JAK2V617F突变在骨髓增殖性肿瘤和急性髓性白血病（M2）中的实验和临床研究
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摘要
【背景与目的】
近年来慢性骨髓增殖性肿瘤(MPN)发病机制的研究取得了重要进展,2005年几个研究小组几乎同时发现,MPN患者造血细胞信号传导分子JAK2基因第1849位鸟嘌呤(G)被胸腺嘧啶(T)所取替,导致JAK2激酶617位缬氨酸(V)错义编码为苯丙氨酸(JAK2V617F),使JAK2分子得到持续活化,此改变可能与MPN的发病有密切的关联,并发现在绝大多数真性红细胞增多症(PV)患者和半数左右原发性血小板增多症(ET)患者及原发性骨髓纤维化(MF)患者中能检测到此突变,而在骨髓增生异常综合症、急性白血病患者中偶尔也能检测到此突变。随着研究的深入,发现此突变有两种类型——纯合型突变和杂合型突变,并发现突变型JAK2V617F与野生型JAK2基因的比例决定MPN的表型。因此,对JAK2V617F突变的研究有利于MPN的诊断,判定疾病的预后和未来靶向治疗靶点的选择,成为MPN研究的热点。近年国内对JAK2V617F突变的研究主要集中于临床检测,未能做到对此突变的进一步基因分型和相对定量研究,针对此我们研究了中国人MPN患者和急性白血病M2型患者中JAK2V617F突变的发病率、基因分型、相对定量情况和临床资料间的关系,总结如下。
【方法】
第一部分第一章选取2005年8月～2008年10月我院门诊就诊MPN患者,按照2001年WHO分类诊断标准选择患者,采集患者外周血5ml,Ficoll-paque分离单个核细胞,运用基因组DNA抽提试剂盒进行DNA的抽提,采用等位基因特异性PCR技术(AS-PCR)扩增JAK2基因,扩增产物进行琼脂糖凝胶电泳,筛选出突变阳性患者PCR产物进行测序证实,收集患者的临床资料、染色体结果及随访结果。
第一部分第二章选取2006年1月到2008年6月间到我院就诊的急性白血病M2型患者及部分我所冻存标本作为研究对象,按照《血液学诊断及疗效标准》选择患者,收集患者的临床资料,采集患者的外周血,运用AS-PCR(同第一部分第一章)扩增JAK2基因,并结合临床资料进行研究。
第二部分和第三部分选取2006年1月到2008年6月间到我院就诊的MPN患者及部分我所冻存标本作为研究对象,按照2001年世界卫生组织(WHO)慢性骨髓增殖性疾病诊断标准选择患者,采集患者外周血10ml,Ficoll-paque分离单个核细胞,Trizol一步法抽提总RNA,应用鼠源逆转录酶将RNA逆转录为互补cDNA,采用ARMS(Amplification-refractory muration sequencing)-PCR技术扩增JAK2基因,扩增产物经琼脂糖凝胶电泳进行基因分型,筛选出突变阳性患者PCR产物进行测序证实,并经毛细管电泳进行JAK2V617F突变转录本水平的相对定量分析,染色体检查按照常规R显带技术进行,同时分析检测结果与临床间的关系。
【结果】
1.运用AS-PCR方法检测了412例MPN患者,根据检测结果结合临床资料显示:共发现277例患者JAK2V617F突变阳性,JAK2V617F突变在原发性血小板增多症(ET)、特发性骨髓纤维化(IMF)及不能分类的骨髓增殖性疾病(MPD-U)中发生率分别为55.9%、66.7%和52.4%,差异无统计学意义(P＞0.05),但均较真性红细胞增多症(PV)中JAK2V617F发生率(95.6%)为低(P＜0.05);JAK2突变型患者的平均年龄高于野生型患者(P＜0.01);JAK2V617F阳性组的白细胞水平及血红蛋白水平均较JAK2V617F阴性组为高(P＜0.05);JAK2V617F阳性组中血管事件发生率高于阴性组(P＜0.05);具有JAK2V617F阳性的MPD-U患者较阴性MPD-U患者更易进展为典型MPN(P＜0.05);301例进行过染色体检查的患者中,18例存在核型异常,未发现核型异常与JAK2V617F突变之间存在相关性。
2.运用AS-PCR方法检测了80例AML-M2患者,根据检测结果结合临床资料显示:80例AML-M2患者中共发现JAK2V617F阳性7例,6例为初诊患者,1例为复发患者,总阳性率8.8%:7例JAK2V617F阳性AML-M2患者形态学上血象、骨髓象呈现出白血病改变特征,并无骨髓增殖性疾病(MPD)症象,免疫分型显示为髓系表达;本院治疗5例JAK2V617F阳性AML-M2患者,4例经2疗程诱导治疗达完全缓解(CR),1例未缓解(NR);5例JAK2V617F阳性AML-M2患者,除1例失访外,其余4例总生存期(OS)中位值为18.5个月。
3.运用ARMS-PCR联合毛细管电泳检测123例MPN患者,结果显示:JAK2V617F阳性90例,其中35例真性红细胞增多症(PV)患者中JAK2V617F阳性35例,阳性率100%(35/35),85例原发性血小板增多症(ET)患者中JAK2V617F阳性53例,阳性率62.4%(53/85),低于PV患者,差别有显著性(P＜0.05);3例慢性骨髓纤维化(IMF)患者中JAK2V617F阳性2例,阳性率66.7%(2/3)。90例JAK2V617F突变患者中共检出纯合突变35例,其中PV患者17例(17/35)占48.6%,ET患者17例(17/85)占20%,低于PV患者,差别有显著性(P＜0.05),IMF患者1例;90例毛细管电泳定量分析显示,35例纯合型突变患者JAK2V617F突变转录本水平为(89.5±6.5)%,55例杂合型突变患者JAK2V617F突变转录本水平为(57.9±6.7)%,较纯合型突变患者为低,差别有显著性(P＜0.05);杂合型PV患者JAK2V617F突变转录本水平为(63.7±6.3)%,高于杂和型ET患者的(54.4±6.1)%,差别有显著性(P＜0.05)。93例患者进行了染色体检查,6例有核型异常,但未发现特异性染色体改变。
4.运用ARMS-PCR联合毛细管电泳检测了131例MPN患者,根据检测结果结合临床资料显示:纯合型JAK2V617F突变ET患者和杂合型JAK2V617F突变ET患者其发病年龄均较野生型患者为高(P＜0.05);MPN患者中年龄与JAK2V617F突变的发生间存在相关性(P＜0.05),≥60岁患者JAK2V617F突变转录本水平高于＜60岁患者(P＜0.05);JAK2V617F突变阳性MPN患者(PV和ET)白细胞和血红蛋白水平均高于阴性患者(P＜0.05),ET患者中纯合型突变者白细胞水平高于杂合型突变者(P＜0.05);JAK2V617F突变在PV中的发生率和JAK2V617F纯合型突变在PV中的发生率均较JAK2V617F突变在ET中的发生率和JAK2V617F纯合型突变在ET中的发生率为高(P＜0.05);JAK2V617F转录本水平均值在PV、ET和IMF患者中无差别。
5.运用ARMS-PCR联合毛细管电泳检测了105例MPN患者,根据检测结果结合临床资料显示:有血栓PV、ET患者年龄、WBC数高于无血栓患者(F＜0.05),WBC≥15×10~9/LET患者血栓发生率高于WBC＜15×10~9/L患者(P＜0.05),JAK2V617F阳性患者血栓发生率高于阴性患者(P＜0.05),有血栓事件者JAK2V617F转录本水平高于无血栓事件者(P＜0.05),JAK2V617F阳性患者血栓事件风险度高于JAK2V617F阴性患者(OR=11.30),突变转录本水平在(60-100)%组患者血栓事件风险度高于JAK2V617F阴性组患者(OR=31.5)。
【结论】
1.JAK2V617F突变与MPN患者年龄、外周血细胞计数及血管事件之间存在关联性,JAK2V617F的检测对判定MPN-U患者疾病转归有提示作用。
2.作为AML发病机制中的Ⅰ类改变JAK2V617F突变可能并非是AML发病的初始事件,而是其发病的额外事件,初诊AML患者出现此改变也并非意味着疾病预后较差。
3.ARMS-PCR可作为JAK2V617突变分型较准确的检测方法,结合毛细管电泳可用于此突变的相对定量分析。
4.ARMS-PCR结合毛细管电泳定性和定量分析JAK2V617F突变可用于MPN的诊断、预后判定和微小残留病的检测。
5.高龄、白细胞计数增高,JAK2V617F突变及高转录本水平MPN患者血栓风险可能较高。
Background and objective
Recently,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.
Result
JAK2V617F 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).
Conclusion
The 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.
近年来慢性骨髓增殖性肿瘤(MPN)发病机制的研究取得了重要进展,2005年几个研究小组几乎同时发现,MPN患者造血细胞信号传导分子JAK2基因第1849位鸟嘌呤(G)被胸腺嘧啶(T)所取替,导致JAK2激酶617位缬氨酸(V)错义编码为苯丙氨酸(JAK2V617F),使JAK2分子得到持续活化,此改变可能与MPN的发病有密切的关联,并发现在绝大多数真性红细胞增多症(PV)患者和半数左右原发性血小板增多症(ET)患者及原发性骨髓纤维化(MF)患者中能检测到此突变,而在骨髓增生异常综合症、急性白血病患者中偶尔也能检测到此突变。随着研究的深入,发现此突变有两种类型——纯合型突变和杂合型突变,并发现突变型JAK2V617F与野生型JAK2基因的比例决定MPN的表型。因此,对JAK2V617F突变的研究有利于MPN的诊断,判定疾病的预后和未来靶向治疗靶点的选择,成为MPN研究的热点。近年国内对JAK2V617F突变的研究主要集中于临床检测,未能做到对此突变的进一步基因分型和相对定量研究,针对此我们研究了中国人MPN患者和急性白血病M2型患者中JAK2V617F突变的发病率、基因分型、相对定量情况和临床资料间的关系,总结如下。
【方法】
第一部分第一章选取2005年8月～2008年10月我院门诊就诊MPN患者,按照2001年WHO分类诊断标准选择患者,采集患者外周血5ml,Ficoll-paque分离单个核细胞,运用基因组DNA抽提试剂盒进行DNA的抽提,采用等位基因特异性PCR技术(AS-PCR)扩增JAK2基因,扩增产物进行琼脂糖凝胶电泳,筛选出突变阳性患者PCR产物进行测序证实,收集患者的临床资料、染色体结果及随访结果。
第一部分第二章选取2006年1月到2008年6月间到我院就诊的急性白血病M2型患者及部分我所冻存标本作为研究对象,按照《血液学诊断及疗效标准》选择患者,收集患者的临床资料,采集患者的外周血,运用AS-PCR(同第一部分第一章)扩增JAK2基因,并结合临床资料进行研究。
第二部分和第三部分选取2006年1月到2008年6月间到我院就诊的MPN患者及部分我所冻存标本作为研究对象,按照2001年世界卫生组织(WHO)慢性骨髓增殖性疾病诊断标准选择患者,采集患者外周血10ml,Ficoll-paque分离单个核细胞,Trizol一步法抽提总RNA,应用鼠源逆转录酶将RNA逆转录为互补cDNA,采用ARMS(Amplification-refractory muration sequencing)-PCR技术扩增JAK2基因,扩增产物经琼脂糖凝胶电泳进行基因分型,筛选出突变阳性患者PCR产物进行测序证实,并经毛细管电泳进行JAK2V617F突变转录本水平的相对定量分析,染色体检查按照常规R显带技术进行,同时分析检测结果与临床间的关系。
【结果】
1.运用AS-PCR方法检测了412例MPN患者,根据检测结果结合临床资料显示:共发现277例患者JAK2V617F突变阳性,JAK2V617F突变在原发性血小板增多症(ET)、特发性骨髓纤维化(IMF)及不能分类的骨髓增殖性疾病(MPD-U)中发生率分别为55.9%、66.7%和52.4%,差异无统计学意义(P＞0.05),但均较真性红细胞增多症(PV)中JAK2V617F发生率(95.6%)为低(P＜0.05);JAK2突变型患者的平均年龄高于野生型患者(P＜0.01);JAK2V617F阳性组的白细胞水平及血红蛋白水平均较JAK2V617F阴性组为高(P＜0.05);JAK2V617F阳性组中血管事件发生率高于阴性组(P＜0.05);具有JAK2V617F阳性的MPD-U患者较阴性MPD-U患者更易进展为典型MPN(P＜0.05);301例进行过染色体检查的患者中,18例存在核型异常,未发现核型异常与JAK2V617F突变之间存在相关性。
2.运用AS-PCR方法检测了80例AML-M2患者,根据检测结果结合临床资料显示:80例AML-M2患者中共发现JAK2V617F阳性7例,6例为初诊患者,1例为复发患者,总阳性率8.8%:7例JAK2V617F阳性AML-M2患者形态学上血象、骨髓象呈现出白血病改变特征,并无骨髓增殖性疾病(MPD)症象,免疫分型显示为髓系表达;本院治疗5例JAK2V617F阳性AML-M2患者,4例经2疗程诱导治疗达完全缓解(CR),1例未缓解(NR);5例JAK2V617F阳性AML-M2患者,除1例失访外,其余4例总生存期(OS)中位值为18.5个月。
3.运用ARMS-PCR联合毛细管电泳检测123例MPN患者,结果显示:JAK2V617F阳性90例,其中35例真性红细胞增多症(PV)患者中JAK2V617F阳性35例,阳性率100%(35/35),85例原发性血小板增多症(ET)患者中JAK2V617F阳性53例,阳性率62.4%(53/85),低于PV患者,差别有显著性(P＜0.05);3例慢性骨髓纤维化(IMF)患者中JAK2V617F阳性2例,阳性率66.7%(2/3)。90例JAK2V617F突变患者中共检出纯合突变35例,其中PV患者17例(17/35)占48.6%,ET患者17例(17/85)占20%,低于PV患者,差别有显著性(P＜0.05),IMF患者1例;90例毛细管电泳定量分析显示,35例纯合型突变患者JAK2V617F突变转录本水平为(89.5±6.5)%,55例杂合型突变患者JAK2V617F突变转录本水平为(57.9±6.7)%,较纯合型突变患者为低,差别有显著性(P＜0.05);杂合型PV患者JAK2V617F突变转录本水平为(63.7±6.3)%,高于杂和型ET患者的(54.4±6.1)%,差别有显著性(P＜0.05)。93例患者进行了染色体检查,6例有核型异常,但未发现特异性染色体改变。
4.运用ARMS-PCR联合毛细管电泳检测了131例MPN患者,根据检测结果结合临床资料显示:纯合型JAK2V617F突变ET患者和杂合型JAK2V617F突变ET患者其发病年龄均较野生型患者为高(P＜0.05);MPN患者中年龄与JAK2V617F突变的发生间存在相关性(P＜0.05),≥60岁患者JAK2V617F突变转录本水平高于＜60岁患者(P＜0.05);JAK2V617F突变阳性MPN患者(PV和ET)白细胞和血红蛋白水平均高于阴性患者(P＜0.05),ET患者中纯合型突变者白细胞水平高于杂合型突变者(P＜0.05);JAK2V617F突变在PV中的发生率和JAK2V617F纯合型突变在PV中的发生率均较JAK2V617F突变在ET中的发生率和JAK2V617F纯合型突变在ET中的发生率为高(P＜0.05);JAK2V617F转录本水平均值在PV、ET和IMF患者中无差别。
5.运用ARMS-PCR联合毛细管电泳检测了105例MPN患者,根据检测结果结合临床资料显示:有血栓PV、ET患者年龄、WBC数高于无血栓患者(F＜0.05),WBC≥15×10~9/LET患者血栓发生率高于WBC＜15×10~9/L患者(P＜0.05),JAK2V617F阳性患者血栓发生率高于阴性患者(P＜0.05),有血栓事件者JAK2V617F转录本水平高于无血栓事件者(P＜0.05),JAK2V617F阳性患者血栓事件风险度高于JAK2V617F阴性患者(OR=11.30),突变转录本水平在(60-100)%组患者血栓事件风险度高于JAK2V617F阴性组患者(OR=31.5)。
【结论】
1.JAK2V617F突变与MPN患者年龄、外周血细胞计数及血管事件之间存在关联性,JAK2V617F的检测对判定MPN-U患者疾病转归有提示作用。
2.作为AML发病机制中的Ⅰ类改变JAK2V617F突变可能并非是AML发病的初始事件,而是其发病的额外事件,初诊AML患者出现此改变也并非意味着疾病预后较差。
3.ARMS-PCR可作为JAK2V617突变分型较准确的检测方法,结合毛细管电泳可用于此突变的相对定量分析。
4.ARMS-PCR结合毛细管电泳定性和定量分析JAK2V617F突变可用于MPN的诊断、预后判定和微小残留病的检测。
5.高龄、白细胞计数增高,JAK2V617F突变及高转录本水平MPN患者血栓风险可能较高。
Background and objective
Recently,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.
Result
JAK2V617F 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).
Conclusion
The 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.
引文
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15 Scott LM, Scott MA, Campbell PJ, et al: Progenitors homozygous for the V617F mutation occur in most patients with polycythemia vera but not essential thrombocythemia. Blood2006; 108: 2435 - 2437.
16 Horn T, Kremer M, Dechow T, et al: Detection of the activating JAK2 V617F mutation in paraffin-embedded trephine bone marrow biopsies of patients with chronic myeloproliferative diseases. JMol Diagn 2006; 8: 299 - 304.
17 Bench AJ, Cross NC, Huntly BJ, et al: Myeloproliferative disorders. Best Pract Res Clin Haematol 2001; 14: 531-551.
18 Bench AJ, Pahl HL: Chromosomal abnormalities and molecular markers in myeloproliferative disorders. Semin Hematol 2005; 42: 196 - 205.
19 Najfeld V, Cozza A, Berkofsy-Fessler W, et al: Numerical gain and structural rearrangements of JAK2, identified by FISH, characterize both JAK2(5/7F>F-positive and -negative patients with Ph-negative MPD, myelodysplasia, and B-lymphoid neoplasms. Exp Hematol 2007; 35: 1668 - 1676.
20 Chen Z, Sandberg AA: Molecular cytogenetic aspects of hematological malignancies: clinical implications. Am JMedGenet2002; 115:130-141.
21 Hsiao HH, Ito Y, Sashida G: De novo appearance of der(l;7)(qlO;plO) is associated with leukemic transformation and unfavorable prognosis in essential thrombocythemia. LeukRes 2005; 29: 1247 - 1252.
22 Bacher U, Haferlach T, Kern W, et al: Conventional cytogenetics of myeloproliferative diseases other than CML contribute valid information. Ann Hematol 2005; 84: 250 -257.
23 Panani AD: Cytogenetic findings in untreated patients with essential thrombocy- themia. In Vivo 2006; 20: 381 -384.
1.Baxter EJ,Scott LM,Campbell PJ,et al.Acquired mutation of the tyrosine kinase JAK2in human myeloproliferative diseases[J].Lancet,2005,365:1054-1061.
2.Panani AD.Cytogenetic and molecular aspects of Philadelphia negative chronic myeloproliferative disorders:clinical implications[J].Cancer Letters,2007,255:12-25
3.费海容,张日,陈苏宁,等.骨髓增殖性疾病137例患者JAK2基因突变的研究.中华内科杂志[J],2007,46(4):271-273
4.Vardiman JW,Harris NL,Brunning RD.The World Health Organization(WHO)classification of the myeloid neoplasms[J].Blood,2002,100:2292-2302
5.Vannucchi AM,Pancrazzi A,Bogani C,et al.A quantitative assay for JAK2V617F mutation in myeloproliferative disorders by ARMS-PCR and capillary electrophoresis[J].Leukemia,2006,20:1055-1060
6.Kralovics R,Passamonti F,Buser AS,et al.A gain-of-function mutation of JAK2 in myeloproliferative disorders[J].N Engl J Med,2005,352:1779-1790
7.Kralovics R,Teo SS,Li S,et al.Acquisition of the V617F mutation of JAK2 is a late genetic event in a subset of patients with myeloproliferative disorders[J].Blood.2006.108:1377-1380.
8.Nussenzveig RH,Swierczek SI,Jelinek J,et al.Polycythemia vera is not initiated by JAK2V617F mutation[J].Experimental Hematology,2007,35:32-38.
9.James C,Ugo V,Le Couedic JPL,et al.A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera[J].Nature,2005,434:1144-1148.
10.Scott LM,Scott MA,Campbell PJ,et al.Progenitors homozygous for the V617F JAK2 mutation occure in most patients with polycythemia vera but not essential thrombocythemia[J].Blood,2006,108:2435-2437.
1.Baxter EJ,Scott LM,Campbell PJ,et al.Acquired mutation of the tyrosine kinase JAK2in human myeloproliferative diseases.Lancet,2005,365:1054-1061.
2.Levine RL,Wadleigh M.Activating mutation in the tyrosine kinase JAK2 in polycythemia vera,Essential thrombocythemia,and myeloid metaplasia with myelofibrosis.Cancer Cell,2005,7:387-397.
3.James C,Ugo V,Le Couedic JPL,et al.A unique clonal JAK2 mutation leading to constitutive signaling causes polycythemia vera.Nature,2005,434:1144-1148.
4.Kralovics R,Passamonti F,Buser A S,et al A gain-of- function mutation of JAK2 in myeloproliferative disorders.N Engl J Med,2005,352:1779-1790.
5.Jaffe ES,Harris NL,Stein H,et al.World Health Organization Classification of Tumours of Haemtopoiesis and Lymphoid Tissues.IARC:Lyon,France,2001,pp1-351.
6.Vannucchi AM,Pancrazzi A,Bogani C,et al.A quantitative assay for JAK2V617F mutation in myeloproliferative disorders by ARMS-PCR and capillary electrophoresis.Leukemia,2006,20:1055-1060.
7.Panani AD.Cytogenetic and molecular aspects of Philadelphia negative chronic myeloproliferative disorders:Clinical implications.Cancer Letters,2007,255:12-25.
8. Scott LM , Scott MA, Campbell PJ et al. Progenitors homozygous for the V617F JAK2 mutation occur in most patients with polycythemia vera but not essential thrombocythemia. Blood,2006, 108 : 2435-2437.
9. Marchioli R, Finazzi G, Landolfi R et al. Vascular and neoplastic risk in a large cohort of patients with polycythemia vera J Clini Oncol, 2005,23:2224-2232.
10. Michiels JJ. Clinical pathological and molecular features of the chronic myeloproliferative disorders: MPD 2005 and beyond. Hematology, 2005,10:215-223.
11. Campbell PJ, Scott LM, Buck G, et al.Defmition of subtypes of essential thrombocythaemia and relation to polycythemia vera based on JAK2V617F mutation status: aprospective study .Lancet, 2005, 366:1945-1953.
1 Elliott MA,Tefferi A.Thrombosis and haemorrhage in polycythaemia vera and essential thrombocythaemia.Br J Haematology,2004,128:275-290.
2 Baxter EJ,Scott LM,Campbell PJ,et al.Acquired mutation of the tyrosine kinase JAK2in human myeloproliferative diseases.Lancet,2005,365:1054-1061.
3 James C,Ugo V,Le Couedic JPL,et al.A unique clonal JAK2 mutation leading to constitutive signaling causes polycythaemia vera.Nature,2005,434:1144-1148.
4 Levine RL,Wadleigh M.Activating mutation in the tyrosine kinase JAK2 in polycythemia vera,Essential thrombocythemia,and myeloid metaplasia with myelofibrosis.Cancer Cell,2005,7:387-397.
5 WHO classification of the chronic myeloproliferative disease(MPN) polycythemia vera, chronic idiopathic myelofibrosis, essential thrombocythemia, and MPN unclassifiable. WHO Classification of Tumors. Tumors of Haemtopoiesis and Lymphoid Tissues. Lyon : IARC; 200l.p.31-42
6 Vannucchi AM , Pancrazzi A , Bogani C , et al. A quantitative assay for JAK2V617F mutation in myeloproliferative disorders by ARMS-PCR and capillary electrophoresis. Leukemia, 2006, 20 : 1055-1060.
7 Campbell P J, Green A R. The myeloproliferative disorders. N Engl J Med, 2006, 355:2452 -2466.
8 Carobbio A, Finazzi G, Guerini V, et al.Leukocytosis is a risk factor for thrombosis in essential thrombocythemia: interaction with treatment, standard risk factors, and JAK2 mutation status. Blood, 2007 ,109; 2310—2313.
9 Kralovics R,Passsmonti F,Buser AS,et al.A gain of function mutation of JAK2 in myeloproliferative disorders.N Engl J Med,2005,352: 1779-1790.
10 Xu X,Zhang Q,Luo J, et al. JAK2 (V617F): prevalence in a large Chinese hospital population. Blood, 2007; 109:339-342.
11 Finazzi G,Rambaldi A,Guerini V,et al.Risk of thrombosis in patients with essential thrombocythemia and polycythemia vera according to JAK2V617F mutation status.Haematologica,2007,92:135-136.
12 Antonioli E, Guglielmelli P, Poli G, et al.Influence of JAK2V617F allele burden on phenotype in essential thrombocythemia.Haematologica, 2008, 93:41-48.
13 Landolfi R,Di Gennaro L,Barbui T, et al.Leukocytosis as a major thrombotic risk factor in patients with polycythemia vera. Blood, 2006, 109:2446-2452
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1 Vardiman JW, Brunning RD, Harris NL: WHO histological classification of chronic myeloproliferative diseases. In: World Health Organization Classification of Tumors: Tumors of the Haematopoietic and Lymphoid Tissues (Jaffe ES, Harris NL, Stein H, et al, eds). Lyon, France: International Agency for Research on Cancer (IARC) Press, 2001 ;pp 17-44.
2 James C, Ugo V, Le Couédic JP, et al: A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature 2005; 434: 1144 - 1148.
3 Kralovics R, Passamonti F, Buser AS, et al: A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 2005; 352: 1779 - 1790.
4 Levine RL, Wadleigh M, Cools J, et al: Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell 2005; 7: 387 - 397.
5 Baxter EJ, Scott LM, Campbell PJ, et al: Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 2005; 365: 1054 - 1061.
6 Jones AV, Kreil S, Zoi K, et al: Widespread occurrence of the JAK2 V617F mutation in chronic myeloproliferative disorders. Blood 2005; 106: 2162 - 2168.
7 Zhao R, Xing S, Li Z, et al: Identification of an acquired JAK2 mutation in polycythemia vera. J Biol Chem 2005; 280: 22788 - 22792.
8 Vannucchi AM, Pancrazzi A, Bogani C, et al: A quantitative assay for JAK2~(V617F) mutation in myeloproliferative disorders by ARMS-PCR and capillary electrophoresis. Leukemia 2006; 20: 1055 - 1060.
9 Najfeld V, Coyle T, Berk PD: Transformation of polycythemia vera to acute nonlymphocytic leukemia accompanied by t(1;3)(p36;q21) karyotype. Cancer Genet Cytogenet 1988; 33: 193-200.
10 Shaffer LG, Tommerup N (eds): ISCN 2005: An International System for Human Cytogenetic Nomenclature (2005). Basel: S Karger, 2005.
11 Chen S, Fei H, Zhang R, et al: Analysis of JAK2_(V617F) mutation in Chinese patients with myeloproliferative disorders. Am J Hematol 2007; 82: 458 - 459.
12 Ohyashiki K, Aota Y, Akahane D, et al: JAK2~(V617F) mutational status as determined by semiquantitative sequence-specific primer-single molecule fluorescence detection assay is linked to clinical features in chronic myeloproliferative disorders. Leukemia 2007; 21: 1097-1099.
13 Kralovics R, Teo SS, Li S, et al: Acquisition of the V617F mutation of JAK2 is a late genetic event in a subset of patients with myeloproliferative disorders. Blood 2006; 108: 1377-1380.
14 Nussenzveig RH, Swierczek SI, Jelinek J, et al: Polycythemia vera is not initiated by JAK2V617F mutation. Exp Hematol 2007; 35: 32 - 38.
15 Scott LM, Scott MA, Campbell PJ, et al: Progenitors homozygous for the V617F mutation occur in most patients with polycythemia vera but not essential thrombocythemia. Blood2006; 108: 2435 - 2437.
16 Horn T, Kremer M, Dechow T, et al: Detection of the activating JAK2 V617F mutation in paraffin-embedded trephine bone marrow biopsies of patients with chronic myeloproliferative diseases. JMol Diagn 2006; 8: 299 - 304.
17 Bench AJ, Cross NC, Huntly BJ, et al: Myeloproliferative disorders. Best Pract Res Clin Haematol 2001; 14: 531-551.
18 Bench AJ, Pahl HL: Chromosomal abnormalities and molecular markers in myeloproliferative disorders. Semin Hematol 2005; 42: 196 - 205.
19 Najfeld V, Cozza A, Berkofsy-Fessler W, et al: Numerical gain and structural rearrangements of JAK2, identified by FISH, characterize both JAK2(5/7F>F-positive and -negative patients with Ph-negative MPD, myelodysplasia, and B-lymphoid neoplasms. Exp Hematol 2007; 35: 1668 - 1676.
20 Chen Z, Sandberg AA: Molecular cytogenetic aspects of hematological malignancies: clinical implications. Am JMedGenet2002; 115:130-141.
21 Hsiao HH, Ito Y, Sashida G: De novo appearance of der(l;7)(qlO;plO) is associated with leukemic transformation and unfavorable prognosis in essential thrombocythemia. LeukRes 2005; 29: 1247 - 1252.
22 Bacher U, Haferlach T, Kern W, et al: Conventional cytogenetics of myeloproliferative diseases other than CML contribute valid information. Ann Hematol 2005; 84: 250 -257.
23 Panani AD: Cytogenetic findings in untreated patients with essential thrombocy- themia. In Vivo 2006; 20: 381 -384.
1.Baxter EJ,Scott LM,Campbell PJ,et al.Acquired mutation of the tyrosine kinase JAK2in human myeloproliferative diseases[J].Lancet,2005,365:1054-1061.
2.Panani AD.Cytogenetic and molecular aspects of Philadelphia negative chronic myeloproliferative disorders:clinical implications[J].Cancer Letters,2007,255:12-25
3.费海容,张日,陈苏宁,等.骨髓增殖性疾病137例患者JAK2基因突变的研究.中华内科杂志[J],2007,46(4):271-273
4.Vardiman JW,Harris NL,Brunning RD.The World Health Organization(WHO)classification of the myeloid neoplasms[J].Blood,2002,100:2292-2302
5.Vannucchi AM,Pancrazzi A,Bogani C,et al.A quantitative assay for JAK2V617F mutation in myeloproliferative disorders by ARMS-PCR and capillary electrophoresis[J].Leukemia,2006,20:1055-1060
6.Kralovics R,Passamonti F,Buser AS,et al.A gain-of-function mutation of JAK2 in myeloproliferative disorders[J].N Engl J Med,2005,352:1779-1790
7.Kralovics R,Teo SS,Li S,et al.Acquisition of the V617F mutation of JAK2 is a late genetic event in a subset of patients with myeloproliferative disorders[J].Blood.2006.108:1377-1380.
8.Nussenzveig RH,Swierczek SI,Jelinek J,et al.Polycythemia vera is not initiated by JAK2V617F mutation[J].Experimental Hematology,2007,35:32-38.
9.James C,Ugo V,Le Couedic JPL,et al.A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera[J].Nature,2005,434:1144-1148.
10.Scott LM,Scott MA,Campbell PJ,et al.Progenitors homozygous for the V617F JAK2 mutation occure in most patients with polycythemia vera but not essential thrombocythemia[J].Blood,2006,108:2435-2437.
1.Baxter EJ,Scott LM,Campbell PJ,et al.Acquired mutation of the tyrosine kinase JAK2in human myeloproliferative diseases.Lancet,2005,365:1054-1061.
2.Levine RL,Wadleigh M.Activating mutation in the tyrosine kinase JAK2 in polycythemia vera,Essential thrombocythemia,and myeloid metaplasia with myelofibrosis.Cancer Cell,2005,7:387-397.
3.James C,Ugo V,Le Couedic JPL,et al.A unique clonal JAK2 mutation leading to constitutive signaling causes polycythemia vera.Nature,2005,434:1144-1148.
4.Kralovics R,Passamonti F,Buser A S,et al A gain-of- function mutation of JAK2 in myeloproliferative disorders.N Engl J Med,2005,352:1779-1790.
5.Jaffe ES,Harris NL,Stein H,et al.World Health Organization Classification of Tumours of Haemtopoiesis and Lymphoid Tissues.IARC:Lyon,France,2001,pp1-351.
6.Vannucchi AM,Pancrazzi A,Bogani C,et al.A quantitative assay for JAK2V617F mutation in myeloproliferative disorders by ARMS-PCR and capillary electrophoresis.Leukemia,2006,20:1055-1060.
7.Panani AD.Cytogenetic and molecular aspects of Philadelphia negative chronic myeloproliferative disorders:Clinical implications.Cancer Letters,2007,255:12-25.
8. Scott LM , Scott MA, Campbell PJ et al. Progenitors homozygous for the V617F JAK2 mutation occur in most patients with polycythemia vera but not essential thrombocythemia. Blood,2006, 108 : 2435-2437.
9. Marchioli R, Finazzi G, Landolfi R et al. Vascular and neoplastic risk in a large cohort of patients with polycythemia vera J Clini Oncol, 2005,23:2224-2232.
10. Michiels JJ. Clinical pathological and molecular features of the chronic myeloproliferative disorders: MPD 2005 and beyond. Hematology, 2005,10:215-223.
11. Campbell PJ, Scott LM, Buck G, et al.Defmition of subtypes of essential thrombocythaemia and relation to polycythemia vera based on JAK2V617F mutation status: aprospective study .Lancet, 2005, 366:1945-1953.
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