EGFR Variant Heterogeneity in Glioblastoma Resolved through Single-Nucleus Sequencing

Abstract

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.

Epidermal Growth Factor Receptor Variant III (EGFRvIII) Positivity in EGFR-Amplified Glioblastomas: Prognostic Role and Comparison between Primary and Recurrent Tumors

“…We did not evaluate the prognostic role of less common EGFR deletion variants like EGFRvII (deletion of exon [14][15] and EGFRvV (C-terminal deletions; refs. 8,60,62,63); however, a previous study based on TCGA data did not observe a different outcome in glioblastoma patients whose tumors carried either of these variants (61).…”

“…Several studies have reported on various other EGFR sequence alterations than EGFRvIII in glioblastomas, including SNVs as well as larger rearrangements/deletions affecting the extracellular or intracellular domains (8,14,(57)(58)(59)(60)(61)(62)(63). We therefore additionally investigated 27 pairs of primary and recurrent glioblastomas for other EGFR gene alterations using targeted next-generation sequencing of tumor DNA.…”

Soluble Urokinase Receptor Is Released Selectively by Glioblastoma Cells That Express Epidermal Growth Factor Receptor Variant III and Promotes Tumor Cell Migration and Invasion*

“…Single cell genome sequencing has revealed extensive variability in the degree of EGFR gene amplification and expression of mutated forms of the EGFR, among different tumor cells in a single GBM (54). Soluble factors released by one cell may affect properties of neighboring cells, including the capacity for migration, invasion, and survival.…”

The Case for a Pre-Cancer Genome Atlas (PCGA)

“…Similarly, single-cell DNA-seq can be achieved by performing single nuclei sorting from fresh frozen tissue. This approach can have even higher resolution to unravel evolutionary relationships among subclonal populations (22,23).…”