Preferentially different mechanisms of inactivation of 9p21 gene cluster in liver fluke–related cholangiocarcinoma

SummaryCholangiocarcinoma in northeast Thailand is associated with liver fluke infection. Mechanisms of inactivation of the p15INK4b, p16INK4a, and p14ARF 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 p14ARF, p15INK4b, and p16INK4a 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 p14ARF, 48.9% for p15INK4b, and 28.3% for p16INK4a. Loss of protein expression of p14ARF, p15INK4b, and p16INK4a was 30.9%, 58%, and 81.5%, respectively. Both p14ARF 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 p14ARF, p15INK4b, and p16INK4a in liver fluke–related cholangiocarcinoma are preferentially different, by which epigenetic event being the main mechanism of p14ARF, whereas p16INK4a and p15INK4b inactivation occurs through genetic and both genetic and epigenetic events, respectively.