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

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

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