Droplet digital PCR (ddPCR) using FFPE DNA to assess methylation status ofMGMTgene among patients withIDHmutant astrocytoma andIDHwild-type glioblastoma

MGMT promoter methylation analysis in formalin-fixed paraffin-embedded (FFPE) tissues can be challenging since the DNA obtained is often fragmented. Bisulfite conversion, which is essential to determine methylation status, further degrades DNA. While conventional methylation-specific PCR (MSP) and pyrosequencing assays have long been used to determine the methylation status of MGMT , this study was designed to determine the utility of one-tube DNA extraction method coupled with a droplet digital PCR (ddPCR) assay, to study the epigenetic changes in the promoter region of the MGMT gene using DNA obtained from FFPE. The FFPE blocks of 30 (n=30) patients with Central Nervous System (CNS) WHO grade 4 tumours, previously tested by MSP (2011–2021) were retrieved; DNA was extracted using one-tube extraction method and bisulfite converted. All converted samples were analyzed for methylation status of the MGMT promoter region with a laboratory designed Methylation-Specific ddPCR (MS ddPCR) using degenerate primers and probes that were labelled with FAM or HEX flurocein dye. Of the 30 cases, 20 cases were MGMT methylated and 10 cases were unmethylated by MS ddPCR. The results of MS ddPCR were then compared with those obtained by MSP and found to be concordant in 93.3% (28/30) of the cases and discordant in 2 cases. The Cohen’s kappa coefficient (κ) was 0.84. The sensitivity, specificity, positive predictive value and negative predictive value of the assay in detecting the methylation status was found to be 95%, 90%, 95% and 90%. The results show that MS ddPCR is a valuable tool to detect the methylation status of MGMT in FFPE with high sensitivity. This method is cost-effective and easy to perform and could be an attractive alternative to the routine method of MSP.