p=0.002). In most cases, the HER2 expression of CTC was correlated to the HER2 gene ampliﬁcation status of the corresponding primary tumours in FISH analysis. CONCLUSIONS: CTCs can be found in the peripheral blood in up to one third of all patients with advanced bladder cancer. At one-year follow-up, patients with preoperatively proof of CTCs showed signiﬁcantly worse outcome. HER2 status in CTCs might be useful in assessing a targeted therapy. Source of Funding: Dr. Michael Rink received an investigational grant sponsored by VFK Berlin GmbH. 962 MOLECULAR ALTERATIONS IN BLADDER CANCER ASSOCIATED WITH CARCINOGEN EXPOSURE AND THEIR PROGNOSTIC IMPACT: A LOS ANGELES COUNTY EXPERIENCE Anirban Mitra*, Xuejuan Jiang, Jose Castelao, Debra Hawes, Denice Tsao-Wei, Shan Shi, Lillian Young, Los Angeles, CA; Ram Datar, Miami, FL; Eila Skinner, John Stein, Susan Groshen, Ronald Ross, Timothy Triche, Donald Skinner, Peter Jones, Los Angeles, CA; Richard Cote, Miami, FL INTRODUCTION AND OBJECTIVES: Urothelial carcinoma (UC) of the bladder develops through multiple cellular alterations. Previous multimarker UC studies do not account for risk factor expo- sure that can inﬂuence clinical outcome. Smoking is the most well established risk factor for UC in USA. This study sought to identify smoking-associated molecular alterations 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 in- cluded. Median follow up was 13.2 yrs. To analyze the clinical and molecular impact of smoking, we introduced novel variables – “smoke exposure” considered smoker status and smoking duration, and “smok- ing intensity” considered number of cigarettes smoked daily in addition to the above smoking parameters. For ex-smokers, the “relative ces- sation measure” considered duration of smoking cessation until diag- nosis and smoking duration. Bax, caspase-3, Apaf-1, Bcl-2, p53, p21, COX-2, VEGF, and E-cadherin 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), E-cad- herin (P=0.002), p21 (P=0.022) and Apaf-1 (P=0.047) expressions, and outcome (P0.001). COX-2 alterations were associated with smoke exposure (P=0.011), smoking duration (P=0.02), and relative cessation measure (P=0.015). All smoking variables were associated with outcome (P0.05). Ex-smokers who smoked longer and quit just before diagnosis had worse prognosis than those who smoked for a shorter duration and quit earlier (P=0.049). Apaf-1 (P=0.005), E- cadherin (P=0.014) and p53 (P=0.032) were univariately associated with outcome. E-cadherin 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=0.03), and both factors combined (P=0.025). Increasing number of altered markers was 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 combined (P=0.018). CONCLUSIONS: The study conﬁrms detrimental effects of smoking on UC prognosis. Apaf-1 was the most prognostic individual marker. Number of alterations was the most robust outcome predictor, independent of standard clinicopathologic and epidemiologic criteria, and can identify patients in need of more aggressive treatment. Source of Funding: NIH/NCI 963 DIAGNOSIS, METASTASIS AND RECURRENCE OF TRANSITIONAL CELL CARCINOMA OF THE BLADDER ARE ASSOCIATED WITH EXPRESSION OF HYALURONIC ACID REGULATORS Mario W. Kramer*, Hannover, Germany; Diogo Escudero, Rooz Golshani, Soum Lokeshwar, Miami, FL; Axel S. Merseburger, Markus A. Kuczyk, Hannover, Germany; Mark S. Soloway, Vinata B. Lokeshwar, Miami, FL INTRODUCTION AND OBJECTIVES: Hyaluronic acid (HA)- synthases 1, 2, 3, HA-receptors RHAMM and CD44 (CD44s, CD44v), and HYAL1 hyaluronidase regulate tumor growth and metastasis and some of these molecules are diagnostic and therapeutic targets. The expression of these molecules has not been measured either simulta- neously or prospectively in any normal or tumor tissues. In this study, we evaluated the expression of these seven molecules for their diag- nostic and prognostic potential in bladder cancer (BCa) and investi- gated their function in BCa cells. METHODS: HAS-1, 2, 3, CD44s, CD44v, RHAMM, HYAL-1 and beta-actin mRNA levels were measured, by qPCR, in prospectively collected 72 bladder tissues, and exfoliated urothelial cells from 136 urine specimens. The expression of these molecules in bladder tissues (n=72) was further conﬁrmed by immunohistochemistry. We examined HAS functions in BCa cells by proliferation, apoptosis and Matrigel invasion assays, following siRNA treatment for HAS-1,2,3. RESULTS: HAS1, 2, 3, HYAL-1 and RHAMM mRNA levels were elevated 6-13-fold in BCa tissues (P0.001), CD44s levels de- creased (3-7-fold; P0.001), and CD44v levels remained unaltered. In multivariate analysis, HYAL-1 levels independently associated with metastasis (chi-square = 4.47; P=0.035; RR=1.32; 81% sensitivity, 91.3% speciﬁcity). Immunohistochemistry showed increased expres- sion of HAS1, 2, 3 and HYAL-1 in BCa tissues, but only HYAL-1 staining independently associated with metastasis. In exfoliated cells from BCa patients’ urine, HAS1, 2, 3 and HYAL-1 mRNA levels in- creased (P0.001) but CD44s levels decreased (P=0.004); CD44v and RHAMM levels remained unaltered. HAS1,2,3 and HYAL-1 mRNA levels had 72.3% sensitivity to detect BCa with HAS2 and HYAL-1 having higher speciﬁcity (79.5-80.7%). HAS2-HYAL-1 combination had higher sensitivity (87.2%) and speciﬁcity (77.1%). False positive HAS2-HYAL-1 inference indicated BCa-recurrence within 6 months (chi 2=11.2; P=0.008; RR=6.9). Down regulation of HAS1 and HAS2 expression by siRNA inhibited BCa cell growth, caused 2-2.5-fold induction of apoptosis and inhibited invasion by 40-50%. CONCLUSIONS: This is the ﬁrst study that simultaneously measured the expression of all HA-family markers in cancer. HYAL-1 expression correlates with BCa-metastasis. HAS2-HYAL-1 combina- tion detects BCa with high accuracy and possibly predicts BCa-recur- rence. Similar to HYAL1 hyaluronidase, HA-synthases promote BCa cell growth, invasion, and survival. Source of Funding: None 964 POLYMORPHISMS IN NITRIC-OXIDE SYNTHASE 2 INFLUENCE URINARY-BLADDER CANCER PATHOGENESIS Charlotta Ryk, Gunnar Steineck, N Peter Wiklund, Tommy Nyberg, Petra de Verdier*, Stockholm, Sweden INTRODUCTION AND OBJECTIVES: We analyzed if inherited genetic variants, polymorphisms, in the nitric oxide synthase 2 (NOS2, also known as iNOS) inﬂuence urinary-bladder cancer pathogenesis METHODS: 359 bladder-cancer patients, selected from a pop- ulation-based patient material, and 164 population controls were geno- typed for the promoter repeat polymorphism (CCTTT)n and the exonic Ser608Leu (C/T) polymorphism. We used fragment analysis, allelic discrimination and DNA sequencing for genotyping. Sequencing was used as quality control. Patient genotypes were combined with infor- mation on tumor stage, grade, progression and cancer-speciﬁc death, using a 5-year clinical follow-up. e374 THE JOURNAL OF UROLOGY Vol. 183, No. 4, Supplement, Monday, May 31, 2010