Whole-exome sequencing identifies recurrent SF3B1 R625 mutation and comutation of NF1 and KIT in mucosal melanoma

Abstract

Mucosal melanomas are a rare subtype of melanoma, arising in mucosal tissues, which have a very poor prognosis due to the lack of effective targeted therapies. This study aimed to better understand the molecular landscape of these cancers and find potential new therapeutic targets. Whole-exome sequencing was performed on mucosal melanomas from 19 patients and 135 sun-exposed cutaneous melanomas, with matched peripheral blood samples when available. Mutational profiles were compared between mucosal subgroups and sun-exposed cutaneous melanomas. Comparisons of molecular profiles identified 161 genes enriched in mucosal melanoma (P<0.05). KIT and NF1 were frequently comutated (32%) in the mucosal subgroup, with a significantly higher incidence than that in cutaneous melanoma (4%). Recurrent SF3B1 R625H/S/C mutations were identified and validated in 7 of 19 (37%) mucosal melanoma patients. Mutations in the spliceosome pathway were found to be enriched in mucosal melanomas when compared with cutaneous melanomas. Alternative splicing in four genes were observed in SF3B1-mutant samples compared with the wild-type samples. This study identified potential new therapeutic targets for mucosal melanoma, including comutation of NF1 and KIT, and recurrent R625 mutations in SF3B1. This is the first report of SF3B1 R625 mutations in vulvovaginal mucosal melanoma, with the largest whole-exome sequencing project of mucosal melanomas to date. The results here also indicated that the mutations in SF3B1 lead to alternative splicing in multiple genes. These findings expand our knowledge of this rare disease.

Fig. 1

Mutational landscape of 19 mucosal melanomas compared with cutaneous melanomas. Important differences in individual genes are shown along with P values. Note in particular the significantly higher frequency of mutations in KIT, NF1, and SF3B1 in mucosal melanomas. Common melanoma genes occur in low frequency as highlighted within the figure. Tissue source and mutational signature for individual samples are presented in the lower panel.

Fig. 2

Molecular classification of melanoma with BRAF (V600), NRAS (G12, G13, Q61), NF1, and KIT mutations. (a) Comparison of all 19 mucosal melanomas with our 135 cutaneous melanomas. (b) The difference in molecular classifications between mucosal melanomas arising in different anatomic sites.

Fig. 3

(a) Pathway analysis of genes enriched in mucosal melanoma. (b) Spliceosome pathway genes found in mucosal melanoma were enriched when compared with cutaneous melanomas. (c) Pathway visualization of the spliceosome pathway with mutated genes shown in red.

Fig. 4

(a) Number and location of SF3B1 mutations found in the literature and cBioPortal, followed by the proportion of each cancer’s subtype. Highlighted in pink is the current study representing the largest percentage SF3B1 mutations of any cohort to date. (b–e) Analysis of differentially spliced genes identified in SF3B1-mutant uveal melanoma and breast cancer. (b) CRNDE, (c) RPL31, (d) ABCC5, and (e) TMEM14C were validated using quantitative reverse-transcription PCR in three SF3B1 wild-type (blue) and two SF3B1 R625 (red) mucosal melanoma PDX samples. Uveal melanoma cell lines were used as controls, MP41 (SF3B1 wild-type) and Mel202 (SF3B1 R625G). Unpaired two-tail parametric t-tests were used to determine significance between mutant and wild-type. *P value less than 0.05 was considered statistically significant. Schematic representations of the splicing events is illustrated above each graph. Exons are represented as boxes, with lines representing splicing events occurring in SF3B1 wild-type (blue) and SF3B1 mutant (red). ***P value of less than 0.01.