Abstract
To reveal the clonal architecture of melanoma and associated driver mutations, whole genome sequencing (WGS) and targeted extension sequencing were used to characterize 124 melanoma cases. Significantly mutated gene analysis using 13 WGS cases and 15 additional paired extension cases identified known melanoma genes such as BRAF, NRAS, and CDKN2A, as well as a novel gene EPHA3, previously implicated in other cancer types. Extension studies using tumors from another 96 patients discovered a large number of truncation mutations in tumor suppressors (TP53 and RB1), protein phosphatases (e.g., PTEN, PTPRB, PTPRD, and PTPRT), as well as chromatin remodeling genes (e.g., ASXL3, MLL2, and ARID2). Deep sequencing of mutations revealed subclones in the majority of metastatic tumors from 13 WGS cases. Validated mutations from 12 out of 13 WGS patients exhibited a predominant UV signature characterized by a high frequency of C->T transitions occurring at the 3′ base of dipyrimidine sequences while one patient (MEL9) with a hypermutator phenotype lacked this signature. Strikingly, a subclonal mutation signature analysis revealed that the founding clone in MEL9 exhibited UV signature but the secondary clone did not, suggesting different mutational mechanisms for two clonal populations from the same tumor. Further analysis of four metastases from different geographic locations in 2 melanoma cases revealed phylogenetic relationships and highlighted the genetic alterations responsible for differential drug resistance among metastatic tumors. Our study suggests that clonal evaluation is crucial for understanding tumor etiology and drug resistance in melanoma.
Highlights
The incidence of invasive melanoma in the United States in 2013 is estimated to be 76,690, with approximately 9,480 deaths [1]
All 13 whole genome sequencing (WGS) patients, from whom 15 tumors and 13 sets of normal PBMC were included in this study, had stage IV melanoma
We included capture probes corresponding to all putative somatic single nucleotide variants (SNVs) and small insertions/deletions that overlap with coding exons, splice sites, and RNA genes, a number of high-confidence SNVs and indels in noncoding conserved or regulatory regions, and non-repetitive regions of the human genome
Summary
The incidence of invasive melanoma in the United States in 2013 is estimated to be 76,690, with approximately 9,480 deaths [1]. Work in the last decade has identified a number of common and/or driver mutations in melanoma and helped to elucidate the pathways determining melanoma oncogenesis, proliferation, and metastasis. These discoveries have led to the development of inhibitors for BRAF and KIT (C-Kit) signaling, some of which have shown benefit in melanoma patients [4,5]. The driver mutations in BRAF and NRAS that have been identified cannot fully explain melanoma oncogenesis, as these same mutations have been found at similar rates in benign nevi, or moles [6,7] These common, benign skin lesions infrequently undergo malignant transformation into melanoma, but invariably remain in their growth-arrested state. The mutational variants identified by WGS can provide insight into this process
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