Pyrosequencing versus methylation-specific PCR for assessment of MGMT methylation in tumor and blood samples of glioblastoma patients

Abstract

Circulating biomarkers in blood may provide an interesting alternative to risky tissue biopsies in the diagnosis and follow-up of glioblastoma patients. We have assessed MGMT methylation status in blood and tissue samples from unresected glioblastoma patients who had been included in the randomized GENOM-009 trial. Paired blood and tissue samples were assessed by methylation-specific PCR (MSP) and pyrosequencing (PYR). After establishing the minimum PYR cut-off that could yield a significant difference in overall survival, we assessed the sensitivity, specificity, positive predictive value and negative predictive value (NPV) of the analyses. Methylation could be detected in cfDNA by both MSP and PYR but with low concordance with results in tissue. Sensitivity was low for both methods (31% and 38%, respectively), while specificity was higher for MSP in blood than for PYR in plasma (96% vs 76%) and NPV was similar (56 vs 57%). Concordance of results in tissue by MSP and PYR was 84.3% (P < 0.001) and correlated with outcome. We conclude that detection of cfDNA in the blood of glioblastoma patients can be an alternative when tumor tissue is not available but methods for the detection of cfDNA in blood must improve before it can replace analysis in tumor tissue.

MRI-Based Deep-Learning Method for Determining Glioma MGMT Promoter Methylation Status

“…TCGA uses the Infinium Methylation Assay (https://www.illumina.com/science/technology/microarray/ infinium-methylation-assay.html) to determine MGMT promoter methylation status. [40][41][42] Infinium Methylation Assays are an immunofluorescence method that uses next-generation highthroughput microchip arrays and probes. While these methods have been reported to be more sensitive and specific than the most widely available clinical assays, they require pre-existing probes to detect specific methylation sites.…”

Liquid Biopsy of Methylation Biomarkers in Cell-Free DNA

“…Pyrosequencing is the most commonly used quantitative approach and provides real-time quantitative data on the methylation status of multiple CpGs within an amplicon. Several articles have reported that it can detect cfDNA in certain tumor types [37][38][39]. However, due to its sensitivity, it can only detect methylated alleles of not less than 5% at most and its application in the detection of tumor cfDNA methylation is limited.…”

Extent and prognostic value of MGMT promotor methylation in glioma WHO grade II

“…Sanger sequencing of the Cytosine-Guanine dinucleotide (CpG) sites 74-98 within the MGMT promotor region as previously described 9,10 . Methylation of each individual CpG site was defined as ratio of cytosine/thymine peak > 50%.…”