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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

Dissecting mutational mechanisms using subclonal mutations.(a) Overall sequence context surrounding C->T transitions in MEL9 and MEL10. (b) Density plots showing the founding clone and subclone in MEL9 and MEL10. (c) Mutation context analysis of the founding clones detected a UV mutation signature in both MEL9 and MEL10. (d) Mutation context analysis of secondary clones detected a UV signature in MEL10 but not MEL9.
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pone-0111153-g004: Dissecting mutational mechanisms using subclonal mutations.(a) Overall sequence context surrounding C->T transitions in MEL9 and MEL10. (b) Density plots showing the founding clone and subclone in MEL9 and MEL10. (c) Mutation context analysis of the founding clones detected a UV mutation signature in both MEL9 and MEL10. (d) Mutation context analysis of secondary clones detected a UV signature in MEL10 but not MEL9.

Mentions: It has been shown that DNA damage caused by UV light often leads to the formation of covalent links between two adjacent pyrimidine residues [11]. As a result, C->T mutations in melanoma samples often occur at dipyrimidine sequences. Our analysis of 13 WGS melanoma cases showed that 12 cases had greater than 89.9% of C->T mutations occurring at the 3′ base of a pyrimidine dinucleotide, supporting previous findings [11]. However, MEL9, the hypermutated tumor, lacks this signature and has only 59.5% C->T occurring at the 3′ base of a dipyrimidine, comparable to 53% expected by chance (Figure 4). We reasoned that the UV signature in MEL9 might be masked by a large number of subsequent mutations arising from some other mechanism. One candidate was that these mutations were the result of a DNA repair defect (e.g., S418F and G1134R in MSH6, G2569S in BRCA2, or G648E in ERCC6). To test this hypothesis, we independently analyzed mutations from the founding and subclonal populations described above. (Figure 4). Strikingly, these two subclones in MEL9 exhibited two very distinct phenotypes. The founding clone exhibited a classic UV-damage phenotype with an abundance of C->T transitions and a disproportionately higher number of pyrimidine bases preceding the mutated cytosine bases (Proportion test P = 1.60×10−10). (Figure 4). In contrast, the subclone exhibited a typical pyrimidine base frequency preceding the mutated C base (59.5%, P = 0.17); interestingly this subclone had a significantly higher frequency of pyrimidine bases following the mutated C base (P = 1.72×10−40), consistent with findings in another hypermutated melanoma reported by Berger et al. [19]. Our hypothesis is that UV-driven mutations in the originating, founding clone of MEL9 damaged a DNA-repair gene and spurred a massive deficit in DNA repair. The resulting large number of mutations, occurring later than the UV damage, make up the lower-VAF subclone. The mutation context observed in the secondary clone of MEL9 does not match the patterns expected from defects in MSH2 and MSH6, and it may be attributable to another repair pathway. As a control, we also dissected the mutation spectrum in the founding clone and subclone of MEL10. The subclone for MEL10 also has a larger number of mutations, (Figure 4) but both show a typical UV-signature, with a significant number of Cytosine and Thymine bases preceding C->T transition sites (P-value for founding clone  = 4.01×10−21, P-value for secondary clone  = 3.83×10−65).


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)

Dissecting mutational mechanisms using subclonal mutations.(a) Overall sequence context surrounding C->T transitions in MEL9 and MEL10. (b) Density plots showing the founding clone and subclone in MEL9 and MEL10. (c) Mutation context analysis of the founding clones detected a UV mutation signature in both MEL9 and MEL10. (d) Mutation context analysis of secondary clones detected a UV signature in MEL10 but not MEL9.
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pone-0111153-g004: Dissecting mutational mechanisms using subclonal mutations.(a) Overall sequence context surrounding C->T transitions in MEL9 and MEL10. (b) Density plots showing the founding clone and subclone in MEL9 and MEL10. (c) Mutation context analysis of the founding clones detected a UV mutation signature in both MEL9 and MEL10. (d) Mutation context analysis of secondary clones detected a UV signature in MEL10 but not MEL9.
Mentions: It has been shown that DNA damage caused by UV light often leads to the formation of covalent links between two adjacent pyrimidine residues [11]. As a result, C->T mutations in melanoma samples often occur at dipyrimidine sequences. Our analysis of 13 WGS melanoma cases showed that 12 cases had greater than 89.9% of C->T mutations occurring at the 3′ base of a pyrimidine dinucleotide, supporting previous findings [11]. However, MEL9, the hypermutated tumor, lacks this signature and has only 59.5% C->T occurring at the 3′ base of a dipyrimidine, comparable to 53% expected by chance (Figure 4). We reasoned that the UV signature in MEL9 might be masked by a large number of subsequent mutations arising from some other mechanism. One candidate was that these mutations were the result of a DNA repair defect (e.g., S418F and G1134R in MSH6, G2569S in BRCA2, or G648E in ERCC6). To test this hypothesis, we independently analyzed mutations from the founding and subclonal populations described above. (Figure 4). Strikingly, these two subclones in MEL9 exhibited two very distinct phenotypes. The founding clone exhibited a classic UV-damage phenotype with an abundance of C->T transitions and a disproportionately higher number of pyrimidine bases preceding the mutated cytosine bases (Proportion test P = 1.60×10−10). (Figure 4). In contrast, the subclone exhibited a typical pyrimidine base frequency preceding the mutated C base (59.5%, P = 0.17); interestingly this subclone had a significantly higher frequency of pyrimidine bases following the mutated C base (P = 1.72×10−40), consistent with findings in another hypermutated melanoma reported by Berger et al. [19]. Our hypothesis is that UV-driven mutations in the originating, founding clone of MEL9 damaged a DNA-repair gene and spurred a massive deficit in DNA repair. The resulting large number of mutations, occurring later than the UV damage, make up the lower-VAF subclone. The mutation context observed in the secondary clone of MEL9 does not match the patterns expected from defects in MSH2 and MSH6, and it may be attributable to another repair pathway. As a control, we also dissected the mutation spectrum in the founding clone and subclone of MEL10. The subclone for MEL10 also has a larger number of mutations, (Figure 4) but both show a typical UV-signature, with a significant number of Cytosine and Thymine bases preceding C->T transition sites (P-value for founding clone  = 4.01×10−21, P-value for secondary clone  = 3.83×10−65).

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