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Absolute quantification of somatic DNA alterations in human cancer.

Carter SL, Cibulskis K, Helman E, McKenna A, Shen H, Zack T, Laird PW, Onofrio RC, Winckler W, Weir BA, Beroukhim R, Pellman D, Levine DA, Lander ES, Meyerson M, Getz G - Nat. Biotechnol. (2012)

Bottom Line: We used ABSOLUTE to analyze exome sequencing data from 214 ovarian carcinoma tumor-normal pairs.This analysis identified both pervasive subclonal somatic point-mutations and a small subset of predominantly clonal and homozygous mutations, which were overrepresented in the tumor suppressor genes TP53 and NF1 and in a candidate tumor suppressor gene CDK12.ABSOLUTE will facilitate the design of clinical sequencing studies and studies of cancer genome evolution and intra-tumor heterogeneity.

View Article: PubMed Central - PubMed

Affiliation: The Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA. scarter@broadinstitute.org

ABSTRACT
We describe a computational method that infers tumor purity and malignant cell ploidy directly from analysis of somatic DNA alterations. The method, named ABSOLUTE, can detect subclonal heterogeneity and somatic homozygosity, and it can calculate statistical sensitivity for detection of specific aberrations. We used ABSOLUTE to analyze exome sequencing data from 214 ovarian carcinoma tumor-normal pairs. This analysis identified both pervasive subclonal somatic point-mutations and a small subset of predominantly clonal and homozygous mutations, which were overrepresented in the tumor suppressor genes TP53 and NF1 and in a candidate tumor suppressor gene CDK12. We also used ABSOLUTE to infer absolute allelic copy-number profiles from 3,155 diverse cancer specimens, revealing that genome-doubling events are common in human cancer, likely occur in cells that are already aneuploid, and influence pathways of tumor progression (for example, with recessive inactivation of NF1 being less common after genome doubling). ABSOLUTE will facilitate the design of clinical sequencing studies and studies of cancer genome evolution and intra-tumor heterogeneity.

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ABSOLUTE method validation and comparison a-d Performance of ABSOLUTE and ASCAT on 4 validation assaysRMSE: root mean squared error. P-values were calculated on the squared errors using the paired one-sided Wilcoxon test (*: P < 0.05, **: P < 0.001). See Supplementary Note 1 for the ASCAT2.1 protocol.a, FACS-based ploidy measurements vs. inferred ploidy estimates for 37 primary tumor samples.Dashed line indicates y=x.b, SKY-based ploidy measurements vs. inferred ploidy estimates for 33 cancer cell-lines. Dataare displayed as in a.c, Estimated purity of the 33 cell lines shown in (b) Dashed horizontal line indicates the truepurity (1.0).d, Cancer-normal DNA mixing experiment results for two cell lines. DNA from each cancercell line was mixed with DNA from the matched B-lymphocyte in varying proportions (x-axis).(top) predicted vs. true DNA mixing fractions compared to the y=x line (dashed). (bottom)predicted cancer cell-line ploidy vs. mixture purity. The copy-profile of several samples wasmisinterpreted (x's); these points were not included in the RMSE calculations. Ploidy estimateswere generally consistent with previous SKY analysis of these cell lines: http://www.path.cam.ac.uk/∼pawefish/cell%20line%20catalogues/breast-cell-lines.htm.e, Leukocyte methylation signature enrichment in tumors of histologicaly underestimatedpurity. HGS-OvCa samples are shown grouped according to the indicated histological purityestimates (x-axis)33. Black horizontal lines indicate the median purity of each group, asestimated by ABSOLUTE (y-axis). The color of each point corresponds to the degree to whichthat sample's methylation profile resembled that of purified leukocytes (Online Methods).
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Figure 2: ABSOLUTE method validation and comparison a-d Performance of ABSOLUTE and ASCAT on 4 validation assaysRMSE: root mean squared error. P-values were calculated on the squared errors using the paired one-sided Wilcoxon test (*: P < 0.05, **: P < 0.001). See Supplementary Note 1 for the ASCAT2.1 protocol.a, FACS-based ploidy measurements vs. inferred ploidy estimates for 37 primary tumor samples.Dashed line indicates y=x.b, SKY-based ploidy measurements vs. inferred ploidy estimates for 33 cancer cell-lines. Dataare displayed as in a.c, Estimated purity of the 33 cell lines shown in (b) Dashed horizontal line indicates the truepurity (1.0).d, Cancer-normal DNA mixing experiment results for two cell lines. DNA from each cancercell line was mixed with DNA from the matched B-lymphocyte in varying proportions (x-axis).(top) predicted vs. true DNA mixing fractions compared to the y=x line (dashed). (bottom)predicted cancer cell-line ploidy vs. mixture purity. The copy-profile of several samples wasmisinterpreted (x's); these points were not included in the RMSE calculations. Ploidy estimateswere generally consistent with previous SKY analysis of these cell lines: http://www.path.cam.ac.uk/∼pawefish/cell%20line%20catalogues/breast-cell-lines.htm.e, Leukocyte methylation signature enrichment in tumors of histologicaly underestimatedpurity. HGS-OvCa samples are shown grouped according to the indicated histological purityestimates (x-axis)33. Black horizontal lines indicate the median purity of each group, asestimated by ABSOLUTE (y-axis). The color of each point corresponds to the degree to whichthat sample's methylation profile resembled that of purified leukocytes (Online Methods).

Mentions: We validated the purity and ploidy predictions made by ABSOLUTE on Affymetrix SNP microarray data using several approaches: (i) direct measurement of ploidy of 37 TCGA ovarian carcinoma samples by fluorescence-activated cell sorting 33 (Fig. 2a); (ii) Measurement of ploidy for 33 NCI60 cell lines based on spectral karyotyping34 (Fig. 2b,c); and (iii) DNA-mixing experiments, in which cancer cell lines were mixed with paired normal B-lymphocyte-derived DNAs in varying mass proportions (Fig. 2d, Online Methods). We also evaluated a related computational method, ASCAT30, on these data (Fig. 2a-d, Supplementary Note 1). Although the results were broadly concordant with our estimates, ABSOLUTE achieved significantly more accurate results (Fig. 2a-d) on our validation data. Notably, we observed an apparent bias by ASCAT to underestimate cancer cell fraction (Fig. 2 c,d), consistent with previous reports applying ASCAT in similar mixing experiments based on Illumina SNP arrays30, (Figure S4), suggesting that the bias is independent of measurement platform.


Absolute quantification of somatic DNA alterations in human cancer.

Carter SL, Cibulskis K, Helman E, McKenna A, Shen H, Zack T, Laird PW, Onofrio RC, Winckler W, Weir BA, Beroukhim R, Pellman D, Levine DA, Lander ES, Meyerson M, Getz G - Nat. Biotechnol. (2012)

ABSOLUTE method validation and comparison a-d Performance of ABSOLUTE and ASCAT on 4 validation assaysRMSE: root mean squared error. P-values were calculated on the squared errors using the paired one-sided Wilcoxon test (*: P < 0.05, **: P < 0.001). See Supplementary Note 1 for the ASCAT2.1 protocol.a, FACS-based ploidy measurements vs. inferred ploidy estimates for 37 primary tumor samples.Dashed line indicates y=x.b, SKY-based ploidy measurements vs. inferred ploidy estimates for 33 cancer cell-lines. Dataare displayed as in a.c, Estimated purity of the 33 cell lines shown in (b) Dashed horizontal line indicates the truepurity (1.0).d, Cancer-normal DNA mixing experiment results for two cell lines. DNA from each cancercell line was mixed with DNA from the matched B-lymphocyte in varying proportions (x-axis).(top) predicted vs. true DNA mixing fractions compared to the y=x line (dashed). (bottom)predicted cancer cell-line ploidy vs. mixture purity. The copy-profile of several samples wasmisinterpreted (x's); these points were not included in the RMSE calculations. Ploidy estimateswere generally consistent with previous SKY analysis of these cell lines: http://www.path.cam.ac.uk/∼pawefish/cell%20line%20catalogues/breast-cell-lines.htm.e, Leukocyte methylation signature enrichment in tumors of histologicaly underestimatedpurity. HGS-OvCa samples are shown grouped according to the indicated histological purityestimates (x-axis)33. Black horizontal lines indicate the median purity of each group, asestimated by ABSOLUTE (y-axis). The color of each point corresponds to the degree to whichthat sample's methylation profile resembled that of purified leukocytes (Online Methods).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4383288&req=5

Figure 2: ABSOLUTE method validation and comparison a-d Performance of ABSOLUTE and ASCAT on 4 validation assaysRMSE: root mean squared error. P-values were calculated on the squared errors using the paired one-sided Wilcoxon test (*: P < 0.05, **: P < 0.001). See Supplementary Note 1 for the ASCAT2.1 protocol.a, FACS-based ploidy measurements vs. inferred ploidy estimates for 37 primary tumor samples.Dashed line indicates y=x.b, SKY-based ploidy measurements vs. inferred ploidy estimates for 33 cancer cell-lines. Dataare displayed as in a.c, Estimated purity of the 33 cell lines shown in (b) Dashed horizontal line indicates the truepurity (1.0).d, Cancer-normal DNA mixing experiment results for two cell lines. DNA from each cancercell line was mixed with DNA from the matched B-lymphocyte in varying proportions (x-axis).(top) predicted vs. true DNA mixing fractions compared to the y=x line (dashed). (bottom)predicted cancer cell-line ploidy vs. mixture purity. The copy-profile of several samples wasmisinterpreted (x's); these points were not included in the RMSE calculations. Ploidy estimateswere generally consistent with previous SKY analysis of these cell lines: http://www.path.cam.ac.uk/∼pawefish/cell%20line%20catalogues/breast-cell-lines.htm.e, Leukocyte methylation signature enrichment in tumors of histologicaly underestimatedpurity. HGS-OvCa samples are shown grouped according to the indicated histological purityestimates (x-axis)33. Black horizontal lines indicate the median purity of each group, asestimated by ABSOLUTE (y-axis). The color of each point corresponds to the degree to whichthat sample's methylation profile resembled that of purified leukocytes (Online Methods).
Mentions: We validated the purity and ploidy predictions made by ABSOLUTE on Affymetrix SNP microarray data using several approaches: (i) direct measurement of ploidy of 37 TCGA ovarian carcinoma samples by fluorescence-activated cell sorting 33 (Fig. 2a); (ii) Measurement of ploidy for 33 NCI60 cell lines based on spectral karyotyping34 (Fig. 2b,c); and (iii) DNA-mixing experiments, in which cancer cell lines were mixed with paired normal B-lymphocyte-derived DNAs in varying mass proportions (Fig. 2d, Online Methods). We also evaluated a related computational method, ASCAT30, on these data (Fig. 2a-d, Supplementary Note 1). Although the results were broadly concordant with our estimates, ABSOLUTE achieved significantly more accurate results (Fig. 2a-d) on our validation data. Notably, we observed an apparent bias by ASCAT to underestimate cancer cell fraction (Fig. 2 c,d), consistent with previous reports applying ASCAT in similar mixing experiments based on Illumina SNP arrays30, (Figure S4), suggesting that the bias is independent of measurement platform.

Bottom Line: We used ABSOLUTE to analyze exome sequencing data from 214 ovarian carcinoma tumor-normal pairs.This analysis identified both pervasive subclonal somatic point-mutations and a small subset of predominantly clonal and homozygous mutations, which were overrepresented in the tumor suppressor genes TP53 and NF1 and in a candidate tumor suppressor gene CDK12.ABSOLUTE will facilitate the design of clinical sequencing studies and studies of cancer genome evolution and intra-tumor heterogeneity.

View Article: PubMed Central - PubMed

Affiliation: The Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA. scarter@broadinstitute.org

ABSTRACT
We describe a computational method that infers tumor purity and malignant cell ploidy directly from analysis of somatic DNA alterations. The method, named ABSOLUTE, can detect subclonal heterogeneity and somatic homozygosity, and it can calculate statistical sensitivity for detection of specific aberrations. We used ABSOLUTE to analyze exome sequencing data from 214 ovarian carcinoma tumor-normal pairs. This analysis identified both pervasive subclonal somatic point-mutations and a small subset of predominantly clonal and homozygous mutations, which were overrepresented in the tumor suppressor genes TP53 and NF1 and in a candidate tumor suppressor gene CDK12. We also used ABSOLUTE to infer absolute allelic copy-number profiles from 3,155 diverse cancer specimens, revealing that genome-doubling events are common in human cancer, likely occur in cells that are already aneuploid, and influence pathways of tumor progression (for example, with recessive inactivation of NF1 being less common after genome doubling). ABSOLUTE will facilitate the design of clinical sequencing studies and studies of cancer genome evolution and intra-tumor heterogeneity.

Show MeSH
Related in: MedlinePlus