During the past few decades, the landscape of available therapeutic interventions for cancer treatment has widely expanded, boosted mainly by immunotherapy progress and the precision oncology paradigm. The extensive use of pre-clinical models in cancer research has led to the discovery of new effective treatment options for patients. Despite the notable advancements, some cancer types have found minor benefits from the use of precision-oncology interventions. Characterized by a heterogeneous molecular landscape, bladder cancer is one of the most frequent cancer types in which standard-of- care treatments involve surgical operations accompanied by broad-spectrum chemotherapy. My research stems from the need for precision oncology interventions in bladder cancer and specifically focuses on the development of computational frameworks to guide the discovery of new therapeutic opportunities. This work first introduces the exploration of possible therapeutic interventions in 9p21.3 depleted bladder tumors through the analysis of an in-house large High-Content Drug Screening that tested 2,349 compounds. By combining cell count changes and morphological quantitative features extracted from fluorescence images, we nominate cytarabine as a putative candidate eliciting specific cytotoxic effects in an engineered 9p21.3 depleted bladder cancer model compared to an isogenic wild-type clone. Focusing on the development of computational methodologies to nominate robust context-specific vulnerabilities, I further describe PRODE (PROtein interactions informed Differential Essentiality), an analytical workflow that integrates protein-protein interaction data and Loss of Function screening data. I extensively tested PRODE against the most commonly used and alternative methodologies and demonstrated its superior performance when classifying reference essential and context-essential genes collected from experimental and literature sources. Furthermore, we applied PRODE to a real case scenario, seeking essential genes selectively in the context of HER2+ Breast Cancer tumors. Finally, I report the computational analyses performed on Patient-Derived Organoids (PDOs) established from a bladder cancer cohort. PDOs are demonstrated as informative models when assessing the therapeutic sensitivity of patients to drugs. Overall, this research highlights novel precision-oncology applications by ad-hoc computational analyses that address key open technical and biological challenges in the field of bladder cancer and beyond.

No / Objectives Previous studies have demonstrated that intravesical administration of apaziquone (EOquin) has ablative activity against superficial bladder cancer marker lesions with 8 out of 12 complete responses recorded. We present a comparison between the rates of tumor recurrence before and after treatment with apaziquone. Methods The rate of tumor recurrence after treatment with apaziquone was compared with each patient's historical record of recurrences obtained from a retrospective analysis of the patients' case notes. The time to each recurrence event before apaziquone treatment and the time to the first recurrence after apaziquone treatment were recorded, and the data were analyzed using a population-averaged linear regression model using Stata Release, version 9.2, software. Results Of the eight complete responses obtained in the Phase I study, tumor recurrence occurred in 4 patients and the remaining 4 patients remained disease free after a median follow-up of 31 months. The time to the first recurrence after apaziquone treatment was significantly longer (P <0.001) compared with the historical pattern and recurrence interval before apaziquone. Before apaziquone instillation, the mean ± SE recurrence rate and tumor rate per year was 1.5 ± 0.2 and 4.8 ± 1.2, respectively, and these decreased to 0.6 ± 0.25 and 1.5 ± 0.8, respectively, after apaziquone treatment (P <0.05). Conclusions The results of this study indicate that early recurrences after treatment with apaziquone are infrequent and the interval to recurrence is significantly greater compared with the historical recurrence times for these patients. Larger prospective randomised trials are warranted to confirm these results. Aapaziquone (EOquin, USAN, E09, 3-hydroxy-5-aziridinyl-1-methyl-2[indole-4,7-dione]¿prop-¿-en-¿-ol) belongs to a class of anticancer agents known as bioreductive drugs that require metabolism by cellular reductases to generate a cytotoxic species.1 Although it is chemically related to mitomycin C, apaziquone has a distinctly different mechanism of action and preclinical activity profile.1 and 2 The initial optimism generated by its preclinical activity profile rapidly evaporated after the demonstration that intravenously administered apaziquone was clinically inactive against a range of solid tumors in Phase II clinical trials.3 and 4 Several possible explanations were considered for its lack of efficacy, but poor drug delivery to the tumor because of the rapid pharmacokinetic elimination of apaziquone in conjunction with relatively poor penetration through avascular tissue was considered to be the principal reason.5 On the basis of the rationale that intravesical administration would circumvent the problem of drug delivery and any apaziquone absorbed into the blood stream would be rapidly cleared,6 a Phase I-II clinical pilot study of intravesical administration of apaziquone to superficial bladder tumors was established.7 The results of that trial demonstrated that intravesically administered apaziquone has ablative activity against superficial bladder transitional cell carcinoma (TCC) marker lesions.7 These results were confirmed and extended in a Phase II clinical trial of 47 patients with superficial bladder TCC, in which complete responses were obtained in 67% of patients.8 Because all the enrolled patients in the original trial7 had had multiple recurrences after previous intravesical chemotherapy and/or immunotherapy, the purpose of the present study was, first, to report the recurrences that occurred after apaziquone treatment and, second, to study the effect of apaziquone instillation on the recurrence rate by statistically comparing these results with the historical pattern of recurrences for each patient before treatment with apaziquone.

Objectifs: Les données provenant des centres de soins tertiaires suggèrent que le taux de mortalité péri-opératoire (MPO) après cystectomie notés pour les patients âgés (septuagénaires et octogénaires) n’excède pas celle des patients plus jeunes. Toutefois, les données provenant de la communauté démontrent un phénomène inverse. Spécifiquement, la MPO est plus élevés chez les ainés. Dans cette thèse nous allons présenter une réévaluation contemporaine du taux de MPO après cystectomie. Méthodes: Entre 1988 et 2006, 12722 cystectomies radicales pour le carcinome urothéliale de la vessie ont été enregistrées dans la banque de données SEER. Le taux de MPO a été évalué dans les analyses de régression logistique univariées et multivariées à 90 jours après cystectomie radicale. Les covariables incluaient: le sexe, l’ethnie, l’année de chirurgie, la région d’origine du patient ainsi que le grade et le stade de la tumeur. Résultats: Parmi tous les patients, 4480 étaient des septuagénaires (35.2%) et 1439 étaient des octogénaires (11.3%). Le taux de MPO à 90 jours était de 4% pour la cohorte entière vs. 2% pour les patients moins de 69 ans vs. 5.4% pour les septuagénaires vs. 9.2% pour les octogénaires. Dans les analyses de régression logistiques multivariées, les septuagénaires (OR=2.80; <0.001) et les octogénaires (OR=5.02; <0.001) avaient reçu un taux de MPO plus augmenté que les patients moins de 70 ans après une cystectomie radicale. Conclusion: Cette analyse épidémiologique basée sur les donnés le plus contemporaines démontre que l’âge avancée représente un facteur de risque pour un taux de MPO plus élevé. / Objective Data from tertiary care centers suggest that the perioperative mortality (POM) after radical cystectomy (RC) is not different in septuagenarian or octogenarian patients, compared to younger individuals. Conversely, population-based data state otherwise. We revisited this topic in a large contemporary population-based cohort. Methods Between 1988 and 2006, 12722 radical cystectomies were performed for urothelial carcinoma of the urinary bladder (UCUB) in 17 Surveillance, Epidemiology and End Results (SEER) registries. Of those 4480 were aged 70-79 and 1439 were 80 years and older. Univariable and multivariable logistic regression models tested 90-day mortality (90dM) after radical cystectomy. Covariates consisted of gender, race, year of surgery, SEER registry, histological grade and stage. Results Of all 12722 patients, 4480 (35.2%) were septuagenarian and 1439 (11.3%) were octogenarian. The overall 90dM rate was 4% for the entire population, 2% for patients aged 69 years or younger, 5.4% for septuagenarian patients and 9.2% for octogenarian patients. In multivariable logistic regression analyses, septuagenarian (OR= 2.80; <0.001) and octogenarian (OR= 5.02; <0.001) age increased the risk of 90dM after RC. Conclusions In this population-based analysis, POM was between 3 and 5-fold higher respectively in septuagenarian and octogenarian patients which is higher in tertiary care centers. This information needs to be included in informed consent considerations, specifically if RC will not be performed at a tertiary care center.

O câncer de bexiga (CB) destaca-se como o segundo tipo de doença mais comum do trato geniturinário e o décimo com maior incidência no Brasil, sendo responsável por cerca de 4020 óbitos por ano. Os dados refletem a baixa eficácia dos tratamentos disponíveis. Assim, novas moléculas e sistemas de liberação estão sendo investigados para terapia intravesical do CB. Nesta linha, as oleorresinas de Copaifera e compostos fenólicos como a curcumina (CM) e demetoxicurcumina (DCM) presentes no extrato de Curcuma longa, têm sido alvo de pesquisas por apresentarem propriedades antitumoral, antiproliferativa e proapoptótica. Contudo, a administração desses compostos em sua forma livre torna-se inviável devido à sua alta lipossolubilidade e instabilidade. Dessa forma, o uso de oleorresina de Copaifera duckei (O-Cd) no preparo de carreadores lipídicos nanoestruturados (CLN) para a encapsulação do extrato de C. longa (ECL) poderia ser uma estratégia terapêutica eficaz, além de transpor algumas barreiras da terapia intravesical como a baixa permeação de moléculas no urotélio e seu baixo tempo de residência na bexiga. Assim, os objetivos do presente estudo foram preparar e caracterizar CLN (com O-Cd) e CLN imperfeito (sem O-Cd, CLNimp) para a encapsulação de ECL, e avaliar a citotoxicidade desses sistemas carreadores em células de CB. Os CLN e CLNimp, preparados pelo método de emulsão à quente e sonicação, foram caracterizados quanto ao seu diâmetro, índice de polidispersão (PdI), potencial zeta (PZ), cristalinidade, eficiência de encapsulação (EE) por CLAE, capacidade de encapsulação (CE), e morfologia. Além disso, avaliou-se a citotoxicidade dos CLNs com e sem ECL em células RT4 e foram conduzidos estudos de uptake celular. Obteve-se carreadores com diâmetro ao redor de 200 nm, baixo PdI (?0,3) e PZ negativo (~-20 mV). O ECL foi caracterizado e quantificado por CLAE, sendo verificado uma pureza de 97,90% para CM e 99,29% para DCM. Ambas moléculas foram encapsuladas nos dois CLNs com alta EE (~70%). A encapsulação do ECL não alterou de forma significativa as propriedades físicas dos CLNs. Os dois tipos de CLNs apresentaram baixo índice de recristalização (~60%), forma esférica e superfície lisa. Os CLN e CLNimp foram estáveis no período de 420 e 270 dias, respectivamente. Já os CLN-ECL e CLNimp-ECL foram estáveis por 90 dias. O IC50 dos CLN sem ECL (0,019x1012 partículas/mL) foi menor que o IC50 dos CLNimp sem ECL (>0,061x1012 partículas/mL). O encapsulamento do ECL pelos CLNs não alterou significativamente seu IC50 nos períodos de incubação avaliados. Já o encapsulamento do ECL pelos CLNimp diminuiu o IC50 (19,15 ?g/mL) (0,047x1012 partículas/mL) em relação ao CLNimp vazio. Observou-se maior uptake celular dos carreadores com ECL em relação ao ECL livre. Além disso, foi observada fragmentação do núcleo das células que internalizaram CLN-ECL. As interessantes propriedades físicas e biológicas apresentadas pelos sistemas CLN vazio e CLNimp-ECL indicam seu potencial terapêutico e os tornam promissores para futura terapia do câncer de bexiga. / Bladder cancer (BC) stands out as the second most common genitourinary disease type and the tenth one with the highest incidence in Brazil, being responsible for about 4020 deaths per year. The data reflect the low efficacy of the available treatments. Thus, new molecules and release systems are being investigated for intravesical BC therapy. In this line, Copaifera oleoresins and phenolic compounds such as curcumin (CM) and demethoxycurcumin (DCM) present in Curcuma longa extract, have been the subject of research because they have antitumor, antiproliferative and proapoptotic properties. However, administration of these compounds in their free form becomes infeasible due to their high liposolubility and instability. Therefore, the use of Copaifera duckei oleoresin (Cd-O) in the preparation of nanostructured lipid carriers (NLC) for the encapsulation of C. longa extract (CLE) could be an effective therapeutic strategy, in addition to transposing some barriers of intravesical therapy such as the low permeation of molecules in the urothelium and its low residence time in the bladder. Thus, the aims of the present study were to prepare and characterize NLC (with Cd-O) and imperfect NLC (without Cd-O, NLCimp) for the encapsulation of CLE, and to evaluate the cytotoxicity of these carrier systems in BC cells. The NLC and NLCimp, prepared by the hot emulsion and sonication method, were characterized by their diameter, polydispersity index (PdI), zeta potential (ZP), crystallinity, encapsulation efficiency (EE) by HPLC, encapsulation capacity (EC) and morphology. In addition, the cytotoxicity of NLCs with and without CLE was evaluated in RT4 cells and cell uptake studies were conducted. Carriers with diameter about 200 nm, low PdI (<0.3) and negative ZP (~-20 mV) were obtained. ECL was characterized and quantified by HPLC with a purity of 97.90% for CM and 99.29% for DCM. Both molecules were encapsulated in the two NLCs with high EE (~70%). The encapsulation of CLE did not significantly alter the physical properties of NLCs. Both NLCs presented low recrystallization index (~60%), spherical shape and smooth surface. The NLC and NLCimp were stable over 420 and 270 days, respectively. On the other hand, NLC-CLE and NLCimp-CLE were stable for 90 days. The IC50 of NLC without CLE (0.019x1012 particles/mL) was lower than the IC50 of NLCimp without CLE (> 0.061x1012 particles/mL). The encapsulation of CLE by NLCs did not significantly alter its IC50 in the incubation periods evaluated. The encapsulation of CLE by the NLCimp decreased its IC50 (19.15 ?g/mL) (0.047x1012 particles/mL) in relation to the empty CLNimp. It was observed higher cellular uptake of the carriers with CLE in relation to free CLE. In addition, fragmentation of the nucleus of the cells that internalized NLC-CLE was observed. The interesting physical and biological properties presented by empty NLC and NLCimp-CLE systems indicate their therapeutic potential and make them promising for future bladder cancer therapy

L'objectif de cette thèse est d'évaluer les performances d'une méthode d'imagerie optique non-invasive pour la détection de précancers et cancers précoces de la vessie, à l'aide d'une analyse de lumière laser rétro-diffusée. L'analyse de la distribution spatiale de la lumière à la surface de fantômes multi-couches imitant l'épithelium de vessie avec différentes propriétés d'absorption et de diffusion nous a permis de montrer les modifications de ces propriétés optiques entraînent des changements de la taille de la surface du spot de lumière rétro-diffusée, mesurables par une caméra vidéo. La méthode développée est également sensible à l'accumulation d'un photosensibilisateur et est applicable aussi bien pour des études en réflectance diffuse qu'en fluorescence induite. Les paramètres optiques des fantômes synthétiques tri-couches imitant différents états des épithéliums de vessie ont été calculés à partir de la théorie des ondes électromagnétiques appliquée aux diffuseurs sphériques sans et avec une couche. Ces paramètres ont servi comme entrées aux simulations de Monte Carlo qui ont permis d'obtenir les matrices des distributions d'intensité de réflectance diffuse. Notre étude démontre que les mesures en imagerie de réflectance diffuse non-polarisée permettent de fournir des informations utiles au diagnostic tissulaire / The present thesis aimed to evaluate the performance of non-invasive optical method for bladder pre- and early- cancer detection by means of diffuse-reflected laser light analysis. The analysis of light distribution at the surface of multi-layered bladder phantoms with different scattering and absorption properties showed that the changes in the optical properties lead to increase or decrease of the diffuse-reflected light spot area, detectable by a video camera. It was also determined, that the presented method is capable of detection of the photosensitizer accumulation, and can be applied for both (diffuse-reflected laser and fluorescence) studies simultaneously. The calculations for spherical and ?coated?-spherical tissue scatterers, based on the electromagnetic wave theory, allowed for obtaining optical parameters of three-layered biological phantoms and of bladder tissues at different states. These parameters served as inputs for Monte Carlo simulations, which provided us with matrices of diffuse-reflected light distributions. The study showed that the measurements of non-polarized back-scattered laser light can provide useful information on the tissue state

Le cancer de vessie représente la sixième cause de mortalité par cancer en France. Son incidence a augmenté ces 20 dernières années, mais les taux de survie restent inchangés. La carcinogénèse vésicale fait intervenir différents mécanismes moléculaires qui agissent en réseau comme c’est le cas dans de nombreux cancers. Le développement récent de nouveaux traitements prenant spécifiquement pour cible certaines voies de signalisation apportent de nouveaux espoirs thérapeutiques. Nous nous sommes intéressés dans ce travail à trois axes de recherche pour tenter d’identifier, dans les carcinomes urothéliaux, de nouveaux marqueurs pronostiques moléculaires et de nouvelles cibles thérapeutiques potentielles: l’angiogénèse, la voie de signalisation Hedgehog et les microARNs. Nous avons choisi la RT-PCR quantitative en temps réel à grande échelle permettant d’évaluer le niveau d’expression de nombreux gènes, avec une quantification précise et reproductible des transcrits. L’expression de ces gènes a été corrélée aux données de suivi clinique afin d’identifier de nouveaux biomarqueurs moléculaires prédictifs de l’évolution des tumeurs de vessie.Nous avons ainsi pu démontrer que les niveaux d’expression de certains de ces gènes variaient de façon significative dans les tumeurs de vessie, confirmant le rôle de l’angiogénèse dans la carcinogénèse urothéliale, et plus particulièrement de la voie du VEGF, et suggérant une implication majeure de la voie de signalisation Hedgehog et des microARNs. Par ailleurs, nous avons également pu identifier plusieurs biomarqueurs ayant une valeur pronostique en terme de survie globale dans les tumeurs infiltrantes. C’est le cas du VEGF, qui semble être un biomarqueur moléculaire particulièrement intéressant puisqu’il existe des thérapies ciblées spécifiquement dirigées contre ce ligand ou ses récepteurs avec plusieurs essais cliniques actuellement en cours dans le cancer de vessie. C’est également le cas d’une signature moléculaire associant 3 miARNs (miR-9, miR-182 et miR-200b) ayant une valeur péjorative dans les tumeurs infiltrantes, ouvrant la voie vers de nouvelles stratégies thérapeutiques.L’ensemble de ces études confirment l’intérêt majeur d’une meilleure compréhension des bases moléculaires de la carcinogénèse urothéliale vésicale débouchant sur l’utilisation rationnelle de nouvelles thérapies ciblées dans le cancer de vessie, avec l’espoir d’en améliorer la prise en charge et l’évolution. / Bladder cancer is the sixth cause of cancer mortality in France and its incidence is increasing since the last 20 years, with no improvement in survival outcomes. Bladder carcinogenesis involves different molecular mechanisms such as in many cancers. The recent development of new targeted therapies targeting signaling pathways provides new therapeutic hopes.In this work, we choose to study three molecular pathways in order to identify new prognostic markers and new therapeutic targets in urothelial carcinoma: angiogenesis, Hedgehog signaling pathway, and microRNAs. Real-time quantitative RT-PCR was performed to measure simultaneously expression levels of several genes with precise and reproductible RNA quantification. Our results were correlated with clinical outcomes to identify new molecular markers associated with bladder cancer evolution and to guide the potential use of targeted therapies.We were able to demonstrate that expression levels of several transcripts differ significantly in bladder tumors as compared to normal bladder and that some of them may have a prognostic implication. This is the case of VEGF, which appears to be an interesting molecular marker since there are targeted therapies specifically targeting the pathway and several ongoing trials in bladder cancer. The Hedgehog pathway also appears to be altered in bladder tumors, with a ligand-dependent activation. Then, we were able to identify several deregulated microRNAs and describe a molecular 3 miRNA-signature (miR-9, miR-182 and miR-200b) having a prognostic value in muscle-invasive bladder tumors. All these studies confirm the major interest of molecular biology and new targeted therapies in the treatment of bladder cancer, with the hope of improving management and evolution.

Les rapides progrès technologiques en matière de techniques de biologie à grande échelle, comprenant notamment les microarrays, conduisent en 2006 au développement d’une nouvelle génération de puces à très haute résolution, capables de cibler à la fois tous les gènes du transcriptome humain, mais également tous les exons de ces gènes pris individuellement. L’avènement de cette puce, communément appelée puce exon, permit d’obtenir une mesure précise des changements transcriptomiques affectant les cellules cancéreuses, en offrant la possibilité de prendre en compte l’expression relative de différents exons d’un même gène.L’épissage alternatif et la transcription alternative sont les deux principaux mécanismes biologiques à l’origine de l’existence de plusieurs transcrits pour un même gène. Ces processus biologiques ont été mis en évidence depuis longtemps mais leur régulation dans les cellules normales ainsi que leurs dérégulations dans les cancers sont encore mal caractérisées de par la complexité des mécanismes impliqués. Par leur design, les puces exons permettent de mettre en évidence la présence de variations d’expression entre plusieurs transcrits potentiels d’un même gène, ouvrant ainsi la voie à une meilleure compréhension de ces processus biologiques.A partir d’un important jeu de données d’échantillons de cancers de la vessie dont le profil transcriptomique fut obtenu par puces exons, nous nous sommes intéressés à l’étude des changements d’épissage alternatif et à l’utilisation de promoteurs alternatifs dans les tumeurs de vessie. L’utilisation d’outils statistiques et mathématiques dédiés à l’analyse de ces puces nous a permis dans un premier temps d’identifier de nombreux gènes dont l’expression relative des différents transcrits est spécifiquement dérégulée dans les tumeurs de vessie. Ces transcrits constituent une nouvelle source pour l’identification de cibles thérapeutiques spécifiques des tumeurs. Nous avons pu montrer qu’avec une approche ciblée sur les changements d’expression relative de transcrits alternatifs d’un même gène, il était possible de constituer un panel de potentiels marqueurs tumoraux permettant le développement de nouveaux tests urinaires utiles à la détection des cancers de vessie et à la surveillance des patients.Par une analyse non supervisée des profils d’exons potentiellement dérégulés, nous avons pu observer une stratification des tumeurs similaire à celle observée par l’étude des profils géniques issus de puces classiques, confirmant alors l’existence d’un sous groupe de tumeurs de vessie présentant des caractéristiques transcriptomiques propres. Nous avons pu associer à ce sous-groupe de mauvais pronostic, une signature d’inclusion différentielle de certains exons. Cette signature impliquant 19 gènes permet d’identifier précisément ces tumeurs de manière très spécifique et constitue par conséquent un outil puissant utilisable en clinique.L’étude ciblée d’une voie de signalisation fréquemment dérégulée dans les cancers nous a permis de mettre en évidence une dérégulation globale de l’expression relative des transcrits alternatifs de gènes impliqués dans la prolifération cellulaire, et d’en identifier de probables régulateurs. Enfin, L’analyse des données de puces exons à la lumière des données de méthylation de l’ADN nous a permis d’identifier un mécanisme épigénétique régulant l’utilisation de promoteurs alternatifs dans un sous-groupe de tumeurs de vessie.L’ensemble des résultats obtenus par l’analyse de ces puces exons a par conséquent permis de caractériser à l’échelle du transcrit les dérégulations spécifiques des tumeurs de vessie, et d’en identifier certains mécanismes. Ces dérégulations permettent non seulement d’identifier spécifiquement plusieurs sous-groupe de tumeurs dont un de mauvais pronostic, mais offrent également de nouvelles possibilités quant-à la recherche de marqueurs urinaires pour la surveillance des patients. / The development of microarray technology in the late 1990’s served as an essential tool to comprehend the scope of transcriptomic deregulations occurring in cancer cells. Signals generated from the first generation of transcriptomic microarrays gave simultaneous measures of expression from a large number of genes, therefore enabling to identify candidate genes involved in cancer progression and putative therapeutic targets. In 2006, through a fast de- velopment of high-throughput technologies, the available large scale analysis tools became enriched with a new generation of high resolution microarrays measuring expression signals both at the gene-level and at the exon-level of each gene. The advent of this high-resolution microarray, commonly called exon array, provided the opportunity to get a more accurate meas- ure of transcriptomic changes affecting cancer cells by enabling to consider relative expression changes of the exons from a same gene.Alternative splicing and alternative transcription are the two main biological mechanisms accounting for the production of several transcripts from a same gene. Although these bio- logical processes have been known for a long time, their regulation in normal cells and their deregulation in cancer still remain challenging to well-characterize, mainly due to the complex- ity of the involved mechanisms. Through their design, exon arrays enable to identify variable expression patterns within several potential transcripts of a same gene, therefore bringing new insight into these biological processes.Based on a large dataset of bladder cancer samples that were profiled on exon arrays, we focused on the study of alternative splicing changes and alternative promoter usage in bladder tumours. Analysis of these exon arrays through the use of adapted statistical and mathemat- ical tools initially resulted in the identification of numerous genes showing differential relative expression patterns of their transcripts between cancer and normal samples. These transcripts represent a new opportunity to define tumour-specific therapeutic targets. We demonstrated that using an approach targeted on relative expression changes of transcripts from a same gene, it was possible to build up a panel of potential tumour-specific markers enabling the development of new urinary test to detect bladder cancer and monitor its evolution.Through an unsupervised analysis of putatively deregulated exon profiles, we observed that the partitioning of bladder tumours was similar to the classification resulting from the study of classical gene microarray expression profiles, consequently confirming the existence of a bladder subgroup with peculiar transcriptomic properties. For this subgroup of bad prognosis, we established a signature based on the differential alternative inclusion of several exons. This signature relates to 19 genes and enables to accurately identify tumours from this subgroup, therefore providing a powerful tool to be used in clinical practice.By studying a specific pathway often deregulated in cancer, we highlighted an overall dereg- ulation of the relative expression of alternative transcripts from genes involved in cell prolifer- ation, and identified potential actors involved in the underlying regulatory process. Eventually, the analysis of exon arrays in the light of DNA methylation array data enabled us to identify an epigenetic mechanism regulating the use of alternative promoters in a subgroup of bladder tumours.Together, the results obtained from exon array analysis consequently provided a character- ization at the transcript level of bladder tumour specific deregulations and brought insight into the underlying mechanisms. The highlighted deregulations not only allow to accurately identify two subgroups of tumours, of which one has a bad prognosis, but also offer new possibilities regarding the definition of urinary markers for patient monitoring.

Background: At the time of diagnosis, approximately 75% of all bladder cancers are Non-Muscle Invasive Bladder Cancers (NMIBC) - the standard treatment for these cancers is a Transurethral Resection of the Bladder Tumour (TURBT). Although, the vast majority of these cancers are not life-threatening, they have a high risk of recurrence (and progression, particularly in higher risk NMIBC), despite the use of adjuvant intravesical chemotherapy. Consequently, patients are kept on long term cystoscopic surveillance with endoscopic removal if recurrences are detected - this impacts on patients' quality of life and contributes to the high cost for the healthcare provider. Aims: The fundamental aim of this series of clinical studies, spanning 12 years, was to identify and implement, means of improving the efficiency in both processing and operating on patients with NMIBC to not only reduce recurrence, but also to reduce the duration of follow up and repeat operations. It was an evolutionary process where the findings in the preceding studies formed the basis of the subsequent one - while the aim of the individual studies were different, there was a clear link to the essential principles, thus forming a coherent collection of studies. Methods and results: The project was carried out in 3 phases (with 2 or 3 main studies in each phase, augmented by 1 to 2 linked studies - making the entire submission for PhD by publications a series of 12 studies, to date): Phase 1 (5 studies in this phase): The aim was to demonstrate the natural history of non-invasive bladder cancer and identify sub-categories of patients who could be discharged from surveillance at 5 years. This was initially achieved by evaluating a prospectively maintained cohort of non-invasive bladder cancer patients diagnosed between 1978 and 1984 at the Western General Hospital, Edinburgh. This study identified the importance of the recurrence rate at the first follow up cystoscopy (RRFFC) as an essential prognostic marker. This finding was further validated using 2 separate cohorts from a different Centre (the Royal Infirmary, Edinburgh) managed in the 80s and the 90s, respectively. The data confirmed that over the decades, recurrence patterns do change, possibly as a result of differing techniques and improvements in optics and instruments; however, what remained the same was the prognostic value of the RRFFC. Phase 2 (3 studies in this phase): The early recurrence was deemed to be the result of missed and tumours left behind at the initial TURBT, i.e. a marker of quality. However, RRFFC was only known 3 months after the initial surgery. Since the RRFFC was such an important prognostic factor, the aim of this phase was to determine the surgical factors contributing to the quality of TURBT and subsequently implement changes to the principles in carrying out the surgery to improve this quality. This was achieved by prospective collection of information regarding all patients undergoing TURBT for new bladder cancers, recording the tumour features, surgeon experience, if the resection was deemed to have been complete or not, and the pathological results. We identified that the detrusor muscle in the resected specimen and the experience of the surgeon were independent determinants of TURBT quality. This finding was validated in a further study using cohorts from another time period and another Centre - this allowed me to develop the concept of Good Quality White Light TURBT (GQWLTURBT) as the benchmark for the white light TURBT. Phase 3 (4 studies in this phase): Photodynamic Diagnosis assisted TURBT (PDDTURBT) was demonstrated in randomised controlled trials as a technique that reduces the recurrences in NMIBC. In the absence of evidence with this technique in the 'real life' setting nor comparisons with standardised, benchmarked white light TURBT technique, we performed a prospective controlled study comparing PDD-TURBT and GQ-WLTURBT, evaluating early and delayed recurrence rates in 2 separate studies. I also performed a multicentre UK study on the outcomes with PDD-TURBT and collaborated with other experts in Europe in producing a review article around Photodynamic Diagnosis and the cost effectiveness of this technique. Summary: This coherent series of studies has contributed to knowledge in bladder cancer surgery by, among others: (a) mapping the individual patient natural history of non-invasive bladder cancer; (b) confirming the importance of early recurrence as a strong prognostic indicator; (c) identifying predictors of this early recurrence and the quality of TURBT; (d) introducing the concept of the benchmark Good Quality White Light TURBT and (e) demonstrating the benefits of photodynamic diagnosis within a 'real life' setting.

Contexte. La chloration est le traitement le plus utilisé pour désinfecter l’eau distribuée à la population. Lors du traitement, des sous-produits de chloration (SPCs) se forment par réaction de la matière organique naturelle présente dans l’eau avec le chlore. Les produits les plus présents sont les trihalométhanes (THMs), les acides haloacétiques (HAAs). Des études toxicologiques (animales et in vitro) ont montré la génotoxicité et/ou carcinogénicité et la reprotoxicité de certains SPCs. Nous avons mené deux études évaluant les effets sur la santé humaine de l’exposition aux SPCs, dans le domaine du cancer et de la reproduction. Association entre exposition aux THMs et risque de cancer de la vessie : une analyse poolée de 3 études cas-témoins européennes. Cette analyse a inclus 2381 cas et 3086 témoins issus de 3 études cas-témoins (France, Espagne, Finlande). L’exposition environnementale aux SPCs a été mesurée par la concentration en THMs estimée rétrospectivement dans les réseaux de distribution d’eau au cours des 40 années de la fenêtre d’exposition. Les usages de l’eau connus sont l’ingestion, les douches et les bains, la fréquentation de piscine (étude espagnole seulement). Une relation croissante a été observée entre le niveau environnemental de THMs, la durée d’exposition à une eau de surface chlorée et le risque de cancer de la vessie, chez les hommes uniquement. Aucune association n’a été observée avec l’exposition via l’ingestion d’eau du robinet. L’exposition via les douches, les bains et la piscine est apparue liée au risque de cancer de la vessie. Trois études cas-témoins nord-américaines ont été intégrées dans une méta-régression. Aucune spécificité européenne de la relation dose-réponse n’a été mise en évidence. Une relation dose-réponse globale incluant 4351 cas et 7055 témoins a été estimée. Association entre exposition aux SPCs et risque de prématurité et de retard de croissance intra-utérin (RCIU). Cette étude est issue de la cohorte bretonne Pélagie (3400 femmes enceintes recrutées en début de grossesse entre 2002 et 2006). L’exposition pendant la grossesse a été mesurée à l’aide de 2 indicateurs : la concentration en THMs de l’eau distribuée dans les réseaux et le dosage d’un biomarqueur urinaire (acide trichloroacétique) pour un sous-échantillon de femmes (étude cas-témoins nichée). Les niveaux dans les réseaux proviennent de la base de données réglementaire SISE-Eaux. Les usages de l’eau du robinet par les femmes pendant la grossesse ont été collectés par questionnaire (quantité d’eau du robinet bue, fréquentation de la piscine, fréquence et durée des douches et des bains). Notre étude suggère une association entre l’exposition prénatale aux SPCs et le risque de RCIU. Aucune association n’est observée avec le risque de prématurité. / Background. Chlorine is the most widely used disinfectant for drinking water treatment. During treatment process, the natural organic matter present in water reacts with chlorine to form disinfection by-products (DBPs), such as trihalomethanes (THMs) and haloacetic acids (HAAs), the most occurring ones. Several identified DBPs were recognized carcinogens or fetotoxic in animals. We conducted two epidemiological studies to assess the health effects of human exposure to DBPs regarding cancer and reproduction. Association between THM exposure and the risk of bladder cancer: a pooled analysis of 3 European case-control studies. The study included 2381 cases and 3086 controls from 3 case-control studies (France, Finland, Spain). Environmental exposure to SPCs was measured by THM concentrations in water distribution systems, retrospectively estimated through the 40-year exposure window. Information about water uses (tap water ingestion, showers and baths, swimming-pool attendance) was available. An increased risk of bladder cancer was observed, in men only, with increasing THM concentrations in water systems and duration of exposure to chlorinated surface water. No association was observed with exposure through tap water ingestion. Exposure through showers, baths and swimming in pools (available in Spanish study only) was associated with an increased risk of bladder cancer. Three North-American case-control studies were joined to the three European ones into a meta-regression. No specific dose-response relationship was identified for European studies. A global dose-response relationship was consequently estimated, including 4351 cases and 7055 controls. Association between DBP exposure and the risk of preterm birth and Intra-Uterine Growth Retardation (IUGR). This study was conducted within the PELAGIE cohort (3400 pregnant women included in early pregnancy, in Brittany, France, between 2002 and 2006). Exposure during pregnancy was measured by 2 indicators: THM concentrations in water distribution systems and a biomarker of HAA ingestion, the level of trichloroacetic acid in maternal urine at inclusion (in a nested case-control study). The regulatory database SISE-Eaux was used to estimate THMs concentrations in the water distribution systems. Water uses during pregnancy (ingested tap water, swimming-pool attendance, showers and bath frequencies and duration) were collected with questionnaires. Our results suggest an association between prenatal exposure to DBPs (THMs and HAAs) and the risk of IUGR. No association was found with the risk of preterm birth.

Les travaux présentés dans cette thèse concernent le développement d'un outil d'analyse multiparamétrique pour la quantification de biomarqueurs urinaires du cancer de la vessie.La première partie des travaux de recherche a pour objectif la sélection de marqueurs pour le diagnostic et la récidive des tumeurs urothéliales. Une première étude a permis l'évaluation de la sélectivité de marqueurs candidats dans des échantillons urinaires de patients atteints de cancer de la vessie. Cinq des vingt marqueurs initiaux ont été sélectionnés pour leur performance diagnostique, définissant le Panel 1 : VEGF, MMP9, IL8, PTGS2 et EN2. Une seconde étude a été réalisée afin d'évaluer le potentiel de marqueurs et de paramètres cliniques pour le diagnostic de la récidive des tumeurs urothéliales. Les échantillons urinaires évalués provenaient donc de patients présentant une récidive du cancer de la vessie et de patients ne présentant pas de récidive. Le Panel 2 a ainsi été défini, basé sur le modèle de régression multiple le plus performant. Il comprend les paramètres cliniques et moléculaires suivants : nombre de récidives antérieures, nombre de thérapies par BCG, stade de la tumeur au moment du diagnostic, CDH1, IL8, ErbB2, IL6, EN2 et VEGF.La seconde partie concerne le développement d'un test multiparamétrique pour la quantification des marqueurs sélectionnés. Il s'agit d'une plateforme automatisée, à haut-débit et sous un format de plaque de microtitration 96-puits. La méthode de quantification choisie est un immunoessai de type sandwich sous la forme de puce à protéines. Le développement de la plateforme a débuté avec le Panel 1 dont trois des cinq marqueurs (VEGF, MMP9 et IL8) ont été intégrés avec succès. Suite à la seconde étude de sélection de marqueurs, le développement de l'immunoessai multiparamétrique a été orienté vers le Panel 2. À l'exception du marqueur EN2, nécessitant une configuration d'immunoessai différente, tous les marqueurs du Panel 2 ont pu être intégrés à la plateforme / The work reported in this thesis focuses on the development of a multiplex analytical tool for the quantification of selected bladder cancer urinary biomarkers.The aim of the first part of this work is the selection of urinary biomarkers for the diagnosis and recurrence of urothelial tumors. A first study evaluated the selectivity of candidate markers in urine samples of bladder cancer patients. Five of the twenty initial markers were selected for their diagnostic performance. They define Panel 1: VEGF, MMP9, IL8, PTGS2 and EN2. A second study was then conducted to assess the potential of urinary markers and clinical parameters for the diagnosis of bladder cancer recurrence. Two types of urine samples were thus evaluated: samples from recurrent bladder cancer patients and samples from bladder cancer patients without recurrence. Panel 2 was then defined based on the best performing multivariate regression model. It includes the following clinical and molecular parameters: number of past recurrences, number of BCG therapies, tumor stage at diagnosis, CDH1, IL8, ErbB2, IL6, EN2 and VEGF.The second part involves the development of a multiplex test for the quantification of the selected markers. It is a high-throughput automated platform in a 96-well microtiter plate format. It was designed as a multiplex sandwich immunoassay based on a protein microarray. The platform development began with Panel 1 for which three of the five markers (VEGF, MMP9 and IL8) were successful integrated into a multiplex immunoassay. The end of the second marker selection study marked the development transition from Panel 1 to Panel 2. With the exception of EN2, requiring a different immunoassay configuration, all the Panel 2 markers were integrated into the platform