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

通过尿路上皮细胞富集和 DNA 流式细胞术早期检测膀胱癌

• 批准号: 10570645
• 负责人: Jonathan Carl Dudley
• 金额: $ 27.96万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2028-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10570645
• 关键词: Address; Aneuploidy; Award; Benign; Biological Assay; Biological Markers; Biometry; Biopsy; Biotechnology; Bladder; Bladder Neoplasm; Blood; Cancer Detection; Cancer Patient; Cancerous; Cell Size; Cells; Characteristics; Clinical; Clinical Trials; Clonal Expansion; Complex; Cytolysis; DNA; Data; Detection; Diagnostic; Diagnostic Procedure; Disease; Early Diagnosis; Excision; Flow Cytometry; Future; Gene Mutation; Goals; Graph; Hematopoiesis; Hematuria; Hospitals; Laboratories; Leukocytes; Malignant Neoplasms; Malignant neoplasm of urinary bladder; Medical Oncology; Mentorship; Methodology; Methods; Microscopy; Molecular; Mutation Detection; Nature; Noise; Non-Invasive Detection; Pathology; Patient-Focused Outcomes; Patients; Performance; Physicians; Population; Predisposition; Procedures; Prospective Studies; Research; Research Design; Retrospective cohort study; Sampling; Scientist; Screening for cancer; Sensitivity and Specificity; Solid Neoplasm; Specificity; Specimen; Testing; Training; Transitional Cell Carcinoma; Tumor Pathology; United States; Urine; Urology; Urothelial Cell; Urothelium; Work; cancer biomarkers; cancer cell; cell type; cost; deep sequencing; detection assay; diagnostic tool; improved; improved outcome; neoplastic cell; next generation sequencing; novel; performance tests; programs; screening; skills; specific biomarkers; tumor DNA; urinary

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.

项目概要 膀胱癌是美国第六大常见癌症，根据一些研究， 每个患者一生中治疗费用最高的癌症。但目前的筛查检测灵敏度低、 成本高，或需要侵入性手术，这给早期发现这种疾病带来了障碍。在之前的工作中，我领导 一个团队表明，大量尿液 DNA 的下一代测序可以作为一种敏感且特异的方法 膀胱癌的无创检测方法我现在寻求在这项工作的基础上解决以下三个问题： 在筛查中使用泌尿系肿瘤 DNA 作为生物标志物的主要障碍：1) 大多数患者 转诊膀胱癌筛查时出现血尿或尿液中有血。这导致稀释 肿瘤 DNA 被白细胞 DNA 识别，并且基于测序的检测的灵敏度降低。 2) 批量测序 尿液 DNA 可以检测不会引起膀胱肿瘤的细胞类型的突变（例如克隆性突变） 造血作用），从而降低测定特异性。 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。该提案旨在克服所有三个障碍，创造一个 用于膀胱癌筛查的实用、高性能诊断工作流程。 在目标 1 中，我将改进一种从排泄尿液中富集尿路上皮细胞的新方法，称为 Cell 按大小和选择性裂解富集 (CESSL)。使用已知膀胱患者的尿液样本 肿瘤，然后我将研究肿瘤细胞富集的可实现程度和 CESSL 的普遍性 使用测序和显微镜。从尿液中去除非尿路上皮细胞将提高敏感性和 筛选人群中下游检测的特异性。 在目标 2 中，我将开发一种流式细胞术检测方法，用于检测尿液中的膀胱肿瘤细胞 非整倍体和/或整体低甲基化的存在，这两种 DNA 畸变存在于 >90% 的 膀胱肿瘤和癌症高度特异性。然后我将确定分析和临床表现 该测定的特点，以及 CESSL 对其性能的影响。 该提案将在伯特·沃格尔斯坦（Bert Vogelstein）的指导下在约翰·霍普金斯医院进行， 医学博士。它将由一个科学顾问委员会指导，该委员会包括病理学、泌尿学、肿瘤内科、 生物统计学和流式细胞术。通过完成这个提案，我将开发基于细胞的新技能 诊断方法、诊断研究设计和实验室管理。我的目标是成为 独立实验室的医师科学家，开发新型分子和细胞诊断 实体瘤病理标本工具并评估其在临床试验中的效用。顺利完成 这项研究将产生新的方法，我可以在未来改进并应用于其他病理样本类型 实验室，以及启动膀胱癌检测前瞻性研究所需的数据 筛选，构成 R01 申请的基础。