Background:Biliary tract cancers (BTC) are relatively rare malignant tumours with poor prognosis. It is known from other solid neoplasms that antitumour inflammatory response has an impact on tumour behaviour and patient outcome. The aim of this study was to provide a comprehensive characterisation of antitumour inflammatory response in human BTC.Methods:Tumour-infiltrating T lymphocytes (CD4+, CD8+, and Foxp3+), natural killer cells (perforin+), B lymphocytes (CD20+), macrophages (CD68+) as well as mast cells (CD117+) were assessed by immunohistochemistry in 375 BTC including extrahepatic (ECC; n=157), intrahepatic (ICC; n=149), and gallbladder (GBAC; n=69) adenocarcinomas. Overall and intraepithelial quantity of tumour-infiltrating immune cells was analysed. Data were correlated with clinicopathological variables and patient survival.Results:The most prevalent inflammatory cell type in BTC was the T lymphocyte. Components of the adaptive immune response decreased, whereas innate immune response components increased significantly in the biliary intraepithelial neoplasia – primary carcinoma – metastasis sequence. BTC patients with intraepithelial tumour-infiltrating CD4+, CD8+, and Foxp3+ T lymphocytes showed a significantly longer overall survival. Number of total intraepithelial tumour-infiltrating Foxp3+ regulatory T lymphocytes (HR: 0.492, P=0.002) and CD4+ T lymphocytes (HR: 0.595, P=0.008) were tumour grade- and UICC-stage-independent prognosticators. The subtype-specific evaluation revealed that the tumour-infiltrating lymphocytic infiltrate is a positive outcome predictor in ECC and GBAC but not in ICC.Conclusion:Our findings characterise the immune response in cholangiocarcinogenesis and identify inflammatory cell types that influence the outcome of BTC patients. Further, we show that BTC subtypes show relevant differences with respect to density, quality of inflammation, and impact on patient survival.

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The molecular mechanisms underlying the genesis of cholangiocarcinomas (CCs) are poorly understood. Epigenetic changes such as aberrant hypermethylation and subsequent atypical gene expression are characteristic features of most human cancers. In CC, data regarding global methylation changes are lacking so far. We performed a genome-wide analysis for aberrant promoter methylation in human CCs. We profiled 10 intrahepatic and 8 extrahepatic CCs in comparison to non-neoplastic biliary tissue specimens, using methyl-CpG immunoprecipitation (MCIp) combined with whole-genome CpG island arrays. DNA methylation was confirmed by quantitative mass spectrometric analysis and functional relevance of promoter hypermethylation was shown in demethylation experiments of two CC cell lines using 5-aza-2 0 deoxycytidine (DAC) treatment. Immunohistochemical staining of tissue microarrays (TMAs) from 223 biliary tract cancers (BTCs) was used to analyze candidate gene expression at the protein level. Differentially methylated, promoter-associated regions were nonrandomly distributed and enriched for genes involved in cancer-related pathways including Wnt, transforming growth factor beta (TGF-b), and PI3K signaling pathways. In CC cell lines, silencing of genes involved in Wnt signaling, such as SOX17, WNT3A, DKK2, SFRP1, SFRP2, and SFRP4 was reversed after DAC administration. Candidate protein SFRP2 was substantially down-regulated in neoplastic tissues of all BTC subtypes as compared to normal tissues. A significant inverse correlation of SFRP2 protein expression and pT status was found in BTC patients. Conclusion: We provide a comprehensive analysis to define the genome-wide methylation landscape of human CC. Several candidate genes of cancer-relevant signaling pathways were identified, and closer analysis of selected Wnt pathway genes confirmed the relevance of this pathway in CC. The presented global methylation data are the basis for future studies on epigenetic changes in cholangiocarcinogenesis. (HEPATOLOGY 2014;59:544-554)