PROJECT SUMMARY/ABSTRACT Muscle-invasive bladder cancer (MIBC) is optimally treated with neoadjuvant chemotherapy followed by radical cystectomy (RC), whereby ~35% of patients will have a pathologic complete response (pCR). Given the morbid, complicated, and expensive nature of RC and the well-established pCR rate, there is a groundswell of interest in RC avoidance for patients achieving pCR. However, identifying pCR clinically (as opposed to pathologically) is an inaccurate process. In published studies, patients who avoid RC after being deemed clinical complete responders have a 25-60% likelihood of recurrence, metastasis, or bladder cancer mortality. Better tools to assess residual disease status are needed. To address this need, we developed a urine biopsy test which we call UTeRD (Urine Test for Residual Disease). In UTeRD, DNA is isolated from urine and subjected to next generation sequencing to detect point mutations in a targeted panel of genes. Using UTeRD, most mutations in tumor tissue are detectable as mutations in urine. Further, presence or absence of residual MU after completion of chemotherapy strongly associates with residual disease or pCR at the time of RC, respectively. Therefore, UTeRD could be used after neoadjuvant therapy to better identify patients for RC avoidance. Although UTeRD performs well in distinguishing patients with pCR from residual disease, the negative predictive value (NPV) of UTeRD is only 76%, some urine samples were nondiagnostic, and a urinary DNA preservation protocol needs to be developed in order for the test to be widely adopted. Pre-analytical factors and methodology improvements which we believe will increase the NPV and decrease nondiagnostic rates will be studied in Aim 1. In Aim 2, we will determine if urine preservatives can be used to facilitate shipping to a centralized lab without loss of fidelity of the test. Lastly, in Aim 3, we will determine if the absence of mutations from a urine biopsy is associated with pCR regardless of the pre-surgical therapy. To answer this question, samples obtained on 5 prospective MIBC clinical trials from multiple institutions will be studied using the optimized protocols identified through this research. The research team is comprised of a urologist, medical oncologists, a radiation oncologist, a statistician, a computational biologist who are experts in their fields. The skills and contributions of the team are complimentary and will culminate in the development of a unique and robust biomarker that addresses a significant clinical need using a one-of-a-kind sample cohort. UTeRD may enhance the ability of a bladder cancer clinician to answer highly relevant clinical question, namely, “Does this patient have residual disease after pre- surgical therapy, and therefore, will he/she benefit from RC?”

PROJECT NARRATIVE (RELEVANCE TO PUBLIC HEALTH) Radical cystectomy is a morbid, expensive, and complicated procedure which requires urinary diversion, usually with a urostomy. Because the response rates to pre-surgical chemotherapy and other therapies is high, many patients achieve complete response and therefore may be cured without radical cystectomy. Our intention is to further develop UTeRD, a urine biopsy test, for the purpose of distinguishing responders and nonresponders so that responders can be accurately detected and be triaged into radical cystectomy avoidance algorithms.