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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

Overview of subclonal landscape in melanoma (MEL1, 8, 9, and 10) and their associated driver mutations.Two plots are shown for each case: kernel density (top), followed by the plot of tumor variant allele frequency by sequence depth for sites from selected copy number neutral regions (see Methods). Data shown are from chromosomes 1, 3, 4, 6, 10, and 13 for MEL1, from chromosomes 1, 2, 5, 12, and 20 for MEL8, from chromosomes 1, 3, 4, 6, 7, 12, and 13 for MEL9, and from chromosomes 6, 7, 10, 13, and 15 for MEL10. The data show evidence of two clusters in MEL1, MEL8, MEL9 and MEL10 with the majority of mutations from the lower allele frequency clusters. Mutations detected in significantly mutated genes in this study and genes implicated in Hodis et al. [18] were labeled.
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pone-0111153-g003: Overview of subclonal landscape in melanoma (MEL1, 8, 9, and 10) and their associated driver mutations.Two plots are shown for each case: kernel density (top), followed by the plot of tumor variant allele frequency by sequence depth for sites from selected copy number neutral regions (see Methods). Data shown are from chromosomes 1, 3, 4, 6, 10, and 13 for MEL1, from chromosomes 1, 2, 5, 12, and 20 for MEL8, from chromosomes 1, 3, 4, 6, 7, 12, and 13 for MEL9, and from chromosomes 6, 7, 10, 13, and 15 for MEL10. The data show evidence of two clusters in MEL1, MEL8, MEL9 and MEL10 with the majority of mutations from the lower allele frequency clusters. Mutations detected in significantly mutated genes in this study and genes implicated in Hodis et al. [18] were labeled.

Mentions: We took hundreds of validated somatic mutations with read depths of hundreds to thousands from capture validation and applied the SciClone algorithm (https://github.com/genome/sciclone) to cluster mutations with similar allelic fractions. These clusters are indicative of distinct subclonal populations of tumor cells. Multiple subclones were observed in the majority of 15 WGS tumors (Table 2). Due to the high mutation rate and complex copy number landscape in melanoma, the boundaries of some clusters could not be clearly separated using genome-wide data. We then selected “stable” genomic regions based on LOH and CNV analyses using VarScan 2 [38] and used somatic mutations from these regions for plotting (Figure 3 and Table S12a in File S2). In MEL1, two distinct clusters at 36.7% and 21.7% Variant Allele Frequency (VAF)s were identified. The majority of mutations were from the 21.7% VAF cluster. MEL8 displayed a similar pattern as MEL1, with one cluster at 37.8% VAF and another cluster at 23.4%. The hypermutated MEL9 tumor has the founding cluster at 46.8% VAF and the secondary but dominant cluster centered at 19.8%, suggesting that a massive mutation expansion took place in the 17.6% VAF cluster. Likewise, MEL10 had two clusters, centered at 30.6% and 19.5% VAFs respectively (Figure 3). These estimates of tumor heterogeneity represent a lower bound, and it is possible that additional subclone(s) were present in these samples but not detected. Our results demonstrate that melanoma is a disease characterized by significant intra-tumor heterogeneity.


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)

Overview of subclonal landscape in melanoma (MEL1, 8, 9, and 10) and their associated driver mutations.Two plots are shown for each case: kernel density (top), followed by the plot of tumor variant allele frequency by sequence depth for sites from selected copy number neutral regions (see Methods). Data shown are from chromosomes 1, 3, 4, 6, 10, and 13 for MEL1, from chromosomes 1, 2, 5, 12, and 20 for MEL8, from chromosomes 1, 3, 4, 6, 7, 12, and 13 for MEL9, and from chromosomes 6, 7, 10, 13, and 15 for MEL10. The data show evidence of two clusters in MEL1, MEL8, MEL9 and MEL10 with the majority of mutations from the lower allele frequency clusters. Mutations detected in significantly mutated genes in this study and genes implicated in Hodis et al. [18] were labeled.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111153-g003: Overview of subclonal landscape in melanoma (MEL1, 8, 9, and 10) and their associated driver mutations.Two plots are shown for each case: kernel density (top), followed by the plot of tumor variant allele frequency by sequence depth for sites from selected copy number neutral regions (see Methods). Data shown are from chromosomes 1, 3, 4, 6, 10, and 13 for MEL1, from chromosomes 1, 2, 5, 12, and 20 for MEL8, from chromosomes 1, 3, 4, 6, 7, 12, and 13 for MEL9, and from chromosomes 6, 7, 10, 13, and 15 for MEL10. The data show evidence of two clusters in MEL1, MEL8, MEL9 and MEL10 with the majority of mutations from the lower allele frequency clusters. Mutations detected in significantly mutated genes in this study and genes implicated in Hodis et al. [18] were labeled.
Mentions: We took hundreds of validated somatic mutations with read depths of hundreds to thousands from capture validation and applied the SciClone algorithm (https://github.com/genome/sciclone) to cluster mutations with similar allelic fractions. These clusters are indicative of distinct subclonal populations of tumor cells. Multiple subclones were observed in the majority of 15 WGS tumors (Table 2). Due to the high mutation rate and complex copy number landscape in melanoma, the boundaries of some clusters could not be clearly separated using genome-wide data. We then selected “stable” genomic regions based on LOH and CNV analyses using VarScan 2 [38] and used somatic mutations from these regions for plotting (Figure 3 and Table S12a in File S2). In MEL1, two distinct clusters at 36.7% and 21.7% Variant Allele Frequency (VAF)s were identified. The majority of mutations were from the 21.7% VAF cluster. MEL8 displayed a similar pattern as MEL1, with one cluster at 37.8% VAF and another cluster at 23.4%. The hypermutated MEL9 tumor has the founding cluster at 46.8% VAF and the secondary but dominant cluster centered at 19.8%, suggesting that a massive mutation expansion took place in the 17.6% VAF cluster. Likewise, MEL10 had two clusters, centered at 30.6% and 19.5% VAFs respectively (Figure 3). These estimates of tumor heterogeneity represent a lower bound, and it is possible that additional subclone(s) were present in these samples but not detected. Our results demonstrate that melanoma is a disease characterized by significant intra-tumor heterogeneity.

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