正常核型急性髓系白血病NPM1和FLT3-ITD基因突变的研究

目的:探讨正常核型急性髓系白血病(AML)患者中NPM1基因与FLT3基因内部串联重复(ITD)突变的发生率,并了解其临床特征。方法:收集40例正常核型AML患者初诊时骨髓单个核细胞,采用基因组DNA-PCR方法分别扩增其NPM1和FLT3基因,直接测序法分析NPM1第12外显子突变,琼脂糖电泳分析FLT3-ITD突变。结果:40例正常核型AML患者中共检出NPM1突变15例(37.5%),FLT3-ITD突变8例(20%),4例(10%)同时存在NPM1和FLT3-ITD基因2种突变。单纯NPM1或FLT3-ITD突变的AML患者初诊时表现为高白细胞数、高骨髓原始细胞比例(P0.05)。结论:NPM1和FLT3-ITD突变是正常核型AML患者常见的分子学异常,且与初诊时高外周血白细胞数、高骨髓原始细胞比例相关。     

正常核型急性髓系白血病NPM1和FLT3-ITD基因突变的研究

目的:探讨正常核型急性髓系白血病(AML)患者中NPM1基因与FLT3基因内部串联重复(ITD)突变的发生率,并了解其临床特征。方法:收集40例正常核型AML患者初诊时骨髓单个核细胞,采用基因组DNA-PCR方法分别扩增其NPM1和FLT3基因,直接测序法分析NPM1第12外显子突变,琼脂糖电泳分析FLT3-ITD突变。结果:40例正常核型AML患者中共检出NPM1突变15例(37.5%),FLT3-ITD突变8例(20%),4例(10%)同时存在NPM1和FLT3-ITD基因2种突变。单纯NPM1或FLT3-ITD突变的AML患者初诊时表现为高白细胞数、高骨髓原始细胞比例(P0.05)。结论:NPM1和FLT3-ITD突变是正常核型AML患者常见的分子学异常,且与初诊时高外周血白细胞数、高骨髓原始细胞比例相关。     

急性髓系白血病患者miRNA-181b表达特点及预后意义

目的 探讨微小RNA-181b(miR-181b)在急性髓性白血病(AML)中的表达特点及预后意义.方法 采用实时定量逆转录-聚合酶链反应(RT-PCR)检测158例初诊AML患者及20例健康供者骨髓单个核细胞中miR-181b的表达水平.同时采用基因组DNA-PCR结合测序方法检测158例AML患者核磷蛋白1(NPM1)基因第12号外显子突变和fms样酪氨酸激酶3(FLT3)基因内部串联重复(ITD)突变(FLT3-ITD).结果 AML中miR-181b表达水平较正常对照组显著升高(Z =-2.386,P=0.017),各FAB亚型中M1、M5及M6型miR-181b表达水平较对照组明显升高(P＜0.05).miR-181b高表达与低血红蛋白、高乳酸脱氢酶及NPMI野生型有关.miR-181b高表达完全缓解率较低(x2 =7.717,P=0.005)、总生存期较短(P＜0.05).结论 miR-181b在AML某些亚型中高表达,miR-181b高表达是AML患者不利的预后因素.     

正常核型急性髓系白血病NPM1基因突变的临床研究

目的探讨正常核型急性髓系白血病（AML）患者核仁磷酸蛋白1（NPM1）基因突变发生情况,并了解其临床特征及预后。方法采用基因组DNA-PCR方法检测123例初发AML患者NPM1基因及正常核型AML患者FMS样酪氨酸激酶3（FLT3）基因,直接测序法检测AML患者NPM1基因第12外显子的突变情况,琼脂糖电泳分析正常核型AML患者FLT3基因内部串联重复（ITD）突变。结果 123例AML患者中检出NPM1突变24例（19.5%）,其中A型突变22例、B型突变1例、D型突变1例。57例正常核型中FLT3-ITD阳性10例,其中5例同时发生NPM1和FLT3-ITD两种突变。NPM1突变在正常核型中的发生率为40.3%（23/57）,显著高于异常核型的2.1%（1/47）（P〈0.01）。正常核型中NPM1基因突变者发病年龄高、缓解率高,但合并FLT3-ITD突变者缓解率低。结论 NPM1基因突变是AML尤其是正常核型AML患者常见的分子学异常,NPM1基因突变检测对指导AML患者治疗及评估预后有重要意义。     

急性髓细胞白血病患者NPM1与FLT3基因突变的研究

目的 探讨急性髓细胞白血病（AML）患者中NPM1基因与FLT3基因内部串联重复（ITD）突变的发生率,并了解其临床特征及预后。方法 收集86例成人AML患者初诊时骨髓单个核细胞,采用基因组DNA-PCR方法分别扩增其NPM1和FLT3基因,毛细管电泳方法分析NPM1第12外显子突变,琼脂糖电泳分析FLT3-ITD突变,随访判断其预后。结果 86例AML患者中共检出NPM1基因突变29例（33．7％）,FLT3-ITD突变15例（17,4％）。两种突变在50例染色体正常核型AML中的发生率分别为46．0％和24．0％,明显高于异常核型AML患者。7例中6例NPM1^＋／FLT3-ITD^＋双阳性AML患者表现为正常核型,外周血白细胞均〉50×10^9几。单纯NPM1^＋或FLT3-ITD^＋AML患者初诊时也表现为高白细胞（P〈0.05）,NPM1^＋AML患者CD34表达率较低（P〈0．001）、临床完全缓解（CR）率略高（66.7％、53.3％,P〉0.05）、总生存率（OS）高（P〉0.05）;而FLT3-ITD^＋AML患者CR率略低（50．0％、58．8％,P〉0.05）、OS低（P〉0.05）。NPM1^＋／FLT3-ITD^-、NPM^-／FLT3-ITD^-、NPM^＋／FLT3-ITD^＋、NPM1-／FLT3-ITD^＋四组患者的CR率分别为66．7％、62．5％、50．0％、42．9％（P〉0．05）,各组间OS差异无统计学意义（P〉0．05）。结论 NPM1和FLT3-ITD突变是AML（尤其正常核型AML）患者常见的分子学异常,且与其临床特点、疗效及预后有一定相关性。     

FLT3-ITD突变高等位基因比率在急性髓系白血病中的预后意义

目的:探讨FLT3-ITD突变高等位基因比率在急性髓系白血病(AML)患者中的预后意义。方法:筛选100例检测到FLT3-ITD等位基因比率的AML初诊患者的临床资料,对其临床特征及预后因素进行统计学分析。结果:100例患者初诊时中位白细胞计数为80.1×10~9/L(2×10~9/L～326×10~9/L),中位骨髓原始细胞比例数为68.5%(21%～95%),细胞遗传学检查示正常核型66例。将FLT3-ITD等位基因比率阈值设置在1.0,分为低等位基因比率组(1.0,64例)和高等位基因比率组(≥1.0,36例)进行分析,这些患者的初次完全缓解(CR1)率总体为70.0%,低等位基因比率组和高等位基因比率组间CR1率差异有统计学意义(82.8%vs 47.2%,P0.001)。在接受异基因造血干细胞移植(allo-HSCT)的65例患者中,低等位基因比率组的无复发生存(RFS)和总生存(OS)明显优于高等位基因比率组(RFS:P=0.007,OS:P=0.014)。而对于高等位基因比率组患者,allo-HSCT与化疗比较,未能明显改善其RFS及OS(RFS:P=0.539,OS:P=0.579)。在未进行allo-HSCT的患者中,低等位基因比率组和高等位基因比率组间RFS和OS差异无统计学意义(RFS:P=0.538,OS:P=0.854)。伴有其他突变(CEBPA、NPM1、DNMT3A)的FLT3-ITD高等位基因比率患者的CR1、OS、RFS,与低等位基因比率患者比较差异无统计学意义(P0.05)。对患者的预后因素进行分析发现,CR1、外周白细胞计数及allo-HSCT在单因素、多因素分析中均对RFS、OS有影响。FLT3-ITD高等位基因比率的AML患者能检测到13号染色体长臂杂合性丢失(LOH),且等位基因比率值越高,LOH检测到的频率越高。结论:FLT3-ITD突变高等位基因比率的AML患者具有较高的白细胞计数,多合并NPM1突变、CR1率低及OS、RFS短的特征。对于不同化疗效果均差,即使进行allo-HSCT也未能明显改善其OS及RFS。高等位基因比率患者存在LOH状态,等位基因比率值越高,LOH频率越高。     

维甲酸联合亚砷酸治疗伴FLT3-ITD突变的APL患者的预后分析

目的:探讨急性早幼粒细胞白血病伴FLT3-ITD患者以维甲酸(ATRA)联合三氧化二砷(ATO)为基础治疗方案下的预后影响。方法:回顾性分析101例初诊的成年急性早幼粒细胞白血病患者的临床资料并进行随访,患者初诊时被随机分为接受ATRA+ATO组(55例)及ATRA+化疗组(46例),使用PCR扩增,琼脂糖凝胶电泳检测FLT3-ITD基因突变,比较FLT3-ITD突变在2组的疗效及预后。结果:FLT3-ITD突变率24.75%(25/101),且FLT3-ITD突变与高白细胞计数、高血红蛋白水平、PML-RARA S亚型、Sanz危险分层高危有关(均P0.05)。2组FLT3-ITD阳性患者均诱导死亡率高、缓解率低,差异无统计学意义(均P0.05)。ATRA+化疗组中FLT3-ITD突变患者与FLT3-ITD未突变患者3年无复发生存率分别为68.6%、91.2%(P=0.048),ATRA+ATO组中FLT3-ITD突变患者与FLT3-ITD未突变患者3年无复发生存率分别为90.9%、100.0%(P=0.638);ATRA+化疗组中FLT3-ITD突变患者与FLT3-ITD未突变患者3年总生存率分别为80.8%、97.1%(P=0.077),ATRA+ATO组中FLT3-ITD突变患者与FLT3-ITD未突变患者3年总生存率分别为92.9%、97.6%(P=0.431)。结论:FLT3-ITD突变在ATRA+化疗治疗方案下复发率高,预后不良,诱导和巩固治疗前2个周期加入ATO治疗可能克服FLT3-ITD突变的影响,降低复发率,改善患者预后。     

急性髓系白血病复发前后FLT3突变的变化

目的:分析酪氨酸激酶受体基因FLT3突变在急性髓系白血病(AML)复发前后的变化及其对预后的影响。方法:22例在初诊及复发后均行FLT3-ITD/TKD检测的AML患者中,初诊时FLT3-ITD/TKD突变阳性者(FLT3+组)与阴性者(FLT3-组)各11例。分析2组患者临床基本特征、复发后FLT3突变转阳率、二次缓解率、无病生存(DFS)及总生存(OS)。结果:FLT3+组复发后,FLT3突变转阳率为90.9%(10/11),二次缓解率为9.1%(1/11);FLT3-组复发后,FLT3突变转阳率为27.3%(3/11),二次缓解率为27.3%(3/11)。2组间二次缓解率比较差异无统计学意义(P=0.586)。FLT3+组中位DFS为5(2~22)个月,FLT3-组中位DFS为5(3~19)个月,差异无统计学意义(P=0.531)。FLT3+组中位OS为12(7~33)个月,FLT3-组中位OS为15(6~40)个月,差异无统计学意义(P=0.208)。结论:AML伴有初诊FLT3+患者预后较差,该突变具有不稳定性,因而作为微小残留病的监测指标需慎重。     

急性髓细胞白血病遗传学特征与临床特征分析

目的 :探讨急性髓细胞白血病(AML)患者的分子遗传学特征及其与临床特征的关联性。方法 :采用骨髓短期培养和G显带技术对103例AML患者进行染色体核型分析,并通过PCR技术对其基因突变、融合基因等进行检测。结果:在103例患者样本中所检测到的FMS样酪氨酸激酶3基因的内部串联重复(FLT3-ITD)突变和酪氨酸激酶结构域点突变(FLT3-TKD)、核磷蛋白1(NPM1)基因突变、神经母细胞瘤RAS癌(NRAS)基因突变、异柠檬酸脱氢酶1(IDH1)基因突变、CCAAT/增强子结合蛋白α(CEBPA)基因突变、人类原癌基因C-KIT突变的发生率分别为14.3%、3.3%、14.3%、12.4%、5.6%、10.6%和18.8%;NPM1突变阳性患者较阴性患者骨髓幼稚细胞比例高(P=0.0105),FLT3-ITD突变阳性患者较阴性患者的病死率低(P=0.0285),而C-KIT突变阳性患者较阴性患者的病死率高(P=0.0255)。结论:AML患者的分子遗传学特征细化了基于核型的AML危险度分层,其中FLT3-ITD、NPM1、C-KIT突变与患者的临床特征有关联。     

WT1基因及蛋白在急性髓性白血病的表达及临床意义

目的探讨WT1基因及其蛋白产物在急性髓性白血病（AML）的表达及临床意义。方法采用RT-PCR及免疫组化的方法检测初治及复发的AML患者、正常志愿者骨髓或外周血单个核细胞中WT1基因及其蛋白产物的表达情况,并结合临床观察可盯1基因及蛋白产物表达与疗效的关系。结果 WT1基因及蛋白在45例AML初治患者中分别有39例（86．7％）和38例（84．4％）表达阳性,在6例复发患者中两者均表达阳性;AML患者WT1基因与蛋白表达呈正相关（r=0．277,P〈0．05）。初治患者中WT1基因和蛋白表达阳性的初次化疗完全缓解率分别为42．4％和40．6％,表达阴性的则分别为83．3％和85．7％;WT1基因相对表达量与初治AML患者首次化疗的完全缓解率有关（χ2=4．356,P〈0．05）。结论AML患者WT基因及蛋白的表达检测可作为判断其临床预后的一种手段,WT1蛋白的表达可以作为AML免疫治疗的靶点。     

Non-A type nucleophosmin 1 gene mutation predicts poor clinical outcome in de novo adult acute myeloid leukemia: differential clinical importance of NPM1 mutation according to subtype

Mutations of nucleophosmin gene (NPM1) are known to be related to good prognosis in AML patients lacking FLT3 internal tandem duplication (FLT3-ITD). Recently, NPM1 mutations other than type A were reported, but their clinical significance is not well known. Retrospective medical record review of 106 de novo AML patients lacking FLT3-ITD, who received induction chemotherapy from three centers in Korea between 1997 and 2007, was performed. Direct sequencing of NPM1 and RT-PCR for FLT3-ITD was performed on genomic DNA derived from blood samples of patients before induction chemotherapy for detection of mutations. NPM1 mutation was detected in 18 patients, where 13 were type A mutants and 5 were non-type A mutants. CR, CR1-D and OS was not different according to NPM1 mutational status overall. But, non-type A NPM1 mutation was related to shorter CR1-D when compared with NPM1 wild types and NPM1 type A mutation (p = 0.004). OS was shorter in non-type A mutants when compared with NPM1 wild-type patients and NPM1 type A mutants (p = 0.001). The type of mutation of NPM1 is important for prognosis in de novo AML lacking FLT3-ITD. Non-A type NPM1 mutation is a poor prognostic factor.     

Prevalence and prognostic impact of NPM1 mutations in 1485 adult patients with acute myeloid leukemia (AML)

Mutations of the nucleophosmin (NPM1) gene have recently been described in patients with acute myeloid leukemia (AML). To clarify the prevalence as well as the clinical impact of this mutation, we investigated 1485 patients with AML for NPM1 exon 12 mutations using fragment analysis. A 4 bp insert was detected in 408 of 1485 patients (27.5%). Sequence analysis revealed known mutations (type A, B, and D) as well as 13 novel alterations in 229 analyzed cases. NPM1 mutations were most prevalent in patients with normal karyotype (NK) (324 of 709; 45.7%) compared with 58 of 686 with karyotype abnormalities (8.5%; P < .001) and were significantly associated with several clinical parameters (high bone marrow [BM] blasts, high white blood cell [WBC] and platelet counts, female sex). NPM1 alterations were associated with FLT3-ITD mutations, even if restricted to patients with NK (NPM1-mut/FLT3-ITD: 43.8%; versus NPM1-wt/FLT3-ITD: 19.9%; P < .001). The analysis of the clinical impact in 4 groups (NPM1 and FLT3-ITD single mutants, double mutants, and wild-type [wt] for both) revealed that patients having only an NPM1 mutation had a significantly better overall and disease-free survival and a lower cumulative incidence of relapse. In conclusion, NPM1 mutations represent a common genetic abnormality in adult AML. If not associated with FLT3-ITD mutations, mutant NPM1 appears to identify patients with improved response toward treatment.     

Clinical implications of FLT3 mutations in pediatric AML

Activating mutations of the FLT3 gene occur because of an internal tandem duplication of the juxta-membrane domain (FLT3/ITD) or point mutation of the activation loop domain (FLT3/ALM). The presence of FLT3 mutations as well as the allelic ratio of FLT3/ITD (ITD-AR, mutant-wild type ratio) may have prognostic significance. FLT3 mutation status of 630 children with de novo acute myeloid leukemia (AML) treated on CCG-2941 and -2961 was determined, and ITD-AR was calculated for patients with FLT3/ITD. Clinical characteristics and outcomes for patients with FLT3/ALM and FLT3/ITD at varying ITD-ARs was determined and compared with those without FLT3 mutations (FLT3/WT). FLT3/ITD and FLT3/ALM were detected in 77 (12%) and 42 (6.7%) of the patients. Progression-free survival (PFS) was similar in patients with FLT3/ALM and FLT3/WT (51% versus 55%, P = .862). In contrast, PFS at 4 years from study entry for patients with FLT3/ITD was inferior to that of patients with FLT3/WT (31% versus 55%, P < .001). PFS decreased with increasing FLT3/ITD-AR (P < .001), and those with ITD-AR greater than 0.4 had a significantly worse PFS than those with lower ITD-AR (16% versus 72%, P = .001) or with FLT3/WT (55%, P < .001). ITD-AR defines the prognostic significance in FLT3/ITD-positive AML, and ITD-AR greater than 0.4 is a significant and independent prognostic factor for relapse in pediatric AML.     

Distinctive expression of myelomonocytic markers and down-regulation of CD34 in acute myelogenous leukaemia with FLT3 tandem duplication and nucleophosmin mutation

OBJECTIVE: Patients with acute myelogenous leukaemia (AML) show co-existing frequently internal tandem duplications of FLT3 (FLT3-ITD) and mutations of nucleophosmin (NPM1-Mt). We investigated the biological and clinical significance of FLT3-ITD and/or NPM1-Mt in this context. METHODS: We analysed 89 AML patients according to whether NPM1 and FLT3-ITD were single mutants, double mutants, or wild type for both. RESULTS: FLT3-ITD was detected in 19 of 89 patients (21.3%), while NPM1-Mt was detected in 19 of 89 patients (21.3%); eight of 89 patients (9.0%) carried both FLT3-ITD and NPM1-Mt. By multivariate analysis, white blood cell count and peripheral blood blast cell count at diagnosis were significantly higher in patients with FLT3-ITD but not in those with only NPM1-Mt. NPM1-Mt was significantly related to female gender, normal karyotype, and M4 or M5 disease according to French-American-British criteria. In addition, leukaemic blast cells with NPM1-Mt, FLT3-ITD, or both expressed CD34 less frequently than wild-type blasts (P < 0.0001 and P = 0.005 respectively), while myelomonocytic markers such as CD11b and CD14 were expressed more frequently in patients with NPM1-Mt. CONCLUSION: FLT3-ITD may increase potential for cell proliferation to produce a leukaemic population; NPM1-Mt may cause cells to develop along the myelomonocytic lineage. Extensive analyses and detailed experiments will be required to clarify how NPM1 and FLT3 mutations interact in leukaemogenesis.     

High WT1 mRNA expression after induction chemotherapy and FLT3-ITD have prognostic impact in pediatric acute myeloid leukemia: a study of the Japanese Childhood AML Cooperative Study Group

The prognostic value of WT1 mRNA expression in pediatric acute myeloid leukemia (AML) remains controversial. A sample of newly diagnosed (n?=?158) AML patients from the Japanese Childhood AML Cooperative Treatment Protocol, AML 99, were simultaneously analyzed for WT1 expression, cytogenetic abnormalities and gene alterations (FLT3, KIT, MLL, and RAS). WT1 expression (including more than 2,500 copies/??gRNA) was detected in 122 of the 158 (77.8?%) initial diagnostic AML bone marrow samples (median 45,500 copies/??gRNA). Higher WT1 expression was detected in French American British (FAB)-M0, M3, M7 and lower expression in M4 and M5. Higher WT1 expression was detected in AML with inv(16), t(15;17) and Down syndrome and lower in AML with 11q23 abnormalities. Multivariate analyses demonstrated that FLT3-internal tandem duplication (ITD), KIT mutation, MLL-partial tandem duplication were correlated with poor prognosis; however, higher WT1 expression was not. FLT3-ITD was correlated with WT1 expression and prognosis. Furthermore, 74 WT1 expression after induction chemotherapy was analyzed. Higher WT1 expression after induction chemotherapy was significantly correlated with M1 or M2/M3 marrow, FLT3-ITD and poor prognosis. Multivariate analyses in 74 AML patients revealed that FLT3-ITD, MLL-PTD, and KIT mutations were associated with poor prognosis; however, NRAS Mutation, KRAS mutation and high WT1 expression (>10,000 copies/??gRNA) did not show poor prognosis. Our findings suggest that higher WT1 expression at diagnosis does not correlate with poor prognosis, but that WT1 expression after induction chemotherapy is considered to be a useful predictor of clinical outcome in pediatric AML.     

CD25 expression status improves prognostic risk classification in AML independent of established biomarkers: ECOG phase 3 trial, E1900

We determined the prognostic relevance of CD25 (IL-2 receptor-α) expression in 657 patients (≤ 60 years) with de novo acute myeloid leukemia (AML) treated in the Eastern Cooperative Oncology Group trial, E1900. We identified CD25(POS) myeloblasts in 87 patients (13%), of whom 92% had intermediate-risk cytogenetics. CD25 expression correlated with expression of stem cell antigen CD123. In multivariate analysis, controlled for prognostic baseline characteristics and daunorubicin dose, CD25(POS) patients had inferior complete remission rates (P = .0005) and overall survival (P < .0001) compared with CD25(NEG) cases. In a subset of 396 patients, we integrated CD25 expression with somatic mutation status to determine whether CD25 impacted outcome independent of prognostic mutations. CD25 was positively correlated with internal tandem duplications in FLT3 (FLT3-ITD), DNMT3A, and NPM1 mutations. The adverse prognostic impact of FLT3-ITD(POS) AML was restricted to CD25(POS) patients. CD25 expression improved AML prognostication independent of integrated, cytogenetic and mutational data, such that it reallocated 11% of patients with intermediate-risk disease to the unfavorable-risk group. Gene expression analysis revealed that CD25(POS) status correlated with the expression of previously reported leukemia stem cell signatures. We conclude that CD25(POS) status provides prognostic relevance in AML independent of known biomarkers and is correlated with stem cell gene-expression signatures associated with adverse outcome in AML.