Circulating cell-free and extracellular vesicles-derived microRNA as prognostic biomarkers in patients with early-stage NSCLC: results from RESTING study.

Background Factors to accurately stratify patients with earlystage nonsmall cell lung cancer (NSCLC) in diferent prognostic groups are still needed. This study aims to investigate 1) the prognostic potential of circulating cellfree (CF) and extracellular vesicles (EVs)derived microRNA (miRNAs), and 2) their added value with respect to known prognostic factors (PFs). Methods The RESTING study is a multicentre prospective observational cohort study on resected stage IAIIIA patients with NSCLC. The primary endpoint was diseasefree survival (DFS), and the main analyses were carried out separately for CF and EVmiRNAs. CF and EVmiRNAs were isolated from plasma, and miRNAspecifc libraries were prepared and sequenced. To reach the study aims, three statistical models were specifed: one using the miRNA data only (Model 1); one using both miRNAs and known PFs (age, gender, and pathological stage) (Model 2), and one using the PFs alone (Model 3). Fivefold crossvalidation (CV) was used to assess the predictive performance of each. Standard Cox regression and elastic net regularized Cox regression were used. Results A total of 222 patients were enrolled. The median followup time was 26.3 (95% CI 25.4–27.6) months. From Model 1, three CFmiRNAs and 21 EVmiRNAs were associated with DFS. In Model 2, two CFmiRNAs (miR 29c3p and miR8773p) and fve EVmiRNAs (miR181a23p, miR1825p, miR1925p, miR5323p and miR5895p) remained associated with DFS. From pathway enrichment analysis, TGFbeta and NOTCH were the most involved pathways. Conclusion This study identifed promising prognostic CF and EVmiRNAs that could be used as a noninvasive, costefective tool to aid clinical decisionmaking. However, further evaluation of the obtained miRNAs in an external cohort of patients is warranted.