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

Abstract

MGMT promoter methylation status can change in response to several factors, treatment with alkylating therapy being the mechanism more commonly cited in the literature. 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, in a cohort of patients reoperated for recurrent glioblastoma and having previously completed the Stupp protocol. Methylation status of the MGMT promoter gene of a total of 24 pairs of glioblastoma preselected tumor samples was retrospectively analyzed using pyrosequencing and depicted as percentages or categories (hypermethylated, intermediate methylation, unmethylated). Matched samples were compared using Wilcoxon signed-rank test, and log-rank test was used to establish a correlation with survival data. The median value of MGMT promoter methylation status declined after adjuvant treatment from 20.35% to 14.25% (p = 0.346). A significant correlation between methylation in primary samples and overall survival (p = 0.05) and progression-free survival (p = 0.024) was found. Intermediate methylation status at recurrence was linked to greater survival after progression, without reaching statistical significance (post-progression survival [PPS]) (p = 0.217). Although treatment with alkylating chemotherapy was a common feature in all patients of our cohort, switching in both directions was observed when MGMT promoter methylation status was analyzed as a continuous variable. These data suggest that the dynamics of epigenetics may be very complex and not entirely explained by clonal selection influenced by temozolomide.