Feasibility study investigating circulating peripheral mitochondrial DNA as a potential non-invasive biomarker for diagnosis and surveillance of high risk cutaneous melanoma

Background Cutaneous melanoma is the UK’s fifth commonest cancer and is the leading cause of death from skin malignancy.

Our recent work highlights the phenomenon of tumour cells obtaining their energy through mitochondrial transfer from its surrounding mesenchymal stem cells, which confers a survival advantage for the emerging cancer. Others have shown that mitochondrial DNA (mtDNA) mutations predominate in the cancer cells.

Mitochondrial DNA is an attractive choice for screening and surveillance because it is easily distinguished from nuclear DNA, there is a substantially higher DNA copy number per cell (hundreds versus one) and a significantly lower number of possible mutations in the mtDNA.

Such findings suggest the possibility of detecting peripherally circulating mtDNA as a simple and reproducible biomarker for the early detection of recurrence or disease progression.

Aims A feasibility study in melanoma patients to test the following: 1.To identify patients presenting with high-risk primary melanoma and isolate mtDNA from primary tumour and blood samples pre and post-operatively. 2. To detect novel and existing mutations associated with high-risk primary melanoma 3.

Can we detect mtDNA mutation associated with primary tumours in plasma samples taken pre and post-operatively?

Methods We will undertake an exploratory study in patients presenting with high-risk primary melanoma (AJCC II), prior to staging with sentinel node biopsy and in subsequent follow-up. Historical specimens from melanoma patients held in the Norwich Biorepository will also be studied. Preoperative and sequential postoperative blood samples will be collected from 30 high-risk patients over 12 months.

Circulating mtDNA will be compared with mtDNA in the primary melanoma and also from normal white cells from the peripheral blood as internal control. PacBio SMRT Technology analysis will also investigate the frequency of specific mtDNA mutations.

The ratio of tumour to normal mtDNA will be assessed using real-time PCR to determine if melanoma derived mtDNA could be further developed to investigate the frequency of specific mtDNA mutations against patient outcome with melanoma.

Clinical outcomes data (sentinel node status, recurrence-free survival) will be matched against the circulating mtDNA count and mutational burden.

How the results of this research will be used Data generated in this exploratory study will be used as a paradigm to undertake larger phase III investigations of the therapeutic utility of mtDNA to determine clinical decision making for melanoma patients in the early detection and early treatment phases and for the surveillance of high-risk patients in follow-up.