Tumor stage remains the most important prognostic variable in human colorectal cancers and is used to determine the need for adjuvant chemotherapy. However, there is considerable stage-independent variability in clinical outcome. Despite adjuvant treatment, 30-40% of stage III patients will recur and die of their disease. Accordingly, additional prognostic markers are needed to better define the subset of patients who would benefit most from adjuvant chemotherapy. Prognostic and predictive markers would also enable a more selective, tailored and molecularly- targeted treatment approach. We propose to study apoptotic regulatory proteins in >1,000 well characterized stage II and III colon cancers from patients treated in 5-fluorouracil-based adjuvant therapy trials conducted by the North Central Cancer Treatment Group (NCCTG) and the National Cancer Institute of Canada. Some of these trials include untreated control arms enabling us to determine predictive utility. Defects in the regulation of apoptosis have been shown to contribute to tumor progression and metastasis, and can confer resistance to anti-cancer therapies. Two distinct apoptotic signalling pathways have been defined and include the membrane death receptor (DR) pathway and the mitochondrial pathway. Engagement of the mitochondrial pathway results in cytochrome release which can be attenuated by anti-apoptotic Bcl-2 and by inhibitors of apoptosis proteins (lAPs), which bind to and inhibit caspases. The DR pathway is engaged by members of the TNF superfamily which act as natural ligands for specific DRs. DRs mediate apoptosis through their cytoplasmic death domains. Decoy receptors lack death domains but can compete for ligand binding. Both apoptotic pathways involve activation of intracellular cysteine proteases known as caspases that become activated through proteolytic processing and lead to apoptosis. Our preliminary data indicate that apoptotic regulatory proteins display tumor-specific overexpression in human colon cancers. Moreover, our data suggest that negative and positive regulators of the mitochondrial pathway are significantly associated with shorter and longer patient survival rates, respectively. Recent data also suggest that DR4 is overexpressed in colon cancers and can confer prognostic information, as can decoy receptor 3, an inhibitor of Fas signalling. We propose to determine the predictive and prognostic significance of apoptotic regulatory proteins in 1,324 colon cancer patients. Tissue microarrays will be created from paraffin blocks to enable the efficient immunohistochemical analysis of multiple apoptosis-regulating proteins Our study results will then be correlated with previously determined markers including microsatellite instability, allelic loss, and DNA ploidy and multivariate models will be created to determine the most significant markers for incorporation into a prospective adjuvant therapy trial involving 3,750 patients to be conducted by the NCCTG/Intergroup mechanism.
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