Abstract 151: Development of a robust MGMT promoter methylation assay for glioblastoma multiforme
Authors: Avery Benjamin , Englert Neal , Espinoza Celso , Maynor Timothy , Phillips Christopher , Yan Jim , Turi Thomas , Shuster Jeffery , Anderson Steven M. 
Published: September 2020

Abstract
Background: The degree of methylation in the promoter region of the O-6-methylguanine-DNA methyltransferase (MGMT) gene is considered a prognostic biomarker of patient outcomes in Glioblastoma multiforme (GBM). There are a number of different assays currently available for the determination of MGMT gene methylation status, with the assays using different methods and evaluating different CpG sites in the MGMT promoter and in exon 1 to determine methylation status. There is the possibility that these methods may produce variant methylation results and thus lead to different patient treatment decisions based on the test results.
Methods: We have provided MGMT testing services for clinical samples based upon a published assay (Kam-Morgan et al. 2009 AMP Annual Meeting; Hegi et al., 2019, Clin Cancer Res. 25:1809) for more than a decade. Because of the need to change instrumentation we have further optimized the methodology and analytically validated an MGMT methylation assay 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 region is identified, and the corresponding tumor region from unstained slides macro-dissected. DNA was isolated by standard methods and analyzed by methylation specific real-time PCR. 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 following parameters were evaluated: linearity, precision, and concordance with other testing methods. Results: The assay was shown to demonstrate linearity across input DNA ranges of 2.75 ng to 2500 ng DNA isolated from FFPE clinical samples. Assay precision was measured in clinical specimens with multiple replicates, instruments, and operators, over 6 non-consecutive days of testing. Methylation status for each sample was consistent throughout the study and across all variables. Intra-tumor heterogeneity was assessed in serial sections from clinical samples over a distance of approximately 150 µm of tumor.
Results: (methylated or un-methylated) were concordant for &gt;99% of assays. An orthogonal method of determining methylation at the 8 CpG sites was performed by pyrosequencing. Results indicated good correlation of the two methods with 90% agreement in calls between the methods.
Conclusions: The assay described here provides a robust and reproducible measure of MGMT status in GBM tissue samples.
Citation Format: Benjamin Avery, Neal Englert, Celso Espinoza, Timothy Maynor, Christopher Phillips, Jim Yan, Thomas Turi, Jeffery Shuster, Steven M. Anderson. Development of a robust MGMT promoter methylation assay for glioblastoma multiforme [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 151.