Project Summary/Abstract Background The most common presenting symptom in patients with bladder cancer is hematuria. In two large studies, 5-8% of patients with microscopic hematuria (> 3 RBC/hpf) were noted to harbor bladder cancer (BCa). Published guidelines recommend these patients to obtain voided urinary cytology (VUC), in addition to cystoscopic evaluation. Cystoscopy is an invasive, uncomfortable and expensive procedure associated with side effects such as transient voiding symptoms, hematuria, UTI, and stenosis of the urethra, whereas, VUC has limited sensitivity of 25-40% in detecting BCa (specificity is >90%), especially for low-grade and low-stage tumors. While some commercially available urine-based assays for the detection of BCa are available, many suffer from a reduction in assay specificity compared to VUC (e.g., NMP-22 and BTA). Furthermore, as single markers, these assays, including VUC have insufficient predictive power to be applied to the management of individual patients, and importantly, these techniques are complex, and require skillful interpretation. The clinical impact of the proposal is to develop a more reliable and standardize assay that can detect BCa using a voided urine sample. Premise Our central premise is that our bladder cancer (BCa)- associated diagnostic signature may be successfully adopted to a multiplex bead-based immunoassay platform and utilized for the detection of BCa. Specific Aims: Specific Aim #1: To refine our prototype multiplex bead-based immunoassay such that it is enhanced for our ongoing prospective, multicenter validation studies. Significance If validated, the assay will be incorporated in the current R01 and could ultimately lead to a reduction in the need to subject large numbers of patients who do not have BCa to frequent, uncomfortable and expensive cystoscopic examinations. Methodology We will refine and optimize the multiplex assay and then test it in two large cohorts. prior to being incorporated into the current clinical trials associated with the R01. Expected Results Within this project, we will transform our prototype multiplex bead-based immunoassay into the final multiplex bead-based immunoassay that has been analytically and clinically validated and thus poised to undergo external multiple site validation in the prospective clinical trials associated with the current R01 grant.