Prophylactic isolated limb perfusion for localized, high-risk limb melanoma: results of a multicenter randomized phase III trial. European Organization for Research and Treatment of Cancer Malignant Melanoma Cooperative Group Protocol 18832, the World Health Organization Melanoma Program Trial 15, and the North American Perfusion Group Southwest Oncology Group-8593.

PURPOSE: Patients with primary cutaneous melanoma > or = 1.5 mm in thickness are at high risk of having regional micrometastases at the time of initial surgical treatment. A phase III international study was designed to evaluate whether prophylactic isolated limb perfusion (ILP) could prevent regional recurrence and influence survival. PATIENTS AND METHODS: A total of 832 assessable patients from 16 centers entered the study; 412 were randomized to wide excision (WE) only and 420 to WE plus ILP with melphalan and mild hyperthermia. Median age was 50 years, 68% of patients were female, 79% of melanomas were located on a lower limb, and 47% had a thickness > or = 3 mm. RESULTS: Median follow-up duration is 6.4 years. There was a trend for a longer disease-free interval (DFI) after ILP. The difference was significant for patients who did not undergo elective lymph node dissection (ELND). The impact of ILP was clearly on the occurrence-as first site of progression - of in-transit metastases (ITM), which were reduced from 6.6% to 3.3%, and of regional lymph node (RLN) metastases, with a reduction from 16.7% to 12.6%. There was no benefit from ILP in terms of time to distant metastasis or survival. Side effects were higher after ILP, but transient in most patients. There were two amputations for limb toxicity after ILP. CONCLUSION: Prophylactic ILP with melphalan cannot be recommended as an adjunct to standard surgery in high-risk primary limb melanoma.

Adjuvant treatment of melanoma.

Melanomas represent 4% of all malignant tumors of the skin, yet account for 80% of deaths from skin cancer.While in the early stages patients can be successfully treated with surgical resection, metastatic melanoma prognosis is dismal. Several oncogenes have been identified in melanoma as BRAF, NRAS, c-Kit, and GNA11 GNAQ, each capable of activating MAPK pathway that increases cell proliferation and promotes angiogenesis, although NRAS and c-Kit also activate PI3 kinase pathway, including being more commonly BRAF activated oncogene. The treatment of choice for localised primary cutaneous melanoma is surgery plus lymphadenectomy if regional lymph nodes are involved. The justification for treatment in addition to surgery is based on the poor prognosis for high risk melanomas with a relapse index of 50-80%. Patients included in the high risk group should be assessed for adjuvant treatment with high doses of Interferon- α 2b, as it is the only treatment shown to significantly improve disease free and possibly global survival. In the future we will have to analyze all these therapeutic possibilities on specific targets, probably associated with chemotherapy and/or interferon in the adjuvant treatment, if we want to change the natural history of melanomas.

ARE PPARS NOVEL THERAPEUTIC TARGETS TO IMPROVE MELANOMA TREATMENT ?

Cutaneous melanoma is an aggressive malignant tumor of melanocytes, the pigment- producing cells of the epidermis, with a high incidence in developed countries. Despite some major clinical breakthroughs in the last few years, efficient therapies for metastatic melanoma, which portends a very bad prognosis, are still lacking. Among the potential therapeutic targets that have been attracting at-tention in melanoma are the peroxisome proliferator-activated receptors (PPARs). These members - a, ß and 7 - of the nuclear hormone receptor family, which are ligand-gated transcription factors endowed with a multitude of functions besides metabolism homeostasis, have displayed promising antitumor properties in a wide range of cancer cells, including melanoma. However, our knowledge of PPARs' functions in this skin cancer is far from complete, making the usefulness of any of the a, ß or 7 isotype as a therapeutic target uncertain. In this work, we showed that all three PPAR isotypes are expressed in normal melanocytes, in most melanoma cell lines and in primary and metastatic melanomas, and that PPAR/3 and 7 display transcriptional activity in normal melanocytes and melanoma cells. We also showed that the PPAR7 agonist rosiglitazone had anti-melanoma properties largely independent of PPAR7 expression, which was widely varying across the different cell lines and melanoma biopsies we evaluated and was not correlated with cell line stage. Consistent with the general view of PPAR7 as a tumor suppressor gene, we found that, in human samples, PPAR7 was less expressed in melanoma than in normal skin. Transcriptornic profiling of metastatic melanoma cells in which PPAR7 was pharmacologically modulated revealed an association with epithelial-to-mesenchymal transition, though the functional relevance of this finding remains to be determined. Collectively, our results suggests that PPAR7 activity in melanoma is highly complex and that a straightforward picture of PPAR7's role in this skin cancer is difficult to draw. In this study, we also provided compelling evidence that thioredoxin interacting protein (TXNIP) is, in melanoma, a bona fide PPAR7 target gene, the expression of which is repressed by PPAR7 activation. Although TXNIP is mostly known as an inhibitor of the major antioxidant thioredoxin, it has demonstrated a range of biological functions and is generally considered as a tumor suppressor gene. Consistently, we found that TXNIP expression is associated with growth arrest of melanoma cells in vitro and that forced expression of TXNIP strongly impairs cell proliferation. Interestingly, we also discovered that TXNIP favors melanoma cell migration while it diminishes their adhesion. Finally, we provided several lines of evidence that TXNIP may regulate these processes at the transcriptional level as well as by direct protein-protein interactions in the plasma membrane. Altogether, our findings suggest that the PPAR7 target TXNIP may be a double-edged sword in melanoma, hindering tumor growth but promoting invasion and dissemination. Experiments to evaluate the net biological outcome of TXNIP modulation in vivo are ongoing. -- Le mélanome cutané est une tumeur maligne agressive des mélanocytes, cellules de l'épiderme qui produisent la mélanine. Ce cancer présente un taux d'incidence élevé dans les pays développés et est grevé d'un pronostic très sombre une fois qu'il a disséminé. Malgré les importants progrès réalisés ces dernières années, aucune thérapie lie s'est encore montrée véritablement efficace contre le mélanome métastatique. Parmi les cibles thérapeutiques potentielles, nombre de groupes de recherche se sont penchés sur les peroxisome proliferator-activated receptors (PPARs). Ces récepteurs - a, ß et 7 - font partie de la famille des récepteurs nucléaires aux hormones, des facteurs de transcription activés par des ligands et dotés d'une multitude de fonctions en sus de la régulation du métabolisme. Ces protéines ont démontré des propriétés anti-tumorales prometteuses dans une large gamme de cellules cancéreuses, y compris le mélanome. Cependant, nous connaissons encore très mal les fonctions des PPARs dans ce cancer de la peau, rendant l'utilité thérapeutique de l'un des isotypes a, ß ou 7 incertaine. Dans ce travail, nous avons montré que les trois isotypes sont exprimés dans les mélanocytes normaux, dans la plupart des lignées de mélanome ainsi que dans des mélanomes primaires et métastatiques; nous avons aussi montré que PPAR/3 et 7 sont actifs sur le plan transcriptionnel dans les mélanocytes normaux et les cellules de mélanome. La rosiglitazone, un agoniste de PPAR7, a démontré des propriétés anti-mélanome essentiellement indépendantes de l'expression de PPAR7, qui semble très variable dans les lignées et les biopsies que nous avons évaluées; de plus, l'expression de PPAR7 n'est pas corrélée avec le stade de la lignée. En accord avec la vision communément admise de PPAR7 comme étant un gène suppresseur de tumeur, nous avons observé dans des échantillons humains que PPAR7 est moins exprimé dans les mélanomes que dans la peau normale. Une étude transcrip- tomique de cellules de mélanome métastatique a révélé que la modulation phar-macologique de PPAR7 est associée avec la transition épithélio-mésenchymateuse, même si la pertinence fonctionnelle de cette trouvaille reste à déterminer. Collec-tivement, ces résultats suggèrent que l'activité de PPAR/y dans le mélanome est hautement complexe et qu'une image claire du rôle de PPAR7 dans ce cancer est difficile à dessiner. Dans cette étude, nous avons également fourni de solides preuves que la thiore-doxin interacting protein (TXNIP) est, dans le mélanome, un gène cible bona fide de PPAR7 dont l'expression est réprimée par l'activation de PPAR7. Bien que TXNIP soit surtout connu comme un inhibiteur de la thiorédoxine -un anti-oxydant majeur - cette protéine a démontré une large gamme de fonctions biologiques et est généralement considérée comme un gène suppresseur de tumeur. En accord avec cette conception, nous avons trouvé que l'expression de TXNIP est associée avec l'arrêt de croissance des cellules de mélanome in vitro et que l'expression forcée de TXNIP freine considérablement la prolifération cellulaire. Nous avons aussi découvert que TXNIP favorise la migration des cellules de mélanome alors qu'elle diminue leur adhésion. Enfin, nous avons obtenu plusieurs preuves que TXNIP pourrait réguler ces processus tant au niveau transcriptionnel que par des interactions protéine-protéine au sein de la membrane plasmique. En conclusion, nos résultats suggèrent que la cible de PPAR7 TXNIP pourrait être une épée à double tranchant dans le mélanome, freinant la croissance tumorale mais favorisant l'invasion et la dissémination. Des expériences permettant d'évaluer l'effet biologique net de la modulation de TXNIP in vivo sont en cours.

Expression of Chemokine Receptors in Uveal Melanoma

Purpose:Chemokine receptors are transmembrane G coupled proteins that might be involved in the directional metastatic migration of tumor cells to specific organs. CXCR4 and CCR7 have been implicated in the selective metastasis of cutaneous melanoma cells to lung and lymph node, respectively. CCR6 is expressed in metastases from colon, ovarian and thyroid carcinomas to the liver where its ligand, CCL20, is constitutively expressed. As uveal melanomas frequently metastasize to the liver, we hypothesized that specific chemokine receptors and their respective ligands might be involved in metastasis of uveal melanoma to the liver. Methods:Tissue microarrays were constructed using 100 non irradiated primary uveal melanomas and 84 liver metastases, as well as 12 non liver metastases, collected from the files of Jules Gonin Eye Hospital and Pathology Institute, University of Lausanne. Immunohistochemistry was performed using anti-human CXCR4, SDF1, CCR7, CCL21 and CCR6 antibodies. Results:CXCR4 expression was detected in 36% of primary uveal melanomas and in 63% of liver metastases but no expression was found in metastases to other organs, except for one pancreatic metastasis. SDF1 expression was detected in 3% of primary uveal melanomas and in 26% of liver metastases, as well as in pancreas, lymph node and breast metastases. CCR6 expression was observed in the majority of primary uveal melanomas and liver metastases (73 and 88%, respectively). In addition, CCR6 was also detected in 9 metastases to other organs (pancreas, thyroid, lymph node, skin and breast). CCR7 and CCL21 were neither detected in primary uveal melanoma, nor in the metastases. Conclusions:Chemokine receptors CCR6 and CXCR4 are expressed in a large number of primary uveal melanomas and in uveal melanoma metastases to the liver. CCR6 is also expressed in a small number of metastases to other organs. These findings form the basis for further studies on the potential involvement of CXCR4 and CCR6 in the selective metastasis of uveal melanoma to the liver.

The human T cell response to melanoma antigens.

The cornerstone of the concept of immunosurveillance in cancer should be the experimental demonstration of immune responses able to alter the course of in vivo spontaneous tumor progression. Elegant genetic manipulation of the mouse immune system has proved this tenet. In parallel, progress in understanding human T cell mediated immunity has allowed to document the existence in cancer patients of naturally acquired T cell responses to molecularly defined tumor antigens. Various attributes of cutaneous melanoma tumors, notably their adaptability to in vitro tissue culture conditions, have contributed to convert this tumor in the prototype for studies of human antitumor immune responses. As a consequence, the first human cytolytic T lymphocyte (CTL)-defined tumor antigen and numerous others have been identified using lymphocyte material from patients bearing this tumor, detailed analyses of specific T cell responses have been reported and a relatively large number of clinical trials of vaccination have been performed in the last 15 years. Thus, the "melanoma model" continues to provide valuable insights to guide the development of clinically effective cancer therapies based on the recruitment of the immune system. This chapter reviews recent knowledge on human CD8 and CD4 T cell responses to melanoma antigens.

Methylation of CpG island promoters in uveal melanoma.

BACKGROUND: Inactivation of tumour-related genes by promoter hypermethylation is a common epigenetic event in the development of a variety of tumours. AIM: To investigate in primary uveal melanoma the status of promoter methylation of genes thought to be involved in tumour development: p16, TIMP3, RASSF1, RARB, FHIT, hTERT and APC. METHODS: Gene promoter methylation was studied by methylation-sensitive single-strand conformation analysis and dot-blot assay in a series of 23 primary uveal melanomas. All DNA samples were obtained from paraffin-embedded formalin-fixed tissue blocks. RESULTS: hTERT promoter methylation was found with a relatively high frequency (52%). Promoter methylation of p16, TIMP3, RASSF1, RARB, FHIT and APC was a rare event. For none of these genes did promoter methylation exceed 15% of tumour samples, and, for some genes (FHIT and APC), no methylation was found at all. Furthermore, promoter methylation was absent in 39% (9/23) of cases. In only 22% (5/23) of cases was hypermethylation of at least two promoters observed. CONCLUSIONS: Promoter methylation of hTERT is a regular event in uveal melanoma. Hypermethylation of the other genes studied does not seem to be an essential element in the development of this tumour. As promoter methylation of APC, RASSF1 and RARB is often observed in cutaneous melanoma, these results suggest that different epigenetic events occur in the development of cutaneous and uveal melanoma.

Expression of somatostatin receptors in human melanoma cell lines: effect of two different somatostatin analogues, octreotide and SOM230, on cell proliferation

Somatostatin analogues (SAs) are potential anticancer agents. This study was designed to investigate the expression of somatostatin receptors (SSTRs) in melanoma cells and the effect of two SAs on cell proliferation and viability. Eighteen primary and metastatic human cutaneous melanoma cell lines were treated with octreotide and SOM230. Expression of SSTR1, SSTR2, SSTR3 and SSTR5 was assessed by real-time polymerase chain reaction. Proliferation, viability and cell death were assessed using standard assays. Inhibition was modelled by mixed-effect regression. Melanoma cells expressed one or more SSTR. Both SAs inhibited proliferation of most melanoma cell lines, but inhibition was less than 50%. Neither SA affected cell viability or induced cell death. The results suggest that melanoma cell lines express SSTRs. The SAs investigated, under the conditions used in this study, did not, however, significantly inhibit melanoma growth or induce cell death. Novel SAs, combination therapy with SAs and their anti-angiogenic properties should be further investigated.

Ipilimumab-dependent cell-mediated cytotoxicity of regulatory T cells ex vivo by nonclassical monocytes in melanoma patients.

Enhancing immune responses with immune-modulatory monoclonal antibodies directed to inhibitory immune receptors is a promising modality in cancer therapy. Clinical efficacy has been demonstrated with antibodies blocking inhibitory immune checkpoints such as cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) or PD-1/PD-L1. Treatment with ipilimumab, a fully human CTLA-4-specific mAb, showed durable clinical efficacy in metastatic melanoma; its mechanism of action is, however, only partially understood. This is a study of 29 patients with advanced cutaneous melanoma treated with ipilimumab. We analyzed peripheral blood mononuclear cells (PBMCs) and matched melanoma metastases from 15 patients responding and 14 not responding to ipilimumab by multicolor flow cytometry, antibody-dependent cell-mediated cytotoxicity (ADCC) assay, and immunohistochemistry. PBMCs and matched tumor biopsies were collected 24 h before (i.e., baseline) and up to 4 wk after ipilimumab. Our findings show, to our knowledge for the first time, that ipilimumab can engage ex vivo FcγRIIIA (CD16)-expressing, nonclassical monocytes resulting in ADCC-mediated lysis of regulatory T cells (Tregs). In contrast, classical CD14(++)CD16(-) monocytes are unable to do so. Moreover, we show that patients responding to ipilimumab display significantly higher baseline peripheral frequencies of nonclassical monocytes compared with nonresponder patients. In the tumor microenvironment, responders have higher CD68(+)/CD163(+) macrophage ratios at baseline and show decreased Treg infiltration after treatment. Together, our results suggest that anti-CTLA-4 therapy may target Tregs in vivo. Larger translational studies are, however, warranted to substantiate this mechanism of action of ipilimumab in patients.

Characterization of local and systemic immune-modulation in melanoma patients

Le mélanome cutané est un des cancers les plus agressifs et dont l'incidence augmente le plus en Suisse. Une fois métastatique, le pronostic de survie moyenne avec les thérapies actuelles est d'environ huit mois, avec moins de 5% de survie à cinq ans. Les récents progrès effectués dans la compréhension de la biologie de la cellule tumorale mais surtout dans l'importance du système immunitaire dans le contrôle de ce cancer ont permis le développement de nouveaux traitements novateurs et prometteurs. Ces thérapies, appelées immunothérapies, reposent sur la stimulation et l'augmentation de la réponse immunitaire à la tumeur. Alors que les derniers essais cliniques ont démontré l'efficacité de ces traitements chez les patients avec des stades avancés de la maladie, le contrôle de la maladie à long- terme est seulement atteint chez une minorité des patients. La suppression locale et systémique de la réponse immunitaire spécifique anti-tumorale apparaitrait comme une des raisons expliquant la persistance d'un mauvais pronostic clinique chez ces patients. Des études sur les souris ont montré que les vaisseaux lymphatiques joueraient un rôle primordial dans ce processus en induisant une tolérance immune, ce qui permettrait à la tumeur d'échapper au contrôle du système immunitaire et métastatiser plus facilement. Ces excitantes découvertes n'ont pas encore été établi et prouvé chez l'homme. Dans cette thèse, nous montrons pour la première fois que les vaisseaux lymphatiques sont directement impliqués dans la modulation de la réponse immunitaire au niveau local et systémique dans le mélanome chez l'homme. Ces récentes découvertes montrent le potentiel de combiner des thérapies visant le système lymphatique avec les immunothérapies actuellement utilisées afin d'améliorer le pronostic des patients atteint du mélanome. -- Cutaneous melanoma is one of the most invasive and metastatic human cancers and causes 75% of skin cancer mortality. Current therapies such as surgery and chemotherapy fail to control metastatic disease, and relapse occurs frequently due to microscopic residual lesions. It is, thus, essential to develop and optimize novel therapeutic strategies to improve curative responses in these patients. In recent decades, tumor immunologists have revealed the development of spontaneous adaptive immune responses in melanoma patients, leading to the accumulation of highly differentiated tumor-specific T cells at the tumor site. This remains one of the most powerful prognostic markers to date. Immunotherapies that augment the natural function of these tumor-specific T cells have since emerged as highly attractive therapeutic approaches to eliminate melanoma cells. While recent clinical trials have demonstrated great progress in the treatment of advanced stage melanoma, long-term disease control is still only achieved in a minority of patients. Local and systemic immune suppression by the tumor appears to be responsible, in part, for this poor clinical evolution. These facts underscore the need for a better analysis and characterization of immune- related pathways within the tumor microenvironment (TME), as well as at the systemic level. The overall goal of this thesis is, thus, to obtain greater insight into the complexity and heterogeneity of the TME in human melanoma, as well as to investigate immune modulation beyond the TME, which ultimately influences the immune system throughout the whole body. To achieve this, we established two main objectives: to precisely characterize local and systemic immune modulation (i) in untreated melanoma patients and (ii) in patients undergoing peptide vaccination or checkpoint blockade therapy with anti-cytotoxic T- lymphocyte-asisctaed protein-4 (CTLA-4) antibody. In the first and main part of this thesis, we analyzed lymphatic vessels in relation to anti-tumor immune responses in tissues from vaccinated patients using a combination of immunohistochemistry (IHC) techniques, whole slide scanning/analysis, and an automatic quantification system. Strikingly, we found that increased lymphatic vessel density was associated with high expression of immune suppressive molecules, low functionality of tumor-infiltrating CD8+ T cells and decreased cytokine production by tumor-antigen specific CD8+ T cells in the blood. These data revealed a previously unappreciated local and systemic role of lymphangiogenesis in modulating T cell responses in human cancer and support the use of therapies that target lymphatic vessels combined with existing and future T cell based therapies. In the second objective, we describe a metastatic melanoma patient who developed pulmonary sarcoid-like granulomatosis following repetitive vaccination with peptides and CpG. We demonstrated that the onset of this pulmonary autoimmune adverse event was related to the development of a strong and long-lasting tumor-specific CD8+ T cell response. This constitutes the first demonstration that a new generation tumor vaccine can induce the development of autoimmune adverse events. In the third objective, we assessed the use of Fourier Transform Infrared (FTIR) imaging to identify melanoma cells and lymphocyte subpopulations in lymph node (LN) metastasis tissues, thanks to a fruitful collaboration with researchers in Brussels. We demonstrated that the different cell types in metastatic LNs have different infrared spectral features allowing automated identification of these cells. This technic is therefore capable of distinguishing known and novel biological features in human tissues and has, therefore, significant potential as a tool for histopathological diagnosis and biomarker assessment. Finally, in the fourth objective, we investigated the role of colony- stimulating factor-1 (CSF-1) in modulating the anti-tumor response in ipilimumab-treated patients using IHC and in vitro co-cultures, revealing that melanoma cells produce CSF-1 via CTL-derived cytokines when attacked by cytotoxic T lymphocytes (CTLs), resulting in the recruitment of immunosuppressive monocytes. These findings support the combined use of CSF-1R blockade with T cell based immunotherapy for melanoma patients. Taken together, our results reveal the existence of novel mechanisms of immune modulation and thus promote the optimization of combination immunotherapies against melanoma. -- Le mélanome cutané est un des cancers humains les plus invasifs et métastatiques et est responsable de 75% de la mortalité liée aux cancers de la peau. Les thérapies comme la chirurgie et la chimiothérapie ont échoué à contrôler le mélanome métastatique, par ailleurs les rechutes sous ces traitements ont été montrées fréquentes. Il est donc essentiel de développer et d'optimiser de nouvelles stratégies thérapeutiques pour améliorer les réponses thérapeutiques de ces patients. Durant les dernières décennies, les immunologistes spécialisés dans les tumeurs ont démontré qu'un patient atteint du mélanome pouvait développer spontanément une réponse immune adaptative à sa tumeur et que l'accumulation de cellules T spécifiques tumorales au sein même de la tumeur était un des plus puissants facteurs pronostiques. Les immunothérapies qui ont pour but d'augmenter les fonctions naturelles de ces cellules T spécifiques tumorales ont donc émergé comme des approches thérapeutiques très attractives pour éliminer les cellules du mélanome. Alors que les derniers essais cliniques ont démontré un progrès important dans le traitement des formes avancées du mélanome, le contrôle de la maladie à long-terme est seulement atteint chez une minorité des patients. La suppression immune locale et systémique apparaitrait comme une des raisons expliquant la persistance d'un mauvais pronostic clinique chez ces patients. Ces considérations soulignent la nécessité de mieux analyser et caractériser les voies immunitaires non seulement au niveau local dans le microenvironement tumoral mais aussi au niveau systémique dans le sang des patients. Le but de cette thèse est d'obtenir une plus grande connaissance de la complexité et de l'hétérogénéité du microenvironement tumoral dans les mélanomes mais aussi d'investiguer la modulation immunitaire au delà du microenvironement tumoral au niveau systémique. Afin d'atteindre ce but, nous avons établi deux objectifs principaux : caractériser précisément la modulation locale et systémique du système immunitaire (i) chez les patients atteints du mélanome qui n'ont pas reçu de traitement et (ii) chez les patients qui ont été traités soit par des vaccins soit par des thérapies qui bloquent les points de contrôles. Dans la première et majeure partie de cette thèse, nous avons analysé les vaisseaux lymphatiques en relation avec la réponse immunitaire anti-tumorale dans les tissus des patients vaccinés grâce à des techniques d'immunohistochimie et de quantification informatisé et automatique des marquages. Nous avons trouvé qu'une densité élevée de vaisseaux lymphatiques dans la tumeur était associée à une plus grande expression de molécules immunosuppressives ainsi qu'à une diminution de la fonctionnalité des cellules T spécifiques tumoral dans la tumeur et dans le sang des patients. Ces résultats révèlent un rôle jusqu'à là inconnu des vaisseaux lymphatiques dans la modulation directe du système immunitaire au niveau local et systémique dans les cancers de l'homme. Cette recherche apporte finalement des preuves du potentiel de combiner des thérapies visant le système lymphatique avec des autres immunothérapies déjà utilisées en clinique. Dans le second objectif, nous rapportons le cas d'un patient atteint d'un mélanome avec de multiples métastases qui a développé à la suite de plusieurs vaccinations répétées et consécutives avec des peptides et du CpG, un évènement indésirable sous la forme d'une granulomatose pulmonaire sarcoid-like. Nous avons démontré que l'apparition de cet évènement était intimement liée au développement d'une réponse immunitaire durable et spécifique contre les antigènes de la tumeur. Par là- même, nous prouvons pour la première fois que la nouvelle génération de vaccins est aussi capable d'induire des effets indésirables auto-immuns. Pour le troisième objectif, nous avons voulu savoir si l'utilisation de la spectroscopie infrarouge à transformée de Fourier (IRTF) était capable d'identifier les cellules du mélanome ainsi que les différents sous-types cellulaires dans les ganglions métastatiques. Grâce à nos collaborateurs de Bruxelles, nous avons pu établir que les diverses composantes cellulaires des ganglions atteints par des métastases du mélanome présentaient des spectres infrarouges différents et qu'elles pouvaient être identifiées d'une façon automatique. Cette nouvelle technique permettrait donc de distinguer des caractéristiques biologiques connues ou nouvelles dans les tissus humains qui auraient des retombées pratiques importantes dans le diagnostic histopathologique et dans l'évaluation des biomarqueurs. Finalement dans le dernier objectif, nous avons investigué le rôle du facteur de stimulation des colonies (CSF-1) dans la modulation de la réponse immunitaire anti-tumorale chez les patients qui ont été traités par l'Ipilimumab. Nos expériences in vivo au niveau des tissus tumoraux et nos co-cultures in vitro nous ont permis de démontrer que les cytokines secrétées par les cellules T spécifiques anti-tumorales induisaient la sécrétion de CSF-1 dans les cellules du mélanome ce qui résultait en un recrutement de monocytes immunosuppresseurs. Dans son ensemble, cette thèse révèle donc l'existence de nouveaux mécanismes de modulation de la réponse immunitaire anti-tumorale et propose de nouvelles optimisations de combinaison d'immunothérapies contre le mélanome.

Tratamiento de Artritis reumatoide con antagonistas del factor de necrosis tumoral alfa y su asociación con el desarrollo de melanoma cutáneo: revisión sistemática de la literatura y meta-análisis.

Introducción: El tratamiento con antagonistas del factor de necrosis tumoral alfa (anti TNF) ha impactado el pronóstico y la calidad de vida de los pacientes con artritis reumatoide (AR) positivamente, sin embargo, se interroga un incremento en el riesgo de desarrollar melanoma. Objetivo: Conocer la asociación entre el uso de anti TNF y el desarrollo de melanoma maligno en pacientes con AR. Metodología: Se realizó una búsqueda sistemática en MEDLINE, EMBASE, COCHRANE LIBRARY y LILACS para ensayos clínicos, estudios observacionales, revisiones y meta-análisis en pacientes adultos con diagnóstico de AR y manejo con anti TNF (Certolizumab pegol, Adalimumab, Etanercept, Infliximab y Golimumab). Resultados: 37 estudios clínicos cumplieron los criterios de inclusión para el meta-análisis, con una población de 16567 pacientes. El análisis de heterogeneidad no fue significativo (p=1), no se encontró diferencia en el riesgo entre los grupos comparados DR -0.00 (IC 95% -0.001; -0.001). Un análisis adicional de los estudios en los que se reportó al menos 1 caso de melanoma (4222 pacientes) tampoco mostró diferencia en el riesgo DR -0.00 (IC 95% -0.004 ; -0.003). Conclusión: En la evidencia disponible a la fecha no encontramos asociación significativa entre el tratamiento con anti TNF en pacientes con diagnóstico de AR y el desarrollo de melanoma cutáneo.

Mitose como fator prognóstico para biópsia de linfonodo sentinela em melanoma fino

Paciente, sexo feminino, 23 anos, com melanoma extensivo superficial em dorso, Breslow 0,35 mm, Clark II, sem ulcerações e com 2 mitoses / mm². Foi submetida à ampliação de margem e biópsia de dois linfonodos sentinela (axila esquerda). O exame anatomopatológico mostrou micrometástases, no seio subcapsular de ambos. Seguindo a recomendação do American Joint Commitee on Cancer 2009, a paciente foi submetida à linfadenectomia axilar total, sem outros linfonodos metastáticos. A aplicação da dermatoscopia vem permitindo maior precisão diagnóstica de melanoma cutâneo, contribuindo para maior proporção de melanoma fino ao diagnóstico. A taxa mitótica foi incluída como um importante fator prognóstico para melanomas finos pelo American Joint Commitee on Cancer 2009, sugerindo biópsia para esses pacientes

Estudo do perfil epidemiológico do melanoma cutâneo na cidade de Jaú-SP através do registro de base populacional

Pós-graduação em Pesquisa e Desenvolvimento (Biotecnologia Médica) - FMB

Melanoma Staging Where Are We Now?

The seventh version of the American Joint Committee on Cancer (AJCC) Melanoma Staging guidelines, published in 2009, has significant revisions compared with the previous version. The current schema was based on the largest melanoma patient cohort analyzed to date and is the result of a multivariate analysis of 30,946 patients with stages I, II, and III melanoma and 7972 patients with stage IV melanoma. This article summarizes the findings and the new definitions included in the 2009 AJCC Melanoma Staging and Classification. The TNM categories and the stage groupings are defined. Changes in the melanoma staging system are summarized.

MMP19 is upregulated during melanoma progression and increases invasion of melanoma cells

During the progression of cutaneous melanomas, matrix metalloproteinases (MMPs) facilitate the tumour cells to traverse the basement membrane and invade the dermis. In this study, we analysed the expression of MMP19 in the course of melanoma progression. Although MMP19 was absent in melanocytes and melanoma cells of early stages of melanoma development, its expression was strongly upregulated in the neighbouring keratinocytes that may facilitate the vertical outgrowth of melanoma cells. In contrast to early stages, MMP19 was upregulated during the vertical growth phase of melanoma and in metastases. The upregulation of MMP19 in melanoma of Clark levels IV and V correlates with that of MMP2 and also simultaneously with ceased expression of E-cadherin. To reveal whether MMP19 facilitates the invasion of melanomas, we examined adhesion and migratory capacity of selected melanoma cell lines. Melanoma cell lines with low expression of MMP19 exhibited increased adhesion to various substrates and lower migration in comparison with the cell line with higher expression of MMP19. Moreover, ectopic expression of MMP19 could restore the migratory capacity of melanoma cells with low endogenous level of MMP19. These results suggest that the increase of MMP19 expression hallmarks the progression of cutaneous melanoma and might augment melanoma growth by promoting the invasion of tumour cells.

Expression and functional significance of VE-cadherin in aggressive human melanoma cells: Role in vasculogenic mimicry

We recently have introduced the term vasculogenic mimicry to describe the unique ability of aggressive melanoma tumor cells to form tubular structures and patterned networks in three-dimensional culture, which “mimics” embryonic vasculogenic networks formed by differentiating endothelial cells. In the current study, we address the biological significance of several endothelial-associated molecules (revealed by microarray analysis) with respect to expression and function in highly aggressive and poorly aggressive human cutaneous melanoma cell lines (established from the same patient). In a comparative analysis, CD31 was not expressed by any of the melanoma cell lines, whereas TIE-1 (tyrosine kinase with Ig and epidermal growth factor homology domains-1) was strongly expressed in the highly aggressive tumor cells with a low level of expression in one of the poorly aggressive cell lines. Vascular endothelial (VE)-cadherin was exclusively expressed by highly aggressive melanoma cells and was undetectable in the poorly aggressive tumor cells, suggesting the possibility of a vasculogenic switch. Down-regulation of VE-cadherin expression in the aggressive melanoma cells abrogated their ability to form vasculogenic networks and directly tested the hypothesis that VE-cadherin is critical in melanoma vasculogenic mimicry. These results highlight the plasticity of aggressive melanoma cells and call into question their possible genetic reversion to an embryonic phenotype. This finding could pose a significant clinical challenge in targeting tumor cells that may masquerade as circulating endothelial cells or other embryonic-like stem cells.