Circulating extracellular Vesicles-Derived miRNAs for discriminating patients with malignant from benign Bethesda IV/Thy3f thyroid nodules

Background Patients with indeterminate Bethesda IV/Thy3f thyroid nodules mostly undergo diagnostic hemithyroidectomy for a definitive histological diagnosis, yet only ∼20% prove malignant. Thus, there is an urgent need for diagnostic biomarkers. Blood-derived extracellular vesicles (EVs) are non-invasive liquid biopsy-based biomarkers enabling detection of cancer-driven molecular alterations. We now show that blood-derived EV-enclosed miRNAs (EV-miRNAs) can differentiate patients with malignant from benign Thy3f nodules. Methods EVs were isolated by differential centrifugation from plasma of age/gender-matched malignant and benign Thy3f nodule patients (n=20) and RNA extracted immediately using miRNAs’ Micro Kit. EV-miRNAs were identified by an omics feasibility study conducted in our lab and from the literature. PCR was performed using miRCURY LNA miRNA PCR Kit for relative quantification of EV-miRNAs. For normalisation, miR-103a-3p was used. Results Significant overexpression of plasma-derived EV-miRNA miR-146b-5p, miR-222-3p, miR-127-3p, and miR-195-3p was observed in cancer versus benign Thy3f nodule patients (p=0.0098, 0.0002, 0.0052, 0.0071, respectively). No significant difference was observed for miR-21-5p (p= 0.2213). Area under the receiver operating curve (AUC) was 0.731 for miR-146b-5p, 0.860 for miR-222-3p, 0.755 for miR-127-3p, 0.852 for miR-195-3p. A two-miRNA panel of miR-222-3p and miR-195-3p achieved the highest AUC 0.927 (p=0.0009, 95% CI= 0.810-1.000, sensitivity 90.91%, specificity 80.00% at cut-off <0.4697). Adding miR-127-3p to the panel yielded a slightly lower AUC of 0.9182 but demonstrated highest sensitivity and specificity, 90.91% and 100% respectively. Conclusion A plasma EV-miRNA panel of miR-195-3p, miR-222-3p, miR-127-3p shows strong promise as non-invasive biomarker for accurately identifying malignancy. This could improve risk stratification for Thy3f nodule, supporting clinical diagnostic applicability.