Tese (doutorado)—Universidade de Brasília, Faculdade de Ciências da Saúde, 2009. / Submitted by Kelly Marques (pereira.kelly@gmail.com) on 2009-11-16T17:48:39Z No. of bitstreams: 1 2008_AnaMariaQuinteiroRibeiro.pdf: 722506 bytes, checksum: a724ba85f9f6c24dfa6f8951382e7363 (MD5) / Approved for entry into archive by Guimaraes Jacqueline(jacqueline.guimaraes@bce.unb.br) on 2009-11-17T12:23:45Z (GMT) No. of bitstreams: 1 2008_AnaMariaQuinteiroRibeiro.pdf: 722506 bytes, checksum: a724ba85f9f6c24dfa6f8951382e7363 (MD5) / Made available in DSpace on 2009-11-17T12:23:45Z (GMT). No. of bitstreams: 1 2008_AnaMariaQuinteiroRibeiro.pdf: 722506 bytes, checksum: a724ba85f9f6c24dfa6f8951382e7363 (MD5) Previous issue date: 2009 / O melanoma é um câncer que acomete, sobretudo a pele, é o terceiro mais freqüente depois do carcinoma basocelular e do carcinoma espinocelular e se caracteriza clinicamente na maioria das vezes pela presença de mácula hipercrômica irregular. É considerado o mais grave dos cânceres de pele por induzir metástase mais freqüente, apresentar uma alta mortalidade, sobretudo em lesões com diagnóstico tardio e por ser resistente as terapêuticas disponíveis (quimio e radioterapia). A presente pesquisa propõe-se a descrever os fatores prognósticos de pacientes com melanoma cutâneo do Registro de Câncer de Base Populacional de Goiânia (RCBPG), entre janeiro de 1998 a dezembro de 2003, período em que foram registrados 245 casos novos de melanoma. Destes, 185 foram incluídos no estudo por serem portadores de melanoma cutâneo residirem no município de Goiânia. Os sessenta pacientes restantes foram excluídos da pesquisa por apresentarem melanoma com primário desconhecido, melanoma extracutâneo ou por residirem em outro município. Foram coletados, por meio de um questionário, dados do RCBPG dos pacientes que apresentavam diagnóstico apenas de melanoma cutâneo primário. Após a coleta desses dados, foi realizado um estudo descritivo do tipo coorte de base populacional. A importância prognostica de quatro variáveis histológicas (Breslow, Clark, tipo histológico e número de neoplasias primárias) e três variáveis clínicas (sexo, localização da lesão e extensão) foram estudadas no grupo de 185 pacientes com melanoma cutâneo e foram comparados com a literatura mundial. Os resultados do estudo evidenciaram que os homens apresentaram melanomas mais espessos em comparação com as mulheres, dado que se refletiu em uma maior mortalidade e uma menor sobrevida para o sexo masculino. Nos homens, as lesões localizadas no tronco foram mais freqüentes e, nas mulheres, as das extremidades inferiores. A localização na cabeça e pescoço foi mais freqüente em homens e mulheres acima de sessenta anos. O melanoma extensivo superficial foi o tipo histológico mais freqüente. O melanoma nodular, o segundo de maior freqüência (17%), estava associado a tumores mais espessos. O melanoma cutâneo localizado, sem metástase detectada, foi à extensão de maior freqüência. A metástase à distância foi mais freqüente do que a loco-regional para linfonodos. As principais características dos pacientes e do tumor relacionados a pior prognóstico foram: melanomas do tipo histológico nodular (RH: 5,11; p=0,001) tumores com maior espessura (> 1,70 mm) (RH:12,7; p≥0,02) e os localizados nos pés (RH:4,84; p=0,005). Esses fatores contribuíram para um maior número de óbito e uma menor sobrevida dos pacientes. O aumento do nível de Clark apresentou relação direta com maior número de óbito e com menor sobrevida, o que, não se traduziu em uma associação significativa com pior prognóstico. _____________________________________________________________________________________ ABSTRACT / Melanoma is a cancer that affects mostly the skin. It is the 3rd most frequent type of cancer, after basal cell carcinomas and squamous carcinomas and is clinically characterized by the presence of an irregular hyperchromic macula. It is considered the most serious type of skin cancer because it induces metastases more frequently, the mortality rate is high, especially in lesions diagnosed at a later stage, and finally because it is resistant to the therapeutic options currently available (chemotherapy and radiotherapy).This study aims to describe prognostic factors of patients with cutaneous melanoma, listed in the Population-Based Cancer Registry of Goiânia (RCBPG), from January, 1998 to December, 2003. During that period of time, 245 new cases of melanoma were recorded. From these 245 cases, 185 were included in the study as these individuals with cutaneous melanoma lived in the county of Goiânia. The remaining 60 individuals were excluded because they presented with a melanoma with an unknown primary, ocular melanoma, melanoma of the mucosa, genital melanoma, visceral melanoma or because they lived in another county. RCBPG data from the patients with a diagnosis of primary cutaneous melanoma were obtained by means of a questionnaire and served to perform a population-base cohort descriptive study. The prognostic importance of four histological variables (Breslow, Clark, histogenic type and number of primary neoplasms) and three clinical variables (sex, lesion site and extension) were studied in the series of 185 patients with cutaneous melanoma. The results of this study showed that men presented with thicker melanomas, when compared to women. This information translated into higher mortality and shorter survival time for male individuals. Lesions located in the trunk, followed by those in the head and neck were more frequently found in men and lesions in the lower limbs, followed by locations in the head and neck were more commonly found in women. A head and neck location was more frequent in men and women above 60 years of age. Extensive superficial melanomas were the predominant histogenic type. Nodular melanomas were the second more frequent types (17%) and were associated to thicker tumors. The most frequent extension was the in situ cutaneous melanoma. Distant metastases were more frequent than those in loco or regional, for lymph nodes. During the time this study was conducted, no sentinel lymph node biopsies were performed to evaluate micrometastases of melanomas and this may have contributed to the result. The main characteristics of patients and of tumors related to the worst prognosis were: nodular melanomas (RH: 5,11; p=0,001), thicker tumors (>1.70 mm) (RH:12,7; p≥0,02) and those located in the feet (RH:4,84; p=0,005). These factors contributed to more deaths and a shorter survival time of the patients included in this study. The increase in the Clark level showed a direct relationship to a higher number of deaths, with a shorter survival time. However, this was not translated as a negative association with the worst prognosis in this study.

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Submitted by Alice Araujo (alice.caraujo@ufpe.br) on 2018-06-05T18:34:14Z No. of bitstreams: 1 DISSERTAÇÃO Gislayne Azevedo de Campos Alves.pdf: 1123702 bytes, checksum: 3ed66040a85a71bcbdb5019b172d6d0a (MD5) / Made available in DSpace on 2018-06-05T18:34:14Z (GMT). No. of bitstreams: 1 DISSERTAÇÃO Gislayne Azevedo de Campos Alves.pdf: 1123702 bytes, checksum: 3ed66040a85a71bcbdb5019b172d6d0a (MD5) Previous issue date: 2016-06-07 / O melanoma maligno é uma doença com alto índice de mortalidade. No país, há alguns tratamentos disponíveis, que não são financiados pelo Sistema Único de Saúde (SUS), como o realizado com anticorpo monoclonal, no caso específico, o medicamento ipilimumabe, levando os pacientes a recorrerem ao poder judiciário para ter acesso ao medicamento. O tratamento - procedimento cirúrgico ou quimioterápico - é garantido pelo SUS, através dos hospitais habilitados, como Centros de Assistência de Alta Complexidade em Oncologia (CACON) e Unidades de Assistência de Alta Complexidade (UNACON) e não habilitados. Constatou-se que os dois hospitais que mais realizaram estes procedimentos não estão habilitados em CACON/UNACON. Assim, analisou-se, a partir dos dados em documentos públicos, o custo das ações judiciais na compra do medicamento ipilimumabe, utilizado em pacientes com melanoma maligno pelo Estado de Pernambuco, bem como os valores pagos pelo SUS através da Autorização de Internação Hospitalar (AIH) para tratar o melanoma nos pacientes internados nos hospitais deste Estado. A pesquisa se deu nos anos de 2013 a 2015. Dos dezessete processos que tiveram deferimento por via judicial, foi visto que não houve a continuidade do tratamento em 100% destes, haja vista não conseguiram terminar o que foi preconizado na prescrição médica. As causas foram: o óbito ou a substituição do tratamento pela quimioterapia tradicional. Com isso, o custo na compra do ipilimumabe foi menor do que o preconizado nas medidas tutelares, logo o medicamento comprado para um tratamento não terminado foi remanejado para outro paciente, que ganhou a liminar posteriormente. O Estado desembolsa com o ipilimumabe quase o mesmo valor gasto com as AIH, embora a comparação entre o número de pacientes que fizeram tratamento pelo SUS, com os que tiveram acesso ao ipilimumabe por via judicial fosse 142,1 maior; no entanto, é desproporcional o custo. O gasto para atender os pacientes do SUS é 1,26 vezes maior em relação aos pacientes que acessaram as vias judiciais. Ainda foi visto que em quase sua totalidade, os magistrados de Pernambuco não evocaram nenhuma assessoria técnica a fim de obter mais informações sobre o medicamento solicitado, sendo que a concessão da medida liminar fez-se por meio da prescrição médica, além de fundamentar o direito por meio de artigos constitucionais e infraconstitucionais. Observou-se também que não houve reajuste na tabela dos valores pagos por meio das AIH nos anos da pesquisa, verificando que o menor valor pago para o mesmo procedimento cirúrgico neste período foi de R$ 40,38. O estudo aponta que mais profissionais da saúde precisam ser envolvidos no processo da judicialização, esclarecendo ao magistrado se de fato o medicamento aumenta a sobrevida do paciente em relação ao tratamento estabelecido pelo SUS, já que o impacto financeiro é alto em comparação com os benefícios alcançados nos tratamentos preexistentes e ao número de pacientes atendidos. / Cancer, especially malignant melanoma, is a disease with high mortality rate, therefore, the integral health care of patients with this disease is necessary. Treatment of malignant melanoma is guaranteed by SUS, through authorized hospitals, such as Assistance Center in Highly Complex Oncology Care (CACON) and High Complexity Assistance Units (Unacon) and also by not authorized hospitals. Among the main treatments, there are surgical and chemotherapy treatments which are paid to hospitals through the hospital admission authorization (AIH). New treatments are released on the market for malignant melanoma, but they are still not available in the public system, which leads patients with this malignant neoplasm to refer to the judiciary to have access to the drug, this process is called judicialization of health. Thus, this study aims to evaluate the legalization of ipilimumab and its financial impact on the treatment of malignant melanoma in Pernambuco, from January 2013 until December 2015. It was observed that the price of procedures paid to hospitals in Pernambuco was not ajusted in this period, since the minimum price was the same R$ 40,38 for three years. Two of the hospitals that have performed more of these procedures are not authorized as CACON / Unacon. Among the seven authorized hospitals, four hospitals have performed chemotherapy. Of the seventeen cases that have granted access to drug ipilimumab by the courts, it was detected that a percentage could not complete the recommended treatment in the medical prescription, the causes are death or replacement of the treatment by traditional chemotherapy. The ratio of cost of ipilimumab compared to the cost of the traditional chemotherapy procedure paid to hospitals by hospital admission authorization(AIH) can be 75,6 times higher. It was observed that in just one case the magistrate asked for the state manifestation, on the other cases it wasn’t granted defense to public administration to explain why the drug was not supplied. In two court lawsuits the precautionary measure was granted on the same day the case was assigned, although the average time between the process distribution and granting of the precautionary measure was 9.94 days, and the time between early relief and the supply of the drug was 4.11 days. The cost to purchase ipilimumab is higher with the judicialization process comparing to the cost by hospital admission authorization (AIH), both for the treatment of malignant melanoma. Most of the court lawsuits of judiciary do not request information from other health professionals, their decision is only based on medical prescription. Thus, it is necessary to estabilish a greater link between judiciary and executive so that judicialization process does not further encumber health budget.

Le traitement du mélanome métastatique est longtemps resté cantonné à quelques chimiothérapies cytotoxiques à efficacité limitée. Récemment, le développement des immunothérapies et des thérapies ciblées a constitué une avancée majeure dans la prise en charge du mélanome. La découverte de mutations oncogéniques sur les voies de signalisation de la cellule tumorale a permis de définir de nouvelles cibles pour le développement d’ inhibiteurs pharmacologiques. Parmi ces mutations, la mutation BRAF V600E est présente dans 50% des mélanomes. Une des molécules anti-BRAF développée est le vémurafenib. L’efficacité de cette molécule a permis l’allongement de la durée de survie des patients en situation métastatique de 6 à 8 mois. Malheureusement, malgré la réponse initiale, l’échappement est la règle. Les mécanismes moléculaires responsables de la résistance acquise aux inhibiteurs de BRAF sont multiples et conduisent à l’activation de nombreuses voies de signalisation pour suppléer l’inhibition de BRAF. Actuellement la prise en charge des patients résistants aux inhibiteurs de BRAF n’est pas codifiée et s’avère complexe étant donné la multiplicité des mécanismes d’échappement. Dans le cadre de cette nouvelle approche de médecine personnalisée, de nouveaux outils doivent être développés dans le but (i) de tester l’efficacité de nouvelles molécules et (i) de tenter de prédire l’efficacité/la résistance des différents traitements disponibles. L’un de ces outils sont les xénogreffes de tumeurs issues directement des patients (Patient Derived Tumor Xenograft ou PDTX) ou « avatars ». Mon travail a consisté à mettre en place ce type d’outil pour répondre aux questions suivantes : Les modèles animaux développés sont-ils un bon modèle préclinique translationnel ? Permettent-ils d’étudier les modes d’action des nouvelles thérapies ciblées et d’explorer leurs mécanismes de résistance ? Si tel est le cas, peuvent-ils prédire une réponse clinique ? Différents modèles murins de xénogreffes de mélanome ont été mis au point et exploités. Des cellules tumorales puis des fragments de tumeurs ont été greffées en sous-cutané chez la souris immunodéficiente SCID. Les tumeurs ont été caractérisées en histologie et en biologie moléculaire pour montrer leur stabilité dans le modèle. Après plusieurs passages dans différentes générations de souris, les tumeurs conservent leurs mutations particulières. Par ailleurs différents traitements ont été administrés et la réponse à différentes stratégies thérapeutiques a été évaluée. Ce modèle nous a permis de valider l’efficacité des thérapies ciblées antiBRAF (vémurafenib) mais aussi d’autres approches visant les particularités métaboliques du mélanome. Le modèle avatar a permis de tester in vivo une association thérapeutique innovante qui associe un accélérateur de la phosphorylation mitochondriale, le DCA, et une molécule pro-oxydante, l’elesclomol, confirmant l’effet synergique obtenu in vitro. Par ailleurs l’elesclomol a pu être testé in vivo sur une tumeur devenue résistante à l’inhibiteur de BRAF, le vemurafenib. En outre, le modèle avatar constitue un bon modèle préclinique permettant d’aider directement le clinicien dans ses choix thérapeutiques. En effet nous avons réalisé une PDTX tout au long de l’évolution métastatique d’une patiente. Cette PDTX a permis de tester l’efficacité du vemurafenib et de réintroduire ce traitement à la patiente qui a pu bénéficier d’une amélioration clinique notable. 7 autres PDTX de patients ont été développées permettant de renforcer le modèle et de suivre de manière personnalisée le traitement de ces patients.Grâce à ce travail, nous définissons la place que peuvent prendre les avatars dans la prise en charge actuelle du mélanome métastatique. Ces modèles donnent une réponse précoce quant à leur résistance et aux alternatives thérapeutiques de seconde ligne. / For a long time the standard treatment for metastatic melanoma has been confined to a few cytotoxic chemotherapies with limited efficacy. Recently, the development of immunotherapies and targeted therapies represents major headways in melanoma care management. The discovery of oncogenic mutations on the signaling pathways of the tumor cell allowed the definition of new targets for developing inhibitor drugs. Among these mutations, BRAF V600E is seen in 50% of all melanomas. Vemurafenib is one of the anti-BRAF molecules recently developed. It has shown its efficacy with metastatic patients showing a survival increased by 6 to 8 months. Unfortunately, in spite of the initial positive reaction, the development of a resistance mechanism is ineluctable. There are numerous molecular mechanisms responsible for the acquired resistance to BRAF inhibitors which activate numerous signaling pathways to make up for the BRAF inhibition. Today, there are no standards of care for patients who become resistant to BRAF inhibitors. In the framework of this new personalized care approach, new tools must be developed to (i) test the efficacy of new molecules and (ii) try to predict the efficacy/resistance of the various treatments available. One of these tools is the Patient Derived Tumor Xenograft or PDTX, i.e. xenografts from tumors directly harvested on patients.My work consisted in implementing this type of tool to try and bring an answer to the following questions: Are the developed animal models a good tool for preclinical cross-sectional studies? Can they help study the action modes of new targeted therapies and explore their resistance mechanisms? If this is the case, can they predict a clinical response? Various murine models of melanoma xenografts have been designed and used. Tumor cells and later on tumor fragments were grafted subcutaneously in immunosuppressed SCID mice. Tumors were characterized in histology and molecular biology to show the stability of the model. After several passages in various generations of mice, the tumors retain their specific mutations. Furthermore, various treatments have been administered and the answers to the various therapeutic strategies have been evaluated. This model allowed us to validate the efficacy of antiBRAF targeted therapies (vemurafenib) but also other approaches targeting the metabolic characteristics of melanoma. This avatar model enabled to test in vivo an innovating therapeutic combination, associating an accelerator of oxidative phosphorylation, DCA, to a pre-oxidative molecule, elesclomol, validating the synergic effect obtained in vitro. Furthermore, elesclomol was tested in vivo on a tumor which became resistant to BRAF, vemurafenib. Furthermore, PTDX is a good preclinical tool to guide clinicians in their therapeutic choices. In fact we were able to develop a PDTX during the entire metastatic progression of a patient. This PDTX enabled us to test the efficacy of vemurafenib and reintroduce the treatment to the patient, who showed noticeable clinical improvements. Seven other PDTX were developed in order to reinforce the model and follow, in a personalized manner, the treatment of these seven patients. Thanks to this work, we are able to define the role of avatars in the care management of metastatic melanoma. These models give an early answer regarding tumor resistance and alternative second-line therapies.

Cutaneous melanoma is a cancer of melanocytes, the pigment-producing cells in our skin. It is one of the most aggressive human malignancies, constituting only about 2% of all dermatological cancers but being responsible for over 75% of all deaths from skin cancer. It has recently become a major public health problem, as it is now the fifth most common cancer in the United Kingdom after its incidence more than quadrupled in the last three decades. For these reasons, understanding the biological processes that are involved in its development is of great importance for devising novel treatments and for the management of patients in the clinic. The study of the genetic factors that influence melanoma risk can uncover mechanisms that are relevant in the transition from a benign melanocyte to a malignant melanoma. Approximately 10% of all melanoma cases are familial, and about half of these familial cases can be explained by pathogenetic variants in genes such as cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase 4 (CDK4), breast cancer 2 (BRCA2), BRCA1-associated protein-1 (BAP1) and in the promoter of the telomerase reverse transcriptase (TERT). However, about 50% of all familial melanoma cannot be explained by mutations in known genes. In this dissertation, I detail the methodology I followed in an effort to uncover additional high-penetrance melanoma susceptibility genes. I analysed exome and genome sequence data from a total of 184 individuals that belong to 105 melanoma-prone families from the United Kingdom, The Netherlands and Australia that did not have any pathogenetic variants in known susceptibility genes. I applied different gene prioritisation strategies and developed novel software tools in order to devise a list of plausible melanoma susceptibility candidate genes; these analyses suggested that genes regulating telomere function could be influencing melanoma risk. After performing functional experimental analyses, our research team was able to determine that carriers of rare variants in the protection of telomeres (POT1) gene, a member of the shelterin complex that safeguards telomere integrity, are at high risk for developing melanoma. We successfully described the mechanism by which this happens, showing that the variants identified either disrupt POT1 mRNA splicing or abolish the ability of POT1 to bind to telomeres, and lead to increased telomere length in carriers when compared to melanoma cases with wild-type POT1. The main finding of the work described in this dissertation is the identification of telomere dysfunction as an important contributor to the risk of developing melanoma, and possibly other cancers. Our analyses suggest that POT1 is the second most commonly mutated high-penetrance melanoma susceptibility gene reported thus far, and moreover, that rare variants in this gene constitute the first hereditary mechanism for telomere lengthening in humans.

The purpose of this thesis was to investigate the knowledge and perceptions on the sun, risks of prolonged exposure, tanning and beauty of young Canadian women. Conversations with 15 young women from the University of Ottawa were tape-recorded, transcribed, and then analyzed using thematic analysis and theories on gender and beauty. The results were divided into two articles, the first exploring the perception and knowledge young Canadian women have about the sun, tanning and its risks. In the first article, the themes generated were (a) perceptions of benefits and risks of sun exposure; (b) outdoor versus Indoor tanning; (c) conformity; (d) conflicting and ambiguous messaging; (e) self risk and other’s risk and; (f) no UV index awareness. The second article explores how the fifteen interviewees make sense of the sun safety messaging they are exposed to, and how they act on it. The themes identified were: (a) tanning as a social activity; (b) beauty; (c) base tanning; and (d) wearing SPF and reapplication. The overall conclusion to be drawn from this study is that while for the most part the group of women I interviewed was well informed when it came to sun safety and tanning, they still felt the pressure to tan from peers, society and the media. There were times when they shared that they were misinformed on the risks of engaging in harmful tanning practices. Further, the study contributed to finding that the vast majority of the participants admitted to not checking the UV index before going outdoors, either because they did not understand it or because they felt it would not make a difference to their daily practices and behaviours. This contradicted previous literature that emphasized on the connection Canadians often made with the environment and UV index. Impacts, implications, and future research directions are discussed in both articles.

Class of 2006 Abstract / Objectives: To create, deliver, and evaluate a malignant melanoma continuing education (CE) seminar for pharmacists. Methods: A CE program was developed and presented to educate pharmacists about skin cancer prevention, specifically malignant melanoma, and their role in prevention through patient counseling. All practicing pharmacists who attended the CE program were asked to fill out a knowledge indicator and assess their comfort level in counseling patients about sun safety before and after the program. The participants also answered questions addressing how often they counsel patients on sunscreen use, their personal experience with skin cancer, preferred CE format, previous CE attendance, sex, age, practice site, hours worked per week, and years since graduation from pharmacy school. Results: The survey instrument was completed by 84 pharmacists. The average score on the pre-test knowledge indicator was 4.95 ± 0.39 and the average score on the post-test knowledge indicator was 7.81 ± 0.39. This was a significant improvement (p < 0.01). There was a significant increase in participant comfort level when counseling patients about sun safety after attending the CE program (p < 0.01). Personal experience with skin cancer did not have a significant effect on the pre-test knowledge indicator scores, however it was associated with the knowledge indicator change score (p < 0.01). Completion of previous skin cancer CE programs did not have a significant effect on the pre-test knowledge indicator score or the change score. Conclusions: Pharmacists who attended the CE program improved their knowledge indicator scores when tested about malignant melanoma and sun safety. Many participants felt more comfortable counseling patients about sun safety and felt their counseling on sun safety would change as a result of attending this CE program.

Dysregulation of the chemokine receptor 4 (CXCR4) and its primary ligand CXCL12 (SDF-1, stromal cell-derived factor-1), has been implicated in the progression of melanoma and the mechanisms by which the CXCR4/CXCL12 axis has been shown to activate cell cycle progression is via stimulation of the mitogen-activated protein kinase (MAPK) pathway. Given this, we sought to ascertain the potential cooperativity of CXCR4 with established histopathologic prognosticators including the BRAF status in primary cutaneous melanoma. In this IRB approved study, archived tissue samples with diagnosis of primary cutaneous melanoma were retrieved from the Skin Pathology Laboratory at BUSM, Boston, MA and a total of 107 cases identified as meeting criteria for inclusion. Protein expression of CXCR4 and CXCL12 were assessed using commercially available rabbit polyclonal antibodies (Ab2074 and, ab9797 respectively, Abcam, Cambridge, MA, USA). CXCR4 gene expression (mRNA) was measured by semiquantitative RT-PCR with appropriate controls. For IHC, a semi-quantitative scoring (ranging from 0-3) was used and cases with a score of ≥2 (>10%) were considered positive. Molecular analysis for CXCR4 gene expression and BRAF exon 15 mutation status was performed using mRNA semi-quantitative RT-PCR and DNA Sanger sequencing respectively. Univariate analyses of CXCR4 mRNA expression revealed a statistically significant correlation between elevated CXCR4 expression (low ΔCt value) and presence of the BRAF mutation and absence of a host response (p=0.03 and p=0.0003 respectively). Univariate analyses revealed a significant correlation between elevated CXCR4 mRNA (low ΔCt value) and the following: presence of BRAF mutation and absence of a host response (p=0.03 and 0.0003 respectively). CXCR4 mRNA was significantly higher among both AJCC stage 1 and stage 3 compared to stage 2 (p=0.01). Compared with CXCR4 negative samples, univariate analyses of CXCR4 protein showed that the proportion of CXCR4 positives was significantly greater in melanomas with absence of mitoses (p<0.0001), ulceration (p=0.0008) and regression (p=0.02). Patients presenting at shallower stages (AJCC 1-2) exhibited a larger proportion of CXCR4 positives (76.9%, p<0.0001 and 69.0%, p=0.004), while those at deeper stages (AJCC 3-4) exhibited a larger proportion of negatives (75.0%, p=0.002 and 66.7%, p=0.10). In a multivariable analysis, lower odds of CXCR4 protein expression were associated with AJCC stage-3 (OR=0.16, p=0.01), stage-4 (OR=0.17, p=0.04), and mitoses (OR=0.21, p=0.01). Lack of correlation between CXCR4 mRNA and protein expression suggests that further study is required for a more precise understanding of mRNA-protein interaction for CXCR4 in order to identify factors contributing to the lack of concordance. CXCR4 protein appears to be associated with established prognosticators of good clinical outcome as its expression is less frequently observed in melanomas with mitoses, ulceration and depth >2 mm. The association between CXCR4 mRNA and a brisk host response suggests that it may serve as a biomarker for recruiting melanoma patients for immunotherapy. Higher CXCR4 mRNA in patients with a BRAF mutation suggests its utility as a putative therapeutic target.

Targeting the mutant BRAF and immunotherapy are new approaches to the treatment of metastatic malignant melanoma that has significantly improved survival but is associated with significant toxicity and cost. Potent and specific BRAF inhibitors like vemurafenib and dabrafenib are superior to chemotherapy in treatment of BRAF mutant melanomas which represent nearly 50% of all melanomas. A less toxic approach to treatment of malignant melanoma is hence appealing. Delta-tocotrienol (DT3), an unsaturated vitamin E isoform, and simvastatin, an HMG-CoA reductase inhibitor have been shown to have anti-neoplastic properties. We studied the effects of these chemicals in both BRAF-mutated SK-MEL-28 and BRAF-wild type SK-MEL-2 melanoma cells. MTS assays were used to analyze cytotoxicity. SK-MEL-28 and SK-MEL-2 cells were cultured in MEM media containing 10% serum and plated in 96-well culture plates for 48 hours then treated with DT3 (0-80 µM), simvastatin (0-10 µM), or a combination and dosed again at 72 hours. SK-MEL-28 and SK-MEL-2 cells were grown in 60 mm plates and treated with DT3 at concentrations of 30 µM, simvastatin at concentrations of 10 µM and combination of DT3 and simvastatin at concentrations of 10 µM and 2 µM. Cell were lysed with RIPPA buffer with protease and phosphatase inhibitor after 6 hours of treatment. Protein concentration of cell lysates was measured spectrophotometrically (GLO Max Multi+, Promega), using a BCA protein assay kit. The samples were run in SDS PAGE and blotted onto nitrocellulose membranes. Membranes were incubated with antibodies against Hsp 70 (Enzo Life Sciences, Farmingdale, NY), Hsp 90 (Santa Cruz, Dallas, TX), pS6 and pERK (Cell Signaling, Danvers, MA) and pAKT. Using MTS assay, we found that DT3 (IC50 75.2 μM) and simvastatin (IC50 8.3μM) have cytotoxic effects on melanoma cell line SK-MEL-2, but not on the SK-MEL-28 cells DT3 and simvastatin at the concentrations studied (10-80 μM DT3) and (0.625- 10 μM simvastatin). Further studies determined that simvastatin decreased expression of pS6, pERK on SK-MEL-2 and not DT3. However, these effects are different in SK-MEL-28 cells where there is only decrease in expression of pS6; treated SK-MEL-2 cells also show over-expression of Hsp70 suggestive of a rescue effect leading to lesser cytotoxic activity. The selective cytotoxicity observed in wild type BRAF melanoma cell lines by DT3 and simvastatin warrants further research into the potential therapeutic use of these drugs. A differential cytotoxicity is shown by DT3 and simvastatin in malignant melanoma cells with selective more potency in wild type BRAF melanoma compared to mutant BRAF melanoma cells. Further studies will be undertaken to dissect the mechanistic basis of this differential response.