Excerpt: Approximately 1 week after having been shot in the left eye with a foam dart from a toy gun, a 37-year-old woman presented to an optometrist due to persistent pain in the eye.

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Im Rahmen der Dissertation wurde ein Verfahren zur Quantifizierung monosomer Zellpopulationen innerhalb eines disomen Normalgewebes auf Basis der Pyrosequenzierung von Einzelbasenmutationen etabliert und hinsichtlich seiner Genauigkeit untersucht. Dabei liegt ein besonderer Schwerpunkt auf der Entwicklung eines Verfahrens zur Festlegung von Grenzwerten für die Detektion monosomer Population sowie für genetisch heterogene Subpopulationen. Zur Bestimmung der Genauigkeit wurden Mischreihen von DNA zweier Genotypen angefertigt und das Allelverhältnis durch Pyrosequenzierung gemessen. Diese Ergebnisse wurden genutzt, um Grenzwerte für die Detektion von LOH3-positiven Zellen im UMM estzulegen. In diesen Vorversuchen konnte die Anwendbarkeit der Analysemethode für Proben aus UMM sowohl aus Enukleations wie auch aus Feinnadelaspirationspräparaten demonstriert werden. Es wurde dann in einem weiteren Schritt analysiert, wie viele differente Loci für eine korrekte Diskriminierung zweier Genotypen analysiert werden müssen. Hier wurde gezeigt, dass zum einen die Anzahl der untersuchten SNP aber auch das gemessene Allelverhältnis maßgeblichen Einfluss auf die Genauigkeit der Analyse haben. Basierend auf diesen Daten wurde ein Verfahren entwickelt, das aus der gewünschten Genauigkeit eine Berechnung des Umfangs eines zu etablierenden SNP-Panels ermöglichte.

Numerous events during development require the tightly controlled and regulated interaction of cells - from gastrulation in the early embryo to axonal pathfinding and remodeling of synaptic networks. Each of these events is dependent upon signals generated by cell-cell interactions, which are in turn specified by a diverse number of cell adhesion molecules. Many families of cell adhesion molecules have been described, and these fall into the broad categories of cadherins, immunoglobulin superfamily (IgSF) members, selectins, and integrins. Activated Leukocyte Cell Adhesion Molecule (ALCAM) is a member of the IgSF, and controls numerous developmental processes, ranging from hematopoiesis to neuronal targeting. Furthermore, this protein has been implicated in the progression of numerous cancers of diverse origins. Despite the variety of developmental and pathological processes in which ALCAM has been implicated, little is known about how it signals in the cell - few extracellular binding partners have been isolated, and, as of this writing, no cytoplasmic interactors have been identified. The purpose of the work presented in this thesis was to elucidate the mechanisms by which ALCAM influences cell behavior, specifically in uveal melanoma cells, and to determine novel extra- and intracellular ligands. Here, I report the regulation of cadherin-based junctions by ALCAM in uveal melanoma cells, as well as provide evidence for a novel extracellular interaction with L1 cell adhesion molecule, and identify three novel intracellular binding partners.

Uveal melanoma (UM) is a sight and life-threatening malignancy of the human eye. The potential for progress in translational UM research is, however, hampered by the short supply of clinical samples due to its rarity and also the lack of an informative animal model which would allow experimental intervention to dissect the molecular machinery governing tumor development. Towards this end, we aimed to generate a genetically relevant model of human UM in zebrafish that can be used to study the roles of key genetic determinants in tumor initiation and progression in vivo and also establish a valuable resource for future preclinical studies. Given the pervasive role of Gαq proteins in driving UM pathogenesis, we engineered transgenic zebrafish to express oncogenic GNAQQ209P in the melanocyte lineage. This resulted in hyperplasia of uveal (choroidal) melanocytes, but with no evidence of malignant progression nor perturbation of RPE or skin melanocytes. However, combining expression of oncogenic GNAQQ209P with tp53 inactivation resulted in an earlier onset and even more extensive hyperplasia of choroidal melanocytes that then progressed to UM. While NRASQ61L and BRAFV600E potently stimulate ERK1/2-MAPK signalling pathway, immunohistochemical analysis revealed only sporadic immunoreactivity to phosphorylated ERK1/2 in hyperplastic choroidal lesions and also uveal tumors driven by oncogenic GNAQQ209P, in contrast to an abundant immunoreactivity in oncogenic HRASG12V-driven cutaneous tumors. Rather, ubiquitous positive staining for nuclear YAP was observed in GNAQQ209P-driven uveal tumor specimens. In keeping with a lesser role of GNAQ in regulating ERK1/2-MAPK signalling in UM, we showed that downregulation of oncogenic GNAQQ209P/L or inhibition of PLC-β in the majority of human UM cells expressing oncogenic GNAQQ209P/L barely affected ERK phosphorylation. In summary, this study demonstrates the insufficiency of oncogenic GNAQQ209P alone in driving UM development which only became evident with a second genetic hit involving tp53 inactivation. Our findings also demonstrate a weak correlation between oncogenic GNAQ mutations and sustained ERK1/2-MAPK activation, implying that ERK1/2 signalling is unlikely to be instrumental in the maintenance of GNAQQ209P-driven uveal tumors.

Uveal melanoma (UM) is the most common primary cancer of the eye and its prognosis is strongly influenced by the occurrence of metastases, which are both rapidly developing and mostly fatal. The most frequent driver mutations occur in a small number of genes including GNAQ, GNA11, BAP1, CYSLTR2 and SF3B1. Due to a lack of suitable animal models, the mechanism through which mutations in these genes cause or cooperate in UM initiation and progression is still largely unknown. We aimed at generating transgenic strains expressing the human mutant proteins in zebrafish uveal melanocytes, using the kita promoter. We used the binary Gal4/UAS system to express the mutant genes mentioned above. Moreover, we performed xenotransplantation experiments with uveal melanoma human and zebrafish cell lines in optically-clear, immunocompromised, zebrafish larvae. Transplanted fish developed melanoma near the site of transplantation in two weeks and showed metastatic growth within one month of age. This approach could be used for short-term assays in larvae, and be further developed for long-term uveal melanoma studies. In parallel, we performed a chemical screen using a transgenic model previously generated in our laboratory, where oncogenic RAS is expressed under the kita promoter. As adults transgenic kita:RAS develop cutaneous melanoma with high frequency and uveal melanoma with a much lower percentage. Larvae showed an increased number of melanocytes already at 3 days post fertilization (dpf) as the earliest evidence of abnormal melanocyte growth. Using this model we performed a chemical screen based on automated detection of a reduction of melanocytes number caused by any of the 1280 FDA or EMA approved drugs of the Prestwick library. The analysis showed that 55 molecules were able to reduce by 60% or more the number of melanocytes per embryo. We identified clotrimazole, as the best candidate. The molecule is an azole derivative acting on the energetic metabolism of melanoma cells. We further tested two compounds for each of the 5 pharmacological classes, and a farnesyltransferase inhibitor (lonafarnib), that inhibits an essential post-translational modification of HRAS and suppresses the hyperpigmentation phenotype. Combinations of clotrimazole and lonafarnib showed the most promising results in zebrafish embryos, allowing a dose reduction of both drugs. We performed validation of these observations in the metastatic human melanoma cell line A375M, and in normal human epithelial melanocytes (NHEM) as control cells, in order to investigate the mechanism of action of clotrimazole in blocking the proliferation of transformed melanocytes. Viability assay and analysis of energy metabolism in clotrimazole treated cells show that this drug specifically affects melanoma cells in vitro and transformed melanocytes in vivo, having no effects on NHEM or wild type larvae. Similar effects were observed with another hit of the antifungal class, miconazole. Furthermore, we show that the effects of clotrimazole are mediated by the inhibition of hexokinase activity and suggest further testing of clotrimazole in combinatorial treatments. In conclusion, this thesis investigated different possibilities of modeling the rare cancer uveal melanoma in zebrafish, using both transgenic and transplantation approaches, and developed a pipeline for a high-throughput, semi-automated chemical screen in a zebrafish melanoma that identified clotrimazole and miconazole as targeting a metabolic vulnerability in melanoma cells.

A Braquiterapia oftálmica utiliza radionuclídeos, como o Rutênio-106 no tratamento de melanomas uveais e outras neoplasias oculares. Para realização desse procedimento é necessária uma interação da equipe multidisciplinar principalmente entre a Oftalmologia, Oncologia, Radioterapia, Física Médica e Enfermagem, envolvendo estrutura física e profissionais qualificados para garantir os processos e resultados do procedimento. Este estudo tem o objetivo de avaliar os primeiros tratamentos de braquiterapia oftálmica com Rutênio-106 e também estabelecer protocolos e processos para implementação da técnica em um Hospital Geral. Os resultados obtidos para os primeiros 11 casos tratados entre 2015 e 2017, sendo 10 melanomas e 1 hemangioma, evidenciaram baixa toxicidade aguda. Foram analisados 5 pacientes com seguimento de 5 a 13 meses. Houve regressão da lesão em todos os pacientes, com média de 28% no ápice e 12% na base no período de até 12 meses, sendo observado uma regressão progressiva durante o período de avaliação. Os protocolos foram utilizados e validados durante todas as etapas do tratamento. O uso do Ru-106 mostra-se alternativa viável no tratamento de lesões oculares, sendo primordial a capacitação e integração da equipe e seguimento de protocolos para o êxito do tratamento. / Ophthalmic Brachytherapy uses radionuclides, such as Ruthenium-106 in the treatment of uveal melanomas and other ocular tumors. For this procedure is necessary a multidisciplinary interaction between Ophthalmology, Oncology, Radiotherapy, Medical Physics and Nursing, involving physical structure and qualified professionals to guarantee the processes and results of the procedure. This study aims to evaluate the first ophthalmic brachytherapy treatments with ruthenium- 106 and also to establish protocols and processes for the implementation of this service in a General Hospital. The results obtained for the first 11 cases treated between 2015 and 2017, being 10 melanomas and 1 hemangioma, showed low acute toxicity. Five patients were followed up for 5 to 13 months. There was regression of the lesion in all of these patients, with a mean of 28% at the apex and 12% at the base in the period close to 1 year, with a progressive regression during the evaluation period. The protocols were used and validated during all stages of treatment. The use of 106-Ru is a viable alternative in the treatment of ocular tumors, being the team qualification and the correct follow-up of the protocols crucial for the treatment success.

A Braquiterapia oftálmica utiliza radionuclídeos, como o Rutênio-106 no tratamento de melanomas uveais e outras neoplasias oculares. Para realização desse procedimento é necessária uma interação da equipe multidisciplinar principalmente entre a Oftalmologia, Oncologia, Radioterapia, Física Médica e Enfermagem, envolvendo estrutura física e profissionais qualificados para garantir os processos e resultados do procedimento. Este estudo tem o objetivo de avaliar os primeiros tratamentos de braquiterapia oftálmica com Rutênio-106 e também estabelecer protocolos e processos para implementação da técnica em um Hospital Geral. Os resultados obtidos para os primeiros 11 casos tratados entre 2015 e 2017, sendo 10 melanomas e 1 hemangioma, evidenciaram baixa toxicidade aguda. Foram analisados 5 pacientes com seguimento de 5 a 13 meses. Houve regressão da lesão em todos os pacientes, com média de 28% no ápice e 12% na base no período de até 12 meses, sendo observado uma regressão progressiva durante o período de avaliação. Os protocolos foram utilizados e validados durante todas as etapas do tratamento. O uso do Ru-106 mostra-se alternativa viável no tratamento de lesões oculares, sendo primordial a capacitação e integração da equipe e seguimento de protocolos para o êxito do tratamento. / Ophthalmic Brachytherapy uses radionuclides, such as Ruthenium-106 in the treatment of uveal melanomas and other ocular tumors. For this procedure is necessary a multidisciplinary interaction between Ophthalmology, Oncology, Radiotherapy, Medical Physics and Nursing, involving physical structure and qualified professionals to guarantee the processes and results of the procedure. This study aims to evaluate the first ophthalmic brachytherapy treatments with ruthenium- 106 and also to establish protocols and processes for the implementation of this service in a General Hospital. The results obtained for the first 11 cases treated between 2015 and 2017, being 10 melanomas and 1 hemangioma, showed low acute toxicity. Five patients were followed up for 5 to 13 months. There was regression of the lesion in all of these patients, with a mean of 28% at the apex and 12% at the base in the period close to 1 year, with a progressive regression during the evaluation period. The protocols were used and validated during all stages of treatment. The use of 106-Ru is a viable alternative in the treatment of ocular tumors, being the team qualification and the correct follow-up of the protocols crucial for the treatment success.

Les mélanomes oculaires sont des tumeurs rares représentant environ 5% des mélanomes. Les mélanomes oculaires peuvent provenir de deux tissus : l’uvée (~500 cas/an en France) et la conjonctive (~30 cas/an). Les mélanomes uvéaux présentent un très faible taux de mutations somatiques. Ces tumeurs sont également porteuses d’altérations du nombre de copies caractéristiques (gains du 8q, 1q, 6p, pertes du 3, du 1p, du 6q ou du 8p). L’évolution du génome de ces tumeurs durant la progression métastatique est à ce jour mal décrit. Afin d’explorer l’évolution métastatique du mélanome uvéal, nous avons séquencé les exomes de 14 tumeurs primaires et 79 métastases provenant de 24 patients. Il existait une grande proximité génétique entre tumeurs primaires et métastases avec une médiane de 11,5 mutations dans les tumeurs primaires, et 14 dans les métastases. Bien que les mutations SF3B1 et EIF1AX soient des facteurs pronostiques majeurs dans les mélanomes uvéaux, leurs fréquences dans les métastases étaient similaires à celle observée dans les séries historiques de tumeurs primaires. Les métastases présentaient quelques altérations de nombre de copies supplémentaires par rapport aux tumeurs primaires correspondantes. Parmi les altérations du nombre de copies les plus souvent acquises lors du processus métastatique, les gains du 8q étaient présents dans 92% des métastases. Lors de ce travail, nous avons découvert un mélanome uvéal présentant un phénotype hypermuté CpG>TpG chez une patiente ayant présenté une réponse exceptionnelle à une immunothérapie anti-PD1. Ce phénotype hypermutateur a été expliqué par une mutation germinale délétère de MBD4 (Methyl-CpG Binding Domain 4) avec une inactivation bi-allélique dans la tumeur. Deux autres tumeurs hypermutées CpG>TpG porteuses d’une mutation de MBD4 germinale, un mélanome uvéal et un glioblastome, ont été identifiées dans les bases de données publiques. La biologie des mélanomes conjonctivaux et leurs profils génomiques sont mal connus. Nous avons séquencé les génomes de 6 tumeurs, puis procédé à un séquençage ciblé de 47 autres tumeurs. Nous avons montré que ces tumeurs présentent un profil hypermuté C>T induit par l’exposition aux ultra-violets. Ces tumeurs présentaient un profil de mutations proche des mélanomes cutanés avec une fréquence moindre de mutations BRAF (33%), et des mutations plus spécifiques des mélanomes muqueux telles que des mutations activatrices de KIT et SF3B1 dans les mélanomes conjonctivaux non exposés au soleil. Nous avons également identifié des mutations de CTNNB1 dans les tumeurs développées sur des nevi conjonctivaux. L’ensemble de ces travaux illustrent comment la description moléculaire des tumeurs rares permet d’envisager de nouvelles stratégies de médecine de précision. / Ocular melanomas are rare tumors representing about 5% of all melanomas. Ocular melanomas may arise from two tissues: the uvea (~ 500 cases / year in France) and the conjunctiva (~ 30 cases / year). Uveal melanomas have a very low rate of somatic mutations. These tumors also carry specific distinctive copy number alterations (gains of 8q, 1q, 6p, losses of 3, 1p, 6q or 8p). The evolution of the genome of these tumors during metastatic progression has been poorly described.To explore the metastatic evolution of uveal melanoma, we whole-exome sequenced 14 primary tumors and 79 metastases from 24 patients. Primary tumors and metastases presented close genetic profiles with a median of 11.5 mutations in primary tumors, and 14 in metastases. Although SF3B1 and EIF1AX mutations are major prognostic factors in uveal melanomas, their frequencies in metastases were similar to those observed in historical primary tumors. The metastases showed some additional copy number alterations compared to the corresponding primary tumors. Among the alterations acquired during the metastatic process, 8q gains were present in 92% of metastases.Thanks to this work, we found a uveal melanoma with a CpG> TpG hypermutated phenotype in a patient who had an exceptional response to anti-PD1 immunotherapy. This hypermutated phenotype was explained by a deleterious germline mutation of MBD4 (Methyl-CpG Binding Domain 4) with bi-allelic inactivation in the tumor. Two other hypermuted CpG> TpG tumors with germline MBD4 mutation, a uveal melanoma and a glioblastoma, were identified in public databases.The biology of conjunctival melanomas and their genomic profiles have been scarcely described. We sequenced the genomes of 6 tumors and then target-sequenced 47 other tumors. We showed that these tumors had a C> T hypermuted profile induced by ultraviolet exposure. These tumors presented a pattern of mutations close to cutaneous melanomas with a lower frequency of BRAF mutations (33%), and mutations that were more specific of mucosal melanomas such as activating mutations of KIT and SF3B1 in conjunctival melanomas not exposed to the sun. We also identified CTNNB1 mutations in tumors developed on conjunctival nevi.All of these works illustrate how the molecular description of rare tumors opens new avenues for precision medicine.