UICC stage II and III colorectal cancers (CRC) differ fundamentally in prognosis and therapeutic concepts. To analyze differential gene expression between both stages and to establish a relationship between molecular background and clinical presentation, tumor material from 36 unselected consecutive patients presenting with sporadic CRC, 18 UICC stage II and 18 UICC stage III, were laser microdissected to separate epithelial tumor cells. Gene expression levels were measured using U133A Affymetrix gene arrays. Twelve CRC associated signal transduction pathways as well as all 22,000 probe sets were screened for differential gene expression. We identified a signature consisting of 45 probe sets that allowed discrimination between UICC stage II and stage III with a rate of correct classification of about 80%. The most distinctive elements in this signature were the gene GSTP-binding elongation factor (GSPT2) and the transcription factor HOXA9. Differential expression of these genes was confirmed by quantitative real-time polymerase chain reaction (p (HOXA9) 5 0.04, p (GSTP2) 5 0.02). Despite the reliability of the presented data, there was no substantial differential expression of genes in cancer-related pathways. However, the comparison with recently published data corroborates the 45 gene signature showing structural agreement in the direction of fold changes of gene expression levels for our set of genes chosen to discriminate between both stages. ' 2006 Wiley-Liss, Inc.Key words: colorectal cancer; gene expression analysis; gene signature; classification; UICC stage II; UICC stage III In colorectal cancer (CRC), the prognosis and the type of adjuvant treatment is highly dependent upon the stage of disease. The current staging of CRC is predominantly based on clinical and pathological parameters such as local tumor extension, lymph node involvement and distant metastases. 1,2 More than 70% of patients with curative resection of CRC are classified as UICC stage II and III. While UICC stage II CRC is generally considered to be local and less aggressive with 5-year recurrence rates of 20-25%, tumors of stage UICC III are more dynamic by spreading metastases to lymph nodes. This results in a 5-year recurrence rate of more than 40% after radical resection 3 and constitutes the reason for recommendation of adjuvant therapy.