L'analyse de données d'expression montre qu'il est avantageux d'analyser les processus biologiques en termes de modules plutôt que simplement considérer les gènes un par un. Dans ce projet nous avons conduit une analyse non supervisée des données d'expression de gènes de plusieurs cohortes de tumeurs urothéliales en appliquant la méthode d'Analyse en Composantes Indépendantes. Plusieurs études ont démontré les meilleures performances de l'ACI par rapport à l'ACP et les méthodes de clustering, pour obtenir une décomposition plus réaliste des données d'expression en patterns d'expression pertinents et associés avec le phénotype d'intérêt.Les tumeurs urothéliales apparaissent et évoluent selon deux voies distinctes dont la probabilité de progression en cancer musculo-invasif diffère radicalement. Excepté la mutation de FGFR3 dans le groupe le moins agressif, les processus moléculaires sous-jacents n'ont pas été complètement identifiés. Le principal objectif de cette thèse était dédié aux interprétations biologiques des différentes composantes indépendantes pour aider à confirmer et étendre la liste des processus biologiques connus pour être impliqués dans le cancer de vessie.Chaque composante indépendante est caractérisée par une liste de projections de gènes et de contributions pondérées d'échantillons tumoraux . En combinant expertise biologique et comparaison des listes de gènes à des voies existantes et en étudiant conjointement l'association des composantes aux annotations cliniques et moléculaires, nous avons pu différencier les CIscausées par des facteurs techniques, tels que le prélèvement chirurgical de celles ayant des interprétations biologiques pertinentes. De plus, parmi les signaux pertinents biologiquement, cette analyse nous a permis de différencier les signaux provenant du stroma, comme la réponse immunitaire médiée par les lymphocytesB&T, de ceux produits par les tumeurs elles-mêmes, comme les signaux reliés à la prolifération ou à la différenciation. La classification des tumeurs selon leurs contributions à certaines CIs a pu être étroitement associée à des annotations anatomo-cliniques, et a mis en évidence de nouveaux sous-types de tumeur spotentiels, qui suggèrent l'existence de voies de progression supplémentaires dans le cancer de vessie. De façon similaire, l'étude des contributions de groupes de tumeurs basés sur des annotations cliniques ou moléculaires a montré différents niveaux de contamination par le stroma entre les tumeurs mutées et nonmutées pour FGFR3. La reproductibilité des composantes a été étudiée en utilisant des graphes de corrélation. La majeure partie des CIs interprétées a été validée sur trois jeux de données indépendants, et plusieurs d'entre elles ont aussi détectées dans un jeu de données de lignées cellulaires.Une deuxième étude sur le rétinoblastome a montré que nous pouvions tirer partie de l'ACI dans des contextes variés. Le rétinoblastome est initié par la perte des deux alléles du gène suppresseur de tumeur RB1. D'autres évènements génomiques non identifiés sont nécessaires à la progression de la maladie. Nous avons observé une association entre âge des patients et altérations génomiques. Les patients jeunes présentant moins d'altérations que les patients âgés, ces derniers présentant des gains du 1q et des pertes du 16q. Cette séparation des tumeurs selon l'âge est également observée sur les données d'expression, notamment en appliquant l'ACI dont l'une des composantes discrimine les patients selon leur âge. Ces résultats suggèrent l'existence de deux voies de progression dans le rétinoblastome. L'analyse des données à haut débit fournit de nombreuses listes de gènes. Afin de les interpréter, une possibilité est d'extraire les dernières publications groupées par sujets prédéfinis (fonction, localisation,...). / Practice of gene expression data analysis shows that it is advantageous to analyze biologicalprocesses in terms of modules rather than simply consider gene one by one. In this project, we conducted anunsupervised analysis of the gene expression data of several cohorts of urothelial tumors, applying theIndependent Component Analysis method. Several studies demonstrated the outperformance of ICA overPCA and clustering-based methods in obtaining a more realistic decomposition of the expression data intoconsistent patterns of coexpressed genes associated with the studied phenotypes[1, 2, 3, 4].Urothelial tumors arise and evolve through two distinct pathways which radically differ on their probabilityof progression to muscle invasion. Except the mutation of FGFR3 in the less aggressive group, theunderlying molecular processes have not been completely identified. The first and main objective of the PhDthesis was dedicated to the biological interpretation of the different independent components to help toconfirm and extend the list of biological processes known to be involved in bladder cancer.Each independent component (IC) is characterized by a list of gene projections on the one hand and weightedcontributions of tumor samples on the other hand. By combining biological expertise and comparison of theassociated list of genes to known pathways, and jointly studying the association of the components tomolecular and clinical annotations, we have been able to differentiate components that were caused bytechnical factors, such as surgical sampling, from those having consistent biological interpretationin terms of tumor biology. Moreover, among the biologically meaningful signals, this analysis allowed us todifferentiate the signals from stroma of the tumor, like immune response mediated by B- and T-lymphocytes,from the signals produced by the tumors themselves, like signals related to proliferation, or differentiation.The clustering of the tumor samples according to their contributions on some ICs can be closely associated toanatomo-clinical annotations, and highlighted new potential subtypes of tumors which suggest existence ofadditional pathways of bladder cancer progression. Similarly, the study of the contributions of preestablishedgroups of tumors based on clinical or molecular criteria showed different levels of stromacontamination between FGFR3 non-mutated and mutated tumors. The reproducibility of the components wasinvestigated using correlation graphs. The major part of the interpreted ICs was validated on threeindependent bladder cancer datasets, and several of them were also identified in an urothelial cancer celllines data set.A second study about retinoblastoma gave us the occasion to show that we can take advantage ofICA in various contexts. Retinoblastoma is initiated by the loss of both alleles of the RB1 tumor suppressorgene. Although necessary for initiation, other genomic events, that remain to be identified, are needed for theprogression of the disease [5]. We observed, as it was previously described [6], an association between age ofthe patients and levels of genomic aberrations, the younger patients having fewer alterations than the olderpatients, which generally present gain of 1q and loss of 16q. We showed that this tendency of the tumors tobe clustered into two groups of age can also be observed on the expression data by applying ICA whose oneof the component was highly correlated to the age of the patients. These results suggest the existence of twopathways of progression in retinoblastoma.The analysis of high throughput data provides many lists of genes. To interpret them, a possibility isto study the latest related publications grouped by pre-defined group of topics (function, cellular location...).To that aim, in a third study, we introduced a web-based Java application tool named GeneValorization whichgives a clear and handful overview of the bibliography corresponding to one particular gene list [7].

Les tumeurs de vessie suivent deux voies de progression tumorale. La voie des carcinomes in situ (CIS) qui progressent pour envahir la membrane basale puis le muscle, et la voie des tumeurs papillaires de bas grade qui progressent peu mais qui récidivent fréquemment. Environ 65% des tumeurs papillaires de bas grade présentent une mutation du gène FGFR3 et récemment des protéines de fusion FGFR3-TACC3 ont été observées dans les tumeurs de vessie (dans 10% des tumeurs invasives). Le rôle oncogénique du récepteur FGFR3 muté et de FGFR3-TACC3 a été démontré in vivo et in vitro. Cependant, les voies de signalisation du récepteur FGFR3 muté ou de FGFR3-TACC3 sont à l’heure actuelle très peu caractérisées. Dans ce contexte, deux approches ont été mises en place pour caractériser ces voies de signalisation. La première s’appuie sur l’étude de la phosphorylation des protéines p38, AKT et ERK1/2 par le récepteur FGFR3 muté (S249C) ou sauvage dans la lignée cellulaire fibroblastique NIH3T3, et a permis d’identifier les protéines p38 et AKT comme activées par le récepteur FGFR3 muté et nécessaire pour induire la transformation cellulaire. L’étude de l’activation de ces deux voies de signalisation a été réalisée dans des lignées cellulaires dérivées de tumeurs de vessie exprimant le récepteur FGFR3 muté ou FGFR3-TACC3 de manière endogène et a montré que leur activation était dépendante de celle du récepteur FGFR3. De plus nous avons montré que les protéines p38 et AKT sont impliquées dans le maintien d’une boucle de rétro-contrôle positive entre FGFR3 et MYC : l’activation de FGFR3 induit une surexpression de MYC qui en retour promeut l’expression de FGFR3. La seconde approche est basée sur une étude visant à identifier les partenaires protéiques de FGFR3 par spectrométrie de masse après immunoprécipitation de celui-ci qui avait été réalisée précédemment au laboratoire. L’analyse des données a permis l’obtention d’une liste de 60 protéines identifiées comme partenaires protéiques de FGFR3 avec une grande confiance. La construction d’un réseau à partir de cette liste n’a pas été possible (trop peu d’interactions existant entre ces protéines), nous avons donc développé un algorithme (PEPPER) en collaboration avec un étudiant en bio-informatique au laboratoire, Rémy Nicolle, pour proposer un réseau de signalisation de FGFR3.Les deux approches mises en place au cours de cette thèse nous ont permis de mieux caractériser les voies de signalisation du récepteur FGFR3. L’identification d’une boucle de rétrocontrôle entre FGFR3 et MYC a permis de mieux comprendre pourquoi le récepteur FGFR3 possède des propriétés oncogéniques, et de proposer les protéines p38 et AKT comme cibles thérapeutiques potentielles pour traiter les tumeurs de vessie exprimant le récepteur FGFR3 altéré. La construction du réseau de signalisation de FGFR3 via PEPPER donne une vue d’ensemble des voies de signalisation de FGFR3 et ouvre de nouvelles pistes à étudier. / Bladder cancer progression can be divided in two main pathways. The pathway of In Situ Carcinoma (CIS) which progress through an invasion of the basement membrane and then the muscle and the pathway of Ta papillary tumors which change little but recur frequently after tumor resection. Approximately 65% of Ta papillary tumors harboring a FGFR3 mutation and recently FGFR3-TACC3 fusion proteins have been observed in bladder tumors (about 10% of bladder tumors). The oncogenic role of the mutated FGFR3 receptor and of the FGFR3-TACC3 fusion protein has been demonstrated in vivo and in vitro. However signaling pathways activated by the mutated FGFR3 receptor or by the FGFR3-TACC3 fusion protein are currently poorly characterized.In this context, two approaches have been developed to characterize these signaling pathways. The first is based on the study of p38, AKT and ERK1/2 phosphorylation by the mutated receptor (S249C) or the wild type receptor in the NIH3T3 fibroblastic cell line. This study allowed identifying p38 and AKT as activated by the mutated FGFR3 receptor. Moreover, activation of p38 and AKT by the mutated receptor is critical for cell transformation. Study of the activation of these two signaling has been realized in human bladder cancer cell lines endogenously expressing the mutated FGFR3 receptor or the FGFR3-TACC3 fusion protein. Moreover, we showed that p38 and AKT are involved in the maintenance of a FGFR3/MYC feedback positive loop: FGFR3 activation induce MYC over expression which in turns promotes FGFR3 expression. The second approach is based on a study whose aim was to identify FGFR3 proteins partners by mass spectrometry after a FGFR3 immunoprecipitation, which has been previously realized in the lab. Data analyze led to the obtaining of a list of 60 proteins identified has FGFR3 protein partners with a high confidence. Construction of a FGFR3 network with this list was not possible (too little interactions existing between these proteins), so we developed an algorithm (PEPPER) in collaboration with a student in bioinformatics in the lab, Remy Nicolle, to propose a FGFR3 signaling network.The two approaches developed during this thesis allowed us to better characterize the FGFR3 signaling pathways. Identification of a FGFR3/MYC feedback loop allowed us to better understand why the altered FGFR3 has oncogenic properties and to propose p38 and AKT as news promising therapeutic targets, to treat human bladder tumors harboring the altered FGFR3 receptor. Construction of the FGFR3 signaling network with the algorithme PEPPER give an overview of the FGFR3 signaling pathways and open new tracks to explore.

<p>Cancer immunotherapy aims at reversing the immunosuppressive tumor environment and enhancing anti-tumor immunity. This thesis comprises studies on murine models for prostate (TRAMP-C2) and bladder (MB49) cancer with the aim to explore if the introduction of an adenoviral vector expressing CD40 ligand (AdCD40L) can induce anti-tumor immune responses.</p><p>We show in subcutaneous mouse models that AdCD40L treatment suppresses tumor growth. Bladder cancer is known to secrete immunosuppressive IL-10 which may inhibit T cell function. We show that introducing AdCD40L into mouse bladder tumors inhibits IL-10 production and reverses immunosuppression. AdCD40L-transduced mouse prostate cancer cells showed caspase activation and reduced cell viability. Vaccination with CD40L-modified prostate cancer cells induces anti-tumor responses and protects mice against rechallenge with native TRAMP-C2 cells. In order to enhance AdCD40L therapy, we explored the possibility of combining it with the histone deacetylase inhibitor FK228, also known as depsipeptide. We show that FK228 upregulates coxsackie and adenovirus receptor expression and thereby enhances adenoviral-mediated CD40L expression in both murine and human prostate cancer cells. Increasing amounts of FK228 or AdCD40L reduces prostate cancer cell viability, while the combined treatment gives at least an additive therapeutic effect. Moreover, we show that AdCD40L transduction of prostate cancer cells induces endogenous CD40 expression and sensitize them for CD40L-mediated therapy.</p><p>In order to conduct prostate-specific gene therapy, prostate-specific promoters can be used to drive transgene expression. However, there are no reports on prostate-specific promoters that are transcriptionally active in mouse cells. Here we show that by using the two-step transcription activation system (TSTA), we can enhance the activity of a recombinant human promoter sequence and obtain activity in mouse prostate cancer cells as well. This finding paves the way for future studies of prostate-specific gene therapy in immunocompetent mouse models.</p>

<p>This thesis deals with recognition and elimination of tumours by T lymphocytes and their use in adoptive immunotherapy.</p><p>The first tumour-draining lymph node; the sentinel node, is identified by peritumoural injection of a tracer. This is the hypothesised location for the activation of tumour-reactive lymphocytes. Accordingly, proliferation and IFN-γ production in response to autologous tumour extract was detected in sentinel nodes from patients with colon and urinary bladder cancer. Reactivity in metastatic nodes was generally lower or absent, but the non-responsiveness could be subdued in long-term cultures by addition of tumour antigen and IL-2. A novel padlock-probe based method was developed for measuring the T cell receptor Vβ repertoire. Common Vβ gene expansions were detected in tumour-infiltrating lymphocytes and sentinel nodes. Thus, tumour antigens are recognised in sentinel nodes by Th1 lymphocytes, resulting in a clonally expanded cell population that can be further propagated <i>ex vivo</i>.</p><p>Regulatory T cells (Tregs) may contribute to tumour-induced immunosuppression. Immunohistochemical stainings against the pan-T cell marker CD3 and Treg marker FOXP3 was performed on tumour tissue from 20 historical urinary bladder cancer patients. The ratio of FOXP3⁺ to CD3⁺ cells was lower in patients alive 7 years post-cystectomy, suggesting that Tregs in bladder cancer have prognostic implications.</p><p>Lymphocytes were isolated from sentinel nodes from sixteen patients with advanced or high-risk colon cancer. <i>In vitro</i> expansion with addition of autologous tumour extract and IL-2 mainly promoted the outgrowth of CD4⁺ Th1 lymphocytes, which were safely re-transfused to the patients. Four patients responded with complete tumour regression. Survival time in the Dukes’ D patients was significantly increased compared with conventionally treated controls (2.6 versus 0.8 years; p=0.048).</p><p>In conclusion, human solid tumours are recognised in sentinel nodes and <i>in vitro</i> expanded sentinel node-acquired CD4⁺ T lymphocytes seem useful in the treatment of patients with disseminated cancer.</p>

Cancer immunotherapy aims at reversing the immunosuppressive tumor environment and enhancing anti-tumor immunity. This thesis comprises studies on murine models for prostate (TRAMP-C2) and bladder (MB49) cancer with the aim to explore if the introduction of an adenoviral vector expressing CD40 ligand (AdCD40L) can induce anti-tumor immune responses. We show in subcutaneous mouse models that AdCD40L treatment suppresses tumor growth. Bladder cancer is known to secrete immunosuppressive IL-10 which may inhibit T cell function. We show that introducing AdCD40L into mouse bladder tumors inhibits IL-10 production and reverses immunosuppression. AdCD40L-transduced mouse prostate cancer cells showed caspase activation and reduced cell viability. Vaccination with CD40L-modified prostate cancer cells induces anti-tumor responses and protects mice against rechallenge with native TRAMP-C2 cells. In order to enhance AdCD40L therapy, we explored the possibility of combining it with the histone deacetylase inhibitor FK228, also known as depsipeptide. We show that FK228 upregulates coxsackie and adenovirus receptor expression and thereby enhances adenoviral-mediated CD40L expression in both murine and human prostate cancer cells. Increasing amounts of FK228 or AdCD40L reduces prostate cancer cell viability, while the combined treatment gives at least an additive therapeutic effect. Moreover, we show that AdCD40L transduction of prostate cancer cells induces endogenous CD40 expression and sensitize them for CD40L-mediated therapy. In order to conduct prostate-specific gene therapy, prostate-specific promoters can be used to drive transgene expression. However, there are no reports on prostate-specific promoters that are transcriptionally active in mouse cells. Here we show that by using the two-step transcription activation system (TSTA), we can enhance the activity of a recombinant human promoter sequence and obtain activity in mouse prostate cancer cells as well. This finding paves the way for future studies of prostate-specific gene therapy in immunocompetent mouse models.

This thesis deals with recognition and elimination of tumours by T lymphocytes and their use in adoptive immunotherapy. The first tumour-draining lymph node; the sentinel node, is identified by peritumoural injection of a tracer. This is the hypothesised location for the activation of tumour-reactive lymphocytes. Accordingly, proliferation and IFN-γ production in response to autologous tumour extract was detected in sentinel nodes from patients with colon and urinary bladder cancer. Reactivity in metastatic nodes was generally lower or absent, but the non-responsiveness could be subdued in long-term cultures by addition of tumour antigen and IL-2. A novel padlock-probe based method was developed for measuring the T cell receptor Vβ repertoire. Common Vβ gene expansions were detected in tumour-infiltrating lymphocytes and sentinel nodes. Thus, tumour antigens are recognised in sentinel nodes by Th1 lymphocytes, resulting in a clonally expanded cell population that can be further propagated ex vivo. Regulatory T cells (Tregs) may contribute to tumour-induced immunosuppression. Immunohistochemical stainings against the pan-T cell marker CD3 and Treg marker FOXP3 was performed on tumour tissue from 20 historical urinary bladder cancer patients. The ratio of FOXP3+ to CD3+ cells was lower in patients alive 7 years post-cystectomy, suggesting that Tregs in bladder cancer have prognostic implications. Lymphocytes were isolated from sentinel nodes from sixteen patients with advanced or high-risk colon cancer. In vitro expansion with addition of autologous tumour extract and IL-2 mainly promoted the outgrowth of CD4+ Th1 lymphocytes, which were safely re-transfused to the patients. Four patients responded with complete tumour regression. Survival time in the Dukes’ D patients was significantly increased compared with conventionally treated controls (2.6 versus 0.8 years; p=0.048). In conclusion, human solid tumours are recognised in sentinel nodes and in vitro expanded sentinel node-acquired CD4+ T lymphocytes seem useful in the treatment of patients with disseminated cancer.

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No description available.

Background: Prostate cancer is the most common cancer in Sweden with around 10,000 new cases every year. Kidney and bladder cancer are less common with 1,000 and 2,000 new cases annually, respectively. The incidence of these cancer sites is higher in developed, than in developing countries, suggesting an association between lifestyle and cancer risk. The aims of this thesis were to investigate body mass index (BMI), blood pressure, and blood levels of glucose, total cholesterol, and triglycerides as risk factors for prostate, kidney, and bladder cancer. Furthermore, we aimed at assess probabilities of prostate cancer and competing events, all-cause death, for men with normal and high levels of metabolic factors. Material and methods: This thesis was conducted within the Metabolic Syndrome and Cancer project (Me-Can), a pooled cohort study with data from 578,700 participants from Norway, Sweden, and Austria. Data from metabolic factors were prospectively collected at health examinations and linked to the Cancer and Cause of Death registers in each country.  Results: High levels of metabolic factors were not associated with increased risk of prostate cancer, but high levels of BMI and blood pressure were associated with risk of prostate cancer death. The probability of prostate cancer was higher for men with normal levels of metabolic factors compared to men with high levels, but the probability of all-cause death, was higher for men with high levels than for those with normal levels. For both men and women, high levels of metabolic factors were associated with increased risk of kidney cancer (renal cell carcinoma). Furthermore, blood pressure for men and BMI for women were found as independent risk factors of kidney cancer. High blood pressure was associated with an increased risk of bladder cancer for men. Conclusions: High levels of metabolic factors were associated to risk of kidney and bladder cancer and to death from kidney, bladder, and prostate cancer. Compared to men with normal levels, men with high levels of metabolic factors had a decreased probability of prostate cancer but an increased probability of all-cause death. / <p>Ytterligare forskningsfinansiärer: World Cancer Research Fund (2007/09) och Wereld Kanker Onderzoek Fonds (R2010/247)</p> / Me-Can

Electronic medical record databases (e.g. the Clinical Practice Research Datalink, CPRD) are increasingly used in epidemiological research. The CPRD has two formats of data: coded, which is the sole format used in almost all research; and free-text (or ‘hidden’), which may contain much clinical information but is generally unavailable to researchers. This thesis examines the ramifications of omitting free-text records from research. Cases with bladder (n=4,915) or pancreatic (n=3,635) cancer were matched to controls (n=21,718, bladder; n=16,459, pancreas) on age, sex and GP practice. Coded and text-only records of attendance for haematuria, jaundice and abdominal pain in the year before cancer diagnosis were identified. The number of patients whose entire attendance record for a symptom/sign existed solely in the text was quantified. Associations between recording method (coded or text-only) and case/control status were estimated (χ2 test). For each symptom/sign, the positive predictive value (PPV, Bayes' Theorem) and odds ratio (OR, conditional logistic regression) for cancer were estimated before and after supplementation with text-only records. Text-only recording was considerable, with 7,951/20,958 (37%) of symptom records being in that format. For individual patients, text-only recording was more likely in controls (140/336=42%) than cases (556/3,147=18%) for visible haematuria in bladder cancer (χ2 test, p<0.001), and for jaundice (21/31=67% vs 463/1,565=30%, p<0.0001) and abdominal pain (323/1,126=29% vs 397/1,789=22%, p<0.001) in pancreatic cancer. Adding text records reduced PPVs of visible haematuria for bladder cancer from 4.0% (95% CI: 3.5–4.6%) to 2.9% (2.6–3.2%) and of jaundice for pancreatic cancer from 12.8% (7.3–21.6%) to 6.3% (4.5–8.7%). Coded records suggested that non-visible haematuria occurred in 127/4,915 (2.6%) cases, a figure below that generally used for study. Supplementation with text-only records increased this to 312/4,915 (6.4%), permitting the first estimation of its OR (28.0, 95% CI: 20.7–37.9, p<0.0001) and PPV (1.60%, 1.22–2.10%, p<0.0001) for bladder cancer. The results suggest that GPs make strong clinical judgements about the probable significance of symptoms – preferentially coding clinical features they consider significant to a diagnosis, while using text to record those that they think are not.