Rigorous and reproducible mutational analysis of the urinary exosomal DNA

摘要 外泌体由所有细胞释放并在不同细胞群之间携带生物活性分子， 对靶细胞和组织的生物学具有显著影响。我们是第一个发现双重- 外泌体中DNA链，并共同报道外泌体中发现的管腔内DNA片段 外切DNA（exoDNA）覆盖整个基因组并反映起源细胞的突变谱。随后，委员会注意到， 来自癌症患者血清的exoDNA已被用于检测致癌突变。发表的研究 预测患者血清/血浆exoDNA用于筛选可操作的治疗靶标的潜在用途，以及 生物标志物。然而，到目前为止，这种方法缺乏严格的，可重复的，无偏见的分析， 临床应用所需的程序。我们的初步研究强调， 优化用于外泌体收集、外泌DNA分离、扩增、测序的程序， 计算分析我们的首要目标是为客户提供一个快速、灵敏和可复制的管道， 严格选择尿液样本中exoDNA的体细胞变体，以识别驱动突变，新的 可行的治疗靶点和生物标志物。膀胱尿液中exoDNA的初步分析 使用全外显子组测序（WES）的癌症患者揭示了肿瘤中的多个驱动突变， 结果显示，对于膀胱癌，尿exoDNA在以下方面上级优于血清exoDNA： 使用肿瘤DNA鉴定的突变谱的代表性术语。WES数据的质量分析 揭示了可能限制exoDNA预测价值的重大差异， 更严格和可重复的方法。我们假设尿液exoDNA分离和 可以通过逐步优化外泌体中使用的过程和程序来简化分析 收集、DNA分离、全基因组扩增（WGA）和计算分析。我们 研究小组汇集了外来体生物学、生物信息学、临床和 实验泌尿学为了达到指定的目标，我们建议：（1）确定最佳外泌体的程序 从尿液中收集和提取exoDNA;（2）优化全基因组扩增程序，建立 质量控制面板，并确定用于质量分析的最小exoDNA输入;（3）确定 用于快速和可再现地鉴定突变和生物标志物的计算程序， （4）对内部和外部建立的方法进行严格的独立验证 合作者拟议的研究应排除合理怀疑， exoDNA用于膀胱癌中的突变分析，并提供严格和可重复的方法， 最佳的exoDNA分离和分析，可以应用于其他exoDNA来源和癌症类型。

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.