Primary Liver Cancers—Part 1: Histopathology, Differential Diagnoses, and Risk Stratification

Abstract

Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) are the 2 most common primary malignant liver tumors, with hepatocellular and bile ductular differentiation, respectively. This article reviews the key histopathological findings of these 2 primary liver cancers and includes a review of the role of ancillary testing for differential diagnosis, risk stratification according to the American Joint Committee on Cancer (AJCC) staging recommendation, and a review of precancerous lesions. A literature review was conducted to identify articles with information relevant to precancerous precursors, current histopathological classification, ancillary testing, and risk stratification of primary malignant liver tumors. The histomorphology of normal liver, preinvasive precursors, primary malignancies, and morphological variants, and the utilization of ancillary tests for the pathological diagnosis are described. Dysplastic nodules are the preinvasive precursors of HCC, and intraductal papillary neoplasms of bile ducts and biliary intraepithelial neoplasia are the preinvasive precursors of CC. Benign liver nodules including focal nodular hyperplasia and adenomas are included in this review, since some forms of adenomas progress to HCC and often they have to be differentiated from well-differentiated HCC. A number of morphological variants of HCC have been described in the literature, and it is necessary to be aware of them in order to render the correct diagnosis. Risk stratification is still dependent on the AJCC staging system. The diagnosis of primary liver carcinomas is usually straightforward. Application of the appropriate ancillary studies aids in the differential diagnosis of difficult cases. The understanding of the carcinogenesis of these malignancies has improved with the standardization of the pathological classification of preinvasive precursors and studies of the molecular pathogenesis. Risk stratification still depends on pathological staging.

Full text

Review Article
Primary Liver Cancers—Part 1:
Histopathology, Differential Diagnoses,
and Risk Stratification
Kun Jiang, MD, PhD
1,2
, Sameer Al-Diffalha, MD
3
, and Barbara A. Centeno, MD
1,2
Abstract
Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) are the 2 most common primary malignant liver tumors, with
hepatocellular and bile ductular differentiation, respectively. This article reviews the key histopathological findings of these
2 primary liver cancers and includes a review of the role of ancillary testing for differential diagnosis, risk stratification according to
the American Joint Committee on Cancer (AJCC) staging recommendation, and a review of precancerous lesions. A literature
review was conducted to identify articles with information relevant to precancerous precursors, current histopathological
classification, ancillary testing, and risk stratification of primary malignant liver tumors. The histomorphology of normal liver,
preinvasive precursors, primary malignancies, and morphological variants, and the utilization of ancillary tests for the pathological
diagnosis are described. Dysplastic nodules are the preinvasive precursors of HCC, and intraductal papillary neoplasms of bile
ducts and biliary intraepithelial neoplasia are the preinvasive precursors of CC. Benign liver nodules including focal nodular
hyperplasia and adenomas are included in this review, since some forms of adenomas progress to HCC and often they have to be
differentiated from well-differentiated HCC. A number of morphological variants of HCC have been described in the literature,
and it is necessary to be aware of them in order to render the correct diagnosis. Risk stratification is still dependent on the AJCC
staging system. The diagnosis of primary liver carcinomas is usually straightforward. Application of the appropriate ancillary
studies aids in the differential diagnosis of difficult cases. The understanding of the carcinogenesis of these malignancies has
improved with the standardization of the pathological classification of preinvasive precursors and studies of the molecular
pathogenesis. Risk stratification still depends on pathological staging.
Keywords
hepatocellular carcinoma, cholangiocarcinoma, focal nodular hyperplasia, hepatic adenoma, dysplastic nodule, biliary intraepithe-
lial neoplasia, intraductal papillary neoplasm
Received December 27, 2016. Accepted for publication August 23, 2017.
Introduction
Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC)
are the 2 primary malignancies of the liver. Preinvasive precur-
sor lesions include dysplastic nodules (DNs) leading to HCC and
biliary intraepithelial neoplasia (BilIN) and intraductal papillary
neoplasms of the bile ducts (IPN-B) leading to CC.
Normal Liver
The liver is the largest gland in the human body and weighs on
average 1500 g. It is located in the right upper abdomen and
1
Department of Anatomic Pathology, H. Lee Moffitt Cancer Center and
Research Institute, Tampa, FL, USA
2
Department of Oncologic Sciences, Morsani College of Medicine at
University of South Florida, Tampa, FL, USA
3
Division of Anatomic Pathology, Department of Pathology, University of
Alabama School of Medicine, Birmingham, AL, USA
Corresponding Author:
Barbara A. Centeno, Vice Chair, Clinical Services, Department of Anatomic
Pathology, H. Lee Moffitt Cancer Center and Research Institute, 12902
Magnolia Drive, MCC-LAB, Tampa, FL 33612, USA.
Email: barbara.centeno@moffitt.org
Cancer Control
Volume 25: 1–26
ªThe Author(s) 2018
Reprints and permission:
sagepub.com/journalsPermissions.nav
DOI: 10.1177/1073274817744625
journals.sagepub.com/home/ccx
Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License
(http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further
permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage).

midabdomen and extends to the left upper abdomen under the
diaphragm. The blood supply to the liver comes from the
hepatic artery (20%-40%) and the portal vein (60%-80%).
Blood flows out of the liver through the hepatic vein into the
portal vein.
The liver is regulated by a nervous system containing both
afferent and efferent neurons actively involved in numerous
biological and physiological processes. The autonomic ner-
vous system of the liver is essential in the maintenance of
homeostasis and human metabolism. The afferent branch
includes the sensation of nutrition such as lipids, glucose, and
a spectrum of metabolites, which are involved in trigging the
nervous system to monitor necessary physiological changes.
In parallel, the efferent branch is responsible for metabolic
regulation, modulation of fibrosis and biliary function, and
various associated processes. Subsequently, liver functions
as both a sensor and an effector under the influence of neu-
rological signaling.
Microscopically, the liver is composed mostly of hepato-
cytes with bile duct cells and mesenchymal cells. Figure 1
shows the histology of normal liver and ancillary study stain-
ing patterns. Hepatocytes are polygonal cells with eosinophi-
lic granular cytoplasm and round nuclei with 1 or 2 prominent
nucleoli. The hepatocytes are arranged in anastomosing
plates, usually 1 to 2 cells thick and separated by sinusoids
(Figure 1A-B). The sinusoids are lined by endothelial cells
and also contain Kupffer cells, which are specialized macro-
phages. The space of Disse between the sinusoids and the
hepatocytes contains stellate cells, members of the myofibro-
blastic family, causing fibrosis.
The portal triads contain branches of the portal vein, hepatic
artery, bile ducts, vagus nerve, and lymphatics (Figure 1C).
Blood flows into the liver through the portal tracts and into the
sinusoids and exits through the central veins (Figure 1D),
which converge to drain into the hepatic vein.
The bile canaliculi are located between the walls of the hepa-
tocytes. These communicate with the canals of Herring, which
are lined partly by hepatocytes and partly by bile duct. Bile
drains into bile ductules from the canals of Herring, which con-
verge and form larger interlobular bile ducts in the portal triads.
Normal liver will show a normal reticulin framework (Figure
1E). The vascularity will not be increased and focused on portal
tracts (Figure 1F). The periodic acid-Schiff-diastase (PASD)
stains show the portal tracts (Figure 1G). Glutamine synthetase
(GS) is localized to the central vein area (Figure 1H).
Histology of Preinvasive Precursors
Hepatocyte Dysplastic Foci and Nodules
The International Working Party published standardized termi-
nology and criteria for nodular hepatocellular lesions, and this
terminology is still used today. Precancerous lesions include
dysplastic foci and low- and high-grade DNs, which are
detected most often in cirrhotic livers and also livers with
chronic, noncirrhotic disease. Dysplastic foci are less than
1 mm in diameter and consist of groups of hepatocytes with
dysplasia,
1
now called either large cell or small cell change.
Dysplastic nodules may be single or multiple nodules. They are
macroscopically larger than the surrounding cirrhotic nodules
but less than 15 mm in size. Dysplastic nodules are classified
according to international consensus as low grade or high
grade, based on histomorphological features.
1
The most common cytological change found in dysplastic
foci is small cell change.
2
These cells are smaller than the adja-
cent hepatocytes and show an increased nuclear to cytoplasmic
ratio (N/C), mild nuclear atypia, hyperchromasia, and cytoplas-
mic basophilia (Figure 2A-B). This type of dysplasia is consid-
ered precancerous, since it harbors molecular alterations
involved in carcinogenesis, including chromosomal gains and
losses, telomere shortening, and inactivation of cyclin Depen-
dent Kinase Inhibitor 1A (CDKN1A). These areas will also show
an increase in proliferation markers compared to adjacent non-
neoplastic liver.
3-5
The reticulin stain usually shows a preserved
trabecular framework, without decrease in or loss of expression.
Large cell change is seen in the livers of patients with
chronic hepatitis B virus (HBV) or hepatitis B virus (HCV)
infection or cirrhosis of various etiologies. Pathologically, it
is characterized by nuclear and cytoplasmic enlargement, pre-
served N/C, nuclear pleomorphism, hyperchromasia, and mul-
tinucleation. Its exact nature is not clear. In cirrhosis, it usually
occurs diffusely and is more likely a degenerative change. In
HBV, it appears to be precancerous and is associated with
telomere shortening, Cyclin Dependent Kinase Inhibitor 2A
(CDKN2A)- and CDKN1A-regulated checkpoint inactivation,
increased DNA damage, and a higher proliferation index.
Low-grade DNs are virtually indistinguishable from macro-
regenerative nodules and sometimes the 2 terms are used inter-
changeably. Low-grade DNs may have portal tracts and a bile
ductular reaction within the nodule. They have well-defined
borders. The cells are fairly uniform in appearance with min-
imal nuclear atypia, a slight increase in N/C ratio, and no
mitoses. Low-grade DNs may also show Mallory bodies, bile
stasis, clear cell cytoplasmic change, iron or copper deposits, a
slight decrease in cell size, and fatty changes.
6
The architecture
is maintained, and the liver plates remain a single-cell thick.
Reticulin stain shows a normal framework.
High-grade DNs are characterized by small cell change,
hepatic plates up to 3 cells in thickness, and occasional pseu-
dogland formation (Figure 2). Focal decrease in the reticulin
could be seen although it may remain normal (Figure 2C).
Glypican 3 expression is often negative in the DNs (Figure
2D). These nodules may also contain Mallory bodies, glyco-
gen, fat, clear cell change, rare portal tracts, mitoses, cytoplas-
mic basophilia, and bile. It is largely accepted that high-grade
DN are precursors of HCC. In some cases, it may be impossible
to tell a high-grade DN from well-differentiated HCC, espe-
cially in needle biopsies. High-grade DNs may develop sub-
nodules of HCC. A distinguishing feature is that DNs do not
invade adjacent parenchyma. The cells are fairly uniform in
appearance with minimal nuclear atypia, a slight increase in N/
C ratio, and no mitoses.
6
2Cancer Control

Figure 1. Normal liver. Photomicrograph (original magnification, 40; hematoxylin–eosin [H&E] stain) of benign hepatic parenchyma obtained
by excisional biopsy (A), and needle core biopsy (B). Both show the presence of portal tracts (arrow) and 1- to 2-cell thick hepatic plates.
No large vessels, cytologic atypia, mitoses, or necrosis are seen. (C) Portal tract showing bile duct, vein branch, arteriole, nerve, and lymphatic.
(D) Liver showing central vein. (E) Photomicrograph of reticulin special stain in benign liver tissue, which highlights 1- to 2-cell thick plates and
unremarkable portal tract (arrow). (F) Photomicrograph of CD34 immunostain in benign liver tissue, which demonstrates a restricted pattern of
periportal labeling (arrow). (G) Periodic acid-Schiff (PAS)-diastase highlights portal tract and bile duct (arrow). (H) Photomicrograph of
glutamine synthetase immunostain in benign liver, with pericentral labeling (arrow); peripheral lobular areas are not labeled.
Jiang et al 3

Biliary Intraepithelial Neoplasia and Intraductal
Papillary Neoplasia of the Bile Ducts
Biliary intraepithelial neoplasia (BilIN) and intraductal papil-
lary neoplasm of bile duct (IPNB) are precursors of bile duct
carcinomas.
Biliary intraepithelial neoplasia. Biliary intraepithelial neoplasia is
the preinvasive flat precursor lesion of CC, representing the
pathology of multistep cholangiocarcinogenesis.
7
Before
2005, when the term biliary intraepithelial neoplasia was pro-
posed, it was called biliary atypia or dysplasia.
8
Biliary intrae-
pithelial neoplasia is the biliary counterpart of pancreatic
intraepithelial neoplasia. In general, it is composed of flat or
micropapillary dysplastic epithelium. The terminology applies
to lesions in both the intrahepatic and extrahepatic bile duct
systems. Biliary intraepithelial neoplasia is classified into 3
grades according to Zen et al.
8
Biliary intraepithelial neoplasia 1 (low-grade lesion) shows
flat or micropapillary architecture with focal area of stratifica-
tion (up to lower 2/3). The lining cells show basally located
nuclei with mild atypia. No mitoses or loss of cellular polarity
are noted (Figure 3B).
Biliary intraepithelial neoplasia 2 (intermediate-grade
lesion) shows flat, pseudopapillary or micropapillary architec-
ture with luminal surface pseudostratification. The lining cells
show moderate nuclear atypia, focal loss of polarity, and rare
mitotic figures.
Biliary intraepithelial neoplasia 3 (high-grade lesion, car-
cinoma in situ) shows pseudopapillary, micropapillary, or
flat architecture, and occasionally luminal budding and cri-
briform formation. The lining cells show severe nuclear
atypia with diffuse loss of polarity and increased mitotic
figures (Figure 3C).
Gastric foveolar, pyloric, and intestinal metaplasia are infre-
quently seen in BilIN. They are identified in BilIN-2 and -3
compared to those in BilIN-1.
7,9
Biliary intraepithelial neopla-
sia is often found at the surgical resection margin of biliary
tract adenocarcinomas, but identifying BilIN on frozen section
is of no clinical consequence since only invasive cancer will
have an impact on patient outcome.
10
Finally, it is important to
differentiate BilIN from reactive atypia which is usually asso-
ciated with acute inflammation, erosion, ulceration, and almost
universally, stent effect.
Intraductal papillary neoplasm of the bile duct. Intraductal papil-
lary neoplasm of bile ducts is a rare tumor arising in intrahe-
patic or extrahepatic bile ducts. It is considered a mass-forming
precursor of invasive carcinoma. The exact etiology and patho-
genesis of IPNBs are still unclear, but hepatolithiasis and clo-
norchiasis are known as the 2 major risk factors.
11
Intraductal
papillary neoplasm of bile ducts is usually a single or multiple
Figure 2. Dysplastic foci and nodule. (A) and (B) Small cell change with a high N/C, and small, hyperchromatic nuclei. H&E, 20 and 40.
(C) High-grade dysplastic nodule showing retained reticulin framework. Reticulin, 20. (D) Immunohistochemistry for glypican 3 is negative.
20. N/C indicates nuclear to cytoplasmic ratio.
4Cancer Control

gray tan to yellow, friable polypoid lesions. It is a neoplastic
papillary proliferation replacing the normal ductal epithelium
(Figure 4A-B). Histologically, it is composed of papillary
structures that have fine vascular cores (Figure 4C). The papil-
lae are lined by a spectrum of atypical cells (Figure 4D). It has
been reported that it may be associated with foci of invasive
carcinoma, which recommends a very careful macroscopic and
microscopic examination. According to Wan et al, about 40%
to 80%of IPNBs have a component of invasive carcinoma
(tubular or mucinous adenocarcinoma).
11
IPNB is classified in a manner similar to the World Health
Organization classification of pancreatic intraductal papillary
mucinous neoplasm (IPMN): low-grade, intermediate-grade,
and high-grade IPNB, some of which present with an
associated invasive carcinoma. There are 4 subtypes of IPNB
based on the lining epithelium: pancreatobiliary (the most
common type), intestinal (the second most common), gastric,
and oncocytic types. Usually there is high-grade dysplasia
associated with the pancreatobiliary or intestinal types.
12,13
The pancreatobiliary type is often positive for MUC-1 (and
MUC-5AC, cytokeratin [CK]-7, CK-20) but is negative for
MUC-2. The intestinal type consistently expresses MUC-2
and MUC-5AC, CK-7, and CK-20) but not MUC-1. The
gastric type expresses MUC-5AC, CK-7, and CK-20 but is
negative for MUC-1 and MUC-2. The oncocytic type
consistently expresses MUC-6, MUC-5AC, CK-7, and CK-
20) with focal expression of MUC-1 and/or MUC-2.
11,12,14
Finally, IPNB is considered to be a biliary counterpart of
IPMN due to the multiple similar features between both
entities; they are both intraductal neoplastic processes,
radiologically and grossly identifiable, with intraductal
papillary proliferations and are considered precursors of
tubular and mucinous adenocarcinoma.
Liver stem cells and the progenitors of hepatocarcinogenesis.
Human cancer stem cells possess the capacity to self-renew,
to differentiate into multiple malignant cellular lineages, and to
proliferate. They are associated with a poorer prognosis
because of their greater tumorigenicity and chemoresistance.
Recent advances in stem cell biology have enabled our under-
standing and identification of cancer stem cells in solid tumors
as well as putative stem cells in normal solid organs. Recent
studies illustrated that stem cells play important roles in the
carcinogenesis of various types of cancer, including primary
liver tumors.
15,16
Hepatocellular carcinoma and cholangiocarcinoma are
2 distinct types of liver cancers. It has been generally accepted
that the HCC phenotype is derived from hepatocytes and that
the cholangiocarcinoma phenotype is derived from cholangio-
cytes; however, a histopathological intermediate phenotype has
Figure 3. Biliary intraepithelial neoplasia (BilIN). A, Nonneoplastic bile duct lined by columnar epithelium, with basally located nuclei (H&E,
20). B, BilIN 1. Bile duct lined by pseudostratified columnar cells. The nuclei are slightly enlarged and elongated (H&E, 20). C, BilIN 3. The
cells demonstrate loss of polarity, and extend to the luminal surface. The nuclei are enlarged, with vesicular nuclei and nuclear membrane
irregularity (H&E, 20).
Jiang et al 5

been recognized, which appears to arise from hepatic progeni-
tor or cholangiocarcinoma stem cells.
15,17
Hepatic progenitor cells have been identified in HCC and
can be identified with EpCam, and CD133, CD90, CD44, and
CD13.
18
Cytokeratins 7 and 19 are also recognized as
stemness-related markers in HCC, and expression of these pro-
teins in an HCC predicts a worse outcome for the patient.
Durnez et al
18
had analyzed 109 cases of HCCs and found that
28%of the tumors contained cells expressing CK-7 or CK-19
or both, with features of liver stem cells. Remarkably, the
higher recurrence rate of CK-19-positive tumors after trans-
plantation suggests a worse prognosis for these HCCs com-
pared to CK-19-negative tumors.
18
Gene expression analysis
has shown that tumors expressing CK-7 and CK-19 have an
expression pattern similar to that of fetal hepatoblasts. In this
subset, activation of AP-1 transcription factors appears to play
a key role in hepatic carcinogenesis.
15
Hepatocellular carcinoma expressing stemness-related pro-
teins are more aggressive and have a poorer clinical outcome
compared to the conventional HCCs that do not express
stemness-related markers. These tumors demonstrate an
infiltrative growth pattern, vascular invasion, and more intra-
tumoral fibrous stroma. There is a spectrum of morphological
and immunophenotypic features between HCCs with stemness-
related marker expression, scirrhous HCCs, and combined
hepatocellular–cholangiocarcinoma with stem cell features.
Hepatocellular carcinomas with stemness-related marker
expression are associated with increased serum a-fetoprotein
(AFP) levels and a poor prognosis. The workup of HCC should
include markers of stem cellness, such as CK-19, as tumors
expressing these markers have increased chemoresistance, ear-
lier recurrence after surgical and/or locoregional treatment,
increased invasiveness/metastasis, and poor overall
survival.
19,20
Several studies have also documented the existence of cho-
langiocarcinoma stem cells; several cell surface antigens such
as CD24, EpCAM, CD44, CD133, and others have been shown
to label such cholangiocarcinoma stem cells.
17
Of note, ele-
vated expression of stem cell surface markers was associated
with more aggressive behavior.
17
To date, reports showing
possible signaling pathways in cholangiocarcinoma stem cells
are under investigation, whereas distinct and specific pathways
are expected to be present in these stem cells compared to other
cancer cells that have no stem cell properties.
17
The multipotent nature of the liver stem cells have been well
demonstrated by the recent advancements in stem cell investi-
gations introduced above, especially in the cases of combined
hepatocellular–cholangiocarcinomas frequently encountered
Figure 4. Intraductal papillary neoplasm of the bile duct (IPNB) with moderate dysplasia. This case was adjacent to an invasive carcinoma (not
shown here). A, The lumen of the bile duct is completely replaced by a papillary epithelial proliferation (H&E 4). B. The neoplasm exhibits
finger-like, papillary projections (H&E, 10). C. The papillary fronds are lined by columnar, mucin-containing cells (H&E, 20). D, The nuclei are
enlarged, elongated, and pseudostratified (H&E, 40).
6Cancer Control

clinically. In these combined tumors, the identified progenitor
cells merged with HCC components, cholangiocarcinoma com-
ponents, and the mature-appearing hepatocytes within the same
masses.
21
The most likely explanation is that these tumors are
of hepatic stem cell origin, supporting the concept that human
hepatocarcinogenesis could be due to transformation of pro-
genitor cells and that such a process may lead to the develop-
ment of certain mixed hepatocellular–cholangiocarcinomas as
well as DNs.
21
These liver stem cells have the unique potential
to develop into cholangiocarcinoma stem cells through genetic
alteration in gene expression profiles; and it has been con-
firmed that cholangiocarcinoma is of hepatic progenitor cell
origin due to the expression of certain stem-specific cell mar-
kers.
17
Furthermore, microarray analysis has identified unique
gene expression profile between the tumor cells within the
same tumor and further demonstrated the so-called several
“stemness genes” in the subpopulation of tumor cells.
22
The
study further indicated that this minority population of tumor
cells possess extreme carcinogenic potential and provide het-
erogeneity to the cancer stem cell system.
22
These results indi-
cate that both HCC and cholangiocarcinoma may be derived
from common bipotent progenitor cells and that the hepatic
stem and progenitor cells are able to differentiate into both
hepatocytes and cholangiocytes.
Based on the above discussion, it is conceivable that target-
ing therapies for surface molecular markers or specific signal-
ing pathways of cholangiocarcinoma stem cells may be
important in order to improve the clinical outcome of patients
with this lethal disease. However, cancer stem cells show over-
lapping profiles of their markers and signaling pathways with
normal tissue cells; therefore, the side effects of targeted therapy
remain challenging to predict and could outbalance the clinical
benefits. Identification and combating of unique markers exclu-
sively belonging to stem cells are essential to lower the associ-
ated toxicity to normal cells and functions and to maximize the
clinical benefits. It may be required to combine multiple ther-
apeutic strategies to treat these liver primary tumors.
Hepatocellular Carcinoma
Hepatocellular carcinoma encompasses a group of malignant
liver neoplasms that show hepatocellular differentiation.
Histopathology of HCC
Histopathology of HCC, not otherwise specified. In most cases,
HCC is at least suspected or recognizable directly on H&E-
stained sections, due cytological atypia and architectural
abnormalities such as thickened hepatic plates (Figures 5A and
B), endothelial lining (Figure 5C), pseudo-glandular configura-
tion (Figure 5D), and lymphovascular involvement by tumor
(more often seen in resection material) and the absence of
portal tracts and hepatic lobules. Most HCCs grow in trabecu-
lar, nested, solid, or pseudoacinar (Figure 5E) growth patterns.
Aberrant vessels can be identified within the lobules instead of
portal tracts, which is usually a helpful finding in well-
differentiated HCC (Figure 5D). Bile production can be seen
in fair amount of tumor cells and is pathognomonic for hepa-
tocellular differentiation. Well-differentiated HCCs have abun-
dant finely granular eosinophilic cytoplasm, round nuclei with
dispersed chromatin, and prominent nucleoli (Figure 5F). If the
biopsy was indeed obtained from a mass in the liver and
appears hepatocellular on H&E sections, the differential list
includes HCC and its mimickers such as focal nodular hyper-
plasia (FNH), hepatic adenoma (HA), and DNs.
Similar to benign hepatocytes, the tumor cells in HCC can
demonstrate steatotic, clear cell change (Figure 5F), or signif-
icant nuclear inclusions (Figure 5F). Most HCCs present with
these classic histomorphological characteristics at presenta-
tion; a straightforward diagnosis of HCC can usually be made
without significant obstacles. As HCC becomes less well dif-
ferentiated, the amount of cytoplasm generally decreases, the
N/C increases, as well as the progression of nuclear atypia.
Focal and confluent necrosis is frequently appreciated in
resected HCC tumors or in biopsy material of HCCs when
ample tissue was sampled. Several variants of HCCs have been
recognized in the literature, which are discussed in detail in the
following text.
Variants of HCC
Cirrhosis-like HCCs. Cirrhosis-like HCC is an evolving topic; in
this type of HCC, tumor masses may mimic multifocal cirrhotic
nodules in a patient with known liver cirrhosis, but no true
tumor mass can be seen on radiographic or imaging studies.
Grossly, cirrhosis-like tumor nodules are identified, with subtle
color and texture differences from the background cirrhotic
liver parenchyma.
23
Microscopically, tumor appears well to
moderately differentiated, frequently with ballooning, Mallory
bodies, and cholestasis; tumor cells resemble conventional
HCC cells, but a definitive tumor mass is not well appre-
ciated.
23
Cirrhosis-like HCC is an insidious type of HCC and
is usually not clinically suspected due to the lack of radio-
graphic findings; subsequently, they are often incidentally
identified on a staging protocol and in liver explants. Incidental
microscopic findings of HCC in needle cores from routine
biopsy procedures are highly suspicious of cirrhosis-like HCC.
Cirrhosis-like HCC may also be identified in the vicinity of a
dominant nodule of classic HCC.
The HCC with clear cell change. The HCC with clear cell change
is a fairly common variant of HCC that frequently poses a
diagnostic challenge, as it shows morphological similarities
to a spectrum of adenocarcinoma and epithelioid tumors. Sim-
ilar to benign hepatocytes, HCC cells may undergo steatosis/
fatty change, ballooning, and steatohepatitis-like change with
Mallory body formation. The cells show abundant, clear cyto-
plasm (Figure 6A). Generally, the cytomorphology mimics
other neoplasms with clear cell features. The main diagnostic
challenge lies in efficient usage of available tissue in perform-
ing a panel of special stains and immunohistochemical studies
(to be discussed in the later portion of this review) and to
Jiang et al 7

differentiate other types of tumors with clear cell histology,
such as metastatic renal cell carcinoma, neuroendocrine neo-
plasms with prominent clear cells, clear cell melanoma, epithe-
lioid angiomyolipoma, and any other tumors with epithelioid
morphology and clear cytoplasm encountered in liver.
Scirrhous HCC. In recent years, scirrhous HCC has been
accepted as a unique subtype of HCC, which is distinctive from
the better known fibrolamellar carcinoma.
24-27
This type of
HCC arises in background cirrhotic liver, accounting for
approximately 5%of all HCCs. An interesting fact about this
type of HCC is that tumor nodules often develop beneath the
liver capsule. Scirrhous HCCs are not usually suspected
clinically due to its intriguing radiographic finding that mimics
intrahepatic adenocarcinoma, namely, cholangiocarcinoma;
the best method for establishing a correct diagnosis is tissue
evaluation.
24-27
Microscopically, the characteristics of scir-
rhous HCC is prominent intratumoral dense fibrosis inter-
mingled with neoplastic hepatocytes arranged in various
patterns: trabecular, microtubular, micronodular, and pseudoa-
cinar structures with nests (Figure 6B). Classic HCC-like areas
have also been occasionally seen in cases of multifocal scir-
rhous HCCs.
24-27
The peculiar intratumoral dense fibrosis
mimics posttherapeutic changes following neoadjuvant che-
moradiation or chemoembolization treatment. Review of the
clinical history and correlation with histological and laboratory
Figure 5. Photomicrograph (H&E stain) of hepatocellular carcinoma. A, Absence of portal tracts and thickened hepatic plates in HCC, 40.
B, Solid and nodular growth pattern, 100. C, Endothelial wrapping and lining in sinusoidal space in HCC, 200. D, Unaccompanied vessels.
H&E, 200. E, HCC with a tubular and pseudoglandular pattern, 100. F, HCC with large nuclei with prominent nucleoli, and steatotic clear
change, 200. HCC indicates hepatocellular carcinoma.
8Cancer Control

findings is essential in recognizing this rare variant of HCC.
Immunohistochemical studies had revealed a significantly
higher expression of CK-7 and a significantly lower expression
of hepatocyte paraffin 1 (HepPar1) in scirrhous HCC versus
conventional HCC.
24,27
Prognostically, there are no significant
differences in tumor cell proliferative rate and patient survival
have been detected between the patients with scirrhous HCCs
and those with classic HCCs.
25,26
Sarcomatoid HCCs. Sarcomatoid HCC, also named carcinosar-
coma and spindle cell carcinoma in the literature, is a rare
variant of HCC. The reported prevalence including surgical
resection cases has been less than 2%, consisting mostly of
individual case reports and small case series.
28-31
The majority
of reported sarcomatoid HCC cases contain obvious sarcoma-
tous histology, with or without classic HCC, and with coexist-
ing cholangiocarcinoma elements identified in extremely rare
cases.
28-32
Grossly, sarcomatoid HCCs are large tumors, usu-
ally with satellite nodules, involving a cirrhotic liver. Micro-
scopically, tumor masses were composed of irregular,
polygonal, bizarre, and spindle-shaped malignant epithelioid
cells, mixed with various mesenchymal components, such as
rhabdomyosarcoma, chondroid sarcoma, osteoclast-like giant
cells, and more primitive, hepatoblastoma-like features
33-36
;an
unexpected element of cholangiocarcinoma could also be pres-
ent in this variant of tumors.
32
Upon detailed sectioning, a
distinct transition from HCC to sarcomatoid component may
be confirmed. Expression of AFP by both HCC-like and sarco-
matous cells and elevated levels of AFP in the serum have also
been reported in certain cases. Although classic HCC-like
tumor cells express E-cadherin but not vimentin, the mesench-
ymal (sarcomatous) component highly expresses vimentin with
a loss of E-cadherin protein.
28,34,37
Nuclear proliferative mar-
ker Ki67 was also expressed at higher levels in sarcomatous
versus HCC-like tumor cells. Compared to classic HCCs, sar-
comatoid HCCs have a more aggressive clinical behavior and
are associated with an ominous prognosis.
30,31,38
Optimized
management options are currently undetermined.
The HCC with unusual small cell morphology. The authors have
seen rare cases of HCC with an unusual small cell morphology,
some of which were consultation cases sent to our institute for
second opinion or due to a patient’s transfer of care; similar
cases have also been documented in the literature.
35
The pecu-
liar small cell morphology may cause diagnostic error and
adversely impact clinical decision-making. The key problem
is distinguishing these carcinomas from neuroendocrine tumors
or combined HCC with neuroendocrine differentiation, since
their morphological features are suggestive of neuroendocrine
differentiation. Some of the cases we observed were misdiag-
nosed as neuroendocrine tumors at outside institutions due to
the fact that the HCC cells lacked bile pigment, grew in solid
nests without obvious thickened hepatic plates, and the cells
showed a scant to medium amount of slightly eosinophilic,
Figure 6. Variants of HCC. A, HCC, clear cell variant. H&E, 40. B, Scirrhous variant of HCC, 100. C, Small cell variant HCC. H&E, 40.
HCC indicates hepatocellular carcinoma.
Jiang et al 9

finely granular to clear cytoplasm, easily mimicking neuroen-
docrine tumor cells (Figure 6C). Adding to the difficulty when
investigating this variant of HCC is that the neoplastic cells
might occasionally show patchy, faint nonspecific labeling by
synaptophysin or chromogranin, which upon comparison with
the positive control, should be interpreted as negative. This
subtype will express markers of hepatocellular differentiation,
to be discussed in a later section of this review.
This variant of HCC should also be differentiated from HCC
with a focal neuroendocrine component or combined neuroen-
docrine carcinoma and HCC of the liver.
39,40
An immunohis-
tochemically workup will show the HCC component to express
markers of hepatocellular differentiation and not express mar-
kers of neuroendocrine differentiation. The neuroendocrine
component will express markers of neuroendocrine
differentiation.
39,40
Fibrolamellar HCC. Fibrolamellar HCC (FL-HCC) is a rare, yet
unique variant of HCC and remains incompletely understood.
Fibrolamellar HCC affects youth, adolescents, and elderly
patients of both genders. Caucasians are most commonly
affected by this particular type of HCC.
41-43
Opposite to what
has been known for most HCCs, FL-HCC often occurs in non-
cirrhotic livers in patients in their mid-20s and almost always in
the absence of any known risk factors. A series of reports have
stated that patients with FL-HCC have better outcomes than
those burdened with classic HCCs, but these interesting find-
ings likely has resulted from the absence of cirrhosis in these
relatively young patients, considering cirrhosis as an obvious
adverse factor in classic HCC patients.
41-43
The actual outcome
of FL-HCC is similar to that of classic HCC arising in noncir-
rhotic livers. Patients with FL-HCC have a 5-year survival rate
of around 50%. Clinically, serum neurotensin
44
and vitamin
B12 binding molecules (transcobalamin)
45,46
have been con-
sidered laboratory markers for FL-HCC and have been associ-
ated with tumor burden. Serum AFP has been additionally
studied but was found to be elevated in only a minority of
patients with FL-HCC.
47
Radiographically, an occasional cen-
tral scar may be seen in FL-HCC, a frequent finding classically
seen in FNH (a benign entity to be introduced later); however,
radiological investigation demonstrated that the FL-HCC scar
is often calcified and different from that observed with FNH.
Grossly, FL-HCCs are usually single, firm, and well demar-
cated; they are generally larger than classic HCCs, with an
unusual propensity to metastasize to regional lymph nodes.
47-49
The FL-HCCs often show markedly prominent intratumoral
and peritumoral fibrous bands throughout, with a central scar
resembling FNH (see later text and discussion), in additional to
foci of bile pigment and hemorrhagic necrosis. Although it could
occur in both liver lobes, FL-HCC is more commonly seen in
the left lobe of the liver for undermined reasons.
48,49
Microscopically, the following characteristic features
should point to the diagnosis of FL-HCC: plump polygonal
tumor cells with rich eosinophilic granular cytoplasm (caused
by numerous mitochondria), prominent macronucleoli match-
ing the size of lymphocytes, and lastly lamellar fibrosis
(Figure 7A). These histological characteristics occur without
a clinical presentation of liver cirrhosis or underlying liver
disease and appear unique to FL-HCC, as these fibrous bands
encircle and surround clusters and nodules of neoplastic hepa-
tocytes (Figure 7B). The nuclei are enlarged with prominent
nucleoli (Figure 7C-D). Particularly, FL-HCC cells contain the
so-called “pale bodies,” which are in fact composed of fibrino-
gen and albumin (Figure 7E-F); however, these pale bodies are
nonspecific, as they can be seen in other types of HCCs,
especially in scirrhous HCC. Nonetheless, characteristic fibrous
collagen bands, which are thick and homogenous in FL-HCC,
are not present in scirrhous HCC or any sclerotic variant of HCC.
Moreover, FL-HCC has a higher tendency for regional lymph
node metastasis compared to classic HCC and spreads more
frequently than classic HCC to the peritoneum, omentum, and
lung,
41,47,48,50
with remote metastasis involving left supraclavi-
cular lymph nodes and the abdominal wallseen at our institution.
Steatohepatitic HCC. A recent investigation conducted in India
has described a new variant of HCC termed “steatohepatitic
hepatocellular carcinoma (SH-HCC),” which has been con-
nected with metabolic risk factors identified in Indian
patients.
51
Briefly, 101 cases of HCC within explanted livers
from adult patients were inspected for tumor histomorphology
in the context of clinically identifiable metabolic risk factors in
this study. The authors diagnosed SH variant of HCC in 19
(18.8%) of 101 liver explants. Among SH-HCC cases, 17 were
males and 2 were females, ranging from 47 to 65 years (mean
age 54.8 years). Of notice, 9 of the 19 SH-HCC cases were
associated with HCV, 6 of the 19 were associated with non-
alcoholic fatty liver disease (NAFLD), 2 with HBV, 1 with
alcoholic liver disease (ALD), and the last 1 with mixed (HBV
þHCV þALD) infection. No obvious difference in the size,
location, number of lesions, overall tumor differentiation, and
vascular invasion between SH-HCC and conventional HCC
cases was observed.
51
In addition to the loss of reticulin, gly-
pican 3 appears to be a good diagnostic tool, as this immunos-
tain performed in 18 cases showed strong cytoplasmic (11 of
13) and focal canalicular (2 of 13) labeling in 13 SH-HCC
cases.
51
The authors concluded that a comparison of SH-
HCC with non-SH-HCC was statistically significant (P¼
.03) for an SH-HCC association with metabolic risk factors.
Due to its rarity, the clinical course of SH-HCC has not been
well established, but they appear to have better prognoses when
compared to conventional HCCs; among 19 patients with SH-
HCC, 16 were alive and disease free at 24 to 72 months fol-
lowing the diagnoses, compared to 76 patients with conven-
tional HCC where 16 patient deaths had been documented.
51
We encountered similar SH-HCC case(s) in practice, which
possessed morphological features mimicking steatohepatitis in
the setting of NAFLD. Tumor cells showed macrovesicular
steatosis, inflammation, and ballooning degeneration, with fre-
quent Mallory hyaline and globules; microclusters of lympho-
cytes, plasma cells, and foci of neutrophils were seen in the
vicinity of ballooned neoplastic hepatocytes. The clinical
course of such cases(s) is under observation.
10 Cancer Control

Ancillary Studies in Diagnosing and
Differentiating HCCs
Many cases of liver mass lesions, especially liver core biopsies
and wedge specimens, require confirmatory ancillary studies,
including immunohistochemistry and special stains. Before
selecting any ancillary studies, it is essential to determine
whether the biopsy is from a targeted mass or random liver, a
lesion is present in the material, the tissue is sufficient for
ancillary evaluation, and whether it is hepatocellular or some-
thing unexpected or metastatic. The ancillary tools should be
chosen, based on the questions.
Multiple special stains and immunohistochemical studies have
been described to confirm a diagnosis of HCC, the frequently
utilized ones are reticulin, CD34, polyclonal carcinoembryonic
antigen (CEA) (P-CEA), CD10, glypican 3 and AFP; HepPar-1
and arginase 1 should be performed when the hepatocellular
nature of the tumor is in doubt Arginase 1 shows higher sensitivity
and specificity (both approximately 90%) than HepPar-1 and
glypican 3 in confirming hepatocytic differentiation.
27,52,53
An
investigation conducted by Fujiwara et al summarized the useful-
ness of Arginase 1, HepPar and glypican 3 in the study of liver
tumors. It concluded that arginase 1 is the first choice for con-
firming or excluding HCC, due to its high sensitivity (80%-95%)
Figure 7. Histomorphology of FL-HCC. (A) and (B) FL-HCC with thick fibrous collagen bands that encircle and surround neoplastic hepa-
tocytes (H&E, 40 and 100). (C) and (D) Polygonal FL-HCC cells with abundant eosinophilic cytoplasm, large nuclei, and prominent nucleoli
(200 and 400; arrow: pale/hyaline body). (E) and (F) Polygonal cells with hyaline bodies, some of which cause nuclear indentation and nuclear
pseudo-inclusion (arrows, 600). FL-HCC indicates fibrolamellar hepatocellular carcinoma.
Jiang et al 11

and even higher specificity (95%-100%); especially when com-
bined with HepPar-1 and glypican 3, almost all HCCs, including
rare variants, can be diagnosed with confidence. A similar con-
clusion has been drawn by Radwan et al following comparing
arginase 1 and HepPar-1 expression in 50 HCC, 38 metastatic
carcinoma, 12 cholangiocarcinoma and 10 benign liver cases.
53,54
Glutamine synthetase and heat shock protein 70 2 markers
used to differentiate benign from malignant liver nodules. In
HCC, glutamine synthetase (GS) shows diffuse cytoplasmic
staining.
55
In a recent study, GS and HSP 70 were negative
in all adenomas. At least one of the 2 markers was positive in
85% of of very well-differentiated HCC. It should be kept in
mind that GS also labels FNH (map-like pattern), and could lead
to pitfalls initnerpretation.
56,57
Reticulin is the special stain used most frequently when fac-
ing a diagnosis of possible HCC. Reticulin is lost in the majority
of HCC tumors; or it may highlight thickened hepatocyte plates,
usually not easily appreciated on H&E sections (Figure 8A). In
contrast to HCC, the reticulin stain in benign liver, FNH, regen-
erative nodules, and HAs will highlight the hepatic plates,
demonstrating trabecula composed of a single or double layer
of hepatocytes (Figure 1E). Alternatively, HCC may show a
reduction in the intensity of reticulin staining rather than com-
plete loss, with areas of hepatocytes that do not have direct
contact with reticulin fibers. The diagnosis in these cases has
to be made by other cytological features, including atypia and
proliferative rates. An additional diagnostic pitfall is HCC with
steatosis, because benign hepatic tissue with macrovesicular
steatosis has focal and patchy reticulin loss, which could mimic
HCC. In rare cases of well-differentiated HCC, reticulin stain
could be even retained, disfavoring a diagnosis of HCC. There-
fore, other essential markers of HCC, as well as cytological
atypia and proliferative rates, should be investigated to help
characterize the lesion. Radiological studies, combined with
laboratory findings are also crucial in helping reach a final
diagnosis.
In HCCs, immunostaining for CD34 shows a strong diffuse
sinusoidal staining pattern (Figure 8C-D), whereas in benign
liver, stains for CD34 highlight the zone 1 (periportal) sinusoids
focally (Figure 1F). In HCC with a macrotrabecular growth
pattern, a CD34 stain highlights thickened trabeculae, with neg-
ative labeling inside the tumor mass (Figure 8C-D). However,
occasional HCCs do not show this staining pattern, rendering
CD34 less useful versus some of the other available stains.
More than two-thirds of HCC show a canalicular pattern of
expression with P-CEA and CD10. Considered as indicators of
hepatic differentiation, they are more useful in well-
differentiated and moderately differentiated HCCs. Focal and
patchy staining patterns for p-CEA (Figure 8E) and CD10 have
been seen in nearly one-third of HCCs Staining with P-CEA
may be difficult to interpret as sometimes it shows a nonspe-
cific membranous or even cytoplasmic pattern in HCC. It is
important to keep in mind that the absence of canalicular stain-
ing is usually encountered in poorly differentiated HCCs. The
lack of canalicular staining for p-CEA or CD10 does not neces-
sarily exclude a poorly differentiated HCC; it actually
implicates additional immunostaining workup and correlation
with clinical, radiological, and laboratory findings.
AFP has been the so-called “tumor marker” for HCC sev-
eral decades, but immunohistochemistry for AFP is positive
in only about one-third of HCCs. Due to its low positive rates
and the puzzling fact that patients with HCC frequently show
normal serum AFP levels, even when tumors are positive for AFP
by immunostaining, AFP is not utilized routinely as a first-line
choice in the diagnostic workup of HCCs, except in a poorly
differentiated HCC. AFP expression is not specific for HCC, as
it may also be expressed by intrahepatic CC and metstatic adeno-
carcinomas from other organs. Hence, a positive AFP immunos-
tain needs to be interpreted in the context of histomorphology,
additional immunostains, and clinical findings.
HepPar-1, which recognizes a mitochondrial antigen, is
popularly used to confirm hepatocellular differentiation in
diagnosing HCCs. Immunohistochemistry will demonstrate
coarse granular cytoplasmic staining.
53,58
Approximately
10%of HCCs actually show negative HepPar-1 labeling,
59
especially the poorly differentiated tumors. Therefore, a lack
of expression for HepPar-1 does not exclude hepatocytic dif-
ferentiation. Hepatocyte paraffin 1 expression can be focal
instead of diffuse (Figure 8G).
Hepatocyte paraffin 1 also lacks specificity for hepatocytic
differentiation, since expressionhasbeenreportedinmultiple
other types of carcinomas, such as gastric, esophageal, pul-
monary, and colonic adenocarcinomas; hepatoid carcinomas
from the stomach and pancreas, and adrenal cortical carcino-
mas, to name a few.
37,60,61
Arginase immunostain is prefer-
able for the confirmation of hepatocytic differentiation, as it is
superior to HepPar-1 in terms of specificity and
sensitivity.
52,53
In parallel, as another frequently used unique marker for
HCC, glypican 3 shows positive cytoplasmic labeling in
approximately 80%of HCCs, more likely in tumors that arise
in cirrhotic livers and in the setting of chronic HBV infec-
tion,
62,63
with the background benign liver showing negative
staining. Moreover, glypican 3 is usually negative in well-
differentiated HCCs.
62
Since glypican 3 is virtually always
negative in HAs and in FNH, a positive glypican 3 is highly
compatible with HCC. It has been well acknowledged that
while HepPar-1 is often positive in well-differentiated HCC,
glypican 3 is more so in poorly differentiated tumors.
62
How-
ever, like HepPar-1, glypican 3 is not specific for HCCs, as
other tumor types might be labeled by it. Furthermore, extra
caution is needed for interpreting unexpected glypican 3 posi-
tivity associated with significantly inflamed benign hepato-
cytes,
64
macroregenerative nodules, and DNs.
62
Athird
problematic issue with glypican 3 immunostaining is that the
labeling can be very patchy, especially when dealing with nee-
dle biopsy specimens, as up to 50%of the biopsies could be
negative for glypican 3 due to limited amount of tissue.
53
It is
pivotal to keep in mind that a negative labeling by glypican 3
does not exclude HCC.
Recently, arginase 1 has been identified to be a strong diag-
nostic tool for identifying HCC,
52,53
which is positive in both
12 Cancer Control

benign and malignant hepatocytes, and has been recognized to
possess better sensitivity and specificity than glypican 3 or
HepPar-1. A combination of arginase 1, glypican 3, plus
reticulin, CD34, and p-CEA are frequently chosen, coupled
with the H&E staining probably could satisfy diagnosing
almost all HCCs.
53
Finally, a panel of immunostains has been
Figure 8. Photomicrograph (special stains and immunostains) of HCC. A, Thickened hepatic plates shown by reticulin, 40. B, Absence of iron
deposition, Prussian blue, 100. C and D, Diffuse sinusoidal CD34 labeling in HCC, 100. E, P-CEA labeling with a prominent canalicular
pattern, 100. F, Diffuse glutamine synthetase labeling in HCC, 100. G, Focal, patchy labeling of heppar 1, 100. H, glypican 3 immunostain,
100. HCC indicates hepatocellular carcinoma.
Jiang et al 13

speculated to be of value in providing prognostic information,
as identified for CK-19, whose positivity has been demon-
strated in approximately 10%to 15%of HCCs and has been
recognized as an indicator of “stemcellness” and a worse
prognosis.
65
Except for FL-HCC, cytokeratin staining is not widely
utilized for diagnosing classic HCCs; CAM5.2, CK-8, and
CK-18 are expressed in both benign hepatocytes and HCCs.
40
Cytokeratin 7 immunostaining is mostly negative in HCCs but
can be positive in HCCs that are cholestatic or that arise in
young patients and frequently in FL-HCC.
66
Cytokeratin 20 is
generally negative in HCCs. Fibrolamellar HCC tumors
express markers of hepatocellular differentiation including
arginase,HepPar-1,andp-CEA(canalicular pattern). Cyto-
keratin 68 shows granular cytoplasmic signal in majority of
FL-HCC tumors. The positive rate of glypican 3 in FL-HCC
cases is lower than in classic HCCs and are not used as a first-
line choice.
Examination of the benign background liver for disease
grading and staging is equally important for clinical decision-
making. Sections of benign liver parenchyma away from the
HCC should be taken. Investigation of hepatitis activity, hepa-
tocellular injury, steatosis and steatohepatitis, and fibrosis need
to be undertaken to characterize the background liver. Biopsy
tissue cores obtained for the purpose of assessing the nonneo-
plastic liver should be taken as far away as possible from the
HCC or any mass lesion. Also, the margins of resection in a
surgical resection specimen should not be used due to thermal
artifacts and surgery-related changes.
Benign Hepatocellular Nodules
The 2 other most often encountered hepatocellular lesions in
the liver are FNH and HA, both will be discussed in the fol-
lowing text. The difference between HCC (especially well dif-
ferentiated), FNH, and HA can be clinically and
histopathologically challenging. Hepatic adenoma can show
similar radiological and imaging features to HCC, due to aber-
rant arterioles and abundant blood supply, whereas half of the
FNH cases don’t show radiological or imaging evidence for a
scar when the lesions are less than 4 cm in size. An accurate
diagnosis is crucial for clinical management and patient
outcomes.
Focal Nodular Hyperplasia
Focal nodular hyperplasia is a nonneoplastic, benign reactive
nodular lesion frequently encountered clinically, composed of
bland hepatocytes and fibrotic septa containing proliferating
ductules. Focal nodular hyperplasia develops in noncirrhotic
liver parenchyma, likely due to localized shunting of arterial
blood flow. Any condition causing blood shunting could induce
formation of FNH, as seen in rare cases of HCCs where reac-
tive FNH developed in the vicinity of HCCs due to interference
of blood supply. Focal nodular hyperplasia has no malignant
potential based on current scientific literature.
Clinically, FNH occur frequently in young and middle-aged
women, as either single or multiple lesions, the majority were
identified in women between the ages of 20 and 50 years. Focal
nodular hyperplasia could also develop in children and teen-
agers, usually following chemotherapy for other malignancies.
FNA may even develop in liver allografts.
67
The precise etiol-
ogy for FNH development, however, hasn’t been determined
with certainty, although a potential connection with oral con-
traception has been proposed. The background liver should not
be cirrhotic.
Histologically, FNH is composed of nodules of cytologi-
cally benign hepatocytes separated by thin and thick fibrous
bands (Figure 9A) that may coalesce into a larger central scar.
In smaller FNHs, the nodularity may be less developed and
not easily appreciated at low-power view. The hepatocytes do
not show atypia, and a reticulin stain demonstrates a normal 1-
to 2-cell thick plates. Both the periphery and the central
regions of FNH lesion often show large caliber vessels with
thickened muscular walls (Figure 9C-D). The fibrous bands
typically have proliferating bile ductules, mostly located at
the edges of the fibrous septa (Figure 9E-F). Focal nodular
hyperplasias do not have capsules and true portal tracts. A
central scar is usually appreciated if the lesion is larger than
4 cm but is only seen in half of the smaller lesions. Because
FNH lacks portal tracts and sufficient bile drainage, they often
show cholestasis and mild copper accumulation in the areas
abutting the fibrous septa. Occasionally FNH shows balloon-
ing and Mallory hyaline or fatty change; features resembling
clear cell of the SH variant HCC. The overall architecture and
the bland cytology usually allow distinction of these 2 possi-
bilities on H&E staining. In challenging cases, special stains
for GS can be resorted.
In normal liver, GS only stains a delicate rim of pericentral
hepatocytes surrounding the central veins (Figure 1F). In con-
trast, staining in FNH will show an irregular, “map-like” pat-
tern(Figure9G,H);thisspecificpatterncanbeextremely
helpful in diagnosing FNH.
57
On the other hand, a distinct,
diffuse staining pattern can be observed in either HAs or HCCs
(Figure 8F). However, most HAs and some HCCs are entirely
negative by GS staining. Other useful tools in diagnosing FNH
are cytokeratin stains to highlight the proliferating bile duc-
tules, a copper stain to highlight cholestasis, a reticulin stain,
and potentially glypican 3 and Ki-67 index to help rule out
well-differentiated HCC.
55,57
It has been noted that less than 50%of the FNH cases
could be confidently diagnosed on needle core biopsy.
Although this could be due to sampling errors, as multiple
passes would increase the rate of definitive diagnosis, a
confident diagnosis was often hampered by the absence of
essential imaging evidence and laboratory findings. Because
a diagnosis of FNH is often made only after correlating with
the imaging and histological findings, an experienced
pathologist might conclude the findings are consistent with
or suggestive of FNH after correlation with available ima-
ging studies. Therefore, immunostaining for GS should be
utilized to help clarify these challenging cases.
58
Roncalli
14 Cancer Control

et al reviewed the pathobiology of FNH and HA lesions and
proposed a diagnostic algorithm in an effort to increase the
diagnostic accuracy of these frequently challenging entities.
Basically FNH is a nodular polyclonal tumor-like hepatocy-
tic proliferation that does not undergo hemorrhage or malig-
nant transformation; on the other hand, HAs are a
monoclonal proliferation of bland-looking hepatocytes
embedded in 1- to 2-cell thick hepatic plates. Nuclear atypia
and mitoses could be seen in specific variants. The authors
summarized systematically the differential diagnosis between
traditional HAs and atypical HAs and well-differentiated
HCC.
68
For example, FNH carries nonclonal b-catenin activa-
tion without mutations, contributing to hepatocellular hyper-
plasia and regeneration, and mutations in b-catenin has never
been identified in FNHs.
68
In contrast, HAs have been sub-
classified into different groups based on the status of hepato-
cyte nuclear factor 1a1(HNF1A1)b-catenin, and serum
amyloid A (SAA), or C-reactive protein (CRP).
Figure 9. Histopathological features of FNH. (A) and (B) Photomicrograph (20 and 40; H&E stain) shows the multinodular contoured lesion
with adjacent normal liver with portal tracts (arrow heads, A) and focal inflammatory fibrous bands (arrow, B). (C) and (D) Photomicrograph
(H&E stain, 100) shows a septum (arrow) dividing 2 neighboring nodules (C). The septum contains connective tissue and thick-walled vessels
(arrow heads in C and D). (E) and (F), At the interface of the FNH nodule and septa, reactive biliary ductular proliferation is enriched (arrows).
(G) and (H), Glutamine synthetase showing a maplkie-pattern. Peroxidase, 200. FNH indicates focal nodular hyperplasia.
Jiang et al 15

Hepatic Adenoma
Hepatic adenomas are a group of hepatocytic neoplasms with
well-differentiated morphology that are usually benign. Hepa-
tic adenomas are monoclonal neoplasms with unique molecular
signatures and oncogenetic pathways that are distinct from
HCCs. Hepatic adenomas are well-known to occur in young
females of childbearing age, especially in those with a history
of estrogen-based, oral contraceptive pill usage and less often
to occur in men, usually with a history of anabolic steroid use.
The prevalence of HAs is on the rise due to increased use of
imaging modalities, which leads to increased incidental detec-
tion. The annual incidence is around 4 per 100 000 per year in
developed countries; the female predominance has not been
confirmed in Asian patients, seemingly related to lesser use
of oral contraceptives in these countries.
69-71
Most HAs are
solitary and patients are asymptomatic. The presence of mul-
tiple HAs has been termed hepatic adenomatosis.
72
Hepatic
adenomas carry a risk of rupture and bleeding, and some sub-
sets of HAs have the potential to undergo malignant transfor-
mation.
73
Clinically it is very important to correctly diagnose
and manage HAs, especially when HCC is in the differential
list.
73
Hepatic adenomas are characterized by a relatively uni-
form population of hepatocytes arranged in cell plates of3-cell
thick. The pattern may be slightly more irregular compared to
the adjacent liver. The hepatocytes retain a low N/C. The reti-
culin remains intact. The cells may have cytoplasmic contents
similar to normal hepatocytes. Large arterial vessels, unaccom-
panied by bile ducts, are prominent. The genotypic type will
influence the morphological phenotype. Diagnosis of HA can
be challenging in cases with a limited amount of biopsy mate-
rial as well as due to the overlapping histomorphological fea-
tures with FNH and well-differentiated HCC.
A molecular pathology-based classification for HA has been
established, dividing HAs into subtypes based on their mole-
cular characteristics, as determined by the corresponding
immunohistochemical profiles and histomorphological charac-
teristics.
74,75
The established HA subtypes are (1) HAs with
mutated, inactive HNF1A, (HA-H), (2) HAs with activating
mutations in the CTNNB1 gene encoding b-catenin (HA-B),
(3) HA without mutations of HNF1A or b-catenin genes but
with inflammatory features (HA-I, formerly called telangiecta-
tic FNH), and lastly (4) unclassified HAs that have neither
known gene mutations nor a unique histomorphology
(HA-U,
74
the histological features of these adenomas are
shown in Figure 10A-D, respectively).
Hepatic adenoma with HNF1A mutations account for
approximately 30%of HAs. The mutations are somatic in most
cases; but in some cases, 1 mutation may be germ line. There
are differences in presentation and etiology between the
somatic and germ line patients. Hepatic adenoma with HNF1A
mutation with somatic mutations occur mostly in females with
a history of oral contraceptive use. Hepatic adenoma with
HNF1A mutation with germ line mutations also present mostly
in females but occur at an earlier age and patients do not typi-
cally have a history of oral contraceptive use. The HA-H in
these patients are larger on average. Familial hepatic adenoma-
tosis has been well-documented in patients with germ line
mutations of HNF1A and it is also associated with maturity-
onset diabetes mellitus of youth, type 3.
72
Detection of germ
line mutations of HNF1A in family members of patients with
hepatic adenomatosis has been established to identify familial
predisposition for the disease.
Both somatic and germ line HA-H are morphologically
characterized by significant steatosis, without nuclear atypia
or inflammatory features.
74
Expression of liver fatty acid-
binding protein (LFABP) is downregulated in HA-H as a con-
sequence of the HNF1A mutation,
55
therefore, immunohisto-
chemistry for LFABP serves as a translational marker to
identify this subtype of HA, since expression will be lost in
HA-Hcomparedtonormalliver,inwhichitisnormally
expressed.
Hepatic adenomas with activating mutations in the CTNNB1
gene encoding b-catenin account for 10%to 15%of all HAs.
Mutations in this gene are exclusive of HNF1A mutations.
These occur more often in males than in females. Contributing
factors include congenital metabolic disturbances, such as gly-
cogenosis 1 and 3, the consumption of anabolic steroids, or the
use of oral contraceptives in females. Morphologically, they
are characterized by cytological atypia, peliosis hepatis, pseu-
doacinar formations, and are less frequently steatotic. In HA-B,
two b-catenin target genes, GLUL and GPR49, were found to
be overexpressed 42-fold (ranging from 9 to 87) and 35-fold
(ranging from 8 to 57) when compared with nontumor tissues,
respectively. GLUL encodes GS. Immunohistochemistry for
GS and b-catenin is used a translational marker, with strong
and diffuse expression for GS and aberrant nuclear and cyto-
plasmic expression for b-catenin.
74
HAs with activating mutations in the CTNNB1 gene encoding
b-catenin with mutations in exon 3 of the CTNNB1 gene encod-
ing b-catenin (HA-B
ex3
) are associated with a high risk of malig-
nant transformation. Mutations in exons 7 and 8 have also been
identified. This type, referred to as the weak b-catenin activation
type, has a mild activation of the Wnt/b-catenin pathway and
does not have an increased risk of malignant transformation.
76
Immunohistochemcal expression of b-catenin will not usually
show nuclear expression, and GS expression is faint and patchy.
Inflammatory adenomas (HA-I) account for over 50%of
HAs. These occur in both men and women and may be single
or multiple. Obesity, metabolic syndrome, and alcohol con-
sumption are predisposing risk factor. Inflammatory adenomas
are characterized by activation of the interleukin 6/Janus kinase
(JAK)/Signal transducer and activator of transcription protein
(STAT3) pathway, resulting in overexpression of SAA and
CRP. This pathway is activated through mutations in IL6ST
(most common), Fyn Related Src Family Tyrosine Kinase
(FRK), JAK1,STAT3, and Guanine nucleotide-binding protein
(G9s) subunit alpha isoforms short (GNAS).
76
Morphologi-
cally, HA-I are usually difficult to distinguish from the adjacent
parenchyma. They are characterized by bile ductular prolifera-
tions, but the ductular-like proliferations are located in faux
portal tracts. The faux portal bands contain small arterioles
16 Cancer Control

with an associated mononuclear cell infiltrate. Immunohisto-
chemistry for SAA and CRP serves as translational marker of
this subtype. A subset of HA-I may also have mutations in the
CTNNB1 gene encoding b-catenin, either exon 3 or exons 7 and
8 (HA-IB
ex3
and HA-IB
ex7,8
, respectively). These mixed types
may be recognized with immunohistochemistry for b-catenin.
It is important not to mix HA-I with FNH. Findings that favor
an FNH include the fibrous bands, abnormal thick-walled ves-
sels, and a ductular proliferation that is typically patchy and
within the fibrous bands. Finally, attention should be paid to
FNH-like changes that develop around any mass lesion in the
liver that has interfered with blood flow, including metastatic
neoplasms. In these cases, this reactive rim of hepatocytes can be
indistinguishable from an ordinary FNH on needle biopsy.
In a recent report, Nault et al analyzed the expression of 20
genes and sequenced exon regions of the 8 genes, HNF1A,
IL6ST,CTNNB1,FRK,STAT3,GNAS,JAK1,andTelomerase
Reverse Transcriptase (TERT) , in 607 samples of 533 HAs from
411 patients, collected from 28 centers mainly in France from a
14-year span.
76
Based on their molecular data, they classified
HA into 8 subgroups, including a novel subgroup, representing
4%of HA previously classified as HA-U.
76
This subgroup was
characterized by activation of the sonic hedgehog signaling path-
way (HA-SH) and was associated with obesity and bleeding.
This pathway is driven by structural rearrangements of Inhibin
Beta E Subunit (INHBE) producing a highly expressed INHBE_-
Glioma-associated oncogene family zinc finger 1 (GLI1) fusion
protein leading to the constitutive activation of the sonic
hedgehog pathway. This subtype is exclusive of all other sub-
types. Immunohistochemistry for the protein Prostaglandin D2
synthase (PTGDS) serves as a translational marker.
Studies of intra- and intertumoral genetic heterogeneity
showed a molecular subtype field effect. Nault et al
76
correlated
their molecular data with risk factors for HA development, the
risk of bleeding, and malignant transformation. The cumulative
intake of Oral contraceptives (OCP), obesity, and alcohol intake
contributed to estrogen exposure. Body mass intake also con-
tributed. Patients with higher estrogen exposure or high BMI
were more likely to develop HA-I or HA-SH. Patients with
HA-IB
ex3
were more likely to have had androgen exposure. The
frequency of histological hemorrhage was higher in HA-IB
ex7,8
and SA-SH. Features associated with malignant transformation
included TERT promoter mutations, CTNNB1 exon 3 mutations,
a unique nodule at imaging, high alcohol intake, fibrosis in
nontumoral liver, and diabetes type 2. In patients with multiple
HA, the largest HA tended to be associated with CTNNB1 exon 3
mutations; thus, image-guided biopsies in patients with adeno-
matosis can be directed at the largest nodule. Based on these
findings, the molecular profile can be used to guide resection in
female patients. In male patients, the baseline risk of malignant
transformation is higher (41%in females vs 65%in males), and,
therefore, this risk stratification is not as useful.
76
The final subset has no recognized mutations and no specific
morphological features. This subset is referred to as HA-U.
This genotypic–phenotypic classification and risk factors for
HCC are shown in Figure 11.
Figure 10. (A) to (D) Type 1 to 4 hepatocellular adenoma: (1) HNF-alpha mutated; (2) inflammatory type; (3) Beta-catenin-mutated; and lastly;
(4) nonclassifiable HA. HA indciates hepatic adenoma; HNF, hepatocyte nuclear factor.
Jiang et al 17

Atypical Adenoma
Despite all the investigations discussed so far, atypical yet
well-differentiated hepatocellular lesions keep posing diagnos-
tic challenges. In these unusual cases, the utilization of immu-
nohistochemical markers discussed earlier, glypican 3,
Glutamine synthetase (GS), and Heat shock protein 70
(HSP70), could serve as useful tools to help reach the correct
diagnoses. In making a definite diagnosis, the appropriate
approach to these entities is unexceptionally clinical and radi-
ological, as HA but not FNH is usually associated with young
to middle-aged female, history of oral contraceptive usage,
frequently metabolic syndrome (diabetic, hyperlipidemia, obe-
sity), inflammatory syndrome, and possibly alcohol assump-
tion. All these traits require detailed and systematic
investigation. It has been recognized that sporadic adenomas
unrelated to certain clinical traits/diseases are rare. And it has
been proposed that for those cases with definitely equivocal
pathological features, and those that are frequently associated
with incomplete or an absence of radiological and laboratory
information, the term of well-differentiated hepatocellular neo-
plasm of uncertain malignant potential instead of atypical
hepatocellular neoplasm or atypical HA should be used.
68
Histopathological Staging and Risk
Stratification of HCC
The prognosis of patients with HCC is influenced by tumor size,
tumor number, and the presence of angiolymphatic invasion (HCC
staging). The combination of these factors is key in predicting the
clinical course and patient outcomes. Gross finding of large vessel
invasion has a worse prognosis than small vessel invasion, which is
recognized only on microscopy examination. Tumor differentiation
also influences prognosis, as do morphologic variants discussed in
the following texts. After all, the most critical prognostic factor in
HCC is the resectability. Other prognostic indicators include age
(younger patients better), gender (women usually better), the activ-
ity, and the stage of background liver disease(s).
TNM and Anatomic Stage/Prognostic
Groupings
The TNM staging system of the American Joint Committee on
Cancer (AJCC) and the International Union against Cancer
(UICC) has been widely accepted for staging and risk stratifica-
tion of HCC. The T classificationdepends on the number of tumor
nodules,the size of the largest nodule, and the presence orabsence
of blood vessel invasion. Vascular invasion includes either
grossly appreciated or microscopically identified tumor involve-
ment of vessel spaces. Portal vein invasion by HCC is an impor-
tant adverse prognostic factor and should be reported. The eighth
edition of the AJCC is to be implemented on January 1, 2018.
77
Based on AJCC/UICC convention, the designation “T” rep-
resents a primary HCC that has not been treated previously. The
symbol “p” represents the pathologic classification of the TNM,
as opposed to the clinical classification; the scale of symbol p is
based on gross findings and microscopic examination. pT entails
a resection of the primary HCC or biopsy with sufficient material
for evaluating the highest pT category; pN entails removal of
lymph nodes adequate to validate metastatic disease involving
lymph nodes, and pM indicates microscopic examination and
findings on potential metastatic diseases (distant lesions). On the
other hand, clinical classification (cTNM) is usually performed
and determined by the referring clinician prior to any treatment,
frequently during initial evaluation of the patient or when patho-
logic classification is not possible, such as when the pathology
material has not become available for pathologist’s review.
For identification of special cases of pTNM classifications,
the “m” suffix and “y,” “r,” and “a” prefixes are used. The m
suffix indicates the presence of multiple primary tumors in a
single site, recorded as pT(m)NM. The y prefix indicates that
the tumor classification is performed during or after initial
multimodality therapy (ie, neoadjuvant chemotherapy, radia-
tion therapy, or chemoradiation therapy). The cTNM or pTNM
category is identified by a y prefix. The ycTNM or ypTNM
categorizes the extent of actual tumor at the time of that exam-
ination. The r prefix indicates a recurrent tumor when staged
after a documented disease-free interval and is identified by the
r prefix. Finally, the a prefix designates the tumor stage deter-
mined at autopsy: aTNM. The primary tumor classification for
HCC is shown in Table 1. Lymph node status and distant
metastases are also prognostic indicators.
A complete pathologic staging is almost always carried out on
surgically resected primary HCC. Accurate pathologic staging
depends on pathologic documentation of the anatomic extent of
tumor and whether the primary tumor has been completely excised.
In rare cases, when a biopsied or aspirated tumor becomes non-
resectable due to any reason (such as when the patient declines
surgery, it is technically infeasible, or clinically not indicated), or if
the highest T and N categories, or the M1 category of the metastatic
disease has been confirmed microscopically, the abovementioned
criteria for pathologic classification and staging have been satisfied
without surgical resection of the primary tumor.
Cholangiocarcinoma
Cholangiocarcinoma is a malignant adenocarcinoma with evi-
dence of biliary differentiation. Based on the location, cholangio-
carcinomas are divided into intrahepatic and extrahepatic groups.
Klatskin tumor, or the so called “hilar cholangiocarcinoma,” is an
extrahepatic tumor arising in the right or left hepatic duct or at their
junction. Tumors arising from the intrahepatic hepatic ducts
include intrahepatic and perihilar cholangiocarcinomas. In prac-
tice, the precise origin of some larger tumors can be challenging as
there is no clear evidence whether they are intrahepatic or extra-
hepatic, but fortunately all these tumors show histomorphology of
biliary differentiation. The global incidence of intrahepatic cholan-
giocarcinomas, but not extrahepatic types, shows an increasing
trend. Although the exact etiology has not been well established,
cholangiocarcinomas are associated with chronic inflammatory
conditions of the biliary tract and are also associated with chronic
bile stasis. Based on the current understanding, risk factors for
18 Cancer Control

cholangiocarcinoma include chronic viral hepatitis (type C and B),
metabolic syndrome, obesity and alcohol usage.
78
In addition,
parasitic fluke infections, hepatolithiasis and primary sclerosing
cholangitis (PSC) are other identified risk factors.
78
Precursor lesions leading to cholangiocarcinoma include
high-grade BilIN, discussed earlier in this review. Of notice,
high-grade BilIN-3 lesions are more frequent in cirrhotic
livers, usually involving the medium- and large-sized intra-
hepatic branches of the biliary tree, especially in the setting of
chronic hepatitis C and alcohol abuse–related chronic liver
disease.
79
Histopathology
Compared to other types of cancer involving liver, cholangio-
carcinomas are the ones that elicit a marked desmoplastic fibro-
tic reaction. It has been well recognized that
cholangiocarcinomas demonstrate a wide spectrum of growth
patterns (Figure 12A-F). Tumors can be composed of irregular
cystic, branching/glandular tubular structures or as irregular
aggregates of infiltrating glands (Figure 12A-B). Cholangio-
carcinomas do not always show easily identifiable tubular con-
tours; instead, tumor cells often grow in a nodular pattern or
Figure 11. A new nosology of hepatic adenomas. Reprinted from Gastroenterology, 152(4), Nault JC, Couchy G, Balabaud C, et al. Molecular
Classification of Hepatocellular Adenoma Associates With Risk Factors, Bleeding, and Malignant Transformation, 880–894, 2017, with per-
mission from Elsevier.
Jiang et al 19

form sheets of solid nests that mimic neuroendocrine neo-
plasms and/or HCC (Figure 12C). Other tumors may show
copious mucin components, resembling mucinous adenocarci-
noma or colloid tumors (Figure 12D). It should be kept in mind
that for any cholangiocarcinoma, it is common to see a variety
of the abovementioned histomorphological features in the same
lesion. Focal features of sarcomatoid or clear cell morphology
are not uncommon.
Cholangiocarcinoma tumor cells frequently extend along
the portal tracts by growing within the connective tissue, with-
out directly invading into the bile ducts in the vicinity (Figure
13A), even though this pattern of tumor invasion can be seen in
not only cholangiocarcinoma but also other types of tumors,
including metastatic carcinomas. Also, tumor cells can colo-
nize and extend along the bile ducts and intermingle with
native benign bile ducts and adjacent reactive ductules (Figure
13A). It should always be kept in mind that cholangiocarcino-
mas are neurotropic, and tumor cells frequently approach nerve
tracts, with frequent perineural and intraneural involvement, as
shown in Figure 13B. In practice, this pattern can be very
challenging to recognize, especially during frozen section eva-
luation; more so when the primary tumor is small and when
there is no clinical evidence of metastatic disease. Another
malignant behavior of cholangiocarcinomas is lymphovascular
invasion, as shown in Figure 13C. Astute microscopic exam-
ination and extensive tumor sectioning is essential in reaching
such a diagnosis. The usual intraluminal “dirty” necrosis, fre-
quently associated with colorectal primary adenocarcinomas,
are not typically seen in cholangiocarcinomas.
Ancillary Studies
The pathological diagnosis of primary intrahepatic cholangio-
carcinoma still mostly remains one of exclusion, because of a
lack of specific markers. However, a novel RNA platform
using in-situ hybridization for albumin RNA has shown speci-
ficity for primary liver cancers, including CC.
80
However,
since this test is not widely available, diagnosis of primary
intrahepatic cholangiocarcinoma still requires exclusion of all
other possibilities by analyzing histomorphology, immunopro-
files, imaging studies, as well as clinical evidence.
By immunostaining, cholangiocarcinomas are strongly pos-
itive for CK-7 and CAM5.2 and show cytoplasmic labeling by
p-CEA. Mouse monoclonal antibody that recognizes human
EPCAM on cell membrane (MOC31) is positive in around
90%of cases, whereas CK-19 is positive in 70%to 80%of
cases.
59
Interestingly, there have been findings to suggest that
peripheral cholangiocarcinomas are more likely to be CK-7
positive but CK-20 negative, whereas central counterparts tend
to be positive for both CK-7 and CK-20.
81
CD56 also is
reported to be positive in peripheral cholangiocarcinomas,
which does not necessarily mean there is neuroendocrine dif-
ferentiation, if other neuroendocrine markers are negative.
82
One particularly useful immunostain marker for confirming
cholangiocarcinoma is S100P,
83
which is negative in benign
biliary epithelium but usually positive in cholangiocarcinomas,
especially when combined with U3 small nucleolar ribonucleo-
protein protein (IMP3) and a protein that in humans is encoded
by the von Hippel -Lindau tumor suppressor gene (pVHL).
84
In
comparison, HCCs, including all the variants, are predomi-
nantly positive in the described pattern for the immunostain
markers introduced earlier, including arginase, HepPar-1, gly-
pican 3, CD34, CD10, and p-CEA; HCCs are also positive for
AE1/3 and CAM5.2 in almost all cases but remain negative
mostly for CK-7 and CK-20. In contrast, cholangiocarcinomas
are mostly negative for the markers for HCC, except for p-
CEA, which could be positive in various adenocarcinomas,
irrespective of the organs of origin. Rarely, HepPar-1 could
be positive in cholangiocarcinoma cases, with foci of labeling,
but given the negativity by other HCC markers and adenocar-
cinoma histomorphology and immunoprofiles, a diagnosis of
cholangiocarcinoma should be beyond the question; this occa-
sional cellular labeling is not specific. For combined HCC-CC,
please refer to the later section.
In addition to HCC, a metastatic process from another
organ or site is also frequently suspected when facing a
potential cholangiocarcinoma, such as tumors originating
in gastrointestinal luminal sites, especially those from color-
ectal regions. It should be made clear to the entire clinical
team that there are no specific markers that can be depend-
able for diagnosing cholangiocarcinoma. Cholangiocarcino-
mas are usually positive for CK-7, and sometimes for CK-
20, similar to upper gastrointestinal tract and lung adeno-
carcinomas. A positive Thyroid Transcription Factor 1
(TTF-1) or napsin-A immunostain would strongly favor pul-
monary origin; however, occasional lung tumors are nega-
tive for TTF-1 or napsin-A by immunostaining; these cases
require detailed histomorphological evaluation and systemic
radiological and clinical correlation to narrow down the
most likely origin of the tumor. Likewise, the positive
CK-20 labeling and/or focal CDX-2 immunoreactivity does
not necessarily indicate gastrointestinal tract or colorectal
origin. Clinical history, endoscopic investigation, and ima-
ging studies are all pivotal in reaching the correct
conclusion.
Table 1. The T Categories for Hepatocellular Carcinoma.
T Category T Criteria
TX Primary tumor cannot be assessed
T0 No evidence of primary tumor
T1 Solitary tumor 2 cm, or >2 cm with vascular invasion
T1a Solitary tumor 2cm
T1b Solitary tumor >2cm with vascular invasion
T2 Solitary tumor with vascular invasion; or multiple
tumors, none >5 cm in greatest dimension
T3 Multiple tumors, at least one of which is >5 cm
T4 Single tumor or multiple tumors of any size, involving a
major branch of the portal vein or hepatic vein, or
tumor with direct invasion of adjacent organs than the
gallbladder or with perforation of the visceral
peritoneum
20 Cancer Control

Recently, as already mentioned, modified branched DNA
probes for albumin RNA were developed for in-situ hybridiza-
tion of albumin. This probe showed a sensitivity of 99%for
detecting intrahepatic cholangiocarcinoma and 100%sensitiv-
ity for detecting HCC.
80
Carcinomas arising at other sites,
including extrahepatic cholagniocarcinomas, tested negative.
Histopathological Staging and Risk Stratification
of Cholangiocarcinoma
TNM and anatomic stage/prognostic groupings. The TNM staging
system of the AJCC and the UICC applies to all primary carci-
nomas of the intrahepatic bile ducts and combined
hepatocellular–cholangiocarcinoma. It does not apply to hepa-
tic sarcomas or to metastatic tumors of the liver. Pathologic
staging is usually performed after surgical resection of the
primary tumor. Pathologic staging depends on pathologic doc-
umentation of the anatomic extent of disease, whether the pri-
mary tumor has been completely removed. If a biopsied tumor
is not resected for any reason and if the highest T and N cate-
gories or the M1 category of the tumor can be confirmed micro-
scopically, the criteria for pathologic classification and staging
have been satisfied without complete removal of the primary
cancer. The TNM descriptors are as already reported for HCC.
The AJCC eight edition classifies the T category according to
vascular invasion, extent of invasion, and the number of
Figure 12. Histopathological features of intrahepatic cholangiocarcinoma (ICC). (A) and (B), Photomicrograph (20 and 40; H&E stain)
shows an ICC with cystic glandular structures and adjacent benign liver. (C) Photomicrograph (H&E stain, 40) shows a solid, nodular pattern of
ICC. (D) Photomicrograph (H&E stain, 40) shows mucinous adenocarcinoma morphology of ICC. (E) Photomicrograph (H&E stain, 40)
shows a well-differentiated IHCC. (F) Photomicrograph (H&E stain, 100) shows a moderately to poorly differentiated ICC.
Jiang et al 21

tumors. The T category for intrahepatic cholagniocarcinoma is
shown in Table 2.
77
Combined HCC and Cholangiocarcinoma
Combined HCC and cholangiocarcinoma, also named mixed
HCC and cholangiocarcinoma, or biphenotypic HCC, previ-
ously called “collision tumor,” is a unique tumor that is com-
posed of 2 histopathologically distinct components: a part of
the tumor looks and stains like conventional HCC, whereas the
other part looks and stains like classical cholangiocarcinoma.
Based on the literature, these combined tumors account for
approximately 2%to 3%of all HCCs.
85
The 2 components are
intimately intermingled or in direct contact at least with each
other in the same lesion, with a subtle transition zone identified
in some cases. The HCC component is histologically and
immunophenotypically similar to single HCCs, whereas the
cholangiocarcinoma portion demonstrates unequivocal H&E
findings of adenocarcinoma, with glandular and tubular struc-
ture, micronodular and nested pattern, intracellular and extra-
cellular mucin production (can be confirmed with either
mucicarmine or PAS-diastase histochemical stains) as well as
immunophenotypes similar to any given intrahepatic cholan-
giocarcinoma.
86
Diagnostically, the HCC portion of this
Figure 13. Highly invasive behavior and immunohistochemistry of intrahepatic cholangiocarcinoma (IHCC). A, Photomicrograph (100; H&E
stain) shows IHCC gland invading into benign liver, with adjacent reactive bile ducts (arrows for benign ducts). B, IHCC with intraneural and
perineural invasion; arrow: nerve tract (100, H&E stain). (C) Photomicrograph (H&E stain, 40) shows a vascular space containing tumor
embolus. (D) CK-7 immunostain, (100) shows uniform strong labeling in IHCC tumor cells, in contrast to entirely negative CK-20 (E, 100).
(F) Photomicrograph (100) shows occasional nuclear labeling by Caudal Type Homeobox 2, a nuclear transcription factor in intestinal type
epithelium (CDX-2). CK indicates cytokeratin.
22 Cancer Control

combined tumor follows the same histological and immuno-
phenotypical patterns, as discussed above, including sarcoma-
toid HCC.
87
The investigation of the cholangiocarcinoma
portion has additionally been discussed in the aforementioned
text with conventional intrahepatic cholangiocarcinoma.
Differential Diagnoses of
Cholangiocarcinoma Versus Metastatic
Tumor From Other Organ/Sites
Frequently, making a diagnosis of cholangiocarcinoma espe-
cially the intrahepatic ones, instead of a metastatic adenocarci-
noma from another organ or site, can be difficult and
challenging, more so when there are none or limited clinical
and radiological information available for analysis. The clini-
cal, histological, and molecular information are all essential for
decision-making and management planning.
Clinically, cholangiocarcinomas are associated with chronic
biliary inflammation and regeneration, as frequently seen in
PSC, fluke infestation, chronic hepatitis C and hepatitis B viral
infection, profession-related chronic exposure to chemicals,
and pancreatobiliary malfunction. On the other hand, history
of colorectal, gastrointestinal, or gynecological malignancies
as well their stages are helpful in determining the possibility of
dealing with a metastatic tumor. It is very important to compare
the histomorphology of the primaries and the adenocarcinoma
in the liver to see whether there is any similarities; unfortu-
nately in the setting of a comprehensive cancer center, it is not
unusual to face a newly identified intrahepatic adenocarcinoma
with no or minimal history and no previous cases for morpho-
logical comparison.
Histologically, cholangiocarcinoma follows a stepwise car-
cinogenesis process through a precursor lesion: BilIN, ranging
from low to high grade (carcinoma in situ). The presence of
these precursor lesions in the vicinity of an intrahepatic ade-
nocarcinoma, combined with marked desmoplastic reaction
and high-grade cytological atypia, is highly suggestive of cho-
langiocarcinoma, instead of metastatic colorectal adenocarci-
noma; the latter usually demonstrates palisading, pencil-like
nuclei and intraluminal “dirty” necrosis. However, a frequent
obstacle encountered in practice is the small size of the biopsy
material, which inevitably restricts the differential list and lim-
its reaching a definitive diagnosis due to small areas of useful
diagnostic material. In practice, immunohistochemical studies
(such as cytokeratin-7 (CK-7), cytokeratin-19 (CK-19),
cytokeratin-19 (CK-20), CDX-2, and carbohydrate antigen
19-9 (CA-19-9), in an attempt to tell one potential primary
source from another one, these immunophenotypes have been
discussed in the earlier sections in this review. However, none
of these immunophenotypes are specific for telling cholangio-
carcinoma; they are just frequently seen from pancreatobiliary
and cholangiolar primaries. Subsequently, it is extremely
important to correlate radiological, clinical, and histopatholo-
gical findings in order to draw a correct conclusion.
Molecular wise, in recent years, it has been recognized that a
spectrum of genetic alterations are responsible for the initia-
tion, progression, and prognosis of cholangiocarcinoma; the
main culprits include Kirsten rat sarcoma virus oncogene
(KRAS), tumor protein p53 (TP53), a tumor suppressor gene,
mothers Against DPP Homolog 1 gene (SMAD) mutation,
BRAF,andINK4a, the gene encoding the p16 protein,
88-90
while genetic and epigenetic alterations both cause the activa-
tion of oncogenes and/or loss of tumor suppressor functions.
91-
93
However, these genetic alterations cannot be used to predict
site of origin, as these alterations have also been observed in
colorectal adenocarcinoma or malignancies from other sites.
For example, KRAS mutation is also a frequent event observed
in gastrointestinal adenocarcinomas, especially those from col-
orectal regions. The only exception is loss of SMAD expres-
sion, which has been detected exclusively in about 50%of
pancreatobiliary adenocarcinomas.
To summarize briefly, in order to decide whether an intra-
hepatic adenocarcinoma is a primary cholangiocarcinoma or a
metastatic process, morphological and ancillary test results
need to be utilized in combination with the clinical and radi-
ological findings to reach the most likely conclusion regarding
the true nature of the tumor.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to
the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, author-
ship, and/or publication of this article.
References
1. International Working Party. Terminology of nodular hepatocel-
lular lesions. Hepatology. 1995;22(3):983-993.
2. Watanabe S, Okita K, Harada T, et al. Morphologic studies of the
liver cell dysplasia. Cancer. 1983;51(12):2197-2205.
3. Adachi E, Hashimoto H, Tsuneyoshi M. Proliferating cell nuclear
antigen in hepatocellular carcinoma and small cell liver dysplasia.
Cancer. 1993;72(10):2902-2909.
Table 2. The T Categories for Intrahepatic Cholangiocarcinoma.
T Category T Criteria
TX Primary tumor cannot be assessed
T0 No evidence of primary tumor
Tis Carcinoma in situ (intraductal tumor)
T1 Solitary tumor 5 cm without vascular invasion
T1a Solitary tumor without vascular invasion, 5cmor
>5 cm
T1b Solitary tumor >5 cm without vascular invasion
T2 Solitary tumor with vascular invasion or multiple tumors,
with or without vascular invasion
T3 Tumor perforating the visceral peritoneum
T4 Tumor involving the local extrahepatic structures by
direct invasion
Jiang et al 23

4. Marchio A, Terris B, Meddeb M, et al. Chromosomal abnormal-
ities in liver cell dysplasia detected by comparative genomic
hybridisation. Mol Pathol. 2001;54(4):270-274.
5. Plentz RR, Park YN, Lechel A, et al. Telomere shortening and
inactivation of cell cycle checkpoints characterize human hepa-
tocarcinogenesis. Hepatology. 2007;45(4):968-976.
6. Ferrell LD, Crawford JM, Dhillon AP, Scheuer PJ, Nakanuma Y.
Proposal for standardized criteria for the diagnosis of benign, bor-
derline, and malignant hepatocellular lesions arising in chronic
advanced liver disease. Am J Surg Pathol. 1993;17(11):1113-1123.
7. Sato Y, Harada K, Sasaki M, Nakanuma Y. Histological charac-
terization of biliary intraepithelial neoplasia with respect to pan-
creatic intraepithelial neoplasia. Int J Hepatol. 2014;2014:
678260.
8. Zen Y, Adsay NV, Bardadin K, et al. Biliary intraepithelial neo-
plasia: an international interobserver agreement study and pro-
posal for diagnostic criteria. Mod Pathol. 2007;20(6):701-709.
9. Aishima S, Kubo Y, Tanaka Y, Oda Y. Histological features of
precancerous and early cancerous lesions of biliary tract carci-
noma. J Hepatobiliary Pancreat Sci. 2014;21(7):448-452.
10. Matthaei H, Lingohr P, Strasser A, et al. Biliary intraepithelial
neoplasia (BilIN) is frequently found in surgical margins of bili-
ary tract cancer resection specimens but has no clinical implica-
tions. Virchows Arch. 2015;466(2):133-141.
11. Wan XS, Xu YY, Qian JY, et al. Intraductal papillary neoplasm of
the bile duct. World J Gastroenterol. 2013;19(46):8595-8604.
12. Rocha FG, Lee H, Katabi N, et al. Intraductal papillary neoplasm of
the bile duct: a biliary equivalent to intraductal papillary mucinous
neoplasm of the pancreas? Hepatology. 2012;56(4):1352-1360.
13. Ohtsuka M, Shimizu H, Kato A, et al. Intraductal papillary neo-
plasms of the bile duct. Int J Hepatol. 2014;2014:459091.
14. Sclabas GM, Barton JG, Smyrk TC, et al. Frequency of subtypes
of biliary intraductal papillary mucinous neoplasm and their
MUC1, MUC2, and DPC4 expression patterns differ from pan-
creatic intraductal papillary mucinous neoplasm. J Am Coll Surg.
2012;214(1):27-32.
15. Lee JS, Heo J, Libbrecht L, et al. A novel prognostic subtype of
human hepatocellular carcinoma derived from hepatic progenitor
cells. Nat Med. 2006;12(4):410-416.
16. Oishi N, Kumar MR, Roessler S, et al. Transcriptomic profiling
reveals hepatic stem-like gene signatures and interplay of miR-
200c and epithelial-mesenchymal transition in intrahepatic cho-
langiocarcinoma. Hepatology. 2012;56(5):1792-1803.
17. Kokuryo T, Yokoyama Y, Nagino M. Recent advances in cancer
stem cell research for cholangiocarcinoma. J Hepatobiliary Pan-
creat Sci. 2012;19(6):606-613.
18. Durnez A, Verslype C, Nevens F, et al. The clinicopathological
and prognostic relevance of cytokeratin 7 and 19 expression in
hepatocellular carcinoma. A possible progenitor cell origin. His-
topathology. 2006;49(2):138-151.
19. Kim H, Park YN. Hepatocellular carcinomas expressing ‘stem-
ness’-related markers: clinicopathological characteristics. Dig
Dis. 2014;32(6):778-785.
20. Kim H, Choi GH, Na DC, et al. Human hepatocellular carcinomas
with “stemness”-related marker expression: keratin 19 expression
and a poor prognosis. Hepatology. 2011;54(5):1707-1717.
21. Theise ND, Yao JL, Harada K, et al. Hepatic ‘stem cell’ malig-
nancies in adults: four cases. Histopathology. 2003;43(3):263-271.
22. Chiba T, Kita K, Zheng YW, et al. Side population purified from
hepatocellular carcinoma cells harbors cancer stem cell-like prop-
erties. Hepatology. 2006;44(1):240-251.
23. Jakate S, Yabes A, Giusto D, et al. Diffuse cirrhosis-like hepatocel-
lular carcinoma: a clinically and radiographically undetectedvariant
mimicking cirrhosis. Am J Surg Pathol. 2010;34(7):935-941.
24. Matsuura S, Aishima S, Taguchi K, et al. ‘Scirrhous’ type hepato-
cellular carcinomas: a special reference to expression of cytokeratin
7 and hepatocyte paraffin 1. Histopathology. 2005;47(4):382-390.
25. Kurogi M, Nakashima O, Miyaaki H, Fujimoto M, Kojiro M.
Clinicopathological study of scirrhous hepatocellular carcinoma.
J Gastroenterol Hepatol. 2006;21(9):1470-1477.
26. Lee JH, Choi MS, Gwak GY, et al. Clinicopathologic character-
istics and long-term prognosis of scirrhous hepatocellular carci-
noma. Dig Dis Sci. 2012;57(6):1698-1707.
27. Krings G, Ramachandran R, Jain D, et al. Immunohistochemical
pitfalls and the importance of glypican 3 and arginase in the
diagnosis of scirrhous hepatocellular carcinoma. Mod Pathol.
2013;26(6):782-791.
28. Haratake J, Horie A. An immunohistochemical study of sarcoma-
toid liver carcinomas. Cancer. 1991;68(1):93-97.
29. Inoue T, Kudo M, Minami Y, Chung H, Fukunaga T, Kawasaki T.
Case of rapidly progressed sarcomatoid hepatocellular carcinoma
in a young female without risk factor. Liver Int. 2007;27(10):
1428-1430.
30. Lao XM, Chen DY, Zhang YQ, et al. Primary carcinosarcoma of
the liver: clinicopathologic features of 5 cases and a review of the
literature. Am J Surg Pathol. 2007;31(6):817-826.
31. Koo HR, Park MS, Kim MJ, et al. Radiological and clinical fea-
tures of sarcomatoid hepatocellular carcinoma in 11 cases. J Com-
put Assist Tomogr. 2008;32(5):745-749.
32. Papotti M, Sambataro D, Marchesa P, Negro F. A combined
hepatocellular/cholangiocellular carcinoma with sarcomatoid fea-
tures. Liver. 1997;17(1):47-52.
33. Akasofu M, Kawahara E, Kaji K, Nakanishi I. Sarcomatoid
hepatocellular-carcinoma showing rhabdomyoblastic differentiation
in the adult cirrhotic liver. Virchows Arch. 1999;434(6):511-515.
34. Fu Y, Kobayashi S, Kushida Y, et al. Sarcomatoid hepatocellular
carcinoma with chondroid variant: case report with immunohis-
tochemical findings. Pathol Int. 2000;50(11):919-922.
35. Cho MS, Lee SN, Sung SH, Han WS. Sarcomatoid hepatocellular
carcinoma with hepatoblastoma-like features in an adult. Pathol
Int. 2004;54(6):446-450.
36. Dahm HH. Immunohistochemical evaluation of a sarcomatoid
hepatocellular carcinoma with osteoclast-like giant cells. Diagn
Pathol. 2015;10:40.
37. Chu PG, Ishizawa S, Wu E, Weiss LM. Hepatocyte antigen as a
marker of hepatocellular carcinoma: an immunohistochemical
comparison to carcinoembryonic antigen, CD10, and alpha-feto-
protein. Am J Surg Pathol. 2002;26(8):978-988.
38. Pua U, Low SC, Tan YM, Lim KH. Combined hepatocellular
and cholangiocarcinoma with sarcomatoid transformation:
radiologic-pathologic correlation of a case. Hepatol Int. 2009;
3(4):587-592.
24 Cancer Control

39. Yamaguchi R, Nakashima O, Ogata T, Hanada K, Kumabe T,
Kojiro M. Hepatocellular carcinoma with an unusual neuroendo-
crine component. Pathol Int. 2004;54(11):861-865.
40. Han CP, Hsu JD, Koo CL, Yang SF. Antibody to cytokeratin
(CK8/CK18) is not derived from CAM5.2 clone, and anticytoker-
atin CAM5.2 (Becton Dickinson) is not synonymous with the
antibody (CK8/CK18). Hum Pathol. 2010;41(4):616-617; author
reply 617.
41. Ichikawa T, Federle MP, Grazioli L, Madariaga J, Nalesnik M,
Marsh W. Fibrolamellar hepatocellular carcinoma: imaging and
pathologic findings in 31 recent cases. Radiology. 1999;213(2):
352-361.
42. Katzenstein HM, Krailo MD, Malogolowkin MH, et al. Fibrola-
mellar hepatocellular carcinoma in children and adolescents.
Cancer. 2003;97(8):2006-2012.
43. Stipa F, Yoon SS, Liau KH, et al. Outcome of patients with
fibrolamellar hepatocellular carcinoma. Cancer. 2006;106(6):
1331-1338.
44. Ehrenfried JA, Zhou Z, Thompson JC, Evers BM. Expression of
the neurotensin gene in fetal human liver and fibrolamellar carci-
noma. Ann Surg. 1994;220(4):484-489; discussion 489-491.
45. Simonsen K, Rode A, Nicoll A, et al. Vitamin B(1)(2) and its
binding proteins in hepatocellular carcinoma and chronic liver
diseases. Scand J Gastroenterol. 2014;49(9):1096-1102.
46. Allan BJ, Wang B, Davis JS, et al. A review of 218 pediatric cases
of hepatocellular carcinoma. J Pediatr Surg. 2014;49(1):166-171;
discussion 171.
47. Lim II, Farber BA, LaQuaglia MP. Advances in fibrolamellar
hepatocellular carcinoma: a review. Eur J Pediatr Surg. 2014;
24(6):461-466.
48. El-Serag HB, Davila JA. Is fibrolamellar carcinoma different
from hepatocellular carcinoma? A US population-based study.
Hepatology. 2004;39(3):798-803.
49. Torbenson M. Fibrolamellar carcinoma: 2012 update. Scientifica
(Cairo). 2012;2012:743790.
50. Moreno-Luna LE, Arrieta O, Garcia-Leiva J, et al. Clinical and
pathologic factors associated with survival in young adult
patients with fibrolamellar hepatocarcinoma. BMC cancer.
2005;5:142.
51. Jain D, Nayak NC, Kumaran V, Saigal S. Steatohepatitic hepato-
cellular carcinoma, a morphologic indicator of associated meta-
bolic risk factors: a study from India. Arch Pathol Lab Med. 2013;
137(7):961-966.
52. Yan BC, Gong C, Song J, et al. Arginase-1: a new immunohisto-
chemical marker of hepatocytes and hepatocellular neoplasms.
Am J Surg Pathol. 2010;34(8):1147-1154.
53. Fujiwara M, Kwok S, Yano H, Pai RK. Arginase-1 is a more
sensitive marker of hepatic differentiation than HepPar-1 and
glypican-3 in fine-needle aspiration biopsies. Cancer Cytopathol.
2012;120(4):230-237.
54. Radwan NA, Ahmed NS. The diagnostic value of arginase-1
immunostaining in differentiating hepatocellular carcinoma from
metastatic carcinoma and cholangiocarcinoma as compared to
HepPar-1. Diagn Pathol. 2012;7(149):1-12.
55. Nguyen TB, Roncalli M, Di Tommaso L, Kakar S. Combined use
of heat-shock protein 70 and glutamine synthetase is useful in the
distinction of typical hepatocellular adenoma from atypical hepa-
tocellular neoplasms and well-differentiated hepatocellular carci-
noma. Mod Pathol. 2016 Mar;29(3):283-292.
56. Bioulac-Sage P, Balabaud C, Zucman-Rossi J. Subtype classifi-
cation of hepatocellular adenoma. Dig Surg. 2010;27(1):39-45.
57. Bioulac-Sage P, Cubel G, Taouji S, et al. Immunohistochemical
markers on needle biopsies are helpful for the diagnosis of focal
nodular hyperplasia and hepatocellular adenoma subtypes. Am J
Surg Pathol. 2012;36(11):1691-1699.
58. Wennerberg AE, Nalesnik MA, Coleman WB. Hepatocyte paraf-
fin 1: a monoclonal antibody that reacts with hepatocytes and can
be used for differential diagnosis of hepatic tumors. Am J Pathol.
1993;143(4):1050-1054.
59. Chan ES, Yeh MM. The use of immunohistochemistry in liver
tumors. Clin Liver Dis. 2010;14(4):687-703.
60. Fan Z, van de Rijn M, Montgomery K, Rouse RV. Hep par 1
antibody stain for the differential diagnosis of hepatocellular
carcinoma: 676 tumors tested using tissue microarrays and
conventional tissue sections. Mod Pathol. 2003;16(2):
137-144.
61. Kakar S, Muir T, Murphy LM, Lloyd RV, Burgart LJ. Immunor-
eactivity of Hep Par 1 in hepatic and extrahepatic tumors and its
correlation with albumin in situ hybridization in hepatocellular
carcinoma. Am J Clin Pathol. 2003;119(3):361-366.
62. Shafizadeh N, Ferrell LD, Kakar S. Utility and limitations of
glypican-3 expression for the diagnosis of hepatocellular carci-
noma at both ends of the differentiation spectrum. Mod Pathol.
2008;21(8):1011-1018.
63. Yan B, Wei JJ, Qian YM, et al. Expression and clinicopathologic
significance of glypican 3 in hepatocellular carcinoma. Ann
Diagn Pathol. 2011;15(3):162-169.
64. Abdul-Al HM, Makhlouf HR, Wang G, Goodman ZD. Glypican-
3 expression in benign liver tissue with active hepatitis C: impli-
cations for the diagnosis of hepatocellular carcinoma. Hum
Pathol. 2008;39(2):209-212.
65. Uenishi T, Kubo S, Yamamoto T, et al. Cytokeratin 19 expression
in hepatocellular carcinoma predicts early postoperative recur-
rence. Cancer Sci. 2003;94(10):851-857.
66. Klein WM, Molmenti EP, Colombani PM, et al. Primary liver
carcinoma arising in people younger than 30 years. Am J Clin
Pathol. 2005;124(4):512-518.
67. Ra SH, Kaplan JB, Lassman CR. Focal nodular hyperplasia after
orthotopic liver transplantation. Liver transplantation. Liver
Transpl. 2010;16(1):98-103.
68. Roncalli M, Sciarra A, Tommaso LD. Benign hepatocellular
nodules of healthy liver: focal nodular hyperplasia and hepato-
cellular adenoma. Clin Mol Hepatol. 2016;22(2):199-211.
69. Barthelmes L, Tait IS. Liver cell adenoma and liver cell adeno-
matosis. HPB (Oxford). 2005;7(3):186-196.
70. Lin H, van den Esschert J, Liu C, van Gulik TM. Systematic
review of hepatocellular adenoma in China and other regions. J
Gastroenterol Hepatol. 2011;26(1):28-35.
71. Sasaki M, Nakanuma Y. Overview of hepatocellular adenoma in
Japan. Int J Hepatol. 2012;2012:648131.
72. Greaves WO, Bhattacharya B. Hepatic adenomatosis. Arch
Pathol Lab Med. 2008;132(12):1951-1955.
Jiang et al 25

73. Farges O, Dokmak S. Malignant transformation of liver adenoma:
an analysis of the literature. Dig Surg. 2010;27(1):32-38.
74. Zucman-Rossi J, Jeannot E, Nhieu JT, et al. Genotype-phenotype
correlation in hepatocellular adenoma: new classification and
relationship with HCC. Hepatology. 2006;43(3):515-524.
75. Bioulac-Sage P, Rebouissou S, Thomas C, et al. Hepatocellular
adenoma subtype classification using molecular markers and
immunohistochemistry. Hepatology. 2007;46(3):740-748.
76. Nault JC, Couchy G, Balabaud C, et al. Molecular classification
of hepatocellular adenoma associates with risk factors, bleeding,
and malignant transformation. Gastroenterology. 2017;152(4):
880-894 e886.
77. AJCC Cancer Staging Manual. Amin MB. 8th ed. Springer; 2017.
78. Palmer WC, Patel T. Are common factors involved in the patho-
genesis of primary liver cancers? A meta-analysis of risk factors for
intrahepatic cholangiocarcinoma. J Hepatol. 2012;57(1):69-76.
79. Torbenson M, Yeh MM, Abraham SC. Bile duct dysplasia in the
setting of chronic hepatitis C and alcohol cirrhosis. Am J Surg
Pathol. 2007;31(9):1410-1413.
80. Ferrone CR, Ting DT, Shahid M, et al. The ability to diagnose
intrahepatic cholangiocarcinoma definitively using novel
branched DNA-enhanced albumin RNA in situ hybridization
technology. Ann Surg Oncol. 2016;23(1):290-296.
81. Rullier A, Le Bail B, Fawaz R, Blanc JF, Saric J, Bioulac-Sage P.
Cytokeratin 7 and 20 expression in cholangiocarcinomas varies
along the biliary tract but still differs from that in colorectal
carcinoma metastasis. Am J Surg Pathol. 2000;24(6):870-876.
82. Kozaka K, Sasaki M, Fujii T, et al. A subgroup of intrahepatic
cholangiocarcinoma with an infiltrating replacement growth pat-
tern and a resemblance to reactive proliferating bile ductules: ‘bile
ductular carcinoma’. Histopathology. 2007;51(3):390-400.
83. Hamada S, Satoh K, Hirota M, et al. Calcium-binding protein
S100P is a novel diagnostic marker of cholangiocarcinoma. Can-
cer Sci. 2011;102(1):150-156.
84. Schmidt MT, Himmelfarb EA, Shafi H, Lin F, Xu H, Wang HL.
Use of IMP3, S100P, and pVHL immunopanel to aid in the inter-
pretation of bile duct biopsies with atypical histology or suspi-
cious for malignancy. Appl Immunohistochem Mol Morphol.
2012;20(5):478-487.
85. Yeh MM. Pathology of combined hepatocellular-cholangiocarci-
noma. J Gastroenterol Hepatol. 2010;25(9):1485-1492.
86. Radwan NA, Ahmed NS. The diagnostic value of arginase-1
immunostaining in differentiating hepatocellular carcinoma from
metastatic carcinoma and cholangiocarcinoma as compared to
HepPar-1. Diagn Pathol. 2012;7:149.
87. Chin S, Kim Z. Sarcomatoid combined hepatocellular-
cholangiocarcinoma: a case report and review of literature. Int
J Clin Exp Pathol. 2014;7(11):8290-8294.
88. Hsu M, Sasaki M, Igarashi S, Sato Y, Nakanuma Y. KRAS and
GNAS mutations and p53 overexpression in biliary intraepithelial
neoplasia and intrahepatic cholangiocarcinomas. Cancer. 2013;
119(9):1669-1674.
89. Robertson S, Hyder O, Dodson R, et al. The frequency of KRAS
and BRAF mutations in intrahepatic cholangiocarcinomas and
their correlation with clinical outcome. Hum Pathol. 2013;
44(12):2768-2773.
90. Jang S, Chun SM, Hong SM, et al. High throughput molecular
profiling reveals differential mutation patterns in intrahepatic
cholangiocarcinomas arising in chronic advanced liver diseases.
Mod Pathol. 2014;27(5):731-739.
91. Tischoff I, Wittekind C, Tannapfel A. Role of epigenetic altera-
tions in cholangiocarcinoma. J Hepatobiliary Pancreat Surg.
2006;13(4):274-279.
92. Isomoto H. Epigenetic alterations associated with cholangiocar-
cinoma (review). Oncol Rep. 2009;22(2):227-232.
93. Hamilton JP. Epigenetic mechanisms involved in the pathogen-
esis of hepatobiliary malignancies. Epigenomics. 2010;2(2):
233-243.
26 Cancer Control