Development of a Urine Genetic Test for Non-Muscle-Invasive Bladder Cancer There is an urgent unmet need for a test capable of comprehensive genetic characterization of non-muscle-invasive bladder cancer (NMIBC) to guide treatment and for recurrence surveillance. Bladder cancer (BC) is the 6th most common cancer in the US. Approximately 90% of BC cases are urothelial carcinoma and 75% are NMIBC at diagnosis. The treatments and prognoses are very different between NMIBC and muscle-invasive bladder cancer (MIBC). With limited distinguishing molecular features, NMIBC and MIBC are classified based on the morphology and depth of invasion, which is insufficient for reliable outcome prediction or treatment guidance. NMIBC is a heterogeneous subclassification (stages 0 and 1) of urothelial carcinoma and is characterized by high rates of recurrence and disease progression (as high as 40% within 2 years of initial treatment, with roughly 10% of all cases progressing to MIBC). Cancer is a disease of the genome; thus, BC genetic features should be the most definitive for its diagnosis, staging subclassification, and treatment guidance. Urine has been shown to contain both BC tumor cells and cell-free DNA (cfDNA). However, the complexity of the urine matrix and the low concentration of urinary cfDNA have so far precluded the application of urine BC cfDNA analysis for diagnosis and stratification. JBS Science Inc. has developed a novel, robust urine cfDNA isolation method that overcomes these limitations. We thus hypothesize that this cfDNA isolation method will enable the development of a urine genetic liquid biopsy for NMIBC. Such a test will provide comprehensive NMIBC genetic profiling to distinguish NMIBC from MIBC, guide disease management, and monitor recurrence. Two aims are proposed in this phase I feasibility study. Aim 1 is to develop a BC-targeted, hybridization-based NGS assay and data analysis pipeline. Aim 2 is to compare the tissue/urine and tissue/plasma variant concordances. In addition, a preliminary analysis will be performed of the feasibility of the NGS test being capable of distinguishing NMIBC from MIBC. The success of this phase I study will demonstrate the feasibility of developing a urine test for comprehensive NMIBC genetic characterization that can be used for BC diagnosis, recurrence monitoring, and distinguishing NMIBC from MIBC.

Bladder cancer (BC) is the 6th most common cancer in the US. Approximately 90% of BC cases are urothelial carcinoma and 75% are NMIBC at diagnosis. The treatments and prognoses are very different between NMIBC and MIBC. With limited distinguishing molecular features, NMIBC and MIBC are classified based on the morphology and depth of invasion. This project is to develop a noninvasive urine test to provide a comprehensive genetic characterization of non- muscle-invasive bladder cancer (NMIBC) to guide treatment and for recurrence surveillance.