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

项目摘要 膀胱癌是美国第六大常见癌症，根据一些研究， 每个患者一生中要处理的最昂贵的癌症。但目前的筛查试验灵敏度低，灵敏度高 成本高，或需要侵入性程序，对早期发现这种疾病构成了障碍。在之前的工作中，我领导 一个团队展示了对大量尿液DNA的下一代测序可以作为一种敏感和特异的 膀胱癌的非侵入性检测方法。我现在试图通过解决三个问题来加强这项工作 将尿路肿瘤DNA作为生物标志物用于筛查的主要障碍：1)大多数患者 诊断为膀胱癌筛查时出现血尿，或尿液中有血。这就导致了稀释 白细胞DNA对肿瘤DNA的影响以及基于测序的分析的敏感性降低。2)批量测序 尿DNA可能检测到不会导致膀胱肿瘤的细胞类型的突变(例如克隆性 造血学)，从而降低了检测的特异性。3)下一代的高成本和复杂性 测序方法限制了它们的可及性。这项提议寻求克服所有三个障碍，创造一个 实用、高性能的膀胱癌筛查诊断工作流程。 在目标1中，我将提炼一种从排尿中提取尿路上皮细胞的新方法，称为细胞 按大小浓缩和选择性裂解(CESSL)。使用已知膀胱患者的尿液样本 然后，我将研究CESSL对肿瘤细胞浓缩的可实现程度和普适性 使用测序和显微技术。去除尿液中的非尿路上皮细胞将提高敏感性和 用于筛查的人群中下游检测的特异性。 在目标2中，我将建立一种检测尿液中膀胱癌细胞的流式细胞仪检测方法，基于 存在非整倍体和/或全局低甲基化，这两种DNA异常存在于&gt；90% 对膀胱癌和癌症有高度的特异性。然后我将确定分析和临床表现 这个测试的特点，以及CESSL对其性能的影响。 这项提案将在伯特·沃格尔斯坦的指导下在约翰·霍普金斯医院实施， 马里兰州它将由一个科学顾问委员会指导，委员会成员包括病理学、泌尿学、内科肿瘤学、 生物统计学和流式细胞术。通过完成这项建议，我将发展基于细胞的新技能 诊断方法、诊断研究设计和实验室管理。我的目标是成为一名 独立的实验室医生-科学家，开发新的基于分子和细胞的诊断 用于实体肿瘤病理标本的工具，并评估其在临床试验中的用途。成功完成 这项研究将产生新的方法，我可以改进并在未来应用于其他类型的病理样本 实验室，以及启动我们的膀胱癌检测的前瞻性研究所需的数据 筛选，形成R01应用程序的基础。

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.