Abstract INTRODUCTION: Urothelial carcinoma (UC) of the bladder develops through alterations in several cellular processes. Previous UC multimarker studies do not account for risk factor exposure that can influence clinical outcome. Cigarette smoking is the most well established risk factor for UC in the U.S. This study sought to identify molecular alterations associated with smoking and their prognostic value in a population-based cohort. METHODS: 212 UC patients from the Los Angeles County Cancer Surveillance Program, a NCI/SEER cancer registry, were included. Median follow up was 13.2 years. To analyze the biologic and molecular impact of smoking, we introduced novel variables - “smoke exposure” considered smoker status and duration of smoking, and “smoking intensity” considered number of cigarettes smoked daily in addition to the above smoking parameters. For ex-smokers, the “relative cessation measure” considered duration of smoking cessation until diagnosis and duration of smoking. Bax, caspase-3, Apaf-1, Bcl-2, p53, p21, COX-2, VEGF, and ECAD immunohistochemical expressions were analyzed on archival UC sections. Covariate and clinical outcome (overall survival) associations were examined. RESULTS: Stage was associated with p53 (P<0.001), ECAD (P=0.002), p21 (P=0.022) and Apaf-1 (P=0.047), and outcome (P<0.001). Number of cigarettes smoked daily was associated with Bax (P=0.008) expression. COX-2 alterations were associated with smoke exposure (P=0.011), duration of smoking (P=0.020), and relative cessation measure (P=0.015). All smoking variables were associated with clinical outcome (P<0.050). Ex-smokers who smoked longer and quit shortly before diagnosis had a poorer prognosis than those who smoked for a shorter duration and quit earlier (P=0.049). Apaf-1 (P=0.005), ECAD (P=0.014) and p53 (P=0.032) were univariately associated with outcome. ECAD remained prognostic after stratification by smoking (P=0.001). Apaf-1 was the most valuable individual marker, being prognostic after stratification by stage (P=0.029), smoking (P=0.030), and both factors (P=0.025). The number of altered markers was associated with the relative cessation measure (P=0.050). Increasing alterations were univariately associated with worse prognosis (P<0.001). This remained significant after stratifying by stage (P=0.002), smoking (P<0.001), and both factors (P=0.018). CONCLUSION: The study confirms the detrimental effect of smoking on UC prognosis and identifies key molecules that are deregulated by the carcinogenic exposure. Apaf-1, ECAD and p53 were important individual predictors of outcome, with Apaf-1 being prognostic after stratifying for stage and/or smoking. Number of altered markers was the most robust outcome predictor, independent of standard clinicopathologic and epidemiologic criteria, and can be used as a tool to identify patients who are in need of more aggressive treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4702.