Liquid biopsy using circulating miRNAs for the identification of recurrence-associated biomarkers in glioblastoma CNS WHO grade 4.

Introduction: Glioblastoma CNS WHO Grade 4 (GBM) is associated with poor prognosis and high recurrence rates despite the current therapeutic approaches. Conventional imaging, in particular Magnetic Resonance Imaging (MRI), is primarily used during follow-up to detect tumor recurrence but has limited ability to distinguish recurrence from therapy-related changes, such as tissue necrosis and postoperative scar tissue. Liquid biopsy using circulating tumor-specific serological biomarkers, such as miRNAs, provides a gene expression signature for early identification of GBM recurrence. This pilot "proof-of-concept" study investigated the potential of circulating tumor-derived miRNA in whole blood samples for early detection of tumor recurrence. Methods: In a previous whole-transcriptome screening (NGS) study, whole-blood samples (n = 33) from seven patients and tumor biopsies (n = 4) were used to identify biomarkers. Corresponding to tumor surgery and recurrence, miRNA expression was examined in pre-surgical blood samples and tumor tissues. In whole blood, we investigated whether a pattern could be identified indicating post-surgery downregulation, consistent with reduced tumor burden, followed by a return to pre-surgery levels at recurrence. We identified 1-19 miRNA species per patient (a total of 72 miRNAs) showing this pattern. In this manuscript, we present the qRT-PCR validation results. The 45 most promising miRNAs identified by NGS study were selected and quantified using qRT-PCR. In addition, Results: Two normalization approaches (median DCt normalization and unnormalized raw Ct values) showed no systematic bias. Depending on the normalization method, the agreement between NGS and qRT-PCR ranged from 23.6 to 27.9%. Although the concordance between NGS and qRT-PCR was low at the individual-patient level, qRT-PCR identified a common set of 24 miRNAs differentially regulated across patients. Notably, Discussion: Blood-based miRNA profiling may complement imaging in longitudinal monitoring of GBM patients, although clinical benefits have not been confirmed, and validation in a larger independent cohort is required.