Abstract LB-046: Evaluation of an assay for on-demand and easy assessment of MGMT gene promoter methylation in glioblastoma patients
Authors: Filipits Martin , Brandstetter Anita , Preusser Matthias , Hainfellner Johannes , Spiegl-Kreinecker Sabine , Lai Edwin W. , Kocmond Kriszten , Kohlway Andrew , Weidler Jodi , Bates Michael , Corless Christopher 
Published: July 2017

Background: Epigenetic silencing of the O6-methylguanine DNA methyltransferase (MGMT) gene by promoter methylation is observed in 40 to 50% of glioblastomas and is associated with improved overall survival on radiation and/or temozolamide therapy. Currently, pyrosequencing is commonly used to assess the methylation status of the MGMT gene in formalin-fixed, paraffin-embedded (FFPE) samples. We examined the concordance of the recently developed GeneXpert (GX) MGMT Methylation RUO Assay with pyrosequencing in three independent glioblastoma cohorts.

Patients and methods: The GX MGMT Methylation RUO Assay is a research use only, cartridge-based methylation specific PCR assay performed on the GeneXpert® Instrument (Cepheid) that automates DNA purification, bisulfite conversion, and methylation-specific PCR after sample preparation. Cartridge results are reported in less than 4 hours as delta cycle threshold (dCt) measurements (dCt = beta-actin/ACTB Ct - MGMT Ct). The MGMT methylation status was assessed on FFPE tumor blocks from 262 glioblastoma patients obtained from OHSU (n=63), MUV (n=96), and KUL (n=103). Two adjacent sections from each block were prepared, one 4 µm section for H&E staining to determine the % tumor cell content/tumor area and one 4 µm section for lysate preparation and GX testing. Both GX MGMT Methylation testing and pyrosequencing results were correlated with overall survival of the patients in the MUV cohort.

Results: All of the 262 (100%) glioblastoma samples tested yielded valid results for both GX MGMT Methylation testing and pyrosequencing. The cut-offs were set at dCt -6.2 for the GX MGMT Methylation RUO Assay and 8% methylation for pyrosequencing. The concordance rate between GX MGMT Methylation testing and pyrosequencing was 92% (58/63 OHSU samples, 92% (88/96 MUV samples) and 83% (85/103 KUL samples). The overall concordance rate was 88% (231/262 samples). Sensitivity and specificity was 84% (27/32) and 100% (31/31) for OHSU samples, 89% (33/37) and 93% (55/59) for MUV samples, and 70% (31/44/) and 92% (54/59) for KUL samples, respectively. Overall sensitivity was 81% (91/113) and overall specificity was 94% (140/149). Overall survival data were available for 95 MUV patients. Patients with MGMT promoter hypermethylation based on pyrosequencing had a significantly longer overall survival compared to patients without hypermethylation (median 16 vs. 11 months, p = 0.001). Similar results were observed with the GX MGMT Methylation RUO Assay (median 15 vs. 11 months, p = 0.005).

Conclusion: GX MGMT Methylation testing is highly reproducible and shows good concordance with pyrosequencing in three independent cohorts of glioblastoma patients. Our results suggest that standardized, simplified, and on-demand testing of MGMT promoter methylation by the GX MGMT Methylation RUO Assay is feasible.

Citation Format: Martin Filipits, Anita Brandstetter, Matthias Preusser, Johannes Hainfellner, Sabine Spiegl-Kreinecker, Edwin W. Lai, Kriszten Kocmond, Andrew Kohlway, Jodi Weidler, Michael Bates, Christopher Corless. Evaluation of an assay for on-demand and easy assessment of MGMT gene promoter methylation in glioblastoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-046. doi:10.1158/1538-7445.AM2017-LB-046