Liquid Biopsy for Non-Invasive Assessment of MGMT Gene Methylation in Glioblastoma: A Pilot Study

Glioblastoma (GB) is the most common malignant brain tumor in adults, characterized by rapid progression and poor survival despite standard therapies, including surgery, radiotherapy, and temozolomide chemotherapy (CT). Promoter methylation of the O6-methylguanine-DNA methyltransferase (MGMT) gene is an important prognostic biomarker in GB patients, associated with increased sensitivity to CT. Conventional assessment of the promoter gene methylation relies on tumor biopsy. Tissue-based analyses indicate heterogeneous MGMT methylation in tumor, with the majority of tumors being unmethylated (49–59%), and smaller proportions partially methylated (5–20%) or fully methylated (3–30%). Nonetheless this technique is an invasive procedure that limits longitudinal monitoring. In this context, liquid biopsy has emerged as a non-invasive alternative, enabling the detection of genetic and epigenetic alterations in circulating DNA. This approach is particularly valuable when tumor tissue is insufficient, inaccessible, or unavailable, such as in patients who cannot undergo surgery. Although sensitivity and accuracy remain lower than tissue-based analysis, liquid biopsy represents a complementary or alternative method for MGMT methylation assessment and allows dynamic monitoring of MGMT status. To evaluate the feasibility of liquid biopsy as a translational tool for the detection of MGMT promoter methylation in patients with GBM, exploring the potential of liquid biopsy as a non-invasive alternative to tissue-based analysis. In this prospective phase II pilot study, peripheral blood samples from 10 newly diagnosed GBM patients were analyzed. Serum was obtained by centrifugation at 2,500 rpm for 10 minutes and stored at -20°C until analysis. Serum DNA was extracted from 800 µL of serum. MGMT gene methylation was assessed by quantitative Methylation-Specific PCR (qMSP) with primers specific for methylated and unmethylated sequences following bisulfite conversion of DNA. qMSP reactions were performed in triplicate with an intercalating DNA dye. A sample was considered methylated if amplification occurred with the methylated MGMT primers, and unmethylated if amplification occurred only with the unmethylated MGMT primers. Ten patients with a median age of 55.3 years (range: 34–76 years); five males and five females, newly diagnosed GB were evaluated. Of these, nine patients amplified only with primers specific for unmethylated MGMT and were classified as unmethylated, whereas one patient showed amplification with primers for methylated MGMT, indicating partial methylation. In our cohort of 10 patients with GB, although unmethylated MGMT was more frequente, the proportions of unmethylated and methylated samples identified by liquid biopsy were lower than those reported in primary tumors in literature. Preliminary results of our study indicate that MGMT methylation analysis via liquid biopsy is feasible and may provide useful information when tumor tissue is unavailable, eventhough this technique does not yet replace tissue-based analysis. Larger studies with standardized methods are needed to validate this approach.