前体B细胞淋巴母细胞淋巴瘤/白血病美罗华联合化疗治疗后复发伴CD20抗原表达丢失1例报道并文献复习

目的探讨B细胞恶性肿瘤美罗华(rituximab)治疗后复发伴CD20抗原表达丢失患者的生物学特性.方法报道1例前体B细胞淋巴母细胞淋巴瘤/白血病(precursor-B-LBL/ALL)患者美罗华联合化疗治疗后复发时CD20抗原表达丧失,并进行文献复习.结果 1例39岁男性前体B细胞淋巴母细胞淋巴瘤/白血病(pre-B-LBL/ALL)患者,初治时流式细胞(FCAS)检测瘤细胞表达CD19CD20,CD22和CD25,弱表达CD34,而CD10表达阴性;免疫组化染色CD20广泛阳性;核型为92,XXYY,der(15)t(1;15)(q11;q26)×2[15]/46,XY[5].经美罗华联合mBACOD诱导治疗2疗程后获得完全缓解(CR).巩固化疗4疗程后,予美罗华、大剂量环磷酰胺(CTX)联合重组人源化粒细胞集落刺激因子(rh-G-CSF)动员、采集及冷冻保存自体外周血干细胞(PBSCs).最后一疗程化疗4个月后患者复发,复发时流式细胞(FCAS)检测表达CD19CD10,CD22,CD38和CD13,高表达CD34,而CD20,CD23和FMC7均阴性,免疫组化染色偶见CD20阳性细胞;核型转变为46,XY.结论 B细胞恶性肿瘤美罗华治疗后复发患者应重新进行病理组织学、免疫表型和细胞/分子遗传学检测.     

成人急性白血病中CD117与CD34共表达的临床意义

背景与目的:c-kit受体(c-kitreceptor,c-kitR,CD117)是干细胞因子受体。CD117在急性非淋巴细胞白血病(acutenon-lymphoblasticleukemia,ANLL)中高表达,可作为髓系免疫学标记物,对诊断ANLL有一定参考价值。但是,CD117也可在部分急性淋巴细胞白血病(acutelymphoblasticleukemia,ALL)中表达。CD34为造血干(祖)细胞抗原标记物,在ANLL和ALL中均有高表达。本研究旨在探讨CD117和CD34在急性白血病中共表达的临床意义。方法:采用流式细胞术(flowcytometery,FCM)分别检测92例ALL和81例ANLL初诊患者骨髓单个核细胞(BMMNC)CD117的阳性率和阳性细胞水平;比较ALL和ANLL患者CD117/CD34共表达率的差异,并比较ALL患者中CD117和CD117/CD34共表达率的差异。设立20例健康成人为对照组。结果:在ALL和ANLL患者中CD117阳性率分别为15.2%和71.6%,CD117/CD34共表达率分别为5.4%和55.5%,差异有显著性(P<0.001)。ALL患者中CD117表达率和CD117/CD34共表达率分别为15.2%和5.4%,差异有显著性(P=0.029)。结论:CD117可作为急性白血病的MIC分型诊断之髓系免疫学标志,用以协助ANLL的临床诊断;较之CD117表达,CD117/CD34在ALL中的共表达率更低,可籍此协助排除ALL。     

rhG-CSF在急性淋巴细胞白血病患儿CODP方案化疗中的辅助作用

目的探讨基因重组人粒细胞集落刺激因子(rhG-CSF)在急性淋巴细胞白血病患儿CODP方案化疗中的辅助作用。方法选取急性淋巴细胞白血病化疗患儿80例,根据不同治疗方法将患者分为观察组和对照组,各40例。对照组给予CODP(柔红霉素、长春新碱、环磷酰胺、泼尼松)方案化疗,观察组在对照组基础上给予rhG-CSF辅助化疗,比较2组患儿化疗效果。结果观察组完全缓解率为100.0%,对照组完全缓解率92.5%、部分缓解率7.5%,2组患儿化疗总有效率比较差异无统计学意义(P>0.05)。观察组骨髓抑制(白细胞计数、中性粒细胞和血小板计数下降)和口腔感染发生率明显低于对照组,中性粒细胞恢复时间、白细胞恢复时间和发热持续时间亦明显少于对照组,2组比较差异具有统计学意义(P<0.05)。结论 rhG-CSF辅助急性淋巴细胞白血病CODP方案化疗对改善化疗引起的骨髓抑制、降低感染具有重要的临床意义。     

急性淋巴细胞白血病免疫分型的特点及其临床意义

目的为了探讨急性淋巴细胞白血病(ALL)各亚型免疫分型的特点及其临床意义。方法采用CD45/SSC双参数散点图设门,应用三色流式细胞术,对81例ALL的初诊患者骨髓标本进行免疫分型,并对其中45例进行核型分析。结果(1)B-ALL中CD19表达最常见(阳性率为100%),而T-ALL中CD5和CD7表达阳性率最高,均为90%;B-ALL和T-ALL都存在抗原交叉表达的现象;两组患者的完全缓解率(CR率)并无显著差异(P>0.05)。(2)伴髓系抗原表达的急性淋巴细胞白血病(My ALL)比较常见,本组达到39.5%,常累及B淋巴系统(占My ALL的84.4%);各髓系抗原中以CD13表达阳性率最高;此类患者的CR率较高,儿童CR率为72.2%,成人为78.6%。(3)急性杂合性白血病(HAL)的发病率为19.8%,以髓系、B系共同表达者居多;并且CD34表达阳性率较高(81.3%),该类患者CR率较低(儿童和成人分别为50%和40%)。(4)CD34在B-ALL,My ALL和HAL中表达阳性率较高,而T-ALL中少见(P<0.025)。结论免疫分型在诊断特殊类型的ALL(如HAL,My ALL)中具有显著优势;CD19和CD5诊断B-ALL和T-ALL的灵敏度较好,但特异性不高,存在抗原交叉表达;CD34和髓系抗原的表达与CR率无相关性,但在HAL,CD34的表达与CR率成负相关。     

CD4^＋CD25高表达调节性T细胞与儿童急性淋巴细胞白血病的关系

目的检测急性淋巴细胞白血病（ALL）患儿的CD4^＋CD25调节性T细胞（Treg），探讨其在儿童ALL发生、治疗过程中的意义。方法以40例ALL儿童不同治疗阶段的55份外周血为标本，细胞膜表面抗原采用双色或三色单克隆抗体直接标记法，检测细胞核抗原FoxP3时先标记膜表面抗原，固定破膜后再标记胞核抗原，应用多参数流式细胞仪进行检测。结果ALL患儿的CD4^＋CD25Treg同时表达CD62L和胞核抗原FoxP3。ALL标危组治疗不同阶段儿童的CD4^＋CD25高表达T细胞数值分别为：首次诱导缓解组（1．04±0．33）％，维持治疗组（1．60±0．44）％，持续完全缓解组（1．29±0．30）％；ALL中／高危维持治疗组则为（2．24±0．75）％。结论ALL患儿的CD4^＋CD25Treg数值高于健康儿童，并且与疾病的危险度和治疗的效应有一定的关系；CD4^＋CD25Treg水平升高可能是白血病复发的原因之一。     

急性髓系白血病体外细胞耐药与P170表达及染色体核型关系的研究

目的:探讨急性髓系白血病(AML)患者体外细胞耐药与p170表达及染色体核型之间的关系。方法:用MTT分析方法检测阿糖胞苷(Ara-C)、柔红霉素(DNR)、高三尖杉酯碱(HHT)、安吖啶(AMSA)和米托蒽醌(MTZ)体外细胞耐药。间接免疫荧光染色检测p170蛋白表达水平。R显带分析染色体核型。结果:初治患者(138例)、难治患者(8例)、复发患者(14例)、慢性粒细胞白血病AML变(8例)和骨髓增生异常综合征AML变(8例),各组之间的p170蛋白表达水平和细胞耐药率无显著性差异(P >0.05)。p170 表达率≥5 %的患者Ara-C,DNR和MTZ耐药率明显高于p170 表达率<5 %的患者,但HHT和AMSA的耐药检出率二者无显著性差异。p170表达水平与染色体核型异常无明显相关性。t(8;21)/inv(16)患者未发现对Ara-C有耐药。与正常核型组相比,5/7号染色体异常组的HHT和MTZ耐药发生率、+8组的HHT和DNR的耐药发生率、其他染色体异常组的Ara-C,DNR和HHT的耐药发生率明显增高(P <0.05)。结论:AML体外细胞耐药和p170蛋白表达水平与病期无关,p170 表达率和特异染色体核型异常与体外细胞耐药有一定的相关性。     

以吡柔比星为主的联合化疗对初治急性白血病疗效的临床观察

目的:观察吡柔比星(THP)组成的联合化疗对初治急性白血病的缓解率及毒副作用。方法:治疗组:以THP替代标准方案中DA或VDCP中的柔红霉素(DNR),即用TA与VTCP方案(THP20 mg/m2 ~ 30 mg/m2)对初治的20例急性非淋巴细胞白血病(ANLL)及16例急性淋巴细胞白血病(ALL)诱导缓解治疗。对照组:用DA与VDCP分别对初治25例ANLL与18例ALL患者诱导缓解治疗。分别观察其一个疗程的疗效情况及毒副作用情况。结果:两组在ANLL的总有效率(RR)分别是70 %和76 %,在ALL的RR率分别是75 %与77.8 %,其中CR率ANLL分别为60 %与64 %,ALL两组分别为62.5 %与61.1 %,两组间差异均无显著性(P>0.05),毒副作用THP组主要表现轻度的恶心、呕吐,少数有轻度脱发,仅1例发现心脏出现较度毒性,骨髓毒性较重,DNR组有3例发生了轻度的心脏毒性,其余与THP相似。结论:用THP替代标准方案中的DNR对初治的急性白血病疗效显著,无明显严重的毒副作用,与标准的DA与VDCP方案相比疗效无显著差异,毒副作用基本相当。     

急性淋巴细胞白血病免疫分型的特点及其临床意义(英文)

目的为了探讨急性淋巴细胞白血病(ALL)各亚型免疫分型的特点及其临床意义。方法采用 CD45/SSC 双参数散点图设门,应用三色流式细胞术,对81例 ALL 的初诊患者骨髓标本进行免疫分型,并对其中45例进行核型分析。结果 (1)B-ALL 中 CD19表达最常见(阳性率为100%),而 T-ALL 中 CD5和 CD7表达阳性率最高,均为90%;B-ALL 和 T-ALL 都存在抗原交叉表达的现象;两组患者的完全缓解率(CR 率)并无显著差异(P>0.05)。(2)伴髓系抗原表达的急性淋巴细胞白血病(My~+ ALL)比较常见,本组达到39.5%,常累及 B 淋巴系统(占 My~+ ALL 的84.4%);各髓系抗原中以 CD13表达阳性率最高;此类患者的 CR 率较高,儿童 CR 率为72.2%,成人为78.6%。(3)急性杂合性白血病(HAL)的发病率为19.8%,以髓系、B 系共同表达者居多;并且 CD34表达阳性率较高(81.3%),该类患者 CR 率较低(儿童和成人分别为50%和40%)。(4)CD34在 B-ALL,My~+ALL 和 HAL 中表达阳性率较高,而 T-ALL 中少见(P<0.025)。结论免疫分型在诊断特殊类型的 ALL(如 HAL,My~+ ALL)中具有显著优势;CD19和 CD5诊断 B-ALL 和 T-ALL 的灵敏度较好,但特异性不高,存在抗原交叉表达;CD34和髓系抗原的表达与 CR 率无相关性,但在 HAL,CD34的表达与 CR 率成负相关。     

吡柔比星联合化疗治疗急性白血病32例

目的观察吡柔比星（THP）联合化疗方案对急性白血病（AL）的疗效。方法治疗组AL患者32例,其中急性非淋巴细胞白血病（ANLL）患者20例,采用THP联合阿糖胞苷（Ara—C）（TA方案）。急性淋巴细胞白血病（ALL）患者12例,采用THP联合长春新碱（VCR）、泼尼松（Pred）（VTP方案）。对照组36例,其中ANLL患者24例,ALL患者12例,用柔红霉素（DNR）替代THP。结果治疗组有效率87．5％,对照组有效率87．3％（P〉0．05）。治疗组完全缓解率62．5％,对照组完全缓解率47.2％,两组差异有统计学意义（P〈0．05）。治疗组骨髓抑制较对照组明显,感染发生率高（P〈0．05）。结论吡柔比星联合化疗方案可作为治疗急性白血病一线治疗方案,其完全缓解率高,但骨髓抑制明显。     

白血病骨髓基质细胞对白血病细胞屏蔽效应的体外实验研究

背景与目的:肿瘤微环境是影响肿瘤细胞转归的关键因素之一。骨髓微环境能保护白血病细胞,促进白血病细胞耐药、抗凋亡存活,但机制未完全阐明。本研究中我们主要观察骨髓基质细胞促白血病细胞抵抗柔红霉素(daunorubicin,DNR)杀伤的屏蔽效应,旨在探讨骨髓基质细胞增强Jurkat细胞抗凋亡、抗药特性的可能机制。方法:应用Percoll分离正常及白血病性骨髓单个核细胞,体外培养骨髓基质细胞模拟骨髓微环境功能,与白血病细胞Jurkat体外共培养。AnnexinV/PI双标法流式细胞仪检测0.5μmol/LDNR处理后Jurkat细胞凋亡率的变化。PI染色流式细胞仪检测细胞周期分布。结果:共培养后正常骨髓基质细胞抑制DNR诱导的Jurkat细胞凋亡,与单独悬浮培养组比较显著降低[(8.39±4.08)%和(16.02±1.00)%,P<0.05]。白血病骨髓基质细胞对Jurkat细胞的屏蔽效应强于正常骨髓基质细胞[Jurkat细胞凋亡率分别是(5.73±1.78)%和(8.39±4.08)%,P<0.05]。DNR处理正常或白血病骨髓基质细胞共培养组G0/G1期Jurkat细胞比例高于悬浮培养DNR处理组,而正常与白血病骨髓基质细胞屏蔽的Jurkat细胞G0/G1期阻滞现象无显著性差异[(47.96±5.88)%和(39.25±3.04)%,P>0.05]。结论:骨髓基质细胞可能部分通过阻滞白血病细胞于G0/G1期,抑制DNR诱导的白血病细胞     

In vivo uptake of daunorubicin by acute myeloid leukemia (AML) cells measured by flow cytometry

Monitoring of daunorubicin (DNR) concentrations in leukemic cells in blood and bone marrow in vivo of patients with acute myeloid leukemia may yield insight into the interindividual variations of the clinical response to treatment. We evaluated the applicability of flow cytometry for measuring DNR uptake in direct comparison with high performance liquid chromatography (HPLC). In vitro studies revealed good correlations between the mean cellular fluorescence measured by flow cytometry and the cellular DNR concentrations determined with HPLC. In vivo cell measurements were then obtained in 17 evaluable patients during their first remission induction treatment with DNR and cytosine arabinoside. The results indicate that: (a) DNR fluorescence of leukemic blast cells is intermediate between the smaller lymphocytes and the approximately equally large granulocytes; (b) DNR fluorescence of peripheral blast cells and bone marrow blast cells correlate well (p less than 0.001); and (c) patients reaching complete remission show a tendency of higher DNR fluorescence of leukemic blast cells than do partial responders.     

Flow cytometric characterization of acute myeloid leukemia: IV. Comparison to the differentiation pathway of normal hematopoietic progenitor cells.

Gradual increase of CD38 on cells expressing CD34 characterizes the early cell differentiation pathway of normal human hematopoietic progenitors. In this study the coordinated expression pattern of CD34 and CD38 was assessed on leukemic blasts from bone marrow aspirates of 95 patients with newly diagnosed acute myeloid leukemia (AML). Expression was divided into six categories analogous to the differentiation pathway of normal bone marrow. The CD38 antigen was expressed on the leukemic cells of all patients and CD34+ leukemic cells were found in 79 patients (83%). In 93 patients, the leukemic cells were found along the differentiation pathway defined by CD34 and CD38. In 33 of the 93 patients, a part of the CD34+ cells did not express the CD38 antigen (categories 1 and 2). In another 33 patients, all CD34+ cells expressed CD38 (categories 3 and 4). In the remaining 27 patients, only cells were found which dimly expressed CD34 or did not express CD34 (categories 5 and 6). Of the 93 patients, 88 were treated with intensive chemotherapy according to the protocol of the German AML Cooperative Group. Of these, 21 died early and were not evaluable for treatment response. Complete remission was achieved in 14 of 22 patients (64%) in categories 1 and 2, in 19 of 26 patients (73%) in categories 3 and 4, and in 18 of 19 patients (95%) in categories 5 and 6. The event-free survival was significantly longer in patients of categories 5 and 6 compared to patients in categories 1 and 2 (p less than 0.01) and categories 3 and 4 (p less than 0.05), respectively. We conclude that in the majority of AML patients the immunophenotype of leukemic cells follows the early cell differentiation pathways defined by coordinated expression of CD34 and CD38 similar to that of normal hematopoietic progenitors. The presence of cells in the late cell differentiation stages (CD34+/-, CD38 /+) identifies patients with a higher complete remission rate and longer complete remission duration.     

Reciprocal Interactions of Leukemic Cells with Bone Marrow Stromal Cells Promote Enrichment of Leukemic Stem Cell Compartments in Response to Curcumin and Daunorubicin

A predominant challenge in developing curative leukemia therapy is interactions of leukemic cells with the bone marrow stromal microenvironment. We aimed to investigate the role of stromal cells, such as bone marrow mesenchymal stromal cells (BMSCs) and osteoblasts (OBs), in curcumin (CUR) and daunorubicin (DNR) induced apoptosis of acute myeloid leukemia (AML) cells. We used KG1 and U937 as leukemia cell line models and treated them with CUR and DNR. The cells were then co-cultured with BMSCs or a combination of BMSCs and OBs as feeders. After 24 hours of co-culture, BMSCs or OBs were sorted and separated from the leukemia cells and apoptosis levels were analyzed by annexin/propidium iodide (PI) staining on flow cytometry. Potentially involved molecular pathways were analyzed at gene and protein levels by Real time PCR and western blotting, respectively. The results showed AML cells co-cultured with BMSCs plus OBs to be more resistant to drug induced-apoptosis compared to co-culture with BMSCs alone or without co-culture. Expression levels of OPN, CXCL-12, IL-6, STAT-3 and VCAM-1 were also significantly up-regulated in OBs and AML cells, at both mRNA and protein levels after co-culture, with concurrent enrichment of CD34+ AML cells. Our data showed, in a stromal cell niche-based model, that OBs revoke the influence of BMSCs on leukemic cells and promote enrichment of both CD34+ and CD34- leukemic stem cell (LSC) compartments in response to CUR and DNR. Up-regulation of OPN, CXCL-12, IL-6, STAT-3 and VCAM-1 in OBs and AML cells in co-culture might be part of molecular mechanisms that block CUR or CUR+DNR-induced apoptosis and promote enrichment of CD34+ and CD34- LSCs.     

Breast cancer resistance protein in drug resistance of primitive CD34+38- cells in acute myeloid leukemia.

PURPOSE: Acute myeloid leukemia (AML) is considered a stem cell disease. Incomplete chemotherapeutic eradication of leukemic CD34+38- stem cells is likely to result in disease relapse. The purpose of this study was to investigate the role of the breast cancer resistance protein (BCRP/ATP-binding cassette, subfamily G, member 2) in drug resistance of leukemic stem cells and the effect of its modulation on stem cell eradication in AML. EXPERIMENTAL DESIGN: BCRP expression (measured flow-cytometrically using the BXP21 monoclonal antibody) and the effect of its modulation (using the novel fumitremorgin C analogue KO143) on intracellular mitoxantrone accumulation and in vitro chemosensitivity were assessed in leukemic CD34+38- cells. RESULTS: BCRP was preferentially expressed in leukemic CD34+38- cells and blockage of BCRP-mediated drug extrusion by the novel fumitremorgin C analogue KO143 resulted in increased intracellular mitoxantrone accumulation in these cells in the majority of patients. This increase, however, was much lower than in the mitoxantrone-resistant breast cancer cell line MCF7-MR and significant drug extrusion occurred in the presence of BCRP blockage due to the presence of additional drug transport mechanisms, among which ABCB1 and multiple drug resistance protein. In line with these findings, selective blockage of BCRP by KO143 did not enhance in vitro chemosensitivity of leukemic CD34+38- cells. CONCLUSIONS: These results show that drug extrusion from leukemic stem cells is mediated by the promiscuous action of BCRP and additional transporters. Broad-spectrum inhibition, rather than modulation of single mechanisms, is therefore likely to be required to circumvent drug resistance and eradicate leukemic stem cells in AML.     

Purified autologous grafting in childhood acute lymphoblastic leukemia in second remission: evidence for long-term clinical and molecular remissions.

Autologous transplantation is a treatment option for relapsed childhood acute lymphoblastic leukemia (ALL) in second complete remission (CR2) when a suitable donor is not available. In an attempt to prevent relapses originating from graft leukemic contamination, the experimental protocol of in vitro purification of leukapheretic products with monoclonal antibodies (MoAbs), previously reported for adults, was adopted in 11 of 12 consecutive patients (median age, 9 years) with B cell precursor ALL in CR2 after late relapse (median, 37; range, 31-51 months after the onset) enrolled between July 1997 and July 1999 at a single pediatric center. At a median of 12 days after the mobilizing chemotherapy followed by G-CSF, a median of 13.9 (range, 5.9-18.7) x 10(6) CD34+ cells/kg were collected from each patient and a median of 7.5 (range, 4.1-12.6) x 10(6) CD34+ cells/kg underwent the purification procedure. The first step of immunorosetting allowed a one-log reduction of the total cell count, by eliminating more than 90% of the CD11b+ cells; the second step, performed after incubation with anti-CD19 MoAbs, allowed the depletion of 99% (range, 93-100) of the CD19+ cells, kept within the magnetic field of the immunodepletion column, with a median recovery of 73% (range, 55-87) of the collected CD34+ cells. Molecular analysis assessed the in vitro eradication of detectable leukemic cells. A median reinfusion of 5.2 (range, 3.2-9.1) x 10(6) CD34+ cells/kg for each patient (median viability, 90%), after conditioning with the 'TBI-VP16-CY' regimen, allowed prompt engraftment and immunological reconstitution; no patients experienced severe transplant-related toxicity or major infections. One patient relapsed 7 months after transplantation, while 10 patients are alive in clinical and molecular remission, at a median follow-up of 29 months (range, 15-40) (2-year EFS, 89%, s.e. 9). In conclusion, the procedure proved to be reproducible for pediatric purified autografting, highly efficient concerning stem cell recovery and depletion of leukemia-lineage specific cells, and promising in terms of final outcome.     

Acute promyelocytic leukemia. Therapy results and prognostic factors

From December 1976 to July 1986, 34 patients with acute promyelocytic leukemia (APL) were treated with daunorubicin (DNR) alone and simultaneous supportive therapy with low-dose heparin, platelet transfusions, and fresh frozen plasma. Two consecutive maintenance therapy regimens were employed in patients who achieved complete remission (CR): (1) a classical maintenance with methotrexate and 6-mercaptopurine, with DNR plus methyl-GAG re-inductions; (2) from 1982 an intensive sequential combination therapy regimen was administered. CR was achieved in 23 patients (68%). Only one patient had leukemic resistance. Other failures were a consequence of post-chemotherapy complications. A multivariate logistic regression analysis has been performed to evaluate the prognostic importance on response to remission induction of 25 patient and disease characteristics at diagnosis. The significant variables in decreasing order of significance were: serum albumin level, fever at diagnosis, serum creatinine level, and age. The median duration of remission and survival by Kaplan-Meier analysis were projected to be 24 and 25 months, respectively. Relapses occurred in 11 of 23 CR patients. Nine patients remained in the first remission from 5+ to 37+ months. Short-term (CR) and long-term results (duration of remission and survival) in APL treated for induction with DNR alone were similar to those obtained in other subtypes of acute myeloblastic leukemia by intensive combination chemotherapy.     

Human acute myeloid leukemia CD34+/CD38- progenitor cells have decreased sensitivity to chemotherapy and Fas-induced apoptosis, reduced immunogenicity, and impaired dendritic cell transformation capacities

The destruction of cells capable of initiating and maintaining leukemia challenges the treatment of human acute myeloid leukemia. Recently, CD34+/CD38- leukemia progenitors have been defined as new leukemia-initiating cells less mature than colony-forming cells. Here we show that CD34+/CD38- leukemia precursors have reduced in vitro sensitivity to daunorubicin, a major drug used in leukemia treatment, in comparison with the CD34+/CD38+ counterpart, and increased expression of multidrug resistance genes (mrp/lrp). These precursors show lower expression of Fas/Fas-L and Fas-induced apoptosis than CD34+/CD38+ blasts. Moreover, the CD34+/CD38- leukemic subpopulation induces a weaker mixed leukocyte reaction of responding T-lymphocytes than the CD34+/CD38+ leukemic counterpart, either in a MHC-unmatched or MHC-matched settings. This weaker immunogenicity could be linked to lower expression on CD34+/CD38- leukemia precursors of major immune response molecules (MHC-DR, LFA-3, B7-1, or B7-2) than CD34+/CD38+ leukemic cells. Nonetheless, the susceptibility of the immature CD38- precursors to cytotoxicity was not different from the sensitivity of the CD38+ counterpart. Finally, CD34+/CD38- leukemia precursors, in contrast with CD38+ precursors, failed, under appropriate conditions, to differentiate into dendritic cells, a central step for antigen recognition. This is to our knowledge the first demonstration that the very immature phenotype of CD34+/CD38- leukemic progenitors confers both chemotherapy resistance and decreased capacities to induce an immune response. Because the susceptibility of the immature leukemia cells as cytotoxic targets is maintained, our data underline the importance of improving the initial steps of leukemia recognition, more particularly by defining optimal conditions of dendritic cell transformation of the very immature hematopoietic precursors.     

Detection of minimal residual disease in acute myeloid leukemia.

The distinction of clonogenic leukemic cells (CFU-L) and normal myeloid progenitors (GM-CFU) is a problem because both types of cells respond to the same growth factors and their clones resemble each other morphologically in culture. We investigated by means of an indirect enzyme-immunoassay the expression of "early" and "late" differentiation markers on bone marrow cell suspensions, as well as on agar clones in 18 cases of newly diagnosed acute myeloid leukemia (AML) as compared with 13 normal controls. Uncultured AML cells carried only low amounts of "late" myeloid differentiation antigen (CD15) but expressed nearly normal levels when cultured in agar with colony-stimulating factor (CSF). In contrast to normal bone marrow, AML cells were strongly reactive with "early" differentiation markers (CD10, CD20, CD34) and remained so during culture. Normal and leukemic agar clones could be specifically distinguished by CD20- and CD34 antibodies. By means of a double marker technique, it could be shown that "late" myeloid differentiation markers (CD15) and "early" markers (CD10, CD20, CD34) were coexpressed on the same cells only in AML but not in normal bone marrow. Leukemic clones were identified by phenotyping of agar clones in 17 of 19 cases investigated during complete clinical remission (CR) of the disease. A formal proof of the leukemic origin of CD20/CD34 positive clones grown in CR was made possible in four cases either by Southern blot analysis or by a cytogenetic marker. These results demonstrate that AML cells can partially differentiate in vitro in the presence of CSF. A distinction of AML from normal clones, however, is possible by their reactivity with "early" differentiation markers, because this is maintained under the differentiating influence of CSF. The technique described here identifies residual leukemic clones in the majority of AML in CR, which persist at a constant rate and increase 6 months before cytological relapse.     

Effect of thrombopoietin on acute myelogenous leukemia blasts

It has been shown that the expression of c-mp1, which is a specific receptor for thrombopoietin (TPO), is restricted to the surface of megakaryocytes, platelets, human CD34+ progenitor cells and human erythroid/megakaryocytic leukemic cell lines. Recently, however, it has been reported that some acute myelogenous leukemia (AML) blasts expressed c-mp1 on their cell surface and proliferated in response to TPO. We therefore investigated the effect of thrombopoietin on the growth of leukemic blasts from patients with CD7-positive acute myelogenous leukemia (AML), which is a distinct biological and clinical subtype of AML. Significant growth responses of leukemic blasts to TPO were seen in 10/10 CD7+ and 7/20 CD7- AML cases using 3H-thymidine incorporation, while synergistic stimulatory effects of TPO with stem cell factor (SCF), interleukin-3 (IL-3), granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor were observed in both groups. In a leukemic blast colony assay, significant growth response to TPO was observed in 5/6 CD7+ and 4/17 CD7- AML cases examined. Furthermore, the expression of c-mp1 seemed to be higher in CD7+ AML cases than in CD7- cases, suggesting a relationship between the expression of c-mp1 and the proliferative response to TPO. These findings imply that CD7+ leukemic blasts express functional TPO receptors and proliferate in response to TPO. Thus CD7 expression on AML blasts may indicate the involvement of leukemic progenitors at an early stage of multipotent hemopoietic stem cells. In this review, we discuss the effect of TPO on AML blasts, especially in CD7+ AML cases.     

Altered intracellular distribution of daunorubicin in immature acute myeloid leukemia cells

We have used laser-assisted confocal microscopy to evaluate the intracellular distribution of daunorubicin (DNR) in acute myeloid leukemia (AML) cell lines and fresh AML cells according to their differentiation phenotype. In KG1a, KG1, TF-1 and HEL cells, which express the early differentiation marker CD34, DNR was distributed in perinuclear vesicles which could be associated with the Golgi apparatus, as suggested by the distribution of fluorescent probes specific for intracellular organelles. In contrast, U937 and HL-60 cells, which display a more mature phenotype, exhibited nuclear and diffuse cytoplasmic DNR fluorescence. DNR sequestration was not correlated with P-glycoprotein (P-gp) or multidrug resistance protein expression. Furthermore, PSC833, a potent P-gp blocker, had little effect on drug sequestration in CD34⁺ AML cells. We also tested the effect of metabolic inhibitors, cytoskeleton inhibitors and carboxy-ionophores on DNR distribution in both CD34⁻ and CD34⁺ AML cells. However, only non-specific metabolic inhibitors restored nucleic/cytoplasmic distribution in CD34⁺ cells. In these cells, the intracellular distribution of doxorubicin and idarubicin was very similar to that of DNR, while the distribution of methoxymorpholinyl-doxorubicin was nuclear and diffusely cytoplasmic. In fresh AML cells, DNR was also concentrated in the perinuclear region in CD34⁺ but not in CD34⁻ cells. However, DNR sequestration was not observed in normal CD34⁺ cells. Finally, our results show that DNR is sequestered in organelles in CD34⁺ AML cells via an active mechanism which appears to be different from P-gp-mediated transport. Abnormal DNR distribution may account for the natural resistance of immature AML cells to anthracyclines. Int. J. Cancer 71:292-299, 1997. © 1997 Wiley-Liss, Inc.