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Background  The utility of O 6 -methylguanine-DNA methyltransferase (MGMT) gene promoter methylation status as a prognostic marker in patients with glioblastoma (GBM) has been established. However, the number of CpG sites that must be methylated to cause transcriptional silencing remains unclear, and no significant consensus exists on the optimal method of assessing  MGMT  methylation. We developed a new high-performance liquid chromatography (HPLC) method that enables accurate analysis of DNA methylation levels using long PCR products. In the present study, we analyzed the  MGMT  methylation status of 28 isocitrate dehydrogenase–wild-type GBMs treated with temozolomide using ion-exchange HPLC and set the optimal cutoff values.    Results  We designed three primers for separate regions (regions 1–3) that had 21 to 38 CpGs for PCR and validated the  MGMT  promoter methylation status using frozen samples. There was a strong correlation between HPLC and bisulfite sequencing results ( R  = 0.794). The optimal cutoff values for  MGMT  methylation in HPLC were determined to allow differentiation of patient prognosis by receiver operating characteristic curve analysis. The cutoff values were 34.15% for region 1, 8.84% for region 2, and 36.72% for region 3. Kaplan–Meyer curve analysis estimated that the most differentiated prognosis was enabled in the setting of 8.84% methylation of  MGMT  in region 2. Progression-free survival and overall survival were significantly longer for patients in this setting of region 2 methylation ( p  = 0.00365 and  p  = 0.00258, respectively).    Conclusions  The combination of our HPLC method and the original primer setting provides a new standard method for determination of  MGMT  methylation status in patients with GBM and is useful for refining  MGMT -based drug selection.

Assessment of MGMT methylation status using high-performance liquid chromatography in newly diagnosed glioblastoma