Ann Lab Med 2018; 38(2): 147-154  
Clinical Implications of Quantitative JAK2 V617F Analysis using Droplet Digital PCR in Myeloproliferative Neoplasms
Eunyoung Lee, M.D.1, Kyoung Joo Lee, B.S.2, Hyein Park, B.S.2, Jin Young Chung, B.S.1, Mi-Na Lee, M.D.3, Myung Hee Chang, M.D.4, Jongha Yoo, M.D.5, Hyewon Lee, M.D.1, Sun-Young Kong, M.D.1,2,6,*, and Hyeon-Seok Eom, M.D.1,2,*
Center for Hematologic Malignancy1, National Cancer Center, Goyang; Department of System Cancer Science2, Graduate School of Cancer Science and Policy, National Cancer Center, Goyang; Green Cross Laboratories3, Yongin; Division of Oncology-Hematology4, Department of Internal Medicine, National Health Insurance Service Ilsan Hospital, Goyang; Department of Laboratory Medicine5, National Health Insurance Service Ilsan Hospital, Goyang; Department of Laboratory Medicine6, National Cancer Center, Goyang, Korea
Correspondence to: Hyeon-Seok Eom
Center for Hematologic Malignancy, Research Institute and Hospital, National Cancer Center, 323 Ilsan-ro, Ilsandong-gu, Goyang 10408, Korea
Tel: +82-31-920-2402
Fax: +82-31-920-1163
E-mail: hseom@ncc.re.kr
Co-corresponding author: Sun-Young Kong
Department of Laboratory Medicine, Research Institute and Hospital, National Cancer Center, 323 Ilsan-ro, Ilsandong-gu, Goyang 10408, Korea
Tel: +82-31-920-1735
E-mail: ksy@ncc.re.kr
*These authors equally contributed to this work.
Received: March 19, 2017; Revised: June 18, 2017; Accepted: September 27, 2017; Published online: March 1, 2018.
© The Korean Society for Laboratory Medicine. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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.
Keywords: Digital droplet PCR, JAK2 V617F mutation, Myeloproliferative neoplasms



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