【作者】 张玲；

【导师】 徐望红；

【作者基本信息】 复旦大学 ， 公共卫生（专业学位）， 2013， 硕士

【摘要】 研究背景急性髓细胞白血病(acute myeloid leukemia, AML)是多能干细胞或已轻度分化的前体细胞核型发生突变所形成的造血系统的恶性克隆性疾病,包括所有非淋巴细胞来源的急性白血病。AML预后差,复发率高,部分类型AML的预后因素不明。目的通过分析急性白血病常见基因突变对临床治疗效果及生存状况的影响,评估这些基因突变在白血病患者预后中的作用。方法收集上海瑞金医院血液学研究所和浙江大学附属第一人民医院血液学研究所1998-2011年初发急性髓细胞白血病病人1,185例,剔除AML1-ETO, CBF-MYH1和PML-RARa/NPM1-RARa三种基因突变者后,共纳入605例病例。通过病历和检查报告查阅,获得病例就诊时临床信息,包括性别、年龄、白细胞计数资料WBC(×109/L)、髓原始细胞比例BM blasts (%)、FAB分型和治疗缓解情况(CR)。所有病患者均采用基于柔红霉素和阿糖胞苷联合化疗的方案(DA方案),仅47例有合适供体的病例进行了移植治疗。疗效判断根据病人的症状、体征、血象和骨髓象。完全缓解的标准是：骨髓象：原始粒细胞(Ⅰ型+Ⅱ型)或原单+幼单或原淋+幼淋细胞数≤5%,红细胞及巨核细胞系正常。其中M2b中性中幼粒细胞比例在正常范围；M3型中原始粒+早幼粒≤5%；M4型中原粒Ⅰ型+Ⅱ型+原单+幼单≤5%；M6型中原粒Ⅰ型+Ⅱ型≤5%,原红+幼红以及红系比例基本正常；M7中粒、红两系比例正常,原巨及幼巨基本消失；血象：血红蛋白(Hb)≥100克/L(男)或≥90克/L(女性及儿童),中性粒细胞绝对值≥1.5×109/L血小板数≥100×109/L,分类中无白血病细胞;临床表现：无白血病侵润所致的症状和体征,生活正常或接近生活正常。骨髓标本采用R显带或G显带染色体核型分析,并通过相关分子标志物进行确认；采用SNP、全基因测序、多重RT-PCR等方法测定以下候选检测基因：FLT3-ITD/TKD、MLL/PKD、NPM、 c-KIT、NRAS、CEBPA、DNMT3A、IDH1。其中FLT3-TKD, N-RAS, NPMl, IDH1采用chip为基础的母体辅助激光吸收／离子化mass谱检测方法。FLT3-ITD, c-KIT, CEPBA, DNMT3A采用全基因测序法。6个MLL相关融合基因包括MLL-AF9, MLL-AF10, MLL-AF6, MLL-ELL, MLL-ENL和MLL-AF17采用多重RT-PCR的策略。MLL-PTD使用RT-PCR技术检测。所有突变基因采用二分类变量记录,定义缺失值为NA。所有病例均随访至2012年年底,收集生存状况及死因信息。结果FLT3-ITD/TKD、NPM、DNMT3A基因突变与未突变者初发时白细胞计数及骨髓原始细胞比例有显著差别,基因突变组的中位白细胞计数及中位骨髓原始细胞比例高于未突变组(t检验P<0.05)。MLL/PKD、NPM、NRAS、CEBPA突变与未突变者初发化疗的完全缓解率有显著差别(Fisher X2检验P< 0.05)。MLL /PKD突变组和DNMT3A突变组的中位生存期为8.0士2.2周和7.0士2.1周,分别显著低于未突变组的17.0士2.4周和18.0士2.3周。调整年龄、性别、白细胞计数、骨髓原始细胞比例和FAB分型等混杂因素后,与相应的未突变组相比,MLL/PKD突变组和DNMT3A突变组的死亡风险比(HR)分别为1.9(95%CI：1.3-2.7)和1.6(95%CI：1.1-2.4),是显著的不良预后因素。相反,发生NPM突变和CEBPA突变的病人,其预后好于无突变的病人,HR分别为0.6(95%CI：0.4-0.9)和0.6(95%CI：0.5-0.9)。随着上述四个基因危险基因型数量的增加,病例死亡风险呈显著的上升趋势(P<0.05)。结论对急性髓细胞白血病异常基因的分析对临床治疗方案的选择,疾病危险程度预估以及病例预后评价有一定的参考价值。更多还原

【Abstract】 BackgroundAcute myeloid leukemia (AML) is a malignant clonal disease in hematopoietic system caused by karyotype mutation in pluripotent stem cells or mildly differentiated precursor cells. AML include all sources of acute non-lymphocytic leukemia. Some types of AML have low survival and high recurrence rate. However, the prognostic factors remain unknown for these subtypes.ObjectiveThe study was performed to systemically investigate the frequencies of molecular mutations in AML patients and to evaluate the prognostic value of genetic mutations.MethodsA total of 1,185 primary AML patients diagnosed between 1998 and 2011 were collected from Shanghai Institute of Hematology and Zhejiang Institute of Hematology. After excluding those with mutations at AML1-ETO, CBF-MYH1 and PML-RARa/NPM1-RARa genes,605 patients were included in this study. All patients had chemotherapy with daunorubicin and cytarabine (DA program). Only 47 patients were transplanted with marrow from suitable donors. Through reviewing medical records, we obtained clinical information for all patients, including sex, age, white blood cell (WBC) counts (×109/L), blasts in bone marrow (BM) (%) and FAB types and complete remission (CR) status after the first chemotherapy. The effect of chemotherapy was determined according to the patients’symptoms, signs, and results of blood and bone marrow smears. Bone marrow karyotype analysis was conducted by R-banding or G-banding methods and confirmed with related molecular markers. Mutations at genes of FLT3-TKD, N-RAS, NPM1 and IDH1 were examined using chip-based matrix-assisted laser absorption/ionization mass spectroscopy, whereas mutations at genes of FLT3-ITD, c-KIT, CEPBA and DNMT3A were detected by whole genome sequencing methods. Mutations at MLL-PTD gene and six MLL-related fusion genes, i.e. MLL-AF9, MLL-AF10, MLL-AF6, MLL-ELL, MLL-ENL and MLL-AF17 genes were identified using multiplex RT-PCR. The status of gene mutation was recorded as with or without mutations. All patients were followed-up to collect information on survival status and causes of death by the end of 2012.ResultsA significant difference was observed in the medians of WBC counts and BM blasts between AML patients with and without mutation at FLT3-ITD/TKD, NPM or DNMT3A genes (all P values for t tests< 0.05). The CR rates after chemotherapy differed significantly between patients having and having no mutation at MLL/PKD, NPM, NRAS or CEBPA genes (all P values for Fisher X2 tests< 0.05). The median survival time were 8.0 ± 2.2 weeks and 7.0 ±2.1 weeks, respectively, in patients with MLL/PKD mutation or DNMT3A mutation, significantly higher than 17.0 ± 2.4 weeks and 18.0 ± 2.3 weeks among those without mutation. After adjusting for age, sex, WBC counts, BM blasts and FAB type, the hazard ratio (HR) and 95% confidence interval was 1.9 (95%CI:1.3-2.7) for patients with vs. without mutation at MLL/PKD gene and 1.6 (95%CI:1.1-2.4) for those with vs. without mutation at DNMT3A gene. Conversely, mutations at NPM and CEBPA were associated with a favorable prognosis, with HR being 0.6 (95%CI:0.4-0.9) and 0.6 (95%CI:0.5-0.9), respectively. The risk was observed to increase with the increasing number of risk mutations at the four genes (P for trend< 0.0001).Conclusion:Our results suggest that genetic mutation assay may be helpful for selecting an efficient chemotherapy and predicting the prognosis among Chinese AML patients.更多还原