Transcriptomic Profiling of Pleural Effusions: Differences in Malignant and Infectious Fluids.

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.

Performance of SHOX2 and RASSF1A methylation assay in supernatants and matched cell pellets for the diagnosis of malignant pleural effusion.

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.

Sarcomatoid mesothelioma diagnosed in a patient with mesothelioma in situ: a case report on morphologic differences after 9-month interval with details analysis of cytology in early-stage mesothelioma.

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.

Diagnostic value of SHOX2, RASSF1A gene methylation combined with CEA level detection in malignant pleural effusion.

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.

DNA image cytometry ploidy analysis technique improves the detection rate of pleural effusion cytology.

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.

Immunofluorescent and molecular characterization of effusion tumor cells reveal cancer site-of-origin and disease-driving mutations.

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.

Cell HE staining smears and paired cell paraffin sections in detection of epithelial growth factor receptor gene of pleural fluid specimens. / 细胞HEæŸ“è‰²æ¶‚ç‰‡ä¸Žç»†èƒžèœ¡å—æ£€æµ‹æ™šæœŸéžå°ç»†èƒžè‚ºç™Œèƒ¸è ”ç§¯æ¶²æ ‡æœ¬ä¸­è¡¨çš®ç”Ÿé•¿å› å­å—ä½“åŸºå› çªå˜.

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.

Assessment of a panel of miRNAs in serum and pleural fluid for the differential diagnosis of malignant and benign pleural effusion.

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.

Cytomorphology, Immunophenotype, and cytogenetic profile of leukemic serous effusions.

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.

Clinical value of the detection of EGFR gene mutations in pleural effusion cell blocks among patients with non-small-cell lung cancer.

OBJECTIVE: To evaluate the clinical value of epidermal growth factor receptor (EGFR) detection in pleural effusion cell blocks among patients with non-small-cell lung cancer (NSCLC). METHODS: From July 2016 to September 2018, EGFR gene mutations in 40 lung tumor tissue samples and pleural fluid samples from NSCLC patients in Jinhua Municipal Central Hospital were assessed by the amplification refractory mutation system method. The EGFR results of the two types of samples were compared using the paired Chi-square test, and the mutation positive rates in EGFR exons 18, 19, 20 and 21 were compared between the two types of specimens using the four-grid Chi-square test. RESULTS: Among the 40 tissue samples and pleural effusion samples, 21 and 18 cases of EGFR mutations were detected, respectively, and the mutation positive rates were 52.5% and 45%, respectively. The κ value of the consistency test of the two specimens was 0.851. There were no significant differences in the mutation positive rates in EGFR exons 18, 19, 20, and 21 between the two types of specimens. CONCLUSION: The EGFR results of pleural fluid and tissue samples were in good agreement. Therefore, we can use pleural fluid samples to detect EGFR mutations to guide tyrosine kinase inhibitor treatment for NSCLC patients in whom tumor tissue samples cannot be obtained.

Fluorescence in situ hybridization detection of chromosome 22 monosomy in pleural effusion cytology for the diagnosis of mesothelioma.

BACKGROUND: Malignant pleural mesothelioma (MPM) is characterized by mutations in several genes, including cyclin-dependent kinase-inhibitor 2A/p16 in the 9p21 locus, BRCA1-associated protein 1 (BAP1), and neurofibromatosis type 2 (NF2) in the 22q12 locus. Recent studies indicate that fluorescence in situ hybridization (FISH) detects hemizygous loss of NF2 in tissue specimens of MPM. The authors investigated whether NF2 FISH, either alone or in combination with other diagnostic assays (9p21 FISH, methylthioadenosine phosphorylase [MTAP] immunohistochemistry [IHC], and BAP1 IHC), effectively distinguishes MPM cells from reactive mesothelial cells (RMCs) in cell blocks prepared from pleural effusions. METHODS: FISH assays were used to examine the deletion status of NF2 and 9p21, and IHC was used to determine the expression of MTAP and BAP1 in cell blocks from 54 cases with MPM and 18 cases with RMCs. RESULTS: Hemizygous NF2 loss (chromosome 22 monosomy or hemizygous deletion) showed 51.9% sensitivity (48.1% for chromosome 22 monosomy and 3.7% for hemizygous deletion) and 100% specificity in differentiating MPM cells from RMCs. Combinations of NF2 FISH, 9p21 FISH, and BAP1 IHC assays yielded greater sensitivity (98.1%) than any assay alone (9p21 FISH, 61.1%; MTAP IHC, 52.8%; or BAP1 IHC, 60.4%). The level of hemizygous NF2 loss in cell blocks positively correlated with that in corresponding tissues. Furthermore, to overcome cytologic specimen-specific challenges, FISH combined with cytokeratin AE1/AE3 immunofluorescence was necessary in 25.9% of MPM cases for FISH assessment of predominantly scattered MPM cells. CONCLUSIONS: NF2 FISH alone or in combination with other diagnostic assays effectively differentiates MPM cells from RMCs in cell blocks prepared from pleural effusions.

Diagnostic mesothelioma biomarkers in effusion cytology.

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.

[Results of EGFR Mutations Detected in Pleural Effusion and Its 
Clinical Significance in 132 Patients with Advanced Non-small Cell Lung Cancer: 
A Retrospective Study in A Single Center].

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

Effect of malignant-associated pleural effusion on endothelial viability, motility and angiogenesis in lung cancer.

Malignant pleural effusion (MPE) and paramalignant pleural effusion (PPE) remain debilitating complications in lung cancer patients with poor prognosis and limited treatment options. The role of vascular endothelial cells has not been explored in the pleural environment of lung cancer. By integrating MPE and PPE as malignant-associated pleural fluid (MAPF), the current study aimed to evaluate the effect of MAPF on cell proliferation, migration and angiogenesis of HUVEC. First, increased capillaries were identified in the subpleural layer of lung adenocarcinoma. Compatible with pathological observations, the ubiquitous elevation of HUVEC survival was identified in MAPF culture regardless of the underlying cancer type, the driver gene mutation, prior treatments and evidence of malignant cells in pleural fluid. Moreover, MAPF enhanced HUVEC motility with the formation of lamellipodia and filopodia and focal adhesion complex. Tube formation assay revealed angiogenic behavior with the observation of sheet-like structures. HUVEC cultured with MAPF resulted in a significant increase in MAPK phosphorylation. Accompanied with VEGFR2 upregulation in MAPF culture, there was increased expressions of p-STAT3, HIF-1α and Nf-kB. VEGF/VEGFR2 blockade regressed endothelial migration and angiogenesis but not cell proliferation. Our data indicate the angiogenic activities of MAPF on vascular endothelial cells that revealed increased pleural capillaries in lung cancer. Targeting the VEGF/VEGFR2 pathway might modulate the angiogenic propensity of MAPF in future clinical investigations.

Testing for BAP1 loss and CDKN2A/p16 homozygous deletion improves the accurate diagnosis of mesothelial proliferations in effusion cytology.

BACKGROUND: A number of ancillary tests have been developed that aid in the diagnosis of mesothelioma in cytology specimens. The aim of this retrospective study was to determine whether testing for BAP1 and CDKN2A/p16 status in effusion specimens preceding the tissue diagnosis of mesothelioma would improve diagnostic accuracy and allow an earlier diagnosis of malignancy. METHODS: The study cohort included 99 matched cytology fluid specimens from 74 patients with a surgical specimen diagnosis of malignant mesothelioma (67 epithelioid, 7 biphasic, 55 pleural, and 19 peritoneal). BAP1 immunohistochemistry and p16 fluorescence in situ hybridization (FISH) were performed retrospectively. RESULTS: BAP1 or p16 FISH testing revealed a loss in 7 of 18 (39%) samples originally categorized as benign/reactive, 20 of 33 (61%) interpretable samples categorized as atypical, and 10 of 14 (71%) cases suspicious for mesothelioma. In some cases, the diagnosis of mesothelioma could have been made up to 9 months before biopsy. Similarly, loss of BAP1 or p16 was found in 28 of 30 (93%) samples categorized as malignant, with some cases diagnosable up to 6 months before biopsy. Overall, loss of BAP1 and/or CDKN2A/p16 homozygous deletion would change the diagnostic interpretation in 37 of 60 (62%) (P = .07) effusion specimens, particularly in pleural effusions (32 of 48 samples) (P = .002). The sensitivity of morphologic interpretation alone was 30.3%; however, adding testing for BAP1 and p16 resulted in an increase in sensitivity to 68.7%. (P < .0001). CONCLUSION: These findings suggest that routine use of BAP1 immunochemistry and p16 FISH as adjunctive tests improves the diagnostic accuracy of cytology specimens and potentially allows an earlier diagnosis of malignant mesothelioma.

Identification of differentially expressed miRNAs in differentiating benign from malignant pleural effusion.

BACKGROUND: Tuberculosis pleural effusion (TPE) and malignant pleural effusion (MPE) are very common clinical complications. Considering the totally different prognosis and clinical treatment of TPE and MPE, the accurate and non-invasive diagnosis are very critical for patients with pleural effusion to initiate efficient management and treatment. However, effective clinical biomarkers were rarely explored to distinguish benign from MPE. The purpose of this study is to identify potential miRNAs which can probably be used to differentiate malignant pleural effusion from TPE. RESULTS: A total of 23 significantly differentially expressed miRNAs were identified in MPE, with 18 up-expressed and 5 down-expressed. And the target genes of the miRNAs mainly involved in the biology process of nervous system, cancer, immune system and metabolic process etc. Three high confident target genes, AGO4, FGF9 and LEF1 can be regulated by miR-195-5p, miR-182-5p and miR-34a-5p respectively. And these genes participate in the canonical pathway of regulation of the Epithelial-Mesenchymal and the biological functions of apoptosis, growth of tumor and cell proliferation of tumor cell lines. Further, RT-PCR validation results based on 64 collected individuals showed that the expression levels of the three miRNAs were 2-5 times higher in MPE samples, which were consistent with the microarray results. In addition, ROC curve analysis demonstrated that the combination of the three miRNAs can achieve higher AUC of 0.93 (p-value< 0.0001) to differentiate MPE from TPE. CONCLUSIONS: The identified miR-195-5p, miR-182-5p and miR-34a-5p can become potential diagnostic biomarkers for MPE with further evidences.

The utility of exome sequencing for fetal pleural effusions.

OBJECTIVE: We sought to evaluate the performance of exome sequencing (ES) in determining an underlying genetic etiology for cases of fetal pleural effusions. STUDY DESIGN: We examined a prospective cohort series of fetal pleural effusions visualized sonographically between 1 April 2016 and 31 August 2017. Fetal pleural effusions attributed to twin sharing, anemia, or structural anomalies were excluded, as were all cases with a genetic diagnosis established by karyotype or chromosomal microarray analysis. The remaining cases with pleural effusions of unclear etiology were offered ES. ES was performed by clinical sequencing and/or sequencing under the Baylor-Hopkins Center for Mendelian Genomics' (BHCMG) research platform. All cases were evaluated for novel genes or phenotypic expansion of disease-causing genes. RESULTS: ES was performed on six probands affected by pleural effusions. A pathogenic variant was identified in one case (16.7%). Four additional cases had variants of uncertain significance (VUS) in candidate genes of pathological interest. Neither clinical nor candidate genes were evident in the final case. CONCLUSION: ES should be considered in the evaluation of prenatally detected idiopathic pleural effusions when other diagnostic workup for a genetic etiology has failed.

Label-Free Quantitative Proteomics Identifies Novel Biomarkers for Distinguishing Tuberculosis Pleural Effusion from Malignant Pleural Effusion.

PURPOSE: To identify potential protein biomarkers for distinguishing tuberculosis plural effusion (TBPE) from malignant plural effusion (MPE). EXPERIMENTAL DESIGN: Five independent samples from each group (TBPE and MPE) are enrolled for label-free quantitative proteomics analyses. The differentially expressed proteins are validated by western blot and ELISA. Logistic regression analysis is used to obtain the optimal diagnostic model. RESULTS: In total, 14 proteins with significant difference are identified between TBPE and MPE. Seven differentially expressed proteins are validated using western blot, and the expression patterns of these seven proteins are similar with those in proteomics analysis. Statistically significant differences in four proteins (AGP1, ORM2, C9, and SERPING1) are noted between TBPE and MPE in the training set (n = 230). Logistic regression analysis shows the combination of AGP1-ORM2-C9 presents a sensitivity of 73.0% (92/126) and specificity of 89.4% (93/104) in discriminating TBPE from MPE. Additional validation is performed to evaluate the diagnostic model in an independent blind testing set (n = 80), and yielded a sensitivity of 74.4% (32/43) and specificity of 91.9% (34/37) in discriminating TBPE from MPE. CONCLUSION: The study uncovers the proteomic profiles of TBPE and MPE, and provides new potential diagnostic biomarkers for distinguishing TBPE from MPE.