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The incidence of cutaneous melanoma continues to rise rapidly. Problems are faced throughout its management and this thesis addresses them as follows: secondary prevention, prediction of prognosis and early detection of metastatic disease. Secondary prevention has been shown to be an effective strategy for improving survival in other cancers. A study of the rapid access, early detection, pigmented lesion clinic at Mount Vernon Hospital demonstrates for the first time that such an approach improves outcome in melanoma. Refining current prognostic capability in melanoma is essential for improving disease management. Tissue microarray was used to investigate 480 specimens from 120 primary melanomas for novel prognostic markers. Several markers were identified, including MCAM, CD44v3, Nm23 and P-cadherin, which showed a significant bearing on melanoma patient outcome. Their identification reveals valuable markers for predicting outcome and potential targets for therapeutic manipulation. Immunoscintigraphy is the use of radiolabeled tumour-specific antibodies to detect disease. Previously the whole monoclonal antibody LHM2, directed against the high molecular weight melanoma-associated antigen was studied. Whilst being highly melanoma-specific, its use in melanoma detection is limited. Its large size (150 kDa) results in background accumulation in normal organs, reducing image quality and hindering metastasis detection. The single chain Fv fragment (scFv) is the smallest part of the whole monoclonal antibody that maintains full antigen- binding capability. Its potential superiority over whole monoclonal antibodies has been demonstrated however it requires further refinement before routine use in patients. Two methods of improving tumour targeting with scFvs were investigated: use of a cocktail of scFvs binding different epitopes and use of scFv multimers. The use of a scFv cocktail improved tumour targeting in the spleen and muscle. Dimeric scFvs were shown to improve tumour retention whilst maintaining rapid blood clearance, leading to overall improved tumour targeting compared with monomers.

This study examined whether the transforming growth factor-β (TGF-P) signalling pathway switches from a tumour suppressing to a pro-metastatic function during the progression of human squamous cell carcinoma (SCC). Preliminary studies aimed to determine whether the HaCaT model of human SCC together with the in vivo technique of floor of the mouth (FOM) transplantation of cells to athymic mice provided a good model of human SCC. Following FOM inoculation, HaCaT was non-tumorigenic while the remaining cell lines displayed an increasingly aggressive phenotype in vivo confirming that the HaCaT model and FOM inoculation technique were suitable for the study of SCC progression.

Loss of p53 function is a critical event in tumour development. It has been well documented that p53 mutation can lead to an oncogenic gain of function (GOF) associated with poorer prognosis and therapeutic response. Such mutants exert their gain-of-function properties by modifying cells through altered transcriptional activity as well as novel protein-protein interactions, but the molecular details of the mechanisms involved are still not entirely understood. Recent reports indicate that mutant p53 increases TGFβ-dependent metastatic potential of cancer cells via inhibition of p63 function and also promotes invasion through increased integrin (aSpl) and EGFR recycling, which is also linked with suppression of transcriptionally active p63 in normal epithelial cells, lung cancer cells and invasive breast cancer cells. The aim of this study was to investigate the mechanism of mutant p53 GOF in squamous cell carcinoma of the head and neck (SCCHN) cells. To avoid the potentially confounding impact of studying cells from biologically distinct anatomical sub-sites, the studies described here focused upon the most common sub-type of SCCHN -laryngeal squamous cell carcinoma (LSCC). Two hot-spot p53 mutants representing two distinct mutational classes have been chosen for these studies: R17SH (structural mutation) and R273H (contact mutation), both of which have been previously demonstrated to have GOF properties and have been documented to occur in laryngeal cancer. The mechanism of mutant p53 GOF was investigated by identifying protein interactions of mutant p53 and by studying gene expression changes in LSCC cells expressing these p53 mutants. In addition, the functional consequences of mutant p53 expression in LSCC cells were investigated in terms of response to radiation (a primary treatment modality for LSCC) and motility/invasiveness (key determinants of metastatic potential). A number of proteins have been identified in the mutant p53 containing protein complexes in LSCC cells and one of these, HSP70, has been confirmed by immunoprecipitation to selectively interact with the p53-175H mutant. p53-273H-expressing cells and to a smaller extent p53-175H-expressing cells display increased motility. A stable knock-down of the mutant p53 has been shown to reduce the motility of cells suggesting that p53-273H contributes to increased

Malignant melanoma is an aggressive cutaneous cancer originating in melanocytes, the cells which produce skin pigmentation. When treating malignant melanoma prompt diagnosis is paramount, as the depth of vertical invasion (Breslow thickness) is inversely correlated with survival rates. The development of computerised systems for assisted recognition of malignant melanoma can aid enhanced early diagnosis of this disease and help reduce the subjectivity associated with clinical diagnostic procedures. We describe and evaluate key elements of a prototype system for classifying benign pigmented skin lesions and cutaneous malignant melanoma, with emphasis placed on the extraction of existing clinical criteria. The system utilises image processing methods to enable specific features to be defined. Statistical- and intelligence- based classification techniques are then employed to automate identification of significant features, with the aim of reducing feature analysis variability. After lesion boundary determination, a total of 77 parameters for describing the visual appearance (shape asymmetry, border irregularity, colour and differential distribution, and dimension) of pigmented skin lesions are extracted from digital epiluminescence microscopy images and assessed using a double-blind study. A variety of classification methodologies (specifically C5.0 induction, classification and regression induction, and a neural network model) are applied to assess the diagnostic capability of these parameters and to compare system performance against the diagnostic accuracy achieved by two experts using clinical visual inspection methodologies. It is concluded that the parameters generated may have high discriminative value when differentiating between benign and malignant lesions. When using our full feature set on a data set comprising 30 lesions, diagnostic accuracy achieved via the automated classifiers constituted a significant improvement when compared with clinical diagnostic accuracy achieved by our experts.

A key contributor to the development of skin cancer and ageing is damage to the skin caused by an increase in the cellular load of reactive oxygen species (ROS). ROS generation is increased when the skin is exposed to factors, such as ultraviolet radiation (UVR) from the sun. UVR initiates a cascade of biological events including DNA damage, which if allowed to accumulate can accelerate the ageing process. Previously, studies have reported the ROS scavenging capacity of cytosolic antioxidants and demonstrated their ability to abrogate ROS-induced DNA damage. The aim of this study was to further investigate the relative potency of mitochondrial (mt) located antioxidants such as MitoQ (MO) and tiron, because the mitochondria serve as the main cellular location for ROS formation. Using a lesion specific quantitative real-time PCR (OPCR) assay (which follows the principle that DNA lesions block replication of DNA by Taq polymerase) it was found that UVA (9.2 J/cm2", glass filtered TL-09 source) and hydrogen peroxide (H202) (200 IJM) induced widespread mtDNA damage in human dermal fibroblasts (n = 4, P < 0.001). The mt-Iocalised antioxidant tiron was able to offer complete protection (n = 4, P < 0.001) against the deleterious effect of H202. In comparison, treatment with the optimum, sub-lethal dose (as determined by the MTS assay) of 10 cytosolic antioxidants including curcumin and resveratrol were found to provide moderate protection against H20rinduced mtDNA damage. Interestingly, differential responses to UVA were observed, with antioxidants showing a decline in the proportion of protection offered against this potent mutagen, except in the case of tiron; a bi-functional antioxidant with ROS and iron targeting capacity. These findings demonstrate the potential involvement of iron (present in the cytosol and mitochondria) in H202 and UVA-mediated oxidative damage. We sought to further investigate the potency of tiron against ROS-induced nuclear (n) DNA damage using the comet assay. In comparison to a small panel of cytosolic antioxidants which showed a graded response, tiron was able to abrogate ROS-induced nDNA damage in human dermal fibroblasts and primary keratinocytes. It appears that the ability of tiron to detoxify ROS and in particular iron (which in reaction with H202 give rise to the highly reactive hydroxyl radical) enables this antioxidant to inhibit genomic modifications and significantly contribute to cellular protection compared to other antioxidants.

Cutaneous melanoma is an increasing health problem with an incidence that has steadily increased internationally in the last fifty years. When detected early, excision of the primary tumour is usually curative, however, metastatic disease remains extremely resistant to therapy and as yet, there is still no chemotherapeutic agent which offers a consistent effect on survival. Currently, the overriding prognostic indicator is disease stage defined at diagnosis, based on clinico-histological criteria including Breslow depth, mitotic rate and ulceration of the primary tumour; which combined, form the current American Joint Committee on Cancer (AJCC) Melanoma Staging and Classification system. However AJCC staging criteria alone are unable to identify the subset of patients with high risk early stage primary lesions that will eventually progress. The identification of credible biomarkers to refine the risk of disease progression, as well as predict those patients who would benefit from early stratified treatment is therefore fundamental to the enhancement of treatment efficacy-and the reduction in development of systemic disease.