FLT3及C-KIT基因突变在核心结合因子相关急性髓系白血病中的功能及临床研究

发布时间：2017-12-27 21:27

本文关键词：FLT3及C-KIT基因突变在核心结合因子相关急性髓系白血病中的功能及临床研究　出处：《苏州大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的1.系统分析连续5年在苏州大学附属第一医院确诊的176例核心结合因子相关急性髓系白血病(CBF-AML)患者的细胞遗传学及分子生物学特征。2.以两例伴少见的FLT3-TKD基因突变(FLT3 N676K、FLT3 H671Q)的核心结合因子相关急性髓系白血病的研究入手,克隆两种突变体,并采用体外实验对其进行生物学功能的初步研究。3.回顾性分析伴有4号染色体三体异常的t(8;21)AML中C-KIT基因突变的发生及其临床预后。方法1.176例CBF-AML患者均按照江苏省血液研究所染色体分析实验室常规方法制备染色体标本及R显带技术分析染色体核型,核型异常依据《人类细胞遗传学国际命名体制(International System for Human Cytogenetic Nomenclature 2005)》的规定进行描述。应用聚合酶链反应(PCR)扩增基因组DNA及PCR产物双向Sanger DNA测序法分析176例CBF-AML患者KIT、FLT3、JAK2、RAS、CBL基因突变的发生情况。2.通过PCR的方法定点诱导突变获得两种少见的FLT3-TKD基因突变,以慢病毒为表达载体,分别将FLT3 N676K及FLT3 H671Q与其连接。采用慢病毒包装体系包装FLT3 N676K及FLT3 H671Q病毒,并用病毒侵染小鼠原B细胞——BaF3细胞,以构建稳转细胞。通过CCK-8法绘制生长曲线、甲基纤维素集落形成试验及白介素-3(IL-3)撤退试验观察两种FLT3-TKD基因突变对BaF3细胞增殖能力的影响。3.回顾性研究2005年2月至2013年1月145例初治t(8;21)AML患者,分析4号染色体三体异常及C-KIT基因突变对其预后的影响。采用Kaplan-Meier法进行生存分析,COX比例风险模型进行多因素分析。结果1.本中心176例初诊cbf-aml患者的细胞遗传学及分子生物学特征176例cbf-aml患者中t(8;21)/runx1-runx1t1共139例(79%),inv(16)/cbfβ-myh11共37例(21%)。其中,男性101例,女性75例。中位年龄35(11-77)岁。t(8;21)aml患者fab分型以m2型为主,占84.2%,而inv(16)aml患者主要为m4eo,占64.9%。分析两组病例的临床和实验室资料后发现,在初诊时白细胞(wbc)计数、血红蛋白(hb)计数、骨髓原始细胞比例方面,inv(16)aml均高于t(8;21)aml,且差异具有统计学意义(p0.05);而在其他一般临床特征上两组之间均无统计学差异。176例cbf-aml患者中174例(98.9%)核型分析成功,其中84例(48.3%)还伴有其他附加染色体异常。最常见的为性染色体缺失,-y或-x为55例(31.3%);其次是del(9q)有10例(5.7%);22号染色体三体异常10例(5.7%);4号染色体三体异常7例(4.0%);8号染色体三体异常2例(1.1%);染色体核型分析未检出t(8;21)/inv(16)患者有16例,但均可经rt-pcr检测到runx1-runx1t1或cbfβ-myh11融合基因。对176例患者均进行常见的基因突变检测,共73例(41.5%)未检测到基因突变,97例(55.1%)检测到一种基因突变,6例(3.4%)检测到两种及以上的基因突变。c-kit基因突变是最常见的酪氨酸激酶相关的基因突变,占34.7%(61例),其次是flt3基因突变,占12.5%(22例),其中flt3-itd基因突变率为40.9%(9/22),flt3-tkd基因突变率为59.1%(13/22)。在t(8;21)aml中c-kit基因突变发生率(39.6%)明显高于inv(16)aml(16.2%),且差异具有统计学意义(p0.01)。同时检测到两例少见的flt3-tkd基因突变,分别为flt3n676k及flt3h671q。2.两种少见的flt3-tkd的克隆及功能研究通过分子克隆及细胞培养技术,利用携带flt3n676k及flt3h671q慢病毒载体的质粒转染baf3细胞,经体外试验发现,两种flt3-tkd均能促进细胞增殖,可在一定程度上使baf3细胞减少对il-3的依赖,且在集落形成能力上具有明显优势。3.伴有4号染色体三体异常的t(8;21)aml中c-kit基因突变的发生及其临床预本研究共分析145例初治t(8;21)aml患者,其中男性82例,女性63例,中位年龄33(9-76)岁。共46例(31.7%)患者检测到C-KIT基因突变,其中44例突变位于17号外显子,1例位于8号外显子,另外1例同时位于8号、17号外显子。44例C-KIT基因17号外显子突变类型分布为25例D816,17例N822,1例Y823,1例同时位于D816和N822。根据患者的附加染色体异常进行分组,发现t(8;21)AML伴有4号染色体三体异常的患者中C-KIT基因突变率高达91.6%(11/12),明显高于其他患者26.3%(35/133),差异具有统计学意义(P0.01)。生存分析发现伴有4号染色体三体异常的t(8;21)AML患者的三年总体生存(OS)率及无病生存(DFS)率分别为15%、0%,均明显低于其他患者同期OS及DFS(56%、51%)(P0.01)。并且,同时伴有4号染色体三体及C-KIT基因突变的t(8;21)AML患者的中位生存期较不伴有4号染色体三体及C-KIT基因突变的患者明显缩短(中位生存期分别为8.8和23.2个月,P0.01)。利用COX比例风险模型分析影响t(8;21)AML患者预后的主要因素,其中性别、诱导后疾病缓解状态、4号染色体三体异常、C-KIT基因突变、4号染色体三体异常联合C-KIT基因突变均是影响其预后的主要因素。结论1.本实验通过对176例初治CBF-AML患者进行核型分析及常见基因突变检测,发现性染色体异常是最常见的附加染色体异常,C-KIT基因突变是CBF-AML中最常见的基因突变,并发现了两种少见的FLT3-TKD基因突变类型:FLT3 N676K及FLT3H671Q。2.我们成功构建了两种少见的FLT3-TKD基因突变的慢病毒载体,并建立了BaF3稳转细胞,为研究其功能奠定了基础。两种新型FLT3-TKD可使BaF3细胞获得增殖优势,并且在一定程度上减少其对IL-3的依赖。3.本研究发现伴4号染色体三体异常的t(8;21)AML患者中C-KIT基因突变频率明显增高,且生存分析发现伴有4号染色体三体异常的t(8;21)AML患者生存期明显缩短,4号染色体三体异常及C-KIT基因突变是t(8;21)AML患者预后不良的因素。
[Abstract]:Objective 1. to systematically analyze the cytogenetic and molecular biological characteristics of 176 patients with core binding factor related acute myeloid leukemia (CBF-AML) diagnosed in First Hospital Affiliated to Suzhou University in 5 consecutive years. 2., two cases of acute myeloid leukemia associated with rare FLT3-TKD gene mutation (FLT3 N676K and FLT3 H671Q) were cloned, and two kinds of mutants were cloned, and their biological functions were preliminarily studied in vitro. 3. a retrospective analysis of the mutations in the C-KIT gene in t (8; 21) AML with the trisomy 4 chromosome abnormality and its clinical prognosis. Methods 1.176 cases of patients with CBF-AML chromosome are in accordance with the Jiangsu Institute of hematology laboratory analysis of conventional methods of preparation of chromosome specimens and R banding karyotype analysis of chromosome karyotype abnormalities, according to "international human cytogenetics naming system (International System for Human Cytogenetic Nomenclature 2005)" the provisions described. Polymerase chain reaction (PCR) was used to amplify the genomic DNA and PCR products bidirectional Sanger DNA sequencing method to analyze the occurrence of KIT, FLT3, JAK2, RAS and CBL gene mutations in 176 CBF-AML patients. 2., by means of PCR, two rare FLT3-TKD mutations were obtained by site directed mutagenesis. Lentivirus was used as the expression vector, and FLT3 N676K and FLT3 H671Q were connected respectively. FLT3 N676K and FLT3 H671Q virus were packaged by lentivirus packaging system, and virus infected mouse original B cells, BaF3 cells, in order to construct stable cells. The growth curve, methyl cellulose colony formation test and interleukin -3 (IL-3) withdrawal test were drawn by CCK-8 method to observe the effects of two FLT3-TKD gene mutations on the proliferation of BaF3 cells. 3. a retrospective study of 145 patients with t (8; 21) AML from February 2005 to January 2013 was used to analyze the effect of trisomy 4 on chromosome 4 and the prognosis of C-KIT gene mutation. The Kaplan-Meier method was used to carry out the survival analysis and the COX proportional risk model was used for multi factor analysis. Results 1. cases of 176 cases of newly diagnosed cbf-aml patients had cytogenetics and molecular biology characteristics in 176 cases. In cbf-aml patients, t (8, 21) /runx1-runx1t1 had 139 cases (79%), and inv (16) /cbf -myh11 -myh11 37 cases (21%). Among them, 101 were male and 75 were female. The median age was 35 (11-77) years. The Fab typing of T (8; 21) AML patients was mainly m2, accounting for 84.2%, while inv (16) AML patients were mainly M4Eo, accounting for 64.9%. Analysis of clinical and laboratory data of two cases were found in the initial WBC count (WBC), hemoglobin (HB) count and the percentage of bone marrow blast cells, inv (16) AML was higher than that of T (8; 21) AML, and the difference was statistically significant (P0.05); and in its clinical he characteristics between the two groups were not statistically significant. The karyotype analysis was successful in 174 of 176 cbf-aml patients (98.9%), of which 84 (48.3%) had other additional chromosomal abnormalities. The most common sex chromosome deletion, -y or -x for 55 cases (31.3%); the second is del (9q) 10 cases (5.7%); 10 cases of abnormal trisomy 22 (5.7%); 7 cases of abnormal trisomy 4 (4%); trisomy 8 was abnormal in 2 cases (1.1%); karyotype analysis was not detected in t (8; 21) /inv (16) in 16 patients, but can be detected by RT-PCR to runx1-runx1t1 or CBF beta -myh11 fusion gene. A total of 176 patients were tested for common gene mutations. A total of 73 cases (41.5%) did not detect gene mutations, 97 (55.1%) detected one gene mutation, and 6 (3.4%) detected two or more mutations. C-kit gene mutation is the most common tyrosine kinase related gene mutation, accounting for 34.7% (61 cases), followed by FLT3 gene mutation, accounting for 12.5% (22 cases), of which flt3-itd gene mutation rate is 40.9% (9/22), and flt3-tkd gene mutation rate is 59.1% (13/22). The mutation rate of c-kit gene (39.6%) in t (8; 21) AML was significantly higher than that of inv (16) AML (16.2%), and the difference was statistically significant (P0.01). At the same time, two rare flt3-tkd gene mutations were detected, flt3n676k and flt3h671q, respectively. Cloning and functional analysis of 2. two kinds of rare flt3-tkd by molecular cloning and cell culture techniques, by carrying flt3n676k and flt3h671q lentivirus vector was transfected into baf3 cells, the in vitro experiment showed that two kinds of flt3-tkd can promote cell proliferation and can induce baf3 cells to a certain extent, reduce the dependence on IL-3, and has obvious the advantage in the colony forming ability on. 3. the occurrence of c-kit gene mutation in t (8, 21) AML with chromosome 4 abnormalities and its clinical preclinical study. A total of 145 newly diagnosed t (8, 21) AML patients were analyzed, including 82 males and 63 females, and the median age was 33 (9-76) years. A total of 46 cases (31.7%) were detected C-KIT gene mutation, including 44 cases of mutations located in exon 17, 1 were located in exon 8, the other 1 cases located in exon 17, No. 8. 44 cases of exon 17 of C-KIT gene mutations in the type of distribution of 25 cases D816,17 cases N822,1 cases Y823,1 cases and is located in D816 and N822. According to the patient's additional chromosome abnormalities, we found that the mutation rate of C-KIT gene was 91.6% (11/12) in patients with t (8, 21) AML and 4 chromosome trisomy abnormalities, which was significantly higher than that in other patients (26.3%) (35/133). The difference was statistically significant (P0.01). Survival analysis showed that the three year overall survival (OS) and disease-free survival (DFS) rates of T (8, 21) AML patients with chromosome 4 abnormalities were 15% and 0%, respectively, which were significantly lower than those of other patients in the same period OS and DFS (56%, 51%) (P0.01). Moreover, the median survival time of T (8, 21) AML patients with trisomy 4 and C-KIT gene mutation at the same time was significantly shorter than those without chromosome 4 and C-KIT gene mutation (median survival time was 8.8 and 23.2 months, P0.01). COX proportional hazards model was used to analyze the main prognostic factors of T (8, 21) AML. Gender, post induction disease remission, trisomy 4, C-KIT gene mutation, trisomy 4 and C-KIT gene mutation all influence prognosis.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R733.71

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