ABSTRACT
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-É£, 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.
Subject(s)
Lung Neoplasms , Single-Cell Analysis , Tumor Microenvironment , Humans , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Tumor Microenvironment/immunology , Tumor Microenvironment/genetics , Single-Cell Analysis/methods , Male , Female , CD8-Positive T-Lymphocytes/immunology , Aged , Middle Aged , CD4-Positive T-Lymphocytes/immunology , Sequence Analysis, RNA , Biopsy , Pleural Effusion/pathology , Pleural Effusion/genetics , Pleural Effusion, Malignant/genetics , Pleural Effusion, Malignant/pathology , Prospective StudiesABSTRACT
Fetal pleural effusion has been reported to be associated with chromosomal abnormalities, genetic syndromes, obstructive uropathy, lymphatic vessel abnormalities such as Noonan syndrome, RASopathy and congenital lymphatic anomalies, thoracic cavity defects, Rh or ABO incompatibility, non-immune hydrops fetalis, infections, congenital cardiac anomalies, metabolic diseases and hematologic diseases such as α-thalassemia. This review provides an overview of chromosomal abnormalities associated with fetal pleural effusion which is useful for genetic counseling and fetal therapy at prenatal diagnosis of fetal pleural effusion.
Subject(s)
Heart Defects, Congenital , Pleural Effusion , Pregnancy , Female , Humans , Pleural Effusion/genetics , Chromosome Aberrations , Hydrops Fetalis/diagnosis , Hydrops Fetalis/genetics , Prenatal DiagnosisABSTRACT
Fetal pleural effusion has been reported to be associated with chromosomal abnormalities, genetic syndromes, obstructive uropathy, lymphatic vessel abnormalities such as Noonan syndrome, RASopathy and congenital lymphatic anomalies, thoracic cavity defects, Rh or ABO incompatibility, non-immune hydrops fetalis, infections, congenital cardiac anomalies, metabolic diseases and hematologic diseases such as α-thalassemia. This review provides an overview of syndromic and single gene disorders associated with fetal pleural effusion that is useful for genetic counseling and fetal therapy at prenatal diagnosis of fetal pleural effusion.
Subject(s)
Lymphatic Abnormalities , Lymphatic Vessels , Noonan Syndrome , Pleural Effusion , Pregnancy , Female , Humans , Noonan Syndrome/complications , Noonan Syndrome/diagnosis , Noonan Syndrome/genetics , Pleural Effusion/genetics , Prenatal Diagnosis , Hydrops Fetalis/diagnosis , Hydrops Fetalis/genetics , Lymphatic Abnormalities/complications , Lymphatic Abnormalities/geneticsABSTRACT
Fetal pleural effusion has been reported to be associated with chromosomal abnormalities, genetic syndromes, obstructive uropathy, lymphatic vessel abnormalities such as Noonan syndrome, RASopathy and congenital lymphatic anomalies, thoracic cavity defects, Rh or ABO incompatibility, non-immune hydrops fetalis, infections, congenital cardiac anomalies, metabolic diseases and hematologic diseases such as α-thalassemia. This review provides a comprehensive view of specific and non-specific chromosome aberrations associated with fetal pleural effusion which is useful for genetic counseling and fetal therapy at prenatal diagnosis of fetal pleural effusion.
Subject(s)
Heart Defects, Congenital , Pleural Effusion , Pregnancy , Female , Humans , Chromosome Aberrations , Pleural Effusion/genetics , Hydrops Fetalis/diagnosis , Hydrops Fetalis/genetics , Prenatal Diagnosis , Ultrasonography, PrenatalABSTRACT
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.
Subject(s)
Pleural Effusion, Malignant , Pleural Effusion , Humans , Pleural Effusion, Malignant/genetics , Pleural Effusion/genetics , Exudates and Transudates/metabolism , Pleura/metabolism , Gene Expression ProfilingABSTRACT
Fetal hydrops as detected by prenatal ultrasound usually carries a poor prognosis depending on the underlying aetiology. We describe the prenatal and postnatal clinical course of two unrelated female probands in whom de novo heterozygous missense variants in the planar cell polarity gene CELSR1 were detected using exome sequencing. Using several in vitro assays, we show that the CELSR1 p.(Cys1318Tyr) variant disrupted the subcellular localisation, affected cell-cell junction, impaired planar cell polarity signalling and lowered proliferation rate. These observations suggest that deleterious rare CELSR1 variants could be a possible cause of fetal hydrops.
Subject(s)
Heterozygote , Hydrops Fetalis , Mutation, Missense , Humans , Female , Mutation, Missense/genetics , Hydrops Fetalis/genetics , Hydrops Fetalis/pathology , Pregnancy , Pleural Effusion/genetics , Pleural Effusion/pathology , Cadherins/genetics , Exome Sequencing , Cell Polarity/geneticsABSTRACT
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.
Subject(s)
Cell-Free Nucleic Acids , Pleural Effusion, Malignant , Pleural Effusion , Humans , Pleural Effusion, Malignant/diagnosis , Pleural Effusion, Malignant/genetics , Homeodomain Proteins/genetics , Biomarkers, Tumor/genetics , DNA Methylation , Pleural Effusion/diagnosis , Pleural Effusion/genetics , DNAABSTRACT
BACKGROUND: Overlapping morphological features of mesothelial cells have been rendered it difficult to distinguish between reactive and malignant conditions. The development of methods based on detecting genomic abnormalities using immunohistochemistry and fluorescence in situ hybridization have contributed markedly to solving this problem. It is important to identify bland mesothelioma cells on cytological screening, perform efficient genomic-based testing, and diagnose mesothelioma, because the first clinical manifestation of pleural mesothelioma is pleural effusion, which is the first sample available for pathological diagnosis. However, certain diagnostic aspects remain challenging even for experts. CASE PRESENTATION: This report describes a case of a 72-year-old man with a history of asbestos exposure who presented with pleural effusion as the first symptom and was eventually diagnosed as mesothelioma. Mesothelioma was suspected owing to prominent cell-in-cell engulfment in mesothelial cells on the first cytological sample, and the diagnosis of mesothelioma in situ was confirmed by histology. Unexpectedly, sarcomatoid morphology of mesothelioma was found in the second pathology samples 9 months after the first pathological examination. Both the mesothelioma in situ and invasive lesion showed immunohistochemical loss of methylthioadenosine phosphorylase (MTAP) and homozygous deletion of cyclin dependent kinase inhibitor 2A (CDKN2A) on fluorescence in situ hybridization. The patient received medication therapy but died of disease progression 12 months after the diagnosis of the sarcomatoid morphology of mesothelioma. CONCLUSION: Our case suggests that cell-in-cell engulfment can be conspicuous in early-stage mesothelioma with inconspicuous nuclear atypia and few multinucleated cells. In addition, the presence of MTAP loss and CDKN2A homozygous deletion are suspected to be involved in early formation to invasive lesions and/or sarcomatoid morphology. We believe that it is important to consider genetic abnormalities when deciding on individual patient management. Furthermore, cases of mesothelioma, even those of an in situ lesion, with MTAP loss and/or CDKN2A deletion should be carefully followed up or subjected to early treatment.
Subject(s)
Lung Neoplasms , Mesothelioma, Malignant , Mesothelioma , Pleural Effusion , Pleural Neoplasms , Sarcoma , Male , Humans , Aged , In Situ Hybridization, Fluorescence , Homozygote , Lung Neoplasms/diagnosis , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Sequence Deletion , Mesothelioma/diagnosis , Mesothelioma/genetics , Mesothelioma/pathology , Pleural Neoplasms/diagnosis , Pleural Neoplasms/genetics , Pleural Neoplasms/pathology , Pleural Effusion/genetics , Sarcoma/genetics , Biomarkers, Tumor/genetics , Biomarkers, Tumor/analysis , Ubiquitin Thiolesterase/analysis , Ubiquitin Thiolesterase/geneticsABSTRACT
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.
Subject(s)
Pleural Effusion, Malignant , Pleural Effusion , Humans , Pleural Effusion, Malignant/diagnosis , Pleural Effusion, Malignant/genetics , Carcinoembryonic Antigen , Methylation , Pleural Effusion/genetics , Exudates and Transudates , Homeodomain Proteins/geneticsABSTRACT
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.
Subject(s)
Pleural Effusion, Malignant , Pleural Effusion , Humans , Pleural Effusion, Malignant/diagnosis , Pleural Effusion, Malignant/genetics , Pleural Effusion, Malignant/pathology , DNA, Neoplasm/genetics , Pleural Effusion/genetics , Sensitivity and Specificity , Ploidies , Image CytometryABSTRACT
Rationale: Sonographic septations are assumed to be important clinical predictors of outcome in pleural infection, but the evidence for this is sparse. The inflammatory and fibrinolysis-associated intrapleural pathway(s) leading to septation formation have not been studied in a large cohort of pleural fluid (PF) samples with confirmed pleural infection matched with ultrasound and clinical outcome data. Objectives: To assess the presence and severity of septations against baseline PF PAI-1 (Plasminogen-Activator Inhibitor-1) and other inflammatory and fibrinolysis-associated proteins as well as to correlate these with clinically important outcomes. Methods: We analyzed 214 pleural fluid samples from PILOT (Pleural Infection Longitudinal Outcome Study), a prospective observational pleural infection study, for inflammatory and fibrinolysis-associated proteins using the Luminex platform. Multivariate regression analyses were used to assess the association of pleural biological markers with septation presence and severity (on ultrasound) and clinical outcomes. Measurements and Main Results: PF PAI-1 was the only protein independently associated with septation presence (P < 0.001) and septation severity (P = 0.003). PF PAI-1 concentrations were associated with increased length of stay (P = 0.048) and increased 12-month mortality (P = 0.003). Sonographic septations alone had no relation to clinical outcomes. Conclusions: In a large and well-characterized cohort, this is the first study to associate pleural biological parameters with a validated sonographic septation outcome in pleural infection. PF PAI-1 is the first biomarker to demonstrate an independent association with mortality. Although PF PAI-1 plays an integral role in driving septation formation, septations themselves are not associated with clinically important outcomes. These novel findings now require prospective validation.
Subject(s)
Infections , Plasminogen Activator Inhibitor 1 , Pleural Diseases , Humans , Fibrinolysis , Infections/metabolism , Plasminogen Activator Inhibitor 1/analysis , Plasminogen Activator Inhibitor 1/metabolism , Pleura/diagnostic imaging , Pleura/metabolism , Pleural Diseases/diagnostic imaging , Pleural Diseases/metabolism , Pleural Effusion/genetics , Prospective Studies , Tissue Plasminogen Activator/analysis , Tissue Plasminogen Activator/metabolism , UltrasonographyABSTRACT
Introduction: Pleural tuberculosis (PlTB), the most common site of extrapulmonary TB, is characterized by a paucibacillary nature and a compartmentalized inflammatory response in the pleural cavity, both of which make diagnosis and management extremely challenging. Although transcriptional signatures for pulmonary TB have already been described, data obtained by using this approach for extrapulmonary tuberculosis and, specifically, for pleural tuberculosis are scarce and heterogeneous. In the present study, a set of candidate genes previously described in pulmonary TB was evaluated to identify and validate a transcriptional signature in clinical samples from a Brazilian cohort of PlTB patients and those with other exudative causes of pleural effusion. Methods: As a first step, target genes were selected by a random forest algorithm with recursive feature elimination (RFE) from public microarray datasets. Then, peripheral blood (PB) and pleural fluid (PF) samples from recruited patients presenting exudative pleural effusion were collected during the thoracentesis procedure. Transcriptional analysis of the selected top 10 genes was performed by quantitative RT-PCR (RT-qPCR). Results: Reanalysis of the public datasets identified a set of candidate genes (CARD17, BHLHE40, FCGR1A, BATF2, STAT1, BTN3A1, ANKRD22, C1QB, GBP2, and SEPTIN4) that demonstrated a global accuracy of 89.5% in discriminating pulmonary TB cases from other respiratory diseases. Our validation cohort consisted of PlTB (n = 35) patients and non-TB (n = 34) ones. The gene expressions of CARD17, GBP2, and C1QB in PF at diagnosis were significantly different between the two (PlTB and non-TB) groups (p < 0.0001). It was observed that the gene expressions of CARD17 and GBP2 were higher in PlTB PF than in non-TB patients. C1QB showed the opposite behavior, being higher in the non-TB PF. After anti-TB therapy, however, GBP2 gene expression was significantly reduced in PlTB patients (p < 0.001). Finally, the accuracy of the three above-cited highlighted genes in the PF was analyzed, showing AUCs of 91%, 90%, and 85%, respectively. GBP2 was above 80% (sensitivity = 0.89/specificity = 0.81), and CARD17 showed significant specificity (Se = 0.69/Sp = 0.95) in its capacity to discriminate the groups. Conclusion: CARD17, GBP2, and C1QB showed promise in discriminating PlTB from other causes of exudative pleural effusion by providing accurate diagnoses, thus accelerating the initiation of anti-TB therapy.
Subject(s)
Pleural Effusion , Tuberculosis, Pleural , Tuberculosis, Pulmonary , Humans , Tuberculosis, Pleural/diagnosis , Tuberculosis, Pleural/genetics , Exudates and Transudates , Pleural Effusion/diagnosis , Pleural Effusion/genetics , Pleural Effusion/metabolism , Brazil , Butyrophilins , Antigens, CDABSTRACT
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.
Subject(s)
Adenocarcinoma , Lung Neoplasms , Pleural Effusion, Malignant , Pleural Effusion , Adenocarcinoma/pathology , Biomarkers, Tumor/analysis , Biomarkers, Tumor/genetics , Humans , Immunohistochemistry , Lung Neoplasms/pathology , Mutation , Nuclear Proteins/genetics , Pleural Effusion/genetics , Pleural Effusion, Malignant/diagnosis , Pleural Effusion, Malignant/genetics , Sensitivity and Specificity , Transcription Factors/geneticsABSTRACT
OBJECTIVES: The advanced non-small cell lung cancer (NSCLC) patients with pleural effusion have no opportunity for surgery treatment. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are the first-line drugs for these patients with EGFR-sensitive mutation. However, the disease progression and drug update during or after treatment of EGFR-TKIs bring more challenges and puzzles to clinical diagnosis and treatment, which inevitably requires archived pleural cell samples for EGFR re-examination or comparative study. Understanding the DNA quality of archived pleural fluid samples and effectively using archival data of pleural fluid cells are of great significance for tracing the origin of cases and basic medical research. This study aims to evaluate the consistency of EGFR mutant gene expression between the 2 methods, and to explore a reliable way for preserving cytological data and making full use of cytological archival data via cell HE staining smear and cell paraffin section. METHODS: A total of 57 pleural fluid cytology cases in the Department of Pathology of China Aerospace Center Hospital from October 2014 to April 2021 were selected. Tumor cells were detected by cell HE staining smears and immunohistochemical staining for TTF-1 and Napsin A in the paired cell paraffin sections. There were more than 200 tumor cells in cell HE staining smear and the proportion of tumor cells were ≥70% in matched cell paraffin sections. Patients with 2 cell smears (one for cell data retention and the other for DNA extraction) were selected as the research subjects, and 57 pleural fluid samples were enrolled. EGFR gene mutation was detected by amplification refractory mutation system-polymerase chain reaction in 57 paired cell HE staining smears and cell paraffin sections. DNA concentration was 2 ng/µL. Cell HE smear was amplified side-by-side with DNA samples from paired cell paraffin sections. Result determination was according to the requirements of the reagent instructions. The external control cycle threshold (Ct) value of the No. 8 well of the samples to be tested was between 13 and 21, which was considered as successful and reliable samples. When the Ct value of EGFR gene mutation was <26, it was considered as positive; when the Ct value was between 26 and 29, it was critical positive; when the Ct value was equal or more than 29, it was negative. ΔCt value was the difference between mutant Ct value and externally controlled Ct value. The smaller the ΔCt value was, the better the quality of DNA of the detected sample was. RESULTS: Among the 57 pleural effusion samples, 42 patients were hospitalized with pleural effusion as the first symptom, accounting for 73.7% (42/57). EGFR mutation was detected in 37 samples [64.9% (37/57)]. The mutation rate for 19del was 37.8% (14/37) while for L858R was 48.6% (18/37). Females were 56.7% (21/37) of mutation cases. The mutation consistency rate of cell HE staining smear and matched cell paraffin sections was 100%. The ΔCt values of cell HE staining smears were less than those of matched cell paraffin sections. The mutation Ct values of 37 cytological samples were statistically analyzed according to the preservation periods of the years of 2014-2015, 2016-2017, 2018-2019, and 2020-2021. There were significant differences in cell paraffin section in the years of 2014-2015 and 2016-2017 compared with the years of 2018-2019 and 2020-2021, while no significant differences were found in cell HE staining smear. Statistical analysis of externally controlled Ct values of 57 cytological samples showed that there were significant differences between cell HE staining smears and cell paraffin section in the years of 2014-2015 and 2016-2017, compared with the years of 2018-2019 and 2020-2021. The mutational Ct values of 37 paired cell blocks and smears were all <26, and the externally controlled Ct values of 57 paired cell paraffin sections and HE staining smears were all between 13 and 21. CONCLUSIONS: The DNA quality of cell HE smears and matched cell paraffin section met the qualified requirements. Two methods possess show an excellent consistency in detecting EGFR mutation in NSCLC pleural fluid samples. The DNA quality of cell HE staining smear is better than that of cell paraffin sections, so cell HE staining smear can be used as important supplement of the gene test source. It should be noted that the limitation of cell HE staining smears is non-reproducibility, so multiple smears of pleural fluid are recommended to be prepared for multiple tests.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Pleural Effusion , Carcinoma, Non-Small-Cell Lung/drug therapy , DNA Mutational Analysis/methods , ErbB Receptors/genetics , Female , Humans , Lung Neoplasms/drug therapy , Male , Mutation , Paraffin/therapeutic use , Pleural Effusion/genetics , Protein Kinase Inhibitors/therapeutic use , Staining and LabelingABSTRACT
OBJECTIVE: We aimed to study the value of exome sequencing (ES) in severe pleural effusion with nonimmune hydrops fetalis (NIHF) that underwent thoracoamniotic shunt (TAS). METHODS: It was a retrospective study of NIHF that underwent TAS between 2012 and 2020 at Shanghai First Maternity and Infant Hospital. After a detailed assessment, NIHF cases with aneuploidies, infections, and structural anomalies were excluded, and TAS was offered to cases with severe pleural effusion. Quantitative fluorescence polymerase chain reaction (QF-PCR) was conducted to exclude Trisomy 21, 18, and 13 before fetal therapy, and chromosomal microarray analysis (CMA) was offered to all the cases. Before 2019, ES was retrospectively performed using stored fetal DNA extracted from prenatal samples; from 2019 onward, ES was discussed and offered before intrauterine therapies. RESULTS: A total of 18 NIHF cases underwent TAS with negative CMA and continuing pregnancy were included. Fetal hydrops was relieved in 16 cases (88.9%). The median gestational ages at intervention and at delivery were 31.2 (22.0-33.1) weeks and 34.3 (29.7-38.6) weeks, respectively. The neonatal survival rate was 72.2% (13/18), and no causative gene variants were identified from ES in any survivors. Pathogenic or likely pathogenic variants were detected in 3 out of 5 neonatal deaths. If rapid ES could have been available to guide fetal therapy, the neonatal survival rate after TAS would have increased from 72.2% to 86.7%. CONCLUSIONS: Single-gene disorders were one of the major causes of perinatal death in NIHF cases that underwent fetal therapy. Prenatal rapid ES may be of good promise in NIHF to explore precise etiology and guide fetal therapy.
Subject(s)
Cardiovascular Abnormalities , Pleural Effusion , Cardiovascular Abnormalities/complications , China , Exome , Female , Humans , Hydrops Fetalis/genetics , Hydrops Fetalis/surgery , Infant , Infant, Newborn , Pleural Effusion/genetics , Pleural Effusion/surgery , Pregnancy , Retrospective Studies , Ultrasonography, PrenatalABSTRACT
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.
Subject(s)
MicroRNAs , Pleural Effusion, Malignant , Pleural Effusion , Biomarkers, Tumor , Diagnosis, Differential , Humans , MicroRNAs/genetics , Pleural Effusion/diagnosis , Pleural Effusion/genetics , Pleural Effusion, Malignant/diagnosis , Pleural Effusion, Malignant/genetics , Pleural Effusion, Malignant/metabolismABSTRACT
OBJECTIVES@#The advanced non-small cell lung cancer (NSCLC) patients with pleural effusion have no opportunity for surgery treatment. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are the first-line drugs for these patients with EGFR-sensitive mutation. However, the disease progression and drug update during or after treatment of EGFR-TKIs bring more challenges and puzzles to clinical diagnosis and treatment, which inevitably requires archived pleural cell samples for EGFR re-examination or comparative study. Understanding the DNA quality of archived pleural fluid samples and effectively using archival data of pleural fluid cells are of great significance for tracing the origin of cases and basic medical research. This study aims to evaluate the consistency of EGFR mutant gene expression between the 2 methods, and to explore a reliable way for preserving cytological data and making full use of cytological archival data via cell HE staining smear and cell paraffin section.@*METHODS@#A total of 57 pleural fluid cytology cases in the Department of Pathology of China Aerospace Center Hospital from October 2014 to April 2021 were selected. Tumor cells were detected by cell HE staining smears and immunohistochemical staining for TTF-1 and Napsin A in the paired cell paraffin sections. There were more than 200 tumor cells in cell HE staining smear and the proportion of tumor cells were ≥70% in matched cell paraffin sections. Patients with 2 cell smears (one for cell data retention and the other for DNA extraction) were selected as the research subjects, and 57 pleural fluid samples were enrolled. EGFR gene mutation was detected by amplification refractory mutation system-polymerase chain reaction in 57 paired cell HE staining smears and cell paraffin sections. DNA concentration was 2 ng/μL. Cell HE smear was amplified side-by-side with DNA samples from paired cell paraffin sections. Result determination was according to the requirements of the reagent instructions. The external control cycle threshold (Ct) value of the No. 8 well of the samples to be tested was between 13 and 21, which was considered as successful and reliable samples. When the Ct value of EGFR gene mutation was <26, it was considered as positive; when the Ct value was between 26 and 29, it was critical positive; when the Ct value was equal or more than 29, it was negative. ΔCt value was the difference between mutant Ct value and externally controlled Ct value. The smaller the ΔCt value was, the better the quality of DNA of the detected sample was.@*RESULTS@#Among the 57 pleural effusion samples, 42 patients were hospitalized with pleural effusion as the first symptom, accounting for 73.7% (42/57). EGFR mutation was detected in 37 samples [64.9% (37/57)]. The mutation rate for 19del was 37.8% (14/37) while for L858R was 48.6% (18/37). Females were 56.7% (21/37) of mutation cases. The mutation consistency rate of cell HE staining smear and matched cell paraffin sections was 100%. The ΔCt values of cell HE staining smears were less than those of matched cell paraffin sections. The mutation Ct values of 37 cytological samples were statistically analyzed according to the preservation periods of the years of 2014-2015, 2016-2017, 2018-2019, and 2020-2021. There were significant differences in cell paraffin section in the years of 2014-2015 and 2016-2017 compared with the years of 2018-2019 and 2020-2021, while no significant differences were found in cell HE staining smear. Statistical analysis of externally controlled Ct values of 57 cytological samples showed that there were significant differences between cell HE staining smears and cell paraffin section in the years of 2014-2015 and 2016-2017, compared with the years of 2018-2019 and 2020-2021. The mutational Ct values of 37 paired cell blocks and smears were all <26, and the externally controlled Ct values of 57 paired cell paraffin sections and HE staining smears were all between 13 and 21.@*CONCLUSIONS@#The DNA quality of cell HE smears and matched cell paraffin section met the qualified requirements. Two methods possess show an excellent consistency in detecting EGFR mutation in NSCLC pleural fluid samples. The DNA quality of cell HE staining smear is better than that of cell paraffin sections, so cell HE staining smear can be used as important supplement of the gene test source. It should be noted that the limitation of cell HE staining smears is non-reproducibility, so multiple smears of pleural fluid are recommended to be prepared for multiple tests.
Subject(s)
Female , Humans , Male , Carcinoma, Non-Small-Cell Lung/drug therapy , DNA Mutational Analysis/methods , ErbB Receptors/genetics , Lung Neoplasms/drug therapy , Mutation , Paraffin/therapeutic use , Pleural Effusion/genetics , Protein Kinase Inhibitors/therapeutic use , Staining and LabelingABSTRACT
BACKGROUND: Serous effusions (SE) in leukemic patients can be due to infections, therapy, volume overload, lymphatic obstruction or malignancy having implications on treatment and mortality. The objective of the present study is to highlight the spectrum of cytomorphology, immunophenotype, and cytogenetics in leukemic serous effusions (LSE). MATERIALS: Present study is retrospective and descriptive. We reviewed all the SE, which were reported as suspicious or positive of leukemic infiltration from 2016 to 2019 for cytomorphological features. CSF and effusions involved by lymphomas were excluded. Cyto-diagnosis was compared with primary proven diagnosis (by ancillary techniques) and disconcordant cases were analyzed. RESULTS: Out of total 9723 effusions, only 0.4% (n = 40) showed leukemic involvement and included nine cases of AML, three of B-ALL, 13 T-ALL, 2 MPAL, 6 CML, 5CLL, one each of chronic myelomonocytic leukemia and AML with myelodysplasia. The most common site of involvement was the pleural cavity (n = 30), followed by the peritoneal cavity (n = 7) and the pericardial cavity (n = 3). T -ALL (41.9%) was the most common leukemia involving pleural fluid followed by AML (23.3%). CML (42.8%) was the most common leukemia involving the ascitic fluid followed by B-ALL (28.6%). Accurate diagnosis was given on cytomorphology in 72.5% (29/40) cases and 15.0% (6/40) were reported as non-Hodgkin lymphoma. CONCLUSION: Cytology is an effective tool available to make a diagnosis of LSE. Nuclear indentations in large atypical cells and cells with eosinophilic granular cytoplasm with sparse or abundant eosinophils in the background are an important clue in favor of leukemia over lymphoma.
Subject(s)
Cytogenetic Analysis , Exudates and Transudates , Immunophenotyping , Leukemia , Lymphoma , Adolescent , Adult , Aged , Aged, 80 and over , Ascitic Fluid/immunology , Ascitic Fluid/pathology , Child , Child, Preschool , Cytodiagnosis/methods , Cytological Techniques/methods , Diagnosis, Differential , Exudates and Transudates/cytology , Exudates and Transudates/immunology , Female , Flow Cytometry/methods , Humans , Infant , Leukemia/diagnosis , Leukemia/pathology , Lymphoma/diagnosis , Lymphoma/pathology , Lymphoma, Non-Hodgkin/diagnosis , Lymphoma, Non-Hodgkin/pathology , Male , Middle Aged , Pericardial Effusion/genetics , Pericardial Effusion/immunology , Pericardial Effusion/pathology , Pleural Effusion/genetics , Pleural Effusion/immunology , Pleural Effusion/pathology , Retrospective StudiesABSTRACT
Malignant mesothelioma is a rare malignancy with a poor prognosis whose development is related to asbestos fiber exposure. An increasing role of genetic predisposition has been recognized recently. Pleural biopsy is the gold standard for diagnosis, in which the identification of pleural invasion by atypical mesothelial cell is a major criterion. Pleural effusion is usually the first sign of disease; therefore, a cytological specimen is often the initial or the only specimen available for diagnosis. Given that reactive mesothelial cells may show marked atypia, the diagnosis of mesothelioma on cytomorphology alone is challenging. Accordingly, cell block preparation is encouraged, as it permits immunohistochemical staining. Traditional markers of mesothelioma such as glucose transporter 1 (GLUT1) and insulin-like growth factor 2 mRNA-binding protein 3 (IMP3) are informative, but difficult to interpret when reactive proliferations aberrantly stain positive. BRCA1-associated protein 1 (BAP1) nuclear staining loss is highly specific for mesothelioma, but sensitivity is low in sarcomatoid tumors. Cyclin-dependent kinase inhibitor 2A (CDKN2A)/p16 homozygous deletion, assessed by fluorescence in situ hybridization, is more specific for mesothelioma with better sensitivity, even in the sarcomatoid variant. The surrogate marker methylthioadenosine phosphorylase (MTAP) has been found to demonstrate excellent diagnostic correlation with p16. The purpose of this review is to provide an essential appraisal of the literature regarding the diagnostic value of many of these emerging biomarkers for malignant mesothelioma in effusion cytology.