Two convergent ways for a better molecular characterization and prediction to chemoradiation in patients affected by IDH-wildtype glioblastoma

Glioblastoma (GBM) is a challenging brain tumor with a survival rate of 12 to 15 months. Accurate treatment prediction is crucial, especially for chemotherapy and radiotherapy. Temozolomide (TMZ) is effective in GBM cases with MGMT promoter hypermethylation (40%). However, standard PCR-based DNA methylation analysis is time-consuming due to sodium bisulfite treatment. Additionally, MGMT promoter methylation, while important, doesn't fully capture the disease complexity. Altered miRNA expression may contribute to MGMT gene silencing, adding another layer to GBM molecular dynamics. The work is divided into two sections: 1. Investigating specific miRNAs influence on MGMT and prognosis in GBM patients, aiming to validate their associations with the response to TMZ treatment. 2. Introducing the innovative EpiDirect® assay, developed for bisulfite-free DNA methylation analysis. A comparative analysis with existing bisulfite-dependent assays evaluates sensitivity, specificity, and clinical utility. In the first part, MGMT expression, promoter methylation, and miRNA expression in 112 GBMs were examined through immunohistochemistry (IHC). MiRNA analysis revealed significant associations, with specific miRNAs overexpressed or downregulated, correlating with MGMT protein positivity or promoter methylation. Improved survival rates were noted in certain cases. In the second part, EpiDirect® demonstrated high sensitivity and specificity compared to other methods. It identified methylated samples missed by comparator methods and provided results in under 2 hours, emphasizing efficiency. The study highlights the potential of miRNA expression as an alternative method for assessing promoter methylation and regulating MGMT expression. If validated further, miRNA expression could become a diagnostic tool for predicting the efficacy of chemoradiation, enabling more personalized therapeutic strategies. The introduction of EpiDirect® represents a significant breakthrough by eliminating pre- treatment steps, potentially saving time and facilitating methylation analysis in resource-limited settings, encouraging the adoption of advanced molecular techniques.