Homozygous deletion of the p16INK4a and the p15INK4b tumour suppressor genes in a subset of human sporadic cutaneous malignant melanoma

Chromosome 9p21 is frequently deleted in malignant melanoma, and one familial malignant melanoma gene has been linked to 9p21-22. Recently, the cyclin D-dependent kinase inhibitors (CDKIs) p16(INK4a) and p15(INK4b) have been localized within chromosome 9p21, and the presence of p16(INK4a) point mutations has been demonstrated in familial melanoma and melanoma cell lines in vitro. To analyse the role of these CDKIs in sporadic human cutaneous non-metastatic malignant melanoma, we examined 36 primary tumour specimens representing different stages of melanoma progression and their corresponding normal skin samples for the three mechanisms of CDKI inactivation described so far. Homozygous codeletion of the p16(INK4a) and the p15(INK4b) gene was detected by Southern blot analysis in two tumour samples. By direct sequencing of polymerase chain reaction (PCR)-amplified microdissected genomic DNA, no somatic or germline p16(INK4a) point mutations or small deletions were detected in the remaining 34 tumour samples; one individual exhibited the previously described germline codon 148 (Ala-->Thr) polymorphism. In these tumour specimens, comparative semiquantitative reverse PCR analysis of p16(INK4a) transcript levels revealed no evidence for repression of p16(INK4a) gene transcription and thus for p16(INK4a) promoter inactivation by DNA methylation. These results indicate homozygous p16(INK4a) and p15(INK4b) loss to occur in a subset of cutaneous melanomas and suggest, in view of the frequent loss of heterozygosity on chromosome 9p21, the presence of another tumour suppressor gene within this chromosomal region.