Project Summary/Abstract Background The non-invasive diagnosing of bladder cancer in patients with hematuria, the most common clinical presentation of bladder cancer, or in subjects with a history of bladder cancer on tumor surveillance remains a challenge, and as such, these patients require invasive testing. Current urine-based assays are not robust and therefore cannot be used to ‘rule out’ patients who do not require these invasive testing. Previously, we a) identified a bladder cancer-associated diagnostic protein “fingerprint” comprised of 10 biomarkers, b) developed a multiplex immunoassay to query these 10 biomarkers in voided urine samples and c) performed analytical validation of the multiplex immunoassay. Using the multiplex immunoassay, we have generated encouraging preliminary data in 326 subjects (46 cancer) noting a sensitivity and specificity of 93%. Thus, for the first time, we possess an accurate assay that can be used to ‘rule out’ patients who do not require invasive testing. Furthermore, early detection (i.e., detection prior to clinical manifestation) is an important goal for patients at risk for bladder cancer. At presentation, more than 70% of bladder cancer cases are non- muscle invasive bladder cancer (NMIBC), whilst the remaining 30% are muscle invasive bladder cancer (MIBC) or metastatic. When detected early (i.e., NMIBC), the 5-year survival rate is approximately 94%, compared to a 5-year survival rate of ~50% when the disease is detected as muscle invasive bladder cancer (MIBC), and <20% when the disease is metastatic. However, to date, no early detection assay is available. In a small cohort of 20 bladder cancer patients and 20 matched controls from our ongoing R01 studies, we have noted an elevation in our bladder cancer-associated diagnostic protein “fingerprint” as early as 18 months prior to the clinical diagnosis of bladder cancer and an actual positive multiplex immunoassay in all bladder cancer patients 12 months prior to the clinical diagnosis of bladder cancer. Hypothesis: A bladder cancer-associated diagnostic protein “fingerprint” exists that can be leveraged to indicate the presence of bladder cancer in non- invasively obtained urine samples not only at the time of diagnosis, but prior to the clinical presentation and diagnosis of bladder cancer. Specific Aims: 1) To validate the multiplex immunoassay for bladder cancer detection in a large, nested case-control study (n=800) and 2) To evaluate the multiplex immunoassay for early detection in a large, nested case-control study (n=600). Significance This research will open the door for improving on the non-invasive methods for detecting bladder cancer and as such it will have a marked impact on patient care. Methodology Previously, our group has discovered and performed early validation of bladder cancer-associated diagnostic protein” fingerprint’ with extremely encouraging results. In the current proposal, we now seek to test the multiplex immunoassay in ...