Discovery and validation of extracellular vesicle-associated miRNAs as noninvasive detection biomarkers for early-stage non-small-cell lung cancer.

miRNAs in circulating extracellular vesicles (EVs) are promising biomarkers for cancer. However, their diagnostic ability for early-stage non-small-cell lung cancer (NSCLC) is not well known. In this study, the circulating EV miRNAs profiling was initially performed in 36 untreated NSCLC patients and 36 healthy controls by TaqMan Low Density Array (TLDA). Subsequently, we performed quantitative reverse-transcription PCR assay (RT-qPCR) validation in several independent cohorts that included 159 NSCLC patients, 120 age/sex-matched healthy controls and 31 benign nodule patients enrolled from three different clinical centres. In addition, 38 cases of NSCLC were analysed before and after surgery. We demonstrated that miR-520c-3p and miR-1274b were significantly and steadily increased in NSCLC patients in comparison with healthy controls and benign nodule patients (P < 0.001) and decreased markedly after tumour resection (P < 0.001). The areas under the curve (AUCs) of the ROC curve of the two-miRNA panel were 0.857 (95% CI, 0813-0.901; P < 0.0001) and 0.845 (95% CI, 0.793-0.896; P < 0.0001) for NSCLC and NSCLC stage I, respectively. Furthermore, the panel was able to differentiate NSCLC from benign nodules with an AUC of 0.823 (95% CI, 0.730-0.915; P < 0.0001). Furthermore, logistic regression analysis revealed the two-miRNA panel as an independent risk factor for NSCLC (OR = 16.128, P < 0.0001). In conclusion, miR-520c-3p and miR-1274b have biomarker potential for early diagnosis of NSCLC in multiple centres.