OS3.4 Intra-tumour DNA methylation heterogeneity in glioblastoma

Abstract

INTRODUCTION: A hallmark of glioblastoma (GBM) is the cellular and molecular heterogeneity both inter- and intra-tumourally. This adds to the complexity of GBM regarding risk for sampling bias and tumour escape of targeted therapy. Heterogeneity of gene expression within GBM is well documented, but little is known regarding the epigenetic heterogeneity. This could have implications for diagnostics, as DNA methylation can be used for tumour classification, subtyping and determination of the clinically used biomarker MGMT promoter methylation status. We therefore aimed to profile the intra-tumour epigenetic heterogeneity in GBM. MATERIALS AND METHODS: Three biopsies per tumour from spatially separated regions were collected from 10 adult GBM patients. Only samples that were highly fluorescent using Gliolan (5-ALA) were collected and the location was documented with neuronavigation. We then performed genome-wide DNA methylation analysis to determine the methylation level of ∼850 000 CpG sites and compared inter and intra-tumour variation. The tumour content of the biopsies was assessed based on histology by a specialist in clinical neuropathology. RESULTS: Hierarchical clustering of the CpG sites with the most variable methylation clustered the biopsies from each patient respectively together except for the biopsies from one patient. A large number of differentially methylated probes (DMP) existed between the intra-tumour biopsies and some were located in GBM-related genes. All samples were classified as GBM by methylation profiling, but the strength of the classification and the subtype differed within tumours. We also found intra-tumour heterogeneity in the methylation status of the MGMT promoter in one of the patients. CONCLUSIONS: Intra-tumour biopsies did not cluster together for all patients and displayed many DMP demonstrating that intra-tumour DNA methylation heterogeneity exists in GBM. The tumour type (GBM) is classified homogenously within the tumour for all patients, while the strength of the classification and the subtype differs. The observed intra-tumour heterogeneity in DNA methylation needs to be taken into consideration for methylation-based biomarkers and future stratification of GBM subtypes.