Acute myeloid leukemia (AML) is a heterogeneous malignant disease characterized by a collection of genetic and epigenetic changes. As a consequence, AML can evolve towards more aggressive subtypes during treatment, which require additional therapies to prevent future relapse. As we have previously detected double-stranded DNA (dsDNA) in tumor-derived extracellular vesicles (EVs), in this current study we attempted to evaluate the potential diagnostic applications of AML EV-dsDNA derived from primary bone marrow and peripheral blood plasma samples. EVs from plasma of 29 pediatric AML patients (at initial diagnosis or during treatment) were isolated by ultracentrifugation, after which dsDNA was extracted from obtained EVs and analyzed for leukemia-specific mutations using next generation sequencing (NGS) and GeneScan-based fragment-length analysis. In 18 out of 20 patients, dsDNA harvested from EVs mirrored the (leukemia-specific) mutations found in the genomic DNA obtained from primary leukemia cells. In the nanoparticle tracking analysis (NTA), a decrease in EV numbers was observed in patients after treatment compared with initial diagnosis. Following treatment, in 75 samples out of the 79, these mutations were no longer detectable in EV-dsDNA. In light of our results, we propose the use of leukemia-derived EV-dsDNA as an additional measure for mutational status and, potentially, treatment response in pediatric AML.
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Aziz H, Ping CY, Alias H, Ab Mutalib NS, Jamal R (2017) Gene mutations as emerging biomarkers and therapeutic targets for relapsed acute myeloid leukemia. Front Pharmacol 8:897. https://doi.org/10.3389/fphar.2017.00897
Grove CS, Vassiliou GS (2014) Acute myeloid leukaemia: a paradigm for the clonal evolution of cancer? Dis Model Mech 7(8):941–951. https://doi.org/10.1242/dmm.015974
Paietta E (2012) Minimal residual disease in acute myeloid leukemia: coming of age. Hematol Am Soc Hematol Educ Program 2012:35–42. https://doi.org/10.1182/asheducation-2012.1.35
Lane SW, Scadden DT, Gilliland DG (2009) The leukemic stem cell niche: current concepts and therapeutic opportunities. Blood 114(6):1150–1157. https://doi.org/10.1182/blood-2009-01-202606
Schepers K, Pietras EM, Reynaud D, Flach J, Binnewies M, Garg T, Wagers AJ, Hsiao EC, Passegue E (2013) Myeloproliferative neoplasia remodels the endosteal bone marrow niche into a self-reinforcing leukemic niche. Cell Stem Cell 13(3):285–299. https://doi.org/10.1016/j.stem.2013.06.009
Yanez-Mo M, Siljander PR, Andreu Z, Zavec AB, Borras FE, Buzas EI, Buzas K, Casal E, Cappello F, Carvalho J, Colas E, Cordeiro-da Silva A, Fais S, Falcon-Perez JM, Ghobrial IM, Giebel B, Gimona M, Graner M, Gursel I, Gursel M, Heegaard NH, Hendrix A, Kierulf P, Kokubun K, Kosanovic M, Kralj-Iglic V, Kramer-Albers EM, Laitinen S, Lasser C, Lener T, Ligeti E, Line A, Lipps G, Llorente A, Lotvall J, Mancek-Keber M, Marcilla A, Mittelbrunn M, Nazarenko I, Nolte-'t Hoen EN, Nyman TA, O'Driscoll L, Olivan M, Oliveira C, Pallinger E, Del Portillo HA, Reventos J, Rigau M, Rohde E, Sammar M, Sanchez-Madrid F, Santarem N, Schallmoser K, Ostenfeld MS, Stoorvogel W, Stukelj R, Van der Grein SG, Vasconcelos MH, Wauben MH, De Wever O (2015) Biological properties of extracellular vesicles and their physiological functions. J Extracell Vesicles 4:27066. https://doi.org/10.3402/jev.v4.27066
Deregibus MC, Cantaluppi V, Calogero R, Lo Iacono M, Tetta C, Biancone L, Bruno S, Bussolati B, Camussi G (2007) Endothelial progenitor cell derived microvesicles activate an angiogenic program in endothelial cells by a horizontal transfer of mRNA. Blood 110(7):2440–2448. https://doi.org/10.1182/blood-2007-03-078709
Ratajczak J, Miekus K, Kucia M, Zhang J, Reca R, Dvorak P, Ratajczak MZ (2006) Embryonic stem cell-derived microvesicles reprogram hematopoietic progenitors: evidence for horizontal transfer of mRNA and protein delivery. Leukemia 20(5):847–856. https://doi.org/10.1038/sj.leu.2404132
Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO (2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 9(6):654–659. https://doi.org/10.1038/ncb1596
Castillo J, Bernard V, San Lucas FA, Allenson K, Capello M, Kim DU, Gascoyne P, Mulu FC, Stephens BM, Huang J, Wang H, Momin AA, Jacamo RO, Katz M, Wolff R, Javle M, Varadhachary G, Wistuba II, Hanash S, Maitra A, Alvarez H (2018) Surfaceome profiling enables isolation of cancer-specific exosomal cargo in liquid biopsies from pancreatic cancer patients. Ann Oncol 29(1):223–229. https://doi.org/10.1093/annonc/mdx542
Ludwig AK, Giebel B (2012) Exosomes: small vesicles participating in intercellular communication. Int J Biochem Cell Biol 44(1):11–15. https://doi.org/10.1016/j.biocel.2011.10.005
Fais S, O'Driscoll L, Borras FE, Buzas E, Camussi G, Cappello F, Carvalho J, Cordeiro da Silva A, Del Portillo H, El Andaloussi S, Ficko Trcek T, Furlan R, Hendrix A, Gursel I, Kralj-Iglic V, Kaeffer B, Kosanovic M, Lekka ME, Lipps G, Logozzi M, Marcilla A, Sammar M, Llorente A, Nazarenko I, Oliveira C, Pocsfalvi G, Rajendran L, Raposo G, Rohde E, Siljander P, van Niel G, Vasconcelos MH, Yanez-Mo M, Yliperttula ML, Zarovni N, Zavec AB, Giebel B (2016) Evidence-based clinical use of nanoscale extracellular vesicles in nanomedicine. ACS Nano 10(4):3886–3899. https://doi.org/10.1021/acsnano.5b08015
Thakur BK, Zhang H, Becker A, Matei I, Huang Y, Costa-Silva B, Zheng Y, Hoshino A, Brazier H, Xiang J, Williams C, Rodriguez-Barrueco R, Silva JM, Zhang W, Hearn S, Elemento O, Paknejad N, Manova-Todorova K, Welte K, Bromberg J, Peinado H, Lyden D (2014) Double-stranded DNA in exosomes: a novel biomarker in cancer detection. Cell Res 24(6):766–769. https://doi.org/10.1038/cr.2014.44
Hur JY, Kim HJ, Lee JS, Choi CM, Lee JC, Jung MK, Pack CG, Lee KY (2018) Extracellular vesicle-derived DNA for performing EGFR genotyping of NSCLC patients. Mol Cancer 17(1):15–16. https://doi.org/10.1186/s12943-018-0772-6
Yang S, Che SP, Kurywchak P, Tavormina JL, Gansmo LB, Correa de Sampaio P, Tachezy M, Bockhorn M, Gebauer F, Haltom AR, Melo SA, LeBleu VS, Kalluri R (2017) Detection of mutant KRAS and TP53 DNA in circulating exosomes from healthy individuals and patients with pancreatic cancer. Cancer Biol Ther 18(3):158–165. https://doi.org/10.1080/15384047.2017.1281499
Wang L, Li Y, Guan X, Zhao J, Shen L, Liu J (2018) Exosomal double-stranded DNA as a biomarker for the diagnosis and preoperative assessment of pheochromocytoma and paraganglioma. Mol Cancer 17(1):128–126. https://doi.org/10.1186/s12943-018-0876-z
Kahlert C, Melo SA, Protopopov A, Tang J, Seth S, Koch M, Zhang J, Weitz J, Chin L, Futreal A, Kalluri R (2014) Identification of double-stranded genomic DNA spanning all chromosomes with mutated KRAS and p53 DNA in the serum exosomes of patients with pancreatic cancer. J Biol Chem 289(7):3869–3875. https://doi.org/10.1074/jbc.C113.532267
Svennerholm K, Rodsand P, Hellman U, Waldenstrom A, Lundholm M, Ahren D, Biber B, Ronquist G, Haney M (2016) DNA content in extracellular vesicles isolated from porcine coronary venous blood directly after myocardial ischemic preconditioning. PLoS One 11(7):e0159105. https://doi.org/10.1371/journal.pone.0159105
Vaidya M, Bacchus M, Sugaya K (2018) Differential sequences of exosomal NANOG DNA as a potential diagnostic cancer marker. PLoS One 13(5):e0197782. https://doi.org/10.1371/journal.pone.0197782
Hornick NI, Huan J, Doron B, Goloviznina NA, Lapidus J, Chang BH, Kurre P (2015) Serum exosome microRNA as a minimally-invasive early biomarker of AML. Sci Rep 5:11295. https://doi.org/10.1038/srep11295
Boyiadzis M, Whiteside TL (2016) Plasma-derived exosomes in acute myeloid leukemia for detection of minimal residual disease: are we ready? Expert Rev Mol Diagn 16(6):623–629. https://doi.org/10.1080/14737159.2016.1174578
Hong CS, Muller L, Whiteside TL, Boyiadzis M (2014) Plasma exosomes as markers of therapeutic response in patients with acute myeloid leukemia. Front Immunol 5:160. https://doi.org/10.3389/fimmu.2014.00160
Kunz F, Kontopoulou E, Reinhardt K, Soldierer M, Strachan S, Reinhardt D, Thakur BK (2019) Detection of AML-specific mutations in pediatric patient plasma using extracellular vesicle-derived RNA. Ann Hematol 98(3):595–603. https://doi.org/10.1007/s00277-019-03608-y
Fernando MR, Jiang C, Krzyzanowski GD, Ryan WL (2017) New evidence that a large proportion of human blood plasma cell-free DNA is localized in exosomes. PLoS One 12(8):e0183915. https://doi.org/10.1371/journal.pone.0183915
Whiteside TL (2016) Tumor-derived exosomes and their role in cancer progression. Adv Clin Chem 74:103–141. https://doi.org/10.1016/bs.acc.2015.12.005
Li X, Wang X (2017) The emerging roles and therapeutic potential of exosomes in epithelial ovarian cancer. Mol Cancer 16(1):92. https://doi.org/10.1186/s12943-017-0659-y
We thank Dr. Verena Börger and Michel Bremer from the working group of Prof. Bernd Giebel, Institute of Transfusion Medicine, University Hospital of Essen, for introducing us to ultracentrifugation and NTA devices. We also thank Dr. Thomas Herold from the Institute of Pathology and Institute of Neuropathology for providing us access to devices for analyzing the quality of EV-DNA.
The current study is supported by the José Carreras Leukemia Foundation grant DJCLS 18 R-2017 and Stiftung Universitätsmedizin Essen.
Conflict of interest
The authors declare that they have no conflicts of interest.
Informed consent was obtained from all individual participants (or their parents) included in the study. Each patient was consented following institutional review board approval AML-BFM 2004 (3VCreutzig1).
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
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Kontopoulou, E., Strachan, S., Reinhardt, K. et al. Evaluation of dsDNA from extracellular vesicles (EVs) in pediatric AML diagnostics. Ann Hematol 99, 459–475 (2020). https://doi.org/10.1007/s00277-019-03866-w
- Acute myeloid leukemia
- Mutational detection