Plasma-derived extracellular vesicles miR-335–5p as potential diagnostic biomarkersfor fusion-positive rhabdomyosarcoma

bstractBackground Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma, with embryonal (ERMS)and alveolar (ARMS) representing the two most common histological subtypes. ARMS shows poor prognosis, beingoften metastatic at diagnosis. Thus, the discovery of novel biomarkers predictive of tumor aggressiveness representsone of the most important challenges to overcome and may help the development of tailored therapies. In the lastyears, miRNAs carried in extracellular vesicles (EVs), small vesicles of endocytic origin, have emerged as ideal candidatebiomarkers due to their stability in plasma and their tissue specifcity.Methods EVs miRNAs were isolated from plasma of 21 patients afected by RMS and 13 healthy childrens (HC). Weperformed a miRNA profle using the Serum/Plasma Focus microRNA PCR panels (Qiagen), and RT-qPCR for validationanalysis. Statistically signifcant (p<0.05) miRNAs were obtained by ANOVA test.Results We identifed nine EVs miRNAs (miR-483-5p, miR-132-3p, miR-766-3p, miR-454-3p miR-197-3p, miR-335-3p, miR17-5p, miR-486-5p and miR-484) highly upregulated in RMS patients compared to HCs. Interestingly, 4 miRNAs (miR-335-5p,miR-17-5p, miR-486-5p and miR-484) were signifcantly upregulated in ARMS samples compared to ERMS. In the validationanalysis performed in a larger group of patients only three miRNAs (miR-483-5p, miR-335-5p and miR-484) were diferentially signifcantly expressed in RMS patients compared to HC. Among these, mir-335-5p wassignifcant also when compared ARMS to ERMS patients. MiR-335-5p was upregulated in RMS tumor tissues respect to normal tissues (p=0.00202)and upregulated signifcantly between ARMS and ERMS (p=0.04). Furthermore, the miRNA expression correlatedwith the Intergroup Rhabdomyosarcoma Study (IRS) grouping system, (p=0.0234), and survival (OS, p=0.044; PFS, p=0.025).By performing in situ hybridization, we observed that miR-335-5p signal was exclusively in the cytoplasm of cancer cells.Conclusion We identifed miR-335-5p as signifcantly upregulated in plasma derived EVs and tumor tissue of patientsafected by ARMS. Its expression correlates to stage and survival in patients. Future studies are needed to validate miR335-5p as prognostic biomarker and to deeply elucidate its biological role.