Ce travail avait deux objectifs: 1) définir des groupes de patients (pts) susceptibles de bénéficier d’un conseil génétique par l’identification de facteurs prédictifs de l’existence d’une mutation du gène CDKN2A, un des gènes majeurs de prédisposition au mélanome, dans les familles ne comportant que deux cas (Fam_2 cas). 2) la caractérisation épidémiologique et clinique d’entités particulières du mélanome dans l’objectif secondaire de contribuer à l’identification de gènes de prédisposition à ces entités. Les 2 entités étudiées étaient le mélanome cutané (MC) associé au cancer du rein (CR) et les mélanomes muqueux de la sphère ano-génitale (MMAG).Les populations d’étude sont une collection de 293 pts atteints de MC recrutés de façon consécutive sans connaissance à priori de l'histoire familiale et la collection française MELARISK qui comprend ≥ 3000 sujets prélevés appartenant à des familles à cas multiples de mélanomes ou ayant un MC survenant dans un contexte particulier (association à un autre cancer, topographie rare, survenue avant l’âge de 20 ans, MC multiples sporadiques). Nous avons étudié l'effet de 3 facteurs prédictifs potentiels sur la présence d’une mutation de CDKN2A dans une famille en fonction du nombre de pts atteints dans une famille (2 pts versus ≥3 pts). L’étude a été menée dans 483 familles françaises comprenant 387 Fam_2 cas, et 96 familles avec ≥3 pts atteints de mélanome (Fam_3+ cas). Les facteurs étudiés dans la famille un à un puis conjointement étaient : l’âge médian <50 ans au diagnostic de MC, la survenue de ≥1 cas de MC primitifs multiples (MPM) et la survenue de ≥1 cas de cancer du pancréas (CPCP). La fréquence des mutations était plus élevée dans les Fam_3+ cas (32%) que dans les Fam_2 cas (13%). Alors qu’un âge jeune au diagnostic et la survenue de ≥ 1 MPM étaient associés à la présence de mutations de CDKN2A dans les Fam_2 cas, un âge jeune au diagnostic ainsi que la présence de ≥1 cas de CP était associé significativement aux mutations de CDKN2A dans les Fam_3+ cas. L’étude a montré que les caractéristiques cliniques associées aux mutations de CDKN2A varient, en France, pays de faible incidence de mélanome, en fonction du degré d’agrégation familiale. L’identification de facteurs prédictifs de mutations de CDKN2A dans les Fam_2 cas a contribué à définir des sous-groupes de familles (âge jeune au diagnostic, survenue de MPM) dans lesquels la fréquence des mutations de CDKN2A est supérieure à 20% et auxquels il est légitime de proposer un test génétique. L’analyse des deux séries de pts MM+CR et MMAG a permis d’identifier, en les comparant à la série de MC recrutés de manière consécutive, leurs caractéristiques cliniques et histologiques. Dans ces deux séries, nos résultats ont mis en évidence un contexte de prédisposition héréditaire en partie indépendant de CDKN2A. L’étude de l’association MC et CR chez un même patient a eu deux conséquences pratiques: pour les cliniciens ces résultats suggèrent l’intérêt d’un examen dermatologique en cas de CR et l’intérêt de l’échographie abdominale dans le bilan initial d’un MC pour le dépistage du CR; pour la recherche en génétique, cette série a contribué à l’identification d’une mutation germinale dans le gène MITF qui augmente le risque de développer un MC, un CR ou l’association des deux cancers et qui a des propriétés biologiques intéressantes. L’étude des MMAG a montré que ces mélanomes pouvaient être associés à des MC chez un même malade et/ou survenir dans un contexte familial de mélanome. Le corollaire clinique de ces résultats est que l’examen dermatologique de dépistage ou de surveillance doit être à la fois cutané et muqueux dans un contexte familial de mélanome et qu’en cas de MMAG un examen dermatologique des apparentés doit être proposé comme c’est la règle dans les MC. L’absence de mutation de CDKN2A dans ces localisations muqueuses incite à entreprendre des études génétiques pour identifier les gènes impliqués. / This thesis had two main objectives: 1) To define groups of patients which may benefit from genetic counseling by identifying predictors of mutations of the CDKN2A gene, a major gene predisposing to cutaneous melanoma (CM) in families with only two cases. 2) Epidemiological and clinical characterization of specific entities of melanoma with the secondary objective of contributing to the identification of susceptibility genes for these entities. Coexistence of CM with renal cell carcinoma and mucosal anogenital melanomas were studied.The study populations are a collection of 293 melanoma patients that were ascertained systematically and the French collection MELARISK which is a collection including over 3000 subjects drawn from families with multiple cases of melanoma or melanoma occurring in a particular context (association with another cancer, rare locations, occurrence before the age of 20, multiple sporadic melanomas).We investigated association of three clinical features with the presence of a CDKN2A mutation in a family by extent of CM family clustering (2 versus ≥3 CM patients among first-degree relatives in a family).The study was conducted in 483 French families including 387 families with two melanoma patients, and 96 families with three or more patients with melanoma. The factors examined individually and in a joint analysis in a family were: median age at diagnosis <50 years, ≥1 patient in a family with multiple primary melanomas (MPM) or with pancreatic cancer. The frequency of CDKN2A mutations was higher in F3+ families (32%) than in F2 families (13%). While early age at melanoma diagnosis and occurrence of MPM in ≥1 patient were significantly associated with the risk of a CDKN2A mutation in F2 families, early age at melanoma diagnosis and occurrence of pancreatic cancer in a family were significantly associated with CDKN2A mutations in F3+ families. Thus this study showed that clinical features associated with CDKN2A mutations vary, in France, a country of low incidence of melanoma, according to the degree of familial clustering. Identifying predictors of CDKN2A mutations in families with two melanoma cases has helped to define subgroups of families (early age at CM diagnosis, and/or ≥1 MPM patient) in which the frequency of CDKN2A mutations is above 20% such that these subgroups of F2 families should be offered genetic testing.The analysis of two series of patients, either patients with melanoma coexisting with renal cell carcinoma or patients with anogenital mucosal melanoma identified their clinical and histological features by comparing them to a series of melanomas that were ascertained systematically. In both series, our results suggested a genetic predisposition at least partly independent of CDKN2A. The study of the c renal cell carcinoma; coexistence of CM and renal cancer in the same patient had two practical consequences for clinicians: it suggests the interest of a dermatologic screening visit in patients with renal cell carcinoma and that abdominal ultrasonography or computed tomography scanning performed at the initial workup and during the follow-up of patients with CM may be of value for the early detection of renal cancer. Regarding genetic research, this series has contributed to the identification of a germline mutation in the MITF gene that increases the risk of developing melanoma, renal cancer or both cancers and has interesting biological properties. The study of anogenital melanoma has shown that these melanomas could be associated with cutaneous melanoma in the same patient and it has also shown a high frequency of family history of melanoma associating mucosal and CM suggesting a shared genetic predisposition. Consequently dermatological screening or monitoring must include examination of both skin and mucosa in families with multiple cases of CM; and in case of a mucosal melanoma, a dermatological examination should be offered to relatives. The genetic mechanism has to be identified

Les cellules Natural Killer (NK) sont de grands lymphocytes granuleux capables de rapidement éliminer des cellules tumorales et des cellules infectées par des virus sans immunisation au préalable. Au cours de ma thèse, j’ai analysé plusieurs paramètres impliqués dans la reconnaissance et la lyse des cellules de mélanome par les NK. J’ai montré à partir d’analyses ex vivo que les NK sanguines de patients atteints de mélanome métastatique (stade III-IV) présentent un faible potentiel lytique. Cependant, de telles NK provenant de patients mélanomes de tout stade clinique activées in vitro par de l’IL-2 lysent efficacement des lignées de mélanome métastatique. L’analyse du phénotype de NK circulantes de patients stade IV a montré une diminution de l’expression du récepteur activateur NKp46/NCR1 comparé aux NK de donneurs sains. J’ai également montré une corrélation positive entre l’expression du NKp46 à la surface des NK et la durée du stade IV. Pour caractériser les NK infiltrant le mélanome, J’ai analysé ex vivo les NK infiltrant des ganglions métastatiques (GG) provenant de 25 patients en stade III. Les GG de patients mélanomes contiennent une population unique de NK CD56brightCD16+ représentant 50% des NK dans ces GG qui expriment fortement les récepteurs NK NCR, NKG2D, KIRs et produisent une plus forte proportion de perforin comparée aux NK CD56brightCD16- ganglionnaires. Les NK immunsélectionnées à partir de GG et activées avec de l’IL-2 ou de l’IL-15 lysent rapidement et efficacement des lignées cellulaires de mélanome. Elles sont caractérisées par des capacités lytiques supérieures aux NK sanguines. De plus, afin d’évaluer l’impact des NK au cours du mélanome, j’ai analysé in situ les NK infiltrant des ganglions sentinelles positive et négatif ainsi que des tumeurs cutanées primaires. Les NK sont faibles dans les GS ; cependant nous avons montré que le nombre de NK infiltrant ces ganglions sentinelles est associé à une plus forte rechute à cinq ans des patients. Les cellules NK infiltrant les tumeurs cutanées sont présentes préférentiellement dans la zone peritumorale et sont très rares dans la tumeur.Chez les patients atteints de mélanome, les NK sanguines et infiltrant les tumeurs ont des caractéristiques phénotypiques et fonctionnelles différentes. Une meilleure compréhension de telles différences doit être prise en compte, ainsi la biologie des NK et de leur modulation au cours du cancer est nécessaire pour développer une stratégie thérapeutique à base de cellules NK efficace. / Cytotoxic immune effectors can control the development and growth of certain solid tumours. Among these cytotoxic effectors, NK cells are capable of rapidly eliminating tumour cells and virus-infected cells without prior immunization.The objectives of my thesis were to evaluate the potential role of NK cells in the immune response against melanoma. First, I have characterized the functional status of blood NK cells from melanoma patients at different stages of the disease. I showed that ex vivo NK cells from most advanced stage III-IV patients display low lytic potential. However, IL-2-activated NK cells from patients efficiently lyse melanoma cells and that independently to the clinical stage. Moreover, the expression of the activating receptor NKp46/NCR1 by blood NK cells was decreased in stage IV patients compared to healthy donors, and a positive correlation between NKp46 expression by NK cells and the duration of stage IV was found. I have also characterized ex vivo NK cells infiltrating metastatic lymph nodes (M-LN) from stage III melanoma patients. I have identified in M-LN a unique subpopulation of mature CD56brightCD16+ NK cells that expressed higher NCR, NKG2D, KIRs, and perforin levels than CD56brightCD16- NK cells counterpart. NK cells from M-LN activated with IL-2 or IL-15, rapidly lysed metastatic melanoma cell lines with higher efficiency than autologous blood NK cells. Finally, to determine if NK cells display a prognostic value, I analysed by immunohistochemistry NK cells and other immune cells infiltrating positive and negative sentinel lymph nodes (SLN). SLN are characterized by high densities of macrophages and endothelial cells, even higher in SLN+. Few NK cells and Granzyme B+ cells infiltrate SLNs while CD8+ T cells are numerous. Moreover, numbers of NK cells in SLN correlated with higher rate of 5 year-relapse of patients. Compared to SLN, primary cutaneous melanomas contain high numbers of NK cells that are preferentially localized in the periphery of the tumour and are not related to the Breslow. My findings showed that in melanoma patients, circulating and tumour infiltrating NK cells display unique phenotypic and functional characteristics, indicating that tumour may alter their function. However, they respond to cytokine activation and acquire antitumor lytic potential. In the new landscape of melanoma treatment, NK cells are worthy to be considered for combined treatment with BRAF inhibitors.

Le mélanome est la forme la plus agressive des cancers de la peau. Si sa prise en charge à des stades précoces est de bon pronostic, l’espérance de vie des patients chute dramatiquement pour les stades métastatiques. Malgré les avancées thérapeutiques spectaculaires récentes, le problème majeur réside dans la résistance aux traitements et la récidive et le défi principal est désormais de tendre vers un contrôle efficace et durable. Les anticorps monoclonaux (AcM) ont la capacité de cibler et éliminer spécifiquement la cellule tumorale tout en recrutant des cellules du système immunitaire, permettant de développer et/ou renforcer l’immunité de l’hôte avec le développement d’une réponse immune anti-tumorale de type vaccinale. Dans un modèle de tumeur solide de mélanome murin après greffe sous-cutanée des cellules B16F10, nous avons étudié le potentiel immunomodulateur de l’AcM TA99 qui cible un antigène de surface TYRP-1 surexprimé dans les mélanocytes tumoraux. Nos résultats montrent qu’environ 30% des souris sont protégées sur le long-terme et présentent une réponse immunitaire humorale et cellulaire mémoire. Par ailleurs, l’analyse de l’infiltrat immunitaire chez les souris qui échappent au traitement par l’AcM TA99 et qui développent une tumeur à plus ou moins long terme, montre une surexpression de PD-1 et Tim3 associée à une perte de fonctionnalité des cellules effectrices au sein de la tumeur. Ce même phénotype a été observé sur des biopsies de patients atteints de mélanome métastatique. Nous montrons aussi dans le cadre de ce travail que, le blocage de l’axe PD1/PD-L1 par inoculation d’un AcM anti-PD1 au moment de l’échappement, potentialise la réponse immunitaire anti-tumorale et entraîne une augmentation de la survie. Cependant, l’absence de régression complète suggère la mise en place d’autres voies immunosuppressives. En effet nous avons observé une surexpression des ectonucleotidases CD39 et CD73 dans le micro-environnement tumoral suggérant l’implication de l’adénosine dans la résistance au traitement de l’AcM TA99 plus l’α-PD-1. Ce résultat ouvre des perspectives intéressantes pour le blocage concomitant la voie de l’adénosine et de l’axe PD1/PD-L1. Une autre stratégie a consisté à améliorer les effets immunomodulateurs précoces de l’AcM TA99 en le combinant avec l’oxaliplatine, chimiothérapie favorisant la mort immunogénique. Bien que les combinaisons thérapeutiques testées dans cette étude montrent des effets in vivo encourageants avec un délai significatif dans la survie globale, aucune augmentation significative de la réponse anti-tumorale sur le long terme n’a pu être observée, suggérant la mise en place d’autres voies immunosuppressives non redondantes ou des stratégies de combinaisons non adaptées. Une analyse dynamique approfondie, tant phénotypique que fonctionnelle, des différents acteurs cellulaires du micro-environnement tumoral sera une étape clé dans la mise en place de combinaisons pertinentes en association avec l’AcM TA99. Ce travail prend d’autant plus d’intérêt qu’un essai clinique de phase I (IMC-20D7S) utilisant le flanvotumab (équivalent humain de l’AcM TA99) réalisé chez 27 patients atteints de mélanome métastatique, montre des effets cliniques intéressants sans effets secondaires sévères, ouvrant la voie au développement de combinaisons thérapeutiques associées à cet AcM. / Melanoma is the most aggressive form of skin cancer. Although early management is of good prognosis, the survival of patients decrease dramatically for metastatic stages. Despite the recent spectacular therapeutic advances, the major problem lies in resistance to treatment and relapse and the main challenge now is to develop an effective and sustainable control. Monoclonal antibodies (mAbs) have the ability to specifically target and eliminate tumor cells while recruiting cells from the immune system, to develop and / or enhance the immunity of the host with the development of a vaccinal immune response. In a solid tumor model of murine melanoma after subcutaneous transplantation of B16F10 cells, we investigated the immunomodulatory effect of TA99 mAb targeting a TYRP-1 surface antigen overexpressed in tumor melanocytes. Our results showed that about 30% of mice are protected in the long term and have an antitumoral humoral and cellular immune response. Moreover, the analysis of the immune infiltrate in mice that escape to the treatment with TA99 mAb and develop a tumor, shows an overexpression of PD-1 and Tim3 associated with a loss of effector cell functions within the tumor. This same phenotype has been observed in biopsies of patients with metastatic melanoma. Thus, blocking the PD-1 / PDL-1 axis by inoculation of an anti-PD1 mAb at the time of tumor escape potentiates the anti-tumor immune response and results in increased survival. However, the absence of complete regression suggests the establishment of other immunosuppressive pathways. Indeed we have observed an overexpression of CD39 and CD73 ectonucleotidases in the tumor microenvironment suggesting the involvement of adenosine in the resistance mechanisms observed and opening interesting perspectives for the concomitant blocking of this pathway and the PD1 / PDL-1 axis. Another strategy has been to improve the early immunomodulatory effects of TA99 mAb by combining it with oxaliplatin, a chemotherapy that promotes immunogenic death. Although the therapeutic combinations tested in this study showed encouraging in vivo effects with a significant delay in overall survival, no significant increase in the long-term anti-tumor response was observed, suggesting the establishment of other non-redundant immunosuppressive mechanisms or unsuitable combinations strategies. Both phenotypic and functional analysis of the different cellular actors of the tumor microenvironment will be a key step in the implementation of relevant combinations in association with the TA99 mAb. This work is highlighted by a phase I clinical trial (IMC-20D7S) using flanvotumab (human equivalent of mAb TA99) in 27 patients with metastatic melanoma that shows interesting clinical outcome without severe side effects, opening the way for the development of therapeutic combinations associated with this mAb.

Une tumeur est composée de plusieurs sous populations cellulaires. L’une d’entre elles, celle des cellules souches tumorales, est à l’origine du développement des tumeurs. Une des propriétés majeures de ces cellules est la capacité d’entrer dans un état de quiescence. De ce fait, elles sont résistantes aux thérapies anticancéreuses conventionnelles qui visent les cellules cyclantes et peuvent ainsi persister pendant de nombreuses années. Ce phénomène est appelé dormance tumorale. L’activation de ces cellules souches tumorales quiescentes conduit à la récidive de la maladie. Le passage de l’état quiescent à l’état activé serait réversible, cependant les mécanismes responsables ne sont pas encore connus. Notre hypothèse est que les facteurs inflammatoires stimulent la transition des cellules de l’état quiescent à l’état activé. Dans ce but, nous avons étudié les effets de la principale cytokine pro-inflammatoire, le TNF, sur le compartiment des cellules souches de mélanome et leur activation. Pour cela, nous avons utilisé un système d’expression, inductible par la tétracycline, qui nous a permis d’identifier et d’étudier les cellules quiescentes H2B-GFP positives et cela dans les modèles in vitro des mélanosphères et des équivalents de peaux humaines reconstruites, afin de se rapprocher de l’organisation tumorale in vivo. Grâce à des tests fonctionnels, comme la formation de mélanosphères et de colonies, et diverses techniques telles que la cytométrie en flux, la microscopie à fluorescence et l’analyse de l’expression de gènes au niveau protéique, nous avons mis en évidence que les cellules H2B-GFP positives (« label retaining cells ») au sein des mélanosphères montrent un enrichissement en marqueurs de cellules souches du mélanome (ABCB5, VEGFR). De plus, nous avons montré que le TNF agit sur le compartiment des cellules souches. En effet, un traitement au TNF augmente le pourcentage de cellules exprimant des marqueurs de cellules souches de mélanome, inhibe la différenciation des cellules de mélanome (inhibition de l’expression de Melan-A dans les mélanosphères et diminution de la pigmentation des équivalents de peau), active les cellules souches quiescentes et induit des effets qui perdurent après le retrait du TNF. Notre étude a montré que ces effets seraient causés par une activation des voies PI3K/Akt et NF&#954;B par le TNF. Un grand nombre de données suggérant qu’une sous-population de cellules cancéreuses est capable d’entrer en quiescence en réponse à une thérapie anticancéreuse, nous avons également étudié les effets de la première thérapie ciblée du mélanome : le vemurafenib, sur le compartiment des cellules souches. Nos résultats ont montré que le vemurafenib augmente le compartiment des cellules souches de mélanome (augmentation du nombre de mélanosphères formées et du pourcentage de cellules exprimant un marqueur de cellules souches de mélanome : ABCB5) et induit leur quiescence (augmentation du pourcentage de cellules H2B-GFP+ et en phase GO du cycle cellulaire). Nous avons également montré que le vemurafenib stimule l’activation de protéines régulant la quiescence des cellules souches.Nous espérons que nos recherches apporteront de nouvelles connaissances sur les mécanismes qui contrôlent l’activation des cellules souches cancéreuses quiescentes et offrir de nouvelles perspectives pour le traitement du cancer. / Accumulating data suggest that both cancer development and recurrence depend on the ability of resistant tumor cells to adopt a quiescent or dormant phenotype following treatment. These dormant cells reside in various tissues of patients in complete remission without any clinical manifestation until they reactivate and cause tumor recurrence. Mechanisms that control the activation of quiescent tumor cells remain poorly understood, however, the tumor microenvironment, cellular interactions and various diffusible factors appear essential. Herein, our goal is to decipher whether a major pro-inflammatory cytokine, Tumor Necrosis Factor (TNF) contributes to the quiescence/activation phenotypic switch in melanoma. For this purpose, we used a 3D melanosphere and the in vivo-like skin equivalent models in which to reconstitute the in vivo-relevant cellular heterogeneity and tumor organization and an inducible histone 2B coupled to the GFP (H2B-GFP) expression system to identify the quiescent cell compartment and to monitor the TNF-induced changes. Our results suggest that TNF increases the proportion of H2B-GFP-positive, label retaining cells (LRC) in melanospheres. The LRCs were enriched in melanoma stem cell markers, ABCB5 and VEGFR and this was upregulated by TNF. Furthermore, TNF increases the number of melanospheres, and in skin equivalents, the presence of TNF seems to inhibit the differentiation of melanoma cells and increase the stem cell compartment. This effect appears to be governed by the activation of the PI3K / Akt pathway. In conclusion, these data show that inflammatory environment induced by TNF, activates melanoma quiescent stem cells and increases the compartment of stem cells in skin equivalents by preventing their differentiation. Therefore, the control of inflammation and signaling pathways involved in the maintenance of tumor dormancy during the treatment of the original tumor would be a good therapeutic strategy in the fight against cancer recurrences.A lot of data suggest that a cancer cell subpopulation is able to enter quiescence in response to cancer therapy, therefore we have also studied the effects of the first targeted therapy of melanoma: vemurafenib, on the stem cell compartment. Our results show that vemurafenib increases the number of melanospheres and the percentage of ABCB5+ cells. So vemurafenib increases the melanoma stem cell compartment. Vemurafenib increases also the percentage of H2B-GFP + cells and the percentage of cells in the GO phase of the cell cycle, so induces quiescence of melanoma cells. We also showed that vemurafenib stimulates activation of proteins regulating quiescence of stem cells.We hope that our research will provide new knowledge about the mechanisms that control the activation of quiescent cancer stem cells and provide new perspectives for the treatment of cancer.

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Its incidence has increased globally in the last decades. There are few existing incidence studies focusing on individuals under the age of 40. Objectives Verify incidence and trends for non-melanoma skin cancer as well as for multiple skin malignant neoplasms in populations under the age of 40 in Goiânia, Central-west Brazil, 1988-2009. Methodology A descriptive epidemiological study is reported herein on melanoma skin cancer (MSC) and on non-melanoma skin cancer (NMSC). Incident cases were obtained from the Population-Based Cancer Registry (PBCR) of Goiânia and from its sources. Incident cases with single- or multiple-lesions were identified. The existence of genetic syndromes and/or transplants (in the cases of multiple lesions) was investigated. Frequency, incidence and trends were analyzed for NMSC as well as for single- and multiple- lesion cases. Results: From 1988 to 2009, 1995 skin cancer were identified in 1688 pacients, of which 1614 were basal cell carcinoma (BCC), 222 were squamous cell carcinoma (SCC), and 159 MSC. Analysis revealed 1524 (90.3%) single-lesion cases and 164 (9.7%) multiple-lesion cases. Of these, 10% presented association with genetic syndromes, with an average of 5.4 lesions, while for non-associated cases the average was 2.6 lesions. BCC incidence increased from 1.70 to 3.69 in men and from 2.38 to 6.14 per 100,000 in women in the period. Significant increasing trends were verified for men with APC = 2.8% and for women with APC = 3.9%. BCC incidence trends revealed increases per age group (in men starting from the age of 30 and in women starting from the age of 25) and per topography (in head and neck and members in men, and for all anatomical regions in women). Regarding SCC, non-significant decreasing trends were verified for both genders. Conclusions: Non-melanoma skin cancer in young individuals is not rare. BCC incidence has increased in the population under the age of 40 in Goiânia, Central-west Brazil, especially among women. Approximately 10% of cases are multiple-lesion type, of which for 90% no association was established with genetic syndromes or transplants. / Introdução O câncer de pele é a neoplasia maligna mais comum da população caucasiana mundial. A sua incidência tem aumentado globalmente nas últimas décadas. Há poucos estudos de incidência em pessoas com menos de 40 anos. Objetivos Verificar a incidência e a tendência do câncer de pele não-melanoma e neoplasias malignas múltiplas de pele em população com menos de 40 anos em Goiânia, no centro oeste do Brasil, de 1988 a 2009. Metodologia Estudo epidemiológico descritivo de câncer de pele melanoma (MC) e não melanoma (CPNM). Os casos incidentes foram obtidos do Registro de Câncer de Base Populacional de Goiânia e de suas fontes. Foram identificados os casos incidentes com lesões únicas e múltiplas. Investigou-se a existência de síndromes genéticas e ou transplante nos casos com lesões múltiplas. Analisou-se a freqüência, incidência e a tendência da incidência dos CPNM e dos casos com lesões únicas e múltiplas. Resultados: De 1988 a 2009, 1995 tumores de pele foram identificados em 1688 pacientes, sendo 1614 carcinomas basocelular (CBC), 222 carcinomas espinocelular (CEC) e 159 MC. Encontrou-se 1524 (90,3%) pacientes com lesões únicas e 164 (9,7%) com múltiplas. Destes, 10% apresentavam associação com síndrome genética, com média de 5,1 lesões, enquanto que nos não associados a média foi de 2,6 lesões. A incidência de CBC aumentou em homens de 1,70 para 3,69 e em mulheres de 2,38 para 6,14 por 100.000 no período. Houve tendência de aumento significativo nos homens APC 2,8% e nas mulheres APC 3,9%. A tendência da incidência do CBC mostrou aumento por grupo etário em homens a partir dos 30 anos e nas mulheres a partir dos 25 anos, e por topografia na cabeça e pescoço e membros nos homens, e em todas as regiões anatômicas nas mulheres. Quanto ao CEC, houve tendência de queda em ambos os sexos, não significativa. Conclusão: O câncer de pele não melanoma em jovens não é raro. A incidência de CBC tem aumentado na população com menos de 40 anos em Goiânia no centro oeste do Brasil, principalmente em mulheres. Cerca de 10% dos casos são de lesões múltiplas, sendo que em 90% destes não havia associação com síndrome genética e ou transplante.

O número total de nevos melanocíticos (NM) é um dos mais importantes fatores de risco para o desenvolvimento do melanoma cutâneo (MC); além disso, em uma significativa fração dos casos os NM podem ser também precursores desta neoplasia. CDKN2A é o gene supressor tumoral mais implicado na gênese do melanoma. As mutações desse gene ocorrem em aproximadamente 40% dos casos de melanoma familiar, enquanto o silenciamento não mutacional ocorre em até 75% dos melanomas esporádicos. Ele codifica a proteína p16, que tem sua expressão progressivamente diminuída com a evolução do melanoma, sendo bem expressa nos NM e pouco expressa nos melanomas avançados. O presente estudo avaliou as alterações na expressão imunoistoquímica (IHQ) da proteína p16 nos melanócitos dos NM expostos a duas doses eritematosas mínimas (DEM) de radiação ultravioleta B (RUVB), ou a trauma mecânico controlado. Trinta e sete NM tiveram metade de sua superfície exposta à RUVB enquanto 44 NM foram parcialmente dermoabradidos. A expressão IHQ da p16 estava significativamente diminuída no lado irradiado dos NM, comparada com o lado controle. Não houve diferença estatisticamente significativa na expressão IHQ da proteína p16 nos NM dermoabradidos.

The p300 transcriptional coactivator has been implicated in the development of a large number of malignancies; however, the precise mechanism of p300-associated tumorigenesis remains unclear. Here, we demonstrate the functional impact of p300 in human melanomas using both genetic and chemical approach. Depletion of p300 in human melanoma cells was associated with cellular growth arrest and senescence. Microarray analysis identified the Microphthalmia-associated transcription factor (MITF), a critical lineage-specific transcription factor in melanocytes and melanomas, as a major downstream target of p300 in human melanoma. Ectopic expression of MITF in p300-depleted melanoma cells allowed rescue of the p300-silencing phenotype, suggesting a critical regulatory axis involving p300 and MITF. Chromatin immunoprecipitation studies revealed direct regulation of MITF transcription through p300 acetylation of proximal regulatory domains. Critically, we identified that Forkhead Box M1 (FOXM1), a potent pro-proliferation transcription factor, is a target of the p300-MITF signaling axis. Further evaluation of p300 regulation of melanoma cell growth was performed using a highly selective p300/CBP HAT inhibitor, 228-1. Inhibition of p300/CBP histone acetyltransferase (HAT) activity was found to significantly inhibit proliferation of multiple melanoma lines in an MITF-dependent fashion. Together, these data support the role of p300 as a promising therapeutic target in human melanoma and suggest particular therapeutic efficacy of small molecule inhibitors of p300 HAT activity in tumors expressing high levels of MITF. / 2018-12-14T00:00:00Z

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.

Metastatic melanoma is unusually lethal with a ten year survival rate of less than 10%. Conventional DNA-damaging agents produce little improvement in patient survival. Vemurafenib (Zelboraf), a targeted therapeutic that inhibits the oncogenic BRAF demonstrates significant survival benefit. Unfortunately, it is now evident that there is both intrinsic and acquired resistance. Consequently, new strategies for sensitizing melanomas to vemurafenib are needed. Melanoma resistance to therapy is fueled in part by the integrins, the major cell surface adhesion receptors which are highly over-expressed in melanoma. Both integrin antagonists and agents that engage defective integrins increase the sensitivity of melanomas to chemotherapy. Our laboratory has identified a novel peptide, denoted vinculin activating peptide or VAP that targets integrins from within the cell and brings aberrant integrin function intact. VAP sensitizes melanoma to dacarbazine in vitro and in vivo. The effect VAP has on overcoming resistance to targeted therapies like vemurafenib, as well as the mechanism for its effects are not well understood. The goals of this project are to determine if VAP can be employed to improve sensitivity and/or overcome resistance to vemurafenib and to identify the cell surface target of VAP. Our results show that VAP not only improves melanoma sensitivity to vemurafenib but also decreases intrinsic resistance to this promising drug. In addition, we present evidence that β1 and β3 integrins are the target of VAP's effects. Since peptide-based therapies are not stable in the clinic, we explored another integrin binding partner, kindlin-2. We found that kindlin-2 is over expressed in resistant melanomas. The inhibition of kindlin-2 increases β1 integrin activation and decreases β3 integrin functions. Agents that bring aberrant β1 and β3 integrin function intact can be employed to improve sensitivity and overcome resistance to vemurafenib suggesting that combinatorial therapies that employ vemurafenib and integrin-based agents might be efficacious in combatting resistance in melanoma patients.

Melanoma is one of the fastest growing cancers in the world, and can affect patients earlier in life than most other cancers. Therefore, it is imperative to be able to identify patients at high risk for melanoma and enroll them in screening programs to detect the cancer early. Electronic health records collect an enormous amount of data about real-world patient encounters, treatments, and outcomes. This data can be mined to increase our understanding of melanoma as well as build personalized models to predict risk of developing the cancer. Cancer risk models built from structured clinical data are limited in current research, with most studies involving just a few variables from institutional databases or registries. This dissertation presents data processing and machine learning approaches to build melanoma risk models from a large database of de-identified electronic health records. The database contains consistently captured structured data, enabling the extraction of hundreds of thousands of data points each from millions of patient records. Several experiments are performed to build effective models, particularly to predict sentinel lymph node metastasis in known melanoma patients and to predict individual risk of developing melanoma. Data for these models suffer from high dimensionality and class imbalance. Thus, classifiers such as logistic regression, support vector machines, random forest, and XGBoost are combined with advanced modeling techniques such as feature selection and data sampling. Risk factors are evaluated using regression model weights and decision trees, while personalized predictions are provided through random forest decomposition and Shapley additive explanations. Random undersampling on the melanoma risk dataset shows that many majority samples can be removed without a decrease in model performance. To determine how much data is truly needed, we explore learning curve approximation methods on the melanoma data and three publicly-available large-scale biomedical datasets. We apply an inverse power law model as well as introduce a novel semi-supervised curve creation method that utilizes a small amount of labeled data. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection