The utility of O6-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. 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). 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.

中文翻译：

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使用高效液相色谱法在新诊断的胶质母细胞瘤中评估 MGMT 甲基化状态

O6-甲基鸟嘌呤-DNA 甲基转移酶 (MGMT) 基因启动子甲基化状态作为胶质母细胞瘤 (GBM) 患者预后标志物的效用已经确立。然而，必须被甲基化以引起转录沉默的 CpG 位点的数量仍不清楚，并且在评估 MGMT 甲基化的最佳方法上没有明显的共识。我们开发了一种新的高效液相色谱 (HPLC) 方法，可以使用长 PCR 产物准确分析 DNA 甲基化水平。在本研究中，我们使用离子交换 HPLC 分析了用替莫唑胺处理的 28 种异柠檬酸脱氢酶 - 野生型 GBM 的 MGMT 甲基化状态，并设置了最佳截止值。我们为不同的区域（区域 1-3）设计了三个引物，这些引物具有 21 到 38 个用于 PCR 的 CpG，并使用冷冻样品验证了 MGMT 启动子甲基化状态。HPLC 和亚硫酸氢盐测序结果之间存在很强的相关性 (R = 0.794)。确定了 HPLC 中 MGMT 甲基化的最佳截断值，以通过接受者操作特征曲线分析来区分患者的预后。区域 1 的截止值为 34.15%，区域 2 的截止值为 8.84%，区域 3 的截止值为 36.72%。 Kaplan-Meyer 曲线分析估计，在区域 2 中 MGMT 甲基化率为 8.84% 的情况下，最分化的预后成为可能。在这种区域 2 甲基化情况下，患者的无生存期和总生存期显着更长（分别为 p = 0.00365 和 p = 0.00258）。