Preoperative differentiation of benign from malignant indeterminate thyroid nodules (ITN) remains one of the most challenging issues in endocrine oncology. Most patients with an ITN will undergo diagnostic surgery with around 80% receiving a benign diagnosis on final histology. Liquid biopsy-based analysis of circulating extracellular vesicles (EVs) presents a promising diagnostic modality. The objectives of this study is to measure the plasma levels of circulating EVs and identifying differentially expressed EV miRNAs in patients with benign versus malignant ITNs

To quantify EVs: EVs where isolated by differential centrifugation from plasma of 28 patients with ITN (14 benign and 14 cancers on histology) and 16 matched healthy controls (HCs). The total and subpopulations of cancer related EVs (CXCR7+, CD147+, SDC4+, EpCAM+) were measured using a scatter calibrated flow cytometer. For miRNA sequencing: plasma EVs were isolated, and EV-RNA extracted using exoRNeasy Midi kit (Qiagen) from 3 HCs, 6 benign and 9 cancer patients. Sequencing was performed using DNBseq-G400 sequencer (BGI).

The total plasma EV levels as well as CXCR7+, CD147+, SDC4+, EpCAM+ EVs were significantly higher in patients with ITN (both cancer and benign) compared to HCs (p<0.0001 for all). Two miRNAs (hsa-miR-195-3p, hsa-miR-619-5p) were significantly upregulated (p<0.0001) and 4 were downregulated: hsa-miR-3176, hsa-miR-205-5p, a novel miRNA: hsa-miR208-3p (p<0.0001 for all 3), and hsa-let-7i-3p (p=0.03) in patients with malignant versus benign ITNs

Circulating EVs, especially EV miRNAs, have a high diagnostic value and may improve the diagnostic strategy for patients with ITNs