Background: Current approaches to assessing clinical grade and stage in upper tract urothelial carcinoma (UTUC) have well-known limitations, sometimes leading to suboptimal assignment of therapy. Therefore, a urinary DNA (uDNA) next generation sequencing (NGS) biomarker test was developed for the purpose of enhanced clinical staging and grading using urine collected from patients with UTUC who underwent RNU.Hypotheses: (i) Mutations in UTUC tumor tissue (MT) are detectable in uDNA. (ii) Persistence of MU in the post-neoadjuvant uDNA will correlate with the presence of residual disease in the pathologic RNU specimen. (iii) Mutations which are specific to high stage or high grade disease will be detectable in the uDNA and correlate with pathological stage and grade of UTUC.Aims: (i) To correlate presence of MT with mutations in uDNA (MU). (ii) To correlate MU persistence status with nonresponder status. (iii) To correlate MU to pathological stage and grade in RNU specimens.Methodology: uDNA will be deep sequenced from 145 patients using a panel of 50 relevant genes. All patients underwent RNU ± neoadjuvant chemotherapy. Biomarker performance will be characterized against well-annotated benchmarks. pCR status and grade and stage of disease will be correlated to biomarker readouts using Fisher’s exact test. Preliminary data: uDNA was sequenced in 18 patients (12 with muscle-invasive bladder cancer, 6 with UTUC). The biomarker detected 61% of known mutations, had a 94% accuracy in assigning responder status, and identified high grade UTUC in 5/6 patients.Applicability to patients with urothelial cancer: Successful development of this biomarker test will significantly enhance the clinician’s capacity to accurately stage and grade UTUC extent.