SpringerLink
Log in

Acute promyelocytic leukemia derived extracellular vesicles conserve PML-RARα transcript from storage-inflicted degradation: a stable diagnosis tool in APL patients

  • Original Article
  • Published:
Annals of Hematology Aims and scope Submit manuscript

Abstract

The early death, which is more common in acute promyelocytic leukemia (APL) patients rather than other types of acute myelocytic leukemia (AML) highlights the importance of appropriate diagnostic method for early detection of this disease. The low sensitivity of the conventional methods, low tumor burden in some patients, and the need for bone marrow sampling are some of the diagnostic challenges on the way of proper detection of APL. Given these, we aimed to compare the efficacy of extracellular vesicles (EVs), as a diagnostic tool, with the existing methods. RT-PCR, qPCR, and flow cytometry were applied on EVs and their corresponding associated cellular component collected from 18 APL new cases, 23 patients with minimal residual disease (MRD), and NB4 cell line. RT-PCR results were positive in both cellular and vesicular components of all new cases, NB4 cells, and EVs in contrary to MRD cases. Normalized copy numbers (NCN) of PML-RARα were 5100 and 3950 for cell and EVs, respectively (p < 0.05). There was a significant difference in the NCN of PML-RARα between cells and EVs in BM samples. Investigating the effect of storage at room temperature revealed that PML-RARα level was retained near to the baseline level in EVs, but there was a significant reduction in its copy number in the cellular component during 7 days. Taken together, given to the acceptable stability, EVs could be introduced as a non-invasive liquid biopsy that alongside existing methods could remarkably change the paradigm of APL diagnostic approaches.

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

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price includes VAT (Canada)

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

References

  1. Bally C, Fadlallah J, Leverger G, Bertrand Y, Robert A, Baruchel A, Guerci A, Recher C, Raffoux E, Thomas X (2012) Outcome of acute promyelocytic leukemia (APL) in children and adolescents: an analysis in two consecutive trials of the European APL Group. J Clin Oncol 30(14):1641–1646

    Article  CAS  PubMed  Google Scholar 

  2. Rodriguez-Rodriguez S, Demichelis-Gómez R, Diaz-Huizar MJ, Guerrero-Torres L, Pomerantz A, del Pilar Ortiz-Vilchis M, Aguayo A (2017) Cost-effectiveness of the regimen proposed by the international consortium on acute promyelocyticleukemia in the treatment of newly diagnosed patients with acute promyelocytic leukemia. Am Soc Hematology

  3. Fenaux P, Tallman MS, Estey EH, Löwenberg B, Naoe T, Lengfelder E, Döhner H, Burnett AK, Chen SJ, Mathews V (2019) Management of acute promyelocytic leukemia: Updated recommendations from an expert panel of the European LeukemiaNet

  4. Grimwade D, Jovanovic JV, Hills RK, Nugent EA, Patel Y, Flora R, Diverio D, Jones K, Aslett H, Batson E (2009) Prospective minimal residual disease monitoring to predict relapse of acute promyelocytic leukemia and to direct pre-emptive arsenic trioxide therapy. J Clin Oncol 27(22):3650–3658

    Article  CAS  PubMed  Google Scholar 

  5. Hokland P, Ommen HB (2011) Towards individualized follow-up in adult acute myeloid leukemia in remission. Blood 117(9):2577–2584

    Article  CAS  PubMed  Google Scholar 

  6. Tobal K, Moore H, Macheta M, Yin JL (2001) Monitoring minimal residual disease and predicting relapse in APL by quantitating PML-RARα transcripts with a sensitive competitive RT-PCR method. Leukemia 15(7):1060

    Article  CAS  PubMed  Google Scholar 

  7. Wintrobe MM (2008) Wintrobe’s clinical hematology, vol 1. Lippincott Williams & Wilkins

    Google Scholar 

  8. Marrugo-Ramírez J, Mir M, Samitier J (2018) Blood-based cancer biomarkers in liquid biopsy: a promising non-invasive alternative to tissue biopsy. Int J Mol Sci 19(10):2877

    Article  PubMed Central  CAS  Google Scholar 

  9. Sasso L, Hosamuddin H, Emanueli C (2017) Extracellular vesicles at the cross-line between basic science and clinical needs. Microcirculation 24(1):e12333

    Article  Google Scholar 

  10. van Niel G, D’Angelo G, Raposo G (2018) Shedding light on the cell biology of extracellular vesicles. Nat Rev Mol Cell Biol 19(4):213

    Article  PubMed  CAS  Google Scholar 

  11. Zhang W, **a W, Lv Z, **n Y, Ni C, Yang L (2017) Liquid biopsy for cancer: circulating tumor cells, circulating free DNA or exosomes? Cell Physiol Biochem 41(2):755–768

    Article  PubMed  CAS  Google Scholar 

  12. Kahlert C, Melo SA, Protopopov A, Tang J, Seth S, Koch M, Zhang J, Weitz J, Chin L, Futreal A (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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Caivano A, Laurenzana I, De Luca L, La Rocca F, Simeon V, Trino S, D’Auria F, Traficante A, Maietti M, Izzo T (2015) High serum levels of extracellular vesicles expressing malignancy-related markers are released in patients with various types of hematological neoplastic disorders. Tumor Biology 36(12):9739–9752

    Article  CAS  PubMed  Google Scholar 

  14. Yang C, Yang H, Liu J, Zhu L, Yu S, Zhang X (1815) Gao L (2019) Focus on exosomes: novel pathogenic components of leukemia. American journal of cancer research 9(8):1815

    Google Scholar 

  15. 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

    Article  CAS  PubMed  Google Scholar 

  16. Kontopoulou E, Strachan S, Reinhardt K, Kunz F, Walter C, Walkenfort B, Jastrow H, Hasenberg M, Giebel B, von Neuhoff N (2020) Evaluation of dsDNA from extracellular vesicles (EVs) in pediatric AML diagnostics. Ann Hematol 1–17

  17. Cai J, Han Y, Ren H, Chen C, He D, Zhou L, Eisner GM, Asico LD, Jose PA, Zeng C (2013) Extracellular vesicle-mediated transfer of donor genomic DNA to recipient cells is a novel mechanism for genetic influence between cells. J Mol Cell Biol 5(4):227–238

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 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-β1. Haematologica 96(9):1302–1309

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Boyiadzis M, Whiteside TL (2015) Information transfer by exosomes: a new frontier in hematologic malignancies. Blood Rev 29(5):281–290

    Article  CAS  PubMed  Google Scholar 

  20. Sharifi H, Shafiee A, Molavi G, Razi E, Mousavi N, Sarvizadeh M, Taghizadeh M (2019) Leukemia-derived exosomes: bringing oncogenic signals to blood cells. J Cell Biochem 120(10):16307–16315

    Article  CAS  PubMed  Google Scholar 

  21. Li Y, Zhao J, Yu S, Wang Z, He X, Su Y, Guo T, Sheng H, Chen J, Zheng Q (2019) Extracellular vesicles long RNA sequencing reveals abundant mRNA, circRNA, and lncRNA in Human blood as potential biomarkers for cancer diagnosis. Clin Chem 65(6):798–808

    Article  CAS  PubMed  Google Scholar 

  22. Peng M, **e Y, Li X, Qian Y, Tu X, Yao X, Cheng F, Xu F, Kong D, He B (2019) Resectable lung lesions malignancy assessment and cancer detection by ultra-deep sequencing of targeted gene mutations in plasma cell-free DNA. J Med Genet 56(10):647–653

    Article  CAS  PubMed  Google Scholar 

  23. Théry C, Witwer KW, Aikawa E, Alcaraz MJ, Anderson JD, Andriantsitohaina R, Antoniou A, Arab T, Archer F, Atkin-Smith GK (2018) Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles 7(1):1535750

    Article  PubMed  PubMed Central  Google Scholar 

  24. Pospichalova V, Svoboda J, Dave Z, Kotrbova A, Kaiser K, Klemova D, Ilkovics L, Hampl A, Crha I, Jandakova E (2015) Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer. J Extracell Vesicles 4(1):25530

    Article  PubMed  Google Scholar 

  25. Van Dongen J, Macintyre E, Gabert J, Delabesse E, Rossi V, Saglio G, Gottardi E, Rambaldi A, Dotti G (1901) Griesinger F (1999) Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease. Leukemia 13(12):1901

    Article  CAS  Google Scholar 

  26. Cassinat B, Zassadowski F, Balitrand N, Barbey C, Rain J, Fenaux P, Degos L, Vidaud M, Chomienne C (2000) Quantitation of minimal residual disease in acute promyelocytic leukemia patients with t (15; 17) translocation using real-time RT-PCR. Leukemia 14(2):324

    Article  CAS  PubMed  Google Scholar 

  27. Ghaffari S, Rostami S, Bashash D, Alimoghaddam K, Ghavamzadeh A (2006) Real-time PCR analysis of PML-RARα in newly diagnosed acute promyelocytic leukaemia patients treated with arsenic trioxide as a front-line therapy. Ann Oncol 17(10):1553–1559

    Article  CAS  PubMed  Google Scholar 

  28. Santamaría C, Chillón MC, Fernández C, Martín-Jiménez P, Balanzategui A, Sanz RG, San Miguel JF, González M-G (2007) Using quantification of the PML-RARα transcript to stratify the risk of relapse in patients with acute promyelocytic leukemia. Haematologica 92(3):315–322

    Article  PubMed  Google Scholar 

  29. Beillard E, Pallisgaard N, Van der Velden V, Bi W, Dee R, van der Schoot E, Delabesse E, Macintyre E, Gottardi E, Saglio G (2003) Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patients using ‘real-time’quantitative reverse-transcriptase polymerase chain reaction (RQ-PCR)–a Europe against cancer program. Leukemia 17(12):2474

    Article  CAS  PubMed  Google Scholar 

  30. Biccler JL, Østgård LSG, Severinsen MT, Marcher CW, Møller P, Schöllkopf C, Friis LS, Bøgsted M, Jakobsen LH, El-Galaly TC (2018) Evolution of relative survival for acute promyelocytic leukemia patients alive at landmark time-points: a population-based study. Leukemia:1

  31. Abaza Y, Kantarjian H, Garcia-Manero G, Estey E, Borthakur G, Jabbour E, Faderl S, O’Brien S, Wierda W, Pierce S (2017) Long-term outcome of acute promyelocytic leukemia treated with all-trans-retinoic acid, arsenic trioxide, and gemtuzumab. Blood 129(10):1275–1283

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Hong C-S, 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  PubMed  PubMed Central  CAS  Google Scholar 

  33. Hong CS, Muller L, Boyiadzis M, Whiteside TL (2014) Isolation and characterization of CD34+ blast-derived exosomes in acute myeloid leukemia. PloS one 9(8):e103310

    Article  PubMed  PubMed Central  Google Scholar 

  34. Kucharzewska P, Belting M (2013) Emerging roles of extracellular vesicles in the adaptive response of tumour cells to microenvironmental stress. Journal of Extracellular Vesicles 2(1):20304

    Article  Google Scholar 

  35. Lv L-H, Wan Y-L, Lin Y, Zhang W, Yang M, Li G-L, Lin H-M, Shang C-Z, Chen Y-J, Min J (2012) Anticancer drugs cause release of exosomes with heat shock proteins from human hepatocellular carcinoma cells that elicit effective natural killer cell antitumor responses in vitro. J Biol Chem 287(19):15874–15885

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Fang Y, Garnier D, Lee TH, D’Asti E, Montermini L, Meehan B, Rak J (2016) PML–RARa modulates the vascular signature of extracellular vesicles released by acute promyelocytic leukemia cells. Angiogenesis 19(1):25–38

    Article  CAS  PubMed  Google Scholar 

  37. Kang K-W, Jung J-H, Hur W, Park J, Shin H, Choi B, Jeong H, Kim DS, Yu ES, Lee SR (2018) The Potential of exosomes derived from chronic myelogenous leukaemia cells as a biomarker. Anticancer Res 38(7):3935–3942

    Article  CAS  PubMed  Google Scholar 

  38. Schuurhuis G, Heuser M, Freeman S, Béné M, Buccisano F, Cloos J, Grimwade D, Haferlach T, Hills R, Hourigan C (2018) Minimal/measurable residual disease in AML: a consensus document from the European LeukemiaNet MRD Working Party

  39. Milani G, Lana T, Bresolin S, Aveic S, Pastò A, Frasson C, Te Kronnie G (2017) Expression profiling of circulating microvesicles reveals intercellular transmission of oncogenic pathways. Mol Cancer Res 15(6):683–695

    Article  CAS  PubMed  Google Scholar 

  40. Ghavamzadeh A, Alimoghaddam K, Rostami S, Ghaffari SH, Jahani M, Iravani M, Mousavi SA, Bahar B, Jalili M (2011) Phase II study of single-agent arsenic trioxide for the front-line therapy of acute promyelocytic leukemia. J Clin Oncol 29(20):2753–2757

    Article  CAS  PubMed  Google Scholar 

  41. Grimwade D, Coco FL (2002) Acute promyelocytic leukemia: a model for the role of molecular diagnosis and residual disease monitoring in directing treatment approach in acute myeloid leukemia. Leukemia 16(10):1959–1973

    Article  CAS  PubMed  Google Scholar 

  42. Boddu P, Jorgensen J, Kantarjian H, Borthakur G, Kadia T, Daver N, Alvarado Y, Pemmaraju N, Bose P, Naqvi K (2018) Achievement of a negative minimal residual disease state after hypomethylating agent therapy in older patients with AML reduces the risk of relapse. Leukemia 32(1):241–244

    Article  CAS  PubMed  Google Scholar 

  43. van der Velden VH, Boeckx N, Gonzalez M, Malec M, Barbany G, Lion T, Gottardi E, Pallisgaard N, Beillard E, Hop W (2004) Differential stability of control gene and fusion gene transcripts over time may hamper accurate quantification of minimal residual disease–a study within the Europe Against Cancer Program. Leukemia 18(4):884–886

    Article  PubMed  CAS  Google Scholar 

  44. Opitz L, Salinas-Riester G, Grade M, Jung K, Jo P, Emons G, Ghadimi BM, Beißbarth T, Gaedcke J (2010) Impact of RNA degradation on gene expression profiling. BMC Med Genomics 3(1):36

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  45. Prezeau N, Silvy M, Gabert J, Picard C (2006) Assessment of a new RNA stabilizing reagent (tempus blood RNA) for minimal residual disease in onco-hematology using the EAC protocol. Leuk Res 30(5):569–574

    Article  CAS  PubMed  Google Scholar 

  46. Ge Q, Zhou Y, Lu J, Bai Y, **e X, Lu Z (2014) miRNA in plasma exosome is stable under different storage conditions. Molecules 19(2):1568–1575

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Kalra H, Adda CG, Liem M, Ang CS, Mechler A, Simpson RJ, Hulett MD, Mathivanan S (2013) Comparative proteomics evaluation of plasma exosome isolation techniques and assessment of the stability of exosomes in normal human blood plasma. Proteomics 13(22):3354–3364

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors would like to thank their dear colleagues from Hematopoietic Stem Cell and Bone Marrow Transplantation Research Center (HSCT), Shahid Beheshti University of Medical Sciences, Mr. Amin mirzaeian, Miss Bentolhoda Kohestani, and Miss Shaghayegh Shahsavan for their cooperation in data gathering. This article has been extracted from the thesis written by Mr. Mohieddin Barzegar in School of Allied Medical Science, Shahid Beheshti University of Medical Sciences (Registration No: 15919).

Funding

Funding was done by Shahid Beheshti University of Medical Science Registration No: 15919.

Author information

Authors and Affiliations

  1. Laboratory Hematology and Blood Banking, School of Allied Medical Science, Shahid Beheshti University of Medical Sciences, Darband St, Qods Sq, Tehran, Iran

    Mohieddin Barzegar, Mehdi Allahbakhshian Farsani, Mohammad Rafiee, Vahid Amiri & Mohammad Hossein Mohammadi

  2. HSCT Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran

    Mehdi Allahbakhshian Farsani, Sayeh Parkhihdeh & Mohammad Hossein Mohammadi

  3. Department of Hematology and Blood Transfusion, School of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran

    Fariba Rad

  4. Cellular and Molecular Research Center, Yasuj University of Medical Sciences, Yasuj, Iran

    Fariba Rad

Authors
  1. Mohieddin Barzegar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mehdi Allahbakhshian Farsani
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mohammad Rafiee
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Vahid Amiri
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sayeh Parkhihdeh
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Fariba Rad
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Mohammad Hossein Mohammadi
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Barzegar carried out the experiments, collected the samples, did the statistical analysis, and wrote the first version of the manuscript; Parkhihdeh, Rafiee, and Amiri drafted the manuscript and analyzed the data; Mohammadi, Allahbakhshian Farsani, and Rad conceived of the study, designed and coordinated it, and finalized the manuscript. All authors gave final approval for publication.

Corresponding author

Correspondence to Mohammad Hossein Mohammadi.

Ethics declarations

Ethical approval

The study was approved ethically by committee of ethics in by Shahid Beheshti University of Medical Science.

Informed consent

We have signed consent from participants.

Conflict of interest

The authors declare no competing interests.

Additional information

Publisher’s note

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

Supplementary Information

ESM 1

(DOCX 692 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Barzegar, M., Farsani, M.A., Rafiee, M. et al. Acute promyelocytic leukemia derived extracellular vesicles conserve PML-RARα transcript from storage-inflicted degradation: a stable diagnosis tool in APL patients. Ann Hematol 100, 2241–2252 (2021). https://doi.org/10.1007/s00277-021-04579-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00277-021-04579-9

Keywords

Search

Navigation