Search

Supplementary file containing Supplementary Methods (pertaining to reagents, imaging, immunoblotting, cellular studies, microarray data, NF-κB ELISA, PCR, cell culture, histology, cytology, flow cytometry, constructs, and cellular Assays), 4 Supplementary Tables (pertaining to number of experimental mice, PCR primers, antibodies, and lentiviral shRNA pools), and 7 Supplementary Figures (described in the main and Supplementary text).

Although oncogenic activation of NFκB has been identified in various tumors, the NFκB–activating kinases (inhibitor of NFκB kinases, IKK) responsible for this are elusive. In this study, we determined the role of IKKα and IKKβ in KRAS-mutant lung adenocarcinomas induced by the carcinogen urethane and by respiratory epithelial expression of oncogenic KRASG12D. Using NFκB reporter mice and conditional deletions of IKKα and IKKβ, we identified two distinct early and late activation phases of NFκB during chemical and genetic lung adenocarcinoma development, which were characterized by nuclear translocation of RelB, IκBβ, and IKKα in tumor-initiated cells. IKKα was a cardinal tumor promoter in chemical and genetic KRAS-mutant lung adenocarcinoma, and respiratory epithelial IKKα-deficient mice were markedly protected from the disease. IKKα specifically cooperated with mutant KRAS for tumor induction in a cell-autonomous fashion, providing mutant cells with a survival advantage in vitro and in vivo. IKKα was highly expressed in human lung adenocarcinoma, and a heat shock protein 90 inhibitor that blocks IKK function delivered superior effects against KRAS-mutant lung adenocarcinoma compared with a specific IKKβ inhibitor. These results demonstrate an actionable requirement for IKKα in KRAS-mutant lung adenocarcinoma, marking the kinase as a therapeutic target against this disease.Significance: These findings report a novel requirement for IKKα in mutant KRAS lung tumor formation, with potential therapeutic applications. Cancer Res; 78(11); 2939–51. ©2018 AACR.

Background: It has been postulated that when the intrapleural tumour burden is high, the resultant obliteration of normal mesothelial cell surface of the pleura results in reduction in pleurodesis success rate. Aims: to assess the hypothesis that tumour burden is associated with higher pleurodesis failure, and that tumour type can affect pleurodesis outcomes Methods: Review of recorded video footage of local anaesthetic thoracoscopy (LAT) procedures of 45 patients with proven MPE was conducted by 2 independent assessors blinded to the patient medical records. Abnormalities were assessed according to the presence or absence of; nodules, lymphangitis, inflammation, and adhesions on each of the parietal, visceral and diaphragmatic surfaces. A macroscopic score was developed by adding the number of abnormalities in each pleural surface to produce a total score for tumour burden which was correlated with tumour type and pleurodesis outcome Results: In both mesothelioma (n=21) and non-mesothelioma (n=24), there were no significant differences between the tumour burden score and the outcome of pleurodesis (p=0.188 and 0.173 respectively). The rate of pleurodesis success was higher in the non-mesothelioma group (n=16; 66.7%) compared to the mesothelioma group (n= 9; 42.9%) with no significant difference between both groups (p=0.11) Conclusion: We found no relationship between tumor burden and pleurodesis outcome. Further prospective evaluation in a larger cohort is underway. Consistent with the reported literature, we found that mesothelioma has a high failure rate of pleurodesis compared to non-mesothelioma patients

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 Plasminogen-Activator Inhibitor-1 (PAI-1) and other inflammatory and fibrinolysis-associated proteins as well as to correlate these with clinically important outcomes. Methods: We analysed 214 pleural fluid samples from the PILOT study, a prospective observational pleural infection study, for inflammatory and fibrinolysis-associated proteins using the Luminex platform. Multivariate regression analyses were utilised to assess association of pleural biological markers with septation presence and severity (on ultrasound), and clinical outcomes. Results: PF PAI-1 level was the only protein independently associated with septation presence (p=

Background: Thoracoscopy is the “gold standard” diagnostic modality for investigation of suspected pleural malignancy. It is postulated that meticulous assessment of the pleural cavity may be adequate to indicate malignancy through the macroscopic findings of nodules, pleural thickening, and lymphangitis. We attempted to critically assess this practice, by precisely defining objective macroscopic criteria which might differentiate benign from malignant pleural diseases according to intrapleural pattern and anatomical location, and thereby to explore the predilection of abnormalities to specific sites on pleural surfaces. Methods: A structured review of recorded video footage from medical thoracoscopy procedures in 96 patients was conducted by 2 independent assessors. Abnormalities were scored on agreed, objective criteria for the presence of nodules, lymphangitis and inflammation on each of the costoparietal, visceral and diaphragmatic surfaces. The costoparietal pleura was divided into 6 levels (apical, middle, and inferior surfaces of the lateral and posterior parietal pleura). The anterior surface of the costoparietal pleura was excluded from analysis after interim review as this surface was rarely seen. Results: In the benign group, inflammation was the predominant finding in 65% (n = 33; costoparietal), 44% (n = 21; visceral), and 42% (n = 15; diaphragmatic). Nodules were detected in 24% (n = 12; costoparietal), 8% (n = 4; visceral), and 8% (n = 3; diaphragmatic). The most affected surfaces with inflammation were the middle lateral (60%) and the inferior lateral (57.8%) parts of the costoparietal pleura. In the malignant group, nodules were the predominant finding according to surface in 73% (n = 33; costoparietal), 32% (n = 13; visceral) and 48% (n = 17; diaphragmatic). Inflammation was detected in 44% (n = 20; costoparietal), 25% (n = 10; visceral), and 29% (n = 10; diaphragmatic). The most affected surfaces with nodules were the middle lateral (67.4%) and inferior lateral (66.7%) costoparietal pleural surfaces. Conclusion: This is the first detailed, anatomical description of abnormalities in the pleural space during thoracoscopy. While nodules were the predominant pattern in malignant pleural effusion, they were detected in 24% of benign diagnoses. Detection of nodules in >1 area of the costoparietal pleura was in favor of a malignant diagnosis. Inflammation was the predominant pattern in benign pleural effusion. Our results suggest that macroscopic nodules in malignant diagnoses have a predilection for the middle and inferior surfaces of the lateral costoparietal pleura.

Pleural biopsy findings offer greater diagnostic sensitivity in malignant pleural effusions compared with pleural fluid. The adequacy of pleural biopsy techniques in achieving molecular marker status has not been studied, and such information (termed "actionable" histology) is critical in providing a rational, efficient, and evidence-based approach to diagnostic investigation.What is the adequacy of various pleural biopsy techniques at providing adequate molecular diagnostic information to guide treatment in malignant pleural effusions?This study analyzed anonymized data on 183 patients from four sites across three countries in whom pleural biopsy results had confirmed a malignant diagnosis and molecular profiling was relevant for the diagnosed cancer type. The primary outcome measure was adequacy of pleural biopsy for achieving molecular marker status. Secondary outcomes included clinical factors predictive of achieving a molecular diagnosis.The median age of patients was 71 years (interquartile range, 63-78 years), with 92 of 183 (50%) male. Of the 183 procedures, 105 (57%) were local anesthetic thoracoscopies (LAT), 12 (7%) were CT scan guided, and 66 (36%) were ultrasound guided. Successful molecular marker analysis was associated with mode of biopsy, with LAT having the highst yield and ultrasound-guided biopsy the lowest (LAT vs CT scan guided vs ultrasound guided: LAT yield, 95%; CT scan guided, 86%; and ultrasound guided, 77% [P = .004]). Biopsy technique and size of biopsy sample were independently associated with successful molecular marker analysis. LAT had an adjusted OR for successful diagnosis of 30.16 (95% CI, 3.15-288.56; P = .003) and biopsy sample size an OR of 1.18 (95% CI, 1.02-1.37) per millimeter increase in tissue sample size (P .03).Although previous studies have shown comparable overall diagnostic yields, in the modern era of targeted therapies, this study found that LAT offers far superior results to image-guided techniques at achieving molecular profiling and remains the optimal diagnostic tool.

Intro: The pandemic has put a strain on healthcare services and resources. Overall procedure capacity was reduced and cancer diagnostic pathways were disrupted. The procedural urgency associated with symptomatic patients with often undiagnosed malignant pleural effusion means that nasopharyngeal PCR testing to exclude COVID-19 prior to procedures can often be impractical or logistically challenging Aim: To assess two hypotheses; that SARS-CoV-2 could not be detected in pleural fluid samples from patients with no clinical features of COVID-19 and that pleural procedures can be conducted safely using a pragmatic infection control strategy in the context of a respiratory pandemic Methods: 24 patients completed a screening questionnaire covering symptoms, travel and contacts. If there was no clinical evidence of COVID-19 infection, the day case intervention could proceed as planned, otherwise a negative nasopharyngeal PCR swab within 48 hours was required. Pleural fluid specimens were prospectively collected for the Oxford Radciffe Biobank study and promptly processed and stored using pre-specified protocols. Viral RNA extraction was performed using the QIAamp UltraSens kit (Qiagen, Germany) Result: All patients had no clinical evidence of COVID-19 infection and therefore none required nasopharyngeal PCR swabs. qPCR assays did not detect SARS-CoV-2 virus in pleural fluid of any of the patients. Conclusion: In the absence of clinical evidence of COVID-19, SARS-CoV-2 is unlikely to be detected in pleural fluid. In the face of an ongoing pandemic, urgent outpatient pleural procedures can be safely continued, so as not to negatively impact the care for non-COVID patients

Lung adenocarcinoma (LADC) is the leading cause of cancer death worldwide. Nevertheless, syngeneic mouse models of the disease are sparse, and cell lines suitable for transplantable and immunocompetent mouse models of LADC remain unmet needs. We established multiple mouse LADC cell lines by repeatedly exposing two mouse strains (FVB, Balb/c) to the tobacco carcinogens urethane or diethylnitrosamine and by culturing out the resulting lung tumours for prolonged periods of time. Characterization of the resulting cell lines (n = 7) showed that they were immortal and phenotypically stable in vitro, and oncogenic, metastatic and lethal in vivo. The primary tumours that gave rise to the cell lines, as well as secondary tumours generated by transplantation of the cell lines, displayed typical LADC features, such as glandular architecture and mucin and thyroid transcription factor 1 expression. Moreover, these cells exhibited marked molecular similarity with human smokers’ LADC, including carcinogen-specific Kras point mutations (KrasQ61R in urethane- and KrasQ61H in diethylnitrosamine-triggered cell lines) and Trp53 deletions and displayed stemness features. Interestingly, all cell lines overexpressed proliferin, a murine prolactin orthologue, which functioned as a lung tumour promoter. Furthermore, prolactin was overexpressed and portended poor prognosis in human LADC. In conclusion, we report the first LADC cell lines derived from mice exposed to tobacco carcinogens. These cells closely resemble human LADC and provide a valuable tool for the functional investigation of the pathobiology of the disease.

Malignant pleural effusions (MPE) are common and associated with disabling symptoms, and large hospitalisation costs [1]. Current management of recurrent MPE is symptomatic, with chest drain and talc pleurodesis, or indwelling pleural catheter (IPC) insertion. IPCs are an increasingly attractive option in patients wishing to avoid hospitalisation as they reduce initial length of stay [2, 3], but require repeated domiciliary drainage. Other patients opt for talc pleurodesis because, if successful, it represents a one-time definitive procedure, but it has a modest failure rate (20–30%) [4]. In the case of talc pleurodesis failure, IPC insertion is usually advocated with the consequence that a subset of patients with IPC in situ will have previously received ipsilateral intrapleural talc. IPCs are also increasingly being used in patients with recurrent benign pleural effusions (non-MPE) [5, 6]., Prior talc pleurodesis does not result in worsened outcomes from subsequent indwelling pleural catheter use, and patients should not be dissuaded from choosing talc as a primary treatment for recurrent pleural effusion. http://ow.ly/qAAC30mYmr3

Background Pleural infection is a common and severe disease with high morbidity and mortality worldwide. The knowledge of pleural infection bacteriology remains incomplete, as pathogen detection methods based on culture have insufficient sensitivity and are biased to selected microbes. We designed a study with the aim to discover and investigate the total microbiome of pleural infection and assess the correlation between bacterial patterns and 1-year survival of patients. Methods We assessed 243 pleural fluid samples from the PILOT study, a prospective observational study on pleural infection, with 16S rRNA next generation sequencing. 20 pleural fluid samples from patients with pleural effusion due to a non-infectious cause and ten PCR-grade water samples were used as controls. Downstream analysis was done with the DADA2 pipeline. We applied multivariate Cox regression analyses to investigate the association between bacterial patterns and 1-year survival of patients with pleural infection. Findings Pleural infection was predominately polymicrobial (192 [79%] of 243 samples), with diverse bacterial frequencies observed in monomicrobial and polymicrobial disease and in both community-acquired and hospital-acquired infection. Mixed anaerobes and other Gram-negative bacteria predominated in community-acquired polymicrobial infection whereas Streptococcus pneumoniae prevailed in monomicrobial cases. The presence of anaerobes (hazard ratio 0·46, 95% CI 0·24–0·86, p=0·015) or bacteria of the Streptococcus anginosus group (0·43, 0·19–0·97, p=0·043) was associated with better patient survival, whereas the presence (5·80, 2·37–14·21, p Interpretation Pleural infection is a predominantly polymicrobial infection, explaining the requirement for broad spectrum antibiotic cover in most individuals. High mortality infection associated with S aureus and Enterobacteriaceae favours more aggressive, with a narrower spectrum, antibiotic strategies. Funding UK Medical Research Council, National Institute for Health Research Oxford Biomedical Research Centre, Wellcome Trust, Oxfordshire Health Services Research Committee, Chinese Academy of Medical Sciences, and John Fell Fund.

Background Thoracoscopy is the ‘gold standard’ diagnostic modality for investigation of suspected pleural malignancy.1 It is postulated that meticulous assessment of the pleural cavity may be adequate to diagnose malignancy through finding of nodules, pleural thickening and lymphangitis.2 Given the increased uptake of local anaesthetic thoracoscopy (LAT) recently, we attempted to define precise, objective criteria to differentiate benign from malignant pleural diseases according to the pattern, anatomical site and exploring predilection of abnormalities to specific sites on the pleural surfaces. Methods A structured review of recorded video footage from LAT procedures in 96 patients was conducted by 2 independent assessors. Abnormalities were scored according to the presence or absence of nodules, lymphangitis, inflammation on each of the parietal, visceral and diaphragmatic surfaces, respectively. The parietal pleura was divided into 6 levels (apical, middle, and inferior of the lateral surface and apical, middle, and inferior of the posterior surface). The anterior surface of the parietal pleura was excluded due to difficulty of assessment. Results In the benign group, inflammation was the predominant finding in 65%(n=33; parietal), 44%(n=21; visceral) and 42%(n=15; diaphragmatic). Nodules were detected in 24%(n=12; parietal), 8% (n=4; visceral) and 8%(n=3; diaphragmatic). The most affected surfaces with inflammation were the middle lateral (60%) and the inferior lateral (57.8%) parts of the parietal pleura. In the malignant group, nodules were the predominant finding in 73%(n=33; parietal), 32%(n=13; visceral) and 48%(n=17; diaphragmatic). Inflammation was detected in 44%(n=20; parietal), 25%(n=10; visceral) and 29%(n=10; diaphragmatic). The most affected surfaces with nodules were the middle lateral (67.4%) and the inferior lateral (66.7%) parts of the parietal pleura. Conclusion This study suggests that macroscopic assessment at LAT can differentiate between benign and malignant pleural disease, with the predominance of inflammation and nodules, respectively. In addition, pleural abnormalities appear to have an anatomical predilection, emphasising the importance of inspection and sampling of these areas. The finding that nodules were seen in approximately 1 in 4 benign patients on the parietal pleura is of interest and warrants further exploration in a larger cohort. References Rahman NM. Thorax 2010; 65: ii54–ii60. Boutin C. Endoscopy 1980; 12(04): 155–160.

Background It has been postulated that when the intrapleural tumour burden is high, the resultant obliteration of normal mesothelial cell surface of the pleura results in reduction in pleurodesis success rate.1 This effect has been suggested in the context of worse pleurodesis outcomes in malignant pleural effusion (MPE) secondary to lung cancer or mesothelioma, with more favourable outcomes seen in breast and ovarian cancer.2 This study therefore aimed to assess the hypothesis that tumour burden is associated with higher pleurodesis failure, and that tumour type can affect pleurodesis outcomes. Methods A structured, retrospective review of recorded video footage of local anaesthetic thoracoscopy (LAT) procedures of 45 patients with proven MPE was conducted by 2 independent assessors blinded to the patient medical records. Abnormalities were assessed according to the presence or absence of; nodules, lymphangitis, inflammation, and adhesions on each of the parietal, visceral and diaphragmatic surfaces. A macroscopic score was developed by adding the number of abnormalities in each pleural surface to produce a total score for tumour burden. Two separate assessors correlated the findings with tumour type and pleurodesis outcome from the patient`s clinical records. Results In both mesothelioma (n=21) and non-mesothelioma (n=24), there were no significant differences between the tumour burden score and the outcome of pleurodesis (p=0.188 and 0.173 respectively). The rate of pleurodesis success was higher in the non-mesothelioma group (n=16; 66.7%) compared to the mesothelioma group (n= 9; 42.9%) with no significant difference between both groups (p=0.11) Conclusion In this study, we found no relationship between tumor burden and pleurodesis outcome. While the blinded and detailed analysis of LAT video footage is an important strength of this study, it has significant limitations, namely small numbers and retrospective methodology. Nonetheless it adds important insights that would go against our assumed understanding of this relationship between tumour burden and pleurodesis success. Further prospective evaluation in a larger cohort is underway. Consistent with the reported literature, we found that mesothelioma has a high failure rate of pleurodesis compared to non-mesothelioma patients. References Rodriguez-Panadero F. Respiration 2012; 83(2): 91–98; Bielsa S. Lung 2011;189(2): 151–155.

Background Pleural infection (PI) is a common and complicated disease. Empirical antibiotic usage has been correlated to poor clinical outcomes. Although the identification of the pathogen is essential for successful treatment, conventional culture-based pathogen detection techniques fail in approximately 40% of cases. Therefore, the bacteriology of PI remains incomplete. Next generation sequencing (NGS) is a molecular-based methodology which could be applied to metagenomics studies and improve pathogen recognition. Aim To investigate and characterise the bacterial patterns of PI. Methods Pleural fluid specimens from the ‘Pleural Infection Longitudinal Outcome Study’1 (PILOT, n=243) were subjected to bacterial DNA extraction followed by 16S rRNA NGS. The DADA2 and Phyloseq R packages were used for the analysis of the data. Results We identified 363 distinct species of bacteria, with various abundances among the samples. Diverse patterns between monomicrobial and polymicrobial PI were detected. 131 (54%) samples had one pathogen with abundance over 50% and 89 (36%) samples had at least three pathogens with relative abundance over 10%, suggesting a polymicrobial infection. Discussion We developed a methodology to extract bacterial DNA from pleural fluid specimens derived from patients with PI and the quality was satisfactory to be used for NGS. 16S rRNA gene NGS has the potential to detect the total microbiome of pleural fluid samples1 from complex PI. Funding National Institute for Health Research (NIHR) Oxford Biomedical Research Centre (BRC). Reference Corcoran, J.P., et al. Prospective validation of the RAPID clinical risk prediction score in adult patients with pleural infection: the PILOT study. Eur Respir J ( 2020).

Systemic inflammatory diseases are a heterogeneous family of autoimmune chronic inflammatory disorders that affect multiple systems within the human body. Connective tissue disease (CTD) is a large group within this family characterised by immune-mediated inflammation of the connective tissue. This group of disorders are often associated with pleural manifestations. CTD-induced pleuritis exhibits a wide variety of symptoms and signs including exudative pleural effusions and chest pain. Accurate estimation of prevalence for CTD-related pleuritis is challenging as small effusions are asymptomatic and remain undetected. Rheumatoid arthritis and systemic lupus erythematosus are frequent CTDs and present with pleural pathology in approximately 5–20% and 17–60% of cases, respectively. By contrast, pleural involvement in systemic sclerosis, eosinophilia–myalgia syndrome, mixed connective tissue disease, ankylosing spondylitis, polymyositis and dermatomyositis syndrome is rare. Clinical management depends on the severity of symptoms; however, most effusions resolve spontaneously. In this review we discuss the pathophysiological mechanisms and the clinical considerations of CTD-induced pleuritis., Chronic inflammatory diseases could cause pleural pathology. Clinical management depends on the severity of symptoms, but most effusions resolve spontaneously. https://bit.ly/333euHb

Background Most patients with malignant pleural mesothelioma (MPM) seek treatment with malignant pleural effusion (MPE). In vitro evidence suggests that MPE may not be a simple bystander of malignancy, but rather potentially has biological properties that improve cancer cell survival and promote cancer progression. If this is the case, MPE management may need to shift from current symptomatic strategies to aggressive fluid removal to impact survival. Research Question Is there an association between pleural fluid exposure and survival in MPM? Study Design and Methods Data from 761 patients who received a diagnosis of MPM between 2008 and 2018 were collected from patient medical records in three UK pleural units. Data included factors previously identified as influencing prognosis in MPM. Medical imaging was reviewed for presence, size, and duration of pleural effusion. Time-dependent covariate analysis of pleural fluid exposure and survival (model included weight loss, serum albumin, hemoglobin, MPM subtype, performance status, chemotherapy, and age) and multivariate Cox regression analysis of pleurodesis and survival were conducted. Results Median overall survival was 278 days (interquartile range, 127-505 days; 95% CI, 253-301 days). Pleural fluid exposure duration showed no association with survival (hazard ratio, 1.0; 95% CI, 1.0-1.0). Median survival was 473, 378, and 258 days with complete, partial, and no pleurodesis (P = .008). Interpretation Pleurodesis success seems to be associated with improved survival; however, it is unclear whether duration of MPM exposure to pleural fluid is associated with survival within the limitations of this retrospective study. Future prospective studies are required to assess this potentially important mechanism.

Background: Pleural infection (PI) is a common and complex disease which can be life threatening for immunocompromised and elderly populations. Prior antibiotic use and special bacterial nutritional requirements hamper the accuracy of bacterial identification using current clinical culture-based techniques. Consequently, PI microbiology remains unclear. Next generation sequencing (NGS) has the potential to improve identification of the total bacterial population of a complex sample. Aim: To discover and characterise the microbial patterns of PI using NGS and bioinformatics techniques. Methods: Pleural fluid samples from the “Pleural Infection Longitudinal Outcome Study” (PILOT, ISRCTN50236700, n=243) underwent bacterial DNA extraction followed by 16S rRNA NGS using Illumina MiSeq. Data were analysed with DADA2 and Phyloseq R packages. Results: Analysis showed diverse microbiological patterns for PI as 391 different pathogens were identified up to the genus level. 131 (54%) samples had one pathogen with relative abundance over 50% and 89 (36%) samples had at least three pathogens with relative abundance over 10%, suggesting a polymicrobial infection. Streptococcus pneumoniae was detected in 40 (16%) and Staphylococcus aureus in 20 (8%) samples. Discussion: We established a methodology to extract bacterial DNA from patients with PI and used it as a template to apply NGS. 16S rRNA gene NGS provides a robust method to investigate the bacteriological patterns in pleural fluid of patients with PI. Funding: National Institute for Health Research, Oxford Biomedical Research Centre

Malignant pleural mesothelioma(MPM) presents with malignant pleural effusion(MPE) in most patients. There is pre-clinical evidence that MPE may not be a simple bystander of malignancy,but potentially has biological properties increasing cancer cell proliferation.If this is the case,management of MPE may need to shift from current symptomatic strategies to aggressive fluid removal to impact on survival. Aim: to analyse the association of pleural fluid exposure(duration of effusion,& pleurodesis success)with survival in MPM. Method:Data on 761 patients diagnosed with MPM between 2008-2018 was collected from patients’ medical records in 3 UK pleural units.The medical images were reviewed for presence,size & duration of pleural effusion in order to analyse pleural fluid exposure(total number of days pleural effusion was present overall) & pleurodesis effects. Results: The median overall survival was 278 days(IQR 378).Median survival: 473,378,258 days (p 0.0006) with complete, partial,& no pleurodesis respectively.With time-dependent analysis,pleurodesis success remained a significant influencing factor on survival (p 0.002). There was no association between pleural fluid exposure time & survival (p 0.2) with time-dependent covariate analysis(model included serum albumin,MPM subtype,performance status,presence of symptoms at diagnosis,chemotherapy received). Conclusion: Pleurodesis success is associated with improved survival,however given the limitations of this retrospective study,it is unclear whether duration of MPM exposure to pleural fluid correlates with survival.Future prospective studies are required to assess this potentially important mechanism.

IntroductionThe rising incidence of pleural disease is seeing an international growth of pleural services, with physicians performing an ever-increasing volume of pleural interventions. These are frequently conducted at sites without immediate access to thoracic surgery or interventional radiology and serious complications such as pleural bleeding are likely to be under-reported.AimTo assess whether intercostal vessel screening can be performed by respiratory physicians at the time of pleural intervention, as an additional step that could potentially enhance safe practice.MethodsThis was a prospective, observational study of 596 ultrasound-guided pleural procedures conducted by respiratory physicians and trainees in a tertiary centre. Operators did not have additional formal radiology training. Intercostal vessel screening was performed using a low frequency probe and the colour Doppler feature.ResultsThe intercostal vessels were screened in 95% of procedures and the intercostal artery (ICA) was successfully identified in 53% of cases. Screening resulted in an overall site alteration rate of 16% in all procedures, which increased to 30% when the ICA was successfully identified. This resulted in procedure abandonment in 2% of cases due to absence of a suitable entry site. Intercostal vessel screening was shown to be of particular value in the context of image-guided pleural biopsy.ConclusionIntercostal vessel screening is a simple and potentially important additional step that can be performed by respiratory physicians at the time of pleural intervention without advanced ultrasound expertise. Whether the widespread use of this technique can improve safety requires further evaluation in a multi-centre setting with a robust prospective study.

ImportancePleural effusions frequently signal disseminated cancer. Diagnostic markers of pleural malignancy at presentation that would assess cancer risk and would streamline diagnostic decisions remain unidentified.ObjectiveThe present study aimed at identifying and validating predictors of malignant pleural effusion at patient presentation.DESIGN, SETTING, AND PARTICIPANTSA consecutive cohort of 323 patients with pleural effusion (PE) from different etiologies were recruited between 2013-2017 and was retrospectively analyzed. Data included history, chest X-ray, and blood/pleural fluid cell counts and biochemistry. Group comparison, receiver-operator characteristics, unsupervised hierarchical clustering, binary logistic regression, and random forests were used to develop the malignant pleural effusion detection (MAPED) score. MAPED was validated in an independent retrospective UK cohort (n= 238).Main Outcomes and MeasuresThe outcome was diagnostic of pleural effusion in patients, and the clinical and laboratory indicators available of the patient were measured.ResultsFive variables showed significant diagnostic power and were incorporated into the 5-point MAPED score. Age > 55 years, effusion size > 50% of the most affected lung field, pleural neutrophil count < 2,500/mm3, effusion protein > 3.5 g/dL, and effusion lactate dehydrogenase > 250 U/L, each scoring one point, predicted underlying cancer with the area under curve(AUC) = 0.819 (sensitivity=82%, specificity=74%,P< 10-15) in the derivation cohort. The AUC and net reclassification improvement (NRI) of MAPED score and cytology were not significantly different. However, the integrated discrimination improvement (IDI) of The MAPED score displayed a slight increment(PConclusionsThe MAPED score identifies malignant pleural effusions with satisfactory accuracy and can be used complementary to cytology to streamline diagnostic procedures.

Malignant pleural mesothelioma (MPM) is an aggressive cancer, associated with poor prognosis. We assessed the feasibility of patient-derived cell cultures to serve as an ex vivo model of MPM. Patient-derived MPM cell cultures (n=16) exhibited stemness features and reflected intratumour and interpatient heterogeneity. A subset of the cells were subjected to high-throughput drug screening and coculture assays with cancer-specific cytotoxic T cells and showed diverse responses. Some of the biphasic MPM cells were capable of processing and presenting the neoantigen SSX-2 endogenously. In conclusion, patient-derived MPM cell cultures are a promising and faithful ex vivo model of MPM.

Summary Background The prevalence of malignant pleural effusion is increasing worldwide, but prognostic biomarkers to plan treatment and to understand the underlying mechanisms of disease progression remain unidentified. The PROMISE study was designed with the objectives to discover, validate, and prospectively assess biomarkers of survival and pleurodesis response in malignant pleural effusion and build a score that predicts survival. Methods In this multicohort study, we used five separate and independent datasets from randomised controlled trials to investigate potential biomarkers of survival and pleurodesis. Mass spectrometry-based discovery was used to investigate pleural fluid samples for differential protein expression in patients from the discovery group with different survival and pleurodesis outcomes. Clinical, radiological, and biological variables were entered into least absolute shrinkage and selection operator regression to build a model that predicts 3-month mortality. We evaluated the model using internal and external validation. Findings 17 biomarker candidates of survival and seven of pleurodesis were identified in the discovery dataset. Three independent datasets (n=502) were used for biomarker validation. All pleurodesis biomarkers failed, and gelsolin, macrophage migration inhibitory factor, versican, and tissue inhibitor of metalloproteinases 1 (TIMP1) emerged as accurate predictors of survival. Eight variables (haemoglobin, C-reactive protein, white blood cell count, Eastern Cooperative Oncology Group performance status, cancer type, pleural fluid TIMP1 concentrations, and previous chemotherapy or radiotherapy) were validated and used to develop a survival score. Internal validation with bootstrap resampling and external validation with 162 patients from two independent datasets showed good discrimination (C statistic values of 0·78 [95% CI 0·72–0·83] for internal validation and 0·89 [0·84–0·93] for external validation of the clinical PROMISE score). Interpretation To our knowledge, the PROMISE score is the first prospectively validated prognostic model for malignant pleural effusion that combines biological and clinical parameters to accurately estimate 3-month mortality. It is a robust, clinically relevant prognostic score that can be applied immediately, provide important information on patient prognosis, and guide the selection of appropriate management strategies. Funding European Respiratory Society, Medical Research Funding-University of Oxford, Slater & Gordon Research Fund, and Oxfordshire Health Services Research Committee Research Grants.

Background and Objective: Chemical pleurodesis is performed for patients with MPE with a published success rate of around 80%. It has been postulated that inflammation is key in achieving successful pleural symphysis, as evidenced by higher amounts of pain or detected inflammatory response. Patients with mesothelioma are postulated to have a lower rate of successful pleurodesis due to lack of normal pleural tissue enabling an inflammatory response. Methods: The TIME1 trial data set, in which pleurodesis success and pain were co‐primary outcome measures, was used to address a number of these assumptions. Pain score, systemic inflammatory parameters as a marker of pleural inflammation and cancer type were analysed in relation to pleurodesis success. Results: In total, 285 patients were included with an overall success rate of 81.4%. There was a significantly higher rise in CRP in the Pleurodesis Success group compared with the Pleurodesis Failure group (mean difference: 19.2, 95% CI of the difference: 6.2–32.0, P = 0.004) but no significant change in WCC. There was no significant difference in pain scores or analgesia requirements between the groups. Patients with mesothelioma had a lower rate of pleurodesis success than non‐mesothelioma patients (73.3% vs 84.9%, χ2 = 5.1, P = 0.023). Conclusion: Change in CRP during pleurodesis is associated with successful pleurodesis but higher levels of pain are not associated. Patients with mesothelioma appear less likely to undergo successful pleurodesis than patients with other malignancies, but there is still a significant rise in systemic inflammatory markers. The mechanisms of these findings are unclear but warrant further investigation.

Introduction The chest radiograph is used in clinical practice to guide decision-making in the treatment of pleural infection. This outcome was used as the primary endpoint in the randomised second Multicentre Intrapleural Sepsis Trial (MIST-2), defined as the change in area of pleural opacity, measured as the percentage of the ipsilateral hemithorax occupied by effusion, from day 1 to day 7 [1]. The value of this radiographic outcome measure as a surrogate for predicting clinically important outcomes, e.g. time in hospital (LOS), surgery at 3 months and 3-month mortality, has not been directly addressed. Methods Retrospective analyses were conducted using the prospectively collected data from the MIST-2 database (n=210). Regression analyses were modelled with number of days in hospital (linear), and surgery or death at 3 months, both individually and as a combined outcome (yes/no; logistic), as dependent variables. The independent variables were absolute change in chest radiograph opacity (MIST-2 primary endpoint) and relative change, which is more clinically applicable in daily practice (a secondary endpoint in MIST-2). Each of the analyses was corrected for day 1 radiograph appearance to account for baseline variability. SPSS v25 was used for all analyses. Results Absolute and relative change in chest radiograph opacity were associated with hospital LOS and either surgery or death at 3 months (combined outcome) with strong statistical significance (p≤0.01). Analysing the components of the combined outcome individually, absolute and relative change were associated with surgery at 3 months (p≤0.01 and p=0.021 respectively). Absolute and relative change in chest radiograph to death at 3 months alone was borderline significant (p=0.089) and non-significant (p=0.16) respectively. Conclusion These findings demonstrate that change in chest radiograph during the course of treatment of pleural infection is a robust and clinically important surrogate endpoint which appears to predict meaningful outcomes. Although surgery may be decided upon solely on the basis of the radiograph (which would explain this result), change in x-ray appearance predicts other important outcomes (length of stay). This data supports its clinical utility, and suggests its robust use as a research outcome measure.

BackgroundPleural infection (PI) is a major global disease with an increasing incidence, and pleural fluid (PF) drainage is essential for the successful treatment. The MIST2 study demonstrated that intrapleural administration of tissue plasminogen activator (t-PA) and DNase, or t-PA alone increased the volume of drained PF. Mouse model studies have suggested that the volume increase is due to the interaction of the pleura with the t-PA via the monocyte chemoattractant protein 1 (MCP-1) pathway. We designed a study to determine the time frame of drained PF volume induction on intrapleural delivery of t-PA±DNase in humans, and to test the hypothesis that the induction is mediated by the MCP-1 pathway.MethodsData and samples from the MIST2 study were used (210 PI patients randomised to receive for 3 days either: t-PA and DNase, t-PA and placebo, DNase and placebo or double placebo). PF MCP-1 levels were measured by ELISA. One-way and two-way analysis of variance (ANOVA) with Tukey’s post hoc tests were used to estimate statistical significance. Pearson’s correlation coefficient was used to assess linear correlation.ResultsIntrapleural administration of t-PA±DNase stimulated a statistically significant rise in the volume of drained PF during the treatment period (days 1–3). No significant difference was detected between any groups during the post-treatment period (days 5–7). Intrapleural administration of t-PA increased MCP-1 PF levels during treatment; however, no statistically significant difference was detected between patients who received t-PA and those who did not. PF MCP-1 expression was not correlated to the drug given nor the volume of drained PF.ConclusionsWe conclude that the PF volume drainage increment seen with the administration of t-PA does not appear to act solely via activation of the MCP-1 pathway.

Background: Malignant pleural mesothelioma (MPM) is an incurable and aggressive malignancy, which is typically presented with malignant pleural effusion (MPE), bearing severely comprised quality of life and short survival. Since management of MPE remains symptomatic and targeted MPM treatments are not available the development of translational models to improve patients’ stratification are desperately needed. Aim: To establish a panel of patient derived MPM cell lines which can serve as a faithful model for the discovery of novel mutations and the development of targeted treatments. Methods: MPE samples from MPM patients were collected and cultured to establish a pure cancer cell population. To cross-examine the genomic background of the MPM cells between different passages, a proportion of the cells was kept from each passage. Results: We established a panel of 18 primary personalised mesothelioma cell (PMC) lines. Phenotypically PMC lines exhibited pleomorphic, atypical and often multiple nucleoli. In vitro, the cells were immortal and able to form colonies and tumour-spheres. A high-throughput drug screening assay was used to identify the cluster of the most potent anticancer drugs, demonstrating a heterogeneous response for each PMC line. Genome profiling revealed no major differences for important cancer genes among the passages. T cells from the MPEs were isolated, cultured and examined regarding their cellular and molecular phenotype. Conclusions: We developed a methodology to establish patient derived MPM cell lines which could potentially be used as tools to refine patients’ management.

Lung cancer and chronic lung diseases impose major disease burdens worldwide and are caused by inhaled noxious agents including tobacco smoke. The cellular origins of environmental-induced lung tumors and of the dysfunctional airway and alveolar epithelial turnover observed with chronic lung diseases are unknown. To address this, we combined mouse models of genetic labeling and ablation of airway (club) and alveolar cells with exposure to environmental noxious and carcinogenic agents. Club cells are shown to survive KRAS mutations and to form lung tumors after tobacco carcinogen exposure. Increasing numbers of club cells are found in the alveoli with aging and after lung injury, but go undetected since they express alveolar proteins. Ablation of club cells prevents chemical lung tumors and causes alveolar destruction in adult mice. Hence club cells are important in alveolar maintenance and carcinogenesis and may be a therapeutic target against premalignancy and chronic lung disease., eLife digest The deadliest form of lung cancer is called lung adenocarcinoma, or LUAD. Tobacco chemicals often cause the disease by damaging the genetic information of lung cells. The damage leads to harmful changes in the DNA sequence which prompt the cells to form tumors. For instance, the most common of these changes takes place in a gene called KRAS. However, it is still unclear exactly which type of lung cells are more likely to develop into a tumor. In the lungs, airway epithelial cells cover the inside of the passages that bring the air inside little sacks called alveoli, which are lined by alveolar cells. Previous studies have used genetic methods to switch on the KRAS mutation in different compartments of the mouse lung. This showed that groups of airway cells, of alveolar cells, and of a class of cells located at the junction between airways and alveoli could all give rise to cancer. However, these experiments did not examine how tobacco chemicals could give rise to tumors in different groups of lung cells. Here, Spella et al. triggered LUAD in adult mice by exposing them to the toxic chemicals found in tobacco smoke, but without making any change to the KRAS gene. These mice also had genetically engineered reporters that could be used to deduce where the resulting tumors came from. DNA sequencing showed that the airway epithelial cells gained KRAS mutations after the chemical treatment. When the airway epithelial cells were experimentally removed before the treatments with tobacco chemicals, these mice did not get LUAD tumors. Spella et al. also observed that the tobacco-induced tumors came from the epithelial cells in the airways, and not from the cells in the alveoli. Moreover, when the lung was damaged, airway cells could move to the alveoli and start adopting the identity of alveolar cells, thereby replenishing this population. Together, these experiments imply that tobacco-induced LUAD starts in the airway epithelial cells. These findings suggest that airway epithelial cells could be targeted to stop lung cancer early on. Further studies should also examine how airway epithelial cells can transition to look more like alveolar cells when the lungs get harmed.

Introduction and objectives: Malignant pleural effusion (MPE) is a rapidly rising healthcare burden and critically hampers the patients9 survival and quality of life. Current treatments aim to palliate symptoms and talc pleurodesis remains a standard therapeutic modality. There is little high quality data in prediction of successful pleurodesis and therapeutic biomarkers are desperately needed. Aim: To identify and validate novel therapeutic biomarkers for successful pleurodesis in MPE. Methods: Clinical data and pleural fluid from MPE patients prior to treatment were prospectively collected as part of the randomised TIME2 trial. Patients were classified based on the treatment outcome (success/failure based on study datasets). Pleural fluid on enrolment (Success n=15, Failure n=11) were assessed with mass spectrometry profiling after depletion of the 12 most abundant proteins. Full protein profile analysed with R software and ELISA technique was performed for the validation of the results. Results: Mass spectrometry identified 1,154 proteins, 97 of which were statistically different (two tailed t-test, p Conclusions : Based on a unique biobank and clinical database, we have identified 2 therapeutic biomarkers that may stratify patients9 management. These results require validation which is underway using a retrospective (TIME1 trial) and prospective (SIMPLE study) datasets.