INK4a-ARF alterations and p53 mutations in hepatocellular carcinomas

The INK4a-ARF (CDKN2A)- locus on chromosome 9p21 encodes for two tumour sup pressor 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 alterat ions of p14(ARF), p16(INC4a) and p53. After microdissection, DNA of 71 hepa tocellular carcinomas was analysed for INK4-ARF inactivation and p53 mutati on by DNA sequence analysis, methylation-specific PCR (MSP), restriction-en zyme related polymerase chain reaction (RE-PCR), mRNA expression and immuno histochemistry. In addition, microdeletion of p14(ARF) and p16(INC4a) were assessed by differential PCR. Inactivation of p14(ARF) was found in 11/71 c ases (15%), alterations of p16(INK4a), occurred in 47/71 carcinomas (66%), which correlated with loss of mRNA transcription. Five tumours (7%) had hom ozygous deletions of the INK4a-ARF locus. We failed to detect specific muta tions of both exons. P16(INK4a) methylation with an unmethylated p14(ARF) p romotor 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 es tablish alterations of the INK4a-ARF locus or p53 status as independent pro gnostic factor in these tumours. Our data indicate, that p14(ARF) methylati on occurs independently of p16(INK4a) alterations in a subset of HCC togeth er with wild type p53. The INK4a-ARF-/p53-pathway was disrupted in 86% of H CC, either by p53 mutations or by INK4a-ARF inactivation, and may have co-o perative effects in hepatocarcinogenesis.