Abstract: This proposal will test the hypothesis that differences in patterns of gene expression determine the differing biologic behaviors between colon cancers that are curable with primary surgical therapy and those that ultimately metastasize to the liver and kill. Additionally, we hypothesize that in colon cancer primary tumors, only a minority of cells will be “prometastatic”, that is competent to give rise to liver metastases, and that assays of whole primary tumor lysates may thus fail to distinguish the crucial presence or absence of this “prometastatic” subpopulation. To test these hypotheses, Dr. Sanford Markowitz and his colleagues in the cancer genetics program at the Case Western Reserve University-NCI designated Comprehensive Cancer Center have forged a collaboration with Eos Biotechnology, Inc., a leader in gene expression array technology. The collaboration aims to establish an accurate molecular classification of colon cancer by focusing on a unique collection of surgically resected colon cancer liver metastases, all of whose cells have in vivo demonstrated metastatic ability. Using the Affymetrix human 40K GeneChip expression array technology, these investigators will generate a comprehensive description of global gene expression of these liver metastases. Comparing these liver metastases versus control nonmetastatic colon cancers, that were all cured by surgical excision, will specify a set of metastases specific genes whose expression defines a “metastatic signature.” The goal of identifying those colon cancer primary tumors that can metastasize will be achieved by showing that they bear “prometastatic” cells recognizable by in situ hybridization assay of “metastatic signature” genes. Project aims are: i) To elucidate the “metastatic signature” by comparing on Affymetrix arrays colon cancer liver metastases versus non-metastatic colon cancer primary tumors. ii) To identify among metastases signature genes those specifying early metastatic events detectable by array analyses of colon cancer primary tumors that did metastasize. iii) To use in situ hybridization to confirm the metastatic signature of liver metastases arises from colon epithelial cells. iv) To use in situ hybridization to detect expression of metastatic signature genes by prometastatic cells in colon cancer primary tumors that are simultaneous with lever metastases or are precursor of liver metastases relapse. v) To use in situ hybridization to determine the areas of maximum concentration of “prometastatic” cells in colon cancer primary tumors. vi) To validate the metastatic signature and show it has prognostic power in an independent validation archive of 350 colon cancers. vii) To develop immunohistochemical assays for detection of the metastatic signature.