研究背景：高嗜酸性粒细胞综合征(hypereosinophilic syndrome， HES)是一种以不明原因的嗜酸性粒细胞持续增多累及多系统多脏器的血液系统疾病。该病中青年男性发病多见(男：女=9: 1，发病中位年龄为20-50岁)，增多的嗜酸性粒细胞主要浸润心脏〓、肺以及神经系统〓，严重者可致心肌梗死或成人呼吸窘迫综合征(ARDS)〓，甚至猝死〓，往往预后不良〓。由于HES临床表现多样化，症状与嗜酸性粒细胞波动而变异大〓，因此极易导致误诊或漏诊〓。新近国外研究表明，部分HES患者(56%)嗜酸性粒细胞存在FIP1L1-PDGFRA融合基因，并认为该融合基因不仅是恶性克隆性HES发病的分子基础〓而且可作为克隆性HES诊断的分子标志。国外学者也发现，服用小剂量甲磺酸伊马替尼(商品名格列卫)可使HES患者达到完全血液学及部分分子遗传学缓解〓；同时体内外实验显示〓，信号传导与转录激活因子5 (STAT5)蛋白在FIP1L1-PDGFRA阳性细胞中存在过度激活，且随着格列卫对磷酸化PDGFRA蛋白的抑制而表达下调。本课题以4例HES患者为研究对象，综合观察、分析患者的临床特点，确定是否存在FIP1L1-PDGFRA融合基因，并研究Janus酪氨酸激酶/信号传导与转录激活因子(JAK/STATs)途径的活化状态。对1例FIP1L1-PDGFRA阳性HES患者，我们用小剂量格列卫治疗，从临床症状、嗜酸性粒细胞变化、FIP1L1-PDGFRA融合基因拷贝数及JAK/STATs信号蛋白变化等多方面观察格列卫的治疗效果，以进一步揭示HES的发病机制及格列卫治疗HES的分子机理。 第一部分 高嗜酸性粒细胞综合征临床特点分析 目的：分析我院收治的4例HES患者的临床特点，并分析科外误诊的主要原因，以加深对该病的认识，提高HES的诊断率。方法：按Chuisid标准临床诊断HES；完善白细胞计数分类(瑞-姬氏染色分类)及心、肺、神经系统及肝脾等脏器的辅助检查，分析HES脏器受累情况及与FIP1L1-PDGFRA基因表型之间的关系；观察常规治疗(强的松+羟基脲)对HES的疗效。结果：4例HES患者均符合Chuisid提出的诊断标准，但均曾不同程度的被误诊。所有患者均有不同程度白细胞总数增高，外周血中嗜酸性粒细胞增多；肝脾淋巴结肿大最为常见(100%)；FIP1L1-PDGFRA基因阳性的HES患者更易出现心脏受累；4例HES患者予常规治疗，其中1例HES病情得以有效控制(25%)。结论：HES临床表现多样化，肝脾淋巴结肿大常见；自动血细胞计数仪(三分类及五分类)易将嗜酸性粒细胞误判为中性粒细胞是导致临床误诊的重要原因。血细胞涂片染色显微镜下分类计数可避免对HES的漏诊；HES的临床表现与FIP1L1-PDGFRA基因表型存在一定的相关性；强的松联合羟基脲治疗仅对少部分HES患者有效。 第二部分 高嗜酸性粒细胞综合征FIP1L1-PDGFRA融合基因鉴定及JAK/STATs信号途径分析 目的：证实中国人种的HES患者是否存在FIP1L1-PDGFRA融合基因，以确定该融合基因对克隆性HES诊断是否具有普遍性意义；观察JAK/STATs全信号途径在HES患者嗜酸性粒细胞中的表达激活状况。进一步揭示HES嗜酸性粒细胞增殖的分子机制。方法：HES诊断根据Chusid标准确立；抽提患者粒细胞总RNA并逆转录为cDNA，再用筑巢式聚合酶链反应(Nested-PCR)扩增目的基因，同时以1例嗜酸粒细胞性胃肠炎(ECG)及1例健康志愿者作为阴性对照；对阳性PCR产物进行直接核苷酸测序；用Western印迹技术分别检测HES患者粒细胞中JAK2、STAT3、STAT5及磷酸化STAT5 (P-STAT5)蛋白表达水平；结果：4例HES中的3例存在异常的FIP1L1-PDGFRA融合基因，1例ECG患者及1例健康志愿者均无此融合基因；融合基因在PDGFRA基因上的断裂点均位于12号外显子，而在FIP1L1基因上的断点不固定，分别位于8a号外显子，8a号内含子和8号外显子；FIP1L1-PDGFRA融合基因阳性的3例HES患者均有JAK2、STAT3、STAT5及P-STAT5蛋白表达的显著上调，而1例FIP1L1-PDGFRA阴性的HES患者上述蛋白表达几乎阴性；结论:中国人种的HES存在FP1L1-PDGFRA融合基因，其发生率约75%。FIP1L1-PDGFRA融合基因的存在证实了这部分HES患者辱恶性克隆性疾病。FIP1L1-PDGFRA基因的下游靶标-JAK/STATs全信号蛋白通路存在过度激活，可能是具恶性克隆性质的HES区别于良性HES的分子“标志物”。 第三部分 高嗜酸性粒细胞综合征患者对甲磺酸伊马替尼分子靶向治疗的临床、分子遗传学反应 目的：确定小剂量甲磺酸伊马替尼(商品名格列卫)治疗HES的临床疗效及分子遗传学反应。方法:对1例FIP1L1-PDGFRA融合基因阳性的HES患者口服小剂量格列卫治疗，观察患者临床反应，同时用筑巢逆转录-聚合酶链反应(RT-PCR)及实时定量RT-PCR监测格列卫治疗前，治疗后10天、30天、60天患者粒细胞的FIP1L1-PDGFRA融合基因表达及拷贝数的动态变化， Western印迹技术检测相应时间点JAK2、STAT3、STAT5及P-STAT5蛋白的表达水平并进行比较。结果:1例FIP1L1-PDGFRA(+)的HES患者服用小剂量格列卫(100㎎/d)后，3天后即达到完全血液学缓解(血象及嗜酸性粒细胞比例恢复正常，症状体征消失)；服药30天后FIP1L1-PDGFRA融合基因的表达量明显减低，60天后融合基因表达阴性；实时定量RT-PCR结果显示治疗前，治疗后 10d、30d、60d FIP1L1-PDGFRA mRNA 拷贝数分别为1.01×10⁰， 1.04×10⁻¹，1.08×10⁻³，<1.0×10⁻⁴，呈指数级下降趋势；相应的JAK2、STAT3、STAT5及P-STAT5蛋白表达呈时间依赖性下调， 60天后表达均阴性。结论：小剂量格列卫治疗HES具有明显临床疗效，且可使FIP1L1-PDGFRA(+)的HES患者达到完全的血液学及分子遗传学缓解。 关键词 HES，临床特点，FIP1L1-PDGFRA融合基因， JAK/STATs信号途径，格列卫，融合基因拷贝数

 Background: Hypereosinophilic syndrome is a rare hematology disease characterized by an unexplained eosinophilia with multi-organs involvement. There is an overwhelming male preponderance of 91% to 92%, mostly of middle-aged men(male:female=9:1,median ages are 20-50 years old). Major tissues infiltrated by increased eosinophil include the heart, lungs and nervous system. There may be have serious complications of myocardial infarction and adult respiratory distress syndrome or even sudden death in HES patients, which indicate poor prognosis. HES is easily misdiagnosed as other diseases because of its great clinical heterogeneity and fluctuation of eosinophil count. Recently, It have been showned that abnormal FIP1L1-PDGFRA fusion gene is the molecular basis of pathogenesis of some HES patients(56%), and moreover the FIP1L1-PDGFRA gene may be a molecular mark of malignant HES. Some datas have shown that the HES patients with low-dose imatinib treatment can achieve complete hematologic remisssion and part molecular genetics remission. Both In vitro and vivo, the experiment results revealed that excessive activation of STAT5 in the FIP1L1-PDGFRA-positive cells, and it was downregulated with the inhibition of phosphylated PDGFRA by imatinib.In our study,We observed and analyzed the clinical features of 4 HES patients,and indentified the FIP1L1-PDGFRA fusion gene and detected whether JAK/STATs signal proteins were activated or not. Low-dose imatinib was given to one of HES patient with FIP1L1-PDGFRA fusion, and the effect was assessed systematically based on the dynamic changes of clinical syndromes, eosinophil counts, the copies of FIP1L1-PDGFRA gene and the expression level of JAK/STATs proteins. The aim was to further revealing the pathogenesis of HES and the molecule mechanism of imatinib treatment on HES. Part Ⅰ The Clinical features and laboratory findings of HES Objective: To observe clinical manifestations of 4 HES patients, analyze the major cause of misdiagnosis , and to improve the understanding and avoid the misdiagnosis of HES. Methods: The diagnosis of HES was based on the criteria proposed by Chusid et al. WBC count, classification by Wright-Giemsa stain and laboratory examinations including heart, lung and nerve system were performed. The correlations between clinical phenotype and FIP1L1-PDGFRA gene were identified, and the effect of routine therapy of predine and hydroxyurea was observed. Results: The 4 HES patients met all the criterias for diagnosis of HES which purposed by Chusid in 1975, but all patients were ever misdiagnosed. All 4 HES patients have leukocytosis because of eosinophilia in peripheral blood and all patients have hepatosplenomegaly(100%). The patients with FIP1L1-PDGFRA fusion gene were susceptible to cardiac involvement. All 4 patients were treated with routine therapy and only one HES patient's syndromes were effectively controlled(25%). Conclusion: The HES is of great clinical heterogeneity. Hepatosplenomegaly wasa commom manifestation.Automated analysis of blood cells was the important factors of HES misdiagnosis because it classified eosinophil as neutrophil wrongly. The manual examination and cell sorts of blood smear by microscope was assist to different HES from other diseases. Some relevances between the FIP1L1-PDGFRA gene with clinical phenotype were established. The combine treatment of predisine and hydroxyurea was effective on some HES patients. Part Ⅱ Detection of FIP1L1-PDGFRA fusion and JAK/STATs signal proteins in hypereosinophilic syndrome Objective: To determine whether FIP1L1-PDGFRA fusion gene exists in our HES patients, and confirm the universal significance of FIP1L1-PDGFRA gene in HES; to observe whether JAK/STATs signal pathway are involved in HES patients, and further reveal the pathogenesis of eosinophil proliferation. Methods: The diagnosis of HES was based on the criteria proposed by Chusid et al. The cDNA was synthesized by reverse transcript from granulocytes total RNA of HES . The fusion gene was amplified by Nested-PCR assay and the positive PCR fragments were , sequenced directly. One health volunteer and one ECG patient was as negative controls. The JAK2、 STAT3、 STAT5 and P-STAT5 proteins in granulocytes lysates of HES were detected by Western blotting. Results: 3 of 4 HES patients had the FIP1L1-PDGFRA fusion, while one health volunteer and one ECG patient both have not the fusion gene. The break points in PDGFRA were all located at exon 12 ,while in FIP1L1, the break point was highly variable, which was located at exon 8a 、 intron 8a and exon 8, respectively. Western blot assay showed JAK2,STAT3, STAT5 and P-STAT5 proteins exhibited a upregulated expression in 3 HES patients with FIP1L1-PDGFRA fusion, while all of these proteins expression were almost negative in one case of FIP1L1-PDGFRA negative HES. Conclusion: The FIP1L1-PDGFRA fusion also exists in Chinese HES patients(75%) and it indicates that HES is a malignant hematology disease. And moreover, the activation of JAK/STATs pathway, a down-stream target of the FIP1L1-PDGFRA fusion, is a molecular marker that differentiates malignant HES from benign HES. Part Ⅲ The clinical and molecular genetic response to imatinib in hypereosinophilic syndrome Objective: To confirm the clinical and cytogenetic effect of imatinib treatment on hypereosinophilic syndrome. Methods: One patient with FIP1L1-PDGFRA fusion was administered with low-dose imatinib. The dynamic monitor of FIP1L1-PDGFRA fusion was performed by both Nested RT-PCR analysis and Real-time quantitative RT-PCR(RQ-RT-PCR), and the expressions of JAK2,STAT3,STAT5 and P-STAT5 were detected by western blotting before treatment and 10 days,30days and 60 days after accepting therapy respectively. Results: The continuous hematologic remission (normalization of leukocyte and eosinophil counts and disappearance of clinical symptoms) was observed in one HES patient with FIP1L1-PDGFRA fusion after 3 day of low-dose imatinib therapy(100㎎/d). The amount of FIP1L1-PDGFRA transcripts in peripheral blood leukocytes was undetectable on the 30 days and turned into negative on the 60 days after therapy. Real-time quantitative RT-PCR showed that the copy of FIP1L1-PDGFRA was 1.01×10⁰,1.04×10⁻¹,1.08×10⁻³ and undetectable(＜10⁻⁴) corresponding to pre-imatinib treatment and 10 day, 30 day, 60 day post-imatinib therapy respectively. JAK2,STAT3,STAT5 and P-STAT5 expressions were all down-regulated in a time-dependent way, which were all negative after 60 day of therapy. Conclusion: Low-dose imatinib exerts significant clinical effects on HES patient and can induce complete hematologic and molecular genetics remission in FIP1L1-PDGFRA positive HES. KEY WORDS Hypereosinophilic syndrome, clinical manifestation, FIP1L1-PDGFRA fusion, JAK/STATs signal pathway, Imatinib, the copy of fusion gene