原发性血小板增多症患者的临床及分子生物学特点研究

【作者】 付荣凤；

【导师】 杨仁池；

【作者基本信息】 北京协和医学院 ， 内科学（专业学位）， 2014， 博士

【摘要】 研究背景：原发性血小板增多症(Essential Thrombocythemia, ET)是经典ph(Philadelphia)染色体阴性的骨髓增殖性肿瘤(Myeloproliferative Neoplasms,MPN)中最常见的亚型。血栓栓塞和出血倾向是ET最常见的并发症。传统的危险度分层将年龄≥60岁或既往血栓史列为高危组。近期国际MPN研究和治疗工作组提出了一种新的预测ET患者血栓栓塞的风险模型,即IPSET-血栓模型。研究目的：分析中国ET患者的临床及分子生物学特点,分析其血栓形成危险因素,验证IPSET-血栓模型在中国ET患者的可行性,并评价此模型对治疗的指导意义。研究方法：回顾性分析1982年至2012年我院诊断的ET患者(年龄≥18岁),对患者病历资料进行重新评估,所有患者均符合2008年WHO诊断标准；COX比例风险模型筛选血栓形成的独立危险因素,并用C统计值评价模型的检验效能；乘积极限法(K-M曲线)绘制并比较不同危险组的生存曲线。研究结果：纳入患者共970例,中位随访时间为49(0-360)个月。中国ET患者临床特点与欧美患者相似。其中746例检测了JAK2V617F突变,阳性患者380例(50.9%),阴性患者366例(49.1%)。随访过程中99例(10.2%)发生血栓并发症,5年、10年及15年累积血栓发生率分别为9%、22%及35%。COX模型多因素分析表明年龄>60岁[风险比(HR)=1.949]、既往血栓史(HR=2.484)、JAK2V617F突变(HR=1.719)及心血管危险因素(HR=1.877)为血栓栓塞并发症的独立危险因素,结果与IPSET-血栓模型一致。COX模型中以上危险因素的C值为0.714(95%可信区间:0.655-0.772),高于传统分层模型的C值[0.647(95%可信区间：0.577-0.717)]。根据IPSET-血栓模型将ET患者分为低危、中危及高危组,三组无血栓生存预后有统计学差异(P<0.001)。治疗方面,在中危及高危组中,接受减低血小板治疗的患者的血栓发生率均低于组内未治疗患者(P<0.05),而在低危组患者中,差异没有统计学意义(P=0.446)。减低血小板的药物可能通过减低病程中白细胞数来达到预防血栓发生的作用。结论：我们在中国ET患者中首次验证了IPSET-血栓模型的预后指导意义,并为IPSET-血栓模型指导治疗提供了依据。研究背景：获得性体细胞突变JAK2V617F见于约50%-60%的原发性血小板增多症(Essential Thrombocythemia, ET)患者。近期在约50%的JAK2V617F阴性ET患者中发现了钙网蛋白基因(Calreticulin, CALR)突变,在ET诊断中有重要价值。但此突变尚未在亚洲患者中报道,且其在指导治疗方面的意义尚不明确。研究目的：评估JAK2V617F、MPL和CALR基因突变在中国ET患者中的发生率,并探讨其在ET的诊断、判断预后及治疗中的意义。研究方法：回顾性分析1990年至2013年我院诊断的436例ET患者的临床资料,并在所有患者中检测JAK2V617F、MPL及CALR基因突变。JAK2V617F突变采用实时定量PCR法检测,采用Sanger双向测序检测MPL基因10号外显子及CALR基因9号外显子突变,并将每种CALR基因突变亚型行TA克隆后测序。COX比例风险模型筛选血栓形成的独立危险因素,乘积极限法(K-M曲线)绘制并比较不同危险组的生存曲线。研究结果：中国ET患者的MPL突变频率(1.4%)低于欧美ET患者,但JAK2V617F突变(55.1%)及CALR突变率(22.7%)与欧美患者相似。研究发现了5种新的CALR基因插入及缺失突变。两例患者同时存在JAK2V617F及CALR突变,其中一例CALR突变为新发现的9号外显子点突变(c.997C>T),并证实此点突变为获得性体细胞突变。与JAK2V617F突变患者相比,CALR突变患者的白细胞数、中性粒细胞数、血红蛋白含量及内源性红系集落形成(EEC)阳性率较低,而血小板数较高,差异均有统计学意义(P值均<0.01)。无JAK2/MPL/CALR突变患者的白细胞数、血红蛋白含量及EEC阳性率均介于JAK2V617F突变及CALR突变患者之间。Cox模型多因素分析显示JAK2V617F突变是诊断时(HR=1.836,P=0.040)及随访过程中(HR=2.678,P=0.023)血栓形成的独立危险因素,而CALR基因突变并不是血栓危险因素。K-M曲线显示CALR突变患者的无血栓生存率高于JAK2V617F突变患者(P=0.014),这种差异可能与Ⅱ型CALR突变患者的血栓发生率(7.9%)显著低于JAK2V617F突变患者(30.0%)有关(P=0.004)。JAK2V617F突变患者的骨纤转化率与CALR突变患者无显著差异(P=1.000),但CALR突变患者的骨髓网状纤维沉积分级高于JAK2V617F突变患者(P=0.003),这种现象主要由于I型CALR突变患者中I级网状纤维增高引起。在同一患者接受减低血小板治疗前与治疗后的配对样本中分别检测CALR基因突变,发现减低血小板治疗并不能完全消除CALR突变克隆。联合应用抗血小板及减低血小板的治疗降低了JAK2V617F患者的血栓发生率(P=0.034),但未减少CALR突变患者的血栓发生(P=1.000)。结论：我们在中国ET患者中证实了CALR基因突变在ET的诊断和判断预后中的价值,并发现JAK2V617F和CALR基因突变与临床特点及预后有关。本研究为基于分子生物学指标的治疗策略提供了初步依据。研究背景：儿童原发性血小板增多症(essential thrombocythemia,ET)罕见,由于样本量小,临床特点并未完全明确,因此需要较大样本量研究来评估。研究目的：评价儿童ET患者的症状特点、血栓出血并发症、疾病转化率及治疗策略。研究方法：回顾性分析1986年至2012年于我院诊断为ET的70例儿童患者(年龄≤18岁),对患者的临床特点和实验室检查进行分析。研究结果：中位诊断年龄为12(2-18)岁,伴有明显的男性倾向(男/女=1.4)与成人ET的女性倾向不同。多数患儿(54.3%)就诊时无血小板增多相关症状,有无临床症状与血小板数有关(P=0.002)。儿童ET的JAK2V617F阳性率(18.8%)低于成人ET。与成人不同的是,JAK2V617F突变阳性与阴性患儿的白细胞数、中性粒细胞数、血红蛋白水平及血小板数无显著差异(P均>0.05)。多数患儿(82.6%)的血小板增多相关症状可通过药物缓解。儿童ET患者使用羟基脲也存在耐药或不耐受,但长期使用未发现严重副作用,无一例继发其它恶性肿瘤。ET患儿的血栓发生率低(2.9%)于成人ET,且2例血栓患儿均为女性,其中一例为腹腔静脉血栓导致门静脉海绵样变并合并骨髓纤维化,在儿童ET中是首次报道。与成人ET一样,儿童ET转化为骨髓纤维化的比例较低(2.9%),病程中白细胞数持续升高可能是骨纤转化的前兆。结论：儿童ET在临床转归及分子生物学特点上不同于成人ET。研究背景：儿童原发性血小板增多症(Essential Thrombocythemia,ET)的JAK2V617F阳性率低于成人患者,多数未发现分子克隆标志,发病机制仍不清楚。研究目的：检测儿童ET的突变位点并推测克隆演变,为揭示儿童ET的发病机制提供依据。研究方法：采用高通量目标区域捕获测序技术在20例儿童ET患者中检测与血液肿瘤相关的55个基因突变并进行验证,验证为真阳性的所有位点在100个正常人中检测排除多态性位点。根据基因突变负荷预测克隆演变过程。研究结果：平均测序深度为384×,平均可覆盖靶区域的99.6%,验证结果显示准确率达93.8%。最终确定16种疾病相关点突变,这些突变在100个正常人DNA样本中均未发现。共涉及突变基因11个,累及患者13例,其余7例患儿未发现疾病相关基因突变。与成人不同的是,经自身正常组织对照证实多数突变位点(56.3%)为生殖细胞突变,可能是儿童ET发病提早的重要机制之一。JAK2V617F阳性患者6例,阳性率(18.8%)低于成人ET。所有20例患儿均未发现CALR基因和MPL基因突变。JAK246/1单体型基因型显示,12例为(60%)CG杂合子,8例(40%)为CC纯合子,无GG纯合子。多种基因可以和JAK2V617F同时发生,包括ASXL1、U2AF1、NRAS和IDH1。突变基因涉及多种功能,其中JAK-STAT信号转导通路基因(45%)与表观调节基因(25%)最常累及,两类基因可合并突变(20%)。基于突变负荷的克隆性分析显示JAK2V617F突变可能是发生于表观调节基因突变后的晚期事件。结论：儿童ET的突变谱不同于成人,JAK2V617F突变率低,且无CALR突变,可能存在与成人不同的发病机制。生殖细胞突变可能在儿童ET提前发病机制中起重要作用。JAK2V617F可能并不是驱动基因,而是其它基因突变累积到一定程度的继发改变。更多还原

【Abstract】 Background:Essential thrombocythemia (ET) is the most common type of Philadelphia chromosome negative myeloproliferative neoplasms characterized by increased risk of vascular events (thrombosis and hemorrhage). Traditionally, advanced age≥60and history of thrombosis are regarded as predictive risk factors for thrombosis. Recently, a new International Prognostic Score of thrombosis for ET (IPSET-thrombosis) was proposed to predict thrombotic events.Objectives:To analyze clinical and biological characteristics of ET, and to evaluate the prognostic value and clinical implication of the IPSET-thrombosis model in a large cohort of Chinese ET patients.Methods:We retrospectively evaluated the characteristics and risk factors for thrombosis in Chinese patients diagnosed with ET in our hospital from1982to2012. Their clinical data were carefully reassessed, and all of them met the2008WHO criteria. Cox proportional hazards regression was employed to carry out univariate survival analysis, and factors that were significant were forced to multivariate survival analysis. The C-index was calculated based on Receiver Operating Characteristic curve using prognostic index. Thrombosis-free survival was estimated by Kaplan-Meier method, and log-rank test was used to compare thrombosis-free survival data.Results:A total of970ET patients were enrolled. The median follow-up was49months (range,0-360). Chinese ET patients had similar clinical characteristics as Caucasian patients. JAK2V617F mutation was investigated in746patients among whom380(50.9%) were positive. During follow-up,99(10.2%) patients experienced at least one major thrombotic event. The5-,10-, and15-yr cumulative rates of thrombotic events were9.0%,22.0%, and35.0%, respectively. Similar to the IPSET-thrombosis study, our multivariate analysis revealed age>60(HR=1.949), previous thrombosis (HR=2.484), JAK2V617F mutation (HR=1.719), and cardiovascular risk factors (HR=1.877) as independent risk factors for thrombosis. We confirmed that the above risk factors in IPSET-thrombosis, when compared with traditional risk factors (e.g., age≥60and previous thrombotic events), were more predictive of thrombotic events (C-index0.714vs.0.647). Classification by IPSET-thrombosis risk groups revealed different cumulative thrombosis-free survival (P<0.001). For treatment, patients in the intermediate-and high-risk group derived clinical benefit from cytoreductive agents (P<0.05), but those in the low-risk group did not (P=0.446). The lower risk of thrombosis on cytoreductive therapy was related to decrease in leukocyte count during the disease course.Conclusions:We validate the reproducibility of IPSET-thrombosis in Chinese ET patients and provide key clinical implications. Background:Somatic JAK2V617F mutation exists in about50-60%of patients with essential thrombocythemia (ET). Recently, discovery of calreticulin (CALR) mutations, which were found in about half of ET patients with wild type JAK2, fills the molecular diagnostic gap. However, the prevalence of CALR mutations has not been reported in Asian patients, and its therapeutic implications have not yet been evaluated.Objectives:We studied profiles of specific mutations in Chinese ET patients to provided additional details on the utility of CALR mutations in the diagnosis, prognosis and treatment of ET.Methods:We retrospectively evaluated the clinical characteristics in436Chinese patients diagnosed with ET in our hospital from1990to2013. Mutations in JAK2V617F, MPL exon10and CALR exon9were analyzed in all of the patients. Real-time quantitative PCR assay was performed to detect JAK2V617F mutation. Mutations in CALR and MPL were assessed by bidirectional sequencing. Each type of deletions or insertions in CALR was cloned by the TA cloning system. Cox proportional hazards regression was employed to carry out univariate survival analysis, and factors that were significant were forced to multivariate survival analysis. Thrombosis-free survival was estimated by Kaplan-Meier method, and log-rank test was used to compare thrombosis-free survival data.Results:Compared with Caucasian patients, Chinese patients had a lower frequency of MPL mutations (1.4%) but comparable JAK2V617F (55.1%) and CALR mutations (22.7%). We found5new types of deletions or insertions in CALR. Two patients harbored both JAK2V617F and CALR mutations; one of whom had a novel point mutation in CALR exon9(c.997C>T). Absence of the point mutation in T cells confirmed the mutation to be somatic. Compared with V617F+patients, CALR-mutated patients displayed lower leukocyte count, granulocyte count, hemoglobin level, lower rate of positive EECs but higher platelet count (P<0.01for each comparison). Triple-negative patients had intermediate leukocyte count, hemoglobin level, platelet count and intermediate rate of positive EECs between V617F+and CALR-mutated patients. In multivariate analysis, JAK2V617F mutation was a risk factor regardless of whether thrombosis occurred at diagnosis (Hazard ratio=1.836; P=0.040) or during follow-up (Hazard ratio=2.678; P=0.023), but CALR mutation was not at either time. Kaplan-Meier analysis revealed a more favorable thrombosis-free survival in CALR-mutated patients than in V617F+cases (P=0.014), and the lower frequency of thrombosis in CALR-mutated patients was most likely due to the low thrombotic rate in type2CALR-mutated patients (7.9%). Although no difference concerning fibrotic transformation was found between CALR-mutated and V617F+ET (P=1.000), CALR-mutated patients had a higher degree of reticulin deposition (P=0.003). The difference was mostly ascribed to the higher rate of grade1reticulin deposition in type1CALR-mutated patients compared with V617F+patients. Screening for CALR mutations in paired samples before and after treatment indicated that CALR-mutated clones were not completely eliminated by cytoreductive treatment. Combination use of antiplatelet and cytoreductive agents decreased the risk of thrombosis in V617F+patients (P=0.034), but not in CALR-mutated cases (P=1.000). Conclusions:We confirm the value of CALR mutations in the diagnosis and prognosis of ET in Chinese patients and show the clinical correlates of type1and type2CALR muations. The present study provide new evidence for making treatment strategies based on molecular markers. Background:Essential thrombocythemia (ET) is extremely rare in children, and the clinical course of childhood ET remains unclear because of the small sample number. Further study with a large sample size is needed.Objectives:To evaluate the clinical manifestations, complications, treatment strategies and long-term outcomes in children diagnosed with ET.Methods:A cohort of70children diagnosed with ET (age≤18) were retrospectively evaluated.Results:The median age was12years (range2-18years) with a male predominance (male/female ratio,1.4). The gender distribution was different from adult ET patients. Most of children (54.3%) did not display thrombocytosis-related symptoms at diagnosis, and the presence of symptoms was positively related to platelet count (P=0.002). Compared with adult patients, children with ET had a lower frequency of JAK2V617F mutation (18.8%). Different from adult patiens, blood cell counts were not significantly different between patients with and without JAK2V617F mutation in childhood ET (P>0.05). Most of the symptoms related to thrombocytosis (82.6%) responded to antiplatelet or cytoreductive drugs. Resistance/intolerance existed in children receiving hydroxyurea, but none of them suffered from severe side effects or developed second malignancies during hydroxyurea treatment. Compared with adult patients, children with ET had a lower risk of thrombosis (1.4%). We reported the first case of ET with development of cavernous transformation of portal vein (CTPV) and transformation to overt fibrosis in the same child less than14years old. Two children (2.9%) transformed to overt fibrosis during follow-up, and increase in leukocyte count might predict the occurance of disease transformation.Conclusions:Clinical and molecular features in children with ET are different from adult ET patients. Background:Compared with adult patients, children with essential thrombocythemia (ET) had a lower frequency of JAK2V617F mutation. Most of the children did not harbor a well-known genetic marker, and the pathogenesis of childhood ET remains unclear.Objectives:To investigate molecular features and clonal evolution of childhood ET, and to provide primary evidence on understanding the pathogenesis of childhood ET.Methods:High-throughout targeted capture exome sequencing was used to explore mutations in55genes which were all related to hematologic neoplasms. All of the positive mutations were evaluated in100normal controls to exclude polymorphism. We speculated the clonal evolution based on allele frequency.Results:The mean depth was384×, with a mean coverage of99.6%. Validations of the mutations by Sanger sequencing revealed an accuracy of93.8%. Finally,16mutations were found in11genes and in13patients. All of the16mutations were not found in healthy controls. Different from adult ET, most of the mutations in children were germline mutations (56.3%), which might be one of the important mechanisms by which the onset of the disease occurred earlier in children. JAK2V617F was positive in6patients (18.8%), and the rate was lower than that in adult ET. None of the children displayed CALR-or MPL-mutated. JAK246/1haplotype was found in12children (60%). A list of mutations accompanied with JAK2V617F mutation, including mutations in ASXL1, U2AF1, NRAS and IDH1. Genes in the JAK-STAT pathway (45%) and with epigenetic functions were mostly involved (25%). Concurrent mutations in the above two groups of genes represented20%of the children. Clone evolution analysis based on allele frequency revealed that JAK2V617F might not be the driver mutation, but was an event secondary to other mutations. Conclusions:The mutational profile of childhood ET was different from adult ET. Germline mutations might be one of the important mechanisms by which the onset of the disease occurred earlier. JAK2V617F might be a secondary event.更多还原