Project Summary/Abstract Background Microscopic hematuria (blood in urine) may occur in up to 10% of the general population and results in costly evaluation to ensure it is of no consequence, i.e., no bladder cancer (BCa) is present since hematuria is typically the initial sign of BCa. Furthermore, 75% of patients with newly diagnosed BCa have non- muscle-invasive disease (NMIBC), which has a very high recurrence rate (>70%). Because of this, it is recommended that the patients have adjuvant intravesical bacillus Calmette-Guerin (BCG) instillation to reduce this risk. Despite BCG treatment, up to 50% of patients fail to respond and 20% progress to muscle invasive bladder cancer (MIBC) mandating more radical treatment (i.e., cystectomy - removal of the urinary bladder). Moreover, the delay in radical treatment can negatively impact survival rates, hence, a test that could predict treatment response to intravesical BCG, ensuring the right patient, gets the right treatment at the right time is urgently required. To date, no such test is available. There exists an unmet clinical need for reliable biomarkers to a) ‘rule out’ which patients with microscopic hematuria do not require further evaluation and b) predict which patients will respond to BCG. Hypothesis Our central hypothesis is that a molecular profile exists that is specifically associated with BCa that a) can be utilized to indicate the presence of BCa in non-invasively obtained urine samples and b) can be utilized to predict response to BCG. This hypothesis will be tested by completing the following Specific Aims: 1) To validate the multiplex immunoassay for BCa detection in subjects presenting with microscopic hematuria, 2) To evaluate the cost benefit, cost utility and cost effectiveness of the multiplex immunoassay in subjects with hematuria and a history of BCa on tumor surveillance and 3) To validate prospectively the urine-based multiplex immunoassay to predict BCG response in subjects with intermediate/high risk NMIBC. Significance This research will open the door for improving on the non-invasive methods for detecting BCa and predicting treatment response as such it will have a marked impact on patient care. Methodology Our group has developed and tested a multiplex immunoassay towards a BCa signature with extremely encouraging results in subjects evaluated for gross hematuria and a history of BCa on tumor surveillance. In the current proposal, we now seek to test the multiplex assay in two additional independent cohorts: intermediate/high-risk patients (AUA microscopic hematuria guidelines 2020) presenting with microscopic hematuria for early BCa detection and patients with intermediate/high risk NMIBC treated with intravesical BCG to predict treatment response. Furthermore, we will generate Markov models and using data collected from the parent R01 to assess cost benefit, cost utility and cost effectiveness of the multiplex immunoassay. Expected Results The validation of the multiplex immunoassay will reduce the need to subject large numbers of patients who do not have BCa to frequent, uncomfortable and expensive cystoscopic examinations. Specifically, we would anticipate a reduction in the number of cystoscopies/year by 500,000, which would translate into cost savings of $225 million/year.

Project Narrative The project seeks to validate a more reliable and accurate non-invasive assay to be used to detect bladder cancer and predict response to therapy and which, over the long-term, could have a profound impact on care of bladder cancer patients leading to improved care and reduced healthcare costs.