XLSX file - 424KB, 9 tables containing segmented copy number, SCNAs, chromosomal rearrangements and whole-exomes sequencing data for BT325 and BT340. Table S1: EGFR alterations in TCGA samples with matching RNA and DNA sequencing. Table S2: Genes identified in the chromosome 6-9 amplicon in BT340. Table S3: BT325 segmented copy number data. Table S4: BT340 segmented copy number data. Table S5: BT340 copy number aberrations. Table S6: BT340 chromosomal rearrangements. Table S7: BT325 copy number aberrations and chromosomal rearrangements (each color denotes segments that are joined in one derivative chromosome). Table S8: Somatic SNVs and short insertion/deletions detected in BT325 from whole-exome sequencing data. Table S9: Somatic SNVs and short insertion/deletions detected in BT340 from whole-exome sequencing data.
ARTICLE ABSTRACT
Glioblastomas (GBM) with EGFR amplification represent approximately 50% of newly diagnosed cases, and recent studies have revealed frequent coexistence of multiple EGFR aberrations within the same tumor, which has implications for mutation cooperation and treatment resistance. However, bulk tumor sequencing studies cannot resolve the patterns of how the multiple EGFR aberrations coexist with other mutations within single tumor cells. Here, we applied a population-based single-cell whole-genome sequencing methodology to characterize genomic heterogeneity in EGFR-amplified glioblastomas. Our analysis effectively identified clonal events, including a novel translocation of a super enhancer to the TERT promoter, as well as subclonal LOH and multiple EGFR mutational variants within tumors. Correlating the EGFR mutations onto the cellular hierarchy revealed that EGFR truncation variants (EGFRvII and EGFR carboxyl-terminal deletions) identified in the bulk tumor segregate into nonoverlapping subclonal populations. In vitro and in vivo functional studies show that EGFRvII is oncogenic and sensitive to EGFR inhibitors currently in clinical trials. Thus, the association between diverse activating mutations in EGFR and other subclonal mutations within a single tumor supports an intrinsic mechanism for proliferative and clonal diversification with broad implications in resistance to treatment.Significance: We developed a novel single-cell sequencing methodology capable of identifying unique, nonoverlapping subclonal alterations from archived frozen clinical specimens. Using GBM as an example, we validated our method to successfully define tumor cell subpopulations containing distinct genetic and treatment resistance profiles and potentially mutually cooperative combinations of alterations in EGFR and other genes. Cancer Discov; 4(8); 956–71. ©2014 AACR.See related commentary by Gini and Mischel, p. 876This article is highlighted in the In This Issue feature, p. 855
