Massively parallel sequencing analysis of benign melanocytic naevi

Abstract

Aims: Melanocytic naevi are benign lesions of the skin or mucosa that may constitute non-obligate precursors of malignant melanoma, particularly when they show lentiginous and dysplastic features. The aim of this study was to investigate the repertoire of somatic genetic alterations in melanocytic naevi.

Methods and results: DNA extracted from 12 melanocytic naevi and DNA from matching normal tissue were separately microdissected and subjected to targeted massively parallel sequencing of ≥300 cancer genes. A median of 5.5 (range 1-12) non-synonymous somatic mutations were detected, with 10 cases harbouring mutually exclusive BRAF V600E (6/12) or NRAS (4/12) clonal hotspot mutations. One of the two cases lacking BRAF and NRAS mutations was a dysplastic naevus harbouring an HRAS Q61L hotspot mutation. Analysis of the laser-capture microdissected components of a naevus synchronously diagnosed with in-situ and invasive malignant melanoma revealed a truncal, clonal BRAF V600E mutation, and the acquisition of a CDKN2A homozygous deletion in the invasive component, in conjunction with additional clonal mutations affecting NF2, FAT4 and KDR in both in-situ and invasive malignant components.

Conclusion: Melanocytic naevi harbour recurrent BRAF V600E or NRAS hotspot mutations with low mutational burdens. Our findings also show that progression from naevi to malignant melanoma may be driven by the acquisition of additional genetic alterations, including CDKN2A homozygous deletions.

Keywords: melanocytic naevi; melanoma.

Figure 1:. Morphologic features of melanocytic nevi.

Representative micrographs of melanocytic nevi included in this study. A) SKIN3T. B) SKIN5T. C) SKIN6T. D) SKIN7T. E) SKIN8T. F) SKIN9T. G) SKIN11T. H) SKIN13T. I) SKIN14T. J) SKIN15T. K) SKIN16T.

Figure 2:. Repertoire of non-synonymous somatic mutations detected by targeted capture massively parallel sequencing in melanocytic nevi.

Heatmap indicating the non-synonymous somatic mutations identified in the SKINs analyzed. Each column represents a sample; mutated genes are reported in rows. The types of somatic mutations identified are color-coded according to the legend. Loss of heterozygosity (LOH) is identified by a diagonal bar. Genes significantly mutated in melanomas are highlighted with an asterisk on the right side of the heatmap.

Figure 3:. Comparison of somatic genetic alterations in melanocytic nevi to that of previously reported sequencing studies in nevi.

A) Heatmaps displaying recurrent and/ or shared non-synonymous somatic mutations between the nevi reported in this study (n=12; left) to that of dysplastic nevi reported by Melamed et al (n=15; middle) and globular and reticular nevi reported by Stark et al (n=12 and n=18, respectively; right). Each column represents a sample; mutated genes are reported in rows. The types of somatic mutations identified are color-coded according to the legend. B) Frequency plots and Fisher’s exact test corrected for multiple testing comparing copy number gains and losses between the melanocytic nevi from this study (n=12) and the combined cohort of nevi (n=30; left), globular nevi (n=12; middle), or reticular nevi (n=18; right) from Stark et al. The frequency of gains (green bars) or losses (purple bars) for each gene is plotted on the y-axis according to genomic location (x-axis). Inverse Log₁₀ values of the Fisher’s exact test p-values are plotted according to genomic location. All statistical tests were two-sided.

Figure 4:. Malignant transformation of a nevus to malignant melanoma in situ to invasive melanoma.

A) Representative micrographs of SKIN23T representing a benign nevus (* in top panel) and invasive melanoma (** in top panel) and malignant melanoma in situ (bottom panel). B) The results of the targeted capture MPS analysis showing cancer cell fractions (CCFs) which were based on an integrative analysis of mutant allele fractions, tumor cell content, ploidy, and local copy number using ABSOLUTE and reported for the non-synonymous and promoter somatic mutations identified in a given lesion. CCFs are color-coded according to the legend. Clonal mutations are highlighted in orange boxes. Genes significantly mutated in melanomas are highlighted with an asterisk on the right side of the heatmap. C) Heatmap illustrating the repertoire of copy number alterations as defined by targeted capture MPS. Samples are represented as columns and chromosomes are represented along the y axis. Absolute copy number was defined by ABSOLUTE and are color-coded according to the legend to the right of the heatmap. D) Phylogenetic tree depicting the evolution of the progression from benign nevus to invasive melanoma. The colored branches represent each of the subclones identified, and selected somatic genetic alterations that define a given clone are illustrated along the branches.