for adult HCC. In childhood, pediatric metabolic
conditions play a major role. About one-third of pediatric cases of HCC are associ-
ated with cirrhosis, while liver cirrhosis is present in two-thirds to 90% of adult-
onset HCC. The list of pediatric metabolic conditions may be not complete but
illustrates most of these conditions. This list includes alpha-1-antitrypsin (AAT)
deficiency, hereditary tyrosinemia 1, glycogen storage diseases types I, III, and IV,
genetic hemochromatosis, Wilson’s disease, acute intermittent porphyria, progres-
sive familial intrahepatic cholestasis type 2, mitochondrial electron transport
chain (ETC) disorders, and transaldolase deficiency. However, in some of the
above conditions, HCC does not occur at the pediatric age. The most prominent
disease in this category of predisposing conditions to HCC is AATD.

Alpha-1-antitrypsin deficiency
AATD (Laurell-Eriksson-Syndrome) is the result of a gene mutation, commonly
found in Caucasians. AAT expression is regulated by the SERPINA1 (SERine
Proteinase INhibitor) gene which expresses codominant alleles. The SERPINA1
gene is situated on the protease inhibitor (Pi) locus. It is on the long arm of chro-
mosome 14 (14q31–32). The three most common alleles are the normal M allele,
the S allele and the Z allele. In the setting of the S allele, AAT plasma levels are
about 60% of normal, while in the setting of the Z allele AAT plasma levels are
about 10–15% (113, 114). The nomenclature of SERPINA1 alleles is based on
isoelectric focusing-determined migration of the protein variants that were identi-
fied long before the gene was known. The coding of the alleles was based on
migration in gel electrophoresis using the prefix PI (protease inhibitor) serving as
18 Sergi C M

an alias for the gene (115). The normal AAT protein (the M protein) migrates to
the middle of the isoelectric field, while the abnormal AATD protein (the Z vari-
ant) migrates slowly. Null alleles are pathogenic alleles that result in either no
mRNA product or no protein product. Homozygosity for the Z mutant allele is
accompanied by abnormal retention of altered AAT molecules in the liver, which
may lead to neonatal hepatitis, liver disease in children, and liver disease with the
development of HCC in young adults (116). AAT is an acute-phase protein with
strong inhibitory activity towards proteolytic enzymes, mainly elastase but also
trypsin, chymotrypsin, and thrombin. Lack of AAT is also associated with higher
risk of chronic obstructive pulmonary disease (COPD), which is characterized
clinically by lung emphysema, chronic bronchitis and/or persistent airflow
obstruction in young and middle-aged adults, especially smokers. Adult-onset
AATD-determined liver disease manifests with fibrosis and cirrhosis. It may occur
in the absence of a history of childhood (or neonatal) liver disease. The risk for
HCC is increased in individuals with AATD. The hepatocytes are the main source
of AAT.
The SERPINA1 gene is closely controlled by different cytokines, including
interleukin-1 (IL-1), tumor necrosis factor α (TNFα), and the interleukin-6 family
of cytokines (interleukin-6, leukemia inhibitory factor, oncostatin M). Genetic
mutations of SERPINA1 cause disturbance in protein structure, resulting in
polymerization and intracellular accumulation of the protein. The point muta-
tions accountable for the molecular instability of the protein occur in the axes and
sliding regions of the protein. They are located in the areas involved in the move-
ment of the reactive loop (117). These mutations allow for the spontaneous open-
ing of the main β-pleated sheet of the molecule. It leads to a swift insertion into
the sheet of the reactive loop of the subsequent AAT molecule. As a result, loop-
sheet polymers are formed. Individuals who are either homozygous for most
common mutated variant (– Z), or two other rare variants (Siiyama and M-Malton/
M-Cagliari), show protein aggregation (113, 114, 118). The histological hallmark
of liver disease in AATD is the identification of AAT-containing globules which are
positive to diastase-resistant PAS-D staining in periportal hepatocytes. The link
between AAT and liver malignancy has been explored from several aspects, for
example, AAT polymorphisms, elevated serum levels and changes in the content
of oligosaccharides of AAT. The impossibility to extrude AAT from the ER leads to
the accumulation of polymers in the ER of hepatocytes, leading to damage of the
hepatocytes by the gain-of-function mechanism. This mechanism, by which accu-
mulation of protein damages the cell, is crucial. There is evidence from investiga-
tions using mice transgenic for the Z mutant of the human AAT (AAT-Z) gene in
which hepatitis, and then HCC, developed (119). Marcus et al. recognized an
AAT-Z altered regulation of several genes, including cyclin D1 and MCAM, both
of which led to cell proliferation and tumorigenesis (120). Genetically engineered
fibroblast cell lines arising from the skin of individuals with PiZZ phenotype with
or without liver disease indicate that there is a hold-up in ER degradation of
A1AT-Z (121). The key to liver carcinogenesis seems to reside in inefficient degra-
dation of A1AT-Z in the ER. Variation in the function of mechanisms of intracel-
lular protein degradation and/or variation in the pathways of signal transduction
activated to protect the cell from protein mis-localization and/or aggregation seem
to result in abnormal cell metabolism of hepatocytes, predisposing to liver carci-
nogenesis (122). It is known that terminally misfolded proteins are transported in
 Liver Cancer in Children and Adolescents 19

a selective manner from the ER into the cytosol. These molecules are subsequently
ubiquitinated and degraded by the proteasome in a process called ER-associated
degradation (ERAD). Lack of efficient protein degradation or accumulation of
misfolded proteins in the ER overwhelms the degradation machinery of the cell,
and several ER response pathways are activated. These pathways include the pro-
teasomal degradation pathway and autophagy (118, 123, 124). While the protea-
some is accountable for degrading soluble forms of the Z variant protein (125),
autophagy is specific for the removal of insoluble polymers and aggregates.
Teckmann et al. found that retention of AAT-Z in the ER is associated with a
marked autophagic response (126, 127). The diagnosis of AATD relies on the
serum concentration of AAT by radial immunodiffusion or nephelometry with
molecular genetic confirmation of the variants by screening for the SERPINA1
gene. In conjunction with the periodic pulmonary function tests, children and
young adults with AATD and established liver disease should undergo periodic
(annually or half-annually) ultrasound examination of the liver to monitor for
fibrotic changes and HCC occurrence.

Hereditary tyrosinemia I
Hereditary tyrosinemia I (HT1, OMIM 276700) is the result of an abnormality of
the tyrosine catabolic pathway due to deficiency of the enzyme fumarylacetoace-
tate hydrolase. It is an autosomal recessive disorder. In the liver, there is an accu-
mulation of toxic metabolites (maleylacetoacetate, fumarylacetoacetate, and
succinylacetone) which lead to hepatic and renal manifestations of the disease and
carcinogenesis. HCC occurs commonly in HT1, with a prevalence of 14–75%
(128, 129). Clinically, the spectrum is widely heterogeneous and includes acute or
chronic liver disease, glomerulosclerosis, hypophosphatemic rickets due to renal
tubular dysfunction, neurological porphyria-like crisis, hypertrophic cardiomy-
opathy, hypoglycemia due to hyperinsulinism, and failure to thrive. Currently,
treatment of HT1 is inhibition of the formation of toxic metabolites by nitisinone
[2-(2-nitro-4-trifluoromethyl-benzoyl)-1,3-cyclohexanedione; NTBC] and the
reduction of tyrosine levels by dietary treatment. Heat shock proteins and anti-
apoptotic proteins have been found to be altered in HT1. Presumably, there is a
mechanism whereby transformed cells have a survival advantage in murine mod-
els of HT1 (130, 131). There is a small risk of liver cancer in NTBC treated HT1
patients because this compound blocks the enzyme parahydroxyphenyl-pyruvic
acid dioxygenase, which is involved in the tyrosine degradation pathway, thus
preventing the accumulation of fumarylacetoacetate and succinylacetone. On the
other hand, animal models have revealed that NTBC treatment is unsuccessful in
normalizing tyrosinemia-induced changes in the expression of transcripts encod-
ing proteins involved in signal transduction, protein turnover, cell growth, and
cell proliferation (132). In one study on pediatric patients who underwent OLT,
HCC was found in 12 (75%) liver explants, but in another Iranian study, HCC
was present in just 5 (23%) of the 22 liver explants (133). Screening is recom-
mended in HT1 patients, using regular measures of AFP and imaging. Surveillance
typically includes ultrasound, but in the event a suspicious lesion is found, MRI
could be used for characterization and staging of lesions, as it is more sensitive
compared to ultrasound.
20 Sergi C M

Progressive familial intrahepatic cholestasis type 2

Progressive familial intrahepatic cholestasis type 2 (PFIC 2) is associated with a
mutation in the ABCB11 gene, resulting in a deficiency of bile salt export pump
(BSEP), a membrane acid transporter of bile canaliculi. PFIC2 represents a spe-
cific, previously unrecognized risk for HCC in infants. In BSEP deficiency, there is
poor excretion of bile salts through the canalicular membrane, resulting in con-
stant exposure of hepatocytes to bile salts, leading to chronic inflammation and
carcinogenesis (90, 91, 108, 134–142). HCC occurs in 5–15% of pediatric
patients with BSEP deficiency, mostly in the second year of life (143–146). It has
also been demonstrated that children harboring a D482G mutation have less
severe disease and portal hypertension, while HCC occurs most often in children
with non-D482G mutations (145). In a European study investigating a cohort of
128 children, single-strand conformation polymorphism (SSCP) analysis and
Sanger sequencing of ABCB11 gene identified high risk of HCC (38% vs. 10%) in
children with the presence of two protein-truncating mutations (146). Interestingly,
studies of humans affected by BSEP and Mdr2 knock-out mice revealed that very
few somatic mutations accumulate over time in cancer genes, contrary to what
occurs in adults with HCC as well as other hepatocellular neoplasms where sev-
eral mutations progressively accumulate over a period of time. BSEP individuals
and animal models show massive gene amplification that affects components of
signal transduction pathways. These include the ErbB, activators of c-Jun-N
terminal kinases ( JNK), the PI3K/Akt and the mitogen-activated protein kinase
(MAPK) signaling pathways, among others (147, 148). Another study showed
that BSEP expression is significantly decreased in HCC patients and is associated
with alteration of the nuclear regulatory receptor farsenoid-X receptor (FXR),
with an increase in FXR-α1/FXR-α2 ratio (149). HCC has also been reported in a
new variant of PFIC with marked cholestasis, the so-called TJP2 (tight junction
protein 2) deficiency. Protein-truncating mutations in the TJP2 gene result in fail-
ure of protein localization and related disruption of tight junction structure lead-
ing to severe cholestatic disease of the liver. Claudin (CLDN1) is unable to localize
normally to cholangiocyte borders and biliary canaliculus margins, despite nor-
mal protein levels. Exposure of canalicular and cholangiocyte membranes to high
concentrations of detergent bile acids due to TJP2 deficiency leads to a disintegra-
tion of the hepatobiliary structure, with progressive liver injury and malignant
transformation (150, 151). Lastly, HCC has been sporadically reported in multi-
drug resistance protein-3 (MDR3) deficiency (PFIC-3), but less frequently than
BSEP deficiency (152).

Glycogen storage disorders

Glycogen storage disorders (GSD) may also be associated with HCC. This neo-
plasm may develop from adenomas, suggesting that there is an adenoma-
carcinoma sequence in GSD. GSD Ia, or glucose-6-phosphatase deficiency, may
cause HCA in 16–75% of patients with GSD Ia also harboring HCA. GSD
Ia-associated HCA occurs in the second and third decades of life and differs from
sporadic HCA in that they are greater in number, involve both the right and left
liver, and show no gender predisposition. Good metabolic control influences
regression in size and number of HCA in GSD Ia patients. Malignant conversion
 Liver Cancer in Children and Adolescents 21

of HCA to HCC may occur on rare occasions (153–161). In GSD-Ia HCAs, chro-
mosomal peculiarities are present in 60% of tumors, which is similar to the rate
observed in HCA seen in the general population. However, it seems that a simul-
taneous gain of chromosome 6p and loss of 6q are present in GSD-Ia HCA only.
Moreover, GSD-Ia HCAs show reduced expression of insulin-like growth factor-2
receptor (IGF2R) and large tumor suppressor kinase-1 (LATS1), which are candi-
date tumor suppressor genes at 6q in more than 50% of the tumors. This data
suggests a potential but still inexplicable role of 6q in oncogenic transformation of
such GSD-Ia HCAs (162). Type III GSD (debrancher enzyme deficiency, or Forbes
disease, or limit dextrinosis) also presents with HCA, but liver enzymes are
increased due to hepatocellular injury, and there is evidence of ballooning cell
change and fibrosis. GSD-III HCAs occur in 4–25% of children. Malignant onco-
genic transformation of HCA occurs almost always after liver cirrhosis (156, 163).
Rarely, HCC can occur in GSD-IV (brancher enzyme deficiency also known as
Andersen’s disease or amylopectinosis) (164).

Wilson’s disease
Wilson’s disease (WD) is characterized by copper accumulation and seems to be
protective against tumorigenesis, but HCC has been described (165). A Dutch
study targeting 140 adults with WD for over 15 years (166) reported an estimated
annual risk of HCC of 9 in 10,000 (0.09%). However, another multicenter
European study of 1,186 WD patients showed a prevalence rate of 0.67% (167).
Other predisposing conditions to HCC include genetic hemochromatosis, acute
intermittent porphyria, progressive familial intrahepatic cholestasis type 2, mito-
chondrial electron transport chain (ETC) disorders, glycogen storage diseases
types I, III, and IV, and transaldolase deficiency (168). Transaldolase deficiency is
a genetic disease caused by mutations in the transaldolase gene (TALDO1), which
leads to abnormally low levels of the transaldolase enzyme. Transaldolase is a
metabolic enzyme involved in the pentose phosphate pathway.

CHOLANGIOCELLULAR CARCINOMA

Cholangiocellular carcinoma (CCA) is very rare in childhood and youth, account-

ing for less than 1% of all malignancies in these age groups. CCA arises from the
epithelial lining of the bile ducts and harbors a poor prognosis. In a review of the
scientific literature and the “Surveillance, Epidemiology, and End Results” (SEER)
Program Database, Newsome et al. found 22 individual cases of pediatric CCA
published between 1946 and January 2016 (169). Details of these cases can be
found in Tables 2 and 3 of the SEER analyses (170–189). Briefly, the patients had
a median age of diagnosis of 15 years (range 3–18 years), of which 20 (91%) had
one or more gastrointestinal comorbidities with congenital distortions of the biliary
tract and primary sclerosing cholangitis (PSC) being the most prevalent. PSC is a
long-term progressive hepatobiliary disease with involvement of the liver, gallblad-
der, and extrahepatic biliary tract characterized by inflammation and scarring of
the bile ducts with potential evolution to liver cirrhosis and liver failure. Abdominal
pain, jaundice, and pruritus are the most common clinical signs. In eight patients,
22 Sergi C M

cancer antigen (CA) 19–9 was reported, and it was increased (>129 U/mL) in
four of them (50%). The SEER database program analysis and the scientific lit-
erature evidence showed that after a median follow-up time of 8 months (range
0–60 months), 12 (54.6%) patients had died, 9 (40.9%) patients were alive, and
1 (4.6%) had no reported survival data. Most pediatric cases had underlying risk
factors for CCA, such as PSC, while in adults only an estimated 30% of CCAs
occurred in patients with an underlying risk factor (108, 190–194). In adults,
underlying risk factors include bile duct cysts, primary sclerosing cholangitis
(specifically with inflammatory bowel disease), Caroli Disease, and hepatolithia-
sis. Choledocholithiasis, cholangitis, hepatitis C (HCV), hepatitis B (HBV) and
cirrhosis have been considered potential risk factors as well (108, 169). There is
a potential association of these tumors with viral infections (hepatitis B virus,
hepatitis C virus) and alcohol use, which has been seen increasingly when com-
pared with classic textbook associations of liver flukes (Opisthorchis viverrinii and
Clonorchis sinensis) and primary sclerosing cholangitis. Other chronic inflamma-
tory processes of the biliary tract have also been associated with CCA. In addi-
tion, congenital disorders such as BA and PIFC have been identified as
comorbidities. Congenital malformations of the biliary tree, such as choledochal
cysts and Caroli disease, may present with obstructive cholestasis; however,
patients may be asymptomatic and diagnosed later in life.

Congenital biliary dilatation

Congenital biliary dilatation (CBD) has been described as an important cause
for the development of CCA in younger patients, mostly teenagers, but occa-
sionally in younger children as early 3 years of age (178). The etiology of CCA
from CBD is thought to be the reflux of pancreatic juice into the biliary system
duct via a pancreatico-biliary mal-junction (PBMJ). Oncologic transformation
can occur in the biliary tree anywhere where the biliary epithelial cells can be
influenced by both bile stasis and pancreatic secretion. The first reported malig-
nancy associated with CBD was reported in 1994 (195, 196). The risk of malig-
nant transformation of the biliary epithelium is between 2.5% and 28% (193,
194, 197–205). Malignant transformation is accompanied by genetic mutations
(206). A preoperative diagnosis of CCA associated with CBD remains difficult
to establish and involves identifying either elevated or polypoid lesions in the
bile duct. CCA is therefore frequently diagnosed during laparotomy. Once a
diagnosis of CBD is made, radical surgery in early-stage disease combined with
thorough, intraoperative frozen tissue examination are needed. Adjuvant che-
motherapy for tumor-positive surgical margins has been advocated, but remains
controversial.

Ductal plate malformation

Other conditions associated with CCA are ductal plate malformation (DPM)-
related lesions. An abnormal arrest of the remodeling of the ductal plate during
biliary system development is called DPM of the liver (207–216). Polycystic liver
disease (PLD) is the representative entity resulting from a DPM, which can be
both morphologic mimickers and precursor lesions of neoplastic lesions (217).
 Liver Cancer in Children and Adolescents 23

DPM may have several overlapping entities, which are mostly grouped into:
(i)  Von Meyenburg complexes (VMCs), small nonhereditary nodular cystic
lesions; (ii) isolated polycystic liver disease (PCLD); and (iii) autosomal dominant
polycystic kidney disease (ADPKD), which shows multiple cysts in both kidney
and liver (218, 219). An arrest in the development of the intrahepatic biliary sys-
tem and cystic changes of the bile ducts result from disrupted TGF-β, Notch, and
Wnt signaling or ciliopathy (220). Both PCLD and ADPKD show multiple large
cysts replacing normal hepatic parenchyma. Hepatic failure, and complications
associated with cysts, such as infection, hemorrhage or rupture, are frequent, but
PCLD and ADPKD are not risk factors for CCA in general. VMC is a sporadic,
small (diameter less than 0.5 cm), usually singular DPM lesion, also called biliary
micro-hamartoma. Although it is often an incidental finding in the adult liver, it
is rarely encountered in childhood. Multiple VMCs may occur with multiple
cysts, which are grossly visible and diffusely located along the biliary tree.
Histologically, VMCs consist of irregularly dilated small bile ducts embedded in
the fibrous stroma with intraluminal inspissated bile secretion. VMCs are consid-
ered a part of the spectrum of adult PCLD, but there is no known genetic back-
ground such as ADPKD or PCLD (221). There is a large body of knowledge that
VMC has more than a potential role as a preneoplastic lesion for CCA. This
knowledge is based on several studies that have reported CCA arising from VMCs,
suggesting malignant transformation from VMC to CCA, CCA having histologic
similarities with VMC and DPM, and histologic similarity of CCA and VMC in
K-ras and p53 mutated animal models inducing CCA (222–232). Most recently,
the CCA with DPM pattern seems to have well-defined ARID1A genetic altera-
tions (233).

Pathology and differential diagnosis

It remains a true challenge for pathologists to distinguish between diffuse-type
VMC and CCA. Anatomical locations are used to classify CCA. In the diagnostic
routine, we distinguish intrahepatic from extrahepatic CCA. Intrahepatic CCAs
arise from the biliary system in the liver (bile ducts and segmental bile ducts),
while extrahepatic CCAs arise from perihilar bile ducts (including the right, left
and common hepatic ducts) or the extrahepatic bile duct (the common bile duct).
Grossly, the tumor may present in the form of a mass or having a periductal infil-
trating pattern. Sometimes, the mass-forming and periductal infiltrating patterns
are mixed. The mass-forming type of CCA represents the vast majority. Histologically,
CCA appears as a moderately differentiated gland, or tubule-shaped atypical cell
proliferation on low power and is frequently accompanied by prominent desmo-
plasia (fibrotic stroma). The stroma is particularly evident at the center of the mass.
Unusual growth patterns may also occur, and include trabecular, nested, adeno-
squamous, squamous, mucinous and signet cell patterns, among others (234). In
the trabecular growth pattern, polygonal cells with eosinophilic cytoplasm arranged
in thick trabeculae are seen. Immunohistochemical investigation is crucial in most
cases to confirm a diagnosis of CCA. CCA are typically positive for keratin 7 (CK7)
and keratin 19 (CK19), biliary type of intermediate filaments of the cytoskeleton,
carcinoembryonic antigen (CEA), and EMA. Epithelial cells forming bile ducts
express CK-7 and CK-19 in addition to CK-8 and CK-18. The latter are also
24 Sergi C M

present in normal adult hepatocytes. Patients are almost always negative for keratin
20 (CK20) of the intermediate filaments of the cytoskeleton. In our past investiga-
tions on liver development (209) and ductal plate malformation of the liver (208,
210), we used three monoclonal antibodies against CK-7 (OV-TL 12/ 30, 1:50,
Dako Corporation, Hamburg, Germany), CK-19 (RCK 108, 1:100, Dako
Corporation, Hamburg, Germany), and a mixture of cytokeratin epitopes (AE 1/3,
1:2, Linaris, Camon, Wiesbaden, Germany). AE 1 reacts with specific group A
acidic keratins with a molecular weight of 40,000–50,000 (CK 10, 14, 15, 16, 19),
while AE 3 recognizes all eight group B basic keratins with a molecular weight of
58,000 – 67,000 (CK 1–8). A mixture of monoclonal antibodies AE 1 and AE 3
should theoretically stain both biliary epithelial cells and hepatocytes because AE 1
reacts with CK 19, and AE 3 reacts with both CK 7 and CK 8. However, we found
that in formalin-fixed tissue, the AE 113 cocktail reacts only with biliary cells. This
is probably due to formaldehyde fixation of the tissue, as previously shown
(235–237). The epitope recognized by AE 3 is altered by formalin fixation, making
it less available for binding. Furthermore, it differs from other cytokeratin-related
epitopes because it can only partially be ‘restored’ by antigen-unmasking tech-
niques. In CCA, there is no immunohistochemical detection for CDX2 or Caudal
Type Homeobox 2, whose gene is a component of the caudal-related homeobox
transcription factor family. CDX2 is expressed in the nuclei of intestinal epithelial
cells, and diseases associated with CDX2 include Ampulla of Vater adenocarci-
noma and mucinous adenocarcinoma of the ovary. Among CDX2 related pathways
are transcriptional regulatory networks in embryonic stem cell and incretin synthe-
sis, secretion, and inactivation. Ultrastructurally, CCC tumor cells show cytokera-
tin filaments, an intracytoplasmic lumen, tight junctions at the apices, and
desmosomes at the lateral surfaces of neighboring cells. All these features are char-
acteristics of adenocarcinoma cell origin.
The 8th edition of the Staging Manual of the American Joint Committee on
Cancer (AJCC) has different staging systems for intrahepatic, perihilar, distal
bile duct, and HCC (238). AJCC staging is based on vascular invasion, number
of tumor nodules, perforation of the visceral peritoneum, the involvement of
local extrahepatic tissues and the occurrence of periductal infiltration. In the
differential diagnoses in childhood and youth, it is rare to see metastatic colorec-
tal carcinoma, although I came across one such case in my practice; the tumor
suppressor gene, TP53, was deleted. The major differential diagnosis is with
HCC; particularly with unusual growth patterns such as trabecular and nested
type, careful consideration is necessary. The lack of nucleoli, the lack of bile
production, and the presence of prominent (central) fibrosis is suggestive of
CCA. A nested growth pattern would also suggest a neuroendocrine carcinoma,
but in this case, I would expect to see granular or “salt-and-pepper” (“stippled”)
chromatin, although nucleoli can also be seen in NET of high grade. In this set-
ting, the pathologist should consider abundant apoptosis, mitotic figures and
tumor necrosis, which may be particularly prominent in neuroendocrine carci-
nomas. The distinction of CCA from metastatic gall bladder, pancreatic or upper
gastrointestinal ­adenocarcinomas requires clinical correlation, although such
tumors are p ­ ractically non-existent in childhood and youth. Neuroendocrine
carcinomas and neuroendocrine tumors are often positive for neuroendocrine
 Liver Cancer in Children and Adolescents 25

markers, including chromogranin A and synaptophysin (199, 239). A note

should be given to the precursor lesions for CCA, including biliary intraepithe-
lial neoplasia (BilIN I-III) and intraductal papillary mucinous neoplasm of the
biliary tract. These two pre-invasive lesions of carcinogenesis of the biliary tract
are similar to two precursor lesions of the pancreas: pancreatic intraepithelial
neoplasia (PanIN) and intraductal papillary mucinous neoplasm (IPMN) of the
pancreas (240).

CONCLUSION

In regard to hepatocellular lesions, although diagnosis is typically made using

diagnostic imaging, anatomical pathology and histology with ancillary studies
still play an enormous role in the 21st century. Currently, OLT for unresectable
HCC can be curative. Risk factors for recurrence should be considered for HCC
only, taking into account factors found in metabolic conditions predisposing to
the development of primary neoplasms of the liver. Children and adolescents
with underlying metabolic conditions should have a regular follow-up in
­adulthood. CCA is very rare in pediatrics but is not impossible and must be con-
sidered by pediatricians and pediatric pathologists, as well as the risk factors for
this tumor.

Acknowledgement: Dr. Sergi’s research has been funded by the generosity of

the Stollery Children’s Hospital Foundation and supporters of the Lois Hole
Hospital for Women through the Women and Children’s Health Research
Institute (WCHRI, Grant ID #: 2096), Hubei Province Natural Science Funding
for Hubei University of Technology (100-Talent Grant for Recruitment Program
of Foreign Experts Total Funding: Digital PCR and NGS-based diagnosis for
infection and oncology, 2017–2022), Österreichische Krebshilfe Tyrol
(Krebsgesellschaft Tirol, Austrian Tyrolean Cancer Research Institute, 2008),
Austrian Research Fund (Fonds zur Förderung der wissenschaftlichen Forschung,
FWF, Grant ID L313-B13), Canadian Foundation for Women’s Health, Cancer
Research Society (von Willebrand factor gene expression in cancer cells),
Canadian Institutes of Health research (Omega-3 Fatty Acids for Treatment of
Intestinal Failure Associated Liver Disease: A Translational Research Study), and
the Saudi Cultural Bureau, Ottawa, Canada. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the
manuscript.

Conflict of Interest: The author declares no potential conflicts of interest with

respect to research, authorship and/or publication of this article.

Copyright and permission statement: The author confirms that the ­materials
included in this chapter do not violate copyright laws. Where relevant, a­ ppropriate
permissions have been obtained from the original copyright holder(s). All original
sources have been appropriately acknowledged and/or referenced.
26 Sergi C M

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 2
Hepatocellular Carcinoma in Adults
Aducio L. Thiesen
Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, AB,
Canada
Author for correspondence: Aducio L. Thiesen, Department of Laboratry Medicine and
Pathology, University of Alberta Hospital, Edmonton, Alberta, Canada.
Email: [email protected]
Doi: https://doi.org/10.36255/exonpublications.livercancer.2021.ch2

Abstract: Hepatocellular carcinomas are the most common primary neoplasia

of the liver. The global distribution of hepatocellular carcinoma is related to the
prevalence of hepatitis C in the population. Other major etiologic factors include
alcoholic liver disease and nonalcoholic steatohepatitis. The majority of cases
are discovered when screening patients with either chronic hepatitis or cirrho-
sis, but occasional incidental cases have been reported. Molecular markers and
associated gene alterations are a work in progress. Serological markers and radi-
ology are used to detect the disease in high-risk populations, and to monitor
response to therapy in the affected patients. Even though radiologic features are
specific, tissue diagnosis may be required, particularly for atypical and smaller
lesions. Ancillary studies including reticulin stain and immunohistochemistry
are important for confirmatory diagnosis. Liver transplantation is curative
for hepatocellular carcinoma, but due to limitations in organ availability,
palliative care is required, which mostly includes chemoembolization and
radio-ablation.

Keywords: alpha fetoprotein; BCLC staging; CLIP staging; hepatocellular carci-

noma; Okuda staging

In: Liver Cancer. Sergi CM. (Editor). Exon Publications, Brisbane, Australia.
ISBN: 978-0-6450017-2-3; Doi: https://doi.org/10.36255/exonpublications.livercancer.2021
Copyright: The Authors.
License: This open access article is licenced under Creative Commons Attribution-NonCommercial
4.0 International (CC BY-NC 4.0) https://creativecommons.org/licenses/by-nc/4.0/

39
40 Thiesen A L

INTRODUCTION

Malignancies that arise from hepatocytes are denominated hepatocellular carci-

nomas. Hepatoma is another term; however, this old terminology implies a
benign process and therefore, to avoid confusion, this term should not be used.
Hepatocellular carcinomas are malignant tumors and have the capacity to
metastasize and cause death. The most common sites of metastases are lungs,
abdominal lymph nodes and bones (1). Since hepatocytes are the most a­ bundant
cells within the liver, hepatocellular carcinoma is the most common primary
liver disease in adults (85% of the malignancies of the liver). It is the 6th most
common malignancy worldwide with 841,080 cases in 2018 and predicted
to  be 1,361,836 in 2040 with significant mortality and morbidity (2). The
­incidence of hepatocellular carcinoma is directly related to geography and
­prevalence of infectious diseases. The highest rates of disease are found in Asia
and southern Africa (up to 150 per 100,000), mostly due to the high preva-
lence of infectious hepatitis. In the USA, the prevalence is much less but still
significant (6 per 100,000), typically affecting Asian men (3). The epidemiol-
ogy is similar in Canada with a reported prevalence of 6.8 per 100,000; this
significant rise (3x) since the 1980s is likely related to the expansion of intra-
venous drug abuse and consequent increase in cases of hepatitis C (4). In
Europe, the rate ranges from 3 to 6 per 100,000 individuals depending on the
availability of treatment and prevention of infectious hepatitis, and abuse of
alcohol (5). Hepatocellular carcinoma is the second leading cause of cancer
mortality worldwide. Screening programs and curative therapy for infectious
hepatitis have contributed to a decrease in mortality but alcohol abuse and
metabolic diseases including diabetes and obesity have surpassed the initial
benefit (6, 7).

ETIOLOGY AND PATHOGENESIS

The hepatocytes are active metabolic cells involved in many cell functions and are
subject to a large number of insults that may result in abnormal proliferation (8).
Exhaustion of their regenerative capacity is typically exemplified by cirrhosis.
Thus, the etiologic factors involved in the cirrhotic process are also involved in
tumorigenesis of hepatocellular carcinoma. Chronic hepatitis B and C are by far
the most common causes of hepatocellular carcinoma, followed by alcoholic liver
disease and nonalcoholic steatohepatitis. (9–11). Hepatitis B DNA levels in excess
of 200,000 IU/mL (1,000,000 copies/mL) have been reported to increase the inci-
dence of hepatocellular carcinoma to 1,152 per 100,000 individuals (12, 13).
Other etiologic factors in adults include aflatoxin-contaminated food, diabetes,
obesity, and hemochromatosis (14, 15). In some instances, hepatocellular carci-
noma is detected incidentally during routine image examination. These tumors
are considered sporadic and are result of gene mutations that occur during a per-
son’s lifetime.
Genetic susceptibility to hepatocellular carcinoma has been demonstrated
in  animal models, but not established in humans. Family clustering has been
 Hepatocellular Carcinoma in Adults 41

reported in some Chinese families and Alaskan natives, but infectious hepatitis B
is a requirement for HCC development in this particular population (16, 17).
Other etiologic factors include conditions associated with congenital, genetic,
and metabolic conditions and are directly related to the capacity of these patholo-
gies to develop cirrhosis. Such examples include hemochromatosis, Wilson’s dis-
ease, alpha-1-antitrypsin deficiency and Budd-Chiari syndrome.
Alcoholic liver disease is a risk factor that illustrates regeneration as the key
factor in tumorigenesis. Active intake of alcohol causes hepatocyte injury with
minimal regeneration, which clinically is a low risk factor for hepatocellular car-
cinoma. In contrast, alcohol abstinence results in significant regeneration and a
higher risk of hepatocellular carcinoma (18).
Once the hepatitis B virus is integrated into the hepatocytes, transactivation of
proto-oncogenes, activation of growth factors, and inactivation of tumor suppres-
sor genes may result in abnormal proliferation (19). The hepatitis C virus damages
double-stranded DNA, increasing the frequency of mutations in genes such as
immunoglobulin genes, BCL-6, TP53, and β-catenin, causing abnormal prolifera-
tion (20). Other common mutations encountered in hepatocellular carcinoma
include: telomerase promoter mutations (30 to 60% of HCC) (21); TP53 muta-
tions (18 to 50%) (22) classically associated to exposure to aflatoxin; beta-catenin
(18 to 40%); AXIN1 and AXIN2 genes; chromatin remodeling pathway (ARID1A
and ARID2); Janus kinase (JAK)/signal transducers and activators of transcription
(STAT) pathway (JAK1, IL6R, and IL6ST); genes involved in ubiquitination
(KEAP1); genes involved in RAS/MAPK signaling (RPS6KA3); and genes involved
the oxidative stress pathway (NFE2L2) (23).

NATURAL HISTORY AND PRE-NEOPLASTIC LESIONS

At the cellular level, hepatocellular carcinomas are thought to arise from a dys-
plastic focus (less than 1 mm) that develops into either a low-grade or a high-
grade dysplastic nodule. The high-grade dysplastic nodule, more frequently than
the low-grade dysplastic nodule, progresses to hepatocellular carcinoma (24).
Since the primordial lesion (dysplastic focus) is less than 1 mm, it is not identifi-
able by radiologic studies and usually observed in hepatectomy specimens as focal
cytological atypia within the hepatocytes. Morphologically, the atypia within the
hepatocytes is described as small and large cell changes. The small cell change is
characterized by increased nuclear to cytoplasmic ratio, hyperchromasia and
cytoplasmic basophilia, giving the impression of crowding. Small change atypia is
associated with high-grade dysplastic nodules and a higher risk of developing of
hepatocellular carcinoma. The large cell change is characterized by enlarged cells
with larger nuclei and cytoplasm, but the nuclear to cytoplasmic ratio is pre-
served; frequent multinucleation, nuclear polymorphism and hyperchromasia are
common. Large cell changes are the predominant alteration in low grade dysplas-
tic nodules and indicate a benign feature (25). The biggest diagnostic challenge is
to differentiate a high-grade dysplastic nodule from a well-differentiated hepato-
cellular carcinoma. Reticulin stain with delineation of the hepatic plates is a reli-
able ancillary technique in this context. Preservation of the hepatic plate is typical
42 Thiesen A L

of a high-grade dysplastic nodule, whereas expansion of the hepatic plates is typi-

cal of hepatocellular carcinoma. Other morphologic criteria for hepatocellular
carcinoma include increased cell nuclei with consequent increased nuclear to
cytoplasmic ratio, pseudogland formation and unpaired arteries with absence of
portal triads (Figure 1), and sinusoidal capillarization appreciated with CD34
immunohistochemistry and stromal invasion without ductular reaction at the
periphery of the nodules (26). The size of the lesion may also be helpful, espe-
cially radiologically, with lesions larger than 1 cm and less than 3 cm usually
­classified as dysplastic nodules, and lesions more than 3 cm as hepatocellular
carcinoma (27).
Other preneoplastic lesions include clonal proliferation of hepatocytes forming
hepatocellular adenomas. These lesions are characterized morphologically by well
circumscribed proliferation of bland hepatocytes with intact hepatic plates and
absence of portal triads. The real differential in this situation includes mostly a well
differentiated hepatocellular carcinoma. Ancillary studies such as immunohisto-
chemistry for CD34 demonstrating complete capillarization of the sinusoids,
immunohistochemistry for Glypican-3 and reticulin stain (Figure 2) are helpful for
differential. Hepatocellular adenomas are occasionally linked to oral contraceptives
(28). Other possible etiologies include the use of clomiphene (used as hormonal
treatment for infertility issues) (29), methyltestosterone (anabolic steroid) (30)

Figure 1.  Hematoxylin and Eosin stain 200X. On the left, normal hepatocytes with normal
nuclear cytoplasmic ratio as compared to hepatocellular carcinoma (on the right) with
increased nuclear cytoplasmic ratio, organized into thick trabeculae and few
pesudoglandular structures with bile.
 Hepatocellular Carcinoma in Adults 43

Figure 2.  Reticulin stain 200X. On the left, normal hepatocytes with one to two cell thick
hepatic plates as compared to hepatocellular carcinoma (on the right) with distended
hepatic plates.

and danazol (synthetic androgen used for treatment of endometriosis) (31). At the
molecular level, hepatocellular adenomas are subdivided based on HNF1a muta-
tions (steatotic adenomas), IL-6 ⁄ STAT3 mutations (inflammatory adenomas), and
β-catenin mutations (subgroup of inflammatory adenomas and unclassified adeno-
mas with cytological atypia) (32). Prognostic implications justify such subtyping
with inflammatory adenomas being frequently associated with metabolic syn-
drome, liver steatosis and alcohol exposure (33), and the β-catenin mutated adeno-
mas immunophenotypically represented by nuclear staining associated with a
higher risk of malignancy. Extensive literature and classification of hepatocellular
adenomas is available, but the key is the identification of the lesions with great risk
of progression to hepatocellular carcinoma.

CLINICAL CHARACTERISTICS, DIAGNOSIS, AND

DIAGNOSTIC TESTS

Hepatocellular carcinoma usually affects the younger population. This is most

likely related to significantly higher viral infection rate in individuals of repro-
ductive age. Males are more affected than females, and males are also more
frequently involved with alcohol abuse (34). Most patients are asymptomatic.
44 Thiesen A L

However, symptoms related to predisposing risk factors (cirrhosis and viral infec-
tions) are frequent, such as ascites and esophageal varices. Paraneoplastic syn-
dromes may be occasionally associated with hepatocellular carcinoma, such as
hypoglycemia (35), erythrocytosis (36), hypercalcemia (37) diarrhea (38) and
cutaneous lesions (39). Other less common presentations include fever, infec-
tions, obstructive jaundice, and hemorrhage due to tumor rupture. Lymph node
involvement is not common, and 10 to 15% of the cases present with advanced
disease and metastases to lung, bone, and adrenal gland (40, 41).
Serological markers of the disease include alpha fetoprotein (AFP), heat shock
protein, human cervical cancer oncogene, human telomerase reverse transcriptase
mRNA, certain cytokines and microRNAs. AFP is by far the most used due to its
availability, but screening limitations are significant due to its low sensitivity (41
to 65%). Pregnancy and primary liver and gastrointestinal disease also raise the
levels of AFP, giving false positive results (42). The most accepted cut-off value is
a serum concentration of 20 ng/mL (43). However, the most valuable indication
of AFP is in the follow-up of previously treated hepatocellular carcinoma to detect
the risk of recurrent disease (44). Three different glycoforms of AFP have been
identified: AFP-L1, AFP-L2 and AFP-L3. The latter has been reported to be associ-
ated with more aggressive liver disease and consequently worse prognosis (45, 46).
Glypican-3, a plasma membrane protein involved in the interaction with growth
factors, has been suggested as an adjuvant serologic marker in addition to AFP to
increase the sensitivity of detecting hepatocellular carcinoma (47, 48). Other liver
enzymes have also been proposed in association with AFP to promote screening
for hepatocellular carcinoma such as gamma-glutamyl transferase, Alpha-l-
fucosidase and Des-gamma-carboxyprothrombin. Alternatively, instead of protein
levels, mRNA levels of AFP and GGT may also be helpful prognostic factor after
initial therapy for HCC (49, 50).
Additional serological markers in conjunction with AFP have been proposed
to increase the sensitivity of detection such as alpha-L-fucosidase (AFU) and
transforming growth factors alpha and beta (TGF-α and TGF-β) (51)
Radiologic diagnosis of hepatocellular carcinoma has significant implications
for treatment options, with dysplastic nodules conservatively followed up based
on the fact that one third of these lesions may progress to hepatocellular carci-
noma; and early hepatocellular carcinoma should be treated with possible curative
options such as ablation, resection and transplant (52). The image modality typi-
cally used is ultrasonography, likely due to the availability, but the sensitivity of
65% and specificity of 90% justify the use of more advanced image studies (53).
In referral centers, the image protocol includes four-phase multidetector com-
puted tomography (CT) or dynamic contrast enhanced magnetic resonance imag-
ing (MRI), complementing each other, when suspicious features are noted (54).
The distinction and detection of hepatocellular carcinoma is based on changes of
vascularization including the presence of unpaired arteries and capillarization of
the sinusoids that can be observed by image studies (55, 56). The definite radio-
logic criteria for the diagnosis of hepatocellular carcinoma are defined by contrast
hyperenhancement at the arterial phase and hypoenhancement at the venous
phase for lesions larger than 2 cm (57, 58). This criterion is highly specific and has
replaced biopsy since 2000 (59). For lesions less than 2 cm, there is intense debate
regarding the need of a biopsy, or not to define the lesion. Lesions less than 1 cm
tend to be biopsied but there is variation in practice among centers (60).
 Hepatocellular Carcinoma in Adults 45

When atypical nondiagnostic inconclusive features are noted by radiology, a

biopsy is indicated. Most are image-guided targeted biopsies of the lesions, but a
biopsy away from the lesion may be useful to assess other conditions, for example
cirrhosis, that may increase the risk of hepatocellular carcinoma. Biopsy is pre-
ferred over fine needle aspiration due to the low sensitivity (67%) of the cytology
specimen, although complications such as seeding is obviously higher in the
more invasive biopsy procedure (61). The risk of seeding is small but consider-
able, being estimated at 2.7% (62). The hallmark of a neoplastic proliferation
within a biopsy is the lack of portal triads. Features of hepatocellular differentia-
tion such as bile production and canaliculi are important when distinguishing a
metastatic lesion (63). Reticulin stain as previously commented highlights thick-
ening of the hepatic plates (more than 3 cell thick) and the cytologic features will
define the histologic grade of hepatocellular carcinoma. The best differential
between hepatocellular carcinoma and hepatic adenoma is immunohistochemi-
cal stains for glypican-3 or AFP. However, AFP stain in some laboratories has a lot
of background, making it very difficult to interpret. Glypican-3 may also be focal
within the lesion and the representative core biopsy may be completely negative.
By morphologic analysis, hepatocellular carcinoma may be classified as well-,
moderately, and poorly differentiated, based on resemblance to normal hepato-
cytes. Well-differentiated tumors have slightly enlarged nuclei and increased
nuclear to cytoplasmic ratio and may be very difficult to differentiate from normal
hepatocytes. Moderately differentiated tumors have slightly thicker hepatic plates
and larger cells. Poorly differentiated tumors are heterogeneous and have
­characteristics of an immature tumor with no typical differentiation or matura-
tion, requiring immunohistochemical markers to obtain adequate and conclusive
diagnosis (64).
Immunohistochemical markers are widely used in hepatobiliary pathology.
The most useful marker is HEP PAR-1, a marker of hepatocellular differentiation
but not useful for distinction between benign and malignant hepatocytes.
Glypican- 3 (Figure 3) and arginase-1 are specific markers for malignancy trans-
formation (65). Supportive markers such as CD34 display typical capillarization
of the sinusoids (Figure 4), and CD10 and pCEA demonstrate typical canalicular
pattern. In addition, as an initial panel to exclude other possibilities, cytokeratins
such as CK7 and CK20 are routinely used with the typical immunophenotype for
HCC consisting of negativity for both markers.
Many histologic variants of hepatocellular carcinoma have been described and
some have no clinical significance and may be confused with other entities. Such
examples include clear cell hepatocellular carcinoma mimicking a metastatic clear
cell renal cell carcinoma, pseudoglandular hepatocellular carcinoma mimicking a
metastatic adenocarcinoma and scirrhous hepatocellular carcinoma mimicking a
cholangiocarcinoma (66, 67). In other situations, recognition of a particular his-
tologic variant is significant as in the case of diffuse cirrhosis-like hepatocellular
carcinoma that radiologically mimics cirrhosis, and for that reason is usually
missed (68). The same may be said of other variants such as giant cell variant and
combined hepatocellular carcinoma and cholangiocarcinoma recognized as
tumors with worse prognosis (66). Other variants are presumed to have better
prognosis such as pedunculated hepatocellular carcinoma and fibrolamellar hepa-
tocellular carcinoma (69). Finally, some variants are related to adjuvant therapy as
ablated HCC causing significant necrosis (70).
46 Thiesen A L

Figure 3.  Immunohistochemistry for Glypican- 3 200X. On the left, normal hepatocytes with
no staining (brown pigmentation) as compared to hepatocellular carcinoma (on the right)
with cytoplasmic and few nuclei staining positive (brown discoloration).

Figure 4.  Immunohistochemistry for CD34 200X. On the left, normal hepatocytes with no
staining (brown discoloration) of the sinusoids as compared to hepatocellular carcinoma
(on the right) with positivity within the sinusoids indicating capillarization.
 Hepatocellular Carcinoma in Adults 47

STAGING

After diagnosis, staging of disease is required for adequate prognostication and

subsequent therapy. The current staging systems include the pathologic tumor-
node-metastasis (pTNM) (Table 1), the Okuda and the Cancer of the Liver Italian
Program (CLIP) (Table 2), and the Barcelona Clinic Liver Cancer (BCLC) (71)
(Table 3). The pTNM staging system is purely morphology-based and the other
systems are mixed, using both morphology and clinical parameters to predict
prognosis. The pTNM system fails to consider the residual function of the liver
tissue and therefore does not predict behavior and prognosis especially in patients
treated with partial resection (72). In contrast, the other staging systems take into

TABLE 1 The pTNM classification and staging (2017)

Tumor
T0 No tumor
T1 Solitary tumor smaller or larger than 2 cm without vascular invasion
T1a Solitary tumor smaller or equal to 2 cm
T1b Solitary tumor larger than 2 cm without vascular invasion
T2 Solitary tumor larger than 2 cm with vascular invasion; or multiple tumors, none
larger than 5 cm
T3 Multiple tumors, at least one of which is larger than 5 cm
T4 Single tumor or multiple tumors of any size involving a major branch of the portal
vein or hepatic vein; Tumor(s) with direct invasion of adjacent organs other than
the gallbladder; Perforation of visceral peritoneum
Node
N0 No nodal metastasis
N1 Regional lymph node metastasis
Metastasis
M0 No distant metastasis
M1 Distant metastasis
Stage
IA T1AN0M0
Stage IB T1BN0M0
Stage II T2N0M0
Stage IIIA T3N0M0
Stage IIIB T4N0M0
Stage IVA Any T, N1, M0
Stage IVB Any T, Any N, M1
48 Thiesen A L

TABLE 2 The Okuda and the CLIP staging systems

Okuda
Parameters Criteria Pointsa
Tumor size by imaging >50% 1
Cross-sectional area <50% 0
Ascites Present 1
Absent 0
Serum Albumin (mg/dl) >3 0
<3 1
Serum total bilirubin (mg/dl) <3 0
>3 1
CLIP
Child-Pugh class A 0
B 1
C 2
Tumor morphology Single nodule <50% area 0
Multiple nodules <50% area 1
Massive >50% area 2
AFP (ng/mL) <400 0
≥400 1
Portal vein thrombosis No 0
Yes 1
aTotal
points = Stage.

consideration of the background liver function, predicting prognosis consider-

ably better, but are not suitable for orientating therapy. The Okuda staging sys-
tem is efficient in identifying patients who have very poor prognosis (Stage III)
but fails to distinguish different prognosis in patients grouped as Stage I and II
(73, 74). The CLIP staging system predicts survival more accurately in those
heterogenous group staged as I and II in the Okuda staging system but also fails
in orienting adequate therapy (75). Finally, the BCLC staging system identifies
early HCC for aggressive therapy but fails again to orientate adequate therapy in
the more advanced stages. As illustrated in Tables 2 and 3, both CLIP and the
BLCC staging systems use Child-Pugh class when staging HCC. This scoring
system is based on laboratory tests such as serum albumin, serum bilirubin and
prothrombin time as well as the presence or absence of ascites and hepatic
encephalopathy (76, 77).
 Hepatocellular Carcinoma in Adults 49

TABLE 3 The BCLC staging system

Performance
Stage Status Tumor Stage Liver Function
A= Early A1 0 Single, <5 cm No portal HTN and normal
bilirubin
A2 0 Single, <5 cm Portal HTN and normal bilirubin
A3 0 Single, <5 cm Portal HTN and elevated bilirubin
A4 0 3 tumors <3 cm Child-Pugh class A-B
B = Intermediate 0 Large multinodular Child-Pugh class A-B
C = Advanced 1 and 2 vascular invasion Child-Pugh class A-B
extrahepatic
spread
D = End-stage 3 and 4 Any Child-Pugh class C

TREATMENT

The therapeutic options for HCC consist of liver transplantation, hepatic resec-
tion, ablation, chemoembolization and chemotherapy. The best available treat-
ment is transplantation removing the entire tumor and replacing the diseased liver
(usually cirrhotic) by a new functional healthy organ. However, due to limitations
regarding availability of donor organs other alternatives should be considered.
Living donor transplantation is an alternative option that has been reported to
have similar results in terms of survival rate (78). Contraindications for liver
transplant include extrahepatic spread, multiple tumors with one of the lesions
larger than 3 cm and a single tumor larger than 5 cm emphasizing the need for
proper staging (79–82). Up to 75% of the patients that received a transplant, pre-
viously had received transarterial chemoembolization (TACE) and one third of
this same population had previous radiofrequency ablation (RFA) preventing pro-
gression of disease and winning time until a donor is available (83). If no evidence
of medical liver disease and/or cirrhosis is documented, the patient may be offered
partial hepatectomy as curative therapy (84). The three-year disease-free survival
in cirrhotic patients treated with partial hepatectomy is 18%, whereas it is 83% in
patients treated by transplantation (85).

CONCLUSION

HCC is a frequent tumor endemic in areas of frequent infectious hepatitis.

Patients with confirmed diagnosis of infectious hepatitis should be screened for
early hepatocellular carcinoma and possible therapy. Early detection involves
50 Thiesen A L

serologic markers, mostly AFP and radiologic studies. Staging systems attempt
to subdivide the cases of hepatocellular carcinoma in subgroups to predict
behavior and prognosis as well as orientate therapy. Curative therapy may be
achieved with either resection and/or transplant. Adjuvant therapy such and
TACE and RFA are offered as palliative care in attempt to win some time until a
donor liver is available.

Conflict of interest: The author declares no potential conflict of interest with

respect to research, authorship and/or publication of this chapter.

Copyright and permission statement: The author confirms that the materials
included in this chapter do not violate copyright laws. Where relevant, appropri-
ate permissions have been obtained from the original copyright holder(s), and all
original sources have been appropriately acknowledged or referenced.

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 3
Non-Alcoholic Fatty Liver Disease
Progression to Non-Alcoholic
Steatohepatitis-Related Primary
Liver Cancer
Utibe-Abasi Udoh1-2 • Juan D Sanabria1-2 • Pradeep K Rajan1-2 •
Moumita Banerjee1-2 • Mathew Schade1-2 • Jacqueline A Sanabria1-2 •
Gary Smith1-2 • Gideon Udoh1-2 • Komal Sodhi1-2 • Sandrine Pierre2 •
Joseph I Shapiro1-2 • Juan R Sanabria1-3
1Department of Surgery, Marshall University Joan Edwards School of Medicine,

Huntington WV, USA; 2Marshall Institute for Interdisciplinary Research, Marshall

University Joan Edwards School of Medicine, Huntington WV, USA; 3Department of
Nutrition and Metabolomics Core Facility, Case Western Reserve University School of
Medicine, Cleveland OH, USA.
Author for Correspondence: Juan R Sanabria, Department of Surgery, Marshall University
Joan Edwards School of Medicine, Huntington WV, USA. Email: [email protected]
Doi: https://doi.org/10.36255/exonpublications.livercancer.2021.ch3

Abstract: Hepatocellular carcinoma is the most common type of primary liver

cancer and constitutes about 90-95% of all hepatic malignancies. It is the sec-
ond and fastest-growing cause of cancer-related mortality worldwide. Although
there is multiplicity in the etiology of hepatocellular carcinoma, accumulating
evidence shows that non-alcoholic fatty liver disease has risen to become the top
etiological factor for hepatocellular carcinoma in the United States and other
developed nations, mainly because of the metabolic disturbances from obesity,
a western epidemic. Non-alcoholic fatty liver disease comprises a spectrum of
hepatic pathologies, ranging from simple steatosis to its inflammatory form,
­non-alcoholic steatohepatitis. With its concomitant increasing liver collagen

In: Liver Cancer. Sergi CM. (Editor). Exon Publications, Brisbane, Australia.
ISBN: 978-0-6450017-2-3; Doi: https://doi.org/10.36255/exonpublications.livercancer.2021
Copyright: The Authors.
License: This open access article is licenced under Creative Commons Attribution-NonCommercial
4.0 International (CC BY-NC 4.0) https://creativecommons.org/licenses/by-nc/4.0/

55
56 Utibe-Abasi U et al.

deposition, non-alcoholic steatohepatitis paves the pathway for hepatocellular

carcinoma development, which may occur with or without established
­cirrhosis. This chapter focuses on the current knowledge related to the epidemi-
ology and cellular mechanisms that underpin the progression of non-alcoholic
fatty liver disease to malignancy. Furthermore, it gives insight into the diagnosis,
treatment options, and future directions for non-alcoholic steatohepatitis-
related tumorigenesis.

Keywords: hepatocellular carcinoma; liver transplantation; non-alcoholic fatty

liver disease; non-alcoholic steatohepatitis; trans-arterial chemoembolization

INTRODUCTION

Cancer is the second leading cause of death worldwide, accounting for one in
every six deaths (1). Hepatocellular carcinoma (HCC) is the second and fast-
est-growing cause of malignancy-related mortality with an estimate of 840,000
new cases every year, contributing to 9.1% of all cancer deaths (2–4). HCC is
a highly lethal malignancy that primarily originates from a background of
end-stage liver disease (ESLD) or cirrhosis, the most significant risk factor for
HCC. ESLD develops from multiple etiologies, such as infectious (hepatitis B
and C virus infection), genetically inherited (α-1 antitrypsin deficiency, hemo-
chromatosis, primarily biliary cirrhosis, Wilson’s disease), toxic (alcohol,
drugs, aflatoxins), and metabolic (non-alcoholic steatohepatitis [NASH]) (2).
The only treatment currently available to intervene in the concomitant mala-
dies from parenchymal dysfunction, portal hypertension, and early-stage
malignancy, is liver transplantation. Although transplantation is a highly suc-
cessful form of therapy with overall survival rates of >65% at five years, this
form of therapy is limited due to scarcity of graft donors. Moreover, most
patients with HCC at presentation are staged with advanced disease, thereby
precluding resection or transplantation, and locoregional therapies such as
trans-arterial chemoembolization (TACE), Y-90, and ablation, are performed
as a bridge for liver transplantation. Systemic therapy for advanced disease
only prolongs survival in terms of weeks. Nevertheless, new forms of immu-
notherapy may increase further overall survival. In brief, HCC still carries a
poor prognosis, evolving as a prominent and increasing global health chal-
lenge (2, 5).
Metabolic disturbances like dyslipidemia from obesity are increasing in the
Western world, with one out of three or four adults being either overweight or
obese. It is estimated that 2.2 billion will be overweight and 1.1 billion obese
by 2030 (6–8). The epidemic of obesity and its metabolic consequences
(hypertension [HTN], hypertriglyceridemia, and insulin resistance) have led
to a sharp rise in non-alcoholic fatty liver disease (NAFLD) and its progressed
inflammatory form NASH, and its sequelae ESLD and HCC (2, 5, 9–11). In
the United States (US), NASH-related HCC is predicted to rise in the next ten
years with an estimated increase of 21% for NAFLD, 63% for NASH, and
137% for HCC by 2030 (2, 12). We aim in this chapter to discuss, at least one
 NAFLD Progression to NASH and HCC 57

of the paths for progression of NAFLD-to-NASH-to-cirrhosis, and to uncover

targets for the prevention, early diagnosis, and treatment of HCC.

EPIDEMIOLOGY

The geographical variation in the incidence of HCC worldwide is due, at least in

part, to the multiplicity of risk factors involved in its genesis and progression.
Most HCC cases occur in sub-Saharan Africa and Eastern Asia (80%, Figure 1),
where the main etiological factors are acquired by infection (hepatitis B virus)
or poisoning (aflatoxin) (1, 3, 13, 14). Although hepatitis C virus is a primary
risk factor in the US, Europe, and Japan (3, 13, 15), this trend is fast changing
due to the decline in hepatitis C virus infection from effective antiviral treatment
and the upsurge of obesity, diabetes and HTN (the metabolic syndrome) (9, 16).
About 80 million people in the US are affected by NAFLD, making it the most
common cause of liver disease in the US, surpassing 5 billion US dollars in
annual cost (9). As mentioned earlier, it is estimated that by the year 2030,
2.2 billion people around the world will be overweight and 1.1 billion will be
obese (6–8). Over the last 40 years, the incidence of HCC in the US has tripled,
increasing its burden from 14 million people (2012) to an estimated burden of
22 million people by 2032 (3). Men are more affected than women in a ratio of
2.4:1 (1, 3, 14), and the age-adjusted incidence of HCC has increased from 1.6
to 4.6 per 100,000 individuals among Native Americans and Alaskan Natives,
followed by African Americans, Caucasians, and Hispanics, with an average
five-year survival of <15% (3, 15, 17). Although the prevalence of NAFLD/
NASH is assumed to be prominent among Hispanics and Caucasians, the distri-
bution of cases among NASH-related HCC patients regarding their ethnic
groups is yet to be validated (9, 18, 19).

Age-standardised rates (World)

per 100 000
≥ 8.4
5.8–8.4
4.7–5.8 Data source: Globocan 2018
3.3–4.7 Not applicable Graph production: International Agency for Research on Cancer
(http://gco.iarc.fr/today)
< 3.3 No data World Health Organization

Figure 1.  Global estimates of hepatocellular carcinoma. Estimated age-standardized incidence

rates in both sexes for liver cancer in 2018 (3).
58 Utibe-Abasi U et al.

NAFLD PROGRESSION TO NASH-RELATED HCC

The hallmark of NASH is an inflammatory response that leads to hepatocellular

damage, and progressive collagen deposition (fibrosis) (20, 21). The molecular
pathogenesis of NASH-related HCC is complex and involves the interplay of genetic,
metabolic, immunologic, and endocrine pathways associated with changes in the
gut microbiota communities in response to an increasing mitochondrial dysfunc-
tion from lipid-originated respiratory chain uncoupling (20, 22). Various hypothe-
ses have been enunciated to explain the detailed cellular mechanisms involved in
the progression from NAFLD to NASH and subsequently to HCC. One such theory
entails that steatosis and insulin resistance are the underlying, initiating factors that
set the stage for the progression of NASH from metabolic oxidative stress (Figure 2).
This theory is termed the ‘two-hit’ hypothesis (20, 23–25).
The ‘two-hit’ hypothesis is a widely accepted paradigm to explain the devel-
opment of NASH from simple steatosis (fatty liver) originated from a fat enriched
diet and sedentary habits (24, 26–28). The first hit involves dysregulated hepatic

Insulin Decreased glucose uptake

resistance

Decreased Insulin clearance Increased lipolysis

Increased Hepatic IR, GNG
and VLDL secretion.

VLDL
FFA
Glucose ROS

β-oxidation

TG FFA, glycerol, GNG

GNG
precursors precursor
Adiponectin
FFA
Insulin

DNL

High fat diet

Figure 2.  Mechanisms of fat accumulation in non-alcoholic steatohepatitis. Insulin resistance

causes an influx of FFAs to the liver, owing to increased lipolysis, especially in the visceral
adipose tissue. Increased de novo lipogenesis and fat from the diet also contribute to the
fatty-acid pool. Both VLDL generation and FFA oxidation are increased and are enough to
prevent intrahepatic lipid accumulation. DNL, de novo lipogenesis; FFA, free fatty acid;
GNG, gluconeogenesis; IR, insulin resistance; ROS, reactive oxygen species; TG, triglycerides;
VLDL, very low-density lipoproteins (23).
 NAFLD Progression to NASH and HCC 59

lipid accumulation (steatosis), aggravated by the further development of an

insulin-resistant status, exposing the cell to oxidative stress, to a second hit that
overwhelms redox defensive mechanisms, leading to hepatocyte/stellate
senescence phenotype and inflammation (29–31). Insulin resistance upregulates
lipolysis, leading to an increase in the level of serum free fatty acid (FFA). The
increase of free fatty acids result in the delivering of triglycerides from the liver
to peripheral organs, aggravating the increased storage of lipids in the liver.
Further accumulation of triglycerides derivatives in the mitochondria drives a
saturation of the β-lipid oxidation process, resulting in the increased production
of reactive oxygen intermediates (ROI) that mediates the second hit (oxidative
stress) with succinate accumulation and uncoupling of the respiratory chain.
Oxidative stress promotes cellular processes such as lipid peroxidation, produc-
tion of proinflammatory substances and mitochondrial damage (20, 32–36).
Alternatively to the two-hit hypothesis, proponents of a multi-parallel hit theory
(20, 37), postulate that NASH develops from a multiplicity of factors that act in
parallel with each other. Such factors include genetic variations, abnormal lipid
metabolism, oxidative and endoplasmic reticulum stress, mitochondrial dys-
function, altered immune responses, and imbalance in gut microbiota. Proponents
of this theory suggest that liver inflammation is the initial cause of fibrosis
­progression in NASH, rather than steatosis (20, 38).
In NASH, metabolic mechanisms appear to be the principal drivers that con-
trol the progression to HCC. Energy balance and cell cycle regulation in the hepa-
tocytes are key processes that interplay between on-off insulin signaling and
lipotoxicity (20). Insulin resistance-induced hyperinsulinemia in turn triggers the
upregulation of insulin and insulin-like growth factor-1 (IGF-1) expression in the
hepatocytes and subsequent binding to their respective receptors (20, 39). Such
binding elicits a signaling cascade through the insulin receptor-substrate 1, which
results in the activation of its downstream target pathways, namely the PI3K and
MAPK pathways. These pathways have been reported to play a role in the tumori-
genesis of HCC from NASH via the induction of cell proliferation and inhibition
of apoptosis in hepatocytes (20, 39–41). Studies have shown that the PI3K path-
way involvement in the progression of HCC is primarily mediated by its action on
cyclin D1-dependent cell cycle, Mdm2/p53-dependent apoptosis, and mTOR-
dependent cell growth (20, 42, 43). The MAPK pathway drives tumor formation
and progression by inducing the transcription of protooncogenes such as c-fos,
and c-jun. Furthermore, MAPK pathway activates the Wnt/β-catenin signaling
cascade, which promotes fibrosis and malignancy in the liver (20, 44). Studies
from our lab have also revealed that Src-phosphorylation at the α1-Na/K-ATPase-
Caveolin-1 complex at caveola activates or amplifies the PI3K pathway and
­promotes hepatocarcinogenesis via the upregulation of survivin and the down-
regulation of the second mitochondria-derived activator of caspases (Smac)/
DIABLO proteins in hepatic parenchymal cells (45).
The α1-Na/K-ATPase-Caveolin-1-Src signaling complex is a novel signaling
pathway, comprising of the α1 isoform of Na/K-ATPase (NKA), the scaffolding
protein Caveolin-1 (CAV-1), and the sarcoma related kinase (Src). This pathway
plays a critical role in regulating and transmitting biological signals from mem-
brane micro-domains named caveolae into the interior of the cell. Such signals
play a key role in regulating cell growth and development. The importance of this
signaling mechanism has been demonstrated in the pathogenesis of the metabolic
60 Utibe-Abasi U et al.

syndrome, as well as in aging and embryonic development (46, 47). For instance,
genetic deletion of a caveolin binding motif (CBM) at the α1-NKA resulted in a
lethal embryonic phenotype in homozygous mice, despite normal NKA protein
expression and ion pumping function (47). This observation indicates that
α1-NKA-CAV-1 interaction is necessary for the proper execution of developmen-
tal signaling pathways. Conversely, chronic activation of the NKA-CAV-1-Src sig-
naling complex promotes pro-inflammatory pathways and tissue fibrosis through
the amplification of reactive oxygen intermediates. This pathway embraces a
vicious feed-forward mechanism, as evident in several disease phenotypes,
including renal fibrosis, uremic cardiomyopathy, and metabolic disorders such as
obesity (46, 48, 49). Although a balanced signaling mechanism through
NKA-CAV-1-Src is essential for normal physiological function, chronic activation
of this signaling mechanism under pathophysiological conditions can further
promote or aggravate disease conditions such as cancer. Recent in vivo and in vitro
data from our lab have shown that dysregulation of this signaling pathway
resulted in an imbalance in Smac/DIABLO-Survivin apoptotic signaling in the
cell via the upregulation of Survivin (anti-apoptotic protein) and a downregula-
tion of Smac/ DIABLO (pro-apoptotic protein), leading to an “oncogenic apop-
totic switch” that favors the development and progression of NASH to HCC (45).
Additionally, inhibition of this pathway by a novel peptide, known as pNaKtide
(developed from N domain of Na/K-ATPase), resulted in the reversal of the
­“oncogenic apoptotic switch”, leading to HCC prevention, tumor regression and
reduction in fibrosis (45). Furthermore, evidence from different backgrounds
enforces the fact that the central pathway controlling energy homeostasis, the
phosphoinositide 3-kinase (PI3K)-AKT-mTOR pathway is involved and mutated
in over 50% of all HCC cases, thus placing cell energy disturbances at the center
of liver tumorigenesis (50).
As mentioned earlier, an excess in the production of ROI in NASH, leads to
an imbalance in the cell’s redox status with progressive respiratory chain disrup-
tion and energy depletion followed by mitochondrial membrane pores opening
and the subsequent leakage of cytochrome C and SMAC, concluding in the acti-
vation of the cell apoptotic cascade (20). Although metabolic stress peaks at the
mitochondria, both insulin resistance and lipotoxicity are linked to several
other cellular mechanisms, such as oxidative and endoplasmic reticulum (ER)
stress, which may also contribute to cell injury and progression of NASH to
HCC (20, 51). Another emerging mechanism that may play a key role in the
progression of NASH to HCC is autophagy. Intracellular organelle/protein
autophagy is critical in cell survival by recycling metabolic components and is
increased during cellular stress. The cell, by this process, removes cytosolic
­non-functional organelles or macromolecules by transporting them into double-
membrane vesicles and delivering them to lysosomes for degradation (20). In
the liver, autophagy suppresses protein aggregation, lipid accumulation, oxida-
tive stress, chronic cell death, and inflammation. In addition, autophagy has
been shown to control adipogenesis and adipose tissue differentiation (20, 52).
Nevertheless, studies have also revealed that autophagy enables the parenchy-
mal cells to tolerate more stress, promoting tumorigenesis (20, 53, 54). Despite
the controversial role of autophagy in promoting or inhibiting NASH progres-
sion to HCC, its cellular role in an energy inefficient metabolism via PI3K/mTOR
pathway remains to be determined (20).
 NAFLD Progression to NASH and HCC 61

DIAGNOSIS AND STAGING OF HEPATOCELLULAR

CARCINOMA

An accurate diagnosis and proper staging assessment are necessary to determine

the optimal treatment method for the individual patient with HCC. A liver tumor
is usually detected by imaging and its histology confirmed by tissue analysis (55).
The various imaging methods include ultrasound (US), which is recommended
every six months as a non-invasive low radiation cost-effective screening tech-
nique on high-risk patients, and computed tomography (CT) and magnetic reso-
nance imaging (MRI), which are complementary techniques that detect and
characterize the different nodules that develop in cirrhosis (Figure 3) (56–59).
Although there are at least seven staging systems for HCC, the Barcelona Clinic
Liver Cancer (BCLC) classification is the most widely and accepted staging system
used (Figure 4) (55, 60, 61). The BCLC classification system (Table 1) includes
guidelines for the treatment of HCC and has been endorsed by the European
Association for the Study of the Liver (EASL), European Organization for Research

Figure 3.  Sensitivity of ultrasound as a surveillance tool for the detection of hepatocellular
carcinoma. Ultrasound is recommended every 6 months in high-risk populations for
monitoring the development of HCC, as well as in cirrhotic patients. Its sensitivity is around
77% and it can be complemented, if needed, with other imaging modalities, such as
computed tomography or magnetic resonance imaging (59).
62 Utibe-Abasi U et al.

HCC
PS 0, Child-Pugh A Okuda 3, PS > 2,
Child-Pugh C

Very early stage (0) Early stage (A) Intermediate stage Advanced stage (C)
Single < 2 cm Single or 3 nodules (B) Portal invasion, Terminal
Carcinoma in situ < 3 cm, PS 0 Multinodular, PS 0 N1, M1, PS 1–2 stage (D)

Single nodules ≤ 3 cm

Portal pressure/bilirubin
Increased Associated

Normal No Yes

Resection Liver transplantation RFA/PEI TACE Sorafenib Symptomatic

(20%); survival <
Curative treatments (30%); 5-yr survival: 40%–70% RCTs (50%); 3-yr survival: 10%–40% 3 mos

Figure 4.  Staging system for hepatocellular carcinoma. The Barcelona group developed a
staging system for HCC that includes five stages, given recommended therapy for each stage
and change of cure and overall survival (61).

TABLE 1 The Barcelona Clinic Liver Cancer (BCLC)

Staging System (60)
BCLC stage ECOG PS Liver function: Child-Pugh Tumor stage
Very early stage (0) 0 A Single ≤2 cm
Early stage (A) 0 A-B Single ≤3, nodules ≤3 cm
Intermediate stage (B) 0 A-B Multinodular
Advanced stage (C) 1-2 A-B Vascular invasion, extrahepatic
spread
Terminal stage (D) 3-4 C Any
Stage 0, A, and B, all criteria should be fulfilled.
Stage C and D at least one criterion should be fulfilled.
ECOGPS, Eastern Cooperative Oncology Group Performance Status.

and Treatment of Cancer (EORTC) and the American Association for the Study of
Liver Diseases (AASLD) (60, 62). This system stages an individual as having very
early, early, intermediate, advanced, and very advanced (terminal) HCC based
on  tumor burden, severity of liver disease and his/her performance status
­matching the recommended evidence-based treatment by the stage of liver tumor
(55, 60).

TREATMENT AND MANAGEMENT OF HCC

The management and treatment of HCC is complex, and still carries a poor prog-
nosis since an advanced stage is diagnosed in over 50% of the cases (63, 64).
 NAFLD Progression to NASH and HCC 63

Albeit great advances have been made in the management and treatment of HCC
patients at various stages of the disease (65). Patients in very early or early stage
of HCC may benefit from curative procedures, mainly surgical resection and liver
transplantation, as well as locoregional therapies such as thermal-related ablative
procedures. In the intermediate stage of the disease, locoregional procedures such
as TACE and Y-90 embolization are performed as a bridge for transplantation, as
a method to downstage tumor burden, or as to a limited time local disease control
(63, 65, 66). Image guided ablative therapy by microwave, radiofrequency or
cryotherapy procedures are used in tumors <4 cm in size with the Intent to cure.
They can be delivered in a percutaneous, laparoscopic, or open manner as the
only procedure, or as a complement to surgery (60, 64).

Liver resection
Surgical resection of the liver is a curative therapy and preferred treatment method
for patients with noncirrhotic or compensated cirrhosis (Child A, Low MELD,
Figure 5), solitary nodules (tumor size <5 cm) and adequate liver function with
no microvascular invasion or disease dissemination (60, 66). The preferred treat-
ment option for patients with very early and early stage of HCC with cirrhosis is
surgical resection if their hepatic function is intact and bilirubin level is (<1 mg/dl
or <17.1 μmol/l) with limited or no portal hypertension (60, 62). Such patients
have a survival rate of about 70% at 5 years, limited by a degree of patients who
undergo liver decompensation after surgery (60, 67, 68). Thus, selection of
patients for liver resection therapy is critical as there is a probability of liver failure
after resection with high mortality rates (60, 68).

Liver transplantation
Liver transplantation is a curative modality for the patient with HCC and border-
line to decompensated cirrhosis (Figure 6) (66). It offers a high survival rate and
the lowest probability of tumor recurrence due to the removal of the fibrotic envi-
ronment (68). Nonetheless, due to the scarcity of graft donors, there is a signifi-
cant patient mortality from dropping off the liver waiting list due to tumor
progression. To overcome longer cadaveric organs offer waiting times, living
non- or related-liver transplantation has been presented as an option (66, 68, 69).

Figure 5.  Liver imaging and pathology specimen after liver resection for hepatocellular
carcinoma. A. A right tri-segmentectomy was performed. B. Tumor was removed, note the
clear margins. C. MRI, showing the large HCC with multiple satellite lesions before resection.
The patient did well after surgery, and she was discharged home 5 days after procedure, with
no evidence for recurrences at 2 years follow up.
64 Utibe-Abasi U et al.

Figure 6.  Liver transplant procedure in a patient with end stage liver disease. A. Liver with
advanced fibrosis is removed. B. Replaced with a liver graft form a deceased donor.
C. Reperfusion showing satisfactory appeaance. D. Classic vascular and biliary
reconstruction performed at the proximal and distal end:end caval anastomosis, porto:portal
and common hepatic to common hepatic arterial reconstruction. In this case, no blood
products were given to the patient during the procedure. The patient was discharged 6 days
after surgery. The patient is alive and doing well 5 years after graft implantation.

Single tumor, not > 5 cm Number of

h
g
Nodules

ur

na
Up to 3 tumors, none > 3 cm

c
tts

tri
el
6

ul n
en
Pi

rc

M apa
tic
Ba

J
5

on co
Pa Fra se
pl is
4

s
Sa rou

m nc

a
B

i
no

na
ul
3

n
ila

a .Si
Pa
M

rc rk o
on Mt
Ba Yo sc

or
ris

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Re lla gh
i
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ew c
Pa

st u
N ran

ur
gi s T
Sa plo
no

el

b
F
ila

tts
Pa

a
1 M

Pi
D
0 Tumor Size
Absence of macroscopic vascular invasion, (cm)
0 1 2 3 4 5 6 7 8 9 10
absence of extrahepatic spread
75–80% 50–75% 35–50%
Expected 5-year survival

Figure 7.  The Milan criteria for liver transplantation in patients with end-stage liver disease and
hepatocellular carcinoma. Patients with end-stage liver disease and hepatocellular carcinoma
are entered into the liver transplant waiting list if they fulfil the Milan criteria, which includes
one tumor <5 cm in major diameter, or a maximum of 3 tumors with the largest <3 cm in
major diameter. In addition, the patient should have no vascular invasion or evidence of
extrahepatic disease. Other groups have explored other criteria with the development of the
metro-ticket concept that evaluates the probability of overall survival after liver
transplantation in patients with tumor burden over the Milan criteria (70).

The ideal candidate for liver transplantation (cadaveric or living-related) are those
who satisfy the Milan criteria (solitary tumor up to 5 cm in size, or 3 tumors
where the largest tumor is up to 3 cm in size, Figure 7) (66, 70). The Milan criteria
showed a low probability of tumor recurrence after transplantation, with over
75% survival in 5 years and over 90% tumor reoccurrence-free survival rate
(66, 68). Nevertheless, this concept has been challenged by several groups, and
many transplant centers offer liver transplantation for patients with HCC outside
of the Milan criteria with acceptable outcomes (66, 71). One other criterion is that
of the University of California at San Francisco, which include single tumors
≤6.5 cm or 2 to 3 tumors ≤4.5 cm, with a total tumor diameter ≤8 cm (66, 71).

Percutaneous ethanol injection

Percutaneous ethanol injection (PEI) involves the imaging guided injection of
ethanol into the tumor to induce coagulation necrosis (Figure 8) (66, 72). The
non-subcapsular, non-perivascular nodules <2 cm are ideal for PEI because of its
 NAFLD Progression to NASH and HCC 65

Figure 8.  Ultrasound-guided percutaneous alcohol injection of hepatocellular carcinoma. A and

B. Under image-guidance by US, administration of ethanol (97%) is performed into the
tumor. C. Noting an enlarging hyperechoic signal. Although the procedure in well-trained
hands is reliable and practical for large scale application, malignant recurrence rates are high.

limited capacity to penetrate the tumor beyond its pseudo-capsule or fibrotic

septa (60, 72). Although it is a cost-effective form of therapy, the recurrence tumor
rate is higher when compared to other locoregional therapies, thus PEI has fallen
into disfavor as the first line therapy for small HCC lesions. PEI has a recurrence-
free survival rate of 77% at one year as compared to 86% in patients treated with
radiofrequency ablation (RFA) (64). Its side effects include post procedural pain,
and it requires several sessions to yield complete treatment (66).

Radiofrequency ablation
Image-guided RFA is indicated in patients with tumor lesions ≤3 cm (single or up
to 3 lesions) or single lesions ≤4 cm, not in proximity to major vascular or biliary
conduits, with intact liver function belonging to Child-Pugh A or B group and
ECOG status 1-2 (72, 73). The heat emanated from high frequency oscillating
electrical currents at the needle tip of the probe transforms dripping NS0.9% into
vapor, resulting in tissue necrosis (Figure 9) (66, 72, 74). However, the thermal
effect is dissipated by the “sink effect” of a vessel in proximity and by the size of
the tumor (72).

Microwave ablation
Microwave ablation is the term that is used to describe tumor destruction by elec-
tromagnetic waves at frequency ≥900 kHz (74). It is one of the treatments of
choice for HCC patients with tumors that are less than 4 cm in size without the
sink effect limitation of RFA (66). The image-guided probe placement, as in RFA
can be achieved percutaneously, laparoscopically, or during open surgery (66, 75).
Contraindications include macrovascular invasion of tumor, main portal vein
destruction, decompensated cirrhosis (Child-Pugh C), biliary obstruction and
proximity to vital structures mitigated by open or laparoscopic techniques (66).

Cryoablation
Cryoablation, unlike RFA or microwave ablation, uses very low temperatures
from a liquid nitrogen source to destroy tissue by alternating freezing and thawing
based on the Joule-Thompson effect (76). As in other ablative methods, the probe
66 Utibe-Abasi U et al.

Figure 9.  Image-guided radiofrequency ablation for hepatocellular carcinoma. Under

ultrasound-guidance, a radiofrequency ablation needle is placed near the center of the
tumor. A. Alternate current at high frequency turns the needle tip temperature into a thermal
injury with a subsequent death zone. B. At 3 months follow-up, the death zone has
contracted.

can be placed under image guidance to induce “ice ball” formation (76, 77).
Indications for percutaneous cryoablation include patients that satisfy the Milan
criteria; that is, those with tumor size <5 cm in diameter or up to 3 tumors <3 cm,
absence of venous thrombosis, Child- Pugh A and B group without significant
coagulopathy (76). The advantages of cryoablation include the ability to visualize
the ice ball, activate cryo-immunology in cancerous cells, absence of severe dam-
age to blood vessels and less severe pain (77). Cryoablation can be used as an
isolated treatment for patients or in combination with other therapies. Cryotherapy
of liver tumors has become unpopular due to major bleeding after probe removal
and the induction of coagulopathy aggravated by thrombocytopenia, liver decom-
pensation and death (66).

Trans-arterial chemoembolization
TACE is currently the standard of care for patients with intermediate–stage HCC
with preserved liver function (78–80). TACE is useful for patients that have a
Child-Pugh score A or B with tumor diameter of >4 cm or four or more tumors as
well as those with single tumor in which it is challenging to carry out liver resec-
tion or locoregional therapies as a result of systemic co-morbidities or anatomical
limitations (81). TACE takes advantages of the dual arterial and portal venous
liver parenchymal blood supply with preferential arterialization not only in cir-
rhotic liver but of HCC. It involves the selective arterial embolization with a gela-
tin mixed with lipiodol (a radiopaque contrast agent) with or without chemotherapy
(doxorubicin, cisplatin or mitomycin C), into the tumor’s feeding blood vessel (66).
The blockage of the arteries supplying the tumor results in tissue necrosis
(Figure 10) (68, 79). In practice, TACE is a recommended therapy for patients
 NAFLD Progression to NASH and HCC 67

Figure 10.  Trans-arterial chemoembolization of the liver for hepatocellular carcinoma. A and
B. Selective arterial embolization of the vessels feeding the tumor in the liver. C. Tumor
visualized by CT scan. The procedure is performed with or without chemotherapy. D and
E. After embolization, angiography showed obliteration of feeding vessels with shrinkage of
the tumor. F. Follow up CT scan after embolization. Note an increase in patient’s ascites.

with unresectable HCC, nonvascular invasion or disease outside the liver (64).
TACE can also be used with drug-eluting beads (DEB-TACE) and evidence exists
that patients who are on DEB-TACE treatments for unresectable HCC have better
performance in comparison to those on conventional TACE (66). In addition,
TACE is being used for the downstaging of tumors in association with systemic
therapy or as bridge for transplantation.

Y-90 radioembolization
Y-90 radioembolization is a locoregional technique that involves a catheter-based
administration of Y-90 microspheres into the hepatic artery, leading to the delivery
of high radiation doses up to 50 to 150 Gy to tumors without affecting the paren-
chymal cells (Figure 11) (82, 83) . This exploits the principle; intrahepatic tumors
derive their major blood supply from the hepatic artery rather than the portal
vein. Y-90 emits beta radiation with an average energy of 0.9 MeV and a mean
penetration range of 2.5 mm (approximately 1,000 cell diameters). The physical
half-life of Y-90 is 64.2 hours, and it decays to stable Zirconium-90 (82, 83).
Microspheres (embedded with Y-90) are of varying sizes, ranging from 20 to 60
microns. In the US, the common available forms of Y-90 include the Y-90 tagged
glass (TheraSphere) and resin (SIR-Spheres) microspheres (82). The main differ-
ence between Y-90 radioembolization and TACE is that Y-90 microspheres are
smaller than TACE particles (20-30 microns compared to 200-500 microns).
Therefore, TACE gives a more significant embolic effect in comparison to Y-90
radioembolization. However, the main mechanism of action of Y-90 is related to
68 Utibe-Abasi U et al.

Figure 11.  Y-90 embolization for hepatocellular carcinoma. A. Selective angiography of the
liver and detection with obliteration of systemic shunts is performed. B, C and
D. Micro-spheres then are embolized to the liver with no vessel occlusion.

radiation effect and not the embolic occlusion of the blood supply to the cancer
cells, thereby making it a more effective therapy (82, 83).

Stereotactic body radiotherapy

Stereotactic body radiotherapy (SBRT) is a recent advancement in high-precision
radiotherapy (84–86). It is utilized as a locoregional therapy for early-stage HCC
and for patients with locally advanced HCC. Through this procedure tumoricidal
doses of radiation are delivered to hepatic tumors without affecting other organs
(Figure 12) (87). Specifically, SBRT makes it possible to effectively deliver high-
dose ablative radiation in 3-6 treatments to targeted HCC while minimizing
­toxicity to the adjoining normal tissues and organs (84–86). Patients that are
unsuitable for conventional locoregional treatments can benefit from SBRT.
Additionally, it can be used in combination with other therapies to improve out-
come and survival (84, 85).

Irreversible electroporation
Irreversible electroporation (IRE) is a nonthermal type of tumor ablation tech-
nique that is not affected by heat sink, which is a common limitation of other
forms of ablation procedures such RFA ablation. IRE procedure involves the
delivery of short pulses of high-frequency energy to create pores in the lipid
bilayer of cancer cells, which leads to cell death through apoptosis. On the other
hand, acellular elements within the treatment region are not affected, resulting
in the preservation of hepatic parenchymal architecture (88, 89). IRE procedure
gives the best results with tumors that are less than 3 cm (88). Currently the
only commercially available system for IRE is NanoKnife®  (AngioDynamics,
 NAFLD Progression to NASH and HCC 69

A) B)
1500

1250

Tumor volume (cm3)

1000 p = 0.002

750

500

250

0
Initial volume Last FU
C) D)
1.0 1.0

Probability of overall survival

0.9 0.9
Probability of local control

0.8 0.8
0.7 0.7
0.6 0.6
0.5 0.5
0.4 0.4
0.3 0.3
0.2 0.2
HCC HCC
0.1 ICC 0.1 ICC
0.0 0.0
0 10 20 30 40 50 0 12 24 36 48 60
Time (months) Time (months)

Figure 12.  Stereotactic body radiosurgery therapy for hepatocellular carcinoma. A. High doses
of focused radiotherapy are applied to liver tumor sparing normal liver parenchyma using
stereotactic body radiosurgery therapy. B. Shows high rates of tumor response (reduction in
tumor size) by RECIST criteria. C and D. The estimated probability of local control and overall
survival for hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) are
displayed respectively (87).

Queensbury, NY, USA). NanoKnife electrodes are contained in a 19-gauge

probe. This setup allows the use of 6 monopolar electrodes simultaneously.
These electrodes can be placed either surgically or percutaneously bracketing
the target region under the guidance of ultrasound or computed tomography.
The system has proprietary algorithms through which electrical energy delivery
can be calculated. It is important that the treatment delivery be accompanied by
general anesthesia, paralysis, and cardiac synchronization to prevent muscle
contractions and arrhythmias (88, 90).

Systemic or targeted therapies

Systemic therapies are indicated for most patients with advanced stage HCC (91).
Systemic therapies using small molecule drugs that target signaling pathways are
particularly useful for treatment of patients who have undergone liver resection or
transplantation and in which locoregional therapies such as TACE have not been
successful (92). Sorafenib, a first-generation tyrosine inhibitor is an orally admin-
istered approved systemic drug for patients with advanced HCC worldwide
(91, 93). The advent of sorafenib (an antiangiogenic, multitarget tyrosine inhibi-
tor) has triggered dynamic research into possible molecular targeted therapy for
HCC. These investigations center primarily on developing small molecules that
target signaling pathways that are involved in cell proliferation and angiogenesis,
which are critical for tumor development, growth, and metastasis (68, 91, 93).
70 Utibe-Abasi U et al.

Molecular targeted signaling pathways that are considered to play key roles in HCC
tumor formation, growth and progression include: mitogen-activated protein
kinases (MAPK) pathway (Ras/Raf/MEK/ERK pathway), the Phosphatidylinositol-
4,5-bisphosphate 3-kinase (PI3K), mammalian target of rapamycin (mTOR) path-
way and angiogenic pathways (68, 93).

Immunotherapy
Currently there is a dynamic expansion in cancer immunotherapy which focuses
mainly on agents that give a prognostic benefit to cancer patients by stimulating
the immune system to mount a response against developing cancers including
HCC. As earlier stated, there is a paucity of therapeutic options for patients with
advanced stage of HCC, therefore it appears that immunotherapy may hold the
key to effective systemic therapy for these patients (94). Immunotherapy strate-
gies for HCC are based on two main principles, namely: (i) the ability to unmask
ongoing immune responses in the liver during the onset or progression of carci-
nogenesis and (ii) the need to elicit new or different immunological responses.
The first strategy is based on pre-existing immune reactivity to cancer develop-
ment and progression held in check by micro-environmental factors. Such factors
include inhibitory receptors on T cells, especially programmed cell death protein 1
(PD-1) and cytotoxic T-lymphocyte associated antigen 4 (CTLA-4); other factors
are immunosuppressive cytokines such as Transforming growth factor beta
(TGF-β) (94). In this strategy, the therapies are not directed towards a specific
biological molecule produced by the cancer cells, but there is a general heighten-
ing/unmasking of immune responses to destroy tumor formation and progression.
On the other hand, antibodies that directly target molecules expressed on HCC
tumor cells, such as alpha-fetoprotein (AFP) or glypican-3 (GPC-3) fall within the
second strategy. These therapies can be enhanced by coupling these antibodies to
effector cells, such as T cells or natural killer cells (94). Additionally, administra-
tion of vaccines and the use of oncolytic viruses can operate through these two
mechanisms, that is, by unmasking already existing immune responses or prompt-
ing de novo T cell responses to substances expressed by HCC tumor cells (94). It
is worth noting that in line with the first strategy, the US Food and Drug
Administration (FDA) has approved the use of nivolumab (a PD-1 inhibitor) as an
immunotherapy for HCC (94, 95).

Diet/lifestyle and management of NASH related HCC

It has been widely reported that lifestyle changes (healthy diet and exercise) can
significantly reduce the formation of NAFLD/NASH and its progression to HCC.
Various studies have shown that regular aerobic exercise and weight loss resolves
fatty deposition, reduce insulin resistance, and improve inflammatory activity in
the liver (96). Interestingly, accumulating data reveals that some of the factors that
are useful in lowering the risk of developing cancer including HCC, include exer-
cise and caffeine (97). A diet consisting of a high intake of vegetable oils, fruits,
vegetables, legumes, cereals and fish, and low consumption of saturated fat and
non-fish meat products (Mediterranean diet), has also been shown to have a pro-
tective effect in the development and progression of HCC. Additionally, dietary
 NAFLD Progression to NASH and HCC 71

antioxidants such as coenzyme Q, vitamins C and E, selenium and certain phyto-

chemicals present in fruit, vegetables, herbs, and medicinal plants as well as coffee
have been shown to be useful in HCC prevention (96, 97).

CONCLUSION

Over the last two decades the incidence of NASH-related HCC has risen exponen-
tially, mainly due to metabolic disturbances promoted by the epidemic of obesity.
HCC has become a major and steadily increasing global health challenge due to a
paucity of biomarkers for its early detection coupled with few treatment options
and a 50–70% recurrence rate after resection or locoregional therapy. Treatment
options that are available for patients in very early or early stage of HCC include
surgical resection, liver transplant and ablation procedures with the intend to
cure. Those patients in the intermediate-stage are often treated with TACE or Y-90
radioembolization. Immuno-strategies are becoming the first line of therapy, fol-
lowed by sorafenib as systemic treatment for patients with advanced stage of the
disease. There is need for basic, translational, and clinical research targeting cel-
lular and molecular pathways that play key roles in cancer development and pro-
gression, aiming for novel and more effective therapies for NASH-related HCC.
One of such pathways is the α1-Na/K-ATPase-CAV-1-Src signaling pathway at the
cell membrane that plays a role in the regulation of embryonic development and
cell growth. Disturbances in this pathway has been shown to splinter the fragile
balance of apoptosis regulator proteins promoting an oncogenic “apoptotic
switch” that favors hepatic cell tumorigenesis. Furthermore, the inhibition of this
pathway may be a putative target for the treatment of HCC.

Conflict of interest: The authors declare no potential conflicts of interest with

respect to research, authorship and/or publication of this chapter.

Copyright and permission statement: The authors confirm that the materials
included in this chapter do not violate copyright laws. Where relevant, appropri-
ate permissions have been obtained from the original copyright holder(s), and all
original sources have been appropriately acknowledged or referenced.

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 4
Gross Dissection of Liver for
Hepatocellular Carcinoma Using
AJCC Cancer Staging Manual
8th Edition: Anatomical and
Practical Considerations
Marla Beach1 • Laura Henao Caviedes1 • Consolato M. Sergi2
1
Department of Laboratory Medicine and Pathology, University of Alberta Hospital and
Stollery Children’s Hospital, Edmonton, AB, Canada; 2Departments of Pediatrics,
Laboratory Medicine and Pathology, Stollery Children’s Hospital, University of Alberta,
Edmonton, AB, Canada.
Author for Correspondence: Marla Beach, Department of Laboratory Medicine and
Pathology, University of Alberta Hospital and Stollery Children’s Hospital, Edmonton, AB,
Canada. Email: [email protected]
Doi: https://doi.org/10.36255/exonpublications.livercancer.2021.ch4

Abstract: Hepatocellular carcinoma is one of the most common causes of cancer-

related death worldwide. In cases of liver resection for hepatocellular carcinoma,
the dissector, whether a pathologist’s assistant, physician, or resident, must have a
clear understanding of both the terminology of liver anatomy and the require-
ments of the corresponding College of American Pathologists Cancer Protocol to
properly orient, describe, dissect, and sample the specimen. This chapter pro-
vides guidance for the gross dissection procedure for the production of a valuable
pathology report, which is of key importance for a patients’ ongoing care.

Keywords: AJCC cancer staging manual; Couinaud segments; gross dissection of

liver; hepatocellular carcinoma; pathology

In: Liver Cancer. Sergi CM. (Editor). Exon Publications, Brisbane, Australia.
ISBN: 978-0-6450017-2-3; Doi: https://doi.org/10.36255/exonpublications.livercancer.2021
Copyright: The Authors.
License: This open access article is licenced under Creative Commons Attribution-NonCommercial
4.0 International (CC BY-NC 4.0) https://creativecommons.org/licenses/by-nc/4.0/

77
78 Beach M et al.

INTRODUCTION

The liver is a solid, asymmetrical organ normally located in the right upper quad-
rant of the abdomen and is found exclusively in vertebrates (1). An exception to
this rule occurs in the case of heterotaxis or Ivemark Syndrome, in which the liver
is located centrally or to the left side of the body (2). The liver synthesizes proteins
and produces biochemicals, which are specifically important for digestion and
growth. The gallbladder is a small sac, which is found on the visceral side of
the  liver. The gallbladder is adherent to the posterior liver along one side (the
gallbladder ‘bed’), and in rare cases, is not visible as it can be entirely intrahepatic (3).
A thorough inspection should be given in cases of biliary atresia with Kasai
porto-enterostomy, because the gallbladder can be very small or absent (4). The
gallbladder stores and delivers bile produced by the liver into the digestive tube.
The commonly used College of American Pathologists (CAP) Cancer Protocols
detail protocol for reporting, designed to produce a standardized, comprehensive
reporting of hepatocellular carcinoma (5). These synoptics are derived from the
AJCC Cancer Staging Manual (8th ed) (6). The main categories of a complete
pathological report, using the CAP synoptic, employs three main categories:
(i) primary tumor (T stage), (ii) regional lymph nodes (N stage), and (iii) distant
metastasis (M stage). As with most pathological specimens, unless a metastasec-
tomy is performed, gross bench considerations are primarily T and N staging.
Therefore, the M stage is not discussed here.

GROSS AND SURGICAL ANATOMY OF THE LIVER

Gross descriptors of liver anatomy, in practice, are generally an ad hoc miscellany

of older anatomical landmarks (7), along with the more functional Couinaud liver
segments (8). Generating and reviewing gross descriptions of liver resections
require an understanding of both.

Surface inspection
Surface inspection of the liver reveals many anatomical landmarks. These include
the falciform ligament, the ligamentum teres (round ligament), porta hepatis, gall-
bladder, and the inferior vena cava (or its groove). Porta hepatis, or the hilum,
contains the veins entering the liver (portal vein), the hepatic artery, and the bile
ducts exiting the liver. The falciform ligament is thin, membranous, and attaches
the liver to the anterior abdominal wall. At the liver’s inferior edge, the falciform
ligament merges with the round ligament (the umbilical vein’s remanent). The
round ligament sits in a grove called the umbilical fissure. Macroscopically, the
Falciform ligament has commonly been used as the border between the right and
left lobes. It grossly sits at a junction where the liver is larger (right lobe) and
smaller (left lobe). This is the anatomical right and left lobes. If inspected from
below (surgeon’s point of view) or posteriorly on the dissection table, four addi-
tional ‘lobes’ are appreciated. At the top, just medial to the IVC groove is a square
to rectangular protrusion designated the caudate lobe (caudate is Latin for ‘tail’,
 Gross Dissection of Liver 79

as if it were the tail of the liver). Below the caudate lobe is the gallbladder (centre
right), and the hilum (centre left). The gallbladder is a soft sac, commonly green
or purple, adjacent to the posterior and inferior liver. The hilum in the resected
liver will likely have multiple sutures, clips, and staples on its structures: portal
vein and hepatic artery entering the liver, and the bile ducts exiting the liver. The
round ligament sits to the left of the hilum and delineates the left anatomical lobe
on the posterior liver surface. The quadrate lobe is not a distinct lobar structure,
rather, it is a four-sided area (hence ‘quad’) bounded by the porta hepatis superi-
orly, the gallbladder laterally, the round ligament medially, and the free edge of the
liver inferiorly.
Three main hepatic veins drain from the liver superiorly; the right, middle and
left hepatic veins. In the resected total liver, careful inspection of the IVC or its
groove will reveal the left and central hepatic veins. Commonly, adjacent to the
IVC to the right, the right hepatic vein is found.

Couinaud segments
Further understanding of blood and bile flow in the liver informs the functional
description of liver segments described by Couinaud (8). This system separates
the liver, based on studies done with corrosion casting into eight independent
segments. The middle hepatic vein level, as it extends from inferior to drain into
the IVC superiorly, divides the functional right and left liver in this system. At the
gross level, an estimation of this border is Cantlie’s line. Cantlie’s line is an imagi-
nary line through the fossa of the inferior vena cava to the gallbladder’s fossa,
extending through to the front of the liver (9). Each segment has its own biliary
drainage and vascular inflow and outflow. In the centre of each segment, there are
branches of the portal vein, hepatic artery, and bile duct. In the periphery of each
liver subsegment, there is a vascular outflow through the hepatic veins. The main
dividing lines of liver segments in the transverse plane are the right and left portal
veins. Dividing lines into the vertical and oblique planes are the three main hepatic
veins (right, middle, and left). The segments are numbered clockwise from 1–8
(or I–VIII) (Figure 1). In this system, Segment 1 corresponds to the caudate lobe.
Segments 2–4 comprise the functional right lobe (to the right of Cantlie’s line), and
segments 5–8 comprise the functional left lobe (to the left of Cantlie’s line). The
only segment that is often subdivided is segment 4, into segments 4a (superior)
and 4b (inferior). The quadrate lobe does not have a corresponding functional
segment, rather is part of segment 4b. The Falciform ligament does not divide the
functional lobes, but the anatomical lobes; Cantlie’s line better approximates func-
tional division of right and left lobes and is the line between the right lobe
­segments 5, 6, 7 and 8, and the left lobe segments 2, 3 and 4.

Adjacent organs and their impressions on the liver

There are several impressions on the surface of the liver, accommodating the adja-
cent organs. These neighboring organs are key because they can be part of the
resection in cases where the adjacent organs are adherent and possibly involved
by invasive tumor. A classic example is a hepatocellular carcinoma directly and
contiguously invading the right colonic flexure. Underneath the right lobe, and to
80 Beach M et al.

Figure 1.  Functional (Couinaud) segments of the liver. A. Anterior surface. B. Posterior surface.
The segments 1-8 are delineated by hashed lines in relation to the anatomical landmarks.

the right of the gallbladder for some, it is essential to recognize two impressions
which are one behind the other and separated by a ridge. The first is the colic
impression, a shallow liver parenchyma compression that is formed by the hepatic
flexure of the large bowel. The second is the renal impression, which lies behind
the colic impression. It accommodates part of the right kidney and part of the
suprarenal gland. Other important impressions include the duodenal and gastric
impressions. The duodenal impression has a distinct, characteristic shape and can
be found medial to the renal impression and lies between the segment 8 and the
 Gross Dissection of Liver 81

neck of the gallbladder. Visible on the left lobe of the liver, on the inferior surface
is the gastric impression. Lastly, while not an impression, rather a point of contact,
is the superior, rounded surface of the right and mid liver, where tumour involve-
ment of the diaphragm is possible.

Grossing
When a surgical procedure is performed to resect a liver mass, the specific proce-
dure needs to be correctly identified. It is important to separate total hepatectomy
from partial hepatectomy and wedge resection. Total hepatectomies are received
in the setting of orthotopic liver transplantation (10). Thorough review of clinical
history will enlighten the dissector as to the surgeon’s intraoperative findings, the
lobes, segments, any structures removed, as well has any history of previous liver
surgeries, diagnoses, or therapies. Particular attention should be paid to the surgi-
cal report, number, location, and size of previously identified tumors, as well as
previous therapies such as resection, and oncological or ablative procedures (11).
The partial hepatectomy is subdivided into: (i) major hepatectomy, when three
segments or more are involved, and (ii) minor hepatectomy, when the resection
involves only one or two segments. Review the clinical information regarding the
type of resection, anatomical lobe, and the segments that were resected. Wedge
resections are small and may have one, two, or three sides with the identifiable
liver capsule.

Preparation of fresh tissue

All liver resections should be prepared in the fresh state to aid in immediate and
complete formalin fixation. Tissue should be weighed and measured in three
dimensions. If a gallbladder is present, this should also be measured. Inking
should be performed. Inking serves two purposes: to mark surgical resection
margin(s) and mark and aid assessment of areas of concern. The surgical resection
margin is where the liver parenchyma was transected by the surgeon and is often
cauterized and may have many surgical staples or sutures. Other areas that can be
inked are roughened areas where the liver was dissected away from other organs
or possible tumor involvement locations. Roughened anatomical areas may
include ‘bare’ areas where the liver was in contact with other organs, or the gall-
bladder bed, for example. While there are normally some roughened areas of the
liver capsule these may also indicate where a tumor may involve or perforates the
liver capsule. As such these can be inked in various colors so that they can be eas-
ily found and assessed after cutting. After weighing, measuring, and inking, the
tissue is serially sectioned and submerged in formalin for fixation.

Gross inspection, description, and sampling

At the gross bench, one proceeds with a structured approach to any specimen (12).
This ensures all findings are captured, and cases are consistent. One common
approach is the outside to inside approach, which is a systematic inspection and
description of an organ from the outer surface (capsule or serosa) to its cut sur-
face’s inside structures and appearance. For the liver, we inspect the outer surface,
82 Beach M et al.

identify the size, shape, landmarks, and attached structures to determine what
was resected. It is imperative to liaise with the surgeon under challenging cases to
ensure the specimen’s proper handling.

Tumor
With regard to the tumor, it is crucial to identify all tumors. The largest five masses
must be described in detail. When multiple tumors are present, care must be
taken not to confuse satellitosis with multiple primary tumors. A satellite nodule
is smaller than the main tumor and will sit within two centimeters from it, sepa-
rated by background liver tissue. All tumors must be described with regards to
their size, location (and structures involved), distance to resection margins, cut
surface/consistency, and presence of hemorrhage or necrosis. For tumor size,
ensure the size is described, three dimensionally, for the largest 5 tumors, in
centimeters.
Regarding the tumor location, it is important to specify which functional
­segment the tumor is found. The functional segments cannot be discerned in the
solid organ with the naked eye, hence the use of anatomical landmarks to help
approximate segments. Figures 1A and 1B demonstrate the approximate func-
tional segments overlaid with the gross appearance and anatomical landmarks on
a total hepatectomy. With lesions that are at, near, or straddle the areas where two
segments meet, it is not uncommon to include both segment numbers in the
description. For example, if a mass is identified on the anterior surface, just to the
left of the Falciform ligament may be arising from segment 2 or 3 and can be
described as located in segment 2/3. Carefully inspect the edges of the tumor(s)
for the involvement of vascular structures and select sections to confirm vascular
invasion. With regards to the tumor extension, it is important to select all events
that apply, including no evidence of primary tumor, tumor confined to the liver,
tumor involving a major branch of the portal vein or the involvement of the
hepatic veins, the involvement of the visceral peritoneum with obvious perfora-
tion, the invasion of the gallbladder, and invasion of the diaphragm, that is, the
direct extension of the tumor into other neighboring organs. If the tumor catego-
ries cannot be assessed, this also needs to be specified in the protocol.
Margins are those areas of the specimen where the surgeon has dissected,
either sharp or blunt, to sever it from the patient. It is essential to recognize the
parenchymal margin, which can be involved by the neoplasm. If the neoplasm
does not involve the parenchymal margin, the invasive carcinoma distance from
the margin in centimeters needs to be stated in the gross description. Additional
margins need to be specified if they are required; for example, if there is an
attached organ involved by the mass, the distance to the margin(s) of that tissue
needs to be recorded.
The cut surface of the mass should be described. This includes color, consis-
tency, and description of the tumor border. Clinical history of ablative, chemo-
therapeutic or similar treatments is important to note. In these cases, the tumor
may be completely or partially necrotic. Inspect the entire tumor and estimate the
percent necrosis. Necrosis in treated liver tumors often appears as homogenous,
crumbly, soft to firm but friable cut surface mottled green/brown. The surround-
ing parenchyma is often white, rubbery and fibrous. If residual tumor is present,
give the size of residual tumor in three dimensions, in centimeters.
 Gross Dissection of Liver 83

Lymph nodes
If resected with part of the hilum, the specimen may include soft tissue, including
a few lymph nodes. Ensure all soft tissue at the hilum is carefully inspected, and
all potential nodes found are submitted for histology. If the gallbladder is present,
also inspect the soft tissue around the cystic duct for any lymph nodes. Other
regional lymph nodes of liver tumors (hepatoduodenal ligament nodes, inferior
phrenic, and caval nodes) may be received separately. The number of submitted
lymph nodes need to be adequately identified. The number of lymph nodes
involved, and the number of lymph nodes examined need to be specified in the
gross description report.

Pathologic staging
With regards to the pathologic stage classification according to the pTNM, AJCC
8th edition (6), three descriptors are considered, including the letter “m” as mul-
tiple primary tumors, the letter “r” as recurrent, and the letter “y” as posttreat-
ment. In the TNM, a solitary tumour of 2 cm or less is classified as pT1, while a
tumor with 2 cm or more without vascular invasion is considered pT1b. A solitary
tumor of 2 cm or less is pT1a. The labeling of pT1 is also used if the tumor size is
more than 2 cm without vascular invasion. The labeling of pT2 regards a solitary
tumor of a diameter of more than 2 cm with vascular invasion or multiple tumors,
but none more than 5 cm. In the case of multiple tumors in which at least one is
more than 5 cm, the labeling of pT3 is used. Finally, pT4 is used when a single
tumor or multiple tumors of any diameter involve a major branch of the portal
vein or hepatic vein, or when tumor(s) with specific direct invasion of adjacent
organs other than the gallbladder or with perforation of visceral peritoneum are
found. The labeling of pN1 is used in case of regional lymph node metastasis,
while pN0 when no regional lymph node metastases are seen. The labeling of
pM1 is used when distant metastasis is seen and is required only if confirmed
pathologically. Additional pathologic findings include fibrosis, cirrhosis, low-
grade dysplastic nodule, high-grade dysplastic nodule, steatohepatitis, iron over-
load or chronic hepatitis. Also report on fibrosis present and indicate the
assessment scale used. The protocol described above applies only to hepatic resec-
tion specimens containing hepatocellular carcinoma, including fibrolamellar
carcinoma. Carcinoma of the intrahepatic bile ducts are staged using a separate
TNM system. This protocol should not be used for combined hepatocellular
carcinoma - cholangiocellular carcinoma, sarcomas, and metastatic tumors.

Sampling for histology

In preparing the cassettes, sections should be prepared from each major tumor
nodule with representative sampling of smaller nodules. The locations and patho-
logic parameters should be provided for the five largest tumors. In the case of
cirrhotic nodules that are larger than the surrounding background level, they
need to be separately sampled, because such nodules may contain dysplastic
changes. Satellite nodules, multifocal primary hepatocellular carcinoma and intra-
hepatic metastasis are obviously assessed as multiple tumors. In the case radiofre-
quency ablation or transarterial chemoembolization has been used, the extent of
84 Beach M et al.

necrosis seen grossly and microscopically is crucial, because it needs it to be cor-

related with the downstaging used on imaging. The tumor needs to be studied
microscopically in its entirety when it is up to 2 centimeters in size. In the case of
larger tumors, an additional section for each one centimeter is recommended and
should be provided to the pathologist. Further sampling is also necessary from the
periphery of the tumor or areas that seem to be viable. Overall, a combination of
gross and microscopic findings determines the extent of necrosis and should be
reported in up to five of the largest tumor nodules. The definition of satellitosis is
not uniform, and no universal definition has been provided. It is improper to label
as satellite any tumor nodule within a vascular structure, which needs to be better
categorized as lymphatic a vascular invasion.

CONCLUSION

Gross dissection of liver resection specimens for hepatocellular carcinoma is an

integral part of a comprehensive, complete pathology report. A systematic
approach to these specimens is key to the proper collection of data elements
needed for the completion of the relevant CAP cancer protocol synoptic report.
Understanding liver anatomy, both traditional anatomical landmarks, and func-
tional liver segments is crucial to translate and correlate gross and clinical findings
to ensure an accurate and reliable pathology report that enables the patients’
ongoing care.

Conflict of Interest: The authors declare no potential conflicts of interest with

respect to research, authorship and/or publication of this chapter.

Copyright and Permission Statement: The authors confirm that the materials
included in this chapter do not violate copyright laws. Where relevant, a­ ppropriate
permissions have been obtained from the original copyright holder(s), and all
original sources have been appropriately acknowledged or referenced.

REFERENCES

1. Polampelli A. What is the liver pathophysiology? J Liver Dis Transplant 2020;9(3):172.

2. Hrusca A, Rachisan AL, Lucian B, Oprita S, Manole S, Cainap S. Ivemark syndrome-a rare entity
with specific anatomical features. Rev Med Chil. 2015;143(3):383–6. https://doi.org/10.4067/
S0034-98872015000300014
3. Monib S, Mahapatra P, Fayez H. Intrahepatic Gallbladder. Eur J Case Rep Intern Med. 2019;6(6).
https://doi.org/10.12890/2019_001123
4. Yamataka,A. Laparoscopic Kasai portoenterostomy for biliary atresia, J Hepatobiliary Pancreat Sci.
2013;20:481–6. https://doi.org/10.1007/s00534-013-0607-1
5. Protocol for the Examination of Specimens From Patients With Hepatocellular Carcinoma. https://
documents.cap.org/protocols/cp-gihepatobiliary-hepatocellular-20-4100.pdf [Accessed on 11 Feb
2021]
6. Amin MB, Edge S, Greene F, Byrd DR, Brookland RK, Washington MK, et al. AJCC Cancer Staging
Manual (8th edition). Springer International Publishing: American Joint Commission on Cancer;
2017.
 Gross Dissection of Liver 85

7. Bismuth, H. Surgical anatomy and anatomical surgery of the liver. World J. Surg. 1982;6:3–9. https://
doi.org/10.1007/BF01656368
8. Strasberg SM. Nomenclature of hepatic anatomy and resections: a review of the Brisbane 2000 system.
J Hepatobiliary Pancreat Surg. 2005;12:351–5. https://doi.org/10.1007/s00534-005-0999-7
9. Cantlie, J. On a new arrangement of the right and left lobes of the liver. Proceedings - Anatomical
Society of Great Britain and Ireland. 1897;32:4–9.
10. Makowka L, Stieber AC, Sher L, et al. Surgical technique of orthotopic liver transplantation.
Gastroenterol Clin North Am. 1988;17(1):33–51.
11. Weledji EP, Ngounou E. The Impact of Segmental Anatomy on Hepatic Oncologic Resections. Curr
Surg Rep. 2016;4(4). https://doi.org/10.1007/s40137-015-0122-1
12. Dayton AS, Ro JY, Schwartz MR, Ayala AG, Raymond AK. Raymond’s Paragraph System: an alternative
format for the organization of gross pathology reports and its implementation in an academic teaching
hospital. Arch Pathol Lab Med. 2009;133(2):298–302. https://doi.org/10.5858/133.2.298
 5
Radiologic-Pathologic Correlation of
Liver Tumors
Eric Lachance1 • Jake Mandziuk2 • Consolato M. Sergi3 • Justin Bateman2 •
Gavin Low1
1
Department of Radiology and Diagnostic Imaging, University of Alberta, Edmonton,
Alberta, Canada; 2Department of Laboratory Medicine and Pathology, University of Alberta,
Edmonton, Alberta, Canada; 3Departments of Pediatrics, Laboratory Medicine and
Pathology, Stollery Children’s Hospital, University of Alberta, Edmonton, AB, Canada.
Author for correspondence: Jake Mandziuk, Department of Laboratory Medicine and
Pathology, University of Alberta, Edmonton, AB, Canada. Email: [email protected]
Doi: https://doi.org/10.36255/exonpublications.livercancer.2021.ch5

Abstract: Radiologic and pathologic features of common and/or critical tumor or

tumor-like diagnoses (lesions) of the liver are discussed within. Hepatocellular
lesions (focal nodular hyperplasia, hepatocellular adenoma, hepatocellular carci-
noma, and hepatoblastoma), biliary lesions (mucinous cystic neoplasm and intra-
hepatic cholangiocarcinoma), vascular mesenchymal lesions (cavernous
hemangioma, epithelioid hemangioendothelioma, and hepatic angiosarcoma),
and metastatic malignancies are the primary focus, although a more comprehen-
sive list of lesions is also provided. Definitions, distributions, gross appearances
and microscopic pathological features are introduced first, followed by radiologic
correlation. Multiple imaging modalities are explored with an emphasis on those
that provide the greatest value for the lesion under evaluation. A common under-
standing of the features of both diagnostic specialties will allow for high-quality
correlation and subsequent high-quality patient care. Representative images high-
lighting important features are also presented.

Keywords: imaging; liver; pathology; radiology; tumors 

In: Liver Cancer. Sergi CM. (Editor). Exon Publications, Brisbane, Australia.
ISBN: 978-0-6450017-2-3; Doi: https://doi.org/10.36255/exonpublications.livercancer.2021
Copyright: The Authors.
License: This open access article is licenced under Creative Commons Attribution-NonCommercial
4.0 International (CC BY-NC 4.0) https://creativecommons.org/licenses/by-nc/4.0/

87
88 Lachance E et al.

INTRODUCTION

This chapter is designed to introduce the reader to the radiologic and pathologic
features of common and/or critical tumor or tumor-like diagnoses (lesions) within
the liver. It is divided into hepatocellular lesions, biliary lesions, vascular mesen-
chymal lesions, and metastatic malignancies. However, a broad differential is
important when approaching liver lesions, and a more comprehensive list of
lesions is provided in Table 1.

TABLE 1 Categorization of Liver Lesions

Hepatocellular
Benign Precursors Malignant
Focal Nodular Hyperplasia Low-Grade Dysplastic Nodule Hepatocellular Carcinoma
Macroregenerative Nodule High-Grade Dysplastic Nodule Hepatoblastoma
Hepatocellular Adenoma
Biliary
Benign Precursors Malignant
Bile Duct Hamartoma Intraductal Papillary Intraductal Papillary Neoplasm with
Neoplasma Associated Invasive Carcinomaa
Bile Duct Adenoma Mucinous Cystic Neoplasma Mucinous Cystic Neoplasm with
Associated Invasive Carcinomaa
Biliary Adenofibroma Cholangiocarcinoma
Solitary Biliary Cyst
Mesenchymal
Benign Intermediate Malignant
Cavernous Hemangioma Inflammatory Myofibroblastic Epithelioid Hemangioendothelioma
Tumor
Infantile Hemangioma Solitary Fibrous Tumor Angiosarcoma
Angiomyolipoma Kaposi Sarcoma Embryonal Rhabdomyosarcoma
Mesenchymal Hamartoma Undifferentiated Embryonal Sarcoma
Other
Metastatic Malignancy
Infection/Abscess
Confluent Hepatic Fibrosis
Hydatid Cyst
Neuroendocrine Neoplasm
Lymphoma
aThe 4th edition of the WHO Classification of Tumors of the Digestive System in 2010 removed the terms biliary
cystadenoma and biliary cystadenocarcinoma, refining into mucinous cystic neoplasms (MCN) and intraductal papillary
neoplasm (IPN), “with associated invasive carcinoma” added, if present. Literature in the interval using the term biliary
cystadenoma and/or cystadenocarcinoma may include MCN and/or IPN, with or without associated invasive carcinoma.
 Radiologic-Pathologic Correlation of Liver Tumors 89

Radiology plays an important and varied role, including screening, diagnosis,

and treatment of liver lesions. Ultrasound plays an increasingly important role in
the diagnosis of liver lesions, in part due to increased utilization of contrast-
enhanced ultrasound (CEUS), with clinical use in Europe, Asia, and Canada
established and increasing use in the United States after FDA approval for abdom-
inal use in 2016 (1). Magnetic resonance imaging (MRI) is also becoming increas-
ingly important, with the ability to perform additional pulse sequence phases (for
example, diffusion-weighted imaging [DWI]) and increased accuracy without
increased exposure to ionizing radiation as in computed tomography (CT).
Additionally, hepatobiliary gadolinium contrast agents such as gadoxetic acid
(Primovist, Eovist) are increasingly used, adding accuracy in some instances com-
pared to conventional extracellular gadolinium contrast agents. Nuclear medicine
tests are utilized in select scenarios and not elaborated on in detail. Dual photon
nuclear imaging tests, such as positron emission tomography with computed
tomography (PET/CT), play an important role in the detection of extrahepatic
(and to a lesser extent, intrahepatic) metastases, and traditional single-photon
emission computed tomography (SPECT) nuclear imaging tests (for example,
Tc-99m sulfur colloid) are largely of historical value. Interventional radiology is
not elaborated on in this chapter but is invaluable in diagnosing liver lesions with
image-guided biopsy and in treatment.
Pathology provides further diagnostic insight into material obtained through
biopsy or surgical resection. Gross appearances guide sampling approaches, and
microscopic examination can lead to a final diagnosis. In challenging cases or in
metastatic disease, additional ancillary studies, including immunohistochemical
(IHC) stains, help determine cell lineage and refine the diagnosis. If present, char-
acteristic molecular alterations can define a diagnosis.

HEPATOCELLULAR LESIONS

Hepatocytes are the main functional epithelial cells of the liver and are arranged
within hepatic plates supported by a fine reticulin fiber meshwork. The following
sections discuss lesions involving hepatocytes and includes focal nodular hyper-
plasia, hepatocellular adenoma, hepatocellular carcinoma, and hepatoblastoma.

Focal nodular hyperplasia

Focal nodular hyperplasia (FNH) (Figure 1) is a non-neoplastic mass-forming
lesion characterized by hyperplastic nodules, fibrous septae, and abnormal blood
vessels within a region of altered hepatic blood flow (2–3). FNH is a solitary mass
in three-quarters of cases with a right-lobe predilection; however, more than fif-
teen masses have been documented distributed throughout the liver (4). Grossly,
FNH is a well-circumscribed and non-encapsulated bulging mass with a central
stellate scar and radiating fibrous septae that create a multinodular appearance.
Diameters are usually less than 5 cm but can exceed 10 cm, and central scars are
present approximately half of the time (5). Microscopically, nodules show cyto-
logically benign hepatocytes arranged in hepatic plates up to two cells thick with
preservation of the reticulin network. Dystrophic vessels are present in the central
90 Lachance E et al.

Figure 1.  Representative images of focal nodular hyperplasia. A. On portal phase CT in liver
windows the isoenhancing lesion with hypoenhancing central scar in segment 2/3 is subtly
appreciated (white arrow to the edge of lesion), subtly bulging the inner contour of the left
lobe, a so-called “stealth” lesion. B and C. On MRI in a different patient, a similarly located
segment 2/3 lesion is isointense with the background liver on the T1 weighted image (B) and
T2 weighted image (C). D and E. Following intravenous gadoxetic acid administration, the
lesion shows homogenous hyperenhancement during the arterial (D) and portal (E) phases.
F. On the hepatobiliary phase at 20 minutes, the lesion shows uniformly higher signal than
the background liver. G and H. A partial hepatectomy from a separate patient shows a
well-circumscribed lesion with a central stellate scar (white arrow) and radiating fibrous
septae, seen grossly (G) and microscopically (H). I. Ductular reaction (black arrow) is present
at the interface of fibrous regions with nodules. Slides are stained with Hematoxylin and
Eosin. Total image magnification: H - 25X; I - 100X.

scar, and a ductular reaction is seen at the interface between the fibrous septae and
hyperplastic nodules. Normal bile ducts and portal tracts are absent. Glutamine
synthetase demonstrates a characteristic ‘map-like’ pattern of staining by immu-
nohistochemistry (6–7).
FNH can be subtle on unenhanced imaging, due to the benign hyperplastic
tissue blending in with adjacent liver parenchyma, classically described as a
“stealth lesion” (8). On ultrasound, the central scar may be indistinctly visualized;
otherwise, FNH echogenicity is varied and nonspecific. However, contrast-
enhanced ultrasound, CT, or MRI is usually diagnostic. The central feeding artery
with centrifugal flow may be seen as the characteristic “spoke-wheel” pattern
on color Doppler, CEUS, and sometimes CT arterial phase, most commonly in
larger lesions (9). FNH is typically a subtle hypoattenuating or isoattenuating
 Radiologic-Pathologic Correlation of Liver Tumors 91

homogenous lesion on unenhanced CT. On multiphasic CT, the arterial phase is

most diagnostic for FNH, with homogenous enhancement and hypoattenuating
central scar, if present. On the portal-venous phase, enhancement is subdued,
appearing mildly hyperattenuating or isoattenuating to the liver with the scar
remaining hypoattenuating. On delayed phase imaging, the scar is typically
hyperenhancing, with the remainder of the lesion isoattenuating. 
MRI provides the most diagnostic characterization of FNH (with a specificity
of 98%) (10). FNH is T1-weighted imaging (T1WI) isointense or slightly hypoin-
tense, and T2-weighted imaging (T2WI) isointense or slightly hyperintense. If
present, the central scar is T1WI hypointense and T2WI hyperintense. Similar to
CEUS and CT, during the arterial phase, FNH demonstrates intense homogenous
enhancement (sparing the central scar), is relatively isointense on portal-venous
phase (hypointense scar), and isointense on delayed phase (hyperintense scar).
Gadoxetic acid-enhanced MRI is sensitive for FNH, showing a high signal on the
hepatobiliary phase at 20 minutes, which aids in differentiation from other arterial
enhancing liver lesions, such as adenomas (11).

Hepatocellular adenoma
Hepatocellular adenoma (HCA) (Figure 2) is a benign hepatocellular neoplasm,
which may be solitary or multiple (2). Liver adenomatosis is defined by 10 or
more HCAs and is diagnosed most frequently in patients with nodules greater
than 5 cm (12). Hemorrhage is the first presentation in 15% of patients and is
associated with lesions greater than 5 cm (13). Grossly, HCAs are non-encapsulated
and soft tan or red-brown masses that occur within a non-cirrhotic liver.
Microscopically, the tumor is composed of benign hepatocytes arranged in plates
up to two cells thick with unpaired arteries and an absence of portal tracts.
Currently, there are four recognized genotypic-phenotypic subtypes: (i) HNF1α-
inactivated HCA with marked steatosis and loss of liver fatty acid-binding protein
expression by IHC, (ii) inflammatory HCA (JAK/STAT3 pathway activation) with
inflammation, sinusoidal dilatation, ductular reaction, and diffuse expression of
C-reactive protein and serum amyloid A by IHC, (iii) beta-catenin activated
inflammatory HCA, and (iv) beta-catenin activated HCA. Beta-catenin activated
HCAs may show atypical cytological and/or architectural features, suggesting
transformation to hepatocellular carcinoma (HCC) (14). Evaluation of IHC stains
may show nuclear beta-catenin expression along with over-expression of gluta-
mine synthetase within these beta-catenin activated HCAs. Careful evaluation of a
reticulin stain is essential to help exclude malignant transformation.
Imaging features of HCA are often varied, with slightly different characteristics
depending on the subtype. Hemorrhage with possible subsequent calcification,
fat, and encapsulation may be seen. Of note, HCAs 5 cm or larger are at higher
risk for both hemorrhage and malignant transformation into HCC, and require
close imaging follow up. Males with HCAs are often treated by prophylactic surgi-
cal resection, particularly for solitary or large lesions, as a result of the increased
incidence of the beta-catenin subtype.
Ultrasound is nonspecific, demonstrating a well-circumscribed lesion with
variable echogenicity, more often hyperechoic. CEUS may increase specificity,
particularly showing centripetal arterial flow, differentiating HCA from FNH,
which typically illustrate a centrifugal arterial flow. Multiphasic contrast-enhanced
92 Lachance E et al.

Figure 2.  Representative images of hepatocellular adenoma. A and B. The large lesion (>5 cm)
in segments 5 and 6 shows signal loss on the opposed phase T1 weighted image (B)
compared to the in-phase T1 weighted image (A) denoting microscopic fat content. C. The
lesion shows heterogeneous high signal on the T2 weighted image. D, E, and F. Following
gadolinium administration, the lesion shows heterogeneous arterial (D) and portal phase (E)
hyperenhancement and delayed phase washout (F). An ancillary finding of an enhancing
capsule is depicted in (E) and (F). G. A partial hepatectomy from a separate patient shows a
tan and poorly defined lesion grossly, with areas of hemorrhage and necrosis. H.
Microscopically the neoplastic lesion (white arrow) shows an absence of portal tracts and is
notably different from the background parenchyma (black arrow). I. Benign hepatocytes are
arranged in cell plates up to two cells thick. Slides are stained with Hematoxylin and Eosin.
Total image magnification: H - 25X; I - 400X.

CT demonstrates a well-circumscribed mass, which is typically isoattenuating on

the unenhanced phase, with heterogeneous arterial phase hyperenhancement,
usually returning to near isoattenuation on portal-venous phase and delayed
phase imaging. Following hyperenhancement, contrast washout (hypoenhance-
ment on the portal-venous and delayed phases) may be seen in both CT and MRI,
mimicking malignant lesions.
On MRI, T1WI and T2WI signal intensities are variable, depending on the
presence of fat, hemorrhage, and calcification. A high signal on fat-saturated
T1WI denotes the presence of intratumoral hemorrhage. Contrast-enhanced
phases are similar to CT, with early arterial enhancement, and are typically
isointense on portal-venous phase imaging. HCAs are predominantly hypoin-
tense on hepatobiliary phase gadoxetic acid imaging, an important distinction
from FNH. Opposed phase T1WI and fat-saturated T1WI are helpful to demon-
strate microscopic fat and macroscopic fat, respectively, a characteristic feature
 Radiologic-Pathologic Correlation of Liver Tumors 93

of HNF1α-inactivated HCAs. Conversely, inflammatory HCAs have a characteris-

tic high signal peripheral rim on T2WI, attributed to sinusoidal dilatation, termed
the “atoll sign” (15).

Hepatocellular carcinoma
Hepatocellular carcinoma (HCC) (Figure 3) is a malignant hepatocellular neo-
plasm, commonly occurring in the setting of underlying chronic liver disease (2).
Traditionally, macroscopic patterns of HCC are described as single or multiple
liver nodules, massive nodules that may involve multiple liver segments, and dif-
fuse forms with multiple small nodules throughout the liver mimicking cirrhosis
(16). Tumor nodules smaller than the main tumor and less than 2 cm away (sepa-
rated by non-neoplastic parenchyma) are classified as satellite nodules and

Figure 3.  Representative images of hepatocellular carcinoma. A. Transverse ultrasound image

of the liver demonstrates a new hyperechoic lesion in a cirrhotic liver, highly suspicious for
HCC. B and C. The lesion in segment 6 shows signal loss on the opposed phase T1 weighted
image (C) compared to the in-phase T1 weighted image (B) denoting microscopic fat
content. D. The lesion shows heterogeneous high signal on the T2 weighted image. E and
F. Following gadolinium administration, the lesion shows heterogeneous arterial
enhancement (E) and portal phase wash-out (F). G. A representative slice of liver from an
explant of a separate patient shows a soft variegated lesion grossly (white arrow), ranging
from green to red-brown, embedded in a firm cirrhotic liver parenchyma. H and I. There is a
loss of normal hepatic architecture with trabecular and pseudoglandular growth patterns (H)
of malignant cells with enlarged vesicular nuclei and eosinophilic cytoplasm (I). Slides are
stained with Hematoxylin and Eosin. Total image magnification: H - 25X; I - 400X.
94 Lachance E et al.

usually represent intrahepatic metastases. Nodules that are far from the primary
tumor may represent either synchronous tumors or intrahepatic metastases (16).
Involvement and spread through intrahepatic portal vein branches are common. 
Grossly, HCC is soft with a variegated appearance ranging from green to light
tan depending on bile and fat content respectively, and foci of hemorrhage or
necrosis. Tumors may have a capsule of fibrotic and inflamed tissue and can be
seen invading large vascular structures. Fibrolamellar and scirrhous subtypes may
appear firmer and more fibrotic compared to conventional HCCs, and these fea-
tures may mimic the central scar of FNH or even cholangiocarcinoma. Fibrolamellar
carcinoma is a distinct lesion affecting younger adults without pre-existing cir-
rhosis or elevated serum AFP. It is typically a large mass with calcification, hemor-
rhage, or necrosis, can mimic FNH with a central scar, commonly metastasizes to
lymph nodes and distant sites, and shows a DNAJB1-PRKACA fusion transcript
(17–18).
Microscopically, HCC shows variable degrees of cytological and architectural
atypia with thickened hepatic plates and disruption/loss of the reticulin frame-
work. Hepatocellular differentiation is often apparent on hematoxylin and eosin
(H&E); however, IHC stains (Hep Par-1 and Arginase-1) are sometimes necessary
for poorly differentiated tumors to establish hepatocellular origin. Furthermore,
careful examination of reticulin stain and identification of abnormal expression of
Glypican-3 IHC may be required in well-differentiated tumors to establish a diag-
nosis of carcinoma. Increased arterial blood flow leads to sinusoidal capillariza-
tion, which can be demonstrated by CD34 IHC. There are four predominant
growth patterns including trabecular (70%), solid (20%), pseudoglandular (10%),
and macrotrabecular with cell plates greater than ten cells in thickness (1%),
although mixed patterns are common (19). Approximately 35% of HCCs show
distinct clinicopathologic subtypes, including steatohepatitic (most commonly in
patients with underlying fatty liver disease), clear cell (cytoplasmic clearing sec-
ondary to glycogen accumulation), scirrhous (fibrosis in the majority of the tumor,
separate small nests of tumor cells in thin trabeculae), and fibrolamellar (large
polygonal hepatocytes with abundant cytoplasm and prominent nucleoli, pale
bodies, thick collagenous bands in parallel orientations, and non-cirrhotic back-
ground liver) (20). Combined hepatocellular cholangiocarcinoma is a rare diag-
nosis with convincing HCC and cholangiocarcinoma morphologies present within
the same tumor. 
Precursor lesions of HCC are commonly identified in hepatectomy specimens
and occasionally in biopsy specimens in patients with advanced chronic liver
disease. These precursor lesions include dysplastic foci (<1 mm) and dysplastic
nodules (approximately 5–15 mm) (21). Dysplastic foci are incidental findings
and include small cell and large cell cytological change or small groups of hepato-
cytes with an otherwise clonal appearance (for example, iron free foci). Dysplastic
nodules can be low- or high-grade, and the high-grade dysplastic nodules can
mimic small (<2 cm) early HCCs. Features that favor the designation of HCC over
a dysplastic nodule include a nodule-in-nodule growth pattern, stromal invasion,
loss of reticulin, and expression of Glypican-3 by IHC. Low-grade dysplastic nod-
ules require distinction from large regenerative nodules, although this distinction
can be challenging and of limited significance in explanted livers (2).
Across imaging modalities, differentiation of small HCCs from regenerative or
dysplastic nodules can be challenging in the setting of cirrhosis. Smaller lesions
 Radiologic-Pathologic Correlation of Liver Tumors 95

are typically homogenous, with larger lesions demonstrating variable heterogene-

ity secondary to the variable presence of fat, fibrosis, necrosis, hemorrhage, and
calcification. The American College of Radiology (ACR) Liver Imaging and
Reporting Data System (LI-RADS) version 2018 provides a validated set of stan-
dardized diagnostic criteria for HCC based on CT/MRI. It has become integrated
into the American Association for the Study of Liver Diseases (AASLD) clinical
practice guidelines (22–23). LI-RADS provides uniformity and improved accu-
racy in diagnostic reporting, including consistency in imaging work-up options
and follow-up time intervals. 
On ultrasound, small HCCs are usually hypoechoic, with larger lesions dem-
onstrating variable echogenicity and heterogeneity. A thin hypoechoic pseudocap-
sule may be seen. Doppler may demonstrate vascularity and arteriovenous
shunting. Increased arterialisation and progressive loss of the portal-venous vas-
cular contribution results in a characteristic pattern on contrast-enhanced ultra-
sound, CT, and MRI of late arterial hyperenhancement with decreased enhancement
relative to liver parenchyma (wash-out) on delayed phases, variably present on
the portal-venous phase (24). Unenhanced CT has limited sensitivity for HCC,
typically hypoattenuating if detected. 
Contrast-enhanced MRI (extracellular or hepatobiliary gadolinium agents)
shows significantly higher sensitivity for HCC than contrast-enhanced CT in the
setting of cirrhosis (82% vs. 66%) with similar specificities (91% vs. 92%) (25).
Approximately 20% of HCCs arise in non-cirrhotic livers, with contrast-enhanced
MRI also demonstrating high sensitivity (91%) and specificity (75–98%) (26).
HCC is usually T1WI isointense to hypointense, and T2WI heterogeneously
intermediate to high signal. Late arterial hyperenhancement is typically homoge-
nous for smaller lesions and more heterogeneous for larger lesions. Portal-venous
phase wash-out is variable depending on size, with smaller lesions typically retain-
ing a degree of perceptible enhancement and larger lesions demonstrating greater
wash-out compared to the adjacent liver. With delayed phases, lesions typically
demonstrate wash-out with residual enhancement in the pseudocapsule, if
present. A recognized pitfall is that small (<2 cm) early HCCs may show a lack of
delayed phase washout, and close imaging follow up of these lesions in the cir-
rhotic liver is required. DWI shows diffusion restriction in areas of high cellularity
and cell membrane density (for example, malignancy) (27). DWI increases the
sensitivity of conventional extracellular contrast-enhanced MRI, especially for
small (<2 cm) HCCs (28). Sensitivity for HCC with gadoxetic acid contrast-
enhanced MRI is improved (95.2%) when including wash-out criteria for the
hepatobiliary phase (29). 

Hepatoblastoma
Hepatoblastoma (HB) is a primary malignant liver neoplasm that recapitulates the
developing fetal/embryonal liver with variable proportions of epithelial and mes-
enchymal elements and is the most common malignant liver tumor in children
(30). HB is frequently a solitary mass; however, poor prognostic features include
multifocality (20%), the involvement of the vena cava or all three hepatic veins
(10%), the involvement of the portal bifurcation or both right and left portal veins
(10%), extrahepatic tumor extension (5%), and tumor rupture (5%) (31). Grossly,
tumors are nodular or bosselated, and cut surfaces depend on tumor components,
96 Lachance E et al.

ranging from soft tan-brown in fetal patterns to gritty whitish speckles of osteoid
in mesenchymal patterns. A variegated appearance can result from cystic degen-
eration, necrosis, or hemorrhage.
Microscopically, HBs are classified according to the International Pediatric
Liver Tumors Consensus Classification of Liver Hepatoblastoma (32). Tumors
can be either purely epithelial or contain a mixture of epithelial and mesenchymal
components (spindle cells, osteoid, or cartilage) (32). Epithelial tumors may
show a single or combination of histologic patterns including: fetal (characterized
by trabecular growth of fetal-type hepatocytes, clear or finely granular cytoplasm
depending on cytoplasmic glycogen content, variable degrees of mitotic activity,
and nuclear pleomorphism), embryonal (primitive tubule formation, angulated
nuclei, and high nuclear-to-cytoplasmic ratios), macrotrabecular (fetal or
embryonal patterns in trabeculae greater than 5 cells thick), small cell
undifferentiated (diffuse sheets or nests of cells with minimal cytoplasm, frequent
apoptosis, mitotic activity, and necrosis), and cholangioblastic (formation of
small ducts). The fetal pattern can be further subclassified into those with
low-mitotic activity (well-differentiated), mitotically active (crowded fetal), and
pleomorphic types. The small cell undifferentiated pattern can be further
subdivided into SMARCB1 (INI1) positive and negative types with the help of
IHC (2). Mixed epithelial and mesenchymal tumors are further subclassified
into those with and without teratoid features. Given the wide variation in
histological patterns, biopsies may not be representative of the tumor overall.
Neoadjuvant chemotherapy can markedly alter the histologic appearance with
changes including fibrosis, necrosis/hemorrhage, and the presence of osteoid-like
tissue (30).
Ultrasound is commonly the first modality used when detecting HB, with
further characterization by CT or MRI. MRI is advantageous in children, given
the lack of radiation, and likely has increased sensitivity and specificity with
gadoxetic acid MRI. Ultrasound appearances are dependent on the underlying
histologic pattern. Generally, these lesions are lobular, well-circumscribed
hypoechoic or heterogeneous lesions. Calcification, necrosis and vascular inva-
sion may be seen. HB is typically heterogeneous and hypoattenuating to the liver
parenchyma on CT. Speckled or amorphous calcification is seen in greater
than 50% of lesions (33). Enhancement is typically less than that of the liver;
however, peripheral arterial enhancement may be observed (33). On MRI,
hepatoblastomas are typically heterogeneously T2WI hyperintense and T1WI
hypointense. On contrast-enhanced MRI, HB is typically hypointense on the
arterial, portal-venous, and delayed phases (34–35). The presence of a liver mass
and elevated serum AFP in a child less than 4 years should alert the radiologist
to the possibility of HB.

BILIARY LESIONS

A network of bile canaliculi and a system of bile ducts located within portal tracts
transports bile into larger intrahepatic ducts, exiting at the liver hilum. The fol-
lowing sections discuss lesions involving bile ducts and includes mucinous cystic
neoplasm and intrahepatic cholangiocarcinoma.
 Radiologic-Pathologic Correlation of Liver Tumors 97

Mucinous cystic neoplasm

Mucinous cystic neoplasm (MCN) is a cyst-forming epithelial neoplasm of the
liver that occurs almost exclusively in females. MCNs generally do not communi-
cate with the biliary tree and show a characteristic subepithelial ovarian-type
stroma, ranging from focal (8%) to diffuse (36%) (36). These two features can
help differentiate it from other cystic neoplasms of the liver (for example, intra-
ductal papillary neoplasm of the bile ducts). MCNs are uncommon hepatic cysts
(11%), frequently solitary, have an average size of 11 cm (range 5–23 cm), located
predominantly in the left lobe (72%), and rarely have associated carcinomas (6%)
(36). Grossly, MCNs are well-demarcated multiloculated cystic lesions with
smooth inner surfaces containing mucinous, hemorrhagic, or serous fluid. Solid
grey-white areas are concerning for invasive carcinoma. Microscopically, cyst
walls are lined by either flattened epithelial cells or columnar/cuboidal cells with
eosinophilic or mucinous cytoplasm and basally oriented nuclei and are delimited
by a fibrous capsule. Low- and intermediate-grade dysplasia is common in MCNs,
and cases must be thoroughly sampled and examined to exclude invasive adeno-
carcinoma, often with associated high-grade dysplasia (37). Estrogen and proges-
terone receptor IHC can be used to highlight the characteristic stroma in certain
cases (2). 
Radiology literature historically and still frequently describes MCNs of the
liver and bile ducts as biliary cystadenomas and biliary cystadenocarcinomas.
Across modalities (ultrasound, CT, MRI), MCNs are typically large, solitary, mul-
tilocular cystic lesions with smooth margins and internal septations (38). Varying
mucin concentrations produce differing signal intensity within each locule of the
multilocular cystic lesion; intermediate to high signal on T2WI, and low to high
signal on T1WI. This may produce a “stained-glass” appearance, more commonly
described in mucinous ovarian epithelial cystic neoplasms. 
The presence of rapid growth, solid nodularity (particularly if enhancing),
coarse calcification, irregular septations, and internal hemorrhage is suspicious
for cystadenocarcinoma, although multiple studies have proven that imaging is
not sufficiently diagnostic (39).

Intrahepatic cholangiocarcinoma
Intrahepatic cholangiocarcinoma (ICCA) (Figure 4) is a primary intrahepatic
malignant neoplasm of epithelial cells with biliary differentiation. Two subtypes
with unique clinicopathological features have been described: (i) large duct ICCA,
arising from large perihilar intrahepatic bile ducts proximal to the right and left
hepatic ducts, and (ii) small duct ICCA arising from small bile ducts or ductules
in a peripheral location. Large duct and small duct subtypes account for 45% and
55% of all ICCAs, respectively (40), with 60% of all ICCAs being greater than
5 cm in size (41).
Grossly, large duct ICCAs often present as mass lesions around the larger cali-
ber bile duct branches with invasion into the surrounding hepatic parenchyma.
Some large duct ICCAs will not form a mass lesion but rather spread along bile
ducts in a diffuse and longitudinal manner (referred to as a “periductal infiltrat-
ing” pattern of growth) (42). They may be found in association with macroscopi-
cally visible intraductal papillary neoplasms of the bile ducts, not discussed within
98 Lachance E et al.

Figure 4.  Representative images of intrahepatic cholangiocarcinoma. A and B. The lesion shows
low signal on the T1 weighted image (A) and intermediate to high signal on the T2 weighted
image (B) with surrounding mild peripheral intrahepatic biliary dilatation. C. The lesion
shows intense diffusion restriction on the b=150 s/mm2 image. D, E, and F. Following
gadolinium administration, the lesion shows heterogeneous arterial (D) and portal phase (E)
enhancement (predominantly in the periphery), with progressive centripetal filling-in,
completely hyperenhancing on the delayed phase (F) at 5 minutes. G. A partial hepatectomy
from a separate patient shows a tan-white sclerosing lesion grossly, with extensive
involvement of a probed large duct. H. Microscopically an invasive adenocarcinoma (white
arrow) is seen arising from a large duct (black arrow). I. Well-formed glands are seen
invading a nerve. Slides are stained with Hematoxylin and Eosin. Total image magnification:
H - 25X; I - 200X.

this chapter. Small duct ICCAs typically present as a peripheral mass-forming

lesion that is white-grey and nodular, often within a background of cirrhosis (62%
of cases) (40). 
Microscopically, large duct ICCAs are invasive adenocarcinomas of tubular or
solid growth patterns with extensive desmoplastic reaction, sclerosis of larger bile
ducts, portal tract involvement, frequent perineural and lymphatic space inva-
sion, and frequent lymph node metastasis. They may be found in association with
microscopically visible biliary intraepithelial neoplasms, which are not discussed
in this chapter. Small duct ICCAs show variable proportions of slit-like lumens in
cord-like growth patterns, or distinct lumens in tubular growth patterns found
replacing the hepatocytes in regenerative nodules. All ICCAs have small-to-
medium-sized cuboidal or columnar cells, eosinophilic or vacuolated cytoplasm,
small nuclei, and variably prominent nucleoli. The diagnosis of adenocarcinoma
is typically readily made by morphology alone; however, poorly differentiated
 Radiologic-Pathologic Correlation of Liver Tumors 99

cases may lack apparent glandular differentiation and require IHC to exclude
other primary neoplasms (for example, poorly differentiated HCC). Combined
hepatocellular cholangiocarcinoma is a rare malignancy composed of both
unequivocal HCC and ICCA components within the same tumor and may occa-
sionally be encountered in biopsy specimens. ICCA does not show a specific mor-
phology or IHC profile. Therefore, careful clinical and radiological correlation is
essential to rule out a metastatic adenocarcinoma from another site.
On imaging, the mass-forming pattern of ICCA demonstrates a
well-circumscribed, often large, lobulated mass frequently with satellite nodules.
The periductal-infiltrating pattern demonstrates growth along bile ducts with an
elongated, branching, spiculated appearance. Capsular retraction, more typical of
the mass-forming pattern, is characteristic but not pathognomonic for ICCA.
Upstream biliary ductal dilatation is typical but not specific.
Mass-forming ICCAs are typically hyperechoic on ultrasound if larger than
3 cm and isoechoic or hypoechoic if smaller (43). A peripheral hypoechoic rim is
seen in approximately 35% of cases (43). On unenhanced CT, they are typically
hypoattenuating (43), and calcification may be present. They are typically hetero-
geneously T1WI hypointense and demonstrate a T2WI hyperintense peripheral
rim with central hypodensity, which may be hyperintense in the setting of internal
necrosis (43). The enhancement pattern on MRI and CT is typically arterial phase
continuous rim-like enhancement (targetoid) with progressive, concentric cen-
tripetal fill-in on the portal-venous phase and persistent enhancement on delayed
imaging (43).
Periductal-infiltrating ICCAs are typically enhancing on CT and MRI (43). Bile
duct imaging, including endoscopic retrograde cholangiopancreatography (ERCP)
and magnetic resonance cholangiopancreatography (MRCP), are highly sensitive
and specific for bile duct narrowing and stricturing, which may be otherwise subtle.
MRCP is 97% sensitive and 98% specific for the presence of obstruction (44).
Differentiating HCC from ICCA is important as locoregional treatments for
HCC (for example, ablative or transcatheter therapy) are not effective for ICCA,
and liver transplantation is a relative contraindication for ICCA due to the high
risk of recurrence (45). A helpful finding is the presence of tumor thrombus, more
commonly associated with HCC, while ICCA more commonly causes venous
occlusion from extrinsic compression.

VASCULAR MESENCHYMAL LESIONS

Fine vascular sinusoids transport blood between hepatocytes from terminal

branches of both the hepatic artery and hepatic portal vein, components of the
portal tracts, to hepatic venules that drain into the hepatic vein. The following
sections discuss lesions involving liver vasculature and includes cavernous hem-
angioma, epithelioid hemangioendothelioma, and hepatic angiosarcoma.

Cavernous hemangioma
Cavernous hemangioma (CH) of the liver is a benign venous malformation with
no malignant potential. CHs are most often solitary and are categorized according
100 Lachance E et al.

to size: small (less than 5 cm), large (5–10 cm), and giant (>10 cm). Frequencies
are approximately 30%, 35%, and 35%, respectively (46). Grossly, CHs are well-
circumscribed, soft, spongy purple-red lesions located subcapsular or deep in the
liver parenchyma. Microscopically, they consist of variably sized large vascular
spaces lined by a bland epithelium and are embedded in a fibrous stroma. Thrombi
may be present, and when extensive, can result in significant sclerosis of the
lesion. Although well-circumscribed grossly, the tumor interface microscopically
is irregular, with separate hemangioma-like vessels located 0.1–2 cm away from
the lesion in 80% of cases (47).
As the most common benign liver tumor, CHs are frequently detected inciden-
tally on imaging and can show a wide variety of atypical appearances. CHs are
typically well-circumscribed and hyperechoic on ultrasound, with a well margin-
ated lobulated contour that is better appreciated in larger CHs. Heterogeneity is
more common with giant CHs, although the lesion periphery typically remains
echogenic (48). A central scar may be seen in a minority of giant CHs, mimicking
FNH, and calcification is rare, sometimes seen in the central scar. No central flow
is demonstrated using color Doppler; however, peripheral vascularity can be
present. A “reverse target” morphology with hyperechoic rim may be seen in a
minority, with metastasis or HCC as the diagnosis of exclusion on a background
of normal or cirrhotic liver parenchyma, respectively (49). Where CEUS is
available, enhancement patterns parallel CT and MRI.
On CT, attenuation and enhancement across phases mirror blood pool (for
example, the aorta), due to the underlying disorganized venous histology, with
more rapid (“flash”) filling and delayed/centripetal filling observed in small and
giant CHs, respectively. The peripheral nodular contour with discontinuous
enhancement is more conspicuous with large and giant CHs. The central scar, if
present, is characteristically non-enhancing. MRI has the greatest sensitivity and
specificity for diagnosing CH. On MRI, CHs are T1WI isointense or hypointense,
with hypointensity typically observed in giant CHs. If present, the central scar is
markedly T1WI hypointense. CHs are typically T2WI hyperintense due to the
long T2 relaxation time in these lesions. MRI contrast enhancement patterns are
similar to those described for CT; however, some CHs show slow enhancement
with fill-in on delayed phase images, and rarely a centrifugal (inside-out) enhance-
ment pattern (50).
Typically reserved for diagnostic clarification in patients unable to undergo
MRI or contrast-enhanced studies, Tc-99m labelled RBC scans demonstrate pho-
topenia or decreased activity relative to liver parenchyma on early dynamic scans
with persistent filling of large and giant CHs on delayed imaging. This differenti-
ates CHs from vascular tumors such as HCC, HCA, and FNH. When combined
with multi-headed SPECT the positive predictive value approaches 100%, with
sensitivity markedly improving in larger lesions (20% in lesions 0.5–0.9 cm;
100% in lesions >1.4 cm) (51).

Epithelioid hemangioendothelioma
Epithelioid hemangioendothelioma (EHE) (Figure 5) of the liver is a malignant
endothelial neoplasm. EHE is frequently multifocal (87%), bilobar (81%), and
arises in the absence of chronic liver disease (52). Grossly, there is nodular or
 Radiologic-Pathologic Correlation of Liver Tumors 101

Figure 5.  Representative images of hepatic epithelioid hemangioendothelioma. A. Transverse

ultrasound image of the liver demonstrates a centrally hyperechoic lesion with a thin
hypoechoic halo (“targetoid” appearance) in the right lobe of the liver with multifocal
peripheral targetoid, heterogeneous and hypoechoic lesions involving both lobes (not
shown). B and C. These lesions show low signal on the T1 weighted image (B) and
intermediate to high signal on the T2 weighted image (C). One lesion in segment 7 at the
subcapsular border is associated with focal capsular retraction (*). D, E, and F. Following
gadolinium administration, the lesions show targetoid peripheral enhancement during the
arterial (D) and portal (E) phases, and centripetal fill-in on the delayed phase (F). G. A
representative slice of liver from a complete total hepatectomy of a separate patient shows
multiple small red-brown lesions (white arrow) scattered throughout the parenchyma
grossly. H and I. Bland epithelioid and spindled cells (I) are embedded in a fibrous and
myxohyaline background (H). Slides are stained with Hematoxylin and Eosin. Total image
magnification: H - 25X; I - 400X.

multinodular growth of firm, gray-white tumor.  Microscopically, epithelioid or

stellate spindled cells are arranged in cords, small nests, or single cells, and
embedded in a fibrous or myxohyaline background. Nuclei show fine chromatin
with small nucleoli, and the eosinophilic cytoplasm can show notable well-defined
intracytoplasmic vacuoles/lumina of variable sizes. The mitotic rate is variable.
WWRT1-CAMTA1 and YAP1-TFE3 translocations are present in approximately
80% and 5% of EHEs, respectively (53). A panel of IHC stains is typically required
to confirm endothelial origin (for example, CD31 and ERG) and to exclude the
possibility of carcinoma, although keratin stains may also be positive. CAMTA1
IHC stain is positive in a significant proportion of cases, given the associated gene
rearrangement (2).
102 Lachance E et al.

EHE is most commonly identified as coalescent, peripheral, target-like hepatic

nodules with capsular retraction on a normal liver parenchyma background.
Calcification is uncommon. Ultrasound is nonspecific; nodule echogenicity is
variable, often hypoechoic (54). Unenhanced CT is also nonspecific, demonstrat-
ing hypoattenuating nodules. A ring or target appearance is common on MRI and
CT, with 2 or 3 rings of differing enhancement/intensity. A 3-ring lesion consists
of a dominant central non-enhancing or delayed enhancing fibrous myxoid
stroma, which may demonstrate necrosis, an enhancing inner peripheral ring of
proliferating tumor cells, and an outer avascular non-enhancing rim of tumor
infiltrating into sinusoids and portal branches (55–57). On T1WI, nodules are
typically markedly hypointense centrally, with a less hypointense peripheral rim.
On T2WI, small nodules are typically hyperintense, with larger lesions demon-
strating a target pattern of central hyperintensity, a thin inner peripheral hypoin-
tense ring, and a hyperintense outer ring (56). Nodules may demonstrate a
“Lollipop” sign; a well-defined, predominantly hypoenhancing nodule represent-
ing the spherical candy portion, and thrombosis within an associated vein, termi-
nating smoothly at the edge or within the lesion rim, representing the stick (58).

Hepatic angiosarcoma
Hepatic angiosarcoma (HAS) is a primary malignant endothelial neoplasm. HAS
is predominantly bilobar (80%) and can show variable patterns of growth, includ-
ing multinodular (40%), solitary (20%), dominant mass with satellite nodules
(20%), and diffusely infiltrating (20%) (59). Grossly, tumors are poorly defined
and heterogenous, with solid gray-white areas and hemorrhagic areas showing
large blood-filled spaces. Microscopically, tumors also display heterogeneity. Well-
formed anastomosing vascular channels alternate with solid sheets of tumor lack-
ing vascular formation. Tumor cells can show high-grade features, including
epithelioid or spindled cells with marked nuclear pleomorphism, hyperchromatic
nuclei, and abundant mitoses. Infarct, necrosis, and fibrosis are common. At the
periphery of the tumor, malignant cells characteristically grow along and replace
endothelial cells present in pre-existing sinusoids. A high index of suspicion is
required in some cases, along with a panel of IHC stains, to make a correct
diagnosis. Epithelioid HAS in particular can mimic carcinomas, melanomas, and
lymphomas by morphology alone, and therefore a panel of IHC stains is generally
recommended, including markers of vascular differentiation such as CD31 and
ERG (keratins and EMA may be positive) (2).
HAS demonstrates a variable appearance on radiologic imaging, reflecting the
underlying pleomorphic histology. HAS is commonly heterogeneous and hyper-
vascular, with multiplicity, multiorgan involvement, rapidly progressive growth,
and a history of exposure to particular carcinogens (for example, vinyl chloride
polymers) pointing toward the underlying diagnosis, which ultimately relies on
histology. Hemorrhage, necrosis, and calcification may be seen. A background of
liver cirrhosis or fibrosis is reported in approximately 40% of patients (60). On
ultrasound, lesions are generally heterogeneous. On unenhanced CT, lesions are
typically hypoattenuating, although heterogeneity may be seen, particularly in the
setting of hemorrhage. On MRI, lesions are heterogeneous and may have an ill-
defined outline. They may be T1WI hypointense but can show high signal with
 Radiologic-Pathologic Correlation of Liver Tumors 103

intratumoral hemorrhage. On T2WI, HAS shows heterogeneous intermediate-to-

high signal intensity. On CT and MRI, HAS enhancement parallels blood pool,
typically bizarre and disordered, with centrifugal and centripetal filling being the
predominantly described progressive enhancement patterns (45, 60–61).
Peripheral arterial enhancement is typically circumferential rather than nodular
and discontinuous, as in CHs. Flash-filling of smaller lesions is also observed. In
a multi-institutional review of 35 cases, arterial phase foci of hyperenhancement
were seen in 89.7%, with none demonstrating portal or hepatic vein invasion,
which may help in distinguishing HAS from HCC (60). Rapid growth on serial
imaging and ancillary findings such as lymph node or extrahepatic organ metas-
tases are recognized features (45).

METASTATIC MALIGNANCIES

Metastatic malignancies are common in the liver (more common than primary
malignancies) and frequently multifocal; however, solitary nodules and diffuse
parenchymal involvement without discrete nodule formation is possible. Tumors
are primarily carcinomas (92%), of which adenocarcinomas account for the
majority (75%), but melanomas (2.4%) and sarcomas (1%) are also found (62).
The most frequent primary sites of origin for adenocarcinomas are colorectal
(46%), pancreas (10%), breast (8%), and lung (4%) (62). Gross and microscopic
appearances differ based on the primary site of origin. Many cases of metastases
involving the liver can be quickly sorted out based on a combination of tumor
morphology/immunophenotype, clinical history, and radiology. Some cases, how-
ever, present a much greater challenge for pathologists, radiologists, and clinicians
alike, and a multidisciplinary team-based approach can be tremendously helpful
in determining the site of origin and/or appropriate management.
The radiologic appearance of metastatic malignancies also varies, with
appearances mimicking those seen at the respective sites of primary malignancies.
Common examples include hypovascular epithelial metastases (for example,
colon, lung, and gastric), hypervascular metastases (for example, renal cell
carcinoma, melanoma, thyroid, and neuroendocrine tumors), and cystic liver
metastases (for example, mucinous or serous primary malignancies from the
ovary, colon, and pancreas).
In comparison to the gold standard of resection or intraoperative ultrasound,
conventional ultrasound has modest sensitivity for metastases (50–76%), improv-
ing with contrast-enhancement (82–86%) (63), showing similar sensitivities to
contrast-enhanced CT and MRI (64–65). Ultrasound appearances are varied with
the following typical appearances: hypoechoic in hypovascular metastases, cen-
trally hyperechoic and peripherally hypoechoic (targetoid) in aggressive metasta-
ses, and hyperechoic in hypervascular metastases.
Unenhanced CT has poor sensitivity for most metastases, with high sensitiv-
ity for calcification, a nonspecific but common finding in mucinous lesions.
Metastases are typically T1WI hypointense and T2WI hyperintense, heteroge-
neous in morphology, and may be associated with an ill-defined outline.
Enhancement characteristics are reflective of the predominant hepatic
arterial  supply, appearing similar on CT and conventional extracellular
­
104 Lachance E et al.

contrast-enhanced MRI, with the latter demonstrating higher sensitivity and

specificity. Arterial enhancement with wash-out on portal-venous and delayed
phases is characteristic for malignant liver lesions (including metastases) with a
sensitivity of 24.5% and specificity of 100% on MRI (66). Cystic metastases are
typically non-enhancing. 
On meta-analysis, gadoxetic acid-enhanced MRI was superior to contrast-
enhanced CT (sensitivity 86.9–100% vs. 51.8–84.6%, specificity 80.2–98.0% vs.
77.2–98.0%) (67). On gadoxetic acid-enhanced MRI, metastases are typically
conspicuously hypointense on delayed phase imaging as a result of the lack of
hepatocytes and biliary ducts. Additional sequences such as DWI may increase
MRI sensitivity and specificity. 18F-fluorodeoxyglucose PET/CT (FDG PET/CT) is
principally utilized for the detection of extrahepatic metastases, particularly in
lymph nodes, with high background liver FDG uptake slightly limiting the detec-
tion of intrahepatic metastases, as seen when evaluating for colorectal cancer liver
metastases, where MRI demonstrated superior sensitivity (84–86%), when com-
pared to FDG PET/CT (72%) (68).

CONCLUSION

As research progresses, diagnostic criteria and diagnoses evolve. Novel tech-

niques develop and characteristic molecular alterations are discovered. With an
expansion of knowledge comes an increasing need for effective communication
and feedback between diagnostic specialties. This chapter serves as a reference
for diagnostic findings in common and/or critical liver lesions. Radiologic
and pathologic correlation will continue to be necessary for providing patients,
their ­families, and their clinical care teams accurate diagnoses to guide future
management.

Conflict of interest: The authors declare no potential conflicts of interest with

respect to research, authorship and/or publication of this article.

Copyright and permission statement: The authors confirm that the materials
included in this chapter do not violate copyright laws. Where relevant, appropri-
ate permissions have been obtained from the original copyright holder(s), and all
original sources have been appropriately acknowledged or referenced.

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doi.org/10.1007/s00330-018-5469-0
 6
Achieving a Cure: The Next Frontier
in Hepatitis B Treatment
Tina Boortalary1 • Brianna Shinn1, 2 • Dina Halegoua-DeMarzio1,2 •
Hie-Won Hann1,2
1
Department of Medicine, Thomas Jefferson University Hospital, Philadelphia, PA, USA;
2
Division of Gastroenterology and Hepatology, Department of Medicine, Thomas Jefferson
University Hospital, Philadelphia, PA, USA
Author for correspondence: Hie-Won Hann, Division of Gastroenterology and
Hepatology, Department of Medicine, Thomas Jefferson University Hospital, Philadelphia,
PA, USA. Email: [email protected]
Doi: https://doi.org/10.36255/exonpublications.livercancer.2021.ch6

Abstract: Since the discovery of the hepatitis B virus (HBV) by Blumberg et al. in
1965, significant progress has been made in understanding the pathogenesis of
the virus and creating an effective vaccine. In the past two decades, several a­ ntiviral
therapies have reduced the incidence of HBV-related hepatocellular carcinoma.
The nucleos(t)ide analogues have succeeded in decreasing the viral load to
­undetectable levels but have been unable to eradicate the virus due to the persis-
tence of covalently closed circular DNA in the hepatocyte nucleus. Despite being
on successful antiviral therapy for multiple years, patients are still at risk of devel-
oping hepatocellular carcinoma. Recently, a number of different drug targets have
been identified that intervene on the viral replication cycle or host immune
system. This chapter discusses the immunopathogenesis of the virus, the
­
­effectiveness of nucleos(t)ide analogues, and recent therapeutic developments. In
light of robust progress achieved in antiviral therapy, the cure for hepatitis B is
likely on the horizon.

Keywords: cccDNA; chronic hepatitis B; hepatocellular carcinoma; hepatitis B

treatment; hepatitis B virus

In: Liver Cancer. Sergi CM. (Editor). Exon Publications, Brisbane, Australia.
ISBN: 978-0-6450017-2-3; Doi: https://doi.org/10.36255/exonpublications.livercancer.2021
Copyright: The Authors.
License: This open access article is licenced under Creative Commons Attribution-NonCommercial
4.0 International (CC BY-NC 4.0) https://creativecommons.org/licenses/by-nc/4.0/

109
110 Boortalary T et al.

INTRODUCTION

Despite the discovery of an effective vaccine approximately 40 years ago, hepatitis B

remains an important public health concern and cause of liver-related morbidity
and mortality. Over 250 million people are carriers of the virus, and nearly one
million deaths occur yearly from complications of cirrhosis and hepatocellular
carcinoma (HCC) (1). Although there is currently no cure for the hepatitis B virus
(HBV), significant progress has been made in understanding its pathogenesis and
hepatocarcinogenesis. This has paved the way for the development of therapies
that have decreased the incidence of HBV-associated HCC significantly.
Our understanding of HBV began in 1965 when Blumberg and colleagues
identified a new and unknown antigen in the blood sample of an Australian
aborigine, subsequently named the “Australia (AU) antigen”(2, 3). This antigen
reacted with an antibody in the serum of a transfusion-dependent patient with
hemophilia. Furthermore, the AU antigen was found to be present in the donor’s
blood, which caused hepatitis in the recipient. This observation confirmed the
antigen to be responsible for post-transfusion hepatitis. The AU antigen later
became known as the Hepatitis B surface antigen (HBsAg). In 1970, Dane et al.,
using electron microscopy, identified the whole virus particle, which was later
named the “Dane particle” (4, 5). In 1972, Magnius et al. identified another solu-
ble viral protein and named it “hepatitis B e antigen (HBeAg) (6). They found that
the presence of HBeAg increased the risk for transmission of the virus. Initial stud-
ies made the serologic diagnosis of hepatitis B possible and paved the way for
more rigorous virologic investigation that ultimately led to the creation of the first
vaccine. For his work on hepatitis B, Blumberg received the Nobel Prize in
Medicine in 1976. Furthermore, Blumberg and his colleague Millman at the Fox
Chase Cancer Center in Philadelphia hypothesized that HBsAg would be able to
provoke an immune response and protect patients from hepatitis B. This finding
was the basis for the creation of the first plasma vaccine, Heptavax B, in 1982 by
Maurice Hilleman and his group at Merck. Heptavax B was derived from the
blood of infected individuals (7). There was concern that viral pathogens could be
transmitted through the vaccine in the era of HIV/AIDS and “non-A, non-B”
hepatitis. Ultimately, this led to the development of a second vaccine in 1986
using recombinant DNA technology. The next generation vaccine was created by
utilizing yeast cells for the transcription of surface antigen coding sequences (8).
It was the first vaccine to use recombinant DNA methods and is still used today.
The recombinant vaccines are highly effective, protecting against chronic
­hepatitis B at a rate of 94–98% for at least 20 years (9, 10). After vaccination, it
has been shown that hepatitis B surface antibody concentrations decline dramati-
cally to less than 10 mIU/mL in more than 50% of patients within 5–10 years
(11–14). Despite the decrease of anti-HBs over time, studies have shown that a
primary series of hepatitis B vaccine remains effective in preventing infection for
more than 20 years (15, 16).
In 1992, the World Health Organization (WHO) recommended that coun-
tries integrate hepatitis B vaccine into their national immunization schedules by
1997 (17). The hepatitis B vaccine was named by the WHO as the first “cancer
vaccine” given its ability to prevent HBV-associated HCC. Due to the knowledge
that patients infected with hepatitis B have a 14–45% risk of developing
 Treatment for Hepatitis B 111

HBV- associated HCC, the hepatitis vaccine has been one of the greatest
­achievements in public health.

HBV LIFE CYCLE AND IMMUNOPATHOGENESIS

HBV is an enveloped, partially double-stranded DNA virus that is a member of the

Hepadnaviridae family. The virus consists of an outer envelope, coated with
HBsAg and an inner nucleocapsid composed of hepatitis B core antigen subunits.
Within the nucleocapsid resides the viral genome, which is covalently cross-
linked to the DNA polymerase. The genome consists of a partially double-
stranded, relaxed circular (rc) DNA and contains four overlapping open reading
frames (ORF) that encode viral polymerase (Pol), surface antigen, nucleocapsid,
X proteins, and other regulatory sequences (18). The replication cycle of the virus
is illustrated by Zeisel et al. and Levrero et al. (Figure 1) (19, 20). The virus uses
sodium taurocholate cotransporting polypeptide (NTCP), a transmembrane trans-
porter predominantly expressed in the liver to gain entry to the host cell (21).
This receptor’s normal function is to aid in bile transport in the enterohepatic
circulation. After being taken in by receptor mediated endocytosis, the virions are

Entry inhibitors
• Lipopeptides, RNA interference,
ed
Targeting rat Arrowhead, Tekmira,
e.g. Myrcludex-B eg NA
Int V D
• Cyclosporin derivatives cccDNA HB Alnylam, GSK

nuclear cccDNA subgenomic RNAs

cap pA
cap pA
cap pA

enveloped pgRNA
RC-DNA cap pA
virion
mature
core protein
RC-DNA
+ P protein
nucleo- immature
capsid RNA
Caspsid inhibitors
nucleocapsid
Novira, Janssen, Abbvie
Inhibitors of HBsAg AssemblyPHARMA, Gilead
release
Replicor plasma
membrane
Polymerase inhibitors
• Nucleoside analogues Immune modulation
• Non-nucleosides • Toll-like receptors agonists,
PD-1
Y Gilead, Roche
“exaustion” • Anti-PD-1 mAb,
“high antigen load” CD8+
BMS, Merck
T cell
B cell • Vaccine therapy:
Transgene, Gilead, Roche
Dysfunctional
Insufficient Innovio, Medimmune, ITS
T-cell response
B-cell response
Current Opinion in Virology

Figure 1.  Hepatitis B replication cycle and potential drug targets. Adapted from Levrero et al.
(20). Used with permission from original copyright holder.
112 Boortalary T et al.

uncoated and the nucleocapsid is taken to the location of the host cell’s nucleus
where the rcDNA is imported through the nuclear pore into the nucleus.
The first step in replication involves the transformation of the rcDNA into a
covalently, closed, circular DNA (cccDNA) mini-chromosome by host cell repair
mechanisms (22). The cccDNA serves as the transcriptional template by utilizing
host RNA polymerase II for the production of a four viral mRNA intermediates:
a “larger-than-genome” pregenomic RNA and shorter, subgenomic transcripts.
The pregenomic RNA (pgRNA) is essential for viral replication given that it
serves as the template for DNA synthesis and for the translation of capsid pro-
teins (HBcAg) and HBV DNA polymerase. Once the viral DNA polymerase is
formed, it attaches to its own transcript forming the pgRNA-polymerase complex.
At the same time, the core particles construct a capsid around the pgRNA-
polymerase complex. Within this complex, viral DNA synthesis takes place
through reverse transcription and the rcDNA is formed. The shorter mRNA is
translated and undergoes additional processing to form proteins that become
HBV surface antigens in the viral envelope (23). In addition to forming new
virions, the capsid containing rcDNA is shuttled back to the nucleus to replenish
cccDNA. Therefore, sources for cccDNA include both the new entry of viral par-
ticles as well as the translocation of newly synthesized rcDNA from the cyto-
plasm to the nucleus (24). Even in the case of HBsAg seroconversion, the
persistence of cccDNA in the nucleus is believed to cause infectivity and poses
as an obstacle for cure (25).

NATURAL HISTORY OF HEPATITIS B VIRUS AND

CARCINOGENESIS

HBV is not directly cytotoxic to liver cells. Liver disease is mediated through inter-
play of HBV replication and the host immune response. During the replication
process, HBsAg is taken up and degraded by antigen presenting cells and dis-
played on MHC class I and class II molecules. A T-lymphocyte mediated response
leads to apoptosis or release of cytokines that can down-regulate viral replication.
The persistence of HBV DNA activates the host immune system leading to the
chronic inflammation implicated in the development of HCC.
The connection between HBV and hepatocarcinogenesis has been firmly estab-
lished since the 1980’s. Although the molecular mechanisms of HBV carcinogen-
esis are not fully understood, it is believed to be due to a multifactorial process
involving chronic liver inflammation, chromosomal instability and disruptions of
cell signaling (26). Studies have shown that HBV DNA integration can lead to
duplications, deletions, and chromosomal translocations of important genes that
code for proteins (P53, Ras, TGF-B, Wnt/β-Catenin, cyclins A and D1) involved
in cell signaling, proliferation and apoptosis (27, 28). The viral genotype is a risk
factor for malignancy, with genotype C having a higher risk of causing HCC
(29, 30). Specific mutations in pre-core regions and open reading frames coding
PreS1/PreS2/S have been established as enhancers of the carcinogenic potential of
HBV (29). HBV protein X is thought to play an important role in regulating cel-
lular transcription, apoptosis pathways, and cellular proliferation (31).
 Treatment for Hepatitis B 113

TREATMENT

The goal of HBV treatment has been to reduce viral load and halt progression of
liver disease leading to HCC. To date, these goals have been achieved by the use
of pegylated interferon (PegIFN) and nucleos(t)ide analogues (NAs) including
lamivudine, entecavir, telbivudine, adefovir, tenofovir disoproxil fumarate and
tenofovir alafenamide. The exact mechanism of PegIFN is not known, however
they are believed to act through immunomodulation and, to a lesser extent, by
exerting a weak antiviral effect by intervening in several steps of the viral replica-
tion cycle. Unlike the NAs, PegIFN has a finite course of treatment. Its use is
limited by a significant adverse effect profile, a requirement for weekly injections,
and a large portion of non-responders. Lamivudine is an oral antiviral drug and
was the first NA approved for the treatment of hepatitis B in 1998. Subsequently,
more NAs including adefovir, entecavir, telbivudine, tenofovir disoproxil fuma-
rate and tenofovir alafenamide became available. The best outcome expected of
current antivirals is the seroconversion of HBsAg, but this clearance is only
achieved in 10% of patients (32, 33).

Prevention of HCC in patients who were treated with

antiviral therapy
While a cure for HBV infection is not available, several studies have shown a
decrease of the development of HCC in patients who were treated with antiviral
therapy. Liaw et al. showed that continuous treatment with lamivudine delayed
clinical progression in those with chronic hepatitis B and advanced fibrosis.
Patients who were assigned to receive lamivudine over placebo saw significantly
lower Child-Pugh scores and decreased rates of HCC at a median duration of
32 months (34). This placebo-controlled study had to be stopped early due to the
significant difference between the control and treatment groups. Hosaka et al.
demonstrated that patients treated with entecavir had lower rates of HCC (3.7%)
compared to non-treated HBV patients (13.7%) within five years (p < 0.001). In
a stratified analysis, entecavir reduced the risk of HCC four-fold in cirrhotic
patients, but was not significant in those without cirrhosis (35). Kim et al. showed
that HCC was decreased in those treated with tenofovir disoproxil fumarate (36).

Prevention of recurrence of HCC with antiviral therapy

Surgical resection is the preferred treatment for patients with local HCC and
­preserved liver function (37). Nonetheless, there is a high rate of recurrence in
those with underlying chronic HBV. Persistent or high hepatitis B viral loads with
or without hepatic inflammation have been associated with HCC recurrence after
resection (37–39). The NAs have been widely used in patients following surgical
resection or ablation of HCC. They have generally showed trends toward improved
liver function and prolonged disease-free survival.
A number of studies from eastern Asia (40–44) and the US (45, 46) have
shown a decrease in HCC recurrence and improved mortality following antiviral
therapy. In 2005, Piao et al. studied outcomes in patients with HBV-related HCC
114 Boortalary T et al.

who were treated with lamivudine. The study demonstrated that compared to an
untreated, matched control group, patients with HCC who were treated with
lamivudine achieved significant improvements in Child-Pugh scores at 24 months
and lower mortality rates from liver failure. However, there was no change in HCC
recurrence or overall survival between the two groups (40). Kuzuya et al. (41),
compared outcomes in patients with HBV-related HCC who received lamivudine
(n = 16) and those who did not (n = 33) following treatment for HCC (resection
or radiofrequency ablation). The lamivudine group had better Child-Pugh scores
at the time of HCC recurrence. Although there was no difference in rates of HCC
recurrence or survival between the groups, patients treated with lamivudine had
better remnant liver function at the time of HCC recurrence and were more likely
to receive curative treatment (41). Kubo et. al. demonstrated that patients treated
with lamivudine after curative resection for HBV-related HCC were more likely to
have improved tumor-free survival compared to those who did not receive lami-
vudine treatment (43). Prolonged disease-free survival and overall survival rates
were reported in another study in which patients received antiviral treatment after
surgery for HBV-related HCC (44). In a large Taiwanese cohort, patients receiving
NAs after liver resection were found to have lower risk of HCC recurrence and
improved survival rates (42). In a two-stage longitudinal study that included a
randomized clinical trial, Yin et al. also demonstrated that patients receiving NAs
after surgery for HBV-related HCC had significantly decreased risk of HCC recur-
rence and HCC-related deaths (47). In studies from the US, Hann et al. demon-
strated an increased median survival time of 80 months in patients receiving NAs
for HBV-related HCC (after local tumor ablation) compared to a median survival
time of 16 months in those without NA treatment (45, 46). Many of these studies
were pooled in a meta-analysis performed by Yuan et al. and showed that the one
and three-year recurrence rates of HCC and disease-free survival were signifi-
cantly better in patients treated with NAs after curative treatment (liver transplant,
hepatectomy, radiofrequency ablation) for HBV-related HCC (48). More recently,
a study with 487 subjects demonstrated that HCC recurrence and HCC-related
death were significantly decreased in patients who received antiviral treatment
after hepatectomy for HCC compared to those who did not (p = 0.28, p = 0.004,
respectively) (49).

Persistent risk for HCC despite successful HBV suppression

Despite successful viral suppression, the risk of HCC persists even a decade after
treatment. A large European study evaluated the risk factors and performance
of  risk scores in entecavir-treated patients. Among the 744 patients studied,
14  patients developed HCC in a median time of approximately 3 years, 12 of
whom (85%) had reached a virological response (HBV DNA level<80 IU/mL)
before the diagnosis of HCC. An association between virologic suppression and
HCC (HR = 0.87; p = 0.87) was not observed (50). A large retrospective study
from Greece showed that virologic remission, defined as undetectable DNA levels
(defined as concentrations <200 IU/ml) did not significantly affect the incidence
of HCC in all patients or those with cirrhosis (51). Despite undetectable HBV
levels, HCC was noted to recur at an average of 4–5 years after treatment, espe-
cially in those with cirrhosis (52). Another study from Japan showed that 13 of
 Treatment for Hepatitis B 115

133 patients with HBV who achieved undetectable viral levels developed HCC.
They identified cirrhosis as an associated risk factor (53). It is important to recog-
nize that “undetectable” is a relative term given the variation in measurements
conducted by different laboratory assays. At the Liver Disease Prevention Center,
Division of Gastroenterology and Hepatology of Thomas Jefferson University
Hospital, long-term follow-up data has demonstrated HCC recurrence in patients
even after a decade of successful viral suppression (54, 55) In our observational
cohort, as of October 2020, 17 patients developed HCC despite having been
treated for 9–19 years (median 13 years) with undetectable HBV DNA for 3–12
years (median 8 years) (Table 1). This is among the longest known follow up stud-
ies on the development of HCC in patients taking antiviral therapy. Recurrence of
HCC has also been noted after the treatment of the initial tumor despite successful
viral suppression. We have observed six patients who, after first HCC treatment,
started antiviral therapy which led to successful HBV suppression for years
(5–11 years) and yet developed subsequent new or recurrent HCC (Figure 2).
Li et al. compared patients who received NA treatment (lamivudine with or with-
out adefovir, n = 43) after hepatectomy for advanced HCC and those who did not
receive NAs (n = 36) (56). There was no difference in the recurrence rate of HCC
or disease-free survival between treatment group and control. Kuzuya et al. also
found that the recurrence rates of HCC were not decreased after the administra-
tion of nucleotide analogues, but more favorable Child-Pugh scores were observed
at the time of HCC reoccurrence (41).

ARE WE CLOSE TO ACHIEVING HBV CURE?

The currently available NAs target the reverse transcription of HBV RNA and have
been proven to delay progression to cirrhosis. However, despite their success in
halting inflammation in the liver, the risk of HCC has persisted. As demonstrated
in Figure 1, NAs suppress HBV replication at one point in the cycle, but do not
eliminate the cccDNA, which remains in the hepatocytes. There are currently a
number of clinical trials, summarized in Table 2, that strategize different
approaches to eradicating HBV (57). Each drug targets a specific point of the viral
replication cycle or acts indirectly on the host immune functions. Targets can
interfere with replication through the following mechanisms: (i) inhibition of viral
entry; (ii) blocking rcDNA entry into the hepatic nucleus; (iii) prevention of
cccDNA formation; (iv) prevention of mRNA transcription; (v) inhibition of cap-
sid formation; (vi) inhibition of reverse transcription; and (vii) inhibition of HBV
release into circulation.

Entry inhibitors
Blocking HBV entry into the hepatocyte can prevent the earliest step of infection.
This is the target of Bulevirtide or formally known as Myrcludex B. The drug
works by inhibiting attachment of viral pre-S1 protein to NTCP, therefore block-
ing the mechanism for entry (58). It has been approved by the European
Commission as the first drug effective for hepatitis D and hepatitis B co-infection.
 116
TABLE 1 Development of HCC in patients with cirrhosis on long-term antiviral therapy

Change in Yrs on Yrs with

Date Child Class Date HCC anti-HBV Tx HBV Age (yr) at Tumor size HBVDNA
Pt StartTx on Tx Dx at HCC Dx DNA(-)* HCC Dx at Dx at HCC Dx Anti-HBV Tx Status
1 4/1998 7/2007 9 3 53 1.1 Junction UD LAM + TDF Alive
Boortalary T et al.

B→A
2 1/1998 B→A 3/2008 10 8 68 2.8 Rt UD LAM + TDF Dead
3 5/1998 A→A 2/2008 10 7 76 1.8 Lt UD LAM + TDF Alive
4 7/2001 B→B 9/2010 9 4 54 2.8 Rt UD LAM + TDF Dead
5 8/2004 B→B 11/2010 16 4 53 3.9 Rt UD LAM + TDF Alive
6 7/2001 B→B 1/2011 10 5 55 2.8 Rt UD LAM + TDF Dead
7 2/2004 A→A 6/2013 9 8 57 2.5 Lt med UD TDF Dead
8 2/1996 A→A 7/2013 17 10 73 1.6 Rt UD TDF Dead
9 8/1997 A→A 6/2014 17 6 54 2.2 Lt lat UD ETV Alive
10 3/2004 B→B 6/2013 9 7 57 2.5 Lt UD TDF Dead
11 7/2001 A→A 6/2014 13 7 54 2.2 Lt UD TDF Alive
12 5/1996 A→A 10/2014 18 10 74 3.4 Rt UD LAM + TDF Dead
13 2/2000 A→A 10/2014 14 12 62 3.8 Rt UD ETV + TDF Alive
14 2/2000 A→A 4/2015 15 12 62 3.4 Rt UD TDF Alive
15 2/2000 B→A 5/2015 15 12 65 3.8 Rt UD TDF Alive
16 12/1998 A→A 8/2017 19 8 64 2.0 Rt UD LAM + TDF Alive
17 2/2008 A→A 6/2019 11 10 57 2.2 Rt UD ETV + TDF Alive
Dx: Diagnosis, ETV: Entecavir, LAM: Lamivudine, Lt: Left, Pt: Patient, Rt: Right, TDF: Tenofovir Disoproxil Fumarate, Tx: Treatment, UD: Undetectable,
*Years with HBV DNA (-) at diagnosis of HCC
 Treatment for Hepatitis B 117

HCC in Chronic HBV

85
80 10 years
75 11 years
5 years
Age (years)

70 3 years Patient 1
5 years Patient 2
65
10 years Patient 3
60 7 years Patient 4
Patient 5
55 5 years Patient 6
50
45
First HCC Second HCC Third HCC
Event

Figure 2.  Development of subsequent new and recurrent HCC while on successful antiviral
treatment. Graph of 6 patients demonstrates long duration time between initial and
subsequent HCC diagnoses.

TABLE 2 Different compounds in development for

treatment of hepatitis B—adapted from
Hepatitis B Foundation (57)
Hepatitis B Cure Watch
Direct Acting Antivirals Phase of Trial
Entry Inhibitors (Bulevirtide) II
Silencing RNA (siRNA) Preclinical, I, II
Capsid or Core Inhibitors Preclinical, I, II
Reverse Transcriptase Inhibitors Approved, I, III
HBsAg Inhibitors I, II
Indirect Acting Antivirals Phase of Trial
Therapeutic Vaccines Preclinical, I, II
Innate Immune Defense Pathway I, II
Host Acting Pathway I, II
Gene Editing Preclinical

Phase 2 data has shown that there is a reduction of viral HDV RNA after b
­ ulevirtide
when combined with tenofovir or peg-interferon-α (59, 60).

Small interfering RNAs

Small interfering RNAs (siRNAs) exhibit their effect by binding and interfering
with mRNA transcripts. Because the HBV has overlapping reading frame, siRNAs
are an appealing approach to eliminate multiple different proteins (61). There are
several agents at various points in clinical trials. ARC-520 was an earlier siRNA
118 Boortalary T et al.

developed against HBV, which showed reductions of serum HBsAg concentrations

when given as monthly injections with oral entecavir in HBeAg positive patients.
There was less of a reduction in HBeAg-negative patients (62). This was likely
because the drug targeted cccDNA-derived pgRNA, the source of HBsAg in
HBeAg-positive patients and not the integrated HBV DNA seen in HBeAg-negative
patients (63). JNJ-3989 and AB-729 were second-generation siRNAs that showed
reductions of HBsAg regardless of HBeAg positivity (64–66).

Capsid inhibitors
HBV core protein is necessary for capsid formation, genome packaging and deliv-
ery of rcDNA into the nucleus. Therefore, core inhibitors or core modulators dis-
rupt the viral lifecycle by inducing the assembly of defective capsids or capsids
that lack genetic material. By this mechanism, they are thought to intervene on
multiple points of the lifecycle, even targeting cccDNA by causing its destabiliza-
tion and preventing its replenishment. There are several different classes of
core protein assembly modulators (CpAMs) including phenylpropenamide which
have normal morphologies but lack genetic material (empty capsids) and
hetero-aryl-dihydropyrimidines (HAP) derivatives, which have aberrant mor-
phologies (67). The first compound studied in humans in phase 1 b study was
NVR3-778. It showed a 1–2 log reduction in HBV DNA levels but no change in
HBsAg levels until the drug was combined with peg-IFN (68).
Core inhibitors are the first viral specific compounds that are capable of target-
ing cccDNA. A recent phase 2 study (69) demonstrated that ABI-H0731, a core
protein inhibitor, not only interfered with viral capsid formation and entry of
rcDNA into the nucleus, but also decreased levels of pgRNA. In that study, the
combination of the ABI-H0731 and entecavir showed greater reductions of viral
DNA load and pgRNA levels by week 24 in treatment naïve, HBeAg+, individuals
compared to entecavir alone (p = 0.0452). When patients were switched from
entecavir to the combination of both, they saw immediate decline in HBV DNA
and pgRNA levels (Figure 3). By week 48, the mean HBV DNA and pgRNA levels
declined 6.3 logs and 3.0 logs from baseline, respectively. The accelerated decline
after the addition of ABI-H0731 was attributed to the reductions in the cccDNA
pool, the only known source of HBV pgRNA (70).

Reverse transcriptase inhibitors

NAs act by interfering with synthesis of viral DNA from pgRNA. Currently six
NAs are available for treatment of HBV. As synthetic compounds, they resemble
endogenous nucleosides that are incorporated into growing DNA strands dur-
ing replication. They halt DNA synthesis and terminate chain elongation by
inhibiting reverse transcriptase (71). Besifovir dipivoxil maleate, a novel drug in
this class was approved for use in Korea after phase III trials (72). It has been
shown to have antiviral efficacy comparable to that of tenofovir disoproxil fuma-
rate (TDF) after 48 weeks of treatment and has been associated with reduced
bone and renal toxicities (73). Lipid conjugates of TDF are also in development
and have been shown to increase activity against HBV and have a better safety
profile (74).
 Treatment for Hepatitis B 119

HBV DNA levels HBV pgRNA levels

0 0
ETV ETV

Mean HBV pgRNA Change from

Mean HBV DNA Change from

–1 ETV+731 ETV+731
–1
ETV to Combo
Baseline (log10 IU/mL)

Baseline (log10 U/mL)

–2 ETV to Combo
–2
–3
–4 –3
9 of 21 patients with
–5 DNA <20 IU/mL
–4
–6
–5
–7 4 of 21 patients with
Study 202 211 Study 202 211 pgRNA <135 U/mL
–8 –6
Baseline 10 20 30 40 50 60 Baseline 10 20 30 40 50 60
Treatment Week Treatment Week

Figure 3.  Study 202 and 211 results of Entecavir alone, Entecavir+ABI-H0731 (Core Protein
Inhibitor), and switch from entecavir to combo on HBV DNA levels and pgRNA levels. Adapted
from Sulkowski et al. (70). Used with permission. The combination of the ABI-H0731 and
entecavir showed greater reductions of viral DNA load and pgRNA levels by week 24 in
treatment naïve, HBeAg+, individuals compared to entecavir alone (p = 0.0452). When
patients were switched from entecavir to the combination of both, they saw immediate
decline in HBV DNA and pgRNA levels.

HBsAg inhibitors
HBsAg release inhibitors interfere with the production of HBsAg, which is
needed for the virus to enter and leave the liver cell. Most of viral antigen in the
blood is HBsAg and circulating levels are almost entirely made of HBV subviral
particles (SVP). These small particles are produced independently of viral repli-
cation and are non-infectious. However, it has been suggested that SVPs can
neutralize antibodies to the virus and suppress other aspects of host immune
function (75, 76). The nucleic acid polymer (NAP), REP 2139, is a synthetic
RNA that is taken up by hepatocytes and suppresses the production of SVPs
derived from integrated HBV DNA or cccDNA. A phase 2 randomized control
trial showed that the addition of NAPs to TDF and pegIFN allowed significantly
more patients to achieve HBsAg levels below 1 IU/mL and HBsAg seroconver-
sion (77). Another method by which HBsAg is inhibited is through interruption
in the intracellular re-localization of the protein to the Golgi apparatus. This is
the mechanism utilized by Benzimidazole, BM601, which decreases HBsAg and
HBV release (78).

cccDNA inhibitors
Directly inhibiting cccDNA would be the optimal HBV therapy, but aside from
the capsid inhibitor (ABI-H0731) that has shown promising results, no drugs
have shown to target cccDNA in clinical trials (79). Potential mechanisms to tar-
get cccDNA may involve inhibiting its formation, transfer into the nucleus, con-
version to cccDNA, or transcription after cccDNA is produced. Several
gene-editing technologies utilize site-specific nucleases to cleave HBV DNA. The
zinc finger nucleases (ZFNs), transcription activator-like effector nucleases
120 Boortalary T et al.

(TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR/

Cas system) have been developed to interrupt cccDNA in vitro (75). After a spe-
cific coding sequence is matched with the desired segment of DNA, a double-
stranded break is made in the genetic material. The host cell’s DNA repair
machinery is used to repair the DNA, introducing mutations that render the gene
ineffective (80). Although the field of gene editing is exciting, a lot of work needs
to be done to eliminate “off target” effects and improve delivery of the large
compounds.

Antivirals targeting host immunity

Immune modulation of the host’s innate and adaptive immune responses is
another area of interest in HBV therapeutic research. Vaccine technology that
aids in treatment rather than prevention is sought. These new vaccines seek to
generate HBV specific T cells responses. However, results have not been very
promising. Toll-Like Receptor agonists are being developed to help recognize
the virus and produce cytokines necessary for activating the innate immune
system (81). Checkpoint inhibitors used in the field of oncology are also being
investigated for the treatment of HBV. They are targeted against proteins such
as programmed cell death protein 1 (PD-1)/programmed death-ligand 1
(PD-L1), and block inhibition of T-cells and restore their activity in recognizing
antigens (82). Another category of therapeutics targets cellular inhibitor of
apoptosis proteins (cIAPs). cIAPs diminish TNF-signaling and apoptosis dur-
ing hepatitis B infection, leading to the persistence of infected hepatocytes and
HBV (83).

CONCLUSION

While significant advancements have been made regarding the understanding

of immunopathogenesis and management of hepatitis B, no curative treatment
for hepatitis B exists. Furthermore, the virus has been shown to re-emerge and
cause significant mortality even at undetectable levels. The ultimate goal of
the new therapies will be to eradicate cccDNA from hepatocytes. The cure for
the virus will likely rely on the combination of multiple therapies, targeting
different aspects of the replication cycle. With the recent advances in drug
development, the treatment for hepatitis B may be very different in the near
future.

Conflict of interest: The authors declare no potential conflicts of interest with

respect to research, authorship and/or publication of this chapter.

Copyright and permission statement: The authors confirm that the materi-
als included in this chapter do not violate copyright laws. Where relevant,
appropriate permissions have been obtained from the original copyright
holder(s), and all original sources have been appropriately acknowledged or
referenced.
 Treatment for Hepatitis B 121

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 7
Locoregional Therapies for Bridging
and Downstaging Hepatocellular
Carcinoma Prior to Liver Transplant
Sandeep A. Ponniah • Andreas G. Zori • Roniel Cabrera
Division of Gastroenterology, Hepatology, and Nutrition, University of Florida,
Gainesville, FL, USA
Author for correspondence: Andreas G. Zori, Division of Gastroenterology,
Hepatology, and Nutrition, University of Florida, Gainesville, FL, USA.
Email: [email protected]
Doi: https://doi.org/10.36255/exonpublications.livercancer.2021.ch7

Abstract: Hepatocellular carcinoma is the most common primary liver malig-

nancy and is a common indication for liver transplantation. To qualify for liver
transplantation, the size and number of tumors must be within established c­ riteria.
The Milan criteria is the most well-established of these criteria, however there is
evidence these criteria can be safely expanded without affecting outcomes. While
awaiting liver transplantation, locoregional therapy can be used as bridging ther-
apy to maintain the tumor burden within criteria. Locoregional therapy can also
be used to decrease tumor burden within transplant criteria, a process called
downstaging. For tumors <3 cm, thermal ablation—most commonly using a
radio-frequency probe—is preferred when feasible and offers tumor control
approaching that of resection. Larger or multifocal lesions are usually treated with
either trans-arterial chemoembolization or yttrium-90 trans-arterial radioemboli-
zation. The choice between these two interventions is generally based on institu-
tional preference as neither has demonstrated survival advantage in the transplant
population. However, single center trials show longer time to progression,
improved downstaging success, and less microvascular invasion in patients treated

In: Liver Cancer. Sergi CM. (Editor). Exon Publications, Brisbane, Australia.
ISBN: 978-0-6450017-2-3; Doi: https://doi.org/10.36255/exonpublications.livercancer.2021
Copyright: The Authors.
License: This open access article is licenced under Creative Commons Attribution-NonCommercial
4.0 International (CC BY-NC 4.0) https://creativecommons.org/licenses/by-nc/4.0/

127
128 Ponniah S A et al.

with trans-arterial radioembolization. More recently stereotactic body radiation

therapy has demonstrated efficacy in patients who are not candidates for other
locoregional therapy or have progressed despite prior locoregional therapy.

Keywords: bridging therapy; downstaging; locoregional therapy; Milan criteria;

transarterial chemoembolization

INTRODUCTION

Therapies for hepatocellular carcinoma (HCC) have evolved over the last several
decades, but liver transplantation remains the only curative option in patients
who are not candidates for resection. Liver transplantation allows for the cure of
both the tumor and any underlying chronic liver disease. However, more than
70% of patients present with advanced disease that does not meet the criteria for
transplantation (1). The first and most widely accepted criteria for liver transplan-
tation in patients with HCC are the “Milan criteria”, developed by Mazzaferro
et  al. (2). The Milan criteria include stage T1 (1 lesion <2 cm) and stage T2
(1 lesion 2–5 cm or up to 3 lesions ≤3 cm), tumors without vascular invasion,
lymph node involvement, or extrahepatic metastases (3). When these criteria are
met, four-year survival after transplant is >80% with recurrence rates below 15%
(2). In the United States, the United Network for Organ Sharing (UNOS) affords
patients who meet stage T2 disease Model for End-Stage Liver Disease (MELD)
“exception points” due to poor survival associated with HCC that is not accounted
for in standard MELD scoring system. Although the amount of HCC exception
points has varied over time and by country, the current standard exception criteria
adopted by UNOS assigns points equal to the mean MELD at transplant minus 3
of all liver transplants recipients (except status 1A/1B, living donor, donation after
cardiac death donor and donors more than 500 miles from recipient hospital) in
the last 180 days within 250 nautical miles of the listing center. Patients who do
not meet the Milan criteria (greater than stage T2) can be eligible for liver trans-
plantation if they receive locoregional therapies that reduce their tumor burden
and maintain it within Milan criteria for 6 months. Lesions that are eligible for
downstaging beyond Milan criteria are discussed in the following sections.

EXPANDED TRANSPLANT CRITERIA

A growing body of literature supports that the Milan criteria can be safely expanded
to allow patients with more advanced disease access to liver transplantation, and
under the right circumstances, they have comparable outcomes to patients that
meet the Milan criteria. As such, modifications to the Milan criteria have been col-
lectively termed “expanded criteria.” In 2001, Yao et al. (4) from the University of
California, San Francisco (UCSF), published a study with expanded criteria
termed the “UCSF criteria”: 1 lesion ≤6.5 cm or up to 3 lesions with the largest
lesion ≤4.5 cm, with a total diameter of 8 cm (Table 1). In this study, patients had
1- and 5-year survival rates that were 90% and 75.2% respectively. In 2007, this
 Locoregional Therapy Prior to Liver Transplant 129

TABLE 1 Selection Criteria for Liver Transplantation

Milan criteria
• Most common eligibility criteria for LT among patients with HCC
• Single lesion ≤5 cm, or up to 3 lesions each ≤3 cm
• No evidence of extra-hepatic metastases or vascular invasion

Expanded criteria
• UCSF: Single lesion ≤6.5 cm or ≤3 lesions with the largest being ≤4.5 cm and a total diameter
≤8 cm
• Up-to-7: 7 cm as the sum of the size of the largest tumor and the number of tumors. No vascular
invasion.
• Toronto criteria: Any tumor size or number. All lesions require a liver biopsy and must NOT
show poor differentiation. No extra-hepatic metastasis, venous/biliary thrombosis OR cancer
related symptoms.

UNOS Criteria for Downstaging

• Single lesion >5 cm but ≤8 cm
• 2-3 lesions each ≤5 cm with total diameter of all lesions ≤8 cm
• 4-5 lesions each ≤3 cm with total diameter of all lesions ≤8 cm
HCC, hepatocellular carcinoma; LT, liver transplant; MC, Milan Criteria; UCSF, University of California in San Francisco

same group from UCSF published a prospective study that showed a 5 year sur-
vival rate of 81%, which was similar to Milan criteria but with the added benefit
of being able to transplant an additional 5–20% of patients initially not included
by Milan criteria (5). Several studies have validated UCSF criteria having similar
rates of survival compared to the Milan criteria (6).
In 2009, Mazzaferro et al. (7) described the Up-to-Seven criteria, where the
sum of the total number of lesions and size in centimeters (cm) of the largest
lesion can be up to 7 if vascular invasion and metastases are absent. In the initial
study of over 1,500 patients, 5-year survival rates in patients that met Up-to-
Seven criteria were similar (71.2%) to those that met the Milan criteria (73.3%) (7).
This lead to the creation of the “Metro Ticket Calculator”, which provides 3- and
5-year overall survival probabilities based on characteristics of HCC lesions (8).
In 2016, the Toronto criteria was proposed, which do not include size or tumor
number restrictions, but based candidacy on absence of extrahepatic disease,
venous thrombi, cancer-related symptoms, and high-risk features on biopsy. In
validating this criteria, patients transplanted outside MC (with the majority being
beyond UCSF criteria but within Toronto criteria), had 5-year survival rates that
were not statistically different to those within MC (68% vs 78%) (9). Other trans-
plant centers outside the USA and Canada such as in Italy, England, Japan,
Australia, New Zealand, and China have similar “expanded” criteria, and have
shown comparable outcomes to the Milan criteria. Although individual centers
may elect to transplant patients outside of Milan criteria, in the United States,
tumor burden must be brought within the Milan criteria with locoregional ther-
apy to qualify for standard MELD points, which is discussed in more detail in the
downstaging section.
130 Ponniah S A et al.

Bridging therapy
Once listed for transplant, HCC patients may experience long waiting times and
growth of tumor beyond the Milan criteria, putting them at risk for dropout from
the waiting list. Waiting times often exceed 1 year and can be as long as 2 years
(10). Dropout rates while awaiting liver transplantation have been noted to be
25% at 6 months, 38% at 12 months and up to 55.1% at 18 months (11). Bridging
therapy is locoregional therapy administered to patients on the liver transplanta-
tion waitlist to prevent tumor progression and decrease dropout rates, acting as a
“bridge” until a suitable donor is obtained. Use of bridging therapy has steadily
increased and is now used for most patients. Patients derive more benefit from
locoregional therapy as the expected liver transplant list increases (12).
Recommendations from the 2010 International Consensus Conference and EASL/
EORTC Clinical Practice Guidelines suggest bridging therapy for all patients with
HCC within the Milan criteria, with wait times >6 months for liver transplantation
(13). In those meeting criteria for the Milan criteria, bridging therapy has decreased
waitlist dropout to 0–10% (12). Risk factors responsible for an increased risk of
dropout include tumors >3 cm or multifocal disease, serum AFP >200 ng/ml,
waitlist time >6 months, and poor response to bridging therapy (12). The data on
survival benefit is discussed later in this chapter.

Downstaging
“Downstaging” involves treating tumors outside of the Milan criteria with locore-
gional therapy to decrease tumor burden, allowing for application of standard
MELD exceptions and ultimately liver transplantation. UCSF first proposed a
downstaging protocol in 2005 where lesions outside the Milan criteria were eligible
for downstaging. Lesions included were: 1 lesion >5 cm and ≤8 cm, 2–3 lesions
each ≤5 cm or 4–5 lesions each ≤3 cm with a total diameter ≤8 cm. In a study from
this group between 2002 and 2012 (14), survival at 5 years post liver transplanta-
tion in patients outside the Milan criteria but who were successfully down staged
was identical to the Milan criteria control group (78%). Similarly in 2018, in a more
recent multi-center study from this same group, 5-year post liver transplant sur-
vival in down staged patients was excellent at 80% (14). Based on these results,
UNOS adopted the UCSF inclusion criteria for downstaging (UNOS-DS) above and
in 2019 finalized the policy verbiage to clearly outline this criterion. Consequently,
patients that are initially outside the Milan criteria but meet UNOS-DS receive auto-
matic approval for MELD exception if they remain within the Milan criteria after
locoregional therapy. Candidates that are outside UNOS-DS who receive downstag-
ing locoregional therapies to the Milan criteria must be referred to the National
Liver Review Board (NLRB) for consideration for MELD exception.
The options for locoregional therapies in downstaging are discussed in detail
below. The decision on optimal locoregional therapy is contingent on multiple
factors, but the Barcelona Clinic Liver Cancer (BCLC) staging system is often used
to help make this decision (8). BCLC staging factors tumor burden, liver function
and patient performance status to stratify patients by risk. Downstaging patients
often have advanced stage disease and based on BCLS, trans-arterial chemoembo-
lization (TACE) is most utilized followed by trans-arterial radioembolization and
ablation (15).
 Locoregional Therapy Prior to Liver Transplant 131

Tumor biology
Over time, the focus of liver transplantation guidelines has shifted beyond tumor
size and number to tumor biology and behavior (16). As a result, UNOS currently
requires a 6-month waiting period prior to granting MELD exception points. This
allows for observation of the tumor to ensure that it does not have aggressive biol-
ogy which rapidly progresses to metastasis and can significantly increase the
chances of post-transplant recurrence. Downstaging is similarly helpful in assess-
ing tumor biology, in that tumors which cannot be brought within transplant
criteria with locoregional therapy confer a poor prognosis. Conversely, tumors
outside of the Milan criteria that respond to downstaging protocols have been
noted with favorable histological changes, including a lack of microvascular inva-
sion, low tumor grading, and lack of satellite lesions (17) which are similarly seen
in patients within the Milan criteria. While histological data, such as degree of
differentiation and microvascular invasion, is helpful in predicting tumor behav-
ior and stratifying risk, it is often not available in pre-transplant settings since in
most cases biopsy is not necessary to make the diagnosis of HCC. Therefore, there
is significant interest in identifying serum markers which can classify tumor biol-
ogy non-invasively.
Serum alpha-fetoprotein (AFP) has been studied extensively as a serum marker
for HCC, and combined with tumor burden prognosticates HCC better than
tumor burden alone (18–20). Absolute AFP >1,000 ng/ml is a strong predictor of
vascular invasion and tumor recurrence. Patients with AFP >1,000 ng/ml had
higher 5-year recurrence rates (53%) compared to patients with AFP 100–1,000
ng/ml (26.8%) and <100 ng/ml (16.2%) (21).
AFP levels have now been adopted by UNOS as a marker for exclusion for liver
transplantation. Patients within the Milan criteria applying for MELD exception
must have an AFP ≤1,000 ng/ml. Patients >1,000 ng/ml can still be granted stan-
dard MELD exception points provided they undergo LRT with drop of AFP <500
ng/ml and remain within this range. If AFP >500 ng/ml after LRT, a review must
be filed with the NLRB.

LOCOREGIONAL THERAPIES

In this section, we discuss the most commonly used locoregional therapies in

bridging and downstaging prior to transplant; TACE, radioembolization with
yttrium-90 (Y-90), thermal ablative therapy and stereotactic body radiation ther-
apy (Table 2). Factors that affect decision-making when formulating an optimal
treatment strategy include tumor stage, performance status, tumor location, sever-
ity of liver disease, organ availability and tumor biology/behavior (22).

Trans-arterial chemoembolization
TACE is one of the most commonly used bridging therapies and involves
­intra-arterial administration of a chemotherapeutic agent, usually doxorubicin,
mitomycin and/or cisplatin followed by an embolizing agent. Lipiodol, an
oily  radiopaque marker used as an emulsifying agent, is coupled to the
 132
TABLE 2 Overview of Locoregional Therapies

Therapy Mechanism Primary Application Complications Advantages Limitations

cTACE Embolic ischemia Larger (>3 cm) tumors PES Studied extensively More systemic toxicity than
augmented Tumors <3 cm not Liver failure can be repeated DEB-TACE. Technical
by emulsified amenable to GI ulcers variability and non-
chemotherapy resection/ablation Liver abscess standardized protocols
Ponniah S A et al.

Renal dysfunction Cannot be used with PVT

DEB-TACE Embolic ischemia Larger (>3 cm) tumors Similar to cTACE More controlled and sustained More costly than cTACE.
augmented by Tumors <3 cm not drug delivery than cTACE Cannot be used with PVT
chemotherapeutic amenable to
drug eluting beads. resection/ablation
TARE Radiation induced Larger (>3 cm) tumors RILD Safe in PVT Requires pre treatment
cell death from Tumors <3 cm not Radiation induced Slower TTP than TACE mapping angiography
Y-90 microspheres, amenable to pneumonitis Outpatient procedure More costly than TACE
minimal embolic resection/ablation Biliary stricturing Requires higher level of
effect Enteritis expertise
Thermal High frequency Smaller (<3 cm) tumors, Thermal injury to adjacent Similar outcomes as surgical Heat sink effect.
Ablation alternating currents ≤3 nodules organs. resection for tumors <3 cm Limited efficacy in tumors
(RFA) induce thermal Improved outcomes Liver capsule rupture. (curative) >3 cm
injury and necrosis combined with TACE Risk of peritoneal seeding Excellent safety profile
for tumors 3-5 cm treating peripheral tumors
SBRT Multiple nonparallel Larger (>3 cm) tumors Few: nausea, vomiting, Alternative BT for patients with Few comparative studies
radiation beams Tumors <3 cm not GI ulcers (rare) decompensated liver disease with other LRTs
delivered in high- amenable to that are not LT candidates or
dose radiation resection/ablation failed other LRTs.
fractions Can treat lesions near adjacent
organs, unlike ablation
No heat sink effect
Spares liver from RILD unlike
TARE
BT, bridging therapy; cTACE, conventional TACE; LRT, locoregional therapy; LT, liver transplant; OS, overall survival; PES, post embolization syndrome; PVT, portal vein thrombosis;
RFA, radiofrequency ablation; TTP, time to progression; RILD, radiation induced liver injury
 Locoregional Therapy Prior to Liver Transplant 133

chemotherapeutic agent and used as a carrier to deliver the drug to the tumor.
Induction of ischemia and tumor necrosis through embolization of the tumor’s
microcirculation is the primary mode of tumor killing, which is then augmented
by the chemotherapeutic agent (23). Trans-arterial embolization can also be done
without a chemotherapeutic component, termed bland embolization, however
this is performed less commonly. Currently, TACE using drug-eluting beads (DEB-
TACE) has become more commonly used than conventional TACE, and as such
in some literature the term TACE is used interchangeably with DEB-TACE. DEB-
TACE involves the injection of embolization beads that are loaded with cytotoxic
chemotherapeutic agents to target a tumor. Contrary to conventional TACE where
there is concern of systemic exposure, drug-eluting beads release the chemothera-
peutic in a controlled and sustained manner, minimizing systemic toxicity and
improving drug delivery to the tumor (24). Both forms of TACE are used for treat-
ing larger (>3 cm) HCC without vascular invasion or extrahepatic spread where
the patient has preserved liver function. Damage to normal liver parenchyma from
arterial embolization is usually mitigated by the liver’s dual blood supply from the
portal vein and the hepatic artery. However, recurrent treatment with TACE
increases risk of complications and some centers recommend no more than 3–4
treatments times a year (1).
The most common complication of TACE, occurring in approximately 50% of
patients, is post-embolization syndrome (25). This occurs less frequently in
patients treated with DEB-TACE than TACE. Post-embolization syndrome con-
sists of a constellation of right upper quadrant pain, nausea, fever, fatigue, ele-
vated liver enzymes, bilirubin and mild to moderate ileus. Typically, symptoms
last 3–4 days and self-resolve in 7–10 days. Because this occurs frequently, in
most centers patients are observed as inpatients following TACE. Despite most
chemotherapy being localized to liver, other systemic complications include nau-
sea, vomiting and very rarely bone marrow suppression.
Although serious complications of TACE remain uncommon, liver failure can
occur as a result of ischemic damage. In a meta-analysis (26), liver failure was seen
in 7.5% of patients with HCC treated with TACE. However, liver decompensation
occurs most often in patients who had impaired liver function prior to TACE
treatment; therefore, patient selection is critical to reduce the risk of decompensa-
tion. Other complications of TACE include gastroduodenal ulcers (3–5%), hepatic
abscesses (2%), bile duct injury including strictures (0.5–2%) and renal dysfunc-
tion (2%). Rare but fatal complications include pulmonary and cerebral lipiodol
embolization. Overall, mortality rates from TACE are low at less than 1%, with
rates between 2–3% in patients with large tumors that develop tumor lysis syn-
drome (26). Small prospective studies and a meta-analysis have compared tradi-
tional TACE to DEB-TACE in the past and have suggested lower rates of
complications with similar tumor control in the latter (27–30). In the PRECISION-V
study (31), the largest comparative randomized controlled trial between both,
DEB-TACE showed less hepatotoxicity, better tumor response, and lower doxoru-
bicin related adverse effects compared to conventional TACE (in patients with
Child-Pugh B, ECOG1, bilobar disease and recurrent disease). In the same study,
less post procedural pain was noted with DEB-TACE. By contrast, two retrospec-
tive studies (32, 33) showed a higher risk of liver and biliary injuries with DEB-
TACE than conventional TACE. While the results are favorable, there still exists
some heterogeneity when comparing conventional TACE to DEB-TACE.
134 Ponniah S A et al.

Overall TACE therapies are safe in most patients. Absolute contraindications for
TACE therapies include decompensated cirrhosis (Child-Turcorrte-Pugh B
score  >8), compromised portal vein flow or thrombus, extensive tumor in both
lobes of the liver, and renal failure. Several other relative contraindications exist
and include but are not limited to: serum bilirubin >2mg/dl, lactate dehydrogenase
>425 U/L, AST >100 units/L, tumor burden >50% of the liver, severe comorbidi-
ties, and untreated esophageal varices with bleeding risk. The ideal population for
TACE therapies is asymptomatic patients with solitary or few HCC tumors, well
compensated liver disease, and without metastatic disease or vascular invasion.
The use of TACE is not standardized and varies in protocol and type of embolic
and chemotherapeutic agent use. Few randomized studies compare TACE to sup-
portive care alone, however two landmark studies by Lo et al. (34) and Llovet
et al. (35) show a clear survival benefit in TACE compared with supportive treat-
ment. One-year survival rates were 57% (34) and 82% (35) compared to 32% and
63% in the control groups respectively. Mean survival was significantly longer
with TACE (28.6 months) compared to supportive care (17.9 months; P = 0.009)
(35). Additionally, in a recent systemic review (25) including 10,000 patients,
TACE showed response rates in >50% of patients, with 1 year survival rates >70%.
Current European Association for the Study of the Liver (EASL) guidelines sup-
port TACE with strong recommendation, citing high quality evidence. When
comparing TACE to transcatheter arterial radioembolization (TARE [discussed
below]), TACE is supported by a much greater number of trials, while TARE is
largely supported by single-center studies with variable patient cohorts (36).
While studies comparing TACE with TARE report no difference in overall sur-
vival, a single treatment with TACE is significantly less expensive than TARE (36).
Several favorable studies have advocated the use of TACE as effective bridging
therapy. Lower dropout rates (3–13%) have been noted by several studies com-
pared to historical data (3). In one of the most favorable studies by Graziadei et al.
(37), no patient experienced tumor progression following locoregional therapy
with TACE. Five-year survival rates after liver transplantation were high at 93%
and tumor recurrence rates were notably low (2%) despite long waiting times
prior to liver transplantation (mean of 178 days). While these studies show posi-
tive results, other studies (38–40) are less favorable and show no benefit in post
liver transplantation survival or tumor recurrence.
Downstaging using TACE has been shown to be effective, however success
rates have been variable in the literature. In the first ever study on locoregional
therapy in 1997, 62% of patients outside of MC were down staged with improved
5-year survival when compared to patients who could not be down staged with
TACE or did not receive TACE (1). Other studies have shown a similar favorable
role of TACE in downstaging patients to MC, however results remain inconsistent
with success rates ranging from 24% (41) to 90% (42).
Despite continued improvement in technique and patient selection, there con-
tinues to be debate regarding the ideal patient population, tumor burden and
tumor biology for TACE in the treatment of HCC.

Transcatheter arterial radioembolization

TARE with Yttrium-90 glass microspheres induces extensive tumor necrosis with
a favorable safety profile. Y-90 is a beta-emitting radioisotope of Yttrium that is
 Locoregional Therapy Prior to Liver Transplant 135

attached to a bead and injected into the hepatic artery, designed to emit radiation
over a very small distance within the liver (2.5 mm). Y-90 undergoes beta decay
and irradiates the nearby tumor causing cell death. In comparison to TACE, this
procedure provides therapeutic effect through radiation instead of embolization.
This results in reduced toxicity and damage to the liver parenchyma and can be
used safely in patients with portal vein thrombosis (43, 44). The half-life of Y-90
is 2.67 days, and almost all radiation is delivered to the tumor within 2 weeks
after treatment. TARE has been used for bridging, downstaging, as a palliative
treatment for advanced disease, and as adjuvant therapy for surgically resected
HCC (45, 46).
Before the use of TARE, visceral angiography mapping with technetium-99
and SPECT-CT is required to detect shunts to the GI tract or lung. If shunts can-
not be reduced to less than 20% of the hepatic artery blood flow or less than 30
Gy radiation dose absorbed to the lungs via embolization or other means, there is
a high risk of toxicity and TARE should not be performed. Contraindications are
similar to TACE, however, unlike TACE, main portal vein thrombosis or obstruc-
tion is not a contraindication (47). TARE is also safe in patients with prior tran-
sjugular intrahepatic portosystemic shunt (48). Overall tolerance and safety are
comparable to TACE, despite fewer published studies involving TARE. However,
post-embolization syndrome is less common and tends to be less severe when it
does occur (49). Complications unique to TARE include radiation-induced liver
disease (RILD), radiation-induced biliary stricture, radiation-induced pneumoni-
tis, and radiation induced enteritis. The frequency and risk factors for each have
not been well studied and are listed in only a few studies. RILD is considered to
be a serious event, with two studies (50, 51) suggesting rates between 4% and
20%, and presents as jaundice, ascites, and manifestations of liver decompensa-
tion 2–8 weeks after treatment. The risk of RILD increases with repeated Y-90
administrations (51). Radiation-induced biliary stricturing is a less common
adverse effect of TARE occurring in less than 10% of patients (52). The frequency
of these complications has decreased with improvement in technique and
increased experience. As with TACE, proceduralists must be cautioned against
inadvertent embolization of the cystic artery to prevent gallbladder necrosis.
Radiation-induced pneumonitis and enteritis may occur if shunts to the gastroin-
testinal tract or lung are detected on pre-procedural evaluation, but rarely occur
if standard precautions are followed. Lastly, a theoretical concern with Y-90 used
as a bridge includes the risk of radiation exposure to the surgical or pathology
team handling the explanted liver, but considering its short half-life, this only
seems to be a consideration if liver transplantation is performed within 4 weeks
of TARE (3).
Because there are limited head-to-head comparisons between TACE and TARE,
there is no consensus regarding when/if TARE should be chosen over TACE.
However, TARE has been used increasingly due to a number of favorable studies.
Lewandowski et al. (53) compared TARE to TACE in T3 staged HCC down staged
to T2 which showed a significantly better response (61% vs 37%) and slower time
to progression (33.3 months vs 18.2 months). TARE-based bridging protocols
have also been associated with fewer pre-transplant locoregional therapy and had
lower rates of microvascular invasion in the explanted liver, which is associated
with risk of post-transplant HCC recurrence. Salem et al. (54) performed random-
ized controlled trials (RCTs) comparing TARE to TACE (PREMIERE trial) in 179
136 Ponniah S A et al.

patients and reported a significantly longer time to progression in TARE than with
TACE. The complication rate was also lower in the TARE group. Ettore et al. (55)
performed TARE in a small cohort of patients (22) with the majority being outside
the Milan criteria, and successfully bridged all patients and down staged 79% of
patients outside Milan criteria. More recently, the TRACE trial (56) compared Y-90
to DEB-TACE and included patients with BCLC A/B, ECOG 1 and segmental por-
tal vein thrombosis. This study showed superior results in the Y-90 group with
slower time to progression, improved survival, and comparable safety profiles
between Y-90 and DEB-TACE. While these results are favorable, few current stud-
ies overall have noted a difference in survival between TARE and TACE. Other
limitations to several favorable TARE trials include single centered studies, small
cohorts of patients, inherent physician biases and heterogeneity in results and
reporting. Lastly, TARE is more costly than TACE and may preclude its use at
some institutions (36).
Overall, TARE is likely to be used preferentially where there is higher level of
expertise, familiarity with the procedure, in those with infiltrative tumors, portal
vein invasion, larger tumors (>2 segments) and those with progression of tumor
despite use of TACE.

Thermal ablation
Multiple ablative techniques including radiofrequency ablation (RFA), microwave
ablation (MWA), and cryoablation use directly applied thermal energy to induce
tumor necrosis. RFA is the most commonly used and most widely studied of these
interventions and therefore the focus of discussion for this section. RFA involves
the insertion of narrow probes under imaging guidance, either ultrasound or
computed tomography, into a targeted liver lesion. The probes can be inserted
percutaneously, usually by an interventional radiologist or laparoscopically by a
surgeon. High-frequency alternating currents move from the electrode to the
lesion, creating a frictional pattern of ion movement which induces heating and
necrosis to the target lesion (57). Tumor cells die as the tissue becomes heated
above 60 degrees Celsius. The ablated area consists of the tumor plus 5–10 mm
boundary of nearby liver parenchyma (58). While there is no absolute tumor size
for RFA, most reported favorable outcomes to suggest smaller tumors <3 cm
(59, 60), less than or equal to 3 nodules, and without major vascular or biliary
structures near the target lesions.
Limitations of RFA involve the duration of treatment and the risk of thermal
injury to nearby anatomical structures. RFA typically takes 16–18 mins to ablate
a 3–4 cm lesion and there is a risk of dissipating heat energy to nearby blood
vessels (>3 mm) termed the “heat sink effect”; the “cooler” vascular flow near
the lesion may absorb heat resulting in incomplete tumor necrosis in the target
lesion.
Additionally, RFA is not suitable for lesions near certain anatomical structures,
namely the dome of the liver, the gallbladder and the biliary tree (60). There is
also a risk of liver capsule rupture (~2%) and resulting peritoneal seeding when
treating peripheral tumors (3). Overall, the major complication rate ranges from
2.4–13.1%, but RFA is still superior to surgical resection (13). Livraghi et al. (61),
in 218 patients with small and early HCC, showed no perioperative mortality in
RFA with lower than previously suggested rates of major complications (1.8%).
 Locoregional Therapy Prior to Liver Transplant 137

Analysis of 5 large series (59) showed major complications to be as low as 4.6%

and mention RFA to be quite safe. Other complications are listed but are relatively
uncommon, such as abscess formation, portal vein thrombosis, thoracic injury,
liver decompensation, and bleeding. As in TACE/TARE, post-procedural abdomi-
nal pain similar to post-embolization syndrome is also a consideration.
Overall, RFA is an effective LRT for bridging therapy to prevent dropout and is
recommended in small early HCC < or equal to 3 cm (13), with less favorable
results in larger tumors. Large studies such as by Lu et al. (62) and Mazzaferro
et al. (63) have shown dropout rates as low as 0–6% when RFA is used as bridging
therapy. In larger tumors >3 cm, combination treatments as a bridge to liver trans-
plantation have shown promise. In a review of 7 RCTs (62), a combination of RFA
and TACE was superior to RFA alone and showed a significant survival advantage.
More recent studies (64, 65) have also shown improved outcomes with combina-
tion treatment than RFA alone when used as a bridge to liver transplantation,
especially in larger tumors between 3–5 cm.
RFA is more limited in downstaging, as these tumors are outside MC and often
>3–4 cm. The mechanism in MWA is similar to RFA and is favored at some cen-
ters as it offers more rapid heating, shorter treatment time and larger ablative
zones without an observed heat sink effect. Cryotherapy has also shown favorable
results in bridging locoregional therapy for small HCC lesions, but is not widely
adopted and has inherent limitations.

Stereotactic body radiation therapy

Stereotactic body radiation therapy (SBRT) is a more recently adopted treatment
modality for bridging in HCC and for inoperable patients. SBRT involves directing
multiple nonparallel radiation beams at narrow target sites through single or few
high-dose radiation fractions. It is an alternative for patients with decompensated
liver disease that may not be candidates for other locoregional therapies (12).
Although HCC is a particularly radiosensitive tumor, care must be taken to deliver
as little radiation to the surrounding parenchyma as possible to reduce the risk of
toxicity and decompensation. Before SBRT, 4-dimensional imaging is used to map
the target site. Contrary to conventional external beam, which delivers small
doses of radiation over several weeks, SBRT delivers very large doses of radiation
per session and can be completed in 1–5 days. A variety of dosing and fraction-
ation protocols have been used, with doses ranging from 24–60 Gy over 3–6 frac-
tions (65). Sessions typically last 30–60 mins. Compared to ablative therapy, SBRT
is advantageous in that it can treat lesions near the dome of the liver sparing lung
parenchyma, near the gallbladder, and near large vessels (3). There is no concern
about the heat sink effect. SBRT can spare large portions of normal liver tissue
from RILD. The adverse effects of SBRT are limited and mostly nausea, vomiting,
abdominal pain, and rarely GI ulcers have been noted (3).
Few studies have compared SBRT to other forms of locoregional therapy, but
results have been favorable, especially when used as a neoadjuvant treatment. The
first study in 2009 (66) showed that SBRT was both safe and effective in those
awaiting liver transplantation that could not tolerate TACE, RFA, and percutane-
ous ethanol injection. A larger study (65) corroborated these results and found
SBRT to be both safe and effective in patients with Child A/B liver cirrhosis and
lesions ≤6 cm. More recently, Sapisochin et al. (67) compared SBRT to TACE or
138 Ponniah S A et al.

RFA as a bridging therapy in 379 patients meeting Toronto extended criteria. They
found similar 5-year survival and dropout rates across all three modalities. A pro-
spective study by Lee et al. (68) evaluated SBRT in decompensated cirrhosis with
Child-Pugh B or C cirrhosis. Patients had either already completed TACE prior to
SBRT or had TACE in combination with SBRT. This study showed favorable results
as the Child-Pugh class remained stable in 30%, and even improved in 22% of
patients, and was associated with an overall improvement in survival. Other stud-
ies (69–71), mostly retrospective in nature, all show comparable outcomes in
local control of disease. These studies suggest that SBRT can be used safely as
bridging therapy when other modalities have failed or are not applicable.
Additionally, SBRT may offer advantages in patients with liver dysfunction who
may not tolerate TACE or RFA (67). Overall, SBRT has now emerged as an effec-
tive LRT for patients with advanced disease with minimal toxicity.

CONCLUSION

Bridging therapy is now the standard of care to prevent waitlist dropout and
decrease HCC recurrence post-transplant. There are no prospective RCTs compar-
ing locoregional therapy modalities before liver transplantation, therefore no sin-
gle bridging modality is recommended over another (13). The literature remains
difficult to analyze due to non-standardized treatment protocols and heteroge-
neous patient populations. Recently, Kulik et al. compared 18 studies (72) and
found that bridging locoregional therapy had no significant impact on survival
rate or HCC recurrence post-transplant. Several studies were found to have impre-
cision regarding inclusion criteria, as some patients were within the Milan criteria,
outside the Milan criteria, or undefined (11). The lack of randomized studies and
potentially biased patient selection in these studies should also be considered;
patients who received locoregional therapy, when compared to those who did not,
likely had more risk factors such as advanced tumors, aggressive tumor biology,
and longer wait times (72). Overall, bridging locoregional therapy is safe and
effective. The type of locoregional therapy selected is based on liver function, size,
the number of tumors, and institutional experience. Ablation is the preferred
modality for smaller tumors with a size ≤3 cm; the type of ablation is expertise-
dependent, but RFA is frequently used. Larger lesions are typically treated with
TACE or Y-90 TARE based on institutional experience and/or preference. SBRT is
a novel approach and has shown promising outcomes for those with liver dys-
function, failed other locoregional therapies, or for those who are no longer liver
transplant candidates.
Like bridging therapy, there remains no consensus on optimal downstaging
strategy. Several factors result in largely varying success rates (~25–90%) (11) as
numerous inconsistencies in the current literature make data interpretation diffi-
cult. Firstly, the definition of downstaging is poorly defined amongst studies.
None of the present studies were randomized controlled trials (11). Some studies
define downstaging as the reduction of tumors to the Milan criteria, others base
downstaging on the complete absence of tumors, and few use explant pathology
to defined success (11). Secondly, there are inconsistencies in locoregional ther-
apy selection, varying tumor burden before locoregional therapy, and differing
 Locoregional Therapy Prior to Liver Transplant 139

criteria to access radiographic response (72). Lastly, there is no universal defini-

tion for the waiting period following downstaging to determine efficacy and tim-
ing for liver transplantation (11). Despite these inconsistencies, there is optimism
in tumor downstaging prior to LT. The recent adoption of UNOS-DS as inclusion
criteria and focus on tumor biology rather than tumor burden alone has increased
access to liver transplantation.
Since no single bridging or downstaging modality is advocated over another,
expert preference often leads to individualized therapy. Additional research on
expanded transplant criteria and organ allocation models would be of benefit.
Further trials studying to define optimal waiting times after downstaging
and standardizing study variables (demographics, treatment protocols, etc.)
would minimize confounding factors to allow for a more accurate data
interpretation.

Conflict of Interest: The authors declare no potential conflicts of interest with

respect to research, authorship and/or publication of this chapter.

Copyright and permission statement: The authors confirm that the materials
included in this chapter do not violate copyright laws. Where relevant, appropri-
ate permissions have been obtained from the original copyright holder(s), and all
original sources have been appropriately acknowledged or referenced.

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 8
Hepatoblastoma
Josef Hager1 • Consolato M. Sergi2
1
Department of Pediatric Surgery, University Hospital of Surgery, Medical University of
Innsbruck, Innsbruck, Austria; 2Departments of Pediatrics, Laboratory Medicine and
Pathology, Stollery Children’s Hospital, University of Alberta, Edmonton, AB, Canada
Author for Correspondence: Josef Hager, Department of Pediatric Surgery, University
Hospital of Surgery, Medical University of Innsbruck, Anichstrasse 35, A-6020 Innsbruck,
Austria. Email: [email protected]
Doi: https://doi.org/10.36255/exonpublications.livercancer.2021.ch8

Abstract: Hepatoblastoma is the most common liver cancer in children aged

3 years and younger. The differential diagnosis of this neoplasm is crucial for the
proper management. Recent additions to protocols of the International Society of
Pediatric Oncology and Children’s Oncology Group have been key in tackling
this oncological disease. This chapter provides an overview of the etiology, patho-
genesis, epidemiology, incidence, symptoms, and therapeutic considerations of
hepatoblastoma. The diagnostic measures necessary from a surgical point of view
and the essential operational and technical considerations for the various stages
of hepatoblastoma are discussed.

Keywords: etiology; diagnosis; hepatoblastoma; risk assessment; therapeutic

considerations

INTRODUCTION

Primary liver tumors are a heterogeneous group of epithelial and mesenchymal

tumors. They make up between 1 and 2% of all pediatric tumors. They are rare in
childhood, and about two-thirds of primary liver tumors are malignant. It has
been estimated that about 37% of primary liver tumors are hepatoblastomas (1).

In: Liver Cancer. Sergi CM. (Editor). Exon Publications, Brisbane, Australia.
ISBN: 978-0-6450017-2-3; Doi: https://doi.org/10.36255/exonpublications.livercancer.2021
Copyright: The Authors.
License: This open access article is licenced under Creative Commons Attribution-NonCommercial
4.0 International (CC BY-NC 4.0) https://creativecommons.org/licenses/by-nc/4.0/

145
146 Hager J and Sergi C M

Hepatoblastoma typically occurs in infants and toddlers. It occurs predominantly

in a unifocal manner in the right liver lobe, but it can be multifocal and develop
in all liver segments. Well-developed hepatoblastoma may mimic hepatocellular
carcinoma. Hematogenous lymph nodal metastases have been reported (2). If
hepatoblastoma is completely resectable, the prognosis is favorable, primarily
because it responds well to adjuvant chemotherapy. If it cannot be removed
completely due to late diagnosis, the prognosis is far less favorable, because
hepatoblastoma cells develop resistance to cytostatics after repeated chemother-
apy, limiting therapeutic success (3). To optimize the treatment of malignant
childhood liver tumors, especially hepatoblastoma, the international cooperative
therapy study “Pediatric Hepatic Malignancy International Therapeutic Trial
(PHITT)” was initiated in 2015. The scope of the study was to examine the genetic
and molecular changes that may enable therapy stratification, as with other
embryonic tumors (4, 5). The implementation of this study began at the end
of  2017 (https://www.birmingham.ac.uk/research/crctu/trials/phitt/investigators.
aspx [accessed on 18 February 2021]).

ETIOLOGY

The etiology of hepatoblastoma, which usually occurs sporadically, has not yet
been clarified. Somatic gene mutations in hepatoblasts and other observations
suggest tumor development is spontaneous (6). It is believed that premature chil-
dren with a very low birth weight (<1,500 g) have an increased risk of developing
malignant tumors, including hepatoblastoma. This fact, which was first reported
in 1997 (7), was confirmed by a worldwide scientific study in 2019. Still, an
explanation for this observation - accidental or causal connection - could not be
established (8). The fact that relatively common conditions such as pre-eclampsia,
fetal distress before or during childbirth, or congenital malformations could play
roles as possible tumor inducers could allegedly be statistically determined in
some studies, but an explanation for these observations is still missing (9).
Regardless of these observations, over the last few decades, various genetic dis-
eases have been found to be risk factors for developing hepatoblastoma. Some of
them include familial adenomatous polyposis (10), Beckwith-Wiedemann syn-
drome (11), and trisomy 18 (Edwards syndrome) (12). A connection between the
occurrence of hepatoblastoma with other genetic diseases such as Li-Fraumeni
syndrome or Prader-Willi syndrome has been discussed, but the link has not yet
been proven satisfactorily (13). Finally, various external and epigenetic influences
have been debated as possible causes for neoplastic development. Smoking before
and/or during pregnancy is an example of this; opinions on this, however, are
equivocal (6, 14).

PATHOGENESIS

Hepatoblastomas develop from degenerate hepatoblasts, which can be differenti-

ated according to the different stages of liver development. Hepatoblastomas are
 Hepatoblastoma 147

classified based on the original histological classification of Ishak and Glunz (15).
Histologically, hepatoblastoma are broadly classified into two types: epithelial and
mixed. Depending on the degree of differentiation, hepatoblastoma cells can be
distinguished into two subtypes: embryonic and fetal. In some cases, both cell
types are present. Embryonic tumor cells are less differentiated whereas the fetal
cells are well-differentiated. Small-cell anaplastic hepatoblastoma is a unique sub-
type; it mainly infiltrates the bile ducts and is considered prognostically unfavor-
able (16). In addition to epithelial components, the mixed hepatoblastoma type
contains mesenchymal stroma such as osteoid, collagen fibers, and rarely, carti-
lage and skeletal muscle cells (17). Liver progenitor cells harbor the ability to
express of keratin 19 (CK19) and/or the epithelial cell adhesion molecule (EpCAM)
(18–21). EpCAM is a transmembrane glycoprotein mediating calcium-indepen-
dent homotypic cell-cell adhesion in the epithelium. This molecule is also involved
in cellular signaling, migration, proliferation, and differentiation. It plays a role in
the event-free survival outcome of patients harboring hepatoblastoma (18, 20–23).
CK19 expression has been correlated with aggressive behavior in hepatoblastoma
[20] and hepatocellular carcinoma (24). Of tremendous importance is that
EpCAM expression is independent of previous cisplatin-based chemotherapy and
can be utilized as a tumor marker and potential target for immunotherapy (18, 19).
Kiruthiga et al. found that more than 90% of tumors with strong expression of
EpCAM showed viable tumor after chemotherapy (19).

EPIDEMIOLOGY

Hepatoblastoma can occur in children of any age but occurs predominantly in

children between 6 months and 3 years of age (2, 3, 6, 25). Children over the age
of 5 rarely develop hepatoblastoma (26), but hepatoblastoma has been observed
in adults (27). There is a male predisposition with a ratio of M:F = 1.6:1.0 (1–3,
25, 28). The probability of hepatoblastoma occurring in an infant or young child
varies between 0.5 and 2 cases per million children per year. The explanation for
this vast difference could be due to differing age groups and the possibility that
the low hepatoblastoma values given in recent individual publications originate
from “old” statistics, and therefore no longer accurately depict current incidence
rates (29, 30). Regional peculiarities supposedly play a subordinate role (25). For
example, in the USA, about 250 children develop hepatoblastoma each year (31),
whereas in Germany, only around 20 children per year (32) and in Great Britain
only 10–15 children per year (https://www.cancerresearchuk.org/sites/default/
files/cs_dt_childhood.pdf) develop such a tumor (33).

SYMPTOMS

Hepatoblastoma can remain asymptomatic for months. Babies born prematurely

and newborns with low birth weight should be screened. In affected children,
painless swelling in the upper right abdominal area occasionally occurs in the
early stages. When sick children begin to suffer from symptoms, it is almost
148 Hager J and Sergi C M

always when the disease has reached an advanced stage. Overall, the complaints
are nonspecific, including nausea, vomiting, weight loss and increasing general
weakness, which can delay development. In this context, osteopenia can develop.
Children with hepatoblastoma can become conspicuous due to osteopenia, and
resultant pathological fractures (34). Very rarely, obstructive jaundice can occur
when the tumor occludes the intrahepatic biliary tract (2). Spontaneous tumor
rupture with extensive intra- or extra-tumor bleeding is extremely rare (35).
Precocious puberty can occur in boys due to the increased formation of human
chorionic gonadotropin (β-hCG) caused by hepatoblastoma (36). Further symp-
toms can occur depending on the site of metastasis. The lungs are most com-
monly affected, with breathing difficulties, coughing fits, and occasional
hemoptysis (2, 3).

DIAGNOSTICS

The diagnosis of hepatoblastoma involves tumor detection and staging. The fol-
lowing diagnostic measures are initially recommended in addition to a clinical
examination of the patient. During the clinical examination, the primary focus
should be on signs of a genetic diseases (for example, macroglossia and hemihy-
pertrophy, among others, which are characteristic features suggesting Beckwith-
Wiedemann syndrome). Most patients affected by this syndrome will require
surgery (37).

Laboratory tests
Laboratory diagnostics for hepatoblastoma include a blood count as standard
(mild anemia, leukocytosis, and thrombocytosis are possible), a liver function test
(SGOT, LDH, AP can be slightly increased) including bilirubin (raised in the case
of bile duct obstruction) and the tumor markers ferritin, CEA (carcinoembryonic
antigen test) and the NSE (neuron-specific enolase), and, if necessary, urinary
catecholamines (to rule out neuroblastoma) and an evaluation of the titer regard-
ing hepatotropic viruses. It is essential to determine the malignancy-associated
tumor markers, namely alpha-1-fetoprotein (AFP) and beta-human chorionic
gonadotropin (β-HCG). AFP is elevated in 80–90% of patients. The typical AFP
values of the respective age group must be observed. Hepatoblastomas with low
AFP (<100ng /ml) are considered aggressive and have an abysmal prognosis (38).
β-HCG is increased in about 20% of patients. However, this does not seem not to
have prognostic significance (39).

Imaging
If a liver tumor is suspected, first contrast-enhanced sonography of the liver is
performed. If the tumor shows increased echogenicity on contrast-enhanced
sonography and pronounced vascular supply on Doppler ultrasound, possible
tumor invasion into one or more hepatic vessels is suspected. This indicates a
malignant process; however, these are not confirmatory evidence of malignancy
(40). Other imaging options, namely magnetic resonance imaging (MRI) or
 Hepatoblastoma 149

computed tomography (CT) of the abdomen with contrast agent, can not only
provide evidence of a malignant tumor of the liver, but also allow for the assess-
ment of the extent of malignancy, and even the relationship of the neoplasm to the
hepatic vessels and the liver segments. Nevertheless, a reliable diagnosis of hepa-
toblastoma cannot be achieved with these examinations either (41). But thanks to
these techniques, apart from particular indications, angiography or liver scintigra-
phy can now be used. A lung CT scan to determine or exclude lung metastases
and skeletal scintigraphy with 99-technetium phosphonate to realize or exclude
possible bone metastases is recommended as a precautionary measure. It remains
to be seen whether FDG-PET/CT performed for the initial diagnosis of a possible
hepatoblastoma is sensible, especially since only a possible correlation between
uptake and tumor-related increased AFP values can be established. It is well
known that FDG-PET/CT is vital during treatment or as part of the follow-up of a
malignant tumor. For example, for hepatoblastoma, the detection of metabolically
active metastases indicates an unfavorable prognosis (42).

Histology
To confirm the diagnosis of hepatoblastoma, histological examination of the
tumor biopsy is the gold standard. In most suspected cases, the biopsy material
can be removed using a percutaneous punch (approximately 3–6 liver cores). If
the tumor is difficult to access percutaneously or is heavily vascularized, a biopsy
is indicated either laparoscopically or via laparotomy. Fine-needle aspiration of a
possible hepatoblastoma for the aspirate’s cytological examination is not regarded
by most oncologists as sufficient for a reliable diagnosis, even if there have been
isolated experiences to the contrary (43). This also applies to a percutaneous
punch biopsy by an interventional radiologist (44). The biopsy material should be
examined both conventionally histologically (as a paraffin preparation) and
immunohistochemically. The diagnosis of hepatoblastoma should always be con-
firmed by a reference pathologist, that is, a pathologist with experience working
for either the Children’s Oncology Group (COG) or the International Society of
Pediatric Oncology (SIOP) or the United Kingdom Children Cancer Study Group
(UKCCSG). According to the guidelines of the (German) Society for Pediatric
Oncology and Hematology (GPOH), children between the ages of 6 months and
3 years of age who have a liver tumor suspected of being hepatoblastoma by imag-
ing and an AFP value of over 1,000 ng/ml or an AFP value that is at least three
times higher than the age norm, biopsy confirming the diagnosis of hepatoblas-
toma is not necessary, especially since in these cases the incriminated liver tumor
is always hepatoblastoma (3). However, this view is not generally approved; on
the contrary, most oncologists require a tumor biopsy to confirm the diagnosis
(19) (Figure 1).
In the case of resected specimens, size, the exterior (solid/cystic), and tumor
necrosis are noted (19). Histology is key in reporting hepatoblastoma, and the
report should include the histological subtype, mitotic activity in 10 high power
fields (HPF) (low mitotic activity when ≤5/10 HPF and high mitotic activity when
>5/10 HPF, presence of extramedullary hematopoiesis, and intratumoral fatty
change [steatosis]). Six major subtypes are recognized, including: pure fetal
epithelial, mixed embryonal and fetal epithelial, macro trabecular, small cell
150 Hager J and Sergi C M

Figure 1.  Histology of hepatoblastoma. A. Slice following partial resection of the liver showing
a hepatoblastoma with grey cut surface and small areas of hemorrhages. B. Microphotograph
showing hepatoblastoma with pure fetal histology and minimal mitotic activity (hematoxylin
and eosin staining, x200 original magnification). C. Microphotograph showing
hepatoblastoma of embryonal type (hematoxylin and eosin staining, x200 original
magnification). D. Microphotograph showing a teratoid hepatoblastoma with ribbons and
nephroblastoma-like tubules and acini (hematoxylin and eosin staining, x50 original
magnification, scale bar: 100 micrometers). E. Microphotograph showing lung metastasis of a
hepatoblastoma with two nodules depicting hemosiderin accumulation (blue) both inside
and at the edges of the tumor clusters. The lung tissue in the middle of the microphotograph
shows the characteristic alveolar pattern. Perls’ Prussian Blue (PPB) has its name from the
19th century German pathologist Max Perls, who introduced this technique in histopathology
to stain iron in the ferric state (e.g., ferritin and hemosiderin (Perls’ Prussian Blue, x50 original
magnification, scale bar: 100 micrometers). F. Microphotograph showing a hepatoblastoma
post chemotherapy exhibiting some cell maturation (right) and some hemorrhage (center).
Some fibrosis is encountered on the left side of the microphotograph (hematoxylin and
eosin staining, x200 original magnification).
 Hepatoblastoma 151

undifferentiated (SCUD), and mixed epithelial and mesenchymal (MEM) with or

without teratoid features. Hepatoblastoma should be assigned a category depend-
ing on the prevalent epithelial subtype (≥60%) demonstrated. In the case of a
hepatoblastoma with 60:40 ratios of two or more components, the tumor should
be classified as a mixed subtype. In the post-chemotherapy pathology report, the
percentage of the viable tumor should be included. This rate should be graded as
0% as no viable tumor, <25% as “low viable tumor”, 25%–50% as “moderate
viable tumor”, and ≥50% as “substantial viable tumor” following an extensive
examination of the pathology specimen. Maturation, cytopathic effects of chemo-
therapy and microvascular invasion (MVI) should be documented. The assess-
ment should also involve the radicality, and the distance of the neoplasm to the
surgical resection margin should be measured microscopically and categorized as
≤0.5 cm, 0.6–1 cm, and >1 cm (19).

THERAPEUTIC CONSIDERATIONS

The current therapeutic approach involves three treatment options: (i) pre- and/
or post-operative chemotherapy, (ii) tumorectomy with possible partial liver
resection, and (iii) liver transplantation (29). The use of chemotherapies, includ-
ing platinum compounds for neoadjuvant and adjuvant treatment of hepatoblas-
tomas resulted in a significant improvement in outcome. It has been shown that
the results concerning patient survival are relatively similar between the three
treatment options, even though the appropriate regimens of the various pediatric
oncological groups around the world are not identical (5). Regardless of this fact,
surgical treatment of hepatoblastoma is of great importance. The aim of the surgi-
cal procedure, which aims to be curative, is complete tumor resection. Thanks to
neoadjuvant chemotherapy and various improvements in surgical techniques and
equipment (vascular exclusion, ultrasound knife, etc.), this goal has been achieved
more and more frequently in recent years; unfortunately, in about 10% of
PRETEXT (“PRETreatment EXTension of disease”) IV children, despite aggressive
neoadjuvant chemotherapy, hepatoblastoma is not completely resectable.
Orthotopic liver transplant must be considered in this setting (45).

Staging
According to the generally accepted view, precise clinical staging is a prerequisite
for accurate risk stratification and adequate therapy planning. The so-called
PRETEXT system of the liver tumor study group of the International Society for
Pediatric Oncology (SIOPEL) is used internationally for this purpose. The
PRETEXT system categorizes tumors according to their extent in pre-therapeutic
imaging and has high prognostic relevance (46). Hepatoblastomas are divided
into four different PRETEXT groups (I-IV), depending on how many of the liver’s
four surgical sectors are affected (Table 1).
Also, other characteristics of the extent of the tumor are recorded in capital
letters: C, infiltration of the caudate lobe; V, invasion of the hepatic veins; P, inva-
sion of the portal vein; F, multifocality; E, extrahepatic tumor extension; N, lymph
node involvement; and M, hematogenous metastasis. To describe the extent of the
152 Hager J and Sergi C M

TABLE 1 PRETEXT classification

PRETEXT Definition
I Three contiguous hepatic sections are free of tumor
II One or two sections have tumor involvement, but two adjoining sections are tumor-free
III Two or three sections have tumor involvement, but no two adjoining sections are
tumor-free
IV All four sections showing tumor involvement

TABLE 2 Children’s Cancer Study Group Staging

Stage Definition
I Hepatoblastoma completely resected
II Evidence of microscopic residual neoplastic disease only
III Evidence of gross residual neoplastic disease or positive lymph nodes or “tumor spillage”
IV Evidence of metastatic disease
Source: https://www.childrensoncologygroup.org/index.php/newly-diagnosed-with-hepatoblastoma-or-hepatocellular-
carcinoma-[accessed on 04 February 2021]

tumor after two cycles of neoadjuvant chemotherapy, the same system is then
used for the new staging as the so-called POSTTEXT system (“post-treatment
extension”) (47). It should be noted that the PRETEXT system is accused of being
an almost “overly precise” stage assessment, which is why stages III and IV should
be checked particularly carefully, taking into account the PHITT criteria (48).
An  additional classification about the risk assessment of hepatoblastoma was
proposed by the Children’s Hepatic Tumors International Collaboration -
­
Hepatoblastoma Stratification (CHIC-HS), taking into account the PHITT
­consideration. This proposal, which is based on the PRETEXT system and includes
the patient’s age and AFP level, classifies hepatoblastomas into groups as very
low-, low-, medium-, and high-risk (4). Alternatively, hepatoblastomas can be
classified according to the Children’s Cancer Study Group of the Children’s
Oncology Group (Table 2). Hepatoblastomas are not staged using the American
Joint Committee on Cancer criteria (49).

Risk assessment and therapy planning

As noted above, tumor diagnosis involves two aspects: accurate determination
of hepatoblastoma followed by staging. Staging is essential for appropriate
therapeutic procedure. In this context, essential criteria are to treat a localized
hepatoblastoma to a minimum and an extensive hepatoblastoma as intensively
 Hepatoblastoma 153

as possible (50, 51). Of the imaging methods, sonography is initially of great

importance. Apart from verifying the tumor, its extent can also be assessed
(50, 51). Also, the vascular supply of the hepatoblastoma can be visualized
utilizing color-coded Doppler sonography, or, in the case of focal liver lesions,
employing contrast-enhanced ultrasound (CEUS) (40, 52). Furthermore,
existing thrombus in the vena hepatica, the portal vein, or the vena cava inferior
can be established (48). To be able to examine hepatoblastoma more closely, the
method of choice is MRI (CT should be avoided because of radiation exposure)
(13, 29). Gadolinium-containing contrast media have proven to be particularly
beneficial for the diagnosis of liver tumors using MRI examinations, in particular
gadolinium-ethoxybenzyl-diethylenetriamine penta-acetic acid (Gd-EOB-DTPA),
because it not only provides a clear distinction between healthy and malignant
liver tissue, in the biliary tract and the hepatic vessels, but also detects satellite
tumors or a multifocal hepatoblastoma (53). In addition, an invasion of the
blood vessels can be detected employing MR angiography for confirmation.
With this method, vascular anomalies can be seen as an incidental finding (54).
As already mentioned, approximately 20% of children with hepatoblastoma
have lung metastases, which is why many oncologists require a multidetector
CT (MDCT) of the lungs when initially investigating suspicion of hepatoblas-
toma (44). Abdominal CT, despite radiation exposure, can be beneficial (55).
Because a CT can be done much faster (short sedation or ON duration) than an
MRI and provides similarly useful information, even though it is supposedly less
accurate. Also, variations in the liver segments can be verified, which is certainly
not insignificant from a surgical and technical perspective (56).

Time of surgical procedure

Primary resection of hepatoblastoma is rarely possible in children. According to
the PHITT study, a prerequisite for simple lobectomy is a small solitary hepato-
blastoma (PRETEXT I and possibly II) with a well-differentiated fetal histological
structure, corresponding to a “very low-risk tumor”, that does not show any dif-
ferentiated fetal tissue. Neoadjuvant therapy (for example, two cycles) is often
necessary to reach tumor clearance (57). According to the new classification, the
parameters are listed for very low, low, intermediate, and high-risk tumors.
Induction chemotherapy is carried out within these groups or their subgroups. By
incorporating PRETEXT stages, age, and AFP level in the stratification of neoad-
juvant chemotherapy regimen patient-specific decision can be made (58).
Hepatoblastomas positively respond to individualized neoadjuvant chemotherapy
with a significant reduction in size in 90% of cases. To monitor effect, imaging
examination of the patient is carried out following the cytostatic treatment (usu-
ally after two cycles) (29). If it turns out that a simple lobectomy can safely remove
a hepatoblastoma, tumor resection is scheduled. If this is not possible, the child
will receive two more chemotherapy blocks. This makes up to 50% of children
with PRETEXT stage IV operable (59). After completing block 4, they are pre-
pared for a possible liver transplant (60).
After another imaging examination of the treatment result, two options are
possible: if there is an improvement, one can opt for a complex or extended liver
resection in selected cases on the assumption that the hepatoblastoma can be
removed (61). If this is not the case, all that remains is a planned liver transplant.
154 Hager J and Sergi C M

An extension of the preoperative chemotherapy is because of the often-occurring

resistance to cytostatic drugs. Radiation therapy is not indicated due to insuffi-
cient tumor sensitivity (56, 57). Whether the patients benefit more from an
extended resection or from a liver transplant is not clearly defined in the PHITT
study, as there is not enough data on this (62). The decision to have an extensive
liver resection is difficult, because this procedure is not always feasible. A rescue
liver transplant should be carried out in such a setting (5). A ruptured hepatoblas-
toma is a therapeutic challenge. It is not discussed here because of only a few
published cases. It should be noted that there are various therapy proposals with
satisfactory results in this regard (63).

Surgical approach
In the conventional surgical approach, a slightly arched, transverse upper abdom-
inal laparotomy is usually chosen, which can be extended in the linea alba in a
T-shape to the xiphoid if necessary. The liver is fully mobilized, the hepatoduode-
nal ligament and the inferior vena cava located. They are looped under and above
the liver. After the associated supplying and draining vessels have been removed,
the incriminated liver segment is resected, partly blunt, partly with an ultrasound
knife or LigaSure™. All crossing vessels are ligated. It should be noted that ana-
tomical resections such as segment resection, lobectomy or extended lobectomy
(­tri-segment resection) are to be preferred to atypical (“wedge”) resections or enu-
cleations, as they usually allow more radical resections and fewer complications.
If the distance to the tumor is large enough, a bleeding-free resection can be made
possible with thorough mattress sutures. There are various discussions about
whether the liver should be pinched out during such an operation. It has been
suggested that some surgical techniques are also critical for postoperative liver
function (64). Avoiding clamping the liver for resection has the advantage that
postoperative function regenerates faster (64). In difficult cases, preoperative
imaging of the arteries supplying the tumor is recommended to evaluate the
tumor supply better. As a primary therapeutic consideration, it is possible in this
context to apply particles loaded with a chemotherapeutic directly into the tumor
(65, 66). As a result, the tumor blood flow can be vastly reduced, and tumor
tissue destruction can be achieved. However, this procedure can lead to severe
complications (67). It is possible to remove up to 80% of the liver tissue through the
operation since the liver can regenerate from the remaining tissue. Because the liver
plays a vital role in producing various proteins that are important for the body,
multiple disorders can temporarily occur after the operation due to tissue loss, for
example, blood clotting disorders, disorders of blood sugar regulation, or a lack
of plasma proteins. In the last few years, liver surgery has seen an incredible
boom, including in children. On one hand this is because of surgical-technical
innovations (for example, minimally invasive partial liver resection) and on the
other hand, because of advances in imaging technologies such as image-guided
three-dimensional reconstructions, intraoperative ultrasound (IOUS), and
indocyanine green (ICG) angiography to detect metastasis (68).
A laparoscopic partial liver resection, even of larger sections, is now a wide-
spread method in adults, especially since various technical aids (for example,
B-mode and Doppler ultrasonography) have been developed for this. According
to the literature, hepatoblastomas can be removed in children in a minimally
 Hepatoblastoma 155

invasive manner, but the problem is the abdominal cavity’s size (65, 66). Although
neoadjuvant chemotherapy makes most hepatoblastomas significantly smaller
and thus safely operable, there are no clear international guidelines for laparo-
scopic approach (69). A method that has been known for a long time and is also
suitable for making hepatoblastoma visible is the three-dimensional tumor recon-
struction based on CT data (68, 69). It is crucial because it enables the tumor and
relevant surrounding anatomical structures to be displayed selectively, thanks to
new software and a virtual operation simulation. The three-dimensional recon-
struction provides information about the neoplasm, its topography, whether a
blood vessel has been infiltrated, and the extent of the infiltration (70, 71).
With IOUS, up to 20% of the patients could have morphologically different
results compared to the preoperative MRI examination results, which make it
necessary to change the surgical procedure in individual cases. These changes in
results mainly concern the relationship between hepatoblastomas and hepatic
veins, which is problematic, concerning imaging (72). Apart from the IOUS, an
operating microscope and fluorescent dyes, which accumulate in the tumor and
make it visible under the operating microscope with special filters, are usually
used for tumor and tumor border imaging. ICG has achieved particular impor-
tance for this. It is a fluorescent, colored, water-soluble compound suitable for
various human medical examinations. It has a high binding affinity for all plasma
proteins. ICG absorbs and fluoresces in the visible and near-infrared light spec-
trum (73). ICG allows the monitoring of liver perfusion. Healthy liver tissue
excretes the preparation via the bile within a few hours. In contrast, ICG is retained
in the tumor tissue (29) and therefore ideal for detecting metastases. ICG is usu-
ally administered intravenously 48–72 hours preoperatively to achieve its visual-
ization in the liver. This procedure is also used in hepatoblastoma patients as it
allows the resection margins to be assessed clearly and enable the identification of
residual tumors (74). An essential criterion in the surgical removal of hepatoblas-
toma is, as in all operations, the avoidance of complications (secondary bleeding
and biliary leaks [bilioma formation or occurrence of bilious peritonitis]). During
this phase, growth factors that are increasingly formed in connection with the
surgical trauma can develop a tumor-promoting effect (75). Postoperative chemo-
therapy usually consists of 1–2 courses after liver resection, and twice after liver
transplantation. There are new considerations for reducing ototoxic preparations
as much as possible (76).

Liver transplantation
Liver transplantation is provided for cases of hepatoblastomas that are nonresect-
able: (i) multifocal hepatoblastomas across all four sectors (PRETEXT IV), since
chemotherapy is unlikely to completely eradicate all intrahepatic metastases;
(ii) Central PRETEXT IV hepatoblastomas with vascular invasion, in which the
neoadjuvant chemotherapy cannot downstage the tumor to a PRETEXT III;
(iii)  Hepatoblastomas (PRETEXT III), which tightly surround or enclose large
­vessels (vena cava inferior, hepatic veins), and (iv) hepatoblastomas that do not
respond to chemotherapy. Also, critical tumor resections may be performed using
the heart-lung machine or ex-situ resection. In some settings, liver transplantation
may produce better long-term results than resection alone from an oncological
point of view (77, 78). If tumor resection cannot be carried out, a so-called
156 Hager J and Sergi C M

“rescue” transplant can be performed, but the prognosis is worse than a primary
liver transplant (79). Otte et al. found that orthotopic (split) liver transplant to
treat hepatoblastomas achieved a 6-year survival rate of 82% in 106 patients,
whereas of the 41 patients who underwent “rescue” liver transplantation, it was
only 30% (80). It should be noted that the prognosis of children who have had a
hepatectomy with orthotopic liver transplantation for hepatoblastoma is as good
as that of children who had conventional resection of smaller tumors (81).
However, it must be noted that liver transplantation is not free from comorbidities
and requires lifelong immunosuppression, which in turn implies side effects.
Against a transplant, it is argued that microscopic residues after a tumorectomy do
not reduce the survival rate of those affected (79). It is expected that the PHITT
study will provide further information (82). Opinions differ on the importance of
post-transplant chemotherapy. Otte et al. compared the relevant results in 147
patients in 2004: 65 received post-transplant chemotherapy, 82 did not. The sur-
vival rates of 77% versus 70% were not statistically significant (80). This means
that post-transplant chemotherapy’s benefit must be weighed against the toxic
risks of the treatment, even though a transplanted liver can withstand adjuvant
chemotherapy (79).
For a liver transplant to treat hepatoblastoma, a living donation is best, from
example from a parent. Until the first year of life, due to the immaturity of the
immune system and immunosuppression, such a transplant can be manageable
without risk for the affected child and during the second year of life with a man-
ageable risk, unlike, for example, blood group compatibility (83). After four
weeks, patients with liver transplants can be switched from tacrolimus to siroli-
mus after completion of chemotherapy, or due to the wound healing disorders
associated with these drugs.

Treatment of lung metastases

About 20% of children with hepatoblastoma have lung metastases at the time of
tumor diagnosis. Up to 50% of these patients can achieve remission with neoad-
juvant chemotherapy (29, 84). However, if lung metastases occur during this
treatment, the prognosis for these children is poor (85). After completion of
neoadjuvant chemotherapy, pulmonary metastases that persist must be surgi-
cally removed (86). The only contraindication is impaired lung function.
Opinions differ as to when this intervention should take place (85, 86). The
majority of authors recommend performing this intervention before the start of
postoperative adjuvant chemotherapy and approximately 2 weeks after resec-
tion of the primary tumor, mostly because the affected children will have recov-
ered from the abdominal procedure by this time (84, 85, 87). There is also no
unanimous opinion with regards to the surgical procedure. Surgical treatment of
the metastases can be carried out thoracoscopically or by means of thoracotomy,
depending on the extent of the findings. Many authors advocate a thoracotomy,
especially since this procedure allows identifying foci that cannot be identified
from image morphology or that lie deep in the parenchyma (29). A thoraco-
scopic procedure (VATS technique) is also used successfully in children. Both
techniques will find lung metastases. The use of ICG is recommended, but only
superficial tumor nodules (up to a depth of 1 centimeter) can be adequately
visualized (88, 89).
 Hepatoblastoma 157

If lung metastases are present on both sides, there is no unanimous approach.

Some centers address the more severely affected lung first, and the less affected
lung the next day. Other centers operate on both lungs on the same day, using
sternotomy. Still, other centers carry out the interventions at a longer time interval
(84, 90). The procedural differences are due to the lack of evidence-based guide-
lines that dictate the appropriate surgical treatment for pulmonary hepatoblas-
toma metastases (82, 84).
Any remaining lung metastases pose a significant problem when a liver trans-
plant is the only option. This is because pulmonary metastases that do not respond
to neoadjuvant chemotherapy and those that cannot be approached surgically are
contraindications for liver transplantation (79, 91). In other words, lung metasta-
ses that persist after neoadjuvant therapy should be surgically removed if possible
and the resection should always be performed before the liver transplant (29, 91).
In exceptional cases, for example, when no living donor is available, this can also
be done shortly after the transplant. In such a case, a liver transplant may be pre-
ferred to resection lung metastases, as this procedure may represent the child’s
only chance of survival (79).

FOLLOW-UP CARE

All children with a treated malignant liver tumor require a follow-up period of at
least 5 years after remission. Regular, initially monthly, later 3-monthly, and then
6-monthly check-ups including liver sonography, chest X-ray, and if necessary, CT
and/or MRI (in the event of an increase in AFP) and laboratory values to rule out
tumor recurrence and to assess the long-term effects of therapy are necessary. In
this context, AFP is of great importance as a tumor marker and thus as an indirect
indicator of therapeutic effectiveness. Normalization of the AFP values can be
expected during the neoadjuvant chemotherapy, and after the removal of hepato-
blastoma. If the AFP values do not normalize, a residual tumor can be assumed. If
they have been regular and then rise, the tumor is likely relapsed (92, 93); how-
ever, some observations show that relapse does not have to be accompanied by an
increase in AFP (94). Chemotherapy-induced changes to cardiac and renal func-
tions, changes in blood parameters, and hearing should be monitored. Attention
must also be paid to the development of a second malignancy.

Treatment of relapses
According to the literature, about 12% of hepatoblastoma patients who have
achieved a complete remission are likely to relapse in the liver and/or lungs. To
achieve remission, chemotherapy (95) and surgical removal of the local recur-
rence or the newly occurring lung metastases is necessary (96). Matsunaga et al.
stated in 2003 that, of the 90 patients (without metastases) in whom the hepato-
blastoma had been fully resected, four had a liver recurrence and eight had lung
metastases. Except for one case with multiple lung metastases, all achieved remis-
sion via medicinal or surgical treatment (97). In liver transplant patients it is less
favorable if additional metastases had initially existed. For example, in 2014,
Yamada et al. reported that about 30% of such the cases relapse (98).
158 Hager J and Sergi C M

A second operation on the liver is often difficult and a complete resection of

the recurrence is not possible. According to the literature, only a palliative proce-
dure is possible in about a third of the cases (98, 99). This means that before
relaparotomy is indicated, it should be mostly clear whether surgical removal of a
hepatoblastoma recurrence in the liver is possible or whether a liver transplant
should be considered (29). Based on this, an extensive liver resection should be
attempted before a liver transplant, but if it turns out intraoperatively that this is
not possible, only a rescue liver transplant is the option; however, as already men-
tioned, it should be noted that the prognosis in this case is poor, although opin-
ions differ (86).
Pulmonary relapses are a big problem (96). They can occur as part of liver
recurrences but can also occur in isolation. To give the affected children a realistic
chance of survival, these metastases should also be surgically removed under che-
motherapy (97). Meyers et al. reported that of the 13 thoracotomies, only four
were long-term survivors (87). Shi et al. succeeded in removing the lung metasta-
ses in 8 out of 10 patients; the operated children had a mean survival rate of
around 18 months at the time of publication (100). Passmore et al. pointed out
that repeated thoracotomies can be useful in lung metastases (101). The role of
other techniques for example radiofrequency ablation of metastases is not yet fully
established (102, 103).

CONCLUSION

Hepatoblastoma typically occurs in infants and toddlers and its etiology has not
yet been clarified. Hepatoblastoma is a challenging diagnosis for clinicians and
pathologists. About 20% of children with hepatoblastoma have lung metastases at
the time of tumor diagnosis. Several histological patterns have been associated
with different prognosis. The current therapeutic approach involves chemother-
apy, tumorectomy with possible partial liver dissection, and liver transplantation.
Chemotherapy-induced changes to vital functions should be monitored. About
12% of hepatoblastoma patients who have achieved a complete remission are
likely to relapse. Regular, follow-up is necessary to monitor long-term effects of
therapy.

Acknowledgment: This research has been funded by the generosity of the Stollery
Children’s Hospital Foundation and supporters of the Lois Hole Hospital for
Women through the Women and Children’s Health Research Institute (WCHRI,
Grant ID #: 2096), Hubei Province Natural Science Funding for Hubei University
of Technology (100-Talent Grant for Recruitment Program of Foreign Experts
Total Funding: Digital PCR and NGS-based diagnosis for infection and oncology,
2017–2022), Österreichische Krebshilfe Tyrol (Krebsgesellschaft Tirol, Tyrolean
Cancer Research Institute, Austria, 2007 and 2009 - “DMBTI and cholangiocel-
lular carcinomas” and “Hsp70 and HSPBP1 in carcinomas of the pancreas”),
Austrian Research Fund (Fonds zur Förderung der wissenschaftlichen Forschung,
FWF, Grant ID L313-B13), Canadian Foundation for Women’s Health (“Early
Fetal Heart-RES0000928”), Cancer Research Society (von Willebrand factor gene
expression in cancer cells), Canadian Institutes of Health Research (Omega-3
 Hepatoblastoma 159

Fatty Acids for Treatment of Intestinal Failure Associated Liver Disease: A

Translational Research Study, 2011–2014, CIHR 232514), and the Saudi Cultural
Bureau, Ottawa, Canada. The funders had no role in study design, data collection,
and analysis, decision to publish, or preparation of the manuscript.

Conflict of Interest: The authors declare no potential conflicts of interest with

respect to research, authorship and/or publication of this chapter.

Copyright and Permission Statement: The authors confirm that the materials
included in this chapter do not violate copyright laws. Where relevant, appropri-
ate permissions have been obtained from the original copyright holder(s), and all
original sources have been appropriately acknowledged or referenced.

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 9
Undifferentiated Embryonal Sarcoma
of the Liver in Adults
Jingyang Huang
Department of Pathology and Laboratory Medicine, University of Alberta,
Edmonton, Canada
Author for Correspondence: Jingyang Huang, Department of Pathology and
Laboratory Medicine, University of Alberta, Edmonton, Canada.
E-mail: [email protected]
Doi: https://doi.org/10.36255/exonpublications.livercancer.2021.ch9

Abstract: Undifferentiated embryonal sarcoma of the liver (UESL) is an aggressive

malignancy that most commonly affects the pediatric age group. This tumor very
rarely occurs in adults and, in such instances, can pose a considerable diagnostic
challenge for the clinicians, radiologists, and pathologists involved. The clinical
presentation is most often non-specific, and the radiology shows a solid and cystic
liver mass which has a considerable differential on imaging. Especially in adult
patients, UESL is a diagnosis of exclusion meaning that all other diagnostic pos-
sibilities must be excluded before the diagnosis of UESL can be confidently made.
From a pathology perspective, this means careful examination of the histology
along with a comprehensive panel of immunohistochemistry for almost all cases
of newly diagnosed UESL. The prognosis of UESL used to be dismal, but with
advances in treatment and the introduction of a multimodality approach, there
has been considerable progress in improving outcomes and survival for patients
with this aggressive tumor. This chapter outlines the clinical, radiological, and
pathological features of UESL. An in-depth discussion is undertaken to describe
the diagnostic approach and the differential diagnosis for this rare and challenging
tumor.

Keywords: diagnosis of exclusion; liver tumor; pediatric liver sarcoma; rare adult
hepatic sarcoma; undifferentiated embryonal sarcoma of the liver

In: Liver Cancer. Sergi CM. (Editor). Exon Publications, Brisbane, Australia.
ISBN: 978-0-6450017-2-3; Doi: https://doi.org/10.36255/exonpublications.livercancer.2021
Copyright: The Authors.
License: This open access article is licenced under Creative Commons Attribution-NonCommercial
4.0 International (CC BY-NC 4.0) https://creativecommons.org/licenses/by-nc/4.0/

165
166 Huang J

INTRODUCTION

Undifferentiated embryonal sarcoma of the liver (UESL) is a rare hepatic mesen-

chymal tumor that occurs predominately in children (1, 2). This tumor is most
common between the ages of 6 and 10 (1). It is an aggressive tumor that has sig-
nificant metastatic potential. Around 60 cases have been reported in adults since
this pediatric tumor was first described. The average age for these adult cases was
24 years and the oldest was 84 years of age (3). Because of the limited tissue
encountered on biopsy specimens of adult patients, undifferentiated embryonal
sarcomas can pose a considerable diagnostic conundrum. Extensive immunohis-
tochemical work-up along with correlation with all available clinical and radio-
logical findings is usually required before a firm diagnosis of UESL can be made.
The diagnosis of an undifferentiated embryonal sarcoma is best considered a
­diagnosis of exclusion in adult patients since the primary purpose of ancillary
testing is to exclude all other possibilities (4–6). Timely detection and surgical
resection along with neoadjuvant therapy are essential in achieving favorable
­outcomes (1, 2). In pediatric patients, the 5-year survival is around 86%, with
surgical resection being the most important aspect of treatment (1, 3).

CLINICAL PRESENTATION

UESL is a rare liver mesenchymal neoplasm that most often occurs in children
between 6 and 10 years of age (1). UESL is the third most common primary liver
malignancy in children after hepatoblastoma and hepatocellular carcinoma (1, 7).
This tumor has been reported in adults, but these cases are exceptionally rare (3).
UESL most commonly occurs in the right lobe of the liver with many patients
being asymptomatic for considerable periods of time (3). Those patients who are
symptomatic may present with non-specific features such as nausea, vomiting,
weight loss, fever, and abdominal pain and distention (1, 2). Serology in most
patients is usually negative but in a minority of cases, elevated liver enzymes, alpha
fetoprotein, and erythrocyte sedimentation rates have been encountered (1, 2).

RADIOLOGY

Imaging will demonstrate a heterogeneous liver mass with solid and cystic
areas (8). There are usually no specific imaging features that can help suggest an
undifferentiated embryonal sarcoma (2). On ultrasound, the tumor may appear
more solid than cystic because of its high water content (7). On CT, UESL will be
a hypodense solid and cystic mass (Figure 1A) (2). On MRI, the cysts within the
mass may show multiple fluid-fluid levels (Figure1B) (9). Due to their cystic char-
acter radiologically, UESLs have been known to be mistaken for benign cystic
entities such as biliary cystadenomas, hydatid cyst disease, or venolymphatic mal-
formations (2, 8, 9). In fact, the radiological appearance of USEL can be quite
misleading to a point where delays in diagnosis or even the initiation of the wrong
treatment have occurred (4, 10).
 Undifferentiated Embryonal Sarcoma of the Liver 167

Figure 1.  Radiology of undifferentiated embryonal sarcoma of the liver (UESL). A. An arterial
phase contrast-enhanced computed tomography from a young adult male with UESL. It
demonstrates a large hypodense mass in the right lobe of the liver. B. Magnetic resonance
image showing a large mass in right lobe of the liver with heterogenous and cystic features
along with fluid-fluid levels.

GROSS FEATURES

Hepatic resection specimens show masses most commonly located in the right
lobe of the liver. Many of these tumors are quite large by the time surgical interven-
tion is undertaken. The tumor commonly has a pseudocapsule and due to its large
size, compresses the adjacent normal liver parenchyma. The tumor itself has a
variegated appearance showing cystic and solid areas that have gelatinous, hemor-
rhagic, and/or necrotic features (Figure 2) (2, 3, 7). Tumors that have been large
enough to rupture through the liver capsule have also been encountered (11–13).

HISTOLOGY

USEL is made of high-grade undifferentiated cells with varying degrees of spin-

dling and myxoid change. Like its gross appearance, this tumor is commonly
heterogeneous histologically, and the morphology will vary considerably depend-
ing on the area of examination. Some areas may appear to be composed of a rela-
tively uniform sheet of undifferentiated cells while other areas demonstrate wildly
pleomorphic anaplastic cells, some with multinucleation and a myxosarcomatous
background (Figure 3A). Due to its high-grade nature, considerable areas of
necrosis are found in the majority of cases and abundant mitoses are easily identi-
fied (Figures 3B) (2–4, 6, 7, 14). Areas of cystic change can often be seen histo-
logically (Figure 3C), which corresponds to this tumor’s cystic appearance on
both gross and radiology. Despite the presence of a pseudocapsule, cords of nor-
mal hepatocytes and bile ducts are commonly entrapped along the periphery of
this tumor (Figure 3D) (2). Periodic acid-Schiff positive diastase (PASD)-resistant
eosinophilic globules are often seen in the tumor stroma or within the cytoplasm
of some of the tumor cells (Figures 3E and F) (2, 3, 6). Around half of UESLs will
have areas of extramedullary hematopoiesis (14) and an extremely rare case with
osteoid formation has been reported (13).
168 Huang J

Figure 2.  Gross features of undifferentiated embryonal sarcoma of the liver (USEL). This gross
photograph of a UESL shows a variegated gelatinous liver mass with a lobulated architecture
and areas of necrosis and hemorrhage. This case also shows the tumor rupturing through
the capsule of the liver.

IMMUNOHISTOCHEMISTRY

There is no immunohistochemistry that is specific for UESL. In fact, the key role
of immunohistochemistry is to rule out other possible tumors in the differential
(2–4). Variable expression of actin, vimentin, CD68, BCL-2, desmin, and CD10 is
commonly seen in UESLs (Figures 4A and B). Alpha-1-antitrypsin and
alpha-1-antichymotrypsin positivity within the eosinophilic globules is com-
­
monly encountered (Figure 4C). (2–4, 7). Weak cytokeratin staining has also been
reported, and a minority of UESLs have positivity for glypican 3 and nuclear posi-
tivity for CD117. As a result, glypican 3 should not be used on its own to rule out
a hepatoblastoma and hepatocellular carcinoma. Rather glypican 3 should be
deployed in a panel of stains and interpreted in conjunction with more reliable
markers such as HepPAR1 and β-catenin. UESLs are usually negative for S100,
MART1, myogenin, ALK-1, Beta-catenin, and HepPar1 (2–4, 7).

ELECTRON MICROSCOPY

Ultrastructually, USELs show electron-dense deposits in lysosomes and dilated

rough endoplasmic reticulum. The lysosomal electron-dense deposits represent
 Undifferentiated Embryonal Sarcoma of the Liver 169

Figure 3.  Histological features of undifferentiated embryonal sarcoma of the liver. A. High-grade
myxomatous areas with considerable pleomorphism and an atypical mitotic figure.
B. Hemorrhagic area with abundant tumor necrosis. C. Hemorrhagic area with cystic change.
D. Benign bile duct along with cords and nests of normal hepatocytes entrapped at the
periphery of the tumor. Tumor pseudocapsule at the right bottom corner of the image.
E. High-grade myxomatous area with PASD-resistant eosinophilic hyaline globules. F. Tumor
cells with PASD-resistant eosinophilic hyaline globules.

the PASD-resistant eosinophilic globules seen histologically (2, 6, 15, 16). Some
tumors in adults demonstrated myofilaments and intermediate filaments, suggest-
ing smooth muscle differentiation (17). Lipoblastic differentiation has also been
reported due to the presence of fat droplets (2, 15).
170 Huang J

Figure 4.  Immunohistochemistry (IHC) of undifferentiated embryonal sarcoma of the liver.

A. Strong diffuse positivity for Vimentin IHC. B. Variable positivity for Desmin IHC.
C. Positivity for Alpha 1-Antitrypsin IHC.

MOLECULAR FINDINGS

The pathogenesis of undifferentiated embryonal sarcoma of the liver is unclear.

Molecular and cytogenetic aberrations found in some UESLs include alterations in
p53 tumor suppressor gene leading to inactivation, and translocation t(11;19)
(q11;q13.3/13.4) and add(19)(q13.4). The break-apart site for the t(11;19) was
on the MALAT1 gene and this same translocation has been detected in some mes-
enchymal hamartomas. In addition, some undifferentiated embryonal sarcomas
have areas that histologically resemble mesenchymal hamartomas which supports
the belief that these two tumors are related. Areas that resemble mesenchymal
hamartoma have also been reported in adult patients (18). As a result, it is believed
that mesenchymal hamartomas could possibly be the precursor for some undif-
ferentiated embryonal sarcomas (2–4, 10, 19).

PROGNOSIS AND TREATMENT

UESL is predominately a pediatric tumor, and thus the literature about prognosis
is predominantly based on data from pediatric patients. Historically, this tumor
 Undifferentiated Embryonal Sarcoma of the Liver 171

had an abysmal outlook before a multimodality treatment approach was insti-

tuted. Nowadays, the 5-year overall survival for children is around 86% (1).
Surgical resection is the most important factor in the treatment of these
patients. As with most tumors, the aim of such surgeries is to obtain negative
margins although positive margins in some studies have not been shown to have
considerable effect on outcome. This unexpected finding may be due to chemo-
therapy eradicating any residual tumor left. In addition, due to the rarity of this
tumor, further data in the future will likely give a clearer picture concerning the
impact of margin status (1).
Nowadays, chemotherapy is commonly used in combination with surgery in
treating UESLs and this approach has been recognized as being a major factor in
improving patient outcomes. The chemotherapy regimen used may vary between
institutions and oncologists since a standardized approach has yet to be agreed
upon (1, 4). In addition, a minority of patients had radiation treatment in the
metastatic setting or in an attempt to prevent tumor recurrence. However,
the effectiveness of radiotherapy is not clear at this time and more studies will be
needed before a consensus can be reached (1). In patients with unresectable
tumors, orthotopic liver transplant has been used and may be of benefit in certain
situations. However, the data on orthotopic liver transplant as a treatment is
­limited (1, 3, 4, 10, 20).
In summary, advances in surgery and the introduction of various chemother-
apy regimens have been essential in achieving favorable outcomes for UESLs.
These favorable outcomes currently apply to pediatric patients because there are
not enough adult cases currently to make firm statistical conclusions. The rarity
of this tumor means that there are still considerable limitations to our understand-
ing of how to treat this tumor and further research in the future will be needed as
more cases are accrued (1, 4).

DIFFERENTIAL DIAGNOSIS

Mesenchymal hamartoma is a benign mesenchymal liver tumor that predomi-

nately occurs within the first 5 years of life. This benign tumor has been known to
occur in adults, but such cases are rare (21). The clinical presentation is usually
an enlarging abdominal mass and depending on the size at presentation, the
patient may have symptoms secondary to mass effect. Radiology usually shows a
mass with solid and/or cystic features. Depending on the time of detection, the
tumor can range considerably in size, with some cases reported measuring 30 cm
(21). Pathology classically shows a bland myxoid background with cystic areas.
Interspersed within this myxoid stromal element are blood vessels and bile ducts
in a disorganized pattern. Islands or cords of normal appearing hepatocytes are
usually present along the circumference of the tumor. Most cases will have areas
of extramedullary hematopoiesis (21). Unlike a UESL, there are no worrisome
histological features such as atypia, pleomorphism, atypical mitoses, or necrosis
(21, 22). As mentioned earlier, mesenchymal hamartomas rarely undergo malig-
nant degeneration into an UESL. There is considerable histological and molecular
evidence that mesenchymal hamartomas and undifferentiated embryonal sarco-
mas are related and share certain pathways in their pathogenesis (21–23).
172 Huang J

Hepatoblastoma is a common differential (Table 1) especially in infants and

children less than 5 years of age. Under the microscope, hepatoblastoma often
looks like normal liver parenchyma in varying stages of embryonic development
(2, 24). Although there are different histological subtypes of hepatoblastomas,
many will have areas that resemble hepatocytes. In addition, most hepatoblasto-
mas will not demonstrate the heterogeneous, myxoid, and/or pleomorphic

TABLE 1 Differential diagnosis for undifferentiated

embryonal sarcoma of the liver
Clinical
Tumor type Presentation Histology Ancillary Testing
Hepatoblastoma Children under 5yo Usually resembles liver HepPAR1, glypican 3,
parenchyma in various and β-catenin
stages of development. positive.
Tend to have a more
uniform histology and
less heterogeneity than
UESL.
Hepatocellular Mean age of pediatric Malignant polygonal Keratins, glypican 3,
carcinoma cases around 11yo hepatocytes in various and HepPAR1
patterns histologically. positive.
Mallory hyaline and
intracellular bile may be
present. Fibrolamellar
hepatocellular carcinoma
is also a common
subtype to be aware of.
Alveolar and Pediatric or Usually a small round blue Desmin, myogenin and
embryonal adolescents cell tumor. Certain myoD1 positive.
rhabdomyosarcoma subtypes can appear Most alveolar RMSs
spindled or myxoid. will show PAX3-
FOXO1 or PAX7-
FOXO1 fusion.
Gastrointestinal Most commonly in Most common is spindled DOG1, CD117
stromal tumors adults (median morphology in a positive. The vast
age 65yo), but can fascicular pattern but majority of GISTs
occur in children can appear epithelioid or will also show c-kit,
mixed. PDGFRA, or SDH
mutations.
Melanoma Most commonly in Can have a variety of S100, HMB45,
adults. Very rare histological appearances. MART1, SOX10
to be a primary Spindled, epithelioid, or positive.
liver tumor. even small round blue
cells. Some cases will
have melanin pigment in
the tumor cells, greatly
aiding in identification.
Table continued on following page
 Undifferentiated Embryonal Sarcoma of the Liver 173

TABLE 1 Differential diagnosis for undifferentiated

embryonal sarcoma of the liver (Continued)
Clinical
Tumor type Presentation Histology Ancillary Testing
Perivascular Usually in adult Spindle to epithelioid cells HMB45, Actin, MART1
epithelioid cell patients with prominent blood positive. Less often
tumor vessels and sometimes desmin and S100
fat. positive.
Angiosarcoma Usually in older Infiltrative atypical CD31, CD34, ERG,
adults spindle cells usually D240, Fli-1
with a vasoformative positive.
architecture.
Leiomyosarcoma Usually in older Compact spindle cell Desmin, Actin,
adults proliferation in a Caldesmon
fascicular pattern and positive.
commonly with an
eosinophilic cytoplasm
reminiscent of normal
smooth muscle.
Depending on grade,
varying degrees of
cytological atypia,
mitotic activity, and/or
necrosis are present.
Undifferentiated Predominately in High-grade pleomorphic Dedifferentiated
pleomorphic older adults. malignancy with liposarcoma
sarcoma, These are soft abundant mitotic activity is MDM2 IHC
myxofibrosarcoma, tissue sarcomas and necrosis. Variable positive and MDM2
dedifferentiated that hardly ever myxoid change present. FISH amplified.
liposarcoma occur as a primary Correlation with
liver tumor. clinical-radiological
findings are
essential in ruling
out a metastasis
from a primary soft
tissue sarcoma.

histology seen in undifferentiated embryonal sarcomas of the liver. Also, some

hepatoblastomas will have mesenchymal components with osteoid, chondroid,
teratoid, or rhabdomyoblastic differentiation. The presence of these components
would strongly argue against an undifferentiated embryonal sarcoma. Although
immunohistochemistry may vary depending on the histological subtype, most
hepatoblastomas are positive for HepPAR1, glypican 3, and β-catenin (2). In most
cases, morphological features should allow for proper distinction from an UESL,
but an immunohistochemical panel maybe necessary in ambiguous cases (2, 24).
Hepatocellular carcinoma (HCC) is the second most common primary malig-
nant liver tumor in the pediatric age group, with hepatoblastoma taking the top
spot (Table 1). A considerable number of these pediatric HCC patients will have
174 Huang J

predisposing factors such as alpha 1 antitrypsin deficiency, tyrosinemia, glycogen

storage disorders, and chronic viral hepatitis (especially Hepatitis B). Most
­pediatric HCCs will morphologically resemble their adult counterparts (25). The
fibrolamellar subtype of HCC is also commonly encountered, especially in young
adults (26). The vast majority of HCCs in pediatric and adult patients will not
have a sarcomatoid morphology (27) making histological distinction from UESL
much easier. In addition, most HCCs will resemble hepatocytes to some degree
and some HCCs will have Mallory hyaline and intracellular bile (2, 3). In addition,
strong positive staining for glypican 3 and especially HepPAR1 should greatly
assist in identifying an HCC in challenging cases (2, 3). As stated previously in the
immunohistochemistry section, a minority of USELs will be positive for glypican
3 IHC.
Rhabdomyosarcoma is in the differential, especially in pediatric patients.
Embryonal rhabdomyosarcomas tend to affect a young age group with around
60% of cases occurring before the age of 6 (28, 29). Alveolar rhabdomyosarcoma
tends to have an older age onset, affecting children and adolescents with around
half of cases occurring after 10 years of age (28, 29). A large portion of rhabdo-
myosarcomas will present as a small round blue cell tumor that has fairly uniform
histology. A minority of cases will demonstrate anaplastic cellular features and
show considerable nuclear pleomorphism. In addition, variants of embryonal
rhabdomyosarcoma can have a myxoid background. In general, the vast majority
of rhabdomyosarcomas will not demonstrate the histological heterogeneity of an
undifferentiated embryonal sarcoma of the liver. Furthermore, rhabdomyosarco-
mas are strongly desmin-positive and will also be positive for one or both skeletal
muscle markers (myogenin and MyoD1). An UESL may show variable desmin-
positivity but will not be positive for myogenin or MyoD1. In addition, the major-
ity of alveolar rhabdomyosarcomas will have the PAX3-FOXO1 or PAX7-FOXO1
fusion which is not found in UESLs (30, 31).
Gastrointestinal stromal tumors (GISTs) most often occur in adults (32) and
are usually easily identified because of their histology and positivity for CD117
and DOG1 IHC. GISTs primary to the liver are quite rare (33) when compared to
their much more common counterparts in the digestive tract (28). Since GISTs
have a well-known propensity to spread to the liver, most GISTs encountered on
a liver biopsy will be a metastatic deposit (34, 35). Most cases of GIST will be the
spindle cell type, composed histologically of a relatively uniform sheet of spindle
cells. A minority of GISTs will be of the epithelioid cell type which can appear
morphologically epithelioid, sclerosing, or sarcomatous. A minority of GISTs will
be of the mixed type, meaning that epithelioid and spindle cells are both present
within the same tumor (34, 36, 37). Immunohistochemistry for CD117 and
DOG1 will, in most cases, resolve any diagnostic dilemma (2, 38). Very rarely will
a GIST be negative for both DOG1 and CD117. Molecular testing for GIST muta-
tions (c-kit, PDGFRA, SDH) may need to be undertaken in cases where GIST is
still suspected but the IHC is non-contributory.
Melanoma is well known to be a great mimicker of any tumor histologically.
Primary melanoma of the liver is quite rare, with metastatic melanoma being by
far more common (39). When dealing with most sarcomatous appearing lesions,
the differential should always include melanoma and the appropriate immunohis-
tochemical stains should be done to exclude the possibility. S100 is a good stain
to use when screening a sarcomatous lesion for the possibility of a
 Undifferentiated Embryonal Sarcoma of the Liver 175

melanoma  (3,  40, 41). Additional follow-up stains such as SOX10, MART-1,
MITF, and HMB45 are often used in conjunction with S100 in cases that need
further workup. It should be noted that before diagnosing melanoma in the liver,
one should always consider the possibility of a perivascular epithelioid cell tumor
(PECOMA), because PECOMAs can also be positive for S100, MART1, MITF, and
HMB45. As a result, appropriate myoid markers such as smooth muscle actin and
desmin along with detailed examination of the histology should allow for proper
distinction between a PECOMA and a melanoma (42, 43).

OTHER SARCOMAS

Myxofibrosarcoma or undifferentiated pleomorphic sarcoma (UPS) can have areas

that closely resemble an undifferentiated embryonal sarcoma of the liver. However,
myxofibrosarcomas and UPSs usually affect patients over 50 years of age and
rarely occurs in children or young adults. In addition, the vast majority of these
types of sarcomas occur in the soft tissue and do not present as a primary liver
mass. Clinical presentation, age, past medical history of sarcomas, and adequate
radiology are essential in ruling out a soft tissue sarcoma involving, extending, or
metastasizing to the liver. Immunohistochemistry is of limited use in excluding a
myxofibrosarcoma or UPS since these soft tissue sarcomas are usually negative for
most immunohistochemistry and, like UESL, are diagnoses of exclusion (41).
Dedifferentiated liposarcomas are most commonly encountered as an intra-
abdominal/retroperitoneal mass. The histology can be quite varied, but an undif-
ferentiated pleomorphic sarcoma-like morphology is considered the classic
histology. Similar to UPS, this tumor usually presents in older adults and clinical-
radiological investigations will often help to exclude this tumor as a diagnostic
possibility. Dedifferentiated liposarcomas are MDM2 amplified and tests for
MDM2 (that is, immunohistochemistry or fluorescent in situ hybridization) can
be used in cases that require further work-up (44).
Angiosarcomas and epithelioid hemangioendotheliomas are rare mesenchy-
mal tumors of the liver. Primary angiosarcomas of the liver usually occur in
patients over 50 years of age while hemangioendotheliomas of the liver classically
occurs in middle aged adults (45, 46). Most epithelioid hemangioendotheliomas
(EHE) are easily recognized because of its myxochondroid matrix and the vacuo-
lated endothelial cells arranged in nests or cords (2). Malignant variants of EHEs
more closely resemble angiosarcomas and have high-grade infiltrative spindle
cells that vaguely try to form vessels. Many angiosarcomas will show vasoforma-
tive areas and are not as pleomorphic or myxoid as an UESL, making the distinc-
tion easy. In difficult cases or on limited biopsy material, a panel of vascular
markers (CD31, CD34, D2–40, ERG, and FLI-1) would be of considerable
­assistance (3).
Leiomyosarcomas are malignant tumors of smooth muscle. This tumor rarely
occurs in the liver and the vast majority of primary liver cases happens in adults,
mostly older adults. Most leiomyosarcomas in the liver will be a metastatic deposit
from a primary elsewhere, so sufficient clinical-radiological information is
­essential. Leiomyosarcomas that are not high-grade are usually easily recognized
histologically because of its close resemblance to normal smooth muscle.
176 Huang J

Higher-grade tumors can be more difficult to identify and, in such cases, immu-
nohistochemistry will be necessary. Positivity for actin, desmin, and caldesmon is
usually sufficient to rule out other possibilities in the differential (3, 47).

CONCLUSION

UESL is an aggressive malignancy that does occur in adults but is very rare. It
presents clinically most often with non-specific symptoms of an abdominal mass
with imaging features of a solid and cystic liver tumor. UESL is a diagnosis of
exclusion in pathology, meaning that all other differentials must be reasonably
excluded before a diagnosis of UESL can be confidently made. The pathogenesis
and origin of UESL is unclear at this time, but molecular aberrations in p53 and
translocation t(11;19) have been found. In terms of treatment, considerable prog-
ress has been made by implementing a multimodality approach which has led to
much more favorable outcomes for this high-grade liver tumor.

Acknowledgements: The author acknowledges Dr. Sarat Gaedde (Radiologist at

the Cross Cancer Institute in Edmonton) in helping to provide and interpret the
radiology images for this chapter.

Conflict of interest: The author declares no potential conflicts of interest with

respect to research, authorship and/or publication of this chapter.

Copyright and permission statement: The author confirms that the materials
included in this chapter do not violate copyright laws. Where relevant, appropri-
ate permissions have been obtained from the original copyright holder(s), and all
original sources have been appropriately acknowledged or referenced.

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 Index 179

Index

A Checkpoint inhibitors, 120

Children, 1
AB-729, 118
Children’s Cancer Study Group
ABI-H0731, 118
Staging, 152
Adefovir, 113
Children’s Oncology Group, 8
Adolescents, 1
Cholangiocarcinoma, 21, 97
Adults, 39
Cisplatin, 10
Aflatoxins, 2, 57
Clinical characteristics, 43
AJCC Cancer Staging Manual, 77
CLIP staging systems, 48
Alagille syndrome, 4
Congenital biliary dilatation, 22
Alpha-1-antitrypsin deficiency, 17
Congenital portosystemic shunts, 3
Antiviral, 113, 116, 117
Core protein inhibitor, 119
ARC-520, 117
Couinaud segments, 79
Autoimmune hepatitis, 4
Cryoablation, 65
Cure, 109
B
Barcelona Clinic Liver Cancer score, 8 D
BCLC staging system, 49
Diagnostic tests, 43
Besifovir dipivoxil maleate, 118
Diagnostics, 148
Bevacizumab, 10
Diet, 70
Biliary atresia, 3
Differential diagnosis, 6, 23, 171
Biliary lesions, 96
Downstaging, 127, 129, 130
Bridging therapy, 127, 130
Doxorubicin, 10
Budd-Chiari syndrome, 4
Drug targets, 111
Ductal plate malformation, 22
C
Cabozantinib, 10 E
Capecitabine, 10
Electron microscopy, 168
Capsid inhibitors, 118
Entecavir, 113, 119
Carcinoma of the Liver, 1
Entry inhibitors, 115
Cavernous hemangioma, 99
Epidemiology, 57, 147
cccDNA inhibitors, 119
Epithelioid hemangioendothelioma, 100
CD34, 46
180 Index

Erlotinib, 10 I
Etiology, 40, 146
Ifosfamide, 10
Everolimus, 10
Imaging, 5, 148
Expanded criteria, 127, 129
Immunohistochemistry, 168
Immunopathogenesis, 111
F Immunotherapy, 70
Fibrolamellar carcinoma, 13 International Society of Pediatric
Fluorouracil (5FU), 10 Oncology, 8
Focal nodular hyperplasia, 11, 89 Irinotecan, 10
Follow-up, 11, 157 Irreversible electroporation, 68
Fresh tissue, 81
J
G JAG-1, 4
Gemcitabine, 10 JAGGED-1, 4
Global estimates, 57 JNJ-3989, 118
Glycogen storage disorders, 20
Glypican-3, 46 K
Gross anatomy, 78
Keratin, 13, 16
Gross dissection, 77
Kidney, 7, 23, 80
Gross features, 167
Gross inspection, 81
L
H Laboratory tests, 148
Lamivudine, 113
HBsAg inhibitors, 119
Lifestyle, 70
HBV life cycle, 111
Lipid conjugates, 118
Hematoxylin and eosin stain, 42
Lipid-rich hepatocellular
Hepatic angiosarcoma, 102
carcinoma, 13
Hepatitis B, 109
Liver lesions, 88
Hepatoblastoma, 145
Liver resection, 63
Hepatocellular adenoma, 11, 91
Liver transplantation, 63, 129, 155
Hepatocellular carcinoma, 2, 39, 93
Liver tumors, 2, 87
Hepatocellular lesions, 89
Locoregional therapies, 127, 131
Hereditary tyrosinemia I, 19
Lung metastases, 156
Histology, 149, 167
Lymph nodes, 83
Host immunity, 120
 Index 181

M Q
Management, 62 Quadrant, 78
Metastatic malignancies, 103 Quadrate lobe, 79
Micrographs, 7
Microwave ablation, 65 R
Milan criteria, 64, 129
Radiofrequency ablation, 64
Mitomycin, 10
Radiologic-pathologic correlation, 87
Molecular findings, 170
Radiology, 89, 166
Morphology, 6
Reticulin stain, 43
Mucinous cystic neoplasm, 97
Reverse transcriptase inhibitors, 118
Risk assessment, 152
N
Natural history, 112 S
Non-alcoholic fatty liver disease, 55
Sampling, 81, 83
Non-alcoholic steatohepatitis, 55
Sarcoma of the liver, 165
Nucleos(t)ide analogues, 109
Sarcomas, 175
Selection criteria for liver
O
transplantation, 129
Okuda system, 8, 48 Small interfering RNAs, 117
Oxaliplatin, 10 Sorafenib, 10
Staging, 8, 47, 61, 83, 151
P Stereotactic body radiation therapy, 137
Stereotactic body radiotherapy, 68
Pathogenesis, 40, 146
Surface inspection, 78
Pediatric metabolic conditions, 17
Surgical anatomy of the liver, 78
Pegylated interferon, 113
Surgical approach, 154
Percutaneous ethanol injection, 64
Symptoms, 5, 147
Pregenomic RNA (pgRNA), 112
Systemic therapies, 69
Pre-neoplastic lesions, 41
PRETEXT, 8, 152
T
Prevention, 113, 114
Primary liver cancer, 55 Targeted therapies, 69
Prognosis, 170 Telbivudine, 113
Progressive familial intrahepatic cholestasis Temozolomide, 10
type, 2, 20 Tenofovir alafenamide, 113
182 Index

Tenofovir disoproxil fumarate, 113 V

Therapeutic considerations, 151
Vascular endothelial growth factor, 10
Therapy planning, 152
Vascular mesenchymal lesions, 99
Thermal ablation, 136
Vincristine, 10
Time of surgical procedure, 153
Tivantinib, 10
TNM classification, 47
W
Trans-arterial chemoembolization, Wilson’s disease, 21
66, 131 Wnt/β-Catenin, 112
Transcatheter arterial
radioembolization, 134 X
Treatment of relapses, 157
X-ray imaging, 14
Treatment, 8, 49, 62, 109, 113, 170
Treatment-response, 111
Tumor biology, 131 Y
Tumor node metastasis, 8 Y-90 radioembolization, 67
Tumor, 82
Z
U Z allele, 17
Undifferentiated embryonal sarcoma Zellballen growth pattern, 16
of the liver, 165 Zinc finger nucleases, 119
UNOS criteria, 129 Zonal, 7

Doi: https://doi.org/10.36255/exonpublications.livercancer.2021.index
 Liver Cancer
Consolato M. Sergi, MD, PhD, MPH, FRCPC, FCAP
Editor

Consolato M. Sergi, MD, PhD, MPH, FRCPC, FCAP,

is a full professor of pathology and adjunct
professor of pediatrics at the University of Alberta,
Edmonton, Canada. He is also a consultant for
Standards and Guidelines in Carcinogenesis of
Chemical Compounds published by the World
Health Organization/International Agency on
Research on Cancer (WHO/IARC monographs), Lyon, France.
His research interests include hepatic tumors, metabolic diseases,
cholangiopathies, organ transplantation, and gut/bile microbiome
using cell lines, animal models, and clinical samples. He identified
the role of apoptosis in ductal plate malformation of the liver,
characterized sialidosis, and found two new genes, WDR62, which
encodes a centrosome-associated protein (Nat Genet 2010) and
OTX2, mutations of which can contribute to dysgnathia (J  Med
Genet 2012). Professor Sergi has published more than 300 research/
review articles and several books and book chapters. He has
supervised and mentored many PhD students and clinical fellows.
He is also on the editorial boards of several scientific journals.