Phd in INNOVATIVE BIOMEDICAL TECHNOLOGIES IN CLINICAL MEDICINE

Thesis title: Liquid biopsy in advanced papillary thyroid cancer: a potential tool for evaluating disease progression and response to treatment

Background and Aims: MicroRNAs (miRNAs) play a key role in post-transcriptional regulation and some of them are proving to be promising diagnostic biomarkers of papillary thyroid cancer (PTC). The aims of this study were to analyze the potential role of circulating free miRNAs as biomarkers of disease progression in PTC patients and to investigate if both circulating and exosomal miRNAs can be useful in monitoring the response to treatment of PTC subjects undergoing tyrosine kinase inhibitor (TKI) therapy. Methods: We enrolled 27 PTCs classified as excellent response (n=14), and structural incomplete response (n=13), from whom we collected two blood samples after surgery, and 10 PTCs undergoing TKI therapy, for whom serum samples were collected before the start of therapy and periodically during follow-up visits. We extracted RNA from sera and quantified it by Qubit® 3.0 Fluorometer. The expression levels of miR-146a-5p and miR-221-3p were quantified by digital PCR. Moreover, after isolation of exosomes from serum samples, we extracted RNA and analyzed the expression levels of miR-24-3p, miR-146a-5p, miR-181a-5p, miR-382-5p and miR-485-3p using qRT-PCR. In the in vitro study, we analyzed the expression levels of miR-146a-5p in the culture media of two PTC cell lines, TPC-1 and K1, 24 hours after treatment with a cytostatic, a cytotoxic or a selective RET inhibitor drug. Results: We found that a fold-change cut-off of 2 for circulating free miR-146a-5p can discriminate between patients with a progressive disease and patients with a stable or absent disease with a sensitivity and specificity of 92 and 100%, respectively. A fold-change cut-off of 2,2 for miR-221-3p showed a sensitivity and specificity of 75 and 95%, respectively. Moreover, during the TKI treatment, levels of miR-146a-5p and miR-122-3p increased significantly in conditions of disease stability and partial response to the drug and decreased in case of disease progression. The increase in miRNA levels was probably due to the effect of the drug on cell death, as demonstrated by in vitro experiments. In contrast, the exosomal miRNAs levels showed an opposite trend, increasing during disease progression and decreasing during disease response to TKI. Conclusions: Circulating free miRNAs, and in particular miR-146-5p, appeared to be useful as biomarkers of disease progression in patients with PTC. In addition, circulating free miRNAs seem to be useful for monitoring the efficacy of TKI treatment in patients with advanced PTC, as they reflect drug-induced cell damage. Finally, exosomal miRNAs, and in particular miR-181a-5p, appear to be useful for monitoring response to TKI treatment, as they reflect the percentage of actively proliferating tumor cells.