Analysis of G(1)/S checkpoint regulators in metastatic melanoma

We have analyzed the expression of the CDKNIA (p21(CIP1)), CDKNIB (p27(Kip1 )), TP53, RBI and MDM2 proteins and tumor cell proliferation by immunohisto chemical staining in 59 cases of metastatic melanoma. The genomic status of the CDKN2A (INK4-ARF, p16/p14(ARF)), CDKN2B (p15) and CDKN2C (P18) genes w as determined by PCR-SSCP (single-strand conformation polymorphism) in 46 o f these cases. These results were correlated with various clinico-pathologi cal parameters, including the outcome of combined chemoimmunotherapy. We fo und positive correlations between the expression of CDKNIA and MDM2 (r = 0. 5063, P = 0.001), between the expression of CDKNIB and RBI (r = 0.5026, P = 0.001), and between RE I expression and tumor cell proliferation (0.5564, P < 0.001). Two mutations in the CDKN2A (p16) gene were detected, including a novel base change AAC-->ATC (Asn to Ile) at codon 71, that also changes the codon 85 of the alternative reading frame gene p14(ARF) from CAA to CAT (Gln to His). Homozygous deletion at exon 2 of the CDKN2A (INK4-ARF) gene was detected in six cases. In seven cases, the 540C-->G polymorphism in the 3'UTR of the CDKN2A (p16) gene was found in linkage disequilibrium with th e 74C-->A polymorphism in intron I of the CDKN2B gene (P < 0.0001). These c ases had significantly lower expression of the TP53 protein (P = 0.0032). B oth 540C-->G and 580C-->T polymorphisms in the 3'UTR of the CDKN2A (p16) ge ne were associated with significantly shorter progression time from primary to metastatic disease (P = 0.0071). We conclude, that although none of the analyzed cell cycle regulators could be singled out as a major prognostic factor, G(1)/S checkpoint abnormalities remain one of the most significant factors in the development of malignant melanoma. (C) 2000 Wiley-Liss, Inc.