Melting Point Assay for the JAK2 V617F Mutation, Comparison with Amplification Refractory Mutation System (ARMS) in Diagnostic Samples, and Implications for Daily Routine

Background: Reliable detection of the JAK2 V617F mutation is a major criterion in the diagnosis of BCR/ABL-negative myeloproliferative neoplasms such as polycythemia vera, essential thrombocythemia, and primary myelofibrosis. A multitude of methods has been applied to both qualitative and quantitative assessment of the mutational status of patients, without defining a gold standard for the daily diagnostic routine so far. Methods: We developed a melting point assay to be used on a Rotor-Gene (R) thermal cycler machine, using asymmetric primer concentrations. A human erythroleukemia cell line (HEL) was used as a positive control in a 3-fold lower concentration than the negative control because of the gene amplification of the mutated JAK2 kinase in this cell line. Routine samples from both blood and bone marrow were processed. Additionally, samples were analyzed using an amplification refractory mutation system (ARMS). Results: The sensitivity of the melting point approach was a 5% mutational load. Of 314 bone marrow or blood DNA samples tested, 101 were ARMS positive, and of these, 90 samples tested positive in the melting point assay. Most of the patients had a mutational load between 20% and 50%. No patient had a JAK2 V617F burden higher than 80%. There was no significant difference in the source (bone marrow versus blood), sex, and patient age. Conclusions: We present a reliable and feasible approach for quantitative assessment of the JAK2 V617F status from both blood and bone marrow. A homozygotic mutated cell line or plasmids should be used for dilution standards. We recommend combining this assay with ARMS PCR for result confirmation and higher overall sensitivity.