Intratumoral heterogeneity of receptor tyrosine kinases EGFR and PDGFRA amplification in glioblastoma defines subpopulations with distinct growth factor response

TLDR

Glioblastoma exhibits extensive intratumoral heterogeneity, including variable amplification of receptor tyrosine kinases, yet the functional impact of multiple RTK amplifications on therapeutic resistance remains unclear. This study aimed to map the heterogeneity of RTK amplification and assess functional dependence on EGFR and PDGFRA within GBM tumor cell subpopulations. Using dual‑color FISH on eight EGFR‑/PDGFRA‑amplified tumors and in‑vitro ligand‑stimulation and inhibitor assays on derived cell lines, the authors identified distinct subpopulations amplified for a single RTK and demonstrated genotype selection and the necessity of simultaneous EGFR/PDGFR blockade to suppress PI3K signaling. Analysis of TCGA data revealed that 34 of 463 GBMs (~7%) harbored independent focal amplification of two or more RTKs, predominantly EGFR and PDGFRA, and that 43% of PDGFRA‑amplified tumors also amplified EGFR or MET; FISH and functional studies confirmed that single‑RTK–amplified subpopulations dominate, are clonally related, and require dual inhibition to abrogate PI3K activity.

Abstract

Glioblastoma (GBM) is distinguished by a high degree of intratumoral heterogeneity, which extends to the pattern of expression and amplification of receptor tyrosine kinases (RTKs). Although most GBMs harbor RTK amplifications, clinical trials of small-molecule inhibitors targeting individual RTKs have been disappointing to date. Activation of multiple RTKs within individual GBMs provides a theoretical mechanism of resistance; however, the spectrum of functional RTK dependence among tumor cell subpopulations in actual tumors is unknown. We investigated the pattern of heterogeneity of RTK amplification and functional RTK dependence in GBM tumor cell subpopulations. Analysis of The Cancer Genome Atlas GBM dataset identified 34 of 463 cases showing independent focal amplification of two or more RTKs, most commonly platelet-derived growth factor receptor α (PDGFRA) and epidermal growth factor receptor (EGFR). Dual-color fluorescence in situ hybridization was performed on eight samples with EGFR and PDGFRA amplification, revealing distinct tumor cell subpopulations amplified for only one RTK; in all cases these predominated over cells amplified for both. Cell lines derived from coamplified tumors exhibited genotype selection under RTK-targeted ligand stimulation or pharmacologic inhibition in vitro. Simultaneous inhibition of both EGFR and PDGFR was necessary for abrogation of PI3 kinase pathway activity in the mixed population. DNA sequencing of isolated subpopulations establishes a common clonal origin consistent with late or ongoing divergence of RTK genotype. This phenomenon is especially common among tumors with PDGFRA amplification: overall, 43% of PDGFRA-amplified GBM were found to have amplification of EGFR or the hepatocyte growth factor receptor gene (MET) as well.