Rigorous and reproducible mutational analysis of the urinary exosomal DNA

摘要 外切体由所有细胞释放，并在不同的细胞群之间携带生物活性分子， 对目标细胞和组织的生物学有重大影响。我们的团队是第一个发现双重- 在外体中发现滞留的DNA，并报告在外体中发现的腔内DNA片段 (ExoDNA)覆盖整个基因组，并反映起源细胞的突变概况。后来， 癌症患者血清中的外源DNA已被用于检测致癌突变。已发表的研究 预测患者血清/血浆外源DNA可能用于筛选可操作的治疗靶点和 生物标志物。然而，到目前为止，这种方法缺乏严谨、可重复和不偏不倚的分析 临床应用所需的程序。我们的初步研究强调了系统地 优化外切体收集、外切DNA分离、扩增、测序和 计算分析。我们的首要目标是为以下项目生成快速、敏感且可重复的管道 从尿样中严格选择外源DNA的体细胞变异，以确定驱动突变，新 可操作的治疗靶点和生物标记物。膀胱尿液中外源DNA的初步分析 癌症患者使用完整外显子组测序(WES)发现肿瘤中存在多个驱动因素突变 结果表明，对于膀胱癌，尿液中的外源DNA优于血清外源DNA。 使用肿瘤DNA鉴定的突变图谱的表示术语。WES数据的质量分析 发现了可能限制外源DNA的预测价值并促使其发展的显著差异 更严谨和可重复的方法。我们假设尿液外源DNA的分离和 通过逐步优化外切体中使用的过程和程序，可以简化分析 收集、DNA分离、全基因组扩增(WGA)和计算分析。我们的 调查小组汇集了外显体生物学、生物信息学、临床和医学领域的领先专家 实验泌尿学。为了达到指定的目标，我们建议：(1)确定最佳外切体的程序 从尿液中收集和提取外源DNA；(2)优化全基因组扩增程序，建立 质量控制小组，并确定用于质量分析的最小外源DNA输入； 用于快速和可重复地识别突变和生物标志物的计算程序 外部DNA；(4)对已建立的内部和外部方法进行严格的独立验证 合作者。建议的研究应排除合理怀疑，确定尿液的有效性。 ExoDNA用于膀胱癌的突变分析，并提供严格和可重复性的方法 优化的外源DNA分离和分析，可应用于其他外源DNA和癌症类型。

Abstract Exosomes are released by all cells and carry bioactive molecules between diverse cell populations, with significant impact on the biology of target cells and tissues. Our group was the first to identify double- stranded DNA in exosomes and to report that collectively, intraluminal DNA fragments found in exosomes (exoDNA) cover the entire genome and reflect the mutational profiles of the cells of origin. Subsequently, exoDNA from the sera of cancer patients have been used to detect oncogenic mutations. Published studies predict potential use of patient serum/plasma exoDNA for screening for the actionable therapy targets and biomarkers. However, to date, this methodology lacks rigorous, reproducible, and unbiased analytical procedures required for clinical application. Our preliminary studies underscore the need for systematic optimization of the procedures for exosome collection, exoDNA isolation, amplification, sequencing, and computational analysis. Our overarching goal is to generate a rapid, sensitive, and reproducible pipeline for rigorous selection of somatic variants in exoDNA from urine samples, to identify driver mutations, new actionable therapy targets, and biomarkers. Preliminary analysis of exoDNA from the urines of bladder cancer patients using whole exome sequencing (WES) revealed multiple driver mutations in the tumors and in urinary exoDNA and showed that for bladder cancer, urinary exoDNA was superior to serum exoDNA in terms of representation of mutational profiles identified using tumor DNA. Quality analysis of the WES data revealed significant discrepancies that could limit the predictive value of exoDNA and urge the development of more rigorous and reproducible methodologies. We hypothesize that urine exoDNA isolation and analysis could be streamlined by stepwise optimization of processes and procedures used in exosome collection, DNA isolation, whole genome amplification (WGA), and computational analysis. Our investigative team brought together leading experts in exosome biology, bioinformatics, clinical and experimental urology. To attain designated goals, we propose to: (1) Identify procedures for optimal exosome collection and exoDNA extraction from urine; (2) Optimize whole genome amplification procedures, establish quality control panels and determine the minimal exoDNA input for quality analyses; (3) Determine computational procedures for rapid and reproducible identification of mutations and biomarkers using exoDNA; (4) Perform rigorous independent validation of established methodologies internally and by outside collaborators. The proposed studies should establish beyond reasonable doubt the validity of urinary exoDNA for mutational analysis in bladder cancer and provide rigorous and reproducible methodologies for optimal exoDNA isolation and analysis that can be applied for other exoDNA sources and cancer types.