Development of methylation based prognostic biomarkers to stratify risk or recurrence in early rectal cancer.
Lead Research Organisation:
University of Birmingham
Department Name: Cancer Sciences
Abstract
Rectal cancer affects 10,000 new patients and causes 4,700 deaths each year in England and Wales. The National Bowel Cancer Screening Programme aims to diagnose cancer at a much earlier stage when treatment is more effective. Approximately half of all screen-detected cancers are ‘early‘ and have not penetrated through the bowel wall. The standard treatment involves radical surgery to remove the rectum. Though it offers high rates of cure, it may be complicated by a range of significant adverse effects and frequently results in poor functional outcome such as incontinence, impotence and permanent stomas. Local treatment is likely to be much safer and functionally far superior without substantially compromising cancer survival. Patient selection for local treatment can be improved if we can identify and exclude the small proportion where the cancer appears localised to the bowel wall but in fact, has already spread to the lymph glands. This study aims to identify these cancers more precisely by looking at the behavioural pattern of rectal cancer at a molecular level.
Technical Summary
Background
Bowel cancer screening will change how rectal cancer presents in the UK. Pilot studies demonstrate that half of screen-detected cancers are early (pT1-2,N0M0). Local excision, with radical salvage therapy in the event of recurrence, could be safer and functionally superior to conventional radical surgery. A predictive biomarker indicating ‘high risk‘ of recurrence following local excision could be used to optimise patient selection and so reduce the requirement for late salvage.
We hypothesise that ‘high risk‘ tumours can be identified at a molecular level using epigenetic signatures. Our aim is to develop a panel of functional methylation biomarkers that can discriminate ‘high‘ from ‘low risk‘ early rectal tumours. Our preliminary studies have identified 30 candidate biomarkers. We now wish to select a panel of five for validation in two large patient populations.
METHODS
A) Selection of methylation biomarker
1) Evaluation of gene expression using real-time RT-PCR and selective IHC
Only those candidate markers exhibiting differential expression in node-negative and node-positive rectal tumours will be selected for subsequent analysis.
2) Validation of methylation using Pyrosequencing
We shall confirm the presence of discriminatory methylation values at the candidate sites in node-positive and node-negative rectal tumours using Pyrosequencing.
B) Mechanisms of epigenetic gene silencing
We hypothesise that the ‘high risk‘ phenotype is mediated by a particular epigenetic silencing mechanism. Examination of the histone modifications at silenced loci will identify the dominant pathway. Markers involved in these mechanisms will be determined by IHC (DNMT, SIRT1) and carrier chromatin immunoprecipitation (H3K27me3, H3K27me3).
C) Validation of biomarkers
The panel of biomarkers derived from Stage A and B will be evaluated in:
(a) A large cohort of locally excised rectal tumours where outcome i.e. recurrence is known.
(b) Screen-detected early rectal tumours to determine whether the prevalence of high risk markers is significantly different in early stage symptomatic and screen detected tumours.
(C) Colonic tumours using tissue microarray immunohistochemistry, supervised by Prof. P Quirke, Leeds
FUTURE STUDIES
The ability of the methylation-based biomarkers to inform clinical decision-making will be prospectively evaluated in the Cancer Research UK funded multicentre randomised phase II trial of radical surgery versus short course pre-operative radiotherapy and delayed local excision (TREC) which will launch in 2010.
Bowel cancer screening will change how rectal cancer presents in the UK. Pilot studies demonstrate that half of screen-detected cancers are early (pT1-2,N0M0). Local excision, with radical salvage therapy in the event of recurrence, could be safer and functionally superior to conventional radical surgery. A predictive biomarker indicating ‘high risk‘ of recurrence following local excision could be used to optimise patient selection and so reduce the requirement for late salvage.
We hypothesise that ‘high risk‘ tumours can be identified at a molecular level using epigenetic signatures. Our aim is to develop a panel of functional methylation biomarkers that can discriminate ‘high‘ from ‘low risk‘ early rectal tumours. Our preliminary studies have identified 30 candidate biomarkers. We now wish to select a panel of five for validation in two large patient populations.
METHODS
A) Selection of methylation biomarker
1) Evaluation of gene expression using real-time RT-PCR and selective IHC
Only those candidate markers exhibiting differential expression in node-negative and node-positive rectal tumours will be selected for subsequent analysis.
2) Validation of methylation using Pyrosequencing
We shall confirm the presence of discriminatory methylation values at the candidate sites in node-positive and node-negative rectal tumours using Pyrosequencing.
B) Mechanisms of epigenetic gene silencing
We hypothesise that the ‘high risk‘ phenotype is mediated by a particular epigenetic silencing mechanism. Examination of the histone modifications at silenced loci will identify the dominant pathway. Markers involved in these mechanisms will be determined by IHC (DNMT, SIRT1) and carrier chromatin immunoprecipitation (H3K27me3, H3K27me3).
C) Validation of biomarkers
The panel of biomarkers derived from Stage A and B will be evaluated in:
(a) A large cohort of locally excised rectal tumours where outcome i.e. recurrence is known.
(b) Screen-detected early rectal tumours to determine whether the prevalence of high risk markers is significantly different in early stage symptomatic and screen detected tumours.
(C) Colonic tumours using tissue microarray immunohistochemistry, supervised by Prof. P Quirke, Leeds
FUTURE STUDIES
The ability of the methylation-based biomarkers to inform clinical decision-making will be prospectively evaluated in the Cancer Research UK funded multicentre randomised phase II trial of radical surgery versus short course pre-operative radiotherapy and delayed local excision (TREC) which will launch in 2010.