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Droplet digital PCR for quantification of PML-RARα in acute promyelocytic leukemia: a comprehensive comparison with real-time PCR

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Abstract

Real-time quantitative PCR (qPCR) has been widely implemented for molecular testing, but there are still some inherent limitations that hamper its usefulness. Droplet digital PCR (ddPCR), which can provide direct, standards-free quantification, has recently received increasing attention. In our study, a comprehensive comparison of ddPCR with qPCR in relation to the quantification of PML-RARα was performed to evaluate the diagnostic potential of ddPCR. Results showed that ddPCR displayed significant concordance with qPCR in the detection of PML-RARα in clinical samples, but showed advantages over qPCR in terms of precision, limit of detection (LOD), and other basic performance parameters. A study of the feasibility of duplexing also indicated that ddPCR could simultaneously quantify the target PML-RARα and the clinical common reference gene ABL in a reaction, in contrast to qPCR. Moreover, ddPCR was more tolerant than qPCR of inhibition, and was shown to be able to quantify inhibition-prone samples. Another advantage of using ddPCR in clinical applications is that it will yield accurate results for patients with PML-RARα levels that fluctuate around the LOD of qPCR. Therefore, ddPCR is considered to have the potential to become a reliable alternative technique for quantifying PML-RARα.

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References

  1. Cicconi L, Fenaux P, Kantarjian H, Tallman M, Sanz MA, Lo-Coco F. Molecular remission as a therapeutic objective in acute promyelocytic leukemia. Leukemia. 2018;32(8):1671–8.

    Article  CAS  PubMed  Google Scholar 

  2. Lo-Coco F, Ammatuna E. The biology of acute promyelocytic leukemia and its impact on diagnosis and treatment. Hematol Am Soc Hematol Educ Program. 2006;156-61:514.

    Google Scholar 

  3. Lo Coco F, Diverio D, Falini B, Biondi A, Nervi C, Pelicci PG. Genetic diagnosis and molecular monitoring in the management of acute promyelocytic leukemia. Blood. 1999;94(1):12–22.

    PubMed  Google Scholar 

  4. Sanz MA, Lo-Coco F. Modern approaches to treating acute promyelocytic leukemia. J Clin Oncol. 2011;29(5):495–503.

    Article  PubMed  Google Scholar 

  5. Grimwade D, Jovanovic JV, Hills RK, Nugent EA, Patel Y, et al. Prospective minimal residual disease monitoring to predict relapse of acute promyelocytic leukemia and to direct pre-emptive arsenic trioxide therapy. J Clin Oncol. 2009;27(22):3650–8.

    Article  CAS  PubMed  Google Scholar 

  6. Bhat S, Emslie KR. Digital polymerase chain reaction for characterisation of DNA reference materials. Biomol Detect Quantif. 2016;10:47–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Waggoner J, Ho DY, Libiran P, Pinsky BA. Clinical significance of low cytomegalovirus DNA levels in human plasma. J Clin Microbiol. 2012;50(7):2378–83.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Pinheiro LB, Coleman VA, Hindson CM, Herrmann J, Hindson BJ, Bhat S, et al. Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification. Anal Chem. 2012;84(2):1003–11.

    Article  CAS  PubMed  Google Scholar 

  9. Huggett JF, Whale A. Digital PCR as a novel technology and its potential implications for molecular diagnostics. Clin Chem. 2013;59(12):1691–3.

    Article  CAS  PubMed  Google Scholar 

  10. Bizouarn F. Introduction to digital PCR. Methods Mol Biol. 2014;1160:27–41.

    Article  CAS  PubMed  Google Scholar 

  11. Albano F, Zagaria A, Anelli L, Coccaro N, Tota G, et al. Absolute quantification of the pretreatment PML-RARA transcript defines the relapse risk in acute promyelocytic leukemia. Oncotarget. 2015;6(15):13269–77.

    Article  PubMed  PubMed Central  Google Scholar 

  12. Brunetti C, Anelli L, Zagaria A, Minervini A, Minervini CF, et al. Droplet digital PCR is a reliable tool for monitoring minimal residual disease in acute promyelocytic leukemia. J Mol Diagn. 2017;19(3):437–44.

  13. He HJ, Almeida JL, Lund SP, Steffen CR, Choquette S, Cole KD. Development of NIST Standard Reference Material 2373: genomic DNA standards for HER2 measurements. Biomol Detect Quantif. 2016;8:1–8.

  14. White H, Deprez L, Corbisier P, Hall V, Lin F, et al. A certified plasmid reference material for the standardisation of BCR-ABL1 mRNA quantification by real-time quantitative PCR. Leukemia. 2015;29(2):369–76.

    Article  CAS  PubMed  Google Scholar 

  15. Clinical and Laboratory Standards Institute, Tholen DW, Linnet K, Kondratovich M, Armbruster DA, Garrett PE, Jones RL, Kroll MH, Lequin RM, Pankratz TJ, Scassellati GA, Schimmel H, Tsai J. Protocols for determination of limits of detection and limits of quantitation. Approved guideline EP17-a. Wayne: Clinical and Laboratory Standards Institute; 2004.

  16. Milosevic D, Mills JR, Campion MB, Vidal Folch N, Voss JS, et al. Applying standard clinical chemistry assay validation to droplet digital PCR quantitative liquid biopsy testing. Clin Chem. 2018.

  17. Antonelli G, Padoan A, Aita A, Sciacovelli L, Plebani M. Verification of examination procedures in clinical laboratory for imprecision, trueness and diagnostic accuracy according to ISO 15189:2012: a pragmatic approach. Clin Chem Lab Med. 2017;55(10):1501–8.

    CAS  PubMed  Google Scholar 

  18. Dingle TC, Sedlak RH, Cook L, Jerome KR. Tolerance of droplet-digital PCR vs real-time quantitative PCR to inhibitory substances. Clin Chem. 2013;59(11):1670–2.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Bhat S, McLaughlin JL, Emslie KR. Effect of sustained elevated temperature prior to amplification on template copy number estimation using digital polymerase chain reaction. Analyst. 2011;136(4):724–32.

    Article  CAS  PubMed  Google Scholar 

  20. Cao L, Cui X, Hu J, Li Z, Choi JR, Yang Q, et al. Xu F. Advances in digital polymerase chain reaction (dPCR) and its emerging biomedical applications. Biosens Bioelectron. 2017;90:459–74.

  21. Cao Y, Raith MR, Griffith JF. Droplet digital PCR for simultaneous quantification of general and human-associated fecal indicators for water quality assessment. Water Res. 2015;70:337–49.

    Article  CAS  PubMed  Google Scholar 

  22. Huggett JF, Cowen S, Foy CA. Considerations for digital PCR as an accurate molecular diagnostic tool. Clin Chem. 2015;61(1):79–88.

    Article  CAS  PubMed  Google Scholar 

  23. Sanders R, Huggett JF, Bushell CA, Cowen S, Scott DJ, Foy CA. Evaluation of digital PCR for absolute DNA quantification. Anal Chem. 2011;83(17):6474–84.

    Article  CAS  PubMed  Google Scholar 

  24. Morisset D, Stebih D, Milavec M, Gruden K, Zel J. Quantitative analysis of food and feed samples with droplet digital PCR. PLoS One. 2013;8(5):e62583.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Cao Y, Griffith JF, Dorevitch S, Weisberg SB. Effectiveness of qPCR permutations, internal controls and dilution as means for minimizing the impact of inhibition while measuring Enterococcus in environmental waters. J Appl Microbiol. 2012;113(1):66–75.

  26. Schumacher JA, Scott Reading N, Szankasi P, Matynia AP, Kelley TW. A novel approach to quantitating leukemia fusion transcripts by qRT-PCR without the need for standard curves. Exp Mol Pathol. 2015;99(1):104–8.

    Article  CAS  PubMed  Google Scholar 

  27. Grimwade D, Jovanovic JV, Hills RK. Can we say farewell to monitoring minimal residual disease in acute promyelocytic leukaemia? Best Pract Res Clin Haematol. 2014;27(1):53–61.

    Article  PubMed  Google Scholar 

  28. Coccaro N, Anelli L, Zagaria A, Casieri P, Tota G, et al. Droplet digital PCR is a robust tool for monitoring minimal residual disease in adult Philadelphia-positive acute lymphoblastic leukemia. J Mol Diagn. 2018;20(4):474–82.

    Article  PubMed  Google Scholar 

  29. Verhaegen B, De Reu K, De Zutter L, Verstraete K, Heyndrickx M, Van Coillie E. Comparison of droplet digital PCR and qPCR for the quantification of Shiga toxin-producing Escherichia coli in bovine feces. Toxins (Basel) 2016;8(5).

  30. Jones GM, Busby E, Garson JA, Grant PR, Nastouli E, Devonshire AS, et al. Digital PCR dynamic range is approaching that of real-time quantitative PCR. Biomol Detect Quantif. 2016;10:31–3.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Vynck M, Trypsteen W, Thas O, Vandekerckhove L, De Spiegelaere W. The future of digital polymerase chain reaction in virology. Mol Diagn Ther. 2016;20(5):437–47.

    Article  CAS  PubMed  Google Scholar 

  32. Huggett JF, Foy CA, Benes V, Emslie K, Garson JA, et al. The digital MIQE guidelines: minimum information for publication of quantitative digital PCR experiments. Clin Chem. 2013;59(6):892–902.

    Article  CAS  PubMed  Google Scholar 

  33. Kuypers J, Jerome KR. Applications of digital PCR for clinical microbiology. J Clin Microbiol. 2017;55(6):1621–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Hall Sedlak R, Jerome KR. The potential advantages of digital PCR for clinical virology diagnostics. Expert Rev Mol Diagn. 2014;14(4):501–7.

    Article  CAS  PubMed  Google Scholar 

  35. Basu AS. Digital assays part I: partitioning statistics and digital PCR. SLAS Technol. 2017;22(4):369–86.

    PubMed  Google Scholar 

  36. Song Q, Gao Y, Zhu Q, Tian Q, Yu B, et al. A nanoliter self-priming compartmentalization chip for point-of-care digital PCR analysis. Biomed Microdevices. 2015;17(3):64.

    Article  CAS  PubMed  Google Scholar 

  37. Sinha M, Mack H, Coleman TP, Fraley SI. A high-resolution digital DNA melting platform for robust sequence profiling and enhanced genotype discrimination. SLAS Technol. 2018;23(6):580–91.

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Acknowledgements

This study was supported by the National Natural Science Foundation (grant numbers 81201349, 81000775); Young Medical Key Talents in Jiangsu Province (grant numbers QNRC 2016686, 2016687); Frontier and Key Technical Innovation Projects of Nantong (grant number MS22015049); and the Nantong Science and Technology Plan Project (MS12017008-3).

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Authors and Affiliations

  1. Department of Laboratory Medicine, Affiliated Hospital of Nantong University, No 20, ** Yu, Huimin Wang & Rongrong **g

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  1. Dandan Yuan
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  2. Ming Cui
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  3. Shu** Yu
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  4. Huimin Wang
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  5. Rongrong **g
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Correspondence to Huimin Wang or Rongrong **g.

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The authors declare that all individual participants from whom the blood samples were obtained gave their informed consent, and that the studies were approved by the ethics committee of the Affiliated Hospital of Nantong University and were performed in accordance with ethical standards.

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The authors declare no conflict of interest.

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Yuan, D., Cui, M., Yu, S. et al. Droplet digital PCR for quantification of PML-RARα in acute promyelocytic leukemia: a comprehensive comparison with real-time PCR. Anal Bioanal Chem 411, 895–903 (2019). https://doi.org/10.1007/s00216-018-1508-6

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  • DOI: https://doi.org/10.1007/s00216-018-1508-6

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