WT1基因及CD34在急性白血病中的表达及意义

目的研究WT1基因与CD34在急性白血病（AL）中表达的相关性及其临床意义。方法采用实时定量RT-PCR方法检测92例初治AL患者骨髓细胞WT1基因的表达,同时应用流式细胞仪测定骨髓细胞CD34的表达。结果初治AL患者WT1基因、CD34表达的阳性率分别为67．4％（62／92）、44．6％（41／92）,WT1基因、CD34阳性表达者的缓解率显著低于阴性表达者（P〈0．01）;WT1基因与CD34表达呈正相关（rn=0．5304,χ^z=25．88,P〈0．05）;WT1^＋CD34^＋、WT1^＋CD34、WT1-CD34^-AL患者第一次缓解率比较有统计学差异（P〈0．01）。结论WT1基因、CD34在AL患者骨髓细胞中的表达呈正相关,且阳性表达者的缓解率低、疗效差、预后不良。     

急性髓系白血病患者中期因子基因表达与化疗耐药及预后的关系

目的：探讨中期因子（Midkine，MK）与急性髓系白血病（AML）患者预后的关系。方法：采用半定量逆转录一聚合酶链反应方法检测65例初治及复发AML患者、15例缓解期患者及15例正常人骨髓单个核细胞（MNCs）MK、mdr-1、bcl-2mRNA的表达，并采用蛋白印迹（Westernblot）法检测20例AML患者和5例正常人MNCs孵育24h后，培养基中MK的蛋白表达。结果：65例初治及复发AML患者中25例MK基因表达阳性，15例正常人MK基因表达均阴性。MK基因表达阳性患者的完全缓解（CR）率明显低于表达阴性者（分别为63．16％和93．55％，P〈0．05）；MK基因表达阳性患者的复发率（100％）明显高于表达阴性者（40．00％），P〈0．05。MK与bcl-2基因表达呈正相关（r=0．556，P〈O．01）。结论：AML患者白血病细胞可以产生MK，且MK阳性率的高低与AML病期相关，MK过度表达可能导致临床化疗耐药，是影响AML患者近期预后的重要因素之一。     

ZO-1基因甲基化状态在非霍奇金淋巴瘤骨髓检测中的临床意义

目的探讨紧密连接蛋白-1（ZO-1）基因启动子区甲基化状态在非霍奇金淋巴瘤（NHL）检测中的临床意义。方法采用甲基化特异性PCR方法（MS-PCR）分析10例非血液系统肿瘤者骨髓及45例NHL患者骨髓标本的ZO-1基因启动子区甲基化状况。结果ZO-1基因在10例良性血液病及正常人中呈完全非甲基化状态,在39例初治、复发、未完全缓解的淋巴瘤患者中甲基化阳性率53．85％（P〈0．05）。在39例初治或复发或未达到完全缓解的NHL患者中28例Ⅲ、Ⅳ期的NHL患者ZO-1基因甲基化阳性率64．29％,11例Ⅰ、Ⅱ期的NHL患者ZO-1基因甲基化阳性率27．27％（P〈0．05）。初治、复发患者28例中甲基化阳性16例（57．14％）,经治疗达到部分缓解的11例患者中甲基化阳性5例（45．45％）,临床缓解的6例患者均为完全非甲基化。结论ZO-1基因启动子区高甲基化与疾病分期及缓解明显相关,它可以作为判断NHL进展和评价预后的辅助指标,并以此指导临床治疗。     

初治急性白血病患者肺耐药蛋白和多药耐药相关蛋白基因表达与预后的关系

目的 探讨肺耐药蛋白（1rp)基因和多药耐药相关蛋白（mrp)基因表达与初治急性白血病（AL）患者化疗效果的关系。方法 用逆转聚合酶链反应方法检测58例初治AL患者骨髓单个核细胞中lrp和mrp的mRNA的表达。结果 初治急性淋巴细胞白血病（ALL）患者lrp和mrp阳性表达率分别为15．0％和40．0％, 初治急性非淋巴细胞白血病（ANLL）患者lrp和mrp阳性表达率分别为15．8％和42．1％;在ALL和ANLL组,lrp和mrp基因阳性与阴性患者的首次完全缓解（CR）经统计学分析,差异无显著性（P＞0．05）,而lrp和mrp基因双阳性与双阴性患者的首次CR经统计学分析,差异有显著性（P＜0．05）;lrp和mrp基因两者无相关性。结论 lrp和mrp单独作为预测初治AL患者化疗效果的敏感性较差,而两者联合检测则是预示原发耐药的良好指标。     

肺耐药蛋白在急性白血病中的表达及意义

目的 探讨急性白血病（AL）患者骨髓单个核细胞肺耐药蛋白（LRP）的表达及意义。方法 采用LRP单克隆抗体、流式细胞技术分别测定15例单纯缺铁性贫血患者（对照组）和65例AL患者（AL组）LRP的表达率。结果 LRP的表达率在初治和复发／难治急性淋巴细胞白血病（ALL）患者分别高于初治和复发／难治急性髓细胞白血病（AML）患者（P均〈0．05）;AML中M5亚型表达率高于M3亚型（P〈0．05）。初治及复发／难治LRP（＋）者缓解率明显低于LRP（-）者（P〈0．05）。结论 LRP的表达与AL患者临床预后密切相关,化疗前检测LRP表达率有助于个体化治疗和预测疗效。     

联合定量检测急性髓系白血病患者MDR1和WT1基因表达及临床意义

目的 联合定量检测初治急性髓系白血病(AML)患者MDR1及WT1基因的表达水平,探讨MDR1与WT1的表达量在AML耐药中作用及相互关系.方法 构建实时荧光定量PCR检测WT1和MDR1基因表达水平技术,并检测63例初治AML患者WT1和MDR1基因的表达量及与临床疗效的关系.结果 63例初治AML患者MDRI基因表达(拷贝/μg RNA)的中位数为2863(413～3 410 000),WT1基因表达为52 700(7731～1 020 000).10例正常对照组MDR1和WT1基因表达水平为337(125～840)和849(635～1402).初治AML患者MDR1和WT1基因表达水平均显著高于正常对照组(P＜0.001).且MDR1与WT1基因表达水平呈相关性(P=0.004);FAB各亚型中MDR1和WT1基因表达水平差异无统计学意义(P＞0.05);遗传学危险度分级的高、中、低危3组的WT1和MDR1基因表达水平差异无统计学意义(P＞0.05).FLT3-ITD突变阳性组MDR1基因表达水平与突变阴性组相比差异无统计学意义(P＞0.05).FLT3-ITD突变阳性组的WT1基因表达水平显著高于阴性组(P＜0.05).AML患者中WT1和MDR1基因共同高表达组完全缓解率显著低于共同低表达组(P＜0.05).结论 AML患者MDR1和WT1基因表达水平存在相关性;联合检测AML患者MDR1和WT1基因表达量更能早期预测预后.     

急性髓系白血病患者骨髓单个核细胞中W T1表达及其与疗效、预后的关系

目的：观察急性髓系白血病（AML）患者骨髓单个核细胞中Wilms基因（WT1）的表达,并分析其表达与疗效、预后的关系。方法选择AML患者56例、非肿瘤患者28例。采用定量PCR法检测骨髓单个核细胞中WT1表达,并分析其表达与患者性别、年龄、外周血白细胞数量、骨髓原幼细胞比例、LDH水平的关系。首次诱导化疗后,观察不同WT1表达水平与AML患者的疗效、无事件生存（ EFS）时间、总生存（ OS）时间的关系。结果 AML患者WT1表达水平中位数为2875拷贝／10000 ABL拷贝,高表达阳性率为85．7％;非肿瘤患者WT1表达水平中位数为8拷贝／10000 ABL拷贝,无高表达患者。 WT1表达水平不同的AML患者性别、年龄、外周血白细胞数量、骨髓原幼细胞比例、LDH水平比较差异无统计学意义。 WT1表达正常AML患者首次诱导化疗后完全缓解率、中位EFS时间与WT1高表达AML患者比较,P均＞0．05。 WT1表达正常AML患者中位OS时间为29．5个月,WT1高表达AML患者为19个月,两者比较P均＜0．05。结论 AML患者WT1表达水平明显高于非肿瘤患者;AML患者WT1表达水平与患者的疗效、预后可能有关。     

慢性粒细胞白血病患者WT1基因表达及其临床意义

目的探讨慢性粒细胞白血病（CML）患者骨髓细胞中Wilms瘤抑癌基因（WT1）的表达水平及其临床意义。方法建立实时定量RT-PCR方法,采用Light Cycler PCR仪检测了46例（109份骨髓细胞cDNA标本）CML患者和23例非白血病患者骨髓细胞中WT1及内参β胆色原脱氢酶（GAPDH）的表达水平,以WT1N=（WT1拷贝数／GAPDH拷贝数）×10^4计算WT1表达水平。结果23份CML加速期与22份CML急变期患者骨髓细胞中WT1N的中位表达水平分别为103．71和129．44,明显高于64份CML慢性期和对照组（分别为3．44和1．47,P〈0．01）,对照组与CML慢性期之间WT1基因表达差异无统计学意义;CML加速期与急变期之间WT1基因表达差异也无统计学意义（P〈0．05）。WT1基因表达水平与BCR／ABL融合基因表达水平具有一定的相关性。对其中7例CML患者行异基因骨髓移植前后动态检测WT1上升可提示白血病复发。结论CML患者加速急变期骨髓细胞中WT1基因表达升高,具有参考意义。     

抗凋亡蛋白XIAP在急性白血病中的表达及临床意义

目的本研究通过检测抗凋亡蛋白XIAP在急性白血病（AL）中的表达,探讨XIAP在AL中的临床意义。方法应用SP-免疫组织化学方法检测78例AL患者初治组、缓解组、复发组及25例正常人（对照组）中XIAP的表达情况。结果（1）XIAP在初治AL患者骨髓中表达水平及表达强度均高于缓解组（P〈0．05,P〈0．008）和对照组（P〈0．05,P〈0．008）,与复发组相比差异均无显著性（P〉0．05）;（2）XIAP高表达患者完全缓解率低于XIAP低表达患者（P〈0．05）。结论XIAP过度高表达和急性白血病的发病和复发相关,XIAP可作为预后不良的指标。     

复发/难治AML患者sorcin基因表达及意义

目的研究复发／难治急性髓细胞白血病（AML）患者骨髓可溶性耐药相关钙结合蛋白（sorcin）基因表达及意义。方法采用逆转录聚合酶链反应（RT—PCR）检测65例AML患者（观察组）,27例非白血病患者和健康人（对照组）sorcin基因表达水平。结果观察组sorcin基因表达水平高于对照组（P〈0．001）,其中复发／难治者高于初诊者和完全缓解（CR）者;AML各亚型sorcin基因过度表达存在差异,以M,阳性表达率最高;观察组临床耐药者sorcin基因表达显著高于非临床耐药者（P〈0．001）,且CR率显著低于非临床耐药者（P〈0．001）。结论sorcin基因过度表达与AML患者临床耐药密切相关,是影响预后的重要因素;sorcin可作为检测AML临床耐药和判断预后的指标之一。     

Heme oxygenase-1 plays a crucial role in chemoresistance in acute myeloid leukemia

Abstract

Objectives

The heme oxygenase-1 (HO-1) gene may contribute to the development of acquired chemoresistance in solid tumor cells, but its function in acute myeloid leukemia (AML) remains unclear. Therefore, we investigated whether the expressions of HO-1 mRNA and protein were associated with AML chemoresistance.

Methods

Bone marrow or peripheral blood was obtained from newly diagnosed (n = 26), relapsed (n = 10), and completely remitted (n = 18) patients with AML (M3 exclusion) and healthy donors (n = 10). Small interfering RNA was used to stably silence HO-1 gene expression in AML cell lines. The expressions of HO-1, hypoxia inducible factor-1ɑ (HIF-1ɑ), glucose transporter-1 (GLUT1) mRNA and proteins were measured by quantitative real-time PCR and Western blot. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis induction was analyzed by flow cytometry.

Results

The drug-resistant AML cell line HL-60R was significantly less sensitive to cytarabine and daunorubicin than HL-60 cells. HO-1 mRNA and proteins were highly expressed in HL-60R cells. However, down-regulating HO-1 significantly enhanced the sensitivity of HL-60R to chemotherapy, and the expressions of HIF-1ɑ and GLUT1 mRNA and proteins decreased. Meanwhile, the expressions of caspase-3 and caspase-8 proteins increased, while that of bcl-2 decreased. Overexpressions of HO-1, HIF-1ɑ, and GLUT1 were associated with poor response of AML to chemotherapy.

Conclusions

Overexpressions of HO-1, HIF-1ɑ, and GLUT1 might be involved in the chemoresistance of AML. HO-1 is a potential target to overcome the drug resistance of AML.     

Prognostic relevance of Wilms tumor 1 (WT1) gene Exon 7 mutations in-patient with cytogenetically normal acute myeloid leukemia

This study aimed to assess the prognostic influences of Wilms tumor 1 (WT1) gene mutations in cytogenetically normal acute myeloid leukemia (CN-AML) among Egyptian patients. Exon 7 of WT1 was screened for mutations in samples from 82 CN-AML patients out of 203 newly diagnosed AML patients, using a high-resolution capillary electrophoresis. Seven out of 82 AML patients (8.3 %) harbored WT1 mutations. There was no significant difference between the mutant WT1 and wild type AML patients as regard age, sex, French–American–British subtypes and the prevalence of success of induction remission therapy (P < 0.5). AML patients with mutant WT1 had shorter overall survival as compared to those patients with wild WT1 (HR = 1.38; 95 % CI 4.79–6.86; P = 0.004). In conclusion, CN-AML patients with WT1 gene mutation have poor clinical outcome. We recommend testing the WT1 mutations as part of molecularly based risk assessment and risk-adapted treatment stratification of patients with CN-AML.     

Expression of four major WT1 splicing variants in acute and chronic myeloid leukemia patients analyzed by newly developed four real-time RT PCRs

Although the mechanism of action of leukemic oncogene Wilms' tumor gene 1 (WT1) remains unclear, WT1 has already been used in monitoring of patients with acute myeloid leukemia (AML) and it is being tested for immunotherapy. More detailed understanding of the role of WT1 in leukemia may improve its utilization. At least 36 isoforms may be produced. Four major variants denoted as -5/-KTS, -5/+KTS, +5/-KTS and +5/+KTS are produced by combining splicing of exon 5 and KTS sequence. In this study, we report applicability of newly developed real-time RT PCRs enabling for the first time full quantification of the four major WT1 splicing variants. Following careful optimization and testing of quantification reliability of four assays, we analyzed 34 samples of patients with AML and 12 samples of patients with chronic myeloid leukemia (CML) at the time of diagnosis. Analyses of five more CML patients provided insight into WT1 variants expression kinetics. We found predominance of +5/+KTS in both diagnoses. Comparison of WT1 variant expression in AML and CML patients' groups differing in response to therapy suggested possible importance of particular WT1 variant levels as markers of further disease course.     

Significant co-expression of WT1 and MDR1 genes in acute myeloid leukemia patients at diagnosis

A high expression of Wilms' tumor gene (WT1) in acute myeloid leukemia (AML) seems to correlate with a poor outcome and its increased levels can be predictive of an impending relapse. WT1 has been shown in vitro to interact with the promoter of the MDR1, a gene involved in the multidrug resistance phenomenon. The aim of this study was to measure, by real-time polymerase chain reaction, levels of WT1 and MDR1 expression, in order to find a possible association between these genes, in a series of 50 newly diagnosed AML cases. Twenty-five percent of patients carried very high (>75 degrees percentile) MDR1- and 23.3%WT1-mRNA levels. Interestingly, high levels of WT1 were significantly correlated with correspondent high levels of MDR1 gene. Nevertheless, the co-expression of these genes did not significantly influence the complete response rate to the induction therapy. Reported data confirm the existence of a co-expression of WT1 and MDR1 genes even in vivo; this may be relevant because one consequence could be the positive selection by chemotherapeutic regimens of cells with higher MDR1 levels already present before treatment. Thus, the association between these genes could suggest avoiding the use of drugs involved in the multidrug resistance (MDR) phenomenon in patients carrying high levels of WT1 at diagnosis.     

Analysis of mutational status, SNP rs16754, and expression levels of Wilms tumor 1 (WT1) gene in acute promyelocytic leukemia

Overexpression, polymorphisms, and mutations of the WT1 gene have been reported in several human tumors including acute myeloid leukemia (AML) and variably correlated with prognosis. Acute promyelocytic leukemia (APL) represents the AML subset disclosing higher WT1 expression levels; however, no WT1 studies specifically focused on APL have been conducted. We screened for the presence of mutations, SNP rs16754, and expression levels of WT1 gene in 103 adult patients with newly diagnosed APL. Fms-like tyrosine kinase (FLT3) mutations were analyzed as well. WT1 mutations were identified in four (4?%) patients. At least one copy of the minor SNP rs16754 allele (WT1 ^AG or WT1 ^GG) was detected in 30 (29?%) patients. Six patients (6?%) were homozygous for the minor allele (WT1 ^GG ) and this genotype was associated with higher WT1 mRNA copies (p?=?0.018). FLT3 mutations were found in 37?% of patients and correlated with high WT1 mRNA expression (p?=?0.004). Patients heterozygous or homozygous for the minor allele and patients homozygous for major (WT1 ^AA) allele did not differ in terms of presenting features. In adult APL, WT1 gene mutational and polymorphic profile shows similarities with pediatric AML rather than with adult AML.     

AML1-ETO rapidly induces acute myeloblastic leukemia in cooperation with the Wilms tumor gene, WT1

AML1-ETO, a chimeric gene frequently detected in acute myelogenous leukemia (AML), inhibits the differentiation of myeloid progenitors by suppressing genes associated with myeloid differentiation and increases the replating ability of clonogenic myeloid progenitors. However, AML1-ETO alone cannot induce AML and thus additional genetic events are required for the onset of AML. The Wilms tumor gene (WT1), which has been identified as the gene responsible for Wilms tumor, is expressed at high levels in almost all human leukemias. In this study, we have generated transgenic mice (WT1-Tg) that overexpress WT1 in hematopoietic cells to investigate the effects of WT1 on AML1-ETO-associated leukemogenesis. AML1-ETO-transduced bone marrow (BM) cells from WT1-Tg mice exhibited inhibition of myeloid differentiation at more immature stages and higher in vitro colony-forming ability compared with AML1-ETO-transduced BM cells from wild-type mice. Most importantly, all of the mice that received a transplant of AML1-ETO-transduced BM cells from the WT1-Tg mice rapidly developed AML. These results demonstrate that AML1-ETO may exert its leukemogenic function in cooperation with the expression of WT1.