Melting Point Assay for the JAK2 V617F Mutation, Comparison with Amplification Refractory Mutation System (ARMS) in Diag
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TECHNOLOGY REPORT
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Melting Point Assay for the JAK2 V617F Mutation, Comparison with Amplification Refractory Mutation System (ARMS) in Diagnostic Samples, and Implications for Daily Routine Sebastian Ochsenreither, Mark Reinwald, Eckhard Thiel and Thomas Burmeister Department of Hematology and Oncology, Charite´ Berlin, Campus Benjamin Franklin, Berlin, Germany
Abstract
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 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.
Background The diagnostic algorithm in BCR/ABL-negative myeloproliferative neoplasms (MPNs) has changed fundamentally with the discovery of the activating somatic point mutation 1849G>T (translating to the valine-to-phenylalanine amino acid exchange V617F) in the JH2 pseudokinase domain of the JAK2 gene in 2005.[1-5] Positivity of the mutation is now a major criterion in the diagnosis of MPN because of its high specificity for clonal myeloid lineage proliferation.[6] Because homozygosity is occasionally found for the V617F mutation due to mitotic recombination, especially in patients with polycythemia
vera (PV), the mutational burden in a patient reflects both the number of mutated alleles in the neoplastic clone and the size of this clone in general.[1,2,4] The impact of mutational burden on the clinical appearance has been shown in the context of PV, essential thrombocythemia (ET), and primary myelofibrosis (PMF).[7-9] There
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