Nucleic acids in pleural fluid for the etiological diagnosis of pleural effusion

Pleural effusion is a frequent clinical manifestation of various underlying conditions and is defined as the accumulation of fluid within the pleural space. The annual incidence of pleural diseases is estimated to exceed 350 cases per 100,000 individuals. Although pleural effusion can result from a wide range of causes, the vast majority of cases in adults are attributable to four main etiologies: heart failure (transudative effusion), cancer (malignant pleural effusion; MPE), pneumonia (parapneumonic pleural effusion; PPE), and tuberculosis (tuberculous pleural effusion; TPE) (1). Pleural fluid is typically obtained through thoracentesis, a minimally invasive procedure that enables diagnostic analysis of the fluid. It remains the cornerstone for identifying the underlying cause of a pleural effusion.

Nucleic acids in pleural fluid may serve as novel biomarkers for the etiological diagnosis of pleural effusion. Understanding the genetic changes occurring in different types of pleural effusion is essential. In this context, the study by Zamora-Molina et al. identified significant differences in gene expression among transudative effusions, MPE and PPE, contributing to a better understanding of the underlying pathophysiological mechanisms and the identification of promising new biomarkers (2).

Numerous researchers have evaluated the diagnostic accuracy of various nucleic acid-based biomarkers. The first step in the etiological diagnosis of pleural effusion is differentiating between transudates and exudates. Pleural effusions may develop as a consequence of systemic conditions that disrupt the balance between capillary hydrostatic and oncotic pressures, leading to the formation of a transudate. Common causes include congestive heart failure, hepatic cirrhosis, and nephrotic syndrome. Alternatively, pleural effusions may result from inflammatory or neoplastic processes that increase capillary permeability, producing an exudative effusion. The most common exudative pleural effusions are MPE, PPE, and TPE. Chan et al. observed that cell-free DNA concentration (cfDNA), measured in pleural fluid via quantitative polymerase chain reaction (qPCR) of the β-globin gene, is found in higher concentrations in exudative pleural effusions and allows differentiation between exudates and transudates, achieving a very high diagnostic accuracy, with an area under the receiver operating characteristic curve (AUC) of 0.95 (3).

Similar results were reported by Santotoribio et al., who also observed that this parameter could be used as a biomarker for the diagnosis and management of PPE (4). Parapneumonic effusion can occur at any age and it represents the most common cause of pleural effusion in the pediatric population (5). PPE typically begins as simple effusions (pleural fluid without signs of infection), then progresses to complicated effusions (infected pleural fluid), and may eventually develop into empyema (purulent pleural fluid). The distinction between uncomplicated and complicated parapneumonic effusions is essential, and pleural fluid pH is commonly used for this purpose, as it helps predict disease severity and guide clinical management, with complicated cases often requiring chest tube drainage (6). In this context, it has been proposed that the concentration of cfDNA in pleural fluid, measured by qPCR of the β-globin gene, could serve as an acute-phase inflammatory biomarker to differentiate between both types of effusion and improve diagnostic accuracy (4).

16S ribosomal RNA (16S rRNA) gene sequencing has been used for the diagnosis/identification of bacteria in pleural fluid. This gene encodes a highly conserved subunit of the bacterial ribosome but contains variable regions that allow for the discrimination between different bacterial species. The amplification of the 16S rRNA gene using PCR, followed by sequencing, enables the detection of bacteria even in samples with low bacterial load or in cases where conventional cultures are negative, such as in patients who have received prior antibiotic treatment (7). In a study conducted by Lampejo et al., 16S rRNA sequencing yielded positive results in 31% of pleural fluid samples with negative cultures (8). Other studies explored a promising approach based on the development of syndromic PCR panels for confirming pleural infections and guiding appropriate antibacterial therapy. Kommedal et al. developed a syndromic PCR panel targeting pathogens commonly involved in community-acquired pleural infections, including Haemophilus influenzae, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Aggregatibacter aphrophilus, Fusobacterium nucleatum, Streptococcus intermedius, and species of the Enterobacteriaceae genus. This panel was evaluated against traditional cultures and 16S rRNA sequencing. The sensitivity of the PCR for the target bacteria was 99.5%, outperforming both culture and 16S rRNA sequencing (21.6% and 92.4%, respectively), thus highlighting its utility for the rapid diagnosis of PPE (9). Similar findings were reported by Jacobson et al., who assessed the clinical utility of multiplex qPCR for diagnosing pediatric PPE caused by S. pneumoniae, S. pyogenes, S. aureus, and H. influenzae. The assay demonstrated an overall sensitivity of 97.8% for bacterial genus detection. Notably, among 76 culture-negative cases, multiplex qPCR successfully identified the causative pathogen in 68. Validation using control samples confirmed a specificity of 100% (10). It is important to recognize that multiplex qPCR for bacterial genus detection in pleural fluid may carry the risk of overlooking pathogens not included in the panel; therefore, it should be considered only as a complement to conventional bacterial culture (11).

In relation to tuberculous effusion, the most sensitive biomarker in pleural fluid for the diagnosis of TPE is adenosine deaminase (ADA). An increase in pleural fluid ADA, usually greater than 35–40 U/L, allows prioritization of specific complementary tests for the diagnosis of TPE (12). Nucleic acid amplification tests (NAATs) have reappeared as potential diagnostic techniques for the aetiological diagnosis of TPE in recent years. Wen et al. conducted a comprehensive review evaluating the diagnostic performance of several NAAT platforms—namely Xpert MTB/RIF, Xpert MTB/RIF Ultra, loop-mediated isothermal amplification (LAMP), FluoroType MTB, and various in-house assays—against the standard of M. tuberculosis culture and pleural biopsy. Although these methods enable rapid and specific detection, their clinical utility is limited by the characteristically low bacillary load in pleural fluid. Notably, the Xpert Ultra assay demonstrated superior sensitivity compared to earlier platforms. The review further indicated that combining NAATs with biomarkers such as ADA enhances overall diagnostic accuracy, though large-scale, multicenter validation studies are still needed to establish standardized protocols (13).

Malignant effusion is the result of tumor involvement of the pleura, either by primary pleural tumor cells (as in mesothelioma) or by metastatic tumor cells originating from other tissues. Most pleural exudates are of malignant origin, with lung cancer being the most common cause. MPE occurs in approximately 50% of patients with metastatic tumors and is associated with a poor prognosis. Differentiating between malignant and benign effusions is crucial for both treatment and prognosis. The gold standard diagnostic tests are pleural fluid cytology and pleural biopsy. However, cytology has low sensitivity and is subjective, while biopsy offers high sensitivity and specificity but is invasive and technically complex. In contrast, tumor markers in pleural fluid are objective and represent a minimally invasive technique with high accuracy for the diagnosis of MPE (14). In relation to the investigation of various nucleic acid biomolecules in pleural fluid as potential tumor markers for diagnosis of MPE, extracellular microRNAs (miRNAs) stand out. miRNAs are small sequences (~22 nucleotides) that regulate gene expression by binding to messenger RNA (mRNA) to inhibit its translation or promote its degradation. miRNAs can be classified according to their mode of transport into: a) free or non-vesicular miRNAs: circulating freely in plasma or pleural fluid, bound to carrier proteins (mainly Argonaute 2) or lipoproteins (High density lipoproteins and low-density lipoproteins cholesterol); and b) exosomal miRNAs: encapsulated in exosomes, extracellular vesicles with lipid membranes (15). Several free miRNAs in pleural fluid have been proposed as biomarkers for MPE. Among them, miR-21, miR-24, miR-30d, miR-34a-5p, miR-182, miR-182-5p, and miR-195-5p are overexpressed in patients with MPE compared to benign effusions, whereas miR-22, miR-29c, miR-134, miR-145, miR-185, and miR-198 are underexpressed (16-22). Regarding exosomal miRNAs identified in pleural fluid, several have been reported to be overexpressed in patients with MPE, including miR-141, miR-141-3p, miR-182, miR-182-5p, miR-200a, miR-200b, miR-200c, miR-200c-3p, miR-203a-3p, miR-210, miR-375, miR-889, and miR-1246. In contrast, miR-145-5p is underexpressed (23-28). Table 1 provides a comparative summary of miRNAs reported in the literature for the diagnosis of MPE. It includes 13 published studies, which represent nearly all the available research on miRNAs in pleural fluid, a relatively limited number compared to the extensive literature on conventional tumor markers such as carcinoembryonic antigen (CEA). Another important consideration is the sample size of these studies: only two included ≥100 patients (20,28). Moreover, most of these studies focused on MPEs secondary to lung cancer, which limits their clinical utility for distinguishing malignant from benign effusions, since MPEs can result from various cancer types. Among the studies reviewed, the work by Zhao et al. (28) stands out for including the largest patient cohort and one of the most balanced case–control group distributions. However, it is worth noting that 40 of the 49 MPE cases in that study were caused by lung cancer. The authors also employed two different methodologies for analyzing exosomal miR-182-5p, observing slight variations depending on the internal reference used. Exosomal miR-182-5p in pleural fluid demonstrated moderate diagnostic accuracy for identifying MPE (AUC =0.78–0.80), similar to that of traditional tumor markers such as CEA (14,29). These findings highlight the potential of exosomal miR-182-5p, as well as other exosomal miRNAs, as diagnostic tools for MPE. Nonetheless, it is important to acknowledge that the quantification of miRNAs still presents an unfavorable cost-effectiveness ratio, which currently limits their widespread implementation in clinical laboratories.

Biomarkers in pleural fluid used for the diagnosis of MPE require high specificity to minimise false-positive results. However, this high specificity often comes at the cost of reduced sensitivity. To overcome this limitation, several authors have proposed combining multiple biomarkers to enhance diagnostic sensitivity without compromising specificity. Pleural fluid CEA may be measured easily and quickly in automated analyzers, being a minimally invasive technique with higher sensitivity for diagnosis of MPE (14,29). The combination of exosomal miR-182-5p and CEA in pleural fluid significantly improved diagnostic accuracy, yielding an AUC of 0.91 (28).

In malignant pleural mesothelioma (MPM), a significant overexpression of miR-210 and miR-139-5p has been observed in pleural fluid, while miR-143 and miR-200c are underexpressed. The combination of miR-143, miR-210, and miR-200c constitutes a diagnostic signature capable of distinguishing MPM from MPE due to lung adenocarcinoma and benign pleural effusions, with an AUC of 0.98 and 0.92, respectively (30).

Additionally, the mRNA in pleural fluid has been used for diagnosis of MPE as the model developed by Hsu et al. based on the quantification of mRNA of four genes (DUSP6, MDM2, RNF4, WEE1) (31); or mRNA encoding lipocalin-2 identified by Hydbring et al. that showed high accuracy for the diagnosis of MPE (AUC =0.99) (24). Other nucleic acid biomolecules have been associated with MPE such as the fragmentation pattern of cfDNA by containing much longer DNA fragments in pleural fluid, with sizes exceeding 500 base pairs, compared to plasma (32); moreover, the DNA integrity index, calculated by quantitative PCR of the ratio of two ALU sequences of different lengths, was significantly higher in MPE compared to benign effusions (1.2 vs. 0.8; P<0.001), even in MPM and cases with negative pleural cytology (33); and the analysis of cfDNA methylation in various loci has been employed as a marker of malignancy in pleural fluid (34), as well as various long non-coding RNAs (lncRNAs) such as lncRNA MALAT1, lncRNA H19, and lncRNA CUDR (35).

Finally, it is important to highlight the diagnostic and therapeutic utility of liquid biopsy in pleural fluid for the detection of driver mutations in circulating tumor DNA (ctDNA). ctDNA may be present in the pleural fluid of patients with MPE either freely in the supernatant, within exosomes or extracellular vesicles, or in the cellular pellet of the pleural fluid. Numerous studies have demonstrated a high concordance between ctDNA mutations detected in pleural fluid and those found in primary tumor biopsies. One of the most extensively studied genes in pleural fluid is the epidermal growth factor receptor (EGFR) gene, as it is the most common therapeutic target in non-small cell lung cancer (NSCLC), a frequent cause of MPE. The molecular analysis of this gene is particularly relevant because a substantial proportion of NSCLC patients harbor activating mutations in the tyrosine kinase domain of the EGFR gene. Tyrosine kinase inhibitors are among the recommended treatments for these patients. Additionally, it has been observed that NSCLC patients with EGFR mutations who are treated with tyrosine kinase inhibitors have a longer progression-free survival compared to those with wild-type EGFR. However, resistance often develops, commonly due to secondary mutations in the EGFR gene, with the T790M substitution being the most frequently observed (36).

In conclusion, several studies have proposed nucleic acid biomolecules in pleural fluid as potential biomarkers for the etiological diagnosis of pleural effusions, with miRNAs standing out as particularly promising markers in the context of MPE. miRNAs may complement pleural cytology, aid in the differential diagnosis of tumor histological subtypes, and provide prognostic information. The combination of pleural fluid CEA and miRNAs is recommended to enhance diagnostic accuracy. Furthermore, liquid biopsy of pleural fluid enables the detection of actionable genetic alterations (such as EGFR mutations in NSCLC), facilitating targeted therapy decisions. Beyond malignancy, nucleic acid-based biomarkers, such as the concentration of cfDNA in pleural fluid, measured by qPCR of the β-globin gene, have demonstrated clinical utility in the context of PPE. Despite these promising findings, clinical implementation requires standardization of sample collection and analysis methods, as well as validation in large, multicenter studies.

Acknowledgments

None.

Footnote

Provenance and Peer Review: This article was commissioned by the Editorial Office, Translational Lung Cancer Research. The article has undergone external peer review.

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