Clinical Performance of JAK2 V617F Mutation Detection Assays in a Molecular Diagnostics Laboratory: Evaluation of Screening and Quantitation Methods

Milena Cankovic, PhD; Lisa Whiteley; Robert C. Hawley, MD; Richard J. Zarbo, MD, DMD; Dhananjay Chitale, MD


Am J Clin Pathol. 2009;132(5):713-721. 

In This Article

Abstract and Introduction


The presence of the JAK2 V617F mutation is now part of clinical diagnostic algorithms, and JAK2 status is routinely assessed when BCR/ABL− chronic myeloproliferative neoplasms (MPNs) are suspected. The aim of this study was to evaluate performance of 3 screening and 1 quantitative method for JAK2 V617F detection. For the study, 43 samples (27 bone marrow aspirates and 16 peripheral blood samples) were selected. The screening assays were the JAK2 Activating Mutation Assay (InVivoScribe, San Diego, CA), JAK2 MutaScreen kit (Ipsogen, Luminy Biotech, Marseille, France), and a home-brew melting curve analysis method. Ipsogen's JAK2 MutaQuant assay was used for quantification of mutant and wild-type alleles. The limit of detection was 1% for the kit-based screening methods and 10% for the melting curve method. The JAK2 MutaQuant assay demonstrated analytic sensitivity of 0.01%. All 4 methods detected cases of BCR/ABL− MPNs and gave negative results with BCR/ABL+ chronic myelogenous leukemia, multiple myeloma, myelodysplastic syndrome, and normal cases.


Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell malignancies characterized by hypersensitivity of hematopoietic progenitors to numerous cytokines and by clonal proliferation of one or several myeloid lineages.[1,2] In addition to thrombotic and hemorrhagic complications, leukemic transformation can occur.[3] Typically, the classic MPNs encompass 4 related entities: chronic myelogenous leukemia (CML), polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF).

A point mutation in the Janus kinase 2 gene (JAK2) was identified in several MPNs,[4–7] most frequently in PV (65%–97%), ET (23%–57%), and PMF (35%–57%). It is also infrequently present (3%–5%) in myelodysplastic syndrome, chronic myelomonocytic leukemia, and other atypical chronic myeloid disorders.[8] The common occurrence of this mutation in BCR/ABL− MPNs can be used as a unique molecular marker for distinguishing PV, ET, and PMF from reactive hematopoietic disorders.[9] Also, JAK2 mutation has not been detected in Philadelphia chromosome+ CML, and these mutations are currently considered exclusive.

JAK2 is a cytoplasmic tyrosine kinase that mediates growth factor receptor signaling. The V617F mutation is a G>T transversion at nucleotide 2343, resulting in substitution of phenylalanine for valine (V617F) in the JAK2 protein. This point mutation at amino acid residue 617 appears to cause constitutive activation of the JAK2 tyrosine kinase owing to loss of autoinhibitory control.[10] The mutant has enhanced kinase activity, and when overexpressed together with the erythropoietin receptor in cells, it causes hyperactivation of erythropoietin-induced cell signaling. This gain-of-function mutation of JAK2 may explain the hypersensitivity of PV progenitor cells to growth factors and cytokines. The annual incidence of MPNs is 0.5 to 6.5 per 100,000 people,[11] compared with about 10% of healthy subjects with no clinical and hematologic abnormalities harboring the JAK2 V617F mutation. Recent observations suggest that this mutation occurs at the level of the common myeloid progenitor or upstream at the level of a lymphomyeloid multipotent progenitor cell.[4,12,13]

Identification of a JAK2 V617F mutation establishes the presence of a clonal disorder[14] and is an important diagnostic marker for these disorders.[9] Since the discovery of the JAK2 V617F mutation in 2005, a number of different JAK2 mutation detection protocols became available.[4,15,16] Not all clonal cells express the JAK2 V617F point mutation, and granulocyte enrichment has been suggested for mutation detection assays. Because granulocyte separation is expensive and labor-intensive, with added loss of cells during the processing, testing of unfractionated whole blood and bone marrow samples is a more practical approach for most clinical molecular laboratories. For that reason, a sensitive system must be used for mutation detection, estimation of allele burden, and minimal residual disease (MRD) monitoring. The variety of available techniques provides flexibility of method design, which prompts a need for comparative analysis of these methods.[17,18] The goal of this study was to evaluate several currently available testing methods and to select a simple, preferably kit-based assay for use in our clinical molecular diagnostics laboratory. We report our experience with 4 different fluorescent polymerase chain reaction (PCR)-based assays (3 qualitative and 1 quantitative) for the detection of the JAK2 V617F point mutation.


Comments on Medscape are moderated and should be professional in tone and on topic. You must declare any conflicts of interest related to your comments and responses. Please see our Commenting Guide for further information. We reserve the right to remove posts at our sole discretion.
Post as: