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

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

Abstract

Background: Methylation of the O(6)-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.

Method: We 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.

Results: When 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.

Conclusion: In 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.

© 2012 Håvik et al; licensee BioMed Central Ltd.

Figures

Figure 1
Figure 1
Overall survival in primary glioblastoma patients treated with standard radiotherapy and concomitant temozolomide. (A) Methylation status based on results from qMSP. Blue line; methylated, red line; unmethylated. (B) Methylation status based on results from pyrosequencing. Blue line; methylated, red line; unmethylated. (C) Methylation status based on both qMSP and pyrosequencing results. Blue line; methylated in both methods, red line; unmethylated in both methods, green line; methylated in pyrosequencing assay and unmethylated in qMSP assay. Abbreviation: qMSP, quantitative methylation specific PCR
Figure 2
Figure 2
Gene expression level of MGMT is associated with promoter DNA methylation status. MGMT gene expression in methylated (blue box plots) and unmethylated (red box plots) tissue samples analyzed by two different primer/probe sets (Assay 1; Hs00172470_m1 and assay 2; Hs01037698_m1). (A) Methylation status based on results from qMSP. (B) Methylation status based on results from pyrosequencing. Abbreviation: qMSP, quantitative methylation specific PCR
Figure 3
Figure 3
Scatter plots of MGMT gene expression quantity in methylated and unmethylated samples. Red color indicates samples with negative methylation status by qMSP and pyrosequencing, blue color indicates samples with positive methylation status by qMSP and pyrosequencing, and green color indicate samples with non-concordant methylation status in the qMSP and pyrosequencing analysis. Circles indicate samples with loss of 10q26, whereas dots represent samples without loss of this region. Plots are based on the normalized gene expression detected by primer/probe set Hs00172470_m1. The plots based on the normalized gene expression detected by the other primer/probe set, Hs01037698_m1, were similar (data not shown). Abbreviation: qMSP, quantitative methylation specific PCR

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