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Clonality and evolutionary history of rhabdomyosarcoma.

Chen L, Shern JF, Wei JS, Yohe ME, Song YK, Hurd L, Liao H, Catchpoole D, Skapek SX, Barr FG, Hawkins DS, Khan J - PLoS Genet. (2015)

Bottom Line: Intriguingly, we find that loss of heterozygosity of 11p15.5 and mutations in RAS pathway genes occur early in the evolutionary history of the PAX-fusion-negative-RMS (PFN-RMS) subtype.We discover several early mutations in non-RAS mutated samples and predict them to be drivers in PFN-RMS including recurrent mutation of PKN1.Our findings provide information critical to the understanding of tumorigenesis of RMS.

View Article: PubMed Central - PubMed

Affiliation: Genetics Branch, Oncogenomics Section, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
To infer the subclonality of rhabdomyosarcoma (RMS) and predict the temporal order of genetic events for the tumorigenic process, and to identify novel drivers, we applied a systematic method that takes into account germline and somatic alterations in 44 tumor-normal RMS pairs using deep whole-genome sequencing. Intriguingly, we find that loss of heterozygosity of 11p15.5 and mutations in RAS pathway genes occur early in the evolutionary history of the PAX-fusion-negative-RMS (PFN-RMS) subtype. We discover several early mutations in non-RAS mutated samples and predict them to be drivers in PFN-RMS including recurrent mutation of PKN1. In contrast, we find that PAX-fusion-positive (PFP) subtype tumors have undergone whole-genome duplication in the late stage of cancer evolutionary history and have acquired fewer mutations and subclones than PFN-RMS. Moreover we predict that the PAX3-FOXO1 fusion event occurs earlier than the whole genome duplication. Our findings provide information critical to the understanding of tumorigenesis of RMS.

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Inferring evolutionary history for a typical rhabdomyosarcoma sample RMS2110.(a) A circos plot of tumor genome RMS2110 (before normal cell contamination correction). Gene symbols indicate genes with nonsynonymous mutations. Tracks from outermost to innermost depict chromosome banding, copy number (the height of bars denotes copy number), lesser allele fraction (the height of green bars represents the fraction of lesser allele at each genomic location, valued 0∼0.5), loss of heterogeneity status (each dot represents the probability of loss of heterozygosity for the adjacent segment), intensity of heterozygous (orange bar) and homozygous single nucleotide variants (blue bar), junctions or chromosomal rearrangement (grey lines). (b) Discrepancy between expected and observed status of allelic copy number suggests the existence of subclone(s) in tumor. Red dots denote chromosome segments, whereas blue crosses denote statuses of copy number and lesser allele fraction that leads to an integer allelic copy number. If there are tumor subclones with copy number status different from the major clone, the red dots will deviate from the blue crosses. (c) Observed VAF distribution of somatic mutations on chromosomes without aneuploidy. The normal-mixture like distribution suggest the existence of a minor subclone with VAF<0.5. (d) A scatter plot showing coverage (horizontal axis) and VAF (vertical axis) for somatic mutations (sSNV) on each chromosome.
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pgen.1005075.g001: Inferring evolutionary history for a typical rhabdomyosarcoma sample RMS2110.(a) A circos plot of tumor genome RMS2110 (before normal cell contamination correction). Gene symbols indicate genes with nonsynonymous mutations. Tracks from outermost to innermost depict chromosome banding, copy number (the height of bars denotes copy number), lesser allele fraction (the height of green bars represents the fraction of lesser allele at each genomic location, valued 0∼0.5), loss of heterogeneity status (each dot represents the probability of loss of heterozygosity for the adjacent segment), intensity of heterozygous (orange bar) and homozygous single nucleotide variants (blue bar), junctions or chromosomal rearrangement (grey lines). (b) Discrepancy between expected and observed status of allelic copy number suggests the existence of subclone(s) in tumor. Red dots denote chromosome segments, whereas blue crosses denote statuses of copy number and lesser allele fraction that leads to an integer allelic copy number. If there are tumor subclones with copy number status different from the major clone, the red dots will deviate from the blue crosses. (c) Observed VAF distribution of somatic mutations on chromosomes without aneuploidy. The normal-mixture like distribution suggest the existence of a minor subclone with VAF<0.5. (d) A scatter plot showing coverage (horizontal axis) and VAF (vertical axis) for somatic mutations (sSNV) on each chromosome.

Mentions: We illustrate our method in more detail using one PAX-fusion negative RMS tumor (RMS2110). Genome wide, this tumor had 3,889 somatic mutations including oncogenic mutation KRAS G13D and FGFR4 V550L [23]. Using the procedure described in the previous section, we estimated that this sample had 12% normal cell contamination (S1 Table). Copy number analysis showed that RMS2110 had large-segment allelic imbalance (including copy neutral LOH) and copy number alteration on eleven chromosomes (Fig. 1A).We employed the combination of somatic mutations, allelic imbalance and copy number alterations to dissect tumor clones that have unique genomic profiles. First, VAF of somatic mutations was determined to find subclonal mutations, based on the fact that a subclonal mutation (present only in a part of the tumor cells) usually has lower VAF than full-clonal mutations (present in all tumor cells) [2]. A typical scenario is on chromosomes without aneuploidy or allelic imbalance, where heterozygous full-clonal mutations have VAF equal to 0.5 while subclonal mutations have VAF<0.5. Analysis of RMS2110 showed that more than half of the chromosomes are without aneuploidy or allelic imbalance (Fig. 1A and 1B). On these chromosomes, a small portion of somatic mutations have observed VAF significantly lower than others indicating the presence of subclones (Fig. 1C, where there are two distinct VAF clusters, one is centered on VAF = 0.5 and the other is centered on VAF = 0.2; for individual chromosomes see Fig. 1D). Using a clustering algorithm with cluster-number-selection procedure (S1 Text) we can identify subclonal mutations as those with lower VAF. Given the depth of sequencing coverage, we estimated that we could detect subclonal mutations with VAF as low as 0.1 (S6 Fig.). Second, allelic copy number status is used to detect subclonal copy number alterations, based on the fact that subclones of different copy number will result in a non-integer allelic copy number for the whole tumor sample. The joint status of total copy number and lesser allele fraction (LAF—the ratio between the less allelic copy number and total copy number, estimated by germline single nucleotide variants, see Methods) reflects whether the allelic copy number is an integer—whether the observed allelic copy number (red dots) is on the expected position (blue crosses) in Fig. 1D. Therefore our approach predicts the subclonal copy number alterations and the fraction of tumor cells possessing these changes (S1 Text).


Clonality and evolutionary history of rhabdomyosarcoma.

Chen L, Shern JF, Wei JS, Yohe ME, Song YK, Hurd L, Liao H, Catchpoole D, Skapek SX, Barr FG, Hawkins DS, Khan J - PLoS Genet. (2015)

Inferring evolutionary history for a typical rhabdomyosarcoma sample RMS2110.(a) A circos plot of tumor genome RMS2110 (before normal cell contamination correction). Gene symbols indicate genes with nonsynonymous mutations. Tracks from outermost to innermost depict chromosome banding, copy number (the height of bars denotes copy number), lesser allele fraction (the height of green bars represents the fraction of lesser allele at each genomic location, valued 0∼0.5), loss of heterogeneity status (each dot represents the probability of loss of heterozygosity for the adjacent segment), intensity of heterozygous (orange bar) and homozygous single nucleotide variants (blue bar), junctions or chromosomal rearrangement (grey lines). (b) Discrepancy between expected and observed status of allelic copy number suggests the existence of subclone(s) in tumor. Red dots denote chromosome segments, whereas blue crosses denote statuses of copy number and lesser allele fraction that leads to an integer allelic copy number. If there are tumor subclones with copy number status different from the major clone, the red dots will deviate from the blue crosses. (c) Observed VAF distribution of somatic mutations on chromosomes without aneuploidy. The normal-mixture like distribution suggest the existence of a minor subclone with VAF<0.5. (d) A scatter plot showing coverage (horizontal axis) and VAF (vertical axis) for somatic mutations (sSNV) on each chromosome.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4358975&req=5

pgen.1005075.g001: Inferring evolutionary history for a typical rhabdomyosarcoma sample RMS2110.(a) A circos plot of tumor genome RMS2110 (before normal cell contamination correction). Gene symbols indicate genes with nonsynonymous mutations. Tracks from outermost to innermost depict chromosome banding, copy number (the height of bars denotes copy number), lesser allele fraction (the height of green bars represents the fraction of lesser allele at each genomic location, valued 0∼0.5), loss of heterogeneity status (each dot represents the probability of loss of heterozygosity for the adjacent segment), intensity of heterozygous (orange bar) and homozygous single nucleotide variants (blue bar), junctions or chromosomal rearrangement (grey lines). (b) Discrepancy between expected and observed status of allelic copy number suggests the existence of subclone(s) in tumor. Red dots denote chromosome segments, whereas blue crosses denote statuses of copy number and lesser allele fraction that leads to an integer allelic copy number. If there are tumor subclones with copy number status different from the major clone, the red dots will deviate from the blue crosses. (c) Observed VAF distribution of somatic mutations on chromosomes without aneuploidy. The normal-mixture like distribution suggest the existence of a minor subclone with VAF<0.5. (d) A scatter plot showing coverage (horizontal axis) and VAF (vertical axis) for somatic mutations (sSNV) on each chromosome.
Mentions: We illustrate our method in more detail using one PAX-fusion negative RMS tumor (RMS2110). Genome wide, this tumor had 3,889 somatic mutations including oncogenic mutation KRAS G13D and FGFR4 V550L [23]. Using the procedure described in the previous section, we estimated that this sample had 12% normal cell contamination (S1 Table). Copy number analysis showed that RMS2110 had large-segment allelic imbalance (including copy neutral LOH) and copy number alteration on eleven chromosomes (Fig. 1A).We employed the combination of somatic mutations, allelic imbalance and copy number alterations to dissect tumor clones that have unique genomic profiles. First, VAF of somatic mutations was determined to find subclonal mutations, based on the fact that a subclonal mutation (present only in a part of the tumor cells) usually has lower VAF than full-clonal mutations (present in all tumor cells) [2]. A typical scenario is on chromosomes without aneuploidy or allelic imbalance, where heterozygous full-clonal mutations have VAF equal to 0.5 while subclonal mutations have VAF<0.5. Analysis of RMS2110 showed that more than half of the chromosomes are without aneuploidy or allelic imbalance (Fig. 1A and 1B). On these chromosomes, a small portion of somatic mutations have observed VAF significantly lower than others indicating the presence of subclones (Fig. 1C, where there are two distinct VAF clusters, one is centered on VAF = 0.5 and the other is centered on VAF = 0.2; for individual chromosomes see Fig. 1D). Using a clustering algorithm with cluster-number-selection procedure (S1 Text) we can identify subclonal mutations as those with lower VAF. Given the depth of sequencing coverage, we estimated that we could detect subclonal mutations with VAF as low as 0.1 (S6 Fig.). Second, allelic copy number status is used to detect subclonal copy number alterations, based on the fact that subclones of different copy number will result in a non-integer allelic copy number for the whole tumor sample. The joint status of total copy number and lesser allele fraction (LAF—the ratio between the less allelic copy number and total copy number, estimated by germline single nucleotide variants, see Methods) reflects whether the allelic copy number is an integer—whether the observed allelic copy number (red dots) is on the expected position (blue crosses) in Fig. 1D. Therefore our approach predicts the subclonal copy number alterations and the fraction of tumor cells possessing these changes (S1 Text).

Bottom Line: Intriguingly, we find that loss of heterozygosity of 11p15.5 and mutations in RAS pathway genes occur early in the evolutionary history of the PAX-fusion-negative-RMS (PFN-RMS) subtype.We discover several early mutations in non-RAS mutated samples and predict them to be drivers in PFN-RMS including recurrent mutation of PKN1.Our findings provide information critical to the understanding of tumorigenesis of RMS.

View Article: PubMed Central - PubMed

Affiliation: Genetics Branch, Oncogenomics Section, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
To infer the subclonality of rhabdomyosarcoma (RMS) and predict the temporal order of genetic events for the tumorigenic process, and to identify novel drivers, we applied a systematic method that takes into account germline and somatic alterations in 44 tumor-normal RMS pairs using deep whole-genome sequencing. Intriguingly, we find that loss of heterozygosity of 11p15.5 and mutations in RAS pathway genes occur early in the evolutionary history of the PAX-fusion-negative-RMS (PFN-RMS) subtype. We discover several early mutations in non-RAS mutated samples and predict them to be drivers in PFN-RMS including recurrent mutation of PKN1. In contrast, we find that PAX-fusion-positive (PFP) subtype tumors have undergone whole-genome duplication in the late stage of cancer evolutionary history and have acquired fewer mutations and subclones than PFN-RMS. Moreover we predict that the PAX3-FOXO1 fusion event occurs earlier than the whole genome duplication. Our findings provide information critical to the understanding of tumorigenesis of RMS.

Show MeSH
Related in: MedlinePlus