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Title: Investigation of differentially expressed genes that contribute to therapeutic effects of bevacizumab in BRAF mutant melanoma
Author: Coupe, Nicholas
ISNI:       0000 0004 9356 658X
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2020
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Malignant melanoma is a highly aggressive and often lethal disease. In 2012, patient outcomes were poor after resection of high-risk stage II and III melanoma, and vascular endothelial growth factor (VEGF) has been implicated in melanoma progression. The phase III AVAST-M clinical trial (recruiting between 2007-2012) randomized patients with high risk melanoma to 1 year of adjuvant bevacizumab (anti-VEGF antibody) or observation. Patients treated with bevacizumab experienced superior disease-free survival (DFS), and BRAFV600E mutation was unexpectedly predictive of benefit. The aim in this project was to investigate this novel association between BRAFV600E and VEGF, based on the hypothesis that BRAFV600E melanomas are dependent on VEGF and more sensitive to its inhibition. VEGF and CD31 expression were quantified in tissue microarrays (TMAs) and whole mount sections of AVAST-M melanomas. Although no differences were identified between BRAFV600E and wildtype (WT) tumours from TMAs, there was a trend towards higher VEGF and CD31 expression in BRAFV600E whole mounts. In three independent transcriptomic datasets including NanoString analysis of melanomas from the AVAST-M clinical trial, VEGF itself was not differentially expressed, but all identified receptor kinase ROR2 as overexpressed in BRAFV600E melanomas. Differential ROR2 expression was confirmed in a BRAFV600E/WT isogenic melanoma model. ROR2 expression decreased with pharmacological MEK inhibition, supporting regulation by MAPK signalling. In vitro, ROR2 expression associated with invasive and proliferative phenotypes, with novel evidence that ROR2 regulates VEGF secretion in BRAFV600E mutant melanoma cells. The putative ROR2 ligand Wnt5a was also identified as regulated by MAPK signalling, and MEK inhibition of BRAFV600E mutant ROR2-overexpressing cells was shown to suppress Wnt5a expression and VEGF secretion. An exploratory transcriptomic analysis of bevacizumab treated BRAFV600E/WT isogenic melanoma xenografts identified gene expression changes in the tumour and stroma, that were mutually exclusive between BRAFV600E and WT tumours, both basally and after bevacizumab treatment, and included upregulation of ROR2 in the tumours and Wnt5a in the stroma of BRAFV600E xenografts. Finally, as a predictive biomarker in AVAST-M patient samples, patients with melanomas containing high ROR2 expression trended towards a prolonged DFS after treatment with bevacizumab. In summary, this work identifies Wnt5a-ROR2 signalling as upregulated in BRAF mutant melanomas, contributing to increased VEGF secretion and potentially also to bevacizumab response.
Supervisor: Macaulay, Val ; Middleton, Mark Sponsor: Cancer Research UK
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Oncology ; Melanoma