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Summary: Cholangiocarcinoma in northeast Thailand is associated with liver fluke infection. Mechanisms of inactivation of the p15 INK4b , p16 INK4a , and p14 ARF have been reported in many human cancers but have not hitherto been studied in liver fluke–related cholangiocarcinoma, particularly genetic and epigenetic effects on protein expression. We investigated loss of heterozygosity and microsatellite instability and performed fine mapping of the chromosomal region 9p21-pter in 94 microdissected cholangiocarcinoma samples using polymerase chain reaction based–microsatellite markers. Methylation and protein expression of p14 ARF , p15 INK4b , and p16 INK4a was determined using methylation-specific polymerase chain reaction and immunohistochemistry, respectively. Genetic and epigenetic alterations, including loss of protein expression, were correlated with clinicopathological data. Fine mapping at 9p21-pter showed a distinctive region between D9S286 and D9S1752 of common loss. Methylation frequency was 40.2% for p14 ARF , 48.9% for p15 INK4b , and 28.3% for p16 INK4a . Loss of protein expression of p14ARF, p15INK4b, and p16INK4a was 30.9%, 58%, and 81.5%, respectively. Both p14 ARF methylation and allelic loss at 9p21 were associated with loss of p14ARF expression. Poor prognosis was associated with loss of p16INK4a expression. In conclusion, mechanisms of inactivation of p14 ARF , p15 INK4b , and p16 INK4a in liver fluke–related cholangiocarcinoma are preferentially different, by which epigenetic event being the main mechanism of p14 ARF , whereas p16 INK4a and p15 INK4b inactivation occurs through genetic and both genetic and epigenetic events, respectively. [Copyright &y& Elsevier]

Abstract: Cholangiocarcinoma is a crucial health problem in Northeast Thailand where liver fluke infection is endemic. However, molecular genetic and epigenetic mechanisms involved in carcinogenesis of this cancer remain unclear. We attempted to study genetic and epigenetic alterations of hMLH1 gene in 65 intrahepatic cholangiocarcinoma using polymerase chain reaction (PCR) based microsatellite marker D3S1611 and methylation-specific PCR, respectively. Of 65 cases, 29 (44.6%) showed hypermethylation of hMLH1 promoter. Loss of heterozygosity (LOH) of hMLH1 was detected in 12 of 51 informative cases (23.5%). Five out of 29 (17.2%) methylated cases demonstrated LOH. Aberrant methylation of hMLH1 promoter was significantly associated with poorly differentiated type (P=0.013). Our study suggests that both genetic and epigenetic mechanisms cause the inactivation of hMLH1 where epigenetic is a major event resulting in mismatch repair deficiency and contributing to carcinogenesis of liver fluke related cholangiocarcinoma. Since, gene silencing by methylation is an early event in carcinogenesis, promoter hypermethylation of hMLH1 may be a molecular targeted therapy and prevention of liver fluke related cholangiocarcinoma. [Copyright &y& Elsevier]