The neural crest is a transient, multipotent, cell population that gives rise to several important tissues during embryonic development, including the craniofacial skeleton, peripheral nervous system, and melanocytes. The neural crest arises from the ectoderm, along with the skin and central nervous system. This process of specification is dependent on a gene regulatory network (GRN) which is made up of transcription factors that regulate each other. While we know many of the members of this GRN, the direct connections among the members are largely unsolved. Breakdown of this GRN can lead to birth defects, such as cleft lip and palate, and cancer of neural crest derivatives, such as melanoma, thus understanding the intricate details of this network is important. The transcription factor Tfap2a is an important member of the GRN, as loss of tfap2a and its paralog tfap2c leads to loss of pre-migratory neural crest and all neural crest derivatives. Despite its importance in this network little is known about how its expression is regulated. We hypothesized that, due to its importance in this network, it will have multiple enhancers that drive its expression in the neural crest. We have identified two neural crest enhancers of tfap2a. We found that one of these enhancers is responsive to WNT signals and is maintained by forming a positive feedback loop with Sox10. Our results suggest that this enhancer is important for both induction and maintenance of tfap2a expression in the neural crest. Tfap2 paralogs are important at several different stages throughout neural crest lineage specification. However, the only direct target of Tfap2a that has been identified is sox10. Thus, we wanted to determine the direct targets of Tfap2 in this network. Through the integration of several data sets, including ATAC-seq and expression profiling of tfap2a/c double mutants, we have identified several direct targets including sox9b and alx1. Melanoma is cancer of the melanocytes, a neural crest derivative. Recent studies have shown that melanoma and the neural crest share genetic similarities. TFAP2A expression is decreased in metastatic melanoma compared to primary tumors, thus we wanted to investigate the mechanism of TFAP2A in metastatic melanoma. We found that the promoter of TFAP2A is hypermethylated in some metastatic melanoma tumors. This was confirmed by samples in the TCGA database. Hypermethylation of the promoter contributes to the downregulation of TFAP2A in metastatic melanoma. In conclusion, we have further illuminated the connections among transcription factors in the GRN important for neural crest lineage specification. Further, we have identified a new mechanism regulating TFAP2A expression in metastatic melanoma. Together, these studies reveal regulatory mechanisms of TFAP2A gene expression.

Le mélanome métastatique est resté longtemps synonyme de pronostic catastrophique en raison de la faible efficacité des traitements disponibles. Depuis quelques années, de nombreuses innovations thérapeutiques ont révolutionné la prise en charge de ces patients, avec l’autorisation de mise sur le marché de traitements modulateurs de l’immunothérapie d’une part et de thérapies ciblant la voie des MAP kinases destinées aux patients porteurs de la mutation BRAF (présente dans environ 50% des mélanomes) d’autre part.Les inhibiteurs de BRAF et de MEK sont à l’origine de réponses spectaculaires, mais le contrôle de la maladie est généralement limité dans le temps, avec l’apparition de résistances au traitement entraînant une progression de la maladie. Etablir des stratégies thérapeutiques permettant de retarder ou de contourner ces résistances s’avère donc primordial. Pour ce faire, des biomarqueurs ou outils prédictifs de la réponse, in vitro et in vivo, peuvent contribuer à mieux stratifier les patients, et personnaliser leur prise en charge.Dans une première partie, nous avons comparé l’apport respectif d’un traceur de prolifération cellulaire, la 18F-FLT, et du traceur de consommation de glucose le 18F-FDG, dans l’évaluation thérapeutique d’une xénogreffe de mélanome BRAF muté traité par un inhibiteur de BRAF. Nous avons confirmé la place prépondérante du 18F-FDG, et étayé l’intérêt des index volumiques pour le suivi thérapeutique, notamment avec la 18F-FLT.Dans une seconde partie nous avons évalué in vivo la capacité d’un schéma d’administration intermittente à retarder l’apparition de résistance par rapport à un schéma d’administration continue dans des xénogreffes BRAF mutées traitées par une combinaison d’inhibiteurs BRAF/MEK. Dans nos modèles expérimentaux, il ne semblait pas y avoir de supériorité de l’administration intermittente. Toutefois nos expérimentations n’ont pas permis de trancher de façon formelle la question.Dans une troisième partie, nous avons voulu reproduire le schéma d’administration intermittent ex vivo en utilisant la technique d’histocultures. Nous avons par ailleurs évalué la possibilité pour des histocultures issues de tumeurs de patients de prédire la réponse aux thérapies ciblées chez ces mêmes patients. Les histocultures semblent un outil fiable pour guider les choix thérapeutiques. La documentation des modifications génétiques et de l’hétérogénéité moléculaire du mélanome pourrait également inciter à adapter la stratégie en fonction de l’agressivité présumée du mélanome chez un patient donné / Metastatic melanoma has long been synonymous of dismal prognosis, due to the weak efficacy of available treatments. Numerous therapeutic innovations have profoundly modified the management of these patients, with marketing authorizations of therapies targeting the MAP Kinases pathway for patient with BRAF mutated melanoma (about 50% of patients) on the one hand and immune checkpoint inhibitors on the other hand.BRAF and MEK inhibitors result in dramatic responses, but disease control is generally short-lived, with onset of drug resistance leading to disease progression. Designing therapeutic strategies allowing delaying or bypassing this resistance phenomenon is of primary importance. Thus, in vitro and vivo biomarkers or tools predictive of response could help stratifying patients, and personalize each patient’s management.In the first part, we compared the respective value of a proliferation tracer, 18F-FLT, and of the glucose consumption tracer, 18F-FDG for therapeutic evaluation of a BRAF mutated melanoma xenograft, treated by a BRAF inhibitor. We confirmed the predominant role of 18F-FDG, and backed up the potential interest of volumetric parameters for therapeutic follow-up, especially with 18F-FLT.In a second part we assessed in vivo the ability of an intermittent schedule to delay resistance onset, compared with a continuous schedule in BRAF mutated xenografts treated by a BRAF/MEK inhibitors combination. In our experimental models, there did not seem to be any superiority for the intermittent schedule. However our experiments did not allow us to draw any final conclusions on the subject.In a third part, we tried to reproduce ex vivo the intermittent schedule with histocultures device. We also assessed the possibility for histocultures with patient derived tumours to predict response to targeted therapies in the same patients. Histocultures appear as a relevant tool to guide therapeutic choice. Determination of genetic modifications and molecular heterogeneity may also prompt tailoring therapeutic strategy depending on the supposed aggressiveness of a melanoma in a given patient

Metastatic melanoma is among the most aggressive forms of cancer for which there are no effective therapies. Emerging evidence indicates that melanomas can be sensitized to chemotherapy by increasing the function of integrin transmembrane adhesion receptors. Current integrin therapies work by targeting the extracellular domain, resulting in complete gains or losses of integrin function that lead to toxicity.An attractive alternative approach is to target proteins from inside the cell, such as vinculin, that associate with the integrin cytoplasmic domains and regulate its ligand binding properties. The work presented in this thesis describes a novel reagent, denoted vinculin activating peptide or VAP, which increases integrin activity from within the cell as measured by elevated: (1) numbers of active integrins, (2) adhesion of cells to extracellular matrix ligands, (3) numbers of cell-matrix adhesions, and (4) downstream signaling. The effects of VAP are dependent on both integrins and a key regulatory residue A50 in the vinculin head domain. I further show that VAP dramatically increases the sensitivity of melanomas to chemotherapy in clonal growth assays and in vivo mouse models of melanoma. Finally, we demonstrate that the increase in chemosensitivity results from increases in DNA damage-induced apoptosis by a mechanism that requires both p53 and β1 integrin. Collectively these findings demonstrate that integrin function can be manipulated from within the cell and validate integrins as a new therapeutic target for the treatment of chemoresistant melanomas.

Le mélanome représente 8000 nouveaux cas par an et plus d'un millier de décès. Dés l'apparition de métastases, le pronostic devient très mauvais en raison de l'inefficacité de tous les traitements. Enfin, bien que des résultats encourageants aient été obtenus récemment avec des thérapies ciblées, ces réponses restent insuffisantes. L'identification de nouvelles molécules candidates est donc un élément essentiel pour le traitement du mélanome. Pendant la 1ère partie de ma thèse, je me suis intéressé à la metformine. Nous avons observé une diminution de l’invasion des cellules de mélanome dépendante de l'activation de l’axe AMPK/ p53 en réponse à la metformine. Ainsi, nous avons démontré pour la 1ère fois que la metformine peut être utilisée pour inhiber l'invasion des cellules de mélanome. Ensuite, je me suis intéressé à d'autres antidiabétiques, les TZD. Grâce à un criblage structure / activité, nous avons identifié une nouvelle molécule, le HA15, dérivé des TZD et ayant une très forte activité anti-mélanome. Nous avons montré que le HA15 induit un stress du RE (UPR), par l'intermédiaire de la protéine chaperon Bip, conduisant à l'activation de l'apoptose et de l'autophagie, entraînant ainsi la mort des cellules. Cette étude a permis d’identifier une nouvelle molécule efficace contre le mélanome et renforce l'idée que l'UPR pourrait être une cible thérapeutique pour le traitement du mélanome. / Melanoma accounts for 8000 new cases each year and more than a thousand deaths. If diagnosed early enough, wide surgical excision may be sufficient to treat. However, at the onset of metastasis, the prognosis becomes very bad due to the ineffectiveness of all treatments. Finally, although encouraging results were recently obtained with B-Raf inhibitors and immunotherapy, these responses are insufficient. So identification of new candidate molecules is an essential element for melanoma treatment. During the first part of my PhD, I was interested in metformin, the most commonly used molecule for the treatment of type 2 diabetes mellitus. It had been shown in our laboratory that metformin reduces melanoma cells viability in vitro and in vivo. Melanoma is a highly metastatic cancer, so I focused on the potential effect of metformin on melanoma cells invasion. We observed a decrease of melanoma cells invasion dependent on the activation of AMPK / p53 axis in vitro and in vivo. So we have shown for the first time that metformin can be used to inhibit melanoma cells invasion.During the second part of my PhD, I have been interested in other antidiabetic drugs, thiazolidinediones (TZD). Through a structure / activity screening we identified a molecule, HA15, derived from TZD and having a very strong anti-melanoma activity in vitro and in vivo. We showed that HA15 induced unfolded protein response (UPR), via the chaperone protein Bip, leading to autophagy and apoptosis activation, and resulting in cell death. This study identified a new molecule potentially effective against melanoma and reinforces the idea that UPR could be a therapeutic target for melanoma treatment.

Doctor of Philosophy (PhD) / The incidence of melanoma has risen dramatically during the past three decades, yet there has been little improvement in effective treatments for this intractable and aggressive disease. Melanoma tumours are notoriously resistant to apoptosis, a cell suicide program that is activated by most cancer therapies. This thesis explores the role of the melanoma susceptibility gene product p14ARF in promoting cell cycle arrest and apoptosis, in order to resolve the impact of this tumour suppressor in melanomagenesis and melanoma susceptibility. The p14ARF tumour suppressor gene is mutated in almost half of all cancers, and germline mutations in p14ARF confer a greatly increased risk of developing melanoma. The primary function of p14ARF is to relay oncogenic signals to p53, a central regulator of cellular response to stress. There is conflicting evidence regarding the role of p14ARF in promoting apoptosis. Much of the current evidence is based on murine studies, which may not translate accurately to humans due to important differences in animal physiology and the primary sequence and functions of the mouse and human ARF proteins. Furthermore, results from previous studies are often compounded by supra-physiological expression of p14ARF, and are complicated by the fact that p14ARF shares its genomic sequence with the p16INK4a tumour suppressor gene. This study demonstrates that p14ARF expression in human cancer and primary cell lines promotes rapid p53-dependent cell cycle arrest, rather than apoptosis. As p14ARF expression did not induce apoptosis, we investigated if p14ARF could modulate the sensitivity of a cell to apoptosis induced by cytotoxic agents. Using a p14ARF-inducible U2OS osteosarcoma cell line model, we examined the impact of p14ARF expression on the apoptotic response of the cell to a panel of thirteen cytotoxic agents. p14ARF expression increased apoptosis caused by a sub-set of agents, including trichostatin A, sodium butyrate, DRB, Adriamycin and UVB radiation. p14ARF-mediated chemosensitivity was p53- and caspase-dependent, and involved the loss of mitochondrial potential. While loss of mitochondrial potential was dependent on p53, it was not blocked by caspase inhibition, demonstrating that caspases play a role downstream of mitochondrial depolarisation. Inhibition of individual components of the apoptotic program showed that p14ARF-mediated chemosensitivity was not strictly dependent on the pro-apoptotic Bax or Fas proteins. We also investigated whether p14ARF could sensitise melanoma to chemotherapeutics in vivo. We investigated the expression level of p14ARF, p16INK4a and MITFm and mutation status of B-RAF, N-RAS and PTEN in melanomas from 30 patients that had undergone isolated limb infusion - a palliative therapeutic strategy that results in much higher response rates than systemic treatment. Expression of p14ARF did not predict response to the drugs actinomycin D and melphalan . Instead, high expression of p16INK4a and presence of activating N-RAS mutation were independent predictors of response to high doses of these chemotherapeutic drugs. This work suggests that p14ARF analogues may be beneficial adjuncts in cancer therapy, but are unlikely to be effective as single agents. Additionally, p14ARF mimetics will only be effective in tumours with intact p53 signalling. Melanomas frequently carry functional p53, and may be susceptible to this mode of treatment providing the apoptotic pathway downstream of p53 is intact or can be restored.

La galectine-3 (Gal-3), protéine de type lectine, de 29-35 kDa, étudiée comme marqueur d’aggressivité dans les gliomes, présente des caractéristiques biologiques importantes justifiant son étude dans le domaine du mélanome. En effet, la Gal-3 est une protéine qui peut se lier à la laminine, tout comme l’intégrine α6/β1 dont l’expression est réduite dans le mélanome. L’expression de cette intégrine peut d’ailleurs être modulée par la Gal-3 comme récemment montré dans des lignées cellulaires de cancer du sein (BT-549) et de glioblastome (U373). Le mélanome, véritable problème de santé publique qui est susceptible d’atteindre 1 individu sur 75 dans nos contrées, reste un tumeur mal comprise avec des évolutions parfois incertaines, et des traitements dont l’efficacité est limitée. Le diagnostic histologique du mélanome lui-même peut parfois représenter une difficulté pour le clinicien et l’expert pathologiste ou dermatopathologiste. La couleur (hyperpigmentation d’un lésion pigmentée), dont l’évaluation d’ailleurs reste subjective à défaut de standardisation, ne peut à elle seule signer la malignité d’une lésion pigmentée. Globalement l’évolution d’un patient est prédite par l’indice de Breslow qui traduit en mm l’épaisseur de la tumeur. Si cet indice dépasse 1mm, le risque métastatique augmente, justifiant la réalisation de bilans extensifs de suivi. Ceci dit, certains mélanomes épais peuvent ne pas présenter de caractéristiques d’aggressivité, alors que des mélanomes fins sont parfois mortels. L’identification de marqueurs moléculaires est donc impérative, tant pour développer des stratégies thérapeutiques ciblées, que pour affiner le diagnostic et le pronostic d’un patient. Après avoir mis en évidence par immunohistochimie une expression de Gal-3 par les mélanocytes, nous avons démontré une surexpression de cette protéine par les mélanocytes tumoraux. Nous avons démontré également sur des lésions primitives qu’à l’aggressivité mesurée selon l’indice de Breslow correspondait une diminution de cette surexpression. Cette observation a pu être confirmée par un modèle de greffe orthotopique chez la souris nude. Nous nous somme intéressés par la suite à la détection de la protéine dans le sérum, et nous avons constaté, un taux élevé de Gal-3 dans le sérum de patients en stade métastatique avancé, ce taux élevé pouvant s’expliquer tant par la charge tumorale que par la présence d’une inflammation, d’ailleurs bien connue chez le patient cancéreux en stade avancé. Le rôle antiapoptotique de la Gal-3 nous a alors amené à préciser la valeur prédictive et pronostique de cette protéine. L’hypothèse d’une potentielle action bénéfique sur la réponse immunitaire des patients atteints de mélanome qui ont été vaccinés a été rejetée. La Gal-3 sérique s’est révélée comme facteur de mauvais pronostic chez les patients métastatiques, et une analyse multivariée avec la définition d’une valeur « cut-off » de 10 ng/ml a permis de montrer une valeur pronostique indépendante, supérieure à la S100B et à la CRP.

Because of the failure so far to find effective treatment for patients with advanced stages of melanoma, increasing efforts have been made to find prognostic factors identifying patients in the risk zone for development of metastasis. This thesis investigates the prognostic powers of a few selected serological and immunohistochemical biomarkers. In the first and second study, patients operated on for localized malignant melanoma were investigated regarding the prognostic impact of angiogenic serological markers and circulating levels of S100. We concluded that the S100 assays, especially S100BB, are potential biomarkers in patients with malignant melanoma, correlated to both survival and disease free survival. However, no such conclusion could be drawn from the first study, where we found no correlation to survival and investigated angiogenic markers. In the third and fourth study four new potential immunohistochemical biomarkers where investigated in collaboration with the Swedish Human Protein Atlas Program, and those where TRP-1, galectin-1, DLG5 and syntaxin-7. We found that TRP-1 correlated inversely with tumor stage and galectin-1 correlated to Ki-67. DLG5 showed a significant inverse correlation to Ki67 and the expression of STX7 was inversely correlated to tumor stage, suggesting that decreased expression is associated with more aggressive tumors. None of the investigated markers in study III and IV correlated with disease free survival or overall survival. In the fifth and last study, we examined the expression of SOX10, a transcription factor, in different melanocytic lesions. Also, a proliferation assay was carried out in a human melanoma cell line. The results reveal the presence of SOX10 in different melanocytic lesions, with a weak inverse correlation to survival and a significant inverse correlation to T-stage. A significant decrease in proliferation rate for SOX10 silenced cells was found and our data also suggests an increased migratory response in SOX10 silenced cells.

El nevus displásico (ND), factor de riesgo de melanoma maligno (MM), ha sido fuente de controversia en diferentes sentidos desde su descripción, desde el nombre asignado, los criterios clínicos e histológicos que la caracterizan hasta la gradación de los mismos en tres grados de atipia: leve, moderado y severo. Se ha evaluado en primer lugar si el grado de atipia de los ND tiene alguna relación con el riesgo de padecer melanoma mediante un estudio de datos clínicos y en segundo lugar, buscar un marcador que permita realizar una clasificación del grado de atipia de los nevus displásicos basada en criterios objetivos mediante el estudio de la expresión del marcador de proliferación Ki-67 y el marcador de migración ChAT (Acetil-colina transferasa).En la primera parte del estudio se revisaron los informes de 20.275 nevus recibidos entre 1989 y 1996, constatándose que 6.275 eran ND que pertenecían a 4.481 pacientes. Los pacientes fueron clasificados con respecto a su lesión con mayor grado de atipia y se revisó sus datos clínicos con respecto a la historia de melanoma. Se calculó la Odds Ratio (OR) como medida de asociación entre los grados de atipia de los ND e historia de melanoma. En la segunda parte del estudio se realizó estudio inmunohistoquímico con doble marcaje para HMB-45 y Ki-67 en 36 ND con atipia leve, 36 ND con atipia moderada, 36 ND con atipia severa, 18 nevus melanocíticos benignos (NMB) y 18 MM in-situ, evaluándose la proporción de células melanocíticas de la unión dermo-epidérmica positivas para Ki-67. Así mismo se realizó estudio inmunohistoquímico para ChAT en 30 ND con atipia leve, 30 con atipia moderada y 30 con atipia severa.Los resultados pusieron de manifiesto que 2.504 pacientes presentaban ND con atipia leve como nevus con mayor grado de atipia, 1.657 ND con atipia moderada y 320 ND con atipia severa. La revisión de sus datos clínicos puso de manifiesto que 142 pacientes del grupo de atipia leve, 133 pacientes del grupo con atipia moderada y 63 pacientes del grupo con atipia severa tenían historia de melanoma (c2= 59,89; p<0,001). El estudio de la asociación del grado de atipia con historia de melanoma dio como resultado una OR de 4,08 (2.91-5,7) para los ND con atipia severa versus ND con atipia leve, 2,81 (2-3,95) para los ND con atipia severa vs ND con atipia moderada y 1,45 (1,13-1,87) para ND con atipia moderada vs leve. El estudio inmunohistoquímico con doble marcaje con HMB-45 y Ki-67 mostraron diferencias significativas entre ND vs NMB vs MM (p<0,0001) y entre ND con atipia severa vs ND con atipia leve y moderada (p<0,0003). No se observaron diferencias significativas en el estudio mediante ChAT.En conclusión, la gradación de la atipia citológica de los ND se correlaciona con el riesgo de presentar historia de melanoma y el estudio del índice de proliferación Ki-67 puede ser de ayuda para diferenciar entre NMB, ND y MM, así como para distinguir entre ND con alto y bajo grado de atipia (leve y moderada). / Classification of the different aspects of dysplastic nevus (DN), a risk factor of malignant melanoma (MM), has been source of controversy from its decription, its assigned name and clinical and histological criteria, by which it is chacarcterized, to the ranking of the same into three grades of atypia: mild, moderate, and severe. In the first place, through a study of clinical data, it has been assessed if the grade of DN atypia has some relationship to the risk of suffering from MM; and, in the second place, through studies of the proliferation marker Ki-67 and the ChAT migration marker (acetyl choline transferase), a marker has been searched for, which will allow the classification of the grade of atypia for DN, based on objective criteria. In the first part of the study, reports on 20,275 nevi received between 1989 and 1996 were examined, verifiying that 6,275 of them were DN belonging to 4,481 patients. The patients were classified with respect to their lesions, having the greatest grade of atypia, and their clinical data was also examined, with respect to their history of melanoma. The Odds Ratio (OR) was calculated as a measure of association between the grades of DN aypia and the history of melanoma. In the second part of the study, an immunohistochemical study was conducted, with double staining for HMB-45 and Ki-67 on 36 DN with mild atypia, 36 DN with moderate atypia, 36 DN with severe atypia,18 BMN and 18 MM in-situ, evaluating the proportion of melanocytic cells in the dermo-epidermic junction, showing positive for Ki-67. In this way, an immunohistochemical study was also carried out for ChAT in 30 DN with mild atypia, 30 with moderate atypia and 30 with severe atypia.Results made clear that 2,504 patients presented DN with mild atypia as the highest presenting grade of atypia, 1,657 DN presented with moderate atypia, and 320 presented DN with severe grade of atypia. The review of the clinical data made clear that there were 142 patients in the group having mild atypia, 133 having moderate atypia, and 63 with sever atypia, who all had personal histories of MM (c2= 59,89; p<0,001). The study of the association of the grade of atypia with the history of MM gave as its result an OR of 4.08 (2.91-5.7) for the DN with severe atypia: DN with mild atypia; 2.81 (2-3.95) for the DN with severe atypia: DN with moderate atypia; and 1.45 (1.13-1.87) for DN with moderate atypia : DN with mild atypia. The immunohistochemical study with double staining for HMB-45 and Ki-67 showed significant differences between DN:BMN:MM (p<0.0001) and between DN with severe atypia : DN with mild and moderate atypia (p<0.0003). No significant differences were observed in the study using ChAT. In conclusion, the ranking of the cytological atypia of DN is correlated to the risk of presenting a history of melanoma, and the study of the proliferation rate of Ki-67 can help in the differentiation between BMN, DN, and MM, such as between DN with severe and mild+moderate atypia.

La incidencia del melanoma cutáneo va en aumento en un gran número de países. El diagnóstico precoz es muy importante, ya que el tratamiento más eficaz para el melanoma es la escisión tumoral.La biopsia del ganglio centinela (BGC) es una técnica bien establecida y una herramienta útil para el estadiaje y manejo de pacientes con melanoma maligno cutáneo. La BGC, introducida por Morton y colaboradores, es un procedimiento implantado en múltiples centros, permitiendo el estadiaje anatomopatológico de los ganglios linfáticos regionales.El ganglio centinela (GC) presenta metástasis de melanoma en el 20% de los pacientes con esta patología. En nuestro estudio, la biopsia de ganglio centinela fue positiva en catorce pacientes (28%).Sin embargo, la selección de pacientes para la realización de la biopsia de ganglio centinela y la linfadenectomía selectiva en caso de BGC positiva son temas controvertidos.Recientemente, el Comité Americano sobre Cáncer (AJCC) ha modificado los criterios de estadiaje de los pacientes con melanoma y recomienda la biopsia de ganglio centinela en la mayoría, dado que las metástasis ganglionares microscópicas representan el principal factor predictivo de la supervivencia.Por este motivo, en esta tesis se han recogido las diferentes variables que se han ido identificando en la literatura como predictivas de la supervivencia y de la afectación metastásica de los ganglios linfáticos regionales de esta neoplasia, en un intento de conocer su capacidad de predecir la afectación metastásica en el ganglio centinela. El objetivo de este estudio es desarrollar un modelo pronóstico, basado en datos clínicos del paciente y en características histológicas e inmunohistoquímicas del melanoma primario para evaluar la probabilidad de micrometástasis en GC en estos pacientes. Para ello, esta tesis incluye 50 pacientes sometidos a biopsia de ganglio centinela entre Enero de 1.999 y Agosto de 2.005. Las variables clínicas analizadas han sido la edad, el sexo y la localización del melanoma primario.La evaluación histológica del tumor primario ha incluido características histopatológicas como: Índice de Breslow, nivel de invasión de Clark, fase de crecimiento, tipo de melanoma, índice mitósico, ulceración, invasión vascular angiolinfática, inflamación linfocitaria peritumoral, signos de regresión, neurotropismo, microsatelitosis, afectación anexial y restos de nevus.La expresión de marcadores de diferenciación melanocítica (HMB45, Melan A) y de marcadores de progresión (ki67, p53, p16) se han determinado por técnicas inmunohistoquímicas.El examen histológico del ganglio centinela se ha realizado tras tinción de hematoxilina-eosina e inmunohistoquímica (HMB45).El análisis estadístico ha demostrado que el Índice de Breslow (P=0,019), el índice mitósico (P=0,033) y el porcentaje de ki67 (P=0,022) son factores predictivos independientes de la presencia de metástasis en ganglio centinela. Nuestros datos apoyan la relevancia conocida del espesor de Breslow como el principal factor a considerar en la selección de los pacientes para la realización de la BGC.La utilización de la BGC en pacientes con melanomas delgados (Índice de Breslow ? 1,0 mm) no se realiza de manera rutinaria. Aunque en el presente trabajo ninguna variable estudiada ayuda a predecir la presencia de metástasis de melanoma en GC en el subgrupo de melanomas delgados, consideramos que se han de continuar investigando diferentes factores de riesgo que puedan predecir la positividad del ganglio centinela en este grupo. Si bien nuestro estudio no ha demostrado una asociación de las otras variables analizadas con el resultado de la biopsia de ganglio centinela, el reducido tamaño muestral de este trabajo puede contribuir a que alguna asociación detectada no haya alcanzado la significación estadística, requiriéndose de la replicación del mismo con mayor número de casos. / Melanoma incidence is rising worlwide. Early diagnosis is very important, as the most effective treatment for melanoma still consists of excision of the tumor before onset of the metastatic phase.Sentinel lymph node biopsy is a well-established technique and a precise tool for staging and managing patients with cutaneous malignant melanoma. Introduced by Morton and colleagues, the sentinel lymph node biopsy procedure is now widely available, and marked enhances our ability to pathologically stage the regional nodes.The sentinel node is the site of metastatic melanoma in approximately 20% of melanoma patients. In our study, sentinel node biopsy was positive for tumor in fourteen patients (28%). It is customary to perform a complete dissection of the regional lymph nodes if tumor is present in the sentinel node. But, the selection of patients for sentinel lymph node biopsy and selective lymphadenectomy for histologically positive sentinel lymph nodes are areas of debate. The American Joint Committee on Cancer has recently modified staging criteria for primary melanoma patients and recommends sentinel lymph node biopsy in many, because microscopic nodal metastasis represents the most important factor predicting survival.Because sentinel node status is the most important prognostic factor for melanoma, this analysis was performed to determine if sentinel lymph node dissection status can be predicted using other prognostic factors. The objective of this study is to develop a prognostic model, based on clinical and primary tumor histological and immunohistochemical characteristics, to estimate the probability of micrometastasis in the sentinel lymph node in patients with malignant melanoma.To correlated pathological features in melanoma with sentinel node metastasis this study includes and reviews patients submitted to sentinel node biopsy between January 1999 and August 2005.A total of 50 patients are evaluated in the statistical analysis. Reported clinical, histological and immunohistochemical features are assessed for predictive value by univariate and multivariate logistic regression.Clinical attributes recorded include age, sex and location of the primary melanoma.Histological evaluation of the primary tumor include histopathologic characteristics, some of then are historically been associated with an increased risk of recurrence and mortality like: Breslow thickness, Clark's level of invasion, growth phase, type of melanoma, mitotic rate, ulceration, angiolymphatic invasion, tumor infiltrating lymphocytes, regression, neurotropism, microsatellites, anexial involvement and pre-existing nevus.Expression of melanocytic differentiation markers (HMB45, Melan A), and melanoma progression markers (ki67, p53, p16) were assessed by immunohistochemistry.Histological examination of sentinel node consisted of hematoxylin-eosin and immunohistochemical staining (HMB45).Statistical analysis shows that Breslow thickness (P=0,019), mitotic rate (P=0,033) and ki67 (P=0,022) are found to be independently predictive of the presence of sentinel node metastasis. Our data supports the known relevance of Breslow thickness as the main factor considered in the selection of patients to be submitted to SLN biopsy.Although the use of this technique (sentinel node biopsy) in patients with thin melanomas (Breslow thickness ? 1,0 mm) is not rutine, risk factors that may predict sentinel lymph node positivity in this patient population are under investigation. Although the present study is unable to identify predictors of metastatic involvement in patients with thin melanoma, efforts to identify predictors of SN tumor involvement should continue.The published data are not sufficient to demonstrate a correlation between the other indicators included and a positive sentinel lymph node biopsy, but the cohort size (small sample size) may lack the statistical power to demonstrate subtle associations. So, the predictive value of some variables for positivity needs to be validated in other populations of melanoma patients.

Las metástasis, diseminación y crecimiento de las células tumorales en órganos distantes, representan el aspecto más peligroso y devastador en el melanoma de úvea. A pesar de los avances en el diagnóstico, técnicas quirúrgicas y terapias adyuvantes, la aparición de metástasis sigue siendo la principal causa de muerte en los pacientes con melanoma de úvea. Hasta un 50% de los pacientes morirán a los 10 años del diagnóstico inicial debido a la aparición de metástasis, principalmente en el hígado. A lo largo de las últimas décadas se han identificado varios parámetros pronósticos metastásicos, clínicos e histopatológicos. Actualmente, las principales líneas de investigaciones se centran en el análisis de las alteraciones genéticas y moleculares que puedan estar asociadas al desarrollo de metástasis en melanoma de úvea y permitan establecer de forma precoz los genotipos-fenotipos más agresivos así como desarrollar nuevas alternativas terapéuticas.Las quimiocinas constituyen un grupo de moléculas de bajo peso molecular con propiedades quimioatrayentes implicadas en un gran número de procesos fisiológicos y patológicos. Se ha visto que en un gran número de neoplasias existe un entramado de quimiocinas y sus receptores cuya producción, expresión y señalización se encuentra alterada. La quimiocina CXCL12, también llamada stromal-derived factor-1(SDF-1), y su receptor transmembrana CXCR4 son un ejemplo de ello. La interacción entre ambas proteínas desencadena diversas funciones biológicas dentro del proceso cancerígeno, tanto a nivel del tumor primario como en la cascada metastásica. Su presencia ha demostrado ser imprescindible para la proliferación y supervivencia celular, la angiogénesis, la adhesión e invasión tumoral y la migración selectiva hacia el hígado de las células metastásicas en un gran número de neoplasias, relacionándose su expresión con una mayor malignidad tumoral y una menor supervivencia de los pacientes.En la presente estudio, se examinará por primera vez la expresión de CXCR4 y CXCL12 en especimenes de melanomas malignos de úvea sugiriendo la posibilidad de que, al igual que en otras neoplasias, las células melanocíticas malignas estén utilizando la unión quimiocina-receptor para promover el crecimiento tumoral local así como estimular la formación de focos metastáticos a distancia. Para ello, también se intentará establecer una correlación del grado de expresión de CXCR4 y CXCL12 con la aparición de metástasis y la supervivencia de los pacientes. Y por último se analizará la relación existente entre la expresión de CXCR4 y CXCL12 y otras variables clínicopatológicas de mal pronóstico conocidas como son la edad, sexo, tipo celular, presencia de círculos vasculares cerrados e infiltración linfocitaria tumoral.