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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 distribution in BRAF, NRAS, CDKN2A, EPHA3, GRIN2A, GRIN2B, PTPRT, and ASXL3.The locations of conserved protein domains are highlighted. Each nonsynonymous substitution, splice site mutation, or indel is designated with a circle at the representative protein position with color to indicate the translational effects of the mutation.
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pone-0111153-g002: Mutation distribution in BRAF, NRAS, CDKN2A, EPHA3, GRIN2A, GRIN2B, PTPRT, and ASXL3.The locations of conserved protein domains are highlighted. Each nonsynonymous substitution, splice site mutation, or indel is designated with a circle at the representative protein position with color to indicate the translational effects of the mutation.

Mentions: After our initial discovery using 13 WGS cases followed by the validation analysis described above, we performed further extension screening in 15 melanoma cases (25 metastatic tumors and matched normal tissue; 6 cases with multiple metastases). 1,209 genes were chosen for screening based on our initial WGS results and mutations and genes reported in several recent genomic studies of melanoma (Tables S8a and S8b in File S2) [13], [15]–[18]. Using mutations identified in all 28 cases, we performed MuSiC [32] analysis to discover genes displaying significantly higher mutation rates than expected based on the background mutation rate. A small group of genes was identified as significant after applying a 5% false discovery rate threshold (Table 1). This group included BRAF and NRAS, which were found to be mutated in 18 and 4 patients of 28, respectively (Figure 2). MEL9, the adrenal gland metastasis that was hypermutated, harbored mutations in both BRAF (H574L) and NRAS (Q61R); these two mutations were found to be present in the same variant allele frequency cluster (see Subclonal architecture in melanoma below). Meanwhile, mutations were not detected in either BRAF or NRAS in MEL6, a lung metastasis, and also four other tumors from the latter discovery group of 15 cases. The SMG list includes other genes known to be potentially involved in cancer. For instance, protein tyrosine kinase EPHA3, known mostly for its role in lung cancer [33], had 7 missense and 3 nonsense mutations, and tumor suppressor CDKN2A harbored one splice site, one nonsense, and one frame-shift indel, respectively (Figure 2). Mutations in a wide variety of protein families were seen in this study, including a large number of non-synonymous mutations in protein tyrosine phosphatases (e.g., PTPRB, PTPRT, and PTPN13), and protein tyrosine kinases (e.g., EPHA7, EPHA3, KIT, FGFR4, FGFR1, and ROS1). Of note, 8 missense and 1 nonsense mutations were found in ASXL3, a member of the polycomb group. The existence of mutations in glutamate receptors was described in prior exome sequencing studies [16], and our data not only confirmed that GRIN2A was mutated in melanoma (5 out of 28 cases) but also showed that GRIN2B was recurrently mutated (Figure 2). In addition, a number of mutations have also been found in other metabotropic glutamate receptors, such as GRM1 and GRM3-8. Specifically, out of 23 nonsynonymous mutations from GRM genes, one nonsense and four missense mutations were from GRM3, previously shown to harbor activating mutations in melanomas [17]. The observed mutation rate was 0.22 to 143 mutations per Mbp in the TCGA dataset compared to 3 to 155 mutations per Mbp in our 15 whole genome sequenced samples. In addition to the similar distribution of mutation rates, we also observed recurrent single nucleotide variants including S225F and G394E in EPHA7 and G114E and R136* in EPHA3 from both datasets. The Comparison of the number of mutations in significant genes between this study and TCGA report [34] is shown in Table S15 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 distribution in BRAF, NRAS, CDKN2A, EPHA3, GRIN2A, GRIN2B, PTPRT, and ASXL3.The locations of conserved protein domains are highlighted. Each nonsynonymous substitution, splice site mutation, or indel is designated with a circle at the representative protein position with color to indicate the translational effects of the mutation.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111153-g002: Mutation distribution in BRAF, NRAS, CDKN2A, EPHA3, GRIN2A, GRIN2B, PTPRT, and ASXL3.The locations of conserved protein domains are highlighted. Each nonsynonymous substitution, splice site mutation, or indel is designated with a circle at the representative protein position with color to indicate the translational effects of the mutation.
Mentions: After our initial discovery using 13 WGS cases followed by the validation analysis described above, we performed further extension screening in 15 melanoma cases (25 metastatic tumors and matched normal tissue; 6 cases with multiple metastases). 1,209 genes were chosen for screening based on our initial WGS results and mutations and genes reported in several recent genomic studies of melanoma (Tables S8a and S8b in File S2) [13], [15]–[18]. Using mutations identified in all 28 cases, we performed MuSiC [32] analysis to discover genes displaying significantly higher mutation rates than expected based on the background mutation rate. A small group of genes was identified as significant after applying a 5% false discovery rate threshold (Table 1). This group included BRAF and NRAS, which were found to be mutated in 18 and 4 patients of 28, respectively (Figure 2). MEL9, the adrenal gland metastasis that was hypermutated, harbored mutations in both BRAF (H574L) and NRAS (Q61R); these two mutations were found to be present in the same variant allele frequency cluster (see Subclonal architecture in melanoma below). Meanwhile, mutations were not detected in either BRAF or NRAS in MEL6, a lung metastasis, and also four other tumors from the latter discovery group of 15 cases. The SMG list includes other genes known to be potentially involved in cancer. For instance, protein tyrosine kinase EPHA3, known mostly for its role in lung cancer [33], had 7 missense and 3 nonsense mutations, and tumor suppressor CDKN2A harbored one splice site, one nonsense, and one frame-shift indel, respectively (Figure 2). Mutations in a wide variety of protein families were seen in this study, including a large number of non-synonymous mutations in protein tyrosine phosphatases (e.g., PTPRB, PTPRT, and PTPN13), and protein tyrosine kinases (e.g., EPHA7, EPHA3, KIT, FGFR4, FGFR1, and ROS1). Of note, 8 missense and 1 nonsense mutations were found in ASXL3, a member of the polycomb group. The existence of mutations in glutamate receptors was described in prior exome sequencing studies [16], and our data not only confirmed that GRIN2A was mutated in melanoma (5 out of 28 cases) but also showed that GRIN2B was recurrently mutated (Figure 2). In addition, a number of mutations have also been found in other metabotropic glutamate receptors, such as GRM1 and GRM3-8. Specifically, out of 23 nonsynonymous mutations from GRM genes, one nonsense and four missense mutations were from GRM3, previously shown to harbor activating mutations in melanomas [17]. The observed mutation rate was 0.22 to 143 mutations per Mbp in the TCGA dataset compared to 3 to 155 mutations per Mbp in our 15 whole genome sequenced samples. In addition to the similar distribution of mutation rates, we also observed recurrent single nucleotide variants including S225F and G394E in EPHA7 and G114E and R136* in EPHA3 from both datasets. The Comparison of the number of mutations in significant genes between this study and TCGA report [34] is shown in Table S15 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