BACKGROUND: Malignant and paramalignant pleural effusions are important complications of many malignancies. The two main management options debated in the literature are: 1) insertion of an indwelling pleural catheter (IPC) to achieve chronic drainage of the effusion, or 2) hospitalization with tube thoracostomy and subsequent chemical pleurodesis (CP) with talc or doxycycline to prevent fluid reaccumulation. We aimed to describe a large series of patients with malignant pleural effusions managed with an IPC, identify and validate factors identified in the literature as predictors of spontaneous pleurodesis in the IPC group and compare the group managed with IPC to patients managed with CP. METHODS: We designed a retrospective cohort study comparing patients with malignant and paramalignant pleural effusions managed either with CP between March 1, 2003 and February 28, 2006 or IPC insertion between May 1, 2006 and April 1, 2009. The CP group was identified through the prescription of talc or doxycycline and the IPC group from the IPC clinic database. Data were collected from paper and electronic records and from the Government of Ontario. RESULTS: We identified 193 consecutive patients with an ECOG performance status of less than 4 (ECOG less than 4 means that the patient is not completely disabled and confined to bed or chair) having undergone IPC insertion and 168 who were managed with CP. None of the variables we tested were significant predictors of spontaneous pleurodesis in the IPC group. Pleural effusion control rates at 6 months were higher in the IPC group than in the CP group (52.7% vs 34.0%, p<0.01) but the rate of freedom from pleural effusion at 180 days and catheter removal at 90 days was not significantly different (25.8% in the IPC group and 34.0% in the CP group p=0.14). Patients in the IPC group had a significantly longer median survival (148 days measured from the date of catheter insertion vs 133 days in the CP group, log-rank p<0.05). CONCLUSION: We found an intriguing possible survival benefit favouring management of malignant or paramalignant effusions with an IPC. Given possible biases due to the design of this study and uncertain explanatory mechanism, this needs to be confirmed in a randomized controlled trial. Quality of life, an important measure of success for these palliative procedures, should also be measured.

Le mésothéliome pleural malin (MPM) est une tumeur primitive de la plèvre, rare, très agressive, avec un pronostic sombre. Nous avons souhaité au cours de ce travail de thèse, identifier de nouveaux biomarqueurs du MPM en testant l’influence de l’inactivation des membres de la famille RASSF/Hippo sur la survie des 448 patients inclus dans l’essai clinique MAPS (IFCT-GFPC-0701). Nous souhaitions également comprendre quelles fonctions et signalisations essentielles à l’homéostasie cellulaire, auxquelles participe la voie de signalisation RASSF/Hippo, sont perturbées lors du processus de transformation des cellules mésothéliales. L’inactivation des membres de la voie a été étudiée par PCR spécifique de méthylation (MS-PCR) et leur influence sur la survie des 448 patients inclus dans l’essai clinique MAPS testée en analyse uni- et multivariée avant d’être validée par boostrap. D’autre part, nous avons mimé in cell, l’inactivation par ARN interférence de plusieurs membres de la voie Hippo dans des cellules de lignées mésothéliales humaines (MSTO-211H, H2452, H28 et H2052). Nous avons pu identifier plusieurs biomarqueurs du MPM : i) la kinase MST1 dont l’inactivation est un facteur de mauvais pronostic, ii) l’amphiréguline dont l’expression cytoplasmique est au contraire un facteur de bon pronostic et enfin iii) le CD44 dont l’expression élevée constitue un outil diagnostique du MPM. Les approches in cell, nous ont permis de démontrer que les altérations de la voie RASSF/Hippo induisent une activité inappropriée de l’effecteur terminal YAP : le moins bon pronostic des patients présentant une inactivation de MST1 s’explique ainsi par le fait qu’en régulant l’activité de YAP, MST1 contrôle la balance apoptose/prolifération et prévient l’invasion et la croissance sans adhésion. En son absence, ces processus cellulaires sont dérégulés. Ce travail démontre l’importance de l’axe CD44/RASSF1A/MST1 dans le contrôle d’une activité appropriée de YAP et de l’homéostasie des cellules mésothéliales. La compréhension des désordres cellulaires induits par la dérégulation de le voie RASSF/Hippo, désigne YAP comme cible thérapeutique potentielle chez les patients atteints de MPM et présentant des altérations de cette voie de signalisation. / Malignant pleural mesothelioma (MPM) is a rare, very aggressive, primary tumor with a poor prognosis. During this thesis, we wanted to identify new biomarkers of MPM by testing the influence of the RASSF/Hippo pathway inactivation on the survival of the 448 patients included in the clinical trial MAPS (IFCT- GFPC-0701). We also wanted to understand which functions and signals essential to cellular homeostasis, linked to RASSF/Hippo signaling pathway, are disturbed during the mesothelial cell transformation process. Inactivation of RASSF/Hippo members was studied by methylation-specific PCR (MS-PCR) and their influence on the survival of the 448 patients included in the MAPS clinical trial tested in uni- and multivariate analysis before being validated by bootstrap. We also mimed in cell, by RNA interference, several members of the Hippo pathway inactivation in human mesothelial cells lines (MSTO-211H, H2452, H28 and H2052). We have identified several biomarkers of MPM: i) MST1 kinase whose inactivation is a factor of poor prognosis, ii) amphiregulin whose cytoplasmic expression is on the contrary a factor of good prognosis and finally iii) CD44 whose high expression is a diagnostic tool for MPM. In cell we demonstrate that RASSF/Hippo pathway alterations induce an inappropriate activity of YAP, one Hippo end effector: the poorer prognosis of patients with inactivation of MST1 is thus explained by the fact that, by regulating YAP activity, MST1 controls the apoptosis/proliferation balance and prevents invasion and growth without adhesion from mesothelial cells. In its absence, these cellular processes are deregulated. This work finally demonstrates the importance of the CD44/RASSF1A/MST1 axis in controlling appropriate YAP activity and mesothelial cell homeostasis. The understanding of the cellular disorders induced by the of the RASSF/Hippo pathway deregulation designates YAP as a potential therapeutic target in patients with MPM and presenting alterations of this signaling pathway.

This thesis consists of 3 studies: 1. Determination of the minimal important difference (MID) of the visual analogue scale for dyspnoea (VASD): Determining the MID of the VASD is essential to interpret the results of trials in patients with malignant pleural effusions (MPEs). Patients undergoing a pleural procedure assessed the change in their VASD and the degree of change in their symptoms on a Likert scale. The mean VASD in patients experiencing a ‘small but just worthwhile’ decrease in their symptoms is the MID for the VASD and was found to be 22mm (95% CI 16 - 27mm). 2. Development of a thoracic ultrasound septation score (TUSS): A TUSS is important for objectively assessing the degree of septation within a pleural effusion. An iterative process was used to demonstrate that degree of septation predicts clinical outcome, to identify candidate factors for inclusion in a TUSS and to determine which factors predicted the degree of septation. The final TUSS consisted of an assessment of the degree of homogeneity of septation distribution and number of septations at the most septated area. 3. Effect of an indwelling pleural catheter (IPC) versus standard care for relieving dyspnoea in patients with MPEs: the TIME2 randomised controlled trial (RCT). The objective of this unblinded RCT was to determine whether IPCs are more effective than chest drains and talc pleurodesis at relieving dyspnoea in patients with MPEs. 106 patients were randomised to either IPC or standard care in a 1:1 ratio. The primary outcome was daily VASD over 42 days post intervention. Dyspnoea improved in both groups with no significant difference in mean dyspnoea in the first 42 days (mean score: IPC 25mm (95% CI 19 – 30), standard care 24mm (95% CI 19 – 29)).

The focus of this thesis is on practice changing clinical studies which impact upon the day to day treatment of patients with pleural infection, answering specific questions on several aspects of patient management. Specific areas of assessment in this thesis include: Assessment of the current evidence for optimal drain size choice in patients with pleural infection; Analysis and statistical modelling of a previous cohort of patients with pleural infection, in order to assess optimal drain size choice in pleural infection; The design, conduct and analysis of a 2 x 2 factorial multi-centre randomised, placebo controlled trial to assess the efficacy of two novel intrapleural agents (tPA and DNase) in aiding drainage in patients with pleural infection (The 2nd Multi-centre Intrapleural Sepsis Trial, referred to from here on as MIST2); Validation work informing the primary outcome measure of MIST2, assessing the relationship between chest radiograph imaging of infected pleural effusion and CT measured volume of pleural fluid using novel digital measurement strategies.

Approximately 50% of all malignant pleural mesotheliomas (MPM) are deficient in argininosuccinate synthetase (ASS1), the rate-limiting enzyme in arginine biosynthesis, and are sensitive to arginine deprivation. This discovery in MPM has been translated into the clinic using the arginine depletor pegylated arginine deiminase (ADI-PEG20), which showed a halving in the risk of disease progression in a randomised phase II study. However, unstudied to date, stromal resistance to ADI-PEG20 may reduce its efficacy. Here, I studied the effect of macrophages, abundant in mesothelioma, on the tumour cytotoxicity of ADI-PEG20. A distinct pro-inflammatory cytokine gene expression signature involved in macrophage recruitment and activation was identified and validated in ADI-PEG20-treated ASS1 negative MPM cell lines. In vivo induction of pro-inflammatory cytokines was also seen in ADI-PEG20-treated patient plasma. Notably, in vitro co-culture experiments demonstrated a significant increase in ASS1 negative MPM cell viability upon co-culture with macrophages in the presence of ADI-PEG20. This was accompanied by a significant increase in ASS1 expression in co-cultured macrophages, with a corresponding increase in argininosuccinate lyase (ASL) expression in co-cultured tumour cells and a doubling in levels of the arginine precursor, argininosuccinate, in cell supernatant. The addition of argininosuccinate to tumour cell media rescued ASS1 negative MPM cells from ADI-PEG20 cytotoxicity, while the macrophage-mediated resistance to ADI-PEG20 was abrogated following ASL knockdown in MPM cells. Finally, xenograft studies demonstrated a significant reduction in tumour volume in mice treated with ADI-PEG20 in combination with macrophage depletion, compared with ADI-PEG20 alone. Collectively, the data indicate that as a result of metabolic 'cross-talk' between macrophages and ASS1 negative MPM cells, macrophages mediate MPM resistance to ADI-PEG20 via the provision of argininosuccinate. My studies provide a rationale for combining ADI-PEG20 with an inhibitor of macrophage recruitment in the treatment of ASS1-deficient mesothelioma.

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Foram estudados 115 pacientes, de ambos os sexos, com idade igual ou superior a 15 anos, residentes na cidade de Manus ou na sua zona rural, portadores de derrame pleural de qualquer etiologia, exceto a traumática, no período de 01/07/84 a 01/07/86. Foram estudados os aspectos etiológicos, clínicos e sócio-econômico dos pacientes, através da aplicação de um questionário padrão, bem como a realização de exames complementares, necessários para a detrminação dea etiologia do derrame pleural. Os dados coletados foram analisados , quando quantitativos, pelo teste "f' de Student, e quando qualitativos pelo teste do "Qui-quadrado", com a utilização da correção de Yates para pequenas amostras; adotando-se o nível de 5% para o alfa. No estudo da correlação utilizou-se o teste de Pearson, com igual nível de significância. O estudo demonstrou que o derrame pleural foi mais frequente no sexo masculino, em brancos, de famílias mais numerosas, de menor poder aquisitivo e de menor cobertura previdenciária que o restante da população manauara. A tuberculose foi a causa, confirmada, de maior frequência, mais comum no sexo masculino e acomentendo a faixa etária mais jovem. / We studied 115 patient, of both sexes, with the same or superior age to 15 years, rinesidents in the city of Manaus or in its rural zone, carriers of pleural effusion of any origin except the traumatic, in the period from 01/07/84 to 01/07/86. They were studied the etiologics aspects , clinicai and socioeconomic of the patients, through the application of a standard questionnaire, as well as the accomplishment of complementai exams, necessary for the determination of the origin of the pleural effusion The collected data were analyzed, when quantitative, for the test "t" of Student, and when qualitative for the test of the "Qui-square", with the use of the correction of Yates for small samples; being adopted the levei of 5% for the alpha. In the study of the correlation the test of Pearson was used, with the same significance levei. The study demonstrated that the pleural effusion was more frequent in the masculine sex, in whites, of more numerous families, of smaller purchasing power and of smaller covering social security than the remaining of the population manauara. The tuberculosis went to cause, confirmed, of larger frequency, more common in the masculine sex and reaching the youngest age group.

Introducción Los enzimas proteolíticos responsables de la degradación de la matriz extracelular han demostrado tener un papel importante en los procesos inflamatorios, aunque han sido poco estudiados en la patogenia de los derrames pleurales (DP) paraneumónicos. Las metaloproteinasas (MMP) son una familia de proteasas que degradan prácticamente todos los componentes proteicos de la matriz extracelular. La actividad de las MMP está controlada por inhibidores tisulares específicos (TIMP). En la patogenia de los DP paraneumónicos se han implicado mecanismos inflamatorios y de alteración del sistema de la fibrinolisis pleural. La respuesta inflamatoria pleural iniciada por el mesotelio provoca un aumento de la permeabilidad vascular y la liberación de mediadores inflamatorios, como la IL-8 y el TNF-?, que favorecerán la formación de DP exudado, la invasión del espacio pleural por células inflamatorias y la liberación de productos de la degranulación del neutrófilo, como la elastasa polimorfonuclear. El sistema de la fibrinolisis esta regulado por los activadores de plasminógeno (PA), tisular PA (t-PA) y urokinasa PA (u-PA), y por los inhibidores de los activadores del plasminógeno (PAI), tipos 1 y 2. Objetivos El objetivo de este trabajo es estudiar diferentes metaloproteinasas y sus inhibidores tisulares en diferentes tipos de DP y evaluar su asociación con los marcadores de activación neutrofílica y con elementos del sistema de la fibrinolisis en los DP paraneumónicos.Método El estudio incluyó 51 DP paraneumónicos (30 empiemas o DP paraneumónicos complicados, 21 DP paraneumónicos no complicados), 28 DP tuberculosos, 30 DP neoplásicos y 30 trasudados. Se determinaron por ELISA, en plasma y líquido pleural, los marcadores de activación neutrofílica (TNF-?, IL-8, elastasa polimorfonuclear), elementos del sistema de la fibrinolisis (t-PA, u-PA, PAI-1, PAI-2), y diferentes MMP (MMP-1, MMP-2, MMP-8, MMP-9) y TIMP (TIMP-1, TIMP-2). Resultados Todos los líquidos pleurales estudiados presentaron concentraciones elevadas de MMP-2 y TIMP-1. Los empiemas y DP paraneumónicos complicados presentaron concentraciones de MMP-1, MMP-8 y MMP-9 significativamente superiores respecto al resto de exudados. En este grupo de DP, no se observó asociación entre los niveles de MMP y TIMP en plasma y en líquido pleural. En los DP paraneumónicos, se observó una asociación positiva de MMP-1, MMP-8 y MMP-9 con los marcadores de activación neutrofílica, u-PA y PAI-1. Además, la MMP-8 en líquido pleural se relacionó con la presencia de paquipleuritis residual en este grupo de derrames pleurales.Conclusiones1. Existen niveles elevados de MMP-2 y TIMP-1 en todos los líquidos pleurales estudiados, lo que sugiere una expresión constitutiva por parte de las células mesoteliales pleurales.2. Las concentraciones en líquido pleural de MMP-1, MMP-8 y MMP-9 son significativamente superiores en los DP paraneumónicos complicados y empiemas. 3. La ausencia de correlación entre las concentraciones de MMP y TIMP en líquido pleural y plasma, en los empiemas y DP paraneumómicos complicados, sugiere una expresión compartimentalizada de las mismas en el espacio pleural. 4. En los DP paraneumónicos existe una íntima asociación positiva de MMP-1, MMP-8 y MMP-9 con los marcadores de activación neutrofílica, u-PA y PAI-1, relacionados con el desarrollo de complicaciones locales en los DP bacterianos.5. La MMP-8, en líquido pleural, se relaciona con la presencia de paquipleuritis residual en los DP paraneumónicos. 6. La asociación de MMP-1, MMP-8 y MMP-9 con los marcadores de activación neutrofílica y el sistema de la fibrinolisis, así como la relación de la MMP-8 con la presencia de complicaciones locales en los DP paraneumónicos, sugiere un papel relevante de estas metaloproteinasas en la fisiopatología de los DP bacterianos. / Introduction: Proteolytic enzymes responsible for breakdown of the extracellular matrix play an important role in inflammatory processes, although they have been little studied in relation to the pathogenesis of exudative pleural effusions (PE). The metalloproteinases (MMPs) are a family of proteases that break down virtually all the protein components of the extracellular matrix. The activity of the MMPs is controlled by specific tissue inhibitors of metalloproteinases (TIMPs). Inflammatory mechanisms and alterations in the balance of pleural fibrinolysis have been implicated in the pathophysiology of infectious PE. The inflammatory response initiated by the mesothelium provokes increased vascular patency and the release of inflammatory mediators, e.g. interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-?), thereby favoring formation of exudative PE, invasion of the pleural space by inflammatory cells and release of neutrophil degranulation products, such as polymorphonuclear elastase (PMN-E). The fibrinolytic system is regulated by two plasminogen activators (PAs), tissue PA (t-PA) and urokinase PA (u-PA), and by plasminogen activator inhibitors (PAIs) types 1 and 2 (PAI-1, PAI-2). Objectives: The aim of this work was to study several metalloproteinases and their tissue inhibitors in PE and to evaluate the association between these enzymes and neutrophilic activation markers and elements of the fibrinolysis system in parapneumonic effusions.Methods: The study included 51 parapneumonic effusions (30 empyema or complicated parapneumonic, 21 non-complicated parapneumonic), 28 tuberculous, 30 malignant and 30 transudates. Neutrophilic activation markers (TNF-?, IL-8, polymorphonuclear elastase), fibrinolytic system variables (t-PA, u-PA, PAI-1, PAI-2), and several MMPs (MMP-1, MMP-2, MMP-8, MMP-9) and TIMPs (TIMP-1, TIMP-2), were determined by ELISA in plasma and pleural fluid. Results: Elevated MMP-2 and TIMP-1 concentrations were observed in all the pleural fluid samples studied. The group of empyema or complicated parapneumonic effusions showed higher MMP-1, MMP-8 and MMP-9 concentrations than the remaining exudates. There was no correlation between MMP and TIMP levels in plasma and pleural fluid in this group of effusions. In parapneumonic effusions, MMP-1, MMP-8 and MMP-9 showed a positive correlation with the neutrophilic activation markers and with u-PA and PAI-1. Moreover, there was a relationship between MMP-8 concentration in pleural fluid and pleural thickening at the end of treatment. Conclusions: 1. Elevated MMP-2 and TIMP-1 levels were found in all the pleural fluid samples studied, suggesting their constitutive expression by mesothelial cells.2. The group of empyema or complicated parapneumonic effusions showed the highest concentrations of MMP-1, MMP-8 and MMP-9 in pleural fluid. 3. In empyema and complicated parapneumonic effussion, no correlation was found between plasma and pleural fluid levels of MMPs and TIMPs, suggesting a compartmentalization of these enzymes in the pleural space. 4. In parapneumonic effusions exists a close positive correlation of MMP-1, MMP-8 and MMP-9 with the neutrophilic activation markers (IL-8, TNF-?, PMN-E), u-PA and PAI-1, all of them related to the developement of local complications in infectious effusions. 5. In parapneumonic effusions, pleural fluid MMP-8 was related to the presence of residual pleural thickening. 6. In parapneumonic effusions, the association of MMP-1, MMP-8 and MMP-9 with neutrophil activation markers and the fibrinolysis system, and also the relation of MMP-8 with the presence of local complications, suggests an important role of this enzymes in the physiopathology of infectious effusions.

El derrame pleural paraneumónico es una causa frecuente de derrame pleural exudado. En la fisiopatología de los DP paraneumónicos se han implicado mecanismos inflamatorios y de alteración en el equilibrio de la fibrinolisis pleural. La respuesta inflamatoria pleural, iniciada por el mesotelio, provoca un aumento de la permeabilidad vascular y la liberación de mediadores inflamatorios, que favorecerá la formación de derrame pleural exudado y la invasión del espacio pleural por células inflamatorias. En este proceso tienen un papel importante diferentes citoquinas proinflamatorias, como la IL-8 y el TNF-&#61537;, así como productos de la degranulación de los neutrófilos, como la elastasa polimorfonuclear. El depósito de fibrina en el espacio pleural contribuye a la aparición de los septos y loculaciones que observamos en los derrames pleurales bacterianos. La presencia de engrosamiento pleural residual está ampliamente estudiada en los derrames pleurales tuberculosos pero ha sido poco estudiada en los derrames pleurales paraneumónicos y empiemas.

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