Intratumor DNA methylation heterogeneity in glioblastoma: implications for DNA methylation-based classification

BACKGROUND: A feature of glioblastoma (GBM) is cellular and molecular heterogeneity, both within and between tumors. This variability causes a risk for sampling bias and potential tumor escape from future targeted therapy. Heterogeneous intratumor gene expression in GBM is well documented, but little is known regarding the epigenetic heterogeneity. Variability in DNA methylation within tumors would have implications for diagnostics, as methylation can be used for tumor classification, subtyping, and determination of the clinically used biomarker O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation. We therefore aimed to profile the intratumor DNA methylation heterogeneity in GBM and its effect on diagnostic properties. METHODS: Three to 4 spatially separated biopsies per tumor were collected from 12 GBM patients. We performed genome-wide DNA methylation analysis and investigated intratumor variation. RESULTS: All samples were classified as GBM isocitrate dehydrogenase (IDH) wild type (wt)/mutated by methylation profiling, but the subclass differed within 5 tumors. Some GBM samples exhibited higher DNA methylation differences within tumors than between, and many cytosine-phosphate-guanine (CpG) sites (mean: 17 000) had different methylation levels within the tumors. MGMT methylation status differed in IDH mutated patients (1/1). CONCLUSIONS: We demonstrated that intratumor DNA methylation heterogeneity is a feature of GBM. Although all biopsies were classified as GBM IDH wt/mutated by methylation analysis, the assigned subclass differed in samples from the same patient. The observed heterogeneity within tumors is important to consider for methylation-based biomarkers and future improvements in stratification of GBM patients.