Abstract
BackgroundThe 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.ResultsWe 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.ConclusionsCompared 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-016-0204-7) contains supplementary material, which is available to authorized users.
Highlights
The deoxyribonucleic acid (DNA) repair protein O6-methylguanine-DNA methyltransferase (MGMT) causes resistance of cancer cells to alkylating agents and, is a well-established predictive marker for high-grade gliomas that are routinely treated with alkylating drugs
receiver operating characteristic (ROC) curves were generated to depict the sensitivity and specificity of MGMT promoter methylation status determined by high resolution melt (HRM), methylation specific polymerase chain reaction (PCR) (MSP), PSQ as well as age
Since the MGMT promoter methylation status analyzed by HRM is most precise in determining the patient’s outcome, we recommend HRM as a feasible and reliable method for routine diagnostics of high-grade glioma patients
Summary
The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) causes resistance of cancer cells to alkylating agents and, is a well-established predictive marker for high-grade gliomas that are routinely treated with alkylating drugs. Patients suffering from high-grade gliomas (notably glioblastoma multiforme, WHO grade IV) have a dismal prognosis (14.6 months median survival and a 2-year survival rate of 26 %) [1]. Their first-line therapy is based on the DNA alkylating agent temozolomide (Temodal®) and ionizing radiation [1, 2]. MGMT is an important predictive marker for high-grade gliomas [10, 11]
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