On the Progression From Early-to Late-Stage Melanoma: A Potential Sequence of Molecular Events Using Data From Droplet Digital PCR and Array Comparative Genomic Hybridization, A Pilot Study

ABSTRACT: Melanoma progression is generally associated with a stepwise increase in genomic complexity. Although the copy number variations (CNVs) known to occur in melanoma are well characterized, the order in which they accumulate during melanomagenesis remains incompletely understood. To explore this, we used droplet digital polymerase chain reaction to calculate absolute copy numbers of RREB1 and CDKN2A , the most commonly amplified and deleted genes in melanoma, in 51 formalin-fixed, paraffin-embedded melanomas. Each sample also underwent chromosomal microarray analysis to determine its CNV burden, which we graded according to the number of the following melanoma-associated CNVs present: RREB1 gain, CDKN2A loss, MYC gain, and MYB loss. In this pilot study, increasing CNV burden was associated with a gradual increase in average RREB1 copy number: from 2.00 in CNV-negative melanomas to 2.68 in melanomas with isolated RREB1 gain; 2.75 in melanomas with RREB1 gain and CDKN2A loss; 2.93 in melanomas with RREB1 gain and MYC gain; and 3.23 in melanomas with RREB1 gain, CDKN2A loss, and MYC gain ( F = 10.43, P < 0.001). A parallel trend was observed for average CDKN2A copy number, which decreased progressively with increasing CNV burden: from 2.02 in CNV-negative melanomas to 1.33 in melanomas with isolated CDKN2A loss; 1.15 in melanomas with CDKN2A loss and MYC gain; and 0.53 in melanomas with CDKN2A loss and MYB loss ( F = 12.01, P < 0.001). These findings support a potential model of stepwise CNV accumulation in melanoma: early RREB1 gain and/or CDKN2A loss, followed by MYC gain, and ultimately MYB loss.