Ann Lab Med.  2018 Mar;38(2):147-154. 10.3343/alm.2018.38.2.147.

Clinical Implications of Quantitative JAK2 V617F Analysis using Droplet Digital PCR in Myeloproliferative Neoplasms

• Lee E
• Lee KJ
• Park H
• Chung JY
• Lee MN
• Chang MH
• Yoo J
• Lee H
• Kong SY
• Eom HS

• KMID: 2403360
• DOI: http://doi.org/10.3343/alm.2018.38.2.147

Abstract

BACKGROUND
JAK2 V617F is the most common mutation in myeloproliferative neoplasms (MPNs) and is a major diagnostic criterion. Mutation quantification is useful for classifying patients with MPN into subgroups and for prognostic prediction. Droplet digital PCR (ddPCR) can provide accurate and reproducible quantitative analysis of DNA. This study was designed to verify the correlation of ddPCR with pyrosequencing results in the diagnosis of MPN and to investigate clinical implications of the mutational burden.
METHODS
Peripheral blood or bone marrow samples were obtained from 56 patients newly diagnosed with MPN or previously diagnosed with MPN but not yet indicated for JAK2 inhibitor treatment between 2012 and 2016. The JAK2 V617F mutation was detected by pyrosequencing as a diagnostic work-up. The same samples were used for ddPCR to determine the correlation between assays and establish a detection sensitivity cut-off. Clinical and hematologic aspects were reviewed.
RESULTS
Forty-two (75%) and 46 (82.1%) patients were positive for JAK2 V617F by pyrosequencing and ddPCR, respectively. The mean mutated allele frequency at diagnosis was 37.5±30.1% and was 40.7±31.2% with ddPCR, representing a strong correlation (r=0.9712, P < 0.001). Follow-up samples were available for 12 patients, including eight that were JAK2 V617F-positive. Of these, mutational burden reduction after treatment was observed in six patients (75%), consistent with trends of hematologic improvement.
CONCLUSIONS
Quantitative analysis of the JAK2 V617F mutation using ddPCR was highly correlated with pyrosequencing data and may reflect the clinical response to treatment.

Keyword

Digital droplet PCR; JAK2 V617F mutation; Myeloproliferative neoplasms

MeSH Terms

Bone Marrow
Diagnosis
DNA
Follow-Up Studies
Gene Frequency
Humans
Polymerase Chain Reaction*
DNA

Figure

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  Fig. 1 Consistency of the expected and measured JAK2 V617F mutation percentages in the range of 0.0001–100% with pilot samples. Eight dilutions were measured in eight independent droplet digital PCR experiments.

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  Fig. 2 Correlation and agreement between (A) the expected and measured JAK2 V617F mutation percentages in the range of 0.0001–100% with pilot samples. (B) pyrosequencing and ddPCR results for quantifiable JAK2 V617F mutation levels in clinical samples.Abbreviation: ddPCR, droplet digital PCR.

• 

  Fig. 3 Representative two-dimensional scatter plots of (A) wild-type JAK2 V617F (green droplets) in a healthy person and (B) heterozygous JAK2 V617F mutation (blue droplets) in a patient with MPN detected by ddPCR and pyrosequencing. Orange droplets indicate a sample heterozygous for the JAK2 V617F mutation and JAK2 wild-type.Abbreviations: MPN, myeloproliferative neoplasm; ddPCR, droplet digital PCR.

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