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Glioblastoma is characterized by widespread genetic and transcriptional heterogeneity, yet little is known about the role of the epigenome in glioblastoma disease progression. Here, we present genome-scale maps of the DNA methylation dynamics in matched primary and recurring glioblastoma tumors, based on a national population registry and a comprehensively annotated clinical cohort. We demonstrate the feasibility of DNA methylation mapping in a large set of routinely collected formalin-fixed paraffin-embedded (FFPE) samples, and we validate bisulfite sequencing as a multi-purpose assay that allowed us to infer a range of different genetic, epigenetic, and transcriptional tumor characteristics. Based on these data, we identified characteristic differences between primary and recurring tumors, links between DNA methylation and the tumor microenvironment, and an association of epigenetic tumor heterogeneity with patient survival. In summary, this study provides a resource for dissecting DNA methylation heterogeneity in genetically diverse and heterogeneous tumors, and it demonstrates the feasibility of integrating epigenomics, radiology, and digital pathology in a representative national cohort, leveraging samples and data collected as part of routine clinical practice.

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