Biomarkers of prognosis and response to vitamin D in cutaneous melanoma

This thesis describes an evaluation of next generation sequencing (NGS) as a tool
to study melanoma genomics and identify prognostic biomarkers using formalinfixed
paraffin-embedded (FFPE) primary tumours. I report the evaluation of different
library preparation protocols as a pre-requisite for whole-genome DNA NGS. I show
that libraries can be generated using small amounts of DNA extracted from primary
melanomas (92% success rate). NGS data from 75 samples are presented. To
ensure the accuracy of the data and in order to develop a robust protocol for copy
number analysis using small FFPE tumours the quality of the data was assessed
and potential biases were explored. I observed copy number changes previously
reported in melanoma which provide some support of the validity of the data when
small DNA input is used. Although, this is work under development, overall the data
show that NGS using small FFPE tumours is likely to provide insight into melanoma
biology.
I report a study where osteopontin was tested as a potential prognostic biomarker in
the plasma of patients with melanoma. The analysis suggests that measurement of
plasma osteopontin is unlikely to serve as a useful blood prognostic marker in earlystage
disease patients, but it remains a possible marker of relapse in patients under
follow up as part of a panel of such markers.
The Leeds group have reported that low vitamin D levels at diagnosis are
associated with thicker tumours and poorer outcome. I report an in vitro study
where melanoma cells were cultured with and without vitamin D. Whole-genome
gene expression data were generated using RNA from two vitamin D sensitive
melanoma cell lines. The differentially expressed genes in response to vitamin D
supported the anti-tumour effects of vitamin D previously reported in cancer
models, providing some evidence for vitamin D as a potential adjuvant therapy.