<p>Bacillus Calmette Guérin (BCG), or attenuated Mycobacterium bovis, is the gold standard of immunotherapy in the clinic to treat superficial bladder cancer. However, setbacks remain due to a high recurrence rate, side effects, and BCG-refractory disease. In this thesis, we explored the use of novel immunotherapeutic agents such as CpG oligodeoxynucleotides (CpG ODNs) or synthetic ODNs containing unmethylated CpG dinucleotides. Since unmethylated CpG motifs are predominant in bacterial but not vertebrate DNA, they function as a “danger signal” leading to a potent immune response.</p><p>To be able to test various immunotherapeutic agents, we optimized subcutaneous (s.c.), metastatic, and orthotopic models using the murine bladder-49 (MB49) cancer cell line. In the orthotopic model, we show that poly-L-lysine promotes MB49 attachment to the bladder leading to 100% tumor take. In addition, Clorpactin (sodium oxychlorosene) potently enhances adenoviral transduction in the bladder.</p><p>Utilizing the MB49 model, we compare CpG ODNs with BCG and demonstrate the increased efficacy of CpG ODNs which could cure both s.c. and aggressive orthotopic bladder cancer. In our model, type B ODNs were most optimal and the antitumor response required T cells in order to induce regression and tumor-specific immunity. We also combined CpG ODNs with adenoviral vectors (Ad) expressing the immunostimulatory molecules CD40L, TRANCE, lymphotactin, IL2 or IL15. However, we show that CpG ODNs are effective as a monotherapy and adenoviral vectors did not enhance the effect.</p><p>AdCD40L was also used to genetically modify human dendritic cells (DCs). AdCD40L-transduced DCs not only had a higher and prolonged expression of the Th1 cytokine IL12 compared to TNFα-matured DCs, but CD40L-activated DCs could also resist the suppressive effects of IL10 and TGFβ. Since TNFα is commonly used in clinical DC vaccination protocols and because tumors often secrete immunosuppressive cytokines, these data have important implications for optimizing cancer immunotherapy.</p>

Bacillus Calmette Guérin (BCG), or attenuated Mycobacterium bovis, is the gold standard of immunotherapy in the clinic to treat superficial bladder cancer. However, setbacks remain due to a high recurrence rate, side effects, and BCG-refractory disease. In this thesis, we explored the use of novel immunotherapeutic agents such as CpG oligodeoxynucleotides (CpG ODNs) or synthetic ODNs containing unmethylated CpG dinucleotides. Since unmethylated CpG motifs are predominant in bacterial but not vertebrate DNA, they function as a “danger signal” leading to a potent immune response. To be able to test various immunotherapeutic agents, we optimized subcutaneous (s.c.), metastatic, and orthotopic models using the murine bladder-49 (MB49) cancer cell line. In the orthotopic model, we show that poly-L-lysine promotes MB49 attachment to the bladder leading to 100% tumor take. In addition, Clorpactin (sodium oxychlorosene) potently enhances adenoviral transduction in the bladder. Utilizing the MB49 model, we compare CpG ODNs with BCG and demonstrate the increased efficacy of CpG ODNs which could cure both s.c. and aggressive orthotopic bladder cancer. In our model, type B ODNs were most optimal and the antitumor response required T cells in order to induce regression and tumor-specific immunity. We also combined CpG ODNs with adenoviral vectors (Ad) expressing the immunostimulatory molecules CD40L, TRANCE, lymphotactin, IL2 or IL15. However, we show that CpG ODNs are effective as a monotherapy and adenoviral vectors did not enhance the effect. AdCD40L was also used to genetically modify human dendritic cells (DCs). AdCD40L-transduced DCs not only had a higher and prolonged expression of the Th1 cytokine IL12 compared to TNFα-matured DCs, but CD40L-activated DCs could also resist the suppressive effects of IL10 and TGFβ. Since TNFα is commonly used in clinical DC vaccination protocols and because tumors often secrete immunosuppressive cytokines, these data have important implications for optimizing cancer immunotherapy.

This thesis comprises studies on murine and human models of bladder carcinoma with the aim to develop novel immunogene therapies. On the basis of the results presented in this thesis, a clinical trial is underway. The potential of activating the immune system to combat cancer has long intrigued immunologists. Research has now been intensified and clinically effective treatments are beginning to materialize. We evaluated the induction of anti-tumor responses by inserting immunomodulating genes into tumor cells with adenovectors. Human biopsies and cell lines were positive for adenovirus attachment receptors, and cell lines were easily transduced. CD40L modified cells efficiently induced maturation of dendritic cell (DC). Phenotypical changes of AdCD40L transduced cells, such as increased apoptotic rate, upregulated MHC-I, Fas and TNFR may further strengthen the anti-tumor response. CD40L modified murine bladder cancer cells activated systemic immunity upon vaccination and in situ injections of AdCD40L inhibited tumor progression. Cytotoxic assays revealed the presence of cytotoxic T cells (CTLs) in vaccinated mice. Many tumors have developed ways to evade the immune system. Bladder carcinoma is associated with immune escape mechanisms like IL10 production. We demonstrated that immunosuppression by IL10 inhibited CTL function and that IL10 suppression may be reverted by AdCD40L therapy. In conclusion, AdCD40L therapy induces systemic immunity and inhibits tumor progression in murine models. The immunological mechanisms involve maturation of nearby DCs and CTL induction. AdCD40L therapy is effective despite immune escape mechanisms, e.g. IL10 secretion. The thesis argues for using AdCD40L immunogene therapy as a treatment of bladder carcinoma.

Epidemiologic data suggest that soy consumption may protect against cancer induction in several tissues in humans, including urothelial carcinoma. Genistein have been reported to regulate genes that are involved in several cellular events. However, the molecular mechanism of genistein -induced upregulation of epithelial membrane protein 2 (EMP2), candidate urothelial tumor suppressor, is not entirely understood. At first, we found that the mRNA and protein expression levels of EMP2 were significantly greater in the normal urothelial tissues and human urothelial cells than those in urothelial bladder carcinoma tissues and urothelial cell carcinoma-derived cell lines. Second, EMP2 knockdown via RNA interference markedly enhanced cell proliferation, colony formation, migration and invasiveness. By contrast, EMP2 overexpression suppressed these malignant behaviors. Third, we showed that genistein-induced inhibition in cell proliferation is associated with an increase in EMP2 expression. Using various deleted EMP2 promoter constructs, we defined that the EMP2 core promoter is enough to observe the genistein-induced upregulation of EMP2 transcriptional activity. Using site direct mutagenesis and chromatin immunoprecipitation assays demonstrated that cyclic-AMP response element binding protein 1 (CREB1) acts as a positive regulator of EMP2 transcription by directly binding to its promoter. These results showed EMP2 suppressed urothelial cell carcinoma-derived cell growth, motility and invasion and for the first time that genistein promoted EMP2 expression in urothelial cell carcinoma-derived cells by inducing EMP2 transcriptional activity via CREB1 binding.

These experiments were done to determine the effect of glibenclamide and diazoxide on the growth of human bladder carcinoma (HTB-9) cells in vitro. Cell growth was assayed by cell counts, protein accumulation, and 3H-thymidine uptake. Glibenclamide added at 75 and 150 μM for 48 hr reduced cell proliferation. Dose-inhibition curves showed that glibenclamide added for 48 hr reduced cell growth at concentrations as low as 1 μM (IC50 = 73 μM) when growth was assayed in the absence of added serum. This μM-effect on cell growth was in agreement with the dose range in which glibenclamide decreased open probability of membrane K(ATP) channels. Addition of glibenclamide for 48 hr also altered the distribution of cells within stages of the cell cycle as determined by flow cytometry using 10-5 M bromodeoxyuridine. Glibenclamide (100 μM) increased the percentage of cells in G0/G1 from 33.6% (vehicle control) to 38.3% (P < 0.05), and it reduced the percentage of cells in S phase from 38.3% to 30.6%. On the other hand, diazoxide, which opens membrane K(ATP) channels in HTB-9 cells, stimulated growth measured by protein accumulation, but it did not increase the cell number. We conclude that the sulfonylurea receptor and the corresponding membrane K(ATP) channel are involved in mechanisms controlling HTB-9 cell growth. However, K(ATP) is not rate-limiting among the signaling mechanisms or molecular switches that regulate the cell cycle.

Introduction: les progrès thérapeutiques pour la prise en charge du carcinome urothélial de la vessie, ou tumeur de vessie infiltrant le muscle (TVIM), sont faibles depuis deux décennies, notamment sur le plan de la chimiothérapie, tant dans les formes localisées que métastatiques. L'identification de nouveaux biomarqueurs pouvant améliorer la prise en charge et le pronostic des patients apparait indispensable, notamment pour prédire la réponse aux traitements systémiques. Notre travail a cherché : (i) à identifier des facteurs biologiques prédictifs de rechute après les chimiothérapies adjuvantes (CA) utilisées actuellement, (ii) à déterminer si les altérations moléculaires, cibles de nouvelles thérapies, identifiées dans la tumeur vésicale sont conservées dans les métastases.Objectifs:(i) Rechercher une association entre la rechute après CA et l'expression de six biomarqueurs, ERCC1, Emmprin, Survivin, MDR1, p53 et topoisomérase IIα, dans la tumeur vésicale et/ou une métastase ganglionnaire(ii) Etudier l'hétérogénéité des mutations de FGFR3 dans les TVIM localement avancéesMatériels et Méthodes: Une cohorte rétrospective de 226 patients traités par cystectomie et CA, et une collection tumorale d'échantillons représentatifs de la tumeur primitive et d'une métastase ganglionnaire ont été constituées. L'expression des six biomarqueurs a été déterminée en immunohistochimie sur ces échantillons. Le statut mutationnel de FGFR3 a été déterminé en SNaPshot sur 84 paires tumeur primitive/métastase ganglionnaire.Résultats: Les survies observées dans la cohorte étaient proches de celles rapportées dans la littérature pour des patients de mêmes stades. La collection tumorale étudiée semble donc représentative des TVIM traitées en France. Aucun des six biomarqueurs n'était retrouvé comme prédictif de rechute après traitement. Une mutation de FGFR3 était retrouvée dans 4 (4,7%) des 84 échantillons appariés sans aucune discordance entre tumeur primitive et métastase ganglionnaire.Conclusions: L'expression de ces six biomarqueurs étudiés en immunohistochimie n'est pas associée à un risque de rechute après CA. Le statut mutationnel de FGFR3 est conservé au cours de la dissémination métastatique, et ses mutations activatrices sont présentes dans près de 5% des TVIM localement avancées. Leur conservation dans le processus métastatique renforce l'intérêt de l'évaluation de nouvelles thérapies ciblant le récepteur FGFR3 muté. Enfin, il est possible de rechercher cette mutation sur la tumeur vésicale ou une métastase avec la même pertinence. / Introduction: In muscle invasive urothelial carcinoma of the bladder (MI UCB), few progresses have been made in the last two decades, particularly in chemotherapy, both in localized and metastatic setting. The identification of new biomarkers that can improve the management and prognosis of patients appears essential. Such biomarkers will be clinically relevant if they can predict the response to systemic treatments. In this thesis, we studied biomarkers in two ways: (i) to identify predictive factors of relapse after cisplatin-based adjuvant chemotherapy (AC), (ii) to confirm that the molecular alterations targeted by new therapies and identified in bladder tumor are present in metastases.Objectives:(i) To examine the relationship between relapse after AC and expression of six biomarkers, ERCC1, Emmprin, Survivin, MDR1 p53 and topoisomerase IIα, in bladder tumor and / or lymph node metastases.(ii) To study the heterogeneity of FGFR3 mutations in locally advanced MI UCBMaterials and Methods: A retrospective cohort of 226 patients treated with cystectomy and AC, and tumor collection of representative samples of the primary tumor and lymph node metastases were constituted. The expression of six biomarkers was determined by immunohistochemistry on these samples and determined by morphometry. FGFR3 mutation status was determined by SNaPshot in 84 paired primary tumors and positive lymph nodes.Results: Survivals observed in the cohort were similar to those reported in the literature for patients of same stages, with tumor collection being representative of the MI UCB treated in France. None of the six biomarkers was found as an independent predictive factor of relapse after AC. FGFR3 mutation was found in 4 (4.7%) of the 84 paired samples with complete concordance between primary tumor and lymph node metastases.Conclusions: The expression of the six studied biomarkers by immunohistochemistry is not associated with risk of relapse following AC. FGFR3 mutation status is maintained during metastases process, and FGFR3 activating mutations are present in about 5% of locally advanced MI UCB. FGFR3 mutation remains a promising therapeutic target in a low subset of patients, and the evaluation of new therapies targeting the mutated FGFR3 receptor are needed. Finally, search for these mutations either in bladder tumor or in metastasis is relevant.

Als viert häufigste Karzinomerkrankung bei Männern und zwölft häufigste bei Frauen in Deutschland ist das BCa weitverbreitet in der Gesellschaft. Dies macht die Erforschung der komplexen, molekularen Mechanismen und die Entwicklung individualisierter Therapiestrategien notwendig. Neben externen Umwelteinflüssen wie der Konsum von Tabak, die Exposition von Chemikalien und chronische Entzündungen spielen auch genetische und molekulare Aberrationen eine relevante Rolle in der Tumorinitiation. Zu den molekularen Aberrationen gehört unter anderem die Dysregulation der miRNA-vermittelten RNA-Interferenz. Interessant für diese Arbeit waren die beiden Moleküle NRP2 und VEGFC, welchen in diversen Tumorentitäten eine onkogene Wirkung zugeschrieben wurde und welche bei vermehrter Expression die Tumorentstehung, -progression und Metastasierung über die Tumor-assoziierte Lymphangiogenese fördern. Um die Rolle von NRP2 und VEGFC im BCa näher zu untersuchen, wurden die Expressionen und die Korrelationen dieser und potentiell interagierender miRNAs in BCa- Geweben mittels in silico-Verfahren und in BCa-Zelllinien mittels qPCR-Analysen näher untersucht. Zur weiteren Analyse wurden anschließend auch die Expressionen und Korrelationen alternativer Targetgene und Hostgene sowie gemeinsame Signalwege von vier ausgewählten miRNAs evaluiert. In den in silico- und qPCR-Analysen zeigten sich NRP2 und VEGFC in den BCa-Geweben und -Zelllinien verglichen zu den gesunden Urothelgeweben und Zelllinien niedriger exprimiert. Diese Ergebnisse stehen im Widerspruch zu der Annahme, dass eine verstärkte Expression beider Targetgene mit der Tumorinitiation einhergeht. Jedoch zeigten sich NRP2 und VEGFC verstärkt exprimiert in allen CDDP-resistenten BCa-Zelllinien. Dies spricht dafür, dass beide Targetgene zur Resistenzbildung beitragen. Auch in den Auswertungen der BC- BET-Datenbank war die erhöhte Expression von NRP2 und VEGFC mit aggressiveren Tumoreigenschaften und einer schlechteren Prognose assoziiert. Diese Assoziation unterstützt die Annahme, dass die zunehmende Expression beider Targetgene mit einer Tumorprogression, Metastasierung und der Rezidivbildung in Verbindung steht. In einem weiteren Schritt wurde nach NRP2/VEGFC-regulierenden miRNAs geschaut. Dabei ergaben sich nach in silico- und Literaturrecherche 33 Kandidaten, wovon nach näherer Betrachtung die vier miRNAs miR-27a, -128, -195 und -338-5p zur weiteren Analyse ausgewählt wurden. Für die ausgewählten miRNAs wurden anschließend die Expressionen in BCa-Geweben mittels TCGA-Datenbank und die zellulären Grundexpressionen mittels qPCR-Analyse bestimmt. Hierbei zeigten sich miR-27a und -128 in den BCa-Geweben signifikant erhöht. MiR-195 hingegen war signifikant erniedrigt. Auch miR-338-5p war erniedrigt, jedoch nicht signifikant. Die Auswertungen der zellulären Grundexpressionen zeigte für miR-128 und miR-195 den Trend zu einer verstärkten Expression in den BCa- Zelllinien. Für miR-27a und miR-338-5p zeigte sich keine relevante Expressionsdifferenz. Um eine Aussage über die Signifikanz der Expressionsdifferenzen zu treffen, ist die Analyse einer größeren Anzahl von BCa- und gesunden Zelllinien notwendig. Die genauere Betrachtung der miRNA-Targetgen-Korrelationen sollte Auskunft über mögliche miRNA-mRNA-Interaktionen geben. Eine negative miRNA-Targetgen-Korrelation spricht für eine Inhibierung der Targetgene durch die miRNA. Eine positive Korrelation spricht für eine miRNA-induzierte Verstäkung der Targetgenexpression. Signifikant negative Korrelationen zeigten sich in den BCa-Geweben für miR-27a/NRP2, miR-128/NRP2 und miR-128/VEGFC. Ebenfalls konnten diese negativen Korrelationen in den CDDP-sensitiven und CDDP-resistenten BCa-Zelllinien für miR-27a/NRP2, miR-128/NRP2 und miR- 128/VEGFC verzeichnet werden. Das inverse Verhältnis in den CDDP-resistenten Zelllinien unterstreicht, dass die reduzierte Expression von miR-27a und miR-128 in einer Hochregulierung von NRP2 und VEGFC resultieren könnte, welche die Tumorprogression und Resistenzentwicklung fördern könnte. Gegensätzlich dazu waren die miR-195/VEGFC- und miR-195/NRP2-Korrelationen in den CDDP-resistenten BCa-Zelllinien positiv. In den BCa-Geweben der TCGA-Datenbank waren sie signifikant bzw. per Trend positiv. Dieses Expressionsmuster spricht dafür, dass miR-195 die Expression der beiden Targetgene induzieren könnte. Während sich miR-338-5p in den BCa-Geweben schwach herabreguliert zeigte, ergab die qPCR-Analyse keine relevante Expressionsdifferenz in den malignen und nicht-malignen Zelllinien. Jedoch war die Zelllinie mit der stärksten Expression, gegensätzlich zu den anderen drei miRNAs, eine gesunde Zelllinie. Auch war miR-338-5p, mit Ausnahme von 5637CDDP, in den beiden anderen CDDP-resistenten Zelllinien herabreguliert. Aufgrund der schwachen und nicht signifikanten Korrelationen von miR-338-5p mit den beiden Targetgenen NRP2 und VEGFC konnte keine klare Schlussfolgerung bezüglich der Rolle von miR-338-5p in der Tumorinitiation und –progression gezogen werden. Weiterhin wurde auch das Verhältnis der intragen liegenden miRNAs zu ihren Hostgenen genauer betrachtet. Dabei waren für miR-128/ARPP21 und miR-338-5p/AATK die Korrelationen jeweils signifikant positiv. Auch zeigten sich ARPP21 und AATK in den BCa- Geweben der TCGA-Datenbank signifikant stärker exprimiert. Das spricht dafür, dass eine Koexpression von miRNA und Hostgen vorliegt. Die Recherche nach weiteren alternativen Targetgenen ergab eine Liste von acht Kandidaten, wovon vier (CPD, EDEM3, EYA4, RELN) eine signifikante Expressionsdifferenz zwischen BCa- und gesunden Urothelgewebe aufwiesen. Diese vier alternativen Targetgene zeigten sich zudem in der Literaturrecherche vielfach in die Karzinogenese und Resistenzbildung diverser Tumorentitäten involviert. Die Korrelationen mit den miRNAs zeigten sich für miR-128/CPD, miR-27a/EDEM3, miR-27a/EYA4, miR-128/EYA4, miR- 27a/RELN, miR-128/RELN und miR-195/RELN signifikant. CPD und EDEM3 zeigten sich in den BCa-Geweben der TCGA-Datenbank höher exprimiert, ebenso wie miR-128 und miR- 27a. Die Ergebnisse der Literaturrecherche und die positiven Korrelationen sprechen dafür, dass miR-128 und miR-27a die Expression von CDP bzw. EDEM3 induzieren und die Tumorinitiation und Tumorprogression fördern könnten. Gegensätzlich dazu stehen die negativen Korrelationen von miR-27a und miR-128 mit NRP2 und VEGFC. Auch die Korrelationen mit EYA4 und RELN zeigten sich jeweils negativ. EYA4 konnte vielfach als Tumorsuppressor identifiziert werden, unter anderem im BCa. Es spricht dafür, dass miR- 27a und miR-128 hier eine onkogene Rolle einnehmen könnten. RELN hingegen wurde in der Literatur vielfach als Onkogen identifiziert. Die negative Korrelation mit miR-27a und miR-128 lässt daher annehmen, dass die beiden miRNAs auch tumorsuppressiv wirken. MiR-195 zeigte jeweils eine positive Korrelation mit NRP2, VEGFC und RELN. Unter der Annahme, dass NRP2, VEGFC und RELN, wie vielfach in der Literatur beschrieben, onkogen agieren, sprechen die positiven Korrelationen dafür, dass miR-195 eine Tumor- fördernde Rolle einnimmt. Die erniedrigte Expression der drei Targetgene und miR-195 in den BCa-Geweben der TCGA-Datenbank widersprechen jedoch dieser Annahme. Abschließend wurden gemeinsame Signalwege der vier ausgewählten miRNAs herausgefiltert. Hierbei ergab sich eine Liste von 77 Signalwegen, welche vielfach in diverse Tumorsignalwege involviert sind, unter anderem in den Signalweg der Karzinogenese des BCa. Insgesamt scheinen miRNAs und ihre Zielgene an den komplexen Mechanismen der BCa- Karzinogenese und -Chemoresistenz beteiligt zu sein, wobei sie sowohl onkogene als auch tumorsuppressive Funktionen ausüben können.

Die Verbesserung der Früherkennung des Urothelkarzinoms ist derzeit der einzige Weg zur Senkung der Mortalität dieser Tumorentität. Bisher fehlen Screeeningparameter für das Blasenkarzinom vergleichbar dem Prostata-spezifischen Antigen (PSA) zur Diagnostik des Prostatakarzinoms. Jedoch scheint auf Grund vielversprechender diagnostischer Ansätze und einer Vielzahl potentieller Urin- und Serummarker die Entwicklung eines Screeningverfahrens in der Zukunft möglich. Zur Bestätigung eines Tumorverdachts (z.B. bei schmerzloser Makrohämaturie) erscheint die ALS (5-Aminolävulinsäure)- Fluoreszenzzystoskopie trotz der aktuellen Diskussionen derzeit als die einzige diagnostische Alternative zur Standardweißlichtzystoskopie. Der klinische Wert eines zukünftigen ausschließlich auf der Laser- induzierten Autofluoreszenz (LIF) basierenden bildgebenden Verfahrens ist derzeit unklar. Vorstellbar wäre auch eine Kombination aus LIF- und ALS- Fluoreszenzzystoskopie mit Erhöhung der Spezifität der Fluoreszenzmethode. Die Ergebnisse der in dieser Habilitation vorgestellten Untersuchungen belegen, dass eine nicht- bzw. minimal- invasive Diagnostik des Blasenkarzinoms möglich ist. Die Kombination von verschiedenen diagnostischen Verfahren würde eine den klinischen Erfordernissen entsprechende Charakterisierung des Tumors ermöglichen. Als Vision für die Zukunft wäre die Möglichkeit einer "optischen Biopsie", d.h. die in vivo Diagnostik ohne die Notwendigkeit der Gewebeentnahme, ein neuer Ansatz und hätte mehrere entscheidende Vorteile: 1. Es findet keine Zerstörung des Urothels bzw. des Karzinoms statt. Bei gleichzeitigem Einsatz schonender Therapieverfahren (z.B. Lasertherapie) bestünde kaum die Gefahr einer Tumorzellstreuung. 2. Es könnte eine unbegrenzte Anzahl von "Biopsien" durchgeführt werden mit möglicher Verbesserung der diagnostischen Sensitivität. 3. Im Gegensatz zur herkömmlichen Endoskopie und Mikroskopie erlaubt der kombinierte Einsatz von optischer Kohärenztomographie (OCT), Ultraschall und konfokaler Laser- Rastermikroskopie (CLSM) erstmals eine dreidimensionale Begutachtung des Gewebes. 4. Die Diagnostik erfolgt in vivo ohne Biopsie-, Fixierungs- oder Färbungs-bedingte Artefakte. 5. Der diagnostische und auch der finanzielle Aufwand (Fixierung, H/E- Färbung, Personal etc.) wären geringer. 6. Die Diagnosestellung erfolgt intraoperativ. Dies hat z.B. den Vorteil, dass bei der Diagnose eines muskelinvasiven Tumors und entsprechender Indikation für die radikale Zystektomie auf eine vorherige TURB verzichtet werden könnte. 7. Durch Automatisierung könnte der diagnostische Prozess objektiviert und beschleunigt werden. Die Ergebnisse wären erstmals vergleichbar und reproduzierbar. Nach Meinung des Autors ist die "optische Biopsie" zur Diagnostik maligner Epithelveränderungen in der Zukunft möglich. Sinnvoll erscheint die Kombination aus einem sensitiven orientierenden Verfahren (z.B. ALA/LIF), einer Staging- Methode mit ausreichender Eindringtiefe (z.B. Sonographie, OCT) und einer Grading- Methode mit mikroskopischer Bildauflösung (CLSM). Am Ende der Entwicklung sollte ein Multisensorendoskop stehen, welches die verschiedenen bildgebenden Verfahren in sich vereint. Der Einsatzbereich eines solchen Gerätekonzepts geht weit über den Bereich der Urologie hinaus und wird die Diagnostik und Therapie von Neoplasien in der Zukunft maßgeblich beeinflussen. / At present, the only way to lower the mortality rate of urothelial carcinoma is the improvement of early detection. Screening parameters like prostate specific antigen (PSA) for the diagnosis of prostate cancer are missing. However, promising potential markers in urine and serum make the development of a screening method in the future possible. In order to confirm the suspicion of bladder cancer (e.g. macrohematuria) currently fluorescence cystoscopy with 5-aminolevulinic-acid (ALA) appears as the only alternative to standard white light endoscopy. To date, the clinical value of a diagnostic method solely based on laser- induced autofluorescence (LIF) is unclear. Possible seems the combination of LIF and ALA-fluorescence cystoscopy. The results of the studies described in this "Habilitation" demonstrate that a non- or minimal- invasive diagnosis of bladder carcinoma is achievable. The combination of different diagnostic methods would be sufficient to characterize a tumor in vivo. As a vision for future "optical biopsies", that means the in vivo diagnosis of cancer without the need for excisional biopsies would have several advantages: 1. There is no destruction of the urothelium or the tumor. Therefore the risk of tumor cell seeding is minimal. 2. One could take an unlimited number of "biopsies" with possible improvement of the diagnostic sensitivity. 3. In contrast to traditional white light endoscopy which allows only two-dimensional surface imaging, the combination of optical coherence tomography (OCT), ultrasound (US) and endoscopic confocal microscopy may enable three-dimensional high resolution imaging of tissue structure at depth. 4. One could study human tissue in real time and in its original in vivo state without artifacts caused by biopsy forceps, cautery or fixation. 5. For hospitals the cost of performing histological studies on excised tissue is a significant expense, which optical techniques may reduce by replacing or reducing the number of biopsies. 6. The diagnosis will be determined in the OR. In case of an muscle- invasive tumor this could spare the patient an invasive transurethral resection (TURB) prior to radical cystectomy. 7. Through automation of the diagnostic process the results were more objective and comparable. In conclusion we believe that the concept of "optical biopsies" is a realistic vision, which could eventually change the way we diagnose and treat diseased tissue. The combination of a sensitive orientating method (e.g. ALA/LIF), a staging method (e.g. OCT, US) and a grading method with microscopic resolution (e.g. CLSM) would be necessary. The end point should be the development of a multi-sensor endoscope, which would have applications in almost all clinical disciplines.

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No. of bitstreams: 2 Dissertação - Mariana Paiva Lopes - 2014 .pdf: 2192084 bytes, checksum: e551c174d821b3b398247680a94c40cd (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-03-12 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The gene for tumor necrosis factor (TNF) is located in the human MHC central region known as class III (Major Histocompatibility Complex). This gene encodes an important pro- inflammatory cytokine produced primarily by macrophages, and it has been associated with several diseases, such as autoimmune and degenerative ones. Studies indicate that TNF polymorphisms may influence their level of expression and activity and therefore could influence susceptibility to tumors. Whereas the bladder urothelium can constantly be the target of inflammatory processes and as the main forms of treatment of bladder tumors are immunotherapy, the genetic profile of an individual can influence susceptibility to this type of injury. In this study, we analyzed the structure and variability of the regulatory region of the TNF gene in Brazilian samples and the results were compared with data obtained by 1000Genomes project. A study of association between polymorphisms of the promoter region of TNF and bladder carcinoma patients in the state of São Paulo in Ribeirão Preto, in our analysis was conducted there was no relationship of the data found with bladder carcinoma. In all evaluated populations we found 15 variation points, found in 11 Brazilian and 15 variation points found in the 1000Genomes data, these variation points are arranged in 20 different haplotypes. These haplotypes with comparisons involving primate sequences indicated that one allele arose before the main speciation and human dispersion. Two strains were defined by haplotype relationships and are probably very old in human evolutionary history, since all populations evaluated showed haplotypes belonging to each of these strains. The frequency of haplotype H01 is the highest among all populations evaluated. However, the haplotype H12, although at reduced frequency compared to H01 is probably the oldest haplotype in part by the second line being shared with other primates. / O gene do Fator de Necrose Tumoral (TNF) humano está localizado no MHC (Complexo Principal de Histocompatibilidade) central, região conhecida como classe III. Este gene codifica uma citocina pró-inflamatória importante, produzida principalmente por macrófagos, que tem sido relacionada com diversas doenças, como as autoimunes e as degenerativas. Estudos indicam que polimorfismos do TNF podem influenciar seu nível de expressão e atividade e, portanto, poderiam influenciar a susceptibilidade a tumores. Considerando que o urotélio vesical pode ser constantemente alvo de processos inflamatórios e que as principais formas de tratamento de tumores vesicais são imunoterápicos, o perfil genético de um indivíduo pode influenciar a susceptibilidade a esse tipo de lesão. Neste estudo analisamos a estrutura e a variabilidade da região regulatória do gene TNF em amostras brasileiras e os resultados foram comparados com dados obtidos pelo projeto 1000Genomes. Foi realizado um estudo de associação entre polimorfismos da região promotora do TNF e o carcinoma vesical em pacientes do estado de São Paulo da cidade de Ribeirão Preto, nas nossas análises não houve relação dos dados encontrados com o carcinoma vesical. Considerando todas as populações avaliadas foram encontrados 15 pontos de variação, 11 encontrados nas amostras brasileiras e 15 encontrados nos dados do projeto 1000Genomes, esses pontos de variação estão arranjados em 20 haplótipos diferentes. Esses haplótipos em conjunto com as comparações envolvendo sequências de primatas indicam que um alelo principal surgiu antes da especiação e dispersão humana. Duas linhagens foram definidas pelas relações haplotípicas e são, provavelmente, muito antigas na história evolutiva humana, já que todas as populações avaliadas apresentaram haplótipos pertencentes a cada uma destas linhagens. A frequência do haplótipo H01 é a mais alta entre todas as populações avaliadas. No entanto, o haplótipo H12, embora com frequência reduzida quando comparado com o H01, provavelmente é o haplótipo mais antigo em parte por esta segunda linhagem ser compartilhada com outros primatas.

Copy number variation (CNV) comprises a recently discovered kind of variation involving deletion and duplication of DNA segments of variable size, ranging from a few hundred basepairs to several million. By altering gene dosage levels or disrupting proximal or distant regulatory elements CNVs create human diversity. They represent also an important factor in human evolution and play a role in many disorders including cancer. Array-based comparative genomic hybridization as well as expression arrays are powerful and suitable methods for determination of copy number variations or gene expression changes in the human genome. In paper I we established a 32K clone-based genomic array, covering 99% of the current assembly of the human genome with high resolution and applied it in the profiling of 71 healthy individuals from three ethnic groups. Novel and previously reported CNVs, involving ~3.5% of the genome, were identified. Interestingly, 87% of the detected CNV regions overlapped with known genes indicating that they probably have phenotypic consequences. In papers II through IV we applied this platform to different tumor types, namely two collections of brain tumors, glioblastoma (paper II) and medulloblastoma (paper III), and a set of bladder carcinoma (paper IV) to identify chromosomal alterations at the level of DNA copy number that could be related to tumor initiation/progression. Tumors of the central nervous system represent a heterogeneous group of both benign and malignant neoplasms that affect both children and adults. Glioblastoma and medulloblastoma are two malignant forms. Glioblastoma often affects adults while the embryonal tumor medulloblastoma is the most common malignant brain tumor among children. The detailed profiling of 78 glioblastomas, allowed us to identify a complex pattern of aberrations including frequent and high copy number amplicons (detected in 79% of samples) as well as a number of homozygously deleted loci. These regions encompassed not only previously reported oncogenes and tumor suppressor genes but also numerous novel genes. In paper III, a subset of 26 medulloblastomas was analyzed using the same genomic array. We observed that alterations involving chromosome 17, especially isochromosome 17q, were the most common genomic aberrations in this tumor type, but copy number alterations involving other chromosomes: 1, 7 and 8 were also frequent. Focal amplifications, on chromosome 1 and 3, not previously described, were also detected. These loci may encompass novel genes involved in medulloblastoma development. In paper IV we examined for the presence of DNA copy number alterations and their effect on gene expression in a subset of 21 well-characterized Ta bladder carcinomas, selected for the presence or absence of recurrences. We identified a number of novel genes as well as a significant association between amplifications and high-grade and recurrent tumors which might be clinically useful. The results derived from these studies increase our understanding of the genetic alterations leading to the development of these tumor forms and point out candidate genes that may be used in future as targets for new diagnostic and therapeutic strategies.