Quantitative analysis of MGMT promoter methylation status changes by pyrosequencing in recurrent glioblastoma

Background: MGMT promoter methylation status can change in response to several factors, including treatment with alkylating agents. Some authors have attempted to quantify these alterations with inconsistent results. This study aims to determine changes in MGMT promoter methylation status by pyrosequencing, which quantitatively yields results, providing percentages of methylation of a given CpG dinucleotide in a cohort of patients reoperated for recurrent glioblastoma and having previously completed the Stupp protocol. Methods A total of 25 pairs of glioblastoma preselected tumor samples were retrospectively analyzed using conventional DNA extraction techniques, bisulfite conversion, and PCR amplification of the MGMT promoter gene. Methylation status was obtained using pyrosequencing of 4 CpG dinucleotides within the enhancer region of the MGMT promoter gene and depicted as percentages or categories (hypermethylated, intermediate methylation, unmethylated). Matched samples were compared using Wilcoxon signed-rank test, and the log-rank test was employed to establish a correlation between survival data and methylation status. Results Median value of MGMT promoter methylation status declined after adjuvant treatment from 22.25–16.15%. (p = 0.872). A correlation without statistical significance between methylation in primary samples and OS was found (p = 0.102). Lower degrees of association were obtained when studying primary methylation and PFS (p = 0.187). Intermediate methylation status at recurrence was more linked to PPS (p = 0.03) Conclusions Switching in both directions was observed when analyzing methylation status as a continuous variable. These data suggest that the dynamics of epigenetics may be very complex and not entirely explained by clonal selection or tumor heterogeneity.