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Title: Cancer risk and biomarker analysis in mismatch repair deficient colorectal carcinoma
Author: Barrow, Emma
ISNI:       0000 0004 2685 0775
Awarding Body: The University of Manchester
Current Institution: University of Manchester
Date of Award: 2009
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Lynch Syndrome is caused by mutations in the DNA mismatch repair (MMR) genes; MLH1, MSH2, MSH6 and PMS2. An accurate estimation of cancer risk for MMR mutation carriers is essential for counselling and screening. United Kingdom (UK) specific cancer risks for MMR mutation carriers and cumulative colorectal cancer risks by decade have not been described to date. Using data from 121 Lynch Syndrome families, the cumulative lifetime risks of Lynch Syndrome spectrum cancers in mutation carriers were calculated, correcting for ascertainment bias. This data provides reassurance that current UK screening guidelines are appropriate. For colorectal cancer, tables of risk by decade were compiled for carriers of the different gene mutations. This novel data represents a useful counselling tool. Despite an increased cancer risk, individuals with Lynch Syndrome lack clear phenotypic characteristics. Mutation carrier identification is desirable as screening reduces mortality. After counselling and assessment of family history against clinical criteria, molecular diagnosis of tumour tissue is performed. Immunohistochemistry (IHe) of the MMR proteins has great potential as a method of identifying the most likely mutation and guiding mutation analyse. However IHC of the MMR proteins is not yet well established and problems have been reported with staining and slide interpretation. IHC slide assessment methods in the reported literature have been qualitative. This thesis describes the development and validation of a robust, semiquantitative scoring technique to be used in the assessment of IHC stained tumour sections from patients with possible Lynch Syndrome. Colorectal tumour sections from 51 MMR mutation carriers were stained with 3,3' Diaminobenzidine (DAB) using antibodies against the MMR proteins. Slide assessment was semiquantified using a 0-12 scale, and was found to be highly sensitive and specific for the identification of mutation carriers. It was found however, that protein expression may occur in the context of known pathogenic mutations, a potential pitfall in the screening process. Two quantitative techniques of slide assessment that could potentially obviate the need for human operator slide analysis were explored. Semiconductor quantum dots (QOs) are quantifiable fluorescent labels that can be used for multiplex staining. Quantitative QO IHC was compared to quantitative DAB IHC. Both are novel methodologies. Tumour sections from 36 mutation carriers were stained using each method, and multispectral analysis of the slides was performed. Quantitative analysis of the DAB stained slides was less sensitive than semi-quantitative analysis in this study, but was sufficiently sensitive to be used as a pre-screen in Lynch Syndrome. However despite the potential advantages of quantitative QD staining, it was less sensitive and specific than quantitative DAB IHC. The development of automated DAB IHC staining and quantitative multispectral slide analysis may enable future high throughput IHC for MMR mutation carrier identification. Around 15% of sporadic colorectal cancers develop along the MSI pathway due epigenetic MLH1 inactivation. The evidence suggests that patients with such tumours do not benefit from 5-Fluorouracil based chemotherapy. The cost effectiveness of identifying patients with such tumours was explored. Potential chemotherapeutic savings are substantial, supporting the introduction of this biomarker into clinical practice. The improved diagnostic accuracy of semi-quantitative IHC slide assessment and the potential for automation of quantitative IHC slide assessment would facilitate the introduction of this service.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (M.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available