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Title: Human endogenous retrovirus K gene expression in cutaneous melanoma
Author: Singh, Sarita
ISNI:       0000 0004 2728 4314
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2012
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Introduction: In recent years, the key environmental and genomic changes that "drive" melanoma have become more clearly elucidated but much remains to be understood. Data published in 2003 demonstrated expression of Human Endogenous Retrovirus K (HERV-K) in melanomas but not in normal tissues, indicating a potential oncogenic role. Furthermore, HERV-K proviruses encode the putative oncogenic proteins Rec and Np9. In this work the feasibility of reliable detection of HERV-K expression from formalin-fixed paraffin-embedded (FFPE) tissue has been determined. Expression has been correlated with melanoma subtype and histological markers of poor outcome. Methods: HERV-K mRNA expression in melanoma cell lines (A375 and WM2664), FFPE primary melanoma (n=39), normal skin (n=31) and benign naevus (n=16) tissues was investigated by a novel real-time quantitative RT-PCR (qRT-PCR). Expressed HERVK env sequences were cloned and sequenced. Results: A375 cells expressed all HERV-K genes and produced putative viral particles. Full-length mRNA was expressed in all primary melanoma and control tissues investigated with no differences in expression levels between the groups (pol: n=47, p=0.267; unspliced env: n=40, p=0.823). No correlation was observed with melanoma subtype or stage (Breslow). A proportion of all tissue types expressed np9 with no differences in expression levels between them (p=0.964). Spliced env was expressed in 8/39 melanoma, compared with 1/16 benign naevus and 0/31 normal skin samples (p=0.003). Expression of rec was found in 9/39 melanomas but not in control samples (p=0.009) nor extra-lesional skin in five rec-positive cases (p=0.06). The rec-positive melanomas were histologically in vertical growth phase and thicker than rec-negative tumours (median Breslow 2.1mm versus 1.05mm, p=0.009). Sequencing of expressed env cDNA clones derived from melanoma and control samples (n=8) revealed expression of multiple HERV-K loci. Conclusions: Gene expression can be studied in FFPE tissue. The expression of potentially oncogenic rec in approximately 25% primary melanomas merits further evaluation.
Supervisor: Screaton, Gavin ; Bunker, Christopher Sponsor: Retsas, Spyros ; Skin Treatment & Research Trust ; Chelsea and Westminster Healthcare Charity ; Westminster Medical School Research Trust
Qualification Name: Thesis (M.D.) Qualification Level: Doctoral