Pleural Cells Research Articles

Validation of a diagnostic flowchart for tuberculous pleurisy in pleural fluid with high levels of adenosine deaminase

IntroductionAdenosine deaminase (ADA) in pleural fluid is a useful marker for diagnosing tuberculous pleurisy. However, recent studies have reported a lower specificity of pleural fluid ADA levels. We previously developed a diagnostic flowchart for patients with pleural fluid ADA ≥40 U/L, incorporating variables such as pleural fluid lactate dehydrogenase <825 U/L, predominant pleural fluid neutrophils or cell degeneration, and a pleural fluid ADA/total protein ratio <14. This flowchart was effective in distinguishing between tuberculous pleurisy and other diseases. Here, we conducted a validation analysis of this flowchart. Materials and methodsWe retrospectively collected data from 458 patients with pleural fluid ADA concentrations ≥40 U/L across eight institutions from January 2019 to December 2023. The diagnostic accuracy rate, sensitivity, and specificity of the diagnostic flowchart were analysed and compared to those in the original study. ResultsEighty-seven patients were diagnosed with tuberculous pleurisy, and 371 patients were diagnosed with other diseases. The diagnostic accuracy, sensitivity, and specificity for diagnosing tuberculous pleurisy were 77.7%, 86.2%, and 75.7%, respectively. Compared with that in the original study, the rate of tuberculous pleurisy was lower (19.0% vs. 44.5%, p < 0.001), but the diagnostic accuracy rates were not significantly different (p = 0.253). On the basis of the findings from this validation study, we have revised the flowchart to enhance its utility. ConclusionThe diagnostic flowchart exhibited high diagnostic accuracy in this validation study, comparable to that in the original study. This validation confirms the effectiveness of the flowchart, even in settings with a low incidence of tuberculosis.

Simultaneous detection of 10 cancer-associated amino acids and polyamines by high-performance liquid chromatography-high resolution mass spectrometry in pleural effusion cells obtained from lung adenocarcinoma patients

The deregulation of amino acid and polyamine metabolism is a hallmark of malignancy that regulates cancer cell proliferation, angiogenesis, and invasion. A sensitive mass spectrometry method was developed to simultaneously quantify 10 cancer-associated metabolites in pleural effusion cells for the diagnosis of malignancy and to complement conventional pleural cytology. Analytes were detected by high-performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) using C8-reversed-phase HPLC for separation and sequential window acquisition of all theoretical fragment ion spectra (SWATH) acquisition for obtaining high-resolution quantitative MS/MS chromatograms. This method was validated and applied to pleural effusion cells from patients with lung adenocarcinoma (LUAD, n = 48) and those from benign controls (n = 23). The range of the above metabolites was 2–200 ng/mL for proline, aspartate, ornithine, creatine, glutamine, glutamate, arginine, citrulline, and spermine and 10–1000 ng/mL for putrescine. The intra-assay and inter-assay coefficient of variation was below 13.70 % for all analytes. The joint detection of these metabolites in pleural effusion cells achieved a clinical sensitivity of 75.0 % and specificity of 95.7 % differentiating LUAD patients from benign controls. This assay enabled the detection of 10 cancer-associated metabolites in pleural effusion cells, and the increased concentration of these metabolites was correlated with the presence of LUAD.

Combined detection of SHOX2 and PTGER4 methylation with serum marker CYFRA21-1 for improved diagnosis of malignant pleural mesothelioma

AimsTo investigate the performance of a combined biomarker approach using the methylation status of the short stature homeobox 2 (SHOX2) and prostaglandin E2 receptor EP4 (PTGER4) genes, along with the...

The impact of different fixatives on immunostaining of lung adenocarcinomas in pleural effusion cell blocks.

Cell blocks (CBs) are widely used for biomarker analyses such as immunostaining. Although immunohistochemistry on formalin-fixed paraffin-embedded tissues is standardized, there are multiple preparation methods and fixatives for cytology. Our objective was to investigate the effect of different common fixatives on the immunoreactivity of pleural effusion CBs with metastatic lung adenocarcinomas. This prospective study included 24 malignant pleural effusions from different patients with lung adenocarcinoma. From each case, four identical CBs were fixed in 10% neutral buffered formalin, PreservCyt, CytoLyt, and CytoRich Red (only 17 of the cases), respectively. Samples containing <100 malignant cells were excluded. All CBs were stained with thyroid transcription factor 1 (TTF-1; clones 8G7G3/1 and SPT24), napsin A, claudin 4, CEA, CK7, and epithelial cell adhesion molecule (EpCAM; clones BS14, Ber-Ep4, and MOC-31). The fraction and intensity of stained cells were evaluated. Of the investigated markers, a significant difference in staining proportion was seen for TTF-1 clone 8G7G3/1 and EpCAM clone MOC-31, especially with cases being negative in CytoLyt (33.3% and 83.3% positive, respectively) and PreservCyt (62.5% and 83.3%) whereas being positive in CytoRich Red (76.5% and 94.1%) and formalin (both 95.8%). A significantly weaker intensity of staining was seen for all alcohol-based fixatives compared to formalin for TTF-1 clone 8G7G3/1, napsin A, and EpCAM clone MOC-31, whereas EpCAM clone Ber-Ep4 was significantly weaker only in PreservCyt compared with formalin. Immunocytochemical expression and concordance with formalin-fixed CBs differ depending on the used fixative as well as the antibody and clone, warranting investigation of the reliability of each biomarker for non-formalin-fixed cytology.

Mesothelioma-Associated Fibroblasts Modulate the Response of Mesothelioma Patient-Derived Organoids to Chemotherapy via Interleukin-6.

Malignant pleural mesothelioma (MPM) remains an incurable disease. This is partly due to the lack of experimental models that fully recapitulate the complexity and heterogeneity of MPM, a major challenge for therapeutic management of the disease. In addition, the contribution of the MPM microenvironment is relevant for the adaptive response to therapy. We established mesothelioma patient-derived organoid (mPDO) cultures from MPM pleural effusions and tested their response to pemetrexed and cisplatin. We aimed to evaluate the contribution of mesothelioma-associated fibroblasts (MAFs) to the response to pemetrexed and cisplatin (P+C). Organoid cultures were obtained from eight MPM patients using specific growth media and conditions to expand pleural effusion-derived cells. Flow cytometry was used to verify the similarity of the organoid cultures to the original samples. MAFs were isolated and co-cultured with mPDOs, and the addition of MAFs reduced the sensitivity of mPDOs to P+C. Organoid formation and expression of cancer stem cell markers such as ABCG2, NANOG, and CD44 were altered by conditioned media from treated MAFs. We identified IL-6 as the major contributor to the attenuated response to chemotherapy. IL-6 secretion by MAFs is correlated with increased resistance of mPDOs to pemetrexed and cisplatin.

The regulation and potential role of interleukin-32 in tuberculous pleural effusion.

The possible protective effect of interleukin-32 (IL-32) in Mycobacterium tuberculosis (Mtb) infection has been indicated. However, few studies have been focused on IL-32 in tuberculosis patients. Additionally, the regulation of IL-32 production has rarely been reported. In the present study, the production, regulation, and role of IL-32 in tuberculous pleurisy (TBP) were investigated. We found that the content of IL-32 in tuberculous pleural effusion (TPE) was higher than the level in the malignant pleural effusion and transudative pleural effusion. The level of IL-32 mRNA in pleural fluid mononuclear cells (PFMCs) was higher than that in peripheral blood mononuclear cells (PBMCs) of patients with TBP, and this difference was mainly reflected in the splice variants of IL-32α, IL-32β, and IL-32γ. Compared with the PBMCs, PFMCs featured higher IL-32β/IL-32γ and IL-32α/IL-32γ ratios. In addition, lipopolysaccharide (LPS), Bacillus Calmette-Guérin (BCG), and H37Ra stimulation could induce IL-32 production in the PFMCs. IL-32 production was positively correlated with the TNF-α, IFN-γ, and IL-1Ra levels in TPE, whereas IFN-γ, but not TNF-α or IL-1Ra, could induce the production of IL-32 in PFMCs. Furthermore, IL-32γ could induce the TNF-α production in PFMCs. Monocytes and macrophages were the main sources of IL-32 in PFMCs. Nevertheless, direct cell-cell contact between lymphocytes and monocytes/macrophages plays an important role in enhancing IL-32 production by monocyte/macrophage cells. Finally, compared with the non-tuberculous pleural effusion, the purified CD4+ and CD8+ T cells in TPE expressed higher levels of intracellular IL-32. Our results suggested that, as a potential biomarker, IL-32 may play an essential role in the protection against Mtb infection in patients with TBP. However, further studies need to be carried out to clarify the functions and mechanisms of the IFN-γ/IL-32/TNF-α axis in patients with TBP.

Buckling forces and the wavy folds between pleural epithelial cells

Cell shapes in tissues are affected by the biophysical interaction between cells. Tissue forces can influence specific cell features such as cell geometry and cell surface area. Here, we examined the 2-dimensional shape, size, and perimeter of pleural epithelial cells at various lung volumes. We demonstrated a 1.53-fold increase in 2-dimensional cell surface area and a 1.43-fold increase in cell perimeter at total lung capacity compared to residual lung volume. Consistent with previous results, close inspection of the pleura demonstrated wavy folds between pleural epithelial cells at all lung volumes. To investigate a potential explanation for the wavy folds, we developed a physical simulacrum suggested by D'Arcy Thompson in On Growth and Form. The simulacrum suggested that the wavy folds were the result of redundant cell membranes unable to contract. To test this hypothesis, we developed a numerical simulation to evaluate the impact of an increase in 2-dimensional cell surface area and cell perimeter on the shape of the cell-cell interface. Our simulation demonstrated that an increase in cell perimeter, rather than an increase in 2-dimensional cell surface area, had the most direct impact on the presence of wavy folds. We conclude that wavy folds between pleural epithelial cells reflects buckling forces arising from the excess cell perimeter necessary to accommodate visceral organ expansion.

Diagnostic analysis of pleural fluid cell blocks using relevant immunohistochemical markers in clinically suspicious cases of malignancy.

Thoracocentesis is a less invasive and expensive procedure than pleural biopsy, enabling sample collection for cytological evaluation. Cell blocks prepared from these samples provide histopathological diagnoses; further, immunohistochemistry (IHC) can be performed for subtyping malignancies, guiding the clinician in choosing the appropriate treatment modality for the patient. We aimed to determine the various histological subtypes of malignancies using cell block IHC and compare the cell block and conventional cytology methods for diagnosing malignant pleural effusion. All pleural fluid samples from the clinically suspicious cases of malignancy collected at the Department of Pathology, Government Medical College Kottayam, India, during 18 months, except duplicate and inadequate samples, were included in this prospective observational study. IHC was performed on cell blocks that were positive for malignancy. This study analyzed 630 samples, of which 121 cell blocks demonstrated the presence of malignancy. Overall, 80%, 13%, and 7% of cases were negative, suspicious, and positive for malignancy based on conventional cytology, and 81%, 0%, and 19% were negative, suspicious, and positive for malignancy based on cell blocks, respectively. Among all malignancies, adenocarcinomas from the lung (56%) were the most common, followed by adenocarcinomas from the breast (6%), squamous cell carcinomas (5%), and adenocarcinomas from the ovary (3%). Cell blocks helped detect more malignancies (19%) than conventional cytology (7%), despite a moderate agreement between both methods. Adenocarcinomas were the most common cause of malignant pleural effusions, and the most frequent site of origin was the lungs. Cell blocks helped diagnose more malignancies than conventional cytology.

Single-cell transcriptomic analysis of human pleura reveals stromal heterogeneity and informs in vitro models of mesothelioma.

The pleural lining of the thorax regulates local immunity, inflammation and repair. A variety of conditions, both benign and malignant, including pleural mesothelioma, can affect this tissue. A lack of knowledge concerning the mesothelial and stromal cells comprising the pleura has hampered the development of targeted therapies. Here, we present the first comprehensive single-cell transcriptomic atlas of the human parietal pleura and demonstrate its utility in elucidating pleural biology. We confirm the presence of known universal fibroblasts and describe novel, potentially pleural-specific, fibroblast subtypes. We also present transcriptomic characterisation of multiple in vitro models of benign and malignant mesothelial cells, and characterise these through comparison with in vivo transcriptomic data. While bulk pleural transcriptomes have been reported previously, this is the first study to provide resolution at the single-cell level. We expect our pleural cell atlas will prove invaluable to those studying pleural biology and disease. It has already enabled us to shed light on the transdifferentiation of mesothelial cells, allowing us to develop a simple method for prolonging mesothelial cell differentiation in vitro.

Analysis of In Situ Fluorescent Probes for Rapid Screening of Epidermal Growth Factor Receptor and KRAS Mutations in Exfoliated Cells from Pleural Fluid in Patients with Lung Cancer

To improve the detection rate of non-small-cell lung cancer (NSCLC) exfoliated cells in pleural effusion, we designed nano-MSN-DNA fluorescent probes that could efficiently bind to mutated oncogenes in tumor cells. Mutated NSCLC cells could be detected directly by fluorescence intensity through confocal microscopy without using conventional polymerase chain reaction (PCR). In addition, the DNA probe was highly permeable in NSCLC cells and was stable in methanol at low temperatures. Using the nano-MSN-DNA fluorescent probes, we detected a significantly higher incidence of epidermal growth factor receptor (EGFR) and KRAS mutations in NSCLC pleural effusions and cells compared to those in normal patients, especially in lung adenocarcinoma cells. EGFR and KRAS mutations were more likely to occur in poorly differentiated and clinically advanced NSCLC, and the mutations enhanced tumor aggressiveness, leading to poor prognosis. The nano-MSN-DNA fluorescent probe was significantly more sensitive than Wright staining for screening pleural fluid exfoliated lung squamous carcinoma and adenocarcinoma cells. Thus, the nano-MSN-DNA fluorescent probe shows great potential for screening exfoliated cells from pleural fluid of patients with lung cancer and guiding targeted therapies.

Comparison of immunohistochemical mesothelial biomarkers in paired biopsies and effusion cytology cell blocks from pleural mesothelioma.

Traditionally, the diagnosis of pleural mesothelioma is based on histological material. Minimally invasive effusion cytology specimens are an alternative that, like biopsies, require ancillary analyses. Validation of immunohistochemical (IHC) analyses on cytology, including the surrogate markers for molecular alterations BAP1 and MTAP, is of interest. IHC for eight different markers was performed on 59 paired formalin-fixed, paraffin-embedded pleural biopsies and pleural effusion cell blocks with mesothelioma. Immunoreactivity in ≥10% of tumour cells was considered positive/preserved. The concordance between histological and cytological materials was assessed. The overall percentage of agreement between the histological epithelioid component in 58 biopsies and paired cell blocks was 93% for calretinin, 98% for CK5, 97% for podoplanin, 90% for WT1, 86% for EMA, 100% for desmin, 91% for BAP1, and 72% for MTAP. For 11 cases with biphasic or sarcomatoid histology, the concordance between cytology and the histological sarcomatoid component was low for calretinin, CK5, and WT1 (all ≤45%). For the whole cohort, loss of both BAP1 and MTAP was seen in 40% while both markers were preserved in 11% of the biopsies for epithelioid histology. The corresponding numbers were 54% and 8%, respectively, for the paired cell blocks. Generally, a high concordance for IHC staining was seen between paired biopsies and pleural effusion cell blocks from mesotheliomas, but the somewhat lower agreement for WT1, EMA, and especially MTAP calls for further investigation and local quality assurance. The lower concordance for the sarcomatoid subtype for some markers may indicate biological differences.

Failure pattern and radiotherapy exploration in malignant pleural effusion non-small cell lung cancer treated with targeted therapy.

Actionable mutations are common in non-small cell lung cancer(NSCLC)with malignant pleural effusion(MPE)(MPE-NSCLC). The pattern of failure in MPE-NSCLC treated with targeted therapy after MPE control remains unclear. We aimed to investigate the failure pattern of such patients in a cohort study and explore the possibility of radiotherapy. Computed tomography scans of 86 patients were reviewed in this study. We classified first pattern of failure after MPE control as initial disease sites only (IF), new distant sites only (NF), or IF and NF detected simultaneously (INF). Patients evaluated suitable for radiotherapy after disease progression were divided into two groups: D group without radiotherapy and RD group with radiotherapy. The Kaplan-Meier method and log-rank test were used for survival analyses. Disease progression after MPE control was observed in 42 patients with complete serial imaging. Median time to any progression was 9.5 months. Rate of the IF, NF and INF were 50%, 17% and 33% for all patients,60%,0% and 40% for patients with MPE recurrence (n=10,23.8%) and 47%, 22% and 31% for patients (n=32,76.2%) without MPE recurrence, respectively. Out of 10 patients(23.8%) with MPE recurrence, 7 patients simultaneous underwent primary tumor progression and 5 MPE were cytologically confirmed in 7 patients with examination. The overall survival (OS )rates at 1, 2, 3 years for the RD group and D group were 88.2%, 50.5%, 21.7% and 80.0%, 20.3%, 0%, respectively; the corresponding MST were 26.1 months and 17.5 months, respectively (χ2 = 4.959, p =0.026). Our data indicates that 50% of patients with actionable mutations MPE- NSCLC after MPE control are likely to fail at their initial sites of disease and the use of radiotherapy may bring OS benefits during the course of their disease. Multicenter RCT is necessary to confirm the result in the future.

Rare Case of Primary Pleural Spindle Cell Sarcoma in a Young Patient

Rare Case of Primary Pleural Spindle Cell Sarcoma in a Young Patient

Role of molecular testing for malignant pleural effusion in targeted therapy for advanced non‐small cell lung cancer

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). 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. 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). Malignant pleural effusion cell blocks are recommended for mutation testing for targeted therapies in patients with NSCLC.

Abstract 4883: Pan cancer transcriptomic response to tumor treating fields (TTFields)

Abstract Introduction: Tumor Treating Fields (TTFields) are electric fields that disrupt cellular processes critical for cancer cell viability and tumor progression, ultimately leading to cell death. TTFields therapy is versatile with multiple mechanisms of action, including: anti-mitotic effects, downregulation of DNA double strand break repair, generation of replication stress, upregulation of autophagy, and induction of immunogenic cell death. In this research we were interested in identifying common responses to TTFields across cancer types. We addressed this by integrating whole transcriptome analyses of cancers from various origins. Methods: Control and TTFields-treated samples examined included gastric, ovarian, pancreatic, non-small cell lung carcinoma, and glioblastoma cell lines, pleural mesothelioma patient-derived cell lines, and a hepatocellular carcinoma animal model. A list of differentially expressed genes (DEGs) was generated from transcriptomics analysis. Gene Set Enrichment Analysis (GSEA) was conducted with the MSigDB, and additional pathway databases. Significantly overlapping pathways were identified using ActivePathways package, and an enrichment map was created according to the number of datasets supporting each pathway. Results: The response to TTFields showed a positive correlation of expression across different cell lines and indications, including in an animal model. Downregulation was seen in cell cycle related pathways - E2F targets, Myc targets, G2M checkpoint, and mitotic spindle - indicative of cell cycle arrest, which was mediated by increased expression of tumor suppressors. Several DNA repair pathways were downregulated, including nucleotide excision repair, base excision repair, and the Fanconi Anemia-BRCA pathway. Downregulation was also evident in nuclear envelope, DNA and RNA synthesis, metabolism of amino acids, translation, organelle organization, and splicing. Upregulation was observed in genes associated with the cytoskeleton, adhesion, extracellular proteins and transporters. In the majority of comparisons, an upregulation in the immune response was also observed. Conclusions: Transcriptomic analysis of datasets from different tumor types revealed common expression patterns and pathways involved in the responses to TTFields. Some of the identified pathways corroborate previously described mechanisms, whereas some reveal new potential mechanisms that require further investigation. Citation Format: Kerem Wainer-Katsir, Gitit Lavy-Shahaf, Hila Fishman, Hila Ena, Roni Frechtel-Gerzi, Antonia Martinez-Conde, Eyal Dor-On, Shiri Davidi, Sara Jacobovitch, Itai Tzchori, Yaara Porat, Lianghao Ding, Michael Story, Renfei Du, Ulf Kahlert, Laura Mannarino, Federica Mirimao, Monica Lupi, Maurizio D’Incalci, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti. Pan cancer transcriptomic response to tumor treating fields (TTFields). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4883.

A Study on Assessment of Diagnostic Yield of Cell Block, FNAC and Invasive Biopsy in Lung Mass with Effusion

BACKGROUND: Accurate diagnosis of histological subtype is of paramount importance in case of lung malignancy as it leads to variation in treatment and assessment of prognosis. Hence the current study was done to establish the yields of different diagnostic techniques to perform tests in institutes with lesser equipment. METHODS: prospective observational study performed in 45 patients who presented with pleural effusions with an underlying lung parenchymal mass in department of respiratory medicine, GEMS&amp;H, Srikakulam, Andhra Pradesh from August 2021 to August 2022 were included in the current study. RESULTS: During the period of 12 months that is from August 2021 to August 2022 we came across 60 lung mass cases among them 45 presented along with pleural effusion. In them Cell block results were suggestive of malignant effusion in 13 out of 45 cases giving a yield of about 29% whereas the yield of FNAC (fine needle aspiration cytology) was 53% (24/45) giving a combined yield of 82% from cytological study. Biopsy was conclusive in all the cases with a yield of 100%. CONCLUSION: The combined yield of cell block and FNAC is high even though in the current study the efficacy of cell block and FNAC is lower to that of biopsy. Core needle biopsy is preferable to traditional cytology procedures, according to the current study, but in locations without the necessary equipment or when a patient is unfit for the treatment, the first line of inquiry should always be pleural fluid cell block and FNAC

Dihydroartemisinin Induced Apoptosis and Synergized With Chemotherapy in Pleural Effusion Lymphoma Cells.

Primary effusion lymphoma (PEL) is a rare aggressive B-cell lymphoma associated with HHV-8. With a median survival of fewer than six months, the prognosis of the disease with current standard therapies is usually dismal. Dihydroartemisinin (DHA) is a derivative of artemisinin, originally designed as an antimalarial drug. Several studies have shown that this compound also demonstrates anti-cancer activity in various types of cancer, including hematologic malignancies. Anti-proliferation activity of DHA on 5 PEL cell lines was assessed by MTT assay. Cell cycle arrest was determined by propidium iodide staining and flow cytometry analysis. DHA-induced PEL apoptosis was shown by annexin V/PI staining and western blotting for cleaved caspases 3, 8, and 9. An inhibitory effect on PEL growth was evaluated in a PEL-xenograft mouse model. A synergistic effect of DHA and doxorubicin combination treatment was shown in vitro. DHA showed anti-proliferative activity on PEL and induced caspase-dependent apoptosis in a time- and dose-dependent manner. DHA-induced cell death appeared to be triggered by increased levels of reactive oxygen species (ROS). N-acetylcysteine treatment inhibited DHA-induced ROS elevation and suppressed expression of cleaved caspases leading to significantly reduced PEL apoptosis. DHA treatment also demonstrated an inhibitory effect on PEL cell growth in an in-vivo xenograft model. Moreover, we found that a combination treatment of DHA and doxorubicin, the standard chemotherapy drug for PEL, demonstrated a synergistic effect on PEL cell lines. DHA is a potentially effective candidate drug for PEL treatment.

Refined Diagnosis of Pleural Effusions by Immunocytochemistry of Cell Blocks.

The main objective of microscopic examination of pleural effusions is to ascertain the presence of malignant cells. Effusions prepared routinely using May-Grünwald-Giemsa (MGG)- and Papanicolaou (PAP)-staining can, in a number of cases, provide inconclusive cytological results regarding malignancy. This report describes the refined diagnosis of such cases based on immunocytochemical analysis of pleural effusion cell blocks. Of the 340 pleural effusions obtained during 2019 at the Department of Clinical Cytology, Gävle Hospital, Sweden, 63 (18.5%) contained atypical cells of undetermined significance or potentially malignant cells. This diagnosis could be refined using Epithelial Cell Adhesion Molecule/EPCAM (BEREP4) immunocytochemical analysis of effusion cell blocks, allowing previously inconclusive effusions to be classified as clearly benign 42/63 (66.7%) or malignant 21/63 (33.3%). Effusions initially diagnosed as clearly malignant (27/340; 7.9%) were all 27 (100%) BEREP4-immuno-stained. Most BEREP4-positive effusions (37/48; 77.1%) were also carcinoembryonic antigen (CEA) positive. The number of BEREP4-positive cells, however, tended to exceed that of CEA-positive cells. The BEREP4 positive effusions were further examined using different monoclonal antibodies, such as Thyroid transcription factor 1 (TTF-1) for primary pulmonary adenocarcinoma, to determine the original site of the primary tumour. Immunohistochemical staining of pleural effusion cell blocks significantly refines the diagnosis of serous pleural effusions, especially in cases where the preliminary diagnosis was atypical cells of undetermined significance or potentially malignant cells. Furthermore, in the cases of malignancy, the origin of the primary tumour could most often be determined.

Deep Learning-Based Classification and Targeted Gene Alteration Prediction from Pleural Effusion Cell Block Whole-Slide Images.

Cytopathological examination is one of the main examinations for pleural effusion, and especially for many patients with advanced cancer, pleural effusion is the only accessible specimen for establishing a pathological diagnosis. The lack of cytopathologists and the high cost of gene detection present opportunities for the application of deep learning. In this retrospective analysis, data representing 1321 consecutive cases of pleural effusion were collected. We trained and evaluated our deep learning model based on several tasks, including the diagnosis of benign and malignant pleural effusion, the identification of the primary location of common metastatic cancer from pleural effusion, and the prediction of genetic alterations associated with targeted therapy. We achieved good results in identifying benign and malignant pleural effusions (0.932 AUC (area under the ROC curve)) and the primary location of common metastatic cancer (0.910 AUC). In addition, we analyzed ten genes related to targeted therapy in specimens and used them to train the model regarding four alteration statuses, which also yielded reasonable results (0.869 AUC for ALK fusion, 0.804 AUC for KRAS mutation, 0.644 AUC for EGFR mutation and 0.774 AUC for NONE alteration). Our research shows the feasibility and benefits of deep learning to assist in cytopathological diagnosis in clinical settings.

Topography of pleural epithelial structure enabled by en face isolation and machine learning.

Pleural epithelial adaptations to mechanical stress are relevant to both normal lung function and parenchymal lung diseases. Assessing regional differences in mechanical stress, however, has been complicated by the nonlinear stress-strain properties of the lung and the large displacements with ventilation. Moreover, there is no reliable method of isolating pleural epithelium for structural studies. To define the topographic variation in pleural structure, we developed a method of en face harvest of murine pleural epithelium. Silver-stain was used to highlight cell borders and facilitate imaging with light microscopy. Machine learning and watershed segmentation were used to define the cell area and cell perimeter of the isolated pleural epithelial cells. In the deflated lung at residual volume, the pleural epithelial cells were significantly larger in the apex (624 ± 247 μm2 ) than in basilar regions of the lung (471 ± 119 μm2 ) (p < 0.001). The distortion of apical epithelial cells was consistent with a vertical gradient of pleural pressures. To assess epithelial changes with inflation, the pleura was studied at total lung capacity. The average epithelial cell area increased 57% and the average perimeter increased 27% between residual volume and total lung capacity. The increase in lung volume was less than half the percent change predicted by uniform or isotropic expansion of the lung. We conclude that the structured analysis of pleural epithelial cells complements studies of pulmonary microstructure and provides useful insights into the regional distribution of mechanical stresses in the lung.