Simple SummaryGlioblastoma is the most malignant brain tumor. To date, there is no curative therapy available. SinceEGFRis an interesting candidate in precision medicine, we performed an integrated molecular analysis in glioblastoma with and withoutEGFRamplification. We found that there are significant molecular differences in glioblastoma, depending on theEGFRamplification state. Analysis of top differences revealed DNA replication and packaging, and chromatin and gene silencing pathways. Targeting these altered pathways may open novel targets in precision medicine.AbstractGlioblastoma IDH wildtype is the most frequent brain tumor in adults. It shows a highly malignant behavior and devastating outcomes. To date, there is still no targeted therapy available; thus, patients’ median survival is limited to 12–15 months. Epithelial growth factor receptor (EGFR) is an interesting targetable candidate in advanced precision medicine for brain tumor patients. In this study, we performed integrated epigenome-wide DNA-methylation profiling of 866,895 methylation specific sites in 50 glioblastoma IDH wildtype samples, comparingEGFRamplified and non-amplified glioblastomas. We found 9849 significantly differentially methylated CpGs (DMCGs) with Δβ ≥ 0.1 andp-value < 0.05 inEGFRamplified, compared toEGFRnon-amplified glioblastomas. Of these DMCGs, 2380 were annotated with tiling (2090), promoter (117), gene (69) and CpG islands (104); 7460 are located at other loci. Interestingly, the list of differentially methylated genes allocated eleven functionally relevant RNAs: five miRNAs (miR1180, miR1255B1, miR126, miR128-2, miR3125), two long non-coding RNAs (LINC00474, LINC01091), and four antisense RNAs (EPN2-AS1, MNX1-AS2, NKX2-2-AS1, WWTR1-AS1). Gene ontology (GO) analysis showed enrichment of “DNA replication-dependent nucleosome assembly”, “chromatin silencing at rDNA”, “regulation of gene silencing by miRNA”, “DNA packaging”, “posttranscriptional gene silencing”, “gene silencing by RNA”, “negative regulation of gene expression, epigenetic”, “regulation of gene silencing”, “protein-DNA complex subunit organization”, and “DNA replication-independent nucleosome organization” pathways being hypomethylated inEGFRamplified glioblastomas. In summary, dissecting the methylomes ofEGFRamplified and non-amplified glioblastomas revealed altered DNA replication, DNA packaging, chromatin silencing and gene silencing pathways, opening potential novel targets for future precision medicine.