# Rigorous and reproducible mutational analysis of the urinary exosomal DNA

> **NIH NIH R01** · UNIVERSITY OF TX MD ANDERSON CAN CTR · 2020 · $672,829

## Abstract

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.

## Key facts

- **NIH application ID:** 9963160
- **Project number:** 5R01CA231465-02
- **Recipient organization:** UNIVERSITY OF TX MD ANDERSON CAN CTR
- **Principal Investigator:** COLIN P.N. DINNEY
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $672,829
- **Award type:** 5
- **Project period:** 2019-07-01 → 2024-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9963160

## Citation

> US National Institutes of Health, RePORTER application 9963160, Rigorous and reproducible mutational analysis of the urinary exosomal DNA (5R01CA231465-02). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/9963160. Licensed CC0.

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