Detection of AML-specific mutations in pediatric patient plasma using extracellular vesicle–derived RNA

Abstract

Despite high remission rates, almost 25% of patients with AML will suffer relapse 3–5 years after diagnosis. Therefore, in addition to existing diagnostic and MRD detection tools, there is still a need for the development of novel approaches that can provide information on the state of the disease. Extracellular vesicles (EVs), containing genetic material reflecting the status of the parental cell, have gained interest in recent years as potential diagnostic biomarkers in cancer. Therefore, isolation and characterization of blood and bone marrow plasma-derived EVs from pediatric AML patients could be an additional approach in AML diagnostics and disease monitoring. In this study, we attempt to establish a plasma EV-RNA-based method to detect leukemia-specific FLT3-ITD and NPM1 mutations using established leukemia cell lines and primary pediatric AML plasma samples. We were successfully able to detect FLT3-ITD and NPM1 mutations in the EV-RNA using GeneScan-based fragment-length analysis and real-time PCR assays, respectively, in samples before therapy. This was corresponding to the gDNA mutational analysis from leukemic blasts, and supports the potential of using EV-RNA as a diagnostic biomarker in pediatric AML.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  1. 1.

    Trino S, Lamorte D, Caivano A, Laurenzana I, Tagliaferri D, Falco G, Del Vecchio L, Musto P, and De Luca L (2018) MicroRNAs as new biomarkers for diagnosis and prognosis, and as potential therapeutic targets in acute myeloid leukemia, Int J Mol Sci 19(2):460

  2. 2.

    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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Paietta E Minimal residual disease in acute myeloid leukemia: coming of age. Hematol Am Soc Hematol Educ Program 2012, 2012:35–42

  4. 4.

    Grove CS, Vassiliou GS (2014) Acute myeloid leukaemia: a paradigm for the clonal evolution of cancer? Dis Model Mech 7:941–951

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    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 16:623–629

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Becker A, Thakur BK, Weiss JM, Kim HS, Peinado H, Lyden D (2016) Extracellular vesicles in cancer: cell-to-cell mediators of metastasis. Cancer Cell 30:836–848

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Keller S, Ridinger J, Rupp AK, Janssen JW, Altevogt P (2011) Body fluid derived exosomes as a novel template for clinical diagnostics. J Transl Med 9:86

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Gould SJ, and Raposo G (2013) As we wait: coping with an imperfect nomenclature for extracellular vesicles, J Extracell Vesicles 2:20389

  9. 9.

    Raposo G, Stoorvogel W (2013) Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol 200:373–383

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Abels ER, Breakefield XO (2016) Introduction to extracellular vesicles: biogenesis, RNA cargo selection, content, release, and uptake. Cell Mol Neurobiol 36:301–312

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    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 24:766–769

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Peinado H, Aleckovic M, Lavotshkin S, Matei I, Costa-Silva B, Moreno-Bueno G, Hergueta-Redondo M, Williams C, Garcia-Santos G, Ghajar C, Nitadori-Hoshino A, Hoffman C, Badal K, Garcia BA, Callahan MK, Yuan J, Martins VR, Skog J, Kaplan RN, Brady MS, Wolchok JD, Chapman PB, Kang Y, Bromberg J, Lyden D (2012) Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET. Nat Med 18:883–891

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Pando A, Reagan JL, Quesenberry P, Fast LD (2018) Extracellular vesicles in leukemia. Leuk Res 64:52–60

    Article  CAS  PubMed  Google Scholar 

  14. 14.

    Szczepanski MJ, Szajnik M, Welsh A, Whiteside TL, Boyiadzis M (2011) Blast-derived microvesicles in sera from patients with acute myeloid leukemia suppress natural killer cell function via membrane-associated transforming growth factor-beta1. Haematologica 96:1302–1309

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Caivano A, Laurenzana I, De Luca L, La Rocca F, Simeon V, Trino S, D’Auria F, Traficante A, Maietti M, Izzo T, D’Arena G, Mansueto G, Pietrantuono G, Laurenti L, Musto P, Del Vecchio L (2015) High serum levels of extracellular vesicles expressing malignancy-related markers are released in patients with various types of hematological neoplastic disorders. Tumour Biol 36:9739–9752

    Article  CAS  PubMed  Google Scholar 

  16. 16.

    Caivano A, La Rocca F, Simeon V, Girasole M, Dinarelli S, Laurenzana I, De Stradis A, De Luca L, Trino S, Traficante A, D’Arena G, Mansueto G, Villani O, Pietrantuono G, Laurenti L, Del Vecchio L, Musto P (2017) MicroRNA-155 in serum-derived extracellular vesicles as a potential biomarker for hematologic malignancies - a short report. Cell Oncol (Dordr) 40:97–103

    Article  CAS  Google Scholar 

  17. 17.

    Huan J, Hornick NI, Shurtleff MJ, Skinner AM, Goloviznina NA, Roberts CT Jr, Kurre P (2013) RNA trafficking by acute myelogenous leukemia exosomes. Cancer Res 73:918–929

    Article  CAS  PubMed  Google Scholar 

  18. 18.

    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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Funding

This study was supported by the José Carreras Leukemia Foundation promotion grant 04 PSG/2017 awarded to Fabienne Kunz and Stiftung Universitätsmedizin Essen grant on cancer exosome research awarded to Dr. Basant Kumar Thakur.

Author information

Affiliations

Authors

Contributions

Conception and design: BKT, FK, EK; collection and assembly of data: FK, EK, KR, MS; data analysis and interpretation: BKT, FK, EK; drafting of manuscript: BKT, FK, EK, SS; manuscript writing: BKT, FK, EK, SS; final approval of manuscript: all co-authors.

Corresponding author

Correspondence to Basant Kumar Thakur.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Informed consent

Informed consent was obtained from all individual participants (or their parents) included in the study. Each patient consented following institutional review board approval AML-BFM 2004 (3VCreutzig1).

Ethical approval

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.

Statement on the welfare of animals

This article does not contain any studies with animals performed by any of the authors.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

ESM 1

(PDF 1047 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kunz, F., Kontopoulou, E., Reinhardt, K. et al. Detection of AML-specific mutations in pediatric patient plasma using extracellular vesicle–derived RNA. Ann Hematol 98, 595–603 (2019). https://doi.org/10.1007/s00277-019-03608-y

Download citation

Keywords

  • Extracellular vesicles
  • RNA
  • Pediatric AML
  • Biomarker