To gain a comprehensive view of the genomic landscape in human melanoma tumours, we sequenced the genomes of 25 metastatic melanomas and peripheral blood obtained from the same patients (Supplementary Table 1). Two tumours (ME015 and ME032) were metastases from cutaneous melanomas arising on glabrous (that is, hairless) skin of the extremities, representing the acral subtype. The other tumours were primarily metastases from melanomas originating on hair-bearing skin of the trunk (the most common clinical subtype). Further, ME009 represented a metastasis from a primary melanoma of a patient with a clinical history of chronic ultraviolet exposure.
We obtained 59-fold mean haploid genome coverage for tumour DNA and 32-fold for normal DNA (Supplementary Table 2). On average, 78,775 somatic base substitutions per tumour were identified, consistent with prior reports3, 4 (Supplementary Table 3). This corresponded to an average mutation rate of 30 per Mb. However, the mutation rate varied by nearly two orders of magnitude across the 25 tumours (Fig. 1). The acral melanomas showed mutation rates comparable to other solid tumour types (3 and 14 mutations per Mb)5, 6, whereas melanomas from the trunk harboured substantially more mutations, in agreement with previous studies3, 7, 8. In particular, sample ME009 exhibited a striking rate of 111 somatic mutations per Mb, consistent with a history of chronic sun exposure.
Top bar plot shows somatic mutation rate of 25 sequenced melanoma genomes, in decreasing order. Middle matrix indicates BRAF and NRAS somatic mutation status, with left-adjacent bar plot indicating total number of mutations in each oncogene as well as percent frequency. Bottom plot displays each tumours somatic mutation spectrum as a percentage of all mutations (right axis). Tumour sample names are indicated at the bottom of the figure, with acral melanomas in red.
In tumours with elevated mutation rates, most nucleotide substitutions were CT or GA transitions consistent with ultraviolet irradiation9. The variations in mutation rate correlated with differences in the ultraviolet mutational signature. For example, 93% of substitutions in ME009 but only 36% in acral melanoma ME015 were CT transitions (Fig. 1); these tumours contained the highest and lowest base mutation rates, respectively (111 and 3 mutations per Mb). Interestingly, the acral tumour ME032 also showed a discernible enrichment of ultraviolet-associated mutations (Fig. 1). Thus, genome sequencing readily confirmed the contribution of sun exposure in melanoma aetiology.
In agreement with prior studies7, 9, we detected an overall enrichment for dipyrimidines at CT transitions. Analysis of intragenic CT mutations yielded a significant bias against such mutations on the transcribed strand for most melanomas, consistent with transcription-coupled repair (Supplementary Fig. 1)3, 7, 10. Most commonly, CT mutations occurred at the 3 base of a pyrimidine dinucleotide (CpC or TpC; Supplementary Fig. 2). In contrast, the CT mutations in sample ME009 (with hypermutation and chronic sun exposure history) more often occurred at the 5 base of a pyrimidine dinucleotide. As expected, the acral tumour ME015 exhibited mutation patterns observed in non-ultraviolet-associated tumour types11, such as an increased mutation rate at CpG dinucleotides relative to their overall genome-wide frequency (Supplementary Fig. 2). These different mutational signatures suggest a complex mechanism of ultraviolet mutagenesis across the clinical spectrum of melanoma, probably reflecting distinct histories of environmental exposures and cutaneous biology.
We detected 9,653 missense, nonsense or splice site mutations in 5,712 genes (out of a total of 14,680 coding mutations; Supplementary Tables 4 and 5), with an estimated specificity of 95% (Supplementary Methods). A mutation of BRAF, BRAFV600E, was present in 16 of 25 tumours (64%), including the acral melanoma ME015. NRAS was mutated in 9 of 25 tumours (36%) in a mutually exclusive fashion with BRAF, with the exception of one non-canonical substitution (NRAST50I) in the hypermutated sample ME009. We also identified 6 insertions and 34 deletions in protein coding exons (Supplementary Table 6), including a 21-base-pair (bp) in-frame deletion involving exon 11 of the KIT oncogene in the acral tumour ME032 (Supplementary Fig. 3). KIT mutations occur in 15% of acral and mucosal melanomas12, and melanoma patients with activating KIT mutations in exon 11 have demonstrated marked responses to imatinib treatment13.
We identified an average of 97 structural rearrangements per melanoma genome (range: 6420) (Supplementary Table 7). In addition to displaying a wide range of rearrangement frequencies, the proportion of intrachromosomal and interchromosomal rearrangements varied widely across genomes. ME029, which harboured the largest number of rearrangements (420), contained only 8 interchromosomal events (Fig. 2a). In contrast, ME020 and ME035 contained 95 and 90 interchromosomal rearrangements, respectively (Fig. 2a). In both cases, the vast majority of interchromosomal rearrangements were restricted to two chromosomes. This pattern is reminiscent of chromothripsis14, a process involving catastrophic chromosome breakage that has been observed in several tumour types15, 16.
a, Circos plots representing four melanoma genomes with notable structural alterations. Interchromosomal and intrachromosomal rearrangements are shown in purple and green, respectively. b, Location of breakpoints associated with ETV1 in melanoma ME032. c, Location of breakpoints associated with PREX2 in melanoma ME032. The red arrow indicates a premature stop codon (E824*). All rearrangements in ETV1 and PREX2 were validated by high-throughput PCR and deep sequencing. d, Confirmation of high-level amplification and rearrangement of PREX2 in ME032 by dual-colour break-apart FISH. The assignment of red and green FISH probes to the PREX2 gene region is delineated as bars. Lack of co-localization of red and green probes is indicative of break-apart.
106 genes harboured chromosomal rearrangements in two or more samples (Supplementary Table 8). Many recurrently rearranged loci contain large genes or reside at known or suspected fragile sites17; examples include FHIT (six tumours), MACROD2 (five tumours) and CSMD1 (four tumours). On the other hand, several known cancer genes were also recurrently rearranged, including the PTEN tumour suppressor (four tumours) and MAGI2 (three tumours), which encodes a protein known to bind and stabilize PTEN. MAGI2 was also found disrupted in recent whole-genome studies of prostate cancer18 and a melanoma cell line7. Rearrangements involving the 5 untranslated region of the ataxin 2-binding protein 1 gene (A2BP1) were observed in 4 tumours. A2BP1 encodes an RNA binding protein whose genetic disruption has been linked to spinocerebellar ataxia and other neurodegenerative diseases. A2BP1 undergoes complex splicing regulation in the central nervous system and other tissues19; in melanoma, these rearrangements may disrupt a known A2BP1 splice isoform or enable a de novo splicing product. Together, these results suggest that chromosomal rearrangements may contribute importantly to melanoma genesis or progression.
An acral melanoma (ME032) harboured the second-largest number of total rearrangements (314; Fig. 2a). We employed high-throughput PCR followed by massively parallel sequencing to successfully validate 177 of 182 events tested in this sample, confirming its high rate of rearrangement. The elevated frequency of genomic rearrangements in acral melanomas has been reported previously20. In comparison, ME032 exhibited one of the lowest base-pair mutation rates of the melanomas examined (22nd out of 25 samples), suggesting that different tumours might preferentially enact alternative mechanisms of genomic alteration to drive tumorigenesis.
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Melanoma genome sequencing reveals frequent PREX2 mutations