Preclinical data indicate that A6B4 integrin signaling through Ras homolog gene family, member A, plays an important role in tumor cell motility. The objective of this study was to determine whether the combined expression of A6B4 integrin and neuroepithelioma transforming gene 1 (Net1), a guanine nucleotide exchange factor specific for Ras homolog gene family member A, is associated with adverse clinical outcome in breast cancer patients. Immunohistochemical expression of each protein was evaluated in a tumor tissue microarray prepared from the primary tumors of 94 node-positive patients with invasive breast carcinoma treated with total mastectomy and doxorubicinbased chemotherapy without radiation with a median follow-up of 12.5 years. Associations between staining results and multiple clinicopathologic variables were investigated. Although there was no significant association between A6B4 integrin or Net1 expression and clinical outcome when each marker was considered individually, coexpression of A6B4 and Net1 was associated with decreased distant metastasis -free survival (P = 0.030). In the subset of patients with hormone receptor -positive tumors, coexpression of A6B4 and Net1 was associated with a decrease in distant metastasis -free and overall survival (P < 0.001 and P = 0.006, respectively). Although an association between human epidermal growth factor receptor 2 expression and coexpression of A6B4 and Net1 (P = 0.008) was observed, coexpression of A6B4 and Net1 (hazard ratio, 1.63; P = 0.02) and lymphovascular invasion (hazard ratio, 2.35; P = 0.02) were the only factors independently associated with the development of distant metastasis in multivariate analysis. These findings suggest that coexpression of A6B4 integrin and Net1 could be a useful biomarker for aggressive disease in node-positive breast cancer patients. (Cancer Epidemiol Biomarkers Prev 2009;18(1):80 -6)