Development of a Robust MGMT Promoter Methylation Assay for Glioblastoma Multiforme

AACR 2020 -- The degree of methylation in the promoter region of the O-6-methylguanine-DNA methyltransferase (MGMT) gene is widely accepted as a prognostic biomarker of patient outcomes in Glioblastoma multiforme (GBM). There are a number of different assays currently available for the measurement of DNA methylation, that assess methylation status by different methods and at different CpG sites in the MGMT promoter and in exon 1. There is the possibility that the various methods may produce different methylation results and thus lead to different patient treatment decisions based on the test results. LabCorp of America Holdings has provided MGMT testing services for clinical samples based upon a published assay. Because of the need to change instrumentation this assay was re-formatted, optimized and analytically validated for possible use as an In Vitro Diagnostic Device. Formalin-fixed-paraffin-embedded (FFPE) specimens from GBM patients were evaluated using standard pathology methods, the tumor regions were identified, and the corresponding tissue tumor region from unstained slides were macro-dissected. DNA was isolated and analyzed by methylation specific real-time PCR (MSP). The copy number of methylated MGMT based on 8 CpG sites was determined and normalized to the copy number of β-Actin gene (ACTB). The MGMT MSP Assay was analytically validated to confirm that assay linearity, precision, accuracy, and intra-specimen variability met pre-defined acceptance criteria. Additional analytical performance characteristics of the assay are currently being evaluated. The re-formatted MGMT MSP Assay provides a robust and reproducible measure of MGMT promoter methylation status in GBM tissue samples.