INK4a-ARF alterations and p53 mutations in hepatocellular carcinomas

The INK4a-ARF (CDKN2A)- locus on chromosome 9p21 encodes for two tumour suppressor proteins, p16^INK4a and p14^ARF, that act as upstream regulators of the Rb-CDK4 and p53 pathways. To study the contribution of each pathway in tumorigenesis of hepatocellular carcinoma (HCC), we analysed the alterations of p14^ARF p16^INC4a and p53. After microdissection, DNA of 71 hepatocellular carcinomas was analysed for INK4-ARF inactivation and p53 mutation by DNA sequence analysis, methylation-specific PCR (MSP), restriction-enzyme related polymerase chain reaction (RE-PCR), mRNA expression and immunohistochemistry. In addition, microdeletion of p14^ARF and p16^INC4a were assessed by differential PCR. Inactivation of p14^ARF was found in 11/71 cases (15%), alterations of p16^INK4a occurred in 47/71 carcinomas (66%), which correlated with loss of mRNA transcription. Five tumours (7%) had homozygous deletions of the INK4a-ARF locus. We failed to detect specific mutations of both exons. P16^INK4a methylation with an unmethylated p14^ARF promotor appeared in 39 cases. Mutations of p53 were found in 30 of 71 HCC (42%), and only one of them harboured p14^ARF inactivation. We failed to establish alterations of the INK4a-ARF locus or p53 status as independent prognostic factor in these tumours. Our data indicate, that p14^ARF methylation occurs independently of p16^INK4a alterations in a subset of HCC together with wild type p53. The INK4a-ARF-/p53-pathway was disrupted in 86% of HCC, either by p53 mutations or by INK4a-ARF inactivation, and may have co-operative effects in hepatocarcinogenesis.