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; 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.

项目摘要 膀胱癌是美国第六大癌症，根据一些研究， 每个患者一生最昂贵的癌症。但是当前的筛选测试的灵敏度很低，高 成本或需要侵入性手术，给早期发现该疾病带来了障碍。在先前的工作中，我领导 一个团队表明，大量尿液DNA的下一代测序可以用作敏感和特定的 无创检测膀胱癌的方法。我现在寻求通过解决这三个工作来建立这项工作 在筛查环境中使用尿肿瘤DNA作为生物标志物的主要障碍：1）大多数患者 转介出存在血尿或尿液中血液的膀胱癌筛查。这导致稀释 白细胞DNA的肿瘤DNA和基于测序测定的灵敏度降低。 2）批量测序 尿液DNA可能检测不会引起膀胱肿瘤的细胞类型的突变（例如克隆 造血），从而降低了测定特异性。 3）下一代的高成本和复杂性 测序方法限制了其可访问性。该建议旨在克服所有三个障碍，创造一个 实用的高性能诊断工作流程，用于膀胱癌筛查。 在AIM 1中，我将完善一种新的方法，以富集尿液中的尿路上皮细胞，称为细胞 按大小和选择性裂解（CESSL）富集。使用已知膀胱患者的尿液样品 然后，我将研究CESSL的肿瘤细胞富集的可实现程度 使用测序和显微镜。从尿液中耗尽非尿皮细胞将提高敏感性和 在筛查的人群中下游测定法的特异性。 在AIM 2中，我将开发一种基于尿液中膀胱肿瘤细胞检测尿液中的流式细胞仪测定法 非整倍性和/或全局降压甲基化的存在，两种DNA畸变存在于90％以上 膀胱肿瘤，高度针对癌症。然后，我将确定分析和临床表现 该测定法的特征以及CESSL对其性能的影响。 该建议将在约翰·霍普金斯医院（Johns Hopkins Hospital）在伯特·沃格斯坦（Bert Vogelstein）的指导下进行 MD。它将以科学顾问委员会为指导，包括病理学，泌尿外科，医学肿瘤学专家， 生物统计学和流式细胞仪。通过完成此建议，我将在基于细胞的基于细胞的方面发展新技能 诊断方法，诊断研究设计和实验室管理。我的目标是成为一个 基于实验室的独立医师科学家，他们发展了新颖的分子和基于细胞的诊断 实体瘤病理标本的工具，并评估其在临床试验中的效用。成功完成 这项研究将产生我可以完善并应用于其他病理样本类型的新颖方法 实验室以及对我们对膀胱癌测定法的前瞻性研究所需的数据 筛选，构成R01应用程序的基础。