Methylation subgroup and molecular heterogeneity is a hallmark of glioblastoma: implications for biopsy targeting, classification and therapy

Background: Intratumoral heterogeneity at the cellular and molecular level is a hallmark of glioblastoma (GB) that contributes to treatment resistance and poor clinical outcome. Little is known regarding epigenetic heterogeneity and intratumoral phylogeny and their implication for molecular classification and targeted therapies. Patients and methods: Multiple tissue biopsies (238 in total) were sampled from 56 newly-diagnosed, treatment-naive GB patients from a prospective in-house cohort and publicly available data and profiled for DNA methylation using the Illumina MethylationEPIC array. Methylation-based classification using the glioma classifier developed by Ceccarelli et al. and estimation of the MGMT promoter methylation status via the MGMT-STP27 model were carried out. In addition, copy number variations (CNVs) and phylogeny were analyzed. Results: Almost half of the patients (22/56, 39%) harbored tumors composed of heterogeneous methylation subtypes. We found two predominant subtype combinations: classic-/mesenchymal-like, and mesenchymal-/pilocytic astrocytoma-like. Nine patients (16%) had tumors composed of subvolumes with and without MGMT promoter methylation, whereas 20 patients (36%) were homogeneously methylated, and 27 patients (48%) were homogeneously unmethylated. CNV analysis revealed high variations in many genes, including CDKN2A/B, EGFR, and PTEN. Phylogenetic analysis correspondingly showed a general pattern of CDKN2A/B loss and gain of EGFR, PDGFRA, and CDK4 during early stages of tumor development. Conclusions: (Epi)genetic intratumoral heterogeneity is a hallmark of GB, both at DNA methylation and CNV level. This intratumoral heterogeneity is of utmost importance for molecular classification as well as for defining therapeutic targets in this disease, as single biopsies might underestimate the true molecular diversity in a tumor.