Le carcinome rénal à cellules claires (ccRCC) est la tumeur du rein la plus fréquente. Il se caractérise par une inactivation fréquente du gène suppresseur de tumeur VHL retrouvée dans 70% des tumeurs conduisant à une transcription des gènes cibles du facteur de transcription HIF dont le VEGF. Il s’agit d’une tumeur agressive métastatique chez 50% des patients. Le sunitinib, un inhibiteur des récepteurs tyrosine kinase anti-angiogénique, est actuellement le plus utilisé en 1ère ligne malgré 30% des patients qui progressent rapidement. L’avènement d’un nouvel anti-angiogénique ciblant MET (cabozantinib) et d’immunomodulateurs (anticorps anti-PD-1, nivolumab) rend cruciale la découverte de facteurs prédictifs de réponse au traitement. Dans une 1ére partie, nous avons étudié une série rétrospective de 98 ccRCC consécutifs pour lesquels nous souhaitions étudiés le statut VHL complet et le corréler à l’expression de PD-L1. De plus, alors que le pronostic est différent entre ccRCC métastatiques synchrones (d’emblée) et métachrones (à distance), leur phénotype n’avait jamais été comparé. Pour cela, nous avons effectué une analyse histologique des principaux facteurs pronostiques, immunohistochimique (CAIX, VEGF, PAR3, PD-1 et PD-L1) et moléculaire (statut complet VHL : délétion, mutation et méthylation du promoteur) corrélée à la survie spécifique. Nous avons démontré que le statut VHL non-inactivé (niVHL) était associé à la présence de métastases synchrones, une composante sarcomatoïde, un infiltrat lymphocytaire dense, une surexpression de VEGF, une expression de PD-L1 et à un mauvais pronostic. Nous avons aussi comparé les phénotypes des ccRCC métastatiques métachrones et synchrones. Ces derniers étaient associés à une  composante sarcomatoïde, une expression cytoplasmique de PAR-3, une surexpression de VEGFA, un statut niVHL et à un mauvais pronostic depuis le diagnostic des métastases. Dans une 2ème partie, nous avons étudié une série rétrospective de 90 ccRCC métastatiques consécutifs traités par sunitinib en première ligne afin d’identifier des facteurs prédictifs de réponse ou de résistance. Nous avons utilisé les mêmes techniques que précédemment avec en plus le statut MET (mutation en NGS et expression en IHC). Les patients ont été classés en résistants primaires, intermédiaires et longs répondeurs en fonction de la durée de leur réponse évaluée par des critères radiologiques (RECIST). Nous avons aussi caractérisé le profil génétique de 73 ccRCC de cette série par CGH array pour lesquels nous disposions de congélation. Les patients résistants primaires avaient plus souvent un mauvais pronostic (score de Heng), des métastases hépatiques, une infiltration de la graisse hilaire. Sur le plan cytogénétique, leurs tumeurs présentaient des altérations génétiques plus nombreuses tant au niveau des gains que des pertes. Parmi ces altérations récurrentes, étaient décrites les gains du 5p, 7p, 8q22.1-qter et la perte de la région 6q21-q25.3. Le modèle de Cox multivarié mettait en évidence 4 facteurs indépendants : le score de Heng, des métastases hépatiques, une infiltration de la graisse hilaire et le gain du 8q qui intégrés dans un nomogramme pronostique avaient un c-index de 0.74 et 0.77 pour la survie sans progression et la survie globale. En conclusion, notre étude a permis d’identifier un sous-type de ccRCC avec un statut niVHL de mauvais pronostic qu’il conviendrait d’étudier de manière plus approfondie sur le plan génomique. De plus, nous avons montré une différence de phénotype entre les ccRCC des patients métastatiques synchrones et métachrones alors que leur prise en charge est actuellement équivalente. Enfin nous avons mis en évidence un nomogramme pronostique dans les ccRCC métastatiques traités par sunitinib en 1ère line. Ce nomogramme s’il est confirmé par une étude prospective plus large pourrait avoir un impact clinique important dans la sélection des patients les plus à même de bénéficier des anti-angiogéniques. / Clear cell renal cell carcinoma (ccRCC) is the most common kidney cancer. It is characterized by frequent inactivation of the tumor suppressor gene VHL found in 70% of tumors leading to the transcription of HIF transcription factor target genes such as VEGF. This is an aggressive tumor with 50% of metastatic patients. Sunitinib, an inhibitor of receptor tyrosine kinase antiangiogenic, is currently the most used in 1st line despite 30% of patients who progress quickly. The advent of a new anti-angiogenic targeting MET (cabozantinib) and immunomodulators (anti-PD-1 antibody, nivolumab) makes crucial discovery of predictors of response to treatment. In the first part, we studied a retrospective study of 98 consecutive ccRCC. We assessed complete VHL status and correlated it with the expression of PD-L1. Moreover, while the prognosis is different between ccRCC synchronous metastatic and metachronous, their phenotype have never been compared. In this purpose, we performed an analysis of the main pathological prognostic factors, immunohistochemical markers (CAIX, VEGF, PAR3, PD-1 and PD-L1) and molecular (VHL status: deletion, mutation and promoter methylation) correlated with specific survival. We demonstrated that non-inactivated VHL tumors (niVHL) were associated with the presence of synchronous metastases, sarcomatoid component, a dense lymphocytic infiltrate, an overexpression of VEGF, an expression of PD-L1 and a poor prognosis. We also compared the phenotypes of metachronous and synchronous metastatic ccRCC. The first ones were associated with sarcomatoid component, cytoplasmic expression of PAR-3 overexpression VEGFA and niVHL status and a poor prognosis even from the diagnosis of metastases. In the second part, we studied a retrospective study of 90 consecutive metastatic ccRCC treated with first line sunitinib to identify predictors of response or resistance. We used the same techniques as above plus the MET status (mutation in Next-Generation sequencing and expression by IHC). Patients were classified as primary-refractory, intermediate and long-term responders depending on the duration of their response as assessed by radiological criteria (RECIST). We also characterized the genetic profile of 73 ccRCC of this series by CGH array for which we had frozen tumor. Primary refractory patients often had poor prognosis (Heng criteria), liver metastases, infiltration of the hilar fat. Cytogenetically, their tumors had many more genetic alterations, both gains as losses. These recurrent alterations were gains of 5p, 7p, 8q22.1-qter and loss of 6q21-q25.3 region. The multivariate Cox model highlighted four independent factors: the score of Heng, liver metastases, infiltration of the hilar fat and gain of 8q which integrated into a prognostic nomogram had a c-index of 0.74 for survival progression-free survival and 0.77 for overall survival. In conclusion, our study identified a subtype of ccRCC with a poor prognosis with niVHL status that should be explored at the genomic level. Furthermore, we showed a phenotype difference between ccRCC synchronous and metachronous metastatic patients whereas their care is currently the same. Finally we have identified a prognostic nomogram in metastatic ccRCC treated with sunitinib in the first line. This nomogram if confirmed by a larger prospective study could have a significant clinical impact in the selection of patients most likely to benefit from anti-angiogenic therapy.

In recent years, tyrosine kinase inhibitors have been more and more used for the targeted cancer therapy, due to their ability to disrupt intracellular signalling pathways associated with the development of tumours. Cabozantinib is the tyrosine kinase inhibitor which has been approved for the treatment of thyroid cancer and it is also effective against several other types of cancer. However, multiple drugs combination is often used in anticancer therapy, which may result in their cytochrome P450-mediated interactions. Although this may affect the therapeutic effect of the drugs and cause adverse effects on the organism, very little is known about the effect of cabozantinib on biotransformation enzymes. Therefore, the effect of cabozantinib not only alone but also in combination with the known cytostatic ellipticine on the expression and the activity of cytochromes P450 1A1, 1A2 and 1B1 in rat liver and kidney in vivo was studied in this work. The gene expression was determined by quantitative PCR, the amount of protein was studied by Western blotting and consecutive immunodetection. The enzyme activity was studied using specific marker reactions, 7-ethoxyresorufin O-deethylation for CYP1A1, 7-methoxyresorufin O-demethylation for CYP1A2 and 17β-estradiol 4-hydroxylation for CYP1B1. Our results...

Cabozantinib is an anticancer drug that inhibit tyrosine kinases which allow signal pathways important for growth and development of tumors. It is used for treatment of medullary thyroid cancer, hepatocellular carcinoma and kidney cancer. The major enzymes of the first phase of biotransformation that metabolize cabozantinib are cytochromes P450. In this thesis it was studied metabolism of cabozantinib and cytochromes P450 that participated on this metabolism. Hepatic microsomes of rat, mouse and rabbit were used for studying metabolism of cabozantinib in this thesis. It was also focused on the impact of particular isoforms of cytochromes P450 on metabolism of cabozantinib in rat microsomes. Time dependence of cabozantinib conversion in hepatic rat microsomes was also studied. Enzyme kinetics of metabolism of cabozantinib in hepatic rat microsomes, as well as impact of cytochromes P450 inhibitors on the metabolism were included. Metabolites were analyzed by high performance liquid chromatography (HPLC) and mass spectrometry. Formation of metabolites of cabozantinib increased over time to 30 minutes of incubation and with some others to 40 minutes of incubation. Up to five different metabolites were detected in experiments (M1, desmethyl cabozantinib, M3, monohydroxy cabozantinib and cabozantinib...

Vandetanib, lenvatinib and cabozantinib are inhibitors of receptor tyrosine kinases approved to treat locally advanced or metastatic thyroid gland, kidney and liver cancers. These multi- kinase inhibitors, inhibit phosphorylation of tyrosine moieties of protein, thus modulate cell signalization in cancer cells. Metabolites of vandetanib, lenvatinib and cabozantinib were detected in vitro as well as in vivo in blood and urine. Cytochromes P450 and flavin monooxygenases were identified as primary enzymes participating in metabolism of these drugs. Literature lacks information regarding pharmacological efficacy of vandetanib, lenvatinib and cabozantinib metabolites. The aim of this diploma thesis was the investigation of pharmacological efficacy of N-oxides of vandetanib, lenvatinib and cabozantinib. The viability measurement under normoxic and hypoxic conditions was employed to determined their efficacy. The expression of enzymes of the first phase of xenobiotics metabolism (CYP 450 1A1, 1B1, 3A4 a CYP 450 oxidoreductase) and receptor tyrosine kinases RET and VEGFR2, as well as mechanism of changes in their expression were investigated using western blotting and flow cytometry. High performance liquid chromatography was utilised to investigate possible metabolism of tyrosine kinase inhibitors and...

<p>Prostate cancer is the second leading cause of cancer-related death among American men. Prostate tumor cells exhibit significant tropism for the bone and once metastasis occurs, survival rates fall significantly. Current treatment options are not curative and focus on symptom management. Immunotherapies are rapidly emerging as a possible therapeutic option for a variety of cancers including prostate cancer, however, variable patient response remains a concern. Chemotherapies, like cabozantinib, can have immune-priming effects which sensitize tumors to immunotherapies. Additionally, lower doses of chemotherapy can be used in this context which can reduce patient side effects. It was hypothesized that a combination of chemotherapy (cabozantinib) and immunotherapy (Interleukin-27 (IL-27)) could treat bone-metastatic prostate cancer and also exert pro-osteogenic effects. IL-27 is a multi-functional cytokine, which promotes immune cell recruitment to tumors, while also promoting bone repair. To test this hypothesis, <i>in vivo</i> experiments were performed where syngeneic C57BL/6J mice were implanted intratibially with TRAMP-C2ras-Luc cells able to form tumors in bone. Immunotherapy was administered in the form of intramuscular gene therapy, delivering plasmid DNA encoding a reporter gene (Lucia), or a therapeutic gene (IL-27). Ultrasound was used to aid gene delivery. Various gene delivery methods were tested and optimized through <i>in vivo</i> studies, with microbubbles in combination with ultrasound (sonoporation) emerging as the best method. Following immunotherapy, the animals received either cabozantinib or a vehicle control by oral gavage. Bioluminescence imaging was used to monitor tumor size over time. Combinatorial therapy inhibited tumor growth and improved survival. Further, RNA sequencing and cytokine arrays were used to investigate the mechanisms involved. Microcomputed tomography and differentiation assays indicated that the combination therapy improved bone health by improving osteoblast differentiation and inhibiting osteoclast differentiation. Our conclusion is that a chemo-immunotherapy approach such as the one examined in this work has potential to emerge as a novel therapeutic strategy for treating bone-metastatic prostate cancer. This approach should enable a significant reduction in chemotherapy-associated toxicity, improving sensitivity to immunotherapy, and simultaneously improving bone quality.</p>

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