Ce travail de thèse a consisté en la conception et la réalisation d’une nano-sonde hybride luminescente visant à permettre la détection précoce du cancer de la prostate. La première partie de ce projet a été consacré à la synthèse, par une voie de chimie click, d’une bibliothèque d’acides 4-triazolyl dipicoliniques substitués en position 4 du triazole par une large gamme de substituants. Ces diacides ont permis d’obtenir les complexes d’europium(III) et de terbium(III) correspondant, qui ont montré d’excellentes propriétés optiques, avec des rendements quantiques de luminescence sous excitation UV pouvant atteindre 60% et 36%, pour les complexes d’europium(III) et deterbium(III) respectivement. D’autre-part, ces fluorophores ont pu être excités efficacement en régime biphotonique, à la fois au travers des transitions S0 ®S1 et S0 ®T1. Sur la base de ces résultats, certains de ces chélates ont été sélectionnés afin de les incorporer dans des nanoparticules de silice. Le procédé d’élaboration par microémulsion inverse s’est révélé efficace pour l’incorporation des complexes électriquement neutres, mais n’a pas permis celle de nanohybrides incorporant des complexes chargés négativement. Ces nanohybrides présentent des propriétés optiques caractéristiques des lanthanides, avec des rendements quantiques allant jusqu’à 30%. La surface de ces nano-objets a ensuite été fonctionnalisée par des groupements amino, qui ont permis le greffage de bras espaceur et d’un vecteur ciblant la PSMA, l’un des signaux du cancer de la prostate, nous donnant ainsi accès à un modèle de nano-sonde luminescente. Un autre volet de ce travail a été dédié à l’étude de nouveaux analogues du NAAG, substrat naturel de la PSMA. Bien que la synthèse des deux composés cibles, sélectionnés parmi une vingtaine de structures par modélisation moléculaire, n’ait pu aboutir, elle a été largement avancée. Enfin, la dernière partie de ce travail décrit les premiers résultats obtenus in vitro et in vivo avec les nanosondes. Ces études ont porté sur l’évaluation de la cytotoxicité des nanoparticules ainsi que sur leur biodistribution chez la souris saine et chez la souris porteuse d’une tumeur prostatique. Cette étude a révélé une élimination rapide des nanoparticules par l’organisme, mais n’a malheureusement pas pu mettre en évidence un marquage des zones tumorales par les nanosondes. / The aim of this project was the design of a luminescent nanoprobe allowing the early prostate cancer detection.The first part of this project was the synthesis, through a click chemistry approach, of a library of 4-triazolyl dipicolinic acid substituted in position 4 of the triazole by a wide range of substitutive groups. These diacids were used to synthesise the corresponding europium(III) and terbium(III) complexes, which showed excellent optical properties, with photoluminescence quantum yield under UV excitation reaching 60% for europium(III) complexes and 36% for terbium(III) complexes. Moreover, these phosphores have been efficiently excited in biphotonic regime through the transitions S0 ®S1 and S0 ®T1. The more interesting chelates were selected for their further embedding into silica nanoparticles. The water-in-oil emulsion process showed a great efficiency for the incorporation of electrically neutral complexes, but did not allow the embedding of negatively charged ones. The resulting nanohybrids showed optical properties typical of lanthanides, presenting photoluminescence quantum yields up to 30%. The nanoparticles surface was then functionalised by amino groups, which were used to graft a spacer then a prostate tumour cells vector, giving us a luminescent nanoprobe model. The third section of this work was devoted to the study of new analogues of NAAG, the natural substrate of the PSMA, one of the prostatic cancer signals. Although the synthesis of the two target compounds, selected from more than 20 structures by molecular modelling, was uncompleted, they are now within easy reach. The last part describes the preliminary results obtained in vitro and in vivo with the nanoprobes. These studies were focused on the nanoparticles cytotoxitcity assessment, depending on the surface functionalisation, and on their distribution, in healthy and in prostatic tumour bearing mice. This study revealed the fast elimination of the nanoparticles by the organism, but did not show any concentration of nanoprobes in tumoral area.

Purpose: To evaluate the validity of the Gleason score after neoadjuvant hormonal treatment as predictor of diseasespecific mortality after radical prostatectomy. Patients and Methods: A total of 2,880 patients with a complete data set and a mean follow-up of 10.3 years were studied; 425 of them (15%) had a history of hormonal treatment prior to surgery. The cumulative incidence of deaths from prostate cancer was determined by univariate and multivariate competing risk analysis. Cox proportional hazard models for competing risks were used to study combined effects of the variables on prostate cancer-specific mortality. Results: A higher portion of specimens with a history of neoadjuvant hormonal treatment were assigned Gleason scores of 8–10 (28 vs. 17%, p < 0.0001). The mortality curves in the Gleason score strata <8 vs. 8–10 were at large congruent in patients with and without neoadjuvant hormonal treatment. In patients with neoadjuvant hormonal treatment, a Gleason score of 8–10 was an independent predictor of prostate cancer-specific mortality; the hazard ratio was, however, somewhat lower than in patients without neoadjuvant hormonal treatment. Conclusion: This study suggests that the prognostic value of the post-radical prostatectomy Gleason score is not meaningfully jeopardized by heterogeneous neoadjuvant hormonal treatment in a routine clinical setting.

Introduction: Targeted biopsy of tumour-suspicious lesions detected in multiparametric magnetic resonance imaging (mpMRI) plays an increasing role in the active surveillance (AS) of patients with low-risk prostate cancer (PCa). The aim of this study was to compare MRI/ultrasound-fusion biopsy (fusPbx) with systematic biopsy (sysPbx) in patients undergoing biopsy for AS. Methods: Patients undergoing mpMRI and transperineal fusPbx combined with transrectal sysPbx (comPbx) as surveillance biopsy were investigated. The detection of Gleason score upgrading and reclassification according to Prostate Cancer Research International Active Surveillance criteria were evaluated. Results: Eighty-three patients were enrolled. PCa upgrading was detected in 39% by fusPbx and in 37% by sysPbx (p = 1.0). The percentage of patients who were reclassified in fusPbx and sysPbx (p = 0.45) were 64 and 59% respectively. ComPbx detected more frequently tumour upgrading than fusPbx (71 vs. 64%, p = 0.016) and sysPbx (71 vs. 59%, p < 0.001) and more patients had to be reclassified after comPbx than after fusPbx or sysPbx alone. Conclusions: The combination of fusPbx and sysPbx outperforms both modalities alone with regard to the detection of upgrading and reclassification in patients under AS. Because a high missing rate of significant PCa still exists in both biopsy modalities, a combination of fusPbx and sysPbx should be recommended in these patients.

Objectives: We compared the transperineal MRI/ultrasoundfusion biopsy (fusPbx) to transrectal systematic biopsy (sys-Pbx) in patients with previously negative biopsy and investigated the prediction of tumour aggressiveness with regard to radical prostatectomy (RP) specimen. Material and Methods: A total of 710 patients underwent multiparametric magnetic resonance imaging (mpMRI), which was evaluated in accordance with Prostate Imaging Reporting and Data System (PI-RADS). The maximum PI-RADS (maxPI-RADS) was defined as the highest PI-RADS of all lesions detected in mpMRI. In case of proven prostate cancer (PCa) and performed RP, tumour grading of the biopsy specimen was compared to that of the RP. Significant PCa (csPCa) was defined according to Epstein criteria. Results: Overall, scPCa was detected in 40% of patients. The detection rate of scPCa was 33% for fusPbx and 25% for sysPbx alone (p < 0.005). Patients with a maxPI-RADS ≥3 and a prostate specific antigen (PSA)-density ≥0.2 ng/mL2 harboured more csPCa than those with a PSA-density < 0.2 ng/mL2 (41% [33/81] vs. 20% [48/248]; p < 0.001). Compared to the RP specimen (n = 140), the concordance of tumour grading was 48% (γ = 0.57), 36% (γ = 0.31) and 54% (γ = 0.6) in fusPbx, sysPbx and comPbx, respectively. Conclusions: The combination of fusPbx and sysPbx outperforms both biopsy modalities in patients with re-biopsy. Additionally, the PSA-density may represent a predictor for csPCa in patients with maxPI-RADS ≥3.

Au début des années 2000, il a été montré que le développement des métastases était fortement limité par un phénomène de dormance cellulaire. En effet, après s’être extravasées dans un organe distant, la plupart des cellules tumorales disséminées qui survivent entrent immédiatement dans un état quiescent réversible mais durable sur le long terme, qui est qualifié de dormant. Si le phénomène de dormance permet de limiter le taux de développement de métastases, il est aussi à l’origine de récidives métastasiques tardives, car les cellules dormantes peuvent persister longtemps et sont résistantes à la chimiothérapie. Il est donc important d’analyser les mécanismes moléculaires régulant la dormance des cellules tumorales. Mais cette analyse est difficile à réaliser in vivo du fait de la relative grande rareté des cellules cancéreuses disséminées. Récemment, mon équipe d’accueil a observé que la clonogénicité in vitro des cellules de cancer de la prostate peut être fortement régulée par un phénomène de dormance. Celle-ci est induite lorsque les cellules sont cultivées à la faible densité cellulaire et dans un milieu légèrement hypertonique comme le DMEM. L’objectif de mon travail de thèse était d’analyser les mécanismes moléculaires régulant la dormance des cellules de cancer de la prostate in vitro.Dans un premier temps, nous avons mis en évidence que la dormance nécessite l’activation conjointe de la voie de signalisation BMP/TGF-ß et d’un stress oxydatif. La faible densité cellulaire joue un rôle capital dans l’établissement de la dormance en pré-activant la voie du BMP/TGF-ß et en sensibilisant la cellule au stress oxydatif. L’amplification de ces effets par l’hypertonicité explique l’induction du phénomène de dormance. Nous avons étendu la portée de ces observations en montrant que les androgènes à des concentrations proches des niveaux physiologiques permettent d’induire un phénomène de dormance très similaire, reposant lui-aussi sur l’activation des voies de signalisation du BMP/TGF-ß et du stress oxydatif. De manière intéressante, nous avons confirmé qu’une fois que la dormance est établie, elle se maintient d’une manière autonome. Ainsi, un traitement transitoire par des androgènes s’avère suffisant pour induire la dormance qui se maintient après la remise en culture dans un milieu dépourvu d’androgènes ajoutés. Par ailleurs, nous avons identifié CDKN1A (p21CIP1/WAF1) comme un régulateur de la dormance principalement régulé par le stress oxydatif. Sa surexpression à faible densité cellulaire suffit à induire un phénomène de dormance caractéristique.En conclusion, nous avons montré que la dormance des cellules de cancer de la prostate est régulée par les voies de signalisation du BMP/TGF-ß et du stress oxydatif qui semblent être interconnectées au travers d’une boucle de régulation auto-entretenue. Les androgènes pourraient constituer des inducteurs de dormance efficaces in vivo pour limiter le développement des métastases, ce que nous voudrions tester dans un modèle de dissémination métastasique simplifié chez la souris obtenu par injection intracardiaque de cellules cancéreuses. Par ailleurs, notre modèle prédit que les CTCs (Cellules Tumorales Circulantes dispersées dans la circulation sanguine) pourraient être induites à entrer en dormance avant même de s’extravaser du milieu sanguin du fait des conditions pro-oxydantes régnant dans ce milieu, ce qui pourrait être testé en étudiant si des inhibiteurs du stress oxydatif et/ou des voies du BMP/TGF-ß favorisent la prolifération de CTCs purifiées et mises en cultures. / In the early 2000s, development of metastases was shown to be strongly limited by a phenomenon of cellular dormancy. Indeed, after extravasation in a distant organ, most of the disseminated cancer cells that survive enter into a reversible quiescent that is called dormant state. The phenomenon of dormancy is also the cause of late metastatic recurrences because dormancy confers long-term cellular survival and resistance to chemotherapy. Thus, it is important to analyze the molecular mechanisms regulating cancer cell dormancy. However, in vivo studies are difficult due to the extreme scarcity of disseminated cancer cells. Recently, T. Tchenio et al observed that clonogenicity of prostate cancer cells was strongly regulated by a dormancy phenomenon in vitro. A dormant state was induced when cells are cultured at low density in a slightly hypertonic medium, such as DMEM. The aim of my thesis was to analyze the molecular mechanisms regulating the dormancy of prostate cancer cells in vitro.We first demonstrated that dormancy required a combined activation of both BMP/TGF-ß and oxidative stress signaling pathways. Low cell density plays a key role in dormancy establishment by priming both signaling pathways. Hypertonicity induced dormancy through further amplifying these signaling pathways. We extended the biological relevance of our observation by showing that a closely related phenomenon could be induced by androgens at concentrations close to physiologic levels. Androgen-induced dormancy also relied on activation of the BMP/TGF-ß and oxidative stress signaling pathways. Interestingly, we observed that once dormancy was established, it was maintained autonomously since a transient treatment with androgen was sufficient to induce a dormant state that was self-sustained after withdrawal of exogenous androgens. Besides, CDKN1A (p21 CIP1/WAF1) was identified as a dormancy regulator modulated by oxidative stress. We showed that its transient overexpression at low cell density was sufficient to induce a dormancy phenomenon that mimicked those induced by hypertonicity or androgens.In conclusion, we showed that dormancy of prostate cancer cells was regulated by BMP/TGF-ß and oxidative stress signaling pathways that seemed to be interconnected through a self-sustained regulation loop. Androgen may constitute an effective inducer of dormancy in vivo to limit metastases development. Therefore, we would like to test its effects in vivo in a simplified model of metastatic dissemination in mouse relying on intracardiac injection of cancer cells. Furthermore, our model allowed us to predict that CTCs (Circulating Tumor Cells dispersed in bloodstream) could enter into a dormant state before their extravasation from blood vessels due to the pro-oxidant conditions in this environment. This prediction could be tested by studying whether oxidative stress and BMP/TGF inhibitors could promote proliferation of purified CTCs in vitro.

Les gènes et les protéines n’agissent pas de manière isolée dans les cellules, mais interagissent plutôt pour faire leurs fonctions dans les processus biologiques. Ces interactions peuvent être représentées sous forme de grands réseaux dans lesquels les nœuds sont des gènes ou des protéines et les arêtes représentent leurs interactions. Diverses approches basées sur la théorie des graphes ont été développées pour extraire la connaissance fonctionnelle contenue dans ces réseaux. Néanmoins, ces méthodes ont été principalement appliquées à des réseaux individuels, en ignorant la diversité des interactions biologiques. Nous déclarons que ces différents types d’interactions peuvent être représentés sous la forme de réseaux multiplexes, c’est-à-dire des ensembles de réseaux partageant les mêmes nœuds, ce qui permet une description plus précise des systèmes biologiques. Cette thèse est focalisée sur le développement de nouveaux algorithmes étendant aux réseaux multiplexes certaines méthodes populaires de la théorie des graphes en biologie computationnelle, ainsi que sur leur application à l’étude des maladies humaines. Du côté des applications, nous nous concentrons sur les maladies liées au vieillissement prématuré, un groupe de maladies génétiques ressemblant à certains aspects du vieillissement physiologique à un âge précoce. Nous avons appliqué nos algorithmes pour détecter les modules associés à plus de 70 syndromes annotés avec un phénotype lié au vieillissement prématuré. Les résultats ont révélé le paysage des processus moléculaires perturbés dans ces maladies, qui peuvent être mis en parallèle avec les caractéristiques du vieillissement physiologique. / Genes and proteins do not act isolated in cells but rather interact to perform their functions in signaling pathways, molecular complexes, or, more generally, biological processes. These interactions can be represented as large networks in which nodes are genes or proteins and edges represent their interactions. Various graph-theory based approaches have been developed to extract the functional knowledge contained in biological networks. Nevertheless, these methods have been mainly applied to individual networks, ignoring the diversity of biological interactions. We state here that these different types of interactions can be represented as multiplex networks, i.e. collections of networks sharing the same nodes, leading to a more accurate description of biological systems. This thesis focuses on the extension from individual to multiplex networks of some of the state-of-the-art guilt-by-association methods in computational biology, and on their application to the study of human diseases. On the application side, we concentrate on premature aging diseases, a group of rare genetic disorders that resemble some aspects of physiological aging at an early age. In this framework, we applied our algorithms to detect the modules associated to more than 70 disorders annotated with at least one premature aging related phenotype. The results revealed the landscape of perturbed molecular processes in premature aging diseases, which can be paralleled with the hallmarks of physiological aging to help identifying common and specific features.

Background Prostate cancer (PCa) is the most frequently diagnosed malignant disease among adult males in the USA and the second leading cause of cancer deaths in men. Due to the lack of diagnostic tools that are able to differentiate highly malignant and aggressive cases from indolent tumors, overtreatment has become very common in the era of prostate specific antigen (PSA) screening. New diagnostic methods to determine biological status, malignancy, aggressiveness and extent of PCa are urgently needed. 1H High Resolution Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy (1H HRMAS MRS) can be used to establish PCa metabolomic profiles while preserving tissue architecture for subsequent histopathological analysis. Immunohistochemistry (IHC), as opposed to conventional histopathology methods, has the potential to provide objective, more accurate and quantitative knowledge of tissue pathology. This diagnostic- accuracy study sought to evaluate a novel approach to quantitatively identify metabolomic markers of PCa by exploring the potential of PCa immunomarkers to quantify metabolomic profiles established by 1H HRMAS MRS. Material and Methods 1H HRMAS MRS was performed on tissue samples of 51 prostate cancer patients using a 14.1 Tesla NMR spectrometer (BRUKER Biospin, Billerica, MA) with a rotor synchronized CPMG pulse sequence. Spectral intensities of 36 regions of interest were measured as integrals of curve fittings with Lorentzian-Gaussian line shapes. Immunohistochemistry (IHC) was carried out following the spectroscopy scan, using three prostate immunomarkers to identify cancerous and benign glands: P504S (Alpha-methylacyl-CoA-racemace), CK903 (high-molecular weight cytokeratin) and p63. The immunostaining quality following 1H HRMAS MRS was evaluated and compared to unscanned sections of the same sample, to verify the stability and accessibility of the proposed immunomarkers. IHC images were automatically and quantitatively evaluated, using a quantitative image analysis program (QIAP), to determine the percentage of cancerous and benign epithelia in the tissue cross- sections. The results of the program were validated by a correlation with the results of a quantitative IHC review and quantitative conventional histopathology analysis performed by an experienced pathologist. Ultimately, spectral intensities and the cancer epithelium percentage, obtained from quantitative immunohistochemistry, were correlated in order to validate PCa metabolomic markers identified by 1H HRMAS MRS. Patient outcomes and incidence of recurrence were determined by retrospective review of medical records five years after initial surgery. Categories of recurrence were correlated to spectral intensities to explore potential metabolomic markers of recurrence in the cohort. Results Immunostainings with P504S and CK903 showed excellent staining quality and accessibility following 1H HRMAS MRS, suggesting these markers to be suitable for the presented quantitative approach to determine metabolomics profiles of PCa. In contrast, the quality of p63 IHC was impaired after previously performed spectroscopy. IHC using the immunomarkers P504S and CK903 on adjacent slides was found to present a feasible quantitative diagnostic method to distinguish between benign and cancerous conditions in prostate tissue. The cancer epithelium percentage as determined by QIAP showed a significant correlation to the results of quantitative IHC analysis performed by a pathologist (p < 0.001), as well as to a quantitative conventional histopathology review (p = 0.001). The same was true for the benign epithelium percentage (p < 0.001 and p = 0.0183), validating the presented approach. Two metabolomic regions showed a significant correlation between relative spectral intensities and the cancer epithelium percentage as determined by QIAP: 3.22 ppm (p = 0.015) and 2.68 ppm (p = 0.0144). The metabolites corresponding to these regions, phosphocholine and citrate, could be identified as metabolomic markers of PCa in the present cohort. 45 patients were followed for more than 12 months. Of these, 97.8% were still alive five years after initial surgery. 11 patients (24.4%) experienced a recurrence during the follow- up time. The categories of recurrence showed a correlation to the spectral intensities of two regions, 2.33 – 2.3 ppm (p = 0.0403) and 1.28 ppm (p = 0.0144), corresponding to the metabolites phosphocreatine and lipids. Conclusion This study introduces a method that allows an observer-independent, quantitative analysis of IHC to help establish metabolomic profiles and identify metabolomic markers of PCa from spectral intensities obtained with 1H HRMAS NMR Spectroscopy. The immunomarkers P504S and CK903 have been found suitable IHC analysis following 1H HRMAS MRS. A prospective in vivo application of PCa metabolite profiles and metabolomic markers determined by the presented method could serve as highly sensitive, non- invasive diagnostic tool. This observer- independent, computer- automated, quantitative analysis could help to distinguish highly aggressive tumors from low-malignant conditions, avoid overtreatment and reduce risks and complications for cancer patients in the future. Further studies are needed to verify the identified PCa metabolomic markers and to establish clinical applicability.:Table of Contents Glossary 1 Introduction 1. 1 Prostate Cancer 1. 2 Detection of Prostate Cancer – State of the Art 1. 2. 1 Prostate- Specific Antigen Test and Digital Rectal Examination 1.2.2 Radiographic Methods in PCa Detection 1.2.3 Transrectal Core Biopsies and Histopathological Analysis 1.2.4 Histopathological Grading of Prostate Cancer: GLEASON Score 1.3 Challenges and Need for New Approaches in PCa Diagnostic Management 2 Scientific Background I: Nuclear Magnetic Resonance,1H HRMAS NMR Spectroscopy and Metabolomic Profiles 2.1 Nuclear Magnetic Resonance 2.1.1 Spin Precession 2.1.2 Magnetic Resonance 2.1.3 Chemical Shift and J- coupling 2.2 Nuclear Magnetic Resonance 2.2.1 Magic Angle Spinning and 1H HRMAS NMR Spectroscopy 2.2.2 MAS Spinning Rates and Spinning Side Bands 2. 3 Metabolomics, Metabolite Profiles and Clinical Utility 3 Scientific Background II: Immunohistochemistry of Prostate Cancer 4 Aims of the Study 5 Material and Methods 5.1 Prostate Tissue Samples and Patient Demographics 5.2 1H HRMAS NMR Spectroscopy 5.2.1 Sample Preparation 5.2.2 Spectroscopy Scan 5.2.3 Data Processing 5.3 Immunohistochemistry 5.3.1 Immunohistochemistry Material and Equipment 5.3.2. Immunohistochemistry Protocol 5. 3. 3 Prostate Immunomarker Stability after 1H HRMAS NMR Spectroscopy 5.3.4 Qualitative IHC Analysis 5. 3.5 Quantitative IHC Analysis 5.3.5.1 Quantitative IHC Slide Review 5.3.5.2 Computer-Automated Quantitative IHC Analysis 5.3 Quantitative Histopathology 5. 4 Identification of Prostate Cancer Metabolomic Markers 5. 5 Patient Outcomes and Recurrence Categories 5.6 Statistical Analysis 6 Results 6. 1 Patient demographics 6. 2 Spectroscopy Results 6. 3 Immunohistochemistry 6. 3. 1 Evaluation of Prostate Immunomarker Stability after 1H HRMAS MRS 6. 3. 2 Qualitative Immunohistochemistry 6. 4 Quantitative Immunohistochemistry 6. 4. 1 Quantitative IHC Slide Review 6. 4. 2 Computer-Automated Quantitative IHC Evaluation using QIAP 6. 5 Quantitative Histopathology 6. 6 Identification of Prostate Cancer Metabolomic Markers using QIAP 6. 7 Patient Outcomes and Recurrence 7 Discussion 8 Summary / Abstract 9 Zusammenfassung 10 References 11 Erklärung über die eigenständige Abfassung der Arbeit 12 Danksagung 13 Lebenslauf und Publikationsverzeichnis Appendix A.1 Immunostaining protocols A.2 Spectral Intensities Measured by 1H HRMAS MRS in 51 Samples A.3 Graphs for Correlations of Spectral Intensities and CaE% determined by QIAP in 34 Additional Regions of Interest / Einführung Prostatakrebs ist eine häufigsten Krebserkrankungen in den USA und die zweithäufigste malignom- assoziierte Todesursache männlicher Patienten weltweit. Seit der Einführung des Prostata- spezifischen Antigen (PSA)- Screeningtests wird diese Krebsart in früheren Stadien diagnostiziert und therapiert, wodurch die Mortalitätsrate in den letzten Jahren deutlich reduziert werden konnte. Da moderne diagnostische Methoden bislang jedoch nicht ausreichend in der Lage sind, suffizient zwischen hochmalignen und weniger aggressiven Varianten dieses bösartigen Krebsleidens zu unterscheiden, werden häufig auch Patienten aggressiv therapiert, deren niedriggradiges Prostatakarzinom keine klinische Relevanz gehabt hätte. Es besteht daher ein großes wissenschaftliches Interesse an der Entwicklung neuer diagnostischer Methoden zur akkuraten Bestimmung von biologischem Status, Malignität, Aggressivität und Ausmaß einer Prostatakrebserkrankung. \\\\\\\"1H High Resolution Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy\\\\\\\" (1H HRMAS MRS) ist eine vielversprechende diagnostische Methode, welche es ermöglicht, metabolomische Profile von Prostatakrebs zu erstellen, ohne die Gewebsstruktur der analysierten Proben zu zerstören. Durch anschließende histopathologische Begutachtung lassen sich die erstellten Metabolitprofile validieren und evaluieren. Im Gegensatz zu konventionellen histopathologischen Methoden können durch immunhistochemische Verfahren dabei objektivere, akkuratere und quantifizierbare histopathologische Erkenntnisse gewonnen werden. Die vorliegende Studie präsentiert einen neuentwickelten diagnostischen Ansatz zur quantitativen Bestimmung von metabolomischen Markern von Prostatakrebs, basierend auf der Durchführung von 1H HRMAS NMR Spektroskopie und quantitativer Immunhistochemie. Material und Methoden Einundfünfzig Gewebsproben von Prostatakrebspatienten wurden mittels 1H HRMAS MRS an einem 14.1 T BRUKER NMR Spektrometer unter Einsatz einer CPMG-Pulssequenz untersucht. Spektrale Intensitäten in 36 Metabolitregionen wurden gemessen. Anschließend wurden die analysierten Gewebeproben mit drei Immunfärbemarkern für sowohl malignes (P504S, Alpha-methylacyl-CoA-racemase) als auch benignes (CK903, High-molecular weight cytokeratin, und p63) Prostatagewebe angefärbt und quantitativ mit Hilfe eines Bildanalyseprogramms (QIAP) ausgewertet. Die Anwendbarkeit und Auswertbarkeit der genannten Immunomarker nach Spektroskopie wurde evaluiert und mit der Färbungsqualität von nicht- gescannten Schnitten verglichen. Die Resultate der automatischen Auswertung durch QIAP konnten durch einen erfahrenen Pathologen in einer quantitativen Analyse der Immunfärbungen sowie konventioneller histologischer Färbungen derselben Gewebsproben validiert werden. Die spektralen Intensitäten aus den Messungen mit 1H HRMAS MRS wurden mit den korrespondierenden Ergebnissen der quantitativen Auswertung der Immunfärbungen korreliert, um metabolomische Marker von Prostatakrebs zu identifizieren. Der klinische Verlauf und die Rezidivrate der Patienten wurden 5 Jahre nach der initialen Prostatektomie retrospektiv bestimmt. Rezidivkategorien wurden erstellt und mit den bestimmten spektralen Intensitäten korreliert, um metabolomische Marker für das Auftreten von Prostatakrebsrezidiven zu identifizieren. Ergebnisse Die Immunfärbungen mit P504S und CK903 zeigten exzellente Qualität und Auswertbarkeit nach vorheriger 1H HRMAS MRS. Beide Marker eigneten sich zur Durchführung von quantitativer Immunhistochemie an spektroskopierten Gewebeproben. Im Gegensatz dazu war die Qualität der Immunfärbungen mit p63 nach Spektroskopie vermindert. Quantitative Immunfärbungen unter Einsatz der Immunmarker P504S und CK903 stellten eine praktikable diagnostische Methode dar, um zwischen malignen und benignem Prostatagewebe zu unterscheiden. Der Anteil von bösartig verändertem Prostatagewebe, bestimmt durch QIAP, korrelierte signifikant mit den Ergebnissen der quantitativen Analyse der Immunfärbungen durch den Pathologen (p < 0.001), sowie mit der quantitativen Auswertung der konventionellen histopathologischen Färbung (p = 0.001). Ebenso ließ sich die Bestimmung des Anteils von benignem Gewebe mit QIAP zu den Ergebnissen der pathologischen Analyse korrelieren (p < 0.001 und p = 0.0183). Für zwei metabolomische Regionen konnte ein signifikante Korrelation zwischen relativen spektralen Intensitäten, bestimmt mit 1H HRMAS NMR Spektroskopie, und dem Anteil von malignem Epithelium in derselben Gewebeprobe, ermittelt durch QIAP, festgestellt werden: 3.22 ppm (p = 0.015) und 2.68 ppm (p = 0.0144). Die zu diesen Regionen korrespondierenden Metaboliten, Phosphocholin und Zitrat, konnten als potentielle metabolomische Marker für Prostatakrebs identifiziert werden. Die retrospektiven Analyse der klinischen Daten der Patienten fünf Jahre nach Prostatektomie ergab eine Überlebensrate von 97.8%. Elf dieser Patienten (24.4%) erlitten ein Rezidiv ihrer Erkrankung. Die bestimmten Rezidivkategorien korrelierten signifikant mit zwei metabolomischen Regionen (2.33 – 2.3 ppm, p = 0.0403 und 1.28 ppm, p = 0.0144), welche zu den Metaboliten Phosphokreatin und Lipiden korrespondierten. Schlussfolgerung Die vorliegende Studie präsentiert einen diagnostischen Ansatz zur objektiven und quantitativen Bestimmung metabolomischer Marker von Prostatakrebs unter Verwendung von 1H HRMAS MRS und Immunhistochemie. P504S und CK903 eignen sich als Immunmarker für quantitative Immunfärbungen nach vorheriger Durchführung von 1H HRMAS MRS. Die Metaboliten Phosphocholin und Zitrat konnten in der vorliegenden Patientenkohorte als potentielle metabolomische Marker für Prostatakrebs identifiziert werden. Eine mögliche in vivo Anwendung der gefundenen metabolomischen Marker könnte als hochsensitives, objektives und nicht- invasives diagnostisches Werkzeug der Prostatakrebsdiagnostik dienen. Der vorliegende untersucherunabhängige, automatisierte und quantitative diagnostischer Ansatz hat das Potential, zwischen hochmalignen und weniger aggressiven Krebsfällen zu unterscheiden und somit unnötige Risiken und Komplikationen für Prostatakrebspatienten zu reduzieren. Weitere Untersuchungen sind notwendig, um die identifizierten metabolomischen Marker zu verifizieren und eine klinische Anwendung zu etablieren.:Table of Contents Glossary 1 Introduction 1. 1 Prostate Cancer 1. 2 Detection of Prostate Cancer – State of the Art 1. 2. 1 Prostate- Specific Antigen Test and Digital Rectal Examination 1.2.2 Radiographic Methods in PCa Detection 1.2.3 Transrectal Core Biopsies and Histopathological Analysis 1.2.4 Histopathological Grading of Prostate Cancer: GLEASON Score 1.3 Challenges and Need for New Approaches in PCa Diagnostic Management 2 Scientific Background I: Nuclear Magnetic Resonance,1H HRMAS NMR Spectroscopy and Metabolomic Profiles 2.1 Nuclear Magnetic Resonance 2.1.1 Spin Precession 2.1.2 Magnetic Resonance 2.1.3 Chemical Shift and J- coupling 2.2 Nuclear Magnetic Resonance 2.2.1 Magic Angle Spinning and 1H HRMAS NMR Spectroscopy 2.2.2 MAS Spinning Rates and Spinning Side Bands 2. 3 Metabolomics, Metabolite Profiles and Clinical Utility 3 Scientific Background II: Immunohistochemistry of Prostate Cancer 4 Aims of the Study 5 Material and Methods 5.1 Prostate Tissue Samples and Patient Demographics 5.2 1H HRMAS NMR Spectroscopy 5.2.1 Sample Preparation 5.2.2 Spectroscopy Scan 5.2.3 Data Processing 5.3 Immunohistochemistry 5.3.1 Immunohistochemistry Material and Equipment 5.3.2. Immunohistochemistry Protocol 5. 3. 3 Prostate Immunomarker Stability after 1H HRMAS NMR Spectroscopy 5.3.4 Qualitative IHC Analysis 5. 3.5 Quantitative IHC Analysis 5.3.5.1 Quantitative IHC Slide Review 5.3.5.2 Computer-Automated Quantitative IHC Analysis 5.3 Quantitative Histopathology 5. 4 Identification of Prostate Cancer Metabolomic Markers 5. 5 Patient Outcomes and Recurrence Categories 5.6 Statistical Analysis 6 Results 6. 1 Patient demographics 6. 2 Spectroscopy Results 6. 3 Immunohistochemistry 6. 3. 1 Evaluation of Prostate Immunomarker Stability after 1H HRMAS MRS 6. 3. 2 Qualitative Immunohistochemistry 6. 4 Quantitative Immunohistochemistry 6. 4. 1 Quantitative IHC Slide Review 6. 4. 2 Computer-Automated Quantitative IHC Evaluation using QIAP 6. 5 Quantitative Histopathology 6. 6 Identification of Prostate Cancer Metabolomic Markers using QIAP 6. 7 Patient Outcomes and Recurrence 7 Discussion 8 Summary / Abstract 9 Zusammenfassung 10 References 11 Erklärung über die eigenständige Abfassung der Arbeit 12 Danksagung 13 Lebenslauf und Publikationsverzeichnis Appendix A.1 Immunostaining protocols A.2 Spectral Intensities Measured by 1H HRMAS MRS in 51 Samples A.3 Graphs for Correlations of Spectral Intensities and CaE% determined by QIAP in 34 Additional Regions of Interest

Deregulation of gene expression caused by genetic or epigenetic changes plays an important role in pathogenesis of cancer. The thesis is a commented collection of ten publications dealing with the molecular biology of tumours. The author has significantly contributed to all of them. All the articles contained in the thesis are linked to the topic of assessment of molecules involved in gene expression regulation (microRNAs) or DNA alterations that affect gene expression (promoter methylation, presence of a fusion gene). MicroRNAs are short single-stranded RNA molecules involved in posttranscriptional regulation of gene expression by triggering mRNA degradation or inhibiting translation. It is a basic mechanism with an impact on all cellular processes including the pathogenesis of various diseases. MicroRNAs can either act as oncogenes by decreasing the expression of tumour-suppressor genes or as tumour-suppressor genes by decreasing the expression of oncogenes. However, the network of microRNA - RNA interactions is much more complex. Our published results that are part of this thesis are focused on colorectal carcinoma (CRC), prostate cancer, head and neck squamous cell carcinoma (HNSCC), gastric cancer and non-small cell lung cancer (NSCLC). In patients with CRC, we demonstrated the prognostic...

Despite advances in cancer diagnosis and therapy, cancer is the second leading cause of death globally. The improvements of cancer treatment are the major challenge in this research. The aim of the thesis was studying of effects of two anticancer drugs ellipticine (Elli) and doxorubicin (DOX) on some cancer and healthy cell lines. Specific consideration was given to expand current knowledge about the metabolism and cytostatic effects of Elli in neuroblastoma cell lines. Another part of this study was focused on mechanisms contributing to the development of ellipticine-resistance in cancer cells and influence of histone deacetylase inhibitors on anticancer therapy was investigated. Moreover, the aim was to develop apoferritin (Apo) nanocarrier suitable for the active transport of cytostatics to cancer cells. Several essential data were found in this doctoral thesis. Anticancer efficiency of Elli depends on the CYP3A4-mediated metabolism in cancer. The CYP3A4 enzyme encapsulated into two nanoparticle forms, liposomes and SupersomesTM , was tested to activate ellipticine to its reactive species forming covalent DNA adducts. The formation of adducts seems to be dependent on concentrations of CYP3A4 in nanoparticle systems. A higher effectiveness of CYP3A4 in SupersomesTM than in liposomes to form...

Background: Factors predicting survival in men with lymph node-positive prostate cancer are still poorly defined. Patients and Methods: 193 prostate cancer patients with histopathologically proven lymph node involvement with a median follow-up of 7.3 years were studied. 94% of patients received immediate hormonal therapy. Kaplan-Meier curves were calculated to evaluate overall survival rates and compared with the log-rank test. Cumulative disease-specific and competing mortality rates were calculated by competing risk analysis and compared with the Pepe-Mori test. Cox proportional hazard models were used to determine the independent significance of predictors of all-cause mortality. Results: Age (70 years or older vs. younger), Gleason score (8–10 vs. 7 or lower) and the number of involved nodes (3 or more vs. 1–2) were identified as independent predictors of all-cause mortality. When patients with 0–1 of these risk factors were compared with those with 2–3 risk factors, all-cause (rates after 10 years 21% vs. 71%, p < 0.0001), disease-specific (12 vs. 37%, p = 0.009) and competing mortality (9 vs. 33%, p = 0.02) differed significantly. Conclusions: Some of the excess mortality in patients with poor-risk lymph node-positive prostate cancer may be attributed to increased competing mortality, possibly caused by an interaction between comorbid diseases and hormonally treated persistent or progressive prostate cancer. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.