<p>Mild hyperthermia (39°C-43°C) has numerous therapeutic benefits as an adjuvant therapy in the treatment of a variety of tumor types. Hyperthermia increases tumor blood flow and vascular permeability, promoting drug delivery and tumor oxygenation. Hyperthermia enhances the uptake and efficacy of numerous chemotherapeutic agents, including cisplatin, resulting in increased cytotoxicity. In addition to these biological responses, hyperthermia can be used as a drug-release trigger for temperature-sensitive nanoparticles, resulting in an improved and more targeted drug delivery system. Cisplatin was chosen because 1) it shows broad spectrum activity against a wide range of heatable cancers (i.e., those in sites such as the pancreas, colon and rectum, cervix and bladder, and 2) the same hyperthermic temperatures that enable temperature-sensitive lipsome-drug release also enhance cisplatin-induced cytotoxicity.</p><p>The role of hyperthermia in enhancing cisplatin delivery and cytotoxicity was investigated at both the cellular and tissue levels. While hyperthermia treatment is applicable to a variety of tumor types, the focus of this work was on bladder cancer. The synergistic effects of hyperthermia and cisplatin were investigated, along with the role of copper transport protein 1 (Ctr1) in this process. In addition, cisplatin was encapsulated within temperature-sensitive liposomes, which were used in combination with hyperthermia for targeted drug delivery. These studies demonstrated that the combination of cisplatin and hyperthermia improved drug delivery, and potentially anti-tumor efficacy, and that targeted delivery was enhanced through incorporation of temperature-sensitive liposomes. As many current methods for administering bladder hyperthermia have drawbacks, such as invasiveness and regional heating, the final aim of this study was to develop and test a less-invasive and more focused preclinical bladder heating device in a rat model. </p><p>Hyperthermia sensitizes cells to the cytotoxic effects of the commonly used chemotherapeutic agent cisplatin by increasing drug accumulation and subsequent platinum-DNA adduct formation. However, the molecular mechanisms underlying this enhancement remain unclear. Understanding the fundamental mechanisms involved in the synergistic interaction is necessary to increase the therapeutic benefits of this combination in the clinic. The synergism between the anti-cancer benefits of cisplatin and the drug delivery benefits of hyperthermia may offer a novel and more effective treatment for many cancer patients. We hypothesized that hyperthermia increases cisplatin accumulation and efficacy in part by modulating the function of Ctr1, a major regulator of cellular cisplatin uptake. To test this hypothesis, we examined the significance of Ctr1 during combined hyperthermia and cisplatin therapies and assessed the importance of cisplatin- and hyperthermia-induced Ctr1 multimerization in enhancing cisplatin cytotoxicity. We observed increased Ctr1 multimerization following hyperthermia treatment (41°C) in vitro, compared to normothermic controls (37°C), suggesting that this may be a mechanism for increased cisplatin uptake in heat-treated cells. The impact of increased Ctr1 multimerization was evaluated by measuring platinum accumulation in wild-type (WT) and Ctr1-/- cells. WT cells contained greater levels of platinum compared to Ctr1-/- cells. A further increase in platinum was observed following hyperthermia treatment, but only in the WT cells. Hyperthermia enhanced cisplatin-mediated cytotoxicity in WT cells with a dose-modifying factor (DMF) of 1.8 compared to 1.4 in Ctr1-/- cells. Our data suggest that heat increases Ctr1 activity by increasing multimerization, resulting in enhanced drug accumulation. Although we recognize that the effect of heat on cells is multi-factorial, our results support the hypothesis that Ctr1 is, in part, involved in the synergistic interaction observed with cisplatin and hyperthermia treatment. </p><p>In addition to assessing cisplatin delivery at the cellular level, we evaluated cisplatin delivery at the tissue level, using novel cisplatin-loaded temperature-sensitive liposomes. We hypothesized that delivering cisplatin encapsulated in liposomes under hyperthermic conditions would improve the pharmacokinetic profiles of cisplatin, increase drug delivery to the tumor, decrease normal tissue toxicity, and enhance the anti-tumor activity of cisplatin. We successfully prepared temperature-sensitive liposomes loaded with cisplatin and demonstrated that heat (42°C) sensitizes cisplatin-resistant cells to the cytotoxic effects of cisplatin in vitro. </p><p>Decreased toxicity was observed in animals treated with the cisplatin liposome (± heat) compared to the free drug treatments. A pharmacokinetic study of cisplatin-loaded temperature-sensitive liposomes and free drug was performed in tumor-bearing mice under normothermic and hyperthermic conditions. Cisplatin half-life in plasma was increased following liposome treatment compared to free cisplatin, and cisplatin delivery to the tumors was greatest in mice that received liposomal cisplatin under hyperthermia. These initial in vivo data demonstrate the potential effectiveness of this cisplatin-loaded liposome formulation in the treatment of certain types of cancer. To assess the anti-cancer efficacy of the liposome treatment, a tumor growth delay study was conducted and demonstrated equivalent efficacy for the cisplatin-loaded temperature-sensitive liposome compared to free drug. </p><p>In addition to the liposome work, we developed and evaluated a novel heating device for the bladder. Despite the evidence that hyperthermia is an effective adjuvant treatment strategy, current clinical heating devices are inadequate, warranting the development of a new and improved system. We induced hyperthermia using ferromagnetic nanoparticles and an alternating magnetic field device developed by Actium Biosystems. Initial preclinical studies in a rat model demonstrated preferential bladder heating. However, our preliminary studies show severe toxicity with the direct instillation of the nanoparticles in the bladder, and further studies are needed to potentially modify the nanoparticle coating, the catheterization procedure, as well as to develop a different animal model.</p> / Dissertation

A central theme within the concept of enzyme-directed bioreductive drug development is the potential to predict tumour response based on the profiling of enzymes involved in the bioreductive activation process. Mitomycin C (MMC) is the prototypical bioreductive drug that is reduced to active intermediates by several reductases including NAD(P)H:quinone oxidoreductase (NQO1) and NADPH cytochrome P450 reductase (P450R). The purpose of our study was to determine whether NQO1 and P450R protein expression in a panel of low-grade, human superficial bladder tumours correlates with clinical response to MMC. A retrospective clinical study was conducted in which the response to MMC of 92 bladder cancer patients was compared to the immunohistochemical expression of NQO1 and P450R protein in archived paraffin-embedded bladder tumour specimens. A broad spectrum of NQO1 protein levels exists in bladder tumours between individual patients, ranging from intense to no immunohistochemical staining. In contrast, levels of P450R were similar with most tumours having moderate to high levels. All patients were chemotherapy naïve prior to receiving MMC and clinical response was defined as the time to first recurrence. A poor correlation exists between clinical response and NQO1, P450R or the expression patterns of various combinations of the 2 proteins. The results of our study demonstrate that the clinical response of superficial bladder cancers to MMC cannot be predicted on the basis of NQO1 and/or P450R protein expression and suggest that other factors (other reductases or post DNA damage events) have a significant bearing on tumour response.

Together, colorectal (CRC) and bladder cancer (BlCa) are responsible for a large percentage of cancer related morbidity and mortality in Western society. A dramatic reduction in patient survival occurs as these cancers progress towards invasive and metastatic disease, from a five year survival rate of about 90% for localised disease to approximately 5-10% for advanced disease involving distant metastasis. A greater understanding of disease progression will lead to enhanced screening, diagnostic and treatment strategies, in turn providing an improved prognosis for the patient. The purpose of this study was to expand the current molecular knowledge of CRC and BlCa by elucidating the role of Mxi1 mutations and MTSS1 expression in CRC and BlCa respectively, and to examine the diagnostic potential of these genes. The Mxi1 coding region for 41 tumours, collected by the South Western Sydney Colorectal Cancer Tumour Bank from 2000-2001, was screened for mutations using Dideoxy Fingerprinting (ddF) and sequencing. Sequence alterations were detected in 34% of tumours. Three different polymorphisms and three mutations were detected. One mutation could possibly affect the tumour suppressor function of Mxi1. The presence of a gene mutation did not correlate to any clinical characteristics and is therefore not a suitable diagnostic marker. Microsatellite instability (MSI) status however, significantly correlated with tumour grade. Expression levels of MTSS1 and an associated gene, MTSS2, were examined in 16 BlCa cell lines, 9 clonally-derived BlCa sublines, and 30 transitional cell carcinomas (TCCs) collected by the Heinrich-Heine University from 1993-2000. Variable gene expression was observed in BlCa cell lines and tumour samples. No significant correlation of MTSS expression and invasive ability was observed for the cell lines or tumour samples. Further studies eliminated promoter methylation and p53 functional status as mechanisms involved in MTSS1 and MTSS2 down-regulation. Functional studies performed on stable MTSS1-expressing BlCa lines found that although migration was increased, cells displayed reduced anchorage-independent growth. The invasive ability of these cells was unchanged confirming that expression does not correlate with invasive ability. These data support the role of MTSS1 as a tumour suppressor and not as a metastasis suppressor gene. Although MTSS1 may not be useful in predicting more invasive disease, its role as a tumour suppressor in cancer may be useful.

Together, colorectal (CRC) and bladder cancer (BlCa) are responsible for a large percentage of cancer related morbidity and mortality in Western society. A dramatic reduction in patient survival occurs as these cancers progress towards invasive and metastatic disease, from a five year survival rate of about 90% for localised disease to approximately 5-10% for advanced disease involving distant metastasis. A greater understanding of disease progression will lead to enhanced screening, diagnostic and treatment strategies, in turn providing an improved prognosis for the patient. The purpose of this study was to expand the current molecular knowledge of CRC and BlCa by elucidating the role of Mxi1 mutations and MTSS1 expression in CRC and BlCa respectively, and to examine the diagnostic potential of these genes. The Mxi1 coding region for 41 tumours, collected by the South Western Sydney Colorectal Cancer Tumour Bank from 2000-2001, was screened for mutations using Dideoxy Fingerprinting (ddF) and sequencing. Sequence alterations were detected in 34% of tumours. Three different polymorphisms and three mutations were detected. One mutation could possibly affect the tumour suppressor function of Mxi1. The presence of a gene mutation did not correlate to any clinical characteristics and is therefore not a suitable diagnostic marker. Microsatellite instability (MSI) status however, significantly correlated with tumour grade. Expression levels of MTSS1 and an associated gene, MTSS2, were examined in 16 BlCa cell lines, 9 clonally-derived BlCa sublines, and 30 transitional cell carcinomas (TCCs) collected by the Heinrich-Heine University from 1993-2000. Variable gene expression was observed in BlCa cell lines and tumour samples. No significant correlation of MTSS expression and invasive ability was observed for the cell lines or tumour samples. Further studies eliminated promoter methylation and p53 functional status as mechanisms involved in MTSS1 and MTSS2 down-regulation. Functional studies performed on stable MTSS1-expressing BlCa lines found that although migration was increased, cells displayed reduced anchorage-independent growth. The invasive ability of these cells was unchanged confirming that expression does not correlate with invasive ability. These data support the role of MTSS1 as a tumour suppressor and not as a metastasis suppressor gene. Although MTSS1 may not be useful in predicting more invasive disease, its role as a tumour suppressor in cancer may be useful.

Together, colorectal (CRC) and bladder cancer (BlCa) are responsible for a large percentage of cancer related morbidity and mortality in Western society. A dramatic reduction in patient survival occurs as these cancers progress towards invasive and metastatic disease, from a five year survival rate of about 90% for localised disease to approximately 5-10% for advanced disease involving distant metastasis. A greater understanding of disease progression will lead to enhanced screening, diagnostic and treatment strategies, in turn providing an improved prognosis for the patient. The purpose of this study was to expand the current molecular knowledge of CRC and BlCa by elucidating the role of Mxi1 mutations and MTSS1 expression in CRC and BlCa respectively, and to examine the diagnostic potential of these genes. The Mxi1 coding region for 41 tumours, collected by the South Western Sydney Colorectal Cancer Tumour Bank from 2000-2001, was screened for mutations using Dideoxy Fingerprinting (ddF) and sequencing. Sequence alterations were detected in 34% of tumours. Three different polymorphisms and three mutations were detected. One mutation could possibly affect the tumour suppressor function of Mxi1. The presence of a gene mutation did not correlate to any clinical characteristics and is therefore not a suitable diagnostic marker. Microsatellite instability (MSI) status however, significantly correlated with tumour grade. Expression levels of MTSS1 and an associated gene, MTSS2, were examined in 16 BlCa cell lines, 9 clonally-derived BlCa sublines, and 30 transitional cell carcinomas (TCCs) collected by the Heinrich-Heine University from 1993-2000. Variable gene expression was observed in BlCa cell lines and tumour samples. No significant correlation of MTSS expression and invasive ability was observed for the cell lines or tumour samples. Further studies eliminated promoter methylation and p53 functional status as mechanisms involved in MTSS1 and MTSS2 down-regulation. Functional studies performed on stable MTSS1-expressing BlCa lines found that although migration was increased, cells displayed reduced anchorage-independent growth. The invasive ability of these cells was unchanged confirming that expression does not correlate with invasive ability. These data support the role of MTSS1 as a tumour suppressor and not as a metastasis suppressor gene. Although MTSS1 may not be useful in predicting more invasive disease, its role as a tumour suppressor in cancer may be useful.

Made available in DSpace on 2014-06-11T19:26:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-05-20Bitstream added on 2014-06-13T19:33:14Z : No. of bitstreams: 1 ramos_pmm_me_botib.pdf: 3067176 bytes, checksum: bb2655e0acf6fb1d0c707110baed9681 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Os padrões anormais de metilação do DNA, especialmente a hipermetilação de genes com provável função supressora de tumor, representam um dos mais promissores marcadores moleculares do câncer por levarem à inativação funcional de genes críticos. Estudos prévios documentaram altos níveis de expressão do gene H19 em carcinoma de bexiga recorrentes. O gene H19 é regulado por imprinting, está localizado em 11p15.5 adjacente ao gene IGF2 (insulin-like growth factor 2 – somatomedin A) e codifica um transcrito não codificador de proteínas (micro RNA miR-675). Uma região que atua de forma coordenada no controle da expressão desses genes, chamada DMR (Differentially Methylated Region), atua na determinação do imprinting recíproco e na expressão mutuamente exclusiva dos genes IGF2 e H19. Ela encontra-se não metilada no homólogo materno e metilada no homólogo paterno e contém sete regiões de ligação da proteína CTCF (proteína bloqueadora do acentuador), que é sensível à metilação do DNA. Um relato prévio da literatura sugeriu que somente o sexto sítio de ligação do fator CTCF apresenta metilação parental específica. Este achado foi correlacionado com o padrão de expressão regulado por imprinting dos genes IGF2 e H19 em câncer de bexiga. No presente estudo, o padrão de metilação alelo-específico do gene H19 foi determinado em duas regiões distintas: no sexto sítio de ligação do fator CTCF contido na DMR e no primeiro éxon do gene H19 utilizando-se três abordagens diferentes: MSRE-PCR-RFLP (Methylation Sensitive Restriction Enzyme - Polymerase Chain Reaction - Restriction Fragment Length Polymorphism), qMSP (quantitative real time Methylation Specific Polymerase Chain Reaction) para o sexto sítio e MSP-CTPP (Methylation Specific Polymerase Chain Reaction with Confontring Two Pair-Primers) para a região do primeiro éxon em 52 amostras de tecidos... / Abnormal patterns of DNA methylation, especially hypermethylation of genes demonstrating tumoral suppressor functions represent one of the most promising molecular markers of cancer because they can lead to functional inactivation of critical genes. Previous studies have documented high levels of H19 gene expression in recurrent bladder carcinomas. The H19 gene is regulated by imprinting, is located at 11p15.5 adjacent to the IGF2 gene (insulin-like growth factor 2 - somatomedin A) and encodes a non-coding transcript (micro RNA miR-675). A Differentially Methylated Region (DMR) region acts in a coordinated manner to control the expression of the IGF2 and H19 genes by determining their reciprocal imprinting and mutually exclusive expression patterns. The H19-DMR contains seven potential CTCF-binding sites. These sites are located upstream to the transcriptional initiation site, and the gamete-specific methylation acts as an insulator by precluding CTCF binding in the paternal allele. A previous literature report have suggested that only the sixth CTCF-binding site shows parental specific methylation. This finding was correlated with the imprinting expression pattern of IGF2 and H19 genes in bladder cancer. In the present study, the allele-specific methylation pattern of the H19 gene was evaluated in two distinct target regions: the sixth CTCF-binding site located in the DMR and the first exon of the H19 gene using three different approaches: MSRE-PCR-RFLP (Methylation Sensitive Restriction Enzyme - Polymerase Chain Reaction - Restriction Fragment Length Polymorphism), qMSP (quantitative real time Methylation Specific Polymerase Chain Reaction) for the sixth CTCF-binding site, and the first exon of the H19 gene was analyzed by MSP-CTPP (Methylation Specific Polymerase Chain Reaction with Confronting Two-Pair Primers) in 52 samples of bladder tumors matched to normal adjacent tissues obtained... (Complete abstract click electronic access below)

Orientador: Cláudia Aparecida Rainho / Banca: Marcos Vinícius de Marcos Gomes / Banca: José Carlos Souza Trndade Filho / Resumo: Os padrões anormais de metilação do DNA, especialmente a hipermetilação de genes com provável função supressora de tumor, representam um dos mais promissores marcadores moleculares do câncer por levarem à inativação funcional de genes críticos. Estudos prévios documentaram altos níveis de expressão do gene H19 em carcinoma de bexiga recorrentes. O gene H19 é regulado por imprinting, está localizado em 11p15.5 adjacente ao gene IGF2 (insulin-like growth factor 2 - somatomedin A) e codifica um transcrito não codificador de proteínas (micro RNA miR-675). Uma região que atua de forma coordenada no controle da expressão desses genes, chamada DMR (Differentially Methylated Region), atua na determinação do imprinting recíproco e na expressão mutuamente exclusiva dos genes IGF2 e H19. Ela encontra-se não metilada no homólogo materno e metilada no homólogo paterno e contém sete regiões de ligação da proteína CTCF (proteína bloqueadora do acentuador), que é sensível à metilação do DNA. Um relato prévio da literatura sugeriu que somente o sexto sítio de ligação do fator CTCF apresenta metilação parental específica. Este achado foi correlacionado com o padrão de expressão regulado por imprinting dos genes IGF2 e H19 em câncer de bexiga. No presente estudo, o padrão de metilação alelo-específico do gene H19 foi determinado em duas regiões distintas: no sexto sítio de ligação do fator CTCF contido na DMR e no primeiro éxon do gene H19 utilizando-se três abordagens diferentes: MSRE-PCR-RFLP (Methylation Sensitive Restriction Enzyme - Polymerase Chain Reaction - Restriction Fragment Length Polymorphism), qMSP (quantitative real time Methylation Specific Polymerase Chain Reaction) para o sexto sítio e MSP-CTPP (Methylation Specific Polymerase Chain Reaction with Confontring Two Pair-Primers) para a região do primeiro éxon em 52 amostras de tecidos... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Abnormal patterns of DNA methylation, especially hypermethylation of genes demonstrating tumoral suppressor functions represent one of the most promising molecular markers of cancer because they can lead to functional inactivation of critical genes. Previous studies have documented high levels of H19 gene expression in recurrent bladder carcinomas. The H19 gene is regulated by imprinting, is located at 11p15.5 adjacent to the IGF2 gene (insulin-like growth factor 2 - somatomedin A) and encodes a non-coding transcript (micro RNA miR-675). A Differentially Methylated Region (DMR) region acts in a coordinated manner to control the expression of the IGF2 and H19 genes by determining their reciprocal imprinting and mutually exclusive expression patterns. The H19-DMR contains seven potential CTCF-binding sites. These sites are located upstream to the transcriptional initiation site, and the gamete-specific methylation acts as an insulator by precluding CTCF binding in the paternal allele. A previous literature report have suggested that only the sixth CTCF-binding site shows parental specific methylation. This finding was correlated with the imprinting expression pattern of IGF2 and H19 genes in bladder cancer. In the present study, the allele-specific methylation pattern of the H19 gene was evaluated in two distinct target regions: the sixth CTCF-binding site located in the DMR and the first exon of the H19 gene using three different approaches: MSRE-PCR-RFLP (Methylation Sensitive Restriction Enzyme - Polymerase Chain Reaction - Restriction Fragment Length Polymorphism), qMSP (quantitative real time Methylation Specific Polymerase Chain Reaction) for the sixth CTCF-binding site, and the first exon of the H19 gene was analyzed by MSP-CTPP (Methylation Specific Polymerase Chain Reaction with Confronting Two-Pair Primers) in 52 samples of bladder tumors matched to normal adjacent tissues obtained... (Complete abstract click electronic access below) / Mestre

Orientador: Wílson Mello Júnior / Resumo: O carcinoma de bexiga urinária é o tipo de câncer mais prevalente no sistema urinário em seres humanos e apresenta altos índices de morbidade e mortalidade. Seu desenvolvimento está relacionado a diversos fatores de risco, como o hábito de fumar, a exposição a agentes químicos industriais e metais pesados e o estresse crônico. As experiências adversas no início da vida estão relacionadas à modulação de eixos hormonais importantes, como o hipotalâmicohipofisário-adrenal (HHA) e hipotalâmico-hipofisário-gonadal (HHG). Alterações na resposta do eixo HHA, componente central da resposta fisiológica ao estresse, levam a desordens fisiológicas, como aumento da proliferação celular, angiogênese e prejuízo da resposta imune, que podem ser responsáveis pelo aumento da susceptibilidade do indivíduo a uma série de doenças na vida adulta, incluindo o câncer. Paralelamente, alterações na fisiologia de hormônios esteroides como andrógenos, estrógenos e glicocorticoides, bem como seus receptores, também têm sido relacionadas ao desenvolvimento de enfermidades como o câncer de bexiga urinária. Em ratos, o comportamento materno funciona como um fator importante na modulação desses eixos hormonais, modificando suas respostas na vida adulta, e estudos recentes têm relacionado os cuidados recebidos por filhotes à incidência de tumores. O objetivo deste estudo foi avaliar a influência do comportamento materno e sua consequente modulação nos eixos HHA e HHG, no desenvolvimento do câncer de bexiga u... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The urinary bladder carcinoma is the most prevalent type of cancer in the urinary tract in humans, with high morbidity and mortality. Its development is related to several risk factors such as smoking, exposure to industrial chemicals and heavy metals and chronic stress. Adverse experiences early in life are related to modulation of important hormonal axes, as the hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-gonadal (HPG). Changes in the response of the HPA axis, a central component of the physiological response to stress, lead to physiological disorders, such as increased cell proliferation, angiogenesis, and impaired immune response, which may be responsible for the increased susceptibility of the individual to a number of diseases in adult life, including cancer. Similarly, changes in the physiology of steroid hormones such as androgens, oestrogens and glucocorticoids and their receptors have also been related to the development of diseases such as bladder cancer. In rats, maternal behavior serves as an important factor in the modulation of hormonal axes, modifying their responses in adulthood, and recent studies have linked the care received by pups and the incidence of tumors. The aim of this study was to evaluate the influence of maternal behavior and its consequent modulation in HPA and HPG axes in the development of bladder cancer. The results showed that rats reared by the negligent mothers had a greater susceptibility to the development of chemica... (Complete abstract click electronic access below) / Mestre

Abstract Bladder cancer when superficial has a good prognosis but it has a high recurrence risk and about 10–15% of the superficial carcinomas will progress into muscle invasive or metastatic type. The most powerful factor for predicting the behavior of bladder carcinoma is the stage of the tumor. Invasion to the lamina propria increases the risk of recurrence and progress to muscle-invasive tumor. Also grade of the tumor and tumor multiplicity associates with high risk for recurrence. New markers are still needed to find those patients who need more and better treatments to avoid the recurrence and progress. The need for new non-invasive markers to diminish the need for frequent cystoscopy in follow-up is also obvious. Gelatinases MMP-2 and MMP-9 are known to associate to tumor invasion and progression. Also their tissue inhibitors TIMP-1 and TIMP-2 take part in these diversified processes and metastasis formation. In the present work the expression and clinical value of gelatinases MMP-2 and MMP-9 and their tissue inhibitors TIMP-1 and TIMP-2 were evaluated in bladder carcinoma. Primary tissue samples of 121 patients were analyzed for expression of MMP-2 and/or MMP-9 using immunohistochemistry. The serum samples of 87 patients who were treated in the Oncology Department of Oulu University Hospital were collected and studied with ELISA. The control group consisted of 44 healthy volunteers. Overexperssion of MMP-2 protein correlated significantly to disease-specific survival and showed an independent prognostic value as a biomarker. High MMP-9 expression instead correlated to favorable overall survival of bladder cancer patients. Circulating proMMP-2, TIMP-2 and MMP-2:TIMP-2 complex levels were lower in cancer patients than in healthy volunteers in control group. High levels of all these three markers correlated with better prognosis in bladder cancer patients.

L’objectif du travail a été d’explorer la valeur pronostique pour les tumeurs de la vessie des polymorphismes de gènes associés à l’inflammation et du microenvironnement tumoral lymphocytaire CD8+. Pour les marqueurs constitutionnels, deux approches ont été conduites concomitamment, l’une explorant de façon globale les gènes associés à l’inflammation, l’autre ciblant un gène inflammatoire d’intérêt, PDL1, impliqué dans des points de contrôles immunologiques. A l’échelle du génome, en utilisant des méthodes statistiques soit classiques soit innovantes (dites multi marqueurs), nous avons démontré que les variants (SNP) dans les gènes TNIP1, CD5 et JAK3 étaient associés au risque de récidive des tumeurs de vessie non invasives du muscle alors que les variants dans les gènes MASP1, AIRE et CD3 étaient associés au risque de progression. Dans un deuxième temps, l’association entre variants dans le gène de PDL1 et pronostic des tumeurs de vessie a été explorée en appliquant une méthode classique « SNP par SNP » et une approche à l’échelle du gène. Nous avons identifié une forte association entre des variants de PDL1 et le pronostic de tumeurs de vessie envahissant le muscle dans une large cohorte prospective, mais sans pouvoir répliquer ce résultat dans une série issue du consortium TCGA.Dans les tumeurs n’envahissant pas le muscle, nous avons développé et évalué une méthode d’évaluation standardisée de l’infiltrat lymphocytaire CD8+, cellules T-cytotoxiques impliquées dans la mort des cellules tumorales. L’analyse morphométrique après double immuno-marquage des cellules tumorales et des lymphocytes CD8+ et numérisation a permis d’estimer de façon séparée le compte des cellules inflammatoires dans la tumeur et le stroma, et d’estimer l’hétérogénéité spatiale intra-tumorale. Nous avons montré que cette hétérogénéité limite les estimations de l’infiltrat CD8+ sur les puces tissulaires (Tissue Micro Array) qui échantillonnent les tumeurs de façon restrictive. Sous cette réserve, nous avons identifié dans les tumeurs n’envahissant pas le muscle une association entre l’infiltrat lymphocytaire CD8+ et le stade tumoral Ta/T1, ainsi qu’avec le risque de récidive des tumeurs T1. A l’avenir, variations génétiques constitutionnelles dans les gènes de l’inflammation et évaluation de l’infiltrat tumoral inflammatoire pourraient être intégrées en vue d’améliorer la prédiction du pronostic des tumeurs vésicales. / The aim of this study was to explore prognostic value for bladder cancer of germline polymorphisms in inflammatory genes and tumor CD8+ lymphocytic microenvironment. For constitutional markers, two approaches were conducted jointly: one genome-based using specific GWAS statistical methods, the other gene-based focusing on PDL1, an inflammatory gene implicated in immune checkpoints. At the genome level, using both standard and innovative statistical methods (multi marker methods Bayesian Lasso and Bayes A) we demonstrated that variants (SNPs) in TNIP1, CD5 and JAK3 were associated with the risk of recurrence of non-muscle invasive bladder cancer (NMIBC) while SNPs in MASP1, AIRE and CD3 were associated with risk of progression. Meanwhile, association between PDL1 and prognosis of NMIBC and muscle invasive BC (MIBC) was explored using classical SNPS by SNP investigations and a gene based approach. We identified a very strong association between PDL1 variants and MIBC prognosis in a large prospective cohort but failed replicating those results in the TCGA consortium series.In non-muscle invasive bladder cancer, we developed and evaluated a standardized counting approach of CD8+ cells, T-cytotoxic lymphocytes implicated in tumor cells death. Morphometric analysis after double immuno-staining of tumor cells and digitalization allowed separate estimation of CD8+ cells in the tumor and stroma compartment and estimation of spatial intra tumoral heterogeneity. We demonstrated that this heterogeneity compromised CD8+ estimation on Tissue Micro Arrays, which sample the tumors in a restrictive manner. Keeping those limitations in mind, we identified an association between CD8+ inflammatory infiltrate and both NMIBC stage and T1 tumours risk of recurrence. In the future, germline variation in inflammatory genes and evaluation of tumor inflammatory infiltrate could be integrated for better prediction of bladder cancer prognosis.