The Clinical Implications of Inconsistently Methylated Results from Glioblastoma MGMT Testing by Replicate Methylation-Specific PCR

Abstract

The methylation status of the promoter of the O-methylguanine DNA methyltransferase gene (MGMT) is an established prognostic and predictive biomarker of glioblastoma (GBM). At the Center for Advanced Molecular Diagnostics, MGMT testing is performed by methylation-specific PCR with multiple replicates, leading to three types of reportable results: methylated, unmethylated, and inconsistently methylated. An inconsistently methylated result is reported when a methylated peak is seen in some but not all of the PCR replicates from a single DNA sample. To better understand the clinical implications of these results, we performed a retrospective review of all MGMT testing at our laboratory over a 5-year period, and correlated test results with outcome and specimen-quality data. This review yielded several novel findings. First, inconsistent MGMT methylation on replicate methylation-specific PCR is not uncommon, composes 12% (58/465) of our GBM results. Second, inconsistently methylated GBM cases are associated with relatively poor overall survival (more similar to unmethylated than to methylated cases). Third and interestingly, there appears to be a dose-response relationship between patient survival and the extent of methylation in inconsistently methylated GBMs. Finally, our analyses of specimen-quality data suggest that a combination of technical factors (eg, small samples) and tumor biology may explain inconsistent MGMT results on replicate methylation-specific PCR testing.

MGMT promoter methylation status testing to guide therapy for glioblastoma: refining the approach based on emerging evidence and current challenges

“…The use of the MSP method for detection of MGMT methylation status is supported by evidence from multiple randomized trials. 2,32,33 First described by Herman et al, 34 the technique is dependent on bisulfite conversion (which converts unmethylated cytosines to uracils), followed by use of primers designed to measure multiple CpG dinucleotides within the MGMT promoter to specifically amplify alleles with either unconverted or converted cytosines, representing methylated and unmethylated sequences, respectively ( Fig. 1).…”

MGMT Promoter Methylation Cutoff with Safety Margin for Selecting Glioblastoma Patients into Trials Omitting Temozolomide: A Pooled Analysis of Four Clinical Trials

“…Distinct tests use different principles (15) and/or interrogate different CpGs that do not all have the same impact on MGMT silencing (26)(27)(28), which is the principle mechanism for sensitizing patients to TMZ. Consequently, cutoffs and corresponding safety margins need to be determined and validated for each assay (17,(29)(30)(31).…”

A novel analytical model of MGMT methylation pyrosequencing offers improved predictive performance in patients with gliomas

“…Given the heterogeneity of the methylation levels of CpG sites in the MGMT promoter [11], Methylation-specific PCR based methods have the drawbacks to reflect the heterogeneity of CpG sites [23]. Moreover, the results of these methods critically depend on the employed PCR protocols [15], and thus lacking stability [40]. The method with microarray chips is usually utilized in large-scale analyses of the methylome of gliomas, it is inconvenient to be used in clinical testing, due to the high cost and complicated data analysis process [11,38].…”