Early detection of bladder cancer through urothelial cell enrichment and DNA flow cytometry

Project Summary Bladder cancer is the sixth most common cancer in the United States, and according to some studies, the most expensive cancer to manage per patient lifetime. But current screening tests have low sensitivity, high cost, or require invasive procedures, presenting a barrier to the early detection this disease. In prior work, I led a team showing that next-generation sequencing of bulk urinary DNA can serve as a sensitive and specific method for the noninvasive detection of bladder cancer. I now seek to build on this work by addressing three major obstacles to the use of urinary tumor DNA as a biomarker in a screening context: 1) Most patients referred for bladder cancer screening present with hematuria, or blood in their urine. This results in the dilution of tumor DNA by leukocyte DNA and decreased sensitivity of sequencing-based assays. 2) Bulk sequencing of urinary DNA may detect mutations in cell types that do not give rise to bladder tumors (e.g. clonal hematopoiesis), thereby decreasing assay specificity. 3) The high cost and complexity of next-generation sequencing methods limits their accessibility. This proposal seeks to overcome all three obstacles, creating a practical, high performance diagnostic workflow for bladder cancer screening. In Aim 1, I will refine a novel method for the enrichment of urothelial cells from voided urine, called Cell Enrichment by Size and Selective Lysis (CESSL). Using urine samples from patients with known bladder tumors, I will then investigate the achievable degree of tumor cell enrichment and generalizability of CESSL using sequencing and microscopy. Depletion of non-urothelial cells from urine will improve the sensitivity and specificity of downstream assays in the population that presents for screening. In Aim 2, I will develop a flow cytometry assay for the detection of bladder tumor cells in urine based on the presence of aneuploidy and/or global hypomethylation, two DNA aberrations that are present in >90% of bladder tumors and highly specific for cancer. I will then determine the analytical and clinical performance characteristics of this assay, as well as the impact of CESSL on its performance. This proposal will be carried out at the Johns Hopkins Hospital under the mentorship of Bert Vogelstein, MD. It will be guided by a scientific advisory board including experts in pathology, urology, medical oncology, biostatistics, and flow cytometry. Through completion of this proposal, I will develop new skills in cell-based diagnostic methods, diagnostic study design, and laboratory management. My goal is to become an independent laboratory-based physician-scientist who develops novel molecular and cell-based diagnostic tools for solid tumor pathology specimens and evaluates their utility in clinical trials. Successful completion of this study will produce novel methods that I can refine and apply to other pathology sample types in my future laboratory, as well as the data necessary to initiate a prospective study of our assay for bladder cancer screening, forming the basis of an R01 application.