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Introduction: Comprehensive next-generation sequencing (NGS) of non-small-cell lung cancer specimens can identify oncogenic driver mutations and their corresponding targeted therapies. Plasma cell-free DNA (cfDNA) genotyping is easy to perform; however, false negatives cannot be overlooked. We explored malignant pleural effusion (MPE), a rich source of cfDNA, as a non-inferior alternative to tumor tissues for genotyping., Methods: We conducted a prospective trial including 39 patients with newly diagnosed stage IV lung adenocarcinoma who presented with MPE. Tissue tests matching hotspot variants, including EGFR, ALK, and ROS1, were compared with the AlphaLiquid100 of PE-cfDNA., Results: Among the 39 PE-cfDNA samples successfully sequenced, 32 (82.1%) had a PE cell-block tumor content of < 10%. Standard tissue or cell-block testing for EGFR, ALK, and ROS1 identified 20 mutations (51.3%), whereas PE cfDNA identified 25 mutations (64.1%). Five EGFR mutations were observed in PE cfDNA but not in Cobas EGFR owing to coverage or insufficient tumor content issues. The overall rate of oncogenic mutations identified in the PE cfDNA was 92.3%, and the mutation distribution was as follows: even with a very low cfDNA input, high detection rates could be achieved. Otherwise, most patients harbored co-mutations. Comparison of pleural fluid NGS with traditional testing revealed differences in accuracy. We also followed up with patients with EGFR-sensitizing mutations who had a treatment response rate of 97.2% after 3 months., Conclusions: Genotyping of MPE supernatant cfDNA is feasible in clinical practice, in addition to plasma and tumor testing, to improve diagnostic yield and extend patients' benefit from targeted therapies., (© 2024. The Author(s).)

Personalized treatment for patients with advanced solid tumors critically depends on the deep characterization of tumor cells from patient biopsies. Here, we comprehensively characterize a pan-cancer cohort of 150 malignant serous effusion (MSE) samples at the cellular, molecular, and functional level. We find that MSE-derived cancer cells retain the genomic and transcriptomic profiles of their corresponding primary tumors, validating their use as a patient-relevant model system for solid tumor biology. Integrative analyses reveal that baseline gene expression patterns relate to global ex vivo drug sensitivity, while high-throughput drug-induced transcriptional changes in MSE samples are indicative of drug mode of action and acquired treatment resistance. A case study exemplifies the added value of multi-modal MSE profiling for patients who lack genetically stratified treatment options. In summary, our study provides a functional multi-omics view on a pan-cancer solid tumor cohort and underlines the feasibility and utility of MSE-based precision oncology., (© 2024. The Author(s).)

Malignant pleural effusion (MPE) from patients with advanced non-small-cell lung cancer (NSCLC) has been proven valuable for molecular analysis; however, simultaneous detection of driver fusions in MPE is still challenging. In this study, we investigated the Idylla™ GeneFusion Panel, a stand-alone test in tissue samples, in the evaluation of ALK , ROS1 , RET and MET ex14 skipping mutations in MPE and compared its performance with routine reference methods (Real-time-based and Next-generation Sequencing-NGS). The inclusion criteria for sample selection were as follows: advanced NSCLC harboring ALK , ROS1 , RET fusions or MET exon-skipping alterations and the availability of MPE collected at diagnosis or disease progression. Molecular alterations have been investigated on tissue by fluorescence in situ hybridization (FISH) or Real-time PCR or NGS. For molecular profiling with the Idylla™ GeneFusion, 200 µL of MPE supernatants combined with 50 µL of RNA Later solution were loaded into the Idylla™ cartridge without cfRNA extraction. The Idylla™ GeneFusion Assay performed on MPEs was able to confirm molecular profile, previously diagnosed with conventional methods, in all cases. Our data confirm that MPE are suitable material for investigating fusion alterations. The Idylla™ GeneFusion, although indicated for investigation of tissue samples, offers the possibility of performing a molecular characterization of supernatants without undertaking the entire cfRNA extraction procedure providing a rapid and reliable strategy for the detection of actionable genetic alterations.

Background: Direct comparison of tumor microenvironment of matched lung cancer biopsies and pleural effusions (PE) from the same patients is critical in understanding tumor biology but has not been performed. This is the first study to compare the lung cancer and PE microenvironment by single-cell RNA sequencing (scRNA-seq)., Methods: Matched lung cancer biopsies and PE were obtained prospectively from ten patients. We isolated CD45 + cells and performed scRNA-seq to compare the biopsies and PE., Results: PE had a higher proportion of CD4 + T cells but lower proportion of CD8 + T cells (False detection rate, FDR = 0.0003) compared to biopsies. There was a higher proportion of naïve CD4 + T cells (FDR = 0.04) and naïve CD8 + T cells (FDR = 0.0008) in PE vs. biopsies. On the other hand, there was a higher proportion of Tregs (FDR = 0.04), effector CD8 + (FDR = 0.006), and exhausted CD8 + T cells (FDR = 0.01) in biopsies. The expression of inflammatory genes in T cells was increased in biopsies vs. PE, including TNF, IFN-ɣ, IL-1R1, IL-1R2, IL-2, IL-12RB2, IL-18R1, and IL-18RAP (FDR = 0.009, 0.013, 0.029, 0.043, 0.009, 0.013, 0.004, and 0.003, respectively). The gene expression of exhaustion markers in T cells was also increased in tumor biopsies including PDCD1, CTLA4, LAG 3, HAVCR2, TIGIT, and CD160 (FDR = 0.008, 0.003, 0.002, 0.011, 0.006, and 0.049, respectively)., Conclusions: There is a higher proportion of naïve T cells and lower proportion of exhausted T cells and Tregs in PE compared to lung cancer biopsies, which can be leveraged for prognostic and therapeutic applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Introduction: DNA extracted from malignant pleural effusion (PE) sediments is the traditional source of tumor DNA for predictive biomarker molecular testing (MT). Few recent studies have proposed the utility of cell-free DNA (cfDNA) extracted from effusion cytology centrifuged supernatants (CCS) in MT. The aim of this study was to assess the feasibility and utility of molecular testing on cfDNA extracted from PE CCS in lung cancer patients., Materials and Methods: The study was of prospective design. All PE CCS were collected and stored. Subsequently, in patients confirmed as primary lung adenocarcinoma (LUAD) and where patient matched effusion sediment/tissue biopsy/plasma was being tested for EGFR mutations, cfDNA extraction and EGFR MT by real-time polymerase chain reaction (qPCR) were performed. Custom panel targeted next-generation sequencing (NGS) (Ion Torrent; Thermo Fisher, Carlsbad, CA) was also performed wherever feasible., Results: Out of 299 PE CCS collected, 20 CCS samples were included in the study. Concordant EGFR mutations were detected in pleural effusion CCS of 10 of 11 (91%) EGFR mutant cases as per qPCR performed on the matched sediment DNA (n = 8), lung biopsy (n = 2), and plasma (n = 1) samples. In 1 positive sample, CCS detected additional EGFR T790M mutation. Among 10 CCS samples also tested by NGS, additional EGFR mutations missed by qPCR were picked up in 2 (2 of 10). Success of mutation detection in CCS cfDNA did not correlate with cfDNA quantity or tumor fraction in sediment., Conclusions: cfDNA from effusion CCS is a reliable and independent source of tumor DNA highly amenable for MT and complement results from other tumor DNA sources for comprehensive mutation profiling in LUAD patients., (Copyright © 2024 American Society of Cytopathology. Published by Elsevier Inc. All rights reserved.)

Background: Malignant pleural effusion (MPE) is a common complication of non-small cell lung cancer (NSCLC). Patients with NSCLC exhibit a high rate of epidermal growth factor receptor (EGFR) mutations. The detection of EGFR mutations is usually time-consuming and costly. This study aimed at identifying potential biomarkers of EGFR mutations in MPE of NSCLC patients by metabolomics., Methods: In total, 58 MPE samples from 30 EGFR mutant and from 28 wild-type NSCLC patients were collected and analyzed by using hydrogen nuclear magnetic resonance (1H NMR) based metabolomics and UPLC-MS/MS based amino acid analysis., Results: Our 1H NMR study showed a significant increase in the lysine levels but a significant decrease in the alanine levels in MPE of NSCLC patients with EGFR-mutant. Twelve amino acids in MPE were further determined by UPLC-MS/MS. It showed that alanine in MPE (6.34 ± 1.88 vs. 8.73 ± 3.68) were significantly decreased and leucine (3.13 ± 0.57 vs. 2.22 ± 0.13), lysine (2.19 ± 0.50 vs. 1.53 ± 0.40), and tyrosine (2.69 ± 0.71 vs. 1.89 ± 0.46) were increased in the EGFR mutation group; leucine (2.19 ± 0.50 vs. 1.53 ± 0.40), methionine (2.19 ± 0.50 vs. 1.53 ± 0.40), and threonine (2.19 ± 0.50 vs. 1.53 ± 0.40) in MPE were significantly lower in the EGRF 19 mutation compared with 21 mutation patients. The area under the receiver operating characteristic curve of 0.851 and 0.931 would be achieved by the logistic model for classification of EGFR-mutant patients from the wild-type controls or the exon 19 from exon 21 mutant patients., Conclusions: Amino acids in MPE are significantly altered and helpful in the diagnosis of EGFR-mutant patients from the wild-type controls or the exon 19 from exon 21 mutant patients with high accuracy, which is worthy of further study.

Background: Recurrent malignant pleural effusion (MPE) resulting from non-small-cell lung cancer (NSCLC) is easily refractory to conventional therapeutics and lacks predictive markers. The cellular or genetic signatures of recurrent MPE still remain largely uncertain., Methods: 16 NSCLC patients with pleural effusions were recruited, followed by corresponding treatments based on primary tumours. Non-recurrent or recurrent MPE was determined after 3-6 weeks of treatments. The status of MPE was verified by computer tomography (CT) and cytopathology, and the baseline pleural fluids were collected for single-cell RNA sequencing (scRNA-seq). Samples were then integrated and profiled. Cellular communications and trajectories were inferred by bioinformatic algorithms. Comparative analysis was conducted and the results were further validated by quantitative polymerase chain reaction (qPCR) in a larger MPE cohort from the authors' centre (n = 64)., Results: The scRNA-seq revealed that 33 590 cells were annotated as 7 major cell types and further characterized into 14 cell clusters precisely. The cell cluster C1, classified as Epithelial Cell Adhesion Molecule (EpCAM)+ metastatic cancer cell and correlated with activation of tight junction and adherence junction, was significantly enriched in the recurrent MPE group, in which Claudin-4 (CLDN4) was identified. The subset cell cluster C3 of C1, which was enriched in recurrent MPE and demonstrated a phenotype of ameboidal-type cell migration, also showed a markedly higher expression of CLDN4. Meanwhile, the expression of CLDN4 was positively correlated with E74 Like ETS Transcription Factor 3 (ELF3), EpCAM and Tumour Associated Calcium Signal Transducer 2 (TACSTD2), independent of driver-gene status. CLDN4 was also found to be associated with the expression of Hypoxia Inducible Factor 1 Subunit Alpha (HIF1A) and Vascular Endothelial Growth Factor A (VEGFA), and the cell cluster C1 was the major mediator in cellular communication of VEGFA signalling. In the extensive MPE cohort, a notably increased expression of CLDN4 in cells from pleural effusion among patients diagnosed with recurrent MPE was observed, compared with the non-recurrent group, which was also associated with a trend towards worse overall survival (OS)., Conclusions: CLDN4 could be considered as a predictive marker of recurrent MPE among patients with advanced NSCLC. Further validation for its clinical value in cohorts with larger sample size and in-depth mechanism studies on its biological function are warranted., Trial Registration: Not applicable., (© 2024 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Background and Objectives : Different cellular and molecular processes are involved in the production of malignant and infectious pleural effusions. However, the underlying mechanisms responsible for these differences or their consequences remain incompletely understood. The objective of this study was to identify differences in gene expression in pleural exudates of malignant and infectious aetiology and establish the possible different biological processes involved in both situations. Materials and Methods : RNA transcriptomic analysis was performed on 46 pleural fluid samples obtained during diagnostic thoracocenteses from 46 patients. There were 35 exudates (19 malignant and 16 infectious effusions) and 11 transudates that were used as a reference control group. Differential gene expression analysis for both exudative groups was identified. An enrichment score using the Human Kegg Orthology database was used for establishing the biological processes associated with malignant and infectious pleural effusions. Results : When comparing malignant exudates with infectious effusions, 27 differentially expressed genes with statistical significance were identified. Network analysis showed ten different biological processes for malignant and for infectious pleural effusions. In malignant fluids, processes related to protein synthesis and processing predominate. In infectious exudates, biological processes in connection with ATP production prevail. Conclusions : This study demonstrates differentially expressed genes in malignant and infectious pleural effusions, which could have important implications in the search for diagnostic or prognostic biomarkers. In addition, for the first time, biological processes involved in these two causes of pleural exudates have been described.

Diagnosis of malignant pleural effusion (MPE) is made by cytological examination of pleural fluid or histological examination of pleural tissue from biopsy. Unfortunately, detection of malignancy using cytology has an overall sensitivity of 50%, and is dependent upon tumor load, volume of fluid assessed, and cytopathologist experience. The diagnostic yield of pleural fluid cytology is also compromised by low abundance of tumor cells or when morphology is obscured by inflammation or reactive mesothelial cells. A reliable molecular marker that may complement fluid cytology for the diagnosis of malignant pleural effusion is needed. The purpose of this study was to establish a molecular diagnostic approach based on pleural effusion cell-free DNA methylation analysis for the differential diagnosis of malignant pleural effusion and benign pleural effusion. This was a blind, prospective case-control biomarker study. We recruited 104 patients with pleural effusion for the study. We collected pleural fluid from patients with: MPE (n = 48), indeterminate pleural effusion in subjects with known malignancy or IPE (n = 28), and benign PE (n = 28), and performed the Sentinel-MPE liquid biopsy assay. The methylation level of Sentinel-MPE was markedly higher in the MPE samples compared to BPE control samples (p < 0.0001) and the same tendency was observed relative to IPE (p = 0.004). We also noted that the methylation signal was significantly higher in IPE relative to BPE (p < 0.001). We also assessed the diagnostic efficiency of the Sentinel-MPE test by performing receiver operating characteristic analysis (ROC). For the ROC analysis we combined the malignant and indeterminate pleural effusion groups (n = 76) and compared against the benign group (n = 28). The detection sensitivity and specificity of the Sentinel-MPE test was high (AUC = 0.912). The Sentinel-MPE appears to have better performance characteristics than cytology analysis. However, combining Sentinel-MPE with cytology analysis could be an even more effective approach for the diagnosis of MPE. The Sentinel-MPE test can discriminate between BPE and MPE. The Sentinel-MPE liquid biopsy test can detect aberrant DNA in several different tumor types. The Sentinel-MPE test can be a complementary tool to cytology in the diagnosis of MPE., (© 2024. The Author(s).)

Background: As an epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), osimertinib has emerged as a standard EGFR-mutation positive treatment for non-small cell lung cancer (NSCLC). However, the efficacy of osimertinib for malignant pleural effusion (MPE) remains understudied. This study aimed to evaluate the impact of osimertinib on time to treatment failure (TTF) and overall survival (OS) in patients with EGFR-mutation positive NSCLC, comparing those with and without MPE., Methods: This retrospective analysis included patients with advanced or recurrent NSCLC treated with osimertinib at our hospital between April 2016 and June 2021. TTF was defined as the duration from osimertinib initiation to discontinuation, and OS as the duration until death, irrespective of the reason., Results: Among 229 patients receiving osimertinib, 84 had MPE before administration, 39 acquired EGFR exon20 T790M mutation following previous EGFR-TKI therapy, and 45 were EGFR-TKI-naive. Among EGFR-TKI-naive patients, median TTF was 14.8 and 19.8 months for those with and without MPE, respectively (hazard ratio [HR] 1.40; 95% confidence interval [CI]: 0.90-2.18; p = 0.12). Median OS was 32.0 and 42.0 months for patients with and without MPE, respectively (HR 1.43; 95% CI: 0.86-2.38; p = 0.16). Among patients with T790M mutation, median TTF was 12.3 and 13.1 months for patients with and without MPE, respectively (HR 1.03; 95% CI: 0.69-1.55; p = 0.88). Median OS for patients with and without MPE was 23.2 and 24.7 months, respectively (HR 1.09; 95% CI: 0.72-1.67; p = 0.68)., Conclusion: Among patients with EGFR-mutation positive NSCLC, the evidence of MPE has little effect on survival with osimertinib., (© 2024 The Authors. Thoracic Cancer published by John Wiley & Sons Australia, Ltd.)

Background: It is difficult to distinguish between malignant pleural effusion (MPE) and benign pleural effusion (BPE). The purpose of this study was to determine the best specimen type by evaluating the DNA methylation status of SHOX2 and RASSF1A in 3 matched PE components., Methods: In total, 94 patients were enrolled, including 45 MPE, 35 BPE, and 14 undefined PE (UPE) with malignancies. PE samples were processed into supernatants, fresh-cell pellets, and formalin-fixed and paraffin-embedded (FFPE) cell blocks, respectively. A quantitative real-time PCR was used to detect the methylation status of SHOX2 and RASSF1A., Results: SHOX2 and RASSF1A methylation levels were significantly higher in the 3 MPE sample types than those of BPE (P < 0.05). The area under the curve using cell-free DNA (cf-DNA) was the highest. The detection sensitivity of SHOX2 and RASSF1A in fresh-cell DNA, cf-DNA and FFPE cell-block were 71.1% (32/45), 97.8% (44/45) and 66.7% (28/42), respectively, with specificities of 97.1% (34/35), 94.3% (33/35), and 96.9% (31/32). Notably, a combination of the cytological analysis and cf-DNA methylation assay showed an increase in positivity rate from 75.6% to 100%., Conclusions: The SHOX2 and RASSF1A methylation assay using cf-DNA, the primary recommended specimen type, can excellently increase the diagnostic sensitivity of MPE. A combination of methylation assay with cytological analysis can be used for auxiliary diagnosis of PE., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Introduction: Immunohistochemistry (IHC) for the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) biomarkers has prognostic and therapeutic value in breast cancer. This study aimed to compare the expression of ER, PR, and HER2 between paired malignant effusions and tissue samples of breast cancer., Methods: Our electronic archive was searched for all effusions diagnosed as breast carcinomas within a pre-defined period (January 2018-October 2021). Next, their cell blocks (CBs) were subjected to ER, PR, HER2 IHC, or in situ hybridization, in addition to EGFR IHC. The expression of hormone receptors (HRs) and HER2 was subsequently compared between tissue and effusion cytology samples derived from the same patients., Results: Only 2/76 (2.6%) of the breast cancer patients analyzed showed a malignant effusion at their initial presentation. ER, PR, and HER2 discordance rates between paired malignant effusions and tissue samples obtained at initial diagnosis were 24.3% (17/73), 40.8% (29/71), and 9.1% (6/66), respectively. The HR-/HER2- status was found more often at effusions compared to paired tissue biopsies obtained at initial diagnosis (30/70 vs. 17/70; p &lt; 0.001). In addition, the HR-/HER2- status was significantly associated with an earlier development of a malignant effusion, when found at initial diagnosis (p &lt; 0.001; log-rank test), first recurrence/metastasis (either solid or effusion) (p = 0.012; log-rank test), effusion samples (p = 0.007; log-rank test), and any tumor sample obtained (p = 0.009; log-rank test). Lastly, EGFR overexpression in the HR-/HER2- effusion samples was significantly associated with a shorter post-effusion survival (p = 0.019; log-rank test)., Conclusion: Serous effusion cytology provides high-quality material for ancillary techniques, especially when CBs are prepared, reflecting cancer heterogeneity., (© 2023 S. Karger AG, Basel.)

Introduction: This study was to investigate the diagnostic value of percutaneous closed pleural brushing (CPBR) followed by cell block technique for malignant pleural effusion (MPE) and the predictive efficacy of pleural fluid carcinoembryonic antigen (CEA) for epidermal growth factor receptor (EGFR) mutations in lung adenocarcinoma patients with MPE., Methods: All patients underwent closed pleural biopsy (CPB) and CPBR followed by cell block examination. MPE-positive diagnostic rates between the two methods were compared. Univariate and multivariate analyses were performed to determine factors influencing the EGFR mutations. Receiver operating characteristic (ROC) curve was used to analyze the predictive efficacy of pleural fluid CEA for EGFR mutations., Results: The cumulative positive diagnostic rates for MPE after single and twice CPBR followed by cell block examination were 80.5% and 89.0%, higher than CPB (45.7%, 54.3%) (P < 0.001). Univariate analysis showed that EGFR mutation was associated with pleural fluid and serum CEA (P < 0.05). Multivariate analysis showed that pleural fluid CEA was an independent risk factor for predicting EGFR mutation (P < 0.001). The area under the curve (AUC) of pleural fluid CEA for EGFR mutation prediction was 0.774, higher than serum CEA (P = 0.043), but no difference with the combined test (P > 0.05)., Conclusion: Compared with CPB, CPBR followed by the cell block technique can significantly increase the positive diagnostic rate of suspected MPE. CEA testing of pleural fluid after CPBR has a high predictive efficacy for EGFR mutation in lung adenocarcinoma patients with MPE, implying pleural fluid extracted for cell block after CPBR may be an ideal specimen for genetic testing., (© 2023 The Authors. The Clinical Respiratory Journal published by John Wiley & Sons Ltd.)

Objective High pleural amylase levels have been reported in patients with malignant pleural effusion; however, the characteristics of this association are uncertain. Therefore, this study investigated the factors, such as cancer type and oncogenic drivers, related to pleural amylase levels in patients with malignant pleural effusion. Methods We retrospectively collected the data of 362 cancer patients [lung adenocarcinoma (n=256), lung squamous carcinoma (n=12), small-cell lung carcinoma (n=32), other lung cancers (n=5), mesothelioma (n=31), and metastatic cancer (n=26)] with malignant pleural effusion at Fukujuji Hospital from January 2012 to October 2022. Pleural amylase levels were compared. Results Pleural amylase levels were significantly higher in patients with lung adenocarcinoma [median 58.6 IU/L (interquartile range (IQR) 33.8-139.3)] than in those with small-cell lung carcinoma [median 37.2 IU/L (IQR 26.3-63.7), p=0.012]. The median pleural amylase level was higher in patients with lung adenocarcinoma than in those with other cancer or histologic types, although the difference was not significant. Pleural amylase levels were higher in epidermal growth factor receptor (EGFR) mutation-positive patients than in EGFR mutation-negative patients [median 95.8 IU/L (IQR 52.7-246.5) vs. median 51.2 IU/L (IQR 27.8-96.9), p<0.001]. The Kaplan-Meier survival curves of pleural amylase ≥75 IU/L were higher than those of pleural amylase <75 IU/L [log-rank test p<0.001, hazard ratio 0.54 (95% confidence interval: 0.41-0.71)]. Conclusion This study demonstrates that pleural amylase levels were elevated in patients with lung adenocarcinoma and EGFR mutations. Furthermore, a high pleural amylase level was associated with a good prognosis.

Malignant pleural effusion (MPE), which is a complex microenvironment that contains numerous immune and tumour signals, is common in lung cancer. Gene alterations, such as driver gene mutations, are believed to affect the components of tumour immunity in the microenvironment (TIME) of non-small-cell lung cancer. In this study, we have shown that pleural CD39 + CD8 + T cells are selectively elevated in lung adenocarcinoma (LUAD) with wild-type epidermal growth factor receptor (EGFR wt ) compared to those with newly diagnosed mutant EGFR (EGFR mu ). Furthermore, these CD39 + CD8 + T cells are more prevalent in MPE with acquired resistance to EGFR-tyrosine kinase inhibitors (AR-EGFR-TKIs). Our analysis reveals that pleural CD39 + CD8 + T cells exhibit an exhausted phenotype while still retaining cytolytic function. Additionally, they have a higher T cell receptor (TCR) repertoire clonality compared to CD39-CD8 + T cells, which is a unique characteristic of LUAD-related MPE. Further investigation has shown that TCR-Vβ clonality tends to be more enhanced in pleural CD39 + CD8 + T cells from MPE with AR-EGFR-TKIs. In summary, we have identified a subset of CD8 + T cells expressing CD39 in MPE, which may potentially be tumour-reactive CD8 + T cells. This study provides new insights into the dynamic immune composition of the EGFR mu tumour microenvironment., (© 2023. The Author(s).)

Background: It is a significant challenge to identify pleural effusion (PE) through differential diagnosis in clinical settings. The present study endeavors to devise a strategy to differentiate between malignant pleural effusion (MPE) and benign pleural effusion (BPE) by detecting gene methylation., Methods: This study recruited 214 patients with PE, among which 104 patients were identified with MPE, while the remaining 110 patients were categorized as having BPE. The methylation levels of short stature homeobox 2 ( SHOX2 ) and RAS association domain family 1, isoform A ( RASSF1A ) genes were analyzed through methylation-specific polymerase chain reaction (MS-PCR)., Results: The methylation status of either SHOX2 or RASSF1A genes was significantly elevated in MPE compared to BPE. The sensitivity and specificity of SHOX2 and RASSF1A methylation in diagnosing PE were 66.3% and 90.9%, respectively. The sensitivity of the combined methylation detection intended to diagnose pulmonary MPE was 73.5% and 52.8% in non-pulmonary MPE ( p < 0.05), suggesting that combined detection of SHOX2 and RASSF1A methylation had high diagnostic value for lung cancer. In comparison to the results of cytology and DNA ploidy detection, methylation detection demonstrated a superior diagnostic efficiency in the diagnosis of lung cancer ( p < 0.05). Additionally, the combined detection of SHOX2 and RASSF1A methylation was more potent in diagnosing BPE and MPE ( p < 0.05), while compensating for the limitations of cytology and DNA ploidy detection., Conclusions: The detection of SHOX2 and RASSF1A methylation can effectively differentiate between BPE and MPE, especially in diagnosing pulmonary MPE.

Background: Primary lung tumors are sometimes resected when either pleural dissemination (PD) or malignant pleural effusion (MPE) exists. This study clarified the prognostic factors for non-small cell lung cancer (NSCLC) with either PD and MPE, or both, detected during or after surgery., Patients and Methods: We examined patients with NSCLC from a multicenter database who had either PD, MPE, or both, detected during or after surgery between 2005 and 2015. Hazard ratios and 95% confidence intervals were estimated using the Cox proportional hazards model adjusted for potential confounding factors., Results: Among 9463 registered patients, PD, MPE, or both, were found in 114 patients with NSCLC during or after surgery. Primary tumor resection and exploratory thoracotomy were performed in 65 and 49 patients, respectively. In univariate analysis, adenocarcinoma, clinically undetected lymph node metastasis (c-N0 or unknown), EGFR mutation, and combination of chemotherapy or tyrosine kinase inhibitors after surgery were better prognostic factors for overall survival (OS), whereas in the multivariate analysis, adenocarcinoma, clinically undetected lymph node metastasis, and EGFR mutation were favorable independent prognostic factors in OS. Additionally, limited to patients with EGFR mutation, patients with primary lung tumor resection showed a significantly better 5-year OS than those with exploratory thoracotomy (86.4 vs. 44.8%; p < 0.001)., Conclusion: Our findings show that surgical resection of primary tumors could improve the prognosis of patients with PD, MPE, or both, detected during or after surgery when the tumors harbor an EGFR mutation., (© 2023. The Author(s).)

Background: Metagenomic next-generation sequencing (mNGS) has become a powerful tool for pathogen detection, but the value of human sequencing reads generated from it is underestimated., Methods: A total of 138 patients with pleural effusion (PE) were diagnosed with tuberculous pleurisy (TBP, N = 82), malignant pleural effusion (MPE, N = 35), or non-TB infection (N = 21), whose PE samples all underwent mNGS analysis. Clinical TB tests including culture, Acid-Fast Bacillus (AFB) test, Xpert, and T-SPOT, were performed. To utilize mNGS for MPE identification, 25 non-MPE samples (20 TBP and 5 non-TB infection) were randomly selected to set human chromosome copy number baseline and generalized linear modeling was performed using copy number variant (CNV) features of the rest 113 samples (35 MPE and 78 non-MPE)., Results: The performance of TB detection was compared among five methods. T-SPOT demonstrated the highest sensitivity (61% vs. culture 32%, AFB 12%, Xpert 35%, and mNGS 49%) but with the highest false-positive rate (10%) as well. In contrast, mNGS was able to detect TB-genome in nearly half (40/82) of the PE samples from TBP subgroup, with 100% specificity. To evaluate the performance of using CNV features of the human genome for MPE prediction, we performed the leave-one-out cross-validation (LOOCV) in the subcohort excluding the 25 non-MPE samples for setting copy number standards, which demonstrated 54.1% sensitivity, 80.8% specificity, 71.7% accuracy, and an AUC of 0.851., Conclusion: In summary, we exploited the value of human and non-human sequencing reads generated from mNGS, which showed promising ability in simultaneously detecting TBP and MPE., (© 2023. BioMed Central Ltd., part of Springer Nature.)

The sensitivity of pleural fluid (PF) analyses for the diagnosis of malignant pleural effusions (MPEs) is low to moderate. Knowledge about the pathobiology and molecular characteristics of this condition is limited. In this study, the crosstalk between stromal cells and tumor cells was investigated in vitro in order to reveal factors that are present in PF which can mediate MPE formation and aid in discriminating between benign and malignant etiologies. Eighteen PF samples, in different proportions, were exposed in vitro to mesothelial MeT-5A cells to determine the biological effects on these cells. Treatment of normal mesothelial MeT-5A cells with malignant PF increased cell viability, proliferation, and migration, and activated different survival-related signaling pathways. We identified differentially expressed miRNAs in PF samples that could be responsible for these changes. Consistently, bioinformatics analysis revealed an enrichment of the discovered miRNAs in migration-related processes. Notably, the abundance of three miRNAs (miR-141-3p, miR-203a-3, and miR-200c-3p) correctly classified MPEs with false-negative cytological examination results, indicating the potential of these molecules for improving diagnosis. Malignant PF produces phenotypic and functional changes in normal mesothelial cells. These changes are partly mediated by certain miRNAs, which, in turn, could serve to differentiate malignant from benign effusions.

Background: Pleural cytology is currently used to assess targetable mutations in patients with advanced lung adenocarcinoma. However, it is fraught with low diagnostic yield., Research Question: Can pleural cell-free DNA (cfDNA) be used to assess targetable mutations in lung adenocarcinoma patients with malignant pleural effusions (MPE)?, Study Design and Methods: Patients with lung adenocarcinoma MPE were recruited prospectively between January 2017 and September 2021. Oncogenic mutations were assessed by treating providers using pleural fluid cytology or lung cancer biopsies. Pleural and plasma cfDNA were used to assess the mutations using next-generation sequencing (NGS)., Results: Fifty-four pleural fluid samples were collected from 42 patients. The diagnostic yield to detect oncogenic mutations for pleural cfDNA, pleural cytology, biopsy, and plasma cfDNA was 49/54 (90.7%), 16/33 (48.5%), 22/25 (88%), and 24/32 (75%), respectively, P < .001. The agreement of mutations in positive samples between pleural cfDNA and pleural cytology was 100%, whereas the agreement of pleural cfDNA with biopsies was 89.4%. The median concentration (interquartile range) of pleural cfDNA was higher than plasma: 28,444 (4,957-67,051) vs 2,966.5 (2,167-5,025) copies of amplifiable DNA per mL, P < .01. Median of 5 mL (interquartile range, 4.5-5) of pleural fluid supernatant was adequate for cfDNA testing., Interpretation: The diagnostic yield of pleural cfDNA NGS for oncogenic mutations in lung adenocarcinoma patients is comparable to tumor biopsies and higher than pleural cytology and plasma cfDNA. The pleural cfDNA can be longitudinally collected, can be readily incorporated in clinical workflow, and may decrease the need for additional biopsies., (Published by Elsevier Inc.)

Objectives: To confirm the predictive value of targeted therapies for oncogenic driver gene mutations detected in malignant pleural effusion (MPE) cell blocks from patients with advanced non-small cell lung cancer (NSCLC)., Methods: For patients with NSCLC whose tumor tissues could not be used to detect oncogenic driver gene status, molecular mutation status in 101 MPE cell blocks was tested using amplification refractory mutation system polymerase chain reaction prior to treatment. Corresponding targeted therapies were adopted based on the detection results., Results: Mutations observed in MPE cell blocks included epidermal growth factor receptor mutation (EGFR) (60.4% [61/101]), anaplastic lymphoma kinase fusion (6.3% [5/80]), and ROS proto-oncogene 1 receptor tyrosine kinase fusion (3% [2/70]). Other mutations that were found in <5% of patients included epidermal growth factor receptor-2, rat sarcoma-filtered germ carcinogenic homologous B1, neuroblastoma RAS viral oncogene homolog, and mesenchymal epithelial transition factor exon 14. The median follow-up time was 23.5 months for the 41 patients with a single EGFR mutation and who received tyrosine kinase inhibitor monotherapy as the first-line treatment; in these patients, the objective response rate was 78% (95% confidence intervals (CI), 62% to 89%), progression-free survival was 10.8 months (95% CI, 8.7 to 13.0 months), and overall survival was 31.7 months (95% CI, 13.9 to 49.4 months)., Conclusions: Malignant pleural effusion cell blocks are recommended for mutation testing for targeted therapies in patients with NSCLC., (© 2023 Wiley Periodicals LLC.)

Aim: To investigate the diagnostic value of combined detection of SHOX2 and RASSF1A gene methylation with carcinoembryonic antigen (CEA) level in diagnosing malignant pleural effusion., Methods: Between March 2020 and December 2021, we enrolled 68 patients with pleural effusion admitted to the Department of Respiratory and critical care medicine of Foshan Second People's Hospital. The study group included 35 cases of malignant pleural effusion and 33 cases of benign pleural effusion. Methylation of the short homeobox 2 genes (SHOX2) and RAS-related region family 1A gene (RASSF1A) in pleural effusion samples were detected by real-time fluorescence quantitative PCR, and the level of carcinoembryonic antigen (CEA) in pleural effusion samples was detected by immune flow cytometry fluorescence quantitative chemiluminescence., Results: SHOX2 or RASSF1A gene methylation was detected in 5 cases in the benign pleural effusion group and 25 patients in the malignant pleural effusion group. The positive rate of SHOX2 or RASSF1A gene methylation in the malignant pleural effusion group was significantly higher than in the benign pleural effusion group (71.4% vs. 15.2%, P < 0.01). Positive CEA (CEA > 5 ng/m) was detected in 1 case in the benign pleural effusion group and 26 patients in the malignant pleural effusion group. The CEA-positive rate in the malignant pleural effusion group was significantly higher than in the benign pleural effusion group (74.3% vs. 3%, P < 0.01). When SHOX2 and RASSF1A gene methylation was combined with CEA detection, 6 cases were positive in the benign pleural effusion group, and 31 patients were positive in the malignant pleural effusion group. The positive rate of combined detection in the malignant pleural effusion group was significantly higher than in the benign pleural effusion group (88.6% vs. 18.2%, P < 0.01). The sensitivity, specificity, accuracy, positive predictive value, negative predictive value, and Youden's index of SHOX2, RASSF1A gene methylation combined with CEA in diagnosing malignant pleural effusion were 88.6%, 81.8%, 85.3%, 83.8%, 87.1% and 0.7 respectively., Conclusion: The combined detection of SHOX2 and RASSF1A gene methylation with CEA level in pleural effusion has a high diagnostic value for malignant pleural effusion., (© 2023. The Author(s).)

Introduction: Tumor-associated macrophages are one of the key components of the tumor microenvironment. The immunomodulatory activity and function of macrophages in malignant pleural effusion (MPE), a special tumor metastasis microenvironment, have not been clearly defined., Methods: MPE-based single-cell RNA sequencing data was used to characterize macrophages. Subsequently, the regulatory effect of macrophages and their secreted exosomes on T cells was verified by experiments. Next, miRNA microarray was used to analyze differentially expressed miRNAs in MPE and benign pleural effusion, and data from The Cancer Genome Atlas (TCGA) was used to evaluate the correlation between miRNAs and patient survival., Results: Single-cell RNA sequencing data showed macrophages were mainly M2 polarized in MPE and had higher exosome secretion function compared with those in blood. We found that exosomes released from macrophages could promote the differentiation of naïve T cells into Treg cells in MPE. We detected differential expression miRNAs in macrophage-derived exosomes between MPE and benign pleural effusion by miRNA microarray and found that miR-4443 was significantly overexpressed in MPE exosomes. Gene functional enrichment analysis showed that the target genes of miR-4443 were involved in the regulation of protein kinase B signaling and lipid biosynthetic process., Conclusions: Taken together, these results reveal that exosomes mediate the intercellular communication between macrophages and T cells, yielding an immunosuppressive environment for MPE. miR-4443 expressed by macrophages, but not total miR-4443, might serve as a prognostic marker in patients with metastatic lung cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Shao, Pei, Chen, Wang, Wang and Zhai.)

Objective: To explore the clinical diagnostic value of DNA image cytometry (DNA-ICM) ploidy analysis in malignant pleural effusion cancer screening, this study analyzed the effect of exfoliated cell smears (ECSs), cell blocks (CBs), and immunochemistry., Method: A total of 830 cases of pleural effusion were considered for the DNA-ICM ploidy analysis. The ECSs were centrifuged, the CBs were formed, and the DNA-ICM ploidy analysis was carried out in the diagnosis of malignant pleural effusion. Immunochemistry and biopsy was applied to differentiate between benign and malignant pleural effusion and to determine the source of the latter. The sensitivity and specificity differences between the three methods alone and in combination were compared., Results: The sensitivity of the DNA-ICM, ECS, and CB methods was 96.28%, 94.93%, and 95.95%, respectively, and the specificity of each method was 86.52%, 87.08%, and 86.14%, respectively. The sensitivity and specificity of the combined diagnosis method were 99.32% and 75.09%, respectively. Among the 22 cases diagnosed as positive in the DNA-ICM ploidy analysis but negative in the ECS and CB analyses, four cases were diagnosed as positive by comprehensive clinical diagnosis., Conclusion: The sensitivity and specificity of DNA-ICM ploidy analysis are high; the positive detection rate of pleural fluid cytology is effectively increased, and the missed detection rate of cell pathologies is effectively reduced. The combination of the three methods significantly improves the specificity and sensitivity of the diagnosis of malignant pleural effusion, and immunochemistry with CBs can be used to accurately analyze the primary tumor site., (© 2022 Wiley Periodicals LLC.)

Malignant pleural effusion (MPE) is a functional 'cold' tumor microenvironment in which the antitumor activity of CD8 + T cells and natural killer T (NKT)-like cells is suppressed and the function of regulatory T (T reg ) cells is enhanced. Using flow cytometry and immunofluorescence staining, we detected a distinct subset of NKT-like cells expressing FOXP3 in MPE. Through single-cell RNA sequencing (scRNA-seq) analysis, we found that the glycolysis pathway and pyruvate metabolism were highly activated in FOXP3 + NKT-like cells. Similar to T reg cells, FOXP3 + NKT-like cells highly expressed monocarboxylate transporter 1 (MCT1) and lactate dehydrogenase B to uptake and utilize lactate, thereby maintaining their immunosuppressive function and hyperlactylation in MPE. Furthermore, we found that MCT1 small molecule inhibitor 7ACC2 significantly reduced FOXP3 expression and histone lactylation levels in NKT-like cells in vitro. In conclusion, we reveal for the first time the altered phenotypic and metabolic features of FOXP3 + NKT-like cells in human MPE., Competing Interests: We declare no competing interests., (© 2022 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Background: Lung adenocarcinoma (LUAD) is a highly malignant tumor with a very low five-year survival rate. In this study, we aimed to identify differentially expressed long-chain non-coding RNA (lncRNAs) and mRNAs from benign and malignant pleural effusion exosomes., Methods: We used gene microassay and quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) to detect and verify differentially expressed mRNAs and lncRNAs in benign and malignant pleural effusion exosomes. Gene Ontology (GO) functional significance and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway significance enrichment analyses were performed to identify the difference in biological processes and functions between different mRNAs. We selected the lncRNA ZBED5-AS1 with an upregulated differential fold of 3.003 and conducted a preliminary study on its cellular function., Results: Gene microassay results revealed that 177 differentially expressed lncRNAs were upregulated, and 215 were downregulated. The top 10 upregulated were FMN1, AL118505.1, LINC00452, AL109811.2, CATG00000040683.1, AC137932.1, AC008619.1, AL450344.1, AC092718.6, and ZBED5-AS1. The top 10 downregulated were TEX41, G067726, JAZF1-AS1, AC027328.1, AL445645.1, AL022345.4, AC008572.1, AC123777.1, AC093714.1, and PHKG1. For the mRNAs, 79 were upregulated, and 123 were notably downregulated. GO analysis revealed that the upregulated differential mRNAs were mainly involved in "cellular response to acidic pH" (biological processes), "endoplasmic reticulum part" (cellular components), and "at DNA binding, cyclase activity" (molecular functions). KEGG pathways were found to be related to V. cholerae infection, Parkinson's disease, and cell adhesion molecules. RT-qPCR showed that ZBED5-AS1 was highly expressed in LUAD tissues, cells, and benign and malignant pleural fluid exosomes. Overexpression of ZBED5-AS1 could significantly promote the proliferation, migration, invasion, and colony formation of LUAD cells, and knockdown had the opposite consequence., Conclusion: The pleural effusion exosomes from patients with LUAD include several improperly expressed genes, and lncRNA-ZBED5-AS1 is a new biomarker that aids in our understanding of the occurrence and progression of LUAD., (© 2022 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Lung and breast cancer are the two most common causes of malignant pleural effusion (MPE). MPE diagnosis plays a crucial role in determining staging and therapeutic interventions in these cancers. However, our understanding of the pathogenesis and progression of MPE at the molecular level is limited. Extracellular Vesicles (EVs) and their contents, including microRNAs (miRNAs), can be isolated from all bodily fluids, including pleural fluid. This study aims to compare EV-miRNA patterns of expression in MPE caused by breast (BA-MPE) and lung (LA-MPE) adenocarcinomas compared to the control group of heart-failure-induced effusions (HF-PE). We conducted an analysis of 24 pleural fluid samples (8 LA-MPE, 8 BA-MPE, and 8 HF-PE). Using NanoString technology, we profiled miRNAs within EVs isolated from 12 cases. Bioinformatic analysis demonstrated differential expression of miR-1246 in the MPE group vs. HF-PE group and miR-150-5p and miR-1246 in the BA-MPE vs. LA-MPE group, respectively. This difference was demonstrated and validated in an independent cohort using real-time PCR (RT-PCR). miRNA-1246 demonstrated 4-fold increased expression (OR: 3.87, 95% CI: 0.43, 35) in the MPE vs. HF-PE group, resulting in an area under the curve of 0.80 (95% CI: 0.60, 0.99). The highest accuracy for differentiating MPE vs. HF-PE was seen with a combination of miRNAs compared to each miRNA alone. Consistent with prior studies, this study demonstrates dysregulation of specific EV-based miRNAs in breast and lung cancer; pleural fluid provides direct access for the analysis of these EV-miRNAs as biomarkers and potential targets and may provide insight into the underlying pathogenesis of tumor progression. These findings should be explored in large prospective studies.

Background: Effective cancer treatment relies on precision diagnostics. In cytology, an accurate diagnosis facilitates the determination of proper therapeutics for patients with cancer. Previously, the authors developed a multiplexed immunofluorescent panel to detect epithelial malignancies from pleural effusion specimens. Their assay reliably distinguished effusion tumor cells (ETCs) from nonmalignant cells; however, it lacked the capacity to reveal specific cancer origin information. Furthermore, DNA profiling of ETCs revealed some, but not all, cancer-driver mutations., Methods: The authors developed a new multiplex immunofluorescent panel that detected both malignancy and pulmonary origin by incorporating the thyroid transcription factor-1 (TTF-1) biomarker. Evaluation for TTF-1-positive ETCs (T-ETCs) was performed on 12 patient samples. T-ETCs and parallel ETCs from selected patients were collected and subjected to DNA profiling to identify pathogenic mutations. All samples were obtained with Institutional Review Board approval., Results: Malignancy was detected in all samples. T-ETCs were identified in 9 of 10 patients who had clinically reported TTF-1 positivity (90% sensitivity and 100% specificity). Furthermore, DNA profiling of as few as five T-ETCs identified pathogenic mutations with equal or greater sensitivity compared with profiling of ETCs, both of which showed high concordance with clinical findings., Conclusions: The findings suggest that the immunofluorescent and molecular characterization of tumor cells from pleural effusion specimens can provide reliable diagnostic information, even with very few cells. The integration of site-specific biomarkers like TTF-1 into ETC analysis may facilitate better refined diagnosis and improve patient care., (© 2022 American Cancer Society.)

Malignant pleural effusions (MPE) are a common terminal pathway for many types of cancer, especially non-small cell lung cancer (NSCLC). However, the phenotype and differentiation status of MPE-infiltrating CD8 + T cells have not yet been systematically addressed. In this study, the surface molecules and cytokine secretion of T cells in MPE and peripheral blood (PB) were analyzed using flow cytometry. We found an increased frequency of CD8 + T cells in MPE compared to PB among lung cancer patients, of which the effector memory subset (Tem, CCR7 - CD45RA - ) and central memory subset (Tcm, CCR7 + CD45RA - ) were upregulated. MPE-derived Tem and Tcm subsets expressed more PD1 or CD39, and there was a greater population of cells in these subsets that co-expressed them. In addition, Tem and Tcm cells from MPE had higher cytokine production than terminally differentiated effector memory cells (TemRA, CCR7 - CD45RA + ) and naïve cells (Tnaive, CCR7 + CD45RA + ). Our results demonstrate that the Tem and Tcm cells in MPE may have advantages in both tumor reactivity and immune functionality. Altogether, these findings help to characterize the phenotype of MPE-derived CD8 + T cells in terms of differentiation and tumor reactivity and reveal their potential as a target for immunotherapy., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Malignant pleural effusion (MPE) represents advanced malignant disease with poor prognosis. To date, pleural effusion cytology remains the best test to diagnose MPE but suffers from limited diagnostic sensitivity and high variation. We report a hexokinase 2-based method (HK2-seq) as a novel diagnostic method for multicancer MPE diagnosis., Methods: HK2-seq employed HK2 as a new metabolic function-associated marker to detect disseminated tumor cells engaging increased glycolysis in pleural effusion from many cancer types. Single-cell sequencing was used to confirm the malignancy of HK2-derived high glycolytic tumor cells (hgTCs) at the single-cell level via surveying genome-wide copy number alterations (CNAs), leading to establishment of definitive MPE diagnosis., Results: In a prospective cohort study including 111 patients with pleural effusion, the HK2 test showed diagnostic sensitivity, diagnostic specificity, positive predictive value, and negative predictive value of 91% (95% CI: 80%-97%), 84% (95% CI: 68%-93%), 90% (95% CI: 79%-96%), and 86% (95% CI: 70%-95%), respectively, in MPE diagnosis across 12 different cancer types. In contrast, pleural effusion cytology exhibits an overall diagnostic sensitivity of 45%. In addition to confirming the tumor origin of hgTCs, single-cell sequencing allowed identification of prognostic or targetable CNAs in hgTCs, especially CNAs found in liquid biopsies but absent in solid biopsies., Conclusions: HK2-seq establishes definitive MPE diagnosis across many cancer types with high diagnostic performance. It has the potential to be used for multicancer detection of circulating tumor cells in blood and other types of body fluids, as well as liquid biopsy-based genomic characterization for informative diagnosis., (© American Association for Clinical Chemistry 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Immune checkpoint inhibitors are still unable to provide clinical benefit to the large majority of non-small cell lung cancer (NSCLC) patients. A deeper characterization of the tumor immune microenvironment (TIME) is expected to shed light on the mechanisms of cancer immune evasion and resistance to immunotherapy. Here, we exploited malignant pleural effusions (MPEs) from lung adenocarcinoma (LUAD) patients as a model system to decipher TIME in metastatic NSCLC., Methods: Mononuclear cells from MPEs (PEMC) and peripheral blood (PBMC), cell free pleural fluid and/or plasma were collected from a total of 24 LUAD patients and 12 healthy donors. Bulk-RNA sequencing was performed on total RNA extracted from PEMC and matched PBMC. The DEseq2 Bioconductor package was used to perform differential expression analysis and CIBERSORTx for the regression-based immune deconvolution of bulk gene expression data. Cytokinome analysis of cell-free pleural fluid and plasma samples was performed using a 48-Plex Assay panel. THP-1 monocytic cells were used to assess macrophage polarization. Survival analyses on NSCLC patients were performed using KM Plotter (LUAD, N=672; lung squamous cell carcinoma, N=271)., Results: Transcriptomic analysis of immune cells and cytokinome analysis of soluble factors in the pleural fluid depicted MPEs as a metastatic niche in which all the components required for an effective antitumor response are present, but conscripted in a wound-healing, proinflammatory and tumor-supportive mode. The bioinformatic deconvolution analysis revealed an immune landscape dominated by myeloid subsets with the prevalence of monocytes, protumoral macrophages and activated mast cells. Focusing on macrophages we identified an MPEs-distinctive signature associated with worse clinical outcome in LUAD patients., Conclusions: Our study reports for the first time a wide characterization of MPEs LUAD microenvironment, highlighting the importance of specific components of the myeloid compartment and opens new perspectives for the rational design of new therapies for metastatic NSCLC., Competing Interests: Competing interests: All the authors with the exception of LA have nothing to disclose. LA is an employee of Takis srl., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Malignant melanotic nerve sheath tumor (MMNST) is an exceedingly rare and aggressive neoplasm of Schwann cell origin that has seldom been described in the cytopathology literature. Herein we present a case of a 60-year-old female with a 3.8 cm presacral mass that was diagnosed as a MMNST. A molecular workup demonstrated TERT promoter -124C > T and TET2 Q891* gene mutations. Within 2 years of her initial diagnosis, she had developed widespread metastasis and pleural effusions. A cytologic workup of the pleural fluid revealed clusters of vacuolated epithelioid cells with enlarged nuclei, prominent nucleoli, and occasional multinucleation. The lesional cells were positive for SOX10, S100-protein, Melan-A, and HMB45, while negative for Calretinin, MOC31, and monoclonal CEA. In this clinicopathologic context, a diagnosis of metastatic MMNST was rendered. Awareness of this entity and its clinical presentation, along with a critical understanding of its molecular findings and that of imitators, is crucial in achieving an accurate diagnosis., (© 2021 Wiley Periodicals LLC.)

Body cavity fluids accumulating in progressive malignancies are potential subjects of regular clinical testing for cancer-related features. Besides the cellular component, the supernatant of the fluid proved to gain diagnostic impact as the cell-free DNA (cfDNA) fraction ideally reflects general molecular features of the related tumorous process, e.g. in lung carcinoma. Thus, malignant pleural effusions can be used for lung cancer genetic profiling and this might remain the only source for testing in critical cases. The cfDNA concentration of the pleural effusion depends on many factors in both benign and malignant conditions. Further to direct pleural metastatic spread, the redirection of tissue lymphatic circulation, tumor angiogenesis, inflammatory processes and other variables may contribute to or enhance the enrichment of the effusion tumor DNA from the earliest stages of carcinogenesis. Our review addresses the traffic of cfDNA in the pleural space and the diagnostic utility of effusion cfDNA from the perspective of the complex pleural pathophysiology., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Malignant pleural effusion (MPE) is indicative of terminal malignancy with a uniformly fatal prognosis. Often, two distinct compartments of tumour microenvironment, the effusion and disseminated pleural tumours, co-exist in the pleural cavity, presenting a major challenge for therapeutic interventions and drug delivery. Clinical evidence suggests that MPE comprises abundant tumour-associated myeloid cells with the tumour-promoting phenotype, impairing antitumour immunity. Here we developed a liposomal nanoparticle loaded with cyclic dinucleotide (LNP-CDN) for targeted activation of stimulators of interferon genes signalling in macrophages and dendritic cells and showed that, on intrapleural administration, they induce drastic changes in the transcriptional landscape in MPE, mitigating the immune cold MPE in both effusion and pleural tumours. Moreover, combination immunotherapy with blockade of programmed death ligand 1 potently reduced MPE volume and inhibited tumour growth not only in the pleural cavity but also in the lung parenchyma, conferring significantly prolonged survival of MPE-bearing mice. Furthermore, the LNP-CDN-induced immunological effects were also observed with clinical MPE samples, suggesting the potential of intrapleural LNP-CDN for clinical MPE immunotherapy., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Differential diagnosis between malignant pleural effusion (MPE) and benign pleural effusion (BPE) remains a clinical challenge., Objective: The aim of the study is to assess the efficacy of the serum and pleural fluid (PF) miRNA panels in distinguishing MPE from BPE., Methods: Fourteen candidate miRNAs which were shown aberrant expression in lung cancer based on previous studies were tested by quantitative real-time PCR (qRT-PCR) in 20 MPE patients and 20 BPE patients. Significantly aberrantly expressed miRNAs were further assessed by qRT-PCR in all patients enrolled in this study. A receiver operating characteristic (ROC) curve was constructed, and the area under the ROC curve (AUC) was calculated to evaluated the diagnostic performance of the miRNAs., Results: miR-21, miR-29c and miR-182 were found to be significantly aberrantly expressed in the serum and PF of MPE patients. The AUCs for the combination of miR-21, miR-29c and miR-182 in serum and PF were 0.832 and 0.89 respectively in distinguishing MPE from infection-associated PE including tuberculous pleurisy and parapneumonia PE, and 0.866 and 0.919 respectively for differentiating MPE from heart failure-associated PE, which were superior to AUC of each individual miRNAs., Conclusions: miR-21, miR-29c and miR-182 in serum and PF could be useful biomarkers for diagnosis of MPE.

The complex interactions among different immune cells have important functions in the development of malignant pleural effusion (MPE). Here we perform single-cell RNA sequencing on 62,382 cells from MPE patients induced by non-small cell lung cancer to describe the composition, lineage, and functional states of infiltrating immune cells in MPE. Immune cells in MPE display a number of transcriptional signatures enriched for regulatory T cells, B cells, macrophages, and dendritic cells compared to corresponding counterparts in blood. Helper T, cytotoxic T, regulatory T, and T follicular helper cells express multiple immune checkpoints or costimulatory molecules. Cell-cell interaction analysis identifies regulatory B cells with more interactions with CD4 + T cells compared to CD8 + T cells. Macrophages are transcriptionally heterogeneous and conform to M2 polarization characteristics. In addition, immune cells in MPE show the general up-regulation of glycolytic pathways associated with the hypoxic microenvironment. These findings show a detailed atlas of immune cells in human MPE and enhance the understanding of potential diagnostic and therapeutic targets in advanced non-small cell lung cancer., (© 2021. The Author(s).)

Background and Objective: Prognostication of malignant pleural effusion (MPE) guides treatment strategies but existing prognostication scores are yet to be validated in Asians. We aimed to evaluate the performance of these scores in an Asian population. A refined score was also proposed based on the impact of EGFR mutation on survival., Methods: Survival and clinical data of histocytologically-confirmed MPE patients from a Hong Kong hospital were analyzed with the LENT, modified-LENT, PROMISE and SELECT (converted from its original model) scores. A refinement of the LENT score for Asians was proposed by inclusion of EGFR status (EGFR-LENT), which was compared with the LENT score and validated in an independent patient cohort., Results: All prognostication scores performed well on risk stratification by Kaplan-Meier curve (log rank p < 0.0001) in 368 MPE patients except for LENT in low-risk group. C-statistics for LENT, modified-LENT, PROMISE and SELECT in predicting 3-month mortality were 0.77, 0.80, 0.80 and 0.82, respectively. The proposed LENT score refinement (EGFR-LENT) improved stratification among low-risk patients; with a higher C-statistic (0.83) in 3-month mortality prediction than LENT (0.77, p = 0.0121), PROMISE (0.80, p = 0.3713), and SELECT (0.82, p = 0.7908) scores. Validation of EGFR-LENT in an independent cohort (124 patients) confirmed good performance in predicting 3-month mortality (C-statistic 0.87, vs 0.79 in LENT, p = 0.0444)., Conclusion: All existing scores had reasonable performance in prognosticating MPE, and LENT score refinement by inclusion of EGFR mutation status improved its performance among Asian MPE patients., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Cancer is a leading cause of death worldwide, and patients may have advanced disease when diagnosed. Targeted therapies guided by molecular subtyping of cancer can benefit patients significantly. Pleural effusions are frequently observed in patients with metastatic cancer and are routinely removed for therapeutic purposes; however, effusion specimens have not been recognized as typical substrates for clinical molecular testing because of frequent low tumor cellularity., Methods: Excess remnant pleural effusion samples (N = 25) from 21 patients with and without suspected malignancy were collected at Stanford Health Care between December 2019 and November 2020. Samples were processed into ThinPrep slides and underwent novel effusion tumor cell (ETC) analysis. The ETC results were compared with the original clinical diagnoses for accuracy. A subset of confirmed ETCs was further isolated and processed for molecular profiling to identify cancer driver mutations. All samples were obtained with Institutional Review Board approval., Results: The authors established novel quantitative standards to identify ETCs and detected epithelial malignancy with 89.5% sensitivity and 100% specificity in the pleural effusion samples. Molecular profiling of confirmed ETCs (pools of 5 cells evaluated) revealed key pathogenic mutations consistent with clinical molecular findings., Conclusions: In this study, the authors developed a novel ETC-testing assay that detected epithelial malignancies in pleural effusions with high sensitivity and specificity. Molecular profiling of 5 ETCs showed promising concordance with the clinical molecular findings. To promote cancer subtyping and guide treatment, this ETC-testing assay will need to be validated in larger patient cohorts to facilitate integration into cytologic workflow., (© 2021 American Cancer Society.)

Approximately 5-7% of non-small cell lung cancer (NSCLC) cases harbor an anaplastic lymphoma kinase (ALK) fusion gene and may benefit from ALK inhibitor therapy. To detect ALK fusion genes, we developed a novel test using reverse transcription polymerase chain reaction (RT-PCR) for the ALK kinase domain (KD). Since ALK expression is mostly silenced in the adult with the exception of neuronal tissue, the normal lung tissue, mesothelial lining, and inflammatory cells are devoid of ALK transcript, making ALK KD RT-PCR an ideal surrogate test for ALK fusion transcripts in lung or pleural effusion. The test was designed with a short PCR product (197 bp) to work for both malignant pleural effusion (MPE) and formalin-fixed, paraffin-embedded (FFPE) NSCLC samples. Using ALK IHC as a reference, the sensitivity of the test was 100% for both MPE and FFPE. The specificity was 97.6% for MPE and 97.4% for FFPE. Two false positive cases were found. One was a metastatic brain lesion which should be avoided in the future due to intrinsic ALK expression in the neuronal tissue. The other one resulted from ALK gene amplification. Due to potential false positivity, subsequent confirmation tests such as fluorescence in situ hybridization or multiplex PCR would be preferable. Nevertheless, the test is simple and inexpensive with no false negativity, making it a desirable screening test. It also offers an advantage over multiplex RT-PCR with the capability to detect novel ALK fusions. Indeed through the screening test, we found a novel ALK fusion partner (sperm antigen with calponin homology and coiled-coil domains 1 like gene, SPECC1L) with increased sensitivity to crizotinib in vitro. In summary, a novel RNA-based ALK KD analysis was developed for ALK rearrangement screening in MPE and FFPE specimens of NSCLC. This simple inexpensive test can be implemented as routine diagnostics., Competing Interests: The authors have declared that no competing interests exist.

Liquid biopsy analyzes the current status of primary tumors and their metastatic regions. We aimed to develop an optimized protocol for single-cell sequencing of floating tumor cells (FTCs) in pleural effusion as a laboratory test. FTCs were enriched using a negative selection of white blood cells by a magnetic-activated cell sorting system, and CD45-negative and cytokeratin-positive selection using a microfluidic cell separation system with a dielectrophoretic array. The enriched tumor cells were subjected to whole-genome amplification (WGA) followed by genome sequencing. The FTC analysis detected an EGFR exon 19 deletion in Case 1 (12/19 cells, 63.2%), and EML4-ALK fusion (17/20 cells, 85%) with an alectinib-resistant mutation of ALK (p.G1202R) in Case 2. To eliminate WGA-associated errors and increase the uniformity of the WGA product, the protocol was revised to sequence multiple single FTCs individually. An analytical pipeline, accurate single-cell mutation detector (ASMD), was developed to identify somatic mutations of FTCs. The large numbers of WGA-associated errors were cleaned up, and the somatic mutations detected in FTCs by ASMD were concordant with those found in tissue specimens. This protocol is applicable to circulating tumor cells analysis of peripheral blood and expands the possibility of utilizing molecular profiling of cancers., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Background: Pleural effusion from patients with advanced non-small cell lung cancer (NSCLC) has been proved valuable for molecular analysis, especially when the tissue sample not available. However, simultaneous detection of multiple driver gene alterations especially the fusions is still challenging., Methods: In this study, 77 patients with advanced NSCLC and pleural effusion were enrolled, 49 of whom had matched tumor tissues. Supernatants, cell sediments, and cell blocks were prepared from pleural effusion samples for detection of driver alterations by a PCR-based 9-gene mutation detection kit., Results: Mutations in EGFR, KRAS, and HER2 were detected in DNA and cfDNA, fusions in ALK was detected in RNA and cfRNA. Compared with matched tumor tissue, the supernatant showed the highest overall sensitivity (81.3%), with 81.5% for SNV/Indels by cfDNA and 80% for fusions by cfRNA, followed by cell blocks (71.0%) and the cell sediments (66.7%). Within the group of treatment-naïve patients or malignant cells observed in the cell sediments, supernatant showed higher overall sensitivity (89.5% and 92.3%) with both 100% for fusions., Conclusions: CfDNA and cfRNA derived from pleural effusion supernatant have been successfully tested with a PCR-based multigene detection kit. Pleural effusion supernatant seems a preferred material for detection of multigene alterations to guide treatment decision of advanced NSCLC., (© 2021 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Pathogenic molecular features gained specific significance in therapeutic decisions in lung carcinoma in the past decade. Initial and follow up genetic testing requres appropriate amounts and quality of tumor derived DNA, but tumor sampling, especially for disease monitoring is generally limited. Further to the peripheral blood (PB), samples from pleural fluid, accumulating in diverse lung processes might serve as an alternative source for cell-free DNA (cfDNA) for genetic profiling. In our study, cfDNA isolated from the pleural effusion and from the PB, and genomic DNA (gDNA) obtained from tissue/cellular samples were analyzed and compared from altogether 65 patients with pulmonary disease, including 36 lung adenocarcinomas. The quantity of effusion cfDNA yield appeared to be significantly higher compared to that from simultaneously collected PB plasma (23.2 vs. 4.8 ng/μl, p < 0.05). Gene mutations could be safely demonstrated from the effusion cfDNA fraction obtained from adenocarcinoma patients, 3/36 EGFR , 9/36 KRAS and 1/36 BRAF gene variants were detected. In this series, 9/13 samples showed an effusion+/plasma-mutational status, while only 1/13 samples presented with the opposite findings (effusion-/plasma+). gDNA analysis from sediment cell blocks from the identical effusion sample was surprisingly ineffective for lung adenocarcinoma profiling due to the low DNA yield. In conclusion, the cell free supernatant of pleural effusions appears to concentrate cancer derived cfDNA and seems to be particularly suitable for serial genotyping of pulmonary adenocarcinoma., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mokánszki, Bádon, Mónus, Tóth, Bittner and Méhes.)

Malignant pleural effusion (MPE) is a frequent complication of malignancies and poses a clinical problem. CD4 + T lymphocytes are the most frequent cell population in MPE. Traditionally, CD4 + T cells are classified into two subsets based on cytokine production profiles, type 1 (Th1) and type 2 (Th2) helper T cells, which exhibit distinct functions. Recently, other T-cell subsets have been added to the Th-cell "portfolio", including regulatory T, Th17, Th9, and Th22 cells. The current review focuses on summarizing the Th-cell phenotypic characteristics, mechanism of Th-cell differentiation, and their pleural space recruitment, based on recent research. We also describe the interplay in MPE among different Th cells, as well as Th cells and lung cancer cells or mesothelial cells. Future research should expand the landscape map of human MPE immune cells, explore the immuno-regulation of B cells, and investigate the communication between macrophages and Th cells in MPE, which may facilitate meaningful advancements in the diagnoses and therapeutics of MPE., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Cell-free DNA (cfDNA) in supernatant of pleural effusion from advanced NSCLC patients has been proved as surrogate sample detecting therapeutic targets as well as tumor mutation burden (TMB). As recently reported, cfDNA in pleural effusion supernatant is superior to plasma in TMB evaluation. It is reasonable to hypothesize that cfDNA profile in pleural effusion (PE) and plasma might be different. It remains to be elucidated why cfDNA in PE supernatant impacts on genetic analysis. Consequently, the approach dealing with cfDNA from PE supernatant might need to be different from that for plasma cfDNA in order to obtain accurate clinical genetic testing result. Methods: Pleural effusion samples from 32 patients with stage IV lung adenocarcinoma were collected. Supernatant and sediment were processed separately to extract Cell-free DNA as well as sediment DNA (PE-S). cfDNA from pleural effusion was analyzed by Agilent 2100 bioanalyzer. Libraries were prepared by 1) direct use of the total cfDNA without fragmentation step (PE-FL) or 2) use of full-length cfDNA fragmented to 150-250bp (PE-F), 3) use of cfDNA fragments enriched to ~167bp (PE-E167) as well as 4) use of cfDNA fragments larger than 500bp enriched (PE-E500). All samples were subjected to targeted next-generation sequencing (NGS) with a panel of 448 cancer-related genes as well as a panel of 10 NSCLC driver genes. Results: cfDNA were successfully extracted from 30 MPE samples. cfDNA displayed distinct profile in supernatant of malignant pleural effusion from that of plasma cfDNA. No statistical difference in detection of hotspot variations between PE-E167 and PE-F by 448-gene or 10-gene panel. While TMB from PE-F samples was significantly higher than that from PE-E167 and PE-FL. Higher TMB from PE-F was resulted from cancer-unspecific variants with low allele frequency (0.1%-1%) which were mainly introduced by long-fragment cfDNA. Similar genetic profile was observed between paired cfDNA of PE-FL and cfDNA of PE-E167. Conclusion: Long-fragment cfDNA in the PE supernatant will introduce low abundant cancer unrelated variants which leads overestimation of TMB. Paired PE-FL and PE-E167 gave comparable outcomes. Direct use of the total cfDNA without fragmentation step (PE-FL) is recommended for library preparation of NGS testing in clinical practice to exclude interference from long fragments of the cfDNA., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Purpose: For anaplastic lymphoma kinase (ALK) gene detection, the centrifugal sedimentation method (CSM) and cell block method (CBM) are commonly used to process samples of bloody pleural effusions (BPEs). However, in practice, the impurity content in the processed samples often affects the results and even leads to the detection failure. The purpose of this study was to establish a cell enrichment method (CEM) by using a disposable membrane cell collector to remove blood and inflammatory cells and enrich lung adenocarcinoma cells in BPE for more efficient RNA extraction and ALK gene detection., Materials and Methods: CEM proposed in this study and the traditional CSM and CBM were used to treat BPE samples collected from 37 lung adenocarcinoma patients. A DeNovix DS-11 ultraviolet spectrophotometer was used to measure the concentration and purity of extracted RNA. Amplification refractory mutation systems (ARMS) and ABI 7500 fluorescence qPCR were used to detect ALK gene. Through statistical analysis, the CEM was compared with the CSM and CBM in RNA concentration, purity, and ALK gene detection results., Results: The concentration of RNA extracted by using the CEM was significantly higher than that extracted by using the CBM and CSM (p < 0.001). The purity of RNA extracted by using the CEM was significantly higher than that by the other 2 methods (p = 0.011, p = 0.005). ALK gene testing with PCR was successful in all the samples using the CEM, but 2 cases by the CSM and 1 case by the CBM failed., Conclusions: Using the disposable membrane cell collector to process BPE of lung adenocarcinoma patients for RNA extraction and ALK gene detection is more effective and successful compared with the traditional methods, and it is suggested to be further applied and popularized in clinical practice., (© 2021 S. Karger AG, Basel.)

Background: The lack of pathological quality control standard in detecting epidermal growth factor receptor (EGFR) gene mutation in malignant pleural effusion leads to confusion in the interpretation of detection results and the clinical use of EGFR-tyrosine kinase inhibitor (TKI). Therefore, it is very important to propose quality control standards and guide the detection of EGFR mutation in pleural effusion. The aim of this study is to retrospectively analyze the results of EGFR gene mutation in pleural effusion sediment section according to strict pathological quality control standards, and the therapeutic effect of EGFR-TKIs guided by this detection results., Methods: From January 2012 to June 2018, the clinical data of patients with pleural effusion collected from Department of Pathology of Peking Union Medical College Hospital were analyzed retrospectively. Among them, 132 patients with relatively complete clinical data and with EGFR gene mutation detection of paraffin-embedded pleural effusion sediment section according to the established quality control standard were included. According to the results of EGFR gene mutation, it was divided into positive group and negative group, and the efficacy of EGFR-TKIs in different groups was compared., Results: After the centrifugation of pleural effusion, the sediment was embedded in paraffin, sectioned, and observed under the microscope after HE staining. If the number of tumor cells ≥100, it met the pathological quality control standard, and it could be used for subsequent EGFR gene mutation detection. EGFR gene mutations were detected in 72 (54.5%) of 132 patients. EGFR-TKIs were used in 69 of 72 mutation positive patients. Of 60 EGFR mutation negative patients, only 15 used EGFR-TKIs. In EGFR mutation positive group, the disease control rate (DCR) was 95.8%, and the median progression-free survival (PFS) was 11 months. In EGFR mutation negative group, the DCR was 0%, and the median PFS was 1 month. The DCR and PFS were significantly different between the two groups (P<0.05)., Conclusions: According to the pathological quality control standards, the embedded section of pleural fluid sediment can be used to detect EGFR gene mutation, and the results can be used to guide the clinical use of EGFR-TKIs.