Brown and darker orange represent sun-exposed tumors and tumors of unknown origin, respectively. has been instrumental in the discovery of new cancer-driver genes. These NGS studies have corroborated the previously identified frequent recurrent somatic mutations in and and revealed new melanoma mutations, including a recurrent mutation in (and that reduce the phosphatases catalytic activity, consequently dysregulating the kinase AURKA and causing chromosome instability4,5. Frequent inactivating mutations were also discovered in the tumor suppressors and and that are likely to enhance melanoma pathogenesis6,7. Furthermore, recent studies have shed light on variants in regulatory regions of the melanoma genome. Recurrent mutations in the promoter, which alter a transcription factorCbinding motif and possibly lead to increased expression of TERT, shield melanoma cells from senescence8,9. NGS has also fostered an increased understanding of the genetics of noncutaneous melanomas, with the discovery of frequent mutations in in uveal melanoma10,11. We report here the results of WES analysis of 213 human melanoma samples, including samples from Solcitinib (GSK2586184) 109 patients that we studied previously3 (Supplementary Data). Matched normal DNA was sequenced and analyzed from 133 of the tumors. We also tested the response of melanoma cell lines to the MEK inhibitor selumetinib (AZD6244), currently in clinical trials, and to the ERK inhibitor SCH772984 and performed protein blot analyses to correlate the effects of specific mutations with drug response. RESULTS Identification of or mutations but remain in a growth-arrested state. In some melanomas, somatic mutations in or are likely to account for initiation of the proliferative state. To comprehensively understand the mutations that lead to malignant transformation, we analyzed genes for evidence of selection and significantly increased mutation burden. We applied the 20/20 rule to identify genes with nonsilent mutations at recurrent positions that constituted 20% or more of all observed mutations or genes with at least 20% inactivating mutations, that is, nonsense, splice-site variant or insertion-deletion (indel) mutations12. The top 40 ranked genes from this analysis are shown in Table 1 (details are also provided in the Supplementary Data). Among those, we identified 11 genes that exhibited statistically significant mutation counts above what was expected on the basis of a driver gene analysis by MutSigCV13 (Fig. 1 and Supplementary Data). Open in a separate window Figure 1 Melanoma mutational landscape (= 213). Top 11 melanoma-driver genes that reach genome-wide significance according to background mutation-frequency estimation. Purple, metastatic melanoma; green, patients over 65 years old; red, mutations at recurrent positions; dark blue, inactivating mutations (nonsense, splice, indel); light blue, predicted harmful mutations. Brown and darker orange represent sun-exposed tumors and tumors of unknown origin, respectively. Mutations in and are marked in light orange and yellow, respectively. Mutation counts correspond to novel mutations that are not found in repositories of common human variants. Table 1 Top mutated genes across the Yale cohort (= 213) and and 3 double mutants (Table 2). Ninety percent of the mutations. Among the tumors with no detectable or mutation, a total of 46.4% (26 of 56) were mutants (Table 2). Of those, more than 80% (21 of 26) either showed loss of heterozygosity (LOH) across Ntn1 the locus or were compound heterozygotes harboring two mutations. Conversely, of the 12 double-mutant melanomas, one-third (4 of 12) showed LOH or compound heterozygosity (Supplementary Data). Table 2 Mutational status of the Yale cohort (= 213) (p.Val600) and (p.Gln61/Gly12/Gly13) and inactivating mutations (stop mutations, splice-site variants, indels) and predicted damaging mutations. WT, tripleCwild-type melanomas. = 1.5 10?10) and occurred in significantly older patients (= 0.017), but they were associated with similar overall patient survival compared to and cause the inherited human developmental disease STAR syndrome15. Early-termination alterations in FAM58A were present in four tumors from male patients. All of the tumors were wild type; two were gene transcript and protein levels17. Mutations in genes involved in chromatin modification or DNA repair were identified, some for the first time in melanoma. The list.Sznol and H.M.K. melanoma. Much of the information is from next-generation sequencing (NGS), particularly exome sequencing, and has been instrumental in the discovery of new cancer-driver genes. These NGS studies have corroborated the previously identified frequent recurrent somatic mutations in and and revealed new melanoma mutations, including a recurrent mutation in (and that reduce the phosphatases catalytic activity, consequently dysregulating the kinase AURKA and causing chromosome instability4,5. Frequent inactivating mutations were also discovered in the tumor suppressors and and that are likely to enhance melanoma pathogenesis6,7. Furthermore, recent studies have shed light on variants in regulatory regions of the melanoma genome. Recurrent mutations in the promoter, which alter a Solcitinib (GSK2586184) transcription factorCbinding motif and possibly lead to increased expression of TERT, shield melanoma cells from senescence8,9. NGS has also fostered an increased understanding of the genetics of noncutaneous melanomas, with the discovery of frequent mutations in in uveal melanoma10,11. We report here the results of WES analysis of 213 human melanoma samples, including samples from 109 patients that we studied previously3 (Supplementary Data). Matched normal DNA was sequenced and analyzed from 133 of the tumors. We also tested the response of melanoma cell lines to the MEK inhibitor selumetinib (AZD6244), currently in clinical trials, and to the ERK inhibitor SCH772984 and performed protein blot analyses to correlate the effects of specific mutations with drug Solcitinib (GSK2586184) response. RESULTS Identification of or mutations but remain in a growth-arrested state. In some melanomas, somatic mutations in or are likely to account for initiation of the proliferative state. To comprehensively understand the mutations that lead to malignant transformation, we analyzed genes for evidence of selection and significantly increased mutation burden. We applied the 20/20 rule to identify genes with nonsilent mutations at recurrent positions that constituted 20% or more of all observed mutations or genes with at least 20% inactivating mutations, that is, nonsense, splice-site variant or insertion-deletion (indel) mutations12. The top 40 rated genes from this analysis are demonstrated in Table 1 (details will also be offered in the Supplementary Data). Among those, we recognized 11 genes that exhibited statistically significant mutation counts above what was expected on the basis of a driver gene analysis by MutSigCV13 (Fig. 1 and Supplementary Data). Open in a separate window Number 1 Melanoma mutational scenery (= 213). Top 11 melanoma-driver genes that reach genome-wide significance relating to background mutation-frequency estimation. Purple, metastatic melanoma; green, individuals over 65 years old; reddish, mutations at recurrent positions; dark blue, inactivating mutations (nonsense, splice, indel); light blue, expected harmful mutations. Brown and darker orange represent sun-exposed tumors and tumors of unfamiliar source, respectively. Mutations in and are designated in light orange and yellow, respectively. Mutation counts correspond to novel mutations that are not found in repositories of common human being variants. Table 1 Top mutated genes across the Yale cohort (= 213) and and 3 double mutants (Table 2). Ninety percent of the mutations. Among the tumors with no detectable or mutation, a total of 46.4% (26 of 56) were mutants (Table 2). Of those, more than 80% (21 of 26) either showed loss of heterozygosity (LOH) across the locus or were compound heterozygotes harboring two mutations. Conversely, of the 12 double-mutant melanomas, one-third (4 of 12) showed LOH or compound heterozygosity (Supplementary Data). Table 2 Mutational status of the Yale cohort (= 213) (p.Val600) and (p.Gln61/Gly12/Gly13) and inactivating mutations (stop mutations, splice-site variants, indels) and predicted damaging mutations. WT, tripleCwild-type melanomas. = 1.5 10?10) and occurred in significantly older individuals (= 0.017), but they were associated with similar overall patient survival compared to and cause the inherited human being developmental disease Celebrity syndrome15. Early-termination alterations in FAM58A were present in four tumors from male individuals. All the tumors were crazy type; two were gene transcript and protein levels17. Mutations in genes involved in chromatin changes or DNA restoration were recognized, some for the first time in melanoma. The list included two SWI/SNF family members, (linked to gastric cancers18) and and crazy type (= 0.006; Supplementary Data). Of the nine mutations, two were nonsense and three were recurrent at position R551C. Four of the seven mutation), and two were compound heterozygotes. The recurrent and additional genes influencing RAS-MAPK signaling28. A search across these genes showed that four linked to Noonan syndrome.
Brown and darker orange represent sun-exposed tumors and tumors of unknown origin, respectively