The number of methylated CpG sites within the MGMT promoter region linearly correlates with outcome in glioblastoma receiving alkylating agents

Abstract

MGMT-promoter methylation is associated with favorable outcome in glioblastoma. The aim of this study was to determine whether the absolute number of methylated Cytosine-Guanine-dinucleotide-(CpG-)sites within the DMR-2 island of the MGMT-promoter may correlate with outcome in a qualitative or quantitative fashion. In a cohort of newly diagnosed glioblastoma patients treated with stereotactic biopsy or open tumor resection plus concomitant chemoradiotherapy, we assessed MGMT-promoter methylation by methylation-specific polymerase-chain-reaction (MSP). Methylation of the CpG-sites 74-98 within the MGMT-promoter region was additionally analysed by Sanger sequencing, and the total number of methylated CpG-sites was correlated with outcome using proportional hazards models. 215 patients with glioblastoma were identified and stratified per MSP (positive: 53%, negative: 47%). Among MSP-positive tumors, hierarchical clustering identified three subgroups with different methylation rates (median: 80% vs. 52% vs. 47%), indicating a site-dependent methylation propagation. The methylation status of a given CpG-site indicated a neighborhood-dependent methylation propagation. Survival was linearly associated with the cumulative number of methylated CpG-sites. This was particularly true in patients who received at least one adjuvant cycle of temozolomide. Notably, all CpG-sites analyzed contributed similarly to effect size; this enabled a further predictive substratification of MSP-positive tumors with median OS ranging from as low as 17.1 months (< 18 methylated CpG-sites) to as high as 26.2 months (≥ 18 methylated CpG-sites) in the overall cohort. All in all, total number of methylated CpG-sites may correlate with outcome in a linear fashion. Such analysis may therefore add further predictive value to conventional methods of determining the MGMT-promoter status.