SUMMARY Upper Tract Urothelial Carcinoma (UTUC) is a rare disease that accounts for 5-10% of all urothelial tumors with an estimated annual incidence in Western countries of almost two cases per 100,000 inhabitants. The 5- year specific survival is < 50% for stage 2-3 patients but < 10% for those with stage 4 disease. This poor survival rate demands improvements in preoperative detection. Diagnosis and preoperative risk-stratification of UTUC patients present distinct challenges given the limitations of current available tools. Although urine cytology has an acceptable specificity to detect UTUC, the sensitivity is relatively low, particularly in low-grade carcinomas and pelvicalyceal tumors. The invasive ureteroscopy (URS) biopsy can enhance the diagnostic accuracy, yet under-grading and under-staging rates remain a concern at 32% and 46%, respectively. Nowadays, clinical decision-making and the prognosis of UTUC relies heavily on TNM stage and pathological grade that is not accurately available until radical nephroureterectomy (RNU) is performed. Major improvements in predictive tools have occurred in recent years and unique parameters have been proposed to distinguish between low- and high- risk tumors. DNA methylation alterations have emerged as potential diagnostic and prognostic factors for urothelial carcinoma at the molecular level. For example, bladder cancer-specific DNA methylation changes can be detected in urine sediments and can be used as non-invasive diagnostic or surveillance markers. Thus, a personalized medicine approach for the diagnosis and prognosis of UTUC is urgently needed. The goal of this study is to develop DNA methylation biomarkers to identify aggressiveness of UTUC. In a preliminary study, we identified differential DNA methylation patterns in a small group of patients who had exhibited DNA markers for UTUC aggressiveness. We hypothesize that bladder cancer DNA methylation marker panels can be used to: 1) characterize UTUC-specific and/or aggressive UTUC specific DNA methylation markers in primary UTUC specimens (Aim 1); 2) validate these markers in urine sediments of UTUC patients for monitoring disease diagnosis, prognosis and recurrence (Aim 2). Results from this proposal will support further independent investigations into the role of DNA methylation markers in UTUC diagnosis and prognosis, improve the standard of care in predictive tools and different parameters have been proposed to distinguish between low- and high- risk tumors and provide UTUC-specific DNA methylation profiles that aid physicians’ decisions.