Made available in DSpace on 2015-08-28T02:03:46Z (GMT). No. of bitstreams: 1 000474248-Texto+Completo-0.pdf: 1288519 bytes, checksum: 76a0f632ccb7c577dfc183ba0e91a44b (MD5) Previous issue date: 2015 / Objectives: To evaluate the characteristics of cell nuclei of melanocytes using digital nuclear morphometric analysis. To evaluate the nuclear morphometric differences between nevi excised in winter or summer; demonstrating the presence of characteristics and irregularities in nuclear morphology caused by solar radiation on nevi. To consider whether there are morphological differences between common acquired nevi, dysplastic nevi and melanomas. To access the potential use of nuclear morphometry to detect cell senescence, apoptosis and other nuclear irregularities of nevi and melanomas in HE stained slides of paraffin embedded specimens.Methodology: Histopathological specimens were subjected to digital image analysis.Results: Through this pilot study, glass slides obtained from biopsies of 30 patients were analyzed; 10 nevi excised in winter, 5 nevi excised in summer, 9 dysplastic nevi and 6 melanomas. Between winter and summer nevi; secondary variables able to perform such a separation were 'variability Area' and % of nuclei with NII higher than 1SD.Conclusion: There are differences of nuclear morphometric characteristics of excised nevi in winter and summer and can be caused by cumulative effects of solar radiation on nevi. There are morphometric differences between common acquired nevi, dysplastic nevi and melanomas. The dysplastic nevi and melanomas were separated by the average area and percentage of nuclei with area above one standard deviation of the mean area. / Objetivos: Avaliar as características dos núcleos celulares de melanócitos através de análise morfométrica nuclear digital e as diferenças morfométricas nucleares entre nevos excisados no inverno e nevos excisados após o verão; para detectar possíveis características e irregularidades na morfometria nuclear provocadas pela radiação solar sobre nevos. Averiguar a possibilidade de identificação de diferenças morfométricas entre nevos adquiridos comuns, nevos displásicos e melanomas e avaliar a possibilidade de detecção de senescência, apoptose e outras irregularidades nucleares em nevos e melanomas por morfometria digital em cortes histológicos corado por HE a partir de blocos de parafina.Metodologia: Os espécimes histopatológicos foram submetidos à análise de imagem digital realizada através de ferramentas de avaliação nuclear digital.Resultados: Neste estudo piloto foram analisados os espécimes de biópsia de 30 pacientes, compostos por: 10 nevos biopsiados no inverno, 5 nevos no verão, 9 nevos displásicos e 6 melanomas malignos. Nos nevos de inverno e nevos de verão; as variáveis capazes de realizar tal separação foram ‘Variabilidade da Área’ e ‘porcentagem de núcleos com NII acima do valor da média+1DP.Conclusão: Foram detectadas diferenças morfométricas nucleares entre nevos excisados no inverno e nevos excisados após o verão, que podem ser provocadas pelos efeitos cumulativos da radiação solar sobre nevos. Foram catacterizadas diferenças morfométricas entre nevos adquiridos comuns, nevos displásicos e melanomas. Os nevos displásicos e melanomas foram separados pelas características de área média e porcentagem de núcleos com área acima de um desvio padrão da média.

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Malignant melanoma results in 132,000 cases worldwide each year with an incidence rate that is increasing faster than for any other skin cancer. In the UK, cutaneous melanoma is the sixth most commonly diagnosed cancer and the second most common in young people aged 15-34 (excluding non-melanoma skin cancer). Furthermore, while less common than NMSC, malignant melanoma accounts for 4% of skin cancer cases and 74% of skin cancer-related deaths. Although early surgical removal of primary tumours is an effective treatment, patients that develop metastatic melanoma have a very poor prognosis (5 year survival rate is only 5%). Elevated expression of a number of DNA repair genes has been reported in primary melanomas that subsequently metastasised when compared to non-recurrent primary tumours. In addition, patients who do not respond to chemotherapy have elevated expression of DNA repair genes. One chemotherapeutic that is effective against a range of other cancers, but not melanoma is cisplatin. Elevated levels of the DNA repair protein ERCC1, which is needed to remove cisplatin-induced DNA damage, has been found to be an indicator of poor prognosis in ovarian and lung cancer. To test our hypothesis that elevated ERCC1 levels account for an increased resistance to cisplatin in melanoma, a xenograft experiment was performed. Our results show that ERCC1 proficient melanoma xenografts initially responded to cisplatin treatment however resistance soon followed. Tumours deficient in ERCC1 however could be cured after only two treatments of cisplatin, indicating a novel method to overcome chemoresistance in metastatic melanoma. The aim of the project was to identify novel compounds to improve therapy of melanoma. To achieve this, in collaboration with Dr Patton we performed a cell culture screen to identify compounds which display specificity against melanoma cell lines. In addition, we sought to identify compounds which would overcome cisplatin resistance. We identified a series of nitrofuran compounds which are potent against melanoma and neuroblastoma cell lines and enhanced the toxicity of cisplatin through an ERCC1 independent pathway. In addition, we showed that melanin pigmentation is protective against nitrofuran toxicity. We have proposed the structure specific endonuclease, ERCC1-XPF, as a drug target to overcome chemoresistance. We collaborated with Professor Walkinshaw to perform an in silico screen for protein-protein interaction inhibitors to disrupt the obligate dimerization between ERCC1 and XPF. In addition we directly inhibited the endonuclease activity by developing XPF endonuclease domain inhibitors and utilised a range of biochemical, molecular biology and cell culture assays to validate ERCC1-XPF inhibitors. Furthermore, we developed an in vitro endonuclease assay for ERCC1-XPF, FEN1 and DNase1 and utilised these to demonstrate compound specificity of our validated ERCC1-XPF inhibitors. In collaboration with MRC Technology we utilised the ERCC1-XPF endonuclease assay to perform a high throughput screen. We characterised hit compounds to demonstrate physical binding and in vitro specificity for ERCC1-XPF. In conclusion, we have discovered new compounds which may prove beneficial for the treatment of malignant melanoma.

MITF is the master melanocyte transcription factor and has a complex role in melanoma. Both gain- and loss-of function mutations in MITF have been identified in melanoma, although its’ role in melanoma development and the effects of targeting MITF are unknown. Using a temperature-sensitive mitf zebrafish mutant I show that low levels of MITF are oncogenic with BRAFV600E in melanoma progression. By pathology and MITF target gene expression, BRAFV600Emitfavc7 tumours are distinct from BRAFV600Ep53M214K tumours, and represent two melanoma subtypes. Melanomagenesis can also be driven independently of BRAFV600E, in a transgenic zebrafish with mutations in mitf and p53, representing a new melanoma model. Abrogating MITF activity in BRAFV600Emitfavc7 melanoma leads to regression of the tumour, characterised by macrophage infiltration and increased apoptosis. This result confirms the dependence on MITF activity in BRAFV600Emitfavc7 melanomas and highlights the role of MITF as a therapeutic target for melanoma. Exome and transcriptome sequencing has been carried out to gain insight into the expression and genomic mutational landscape that is driven by these melanoma transgenic models and results in these genotype-phenotype specific subtypes observed.

Class of 2016 Abstract / Objectives: To test whether ipilimumab therapy affects the endocrine system in patients with Metastatic Melanoma. Methods: A retrospective chart review was performed that included patients with Metastatic Melanoma that had at least one dose of ipilimumab. Results: The primary finding of this study is that 38% of patients used at least 20 mg of prednisone daily or more while on Ipilimumab therapy. Only 33% of patients had endocrine lab values reported. Conclusions: There was not enough data collected to adequately show that Ipilimumab affects the endocrine system. There was also insufficient reporting of appropriate serum levels. More research on the importance of reporting lab values while on ipilimumab therapy needs to be conducted.

This thesis examined the effect and biochemical mechanism by which combined vitamin E and C supplementation may influence tumour cell growth. The study initially addressed the effect of combined vitamin E succinate and Asc supplementation over a nutritional concentration range (5- 20μg/ml) and (25-50μg/ml) respectively, on the in vitro growth of non-malignant LLCMK and malignant BL6 cells. Supplementation of BL6 and LLCMK cells with combined vitamin E succinate and ascorbic acid, resulted in no significant increasing or decreasing trend in LLCMK cell growth, while in BL6 cells a significant decrease in cell growth was observed at all combined vitamin concentrations. It has been suggested that these vitamins may act synergistically to inhibit tumour cell growth through their antioxidant properties in quenching free radicals and lipid peroxidation and furthermore through their modulation of the activities of various enzymes and metabolites in the eicosanoid pathway. This study consequently investigated the effects of combined vitamin E succinate and ascorbic acid supplementation on these parameters. Throughout this study, emphasis was placed on the BL6 melanoma cells, as combined vitamin E succinate and ascorbic acid supplementation did not significantly affect growth or levels of secondary metabolites in the non-malignant LLCMK cells. Combined vitamin E succinate and ascorbic acid supplementation of BL6 cells resulted in a marked but non significant increase in free radical and a significant increase in lipid peroxidation levels. This prooxidant effect was accompanied by a significant decrease in BL6 cell growth, suggesting that the growth inhibitory effects of combined vitainin E succinate and ascorbic acid on BL6 cells in vitro was not mediated through their synergistic antioxidant properties. Vitamin E succinate is a nonphysiological antioxidant in its esterified form, hence cleavage of the succinate group must occur in order for ascorbic acid to interact with the free alcohol, vitamin E. The inability of combined vitamin E succinate and ascorbic acid to reduce free radicals and lipid peroxidation levels within BL6 cells may not be due to their ineffectiveness as antioxidants but rather the presence of other contributing factors which influence the oxidation state within the BL6 cells. Vitamin E is believed to modulate membrane-bound enzymes through membrane stabilization. Furthermore, the stabilizing effect of vitamin E may be enhanced by the ascorbic acid-sparing effect of vitamin E. Hence, this study investigated the effect of combined vitamin E succinate and ascorbic acid in modulating the activity of various enzymes and secondary messengers in the eicosanoid pathway. Supplementation with combined vitamin E succinate (5-20μg/ml) and ascorbic acid (25-50μg/ml) resulted in significant increases in phospholipase A₂, 5-lipoxygenase, cyclooxygenase and adenyl ate cyclase activity, with a significant decrease in BL6 cell growth. The possible synergistic action of these vitamins in terms of modulating membrane-bound enzymes was further substantiated by uptake and cellular distribution studies. Vitamin E succinate and vitamin E in the membrane fraction increased significantly compared to control cultures, while ascorbic acid levels were significantly higher in the stroma fraction when compared to membrane fractions. Consequently, another factor accounting for increased activities of phospholipase A2, 5-lipoxygenase and adenylate cyclase activities as a result of vitamin supplementation in BL6 cells may be an increased availability of Ca²+. Supplementation of BL6 cells with combined vitamin E succinate and ascorbic acid resulted in significant increases in intracellular Ca²+ levels at all combined vitamin groups. Furthermore, this increase in intracellular Ca²+ was positively correlated with cl1anges of the above-mentioned enzyme activities. Within the eicosanoid pathway, the rate of prostaglandin synthesis is regulated by phospholipase A₂ activity and arachidonic acid release, and the net prostaglandin production is dependent on cyclooxygenase activity, hence the effects of combined vitamin E succinate and ascorbic acid on arachidonic acid composition and prostaglandin production within BL6 cells was determined. The percentage arachidonic acid composition of the BL6 cells was elevated and inversely related to cell growth following combined vitamin E succinate and ascorbic acid supplementation. Prostaglandin E₂ and prostaglandin I₂ levels increased significantly, while those of prostaglandin D2 and prostaglandin F₂α increased markedly following supplementation of combined vitamin E succinate and ascorbic acid. These increases in prostaglandin levels were inversely related to BL6 cell growth, suggesting that the prostaglandins were involved in negative regulation of BL6 cell growth. When comparing the levels of prostaglandins, prostaglandin E2 levels were significantly higher when compared to prostaglandin D₂, prostaglandin F₂α and prostaglandin I₂ suggesting that vitamin E₂ succinate and ascorbic acid effects were mediated primarily through an increase in prostaglandin E2. Hence, prostaglandin E2 levels in combined vitamin E succinate and ascorbic acid appeared to be dependent on the amount of precursor present and the activity of its synthetic enzymes. This was confirmed when BL6 cells were supplemented with arachidonic acid. Arachidonic acid had an inhibitory effect on BL6 cell growth and also stimulated prostaglandin E₂ production. Prostaglandin E₂ levels are in turn believed to modulate adenylate cyclase activity in BL6 cells, hence it is reasonable to conclude that adenylate cyclase activity is dependent on prostaglandin E₂ levels. Combined vitamin E succinate and Asc supplementation to BL6 cells resulted in significant increases in adenyl ate cyclase and cyclic adenosine monophosphate, which again correlated with a significant decrease in cell growth. As cyclic adenosine monophosphate has a regulatory role in the cell cycle this study suggested that the effect of combined vitamin E succinate and ascorbic acid supplementation was mediated through the final effect provided by the second messenger, cyclic adenosine monophosphate. This was confirmed when BL6 cells were supplemented with dexamethasone, a phospholipase A₂ inhibitor. This treatment rsulted in combined vitamin E succinate and ascorbic acid having no inhibitory effect on BL6 cell growth. Cyclooxygenase activity, prostaglandin E₂ levels, adenylate cyclase activity and cyclic adenosine monophosphate levels were significantly lower in dexamethasone-treated cells compared to non-treated dexamethasone cultures. The reason for the increased free radical and lipid peroxidation levels in BL6 cells was further investigated. Cyclooxygenase enzymes are believed to generate free radical species during catalytic activity. Analysis of free radical and lipid peroxidation levels following supplementation with dexamethasone revealed markedly lower free radical and significantly lower lipid peroxidation levels in comparison with control cultures and non dexamethasone-treated cultures. These results suggest that the observed increases in free radical and lipid peroxidation levels in BL6 cells supplemented with combined vitamin E succinate and ascorbic acid were indirectly due to the increase in cyclooxygenase activity in these cells.

This thesis examines the effect of combined vitamin E succinate and Asc supplementation on the in vitro growth of a non-malignant monkey kidney (LLCMK) and a malignant melanoma (BL6) cell line, with nutritional concentration ranges of 5-20µg/ml and 25-50µg/ml respectively. Vitamin E and C are thought to interact synergistically to inhibit tumour cell growth by virtue of their antioxidant properties, whereby they quench free radicals and terminate lipid peroxidation. Furthermore vitamin E and C are thought to modulate the biosynthetic pathways in arachidonic acid metabolism at a number of different points. This may also offer a means of regulating tumour cell growth. It is well documented that vitamin E and C are distributed in the lipid and aqueous phases in the cell respectively. However, the cells need to obtain the vitamins from the environment in which they are found in order to exert a growth inhibitory effect. Supplementation of combined vitamin E succinate and Asc on BL6 and LLCMK cells resulted in a significant increase in LLCMK cell growth, and a significant decrease in cell growth was observed in BL6 cells. Vitamin E succinate in its esterified form cannot function as an antioxidant and requires the cleavage of the succinate to become an active antioxidant. The metabolism of vitamin E succinate to form free vitamin E in LLCMK and BL6 cells resulted in the cleavage of the succinate group from the vitamin E molecule in BL6 cells only, thus suggesting that an esterase may be present in BL6 cells. This would allow for a synergistic interaction between the two vitamins. The arachidonic acid cascade generates a family of bioactive lipids that modulate diverse physiological and pathological responses including tumour growth and promotion. The enzyme prostaglandin endoperoxide synthase (PGHS) or cyclooxygenase (Cox) is the key enzyme in the biosynthetic pathway leading to the formation of prostaglandins. Two enzyme isoforms of Cox have been identified, Cox 1 and Cox 2. Supplementation with vitamin E succinate and Asc at a combination 20:25µg/ml respectively resulted in a trend of increasing Cox activity over 12 hours suggesting that vitamin E and Asc have a stimulatory effect on Cox activity in BL6 cells. The inhibitors of Cox 2, dexamethasone, showed a decreasing trend in Cox activity at the 20:25µg/ml combination, while cycloheximide showed an initial stimulatory effect and then a gradual decrease in Cox activity. The elimination of the Cox activity by dexamethasone suggests that transcriptional regulation may be occurring in BL6 cells. We examined by Northern blot analysis whether combined supplementation of vitamin E succinate and Asc caused an elevation of Cox 2 RNA expression in BL6 cells. An inducible effect of Cox 2 was observed after 2 hours of supplementation with a combination of vitamin E succinate and Asc in BL6 cells, however the results are inconclusive and further studies are required to substantiate this finding.

Class of 2013 Abstract / Specific Aims: The purpose of this project was to determine inpatient charges, as well as define the frequency and mortality associated with the various sites of melanoma metastasis. Methods: Data was taken from the national database Agency for Healthcare Research and Quality (AHRQ) Healthcare Cost and Utilization Project (H-CUP) Nationwide Inpatient Sample (NIS) and was collected on patients admitted into hospital with any diagnosis of melanoma with disease progression of distant metastasis. Logistic multivariate regression was used to find odds ration by patient characteristic. Overall charges were assessed using a gamma multivariant regression. Multiariant regression was used to determine other patient demographics. Main Results: Average inpatient charges for stage IV melanoma was $32,296 per patient with a national inpatient total bill of $5.56 billion. The metastatic sites associated with the highest inpatient charges were genitourinary tract (exp B = 1.276), gastrointestinal tract (exp B=1.146), bone (exp B=1.132), lung (exp B=1.097), and lymph (exp b=1.092). The most common sites of melanoma dissemination for in-patient mortality cases were lymph (21.7%), lung and respiratory (19.2%), central nervous system (17.1%), and bone (17.1%). Conclusion: The annual average hospital charges per patient for melanoma with distant metastasis is about $32,000. We suggest that metastases of the genitourinary tract, gastrointestinal tract, bone, lung, and lymphatic system are associated with the highest hospital charges, while metastases to the CNS, bone, liver, lung, GI, and wide dissemination are associated with increased mortality.

M.Tech. / Photodynamic therapy is a treatment that is used for the destruction of certain types of tumours and is emerging as a promising treatment modality in the field of dermatology (Davids et al., 2008). The photochemical interactions of the photosensitizer, light and molecular oxygen producing reactive oxygen species known as ROS, results in damage to organelles within malignant cells and so can lead to tumour destruction (Plaetzer et al., 2008). Melanoma is one of the most common forms of malignancies (Oliveria et al., 2007). Unfortunately, there are limited treatment options available for this disease because chemotherapy and radiation therapy are largely ineffective. Metastatic disease frequently develops even after potentially curative surgery (MacCormack, 2008). Since this metastatic disease is an understudied cancer, and the incidence and mortality is increasing, describing the long term burden of this cancer and identifying factors that contribute to it will facilitate efforts to develop responsive prevention strategies, so that novel therapies such as PDT can be proposed (Oliveria et al., 2007; Pan et al., 2008; Schuitmaker et al., 1996). Numerous worldwide clinical trials have shown that PDT represents an effective and safe modality for various skin disorders, but little research has been done in terms of its effect on malignant melanomas (De Rosa and Bentley, 2000; Kolarova et al., 2008). In order for PDT to be an effective treatment modality it depends on many factors such as the type of photosensitizer utilized its ability to selectively penetrate tumour cells and the duration of the treatment (Robertson et al., 2009). Other important factors include the type of activating light source, its ability to penetrate the desired target and the duration of exposure (Plaetzer et al., 2008). Lastly, the type of target cells and their oxygen status also play an important role in the efficacy iv of PDT (Kolarova et al., 2008). In order for PDT to be completely effective, the resulting damage from the treatment must surpass cellular repair mechanisms and cause direct destruction of cellular pathways through vascular compromise and increase immune response to overcome disease (Pazos and Nader, 2007). Porphyrins are the most studied photosensitizers and their disadvantage is the inability to specifically localize in tumour cells and so they are retained in normal cells for prolonged periods, causing patients to be photosensitive (Braathen et al., 2007). This factor stimulated the development of second generation photosensitizers with improved physical, chemical and spectral properties (Davids et al., 2008). Phthalocyanine compounds are second generation photosensitizers which have shown potential in the PDT treatment of many cancers (Kolarova et al., 2007).

We report a case of malignant melanoma (MM) metastatic to the parotid gland, initially discovered on fine needle aspiration (FNA). The patient presented with a mass in the parotid gland area with previous history only significant for prostatic carcinoma. The initial FNA impression was melanoma. The smears were hypercellular with bloody necrotic background. The cells were epithelioid with mild nuclear atypia. Discrete cytoplasmic pigmentation was seen. No lymphoglandular bodies were noticed. Fragments of benign salivary gland were also identified. The cytological diagnosis of MM triggered onsite thorough physical examination for potential primary, where a scalp pigmented lesion was discovered hidden by overlying covering hair. Our differential diagnosis included melanoma, metastatic carcinoma, and lymphoma. Further work up for melanoma with S100, HMB45, and Mart 1 confirmed our top differential diagnosis. We emphasize thorough physical examination in such circumstances, and the importance of onsite evaluation guiding clinicians looking for primary.