Different expression patterns of mucin c

Different expression patterns of mucin core proteins and cytokeratinsduring intrahepatic cholangiocarcinogenesis from biliary intraepithelial neoplasia and intraductal papillary neoplasm

Different expression patterns of mucin core proteins and cytokeratins

during intrahepatic cholangiocarcinogenesis from biliary

intraepithelial neoplasia and intraductal papillary neoplasm

of the bile duct—an immunohistochemical study

of 110 cases of hepatolithiasis

Yoh Zen1,2, Motoko Sasaki1, Takahiko Fujii1, Tse-Ching Chen3, Miin-Fu Chen4, Ta-Sen Yeh4,

Yi-Yin Jan4, Shiu-Feng Huang4, Yuji Nimura5, Yasuni Nakanuma1,*

1

Department of Human Pathology, Kanazawa University Graduate School of Medicine, Kanazawa 920-8640, Japan

2

Division of Pathology, Kanazawa University Hospital, Kanazawa, Japan

3

Department of Pathology, Chang Gung Memorial Hospital, Lin Kou, Taiwan ROC

4

Department of Surgery, Chang Gung Memorial Hospital, Lin Kou, Taiwan ROC

5

Division of Surgical Oncology, Department of Surgery, Nagoya University Graduate School of Medicine, Nagoya, Japan

See Editorial, pages 249–250

Background/Aims: Two types of neoplastic lesions preceding invasive intrahepatic cholangiocarcinoma (ICC) are identified: a flat-type neoplastic lesion called ‘biliary intraepithelial neoplasia (BilIN)’ and an intraductal papillary neoplasm of the bile duct (IPN-B) Multi-step carcinogenesis has been suggested in both lesions, although phenotypic changes during this process remain unclarified.

Methods: We immunohistochemically examined expression patterns of MUC1, MUC2, MUC5AC, cytokeratin 7 (CK7), and CK20 in BilIN, IPN-B, and ICC in 110 cases of hepatolithiasis.

Results: Thirty-seven cases of ICC in hepatolithiasis were divided into 18 tubular adenocarcinomas with BilIN, 10 tubular adenocarcinomas with IPN-B and nine colloid carcinomas with IPN-B Carcinogenesis via BilIN was characterized by MUC2K/CK7C/CK20Kwith increasing MUC1 expression IPN-B was characterized by the intestinal phenotype (MUC2C/CK20C), and carcinogenesis leading to tubular adenocarcinoma was associated with increasing MUC1 expression and that to colloid carcinoma with MUC1-negativity Pathological stages of tubular adenocarcinoma of ICC with BilIN or IPN-B were more advanced than those of colloid carcinoma with IPN-B Conclusions: Immunophenotypes of MUCs and cytokeratins might characterize three cholangiocarcinogenetic pathways in hepatolithiasis Increased expression of MUC1 in BilIN and also IPN-B is associated with tubular adenocarcinoma, while colloid carcinoma in IPN-B is characterized by MUC1-negativity and less advanced pathologic stages.

q 2005 European Association for the Study of the Liver Published by Elsevier B.V All rights reserved.

Keywords: Intrahepatic cholangiocarcinoma; Dysplasia; Colloid carcinoma; Biliary papillomatosis; PanIN

www.elsevier.com/locate/jhep

0168-8278/$30.00 q 2005 European Association for the Study of the Liver Published by Elsevier B.V All rights reserved

doi:10.1016/j.jhep.2005.09.025

Received 5 June 2005; received in revised form 29 August 2005; accepted 5 September 2005; available online 5 December 2005

* Corresponding author Tel.: C81 76 265 2195; fax: C81 76 234 4229

E-mail address: pbcpsc@kenroku.kanazawa-u.ac.jp (Y Nakanuma)

Abbreviations: BilIN, biliary intraepithelial neoplasia; CK, cytokeratin; ICC, intrahepatic cholangiocarcinoma; IPMN-P, intraductal papillary mucinous neoplasm of the pancreas; IPN-B, intraductal papillary neoplasm of the bile duct; MUC, mucin core protein; PanIN, pancreatic intraepithelial neoplasia

1 Introduction

Intrahepatic cholangiocarcinoma (ICC) is the second

most common malignant tumor of the liver [1,2] Once ICC

shows invasive growth, it aggressively invades the

surrounding tissue and is commonly associated with distant

metastasis, finally resulting in a poor prognosis [2–4] Two

distinct neoplastic lesions preceding invasive ICC have

been identified so far [5] The first neoplastic lesion is a flat

or micropapillary growth of atypical biliary epithelium,

which has been called ‘biliary dysplasia’ [4] ‘Biliary

intraepithelial neoplasia (BilIN)’ was used for these lesions

as one of the precursor lesions of ICC in World Health

Organization’s classification of tumors [6] The other is an

intraductal papillary neoplasm of the bile duct (IPN-B) with

malignant potential, which is histologically characterized by

the prominent papillary growth of atypical biliary

epi-thelium with distinct fibrovascular cores and frequent mucin

over-production [7,8]

BilIN and IPN-B share pathologic characteristics [4–8] :

they usually occur in the large-sized intrahepatic bile ducts

and are rarely present in the septal or interlobular bile ducts.

In addition, both BilIN and IPN-B more frequently arise

during chronic inflammatory biliary diseases, such as

hepatolithiasis, primary sclerosing cholangitis, and

infesta-tion by liver flukes [6–9] Among them, hepatolithiasis is

unique in that both premalignant lesions (BilIN or IPN-B)

can occur, whereas primary sclerosing cholangitis and

parasitic infections are usually associated with only BilIN

and not IPN-B [7] On the other hand, BilIN and IPN-B have

different pathologic characteristics BilIN can be identified

only microscopically (a microscopic lesion), whereas IPN-B

is identifiable on radiologic images or through macroscopic

examination [6,7] When accompanied by invasive lesions,

BilIN is known to progresses to conventional ICC (tubular

adenocarcinoma), whereas IPN-B is associated with colloid

carcinoma (mucinous carcinoma) in addition to

conven-tional ICC [7,8,10]

Multi-step carcinogenesis has been suggested in ICCs

arising from BilIN and IPN-B [4,8] However, little is

known about the phenotypic or genetic changes during their

carcinogenetic pathways, because only a few studies using

both BilIN and IPN-B have been conducted In this study,

we immunohistologically examined the phenotypic changes

of mucin core proteins (MUCs) and cytokeratins (CKs)

during cholangiocarcinogenesis from BilIN and IPN-B in

110 hepatolithiasis cases.

2 Materials and methods

2.1 Case selection and classification of biliary lesions

A total of 110 hepatolithiasis cases were obtained from the liver disease

file of Department of Human Pathology, Kanazawa University Graduate

School of Medicine, and that of Department of Pathology, Chang Gung

Memorial Hospital (Table 1) All were surgically-resected cases (65 cases, left lobectomy; 39 cases, right lobectomy; and six cases, segmentectomy All biliary lesions examined were found in the intrahepatic large bile ducts, which correspond to the first to third branches of the right and left hepatic ducts [11] The biliary epithelial lesions in hepatolithiasis were classifiable into five categories (Table 1); non-neoplastic biliary epithelium, BilIN, ICC with BilIN, IPN-B, and ICC with IPN-B BilIN was histologically defined as a flat or micropapillary proliferation of atypical biliary epithelium showing multilayering, piled-up nuclei, an increased nucleocytoplasmic ratio, a partial loss of nuclear polarity, and nuclear hyperchromasia and pleomorphism In contrast, IPN-B was microscopi-cally defined as a prominent papillary proliferation of atypical biliary epithelium with distinct fibrovascular cores, showing nuclear stratification, piled-up nuclei, and nuclear enlargement In this study, one lesion representative of these categories was chosen from each case of hepatolithiasis: non-neoplastic biliary epithelium (regenerative and hyperplastic changes) was chosen in 10 cases of hepatolithiasis, BilINs in

38 cases, ICC with BilIN in 18 cases, IPN-Bs in 25 cases, and ICC with IPN-B in 19 cases

Next, BilINs and IPN-Bs were graded Based on previously reported criteria, BilINs were classified into three grades: BilIN-1 (corresponding to low-grade dysplasia), BilIN-2 (high-grade dysplasia), and BilIN-3 (carcinoma in situ) [12] Briefly, BilIN-1 showed mild cellular/nuclear atypia such as nuclear membrane irregularity or nuclear enlargement with only a minimal disturbance of cellular polarity BilIN-2 had evident cellular/nuclear atypia, but not enough to suggest overt carcinoma, with a focal disturbance of cellular polarity BilIN-3 showed a diffuse disturbance

of cellular polarity with or without distinct cellular/nuclear atypia corresponding to an overt carcinoma In this study, BilIN-2 (chosen from

17 cases of hepatolithiasis) and BilIN-3 (from nine cases) were grouped together as high-grade BilIN (BilIN-2/3) Eighteen cases of ICC with BilIN were all histologically tubular adenocarcinoma (eight cases, well differentiated; six cases, moderately differentiated; and four cases, poorly differentiated)

Because there are no well-established criteria for grading IPN-B, we graded IPN-B in consideration of World Health Organization’s criteria for intraductal papillary mucinous neoplasm of the pancreas (IPMN-P)[13] IPN-Bs were classified into two subgroups: IPN-B1 (corresponding to benign and borderline lesions of IPMN-P) (chosen from 12 cases of hepatolithiasis) and IPN-B2 (corresponding to carcinoma in situ) (from 13 cases) One IPN-B2 histologically consisted of oncocytic tumor cells (oncocytic type) Out of 19 ICCs with IPN-B, nine were histologically colloid carcinomas, whereas nine were tubular adenocarcinomas (eight cases, well differentiated; and one case, poorly differentiated) The remaining case was oncocytic papillary adenocarcinoma with invasion BilIN-1, BilIN-2/3 and ICC with BilIN were regarded as BilIN lineage, while IPN-B1, IPN-B2 and ICC with IPN-B were regarded as IPN-B lineage

Table 1 Cases analyzed in this study and their clinical features

Number Average age

(range)

Male: female Non-neoplastic

epithelium

10 49.1 (40–60) 6:4 BilIN (total) 38 56.8 (36–83) 17:21

ICC with BilIN 18 61.0 (43–83) 8:10 IPN-B (total) 25 55.7 (35–66) 9:16

ICC with IPN-B 19 59.8 (46–78) 6:13 BilIN, biliary intraepithelial neoplasia; BilIN-1, low-grade BilIN; BilIN-2/

3, high-grade BilIN; IPN-B, intraductal papillary neoplasm of the bile duct; IPN-B, intraductal papillary neoplasm of the bile duct; IPN-B1, low-grade IPN-B corresponding to benign and borderline lesions; IPN-B2, high-grade IPN-B corresponding to carcinoma in situ; ICC, intrahepatic cholangio-carcinoma

2.2 Immunohistochemistry

Immunostainings of MUC1, MUC2, MUC5AC, CK7, and CK20 were

done using the EnVisionC system (Dako Cytomation, Glostrup, Denmark)

Deparaffinized sections used for MUC2, MUC5AC, CK7, and CK20 were

microwaved in 10 mM citrate buffer (pH 6.0) for 20 min After blocking of

endogenous peroxidase and incubation in normal goat serum (1:10; Vector

Laboratories, Burlingame, CA) for 20 min, the deparaffinized sections were

incubated overnight at 4 8C with primary monoclonal antibodies:

anti-MUC1 (clone DF3; 1:100; Toray Fuji Bionics, Tokyo, Japan), anti-MUC2

(clone Ccp58; 1:100; Novocastra Laboratories, Newcastle, UK),

anti-MUC5AC (clone CLH2; 1:100; Novocastra Laboratories), anti-CK7 (clone

OV-TL 12/30; 1:50; Dako Cytomation), and anti-CK20 (clone Ks20.8; 1:50

dilution; Dako Cytomation) The sections were then incubated at room

temperature for 1 h with goat anti-mouse immunoglobulins conjugated to

peroxidase labeled-dextran polymer (EnVisionC; Dako Cytomation) The

reaction products were developed by immersing the section in a 3,30

-diaminobenzidine tetrahydrochloride (DAB) solution containing 0.03%

hydrogen peroxide Nuclei were lightly counterstained with hematoxylin

Expression of MUC1, MUC2, MUC5AC, CK7, and CK20 was

evaluated semiquantitatively into four scores according to the percentage

of positive cells in the individual lesion:—(negative), 0%; 1C (focal), 1%

to 10%; 2C (moderate), 11–50%; 3C (extensive), more than 50%

2.3 Macroscopic growth patterns and pathological stages

of ICC

The growth pattern of ICC was grossly classified as mass-forming,

periductal infiltrating, or intraductal growth [6,14] In addition, colloid

carcinoma was identifiable grossly and histologically Based on TNM

classification[15], ICC cases were classified into five pathological stages;

stage I, T1N0M0; stage II, T2N0M0; stage III, T3N0M0; stage IVA, T4N0M0; and stage IVB, any T with N1 or M1

2.4 Statistical analysis

The Mann–Whitney U test was employed with a significance level of P!0.05

3 Results

3.1 Pathological characteristics

Tumor sizes, macroscopic growth patterns, and patho-logical stages of BilIN, IPN-B, and ICC cases are shown in

Table 2 BilIN was identifiable microscopically, whereas IPN-B showed macroscopic papillary lesions in the saccular

or cystically dilated bile ducts All colloid carcinomas with IPN-B characteristically showed muconodular lesions in contrast to the mass-forming or periductal growth of tubular adenocarcinoma with BilIN or IPN-B No cases of colloid carcinoma showed lymph node or distant metastasis As for pathological stage, colloid carcinomas (with IPN-B) were less advanced than tubular adenocarcinomas with BilIN or IPN-B (P!0.05) ( Table 2 ) The pathological stages of tubular adenocarcinoma with BilIN did not differ from those

of tubular adenocarcinoma with IPN-B.

Table 2

Tumor sizes, macroscopic growth types, and pathological stages of biliary intraepithelial neoplasia, intraductal papillary neoplasm of the bile duct, and intrahepatic cholangiocarcinoma

Number Size (range) (cm) Macroscopic growth type (number of

cases)

Pathological stagea

(I/II/III/IVA/IVB)

ICC with BilIN 18 2.6 (1.0–4.3) Nodular (14), periductal growth (4) 3/6/3/1/5

ICC with IPN-B

Tubular adenocarcinomab 10 3.1 (1.7–4.5) Nodular (8), periductal growth (2) 1/3/3/1/2

BilIN, BilIN-1, BilIN-2/3, IPN-B, IPN-B1, IPN-B2, and ICC, same as footnote ofTable 1; NA, not-analyzed for non-invasive lesions;

a According to the TNM classification (Ref.[15])

b

Including one case of oncocytic papillary adenocarcinoma

c

P!0.05 vs ICC with BilIN and tubular adenocarcinoma (ICC with IPN-B)

Table 3

Incidence of gastrointestinal metaplasia (goblet cell metaplasia, Paneth cell metaplasia, foveolar metaplasia, and pseudopyloric gland metaplasia) in non-neoplastic and neoplastic biliary epithelial lesions

Number Goblet cell

metaplasiaa

Paneth cell metaplasia

Foveolar metaplasiab

Pseudopyloric gland metaplasia

BilIN, BilIN-1, BilIN-2/3, IPN-B, IPN-B1, IPN-B2, ICC, same as footnote ofTable 1

a

P!0.005 in IPN-B lineage (IPN-Bs and ICC with IPN-B) vs non-neoplastic epithelium or BilIN lineage (BilINs and ICC with BilIN)

b

P!0.05 in BilIN lineage or IPN-B lineage vs non-neoplastic epithelium

3.2 Gastrointestinal metaplasia

As shown in Table 3 , intestinal metaplasia (goblet cell

metaplasia or Paneth cell metaplasia) was more frequent in

cases of IPN-B lineage than BilIN lineage or non-neoplastic

epithelium Gastric metaplasia (foveolar or pseudopyloric

gland metaplasia) was similarly observed in BilIN and

IPN-B lineages.

3.3 MUC1, MUC2, and MUC5AC expression

As shown in Figs 1 and 2, MUC1 was expressed mainly

in the apical membrane and occasionally in the cytoplasm of

non-neoplastic and neoplastic biliary epithelial cells The

latter expression was more frequent in invasive ICC MUC1

was more frequently expressed in BilINs (26%), especially

in high-grade lesions, compared to non-neoplastic biliary

epithelium (0%) and IPN-Bs (4%) ( Figs 1–3 ) In all ICC

cases with BilIN, MUC1 was expressed in more than 50% of

tumor cells, whereas 47% of ICC cases with IPN-B were

negative for MUC1 (P!0.001).

MUC2 was mostly expressed in the cytoplasm of biliary

epithelial cells In four cases of non-neoplastic biliary

epithelium (40%), MUC2 was expressed focally MUC2

was more commonly expressed in IPN-Bs (88%) and ICC

with IPN-B (89%) compared to BilINs (21%) and ICC with

BilIN (17%), respectively (P!0.001) ( Figs 1–3 )

Further-more, in most cases of IPN-Bs (64%) and of ICC with

IPN-B (58%), MUC2 was expressed in more than 50% of

the tumor cells.

MUC5AC was expressed in the cytoplasm and apical

membrane of biliary epithelial cells MUC5AC was

expressed in only four cases of non-neoplastic epithelium

(40%), whereas MUC5AC expression was more frequently

observed in BilINs (89%), ICC with BilIN (83%), IPN-Bs

(96%), and ICC with IPN-B (100%) (P!0.001) ( Figs 1–3 ).

No difference in MUC5AC expression was observed

between BilIN and IPN-B lineages.

3.4 Combination of MUC1 and MUC2 expression

We evaluated the combined expression patterns of

MUC1 and MUC2 in each lesion ( Fig 4 ) Non-neoplastic

biliary epithelium showed the MUC1K/MUC2K (60%) or

MUC1K/MUC2C pattern (40%) The most common

immunophenotype in BilINs was MUC1K/MUC2K

(63%) Frequency and degree of the MUC1C/MUC2K

pattern increased step-wise in the BilIN lineage along with

histological grade (BilIN-1, BilIN-2/3 and ICC with BilIN).

In 15 cases of ICC with BilIN (MUC1C/MUC2K), BilIN

around ICC showed the MUC1K/MUC2K (11 cases) or

MUC1C/MUC2K pattern (four cases) ( Fig 5 (A) and (B)).

Similarly, in three cases of ICC with BilIN (MUC1C/

MUC2C), BilIN around ICC showed the MUC1K/

MUC2C(two case) or MUC1C/MUC2C patterns (one

case).

The most common immunophenotype in IPN-Bs was the MUC1K/MUC2C pattern (84%) ( Fig 3 ) In ICC with IPN-B, 17 out of 19 cases could be classified into two immunophenotypes: the MUC1K/MUC2C(47%) or MUC1C/MUC2C pattern (42%) Out of nine colloid carcinomas, eight (89%) showed the MUC1K/MUC2C pattern ( Fig 5 (C) and (D)), while in the remaining case (11%), MUC1 was focally expressed in addition to MUC2 (MUC1C/MUC2C) Tubular adenocarcinomas with

IPN-B showed the MUC1C/MUC2C(seven cases, 70%),

MUC1*

MUC2†

MUC5AC‡

Case ratio (%)

11-50% 1-10% negative 50%<

Non-neoplastic BilIN-1 BilIN-2/3 ICC with BilIN IPN-B1 IPN-B2 ICC with IPN-B

Non-neoplastic BilIN-1 BilIN-2/3 ICC with BilIN IPN-B1 IPN-B2 ICC with IPN-B

Non-neoplastic BilIN-1 BilIN-2/3 ICC with BilIN IPN-B1 IPN-B2 ICC with IPN-B

Fig 1 MUC1, MUC2, and MUC5AC expression in non-neoplastic and neoplastic biliary epithelial lesions MUC1 expression is frequently observed in intrahepatic cholangiocarcinoma (ICC), especially ICC with biliary intraepithelial neoplasm (BilIN), compared to non-neoplastic and non-invasive lesions MUC2 is expressed in most cases

of intraductal papillary neoplasm of the bile duct (IPN-B) MUC5AC expression is observed in most cases of BilIN and IPN-B *, P!0.001 in ICC with BilIN vs BilINs, ICC with IPN-B vs IPN-Bs, and ICC with IPN-B vs ICC with BilIN; †, P!0.001 in IPN-B lineage (IPN-Bs and ICC with IPN-B) vs non-neoplastic epithelium or BilIN lineage (BilINs and ICC with BilIN); ‡, P!0.001 in BilIN lineage or IPN-B lineage vs non-neoplastic epithelium

MUC1C/MUC2K (two cases, 20%), and MUC1K/

MUC2C pattern (one case, 10%) Two cases of

oncocy-tic-type IPN-B (IPN-B2 and ICC with IPN-B) showed the

MUC1C/MUC2C pattern, although MUC2 expression

was only focal.

3.5 CK7 and CK20 expressions

As shown in Fig 6 , in all cases examined except one

(IPN-B1), CK7 was expressed regardless of the degree of

atypia ( Figs 2, 3 and 6 ) In most cases examined in this

study (98%), CK7 was expressed in more than 10% of the

tumor cells, whereas one case of colloid carcinoma (IPN-B

with ICC) showed only a focal expression of CK7 CK20

expression was more commonly observed in IPN-Bs (84%)

and ICC with IPN-B (74%) compared to non-neoplastic

epithelium (10%), BilINs (16%), and ICC with BilIN (17%)

(P!0.001) ( Figs 2, 3 and 6 ).

3.6 Combination of CK7 and CK20 expression

As shown in Fig 7 , in all cases except one (IPN-B1), the

immunophenotype could be classified as the CK7C/

CK20K or CK7C/CK20C The former was common in

BilINs (84%) and ICC with BilIN (83%) ( Fig 2 ), whereas the latter was common in Bs (80%) and ICC with

IPN-B (74%) ( Figs 2 and 3 ) Among 37 ICC cases, the combined immunophenotype of CK7 and CK20 (CK7C/CK20K or

Fig 2 MUC1, MUC2, MUC5AC, CK7, and CK20 expression in a case

of biliary intraepithelial neoplasia (BilIN-2) BilIN-2 is observed on the

epithelium of the intrahepatic large bile ducts with intra-epithelial

extension into peribiliary glands (glandular involvement: arrow) (HE)

MUC1 (arrows), MUC5AC, and CK7 are expressed in tumor cells,

whereas MUC2 and CK20 are not (Original magnification !100)

Fig 3 MUC1, MUC2, MUC5AC, CK7, and CK20 expression in a case

of intraductal papillary neoplasm of the bile duct (IPN-B2) IPN-B2 shows prominent papillary proliferation with mucin hypersecretion (HE) MUC2, MUC5AC, CK7 (arrow), and CK20 (arrows) are expressed in tumor cells, whereas MUC1 is not (Original magnification

!100)

MUC1/MUC2

MUC1-/MUC2+

MUC1-/MUC2-MUC1+/MUC2+

Case ratio (%)

Non-neoplastic BilIN-1 BilIN-2/3 ICC with BilIN IPN-B1 IPN-B2 ICC with IPN-B

Fig 4 Expression patterns of MUC1/MUC2 in non-neoplastic and neoplastic biliary epithelial lesions In the biliary intraepithelial neoplasm (BilIN) lineage, the most common immunophenotype changes from MUC1K/MUC2K to MUC1C/MUC2K with histologi-cal-grade progression In the intraductal papillary neoplasm of the bile duct (IPN-B) lineage, MUC1K/MUC2C is the most common pattern

in IPN-B, and ICC with IPN-B cases can be divided into two major groups: MUC1C/MUC2C or MUC1K/MUC2C

CK7C/CK20C) was the same in invasive ICC lesions and non-invasive BilIN and IPN-B in 36 cases, whereas the remaining case of ICC with BilIN (CK7C/CK20C) showed a different expression pattern (CK7C/CK20K) in BilIN around ICC Out of nine cases of IPN-B with colloid

Fig 5 MUC1 and MUC2 expression in ICC with biliary intraepithelial

neoplasm (BilIN) or intraductal papillary neoplasm of the bile duct

(IPN-B) (A) ICC with BilIN consists of a non-invasive lesion (arrow)

and poorly differentiated tubular adenocarcinoma (B) MUC1 is

expressed in the invasive lesion, but not in the non-invasive lesion

(C) ICC with IPN-B consists of the intraductal papillary proliferation

of tumor cells (arrows) and an invasive colloid carcinoma (*) (D)

MUC2 is expressed in both non-invasive and invasive lesions (A: HE

staining, B: MUC1, C: HE staining, D: MUC2, original magnification

!100 [A,B] and !40 [C,D])

Non-neoplastic BilIN-1 BilIN-2/3 ICC with BilIN IPN-B1 IPN-B2 ICC with IPN-B

CK7/CK20

Case ratio (%)

CK7-/CK20+

CK7+/CK20- CK7-/CK20-CK7+/CK20+

Fig 7 Expression patterns of CK7/CK20 in non-neoplastic and neoplastic biliary epithelial lesions CK7C/CK20K is the most common pattern in non-neoplastic biliary epithelium and the biliary intraepithelial neoplasm (BilIN) lineage, whereas CK7C/CK20C is the most common pattern in the intraductal papillary neoplasm of the bile duct (IPN-B) lineage

Fig 8 Histopathology of a case of intraductal papillary neoplasm of the bile duct (IPN-B1) with a unique immunophenotype (MUC1K /MUC2C/MUC5ACK/CK7K/CK20C) (Original magnification

!100)

CK7

CK20*

Case ratio (%)

Non-neoplastic

BilIN-1

BilIN-2/3

ICC with BilIN

IPN-B1

IPN-B2

ICC with IPN-B

Non-neoplastic

BilIN-1

BilIN-2/3

ICC with BilIN

IPN-B1

IPN-B2

ICC with IPN-B

11-50% 1-10% negative 50%<

Fig 6 CK7 and CK20 expression in non-neoplastic and neoplastic biliary

epithelial lesions All the cases examined except one (low-grade

intraductal papillary neoplasm of the bile duct [IPN-B1]) show CK7

expression CK20 expression is more commonly observed in the IPN-B

lineage *, P!0.001 in the IPN-B lineage (IPN-Bs and ICC with IPN-B) vs

non-neoplastic epithelium or the biliary intraepithelial neoplasm (BilIN)

lineage (BilINs and intrahepatic cholangiocarcinoma (ICC) with BilIN)

carcinoma, seven (78%) had the CK7C/CK20C pattern,

and two (22%) had the CK7C/CK20K pattern Two cases

of oncocytic-type IPN-B (IPN-B2 and ICC with IPN-B)

showed the CK7C/CK20K pattern.

Interestingly, one IPN-B1 showed the CK7K/CK20C

pattern, which was unique among all the cases examined in

this study In addition, this case showed a marked

expression of MUC2 but not MUC1 or MUC5AC That is,

this case was characterized by the immunophenotypes of

CK7(K), CK20(3C), MUC1(K), MUC2(3C), and

MUC5AC(K), which corresponded to the complete

intestinal immunophenotype (CK20C/MUC2C) without

biliary (CK7) or gastric immunophenotypes (MUC5AC).

Indeed, this case histologically resembled a tubulo-villous

adenoma of the intestine ( Fig 8 ).

4 Discussion

It was found in this study that expression pattern of

MUCs and CKs in neoplastic biliary epithelia differed

according to the pathway of cholangiocarcinogenesis, via

BilIN or IPN-B lineage BilIN was associated with tubular

adenocarcinoma, and this pathway was characterized by

MUC2K/CK7C/CK20K with an increased expression of

MUC1 along with the disease’s progression This

immuno-phenotype is characterized by the biliary type (CK7),

however, MUC2 and CK20 reflecting the intestinal type

were rarely expressed in this lineage.

IPN-B was frequently associated with colloid carcinoma

(47%) compared to BilIN as reported previously [7] , though

the remaining ICCs with IPN-B (53%) were tubular

adenocarcinomas During the carcinogenesis from IPN-B,

most of non-invasive neoplastic lesions (85%) were

MUC1K/MUC2C When invasive ICC developed, seven

(70%) of 10 tubular adenocarcinomas with IPN-B were

MUC1-positive (MUC1C/MUC2C), whereas eight (89%)

of nine colloid carcinomas remained MUC1-negative

(MUC1K/MUC2C) That is, MUC1 expression was

frequently associated with the development of tubular

adenocarcinoma, while colloid carcinoma was associated

with negativity for MUC1 As for CKs, the CK7C/CK20C

pattern was almost always observed in the IPN-B-pathway

either to tubular adenocarcinoma or colloid type These

immunophenotypes suggest that tumor cells during this

carcinogenesis retain both biliary (CK7) and intestinal

immunophenotypes (MUC2 and CK20).

In addition, MUC5AC expression (gastric phenotype)

was frequent in both BilIN and IPN-B lineages in which

foveolar (gastric) metaplasia was frequent Goblet cell

(intestinal) metaplasia was frequently observed in IPN-B

lineage with frequent MUC2 expression (intestinal

phenotype), whereas intestinal metaplasia was not

frequent in BilIN lineage with infrequent MUC2

expression These findings were compatible with the

common occurrence of gastric metaplasia in both

lineages, while intestinal metaplasia was more frequent

in IPN-B lineage.

Adsay et al [16] performed a similar immunopathologic study on MUC1 and MUC2 expression in pancreatic intraepithelial neoplasia (PanIN), IPMN-P, pancreatic ductal adenocarcinoma, and colloid carcinoma They proposed two distinct carcinogenetic pathways: PanIN to ductal adenocarcinoma (MUC1-positive pathway) and IPMN-P to colloid carcinoma (MUC2-positive pathway)

[16] These immunophenotypic changes during pancreatic carcinogenesis via PanIN and IPMN-P resemble those during cholangiocarcinogenesis from BilIN and IPN-B, respectively However, there are minor differences between them That is, MUC2 expression was observed in BilIN and ICC with BilIN (21 and 17%) but there was little expression

of MUC2 in PanIN [16,17] MUC2 expression in BilIN might reflect a preceding condition (hepatolithiasis), in that MUC2 expression in the intrahepatic large bile duct is one

of the key events in the formation of stones [18,19] This metaplastic change related to hepatolithiasis may be at least partly responsible for the development of BilIN Apart from this minor difference, the similarity of immunophenotype between BilIN and PanIN and between IPN-B and IPMN-P support the theory that BilIN and IPN-B are biliary counterparts of PanIN and IPMN-P, respectively.

This study suggested three cholangiocarcinogenetic pathways from BilIN and IPN-B to ICC: BilIN progressing

to tubular adenocarcinoma, IPN-B to tubular adenocarci-noma, and IPN-B to colloid carcinoma All of the ICCs associated with BilIN were tubular adenocarcinomas, whereas ICC with IPN-B was either tubular adenocarci-noma or colloid carciadenocarci-noma Interestingly, the pathological stages of colloid carcinoma with IPN-B were less advanced than those of tubular adenocarcinoma with BilIN or IPN-B Recently, Shibahara et al [20] examined mucin-producing bile duct tumors, which were closely related to IPN-B in this study, and reported that patients with tubular adenocarci-noma had a significantly lower rate of survival than those with mucinous (colloid) carcinoma, and that MUC1 expression was closely related to a poor prognosis The present study also showed that increasing levels of MUC1 in BilIN and also IPN-B suggest the development of tubular adenocarcinoma, while MUC1-negativity suggests the development of colloid carcinoma Taken together, colloid carcinoma with MUC1 negativity may have a better prognosis than tubular adenocarcinoma with BilIN or IPN-B which shows an increase in the expression of MUC1 IPMN-P is classified histopathologically into three types

[21–26] ; MUC1K/MUC2C type characterized by dark columnar cells, MUC1K/MUC2K type characterized by clear cells containing abundant intracytoplasmic mucin, and MUC1C/MUC2G type characterized by papillary pro-liferation of compact or oncocytic tumor cells Interestingly, the IPN-B cases in this study could be classified into similar groups based on the MUC1/MUC2 immunophenotypes Out

of 25 cases of IPN-B, 21 and three cases corresponded to the

MUC1K/MUC2C and MUC1K/MUC2K types,

respect-ively The remaining case (oncocytic type) showed mainly

MUC1 expression with only a focal expression of MUC2,

which could correspond to the MUC1C/MUC2G type of

IPMN-P More studies are mandatory to clarify the

clinicopathologic characteristics of IPN-B with different

immunophenotypes.

In this study, we encountered a case of IPN-B with

a unique immunophenotype (MUC1K/MUC2C/

MUC5ACK/CK7K/CK20C) ( Fig 8 ) Histologically,

this tumor closely resembled an intestinal adenoma In

addition to the unusual expression pattern of CK7K/

CK20C, the tumor cells did not express MUC5AC, which

was expressed in all IPN-B cases with or without ICC This

immunophenotype suggested a complete loss of the biliary

immunophenotype and the acquisition of the intestinal, not

gastric, immunophenotype during tumorigenesis However,

a study of similar cases is essential whether this type

belongs to a different lineage of IPN-B or not.

In conclusion, this study revealed three different

expression patterns for MUCs and cytokeratins in neoplastic

biliary epithelia of BilIN and IPN-B with progression to

ICC That is, carcinogenesis via BilIN leading to tubular

adenocarcinoma was characterized by MUC2K/CK7C/

CK20K with an increase in expression of MUC1 IPN-B

was characterized by the intestinal phenotype (MUC2C/

CK7C/CK20C), and carcinogenesis to tubular

adenocar-cinoma was associated with enhanced expression of MUC1

and carcinogenesis to colloid carcinoma with negativity for

MUC1 The pathological stages of colloid carcinoma with

IPN-B were less advanced than those of tubular

adeno-carcinoma of ICC with BilIN or IPN-B Immunophenotypes

of MUCs and cytokeratins might characterize three

cholangiocarcinogenetic pathways in hepatolithiasis

Identi-fication of these pathways with the help of

immunopheno-typing of MUCs and CKs may provide useful information to

clinicians with respect to progression and even prognosis.

Acknowledgements

Supported in part by Grants-in-Aid for Intrahepatic

Calculi from the Ministry of Health and Welfare, Japan.

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