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.
Highlights
Glioblastoma is the most frequent primary brain tumor with a devastating natural history [23]
We describe a large cohort of patients with histologically verified glioblastoma treated at a single academic neuro-oncology center with standard of care including alkylating chemotherapy, where both Methylation-specific polymerase chain reaction analysis (MSP) and Sanger sequencing (Sseq) were applied in parallel for methylguanine-DNA methyltransferase (MGMT) promoter methylation testing and evaluated independently
Biopsied patients were significantly older (63 vs. 58 years, p < 0.01), had worse clinical pretreatment status, and more often deep seated or multifocal tumor location (49.6% vs. 19.0%, p < 0.01). (See figure on page.) Fig. 1 (a) Graphical representation of the MGMT 5′-Cytosine-Guanine dinucleotide (CpG) island methylation patterns detected by Sanger sequencing in 113 MSP-positive and 102 MSP-negative glioblastoma patients
Summary
Glioblastoma is the most frequent primary brain tumor with a devastating natural history [23]. Such methods usually analyze only a small number of the Cytosine-Guanine dinucleotide (CpG) sites within the differentially methylated region-2 (DMR2) island of the MGMT promoter region, and results are typically reported either as ‘methylated’ or ‘unmethylated’ by laboratory core facilities [2, 19] Such approaches may miss the potential role of the patient’s individual CpG methylation pattern, the effect of quantitative differences in methylation, and cases of “grey zone methylation” as recently proposed [13, 24]. It is unclear whether there might be a correlation between outcome and number of methylated CpG sites, those sites which are not analyzed by standard techniques like MSP
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