Program Official

Principal Investigator

Awardee Organization

University Of Tx Md Anderson Can Ctr
United States

Fiscal Year
Activity Code
Early Stage Investigator Grants (ESI)
Not Applicable
Project End Date

Rigorous and reproducible mutational analysis of the urinary exosomal DNA

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 doublestranded 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.


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  • Kugeratski FG, Kalluri R. Exosomes as mediators of immune regulation and immunotherapy in cancer. The FEBS journal. 2021 Jan;288(1):10-35. Epub 2020 Oct 3. PMID: 32910536
  • 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. Unique somatic variants in DNA from urine exosomes of individuals with bladder cancer. Molecular therapy. Methods & clinical development. 2021 May 29;22:360-376. doi: 10.1016/j.omtm.2021.05.010. eCollection 2021 Sep 10. PMID: 34514028
  • Kugeratski FG, McAndrews KM, Kalluri R. Multifunctional Applications of Engineered Extracellular Vesicles in the Treatment of Cancer. Endocrinology. 2021 Mar 1;162. (3). PMID: 33411885
  • Cao S, Wang JR, Ji S, Yang P, Dai Y, Guo S, Montierth MD, Shen JP, Zhao X, Chen J, Lee JJ, Guerrero PA, Spetsieris N, Engedal N, Taavitsainen S, Yu K, Livingstone J, Bhandari V, Hubert SM, Daw NC, Futreal PA, Efstathiou E, Lim B, Viale A, Zhang J, Nykter M, Czerniak BA, Brown PH, Swanton C, Msaouel P, Maitra A, Kopetz S, Campbell P, Speed TP, Boutros PC, Zhu H, Urbanucci A, Demeulemeester J, Van Loo P, Wang W. Estimation of tumor cell total mRNA expression in 15 cancer types predicts disease progression. Nature biotechnology. 2022 Nov;40(11):1624-1633. Epub 2022 Jun 13. PMID: 35697807
  • Kugeratski FG, Hodge K, Lilla S, McAndrews KM, Zhou X, Hwang RF, Zanivan S, Kalluri R. Quantitative proteomics identifies the core proteome of exosomes with syntenin-1 as the highest abundant protein and a putative universal biomarker. Nature cell biology. 2021 Jun;23(6):631-641. Epub 2021 Jun 9. PMID: 34108659
  • Kalluri R, McAndrews KM. The role of extracellular vesicles in cancer. Cell. 2023 Apr 13;186(8):1610-1626. PMID: 37059067
  • Xunian Z, Kalluri R. Biology and therapeutic potential of mesenchymal stem cell-derived exosomes. Cancer science. 2020 Sep;111(9):3100-3110. Epub 2020 Jul 28. PMID: 32639675
  • Kugeratski FG, LeBleu VS, Dowlatshahi DP, Sugimoto H, Arian KA, Fan Y, Huang L, Wells D, Lilla S, Hodge K, Zanivan S, McAndrews KM, Kalluri R. Engineered immunomodulatory extracellular vesicles derived from epithelial cells acquire capacity for positive and negative T cell co-stimulation in cancer and autoimmunity. bioRxiv : the preprint server for biology. 2023 Nov 4. PMID: 37961535