Rapid identification of heterozygous or homozygous JAK2 ^V617F mutations in myeloproliferative neoplasms using melting curve analysis

Background/Purpose: The activating JAK2 mutation with a G-C to T-A transversion at codon 617 (JAK2 ^V617F) is associated with myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis. Here, we report a technical advance in the diagnosis of JAK2 ^V617F in MPNs by melting curve analysis (MCA). Methods: From January through December 2006, we prospectively enrolled 78 patients with PV (n=21), ET (n=32), myelofibrosis (n=5), secondary erythrocytosis (n=4), secondary thrombocytosis (n=2), acute myelocytic leukemia (n=4), chronic myelocytic leukemia (n=8), and myelodysplastic syndrome (n=2). Mutation analysis for JAK2 ^V617F was performed on either bone marrow or peripheral blood cells using allele-specific polymerase chain reaction (AS-PCR) or sequencing and fluorescence resonance energy transfer (FRET) probes with MCA. Results: For the initial 30 samples, the detection rate of JAK2 ^V617F using MCA was comparable to the gold standard of the PCR sequencing methods. However, the turnaround times for MCA and PCR were 2 hours and 2 days, respectively. The detection rate of JAK2 ^V617F was 76.2% for PV (homozygous in 14.3%), 46.9% for ET, 80% for myelofibrosis (homozygous in 20%), and 0% for the other conditions. In PV, patients with homozygous JAK2 ^V617F presented with significantly longer disease durations than heterozygous patients. In ET, there were no differences in the clinical parameters of patients harboring JAK2 ^V617F compared with those with wild-type JAK2. Conclusion: Heterozygous and homozygous JAK2 ^V617F mutations can be identified using the rapid and reliable assay based on FRET probes and MCA. Detection of JAK2 ^V617F can be used to assist in the diagnosis of BCR/ABL-negative MPNs.