High Frequency of p16INK4A Promoter Methylation in NRAS-Mutated Cutaneous Melanoma

The p16INK4A tumor suppressor is often deleted, or otherwise inactivated, in malignant melanoma. To investigate the loss of p16INK4A in greater detail, we analyzed 77 cutaneous melanoma metastases. Of these 56 retained at least one p16INK4A allele, and 21 had biallelic deletions. Using methylation-specific PCR, direct sequencing, and immunohistochemical methods, we analyzed p16INK4A promoter methylation, mutations, and protein expression, respectively. In addition, 14 corresponding primary tumors were analyzed for protein expression. Results were compared to clinicopathological parameters and previously obtained data regarding mutations in proto-oncogenes NRAS and BRAF. Results revealed that p16INK4A promoter methylation was present in 15 of 59 (25%) metastases; nonsynonymous mutations in 9 of 56 (16%) metastases; and protein expression in 12 of 67 (18%) metastases. Protein expression was lost during progression from primary to metastatic tumors, 71% (10 of 14) and 43% (6 of 14) being positive, respectively. However, the genetic and epigenetic alterations of p16INK4A observed could not explain the lack of p16INK4A protein in 27 metastases, indicating the presence of additional inactivating mechanisms for p16INK4A. Interestingly, p16INK4A promoter methylation was significantly overrepresented in NRAS-mutated samples compared to NRAS wild-type samples (P=0.0004), indicating an association between these two events.