Lhotska Halka, Janeckova Karolina, Cechova Hana, Macoun Jaromir, Aghova Tatiana, Lizcova Libuse, Svobodova Karla, Hodanova Lucie, Konecna Dora, Soukup Jiri, Kramar Filip, Netuka David, Zemanova Zuzana
Center of Oncocytogenomics, Institute of Medical Biochemistry and Laboratory Diagnostics, General University Hospital and 1st Faculty of Medicine of Charles University in Prague, U Nemocnice 499/2, 128 00, Prague, Czech Republic.
Department of HLA, Institute of Hematology and Blood Transfusion, U Nemocnice 2094/1, 128 00, Prague, Czech Republic.
Clin Epigenetics. 2025 Jan 29;17(1):16. doi: 10.1186/s13148-025-01822-2.
Glioblastoma is the commonest malignant brain tumor and has a very poor prognosis. Reduced expression of the MGMT gene (10q26.3), influenced primarily by the methylation of two differentially methylated regions (DMR1 and DMR2), is associated with a good response to temozolomide treatment. However, suitable methods for detecting the methylation of the MGMT gene promoter and setting appropriate cutoff values are debated.
A cohort of 108 patients with histologically and genetically defined glioblastoma was retrospectively examined with methylation-specific Sanger sequencing (sSeq) and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) methods. The DMR2 region was methylated in 29% of samples, whereas DMR1 was methylated in 12% of samples. Methylation detected with the MS-MLPA method using probes MGMT_215, MGMT_190, and MGMT_124 from the ME012-A1 kit (located in DMR1 and DMR2) correlated with the methylation of the corresponding CpG dinucleotides detected with sSeq (p = 0.005 for probe MGMT_215; p < 0.001 for probe MGMT_190; p = 0.016 for probe MGMT_124). The threshold for methylation detection with the MS-MLPA method was calculated with a ROC curve analysis and principal components analysis of the data obtained with the MS-MLPA and sSeq methods, yielding a weighted value of 0.362. Thus, methylation of the MGMT gene promoter was confirmed in 36% of samples. These patients had statistically significantly better overall survival (p = 0.003).
Our results show that the threshold for methylation detection with the MS-MLPA method determined here is useful from a diagnostic perspective because it allows the stratification of patients who will benefit from specific treatment protocols, including temozolomide. Detailed analysis of the MGMT gene promoter enables the more-precise and personalized treatment of patients with glioblastoma.
胶质母细胞瘤是最常见的恶性脑肿瘤，预后很差。MGMT基因（10q26.3）表达降低主要受两个差异甲基化区域（DMR1和DMR2）甲基化的影响，与替莫唑胺治疗反应良好相关。然而，检测MGMT基因启动子甲基化的合适方法以及设定适当的临界值仍存在争议。
对108例经组织学和遗传学确诊的胶质母细胞瘤患者进行回顾性研究，采用甲基化特异性桑格测序（sSeq）和甲基化特异性多重连接依赖探针扩增（MS-MLPA）方法。29%的样本中DMR2区域甲基化，而12%的样本中DMR1区域甲基化。使用ME012-A1试剂盒中的探针MGMT_215、MGMT_190和MGMT_124（位于DMR1和DMR2）通过MS-MLPA方法检测到的甲基化与sSeq检测到的相应CpG二核苷酸甲基化相关（探针MGMT_215，p = 0.005；探针MGMT_190，p < ０.001；探针MGMT_124，p = 0.016）。通过对MS-MLPA和sSeq方法获得的数据进行ROC曲线分析和主成分分析，计算出MS-MLPA方法检测甲基化的阈值，加权值为0.362。因此，36%的样本中证实了MGMT基因启动子甲基化。这些患者的总生存期在统计学上有显著改善（p = 0.003）。
我们的结果表明，此处确定的MS-MLPA方法检测甲基化的阈值从诊断角度来看是有用的，因为它可以对将从包括替莫唑胺在内的特定治疗方案中获益的患者进行分层。对MGMT基因启动子的详细分析能够实现胶质母细胞瘤患者更精确和个性化的治疗。
