No description available.

Non-melanoma skin (NMSC) cancer is the most common human malignancy. Cutaneous squamous cell carcinoma (cSCC) and its precursor, actinic keratosis (AK) affect tens of thousands of people each year in the UK. Merkel cell carcinoma is a rare, yet aggressive type of NMSC recently linked with Merkel Cell Polyomavirus (MCPyV). In spite of the clinical burden of NMSC, key molecular regulatory patterns remain largely unknown. The aims of this thesis were to investigate genome-wide genetic, epigenetic and transcriptional changes in AK and cSCC, and assess the prevalence of MCPyV and its effect on methylation in NMSC. Copy-number analysis revealed that AK harbours significantly more genomic aberrations compared to skin, the majority of which occurs on chromosomes 8 and 9. Transcriptional profiling has found 292 and 308 genes as differentially expressed in AK compared to non-sunexposed and sun-exposed skin, respectively, and gene-set enrichment analysis (GSEA) revealed dysregulation of PPAR pathway in this lesion. Expression profiling of cSCC and AK has revealed 346 differentially expressed genes, and GSEA detected dysregulation in several canonical pathways including TGF-β and MAPK pathway. Aberrant methylation in cSCC cell lines occurs in the promoters of many developmental genes. A total of 1085 hyper- and 833 hypomethylated genes were detected in cSCCs, and GSEA revealed dysregulation of critical signalling pathways (WNT, MAPK signalling pathways). Methylation analysis of AK revealed a total of 4194 differentially methylated genes, and implicated FOXF2, PITX2, RUNX1 and SMAD3 transcription factors in this lesions. MiRNA profiling of cSCC and normal skin revealed significant dysregulation of 38 miRNAs including several of viral origin. MCPyV was shown to be common in NMSC, yet MCPyV nor human papillomavirus does not affect cSCC methylation. Taken together, this work provides novel insight into molecular regulation of cSCC oncogenesis, and identifies potential epigenetic targets for functional evaluation in this malignancy.

Indiana University-Purdue University Indianapolis (IUPUI) / Purpose. In the US, melanoma and non-melanoma skin cancer (NMSC) rates have increased substantially in recent decades. While many skin cancer risk factors have been established, the impact of dietary citrus, which is naturally abundant in photocarcinogenic psoralens, remains enigmatic. The purpose of this research was to investigate associations between citrus consumption and risks of melanoma and NMSC, and to conduct a genome-wide study to identify genetic variants that may modify this association. Methods. Participants from the UK Biobank were leveraged for these analyses. Citrus consumption was collected via five rounds of 24-hour recall questionnaires, with complete citrus data available for n=210,126 participants. Ascertainment of melanoma and NMSC cases were identified by international classification of disease codes via linkage with national registries. Logistic regression was used to estimate odds ratios and 95% confidence intervals for the associations between citrus consumption and skin cancer outcomes. Individual citrus products were assessed for independent associations with skin cancer risk, and established skin cancer risk factors were tested for interaction. Joint 2-degree-of-freedom (df) and 1-df tests were used to assess interaction between total citrus consumption and genetic variants. Results. After controlling for covariates, high total citrus consumption was significantly associated with increased melanoma risk, an association primarily driven by orange and orange juice consumption. Skin color was found to be a significant effect modifier for the association between total citrus consumption and melanoma risk, but only before adjusting for multiple comparisons. No significant associations were observed for high total citrus consumption or consumption of any individual citrus products and NMSC risk. Significant associations for half a serving of citrus consumption and NMSC risk were likely due to chance or confounding. Index SNPs on chromosomes 3, 9, and 16 were significant according to the joint 2-df test, and 7 SNPs on chromosome 16 displayed evidence of a citrus-gene interaction. Conclusion. My analyses provide evidence in support of high citrus consumption significantly increasing risk of melanoma, but not NMSC. I also identified SNPs on AFG3L1P that may modify this association. Future research should further explore these associations, particularly for NMSC and to confirm my genetic findings.

No description available.

Non-melanoma skin cancer (NMSC) is the most common cancer in the world with a lifetime risk for development as high as 2 in 3 in Queensland, Australia. Mortality is quite low, representing an approximate 360 deaths in Australia annually but cost of treatment is extremely high, estimated at $232 million each year. Squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) are the two most common forms of NMSC. Although BCC generally do not have the propensity to metastasise, they are highly invasive and can be locally destructive. SCC on the other hand is invasive and has metastatic potential. SCC is generally derived from a precursor lesion, solar keratosis (SK), which is also considered to be a biomarker of BCC, SCC and malignant melanoma. According to one theory, SKs actually represent the first recognisable stage of SCC development and therefore may be indicative of the earliest stage of NMSC development. In addition to these common forms of NMSC, rarer forms such as keratoacanthoma (KA), which spontaneously regress, and SCC in situ, which rarely become invasive, may provide clues into protective mechanisms associated with prevention of development. Like all other cancers, NMSC arises from an accumulation of genetic abnormalities that result in severe cellular dysfunction. A number of genes have been proposed in the development of NMSC, including p53, CDKN2a, Bcl-2 and the Ras family of genes, which are typically associated with proliferative and differentiation processes. Also, a number of genetic disorders that predispose individuals to NMSC have also been identified. Genetic abnormalities in these genes may be a result of somatic mutations that may be promoted by environmental carcinogens. For NMSC, ultraviolet (UV) radiation is the primary environmental stimulus that acts upon skin to generate mutations. UV effects are 2-fold; the first being direct damage produced by UVB radiation and the second being indirect damage as a result of UVA-induced oxidative stress. In addition to mutations of genes that directly result in carcinogenesis, polymorphic variants of genes may also play a role in susceptibility to NMSC. These susceptibility genes may have immunogenic, detoxifying or transcriptional roles that could be involved in increased mutagenesis or activation of cancer causing genes. The purpose of this study was ultimately to identify further molecular based mechanisms associated with the development of non-melanoma skin cancer. Initially, this study aimed to examine the effects of aberrant chromosomal regions on NMSC development and also to identify candidate genes within these regions that may be implicated in the development and progression of NMSC. Also, based on chromosomal and functional implications, a number of candidate genes were assessed using association analysis to determine their involvement in susceptibility to the earliest stages of NMSC development. Implicated susceptibility genes were then further investigated to determine their response to UV radiation. Therefore the methodological approach of these studies was based on three broad technical applications of cytogenetic, association and expression analyses. Previous comparative genomic hybridisation (CGH) studies implicated the 18q chromosomal region in progression of SK to SCC and this region was therefore suspected of harbouring one or more tumour suppressor genes that were associated with a more malignant phenotype. Following on from this analysis, loss of heterozygosity (LOH) analysis was used for further delineation of this region and possibly to implicate candidate genes involved in progression. Additionally, CGH was used to investigate keratoacanthoma to determine aberrant regions that might be involved in progression and also regression of this NMSC. Genes that had potential functional roles in NMSC development and that were located in or near regions implicated by these cytogenetic analyses were further investigated using association analysis. Association analysis was performed using polymerase chain reaction and subsequent restriction enzyme digestion or GeneScan analysis to determine genotype and allele frequencies in an SK affected versus control population for polymorphisms within a number of candidate genes. This population was carefully phenotyped so that not only genotypic factors could be analysed but also their interaction with a number of phenotypic and environmental risk factors. Genes with polymorphisms that did show association with solar keratosis development were then examined functionally. Specifically, gene expression analysis was undertaken to investigate their response to UV radiation. Both UVA only and combined UVA/UVB treatments were used for short term irradiation and also for long term irradiation with recovery to determine differential effects of UV range and dose in human skin. Relative mRNA expression analysis of these genes was performed using quantitative real time reverse transcription polymerase chain reaction to determine if UV radiation imposed gene expression changes in the skin. A combination of these methodologies provided a wide basis for investigation of NMSC. Cytogenetic, association and expression analyses all allow for the identification of molecular risk factors that cause or are associated with NMSC development and progression. These analyses provided diverse results that implicated various molecular mechanisms in the development of NMSC. Cytogenetic analysis is a powerful technique, especially for the identification of a broad range of aberrations throughout the genome. This study employed LOH analysis to investigate an implicated region involved in progression to SCC and to attempt identification of candidate genes that may be involved in this process. LOH analysis was successfully performed on 9 SCCs, 5 SCCs in situ and 2 SKs using 8 microsatellite markers within the 18q region. Polymerase chain reaction (PCR) was used to amplify polymorphic regions of these markers and genotypic composition was determined for normal and cancerous tissue within the specimen. In heterozygote individuals, determined by analysis of normal tissue, the cancerous tissue was examined to determine if alleles within the implicated region had been lost. However, after analysis of multiple different samples, there was no LOH detected in any of the samples examined for this analysis. This does not necessarily reject a role for 18q, or genes within this region, as the localisation of candidate tumour suppressor genes within a small region may indicate a tighter region of involvement than was expected. As such, a more targeted study may further delineate this region and implicate candidate genes in progression of SK to the more malignant phenotype of SCC. Further CGH analysis of keratoacanthoma was also undertaken to identify aberrations associated with development and also regression of this skin cancer. CGH was performed using universal amplification and nick translation to incorporate a fluorescent dye. Differentially labelled normal and tumour DNA were then competitively hybridised to a normal metaphase spread and fluorescence emission indicated either amplification or deletion of specific chromosomal regions. In total, 6 KA samples were analysed, with 2 samples each from evolving, matured and regressing stages of KA development. In general, regressing KAs appeared to be more highly associated with deleted regions than evolving and matured KAs. Specifically, the 15q chromosomal region that was deleted in regressing KAs but amplified in evolving or matured KAs, may be significantly involved in the process of KA regression. Also various candidate genes that were being considered for analysis were located in or near some of these implicated regions, including GSTM1, GSTP1 and SSTR2. As such, these candidate genes were targeted for further investigation. A number of susceptibility genes that were located in or near aberrant regions implicated in NMSC development were investigated using association analysis. These genes included members of the somatostatin receptor family (SSTR1 and SSTR2), members of the glutathione-S-transferase (GST) family (GSTM1, GSTT1, GSTP1 and GSTZ1) and the vitamin D receptor (VDR). Studies detected a number of interesting interactions between genetic, environmental and phenotypic factors in the development of the early stages of non-melanoma skin cancer. Additionally, genes implicated in NMSC development were further investigated using expression analysis to determine response to UV radiation. Association analysis was initially performed on members of the somatostatin receptor family. Somatostatin is a growth inhibiting factor, amongst other things, that mediates its actions through the somatostatin receptors (SSTRs). The presence of these receptors (SSTR1-5) in tumour cells indicates a potential for somatostatin to bind and suppress growth, as well as allowing for therapeutic treatment with somatostatin analogues. Additionally, expression of these receptors in normal tissue, including skin, should allow for potential protection against tumour growth. The genes for SSTR1 and SSTR2 have been shown to contain dinucleotide repeat polymorphisms, and although these polymorphisms may not directly result in altered expression or binding potential, they may be linked to another functional polymorphism that does. Using association analysis the SSTR1 and SSTR2 genes were investigated to determine whether they play a role in the development of solar keratosis. Results showed that there were no significant differences between SSTR1 and SSTR2 polymorphism frequencies in the tested solar keratosis population (P = 0.10 and P = 0.883, respectively) as compared to an unaffected population. Hence, these studies do not support a role for the SSTR1 or SSTR2 genes in solar keratosis development. Further association analysis and subsequent expression analysis was also performed on members of the glutathione-S-transferase family. The GST enzymes play a role in the detoxification of a number of carcinogens and mutagens, including those produced by UV-induced oxidative stress. This study examined the role of GSTM1, GSTT1, GSTP1 and GSTZ1 gene polymorphisms in susceptibility to SK development. Association analysis was performed to detect allele and genotype frequency differences in SK affected and control populations using PCR and restriction enzyme digestion. No significant differences were detected in GSTP1 and GSTZ1 allele or genotype frequencies, however polymorphisms within both genes were found to be in linkage disequilibrium, as previously reported, and a new allelic variant of the GSTZ1 gene was identified. Significant associations between GSTM1 (P = 0.003) and GSTT1 (P = 0.039) genotypes and SK development were detected, with the null variants of both genes conferring an approximate 2-fold increase in risk for solar keratosis development (OR: 2.1; CI: 1.3-3.5 and OR: 2.3; CI: 1.0-5.0 for GSTM1 and GSTT1, respectively). For the GSTM1 gene, this risk was significantly higher in conjunction with high outdoor exposure (OR: 3.4; CI: 1.9-6.3) and although the GSTT1 gene showed a similar trend (OR: 2.9; CI: 1.1-7.7), this did not reach significance. The increased risk of SK development associated with these genes is likely due to a decreased ability of the skin to detoxify mutagenic compounds produced by UV-induced oxidative stress, and hence a decreased ability to protect against carcinogenesis. Implication of the GSTM1 and GSTT1 null variants in solar keratosis development prompted interest in analysis of gene expression changes in response to UV radiation. Due to the high homology of the GSTM1 gene with other GSTM genes, and therefore potential issues with primer specificity, the GSTT1 gene was focussed on for the expression studies. Real time reverse transcription PCR, incorporating SYBR green fluorescence and 18S as a comparative gene, was used to study GSTT1 gene expression changes in response to both UVA and combined UVA/UVB radiation. It was found that only short term UV radiation had an effect on GSTT1 expression changes, whereas no alteration of gene expression was seen after 4 and 12 hours of recovery from long term irradiation between irradiated and matched non-irradiated skin samples. This indicated that changes in gene expression for the GSTT1 gene apparently occur relatively quickly after exposure to UV radiation. Analysis of both UVA only and combined UVA/UVB short term irradiation indicated that an initial decrease in expression, followed by an increase was likely to represent translation into protein and subsequent transcription of mRNA, and in some cases a second decrease indicated further translation. Hence, it appears as though UV radiation does have a significant effect on the expression of at least one GST gene, and that UV radiation in combination with genetic variation of these genes may play a role in the development of NMSC. Finally, association and subsequent expression analysis was also performed on the vitamin D receptor. The hormonal form of vitamin D, 1a25 dihydroxyvitamin D3, has been shown to have numerous cancer-related effects, including antiproliferative, differentiation, proapoptotic and antiangiogenic effects. These effects are mediated through the binding of 1a25 dihydroxyvitamin D3 to the vitamin D receptor and subsequent transcriptional pathways. Polymorphisms within the VDR are known to regulate its transcription and therefore expression, which is linked to the ability of 1a25 dihydroxyvitamin D3 to bind. Association analysis of a 5’ initiation codon variant (Fok I) and two 3’ variants (Apa I and Taq I) was performed in SK affected and control populations. Although the Fok I variant showed no association with SK development, both the Apa I and Taq I variants were found to be associated with SK development (P = 0.043 and P = 0.012, respectively). In particular, the Aa and Tt genotypes were associated with increased risk of SK. These results were however more complicated, as shown by further analysis. This showed that genotypes containing at least one allele that conferred decreased VDR transcription (ie. AA/Aa and Tt/tt) increased risk of SK development by 2-fold in fair skinned individuals (OR: 2.1; CI: 1.2-3.7 and OR: 1.7; CI: 1.1-2.7 for Apa I and Taq I variants, respectively) but also found to decrease the risk of SK development by 2-fold in medium skinned individuals (OR: 0.5; CI: 0.3-1.0 for Apa I variants). Additionally, genotypes containing 2 alleles conferring decreased transcription of the VDR gene were found to further increase the risk for SK development in fair skinned individuals (OR: 2.5; CI: 1.4-4.5 and OR: 2.4; CI: 1.2-5.0 for Apa I and Taq I variants, respectively), indicating a possible additive effect for the alleles. The highly differential association of the VDR gene polymorphisms amongst phenotypes may reflect a combination between the ability of an individual to synthesise 1a25 dihydroxyvitamin D3 with the binding availability of the VDR. To further investigate the role of VDR in NMSC, expression analysis of the VDR gene was undertaken using real time reverse transcription PCR, with SYBR green fluorescence and 18S as a comparative gene, to examine expression pattern changes associated with UV radiation. It was found that short term irradiation, as well as long term irradiation and recovery were associated with gene expression changes. Short term irradiation resulted in patterns indicative of translation and subsequent transcription, whereas long term irradiated samples resulted in reduction of VDR expression that was recovered after an extended period of time. Thus, VDR expression is clearly influenced by UV exposure. It would be very interesting to see more specifically if particular VDR genotypes, which appear to play a role in NMSC risk, also are affected differentially by UV exposure. It is possible that VDR expression is reduced to limit excessive binding of 1a25 dihydroxyvitamin D3, although since both UVA and UVB radiation affect VDR expression, this may not be mediated the effect of 1a25 dihydroxyvitamin D3 but rather a different pathway resulting from a general UV response. In summary, the detection of a number of susceptibility genes involved in SK development and their subsequent expression analysis in response to UV radiation has given further insight into the molecular changes associated with NMSC. In fact, both detoxification genes (GSTM1 and GSTT1) and a transcription related gene (VDR), were found to confer susceptibility to solar keratosis, an early stage skin lesion with tumourigenic potential. This suggests that even the earliest stages of skin cancer are mediated through a wide range of effects. Additionally, expression changes related to these genes indicate that they are associated with the well known environmental carcinogen of UV radiation and that their effects may be mediated through a wide range of pathways. Although implication of the 18q region in SCC progression was not confirmed in this study, it is still likely to play a role in malignant transformation. The implication of this region, as well as the implication of susceptibility genes has vastly increased knowledge into processes associated with NMSC. Although additional analysis can confirm and further implicate these molecular alterations, this study has resulted in a more comprehensive understanding of NMSC that may ultimately be of benefit in terms of prognosis and treatment.

No description available.

No description available.

No description available.

Photodynamic therapy (PDT) is a multimodality cancer treatment available for the palliation or eradication of systemic and cutaneous malignancies. In this thesis, the application of PDT is for the treatment of non-melanoma skin cancer (NMSC). While PDT has a well-documented track record, there are, at this time no significant indicators to suggest the superiority of one treatment regime over the next. The motivation for this work is to provide additional evidence pertaining to PDT treatment variables, and to assist in optimising PDT treatment regimes. One such variable is the treatment light dose. Determining the light dose more accurately would assist in optimising treatment schedules. Furthermore, choice of photosensitiser pro-drug type and application times still lack an evidence base. To address issues concerning treatment parameters, fluorescence spectroscopy – a valuable optical diagnostic technique – was used. Monitoring the in vivo PpIX fluorescence and photobleaching during PDT was employed to provide information pertaining to the progression of treatment. This was demonstrated by performing a clinical study at the Photobiology Unit, Ninewells Hospital and Medical School, Dundee. Two different photosensitiser pro-drugs – either 5-aminolaevulinic acid (ALA) or its methyl ester (MAL) – were investigated and based on the fluorescence and pain data recorded both may be equally suitable for topical PDT. During PDT, surface fluorescence is observed to diminish with time – due to photobleaching – although cancerous cells may continue to be destroyed deep down in the tissue. Therefore, it is difficult to ascertain what is happening at depth in the tumour. This raised the questions; How long after surface PpIX fluorescence has diminished is the PDT treatment still effective and to what depths below the surface is effective treatment provided? In order to address these important questions, a three-dimensional (3D) Monte Carlo radiation transfer (MCRT) model was used to compute the light dose and the ¹O₂ production within a tumour, and the PpIX fluorescence emission from the tumour. An implicit dosimetry approach based on a single parameter – fluorescence photobleaching – was used in order to determine the ¹O₂ generation, which is assumed to be related to tissue damage. Findings from our model recommended administering a larger treatment light dose, advocating an increase in the treatment time after surface PpIX fluorescence has diminished. This increase may ultimately assist in optimising PDT treatment regimes, particularly at depth within tumours.

O trabalho apresentado foi realizado em duas etapas independentes e baseou-se no estudo de diferentes sistemas nanométricos para viabilizar a aplicação da ftalocianina de cloro alumínio (ClAlPc) na terapia fotodinâmica (TFD) para o tratamento do câncer de pele do tipo melanoma. O fármaco fotossensibilizante (FS) utilizado apresenta propriedades físico-químicas que lhe permitem exercer sua atividade fotodinâmica com excelência, sem a interferência do cromóforo endógeno melanina existente nas células melanocíticas. Para driblar sua elevada hidrofobicidade, ClAlPc foi encapsulada em sistemas nanométricos para administração em meio fisiológico. Inicialmente nanopartículas lipídicas sólidas (NLS) foram desenvolvidas por emulsificação direta, após um estudo de elaboração do diagrama de fases. O compritol foi o lipídio sólido escolhido para compor as NLS, com diferentes concentrações de ClAlPc. Todas as formulações desenvolvidas foram devidamente caracterizadas, com tamanho médio entre 100 e 200 nm, baixa polidispersão, potencial zeta adequadamente negativo (~|30| mV), drug loading de ClAlPc entre 76-94% (com pequena redução após 24 meses) e alta eficiência de encapsulação (E.E.). A morfologia arredondada das nanopartículas foi confirmada por microscopia eletrônica de transmissão e de força atômica. A estabilidade das NLS foi de 24 meses. A avaliação da cristalinidade do lipídio revelou a integração da ClAlPc à matriz lipídica da NLS, presença de estruturas polimórficas e grau de cristalinidade adequado, sem alterações após 24 meses. Nos estudos de difusão in vitro, observou-se que ftalocianina encapsulada nas NLS acumulam-se preferencialmente na epiderme e derme do que no estrato córneo, sem traços de permeação do ativo. Foi confirmado o caráter biocompatível das NLS sobre fibroblastos NIH-3T3. A ftalocianina encapsulada nas NLS não foi tóxica na linhagem de melanoma B16-F10 na ausência de luz, porém, apresentou excelente efeito fototóxico (0,75 ?g mL-1 de ClAlPc nanoencapsulada e irradiação entre 0,5 e 2,0 J cm-2), com redução da viabilidade celular de 87%. O segundo sistema de veiculação estudado foram as vesículas cataniônicas (VesCat), que se formam espontaneamente em água com o tensoativo TriCat 12. A obtenção das vesículas contendo ClAlPc envolve uma etapa adicional, para remoção de solvente orgânico, que foi aprimorada, reduzindo o tempo de produção em 55%. As VesCat/ClAlPc obtidas mantiveram suas propriedades físico-químicas e morfologia arredondada (confirmada por microscopia eletrônica de varredura), drug loading de 47% e alta E.E. Os resultados comprovaram que a aplicação desses dois sistemas nanométricos é altamente eficiente para aplicação da TFD no tratamento do câncer de pele do tipo melanoma ou outras doenças cutâneas, apresentando características favoráveis para avanços nos estudos de fase clínica e pré-clínica. / The present work was conducted in two independent steps, which were based on the study of different nanometric systems that make feasible the application of aluminum phthalocyanine chloride (ClAlPc) in the photodynamic therapy (PDT) to the melanoma skin cancer treatment. The photosensitizer (PS) used has physical-chemical properties that allow it to perform its photodynamic activity with excellence, without the interference of the melanin, an endogenous chromophore found in melanotic cells. In order to circumvent the high PS hydrophobicity, ClAlPc was encapsulated into nanosystems to administration in physiological environment. At first, solid lipid nanoparticles (SLN) were developed by direct emulsification process after drawing up phase diagram study. The solid lipid compritol was chosen to make the SLN, produced with different ClAlPc concentrations. The developed samples were properly characterized with mean size between 100-200 nm, low polydispersity, negative zeta potential (~|30| mV), ClAlPc drug loading around 76-94% (with slight decrease after 24 months) and high encapsulation efficiency (EE). The round shape of SLN was confirmed by transmission electron microscopy and atomic force microscopy. The nanoparticles were stable for at least 24 months. The evaluation of lipid crystallinity has proved the ClAlPc integration to SLN lipid matrix, the presence of polymorphic structures and a suitable crystalline degree, without large variations after 24 months. In the in vitro diffusion studies were observed that phthalocyanine conveyed in the nanoparticles accumulates preferably in the epidermis and dermis than in the stratum corneum, without any drug permeation traits. The NLS biocompatibility was confirmed on NIH-3T3 fibroblasts. ClAlPc-loaded NLS did not exhibit toxicity on B16-F10 melanoma cell line in the dark, but it was shown their outstanding phototoxicity effect (0.75 ?g mL-1 of encapsulated ClAlPc and irradiation between 0.5 and 2.0 J cm-2) with cell viability reduction of 87%. The second drug delivery system studied were the catanionic vesicles (VesCat) that are spontaneously obtained by mixing the self-assembly surfactant TriCat 12 in water. The production of ClAlPc-loaded vesicles comprises an additional step (to remove the organic solvent) that was optimized, saving 55% of the production time. The final VesCat/ClAlPc kept their physical-chemical properties and round shape (confirmed by scanning electron microscopy), drug loading of 47% and high EE. Hence, the results have proved the great efficiency of these two nanometric systems applied in the PDT to the treatment of melanoma skin cancer and other cutaneous disease, useful features for further progress towards preclinical and clinical trials.