BACKGROUND, AIMS: Genomewide expression profiling has identified a number of genes expressed at higher levels in colorectal cancer (CRC) than in normal tissues. Our objectives in this study were: 1) to test whether genes were also distinct on the protein level; 2) to evaluate these biomarkers in a series of well-characterized CRCs;, 3) to apply hierarchical cluster analysis to the immunohistochemical data. METHODS: Tissue microarrays (TMAs) comprising 351 CRC specimens from 270 patients were constructed to evaluate the genes Adam10, CyclinD1, Annexinll, NFKB, Casein-kinase-2-beta (CK2B), YB-1, P32, Rad51, c-fos, IGFBP4, Connexin26 (Cx26). In total, 3,797 samples were analyzed. RESULTS: Unsupervised hierarchical clustering discovered subgroups of CRC that differed by tumor stage, survival. KaplanMeier analysis showed that reduced Cx26 expression was significantly associated with shorter patient survival, higher tumor grade (G₁/G₂vs G₃, P = .02), Adam10 expression with a higher tumor stage (pT_1/2vs pT_3/4, P = .04). CONCLUSIONS: Our study highlights the potential of TMAs for a higher-dimensional analysis by evaluating serial sections of the same tissue core (three-dimensional TMA analysis). In addition, it endorses the use of immunohistochemistry supplemented by hierarchical clustering for the identification of tumor subgroups with diagnostic, prognostic signatures.