Rigorous and reproducible mutational analysis of the urinary exosomal DNA

对尿液外泌体 DNA 进行严格且可重复的突变分析

• 批准号: 10669649
• 负责人: COLIN P.N. DINNEY
• 金额: $ 60.5万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10669649
• 关键词: Affinity Chromatography; Alleles; Bioinformatics; Biological; Biological Markers; Biology; Biopsy; Cancer Patient; Capillary Electrophoresis; Cells; Clinical; Collaborations; Collection; Computer Analysis; DNA; DNA sequencing; Data; Detection; Development; Digestion; Dropout; Drops; Genes; Genomics; Goals; Heterogeneity; Institution; Liquid substance; Malignant Neoplasms; Malignant neoplasm of urinary bladder; Methodology; Molecular Sieve Chromatography; Mutate; Mutation; Mutation Analysis; Noise; Nucleic Acids; Oncogenic; Patients; Plasma; Population; Predictive Value; Procedures; Publishing; Quality Control; Reporting; Reproducibility; Research; Sampling; Serum; Signal Transduction; Skeleton; Source; Spectrophotometry; System; Tissues; Ultracentrifugation; Uncertainty; Urine; Urology; Validation; Variant; cancer type; cell free DNA; clinical application; comparative; driver mutation; ds-DNA; exome sequencing; exosome; improved; nano; process optimization; reconstruction; screening; single-cell RNA sequencing; targeted treatment; transcriptome sequencing; tumor; tumor DNA; tumor progression; urinary; whole genome

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.

抽象的 外泌体均由所有细胞释放，并在各种细胞种群之间携带生物活性分子， 对靶细胞和组织的生物学有重大影响。我们的小组是第一个确定双重的人 滞留的DNA在外泌体中，并报告外泌体中发现的腔内DNA片段 （Exodna）覆盖整个基因组，并反映原点细胞的突变特征。随后， 来自癌症患者血清的外射已用于检测致癌突变。已发表的研究 预测患者血清/血浆外埃及纳的潜在使用用于筛查可操作的治疗靶标和 生物标志物。但是，迄今为止，这种方法缺乏严格，可重复和无偏的分析 临床应用所需的程序。我们的初步研究强调了系统的需求 优化外泌体收集，外eDNA隔离，放大，测序和 计算分析。我们的总体目标是生成一条快速，敏感和可重复的管道 从尿液样品中的出埃及纳埃及区中进行了严格选择，以识别驱动器突变，新的 可操作的治疗靶标和生物标志物。从膀胱尿液中的出埃及纳的初步分析 癌症患者使用整个外显子组测序（WES）显示肿瘤中有多个驱动器突变 在泌尿外埃及纳中，表明对于膀胱癌，泌尿外埃及纳在 使用肿瘤DNA鉴定的突变特征的表示条款。 WES数据的质量分析 揭示了可能限制出埃及纳的预测价值并敦促发展的重大差异 更严格和可重复的方法。我们假设尿液外埃及纳分离和 可以通过逐步优化外显子体中使用的过程和过程来简化分析 收集，DNA隔离，整个基因组扩增（WGA）和计算分析。我们的 调查团队汇集了外泌体生物学，生物信息学，临床和 实验泌尿科。为了实现指定目标，我们建议：（1）确定最佳外泌体的程序 从尿液中收集和外埃及纳萃取； （2）优化整个基因组扩增程序，建立 质量控制面板并确定最小外何数输入以进行质量分析； （3）确定 快速且可重复地识别突变和生物标志物的计算程序 出埃及娜； （4）在内部和外部对既定方法进行严格的独立验证 合作者。拟议的研究应确定尿尿的有效性毫无疑问 出埃及纳用于膀胱癌突变分析，并为 最佳的外射离分离和分析可用于其他出埃及区源和癌症类型。

项目成果

Quantitative proteomics identifies the core proteome of exosomes with syntenin-1 as the highest abundant protein and a putative universal biomarker.

• DOI: 10.1038/s41556-021-00693-y
• 发表时间: 2021-06
• 期刊: Nature cell biology
• 影响因子: 21.3

Unique somatic variants in DNA from urine exosomes of individuals with bladder cancer.

• DOI: 10.1016/j.omtm.2021.05.010
• 发表时间: 2021-09-10
• 期刊: Molecular therapy. Methods & clinical development
• 作者: Zhou X;Kurywchak P;Wolf-Dennen K;Che SPY;Sulakhe D;D'Souza M;Xie B;Maltsev N;Gilliam TC;Wu CC;McAndrews KM;LeBleu VS;McConkey DJ;Volpert OV;Pretzsch SM;Czerniak BA;Dinney CP;Kalluri R
• 通讯作者: Kalluri R