Determination of longitudinal Circulating levels of miR-21-5p, miR-23b-3p and miR-34a-5p in plasma of patients with glioblastoma using droplet digital PCR

: Glioblastoma (GBM) is characterized by molecular heterogeneity and rapid progression. Liquid biopsy is increasingly recognised as a promising non-invasive biomarker for GBM diagnosis, prognosis and treatment monitoring. The aim of the study was to explore circulating microRNAs (miRNA) as potential GBM-biomarkers. We developed droplet digital PCR (ddPCR) assays to quantify plasmatic concentrations of miR-21-5p, miR-23b-3p and miR-34a-5p both in healthy volunteers and GBM patients at diagnosis and over follow-up (1, 3, 6 and 12 months after surgery). Furthermore, we investigated the correlations between peripheral miRNA levels and clinical, neuroradiological, pathological features, extent of tumour resection, overall (OS) and recurrence-free (RFS) survivals. Our findings showed that these miRNAs were detectable in all samples, even if with different profiles, showing a similar longitudinal course characterized by a gradual increase 1 and 3 months after surgery, followed by a progressive decrease 6 and 12 months after surgery. miR-34a-5p levels were significantly higher in GBM patients compared to healthy volunteers (AUC = 0.664, p = 0.039; cut-off: 1.25 copies/µL). RFS (7.6 vs. 15.6 months, p = 0.049) and OS (8.2 vs. 24.5 months, p = 0.032) were significantly shorter in patients with miR-34a-5p levels below the mean at diagnosis and 3 months after surgery, respectively. Similarly, OS (13.3 vs. 24.4months, p = 0.024) was significantly shorter in patients with miR-21-5p levels below the mean 6 months after surgery. This study highlights the potential clinical utility of ddPCR-based quantification of plasmatic miRNAs in GBM. Longitudinal analysis revealed consistent dynamic expression patterns for all three investigated miRNAs, with miR-34a-5p and miR-21-5p emerging as potential prognostic biomarkers. Although the diagnostic performance of miR-34a-5p was intermediate and the small cohort size limited definitive conclusions, these preliminary findings support further exploration of these miRNAs as part of a multi-marker panel to enhance diagnostic and prognostic accuracy in GBM. Larger, prospective studies are required to validate these results and to elucidate the biological underpinnings of peripheral miRNA dynamics in the context of GBM pathophysiology and treatment.