MGMT promoter methylation determined by HRM in comparison to MSP and pyrosequencing for predicting high-grade glioma response.
MGMT promoter methylation determined by HRM in comparison to MSP and pyrosequencing for predicting high-grade glioma response.
复制标题
DOI：
10.1186/s13148-016-0204-7
复制
发表时间：
2016
影响因子：
5.7
通讯作者：
Kaina B
中科院分区：
文献类型：
--
作者：
Switzeny OJ;Christmann M;Renovanz M;Giese A;Sommer C;Kaina B
DNA修复蛋白O 6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）导致癌细胞对烷化剂的耐药性，因此是常规使用烷化剂治疗的高级别胶质瘤的公认预测标志物。由于MGMT在表观遗传学上受到高度调节，因此MGMT启动子甲基化状态被用作MGMT沉默的指标，预测胶质瘤治疗的结果。MGMT启动子甲基化通常通过甲基化特异性PCR（MSP）来确定，MSP是一种劳动密集型且易于出错的方法，通常半定量地使用。为了寻找替代方案，我们使用了封闭管高分辨率熔解（HRM）分析，这是一种定量方法，并将其与MSP和焦磷酸测序的预测价值进行了比较。我们通过HRM、MSP和焦磷酸测序分析了具有已知MGMT活性的胶质母细胞瘤细胞系和来自IDH 1野生型高级别胶质瘤患者（WHO III/IV级）的福尔马林固定样本，这些患者接受了放疗和替莫唑胺治疗。比较了表现出甲基化和未甲基化MGMT状态的患者的无进展生存期（PFS）和总生存期（OS）。确定与PFS和OS相关的启动子甲基化截止水平。在多变量考克斯回归模型中，通过HRM而不是MSP分析的高级别胶质瘤的MGMT启动子甲基化被发现是OS的独立预测标志物。单变量Kaplan-Meier分析显示，当使用定量HRM而不是MSP时，PFS和OS在甲基化和非甲基化肿瘤之间具有显著且更好的区分。与MSP和焦磷酸测序相比，HRM方法简单，成本效益高，准确度高，速度快。HRM在定量甲基化水平方面至少等同于焦磷酸测序。与MSP相比，它在预测高级别胶质瘤患者的PFS和OS方面具有上级优势，因此可以推荐常规用于确定胶质瘤的MGMT状态。本文的在线版本（doi：10.1186/s13148-016-0204-7）包含补充材料，可供授权用户使用。
The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) causes resistance of cancer cells to alkylating agents and, therefore, is a well-established predictive marker for high-grade gliomas that are routinely treated with alkylating drugs. Since MGMT is highly epigenetically regulated, the MGMT promoter methylation status is taken as an indicator of MGMT silencing, predicting the outcome of glioma therapy. MGMT promoter methylation is usually determined by methylation specific PCR (MSP), which is a labor intensive and error-prone method often used semi-quantitatively. Searching for alternatives, we used closed-tube high resolution melt (HRM) analysis, which is a quantitative method, and compared it with MSP and pyrosequencing regarding its predictive value. We analyzed glioblastoma cell lines with known MGMT activity and formalin-fixed samples from IDH1 wild-type high-grade glioma patients (WHO grade III/IV) treated with radiation and temozolomide by HRM, MSP, and pyrosequencing. The data were compared as to progression-free survival (PFS) and overall survival (OS) of patients exhibiting the methylated and unmethylated MGMT status. A promoter methylation cut-off level relevant for PFS and OS was determined. In a multivariate Cox regression model, methylation of MGMT promoter of high-grade gliomas analyzed by HRM, but not MSP, was found to be an independent predictive marker for OS. Univariate Kaplan–Meier analyses revealed for PFS and OS a significant and better discrimination between methylated and unmethylated tumors when quantitative HRM was used instead of MSP. Compared to MSP and pyrosequencing, the HRM method is simple, cost effective, highly accurate and fast. HRM is at least equivalent to pyrosequencing in quantifying the methylation level. It is superior in predicting PFS and OS of high-grade glioma patients compared to MSP and, therefore, can be recommended being used routinely for determination of the MGMT status of gliomas. The online version of this article (doi:10.1186/s13148-016-0204-7) contains supplementary material, which is available to authorized users.