The Experimental And Clinical Study On JAK2V617F Mutation In Myeloproliferative Neoplasms And Acute Myeloid Leukemia (M2) Patients

Background and objectiveRecently,the study of pathogenesis on chronic myeloproliferative neoplasms(MPN) has achieved important progress.In early 2005,several independent groups almost simultaneously reported the presence of a substitution of guanine for thymine at position 1849(in exon 14) of JAK2 gene,which is a signal transmission gene,results in phenylalanine instead of valine at amino acide position 617 of JAK2 kinase(JAK2V617F). This change,which developed continuous JAK2V617F activation,had close relation with MPN pathogenesis and could be detected in vast majority of polycythemia vera(PV) patients,nearly half of primary thrombocythemia(ET) and primary myelofibrosis(PMF) patients,but occasionally present in myelodysplastic syndrome(MDS) and acute leukemia (AL) patients.With the investigation thorough,two kinds of mutation have been identified—homozygotic and heterozygotic mutation,and we found the ratio of mutant/wild-type JAK2 mRNA determine MPN phenotype.Therefore,the detection of JAK2V617F mutation in patients with MPN will be advantageous to MPN diagnosis,the prognosis determination and target cancer treatment.However,in domestic,the researches on JAK2V617F mutation have mainly concentrated in clinical examination without genotyping and relatively quantitative assay for the past few years.To clarify the incidence rate,genotype,relatively quantitative level of JAK2 mRNA,which correlated with clinical feature in chinese patients with MPN and acute leukemia(M2),We designed this research.Methods In first chapter of the first part,we choosed 412 patients who attended The First Affiliated Hospital of Soochow University and were diagnosed with MPN(PV,ET or IMF) between August 2005 and October 2008.The diagnostic criteria were made according to WHO criteria.Clinical data and periphery blood samples were collected for studing.Fresh venous blood(5 ml) was collected into tubes containing ethylenediaminetetra-acetic acid (EDTA).Granulocytes were enriched by density-gradient centrifugation with Ficoll-Paque solution.Total cellular DNA was extracted from granulocytes by using DNA extracted kit.AS(Allele Specific)-PCR was used to amplify JAK2 gene.Direct sequencing of PCR production was performed on patients with postive JAK2V617F mutation.At the same time,We also collected patient's clinical data,chromosome changes and the result of following up for studing.In the second chapter of the first part,We selected 12 freezed samples in our laboratory and 68 patients as objects of study,who attended The First Affiliated Hospital of Soochow University and were diagnosed with acute leukemia between January 2006 and June 2008.The diagnostic criteria were made according to the book of Hematology Diagnose and Treatment Effectiveness.The AS-PCR was used to amplify JAK2V617F mutation as mention in first chapter of the first part.Clinical data,chromosome change and the result of following up were collected for studing.In second and third parts,We selected some freezed samples in our laboratory and some patients as objects of study,who attended The First Affiliated Hospital of Soochow University and were diagnosed with MPN(PV,ET or IMF) between January 2006 and June 2008.The diagnostic criteria were made according to WHO criteria.Fresh venous blood (10 ml) was collected into tubes containing ethylenediaminetetra-acetic acid(EDTA). Granulocytes were enriched by density-gradient centrifugation with Ficoll-Paque solution. Total cellular RNA was extracted from granulocytes by using TRIzol reagent.First-strand cDNA synthesis was performed with Moloney Murine Leukemia Virus(M-MLV) reverse transcriptase.ARMS(Amplification-refractory mutation sequencing)-PCR was used to amplify JAK2 gene.The ARMS amplicons were separated by gel electrophoresis for identification of genotype.Direct sequencing of PCR production was performed on patients with postive JAK2V617F mutation.Capillary electrophoresis was use to determine the ratio of mutant/wide-type JAK2 mRNA.Abnormal chromosomes were identified according to the standard R-banding pattern.ResultJAK2V617F mutation was detected in 277 of the 412 patients with MPN by the way of AS-PCR,the frequency of JAK2V617F mutation was similar among essential thrombocythemia(ET),idiopathic myelofibrosis(IMF) and chronic myeloproliferative disorders-unclassified(MPD-U)(P＞0.05),but it was significantly lower than polycythemia vera(PV)(P＜0.05).The presence of JAK2V617F was found to be significantly correlative with advanced age at diagnosis(P＜0.01),higher hemoglobin levels and higher leukocyte counts(P＜0.05).Significant difference was found in complication of vascular events between JAK2V617 positive and negative patients(P＜0.05).JAK2V617F positive MPD-U patients were more progress to typical MPN compared with JAK2V617F negative MPD-U patients.Cytogenetic analysis was performed in 301 of the 412 patients,the association between abnormal karyotype and JAK2V617F was not found.Of 80 de novo AML-M2 patients were examined using the method of AS-PCR,6 at the time of first diagnosis and 1 at relapse were found to have JAK2V617F mutation(8.8%7/80).Morphologically,the whole blood and bone marrow pictures of 7 AML-M2 patients with JAK2V617F mutation presented the disease of acute leukemia instead of the picture of myeloproliferative disorders,Immunophenotypically,bone marrow samples showed myelogenous linage expression.Complete remission was obtained after 2 induction courses in 4 of the 5 AML-M2 patients with JAK2V617F mutation who receive treatment,while one patient had no response to treatment;Follow-up was perform in all 5 patients,with a median survival was 18.5 months in 4 patients.JAK2V617F mutation was detected in 35(100%)of the 35 patients with PV,53(62.4 %)of the 85 with ET and 2(66.7%) of the 3 with IMF by combination of ARMS-PCR and capillary electrophoresis,the difference between PV and ET was significant(P＜0.05).Of 90 JAK2V617F patients examined,17(48.6%17/35) patients with PV and 17(20%17/85) patients with ET and 1(50%1/2) patient with IMF were homozygotes.ET patients showed lower prevalence of homozygote(P＜0.05).A quantitative assay by capillary electrophoresis in 90 MPN patients showed that the mutated mRNA ratio was(89.5±6.5)% (range,70%-100%) in the JAK2V617F homozygote and(57.9±6.7)%(range,48.2% -70%) in the JAK2V617F heterozygote patients.18 PV heterozygote patients showed higher levels of mutated JAK2 mRNA than 36 heterozygote ET patients(P＜0.05). Cytogenetic analysis was performed in 93 of the 123 patients,6 patients exhibited abnormal karyotype,but special chromosomal abnormality were not found.JAK2V617F mutation was detected in 131 patients with MPN by combination of ARMS-PCR and capillary electrophoresis.According to the test results and clinical materials,a higher prevalence of JAK2V617F in either the heterozygote or homozyote status in essential thrombocythemia(ET) was observed in elderly patients with ET (P＜0.05);the presence of JAK2V617F was found to be significantly correlative with advanced age at diagnosis(P＜0.05);the patients whose age≥60 years showed significantly higher JAK2 mutated RNA levels as compared to patients whose age＜60 years(P＜0.05);The presence of JAK2V617F in PV and ET was found to be significantly associated with higher hemoglobin level and higher leukocyte count(P＜0.05),in addition, as compared to heterozygous ET patients,higher leukocyte count was observed in homozygous ET patients(P＜0.05);The frequency of JAK2V617F mutation and the prevalence of homozygote in PV patients were higher than in ET patients(P＜0.05);The diffences of JAK2V617F mRNA levels among PV,ET and IMF were not significant.JAK2V617F mutation was detected in 105 patients with MPN by combination of ARMS-PCR and capillary electrophoresis.According to the test results and clinical materials,PV and ET patients with thrombopoiesis had a higher white cell count and older age(P＜0.05).ET Patients with a white cell count≥1.5×10~9/L had significantly more thrombopoiesis than those with a white cell count＜1.5×10~9/L(P＞0.05).The prevalence of thrombotic complications in JAK2-positive patients was higher than in wild-type patients(P＜0.05).Compared with patients without thrombopoiesis,levels of mutated JAK2 mRNA in patients with thrombopoiesis were higher.A significant increase at risk of thrombopoiesis was seen in the patients carrying JAK2V617F(P＜0.05).The relative risk(odds ratio OR) of thrombopoiesis in patients with mutated mRNA ratio ranged from 60%to 100%was significant higher than that in the patients without JAK2V617F mutation(OR=31.5).ConclusionThe JAK2V617F mutation in MPN was correlative with advanced age,higher leukocyte counts,hemoglobin level and vascular events.The detection of JAK2V617F mutation in MPD-U patients may help to determine the transformation of the disease.JAK2V617F mutation,as a 1-type mutation,might not be an initiated event in the pathogenesis of AML,and its present does not meaned a poor prognosis affair in de novo leukemia.The ARMS PCR technique can be used to detect the frequency and mutational status of JAK2V617F mutation owing to sensitivity.The combination of ARMS-PCR and capillary electrophoresis enables quantitative assay of JAK2V617F mutation,which helps in MPN diagnosis and estimation of minimal residual disease.Older age,increased white cell count,JAK2V617F mutation and higher levels of mutated JAK2 mRNA were probably higher risk factors for thrombopoiesis.