Mutational analysis of the CDKN2 gene in metastases from patients with cutaneous malignant melanoma

Abstract

A Platz', U Ringborg', B LagerlOf, E Lundqvist3, P Sevigny3 and M Inganas3Departments of 'Oncology and 2Pathology, Radiumhemmet, Karolinska Hospital, S-1 71 76 Stockholm; 3Pharmacia Biotech, S-751 82 Uppsala, Sweden.Summary We analysed 26 metastases from 25 patients with sporadic cutaneous malignant melanoma for alterations in the CDKN2 gene by a combined polymerase chain reaction/single-strand conformation polymorphism (PCR/SSCP)/nucleotide sequencing approach. Eleven alterations (one in exon 1, five in exon 2 and five in the 3' non-coding sequence of the exon 3 region) were concordantly and independently detected by both SSCP and nucleotide sequence analysis. Two of the exon 2 changes and the five changes in the non-coding exon 3 region are likely to represent natural polymorphism. Four (15%) of 26 metastases thus had CDKN2 mutations and belonged to 3 (12%) of 25 patients. Semi-quantitative PCR furthermore revealed no sign of homozygous deletions of the CDKN2 exon 2 region. The results support an involvement of the CDKN2 product in the development of a subgroup of sporadic melanomas and encourage the search for alterations in additional genes of the 9p21 region.

Genes involved in cell cycle G1 checkpoint control are frequently mutated in human melanoma metastases

“…Histological examination of the tumour tissue pieces revealed the presence of highly homogeneous tumour cell populations. There was no sign of homozygous deletions involving the investigated gene regions within the limits of the employed PCR method (Platz et al, 1996). The presence of subclones with gene losses may, however, have escaped detection.…”

“…The same samples had previously been analysed for CDKN2A and mutant alleles found (Platz et al, 1996).…”

“…Mutational analyses of both p53 and CDKN2A in tumour samples from sporadic human malignant melanoma have been carried out by several investigators, but conflicting results have been obtained (Volkenandt et al, 1991;Castresana et al, 1993;Florenes et al, 1994;Ohta et al, 1994;Gruis et al, 1995;Platz et al, 1996). No report on mutational alteration of the p15 gene CDKN2B in sporadic human malignant melanoma has yet been published.…”

“…We have recently registered the presence of somatic mutations in the CDKN2A gene in melanoma metastases from 3/25 (12%) of patients with sporadic cutaneous malignant melanoma (Platz et al, 1996). The present report describes the mutational analysis of the same patient samples for CDKN2B exons 1 and 2, for p53 exons 4-9 and for N-ras exon 2, by polymerase chain reaction-single-strand-conformation polymorphism (PCR/SSCP) and their final characterisation by nucleotide sequence analysis.…”

“…Polymerase chain reaction (PCR) and single strand conformation polymorphism analysis (SSCP) Amplification of CDKN2A exon regions and their SSCP analysis has been reported earlier (Platz et al, 1996). The CDKN2B exon 1 and 2 regions were separately amplified.…”