The most important prognostic factor in patient with colorectal cancer is disease stage at the time of diagnosis.  Disease stage is a reflection of the degree of local invasion of the tumour and the extent of distant spread (metastasis). In order to improve the present 50% five-year survival rate for CRC, it will be necessary to devise improved treatments or to target existing therapies more effectively.  Therefore, it is desirable to identify the specific molecular changes in cancer cells associated with their malignant behaviour. The matrix metalloproteinases (MMPs) are a group of proteolytic enzymes, which are known to play multiple roles in tumour growth, invasion and spread.  Their main physiological inhibitors are the tissue inhibitors of metalloproteinases (TIMPs). This study uses immunohistochemistry to determine the presence of all the major MMPs and TIMPs, in a well-characterised series of 249 colorectal cancers.  This work is the largest number of MMPs and TIMPs examined in any published study to date.  I have assessed each of the MMPs and TIMPs separately and correlated the findings with all the known prognostic factors in order to ascertain the significance of this group of molecules in colorectal cancer.  Furthermore, I have collectively evaluated all of the results, using unsupervised hierarchical cluster analysis, thereby identifying two distinct groups of patients with different MMP/TIMP profiles and significantly different survival outcomes.  Comparison of these groupings with existing prognostic factors in multivariate analysis showed the MMP/TIMP profile to be the single most powerful prognostic variable. This is the first study to demonstrate that the MMP/TIMP system produces an aggressive phenotype in colorectal cancer. My findings represent a breakthrough in understanding the molecular characteristics of colorectal cancer and provide the basis for the re-evaluation of the use of MMP inhibitors as anti-cancer drugs.

In this thesis, an <i>in silico</i> investigation of available online microarray data found that normal colon, polyps and CRC have distinct gene expression patterns and that several key inflammation-related genes are up-regulated in polyps. This was also the findings of a pilot microarray study using Affymetrix human GeneChip^® technology on normal colon and polyps biopsied from one individual.  Validation of the microarray data for <i>IL-1B</i> and <i>il-8</i> was performed by RT-PCR and also revealed an overall up-regulation of these genes in polyp compared to normal colon. A CRC case-control study was then used to assess the role of pro-inflammatory single nucleotide polymorphisms (SNPs) in <i>IL-1B (-31 T/C), </i>TNF-A<i> (-308 G/A), TGF-B </i>(-509 C/T) and <i>IL-10</i> (-592 C/A, -1082 G/A) in sporadic CRC in the North East of Scotland.  Their interaction with aspirin use was also investigated. Whilst statistically significant associations were not found between any of the SNPs and CRC alone, a statistically significant halving in risk of CRC (OR, 0.50; 95% CI, 0.27-.097) was found in carriers of the variant <i>IL-10-592</i> A allele who were regular aspirin users. Finally, the frequencies of 2 promoter, one exonic and one 3’-UTR <i>COX-2</i>SNPs were assessed in a Scottish population. The -899 and -197 <i>COX-2</i> promoter SNPs were not identified but the allele frequencies of the exon-3 and 3’UTR <i>COX-2</i> SNPs were similar to those reported in previous Caucasian studies. Further analysis including the <i>COX-2 </i>-765 G>SNP showed that -765 GG homozygotes infected with <i>H. pylori</i> had a significantly increased risk of developing pre-malignant changes of gastric cancer (OR 5.7, 95% CI, 2.3-14.1).  This result is in keeping with previous studies which have suggested that the variant C allele is associated with reduced COX-2 suppression and inflammatory response.

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Colorectal cancer is a major health problem for both men and women today. It is also lone of several site-specific cancers that patients are likely to survive beyondfive years of diagnosis. The majority of patients show good psychological adjustment following the diagnosis and treatment of colorectal cancer. However, the literature suggests that approximately one quarter of patients continue to have clinically significant psychological symptoms at least one year after diagnosis. This study aimed to investigate psychological adjustment in 33 colorectal cancer patients (J -2 years postsurgery); and to explore a series of demographic, disease and treatment, psychological and social factors that may be associated with psychological adjustment. A secondary aim of the study was to explore colorectal cancer patients' perceptions of and satisfaction with, care during diagnosis and treatment. Results showed lower levels of psychological distress (anxiety and depression) than those reported in previous studies with under 10 per cent of clinically significant cases. Levels of psychological distress were associated with age, disease severity and the presence of other concurrent physical health problems. Coping style was found to relate significantly with psychological adjustment. Taking the methodological limitations of this study into consideration, the results are interpreted in the context of the empirical and theoretical literature. Clinical and research implications are also discussed. A larger scale, longitudinal evaluation of the issues is clearly needed.

Introduction: The role of fibroblast growth factor-2 (FGF-2) on colorectal cancer (CRC) cells exposed to chemotherapy has not been studied extensively. This thesis investigated whether FGF-2 mediates chemoresistance in primary (SW480) and metastatic (SW620) colon adenocarcinoma cell lines. Methods: Proliferation assays were used to assess the response of SW480 and SW620 colon cancer cell lines to varying concentrations of FGF-2 and to optimise the dose of 5- FU at which 50% cell death was observed. Cell survival assays were performed following 96 hours exposure to 5-FU ± FGF-2. Levels of chemotherapy induced apoptosis were determined using Caspase-3/7 assay. Expression of anti-apoptotic proteins (Bcl-2 and Bcl-XL) and FGFRs at both protein and gene level were determined to see if these contributed to the difference in chemoprotection observed. Results: At 0.25 ng/ml, FGF-2 did not affect proliferation in either cell lines. 25μM of 5-FU resulted in 50% kill in both cell lines. Significant cell survival was observed when FGF-2 (0.25 ng/ml) pre-treated SW620 cells were exposed to 5-FU (25 μM) compared to cells exposed to 5-FU alone (81% vs 60%, p=0.015). This chemoresistance was associated with attenuation of cellular apoptosis (p=0.04) with no significant change in expression of Bcl-2 and Bcl-XL at gene or protein level. This survival advantage was not seen in SW480 cells (59% vs 55%, p=0.35). There were no observed differences in the expression of FGFR1-4 in either cell lines. Conclusion: FGF-2 offers chemoresistance to SW620 and not to SW480 cells exposed to 5-FU. Both cell lines expressed fgf2 and fgfr1-4 genes, suggesting that fgfr expression does not account for the difference in chemoresistance. FGF-2 offered protection by causing significant reduction in chemotherapy induced apoptosis in SW620 colon cancer cell line; however this was not due to increased expression of anti-apoptotic proteins. The molecular mechanisms for this selective chemoprotection need to be investigated further.

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This thesis sought to define deletions of chromosome lOin sporadic colorectal adenocarcinoma and determine their role in colorectal tumorigenesis. This thesis has shown a region of frequent loss of heterozygosity (LOH) in the region lOql1-21 centring on the marker D10S1790 at lOq21 suggesting this is the most likely locus of a putative tumour suppressor gene in this region. It has also been shown that LOH at this marker (D10S1790) is significantly associated with earlier presentation of patients. Analysis of adenomas revealed a low frequency of LOH in this region with only two of nine severely dysplastic adenomas showing LOH and none of those with mild or moderate dysplasia. This suggests that loss of the putative tumour suppressor gene at this locus is a late event in colorectal tumorigenesis. Furthermore, deletions at the chromosome 17 locus containing the known tumour suppressor gene, pS3, were examined together with pS3 expression by immunohistochemistry, which are also known to occur late in colorectal tumorigenesis. The frequency of LOH at the pS3 locus as well as pS3 expression were similar to previous studies and there was no significant association with losses at lOq suggesting that loss of a putative tumour suppressor gene at 10q occurs independently of loss of p53 function. LOH at lOq21 was also found not to be associated with LOH at the APe gene locus on 5q21-22 which occurs in up to 50% of sporadic colorectal adenocarcinomas. This thesis suggests that loss of the putative tumour suppressor genets) on lOq provides a selective advantage for clonal expansion in the somatic evolutionary process of colorectal tumorigenesis. Elucidation of the tumour suppressor genets) at this site will further help our understanding of the series of genetic mutations that occur in the evolution of colorectal cancer and will contribute to the efforts in the prevention, early detection and treatment of this prevalent disease.

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