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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

基本信息

批准号：
10431912
负责人：
COLIN P.N. DINNEY
金额：
$ 60.45万
依托单位：
UNIVERSITY OF TX MD ANDERSON CAN CTR
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-07-01 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10431912
关键词：
Affinity Chromatography Alleles Bioinformatics Biological Biological Markers Biology Biopsy Cancer Patient Cells Clinical Collection Computer Analysis DNA DNA sequencing Data Detection Development Digestion Dropout Electrophoresis 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 base cancer type cell free DNA clinical application comparative driver mutation ds-DNA exome sequencing exosome improved process optimization reconstruction screening single-cell RNA sequencing targeted biomarker targeted treatment transcriptome sequencing tumor tumor DNA tumor progression urinary whole genome

项目摘要

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.

摘要