Clinical Significance of Cell Cycle and Apoptosis- Related Markers in Biliary Tract Cancer,Cholangiocarcinoma is the second most common primary hepatic tumor, and its incidence is increasing. Prevention of this cancer is very difficult, and only a few risk factors have been so far identified. Cholangiocarcinoma arises from the biliary epithelial cells and can occur anywhere along the biliary tract.,Tumors are divided anatomically into intrahepatic and extrahepatic cholangiocarcinomas, as well as adenocarcinomas of the gallbladder.Despite their similar morphologic features, differences have been found among these subtypes in relation to their clinical course, genetic profiles, and immunophenotypes.,Materials and Methods,Materials and Methods Formalin-fixed and paraffin-embedded tumor and control specimens were retrieved from the archives of the Institute of Pathology, University of Bern, Bern, Switzerland. All tumor and control specimens were reviewed by one pathologist (E.K.).,. The TMA consisted of 128 cholangiocarcinoma cases including 42 intrahepatic and 70 extrahepatic cholangiocarcinomas and 16 gallbladder carcinomas, as well as 29 normal control samples. The 128 patients comprised 70 men and 58 women with a mean SD age of 64 2 years (range, 30-90 years).,Assessment of Behavior Medical charts were available for 57 (44.5%) of 128 patients. Of these 57 patients, 45 (79%) died of the disease, and 6 (11%) were alive with recurrent or metastatic disease. An additional 6 patients (11%) were alive without disease. The median follow-up was 11.5 months (mean SD, 21.05 4.9 months; range, 0-108 months).,Immunohistochemical Staining,The TMA sections were used for immunohistochemical staining with 7 different antibodies. The primary antibodies and their dilutions are listed in Table 1.,Immunohistochemical Staining,. The primary antibody was omitted for negative control experiments. For a positive control experiment, a TMA with various normal tissue samples was stained in parallel. Nuclear immunoreactivity for p53, p16, p21, and p27 was visually scored. A case was considered positive if immunoreactivity was detected in at least 10% of the tumor cell nuclei, on the basis of previous reports. Only for p27, the cutoff was 50% of positive tumor cell nuclei.,For bcl-2 and bax, staining in more than 10% of tumor cells within a tissue spot was required to define positivity.For COX-2, tumor samples were considered positive when more than 20% of the cells were immunoreactive.,Results,The immunohistochemical findings are summarized in Table 2 and Table 3. Representative IHC stainings are shown in Image 1 and Image 2.,Immunohistochemical detection of Ki-67 (A),p53 (B),bcl-2 (C),bax (D) in cholangiocarcinoma.,Immunohistochemical detection of p16 (A),p21,p27,Cholangiocarcinomas vs Normal Control Samples,In comparison with normal tissue (29 specimens), cholangiocarcinoma cases expressed COX-2, p53, bcl-2, and bax significantly more frequently. In more detail, the majority of the tumors (84.0%) were found to be COX-2+, whereas only 12% of the normal tissues (3/25) exhibited a positive reaction (P .005).,Positive p53 nuclear staining was observed in 23.5% of the tumors, whereas no nuclear staining was noted in the normal bile ducts (P = .0225). Regarding bcl-2 and bax immunoreactivity, 23.6% and 41.3% of the cholangiocarcinomas, respectively, showed a positive cytoplasmic reaction. Normal bile ducts remained consistently immunonegative for bcl-2 (P = .0409), whereas only 5% of the control cases showed a positive reaction for bax (P = .0037).,Differences in the Immunophenotype Between Intrahepatic and Extrahepatic Tumors,Distinctive immunophenotypes were observed regarding localization of the neoplasms. Intrahepatic tumors more frequently expressed bcl-2 and p16 (ie, 15/35 43% and 15/33 45%, respectively) than did extrahepatic cholangiocarcinomas, in which bcl-2 and p16 immunostaining was noted in 10 (14%) of 73 and 12 (18%) of 66 cases, respectively (P = .0036 and P = .0133). In contrast, p53 immunoreactivity was restricted to extrahepatic tumors, being found in 20 (32%) of 62 extrahepatic cholangiocarcinomas and 4 (31%) of 13 gallbladder carcinomas, whereas no p53 nuclear staining (0%) was observed in their intrahepatic counterparts (P = .0035).,Prognostic Significance,Regarding survival, patients with p16+ tumors survived significantly longer than patients with p16 tumors (P .001) Figure 1, and loss of p16 expression was associated with reduced survival. The expression of the other markers (COX-2, p53, bcl-2, bax, p21, and p27) did not show any correlation with the survival.,Discussion,Cholangiocarcinoma is a devastating cancer with presently no effective treatment. The primary aim of this study was to examine cholangiocarcinomas from different anatomic sites to identify similarities and differences in the expression of diagnostic and prognostic factors that would eventually provide a guide for developing novel therapeutic strategies for this lethal disease.,