Table 1_Liquid biopsy using circulating miRNAs for the identification of recurrence-associated biomarkers in glioblastoma CNS WHO grade 4.xlsx

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, miR-21 was included as a promising candidate reported in the literature. To address challenges in miRNA normalization, five normalization strategies and unnormalized raw Ct values were compared.</p>Results<p>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, miR-424-3p and miR-362-5p were shared by five of seven patients, followed by miR-1307-3p and miR-22-5p, each shared by four patients.

Interestingly, miR-21 did not show the predefined recurrence-associated pattern in any patient.</p>Discussion<p>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.</p>