Etoposide sensitivity does not predict MLL rearrangements or risk of therapy-related acute myeloid leukemia

Therapy-related acute myeloid leukemia (t-AML) caused by MLL rearrangements (rMLL) can arise from topoisomerase II agents. However, whether rMLL-related leukemogenesis is inextricably linked to drug cytotoxicity remains controversial. We therefore compared (i) rMLL in children with acute lymphoblastic leukemia (ALL) who developed t-AML and those who did not, (ii) epipodophyllotoxin toxicity in patients with t-AML and in controls, and (iii) rMLL in cells sensitive to etoposide and in those resistant to etoposide. In children with ALL, rMLL appeared to be more frequent in children who developed t-AML than in those who did not (seven pairs, P = 0.04), although independent of the cumulative etoposide dose (P = 0.5). Similarly, the frequency of epipodophyllotoxin-related toxicities did not differ between patients with t-AML and controls (26 pairs, P > 0.17). Moreover, in 25 cell lines, etoposide-induced MLL fusions did not differ in sensitive vs. resistant lines at equitoxic concentrations (P = 0.65). Together, these results indicate that epipodophyllotoxin-mediated leukemogenesis is not directly linked to drug cytotoxicity.     

儿童急性白血病MLL基因的检测及临床意义

目的对儿童急性白血病(acute leukem ia,AL)MLL基因重排进行临床和实验研究。方法采用荧光原位杂交(FISH)和多重RT-PCR技术,联合染色体R带核型分析及流式细胞仪免疫表型检测,对27例儿童AL进行分析。结果25例儿童AL中的9例(36.0%)有MLL重排,ALL中的发生率为4.0%,AML中的发生率为3.7%。多重RT-PCR检测到MLL易位产生的融合基因4例,分别为MLL/AF4 2例、MLL/AF9和MLL/AF10各1例;27例白血病患儿进行了染色体核型分析,22例(81.5%)有克隆性染色体异常。在MLL重排中ALL均为B系ALL,AML均为M5b。结论MLL基因重排可能与儿童急性单核细胞白血病和B细胞系ALL有关。     

MLL基因重排急性白血病患儿的临床和生物学特点研究

目的对儿童急性白血病进行混合谱系白血病(mixed lineage leukemia，MLL)基因重排的临床和实验研究：方法应用MLL双色荧光原位杂交(FISH)基因探针进行双色FISH，以13对引物行多重RT—PCR检测融合基冈，并联合染色体R带核型分析及流式细胞仪免疫表型检测，对298例儿童急性白血病中16例含有MLL基因重排患儿进行分析：结果MLL基因重排的白血病在儿童急性白血病中占5.4％，而在婴幼儿白血病中占56．3％；对106例患进行多重RT—PCR，检测到涉及MLL基因重排11例，其中MLL／AF42例，MLL／AF61例，MLL／AF6、MLI／ELL合并MLL／AFX或HOX11各1例，MLL／AF92例，MLL／AF101例，MLI／ELL2例。串联重复dup11q231例，HOX活化1例；27例进行了FISH检测，发现9例有MLL重排，其阳性率为36．0％；16例MLL基因重排患儿中14例(87．5％)有克隆性染色体异常，其中11例累及11号染色体[t(4；11)2例，t(6；11)、t(8；11)、t(7；8；11)、t(9；11)各1例， 112例，11q-3例]；16例MLL基因重排患儿中11例为B系ALL，以B祖细胞或前B细胞白血病为主，5例为急性单核细胞白血病，其中3例有CD7和CD2淋系抗原表达；16例MLL基因重排患儿中8例接受治疗，7例获得完全缓解。结论多重RT—PCR和FISH联合是检测MLL重排最精确和灵敏的方法，MLL基冈重排中包括易位、缺失和重复。MLL基因重排的检测对儿童急性白血病预后判断和治疗方案的选择具有重要意义，并且对WHO分型将其单独列为11q23／MLL白血病提供了有力的依据。     

MEA方案治疗伴有11q23/混合谱系白血病重排的急性髓性白血病17例疗效分析

目的观察米托蒽醌＋足叶乙甙＋阿糖胞苷（MEA）方案对伴11q23／混合谱系白血病（MLL）重排的急性髓性白血病（AML）患者的治疗效果。方法对诊断明确的17例患者按MEA方案治疗1、2个疗程后评价疗效。结果完全缓解率70．6％，总有效率82．4％。MEA方案的主要副作用为骨髓抑制，但均可耐受。结论MEA方案对伴11q23／MLL重排的AML患者疗效较好，可考虑作为其首选治疗方案。     

免疫球蛋白重链和T细胞受体基因重排与急性髓细胞白血病的关系

目的:探讨急性髄细胞白血病(acutemyeloidleukemia,AML)患者的免疫球蛋白重链(IgH)、T细胞受体(TCR)基因重排对疾病预后的影响。方法:用聚合酶链反应(PCR)方法检测IgH、TCR基因重排。结果:38例患者中12例发生IgH基因重排(31.6%),8例TCR基因重排(21.1%)。阳性病例完全缓解率低;缓解期长的病例阳性率低;初发或复发时阳性率高。结论:IgH、TCR基因重排提示AML患者预后不良,需密切随访。     

三例急性髓系白血病患者基因表达谱的演变

目的研究急性髓系白血病（AML）患者初诊与复发时的基因表达谱差异，探讨难治性AML的发病机制。方法应用Agilent Human 1B寡核苷酸基因芯片，动态检测了3例AML-M2a患者初诊、复发时骨髓单个核细胞的基因表达谱差异。结果在检测的20173个基因中，有10个基因在3例患者初诊、复发时共同差异表达，其中7个基冈在复发时均共同上调，3个基因在复发时均表现下调。结论DAPKI等10个基因的表达变化可能与AML-M2a发病和复发有关，这些新基因的发现可能对早期诊断难治性AML具有重要价值，同时为难治性AML提供新的治疗靶点。     

Natural history of chronic myelomonocytic leukemia: gene sequencing identifies multiple clonal molecular abnormalities associated with rapid progression to acute myeloid leukemia

Gene panel sequencing in a CMML patient without any detectable genetic abnormality by conventional genetic studies identified four concurrent somatic mutations in three genes. Gene panel mutation analysis is a rapidly emerging clinical tool to demonstrate the clonality in hematologic malignancies, and to identify the potential targets for therapy.     

T cell receptor alpha-chain gene rearrangements in B-precursor leukemia are in contrast to the findings in T cell acute lymphoblastic leukemia. Comparative study of T cell receptor gene rearrangement in childhood leukemia.

We have analyzed T cell receptor alpha-chain gene configuration using three genomic joining (J) region probes in 64 children with acute lymphoblastic leukemia (ALL). 11 out of 18 T-ALLs were T3 positive; alpha-chain gene rearrangements were demonstrated in only two of 18, indicating that the majority of T-ALLs would have rearrangements involving J alpha segments located upstream of these probes. In contrast, 15 out of 46 B-precursor ALLs showed rearrangements of the alpha-chain gene and J alpha segments located approximately 20-30 kb upstream of the constant region were involved in 13 of these patients. Nine of 15 B-precursor ALLs with rearranged alpha-chain genes had rearrangements of both gamma- and beta-chain genes, whereas the remaining six had no rearrangements of gamma- and beta-chain genes. These findings indicated that alpha-chain gene rearrangement is not specific for T lineage cells and gamma- and/or beta-chain gene rearrangement does not appear essential for alpha-chain gene rearrangement, at least in B-precursor leukemic cells.     

多参数流式细胞术在慢性粒单核细胞白血病、骨髓增生异常综合症及急性单核细胞白血病免疫表型鉴别中的应用及其意义

本研究应用多参数流式细胞术分析慢性粒单核细胞白血病(CMML),骨髓增生异常综合症(DMS)以及急性单核细胞白血病(AML-M5b)的免疫表型特点,探究其在诊断及鉴别诊断上述疾病中的意义和价值。应用多参数流式细胞术比较分析14例CMML患者、48例MDS患者、46例AML-M5b患者及18例正常人骨髓标本的免疫分型特点。结果表明,CMML患者单核细胞比例明显高于MDS、AML-M5b患者及正常人骨髓(P<0.05),后3者之间无显著差别。MDS患者骨髓原始细胞比例明显高于正常骨髓标本(P<0.05),但与CMML患者无明显差别。AML-M5b患者骨髓成熟粒细胞比例较CMML、MDS患者及正常骨髓标本明显减低(P<0.05)。MDS、AML-M5b及CMML患者骨髓CD45/SSC特点与正常骨髓标本均存在一定差异。CMML患者骨髓具有CD2、CD56异常表达及CD14拖尾现象,与MDS、AML-M5b及正常骨髓标本相比差异显著(P<0.05)。MDS与CMML患者骨髓单核细胞CD15表达缺失或减低现象较正常骨髓标本及AML-M 5b患者显著,且CMML患者异常率高于MDS患者(P<0.05),两者粒细胞群均有显著CD13/CD11b/CD16表达规律异常现象,但两者间无显著差异。其他抗原表达呈不同程度异常,无统计学差异。结论 MDS、CMML及AML-M5b骨髓细胞免疫表型均具有各自的特点及不同程度的相似处。多参数流式细胞术综合分析其免疫表型,对于区分CMML、MDS以及AML-M5b具有重要鉴别意义。其中单核细胞比例增高,伴有CD2、CD56异常表达,CD14拖尾现象,CD15缺失或减低及成熟粒细胞CD13-CD11b-CD16表达规律异常,对CMML诊断具有重要意义。     

Acute myelomonocytic leukemia: an unusual variant with both granulocytic and monocytic esterases in the leukemic cells

By using the combination of alpha-naphthyl butyrate esterase and chloroacetate esterase for cytochemical detection of monocytes and granulocytes, respectively, we examined and identified five adult patients with acute myeloid leukemia whose leukemic cells often (20 to 30%) had the characteristics of both monocytes and granulocytes. All five patients were men, 23 to 82 years of age. Two patients had manifestations of preleukemia. One of these two patients had received treatment for lymphoma for 12 months before acute myelomonocytic leukemia was diagnosed. One patient had gum hypertrophy, and two had leukemia cutis. At the time of initial examination, four patients had blood leukocyte counts higher than 80,000/mm3 and one had leukopenia. Four patients received chemotherapy; three responded temporarily but died within 1 year after the myelomonocytic leukemia had been diagnosed. The patient with leukopenia has remained in complete remission for 2 years. With more frequent use of double esterase stains for classification of acute myeloid leukemias, this variant of acute myelomonocytic leukemia should be detected more often and its clinical behavior should be better understood.     

135例急性髓细胞白血病免疫表型特点及意义

目的探讨急性髓系白血病(AML)免疫表型的特点及意义。方法应用流式细胞术,采用CD45/SSC双参数方案对135例AML患者进行免疫表型分析。结果在135例急性髓系白血病中表达较高的抗原依次是MPO(94.8%)、CD33(94.8%)、CD13(90.4%)、HLA-DR(74.1%)、CD117(65.2%)和CD34(61.5%)。CD14仅表达于急性粒-单细胞白血病(M4)和急性单核细胞白血病(M5)。AML伴淋系抗原表达以CD7(32.6%)最为多见,其次为CD56(26.7%)和CD19(10.4%)。共发现4例急性双表型白血病(ABL),同时具有髓系和淋系抗原表达。结论 MPO、CD33、CD13是诊断AML最敏感的抗原。CD7、CD56、CD19在AML诊断及预后判断具有重要意义。免疫表型分析有助于AML的诊断及预后判断。     

High frequency of clonal immunoglobulin or T cell receptor gene rearrangements in acute myelogenous leukemia expressing terminal deoxyribonucleotidyltransferase

Ig and T cell receptor rearrangements have been used as irreversible markers of lineage and clonality in the study of B- and T-lymphoid populations. We have addressed the issue of lymphoid lineage specificity of these rearrangements by analyzing a panel of 25 TdT- acute myelogenous leukemias, 13 TdT+ AMLs, and 4 TdT+ undifferentiated leukemias. We report that while gene rearrangements represent extremely rare events in classical TdT- AML (less than 8%), rearrangements of either the Ig or T beta locus or both were detectable in the majority of the TdT+ AMLs (greater than 60%), and rearrangements of both loci were detectable in all of the TdT+ undifferentiated leukemias. These data demonstrate a significant association between TdT expression and Ig or T beta gene rearrangements even outside the lymphoid lineage, further supporting a role for TdT in Ig and T cell receptor gene assembly. These data also indicate that a coordinated program of lymphoid gene expression involving TdT-CD7-expression and Ig/T beta rearrangements can be activated before myeloid commitment. Whether the activation of this program represents a normal, albeit rare, event in early myelopoiesis or a transformation-related event present only in leukemic cells remains to be determined.     

ID1基因在急性髓系白血病中的表达及其临床意义

目的 探讨分化抑制因子1(ID1)基因在急性髓系白血病(AML)患者中的表达及其临床意义.方法 采用实时定量PCR(RQ-PCR)方法 检测114例初发成人AML患者ID1基因的表达,并分析其临床意义.结果 114例AML患者均检测到ID1基因表达,中位表达水平8525(57～11 233 238);AML不同核型预后组之间ID1基因表达水平有明显差异,预后不良组明显高于预后中等组[中位表达水平分别为36 840(336～11 233 238)和6630(66～1 840798)](P=0.006);ID1基因表达水平与年龄[≥60岁患者明显高于<60岁患者(P=0.002)]和WBC[≥10×109/L患者明显高于<10×109/L患者(P=0.005)]相关;在年龄<60岁AML患者中,第1个疗程结束后未获得完全缓解(CR)患者伴有ID1基因高表达(CR与未CR患者中位表达水平分别为1268和9537,P=0.010).结论 ID1基因高表达多见于AML核型预后不良和高龄患者,与AML不良预后相关;ID1基因可能是成人AML患者不良预后的分子标志.     

Expression and role of amyloid precrusor protein gene in acute myeloid leukemia

OBJECTIVE: To investigate the expression of amyloid precursor protein (APP) gene in acute myeloid leukemia (AML) and its biological behaviour in AML cells. METHODS: The expressions of APP mRNA in 85 AML and 20 nonmalignant hematological diseases patients (as control) were measured by real-time PCR. The expression of APP in AML cell lines was also examined by real-time PCR and Western blot and the results were compared with those in their original subtypes. Small interfering RNAs (siRNAs) targeting APP gene were synthesized and transfected into HL-60 cell by lipofectamine 2000 for 24 h, 48h and 72 h. Cell growth was measured by trypan blue dye exclusion and MTT, differentiation by Wright-Giemsa staining, cell cycle by PI/RNase staining, apoptosis by Annexin V/PI and Hoechst33342 staining. Apoptosis-related protein NF-κB, bcl-2 and Caspase-3 were detected by Western blot after siRNAs transfection for 48 h. Sensitivity to adriamycin was measured by MTT. RESULTS: The expression of APP mRNA among AML subtypes differed significantly (P = 0.019), the highest expression subtype was M(2) with t(8;21) (median 0.1080), followed in order by AML-undefined (0.0467), M(3) (0.0266), M(2a) (0.0221), M(4a) (0.0167), M(5b) (0.0151), and M(4b) (0.0025). APP expression had no significant effect on AML clinical characteristics excepting for subtypes. The expression of APP in Kasumi-1 cells was significantly higher than that of U937 cells (P < 0.05), which was in agreement with APP expression in their original AML subtypes. After siRNAs transfection for 24 h, 48 h, and 72 h, no significant difference in proliferation, differentiation, apoptosis, cell cycle and sensitivity to adriamycin was detected between interfering group and control groups (P > 0.05). CONCLUSIONS: The APP mRNA expression was highest in M(2) with t(8;21) and lowest in M(5b). Down-regulation of APP expression has no significant effects on biological behaviour of HL-60 cells.     

淀粉样前体蛋白基因在急性髓系白血病的表达及其生物学行为研究

目的 探讨淀粉样前体蛋白(APP)基因在急性髓系白血病(AML)细胞的表达及其生物学行为.方法 应用实时定量PCR方法(2-ΔCt)检测85例初诊AML和20例非恶性血液病患者(对照)骨髓细胞APP mRNA的表达,并研究其表达水平对AML患者的临床特征和治疗反应的影响;应用实时定量PCR和Western blot方法检测APP基因和蛋白在AML细胞株的表达,并与AML原代细胞亚型的结果相比较;化学合成针对APP基因的小干扰RNA(siRNA),利用脂质体转染HL-60细胞,下调APP基因表达.RNA干扰24、48、72 h后,采用MTT法及锥虫蓝拒染细胞计数检测细胞增殖;瑞特-姬姆萨染色观察细胞形态;PI染色流式细胞术分析细胞周期;Annexin V/PI双染色流式细胞术及Hoechst染色检测细胞凋亡;RNA干扰48 h,Western blot法检测凋亡相关蛋白NF-κB、bcl-2和caspase-3的表达;MTT法检测细胞对阿霉素的敏感性.结果 APP mRNA表达在AML亚型间差异有统计学意义(P=0.019),伴t(8;21)的M2b表达最高(中位值0.1080),其次是AML-未定型(0.0467)、M3(0.0266)、M2a(0.0221)、M4a(0.0167)、M5b(0.0151)、M4b(0.0025),两两比较分析显示,M2b患者APPmRNA表达显著高于M5b患者(P=0.000).APP mRNA表达水平对AML亚型以外患者临床特征及治疗反应无显著影响(P＞0.05).Kasumi-1细胞APP基因表达显著高于U937细胞(P＜0.05),与AML各亚型原代细胞中的结果相一致.APP-siRNA转染HL-60细胞后,其APP mRNA表达下凋64%,在培养24、48及72 h后检测HL-60细胞增殖、分化、凋亡、细胞周期及对阿霉素敏感性均无明显变化(P＞0.05).结论 伴t(8;21)异位的M2型白血病高表达APP mRNA,单核细胞白血病低表达APPmRNA.APP基因表达沉默对HL-60细胞的生物学作用无显著影响.     

急性髓细胞白血病基因突变研究现状及展望

急性髓细胞白血病(AML)是一组高度异质性的克隆性疾患,具有独特的细胞形态学、免疫表型、细胞遗传学及分子遗传学特征.一些重现性基因突变已被证实是AML诊断分型、危险分层及评估预后的重要因素,并且一些针对突变基因的特异性靶向药物也逐渐进入临床研究.笔者根据目前AML的相关研究进展,对一些与AML预后相关的基因突变进行总结,旨在为理解和探索AML的发病机制拓宽思路.     

急性髓细胞白血病DNMT3A突变的免疫表型特征分析

目的探讨急性髓细胞白血病(AML)患者白细胞表面分化抗原(CD)表达与DNA甲基转移酶3A基因(DNMT3A)突变之间的关系。方法收集264例初发AML患者骨髓样本,用荧光定量PCR检测其融合基因表达水平,流式细胞术检测其CD抗原表达水平,Sanger测序法检测其DNMT3A基因突变,并结合临床特征进行分析。结果 264例AML患者中发现237例DNMT3A野生型;27例(10.2%)患者存在DNMT3A突变。突变类型包括DNMT3A R882H 12例,DNMT3A R882C 15例。在AML各亚型中的DNMT3A突变率分别为M00%(0/5),M114.3%(4/28),M25.0%(3/60),M30%(0/24),M410.7%(9/84)和M517.4%(11/63)。在237例野生型患者中发现AML1-ETO、BCR-ABL、CBFB-MYH、MLL-AF9、PML-RARa融合基因突变分别为19例、3例、19例、2例、18例,但27例突变型患者均未合并上述融合基因。CD抗原分析结果显示,CD14、CD56抗原表达率在野生型和突变型患者差异有统计学意义(P0.05),而其他免疫抗原在两组间差异均无统计学意义(P0.05);在纳入预后分析的36例AML-M5中,DNMT3A野生组的1个疗程缓解率为53.6%(15/28),DNMT3A突变组的1个疗程缓解率为25.0%(2/8),两组间差异无统计学意义(χ2=2.04,P0.05)。其中24例CD56阳性的AML-M5患者在DNMT3A突变组中1个疗程缓解率为25.0%(2/8),明显低于野生组的68.8%(11/16)(P=0.043)。结论 DNMT3A基因突变在AML-M1、M2、M4及M5患者中均有发现;在AML-M5患者中DNMT3A基因突变与CD抗原有一定关联性,原始细胞表达CD56、CD14在DNMT3A基因突变患者中增高,而CD34表达下降;CD56表达阳性的AML-M5患者若伴有DNMT3A基因突变,其预后较差。     

细胞遗传学与荧光原位杂交对急性髓性白血病染色体异常的研究

目的:比较常规细胞遗传学G显带与荧光原位杂交(FISH)对急性髓白血病染色体异常的研究。方法:所有患者初诊时均做常规G显带,根据G显带结果选用相关的特异性探针进行荧光原位杂交。结果:行G显带检测的142例患者中124例获得染色体核型,18例失败。1例正常核型和3例无可供分析的分裂相的患者FISH均检出异常克隆。FISH辨别出2例t(8;21)变异易位。结论:对于有足够可分析分裂相的患者,常规细胞遗传学G显带是检测白血病染色体异常核型的可靠工具。对可疑有变异易位或因染色体形态差难以辨认的核型,或因细胞分裂指数低而分裂相少或无分裂相的患者,FISH检测是重要的补充。