Pleural Samples Research Articles

Le profil bactériologique des pleurésies purulentes au CHU de Marrakech

Prognosis of purulent pleurisy remains severe especially in children despite antibiotic therapy. The bacteriological diagnosis is essential to confirm the diagnosis, adapt antibiotic therapy and guide the etiology. The objective of the study is to identify the bacteria involved in pleural puncture samples and evaluate antibiotic susceptibility among patients hospitalized at the different services at the University Hospital of Marrakech. It is a retrospective study of 6 years (2012-2017), including all pleural puncture (LP) samples taken in clinical care at beds of patients, received at the laboratory and from patients hospitalized at the different services at the University Hospital Center of Marrakesh. Out of 733 Purulent pleurisy completed, 98 were positive (13,3 %). The average age was 30 years old. The bacteriological profile was dominated by gram-positive bacteria (63,2 %). among them, Streptococcus pneumoniae (16,4 %), followed by Staphylococcus aureus (15,4 %), coagulase-negative Staphylococcus (SCN) (11,2 %), Streptococcus spp (14,1 %), Streptococcus haemolytic Group A (5,1 %) and Enterococcus spp (1 %). Enterobacteria accounted for 20,5 %. Pseudomonas aeuroginosa was found in 8,3 % of cases. The documented pleurisy came mainly from the pneumology department (20.5 %) pediatric department (18 %) and the infectious diseases department (11,6 %). The study of antibiotic susceptibility of isolated strains revealed penicillin sensitivity of 68.75 % in Streptococcus pneumoniae isolates. All strains of Staphylococcus aureus were sensitive to meticillin. In Enterobacteria, resistance to C3G was 35 %.The prevention of purulent pleurisy would consist of good management of otorhinolaryngology infections, broncho-pulmonary infections and pneumococcal vaccination of subjects at risk.

ADAM10 mediates malignant pleural mesothelioma invasiveness

Malignant pleural mesothelioma (MPM) is an aggressive cancer with limited therapeutic options and treatment efficiency. Even if the latency period between asbestos exposure, the main risk factor, and mesothelioma development is very long, the local invasion of mesothelioma is very rapid leading to a mean survival of one year after diagnosis. ADAM10 (A Disintegrin And Metalloprotease) sheddase targets membrane-bound substrates and its overexpression is associated with progression in several cancers. However, nothing is known about ADAM10 implication in MPM. In this study, we demonstrated higher ADAM10 expression levels in human MPM as compared to control pleural samples and in human MPM cell line. This ADAM10 overexpression was also observed in murine MPM samples. Two mouse mesothelioma cell lines were used in this study including one primary cell line obtained by repeated asbestos fibre injections. We show, in vitro, that ADAM10 targeting through shRNA and pharmacological (GI254023X) approaches reduced drastically mesothelioma cell migration and invasion, as well as for human mesothelioma cells treated with siRNA targeting ADAM10. Moreover, ADAM10 downregulation in murine mesothelioma cells significantly impairs MPM progression in vivo after intrapleural cell injection. We also demonstrate that ADAM10 sheddase downregulation decreases the production of a soluble N-cadherin fragment through membrane N-cadherin, which stimulated mesothelioma cell migration. Taken together, we demonstrate that ADAM10 is overexpressed in MPM and takes part to MPM progression through the generation of N-cadherin fragment that stimulates mesothelioma cell migration. ADAM10 inhibition is worth considering as a therapeutic perspective in mesothelioma context.

Pleural fluid penetration of moxifloxacin and doripenem: An experimental model of empyema.

OBJECTIVE:This study aimed to evaluate the penetration of moxifloxacin and doripenem into the pleural fluid (PF) using a rabbit model of empyema.METHODS:An empyema was induced using the intrapleural injection of turpentine (1 mL), followed 24 h later by instillation of 5 mL Klebsiella Pneumoniae (ATCC 33495), Fusobacterium nucleatum (ATCC 25586) and Streptokok Pneumoniae (ATCC 6305) into the pleural space. After an empyema was corroborated, Moxifloxacin (25 mg/kg-1) and Doripenem (20 mg/kg-1) were administered intraperitoneally. To determine the levels of antibiotics measured by High-Performance Liquid Chromatography in pleural and blood samples were obtained serially at 8, 24, 48 and 72nd hour.RESULTS:The penetration of both antibiotics into the PF was very good. The penetration rate of doripenem (area under the curve (AUC) for PF/blood (AUCPF/AUCblood) ratio=1.68) was better than moxifloxacin (ratio=0.78). Equalization time between the PF and blood concentration of doripenem was more quickly than moxifloxacin. Peak PF concentration of moxifloxacin was 0,81 μg/mL-1 and occurred 8 h after infusion and then gradually decreased; at the beginning of the blood and pleural fluid concentrations of doripenem were equal. While the pleura concentration was increasing, blood concentration was almost the same. Doripenem reached a peak concentration (0.54 μg/ml) 24 h post-administration.CONCLUSION:Differences were found in the penetration of the two antibiotics. Doripenem had convenient penetration PF compared to moxifloxacin. Due to the differences between human and rabbit pleural thickness, doripenem’s pleural penetration should be examined in infection models in animals with equal pleura thickness and clinical trials.

Diagnostic approach to pleural effusions

The increasing incidence of pleural effusions from a variety of etiologies is associated with significant socioeconomic burden. Effective treatment for pleural effusions first requires a comprehensive diagnostic work up encompassing analysis of pleural fluid chemistry, microbiology studies and invasive sampling, all of which should be incorporated with relevant clinical data to reach a final diagnosis. This review article addresses current diagnostic approaches to pleural effusions.

Quantitative INTERSPECIMEN variability during biphasic pleural fluid cytological sampling in adenocarcinoma effusions.

Given that cytology of adenocarcinoma-induced pleural effusions has a high diagnostic yield, we have comparatively evaluated the cytological information of smears during biphasic sampling of pleural fluid in patients with metastatic pleural adenocarcinoma from various primary sites. We studied 25 male and 21 female patients, aged 59.4 ±17.2 years (mean ± SD) with unilateral malignant effusion of varying magnitude due to confirmed adenocarcinoma from various primary sites. At thoracocentesis we collected two 30-ml samples of pleural fluid, the first at the very beginning of fluid aspiration (S1) and the second just before termination of fluid removal (S2) and recorded the volume of fluid aspirated between the 2 samples. Cytological smears were examined under light microscopy by 3 independent cytologists after Papanicolaou stain. Quantitative assessment of cell types was averaged among 50 visual fields for each smear. In S1 versus S2 the mean number of mesothelial cells was 7.8 ± 4.8 versus 12.1 ± 5.4 (mean ± SD), of lymphocytes 64.6 ± 12.9 versus 85.9 ± 17.4, of neutrophils 8.5 ± 4.4 versus 11.7 ± 5.2, of eosinophils 1.5 ± 0.3 versus 1.7 ± 0.8, and the number of malignant cell aggregates(NMCA) was 11.9 ±4.9 versus 20.7 ± 5.1. The differences in numbers of all cell types including NMCA were statistically significant between S1 and S2 (P < .01). A strong significant linear association between S2/S1 ratio of NMCA and the volume of fluid aspirated between samples was also found (95% confidence interval [CI], 0.209-0.236, P-value < .001). Specimens aspirated before completion of fluid drainage are shown to contain significantly more diagnostic information than those aspirated at the beginning of fluid removal.

Multisensor Imaging-From Sample Preparation to Integrated Multimodal Interpretation of LA-ICPMS and MALDI MS Imaging Data.

Laterally resolved chemical analysis (chemical imaging) has increasingly attracted attention in the Life Sciences during the past years. While some developments have provided improvements in lateral resolution and speed of analysis, there is a trend toward the combination of two or more analysis techniques, so-called multisensor imaging, for providing deeper information into the biochemical processes within one sample. In this work, a human malignant pleural mesothelioma sample from a patient treated with cisplatin as a cytostatic agent has been analyzed using laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). While LA-ICPMS was able to provide quantitative information on the platinum distribution along with the distribution of other elemental analytes in the tissue sample, MALDI MS could reveal full information on lipid distributions, as both modes of polarity, negative and positive, were used for measurements. Tandem MS experiments verified the occurrence of distinct lipid classes. All imaging analyses were performed using a lateral resolution of 40 μm, providing information with excellent depth of details. By analyzing the very same tissue section, it was possible to perfectly correlate the obtained analyte distribution information in an evaluation approach comprising LA-ICPMS and MALDI MS data. Correlations between platinum, phosphorus, and lipid distributions were found by the use of advanced statistics. The present proof-of-principle study demonstrates the benefit of data combination for outcomes beyond one method imaging modality and highlights the value of advanced chemical imaging in the Life Sciences.

Increased micronucleus count predicts malignant behavior in pleural effusion fluid

Background/aim: Micronucleus (MN) frequency is used as a biomarker of chromosomal damage, genome instability, and cancer risk. The aim of this study was to evaluate the diagnostic usefulness of MN frequency to differentiate between malignant and benign pleural effusion samples. Materials and methods: Retrospectively, 78 pleural fluid cytology samples (including 20 cases of benign reactive mesothelial cells, 22 cases of suspicious cytology, and 36 cases of malignant cytology) were examined. The number of micronucleated cells in 1000 well- preserved cells was counted. Statistical tests were performed to compare the study groups. Recover operating characteristics (ROC) curve analysis was performed to suggest a cut-off value for predicting malignant behavior. Results: We evaluated a total of 78 cases of pleural effusion cytology. The number of micronucleated cells was significantly higher in cases with malignant outcome compared to cases with benign outcome. We observed that malignant samples had more micronucleated cells than suspicious ones, and suspicious cases had more micronucleated cells than reactive ones. There was a significant difference among all study groups. In addition, the frequency of MN-containing cells in suspicious cases correlates well with their outcomes. Conclusion: The results of this study reveal that there is an absolute, consistent, and proportional relationship between MN counts and malignancy in cytological samples of pleural effusions. MN scoring may be a helpful diagnostic tool for distinguishing malignant effusions from benign ones, and may be used as an adjunct tool to predict malignant behavior in challenging cases.

A Pilot Feasibility Study in Establishing the Role of Ultrasound-Guided Pleural Biopsies in Pleural Infection (The AUDIO Study)

Pleural infection is a common complication of pneumonia associated with high mortality and poor clinical outcome. Treatment of pleural infection relies on the use of broad-spectrum antibiotics because reliable pathogen identification occurs infrequently. We performed a feasibility interventional clinical study assessing the safety and significance of ultrasound (US)-guided pleural biopsy culture to increase microbiological yield. In an exploratory investigation, the 16S ribosomal RNA technique was applied to assess its utility on increasing speed and accuracy vsstandard microbiological diagnosis. Twenty patients with clinically established pleural infection were recruited. Participants underwent a detailed US scan and US-guided pleural biopsies before chest drain insertion, alongside standard clinical management. Pleural biopsies and routine clinical samples (pleural fluid and blood) were submitted for microbiological analysis. US-guided pleural biopsies were safe with no adverse events. US-guided pleural biopsies increased microbiological yield by 25%in addition to pleural fluid and blood samples. The technique provided a substantially higher microbiological yield compared with pleural fluid and blood culture samples (45%compared with 20%and 10%, respectively). The 16S ribosomal RNA technique was successfully applied to pleural biopsy samples, demonstrating high sensitivity (93%) and specificity (89.5%). Our findings demonstrate the safety of US-guided pleural biopsies in patients with pleural infection and a substantial increase in microbiological diagnosis, suggesting potential niche of infection in this disease. Quantitative polymerase chain reaction primer assessment of pleural fluid and biopsy appears to have excellent sensitivity and specificity.

Thoracoscopic pleural biopsy improves yield of Xpert MTB/RIF for diagnosis of pleural tuberculosis.

Extrapulmonary tuberculosis (EPTB) accounts for ~15% of all TB patients, and TB pleural effusion is the second most common site of EPTB. The diagnosis of pleural TB is challenging due to the pauci-bacillary nature of the disease. Histopathology of thoracoscopically obtained pleural biopsy provides the highest diagnostic yield. The Xpert MTB/RIF assay (Xpert) is a PCR test that can identify both Mycobacterium tuberculosis (MTB) and rifampicin resistance. Currently, there is a lack of clarity regarding the value of Xpert on pleural tissue. We report our experience of using Xpert on thoracoscopic pleural biopsy samples. We retrospectively reviewed the records of patients who underwent thoracoscopy in our institution over a 1-year period. Relevant clinical details; indications; and results of tests on pleural tissue and fluid, including histopathology, mycobacterial cultures and Xpert, were extracted. Of the 156 patients who underwent thoracoscopy, 73 (47%) had TB, 66 (42%) malignancy and 17 (11%) other conditions. Histopathology was diagnostic in all the 73 TB patients (100%). The yields of the microbiological tests against histopathology on thoracoscopic biopsy sample and pleural fluid were: pleural tissue Xpert 45%, pleural tissue culture 39%, pleural fluid culture 17% and pleural fluid Xpert 14%. Pleural tissue provided higher yields than fluid in both Xpert and culture (P < 0.05). Pleural tissue Xpert provided a higher yield than culture and substantially improved yield compared with closed pleural biopsy as we previously reported. Thoracoscopic pleural biopsy results in increased sensitivity on Xpert testing.

Induction of protein citrullination and auto-antibodies production in murine exposed to nickel nanomaterials

Citrullination, or the post-translational deimination of polypeptide-bound arginine, is involved in several pathological processes in the body, including autoimmunity and tumorigenesis. Recent studies have shown that nanomaterials can trigger protein citrullination, which might constitute a common pathogenic link to disease development. Here we demonstrated auto-antibody production in serum of nanomaterials-treated mice. Citrullination-associated phenomena and PAD levels were found to be elevated in nanomaterials -treated cell lines as well as in the spleen, kidneys and lymph nodes of mice, suggesting a systemic response to nanomaterials injection, and validated in human pleural and pericardial malignant mesothelioma (MM) samples. The observed systemic responses in mice exposed to nanomaterials support the evidence linking exposure to environmental factors with the development of autoimmunity responses and reinforces the need for comprehensive safety screening of nanomaterials. Furthermore, these nanomaterials induce pathological processes that mimic those observed in Pleural MM, and therefore require further investigations into their carcinogenicity.

Экспериментальное обоснование нового метода иммунодиагностики туберкулезных плевритов

The current study was perfomed to investigate the representativeness of the basic and antigen induced levels of specific (IFN-y) and non-specific (IL-6) biomarkers for the diagnosis of tuberculous pleural effusion. Ninety-three samples of pleural fluid from patient with tuberculosis (TP) and non-tuberculosis (Non-TP) pleurisy were examined. Induction of the synthesis IFN-gamma by immunocompetent cells in pleural exudate was carried out by the recombinant protein CFP10-ESAT6 contained in the diagnosticum «DIASKINTEST» by our method (Patent RF, № 2664427; 2017). Serum and pleural fluid IFN-y and IL-6 levels were measured by enzyme-linked immunosorbent assay technique (enzyme-am-plified sensitivity immunoassay kits, Vector-Best reagent kit (Russia). Our results suggested that introduction of M. tuberculosis antigens into the pleural fluids samples of TP group caused a significant increase of the IFN-y levels compared to basic level, which were not observed in the Non-TP group. Detection of IFN-gamma antigen-induced level in pleural fluid is a promising method for the diagnosis of tuberculous pleurisy. We found no significant differences in the basic and antigen-induced levels of IL-6 in patients with TP and Non-TP pleurisy.

Cryobiopsy of the Pleura: An Improved Diagnostic Tool.

Medical thoracoscopy (rigid and semirigid pleuroscopy) has revolutionized the approach to the diagnosis of pleural disease by offering a very high diagnostic yield. Rigid pleuroscopy offers the advantages of therapeutic intervention and larger biopsy specimens, whereas semirigid pleuroscopy using a standard biopsy forceps yields smaller and more superficial pleural samples. Cryobiopsy through semirigid pleuroscope in anecdotal studies has been used to overcome these disadvantages. We compared the safety and efficacy of cryobiopsy with conventional forceps biopsy in terms of the specimen size and diagnostic yield. We analyzed data of 139 (87 cryobiopsies and 52 forceps biopsies) patients with undiagnosed pleural effusion who underwent pleuroscopy using a semirigid pleuroscope. A cryoprobe (ERBE, 2.4 mm) was passed through the working channel of the semirigid pleuroscope, the target area of parietal pleura was frozen for an average freezing time of 8 seconds, then the semirigid pleuroscope along with the probe was forcibly withdrawn en bloc avulsing the frozen parietal pleura. Two to 3 samples were taken from each patient. The diagnostic yield was 99% with cryobiopsy and 96% with forceps biopsy. The average specimen size through cryoprobe (13.2±6.7; range, 7 to 35 mm) was significantly larger than with the conventional forceps (6.8±3.3; range, 2 to 15 mm) (P<0.001), and no major complications were noted. Cryobiopsy of the parietal pleura through the semirigid pleuroscope is a safe procedure with a very high diagnostic yield.

Diagnostic value of medical thoracoscopy in malignant pleural effusion induced by non-Hodgkin's lymphoma.

Malignant pleural effusion (MPE) appears in up to 20% of patients with non-Hodgkin's lymphoma (NHL). The present study aimed to assess the efficacy of medical thoracoscopy (MT) in the diagnosis of patients with MPE induced by NHL. Between July 2005 and June 2014, 833 patients with pleural effusions of unknown etiology underwent MT in Beijing Chaoyang Hospital (Beijing, China), where diagnostic thoracocentesis or/and blind pleural biopsy had failed to yield an answer. Demographic, radiographic, thoracoscopic, histological and immunophenotyping data of 10 NHL patients with MPE were then retrospectively analyzed. Under medical thoracoscopy, pleural nodules (in n=6 patients), hyperemia (n=5), plaque-like lesions (n=4), pleural thickening (n=3), cellulose (n=3), ulcer (n=2), adhesion (n=2), and scattered hemorrhagic spots (n=1) were observed on the surface of parietal pleura. Histopathological and immunohistochemical analysis of pleural biopsy samples led to a correct diagnosis of B-cell NHL in 7 patients and T-lymphoblastic NHL in 2 patients. Data from the present study demonstrated that pleural biopsy through MT achieved a definite diagnosis of NHL in 9 out of 10 (90%) patients with MPE induced by NHL. Therefore, MT is a useful method for diagnosing MPE induced by NHL.

Abstract 3715A: Effusions of mesothelioma patients: What's in it for immunotherapy

Abstract Malignant pleural mesothelioma (MPM) is an aggressive cancer with a poor prognosis for which new therapeutic strategies are available. Data derived from a small number of mesothelioma patients suggest that blocking immune checkpoints could offer new treatment opportunities. Gaining more insight in the immunological aspect of the tumor microenvironment (TME) in MPM is essential to develop an effective immunotherapy. In this context, we investigated expression of the immune checkpoint molecules TIM-3, LAG-3, PD-1 and its ligand PD-L1 on cells in human pleural (collected via thoracocentesis, n=6) and ascites (collected via paracentesis, n=5) MPM fluids and identified different subsets of immune cells in the TME using multicolor flow cytometry. Different types of immune cells in the fluids could be detected, with predominant occurrence of CD3+CD4+ T cells, CD64+ macrophages and CD11c+CD303+ dendritic cells (DCs). CD3+CD8+ T cells, CD3-CD56+ natural killer (NK) cells and CD19+ B cells were also present to a lesser extent. While CD4+ and CD8+ T cells and CD64+ macrophages could be detected in all samples, this was not the case for the other cell types. Ascites fluids contained more podoplanin+ (PDPN) tumor cells compared to pleural fluids (average 3.0% vs 0.3%). PD-1+ T cells and NK cells could be detected in all pleural and 80% of the ascites samples. 64% of all samples contained LAG-3-expressing cells, most frequently in pleural fluids. In all samples CD4+ T-cells and NK cells showed TIM-3 expression and in 82% of the samples TIM-3 was also expressed on CD8+ T-cells. In all ascites samples PD-L1 was expressed on DCs, B cells, macrophages and PDPN+ MPM tumor cells. PD-L1 was also expressed on DCs in all pleural samples while in 67%, 83% and 83% of those samples CD19+, CD64+ and PDPN+ cells were PD-L1+, respectively. Based on the mean fluorescence intensity PDPN+ tumor cells showed the highest PD-L1 expression in both fluids. In conclusion, we provide a detailed analysis of a diversity of immune cells present in MPM fluid samples. We are the first to demonstrate TIM-3 and LAG-3 expression in mesothelioma effusions. Statistical analysis is ongoing to investigate whether there are differences between both fluid types and whether there are associations between the cellular composition of the fluids and survival of the patients. The finding of expression of PD-1, PD-L1, TIM-3 and LAG-3 on immune cells in MPM fluids warrants further investigation of the effect of immune checkpoint blockade in MPM, with TIM-3 and LAG-3 as interesting new targets. Citation Format: Elly Marcq, Jorrit De Waele, Jonas van Audenaerde, Eva Lion, Eva Santermans, Niel Hens, Patrick Pauwels, Jan P. van Meerbeeck, Evelien L. Smits. Effusions of mesothelioma patients: What's in it for immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3715A. doi:10.1158/1538-7445.AM2017-3715A

Lung cancer genomic alterations in cell free DNA in pleural effusion compared to plasma.

e23206 Background: Circulating tumor DNA (ctDNA) in plasma has been shown to be informative of the genomic alterations present in lung cancer and has been used to guide tumor treatment and monitor tumor progression. However, patients with lung cancer, even in advanced stage, usually present with small numbers of cancer-related genetic aberrations and low mutation allele frequency (MAF) on average. Malignant pleural effusion is one of common complications for lung cancer patients in advanced stage. Cell free DNA (cfDNA) in pleural effusion may be extensively diluted once entering into the peripheral circulation system. Methods: We implemented targeted capture NGS with a gene panel of 180 lung cancer-relevant genes on 23 paired plasma ctDNA and matched pleural cfDNA samples extracted simultaneously from the same patient with advanced lung cancer (n = 23), including 17 adenocarcinoma. Results: Overall, actionable variations with on label, off-label, or experimental drugs available, were identified in 87% (n = 20) of pleural effusion samples, whereas plasma was 48% (n = 11). Median number of pleural somatic mutations was 4, predominantly located in TP53, EGFR, and ALK; while the median number was 2 in plasma samples. Of that, 39% and 74% of plasma ctDNA and pleural cfDNA samples were observed at an average MAF above 1%, respectively. In addition, 48% and 78% of plasma ctDNA and pleural cfDNA samples were observed with the highest MAF above 1%. The concordance rate for EGFR and ALK alterations with plasma NGS was 83% and 87%, respectively. Conclusions: Our results suggest that pleural effusion-derived cfDNA is more sensitive than plasma for advanced lung cancer patients. With higher MAF and detection rate of actionable variations, pleural effusion-derived cfDNA might guide more precise lung cancer treatment.

Pleural fluid cytology samples in cases of suspected lung cancer: An experience from a tertiary care centre.

Pleural effusion is present in some patients with lung carcinoma. This study was undertaken to evaluate the role of pleural fluid cytology in suspected cases of lung carcinoma. In this prospective study, 86 cases of suspected primary lung carcinoma associated with pleural effusion were included. Cytomorphology of conventional smears was combined with LBC smears and a cytomorphological diagnosis was offered. An immunopanel of CK7, CK5/6, p63, TTF1, calretinin, and CD56 was applied on cell blocks. Additional immunochemical markers were performed based on clinical details and cytomorphology, wherever indicated. The mean age of the patients was 56.8 years. The female to male ratio was 2.1:1. Lung nodule/mass could be detected in 70.9% cases by CECT chest. Cytomorphology was evaluated in all the 86 cases and IHC was used to refine the cytomorphological diagnosis. After IHC along with clinico-radiological details, 61/86 (71%) cases were confirmed as metastasis from lung primary, 5/86 (5.8%) were diagnosed as metastatic adenocarcinoma of extrapulmonary origin, and 6/86 (7%) were metastatic SCC. A diagnosis of adenosquamous carcinoma was suspected in five cases. Other cases included small cell carcinoma (n = 2), large cell carcinoma (n = 2), large cell neuroendocrine carcinoma (n = 3), and mesothelioma (n = 1). One case remained "uncategorized" even after IHC. Immunohistochemistry (IHC) performed on the pleural fluid sample can accurately diagnose and subtype primary lung carcinoma even in cases where radiology failed to identify a definite lung lesion. IHC can further help in delineating primary site in tumors of extrapulmonary origin. © 2017 Wiley Periodicals, Inc. Diagn. Cytopathol. 2017;45:195-201. © 2016 Wiley Periodicals, Inc.

Utility of Macrophage-activated Marker CD163 for Diagnosis and Prognosis in Pulmonary Tuberculosis.

Among infectious diseases, tuberculosis (TB) is a leading cause of death worldwide. Accumulated knowledge has revealed that macrophages are deeply involved in the progression and pathogenesis of TB. We hypothesized that the evaluation of a macrophage activation marker may be useful in the diagnosis and assessment of pulmonary TB. To examine the utility of the macrophage activation marker soluble CD163 (sCD163) as a diagnostic tool and measure of disease severity for pulmonary TB and tuberculous pleurisy. We compared the concentration of sCD163 in serum samples of 180 patients with active pulmonary TB with concentrations in serum samples of 45 age- and sex-matched control subjects. We also measured sCD163 in pleural fluid samples of 100 patients with pleural disease, including 31 patients with tuberculous pleurisy. We found increased serum concentrations of sCD163 in patients with active pulmonary TB compared with those of control subjects (1,643 ± 1,737 ng/ml vs. 533.9 ± 49.3 ng/ml; P < 0.0001). sCD163 levels were also higher in pleural fluid samples of patients with pulmonary TB than in those of patients with non-TB pleurisy (5,239 ± 2,436 ng/ml vs. 2,877 ± 1,191 ng/ml; P < 0.0001). The levels of sCD163 in pleural effusions were significantly higher than serum levels obtained simultaneously from the same patients, particularly for patients with tuberculous pleurisy. Patients with a serum level of sCD163 above 1,300 ng/ml, had a mortality rate that was five times higher than that of patients with a lower sCD163 level (44.6% vs. 6.6%; P < 0.0001 by log-rank test). Microscopic examination of lung and pleural tissue samples showed concordance of enhanced CD163 expression with the presence of caseating granulomas in tissue obtained from patients with TB. The macrophage activation marker CD163 was increased in patients with active pulmonary TB compared with age- and sex-matched control subjects. Increased levels of sCD163 were associated with increased mortality in patients with pulmonary TB. sCD163 also showed promise as a diagnostic tool for tuberculous pleurisy. These results warrant further study of sCD163 as a potentially useful biomarker for the diagnosis and assessment of pulmonary TB. Clinical trial registered with www.umin.ac.jp/ctr/index-j.htm (UMIN000003400).

P3.03-034 Comprehensive Immunophenotyping of the Blood and Pleural Fluid from Patients with Malignant Pleural Mesothelioma by Flow Cytometry: Topic: Mesothelioma Clinical

Malignant pleural mesothelioma (MM) remains an almost universally fatal disease with limited treatment options. Immunotherapy represents a rapidly emerging therapeutic strategy for multiple malignancies including MM. However durable therapeutic responses occur in a minority (∼30%) of patients. Better understanding of the quantities and immunophenotype of circulating and intrapleural immune cells is essential to design and apply personalized immunotherapeutic strategies. We used a comprehensive flow cytometry panel (PLoS One. 2015 Mar 23;10 (3):e0121546) to prospectively characterize circulating and pleural fluid immune cells in patients with MPM (n=12) and normal volunteers (circulating cells only, n=50). Matched blood and pleural samples were available from 11 patients, including samples from 9 patients enrolled into a Phase I study investigating the intrapleural administration of the modified vaccine strain measles virus (MV-NIS), MC1023. Pre- and post-treatments samples were available from 7 patients. The immune cell counts and cell fractions were compared using a false discovery rate (FDR) of 10% and the non-parametric Mann-Whitney test and the Wilcoxon matched-pairs signed rank test (blood versus pleural fluid and different time points) (p ≤ 0.05). Cell counts and immune phenotype of circulating immune cells differ between of patients with MPM differ from normal volunteers (31 of 86 cell types). Patients with MPM had fewer B-lymphocytes, more pro-inflammatory monocytes (CD14+CD16+) and exhausted CD4 and CD8 T-lymphocytes (CD4 and CD8 PD1+TIM3+ double positive cells). As expected we observed characteristic differences between blood and pleural fluid in MPM patients. For example there are a higher numbers of antigen experienced, PD-1 expressing CD4 and CD8-memory cells within the pleural fluid compared to the peripheral blood. The intrapleural administration of a modified vaccine strain measles (MV-NIS) triggered changes in pleural and circulating immune cells. While these changes varied among patients, we observed increased CD80 and CD86 expression on CD14+ monocytes in the pleural fluid down. Comprehensive flow-cytometric immunophenotying of blood and pleural fluid is feasible in patients with MPM. This approach may help to identify patients who may respond favorably to specific immunotherapeutic interventions such as immune checkpoint inhibitors or facilitate longitudinal immune monitoring during clinical trials. Additional data is needed and we are continuing to prospectively analyze samples from patients with MPM.

Cytotoxic T Cells in PD-L1-Positive Malignant Pleural Mesotheliomas Are Counterbalanced by Distinct Immunosuppressive Factors.

PD-L1 immunohistochemical staining does not always predict whether a cancer will respond to treatment with PD-1 inhibitors. We sought to characterize immune cell infiltrates and the expression of T-cell inhibitor markers in PD-L1-positive and PD-L1-negative malignant pleural mesothelioma samples. We developed a method for immune cell phenotyping using flow cytometry on solid tumors that have been dissociated into single-cell suspensions and applied this technique to analyze 43 resected malignant pleural mesothelioma specimens. Compared with PD-L1-negative tumors, PD-L1-positive tumors had significantly more infiltrating CD45+ immune cells, a significantly higher proportion of infiltrating CD3+ T cells, and a significantly higher percentage of CD3+ cells displaying the activated HLA-DR+/CD38+ phenotype. PD-L1-positive tumors also had a significantly higher proportion of proliferating CD8+ T cells, a higher fraction of FOXP3+/CD4+ Tregs, and increased expression of PD-1 and TIM-3 on CD4+ and CD8+ T cells. Double-positive PD-1+/TIM-3+ CD8+ T cells were more commonly found on PD-L1-positive tumors. Compared with epithelioid tumors, sarcomatoid and biphasic mesothelioma samples were significantly more likely to be PD-L1 positive and showed more infiltration with CD3+ T cells and PD-1+/TIM-3+ CD8+ T cells. Immunologic phenotypes in mesothelioma differ based on PD-L1 status and histologic subtype. Successful incorporation of comprehensive immune profiling by flow cytometry into prospective clinical trials could refine our ability to predict which patients will respond to specific immune checkpoint blockade strategies. Cancer Immunol Res; 4(12); 1038-48. ©2016 AACR.

The diagnostic performance of routinely acquired and reported computed tomography imaging in patients presenting with suspected pleural malignancy

ObjectivesContrast-enhanced computed tomography (CT) provides essential cross-sectional imaging data in patients with suspected pleural malignancy (PM). The performance of CT in routine practice may be lower than in previously reported research. We assessed this relative to ‘real-life’ factors including use of early arterial-phase contrast enhancement (by CT pulmonary angiography (CTPA)) and non-specialist radiology reporting. Materials and methodsRoutinely acquired and reported CT scans in patients recruited to the DIAPHRAGM study (a prospective, multi-centre observational study of mesothelioma biomarkers) between January 2014 and April 2016 were retrospectively reviewed. CT reports were classified as malignant if they included specific terms e.g. “suspicious of malignancy”, “stage M1a” and benign if others were used e.g. “indeterminate”, “no cause identified”. All patients followed a standard diagnostic algorithm. The diagnostic performance of CT (overall and based on the above factors) was assessed using 2×2 Contingency Tables. Results30/345 (9%) eligible patients were excluded (non-contrast (n=13) or non-contiguous CT (n=4), incomplete follow-up (n=13)). 195/315 (62%) patients studied had PM; 90% were cyto-histologically confirmed. 172/315 (55%) presented as an acute admission, of whom 31/172 (18%) had CTPA. Overall, CT sensitivity was 58% (95% CI 51–65%); specificity was 80% (95% CI 72–87%). Sensitivity of CTPA (performed in 31/315 (10%)) was lower (27% (95% CI 9–53%)) than venous-phase CT (61% (95% CI 53–68%) p=0.0056). Sensitivity of specialist thoracic radiologist reporting was higher (68% (95% CI 55–79%)) than non-specialist reporting (53% (95% CI 44–62%) p=0.0488). Specificity was not significantly different. ConclusionThe diagnostic performance of CT in routine clinical practice is insufficient to exclude or confirm PM. A benign CT report should not dissuade pleural sampling where the presence of primary or secondary pleural malignancy would alter management. Sensitivity is lower with non-thoracic radiology reporting and particularly low using CTPA.