Development and clinical validation of a novel and NON-PCR based method for the detection of the JAK2V617F mutation in chronic mycloproliferative neoplasms

Myeloproliferative Neoplasms (MPNs) are haematologic disorders of myeloid progenitor cells characterized by the frequent presence of an acquired activating mutation in exon 14 of the Janus kinase 2, consisting in a Valine to Phenilalanine substitution at codon 617 (JAK2V617F). The kinase activity of mutated JAK2 is constitutively activated, inducing uncontrolled cell proliferation and resistance to apoptosis. JAK2V617F is found in 95% of Polycythemia Vera (PV), 50% of Idiopathic Myelofibrosis (IMF), and 20-40% of Essential Thrombocythemia (ET), the three diseases belonging to MPNs, as described in the World Health Organization (WHO) classification (REF). Thus, the JAK2V617F mutation represents the key clonal marker for diagnosis of MPNs. The identification of the JAK2V617F is mandatory in the diagnostic work up of PV, ET, and IMF. Several molecular techniques are currently available but each of them presents some limits. We developed a novel non-PCR molecular method for the identification of the JAK2V617F mutation based on an Allele Specific-Loop mediated AMPlification (AS-LAMP). This innovative technique amplifies DNA targets under isothermal conditions with high specificity, efficiency, and rapidity. The method neither requires a thermal cycler equipment nor gel separation and the DNA amplification reaction is visible to the naked eye, monitorable by turbidimetry and fluorescence. This method was validated on DNA from both cell lines as well as patients with chronic myeloproliferative neoplasms. Results were compared with those obtained by conventional PCR methods. The AS-LAMP allows a rapid and robust identification of DNA samples harboring the JAK2V617F mutation. No false positive or false negative results were registered on clinical samples previously tested by the reference assay ASO-PCR. This new assay proved also remarkably sensitive since the mutated JAK2V617F DNA could be detected down to 0.05% on clinical samples. AS-LAMP is a simple, robust and easily applicable tool for the molecular diagnosis and monitoring of JAK2V617F mutation in chronic myeloproliferative neoplasms.