Potential utility of miRNAs derived from pleural fluid extracellular vesicles to differentiate between benign and malignant pleural effusions.

Abstract

Background: Cytological examination is of suboptimal sensitivity but high specificity for the diagnosis of malignant pleural effusions (MPEs). Pleural fluid extracellular vesicles (PFEVs) are enriched with disease-specific microRNAs (miRNAs) which may improve the diagnostic yield for MPE. Our previous study demonstrated the feasibility of isolating miRNAs from PFEVs and profiling PFEV miRNAs by Nanostring nCounter^® Human v3 miRNA expression assay. Here, we interrogated in a small cohort to evaluate the diagnostic potential of PFEV miRNAs to differentiate between benign pleural effusion and MPE.

Methods: Extracellular vesicles (EVs) from pleural fluids were isolated by two sequential ultracentrifugation steps. PFEVs were extracted and characterised by western blotting analysis, particle analysis by tunable resistive pulse sensing (TRPS) technology, and transmission electron microscopy (TEM). Total RNAs (including miRNAs) were extracted from PFEVs and profiled by the Nanostring nCounter^® 827 probe miRNA expression assay. Differential expression analysis of the miRNA expression assays on PFEV samples was performed using the Bioconductor DESeq2 package.

Results: EVs from pleural fluids were evident by staining of positive EV-associated protein markers, particle size distribution within the expected parameters, and the cup-shaped morphology by TEM. Employing Nanostring nCounter^® Human v3 miRNA expression assay, this proof-of-principle study demonstrated PFEV miRNAs were differentially expressed between benign effusions and malignant effusions [malignant pleural mesothelioma (MPM) or lung adenocarcinoma metastatic to pleura (metLUAD)]. The expression of six miRNAs (hsa-miR-1246, hsa-miR-136-5p, hsa-miR-141-3p, hsa-miR-145-5p, hsa-miR-200c-3p, and hsa-miR-9-5p) significantly differed between benign and malignant effusions, or between MPM and metLUAD, at adjusted P<0.05 and log₂fold change ≥1.0.

Conclusions: The miRNAs identified from this study could be interrogated further for their utility as a single biomarker candidate or to be tested simultaneously in a panel to complement pleural effusion diagnostics. PFEV miRNAs represent a novel bioresource with potential to aid in the diagnosis of pleural effusions. Larger prospective studies are needed to confirm their diagnostic utility.