EGFR variant heterogeneity in glioblastoma resolved through single-nucleus sequencing

Glioblastomas 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 with 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 loss-of-heterozygosity 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 non-overlapping subclonal populations. In vitro and in vivo functional studies show 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.