10104-GEN-3 Quantitative Methylation Analysis of the Glioblastoma MGMT Promoter by BSAS and Its Clinical Significance

Abstract Objective In glioblastoma (GBM), methylation of the O6-methylguanine-DNA methyltransferase (MGMT) gene promoter is a critical predictive biomarker for temozolomide (TMZ) therapy. Conventional qualitative assessment by methylation-specific PCR (MSP) cannot determine the degree of methylation and risks false positives. While quantitative methods like pyrosequencing are now available, the clinical significance of low-level methylation remains debated. This study aimed to investigate the impact of MGMT promoter methylation on gene expression and prognosis in greater detail using Bisulfite Amplicon Sequencing (BSAS), a high-resolution method employing next-generation sequencing (NGS). Methods DNA was extracted from GBM patient tumor tissues and treated with bisulfite. The methylation status of the MGMT promoter region was then quantified at single-base resolution using BSAS. MGMT mRNA expression levels in the same samples were quantified using RNA-sequencing (RNA-seq). Correlations between methylation levels, mRNA expression, and clinical data were statistically analyzed. Results Detailed analysis by BSAS revealed that the direct quantitative correlation between MGMT promoter methylation level and mRNA expression was much weaker than previously thought. Nevertheless, the presence of even a low level of promoter methylation was confirmed to be a powerful, independent prognostic factor significantly correlated with improved overall survival. Conclusion These findings suggest that MGMT promoter methylation may function less as a direct “switch” that significantly suppresses gene expression, and more as a sensitive “biomarker” reflecting a favorable biological state of the tumor. This study highlights the importance of quantitative methylation assessment and suggests a more complex biological role for this key epigenetic mark.