【作者】 程译帜；

【导师】 金洁；

【作者基本信息】 浙江大学 ， 内科学， 2009， 博士

【摘要】 急性白血病(AL)是一组异质性疾病,无论在形态学、细胞遗传学、免疫表型、分子遗传学和临床特征上都存在很大的差异。近几十年来,随着细胞遗传学和分子遗传学的发展,核型分析已经成为临床实践中的一项常规检查项目,对于AL的诊断和预后起重要作用。然而,目前文献报道的数据绝大多数来自于西方国家的统计。近年来,一些研究者已经发现血液肿瘤的疾病性质具有人种差异。但这些研究大多局限于例数较少的病例,大规模的统计屈指可数。特别是针对中国患者群至今没有进行过大样本的系统研究。在这一背景下,我们对十几年来在本院接受细胞遗传学分析的两千多名初发AL患者的染色体性状进行了回顾性研究,对异常类型进行分类并计算人口发生率,进而将这些统计结果与其它国家和地区的报道进行比较,希望得出一个较全面的、反映中国患者群特征的急性白血病细胞遗传学谱。另一方面,作为新兴的表观遗传学和表观基因组学最重要的研究内容,关于CpG岛的甲基化所致抑癌基因转录失活,进而影响肿瘤发生、发展这一问题也成为研究热点。携带AL特有的染色体易位/倒位的动物模型提示,单纯的遗传物质改变不足以诱导血液恶性肿瘤的发生,在很多情况下,表观遗传学修饰一同参与肿瘤的发生、发展和恶化,成为这一过程中不可或缺的因素。然而,尽管抑癌基因的甲基化已经成为白血病细胞中的普遍现象,只有少数基因的甲基化状态已经被确定与患者的临床特征及预后有关。因此,我们对多个基因在AL患者中的甲基化状态进行研究,并分析了基因甲基化与患者临床特征及预后之间的关系。第一部分急性髓细胞白血病的细胞遗传学研究目的:探讨急性髓细胞白血病(AML)的细胞遗传学特征,主要核型异常的发生情况以及不同人种之间的差异。方法:对1994年至2007年在本院血液研究所进行核型分析的1432例初发AML患者的细胞遗传学特征进行回顾性分析,并将本研究结果与目前文献中最具代表性的大样本报道进行比较。结果:在1,432名患者中,有1,293例核型分析成功(90%),其中746例(58%)观察到克隆性异常,异常率与文献报道接近。发生频率最高的染色体异常为t(15;17),见于所有分析成功病例的14%,其次为t(8;21),发生率8%,t(9;22),+21和+8,发生率均为2%。在不同的FAB亚型中,M3患者发生染色体易位的比例最高,为58%;M1患者中发生染色体丢失的比例最高,为18%;而M5患者中发生三倍体的比例最高,为29%。复杂核型最常见于M0亚型,占其总数的17%。在0-60岁之间,AML的总体发病率随年龄增长而上升,从0-9岁的0.3例/100,000人增加到50-59岁的6.2例/100,000人,60岁以后开始随年龄增长而下降,从60-69岁的4.9例/100,000人减少到80岁后的1.4例/100,000人。结论:超过半数的初发AML患者伴有克隆性染色体异常。FAB亚型、AML患者的年龄分布以及常见核型异常的发生率在不同人种之间存在较大差异。第二部分急性淋巴细胞白血病的细胞遗传学研究目的:探讨急性淋巴细胞白血病(ALL)的细胞遗传学特征,主要核型异常的发生情况以及不同人种之间的差异。方法:对1994年至2007年在本院血液研究所进行核型分析的585例初发ALL患者的细胞遗传学特征进行回顾性分析,并将本研究结果与目前文献中最具代表性的大样本报道进行比较。结果:585名ALL患者中共有551例核型分析成功(成功率94%),其中300例(49%)观察到克隆性染色体异常。119例(22%)为高超二倍体(＞50条染色体)。发生频率最高的染色体易位是t(9;22),见于11%的患者,其次是11q23重排和T细胞受体(TCR)异常,发生率均为2%。不伴常见染色体易位的复杂核型见于20名(4%)患者。根据2000年浙江省人口普查的统计数据,我们计算了ALL在各年龄段的发病率。在我们统计的人群中,ALL的发病率有两个年龄峰。第一个年龄峰是15-19岁,发病率为2.4例/100,000人,第二个年龄峰是55-59岁,发病率为2.3例/100,000人。结论:接近半数的初发ALL患者伴有克隆性染色体异常。ALL患者的年龄分布及常见核型异常的发生率在不同人种之间存在较大差异。第三部分急性白血病的DNA甲基化研究目的:探讨抑癌基因DKK3.TMS1、DAPK、ASPP1和WIF1在急性髓细胞白血病(AML)中的甲基化情况,以及DNA甲基化在AML中的潜在预后价值。方法:采用甲基化特异性PCR(MSP)检测71例初发AML骨髓样本中DKK3。TMS1、DAPK、ASPP1和WIF1五个基因的甲基化情况,并结合患者的临床表现及预后数据分析抑癌基因启动子甲基化与AML患者临床特征及预后之间的关系。结果:在71例骨髓样本中,DKK3的甲基化频率为63.4%(45/71),DAPK的甲基化频率为60.6%(43/71),而TMS1、ASPP1和WIF1均未检测到甲基化。卡方分析发现,DKK3和DAPK的甲基化状态有显著相关性(P=0.006)。＞60岁的患者DAPK甲基化频率明显高于≤60岁的患者(87.5%vs.57.1%,P=0.037)。发生DAPK甲基化的患者治疗前外周血WBC计数明显高于不发生DAPK甲基化的患者(57.2×10~9/L vs.11.2×10~9/L,P=0.017)。此外,不发生DAPK甲基化的患者一次诱导缓解率高达92.3%,而发生DAPK甲基化的患者缓解率仅为54.2%(P=0.018)。对于伴预后中等核型且接受常规化疗的患者进行的Kaplan-Meier生存曲线分析发现,发生DAPK甲基化的患者平均无复发生存时间为13.2个月,而DAPK不发生甲基化的患者的平均无复发生存时间为24.8个月,具显著差异(P=0.045)。结论:抑癌基因的启动子甲基化在初发AML中普遍存在。DAPK甲基化是影响AML化疗效果以及无复发生存时间的主要因素之一。更多还原

【Abstract】 Acute leukemia(AL) is a group of heterogeneous diseases in terms of morphology, cytogenetics,immunophenotypes,molecular genetics and clinical characteristics.In the last decades,with the development of cytogenetics and molecular genetics, karyotyping has become one of the routine tests in the clinical practice,and plays an important role in the diagnosis and prognosis of AL.However,the majority of data reported were based on the calculation of the western countries.Recently,ettmic disparities of hematological malignancies were reported.Nevertheless,most of those studies were limited to small numbers of cases.Few large-scale studies are available in the medicine literature,especially for the Chinese population.It was on this background that we started a retrospective study on cytogenetics of more than 2 000 AL patients who received cytogenetic analyses in our institution during the previous decade.We calculated the population incidence of specific chromosome abnormalities and compared the main results with reports from other countries,so as to deliver a global picture of the cytogenetics of the Chinese AL patients.On the other hand,as the most important part of the emerging epigenetics and epigenomics,methylation of CpG island resulting in the inhibited expression of cancer suppressor genes,as well as its impact on the pathogenesis and progression of tumors has become a hot issue.Animal models with AL-specific chromosomal translocations/inversions failed to develop overt leukemia,suggesting that sole genetic change is not enough for the development of hematological malignancies.In most cases,epigenetic modifications might be involved.Though aberrant hypermethylation of tumor suppressor genes is now known as a common phenomenon in the leukemic blasts,the relationship between methylation and prognosis was reported only for several genes.Here we studied the methylation status of five genes in AL samples and analysed the correlation between methylation and patients’ clinical features and prognosis.PartⅠCytogenetics of acute myeloid leukemia:a study based on 1432 patientsObjectives:To study the cytogenetic characteristic of acute myeloid leukemia(AML), the incidences of major chromosome aberrations and the disparity between different races.Methods:We performed a retrospective study on the cytogenetic findings of 1 432 de novo AML patients who received cytogenetic analyses in our institution between 1994 and 2007,and compared the main results with representational large-scale studies from other countries.Results:Of the 1 432 patients,1 293 patients received successful cytogenetic analyses (90%).Clonal abnormalities were detected in 746 cases(58%).The abnormal rate was comparable to previous reports.The most frequent chromosome abnormality was t(15;17),detected in 14%of the successful cases,followed by t(8;21),in 8%,and t(9;22),+21 and +8,each in 2%.For different FAB subtypes,translocation was most common in M3 patients,detected in 58%;deletion was most common in M1,detected in 18%;and trisomy was most common in M5,detected in 29%.Complex karyotypes were most frequent in M0 patients,detected in 17%.The total incidence of AML increased with age between 0 and 60 years,that was from 0.3 case/100 000 inhabitants at 0-9 years to 6.2 cases/100 000 inhabitants at 50-59 years;and decreased with age above 60 years,that was from 4.9 cases/100 000 inhabitants at 60-69 years to 1.4 cases/100 000 inhabitants above 80 years.Conclusion:More than a half of the de novo AML patients harbored clonal chromosome abenations.The distribution of FAB subtypes,age of diagnosis and the incidences of major karyotypic abnormalities showed ethnic disparity.PartⅡCytogenetics of acute lymphoid leukemia:a study based on 585 patientsObjectives:To study the cytogenetic characteristic of acute lymphoid leukemia(ALL), the incidences of major chromosome aberrations and the disparity between different races.Methods:We performed a retrospective study on the cytogenetic findings of 585 de novo ALL patients who received cytogenetic analyses in our institution between 1994 and 2007,and compared the main results with representational large-scale studies from other countries.Results:Of 585 ALL patients,551 patients received successful cytogenetic analyses (94%).Clonal chromosome aberrations were detected in 300 cases(49%).High hyperdiploid(defined as more than 50 chromosomes) was observed in 119 cases(22%). The most frequent chromosomal translocation was t(9;22),detected in 11%cases, followed by 11q23 rearrangements and abnormalities of T-cell receptor(TCR),each in 2%.Complex karyotypes without established translocations were detected in 20 cases (4%).We calculated the age-specific incidences of ALL based on the population of Zhejiang Province.There appeared to be two age peaks for ALL.The first was at 15-19 years,with an incidence of 2.4 cases/100 000 inhabitants.The second was at 55-59 years,with an incidence of 2.3 cases/100 000 inhabitants.Conclusion:Nearly a half of the de novo ALL patients had clonal chromosome aberrations.The age distribution and the incidences of main chromosome abnormalities showed ethnic disparity.PartⅢPredictive value of DNA methylation in patients with acute myeloid leukemiaObjectives:To study the methylation status of DKK3,TMS1,DAPK,ASPP1 and WIF1 in acute myeloid leukemia(AML) and the potential predictive value of’ DNA methylation in AML.Methods:The methylation status of DKK3,TMS1,DAPK,ASPP1 and WIF1 in pretreatment bone marrow samples from 71 patients with de novo AML was measured by methylation-specific polymerase chain reaction(MSP).The correlation between methylation and clinical features as well as prognosis of AML patients was analysed.Results:In 71 samples,the methylation frequency of DKK3 was 63.4%(45/71),and the methylation frequency of DAPK was 60.6%(43/71).The methylation of TMS1, ASPP1 and WIF1 was not detected in any of our samples.Chisquare test showed a significant correlation between the methylation status of DKK3 and DAPK(P=0.006). The methylation frequency of DAPK was significantly higher in patients above 60 years(87.5%vs.57.1%,P=0.037).The mean peripheral WBC count of patients with DAPK methylation was significantly higher than those without(57.2×109/L vs. 11.2×109/L,P=0.017).Besides,the complete remissionrate after one course of induction for patients without DAPK methylation was 92.3%,significantly higher than that for patients with DAPK methylation(54.2%).Kaplan-Meier sruvival curve for patients with intermediate-risk karyotypes who received adequate chemotherapy indicated that the mean Recurrence-free Survival(RFS) of patients with DAPK methylation was significantly shorter than that of patients without DAPK methylation (13.2 months vs.24.8 months,P=0.045).Conclusion:Promoter hypermethylation of tumor suppressor genes was common in de novo AML patients.DNA methylation was one of the most important factors to chemotherapy effects and RFS.更多还原