MGMT promoter methylation in gliomas-assessment by pyrosequencing and quantitative methylation-specific PCR

Annette Bentsen Håvik,Petter Brandal,Torstein Ragnar Meling,Ragnhild A Lothe,Sverre Heim,Guro Elisabeth Lind,Hanne-Sofie Spenning Dahlback,Merete Hektoen,Eirik Helseth,Hilde Honne,David Scheie

doi:10.1186/1479-5876-10-36

Annette Bentsen Håvik, Petter Brandal + Show 9 more

Open Access

https://doi.org/10.1186/1479-5876-10-36

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Abstract

BackgroundMethylation of the O6-methylguanine-DNA methyltransferase (MGMT) gene promoter is a favorable prognostic factor in glioblastoma patients. However, reported methylation frequencies vary significantly partly due to lack of consensus in the choice of analytical method.MethodWe examined 35 low- and 99 high-grade gliomas using quantitative methylation specific PCR (qMSP) and pyrosequencing. Gene expression level of MGMT was analyzed by RT-PCR.ResultsWhen examined by qMSP, 26% of low-grade and 37% of high-grade gliomas were found to be methylated, whereas 97% of low-grade and 55% of high-grade gliomas were found methylated by pyrosequencing. The average MGMT gene expression level was significantly lower in the group of patients with a methylated promoter independent of method used for methylation detection. Primary glioblastoma patients with a methylated MGMT promoter (as evaluated by both methylation detection methods) had approximately 5 months longer median survival compared to patients with an unmethylated promoter (log-rank test; pyrosequencing P = .02, qMSP P = .06). One third of the analyzed samples had conflicting methylation results when comparing the data from the qMSP and pyrosequencing. The overall survival analysis shows that these patients have an intermediate prognosis between the groups with concordant MGMT promoter methylation results when comparing the two methods.ConclusionIn our opinion, MGMT promoter methylation analysis gives sufficient prognostic information to merit its inclusion in the standard management of patients with high-grade gliomas, and in this study pyrosequencing came across as the better analytical method.

Highlights

Methylation of the O6-methylguanine-DNA methyltransferase (MGMT) gene promoter is a favorable prognostic factor in glioblastoma patients
The average MGMT gene expression level was significantly lower in the group of patients with a methylated promoter independent of method used for methylation detection
Primary glioblastoma patients with a methylated MGMT promoter had approximately 5 months longer median survival compared to patients with an unmethylated promoter

Summary

Introduction

Methylation of the O6-methylguanine-DNA methyltransferase (MGMT) gene promoter is a favorable prognostic factor in glioblastoma patients. The DNA repair enzyme O-6-methylguanine-DNA methyltransferase (MGMT) removes methyl groups from the O6-position of guanine and the expression of MGMT is thought to inhibit the cytotoxic effect of TMZ [5,6]. Though several studies indicate that MGMT promoter methylation is a prognostic marker [11], there is no clear consensus as to which detection method should be preferred or what constitutes optimal threshold values for scoring samples as methylation positive. A wide range of reported glioma MGMT methylation frequencies can be seen (Additional file 1: Tables S1 and S2) [11]. To illustrate the variability in methylation frequencies and methylation detection methods, we systemized publications reporting MGMT promoter methylation in a tabular overview (Additional file 1: Tables S1 and S2)

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