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Title: Duplex ratio tests as diagnostic tissue biomarkers in malignant melanoma
Author: Moore, David Allan
ISNI:       0000 0004 2744 4736
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 2013
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Malignant melanoma is a tumour with tendency to metastasise early and with a rapidly increasing incidence. A minority of melanomas and their benign counterparts, naevi, prove difficult to diagnose on histopathology alone, which is currently the gold standard and a small percentage are genuinely histologically ambiguous. Chromosomal Instability is a well described feature of malignant tumours and melanomas have been shown to demonstrate typical patterns of chromosomal instability, in comparison to benign naevi which show minimal DNA copy number change. QPCR based assays called Duplex Ratio Tests (DRTs) have been developed by our laboratory for application on DNA from formalin fixed paraffin embedded samples of melanoma and naevi. The reproducibility and accuracy of the DRTs has been demonstrated and appropriate correction factors for DNA quality have been calculated for the assays, based on the results of 108 diploid samples. As a panel, seven DRT assays were able to differentiate unambiguous cases of melanoma and naevi with sensitivity of 95% and specificity of 97.5% respectively on an opportunity sample of 20 naevi and 40 primary melanomas, when tested by logistical regression. When applied to independent true cohorts of 105 melanomas and 103 naevi the sensitivity of DRTs was 84% and specificity 88%. Logistical regression analysis of DRT scores for 20 non-metastasising primary melanomas and 20 unmatched metastasising primaries showed the DRT assays to correctly predict outcome in more cases (28/40) than clinical stage alone (24/40) and when combined with clinical stage, DRT scores predicted outcome with a sensitivity of 85% and specificity of 70%. BRAF mutation analysis of one series of cases indicated that 95% of the naevi and 27% of the primary melanomas tested showed the V600E mutation. Of the 5 metastasising primary melanomas with the mutation, 3 of the subsequent metastases were also V600E positive. DRTs targeting chromosomal centromeres were also developed as part of this project, although testing these assays against a hybridoma cell line DNA panel demonstrated that centromeric alphoid repeat regions show homology between the targeted chromosomes not previously described, making these assays impractical to develop as part of the DRT panel at this stage. DRT assays therefore show potential to act as molecular biomarkers of melanoma on FFPE specimens and DRTs may have applicability to prognosis in melanoma or to other tumour types if new DRTs to relevant loci are developed.
Supervisor: Saldanha, Gerald; Pringle, James H. Sponsor: Not available
Qualification Name: Thesis (M.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available