转录因子GATA-2基因在ANLL、CML和MDS中的表达

目的 :了解 GATA- 2基因在急性非淋巴细胞白血病 (ANL L )、慢性粒细胞白血病 (CML )和骨髓增生异常综合征 (MDS)中的表达情况。方法 :利用 RT- PCR扩增 ANL L、CML和 MDS等 2 46例患者外周血单个核细胞中 GATA- 2基因。结果 :GATA- 2基因在 ANL L、CML 和 MDS中的表达率分别为 88.4%、81.6 %和 97% ,差异与正常人有高度显著性 (P <0 .0 1)。结论 :绝大多数髓系白血病和 MDS标本表达 GATA- 2基因 ,GATA- 2可能是白血病细胞的增殖过程中所需要的转录因子之一。     

血清bFGF水平与MDS分型及预后的关系研究

目的研究骨髓增生异常综合征(MDS)患者血清碱性成纤维细胞生长因子(bFGF)水平与MDS分型、发展及预后的关系。方法应用双抗体夹心酶联免疫吸附法检测MDS、急性髓细胞白血病(AML)患者和正常人的bFGF水平,分析其与MDS分型及临床因素的关系。结果MDS患者血清bFGF水平明显高于正常,其中RAEB、RAEBT型均明显高于RA、RAS型,RAEB、RAEBT组与急性髓细胞白血病(AML)组无显著差异,bFGF水平与患者的外周血常规、骨髓原始细胞比例有关。结论血清bFGF水平与MDS的分型、发展及预后有关。     

初诊急性白血病患者细胞膜组织因子和血浆F1+2检测的临床意义

目的 探讨初诊急性白血病(AL)各亚型患者白血病细胞膜组织因子(TF)的表达程度和血浆凝血酶原片段1+2(F1+2)浓度及其关系以及上述指标对AL并发弥散性血管内凝血(DIC)的预测和诊断评价意义.方法 用流式细胞术(FCM)对2006年10月至2007年10月中国医科大学附属盛京医院收治的50例AL患者和12例正常人骨髓分离的单个核细胞胞膜TF表达进行检测,酶联免疫吸附试验(ELISA)检测血浆F1+2浓度.结果 急性髓性白血病(AML)-M3白血病细胞膜TF的表达[(48.23±11.59)%]高于AML-M1-2、M4和急性淋巴细胞白血病(ALL)(P＜0.05),与AML-M5(40.51%)比较差异无统计学意义(P＞0.05).AML-M3、M4和M5血浆F1+2浓度均显著高于正常对照组(P＜0.05).合并DIC组的TF表达和血浆F1+2浓度均高于无DIC组,差异有统计学意义(P＜0.01).TF表达程度和血浆F1+2浓度呈一定相关性(r=0.682,P＜0.01).结论 初诊AL患者存在凝血激活,表现为白血病细胞膜TF表达增多,凝血酶生成增加,尤以AML-M3和M5为著.FCM测定TF的膜表达程度对AL凝血异常发生潜在性可起一定预测和提示作用.     

骨髓增生异常综合征骨髓细胞凋亡和TNF-α关系的研究

目的：探讨骨髓增生异常综合征(MDS)骨髓细胞调亡特征以及与肿瘤坏死因子α(TNF—α)的关系。方法：分别采用形态学和dUTP末端缺口标记(TUNEL)方法检测骨髓细胞调亡；采用ELISA方法检测骨髓单个核细胞培养后上清液中TNF—α的浓度，并与缺铁性贫血(IDA)患者进行比较。结果：形态学方法检测骨髓单个核细胞培养后细胞调亡指数，MDS明显高于IDA(P＜o．01)，MDS各分型(RA／RAS、RAEB／RAEB—t)之间细胞调亡指数差异无显著性意义(P＞0．05)。同一个体TUNEL法测得的骨髓细胞调亡指数均高于形态学观测的结果。TUNEL检测MDS细胞调亡指数RA／RAS＞RAEB／RAEB—t，但差异无显著性意义(P＞0．05)。MDS骨髓单个核细胞上清中TNF-α浓度高于IDA组，差异有显著性意义(P＜0．05)；MDS各分型之间RA／RAS显著高于RAEB／RAEB—t(P＜0．05)。结论：MDS骨髓细胞确实凋亡过度，早期MDS重于晚期MDS，并与TNF—α呈线性关系；采用TUNEL法检测凋亡细胞较形态学方法敏感。     

慢性淋巴细胞白血病的生物学特点与预后意义

<正>1概述慢性淋巴细胞白血病(CLL)是B细胞慢性淋巴增殖性疾病(B-CLPD)最常见的一种类型,为成熟B细胞肿瘤,以单克隆、成熟的CD5+B淋巴细胞在外周血、骨髓和肝脾进行性积聚为特征。2008年CLL国际工作组(IWCLL)规定的CLL诊断标准:外周血B淋巴细胞≥5×109/L,持续≥3个月;但如伴有骨髓浸润引起的血细胞减少及典型的形态学、免疫表型特征,不管外周血B淋巴细胞数或淋巴结是否受累,也应诊断为CLL。小淋巴细胞淋巴瘤(SLL)与CLL是同一种疾病,为CLL的非白血病表现,二者统称为CLL/SLL。SLL患者表现为淋巴结肿大、无CLL/SLL骨髓浸润所致的血细胞减少及外周血B细胞5×109/L。     

骨髓增生异常综合征患者骨髓血管新生及与临床特征的关系

目的探讨骨髓增生异常综合征(MDS)患者骨髓血管新生及与临床特征的相关性。方法收集2008年5月至2014年5月该院收治的78例MDS患者以及39例急性非淋巴细胞白血病(ANLL)患者,并于同期随机抽取80例健康体检者作为对照组,采用免疫组织化学法分别对三组的骨髓活检标本进行染色并计数微血管数,同时分析其与临床特征的相关性。结果 MDS组、ANLL组以及对照组的骨髓微血管计数经秩和检验,差异有统计学意义(P0.05),MDS组骨髓微血管计数高于对照组而低于ANLL组。MDS低危组骨髓微血管计数低于高危组(P0.05)。MDS患者骨髓微血管计数与性别、年龄、外周血中的白细胞、血小板以及血红蛋白含量无明显相关性(P0.05),与骨髓涂片中的原始细胞百分比有正相关性(r=0.386,P0.05)。结论 MDS患者骨髓中出现不同程度的血管新生,且骨髓原始细胞百分比越高,血管新生数量越多,对临床治疗MDS具有一定的指导意义。     

血液肿瘤患者FLT3/ITD基因突变的检测及临床意义

目的检测血液肿瘤患者FLT3/ITD基因突变,探讨其突变的临床意义。方法2001—2005年对南方医科大学南方医院血液科332例血液肿瘤患者,采用聚合酶链反应(PCR)方法检测FLT3/ITD基因突变。结果FLT3/ITD基因突变阳性率分别为急性髓性白血病(AML)22.3%(23/103)、慢性髓性白血病急变期(CML-BC)6.5%(2/31)、骨髓增生异常综合征(MDS)5.6%(2/36)和急性淋巴细胞白血病(ALL)2.6%(2/76)。而慢性髓性白血病慢性期(CML-CP)、慢性淋巴细胞白血病(CLL)、多发性骨髓瘤(MM)和非霍奇金淋巴瘤(NHL)均未发现FLT3/ITD基因突变。FLT3/ITD基因突变阳性AML患者外周血WBC计数及骨髓白血病细胞比例显著高于FLT3/ITD基因突变阴性AML患者(P<0.05),FLT3/ITD基因突变阳性AML患者完全缓解后18个月内累计复发率(63.6%)显著高于FLT3/ITD基因突变阴性AML组(27.7%)(P<0.05)。结论FLT3/ITD基因突变检测对血液肿瘤预后有一定意义;FLT3/ITD基因突变AML患者预后差。     

转录因子GATA—2基因在ANLL、CML和MDS中的表达

目的：了解GATA－2基因在急性非淋巴细胞白血病（ANLL）、慢性粒细胞白血病（CML）和骨髓增生异常综合征（MDS）中的表达情况。方法：利用RT－PCR扩增ANLL、CML和MDS等246例患者外周血单个核细胞中GATA－2基因。结果：GATA－2基因在ANLL、CML和MDS中的表达率分别为88．4％、81．6％和97％,差异与正常人有高度显著性（P＜0．01）,结论：绝大多数髓系白血病和MDS标本表达GATA－2基因,GATA－2可能是白血病细胞的增殖过程中所需要的转录因子之一。     

CD117和CD34在急性白血病中的临床意义

目的:观察细胞表面分化抗原CD117和CD34在急性白血病中的表达及意义。方法:采用三色流式细胞术,CD45/SSC双参数散点图设门方法,对34例急性白血病患者[急性淋巴细胞白血病(ALL)9例和急性髓细胞白血病(AML)25例]骨髓白血病细胞的免疫分型及CD117和CD34的表达进行检测。结果:CD117在ALL和AML中表达率分别为0和76.0%(19/25),差异有统计学意义(P=0.000)。CD34在ALL和AML中表达率分别为77.8%(7/9)和60.0%(15/25),差异无统计学意义(P=0.815)。在AML-M3亚型中,CD34表达率为16.7%(1/6),显著低于非M3亚型73.7%(14/19)(P=0.007)。结论:CD117检测有助于ALL与AML的鉴别诊断,CD34检测有助于区分AML-M3亚型和非M3亚型。     

白血病患者超敏C反应蛋白水平与临床意义的研究

目的分析超敏C反应蛋白(hs-CRP)在白血病患者中的临床应用价值。方法对85例急性淋巴细胞白血病(ALL)、急性非淋巴细胞白血病(ANLL)、慢性淋巴细胞白血病(CLL)以及慢性粒细胞白血病(CML)患者进行hs-CRP测定,分析不同种类白血病病人的hs-CRP阳性率,并将4组所得结果和健康志愿者相对比。再对35例急性白血病治疗前、28例完全缓解、8例复发者以及22例健康志愿者收集血清hs-CRP值,并进行比较分析。结果 80例(94.12%)患者的hs-CRP测定结果增高。20例ALL患者中升高19例(95.00%),51例ANLL患者升高48例(94.12%),8例CML患者中升高8例(100.00%),6例CLL患者中升高4例(66.67%)。4组患者的检测结果和对照组(2例,9.09%)之间的差异均存在统计学意义(P0.05)。与对照组相比,ALL组、ANLL组、CML组、CLL组的hs-CRP测量值明显提高(分别为75.21±0.18mg/L,80.28±0.32mg/L,68.26±0.41mg/L,64.48±0.22mg/L),差异有统计学意义(P0.05)。AL初诊者及复发患者hs-CRP明显增高,与完全缓解及正常对照组比较的差异存在统计学意义(P0.05)。初诊患者及复发者比较差异无显著性(P0.05);完全缓解与对照组比较的差异没有统计学意义(P0.05)。结论不同分型、不同阶段的白血病检测均会出现hs-CRP阳性结果,可以把hs-CRP测定作为白血病的筛查手段之一。     

Relationship of deoxycytidine kinase and cytoplasmic 5'-nucleotidase to the chemotherapeutic efficacy of 2-chlorodeoxyadenosine

The agent 2-chlorodeoxyadenosine (2-CdA) has chemotherapeutic activity in hairy cell leukemia (HCL) and in refractory chronic lymphocytic leukemia (CLL). The cytotoxic activity of 2-CdA requires the intracellular accumulation of 2-CdA nucleotides. Deoxycytidine kinase (dCK) and cytoplasmic 5'-nucleotidase (5'-NT) are the principal enzymes that phosphorylate 2-CdA and dephosphorylate 2-CdA 5'-monophosphate, respectively. The net accumulation of 2-CdA nucleotides may therefore depend on both dCK and 5'-NT. The purpose of the present experiments was to determine if there is a relationship between pretreatment levels of dCK and 5'-NT in HCL and in CLL cells, and the clinical outcome of 2- CdA treatment. As measured by a direct immunoassay for dCK in 25 CLL patients, and by a 5'-NT activity assay in 23 patients, mean dCK levels were significantly higher in 2-CdA responders than in nonresponders (P < .01), whereas mean 5'-NT levels were significantly lower in 2-CdA responders than in nonresponders (P < .05). Mean dCK levels were higher in six HCL 2-CdA responders than in one nonresponder, whereas mean 5'- NT levels were lower in the 2-CdA responders than in the nonresponder. These results suggest that both dCK and 5'-NT are determinants of 2-CdA responsiveness.     

WT1 gene expression: useful marker for minimal residual disease in childhood myelodysplastic syndromes and juvenile myelo‐monocytic leukemia?

The WT1 gene is considered to be highly expressed in patients with acute myeloid leukemia (AML), acute lymphoblastic leukemia and chronic myeloid leukemia and is thought to play a key role in maintaining the viability of leukemia cells. However, little is known about the WT1 gene expression levels in pediatric patients with juvenile myelo-monocytic leukemia (JMML) and myelodysplastic syndromes (MDS). We studied WT1 expression in diagnostic bone marrow (BM) and peripheral blood (PB) samples of 90 patients with JMML, low grade MDS, advanced MDS and myelodysplasia-related AML in BM (n = 20) and PB (n = 18) samples of normal healthy volunteer donors.     

骨髓增生异常综合征患者姐妹染色单体分化的研究

目的 验证骨髓细胞姐妹染色体单体分化（SCD）检测法在骨髓增生异常综合征（MDS）诊断和预后判断中的意义。方法 对临床上疑为MDS的50例患者分别进行5′溴脱氧尿嘧啶核苷－姐妹染色单体分化染色法（BrdU-SCD)检测和染色体核型分析。结果 50例中32例（64.0%）SCD延迟,对照组中13例急性髓系白血病（AML）患者全部延迟,非恶性血液病中仅2例（10.5%）延迟,而例正常人中无一例延迟。MDS与AML、正常人、再生障碍性贫血（AA）、其他良性增生性贫血之间的SCD延迟率及平均细胞周期时间的差异具有显著性;而正常人与AA、其他良性增生性贫血之间的差异无显著性。随着MDS病情进展,细胞周期时间逐渐延长。50例中24例（48.0%）检出克隆性核型改变。按FAB分型不能确诊为MDS7例中6例有克隆性异常。SCD正常组和延迟组的病死率分别为11.8%和50.0%（P＜0.05）,白血病转化率分别为5.9%和16.7%（P＞0.05）,中位生存时间分别为12和8个月（P＝0.051）。结论 骨髓细胞SCD检测对MDS具有一定的鉴别诊断和预后判断意义。     

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。     

低增生骨髓增生异常综合征的由来和转归

目的探讨低增生骨髓增生异常综合征（ＭＤＳ）的由来和发展。方法对我院１０年中确诊的２５例低增生ＭＤＳ进行了系统分析,对其中１７例患者进行长期追访。结果（１）低增生ＭＤＳ占同期确诊为ＭＤＳ的２１９例患者中１１．４％,确诊时平均年龄为（４４．８±１４．７）岁。（２）ＦＡＢ分型：难治性贫血（ＲＡ）１１例,难治性贫血伴原始细胞增多（ＲＡＥＢ）１４例。（３）低增生ＭＤＳ很可能是ＭＤＳ患者病程中一个阶段,其骨髓增生活跃和低下可以相互转化,这种转化不但可以发生在同一ＦＡＢ亚型内,也可以发生在不同亚型相互转化时。（４）长期随访的１７例患者中有７例转为急性白血病,占４１．２％,６例为急性粒细胞白血病,１例为急性淋巴细胞白血病;７例中３例转为低增生白血病,４例为增生活跃或极度活跃白血病。（５）１７例患者中７例自低增生ＲＡＥＢ转为急性白血病时间为１～７４个月,中数为２７个月。（６）低增生ＭＤＳ的产生与治疗药物无明显相关。结论低增生ＭＤＳ很可能为ＭＤＳ病程中一个阶段而非一种特殊类型。     

Dysregulation of IL-32 in myelodysplastic syndrome and chronic myelomonocytic leukemia modulates apoptosis and impairs NK function

TNFalpha levels are elevated in the marrows of patients with myelodysplastic syndrome (MDS) and are associated with high rates of apoptosis, which contributes to hematopoietic failure. We observed that exposure of human marrow stroma cell lines HS5 and HS27a to TNFalpha increases levels of IL-32 mRNA. IL-32, in turn, induces TNFalpha. Marrow stroma from patients with MDS expressed 14- to 17-fold higher levels of IL-32 mRNA than healthy controls. In contrast, cells from patients with chronic myelomonocytic leukemia (CMML) expressed only one tenth the level of IL-32 measured in healthy controls. Human KG1a leukemia cells underwent apoptosis when cocultured with HS5 stromal cells, but knockdown of IL-32 in the stromal cells by using siRNA abrogated apoptosis in the leukemia cells. IL-32 knockdown cells also showed dysregulation of VEGF and other cytokines. Furthermore, CD56(+) natural killer cells from patients with MDS and CMML expressed IL-32 at lower levels than controls and exhibited reduced cytotoxic activity, which was unaffected by IL-2 treatment. We propose that IL-32 is a marrow stromal marker that distinguishes patients with MDS and CMML. Furthermore, IL-32 appears to contribute to the pathophysiology of MDS and may be a therapeutic target.     

Coculture of native human acute myelogenous leukemia blasts with fibroblasts and osteoblasts results in an increase of vascular endothelial growth factor levels

Abstract:  Objectives : Angiogenesis seems important in the development of acute myelogenous leukemia (AML). Proangiogenic vascular endothelial growth factor (VEGF) is constitutively secreted by the AML blasts for a subset of patients, but it can also be released by non-leukemic bone marrow cells. Methods : VEGF levels were determined after coculture of native human AML blasts with fibroblast lines, osteoblastic sarcoma cell lines, normal bone marrow stromal cells and normal osteoblasts. Cultures were prepared with leukemic and non-leukemic cells separated by a semipermeable membrane or in direct contact. Results : The non-leukemic cells usually showed higher spontaneous VEGF release than AML cell populations. Coculture of AML blasts with HFL1 fibroblasts caused a supra-additive increase of VEGF levels when the cell populations were cultured separately, and the increase was also observed when cells were cultured in direct contact. An increase was also observed when AML blasts were cultured with osteoblastic sarcoma cells, normal bone marrow stromal cells and normal osteoblasts. Coculture had divergent effects on VEGF mRNA levels both for leukemic and non-leukemic cells, but increased mRNA levels were commonly observed especially for the non-leukemic cells. Cytokine inhibition experiments suggest that IL1 is important for the VEGF-increasing crosstalk, whereas the mechanisms are probably heterogeneous for coculture with osteoblasts. Conclusion : The bi-directional crosstalk via local cytokine networks between AML blasts and non-leukemic cells results in increased local VEGF levels, an observation suggesting that VEGF-targeting antiangiogenic therapy should be considered as a general therapeutic strategy in AML.     

Clinical features of atypical refractory anemia (RA)

Twenty-three patients with bicytopenia or pancytopenia were retrospectively studied. The patients with underlying disorders, blast count of more than 5% on bone marrow (BM) aspirate, blast count of more than 1% on peripheral blood or ringed sideroblast count of more than 15% on BM aspirate were excluded. According to Yoshida's criteria, 23 patients were classified into 6 subtypes [AA (aplastic anemia)1: typical AA, AA2: atypical AA, MDS (myelodysplastic syndrome)3: typical RA (refractory anemia, MDS4-6: atypical RA], and AA1 7 cases; AA2 2 cases; MDS3 5 cases; MDS4 1 case; MDS5 2 cases; MDS6 6 cases. To clarify the clinical features of atypical RA group (MDS4-6), we investigated ferrokinetics, RBC life span, karyotype, serum Epo (erythropoietin) concentration, response to therapy and prognosis. Results were as follows: 1) all three RA patients who were younger than 30 years old were included in atypical RA group, 2) in ferrokinetics study PID (plasma iron disappearance time) values of MDS4 and MDS6 patients ranged between those of AA1 and those of MDS3 patients (5 of 7 patients), 3) two cases who developed leukemia belonged to typical RA group, 4) patients with atypical RA showed response to therapy and their prognosis were better than those with typical RA. These observations suggest that atypical RA have different clinical features from typical RA.     

Serum granulocyte colony-stimulating factor levels in healthy volunteers and patients with various disorders as estimated by enzyme immunoassay

In order to better understand the patho-physiologic role of granulocyte colony-stimulating factor (G-CSF), we estimated its serum levels in healthy persons and patients with various disorders, using a newly developed enzyme immunoassay (Motojima et al). In 49 of 56 normal healthy persons (88%), the levels were beneath the sensitivity of the assay (less than 30 pg/mL), while in the remaining seven healthy persons, the levels ranged from 33 to 163 pg/mL. On the other hand, nine of 11 patients (82%) with idiopathic aplastic anemia (AA), one patient with Fanconi's anemia, six of 12 patients (50%) with myelodysplastic syndrome (MDS), five of 12 patients (42%) with acute leukemia without any blast cells in the blood (M4: one, M5: one, L1: one, and L2: two), six of 18 patients (33%) with chronic myeloid leukemia (CML), one of two patients with chronic lymphoid leukemia (CLL), two of four patients with lung cancer, one patient with cyclic neutropenia, two of seven patients with malignant lymphoma, and four patients with acute infection had G-CSF levels ranging from 46 pg/mL to greater than 2,000 pg/mL. Interestingly, a reverse correlation between blood neutrophil count and serum G-CSF level was clearly demonstrated for aplastic anemia (r = -.8169, P less than .01). Moreover, it was found that the G-CSF level rose during the neutropenic phase of cyclic neutropenia and after chemotherapy or bone marrow transplantation (BMT) in three patients with leukemia; also high G-CSF levels were positively correlated to blood neutrophil counts in some cases of infectious disorders and lung cancer. The cellular sources and the mechanisms for production and secretion of circulating G-CSF were not investigated in this study, but the data presented here strongly indicate that G-CSF plays an important role as a circulating neutrophilopoietin.     

Humoral immune responses against Wilms tumor gene WT1 product in patients with hematopoietic malignancies

Wilms tumor gene WT1 is expressed at high levels in hematopoietic malignancies, such as leukemias and myelodysplastic syndromes (MDS), and in various kinds of solid tumors, including lung cancer, and it exerts an oncogenic function in these malignancies. IgM and IgG WT1 antibodies were measured by means of dot blot assay in 73 patients with hematopoietic malignancies (16 acute myeloid leukemia [AML], 11 acute lymphoid leukemia [ALL], 13 chronic myeloid leukemia [CML], and 33 MDS) and 43 healthy volunteers. Immunoglobulin IgM, IgG, and IgM+IgG WT1 antibodies were detected in 40 (54.8%), 40 (54.8%), and 24 (32.8%), respectively, of the 73 patients with hematopoietic malignancies, whereas 7 (16.2%), 2 (4.7%), and none of the 43 healthy volunteers had IgM, IgG, or IgM+IgG WT1 antibodies, respectively. Furthermore, immunoglobulin isotype class switching of WT1 antibodies from IgM to IgG occurred in conjunction with disease progression from refractory anemia (RA) to RA with excess of blasts (RAEB), and further to RAEB in transformation (RAEB-t) in MDS patients. These results showed that humoral immune responses against the WT1 protein could be elicited in patients with WT1-expressing hematopoietic malignancies, and they suggested that the helper T-cell responses needed to induce humoral immune responses and immunoglobulin isotype class switching from IgM to IgG were also generated in these patients. Our findings may provide new insight into the rationale for elicitation of cytotoxic T-cell responses against the WT1 protein in cancer immunotherapy using the WT1 vaccine.