Validation of a PCR-Based MGMT Promoter Methylation Assay and Comparison With Reference Lab Results
Authors: McEwen Abbye , Smith Christina , Konnick Eric 
Published: September 2019

Abstract

Introduction
Glioblastoma multiforme (GBM) is the most aggressive and most common primary brain tumor. Treatment of GBM and other high-grade gliomas consists of surgical resection followed by radiation and temozolomide (TMZ) chemotherapy. TMZ alkylates purine DNA bases, causing DNA damage and eventually cell death. Alkylation at O6-gauanine can be reversed by the DNA repair enzyme methylguanine methyltransferase (MGMT), inhibiting the desired cytotoxic effect. MGMT is epigenetically silenced by methylation of a CpG island in its promoter. Many high-grade gliomas are hypomethylated at the MGMT promoter. MGMT promoter methylation has emerged as a biomarker for response to alkylating chemotherapy. Multiple clinical trials demonstrate that the patients most likely to benefit from TMZ therapy have a hypermethylated MGMT promoter. MGMT promoter status is now incorporated into adjuvant therapy guidelines. Our institution receives approximately 70 to 80 high-grade glioma resections per year. The goal of this study is to validate a PCR-based MGMT promoter methylation assay and to compare results to those previously obtained at a well-known reference lab.

Methods
We extracted DNA from high-grade gliomas specimens (FFPE) or used commercially available methylated and unmethylated DNA controls. DNA was treated with bisulfite to convert unmethylated cytosine into uracil. Promoter methylation was detected with real-time quantitative PCR using a primer/probe set specific for methylated CpG sites within the MGMT promoter (meMGMT, published in Ida et al 2017) or to a housekeeping gene (COL2A1).

Results
Using control methylated DNA, we demonstrated that our assay consistently detects meMGMT and COL2A1 at DNA inputs greater than 5 ng. Both within- and between-run imprecision was minimal with little variation in crossing thresholds (Cts) for both COL2A1 (%CV at 50 ng input: 7.9% [within-run], 0.3% [between-run]) and MGMT (%CV at 50 ng input: 0.6% [within-run], 0.4% [between-run]). Delta Ct values were consistent across different levels of DNA input. Analysis of 15 cases from our archives (8 positive, 7 negative) demonstrates that our results (delta Ct values) correlate (R2 = 0.9) with the reference lab methylation score (<2 considered unmethylated). Separation between positive and negative cases was good with minimal overlap; positive cases had delta Cts of 0.2 to 7.6, while negative cases had delta Cts of 7.1 to 13.6 or had no meMGMT amplification. ROC curve analysis showed an AUC of 0.96, and using the cutoff of delta Ct <7.6, our assay had 93% (14/15) agreement with the reference lab, a sensitivity of 100%, and a specificity of 71%. The single negative case that was misclassified had a reference lab methylation score of 1.86, near their cutoff of <2. Additional patient samples are pending.

Conclusions
Our PCR-based MGMT methylation assay has excellent analytical sensitivity and low imprecision. Preliminary analysis of patient samples shows that our results are concordant with historic reference lab results.