Exploring stem dynamics and the expansion of genetic and epigenetic clones in colitis-associated neoplasia.

Lead Research Organisation: Queen Mary University of London
Department Name: Blizard Institute of Cell and Molecular

Abstract

Ulcerative colitis is a type of chronic inflammation of the large bowel characterised by recurrent damage (ulceration) to the bowel lining. Patients with ulcerative colitis have an increased risk of developing bowel cancer, as cells in their bowel develop mutations (errors) in their DNA (the molecular blueprint of a cell). We believe these DNA mutations result from the continuous damage and repair of the lining of the bowel. Here we intend to investigate the rate at which these errors expand through the bowel lining. From previous studies we have discovered that the patterns of DNA mutation in colitis patients is different from patients who develop cancer without colitis. We plan to further investigate the patterns of DNA alterations in colitis-associated tumours, in order to improve our understanding of tumour development and growth in this inflammatory condition. We hope that eventually we will be able to use markers of DNA alteration in a clinical setting to identify those patients at high risk of developing cancer. We hope that our research will highlight potential target areas for anti-cancer therapy in colitis patients.

Technical Summary

Background
Patients with ulcerative colitis have an increased risk of colorectal cancer (CRC), however the colitis-associated neoplasia pathway is less clearly understood than sporadic tumours. We have previously identified p53 and K-RAS mutations as gate-keeping lesions in colitis neoplasia, and demonstrated a relatively low frequency of APC mutations. Furthermore we identified ‘mutation negative‘ colitis-associated tumours with no detectable genetic changes in APC, p53, K-RAS or p16 genes. We hypothesise that epigenetic alterations drive tumorigenesis in these lesions. We have previously shown that crypt fission is the mechanism behind the spread of mitochondrial DNA mutations in the normal gut and hypothesise that the increase in the crypt fission rate in colitis may be responsible for widespread clonal expansion of mutated cells in this condition.

Aims and Objectives
1 (i).Use mitochondrial DNA mutation patch size analysis to model the rate of crypt fission in UC compared with normal mucosa data.
1. (ii) To investigate whether stem cell dynamics, including stem cell number and the rate of niche succession, are altered in UC crypts.
2. (i) Examine the genetic and epigenetic mutation burden of the cohort of human colitis-associated tumours with wild-type APC, p53, K-RAS and p16.
2. (ii) Look for protumorigenic epigenetic fields, particularly of p16, in UC neoplasia colectomy specimens


Methodology
1.Using established mtDNA histochemical, sequencing and bio-modelling techniques to assess colitis-colectomy specimens for clonal patch size, clustering of mutated crypts and mathematically model crypt fission rates. We will immunostain and calculate crypt fission rates and compare with wildtype controls.
2.Tissue from mutation negative specimens will undergo molecular profiling using array CGH, microsatellite LOH analysis and image cytometry to determine any frequently detectable chromosomal copy number changes. In addition, using methylation specific-PCR and bisulphite sequencing in individual crypts we will look for epigenetic fields within lesions with known mutation status.
Opportunities of the study
By combining assessment of genetic and epigenetic mutation burden in humans and analysing clonal expansion, we hope to determine the rate at which a stem cell clone expands, crypts divide and the timing and spread of mutations in the colitis dysplasia/carcinoma pathway.
We hope that this will improve our understanding of tumour development in this and other inflammatory conditions as well as searching for molecular markers to identify UC patients who are at high risk of developing tumours. This may pave the way for targeted therapy in those cases.
Histopathology is the current gold standard for indentifying those patients at high risk of progressing to cancer. By examining the tumorigenic burden in morphologically normal crypts, we will be able to assess the reliability of this diagnostic method and in the future possibly use this work to provide more accurate biomarkers of tumourigenesis.

Publications

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