title: Sensitive and accurate quantification of JAK2 V617F mutation in chronic myeloproliferative neoplasms by droplet digital PCR.
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.
authors.affiliation: Department of Hematology, Oncology and Stem Cell Transplantation, University Medical Center Freiburg, Hugstetter Str. 55, 79106, Freiburg, Germany. miguel.waterhouse@uniklinik-freiburg.de.
authors.forename: Miguel
authors.initials: M
authors.lastname: Waterhouse
authors.affiliation: Department of Hematology, Oncology and Stem Cell Transplantation, University Medical Center Freiburg, Hugstetter Str. 55, 79106, Freiburg, Germany.|Core Facility. Department of Hematology, Oncology and Stem Cell Transplantation, University Medical Center Freiburg, Freiburg, Germany.
authors.forename: Marie
authors.initials: M
authors.lastname: Follo
authors.affiliation: Department of Hematology, Oncology and Stem Cell Transplantation, University Medical Center Freiburg, Hugstetter Str. 55, 79106, Freiburg, Germany.|Core Facility. Department of Hematology, Oncology and Stem Cell Transplantation, University Medical Center Freiburg, Freiburg, Germany.|Core Facility Genomics. Department of Hematology, Oncology and Stem Cell Transplantation, University Medical Center Freiburg, Freiburg, Germany.
authors.forename: Dietmar
authors.initials: D
authors.lastname: Pfeifer
authors.affiliation: Department of Hematology, Oncology and Stem Cell Transplantation, University Medical Center Freiburg, Hugstetter Str. 55, 79106, Freiburg, Germany.
authors.forename: Nikolas
authors.initials: N
authors.lastname: von Bubnoff
authors.affiliation: Department of Hematology, Oncology and Stem Cell Transplantation, University Medical Center Freiburg, Hugstetter Str. 55, 79106, Freiburg, Germany.
authors.forename: Justus
authors.initials: J
authors.lastname: Duyster
authors.affiliation: Department of Hematology, Oncology and Stem Cell Transplantation, University Medical Center Freiburg, Hugstetter Str. 55, 79106, Freiburg, Germany.
authors.forename: Hartmut
authors.initials: H
authors.lastname: Bertz
authors.affiliation: Department of Hematology, Oncology and Stem Cell Transplantation, University Medical Center Freiburg, Hugstetter Str. 55, 79106, Freiburg, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Finke
mesh_terms: D000368:Aged; D019943:Amino Acid Substitution; D001853:Bone Marrow / Q000201:enzymology; D004252:DNA Mutational Analysis / Q000379:methods; D005189:False Positive Reactions; D005260:Female; D005456:Fluorescent Dyes / Q000032:analysis; D006801:Humans; D053614:Janus Kinase 2 / Q000097:blood / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D020125:Mutation, Missense; D009196:Myeloproliferative Disorders / Q000175:diagnosis / Q000201:enzymology / Q000235:genetics*; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
chemical_list: D005456:Fluorescent Dyes; C507924:JAK2 protein, human; D053614:Janus Kinase 2
keywords: Digital PCR; JAK2 V617F