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Title: Investigating clonal interactions and field effects in colorectal adenomas
Author: Walther, Viola
ISNI:       0000 0004 7971 8161
Awarding Body: Queen Mary, University of London
Current Institution: Queen Mary, University of London
Date of Award: 2019
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According to the somatic mutation theory of carcinogenesis, tumours are derived from a single mutated cell that clonally expands into a neoplasm. However, studies on familial adenomatous polyposis (FAP) colonic adenomas and some sporadic microadenomas have revealed tumours that are polyclonal in origin - they are derived from more than one clone. This has questioned the current dogma of how colonic tumours are initiated; however the mechanisms of how polyclonality is generated are unknown. Studies using chimeric mice have suggested that polyclonal adenomas arise through crosstalk between unique clones in close physical proximity. In this project, the local microenvironment surrounding human adenomas was characterised by investigating the non-dysplastic crypts in close proximity to adenomas, in particular their mutation burden, DNA damage status, crypt stem cell dynamics and the cellular makeup of the stroma. Immunohistochemistry was used to quantify cell proliferation (Ki67), DNA damage (γH2AX) and Wnt signalling status (nuclear β-catenin) in non-dysplastic crypts, stratified according to their physical distance from the nearest dysplastic crypt. Normal crypts within 250μm of an adenoma displayed increased cell proliferation, DNA damage and Wnt signalling. These effects were associated with an increase in T cell, macrophage and fibroblast infiltrate in the non-dysplastic stroma, however the concentration of intraepithelial CD8 T cells in dysplastic crypts showed a significant decrease. Furthermore, cytochrome c oxidase histochemistry (a marker of mitochondrial DNA (mtDNA) mutations - a proxy for mutation pressure on crypts) was used to demonstrate that crypts neighbouring an adenoma contained a higher mutation burden. Furthermore, the proximity of a crypt to an adenoma also affected stem cell dynamics: using somatic mtDNA mutations to trace clonal lineages, it was found that human intestinal stem cell evolution in adenomas and surrounding normal crypts followed neutral drift dynamics. The effects of an adenoma on gene expression in normal epithelium were investigated using murine organoid cultures. Wild type (WT) organoids when grown in the presence of fibroblasts previously exposed to mutant Apc1322/+ organoids demonstrated a significant upregulation of the MAPK, JAK/STAT and Wnt pathway when compared to WT only. Moreover, Tnf-α, MMP9 and collagen genes were found to be upregulated in exposed WT. To conclude, clonal interactions between dysplastic and non-dysplastic epithelium driving clonal expansion were demonstrated: adenomas create a field effect, dysplastic crypts exert mutagenic pressure, and crypt-to-crypt crosstalk between adenomatous and immune cells takes place leading to a pro-tumourigenic environment. This work has made a significant contribution to the understanding of the initiation of cancer in the human colon.
Supervisor: Not available Sponsor: Cancer Research UK
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available