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

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.

Details matter: the role of genomic location and assay standardization in DNA methylation analyses

“…Although, this illustrates how also the choice of technology can impact on the outcome that is measured, it does not imply that individual methods to DNA methylation are not comparable. Indeed, the variation among studies using different methods was not larger than among studies using the same method [14]. In addition, we have also shown that, if carefully aligned and optimized regarding genomic location and sensitivity, multiple DNA methylation assays can yield comparable results [16] and that DNA methylation markers can be validated in independent patient series using independent technologies [17,18].…”

“…In the review, details of all published MGMT studies are summarized, including the choice of locus-specific analysis. The range of methylated high-grade gliomas stretched from less than 20 to more than 90% [14]. Although, this illustrates how also the choice of technology can impact on the outcome that is measured, it does not imply that individual methods to DNA methylation are not comparable.…”

“…The individual methods have various advantages and disadvantages and the choice of method will obviously affect the end results. This is exemplified by a comprehensive review of the analysis of the methylation status of the MGMT promoter [14]. MGMT recently entered clinical guidelines as a predictive biomarker of treatment with alkylating chemotherapy in elderly patients with glioblastoma [15].…”

A novel truncated form of HMGA2 in tumors of the ovaries

“…MGMT promoter methylation increases the sensitivity of cells to alkylating drugs, as has been shown in a number of cancer types, particularly gliomas (29). Due to the efficacy of MGMT promoter methylation status as a prognostic and predictive tumor marker, this assessment has become one of the most commonly requested analyses for gliomas (30). The present study found this gene to be altered in only 2 borderline tumors out of 184 analyzed tumors of different types, indicating that MGMT promoter methylation is not a common event in ovarian tumorigenesis.…”

Global Hypomethylation (LINE-1) and Gene-Specific Hypermethylation (GSTP1) on Initial Negative Prostate Biopsy as Markers of Prostate Cancer on a Rebiopsy

“…For quantitative analysis of LINE-1 and GSTP-1 methylation we used pyrosequencing which, in contrast to quantitative methylation specific PCR (qMS-PCR) used for GSTP1 analysis in prior studies (20)(21)(22), detects low levels of methylation as methylation in each CpG site is measured independently (29). Pyrosequencing has also been reported to have a higher sensitivity and accuracy than qMS-PCR (30).…”