Best Practice & Research Clinical Gastroenterology 29 (2015) 277e293

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Best Practice & Research Clinical

Gastroenterology

Pathologic classification of cholangiocarcinoma:

New concepts
Yasuni Nakanuma, MD, Councilor of Department *,
Yuko Kakuda, MD, Clinical Fellow of Department
Department of Diagnostic Pathology, Shizuoka Cancer Center, Shizuoka, Japan

a b s t r a c t
Keywords:
Cholangiocarcinoma Herein, we propose a new pathologic classification of chol-
Biliary tract angiocarcinoma (CCA) based on recent progress in studies of
Pancreas preinvasive CCA lesions and the relationship of CCA to hepatic
Preinvasive carcinoma progenitor cells, as well as a new concept with respect to the
Adenocarcinoma pathologic similarities between biliary and pancreatic neoplasms.
Depending on anatomical location, CCA is classifiable as intra-
hepatic (iCCA), perihilar (pCCA), and distal CCA (dCCA). iCCA is
classifiable as the conventional type and the bile ductular type,
whereas pCCA and dCCA mainly present as conventional adeno-
carcinoma. In addition, these three CCAs may present as the
intraductal neoplasm type or rare variants. Bile ductular CCA re-
sembles proliferating bile ductules and expressing hepatic pro-
genitor cell phenotypes. Four types of preinvasive lesions are
proposed: flat, papillary, tubular lesion, and cystic lesion. These
lesions are eventually followed by invasive CCA. Interestingly,
these preinvasive lesions have pancreatic counterparts. This CCA
classification may introduce a new field of CCA research.
© 2015 Elsevier Ltd. All rights reserved.

Introduction

Cholangiocarcinoma (CCA), a primary malignant neoplasm of the hepatobiliary system secondary

to hepatocellular carcinoma, arises from the biliary epithelia (lining epithelia and peribiliary glands)
of the intra and extrahepatic bile ducts [1]. Recently, the incidence of CCA has been increasing

* Corresponding author. Department of Diagnostic Pathology, Shizuoka Cancer Center, Shimo-Nagakubo 1007, Sunto-
Nagaizumi, Shizuoka 411-8777, Japan. Tel.: þ81 55 989 5222; fax: þ81 55 989 5783.
E-mail address: pbcpsc@kenroku.kanazawa-u.ac.jp (Y. Nakanuma).

http://dx.doi.org/10.1016/j.bpg.2015.02.006
1521-6918/© 2015 Elsevier Ltd. All rights reserved.
278 Y. Nakanuma, Y. Kakuda / Best Practice & Research Clinical Gastroenterology 29 (2015) 277e293

worldwide [2,3]. CCA exhibits heterogeneous clinical features, genotypes, and biological behaviors
depending on the anatomical location, growth pattern, and histologic differentiation. CCAs are
usually detected and diagnosed clinically at an advanced stage, and most have a poor prognosis even
after surgical resection [2,3].
CCA is usually classified as intrahepatic (iCCA), perihilar (pCCA), and distal type (dCCA) according to
the anatomical location: dCCA involves the common bile duct, pCCA the perihilar bile duct, and iCCA
the second branches of both the hepatic ducts and more proximal bile ducts [4e9]. CCAs arising in the
hepatic parenchyma are classified as iCCA. Histologically, CCAs are generally adenocarcinomas and
present with various differentiation and phenotypes as well as rare variants. Recently, much progress
has been made in the study of precancerous and preinvasive CCA lesions, including biliary intra-
epithelial neoplasm (BilIN), intraductal papillary neoplasm of the bile duct (IPNB), and mucinous cystic
neoplasm (MCN) [1,4,5,10e14]. In addition, the relationship between iCCA and combined hepatocel-
lular cholangiocarcinoma (HC-CC) with stem cell or hepatic progenitor cell (HPC) features highlights
the participation of HPC in the development and progression of iCCA [15e18]. Furthermore, a new
concept of biliary disease with a pancreatic counterpart suggests that pCCA and dCCA and their pre-
cursors BilIN, IPNB, and hepatobiliary MCN (hbMCN) have pancreatic counterparts such as pancreatic
duct adenocarcinoma and its precursors, pancreatic intraepithelial neoplasm (PanIN), intraductal
papillary mucinous neoplasm (IPMN), and pancreatic MCN [19e21].
In this review, a pathologic classification of CCA that is mainly based on the 2010 World Health
Organization (WHO) classification of tumors of the digestive system is proposed; this classification
considers recent progress in preinvasive CCA lesions, the relationship between iCCA and HC-CC with
respect to hepatic progenitor/stem cells, and the new concept of “biliary diseases with pancreatic
counterparts” [1,4,5,14,19e22].

Anatomy of the biliary tract

The right and left hepatic ducts, including their first branches, and the proximal portion of the
extrahepatic bile duct are collectively called the “perihilar bile ducts” [6,7,23e25]. The intrahepatic bile
ducts proximal to the second branches of the right or left hepatic duct are classified as intrahepatic
large and small bile ducts [23]. The former comprise the second to third branches of the right or left
hepatic bile ducts. The latter comprise the septal and interlobular bile ducts. The interlobular bile ducts
are connected to bile ductules. The septal and interlobular bile ducts are larger and smaller than
100 mm in size, respectively. Bile ductules or canals of Hering are located at the periphery of the portal
tracts, facing the periportal hepatocytes [25]. Peribiliary glands are physiologically distributed around
the intrahepatic large bile ducts and perihilar and distal bile ducts [23].

Background hepatobiliary lesions of invasive cholangiocarcinoma

Although CCAs usually develop in normal livers and biliary tracts, some are associated with pre-
ceding biliary or hepatic diseases and have various etiologies. The pathologies of invasive chol-
angiocarcinoma (ICC) with and without such preceding diseases do not usually differ, although some of
the former may exhibit characteristic clinicopathological features according to the background disease
[2,3,26,27].

Chronic inflammatory biliary diseases

Primary sclerosing cholangitis (PSC) with or without inflammatory bowel disease, particularly ul-
cerative colitis, is a risk factor for CCA [2,3,6]. Clinically undetected iCCA and pCCA or precursor lesions
are occasionally encountered in explanted livers with PSC. Hepatolithiasis, which is not rare in East
Asia, is an independent risk factor for CCA, and approximately 3e5% of patients with hepatolithiasis
eventually develop CCA [8,10,28]. Stone-containing bile ducts exhibit hyperplasia of the lining
epithelium, and not infrequently, they exhibit preinvasive lesions such as BilINs, and occasionally IPNB.
Liver flukes, especially Opisthorchis viverrini and Clonorchis sinensis, are risk factors for CCA [1,29e31].
The presence of parasites in the biliary tree induces a chronic inflammatory response and chol-
angiocyte injury and repair, followed by the development of BilIN and IPNB [1,30,31].
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Biliary tract malformation and developmental disorders

ICC may arise in patients with congenital hepatic fibrosis, congenital segmental or multiple dila-
tation of the intrahepatic bile ducts (Caroli disease), and other biliary malformations such as chol-
edochal cysts [1,31]. Biliaryepancreatic maljunction is known to complicate gallbladder carcinoma and
dCCA [32,33].

Chronic advanced, non-biliary liver diseases

Non-biliary cirrhosis, particularly hepatitis virus-related cirrhosis, is recognized as a cause of iCCA.
In Japan, patients with hepatitis C virus-related cirrhosis have an approximately 1000-fold higher risk
of developing iCCA than the general population [26,33,34]. The development of iCCA also appears to
correlate with hepatitis B virus (HBV) infection in areas where both HBV and iCCA are endemic [1].
Advanced nonalcoholic steatohepatitis/nonalcoholic fatty liver disease is another known factor that is
complicated by iCCA [2,3]. Upon clinical detection, such ICCs are usually of a smaller, mass-forming
(MF) type. HPCs may be involved in cholangiocarcinogenesis related to liver cirrhosis [17,34].

Chronic exposure to toxins and chemicals

Thorotrast deposition in the liver is known to be associated with CCA, although such cases are now
rare [1,31]. Recently, an outbreak of CCA occurred among workers in the offset color proof-printing
department at a printing company in Japan [27,35e37]. Most of the workers were relatively young
and were diagnosed with CCA at 25e45 years of age. These patients were exposed to high levels of
concentrated chemicals, particularly dichloromethane and 1,2-dichloropropane, for long durations.
CCA mainly arose from the intrahepatic large bile ducts and perihilar bile ducts. Precancerous or early
cancerous lesions such as BilIN and IPNBs were observed, as well as non-specific bile duct injuries such
as ductal and periductal fibrosis and luminal dilatation with severe epithelial damages with DNA
damages [36].

Others
A causal association has been reported between some CCA cases and EpsteineBarr virus (EBV)
infection [38].

Gross features and classification

CCA exhibits three basic gross growth patterns. i) CCA arising in the liver parenchyma presents as a
mass lesion. Along the grossly visible bile duct, ii) CCA presents as flat or nodular sclerosing growth of
the affected bile duct, with thickening of the duct wall and surrounding tissue and luminal stenosis. iii)
CCA exhibits intraductal growth (IG) in the dilated bile duct lumen.

Intrahepatic CCA
iCCA is grossly classifiable into MF, periductal infiltrating (PI), and IG types [1,9,39,40]. The MF type
is the most common type (78% of iCCA) [40] and presents as a mass lesion in the hepatic parenchyma,
with a gray to grayish-white, firm, and solid carcinoma. The MF type of iCCA can be quite large; central
necrosis or scarring is quite common, and mucin may be observed on cut surfaces. The MF type is not
infrequently associated with chronic, advanced nonbiliary liver disease. The PI type is the infrequent
type (16% of iCCA) [40] and demonstrates spreading of the carcinoma along the portal tracts with
strictures and thickening of the affected bile ducts and dilatation of the peripheral bile ducts as well as
variable carcinoma infiltration into the hepatic parenchyma. The IG type is the uncommon type (6% of
iCCA) [40] and presents as a polypoid or papillary tumor within the dilated duct lumen, and a majority
of such cases represent the malignant progression of IPNB (see below) [41]. iCCA that arises in the
intrahepatic small bile ducts or bile ductules/progenitor cells is usually the MF type, whereas ICC that
arises in the intrahepatic large bile ducts may present as either the PI or IG type. These three gross types
overlap in various combinations in individual cases (39,40).
Some cases of the IG type present cystic dilatation of the affected bile ducts; such cases used to be
described as biliary cystadenoma/adenocarcinoma (see below) [1,13,30].
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Perihilar and distal CCA

pCCA and dCCA (p/dCCA) have comparable similar gross features; a frequent flat or nodular scle-
rosing type (73% of p/dCCA) [41], and a less frequent intraductal papillary type (27%) [41]. The latter
corresponds to cases of the IG type of iCCA [42], whereas the former shares features with the PI type of
iCCA. The precursor lesions of flat or nodular sclerosing type of p/dCCA and PI type of iCCA might derive
from BilIN, and intraductal papillary type of p/dCCA and IG type of iCCA might derive from IPNB.

Histologic features and classification of CCA

The classification of iCCAs and p/dCCA (pCCA and dCCA) is shown in Tables 1A and 1B.

Intrahepatic CCA

Conventional ICCs (bile duct ICCs). This type is an invasive adenocarcinoma with features of variably
sized tubular structures, acini formation, and micropapillary configurations [1,40]. The adenocarci-
noma is generally well to moderately differentiated and composed of columnar to cuboidal epithelial
cells with clear or slightly eosinophilic cytoplasm that resemble biliary epithelial cells. In addition,
poorly differentiated adenocarcinoma is admixed infrequently, and such cases exhibit solid, cord-like,
or cribriform growth with variable cellular and nuclear pleomorphism. A variable desmoplastic and
inflammatory reaction is always observed. Coagulative necrosis and scar-like changes are not infre-
quent, particularly in the central parts. Mucin production is observed in the lumen, along the luminal
sides, and also in carcinoma cell cytoplasm. The adenocarcinoma frequently exhibits portal venous and,
to a lesser degree, lymphatic infiltration. Against the hepatic parenchyma, the carcinoma may present
compressive growth, although no evident fibrous capsule is formed [15,16]. The intervening hepatic
parenchyma containing the portal tracts is forcibly and secondarily incorporated between or into the
carcinoma tissues. The carcinoma also displays invasion between hepatocytes and appears to infiltrate
the sinusoid. Grossly, this CCA type represents the MF or PI gross type [1,39].
Conventional ICC can be further classified into small and large bile duct types according to the level
or size of the affected bile ducts. The former is characterized grossly by the MF type and the latter is
characterized by evident cancerous large bile duct(s) that display the PI pattern. However, the differ-
entiation of these two types is occasionally arbitrary, particularly in advanced cases, because the small
bile duct type may secondarily involve the large bile duct type (cancerization), thus appearing as the
large bile duct type, and the large bile duct often displays a mass around the cancerous large bile duct
along with progression.

Table 1A
Classification of intrahepatic cholangiocarcinoma (iCCA).

Conventional type (bile duct type)

Small bile duct type
Well, moderately, poorly differentiated adenocarcinoma
Large bile duct type
Well, moderately, poorly differentiated adenocarcinoma
Bile ductular type
Intraductal type
Papillary type
Tubular type
Superficial spreading type
Rare variants
Squamous/adenosquamous cell type
Mucinous/signet ring cell
Clear cell type
Undifferentiated type
Lymphoepithelial type
Other
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Table 1B
Classification of perihilar and distal cholangiocarcinoma (p/dCCA).

Conventional type
Well, moderately, poorly differentiated adenocarcinoma
Intraductal type
Papillary type
Tubular type
Superficial spreading type
Rare variants
Squamous/adenosquamous cell type
Mucinous/signet ring cell
Clear cell type
Undifferentiated type
Lymphoepithelial type
Other

1) Small bile duct type: Histologically, a variable but usually small-sized tubular or acinar adenocar-
cinoma exhibits nodular growth and invades the parenchyma. A small, solid cord-like or cribriform
pattern is also observed in variable combinations. The carcinomatous tubules are usually larger than
the interlobular and septal bile ducts.
2) Large bile duct type: Histologically, the cancerous large bile duct shows the luminal spread of
carcinoma with micropapillary and flat configurations along the affected lumen, as well as carci-
noma cell invasion with a variable-sized tubular or acinar configuration into the duct wall and
surrounding parenchyma. Peribiliary glands and their conduits are also involved. Carcinomas
mainly spread and proliferate in the portal tracts, including the bile duct wall. In the invasive area of
the hepatic parenchyma, the carcinoma histologies vary, and some resemble the small bile duct type
or even bile ductular type. This type of iCCA arises from the intrahepatic large bile duct (distal to the
perihilar intrahepatic bile ducts), including the peribiliary glands, although some cases might have
arisen from small bile duct ICC with secondary involvement of the intrahepatic large bile ducts
(cancerization). In some cases, the original cancerous bile ducts seem to be obliterated directly by
carcinoma growth.

Bile ductular type. Grossly, this type belongs to the MF type. The carcinoma cells display rather ho-
mogeneous growth with well to moderate differentiation and small tubular or acinar patterns or
cord-like structures with a slit-like lumen and arborization [15,16]. They resemble bile ductules or
proliferating reactive bile ductules. A small-cord like pattern with spindle cell features and no visible
lumen is occasionally predominant. The carcinoma cell size is usually small in comparison with that
of conventional ICC. There is extensive replacement of non-neoplastic hepatocytes in the hepatic
lobules or regenerative nodules by infiltrating carcinoma cells. Significant collagen fiber deposition
around or along the carcinoma cells is observed, and carcinoma cells are squeezed sporadically into
fibrous tissues; a fibrous or hyalinous configuration reflecting the preexisting hepatic lobules or
regenerative nodules is recognizable, and fibrotic portal tracts are distributed regularly within the
tumor and fibrotic tissues. This type exhibits widespread replacing growth characterized by i) direct
contact between the hepatocytes and carcinoma cells, ii) no or minimal compression of the sur-
rounding hepatic parenchyma by the carcinoma cells, and iii) the apparent replacement of hepato-
cytes by carcinoma cells [15,16].
Neural cell adhesion molecule (NCAM) and epithelial cell adhesion molecule (EpCAM), a marker of
HPCs [15,16], are characteristically detected mainly on the cell membranes and cytoplasm of bile
ductular type cells.
Ductal plate malformation variant: This type mimics ductal plate malformation (DPM), which is
found in Caroli disease or congenital hepatic fibrosis [5,43]. The carcinoma shows an irregular
configuration and lumen lined by a single columnar or cuboidal layer of carcinoma cells. A fibrous
stroma is sometimes found. These carcinomas appear benign but feature infiltrative growth and
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occasional venous invasion. These features are not infrequently admixed with the bile ductular type,
although some cases are exclusively composed of DPM features [43].

IG type carcinoma. This type is found in the second and higher peripheral branches of the right or left
hepatic ducts, and papillary or polypoid tumors are grossly recognizable in the dilated lumen. See
information about this type in the pCCA and dCCA sections.

Rare variants. See below (C. Rare variants).

Perihilar and distal CCA (p/dCCA)

Conventional type. This type corresponds to a major flat or nodular sclerosing gross type of p/dCCA and
falls within the same category as the large bile duct type of conventional iCCA [40]. In the bile duct wall
and periductal connective tissue, this type exhibits well to moderately differentiated, proliferating
tubular adenocarcinoma cells. Occasionally poorly differentiated tubular adenocarcinoma with cord-
like or solid structures and micropapillary carcinoma are also admixed. There are variable desmo-
plastic and inflammatory reactions. Some cases are associated with marked lymphoplasmacytic
infiltration containing many IgG4-positive plasma cells [44], thus mimicking IgG4-related sclerosing
cholangitis [45,46]. The nodularesclerosing type exhibits marked cancerous thickening of the affected
bile ducts, whereas such changes are rather mild in the flat type. On the luminal surface of the affected
bile duct, they show micropapillary or flat adenocarcinoma. Perineural invasion is commonly observed,
and lymphatic and vascular invasion is variably observed. In addition, intraepithelial lateral spreading
of neoplastic cells around the main tumor is found to some extent in many cases; however, it remains
controversial whether this lesion represents carcinomatous spreading of the main tumor or a preex-
isting atypical and neoplastic biliary lesion (BilIN).

Intraductal growth type. Although most tumors of the bile ducts are predominantly conventional type,
some present an exophytic pattern within the bile ducts. This type of p/dCCA falls within the same IG
type category as iCCA. The following three categories are recognizable.
Papillary type. In the variably dilated bile duct, this type exhibits grossly visible papillary or polypoid
growth [1,4,8]. Histologically, some tumors are well to partially moderately differentiated papillary
neoplasms composed of fine fibrovascular cores covered by a cuboidal or columnar epithelial layer that
corresponds to IPNB (see below), and some display adenocarcinoma invasion foci (IPNB with an
associated invasive carcinoma). If present, the tubular components within the papillary lesion are
usually small. Invasive parts do not infrequently present focal mucinous carcinoma elements, although
ordinary tubular adenocarcinoma is common [4,8].
Some tumors comprise moderately differentiated papillary adenocarcinoma with variable amounts
of differentiated tubular and compact or solid adenocarcinoma as well as invasion into the ductal wall.
This presentation may reflect malignant progression of the above-mentioned well differentiated IPNB
within the ductal lumen; however, tumors may originate as well to moderately differentiated papillary
adenocarcinoma and mainly grow within the ductal wall [42].
Intraductal and intraepithelial spreading around the papillary lesion varies but is always
observed, although the significance of this finding remains speculative, as explained for the con-
ventional type.
Tubular type. Some intraductal polypoid tumors comprise predominantly well to moderately
tubular adenocarcinomas with occasionally admixed small amounts of papillary carcinoma. The
distinction of this type from the above-described papillary type is occasionally arbitrary. Mucin hy-
persecretion is absent, and this type appears as a polypoid lesion or cast in the slightly dilated bile duct.
A majority of these tumors might have derived from intraductal tubular neoplasm of the bile duct
(ITNB) with an associated invasive carcinoma or ITNB itself (see below) [47,48].
Superficial spreading type. This rare type of ICCA exhibits extensive spreading of intraepithelial
neoplastic biliary cells with malignant features on the luminal surface of the intrahepatic and/or
extrahepatic bile duct [40,49]. Although the affected bile ducts display varied luminal dilatation, the
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grossly visible nodular or tumor lesions are usually not identifiable or are inconspicuous along the
biliary tree. These lesions may occasionally exhibit focal invasion into the duct wall and surrounding
tissue. This type may represent an extensive spreading of BilIN3 (see below).

Rare variants
The following variants are only occasionally encountered in iCCA, pCCA, and dCCA.

1) Squamous and adenosquamous cell carcinoma: Both squamous cell and adenocarcinoma com-
ponents are mixed, isolated, or adjoining in adenosquamous cell carcinoma. Squamous carcinoma is
usually well differentiated and keratinizing. Chronic cholangitis, such as hepatolithiasis or liver
fluke infection, provides a background lesion for these variants, and this variant has also been re-
ported to occur in cases of polycystic liver.
2) Mucinous carcinoma/signet ring cell carcinoma: In this type, carcinoma cells float within a
mucinous lake. This type is usually found in the invasive areas of IPNB. Signet ring cell carcinoma is
occasionally encountered in mucinous carcinoma, but pure signet ring cell carcinoma is extremely
rare.
3) Clear cell carcinoma: This type is occasionally observed. Tubules or acini composed of columnar
epithelial cells with abundant clear cytoplasm and eccentric small nuclei are predominant. Other
adenocarcinoma histologies such as a micropapillary configuration are encountered focally in this
type.
4) Undifferentiated carcinoma: This carcinoma has a heterogeneous histology and several sub-
categories. Undifferentiated carcinoma may be observed alone or admixed with foci of conventional
CCA, suggesting transformation of conventional CCA. Coagulative necrosis is frequently seen.
Lymphatic or portal venous emboli are frequent.
i) Sarcomatous type: The spindle cell sarcoma type is common, and sarcomatous cells exhibit a
medullary growth pattern and variably express epithelial phenotypes. Adenocarcinoma or cord-
like growth is usually admixed focally. When a more mature sarcomatous component such as
osteosarcoma or angiosarcoma is found in addition to adenocarcinoma, the preferred term is
carcinosarcoma of the liver or biliary tract.
ii) Anaplastic type: One example of this type is pleomorphic carcinoma with loose cell adhesion, in
which anisocytotic carcinoma cells grow in cord or nest-like patterns. Cohesive nests without
glandular differentiation occur sporadically. Giant cells mingled with carcinoma cells. Growth
may be medullary, and no fibrous stroma is observed.
5) Lymphoepithelioma-like carcinoma: Carcinomas with features of undifferentiated tumors
and intense lymphoid stroma, classified as lymphoepithelioma-like carcinomas (LELCs), have
been reported in the liver. Like nasopharyngeal carcinomas, most LELCs are strongly linked to
EBV infection [38]. The prognosis of LELC-type CCA seems to be better than that of conventional
CCA.
6) Neuroendocrine type: Neuroendocrine neoplasms are very rare, but several cases of mixed ade-
noneuroendocrine carcinoma have been reported in pCCA and dCCA as well as in the gallbladder
[38]. In these cases, the adenocarcinoma is located at the tumor surface of the tumor and the
neuroendocrine compartment comprises the majority of stromal invasion. A reported case of IPNB
with an associated invasive carcinoma displayed foci of neuroendocrine carcinoma in the intra-
ductal papillary area [50,51].

Preneoplastic and preinvasive lesions of CCAs

The 2010 WHO classification proposed three types of preinvasive lesions of the biliary tract: flat-
type (BilIN), papillary-type (IPNB), and cystic type (MCN) (1,22). Recently, ITNB was proposed as
another candidate preinvasive lesion type [47,48]. These three types are important for the devel-
opment and progression of CCA arising from the intrahepatic large bile ducts and perihilar and
distal bile ducts, although preinvasive or preneoplastic lesions of iCCA, MF type remain to be
explored [52].
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BilIN
BilIN is usually encountered in the intrahepatic large bile ducts, perihilar and distal bile ducts, and
peribiliary glands [1,11,12,53]. At present, BilINs cannot be detected via image analysis, and diagnosis
depends entirely on histopathological examination. Compared with normal and hyperplastic biliary
epithelium, BilIN is characterized by abnormal epithelial cells with nuclear multilayering and flat or
micropapillary projections into the duct lumen (Fig. 1). The abnormal cells have an increased nuclear/
cytoplasmic ratio, partial loss of nuclear polarity, and nuclear hyperchromasia. These lesions are
divisible into BilIN-1, -2 and -3 according to cellular and nuclear atypia: BilIN-1 and BilIN-2 correspond
to low-grade and intermediate-grade lesions, respectively, and BilIN-3 corresponds to high-grade le-
sions, including noninvasive carcinoma [1,53]. S100P is frequently and extensively expressed in the
nuclei and cytoplasm of BilIN-3 and invasive carcinoma cells, and it is helpful for grading BilINs [53].
The BilIN series represents a multistep carcinogenesis process, followed by conventional type p/
dCCA and large duct type conventional iCCA. BilIN is also applicable to atypical epithelial and pre-
invasive lesions of the gallbladder [1,23].

IPNB
IPNB is characterized by dilated intrahepatic and extrahepatic bile ducts filled with papillary or
villous biliary neoplasm that covers delicate fibrovascular stalks. The dilated bile ducts are fusiform or
cystic (unilobular or multilocular; Fig. 2) [1,4,10,22]. The height of such lesions usually exceeds 5 mm.
These IPNBs occur in older patients with a male predominance, and the left hepatic lobe is somewhat
frequently affected. Synchronous and dyssynchronous IPNB can develop in the intrahepatic and
extrahepatic biliary tree. Approximately one-third of IPNBs secrete excess mucin in the duct lumen
(mucin-secreting biliary tumors) [1,4,9,10,22,54]. Four neoplastic epithelial phenotypes are known:
intestinal, pancreatobiliary, oncocytic, and gastric types.
IPNBs are classifiable as either IPNB of low- and intermediate-grade intraepithelial neoplasia or
IPNB of high-grade intraepithelial neoplasia based on the degree of cellular and nuclear atypia. The

Fig. 1. Biliary intraepithelial neoplasm (BilIN) [52]. Hematoxylineeosin staining. A: BilIN-1, B: BilIN-2, C: BilIN-3.
 Y. Nakanuma, Y. Kakuda / Best Practice & Research Clinical Gastroenterology 29 (2015) 277e293 285

Fig. 2. Intraductal papillary neoplasm of the bile duct (IPNB) with an associated invasive carcinoma. Arrow indicates focal invasion.
Inset shows a higher magnification image of the invasive lesion (arrow). Hematoxylineeosin staining.

low- or intermediate-grade category includes borderline lesions, whereas the high-grade category
includes verified “noninvasive” carcinomas with cellular/nuclear and structural atypia adequate for a
diagnosis of malignancy. IPNB is not infrequently associated with focal and minimal invasion (IPNB
with an associated invasive carcinoma). Tubular and mucinous adenocarcinoma is found in invasive
areas. IPNB includes biliary papilloma and papillomatosis, which are known to undergo malignant
transformation with invasion and metastasis.

ITNB
This type is another form of polypoid lesion with a predominantly tubular growth pattern that
occurs in the large bile ducts, including the perihilar and distal bile ducts, and is followed by invasive
CCA. ITNB has been only occasionally reported and is characterized by a cast-like growth in the dilated
duct lumen (Fig. 3) [47,48]. Entirely intraductal tumors appear to have favorable outcomes, but me-
tastases can occur when invasive carcinoma is present (ITNB with an associated invasive carcinoma).
Recently, Katabi et al reported ten cases of ITNB [48]. The intraductal portions of those tumors (eight
intrahepatic, one extrahepatic hilar, one common bile duct) were densely cellular, comprising back-to-
back tubular glands and solid sheets with minimal papillary architecture. The cells were cuboidal to
columnar with mild to moderate cytological atypia.

hbMCN
hbMCN is a cyst-forming tumor that usually displays no communication with bile ducts and
comprises cuboidal to columnar epithelium with ovarian-like subepithelial stroma. This tumor type
was previously included in the biliary cystadenoma/adenocarcinoma type for a long time [13,40,55].
This tumor type is a rare type, particularly in the eastern countries (the frequency of MCN was 7% of
IPNB) in comparison with western countries [56]. The lining epithelium is flat or papillary and variably
mucin producing, and secondary neoplastic cysts are found in the neoplastic wall. Most hbMCNs are
unilobular or multilocular and range in size from 2.5 to 28 cm [55]. This entity has been widely
recognized since the recent acceptance of an ovarian-like stroma as a diagnostic prerequisite. hbMCN
presents with either benign and borderline features (MCN with low- or intermediate-grade intra-
epithelial neoplasia) or malignant features (MCN with high-grade intraepithelial neoplasia); the
benign and borderline categories are more common, and malignant transformation (Fig. 4) occurs in
these tumors. The presence of ovarian-like stroma in the MCN wall and luminal communication with
the bile duct lumen in IPNB are recommended factors for differentiation from IPNB with cystic luminal
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Fig. 3. Intraductal tubular neoplasm of the bile duct (ITNB) [46]. A: lower magnification, B: higher magnification.

dilatation. Immunostaining to detect estrogen receptor (ER) and progesterone receptor (PgR) is useful
and mandatory for the identification of ovarian-like stroma.

Others
Precursor or preinvasive lesions of iCCA, particularly those of the MF type, are not well known.
However, hepatic adenofibroma, von Meyenburg complex, bile duct adenoma, and atypical epithelial
lesions of small bile ducts are occasionally reported as candidate preinvasive lesions of peripheral iCCA
[52]. Some of the MF type may derive from hepatocytes [57]. However, more studies are required.
Finally, the correlation between BilIN, IPNB and MCN and their advanced gross features is shown in
Fig. 5. ITNB has only recently been proposed and its advanced form remains unclarified. IPNB may be
followed by intraductal growth type of iCCA and the intraductal papillary type of p/dCCA, while BilIN
may be followed by a flat or nodular sclerosing gross type of p/d CCA and periductal infiltrating of iCCA.
MCN may lead to MCN associated with an invasive carcinoma which may progress to cystic lesion with
a surround nodular carcinomatous lesion, grossly. As for mass-forming type of iCCA, the precursor or
pre-invasive lesions remain to be clarified. As for the frequency of these precursor lesions, BilIN may be
common and IPNB less common, because flat or nodular sclerosing gross types of p/d CCA and peri-
ductal infiltrating of iCCA are common and intraductal growth types of iCCA and the intraductal
papillary type of p/dCCA are less common.

Pathologic classification on new concepts

Novel aspects of the pathologic classification of CCA, as described above, are mainly based on recent
progress in research on preinvasive CCA lesions, the relationship between iCCA and HC-CC with respect
to HPCs, and the new concept of “biliary diseases with pancreatic counterparts” [1,4,19,22].

Relationship between iCCA and HC-CC with respect to HPCs

The bile ductular type of iCCA was proposed as a new subtype of iCCA based on its morphologic
similarities to proliferating and reactive bile ductules [15,16]. Carcinoma cells of this type are also
 Y. Nakanuma, Y. Kakuda / Best Practice & Research Clinical Gastroenterology 29 (2015) 277e293 287

Fig. 4. Mucinous cystic neoplasm (MCN) of the liver. A: intermediate-degree MCN with an associated invasive carcinoma. Hema-
toxylineeosin staining (HE). B: high-grade MCN with an associated invasive carcinoma. Arrow indicates invasion. HE. C: estrogen
receptor (ER) expression on ovarian-like stromal cells. ER immunostaining.

positive for NCAM and the HPC marker EpCAM [15,16,43]. These features resemble HC-CC with stem
cell features, particularly the cholangiolocellular subtype [18]. HPCs exist in bile ductules and/or canals
of Hering and can differentiate into hepatocytes and cholangiocytes [17,18]. Primary liver tumors with
HPC features may develop, and these neoplasms may correspond to the bile ductular type of iCCA.
Interestingly, this type exhibits a rather favorable prognosis after surgical resection, in comparison with
conventional iCCA [5]. Bile ductular iCCA components have been found in small duct iCCA and vice

Fig. 5. Relation between preinvasive or precursor biliary lesions and gross types of cholangiocarcinoma (CCA). IPNB, intraductal
papillary neoplasm of bile duct; BilIN, biliary intraepithelial neoplasm; MCN, mucinous cystic neoplasm; iCCA, intrahepatic CCA; p/
d CCA, perihilar and distal CCA.
288 Y. Nakanuma, Y. Kakuda / Best Practice & Research Clinical Gastroenterology 29 (2015) 277e293

versa [15,16], suggesting that at least some conventional small duct iCCAs may derive from bile
ductular type iCCAs [15,16,22].
According to Komuta et al [18], CCAs originating from topographically heterogeneous chol-
angiocytes retain their original features. Among 51 iCCAs, 31 contained only mucin-producing CCA
features (muc-iCCAs), whereas 20 displayed histological diversity, specifically focal hepatocytic dif-
ferentiation and ductular areas reflecting the HPC phenotype (mixed-ICCs). Clinicopathologically, muc-
iCCAs and hilar CCAs were found in predominantly (peri-)hilar locations. In contrast, mixed iCCAs also
contained foci of hepatocytic differentiation and were found in predominantly peripheral locations.
The immunoreactivity of muc-iCCAs and hilar CCAs differed from that of mixed-iCCAs. S100P and
MUC1 expression were significantly upregulated in hilar CCAs and muc-iCCAs, whereas NCAM1 tended
to be upregulated in mixed-iCCAs. Bile ductular iCCAs retaining features of HPCs may be included
among mixed-iCCAs.

Relationship with pancreatic counterparts

Biliary pathology is under consideration, given the similarities between the biliary tract and
pancreas [19,21].

Biliary diseases with pancreatic counterparts. The biliary tree and pancreas are closely located anatom-
ically and share several physiological functions. Both derive almost simultaneously from the foregut,
and recent animal studies revealed that the biliary tree and pancreas exhibit plasticity to each other
during development [19,21]. Experimental studies using animal models have suggested that the biliary
tract holds some potential for pancreatic differentiation [58,59]. In humans, peribiliary glands are
located around the biliary tract and drain into the bile duct lumen via their own conduits [21]. Inter-
estingly, small amounts of pancreatic exocrine acini are intermingled with these glands, suggesting the
possibility that these glands may be abortive pancreatic exocrine acini that were prevented from
differentiating into fully exocrine pancreatic acini [58]. Recently, biliary tract stem cells were found in
the peribiliary glands and were shown to be able to differentiate into pancreatic cells as well as
cholangiocytes and hepatocytes [57]. Based on these findings, it seems plausible that the biliary tract
has pancreatic features (incomplete pancreas) in addition to a duct system specialized for the drainage
of bile secreted by the hepatic parenchyma. In this context, it is reasonable that some biliary diseases
would have similar pathological features and even biological behaviors as pancreatic diseases
(pancreatic counterparts). One example is IgG4-related sclerosing cholangitis and autoimmune
pancreatitis [21].
Collectively, we propose a hypothesis that some non-neoplastic and neoplastic biliary diseases that
exhibit similarities to corresponding pancreatic diseases could be included in a new disease concept,
“biliary diseases with pancreatic counterparts” (Table 2).

pCCA and dCCA (p/dCCA) and BilIN with respect to invasive duct carcinoma of the pancreas and Pan-
IN. Advanced CCA and preneoplastic or preinvasive neoplasms of the biliary tract show similar
morphological or genetic changes to those of their pancreatic counterparts [4,10,19,21]. Specifically,
invasive duct carcinoma of the pancreas (PDAC) and p/dCCA, including conventional iCCA of large bile

Table 2
Neoplastic biliary diseases with pancreatic counterparts.

Biliary tract Pancreas

a. Conventional cholangiocarcinoma of the perihilar Pancreatic duct adenocarcinoma

and distal bile duct
b. Biliary intraepithelial neoplasm (BilIN)
c. Intraductal papillary neoplasm of the bile duct (IPNB)
i. Involvement of bile duct
ii. Involvement of peribiliary glands
d. Intraductal tubular neoplasm of the bile duct (ITNB)
e. Mucinous cystic neoplasm
Pancreatic intraepithelial neoplasm (PanIN)
Intraductal papillary mucinous neoplasm (IPMN)
i. Main pancreatic duct type
ii. Branch pancreatic duct type
Intraductal tubulopapillary neoplasm (ITPN)
Mucinous cystic neoplasm
 Y. Nakanuma, Y. Kakuda / Best Practice & Research Clinical Gastroenterology 29 (2015) 277e293 289

duct type, share many biological and clinical features. In addition, similar intraepithelial neoplasms
have also reported as preneoplastic and preinvasive lesions in both the biliary tract (BilIN) and
pancreas (PanIN) [11,12,20]. BilIN and PanIN are both followed by conventional invasive adenocarci-
noma, suggesting similar carcinogenic processes in the biliary tract and pancreas.
p/dCCA and PDAC. PDAC and p/dCCA (including the conventional large duct type of iCCA) the most
frequent and significant malignant neoplasms of the pancreas and biliary tract, display similar
histopathological features and biological behaviors [19,21,60]. p/dCCA and PDAC commonly exhibit
an infiltrative growth pattern and nodular-sclerosing growth features without capsule formation,
and histologically are typically well to moderately differentiated tubular and/or micropapillary
adenocarcinomas with abundant desmoplastic reactions. These tumors frequently exhibit
lymphatic, vascular, and perineural invasion. Clinically, both tumors affect elderly adults and are
intractable malignancies that are often resistant to surgical and chemotherapeutic approaches. Both
tumors are frequently detected at an advanced stage and have a poor prognosis despite treatment
with these therapies. p/dCCA and PDAC patients also have similar postoperative survival courses.
Interestingly, patients with p/dCCA and PDAC are known to respond well to TS-1 and gemcitabine
chemotherapy [19,21].
p/dCCA and PDAC also have similar histochemical and immunohistochemical phenotypes [60].
For example, both carcinomas frequently express and secrete neutral and acid mucin. Immuno-
histochemically, these tumors frequently express MUC1, MUC3, and MUC5/6 but not MUC2.
Regarding their cytokeratin (CK) profiles, the normal pancreatic duct and bile duct cells express CK7
and CK19, and PDAC and p/dCCA express the same set of CKs as normal duct epithelium. Carci-
noembyronic antigen, carbohydrate antigen 19-9 and Du-Pan 2 are also frequently expressed in
both adenocarcinomas. Anterior gradient protein-2 and S100P are frequently expressed in ECC and
PDAC, whereas NCAM expression and luminal expression of epithelial membrane antigen are un-
common in these carcinomas. PDX1 and HES1 are frequently and markedly expressed in PDAC and
p/dCCA [60].
BilIN and PanIN. PanIN is known as a precursor to PDAC and exhibits microscopic intraepithelial
proliferative changes [61]. PanINs are classified morphologically into three grades: PanIN-1, PanIN-2,
and PanIN-3. PanINs, and particularly higher grade PanINs (PanIN-3), are more common in the
pancreas with PDAC. p/dCCA is also reported to develop via multi-step carcinogenesis, particularly
hepatolithiasis. Microscopic flat or low-papillary dysplastic epithelial lesions proposed to be BilIN, are
graded into three categories similar to PanIN [1,11,53]. In our previous studies, BilIN and PanIN dis-
played similar mucin core protein (MUC1 and MUC2) and CK (CK7 and CK20) expression patterns,
suggesting that similar phenotypic changes may occur in both BilIN and PanIN [62].

IPNB and IPMN. IPMN and IPNB share many features [1,10,63]. Both are composed of fine fibrovascular
cores lined with cuboidal to columnar neoplastic epithelial cells. Interestingly, the lining epithelia of
IPNB and IPMN display either one or several of four phenotypes: intestinal, gastric, pancreatobiliary,
and oncocytic epithelia. Mucin hypersecretion is frequently observed in both tumors.
Main duct IPMN and its biliary counterparts: Typically, main pancreatic duct-type IPMN is
diffusely dilated. The tortuous and irregular duct is often filled with mucin [63]. The affected IPNB bile
ducts, which display variable fusiform dilatation and occasionally cystic or aneurysmal dilatation,
resemble main pancreatic duct-type IPMN [5]. IPNB is now accepted as a counterpart of main duct type
IPMN of the pancreas [4,19,21].
Branch duct-type IPMN and its biliary counterparts: Branch duct-type IPMNs form multicystic,
grape-like structures [63]. These lesions are also characterized by the intraductal proliferation of
columnar mucin-producing cells, and the most tumors of this type are borderline or adenoma and
mainly present with a gastric phenotype [63]. Recently, IPNBs corresponding to branch duct-type IPMN
have also been reported [30,63e65], and this type is speculated to derive from the peribiliary glands.
Some cases only involve the peribiliary glands; others involve the peribiliary glands and bile ducts.
Cystic and micropapillary lesions involving the peribiliary glands present with gastric or pan-
creatobiliary phenotypes and increased cell proliferation. This type is another cystic papillary tumor of
the liver, and may correspond to the branch type of IPMN [65].
290 Y. Nakanuma, Y. Kakuda / Best Practice & Research Clinical Gastroenterology 29 (2015) 277e293

ITNB: ITNB may also correspond to a pancreatic counterpart, intraductal tubular neoplasm of the
pancreas (adenoma or carcinoma) [47,48]. ITNB is an intraductal neoplasm of the bile duct that exhibits
a predominantly tubular growth pattern resembling the recently described intraductal tubulopapillary
neoplasm of the pancreas. Phenotypically, ITNB is either the pancreatobiliary-type or gastric type. ITNB
may also correspond to a pancreatic counterpart, intraductal tubulopapillary neoplasm of the pancreas
(adenoma or carcinoma). However, analyses of ITNB, particularly those involving genetic data are
inadequate.

hbMCN and pancreatic mucinous cystic neoplasm. Pancreatic mucinous cystic neoplasm (pMCN) is char-
acterized by an “ovarian-type” stroma and a lining epithelium comprising single-layered, columnar,
mucin-producing cells [14,66]. The cysts are lined with mucin-positive columnar epithelial cells.
Morphologically, hbMCN is nearly identical to pMCN [13,56,66]. Stromal cells with nuclear ER or PgR
expression were consistently observed in both hbMCN and pMCN. Patients with hbMCN and pMCN are
relatively young and predominantly female. The clinical, histological, and biological behaviors of hbMCN
are similar to those of pancreatic MCN. Collectively, hbMCNs are a biliary counterpart of pMCN.
Serous neoplasms in the liver, similar to those observed in the pancreas, have also occasionally been
reported [31]. These neoplasms may also represent biliary diseases with pancreatic counterparts.

Summary

Based on recent progress in CCA pathological research that includes preinvasive lesions, the simi-
larities between biliary and pancreatic neoplasms, and the relationship between iCCA and HC-CC, CCA
has been pathologically classified as iCCA, pCCA, and dCCA. Histologically, iCCA has been classified as a
conventional (bile duct) type, bile ductular type, and intraductal type along with rare variants, and p/
d CCA has been classified as a conventional and IG type. This classification may introduce a novel
approach to research and clinical practice related to intrahepatic biliary neoplasms.

Conflict of interest

The authors have no conflict of interest to declare.

Practice points

 CCA is classified according to its anatomical location

 CCA is classified as conventional type and intraductal growth type in addition to rare variants
 Intrahepatic CCA (ductular type) has features of hepatic progenitor cells
 Several preinvasive lesions are proposed: flat type, papillary type, tubular type, and cystic
type
 CCA has pancreatic counterparts

Research agenda

 CCA carcinogenesis requires clarification according to the anatomical classification

 Early detection of flat type preinvasive lesions (BilIN) is needed
 Comprehensive classification of hepatic carcinoma with features of hepatic progenitor cells
is needed
 Comprehensive analysis of biliary neoplasm with reference to pancreatic counterparts is
needed
 Y. Nakanuma, Y. Kakuda / Best Practice & Research Clinical Gastroenterology 29 (2015) 277e293 291

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