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

The methylation status of the promoter of the O6-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.