急性髓系白血病患者骨髓涂片FMS样酪氨酸激酶3、NPM1和c-kit基因检测及临床研究

Detection and clinical research of FMS-like tyrosine kinase-3, NPM1 and c-kit genes in bone marrow slides of patients with acute myeloid leukemia

目的探讨提取贮存骨髓涂片DNA的方法，通过对急性髓系白血病（AML）患者FMS样酪氨酸激酶3（FLT3）、NPM1及c-kit基因突变进行检测，分析三种基因突变与AML临床特征之间的关系。方法收集55例AML患者骨髓涂片，采用聚合酶链反应（PCR）、DNA测序和分子克隆方法对FLT3．内部串联重复（ITD）、NPM1和c-kit基因突变进行检测及分析，记录患者疾病缓解、进展及生存时间。结果实验证实对于低温冻存、未经瑞特染色、未用化学方法固定的骨髓涂片标本及室温贮存、经瑞特染色脱色后的标本均能用苯酚：氯仿：异戊醇法成功提取DNA。从骨髓涂片中提取的DNA可用于PCR、直接测序和分子克隆测序分析。在55例AML患者中，FLT3．ITD阳性10例（18．2％），其中9例为杂合型突变，1例为纯合型突变。FLT3-ITD阳性组较阴性组完全缓解（CR）率低，无事件生存（EFS）和总生存（0s）时间短（P〈0．05）。NPM1基因杂合型突变9例（16．4％），全部为A型突变。10个月以内NPM1突变组患者EFS率比野生组高（P〈0．05），19个月以内NPM1突变组OS率比野生组高（P〈0．05）。9例NPM1突变患者中FLT3-ITD阳性3例，CR率由高到低依次为NPM1＋FLT3-ITD-,NPM1-FLT3-ITD-、NPM1-FLT3-ITD＋、NPM1＋FLT3．ITD＋（P〈0．05），且NPM1-FLT3．ITD＋是影响0s的危险因素（RR=1．250，P=0．005）。55例患者中，c-kit基因突变2例（3．6％），分别为D816H突变型和D816V突变型；c-kit基因突变患者与FLT3-ITD阳性及NPM1突变患者无重叠。结论FLT3-ITD突变为AML患者预后不良的分子标志，NPM1基因突变可能提示预后较好，NPM1-FLT3-ITD＋是影响0s的危险因素。AML中c-kit基因突变率低，未见其与FLT3、NPM1基因突变重叠。

Objective To study the FMS-like tyrosine kinase-3 （FLT3） gene, NPM1 gene and c-kit gene mutations in acute myeloid leukemia （AML） by extracting DNA from the storage of bone marrow slides, and to investigate the relationship between the three gene mutations and clinical features in AML. Methods The bone marrow slides of 55 patients diagnosed with AML were enrolled in this study. The PCR, DNA sequencing and molecular cloning were used to detect and analyse the FLT3-ITD, NPM1 and c-kit gene mutations. Patients＇ remission, progression and survival time were also recorded. Results The DNA was successfully extracted from the bone marrow slides with -20 ℃ frozen storage without Wright stained, chemically fixed, and room temperature storage Wright stained discoloured by phenol ： chloroform ： isoamyl alcohol method, which can be used in PCR, direct sequencing and molecular cloning sequencing analysis. 10 of the 55 cases （18.2 %） were FLT3-ITD positive, including 9 cases with heterozygous mutations and 1 ease with homozygous mutation. FLT3-ITD positive group had lower complete remission （CR） rate, shorter event- free survival （EFS） time and overall survival （OS） time than the negative group （P 〈 0.05）. 9 of the 55 cases （16.4 %） had NPM1 heterozygous gene mutations, all belonging to type A. The EFS rate of the patients with NPM1 mutation was higher in 10 months and the OS rate was higher in 19 months （P 〈 0.05）. 3 of 9 NPM1 mutations patients were FLT3-ITD positive. The CR rates of the four groups after initial remission induction therapy in order were NPM1＋ FLT3-1TD-, NPM1- FLT3-ITD-, NPM1- FLT3-ITD＋, NPM1＋ FLT3-ITD＋ （P 〈 0.05）. Besides, NPM1- FLT3-ITD＋ was a risk factor affecting the OS （RR = 1.250, P = 0.005）. 2 of the 55 cases （3.6 %） had c-kit gene mutations, namely mutant D816H and mutant D816V. The c-kit gene mutations were not found in patients with FLT3-ITD and NPM1 mutations. Conclusions The FLT3-ITD mutation is a poor prognosis molecular marker in AML, and NPM1 mutation is a good factor for the prognosis. NPMI- FLT3-ITD＋ is a risk factor affecting OS. The rate of c-kit gene mutation is low in AML, without the overlap of FLT3 and NPM1 mutations.