Evaluation of circulating microRNAs as diagnostic circulating biomarkers in cutaneous melanoma

Melanoma is the fourth and sixth most common malignancy in men and women, respectively, and the 5-year survival rate critically depends on disease stage (American Cancer Society, 2016). MicroRNAs (miRNAs) are small, non-coding, single-stranded RNAs endogenously produced by the cells, which regulate the expression of hundreds of target genes. Circulating miRNAs actively secreted by tumor cells or released as the consequence of tumor cell death, have been proposed as potential biomarkers in cancer patients because of their stability in body fluids, resistance to endogenous RNase and constant expression in healthy individuals (Mitchell et al., 2008; Chen et al., 2008). In the current study, the expression profiles of a selected panel of circulating miRNAs were analysed in plasma samples derived from patients and healthy donors to identify possible candidate biomarkers for diagnosis, prognosis and/or surveillance of human cutaneous melanoma. The study protocol was approved by local Ethics Committee and conducted in accordance to the principles of the Declaration of Helsinki. Blood samples were collected from melanoma patients (n=30) at different disease stages, and from healthy age- and sex-matched volunteers (n=32). Circulating levels of miRNAs were analysed by quantitative real time PCR and data analysis was carried out using two different strategies for normalization: Global Mean Normalization (GMN) and NormFinder model. The GMN approach and NormFinder algorithm provided 13 and 7 significantly dysregulated miRNAs (p<0.05), respectively, and those that resulted significantly dysregulated after normalizations and the Bonferroni correction were selected. Circulating miR-15b-5p miR-149-3p and miR-150-5p were up- regulated, while miR-193a-3p and miR-524-5p were down-regulated in patients (regardless of disease stage) compared to healthy controls. Linear regression and following receiving operator curves (ROC) analyses were performed to evaluate the diagnostic value of these five selected miRNAs (i.e., the ability to discriminate between cases and controls). The area under ROC curve (AUCs) for individual miRNAs ranged from 0.801 to 0.951. Although the predictive power of all selected miRNAs was clearly demonstrated, miR-150-5p and miR-149-3p gave the best performance (AUCs of 0.9489, 95% CI from 0.8852 to 1.017 and 0.9510, 95% CI from 0.8852 to 1.017, respectively). Noteworthy, predictive performance was further improved when considering the double combination of miR-150-5p and miR-149-3p. The double classifier has indeed an increased area under ROC curve (AUC) of 0.966 (95% CI: 0.938–0.994) with 90% sensitivity, 68% specificity. In conclusion, our pilot study identified five circulating miRNAs, of which three detected for the first time in plasma of melanoma patients. MiR-149-3p, miR-150-5p and miR-193a-3p either alone or as a signature are suitable to be considered as potential diagnostic biomarkers in human melanoma and worthy to be validated in further prospective clinical trials. References Luke J.J. and Ott. P.A. (2014) Drug, healthcare and patient safety. 6: 77–88. Aqeilan R.I., Calin G., Croce C.M. (2010) Cell Death Differ. 17:215–220 Lynam-Lennon N, Maher SG, Reynolds JV. (2009) Biol Rev Camb Philos Soc. 84:55–71 Mitchell PS, Parkin RK, Kroh EM, et al. (2008) Proc Natl Acad Sci. 105:10513–8 Chen X, Ba Y, Ma L, Cai X, Yin Y, Wang K, et al. (2008) Cell Res. 18: 997–1006. Segura MF, Belitskaya-Levy I, Rose AE, et al. (2010) Clin Cancer Res 16: 1577–1586