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

Our plain-English summary of Potential utility of miRNAs derived from pleural fluid extracellular vesicles to differentiate between benign and malignant pleural effusions, by T.M. Chee, C.J. Zahra, K.M. Fong, et al (Translational Lung Cancer Research, 2025).

The study

• Authors: T.M. Chee, C.J. Zahra, K.M. Fong, I.A. Yang, R.V. Bowman
• Journal: Translational Lung Cancer Research
• Published: 2025;14(1):124-138
• Cited by: 15 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

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.

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In plain English

This exploratory study from Brisbane, Australia, asked whether microRNAs, small genetic molecules carried in tiny particles (extracellular vesicles) in pleural fluid, differ between cancerous and non-cancerous fluid build-ups around the lungs. The authors used stored pleural fluid from 29 patients: 11 with malignant pleural mesothelioma, 11 with lung adenocarcinoma spread to the pleura, and 7 with non-malignant conditions, including benign asbestos-related pleural effusions. The article reports no measurement of asbestos exposure. Before correction for multiple testing, 32 microRNAs differed between non-malignant and malignant samples; after correction, six were significant in at least one of four comparisons between diagnostic groups. Only hsa-miR-145-5p remained significant when all malignant samples were compared with non-malignant ones; it was higher in non-malignant fluid. Three were higher with lung adenocarcinoma than with mesothelioma. Another, hsa-miR-136-5p, was higher in mesothelioma than in lung adenocarcinoma. The authors stated that the study was small, not powered to show whether any microRNA could tell these diseases apart, and that storage may have altered microRNA levels. The authors concluded that these microRNAs merit further study in larger prospective studies.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Exploratory, proof-of-principle laboratory study of stored, retrospectively collected pleural fluid samples. Extracellular vesicles were isolated and their microRNAs were profiled with the Nanostring nCounter Human v3 miRNA expression assay.
• Who was studied: Pleural fluid from 29 patients (8 female, 21 male; aged 49 to 99, median 71) held in The Prince Charles Hospital Lung Bank, Brisbane, Australia. The groups were 11 with malignant pleural mesothelioma (MPM), 11 with lung adenocarcinoma metastatic to pleura (metLUAD), and 7 with non-malignant (NM) conditions: parapneumonic effusions, inflammatory pleuritis or benign asbestos-related pleural effusions. Collection dates are not given.
• Exposure: No asbestos or fiber exposure was measured. The study compared microRNA levels in pleural fluid extracellular vesicles across diagnoses. The non-malignant group included benign asbestos-related pleural effusions, but the article does not say how many.
• Main results:
  • Between non-malignant and malignant effusions, 32 microRNAs differed at unadjusted P<0.05 with log2 fold change of at least 1.0. After Benjamini-Hochberg adjustment, six microRNAs were significant across the four comparisons between causes of effusion (malignant vs NM, metLUAD vs NM, MPM vs NM, MPM vs metLUAD). They were not all significant in any single comparison.
  • hsa-miR-145-5p was higher in non-malignant than in malignant effusions (adjusted P=0.003 to 0.03). Log2 fold changes were −1.53 (NM vs malignant), −1.38 (NM vs metLUAD) and −1.71 (NM vs MPM). It was the only microRNA listed for the pooled malignant vs NM comparison, and the only one that differed significantly between MPM and non-malignant samples.
  • Four microRNAs (hsa-miR-1246, hsa-miR-141-3p, hsa-miR-200c-3p and hsa-miR-9-5p) were higher in metLUAD than in non-malignant samples (adjusted P<0.001 to P=0.02). The largest differences were for hsa-miR-1246 (log2 fold change 4.75) and hsa-miR-200c-3p (2.86).
  • hsa-miR-1246, hsa-miR-141-3p and hsa-miR-200c-3p were higher in metLUAD than in MPM samples (adjusted P<0.001 to P=0.005; log2 fold change −7.85, −1.54 and −2.39). hsa-miR-136-5p was higher in MPM than in metLUAD samples (adjusted P=0.02; log2 fold change 1.78).
  • Total RNA recovered from the vesicles varied between the 29 samples, from 1.38 to 14.63 ng per mL of pleural fluid.
• The authors' conclusion: The authors concluded that microRNAs in pleural fluid extracellular vesicles could be useful for studying disease-related differences and could in future help improve diagnosis of pleural effusions. They stated that larger prospective studies are needed to confirm diagnostic utility.
• Limitations the authors state:
  • The study was not powered to determine whether any microRNA or microRNA profile can differentiate between pleural diseases, which limits generalizability.
  • The number of samples was small, and the study did not include the whole spectrum of pleural diseases, such as malignant effusions from breast, stomach, kidney, ovary, colon, lymphoma and other lung cancer subtypes.
  • The small study had limited scope to account for differences in microRNA expression by sex and age.
  • Long-term storage and freeze-thaw cycles could have altered microRNA levels. The authors wrote that fresh samples analysed in real time would ideally be used.
  • The study did not compare microRNAs in the vesicles with other parts of the fluid or with plasma.

Topics

Medical terms in this study, explained in our Lexicon: Pleural Effusion, Transmission Electron Microscopy (TEM), Pleural Mesothelioma, Lung Adenocarcinoma, Pleura, Biomarker.

Related studies

• Optimal isolation of extracellular vesicles from pleural fluid and profiling of their microRNA cargo — T.M. Chee, H.E. O'Farrell, et al. (2023)
• Plasmatic extracellular vesicle microRNAs in malignant pleural mesothelioma and asbestos-exposed subjects suggest a 2-miRNA signature as potential biomarker of disease — T. Cavalleri, L. Angelici, et al. (2017)
• Diagnostic and Prognostic Utility of the Extracellular Vesicles Subpopulations Present in Pleural Effusion — J. Javadi, A. Görgens, et al. (2021)
• Small and Large Extracellular Vesicles Derived from Pleural Mesothelioma Cell Lines Offer Biomarker Potential — T. Ahmadzada, A. Vijayan, et al. (2023)
• Serum Extracellular Vesicle-Derived microRNAs as Potential Biomarkers for Pleural Mesothelioma in a European Prospective Study — E. Casalone, G. Birolo, et al. (2022)
• Extracellular vesicles isolated from adipose tissue-derived mesenchymal stromal cells as carriers for Paclitaxel delivery — A. Marcianti, E. Spampinato, et al. (2025)

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