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Clonal architectures and driver mutations in metastatic melanomas.

Ding L, Kim M, Kanchi KL, Dees ND, Lu C, Griffith M, Fenstermacher D, Sung H, Miller CA, Goetz B, Wendl MC, Griffith O, Cornelius LA, Linette GP, McMichael JF, Sondak VK, Fields RC, Ley TJ, Mulé JJ, Wilson RK, Weber JS - PLoS ONE (2014)

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: The Genome Institute, Washington University in St. Louis, St. Louis, Missouri, United States of America; Department of Medicine, Washington University in St. Louis, St. Louis, Missouri, United States of America; Department of Genetics, Washington University in St. Louis, St. Louis, Missouri, United States of America; Siteman Cancer Center, Washington University in St. Louis, St. Louis, Missouri, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

Mutation pattern, spectrum, and clinical features in 15 metastases from 13 WGS melanoma cases.Mutations found in genes from MAP kinase, PI3K-AKT, RB/TP53 pathways and glutamate receptors are shown. Copy number alterations and structural variants found in BRAF, NRAS, TP53, CDKN2A/2B, and CCND1 are also displayed. The numbers and frequencies of tier 1 transition and transversion events identified in all 15 tumors are shown.
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pone-0111153-g001: Mutation pattern, spectrum, and clinical features in 15 metastases from 13 WGS melanoma cases.Mutations found in genes from MAP kinase, PI3K-AKT, RB/TP53 pathways and glutamate receptors are shown. Copy number alterations and structural variants found in BRAF, NRAS, TP53, CDKN2A/2B, and CCND1 are also displayed. The numbers and frequencies of tier 1 transition and transversion events identified in all 15 tumors are shown.

Mentions: All 13 WGS patients, from whom 15 tumors and 13 sets of normal PBMC were included in this study, had stage IV melanoma. The metastatic samples were from diverse locations including lung, chest wall, brain, lymph node, stomach, small intestine, and adrenal gland (Figure 1). Using an Illumina paired-end sequencing strategy, tumor and normal genomes were sequenced to at least 29.5X-fold and 35X-fold haploid coverage, respectively, with corresponding diploid coverage of 98.84% or better based on concordance with SNP array data (Table S1 in File S1). Candidate somatic changes were predicted using multiple algorithms [21]–[25] and selected for hybridization capture-based validation (Supplementary Materials and Methods). We included capture probes corresponding to all putative somatic single nucleotide variants (SNVs) and small insertions/deletions (indels) that overlap with coding exons, splice sites, and RNA genes (tier 1), a number of high-confidence SNVs and indels in non-coding conserved or regulatory regions (tier 2), and non-repetitive regions of the human genome (tier 3). In addition, we included predicted somatic structural variants (SVs) genome-wide for validation (Methods1). Analysis of the high depth sequencing data resulting from the captured target DNAs of 15 tumor and 13 normal samples (Table S2, S3 in File S1 and Supplementary Materials and Methods in File S1) confirmed 17,361 tier 1 point mutations, with a validation rate of 93.6%, 84 tier 1 indels, and 411 somatic SVs (Tables S4 and S5 in File S2). Seven of 15 tumors had over 1,000 tier 1 SNVs. This is among the highest mutation frequency of known cancers. For comparison, AMLs have a median of 13 tier 1 changes per genome [cite TCGA AML] [26] and metastatic breast cancers have been reported with between 32 (lobular) [27] and 50 (basal-like) [28]. C->T transitions were predominant in all 15 tumors, consistent with a UV damage signature (Figure 1). Notably, MEL9, with 6,795 validated tier 1 point mutations (7.7-fold times the average number from the other 12 cases), exhibited the highest C->T transition rate among 15 tumors. The patterns of point mutations were very similar for the paired metachronous tumors from patients 5 and 13 (Figure 1 and Table S6a in File S2). Notably, we identified 443 tier 1 dinucleotide mutations in 13 WGS cases and among them, an average of 74% (ranging from 68% to 78%) are CC->TT changes, consistent with previous reports [11]. The ratio between dinucleotide and point mutations in Tier 1 ranges from 0.76% to 6.28% while the ratio in Tiers 1–3 ranges from 0.46% to 2.43%, consistent with the higher GC content in the coding sequences. (Table S7 in File S1)


Clonal architectures and driver mutations in metastatic melanomas.

Ding L, Kim M, Kanchi KL, Dees ND, Lu C, Griffith M, Fenstermacher D, Sung H, Miller CA, Goetz B, Wendl MC, Griffith O, Cornelius LA, Linette GP, McMichael JF, Sondak VK, Fields RC, Ley TJ, Mulé JJ, Wilson RK, Weber JS - PLoS ONE (2014)

Mutation pattern, spectrum, and clinical features in 15 metastases from 13 WGS melanoma cases.Mutations found in genes from MAP kinase, PI3K-AKT, RB/TP53 pathways and glutamate receptors are shown. Copy number alterations and structural variants found in BRAF, NRAS, TP53, CDKN2A/2B, and CCND1 are also displayed. The numbers and frequencies of tier 1 transition and transversion events identified in all 15 tumors are shown.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4230926&req=5

pone-0111153-g001: Mutation pattern, spectrum, and clinical features in 15 metastases from 13 WGS melanoma cases.Mutations found in genes from MAP kinase, PI3K-AKT, RB/TP53 pathways and glutamate receptors are shown. Copy number alterations and structural variants found in BRAF, NRAS, TP53, CDKN2A/2B, and CCND1 are also displayed. The numbers and frequencies of tier 1 transition and transversion events identified in all 15 tumors are shown.
Mentions: All 13 WGS patients, from whom 15 tumors and 13 sets of normal PBMC were included in this study, had stage IV melanoma. The metastatic samples were from diverse locations including lung, chest wall, brain, lymph node, stomach, small intestine, and adrenal gland (Figure 1). Using an Illumina paired-end sequencing strategy, tumor and normal genomes were sequenced to at least 29.5X-fold and 35X-fold haploid coverage, respectively, with corresponding diploid coverage of 98.84% or better based on concordance with SNP array data (Table S1 in File S1). Candidate somatic changes were predicted using multiple algorithms [21]–[25] and selected for hybridization capture-based validation (Supplementary Materials and Methods). We included capture probes corresponding to all putative somatic single nucleotide variants (SNVs) and small insertions/deletions (indels) that overlap with coding exons, splice sites, and RNA genes (tier 1), a number of high-confidence SNVs and indels in non-coding conserved or regulatory regions (tier 2), and non-repetitive regions of the human genome (tier 3). In addition, we included predicted somatic structural variants (SVs) genome-wide for validation (Methods1). Analysis of the high depth sequencing data resulting from the captured target DNAs of 15 tumor and 13 normal samples (Table S2, S3 in File S1 and Supplementary Materials and Methods in File S1) confirmed 17,361 tier 1 point mutations, with a validation rate of 93.6%, 84 tier 1 indels, and 411 somatic SVs (Tables S4 and S5 in File S2). Seven of 15 tumors had over 1,000 tier 1 SNVs. This is among the highest mutation frequency of known cancers. For comparison, AMLs have a median of 13 tier 1 changes per genome [cite TCGA AML] [26] and metastatic breast cancers have been reported with between 32 (lobular) [27] and 50 (basal-like) [28]. C->T transitions were predominant in all 15 tumors, consistent with a UV damage signature (Figure 1). Notably, MEL9, with 6,795 validated tier 1 point mutations (7.7-fold times the average number from the other 12 cases), exhibited the highest C->T transition rate among 15 tumors. The patterns of point mutations were very similar for the paired metachronous tumors from patients 5 and 13 (Figure 1 and Table S6a in File S2). Notably, we identified 443 tier 1 dinucleotide mutations in 13 WGS cases and among them, an average of 74% (ranging from 68% to 78%) are CC->TT changes, consistent with previous reports [11]. The ratio between dinucleotide and point mutations in Tier 1 ranges from 0.76% to 6.28% while the ratio in Tiers 1–3 ranges from 0.46% to 2.43%, consistent with the higher GC content in the coding sequences. (Table S7 in File S1)

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: The Genome Institute, Washington University in St. Louis, St. Louis, Missouri, United States of America; Department of Medicine, Washington University in St. Louis, St. Louis, Missouri, United States of America; Department of Genetics, Washington University in St. Louis, St. Louis, Missouri, United States of America; Siteman Cancer Center, Washington University in St. Louis, St. Louis, Missouri, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus