MGMT promoter methylation determined by HRM in comparison to MSP and pyrosequencing for predicting high-grade glioma response

May 10, 2016Clinical epigenetics

Comparing Methods to Detect MGMT Gene Changes for Predicting Response in Aggressive Brain Tumors

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Abstract

Methylation of the MGMT promoter in high-grade gliomas, analyzed by high-resolution melt (HRM) analysis, is an independent predictive marker for overall survival.

High-grade gliomas with methylated MGMT promoters show better overall survival and progression-free survival compared to unmethylated tumors.
HRM analysis provides a more accurate and faster assessment of MGMT promoter methylation than traditional methods like methylation specific PCR (MSP).
A specific cut-off level for MGMT promoter methylation was identified as relevant for predicting patient outcomes.
In a multivariate analysis, only HRM-based methylation status was found to be an independent predictor of overall survival.
Quantitative HRM analysis significantly enhances the discrimination between methylated and unmethylated tumors compared to MSP.

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BACKGROUND: The DNA repair protein O(6)-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.

RESULTS: 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.

CONCLUSIONS: 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.

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MGMT promoter methylation determined by HRM in comparison to MSP and pyrosequencing for predicting high-grade glioma response

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