Abstract

The progression from early- to late-stage melanoma typically involves a gradual loss of genomic integrity. However, little is known about how these alterations contribute to melanomagenesis. We explored this question using droplet digital PCR (ddPCR), an emerging technology for copy number variation (CNV) analysis. We performed ddPCR on 29 melanomas (16 primary; 13 metastatic) for absolute quantitation of DNA copy number in RREB1, CDKN2A, MYC, and MYB. Chromosomal microarray analysis (CMA) was used to confirm the presence of CNVs in these genes. Melanomas were grouped into: (1) no CNVs (N=7); (2) isolated RREB1 gain (N=6); (3) RREB1 gain and CDKN2A loss only (N=5); (4) RREB1 gain, CDKN2A loss, and MYC gain only (N=4); and (5) RREB1 gain, CDKN2A loss, MYC gain and MYB loss (N=1). Average ddPCR-determined RREB1 copy numbers for the groups were 2.00, 2.64, 2.73, 3.14, and 3.21. Notably, group 4 melanomas had a significantly higher average RREB1 copy number (3.14) than group 2 melanomas (2.64) (p < 0.05). The remaining 6 melanomas had isolated CDKN2A loss; no melanomas had isolated MYC gain or isolated MYB loss. In our cohort, RREB1 gain was the most common initiating event in the progression from early- to late-stage melanoma, followed by CDKN2A loss. This suggests that, at least for some cases, genomic changes acquired during melanomagenesis occur in a stepwise fashion: RREB1 gain (and/or CDKN2A loss) before MYC gain and finally MYB loss. These findings augment the growing body of literature surrounding the molecular pathways governing melanomagenesis, and justify further investigation.