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Oncocytic papillary neoplasms of the biliary tract: a clinicopathological, mucin core and Wnt pathway protein analysis of four cases
Pathology (August 2007) 39(4), pp. 413–418
ANATOMICAL PATHOLOGY
Oncocytic papillary neoplasms of the biliary tract: a clinicopathological, mucin core and Wnt pathway protein analysis of four cases MARJAN ROUZBAHMAN*, STEFANO SERRA*, N. VOLKAN ADSAY{, PABLO A. BEJARANO{, YASUNI NAKANUMA§ AND RUNJAN CHETTY*
*Department of Pathology, University Health Network/University of Toronto, Toronto, Canada, {Department of Pathology, The Karmanos Cancer Institute and Wayne State University School of Medicine, Detroit, Michigan, USA, {Department of Pathology, University of Miami School of Medicine/Jackson Memorial Hospital, Miami, Florida, USA, and §Department of Human Pathology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan
Summary Aims: Oncocytic change in papillary neoplasms of the biliary tract is a very uncommon finding with little known about pathogenesis, immunophenotype and prognosis, especially in comparison to similar lesions in the pancreatic ductal system. We report four cases of oncocytic biliary intraductal papillary neoplasms (IPNs), highlighting the clinicopathological characteristics of these tumours, the immunohistochemical profile with regard to Wnt pathway proteins and mucin core protein (MUC) status, and compare these findings with the oncocytic variant of intraductal papillary mucinous neoplasm (IPMN) of the pancreas. Methods: Four cases of oncocytic IPN of the extrapancreatic, biliary tree (two with accompanying invasive carcinomas) were examined for mucin profiles and Wnt signalling proteins. The cases were stained for: b-catenin, c-myc, glutathione synthase kinase (GSK), E-cadherin, cyclin D1, and adenomatous polyposis coli (APC), and MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B and MUC6, using standard immunohistochemistry. Results: The cases occurred in three males and one female, ranging in age from 59 to 81 years. The lesions caused obstructive symptoms related to the biliary tree as well as non-specific abdominal symptoms. Typically, cystic lesions were noted grossly. All four of the IPNs were composed of distinctive oncocytic cells. The invasive carcinomas accompanying two of the cases were also composed of oncocytes. None of the cases showed aberrant expression of the Wnt signalling proteins, although cyclin D1 was markedly overexpressed in all four cases. Three of four cases showed the following mucin profile: MUC3, MUC4, MUC5AC, MUC5B and MUC6 positive. Conclusions: The Wnt pathway proteins (especially b-catenin and E-cadherin) are expressed normally in oncocytic variants of intraductal papillary neoplasms of the biliary tree, and the mucin profile is similar to their counterparts in the pancreas. Key words: Oncocytic papillary neoplasm, oncocytes, biliary tract papilloma, biliary papillomatosis, mucins, Wnt pathway. Received 13 November, revised 18 December 2006, accepted 24 January 2007
INTRODUCTION Intraductal papillary neoplasms (IPNs) are well-defined tumours that have been described in the pancreas and biliary tract.1–3 Solitary or multicentric papillary lesions of the intra- and/or extra-hepatic biliary epithelium that can spread extensively along the biliary tree, characterise these uncommon lesions.4,5 Papillomatosis is the term used for the multicentric lesions.6 Some biliary IPNs show similarity to intraductal papillary mucinous neoplasms (IPMNs) of the pancreas and possess a risk of developing invasive carcinoma.7–9 In fact, a significant proportion of patients with IPNs have an invasive biliary carcinoma component at the time of resection.10 Some biliary cystic tumours, biliary cystadenoma and cystadenocarcinoma, have a luminal communication with the bile duct system. It has been suggested that these tumours could be IPNs with cystic dilatation of the bile duct and mucin retention, rather than true biliary cystic neoplasms.11,12 Some intraductal papillary tumours of the bile ducts produce a large amount of mucin that can cause dilatation of biliary ducts. Patients with these tumours frequently present with abdominal pain, obstructive jaundice and cholangitis.13 Oncocytic change is a well-recognised morphological nuance that is described in many different tumours in a wide variety of locations. In the hepatobiliary system, oncocytic variants of hepatocellular carcinoma and cholangiocarcinoma have been described.14 Oncocytic differentiation in intrahepatic biliary cystadenocarcinoma was first reported by Wolf et al. in 199215 and has been subsequently reported by other investigators.16,17 Activation of the Wnt pathway plays an important role in the development of a wide variety of tumour types and has been explored in IPMN of the pancreas.18 Two key genes involved in this process are adenomatous polyposis coli (APC) and b-catenin. b-catenin is a ubiquitously expressed cytoplasmic protein that has a crucial role in both cadherin mediated cell-cell adhesion and as a downstream signalling molecule in the wingless/Wnt pathway. In addition, downstream proteins such as cyclin D1 and c-myc are also thought to be involved with activation of this pathway.
ISSN 0031-3025 printed/ISSN 1465-3931 # 2007 Royal College of Pathologists of Australasia DOI: 10.1080/00313020701444531
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Mucins are high molecular weight glycoproteins characterised by oligosaccharides attached to serine or threonine residues in the mucin core backbone. Several mucin genes, which are differentially expressed by different cells and organs, have been identified.19 Behaviour and prognosis of tumours has been based on their mucin profiles. We report four cases of oncocytic biliary IPNs, two of which have been previously reported as single case reports,13,17 highlighting the clinicopathological characteristics of these tumours, the immunohistochemical profile with regard to Wnt pathway proteins and mucin core protein (MUC) status, and compare these findings with the oncocytic variant of IPMN of the pancreas.
MATERIALS AND METHODS All H&E stained sections were evaluated and four biliary tree papillary tumours containing large cells with eosinophilic, granular cytoplasm, large nuclei and prominent nucleoli, were regarded as oncocytic. Electron microscopic confirmation of numerous cytoplasmic mitochondria was obtained in two of the four cases. In addition to the special mucin core proteins (MUC3, MUC4, MUC5AC, MUC5B and MUC6), immunohistochemistry was performed on formalin fixed, paraffin embedded tissue using the streptavidin-biotin complex with DAB as chromogen (with blocking of endogenous biotin) for the following: b-catenin, c-myc, glutathione synthase kinase (GSK), Ecadherin, cyclin D1, and adenomatous polyposis coli (APC). Normal tissue in the sections and known cases where these proteins are aberrantly expressed were used as positive controls. Mucins were detected by immunohistochemical staining using antiMUC1 (mouse monoclonal antibody; 1:100; Santa-Cruz Biotech, USA), anti-MUC2 (mouse monoclonal antibody; 1:100; Santa-Cruz Biotech), anti-MUC3 (mouse monoclonal antibody, clone M3.1; 1:10; Santa-Cruz Biotech), anti-MUC4 (rabbit polyclonal antibody; 1:500; a kind gift from Dr Kontani, Shiga Medical College, Japan), anti-MUC5B (mouse polyclonal antibody; 1:50; Novocastra, UK), anti-MUC5AC (mouse monoclonal antibody, clone CLH2; 1:200; Novocastra) and anti-MUC6 (mouse monoclonal antibody, clone CLH2; 1:200; Novocastra). The remaining antibodies used were as follows: APC (monoclonal, 1:2000; Abcam, UK), Axin2 (polycolonal, 1:300; in-house Ontario Cancer
Institute, Canada), pan b-catenin (monoclonal, 1:3000; BD Signal Transduction, USA), c-myc (polyclonal, 1:200; Santa Cruz Biotechnology), cyclin D1 (polyclonal, 1:200; LabVision, USA), E-cadherin (monoclonal, 1:100; Zymed Laboratories, USA), GSK3-b (polyclonal, 1:50; Cell Signaling Technology, USA) and Phospho-GSK3a/b (Ser21/9; polyclonal, 1:50; Cell Signaling Technology). In brief, deparaffinised and rehydrated sections were pre-treated in a microwave oven in EDTA buffer (pH 9) or citrate buffer (pH 6) at 95uC for 20 min for antigen retrieval for the detection of MUC3 and MUC5AC, respectively. Then, the sections were immersed in 0.3% H2O2 in methanol for 20 min to abolish endogenous peroxidase activities. The sections were then immersed in 3% normal goat serum diluted in PBS for 60 min. The sections were then incubated with the primary monoclonal antibody at 4uC overnight. The Envision Plus solution (Dako, Denmark) was then applied for 30 min at room temperature. The reaction products were visualised using 3-39-diaminobenzidine tetrahydrochloride (Sigma, USA) and H2O2. The sections were then lightly counterstained with haematoxylin. There was no positive staining when H2O2 without DAB or DAB without H2O2 was applied. Negative controls included substituting the primary antibody with similarly diluted mouse or rabbit or goat control immunoglobulin G (Dakocytomation, Denmark).
RESULTS The results are summarised in Tables 1 and 2. Case 1 This was a 71-year-old male who presented with abdominal fullness and chest pain in July 1998. Radiological studies, including computed tomography and magnetic resonance imaging, revealed multiple cystic lesions and an adjacent solid lesion in the left hepatic lobe. This patient died and the autopsy showed that the left lateral segment of liver was atrophic and replaced by multiple cystic lesions and an adjacent solid lesion. The largest cyst was 4 cm in diameter. The cysts contained mucinous fluid. The internal surface of the cysts, although mostly smooth, displayed multifocal granular and partly papillary foci. There was a solid, well-circumscribed lesion that had a yellow-green mucinous cut surface adjacent to the cystic
TABLE 1 Wnt pathway proteins in oncocytic IPN of the biliary tree Case 1 (intraductal) b-catenin c-myc GSK APC E-cadherin Cyclin D1
M, Ct Nc Nc Ct M Nc
(80%) (80%) (100%) (100%) (100%) (80%)
Case 1 (invasive) M, Ct Nc Ct M Nc Nc
(80%) (80%) (100%) (100%) (80%) (80%)
Case 2 M, Ct Nc Nc Ct M Nc
Case 3
(80%) (80%) (100%) (100%) (100%) (70%)
M, Ct Nc Nc Ct M Nc
(80%) (100%) (100%) (100%) (100%) (80%)
Case 4 M, Ct Nc Nc, Ct Ct, Nc M Nc
(80%) (80%) (100%) (100%) (100%) (70%)
Ct, cytoplasmic; M, membrane; IPN, intraductal papillary neoplasm; Nc, nuclear. TABLE 2 Mucin profiles in oncocytic IPN of the biliary tree Mucin MUC1 MUC2 MUC3 MUC4 MUC5AC MUC5B MUC6
Case 1 (intraductal) Focal membrane 10% Negative 0% Negative 0% Negative 0% Diffuse cytoplasmic 50% Diffuse cytoplasmic 65% Diffuse cytoplasmic 65%
Case 1 (invasive) Focal cytoplasmic 10% Focal cytoplasmic 5% Negative 0% Negative 0% Diffuse cytoplasmic 50% Diffuse cytoplasmic 60% Diffuse cytoplasmic 65%
Case 2 Negative 0% Diffuse cytoplasmic 40% Diffuse cytoplasmic 40% Diffuse cytoplasmic 50% Diffuse cytoplasmic 60% Diffuse cytoplasmic 70% Focal cytoplasmic 10%
Case 3 Negative 0% Negative 0% Diffuse cytoplasmic 40% Focal cytoplasmic 10% Diffuse cytoplasmic 60% Diffuse cytoplasmic 60% Diffuse cytoplasmic 60%
Case 4 Membrane 30% Negative 0% Diffuse cytoplasmic Diffuse cytoplasmic Diffuse cytoplasmic Negative 0% Diffuse cytoplasmic
50% 50% 65% 70%
ONCOCYTIC PAPILLARY NEOPLASMS OF THE BILIARY TRACT
lesion. Carcinomatous infiltration was seen in the liver hilum and hepatoduodenal ligament. The intrahepatic bile duct of the right lobe was variably dilated and cholestasis was evident. Grossly, there was no direct communication between the multicystic lesions and dilated intrahepatic bile duct. Histologically, there was a papillary proliferation with oedematous fibrovascular cores within the cysts as well as filling intrahepatic bile ducts, suggesting that the cysts were secondarily dilated intrahepatic bile ducts. In some areas, the cysts were lined by multilayered cuboidal to low columnar oncocytic cells rather than papillary structures. The epithelial cells had an eosinophilic, granular cytoplasm, dispersed nuclear chromatin and prominent acidophilic nucleoli in keeping with oncocytic change (Fig. 1). The solid areas were composed of solid nests of tumour and small glandular structures with extra-cellular mucin. Tumour cells in this area were morphologically similar (oncocytic) to the cystic lesion indicating that the invasive lesion arose from the cystic lesion. Case 2 A 59-year-old male presented with an episode of abdominal pain, transient jaundice and dark urine in June 2000. An endoscopic retrograde cholangiopancreatography (ERCP) showed gallstones and he had a laparoscopic cholecystectomy. Intraoperative cholangiography showed thickening of the intrapancreatic portion of bile duct, which was confirmed by post-operative ultrasound and ERCP. Finally the patient underwent a Whipple’s operation to excise the involved area completely. The patient survived and was disease-free after 1 year. Gross examination of the resected specimen confirmed that a 5 cm segment of common bile duct was granular and polypoid, leading to significant decrease in luminal diameter. Histologically, a lesion composed of papillary structures lined by oncocytic columnar cells was identified in the common bile duct, with extension into the cystic duct. There was a moderate to severe degree of dysplasia but no evidence of stromal invasion was noted.
Fig. 1 Intraductal papillary neoplasm of the biliary tree composed of cells characterised by deeply eosinophilic granular cytoplasm. The nuclei have vesicular chromatin and prominent nucleoli and the atypia varied from mild to severe. All cases were composed of such cells.
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Case 3 A 52-year-old male presented with a 2-year history of intermittent biliary obstruction in July 2002. The resected specimen consisted of the gallbladder and common hepatic duct at the junction of the right and left hepatic ducts. The common bile duct contained an exophytic lesion extending into the left hepatic duct. The lesion was 1.7 cm in length and confined to the duct lumen. The gallbladder was grossly unremarkable. Histological examination of the specimen revealed a lesion composed of papillary structures lined by cells with oncocytic features and mild dysplasia. No stromal invasion was detected. Electron microscopy performed from paraffin block showed numerous densely packed mitochondria in epithelial cells. The patient was alive 3 years after the surgery without recurrent disease. Case 4 An 81-year-old woman underwent a cholecystectomy via a laparotomy. Gross examination of the surgical specimen showed a cystic mass, approximately 2.5 cm, adjacent to and compressing the cystic duct, liver and gallbladder. The cystic mass was filled with largely necrotic, variegated tan and red-brown tissue but without involvement of adjacent liver. Microscopic examination of this mass revealed a cystadenocarcinoma composed of oncocytic cells with a tubulopapillary growth pattern. The tumour was confined by a dense fibrous capsule, which was focally calcified. There was metastatic carcinoma in three of the local lymph nodes. No follow-up is available on this patient. Immunohistochemistry Table 2 summarises the immunohistochemical results. b-catenin staining was membranous and cytoplasmic; none of the cases showed nuclear accumulation, thus expression of this protein was regarded as normal (Fig. 2). Similarly, APC expression was cytoplasmic and normal in distribution in three cases and Case 4 showed some nuclear staining in addition to cytoplasmic staining. c-myc was strongly positive (nuclear staining) in all four cases and was noted in both intraductal and invasive components of Case 1. Staining for GSK was nuclear and
Fig. 2 b-catenin staining was normal in distribution, being localised to the cell membrane and cytoplasm.
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Fig. 3 Similarly, E-cadherin displayed crisp membrane decoration of cells. This was evident in the intraductal and invasive components of Cases 1 and 4.
considered as normal in distribution in three cases and Case 4 showed cytoplasmic staining as well as nuclear staining. Intense membrane staining for E-cadherin was noted in all cases, including the invasive components (Fig. 3). Cyclin D1 was markedly up-regulated in the majority of tumour cells (Fig. 4). MUC3, MUC4, MUC5AC, MUC5B and MUC6 appeared to show either focal or diffuse cytoplasmic staining in Cases 2, 3 and 4 (Fig. 5A–C), with the exception of no staining in Case 4 for MUC5B. MUC1 was focally positive in Case 1, which showed membranous staining in the intraductal component and cytoplasmic staining in the invasive component. MUC1 membrane positivity was present in approximately 50% of cells in Case 4. It was negative in the other two cases. MUC2 showed focal granular cytoplasmic staining in the invasive component of Case 1 but it was negative in the intraductal component. There was granular cytoplasmic MUC2 staining in more than 50% of tumour cells in Case 2, but Cases 3 and 4 were negative. Interestingly, there was no difference in the staining between the intraductal and invasive components in Case 4; the invasive component in Case 1 expressed MUC2 but was otherwise identical to the intraductal lesion.
Fig. 5 Three cases were (A) MUC3, (B) MUC4 and (C) MUC5AC positive.
DISCUSSION
Fig. 4 There was marked over-expression of cyclin D1 in all four cases.
Papillary lesions of the biliary tree range from biliary papillomatosis through to intraductal adenocarcinomas, similar to that of the pancreas. Whilst biliary papillomatosis contains benign epithelium, dysplasia of increasing severity may be encountered. Tumours with oncocytic phenotype may occur in a variety of organs, but are extremely rare in the biliary
ONCOCYTIC PAPILLARY NEOPLASMS OF THE BILIARY TRACT
system. Intraductal papillary neoplasms of the extrapancreatic biliary tree are rare tumours and there are only a few reports of the oncocytic variant.13,15,16 The oncocytic variant of IPMN of the pancreas is thought to be slightly different from the other histological variants in terms of behaviour and mucin profile. IPMN have been found to be MUC1 negative and either MUC2 positive or negative. Pancreatic ductal adenocarcinoma, on the other hand, is usually MUC1 positive but MUC2 negative. The MUC2 positive IPMN were thought to have a poorer prognosis than the MUC2 negative cases.19 Adsay and colleagues correlated MUC status with the histological type of epithelium encountered in IPMN.20 The intestinal type of IPMN was MUC2 positive, the pancreaticobiliary type was MUC2 negative but MUC1 positive and those cases with gastric foveolar mucosa were both MUC1 and 2 negative.20 Ito et al. found that MUC1 positive IPMN tended to harbour carcinoma and had a rapid progression and a poor prognosis.21 On the other hand, IPMN expressing MUC5AC alone had a good prognosis.21 Results of MUC staining in the oncocytic variant of IPMN are variable. Luttges et al. have reported focal MUC1 and MUC2 positivity,22 while others have reported little or no MUC2 expression.23 However, both studies do concur that MUC5AC is usually expressed by this variant. Our study confirms that all four oncocytic IPN of the biliary tree express MUC5AC and MUC6, while three of four cases expressed MUC5B and MUC3. One case demonstrated membrane staining of MUC1 and a solitary case was positive for MUC2. From these results it appears that oncocytic IPN of the biliary tree has the same mucin profile as oncocytic IPMN of the pancreas, especially with regards to MUC5AC. MUC1 and MUC2 are expressed in the minority of cases. Obviously, these are merely trends as the numbers of cases are somewhat limited due to the rarity of these tumours. Amaya et al. studied the immunohistochemical expression of mucin core proteins in 11 lesions of biliary papillomatosis from seven patients, and five lesions of biliary papillomatosis with foci of carcinoma from four patients.24 However, it must be remembered that none of these cases were oncocytic. Expression of MUC1 was frequent and marked in biliary papillomatosis alone and especially in those cases with carcinoma.24 Case 1 in our series showed no difference in MUC1/MUC2 positivity between the intraductal lesion and the invasive component: both were positive. MUC2 positivity in the series described by Amaya et al. was frequent and marked in biliary papillomatosis alone compared with cases with an accompanying invasive component and carcinomas alone.24 Focal expression of MUC5AC was observed frequently in the series of Amaya et al., irrespective of associated carcinoma or not.24 A proportion of IPMN of the pancreas were shown to have an abnormal Wnt pathway;18 however, the solitary case of oncocytic IPMN in that series did not show abnormalities of Wnt proteins.18 Abraham et al. studied 14 biliary intraductal papillary neoplasms (including 5 cases with associated invasive cholangiocarcinoma) for genetic alterations in the APC/b-catenin pathway, K-ras oncogene mutations, p53/chromosome 17p alterations, and DPC4/ 18q alterations.7 They demonstrated nuclear accumulation of b-catenin protein in three of 12 cases (25%); however, there were no accompanying b-catenin gene mutations, and allelic loss on 5q was present in only one of 10 informative
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cases.7 In our series, none of the cases showed nuclear accumulation of b-catenin. In addition, APC as well as GSK expression appeared to be normal. E-cadherin also showed a normal membranous pattern of staining. Thus, the Wnt pathway proteins appear to be intact (normal expression) in the four cases in this small series. In this report, two of the intraductal papillary lesions were accompanied by an invasive component. One was composed of two components, intracystic papillary lesion and invasive mucinous carcinoma (Case 1), and the other had a cystadenocarcinoma with a tubulopapillary pattern (Case 4). However, there were no real differences between the immuno-patterns of the invasive and intraductal components. In a recently published study by Zen et al. two types of neoplastic lesions preceding invasive intrahepatic cholangiocarcinoma (ICC) were identified: a flat-type neoplastic lesion called ‘biliary intraepithelial neoplasia (BilIN)’ and an intraductal papillary neoplasm of the bile duct (IPN-B).12 The authors investigated expression patterns of MUC1, MUC2, MUC5AC, cytokeratin 7 (CK7), and CK20 in BilIN, IPN-B, and ICC in 110 cases of hepatolithiasis, and they concluded that increased expression of MUC1 in BilIN and IPN-B is associated with tubular adenocarcinoma, while colloid carcinoma in IPN-B is characterised by MUC1 negativity and less advanced pathological stages.12 In our cases, both mucinous and tubulopapillary carcinomas (Cases 1 and 4) showed positive staining for MUC1, while the other cases which lacked an invasive lesion were negative for this protein. In conclusion, the oncocytic variant of IPN is very uncommon and tends to have a mucin profile similar to its pancreatic counterpart. In the few cases examined, the key proteins in the Wnt pathway are not up-regulated, implying that activation of this pathway has not occurred. Address for correspondence: Dr R. Chetty, Department of Pathology, University Health Network, Toronto General Hospital, 200 Elizabeth Street, Eleventh Floor, Eaton Wing, Room 312, Toronto, ON M5G 2C4, Canada. E-mail: [email protected]
References 1. Abraham SC, Lee JH, Boitnott JK, Argani P, Furth EE, Wu TT. Microsatellite instability in intraductal papillary neoplasms of the biliary tract. Mod Pathol 2002; 15: 1309–17. 2. Aoki S, Okayama Y, Kitajima Y, et al. Intrahepatic biliary papilloma morphologically similar to biliary cystadenoma. J Gastroenterol Hepatol 2005; 20: 321–4. 3. De Carlo A, Altobelli S, Cucci M, Masella M, D’Amico G. Intrahepatic biliary papillomatosis: a case report. Chir Ital 2005; 57: 499–503. 4. Helpap B. Malignant papillomatosis of the intrahepatic bile ducts. Acta Hepatogastroenterol (Stuttg) 1977; 24: 419–25. 5. Stoker TA. Intrahepatic biliary papillomatosis. Br J Surg 1984; 71: 398. 6. Guglielmi A, Caputi Jambrenghi O, Verzillo F, et al. Biliary tract papillomatosis. Minerva Chir 2001; 56: 531–3. 7. Abraham SC, Lee JH, Hruban RH, Argani P, Furth EE, Wu TT. Molecular and immunohistochemical analysis of intraductal papillary neoplasms of the biliary tract. Hum Pathol 2003; 34: 902–10. 8. Shimonishi T, Sasaki M, Nakanuma Y. Precancerous lesions of intrahepatic cholangiocarcinoma. J Hepatobiliary Pancreat Surg 2000; 7: 542–50. 9. Yeung YP, AhChong K, Chung CK, Chun AY. Biliary papillomatosis: report of seven cases and review of English literature. J Hepatobiliary Pancreat Surg 2003; 10: 390–5. 10. Lee SS, Kim MH, Lee SK, et al. Clinicopathologic review of 58 patients with biliary papillomatosis. Cancer 2004; 100: 783–793. 11. Sudo Y, Harada K, Tsuneyama K, Katayanagi K, Zen Y, Nakanuma Y. Oncocytic biliary cystadenocarcinoma is a form of
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13. 14. 15. 16. 17. 18.
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intraductal oncocytic papillary neoplasm of the liver. Mod Pathol 2001; 14: 1304–9. Zen Y, Sasaki M, Fujii T, et al. 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. J Hepatol 2006; 44: 350–8. Lim JH, Yoon KH, Kim SH, et al. Intraductal papillary mucinous tumor of the bile ducts. Radiographics 2004; 24: 53–66; discussion 66–7. Chang A, Harawi SJ. Oncocytes, oncocytosis, and oncocytic tumors. Pathol Annu 1992; 27 (Pt 1): 263–304. Wolf HK, Garcia JA, Bossen EH. Oncocytic differentiation in intrahepatic biliary cystadenocarcinoma. Mod Pathol 1992; 5: 665–8. Bardin RL, Trupiano JK, Howerton RM, Geisinger KR. Oncocytic biliary cystadenocarcinoma: a case report and review of the literature. Arch Pathol Lab Med 2004; 128: e25–8. Spector SA, Bejarano PA, Amortegui JD, Renfrow MR, Livingstone AS. Intraductal oncocytic papillary neoplasm of the extrahepatic biliary tree: first report. Am Surg 2004; 70: 55–8. Chetty R, Serra S, Salahshor S, et al. Expression of wnt signalling pathway proteins in intraductal papillary mucinous neoplasms of the pancreas: a tissue microarray analysis. Hum Pathol 2006; 37: 212–7.
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19. Nakamura A, Horinouchi M, Goto M, et al. New classification of pancreatic intraductal papillary-mucinous tumour by mucin expression: its relationship with potential malignancy. J Pathol 2002; 197: 201–10. 20. Adsay NV, Merati K, Basturk O, et al. Pathologically and biologically distinct types of epithelium in intraductal papillary mucinous neoplasms. Delineation of an ‘intestinal’ pathway of carcinogenesis in the pancreas. Am J Surg Pathol 2004; 28: 839–48. 21. Ito H, Endo T, Oka T, et al. Mucin expression profile is related to biological and clinical characteristics of intraductal papillary-mucinous tumors of the pancreas. Pancreas 2005; 30: 96–102. 22. Luttges J, Zamboni G, Longnecker D, Kloppel G. The immunohistochemical mucin expression pattern distinguishes different types of intraductal papillary mucinous neoplasms of the pancreas and determines their relationship to mucinous noncystic carcinoma and ductal adenocarcinoma. Am J Surg Pathol 2001; 25: 942–8. 23. Terris B, Dubois S, Buisine MP, et al. Mucin gene expression in intraductal papillary-mucinous pancreatic tumours and related lesions. J Pathol 2002; 197: 632–7. 24. Amaya S, Sasaki M, Watanabe Y, et al. Expression of MUC1 and MUC2 and carbohydrate antigen Tn change during malignant transformation of biliary papillomatosis. Histopathology 2001; 38: 550–60.
ANATOMICAL PATHOLOGY
Oncocytic papillary neoplasms of the biliary tract: a clinicopathological, mucin core and Wnt pathway protein analysis of four cases MARJAN ROUZBAHMAN*, STEFANO SERRA*, N. VOLKAN ADSAY{, PABLO A. BEJARANO{, YASUNI NAKANUMA§ AND RUNJAN CHETTY*
*Department of Pathology, University Health Network/University of Toronto, Toronto, Canada, {Department of Pathology, The Karmanos Cancer Institute and Wayne State University School of Medicine, Detroit, Michigan, USA, {Department of Pathology, University of Miami School of Medicine/Jackson Memorial Hospital, Miami, Florida, USA, and §Department of Human Pathology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan
Summary Aims: Oncocytic change in papillary neoplasms of the biliary tract is a very uncommon finding with little known about pathogenesis, immunophenotype and prognosis, especially in comparison to similar lesions in the pancreatic ductal system. We report four cases of oncocytic biliary intraductal papillary neoplasms (IPNs), highlighting the clinicopathological characteristics of these tumours, the immunohistochemical profile with regard to Wnt pathway proteins and mucin core protein (MUC) status, and compare these findings with the oncocytic variant of intraductal papillary mucinous neoplasm (IPMN) of the pancreas. Methods: Four cases of oncocytic IPN of the extrapancreatic, biliary tree (two with accompanying invasive carcinomas) were examined for mucin profiles and Wnt signalling proteins. The cases were stained for: b-catenin, c-myc, glutathione synthase kinase (GSK), E-cadherin, cyclin D1, and adenomatous polyposis coli (APC), and MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B and MUC6, using standard immunohistochemistry. Results: The cases occurred in three males and one female, ranging in age from 59 to 81 years. The lesions caused obstructive symptoms related to the biliary tree as well as non-specific abdominal symptoms. Typically, cystic lesions were noted grossly. All four of the IPNs were composed of distinctive oncocytic cells. The invasive carcinomas accompanying two of the cases were also composed of oncocytes. None of the cases showed aberrant expression of the Wnt signalling proteins, although cyclin D1 was markedly overexpressed in all four cases. Three of four cases showed the following mucin profile: MUC3, MUC4, MUC5AC, MUC5B and MUC6 positive. Conclusions: The Wnt pathway proteins (especially b-catenin and E-cadherin) are expressed normally in oncocytic variants of intraductal papillary neoplasms of the biliary tree, and the mucin profile is similar to their counterparts in the pancreas. Key words: Oncocytic papillary neoplasm, oncocytes, biliary tract papilloma, biliary papillomatosis, mucins, Wnt pathway. Received 13 November, revised 18 December 2006, accepted 24 January 2007
INTRODUCTION Intraductal papillary neoplasms (IPNs) are well-defined tumours that have been described in the pancreas and biliary tract.1–3 Solitary or multicentric papillary lesions of the intra- and/or extra-hepatic biliary epithelium that can spread extensively along the biliary tree, characterise these uncommon lesions.4,5 Papillomatosis is the term used for the multicentric lesions.6 Some biliary IPNs show similarity to intraductal papillary mucinous neoplasms (IPMNs) of the pancreas and possess a risk of developing invasive carcinoma.7–9 In fact, a significant proportion of patients with IPNs have an invasive biliary carcinoma component at the time of resection.10 Some biliary cystic tumours, biliary cystadenoma and cystadenocarcinoma, have a luminal communication with the bile duct system. It has been suggested that these tumours could be IPNs with cystic dilatation of the bile duct and mucin retention, rather than true biliary cystic neoplasms.11,12 Some intraductal papillary tumours of the bile ducts produce a large amount of mucin that can cause dilatation of biliary ducts. Patients with these tumours frequently present with abdominal pain, obstructive jaundice and cholangitis.13 Oncocytic change is a well-recognised morphological nuance that is described in many different tumours in a wide variety of locations. In the hepatobiliary system, oncocytic variants of hepatocellular carcinoma and cholangiocarcinoma have been described.14 Oncocytic differentiation in intrahepatic biliary cystadenocarcinoma was first reported by Wolf et al. in 199215 and has been subsequently reported by other investigators.16,17 Activation of the Wnt pathway plays an important role in the development of a wide variety of tumour types and has been explored in IPMN of the pancreas.18 Two key genes involved in this process are adenomatous polyposis coli (APC) and b-catenin. b-catenin is a ubiquitously expressed cytoplasmic protein that has a crucial role in both cadherin mediated cell-cell adhesion and as a downstream signalling molecule in the wingless/Wnt pathway. In addition, downstream proteins such as cyclin D1 and c-myc are also thought to be involved with activation of this pathway.
ISSN 0031-3025 printed/ISSN 1465-3931 # 2007 Royal College of Pathologists of Australasia DOI: 10.1080/00313020701444531
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Mucins are high molecular weight glycoproteins characterised by oligosaccharides attached to serine or threonine residues in the mucin core backbone. Several mucin genes, which are differentially expressed by different cells and organs, have been identified.19 Behaviour and prognosis of tumours has been based on their mucin profiles. We report four cases of oncocytic biliary IPNs, two of which have been previously reported as single case reports,13,17 highlighting the clinicopathological characteristics of these tumours, the immunohistochemical profile with regard to Wnt pathway proteins and mucin core protein (MUC) status, and compare these findings with the oncocytic variant of IPMN of the pancreas.
MATERIALS AND METHODS All H&E stained sections were evaluated and four biliary tree papillary tumours containing large cells with eosinophilic, granular cytoplasm, large nuclei and prominent nucleoli, were regarded as oncocytic. Electron microscopic confirmation of numerous cytoplasmic mitochondria was obtained in two of the four cases. In addition to the special mucin core proteins (MUC3, MUC4, MUC5AC, MUC5B and MUC6), immunohistochemistry was performed on formalin fixed, paraffin embedded tissue using the streptavidin-biotin complex with DAB as chromogen (with blocking of endogenous biotin) for the following: b-catenin, c-myc, glutathione synthase kinase (GSK), Ecadherin, cyclin D1, and adenomatous polyposis coli (APC). Normal tissue in the sections and known cases where these proteins are aberrantly expressed were used as positive controls. Mucins were detected by immunohistochemical staining using antiMUC1 (mouse monoclonal antibody; 1:100; Santa-Cruz Biotech, USA), anti-MUC2 (mouse monoclonal antibody; 1:100; Santa-Cruz Biotech), anti-MUC3 (mouse monoclonal antibody, clone M3.1; 1:10; Santa-Cruz Biotech), anti-MUC4 (rabbit polyclonal antibody; 1:500; a kind gift from Dr Kontani, Shiga Medical College, Japan), anti-MUC5B (mouse polyclonal antibody; 1:50; Novocastra, UK), anti-MUC5AC (mouse monoclonal antibody, clone CLH2; 1:200; Novocastra) and anti-MUC6 (mouse monoclonal antibody, clone CLH2; 1:200; Novocastra). The remaining antibodies used were as follows: APC (monoclonal, 1:2000; Abcam, UK), Axin2 (polycolonal, 1:300; in-house Ontario Cancer
Institute, Canada), pan b-catenin (monoclonal, 1:3000; BD Signal Transduction, USA), c-myc (polyclonal, 1:200; Santa Cruz Biotechnology), cyclin D1 (polyclonal, 1:200; LabVision, USA), E-cadherin (monoclonal, 1:100; Zymed Laboratories, USA), GSK3-b (polyclonal, 1:50; Cell Signaling Technology, USA) and Phospho-GSK3a/b (Ser21/9; polyclonal, 1:50; Cell Signaling Technology). In brief, deparaffinised and rehydrated sections were pre-treated in a microwave oven in EDTA buffer (pH 9) or citrate buffer (pH 6) at 95uC for 20 min for antigen retrieval for the detection of MUC3 and MUC5AC, respectively. Then, the sections were immersed in 0.3% H2O2 in methanol for 20 min to abolish endogenous peroxidase activities. The sections were then immersed in 3% normal goat serum diluted in PBS for 60 min. The sections were then incubated with the primary monoclonal antibody at 4uC overnight. The Envision Plus solution (Dako, Denmark) was then applied for 30 min at room temperature. The reaction products were visualised using 3-39-diaminobenzidine tetrahydrochloride (Sigma, USA) and H2O2. The sections were then lightly counterstained with haematoxylin. There was no positive staining when H2O2 without DAB or DAB without H2O2 was applied. Negative controls included substituting the primary antibody with similarly diluted mouse or rabbit or goat control immunoglobulin G (Dakocytomation, Denmark).
RESULTS The results are summarised in Tables 1 and 2. Case 1 This was a 71-year-old male who presented with abdominal fullness and chest pain in July 1998. Radiological studies, including computed tomography and magnetic resonance imaging, revealed multiple cystic lesions and an adjacent solid lesion in the left hepatic lobe. This patient died and the autopsy showed that the left lateral segment of liver was atrophic and replaced by multiple cystic lesions and an adjacent solid lesion. The largest cyst was 4 cm in diameter. The cysts contained mucinous fluid. The internal surface of the cysts, although mostly smooth, displayed multifocal granular and partly papillary foci. There was a solid, well-circumscribed lesion that had a yellow-green mucinous cut surface adjacent to the cystic
TABLE 1 Wnt pathway proteins in oncocytic IPN of the biliary tree Case 1 (intraductal) b-catenin c-myc GSK APC E-cadherin Cyclin D1
M, Ct Nc Nc Ct M Nc
(80%) (80%) (100%) (100%) (100%) (80%)
Case 1 (invasive) M, Ct Nc Ct M Nc Nc
(80%) (80%) (100%) (100%) (80%) (80%)
Case 2 M, Ct Nc Nc Ct M Nc
Case 3
(80%) (80%) (100%) (100%) (100%) (70%)
M, Ct Nc Nc Ct M Nc
(80%) (100%) (100%) (100%) (100%) (80%)
Case 4 M, Ct Nc Nc, Ct Ct, Nc M Nc
(80%) (80%) (100%) (100%) (100%) (70%)
Ct, cytoplasmic; M, membrane; IPN, intraductal papillary neoplasm; Nc, nuclear. TABLE 2 Mucin profiles in oncocytic IPN of the biliary tree Mucin MUC1 MUC2 MUC3 MUC4 MUC5AC MUC5B MUC6
Case 1 (intraductal) Focal membrane 10% Negative 0% Negative 0% Negative 0% Diffuse cytoplasmic 50% Diffuse cytoplasmic 65% Diffuse cytoplasmic 65%
Case 1 (invasive) Focal cytoplasmic 10% Focal cytoplasmic 5% Negative 0% Negative 0% Diffuse cytoplasmic 50% Diffuse cytoplasmic 60% Diffuse cytoplasmic 65%
Case 2 Negative 0% Diffuse cytoplasmic 40% Diffuse cytoplasmic 40% Diffuse cytoplasmic 50% Diffuse cytoplasmic 60% Diffuse cytoplasmic 70% Focal cytoplasmic 10%
Case 3 Negative 0% Negative 0% Diffuse cytoplasmic 40% Focal cytoplasmic 10% Diffuse cytoplasmic 60% Diffuse cytoplasmic 60% Diffuse cytoplasmic 60%
Case 4 Membrane 30% Negative 0% Diffuse cytoplasmic Diffuse cytoplasmic Diffuse cytoplasmic Negative 0% Diffuse cytoplasmic
50% 50% 65% 70%
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lesion. Carcinomatous infiltration was seen in the liver hilum and hepatoduodenal ligament. The intrahepatic bile duct of the right lobe was variably dilated and cholestasis was evident. Grossly, there was no direct communication between the multicystic lesions and dilated intrahepatic bile duct. Histologically, there was a papillary proliferation with oedematous fibrovascular cores within the cysts as well as filling intrahepatic bile ducts, suggesting that the cysts were secondarily dilated intrahepatic bile ducts. In some areas, the cysts were lined by multilayered cuboidal to low columnar oncocytic cells rather than papillary structures. The epithelial cells had an eosinophilic, granular cytoplasm, dispersed nuclear chromatin and prominent acidophilic nucleoli in keeping with oncocytic change (Fig. 1). The solid areas were composed of solid nests of tumour and small glandular structures with extra-cellular mucin. Tumour cells in this area were morphologically similar (oncocytic) to the cystic lesion indicating that the invasive lesion arose from the cystic lesion. Case 2 A 59-year-old male presented with an episode of abdominal pain, transient jaundice and dark urine in June 2000. An endoscopic retrograde cholangiopancreatography (ERCP) showed gallstones and he had a laparoscopic cholecystectomy. Intraoperative cholangiography showed thickening of the intrapancreatic portion of bile duct, which was confirmed by post-operative ultrasound and ERCP. Finally the patient underwent a Whipple’s operation to excise the involved area completely. The patient survived and was disease-free after 1 year. Gross examination of the resected specimen confirmed that a 5 cm segment of common bile duct was granular and polypoid, leading to significant decrease in luminal diameter. Histologically, a lesion composed of papillary structures lined by oncocytic columnar cells was identified in the common bile duct, with extension into the cystic duct. There was a moderate to severe degree of dysplasia but no evidence of stromal invasion was noted.
Fig. 1 Intraductal papillary neoplasm of the biliary tree composed of cells characterised by deeply eosinophilic granular cytoplasm. The nuclei have vesicular chromatin and prominent nucleoli and the atypia varied from mild to severe. All cases were composed of such cells.
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Case 3 A 52-year-old male presented with a 2-year history of intermittent biliary obstruction in July 2002. The resected specimen consisted of the gallbladder and common hepatic duct at the junction of the right and left hepatic ducts. The common bile duct contained an exophytic lesion extending into the left hepatic duct. The lesion was 1.7 cm in length and confined to the duct lumen. The gallbladder was grossly unremarkable. Histological examination of the specimen revealed a lesion composed of papillary structures lined by cells with oncocytic features and mild dysplasia. No stromal invasion was detected. Electron microscopy performed from paraffin block showed numerous densely packed mitochondria in epithelial cells. The patient was alive 3 years after the surgery without recurrent disease. Case 4 An 81-year-old woman underwent a cholecystectomy via a laparotomy. Gross examination of the surgical specimen showed a cystic mass, approximately 2.5 cm, adjacent to and compressing the cystic duct, liver and gallbladder. The cystic mass was filled with largely necrotic, variegated tan and red-brown tissue but without involvement of adjacent liver. Microscopic examination of this mass revealed a cystadenocarcinoma composed of oncocytic cells with a tubulopapillary growth pattern. The tumour was confined by a dense fibrous capsule, which was focally calcified. There was metastatic carcinoma in three of the local lymph nodes. No follow-up is available on this patient. Immunohistochemistry Table 2 summarises the immunohistochemical results. b-catenin staining was membranous and cytoplasmic; none of the cases showed nuclear accumulation, thus expression of this protein was regarded as normal (Fig. 2). Similarly, APC expression was cytoplasmic and normal in distribution in three cases and Case 4 showed some nuclear staining in addition to cytoplasmic staining. c-myc was strongly positive (nuclear staining) in all four cases and was noted in both intraductal and invasive components of Case 1. Staining for GSK was nuclear and
Fig. 2 b-catenin staining was normal in distribution, being localised to the cell membrane and cytoplasm.
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Fig. 3 Similarly, E-cadherin displayed crisp membrane decoration of cells. This was evident in the intraductal and invasive components of Cases 1 and 4.
considered as normal in distribution in three cases and Case 4 showed cytoplasmic staining as well as nuclear staining. Intense membrane staining for E-cadherin was noted in all cases, including the invasive components (Fig. 3). Cyclin D1 was markedly up-regulated in the majority of tumour cells (Fig. 4). MUC3, MUC4, MUC5AC, MUC5B and MUC6 appeared to show either focal or diffuse cytoplasmic staining in Cases 2, 3 and 4 (Fig. 5A–C), with the exception of no staining in Case 4 for MUC5B. MUC1 was focally positive in Case 1, which showed membranous staining in the intraductal component and cytoplasmic staining in the invasive component. MUC1 membrane positivity was present in approximately 50% of cells in Case 4. It was negative in the other two cases. MUC2 showed focal granular cytoplasmic staining in the invasive component of Case 1 but it was negative in the intraductal component. There was granular cytoplasmic MUC2 staining in more than 50% of tumour cells in Case 2, but Cases 3 and 4 were negative. Interestingly, there was no difference in the staining between the intraductal and invasive components in Case 4; the invasive component in Case 1 expressed MUC2 but was otherwise identical to the intraductal lesion.
Fig. 5 Three cases were (A) MUC3, (B) MUC4 and (C) MUC5AC positive.
DISCUSSION
Fig. 4 There was marked over-expression of cyclin D1 in all four cases.
Papillary lesions of the biliary tree range from biliary papillomatosis through to intraductal adenocarcinomas, similar to that of the pancreas. Whilst biliary papillomatosis contains benign epithelium, dysplasia of increasing severity may be encountered. Tumours with oncocytic phenotype may occur in a variety of organs, but are extremely rare in the biliary
ONCOCYTIC PAPILLARY NEOPLASMS OF THE BILIARY TRACT
system. Intraductal papillary neoplasms of the extrapancreatic biliary tree are rare tumours and there are only a few reports of the oncocytic variant.13,15,16 The oncocytic variant of IPMN of the pancreas is thought to be slightly different from the other histological variants in terms of behaviour and mucin profile. IPMN have been found to be MUC1 negative and either MUC2 positive or negative. Pancreatic ductal adenocarcinoma, on the other hand, is usually MUC1 positive but MUC2 negative. The MUC2 positive IPMN were thought to have a poorer prognosis than the MUC2 negative cases.19 Adsay and colleagues correlated MUC status with the histological type of epithelium encountered in IPMN.20 The intestinal type of IPMN was MUC2 positive, the pancreaticobiliary type was MUC2 negative but MUC1 positive and those cases with gastric foveolar mucosa were both MUC1 and 2 negative.20 Ito et al. found that MUC1 positive IPMN tended to harbour carcinoma and had a rapid progression and a poor prognosis.21 On the other hand, IPMN expressing MUC5AC alone had a good prognosis.21 Results of MUC staining in the oncocytic variant of IPMN are variable. Luttges et al. have reported focal MUC1 and MUC2 positivity,22 while others have reported little or no MUC2 expression.23 However, both studies do concur that MUC5AC is usually expressed by this variant. Our study confirms that all four oncocytic IPN of the biliary tree express MUC5AC and MUC6, while three of four cases expressed MUC5B and MUC3. One case demonstrated membrane staining of MUC1 and a solitary case was positive for MUC2. From these results it appears that oncocytic IPN of the biliary tree has the same mucin profile as oncocytic IPMN of the pancreas, especially with regards to MUC5AC. MUC1 and MUC2 are expressed in the minority of cases. Obviously, these are merely trends as the numbers of cases are somewhat limited due to the rarity of these tumours. Amaya et al. studied the immunohistochemical expression of mucin core proteins in 11 lesions of biliary papillomatosis from seven patients, and five lesions of biliary papillomatosis with foci of carcinoma from four patients.24 However, it must be remembered that none of these cases were oncocytic. Expression of MUC1 was frequent and marked in biliary papillomatosis alone and especially in those cases with carcinoma.24 Case 1 in our series showed no difference in MUC1/MUC2 positivity between the intraductal lesion and the invasive component: both were positive. MUC2 positivity in the series described by Amaya et al. was frequent and marked in biliary papillomatosis alone compared with cases with an accompanying invasive component and carcinomas alone.24 Focal expression of MUC5AC was observed frequently in the series of Amaya et al., irrespective of associated carcinoma or not.24 A proportion of IPMN of the pancreas were shown to have an abnormal Wnt pathway;18 however, the solitary case of oncocytic IPMN in that series did not show abnormalities of Wnt proteins.18 Abraham et al. studied 14 biliary intraductal papillary neoplasms (including 5 cases with associated invasive cholangiocarcinoma) for genetic alterations in the APC/b-catenin pathway, K-ras oncogene mutations, p53/chromosome 17p alterations, and DPC4/ 18q alterations.7 They demonstrated nuclear accumulation of b-catenin protein in three of 12 cases (25%); however, there were no accompanying b-catenin gene mutations, and allelic loss on 5q was present in only one of 10 informative
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cases.7 In our series, none of the cases showed nuclear accumulation of b-catenin. In addition, APC as well as GSK expression appeared to be normal. E-cadherin also showed a normal membranous pattern of staining. Thus, the Wnt pathway proteins appear to be intact (normal expression) in the four cases in this small series. In this report, two of the intraductal papillary lesions were accompanied by an invasive component. One was composed of two components, intracystic papillary lesion and invasive mucinous carcinoma (Case 1), and the other had a cystadenocarcinoma with a tubulopapillary pattern (Case 4). However, there were no real differences between the immuno-patterns of the invasive and intraductal components. In a recently published study by Zen et al. two types of neoplastic lesions preceding invasive intrahepatic cholangiocarcinoma (ICC) were identified: a flat-type neoplastic lesion called ‘biliary intraepithelial neoplasia (BilIN)’ and an intraductal papillary neoplasm of the bile duct (IPN-B).12 The authors investigated expression patterns of MUC1, MUC2, MUC5AC, cytokeratin 7 (CK7), and CK20 in BilIN, IPN-B, and ICC in 110 cases of hepatolithiasis, and they concluded that increased expression of MUC1 in BilIN and IPN-B is associated with tubular adenocarcinoma, while colloid carcinoma in IPN-B is characterised by MUC1 negativity and less advanced pathological stages.12 In our cases, both mucinous and tubulopapillary carcinomas (Cases 1 and 4) showed positive staining for MUC1, while the other cases which lacked an invasive lesion were negative for this protein. In conclusion, the oncocytic variant of IPN is very uncommon and tends to have a mucin profile similar to its pancreatic counterpart. In the few cases examined, the key proteins in the Wnt pathway are not up-regulated, implying that activation of this pathway has not occurred. Address for correspondence: Dr R. Chetty, Department of Pathology, University Health Network, Toronto General Hospital, 200 Elizabeth Street, Eleventh Floor, Eaton Wing, Room 312, Toronto, ON M5G 2C4, Canada. E-mail: [email protected]
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19. Nakamura A, Horinouchi M, Goto M, et al. New classification of pancreatic intraductal papillary-mucinous tumour by mucin expression: its relationship with potential malignancy. J Pathol 2002; 197: 201–10. 20. Adsay NV, Merati K, Basturk O, et al. Pathologically and biologically distinct types of epithelium in intraductal papillary mucinous neoplasms. Delineation of an ‘intestinal’ pathway of carcinogenesis in the pancreas. Am J Surg Pathol 2004; 28: 839–48. 21. Ito H, Endo T, Oka T, et al. Mucin expression profile is related to biological and clinical characteristics of intraductal papillary-mucinous tumors of the pancreas. Pancreas 2005; 30: 96–102. 22. Luttges J, Zamboni G, Longnecker D, Kloppel G. The immunohistochemical mucin expression pattern distinguishes different types of intraductal papillary mucinous neoplasms of the pancreas and determines their relationship to mucinous noncystic carcinoma and ductal adenocarcinoma. Am J Surg Pathol 2001; 25: 942–8. 23. Terris B, Dubois S, Buisine MP, et al. Mucin gene expression in intraductal papillary-mucinous pancreatic tumours and related lesions. J Pathol 2002; 197: 632–7. 24. Amaya S, Sasaki M, Watanabe Y, et al. Expression of MUC1 and MUC2 and carbohydrate antigen Tn change during malignant transformation of biliary papillomatosis. Histopathology 2001; 38: 550–60.