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Sensitive and accurate quantification of JAK2 V617F mutation in chronic myeloproliferative neoplasms by droplet digital PCR - Annals of Hematology

  • ️Finke, Jürgen
  • ️Tue Mar 01 2016

Abstract

The JAK2 V617F mutation can be detected with a high frequency in patients with myeloproliferative neoplasms (MPN). MPN treatment efficiency can be assessed by JAK2 V617F quantification. Real-time quantitative PCR (qPCR) is widely used for JAK2 V617F quantification. Emerging alternative technologies like digital droplet PCR (ddPCR) have been described to overcome inherent qPCR limitations. The purpose of this study was to evaluate the utility of ddPCR for JAK2 V617F quantification in patient samples with MPN. Sensitivity and specificity were established by using DNA artificial mixtures. In addition, 101 samples from 59 patients were evaluated for JAK2 V617F mutation. Limit of detection was 0.01 % for both qPCR and ddPCR. The JAK2 V617F mutation was detected in 43 out of 59 patients by both PCR platforms. However, in 14 % of the samples, JAK2 V617F mutation was detected only with ddPCR. This 14 % of discrepant samples were from patients shortly after allogeneic stem cell transplantation. Percentage of JAK2 V617F mutation measured by qPCR and ddPCR in clinical samples showed a high degree of correlation (Spearman r: 0.9637 p < 0.001) and an excellent agreement assessed by Bland-Altman analysis. In conclusion, ddPCR is a suitable, precise, and sensitive method for quantification of the JAK 2 V617F mutation.

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References

  1. Levine RL, Wadleigh M, Cools J et al (2005) Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia and myeloid metaplasia with myelofibrosis. Cancer Cell 7:387–397

    Article  CAS  PubMed  Google Scholar 

  2. Kralovics R, Passamonti F, Buser AS et al (2005) A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Eng J Med 352:1779–1790

    Article  CAS  Google Scholar 

  3. Swerdlow SH, Campo E, Harris NL et al (eds) (2008) WHO classification of tumors of haematopoietic and lymphoid tissues, 4th edn. IARC Press, Lyon, pp 40–50

    Google Scholar 

  4. Kiladjian JJ, Cassinat B, Turlure P et al (2006) High molecular response rate of polycythemia vera patients treated with pegylated interferon alpha-2a. Blood 108:2037–2040

    Article  CAS  PubMed  Google Scholar 

  5. Paradanani A, Gotlib JR, Jamieson C et al (2011) Safety and efficacy of TG 101348, a selective JAK2 inhibitor, in myelofibrosis. J Clin Oncol 29:789–796

    Article  Google Scholar 

  6. Merker JD, Jones CD, Oh ST et al (2010) Design and evaluation of a real-time PCR for quantification of JAK2 V617F and wild-type JAK2 transcript levels in the clinical laboratory. J Mol Diagn 12:58–64

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Larsen TS, Pallisgaard N, Møller MB, Hasselbalch HC (2007) The JAK2 V617F allele burden in essential thrombocythemia, polycythemia vera and primary myelofibrosis-impact on disease phenotype. Eur J Haematol 79:508–515

    Article  CAS  PubMed  Google Scholar 

  8. Taly V, Pekin D, Benhaim L et al (2013) Multiplex picodroplet digital PCR to detect KRAS mutations in circulating DNA from the plasma of colorectal cancer patients. Clin Chem 59:1722–1731

    Article  CAS  PubMed  Google Scholar 

  9. Kinz E, Leiherer A, Lang AH, Drexel H, Muendlein A (2015) Accurate quantitation of JAK2 V617F allele burden by array-based digital PCR. Int J Lab Hematol 37:217–224

    Article  CAS  PubMed  Google Scholar 

  10. Bland JM, Altman DG (1999) Measuring agreement in method comparison studies. Stat Methods Med Res 8:135–160

    Article  CAS  PubMed  Google Scholar 

  11. Verstovsek S, Kantarjian H, Mesa RA et al (2010) Safety and efficacy of INCB 018424, a JAK1 and JAK2 inhibitor, in myelofibrosis. N Eng J Med 363:1117–1127

    Article  CAS  Google Scholar 

  12. Fontanelli G, Barate C, Ciabatti E et al (2015) Real-time PCR and droplet digital PCR: two techniques for detection of the JAK2vV617F mutation in philadelphia-negative chronic myeloproliferative neoplasms. Int J Lab Hematol. doi:10.1111/ijlh.12404

    PubMed  Google Scholar 

  13. Lange T, Edelmann A, Siebolts U et al (2013) JAK2 p.V617F allele burden in myeloproliferative neoplasms one month after allogeneic stem cell transplantation significantly predicts outcome and risk of relapse. Haematologica 98:722–728

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

The authors thank Edith März, Daniela Kautz and Sabine Lilli for the technical assistance.

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Authors and Affiliations

  1. Department of Hematology, Oncology and Stem Cell Transplantation, University Medical Center Freiburg, Hugstetter Str. 55, 79106, Freiburg, Germany

    Miguel Waterhouse, Marie Follo, Dietmar Pfeifer, Nikolas von Bubnoff, Justus Duyster, Hartmut Bertz & Jürgen Finke

  2. Core Facility. Department of Hematology, Oncology and Stem Cell Transplantation, University Medical Center Freiburg, Freiburg, Germany

    Marie Follo & Dietmar Pfeifer

  3. Core Facility Genomics. Department of Hematology, Oncology and Stem Cell Transplantation, University Medical Center Freiburg, Freiburg, Germany

    Dietmar Pfeifer

Authors

  1. Miguel Waterhouse

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  2. Marie Follo

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  3. Dietmar Pfeifer

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  4. Nikolas von Bubnoff

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  5. Justus Duyster

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  6. Hartmut Bertz

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  7. Jürgen Finke

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Corresponding author

Correspondence to Miguel Waterhouse.

Ethics declarations

This study was approved by the Ethics Committee of the Albert Ludwigs University, Freiburg, Germany. Written informed consent was obtained from the patients in accordance with the declaration of Helsinki.

Conflict of interest

The authors declare that they have no conflict of interest.

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Supplementary Figure 1

False positive events. A Poisson model was applied to fit the false positive event distribution from 15 wild-type samples. The limit of blank was calculated using the upper 95 % confidence interval. (GIF 21 kb)

High resolution image (TIF 54 kb)

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Cite this article

Waterhouse, M., Follo, M., Pfeifer, D. et al. Sensitive and accurate quantification of JAK2 V617F mutation in chronic myeloproliferative neoplasms by droplet digital PCR. Ann Hematol 95, 739–744 (2016). https://doi.org/10.1007/s00277-016-2623-0

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  • Received: 29 December 2015

  • Accepted: 22 February 2016

  • Published: 01 March 2016

  • Issue Date: April 2016

  • DOI: https://doi.org/10.1007/s00277-016-2623-0

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