Genetic and epigenetic alterations of the INK4a-ARF pathway in cholangiocarcinoma

The INK4a-ARF locus, located on chromosome 9p21, encodes two cell-cycle regulatory proteins, p16^INK4a and p14^ARF, acting through the Rb-CDK4 and p53 pathways. To study the contribution of each pathway in the tumuorigenesis of cholangiocarcinoma, the alterations of p14^ARF, p16^INK4a, p53, and pRb were analysed. After microdissection, DNAs from 51 cholangiocarcinomas were analysed by methylation-specific PCR (MSP), restriction-enzyme related polymerase chain reaction (RE-PCR), microsatellite analysis, mRNA expression, and DNA sequencing. Immunohistochemistry of p14^ARF, p16^INK4a, p53, and pRb was also performed. Promoter methylation of p14^ARF was found in 13/51 cases (25%) and p16^INK4a showed aberrant promoter methylation in 39/51 case (76%) which correlated with loss of mRNA transcription. Two tumours (4%) had homozygous deletion of the INK4a-ARF locus. Specific mutations of both exons were not detected. p14^ARF inactivation appeared in the context of an unmethylated p16^INK4a promoter in eight of 13 cases (61%) of the carcinomas methylated at p14^ARF. Mutations of p53 were found in 19 of 51 tumours (37%), and four of them (21%) harboured p14^ARF inactivation. The pRb protein was detected in 30/51 (59%) tumours examined. The absence of pRB protein did not correlate with any of the examined parameters. Alterations of the INK4a-ARF locus, pRB or p53 status could not be established as independent prognostic factors in these tumours. These findings indicate that the INK4a-ARF locus is frequently inactivated in cholangiocarcinoma of the liver and occurs independently of the status of p53 or pRb.