Exome sequencing of desmoplastic melanoma identifies recurrent NFKBIE promoter mutations and diverse activating mutations in the MAPK pathway

Abstract

Desmoplastic melanoma is an uncommon variant of melanoma with sarcomatous histology, distinct clinical behavior and unknown pathogenesis. We performed low-coverage genome and high-coverage exome sequencing of 20 desmoplastic melanomas, followed by targeted sequencing of 293 genes in a validation cohort of 42 cases. A high mutation burden (median of 62 mutations/Mb) ranked desmoplastic melanoma among the most highly mutated cancers. Mutation patterns strongly implicate ultraviolet radiation as the dominant mutagen, indicating a superficially located cell of origin. Newly identified alterations included recurrent promoter mutations of NFKBIE, encoding NF-κB inhibitor ɛ (IκBɛ), in 14.5% of samples. Common oncogenic mutations in melanomas, in particular in BRAF (encoding p.Val600Glu) and NRAS (encoding p.Gln61Lys or p.Gln61Arg), were absent. Instead, other genetic alterations known to activate the MAPK and PI3K signaling cascades were identified in 73% of samples, affecting NF1, CBL, ERBB2, MAP2K1, MAP3K1, BRAF, EGFR, PTPN11, MET, RAC1, SOS2, NRAS and PIK3CA, some of which are candidates for targeted therapies.

Figure 1. Desmoplastic melanomas have a substantial point mutation burden consistent with UV-radiation induced damage

The 62 tumors are ordered by their mutation burden (top panel) with the mutation types annotated (bottom panel). Dashed line corresponds to the mutation burden observed in sun-exposed non-desmoplastic melanoma: 15 mutations/Mb^,. In the bottom panel, C>T mutations following a purine (*) or a pyrimidine (**) are distinguished. Tumors from patients older than 55 years of age had significantly more mutations as compared to those from younger patients (p=2×10^-3, t-test). Cases from the discovery cohort are marked with a “D”.

Figure 2. Nomination of driver mutations in desmoplastic melanoma

A. The number of specific mutations detected (y-axis) is stratified by those mutations' occurrences across samples (x-axis). For example, 3,364 mutations occurred only once, whereas there were 151 mutations occurring in two samples and a single mutation was observed 9 times, affecting the NFKBIE genetic locus. There was also a secondary hotspot in NFKBIE, 15 basepairs from the more common mutation site, which was mutated in two samples. B. Q-Q plot of loss-of-function burdens compared to expected loss-of-function burdens, calculated as described. Solid and dotted lines correspond to false discovery rates (FDRs) of 1.0 and 0.5, respectively. The most significant genes are labeled. C. Tumor suppressor candidates have an increased proportion of damaging mutations and fully clonal mutant allele frequencies (MAFs), undergoing loss of heterozygosity in some cases. Left panel: Fraction of mutation categories compared to all mutations. Right panel: Normalized MAFs (calculated as described) of candidate mutations compared to all mutations. Red vertical bars indicate average MAFs.

Figure 3. Genetic alterations of CBL, MAP3K1, FBXW7, and NFKBIE

A. Enrichment of damaging mutations of CBL in desmoplastic melanoma. B. Recurrent amplification of MAP3K1. C. Enrichment of damaging mutations of FBXW7 in desmoplastic melanoma. D. Recurrent amplification of NFKBIE.

Figure 4. Recurrent mutations affect the promoter of NFKBIE

Mutations are annotated over the entire NFKBIE genetic locus (top panel) and a zoomed inset (bottom panel) with selected tracks featured as described in the main text.

Figure 5. The mutational landscape of desmoplastic melanoma

A. Tiling plot of genetic alterations (rows) in each sample (columns). Mutations were considered homozygous if their MAFs were 1.5× the sample median. Numbers indicate the percent of samples harboring oncogenic alterations. Purple (*) tiles indicate unique hotspot mutations as discussed. GOF = gain-of-function. B. Pathways affected.