Interleukin-8/CXCR1 Signaling Contributes to the Progression of Pulmonary Adenocarcinoma Resulting in Malignant Pleural Effusion.

Pulmonary adenocarcinoma (PADC) treatment limited efficacy in preventing tumor progression, often resulting in malignant pleural effusion (MPE). MPE is filled with various mediators, especially interleukin-8 (IL-8). However, the role of IL-8 and its signaling mechanism within the fluid microenvironment (FME) implicated in tumor progression warrants further investigation. Primary cultured cells from samples of patients with MPE from PADC, along with a commonly utilized lung cancer cell line, were employed to examine the role of IL-8 and its receptor, CXCR1, through comparative analysis. Our study primarily assessed migration and invasion capabilities, epithelial-mesenchymal transition (EMT), and cancer stem cell (CSC) properties. Additionally, IL-8 levels in MPE fluid versus serum, along with immunohistochemical expression of IL-8/CXCR1 signaling in tumor tissue and cell blocks were analyzed. IL-8/CXCR1 overexpression enhanced EMT and CSC properties. Furthermore, the immunocytochemical examination of 17 cell blocks from patients with PADC and MPE corroborated the significant correlation between upregulated IL-8 and CXCR1 expression and the co-expression of IL-8 and CXCR1 in MPE with distant metastasis. In summary, the IL-8/ CXCR1 axis in FME is pivotal to tumor promotion via paracrine and autocrine signaling. Our study provides a therapeutic avenue for improving the prognosis of PADC patients with MPE.

Pleural effusion supernatant: a reliable resource for cell-free DNA in molecular testing of lung cancer.

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.

Consistency Analysis of Programmed Death Ligand 1 Expression in Non-Small Cell Lung Cancer Between Pleural Effusion and Matched Primary Lung Cancer Tissues by Immunohistochemical Double Staining.

In clinical practice, programmed death ligand 1 (PD-L1) detection is prone to nonspecific staining due to the complex cellular composition of pleural effusion smears. In this study, diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole (AEC) immunohistochemistry double staining was performed to investigate PD-L1 expression in tumor cells from malignant pleural effusion (MPE). MPE was considered as a metastasis in non-small cell lung cancer patients; thus, the heterogeneity between metastatic and primary lung cancer was revealed as well. Ninety paired specimens of MPE cell blocks and matched primary lung cancer tissues from non-small cell lung cancer patients were subjected to PD-L1 and thyroid transcription factor-1(TTF-1)/p63 immunohistochemistry double staining. Two experienced pathologists independently evaluated PD-L1 expression using 3 cutoffs (1%, 10%, and 50%). PD-L1 expression in MPE was strongly correlated with that in matched primary lung cancer tissues (R = 0.813; P < .001). Using a 4-tier scale (cutoffs: 1%, 10%, and 50%), the concordance was 71.1% (Cohen's κ = .534). Using a 2-tier scale, the concordance was 75.6% (1%, Cohen's κ = 0.53), 78.9% (10%, Cohen's κ = 0.574), and 95.6% (50%, Cohen's κ = 0.754). The rates of PD-L1 positivity in MPE (56.7%) were higher than that in lung tissues (32.2%). All 27 discordant cases had higher scores in MPE. The double-staining method provided superior identification of PD-L1-positive tumor cells on a background with nonspecific staining. In conclusion, PD-L1 expression was moderately concordant between metastatic MPE cell blocks and matched primary lung carcinoma tissues, with variability related to tumor heterogeneity. MPE should be considered to detect PD-L1 when histological specimens are unattainable, especially when PD-L1 expression is >50%. PD-L1 positivity rates were higher in MPE. Double staining can improve PD-L1 detection by reducing false-negative/positive results.

Usefulness of thoracic ultrasound in the assessment of removal of indwelling pleural catheter in patients with malignant pleural effusion.

INTRODUCTION: There are no defined criteria for deciding to remove a non-functioning indwelling pleural catheter (IPC) when lung re-expansion on chest X-ray is incomplete. Chest computed tomography (chest CT) is usually used. The objective of this work is to validate the usefulness of chest ultrasound performed by a pulmonologist and by a radiologist compared to chest CT. PATIENTS AND METHODS: Prospective, descriptive, multidisciplinary and multicenter study including patients with malignant pleural effusion and non-functioning IPC without lung reexpansion. Decisions made on the basis of chest ultrasound performed by a pulmonologist, and performed by a radiologist, were compared with chest CT as the gold standard. RESULTS: 18 patients were analyzed, all of them underwent ultrasound by a pulmonologist and chest CT and in 11 of them also ultrasound by a radiologist. The ultrasound performed by the pulmonologist presents a sensitivity of 60%, specificity of 100%, PPV 100% and NPV 66% in the decision of the correct removal of the IPC. The concordance of both ultrasounds (pulmonologist and radiologist) was 100%, with a kappa index of 1. The 4 discordant cases were those in which the IPC was not located on the ultrasound. CONCLUSIONS: Thoracic ultrasound performed by an expert pulmonologist is a valid and simple tool to determine spontaneous pleurodesis and remove a non-functioning IPC, which would make it possible to avoid chest CT in those cases in which lung reexpansion is observed with ultrasonography.

Cytomorphology of metastatic malignant peripheral nerve sheath tumour present in pleural fluid: Case study and literature review.

The cytomorphology of MPNST in effusion specimens is rarely described. In this paper, the detailed cytopathological and immunohistochemical characteristics of metastatic MPNST has been described in pleural effusion. Patients' medical history and the judicious utilization of ancillary studies contribute to ensure precise cytological diagnoses. The cytomorphology of malignant peripheral nerve sheath tumour (MPNST) in effusion specimens can be diagnostically challenging. The author presents detailed cytopathological and immunohistochemical characteristics of a case of metastatic MPNST in pleural effusion.

Reliably making the primary diagnosis of mesothelioma utilizing serous fluid cytology specimens: an institutional experience.

INTRODUCTION: The diagnosis of mesothelioma has historically been challenging, especially on serous fluid cytology (SFC). Distinguishing between reactive and neoplastic mesothelial cells can be difficult on cytomorphology alone. However, additional ancillary tests, such as BRCA1 associated protein-1 immunohistochemistry and fluorescence in situ hybridization for cyclin-dependent kinase inhibitor 2A deletion, can provide a sensitive and highly specific method of proving malignancy. MATERIALS AND METHODS: SFC specimens diagnosed as mesothelioma, suspicious for mesothelioma (SM), and atypical mesothelial cells (AMCs) since 2012 were identified by querying the laboratory information system. Clinical data and pathologic parameters were gathered. RESULTS: One hundred ten cases of mesothelioma, SM, and AMC were identified. Of these, 61 cases had a definitive diagnosis of mesothelioma on SFC. Average age at SFC diagnosis was 67 years (26-87 years), with most patients being male (67%). Out of the 61 cases, 11 cases (18%) had an initial diagnosis of mesothelioma made on SFC specimens, with 5 of these 11 cases being in patients that never received a histologic diagnosis of mesothelioma. Ancillary studies were utilized in all 11 cases. An initial diagnosis of metastatic mesothelioma was made on SFC in 9 cases (15%). For 6 of these 9 cases, the SFC diagnosis was the sole diagnosis of metastatic mesothelioma without a companion histologic diagnosis. In addition, 15 cases were diagnosed as SM, with 11 of these cases following a definitive mesothelioma diagnosis. Thirty-four cases were diagnosed as AMC, with 27 cases following a definitive mesothelioma diagnosis. CONCLUSIONS: The diagnosis of mesothelioma can be reliably made on SFC with the appropriate cytomorphology criteria and/or confirmatory ancillary testing.

Exploratory pilot study to characterize the immune landscapes of malignant pleural effusions and their corresponding primary tumors from patients with breast carcinoma and lung adenocarcinoma.

INTRODUCTION: Malignant pleural effusion (MPE) is a frequent complication of advanced malignancies. In this pilot study, we characterized the immune landscapes of MPEs, compared them to their primary tumor (PT) samples from breast carcinoma (BC) and lung adenocarcinoma (LADC), and tested the utility of multiplexed image technology in cytological samples. MATERIALS AND METHODS: We evaluated the immune contexture of 6 BC and 5 LADC MPEs and their PTs using 3 multiplex immunofluorescence panels. We explored the associations between sample characteristics and pleural effusion-free survival. RESULTS: No MPE samples had positive programmed death-ligand 1 expression in malignant cells, although 3 of 11 PTs has positive programmed death-ligand 1 expression (more than 1% expression in malignant cells). Overall, in LADC samples, cluster of differentiation 3 (CD3)+ T cells and CD3+CD8+ cytotoxic T cells predominated (median percentages for MPEs versus PTs: 45.6% versus 40.7% and 4.7% versus 6.6%, respectively) compared with BC. CD68+ macrophages predominated in the BC samples (medians for MPEs 61.2% versus PTs for 57.1%) but not in the LADC samples. Generally in PTs, CD3+CD8+ forkhead box P3+ T cells and the median distances from the malignant cells to CD3+CD8+Ki67+ and CD3+ programmed cell death protein 1 + T cells correlated to earlier MPE after PT diagnosis. CONCLUSIONS: The immune cell phenotypes in the MPEs and PTs were similar within each cancer type but different between BC versus LADC. An MPE analysis can potentially be used as a substitute for a PT analysis, but an expanded study of this topic is essential.

Immune checkpoint inhibitor plus chemotherapy as first-line treatment for non-small cell lung cancer with malignant pleural effusion: a retrospective multicenter study.

BACKGROUND: Immune checkpoint inhibitors (ICI) combined with chemotherapy are efficacious for treating advanced non-small cell lung cancer (NSCLC); however, the effectiveness of this approach in the malignant pleural effusion (MPE) population is unclear. This study evaluated ICI plus chemotherapy in NSCLC patients with MPE. METHODS: Patients from 3 centers in China with NSCLC and MPE who received ICI plus chemotherapy (ICI Plus Chemo) or chemotherapy alone (Chemo) between December 2014 and June 2023 were enrolled. Clinical outcomes and adverse events (AEs) were compared. RESULTS: Of 155 eligible patients, the median age was 61.0 years old. Males and never-smokers accounted for 73.5% and 39.4%, respectively. Fifty-seven and 98 patients received ICI Plus Chemo or Chemo, respectively. With a median study follow-up of 10.8 months, progression-free survival (PFS) was significantly longer with ICI Plus Chemo than with Chemo (median PFS: 7.4 versus 5.7 months; HR = 0.594 [95% CI: 0.403-0.874], P = 0.008). Median overall survival (OS) did not differ between groups (ICI Plus Chemo: 34.2 versus Chemo: 28.3 months; HR = 0.746 [95% CI: 0.420-1.325], P = 0.317). The most common grade 3 or worse AEs included decreased neutrophil count (3 [5.3%] patients in the ICI Plus Chemo group vs. 5 [5.1%] patients in the Chemo group) and decreased hemoglobin (3 [5.3%] versus 10 [10.2%]). CONCLUSIONS: In patients with untreated NSCLC with MPE, ICI plus chemotherapy resulted in significantly longer PFS than chemotherapy and had a manageable tolerability profile, but the effect on OS may be limited.

Establishing mesothelioma patient-derived organoid models from malignant pleural effusions.

OBJECTIVES: Pleural mesothelioma is a cancer arising in the cells that line the lungs and chest wall with poor survival and poor response to first-line therapy. Organoid models of cancer can faithfully recapitulate the genetic and histopathological characteristics of individualized tumors and have potential to be used for precision medicine, however methods of establishing patient-derived mesothelioma organoids have not been well established in the published literature. MATERIALS AND METHODS: Long-term mesothelioma patient-derived organoids were established from ten malignant pleural effusion fluids. Mesothelioma patient-derived organoids were compared to the corresponding biopsy tissue specimens using immunohistochemistry labelling for select diagnostic markers and the TruSight Oncology-500 sequencing assay. Cell viability in response to the chemotherapeutic drug cisplatin was assessed. RESULTS: We established five mesothelioma patient-derived organoid cultures from ten malignant pleural effusion fluids collected from nine individuals with pleural mesothelioma. Mesothelioma patient-derived organoids typically reflected the histopathological and genomic features of patients' matched biopsy specimens and displayed cytotoxic sensitivity to cisplatin in vitro. CONCLUSION: This is the first study of its kind to establish long-term mesothelioma organoid cultures from malignant pleural effusions and report on their utility to test individuals' chemotherapeutic sensitivities ex vivo.

Metastatic upper tract urothelial carcinoma with nest-like features presenting as malignant pleural effusion.

Metastatic urothelial carcinoma is a rare cause of pleural effusions. We report a case of urothelial carcinoma of the upper urinary tract in an oldest-old male patient, a smoker, with situs inversus totalis, that presented uniquely with malignant pleural effusion at presentation without evidence of a primary tumor on imaging. Cytological smears of the massive left pleural effusion revealed epithelioid neoplastic cells arranged in short cords, small-to-large clusters, and raspberry-like morules, mimicking mesothelioma; cell block preparations highlighted the presence of tubules and nest-like structures. The tumor cells showed a high nuclear-to-cytoplasmic ratio, nuclear grooves, and mitotic figures. Cytomorphologic features coupled with the immunophenotype of neoplastic cells (p63, GATA3, and uroplakin II positive) allowed the diagnosis of metastatic urothelial carcinoma and a possible nested subtype. These findings were supported by a total body computed tomography (CT) showing no evidence of a mass in the bladder or elsewhere in the urinary tract but a concentric parietal thickening of the proximal left ureter, suggesting malignancy. To our knowledge, a malignant effusion as a primary manifestation of urothelial carcinoma with nest-like features originating in the upper urinary tract has never been described previously. Our case focuses on the value of cell block in the working-up of neoplastic effusions by revealing the architectural pattern of an uncommon malignancy and the correlation between cytopathology and imaging gross findings to reach an accurate diagnosis.

Detection of effusion tumor cells under different storage and processing conditions.

BACKGROUND: Circulating tumor cells (CTCs) shed into blood provide prognostic and/or predictive information. Previously, the authors established an assay to detect carcinoma cells from pleural fluid, termed effusion tumor cells (ETCs), by employing an immunofluorescence-based CTC-identification platform (RareCyte) on air-dried unstained ThinPrep (TP) slides. To facilitate clinical integration, they evaluated different slide processing and storage conditions, hypothesizing that alternative comparable conditions for ETC detection exist. METHODS: The authors enumerated ETCs on RareCyte, using morphology and mean fluorescence intensity (MFI) cutoffs of >100 arbitrary units (a.u.) for epithelial cellular adhesion molecule (EpCAM) and <100 a.u. for CD45. They analyzed malignant pleural fluid from three patients under seven processing and/or staining conditions, three patients after short-term storage under three conditions, and seven samples following long-term storage at -80°C. MFI values of 4',6-diamidino-2-phenylindol, cytokeratin, CD45, and EpCAM were compared. RESULTS: ETCs were detected in all conditions. Among the different processing conditions tested, the ethanol-fixed, unstained TP was most similar to the previously established air-dried, unstained TP protocol. All smears and Pap-stained TPs had significantly different marker MFIs from the established condition. After short-term storage, the established condition showed comparable results, but ethanol-fixed and Pap-stained slides showed significant differences. ETCs were detectable after long-term storage at -80°C in comparable numbers to freshly prepared slides, but most marker MFIs were significantly different. CONCLUSIONS: It is possible to detect ETCs under different processing and storage conditions, lending promise to the application of this method in broader settings. Because of decreased immunofluorescence-signature distinctions between cells, morphology may need to play a larger role.

Cell-free DNA methylation analysis as a marker of malignancy in pleural fluid.

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.

First-line treatment of driver gene-negative metastatic lung adenocarcinoma with malignant pleural effusion: Should chemotherapy be combined with an immune checkpoint inhibitor or bevacizumab?

Patients with metastatic lung adenocarcinoma (MLA) and malignant pleural effusion (MPE) without driver gene mutations have a poor prognosis. None of the standard treatment strategies is recommended for such patients. We retrospectively analyzed the efficacy of the first-line treatment for this specific population: standard platinum-based doublet chemotherapy (CT), CT plus an immune checkpoint inhibitor (CT plus ICI), and CT plus bevacizumab (CT plus Bev). A total of 323 eligible patients were enrolled: CT alone (n = 166), CT plus Bev (n = 72), and CT plus ICI (n = 85). Treatment efficacy assessments were performed every two cycles according to the RECIST guidelines. The endpoints were overall survival (OS) and progression-free survival (PFS). Kaplan-Meier (K‒M) curves and the log-rank test were used to compare OS and PFS. p < 0.05 was the threshold of significance (statistical software: SPSS). The median follow-up was 11.4 months (range, 2.1-49.6 months). PFS and OS in the CT plus ICI/CT plus Bev cohort were significantly longer than those in the CT group (PFS: 7.8/6.4/3.9 months, p < 0.0001; OS: 16.4/15.6/9.6 months, p < 0.0001, respectively). CT plus Bev had better PFS and OS than CT plus ICI/CT in PD-L1 < 1% patients (PFS: 8.4/5.0/3.8 months, p < 0.0001; OS: 15.6/12.9/9.3 months, p < 0.0001). Among patients with PD-L1 1-49%, CT plus ICI led to a longer PFS and OS (PFS: 8.9/5.8/4.2 months, p = 0.009; OS: 24.2/18.8/11.5 months, p = 0.03). In the cohort with PD-L1 ≥ 50%, CT plus ICI was still the best first-line treatment (PFS: 19.7/13.8/9.6 months, p = 0.033; OS: 27.2/19.6/14.9 months, p = 0.047). In driver gene-negative MLA with MPE, CT plus Bev or ICI better controlled MPE and significantly prolonged survival compared to CT alone. PD-L1 expression (negative/positive) may be a key factor influencing the choice of CT plus Bev or ICI.

An Updated Contextual Approach to Mesothelial Proliferations in Pleural Effusion Cytology Leveraging Morphology, Ancillary Studies, and Novel Biomarkers.

CONTEXT.­: Pleural effusions are common cytologic specimens that can be leveraged to make diagnoses of malignancy that drive appropriate patient management. However, the overlap in morphologic features of reactive mesothelial proliferations, mesotheliomas, and adenocarcinomas can create diagnostic pitfalls in the cytologic evaluation of pleural fluids. OBJECTIVE.­: To review the morphologic spectrum of benign and malignant mesothelial proliferations in pleural effusions, as well as relevant clinicoradiologic contexts and ancillary tests. DATA SOURCES.­: Existing scientific and clinical literature as of January 2023. CONCLUSIONS.­: We can leverage the knowledge of several overlapping morphologic features, clinicoradiologic scenarios, and immunohistochemical studies to enhance the diagnostic accuracy of pleural effusion cytology to appropriately delineate cases of adenocarcinoma, reactive mesothelial proliferation, and mesothelioma. Earlier diagnosis through cytology, particularly in cases of mesothelioma, may positively impact patient treatment options and prognosis.

Intrapleural injection of brucea javanica oil emulsion provided a long-term benefits in patient with malignant pleural effusion from pleural mesothelioma: A case report.

Malignant pleural mesothelioma (MPM) is a severe form of cancer that originates from mesothelium cells. Around 54-90% of mesotheliomas are associated with pleural effusions. Brucea Javanica Oil Emulsion (BJOE) is the processed oil derived from the seeds of Brucea javanica, which has shown potential as a treatment option for several types of cancer. Here, we present a case study of a MPM patient with malignant pleural effusion who received intrapleural injection of BJOE. The treatment resulted in the complete response of pleural effusion and chest tightness. While the precise mechanisms underlying the therapeutic effects of BJOE for pleural effusion are not yet fully understood, it has demonstrated a satisfactory clinical response without significant adverse effects.

Single-cell RNA sequencing reveals immune microenvironment of small cell lung cancer-associated malignant pleural effusion.

We used 10 × genomics single-cell transcriptome sequencing technology to reveal the tumor immune microenvironment characteristics of small cell lung cancer (SCLC) in a patient with malignant pleural effusion (MPE). A total of 8008 high-quality cells were finally obtained for subsequent bioinformatic analysis, which were divided into 10 cell clusters further identified as B cells, T cells, myeloid cells, NK cells, and cancer cells. Such SCLC related genes as NOTCH1, MYC, TSC22D1, SOX4, BLNK, YBX3, VIM, CD8A, CD8B, and KLF6 were expressed in different degrees during differentiation of T and B cells. Different ligands and receptors between T, B and tumor cells almost interact through MHC II, IL-16, galectin, and APP signaling pathway.

C5a enhances inflammation and chemotaxis of γδ T cells in malignant pleural effusion.

BACKGROUND: The inhibitory effect of γδT17 cells on the formation of murine malignant pleural effusions (MPE) has been established. However, there is limited understanding regarding the phenotypic characterization of γδ T cells in MPE patients and their recruitment to the pleural cavity. METHODS: We quantified γδ T cell prevalence in pleural effusions and corresponding peripheral blood from malignant and benign patients using immunohistochemistry and flow cytometry. The expression of effector memory phenotype, stimulatory/inhibitory/chemokine receptors and cytokines on γδ T cells in MPE was analyzed using multicolor flow cytometry. The infiltration of γδ T cells in MPE was assessed through immunofluorescence, ELISA, flow cytometry and transwell migration assay. RESULTS: We observed a significant infiltration of γδ T cells in MPE, surpassing the levels found in blood and benign pleural effusion. γδ T cells in MPE exhibited heightened expression of CD56 and an effector memory phenotype, while displaying lower levels of PD-1. Furthermore, γδ T cells in MPE showed higher levels of cytokines (IFN-γ, IL-17A and IL-22) and chemokine receptors (CCR2, CCR5 and CCR6). CCR2 expression was notably higher in the Vδ2 subtype compared to Vδ1 cells. Moreover, the complement C5a enhanced cytokine release by γδ T cells, upregulated CCR2 expression in Vδ2 subsets, and stimulated the production of chemokines (CCL2, CCL7 and CCL20) in MPE. In vitro utilizing CCR2 neutralising and C5aR antagonist significantly reduced the recruitment of γδ T cells. CONCLUSIONS: γδ T cells infiltrate MPE by overexpressing CCR2 and exhibit hightened inflammation, which is further augmented by C5a.

Morphological markers of chromosomal instability as predictors of malignancy in pleural effusion.

BACKGROUND: Malignancy in pleural effusion is an indication of poor prognosis. The distinction between malignant cells and reactive mesothelial cells in effusion cytology is sometimes difficult and requires ancillary techniques. Evaluation of morphological indicators of chromosomal instability (CI) like micronuclei (MN), chromatin bridging (CB), nuclear budding (NB), and multipolar mitosis (MM) on routine cytology smears is a promising tool to distinguish malignant from benign ascitic fluids. However, it has been scarcely evaluated in pleural effusions. The present study was conducted to evaluate the diagnostic value of these markers in differentiating between malignant and benign pleural fluids. METHODS: It is a cross sectional study in which a total of 72 pleural fluid samples over a period of 2 years received in the cytology department of the hospital were evaluated. The cytological analysis was done by two independent cytopathologists and interpreted as either malignant or benign. Four morphological markers of CI were counted in the May-Grünwald Giemsa (MGG) stained smears of all the cases and the score was compared with the conventional cyto-morphological diagnosis. RESULTS: Out of 72 cases, there were 42 malignant and 30 benign effusions on cytological examination. The mean score of micronuclei count, nuclear budding, chromatin bridging and multipolar mitosis in malignant effusions were 7.26 ± 2.74, 9.55 ± 5.53, 1.83 ± 1.17, and 2.21 ± 1.62 respectively that was significantly higher than the benign effusions (1 ± 0.71, 1.1 ± 0.86, 0.38 ± 0.50, and 0.15 ± 0.37 respectively) (p < .05). On Receiver operating characteristic (ROC) curve analysis, a cut-off of 5 for the MN count had a sensitivity of 88% and specificity of 100% in detecting malignant pleural effusion [Area under curve (AUC) 95.8%, p < .001]. CONCLUSION: Evaluation of morphological indicators of CI on routine MGG stained smears is a simple and cost-effective method to differentiate between benign and malignant pleural fluids.

Heterogeneity of cancer stem cell-related marker expression is associated with three-dimensional structures in malignant pleural effusion produced by lung adenocarcinoma.

INTRODUCTION: Cancer stem cells have been described in lung adenocarcinoma-associated malignant pleural effusion. They show clinically important features, including the ability to initiate new tumours and resistance to treatments. However, their correlation with the three-dimensional tumour structures in the effusion is not well understood. METHODS: Cell blocks produced from lung adenocarcinoma patients' pleural effusion were examined for cancer stem cell-related markers Nanog and CD133 using immunocytochemistry. The three-dimensional cancer cell structures and CD133 expression patterns were visualized with tissue-clearing technology. The expression patterns were correlated with tumour cell structures, genetic variants and clinical outcomes. RESULTS: Thirty-nine patients were analysed. Moderate-to-strong Nanog expression was detected in 27 cases (69%), while CD133 was expressed by more than 1% of cancer cells in 11 cases (28%). Nanog expression was more homogenous within individual specimens, while CD133 expression was detected in single tumour cells or cells within small clusters instead of larger structures in 8 of the 11 positive cases (73%). Although no statistically significant correlation between the markers and tumour genetic variants or patient survival was observed, we recorded seven cases with follow-up specimens after cancer treatment, and four (57%) showed a change in stem cell-related marker expression corresponding to treatment response. CONCLUSIONS: Lung adenocarcinoma cells in the pleural effusion show variable expression of cancer stem cell-related markers, some showing a correlation with the size of cell clusters. Their expression level is potentially correlated with cancer treatment effects.