The Study On JAK2 V617F Mutation In Myeloproliferative Disorder Patients And Its Targeted Therapy In Vitro

Objective:To detect JAK2 V617F mutation in BCR-ABL negative myeloproliferative disorders (MPD).To study the relationship between JAK2 V617F mutation and clinical characteristics in patients with MPD.To investigate the effect of tyrosine kinase inhibitor Gefitinib on JAK2V617F positive HEL cell line.Methods:The first part of the study:Fifty-six patients with BCR-ABL negative MPD (diagnosed in the provincial hospital affiliated to shandong university) were obtained from 20 patients with polycythaemia vera (PV),26 patients with essential thrombocythaemia (ET) and 10 patients with idiopathic myelofibrosis (IMF).11 control samples were obtained, including 4 BCR-ABL positive Chronic myelogenous leukemia (CML),2 patients with secondary polycythaemia and 5 healthy people.3 ml of bone marrow was collected from every MPD patient, and 5 ml of peripheral blood from every control sample. Mononuclear cells were extracted from all the samples by Lymphocytes Separation Medium. Genomic DNA was extracted by DNA kit.JAK2 V617F mutation of MPD patients was detected by allele-specific polymerase chain reaction (AS-PCR) and DNA-sequencing, and its correlation with clinical characteristics of MPD were analyzed.The second part of the study:Different concentrations of Gefitinib were applied to act on HEL cell. The cell inhibitory rate was detected by methyl thiazolyl tetrazolium(MTT) assay, the apoptotic rate and cell cycle were measured by a flow cytometer.Resultes:The first part of the study:JAK2 V617F mutation was detected in 36 of 56 BCR-ABL negative MPD patients, including 17 (17/20,85%) PV patients,14 (14/26,53.8%) ET patients,5 (5/10,50%) IMF patients. There were significant differences in leukocyte (P=0.018) and platelet counts (P=0.021) in PV, leukocyte counts (P=0.001) and hemoglobin (P=0.007) in ET, leukocyte counts (P=0.026) in IMF between JAK2 V617 positive and negative MPD patients. Significant difference was found in complication of bleeding, thrombosis between JAK2 V617 positive and negative ET patients (P=0.016) but not in PV or IMF patients.The second part of the study.Gefitinib could significantly inhibit the proliferation of the HEL cell line in a dose dependent manner. The IC50 was 5.4μM. Gefitinib could effectively induce apoptosis of HEL cell line in a dose dependent manner. Apart from this, Gefitinib could induce HEL cell to arrest at G0/G1 phase.Conclusion:AS-PCR is a sensitive and reliable technique in detecting JAK2 V617F mutation. It can be used to differentiate between ET, PV, IMF and CML.The clinical characteristics of JAK2 V617F positive MPD patients were different from those without the mutation.Gefitinib has significant effect on the HEL cell line on inhibition of proliferation, cell cycle and inducing of apoptosis.