The molecular pathogenesis of biliary tract cancers is poorly understood. By performing immunohistochemical analysis of more than 125 surgically resected cases of biliary tract carcinoma, we have previously shown altered cell cycle regulatory protein expression in biliary tact cancers . Our current findings also show mutated expression of a large number of cell cycle regulators including UBD, BCL2L2, CDC2, MCM2, and CDKN1C in all subtypes. Similarly, Kang et al.  found that expression of G1-S modulators were commonly mutated in 42 cases of IHC. Total loss of p16, p27, and Rb were detected at rates of in 36%, 31%, 12%, respectively, in cancer specimens. Furthermore, in the above study, even in 7 of 13 cases of biliary dysplasia, without frank carcinoma, abnormal expression of p53, cyclin D1 or p16 was detected. Kim et al.  reported that the mutation of the p53, p16, and K-ras genes occurred at rates of 36%, 31% and 20%, respectively, in GBC. A further finding of the above study was that 100% of GBCs and 80% of adenomas displayed loss of heterozygosity at a minimum of one locus which is consistent with our CGH results. Chang et al.  studied loss of heterozygosity in 32 cases of GBC and 11 cases of dysplasia. Loss of one allele was identified on chromosomes 5q (55%) and 17p (40%) in the dysplastic cases and on chromosomes 3p (52%), 5q (66%), 9p (52%), and 17p (58%) in the carcinomas. Loss of heterozygosity on multiple chromosomes was significantly more frequent in patients with metastatic disease than in cases without metastases. In the current report, we similarly found that segments of 3p and 9p were commonly deleted across all subtypes of biliary cancers. However, we additionally discovered that segments of 6q, 8p, and 14q were commonly deleted across subtypes of biliary cancers
There is increasing evidence that overexpression of tyrosine kinase growth factor receptors such as ErbB-2, epidermal growth factor receptor (EGFR), and Met play important roles in the development of biliary tract carcinomas. Nakasawa et al.  studied tyrosine kinase receptor proteins expression by in 221 biliary tract carcinomas and found that overexpression of ErbB-2 was found in 16% of carcinomas of the gallbladder and a slightly lower percentage of extrahepatic bile duct tumors. ErbB-2 gene amplification was present in 79% of cases. Overexpression of EGFR was found in 8% of tumors and was also associated with a high frequency of gene amplification (77%). Met overexpression was most frequent in IHC (21.4%) but was not associated with gene amplification. Microsatellite instability also appears to be a critical factor in selected cases of biliary carcinogenesis. Roa et al.  performed microsatellite analysis on 59 frozen GBC specimens using 13 different markers. They found evidence of microsatellite instability in equal proportions in early and late cancers, and it was also found in premalignant lesions, indicating that inactivation of mismatch repair genes occurs early in gallbladder carcinogenesis.
In addition to finding that a large proportion of differentially expressed genes in this study involved in cell cycle regulation and apoptosis, we also discovered a disproportionate number of mutated genes that control transcriptional regulation, RNA procession, cellular signaling, or are involved with cytoskeletal structure, extracellular matrix, and cellular adhesion. Differentially expressed genes involved with transcriptional regulation include STAT1, NARG1, HOXC6, and MMP11. Important genes involved with signal transduction with altered expression include CXCL5, ECT2, GPRC5A, MELK, and CKS2. Dysregulated genes involved with cytoskeleton, extracellular matrix and cellular adhesion include ITGA7, LAMB3, CECAM5, KRT6B, and CLDN18.
The findings of the present study will serve as a resource for other investigators in this area as we have indentified many potential targets for therapeutic intervention. As an example, we found that TYMS, which encodes an enzyme that catalyzes 5-fluorouracil, was overexpressed 7.2 – 26.0-fold depending on biliary cancer subtype. TYMS expression is correlated inversely with clinical response to 5-fluorouracil-based chemotherapy and the overexpression may explain the futility of 5-fluorouracil-based chemotherapy for biliary carcinomas .
We also found that a number of genes in the ubiquitin pathway had altered expression in each cancer subtypes. For example, more than 20 ubiquitin-related genes had significantly altered expression IHC. In GBC, UBD was overexpressed more than 200-fold and UBE2C was overexpressed nearly 15-fold. Ubiquitin and ubiquitin-like proteins are signaling messengers that regulate a variety of cellular processes including cell proliferation, cell cycle regulation, DNA repair, and apoptosis. There is accumulating evidence that deregulation of this pathway as a result of mutations or altered expression of ubiquitylating or de-ubiquitylating enzymes as well as of Ub-binding proteins affect crucial mediators of these functions and are underlie the pathogenesis of several human malignancies . A variety of inhibitors of the ubiquitin system are currently being experimentally tested in clinical trials with promising early results . These data suggests these inhibitors may have applicability as adjuvants in treating patients with biliary tract carcinomas.
Another promising target uncovered in this report is STAT-1 which was overexpressed nearly 9-fold in cases of cholangiocarcinoma. The Signal Transducers and Activator of Transcription (STAT) proteins regulate many aspects of cell growth, survival and differentiation. The transcription factors of this family are activated by the Janus Kinase JAK and dysregulation of this pathway has been observed in primary tumors and leads to increased angiogenesis, metastases, enhanced survival of tumors, and immunosuppression [23, 24]. A number of JAK/STAT pathway inhibitors are being tested in pre-clinical studies and their application to cancers of the biliary tract may prove promising .