Lyn激酶在伊马替尼耐药的慢性髓系白血病中的作用

本研究探讨Lyn激酶在伊马替尼耐药的慢性髓系性白血病（CML）中的作用。76例CML患者分为初治组、伊马替尼耐药组和伊马替尼治疗有效组,用Western blot方法检测CML患者骨髓单个核细胞中Lyn蛋白的表达水平,比较各组患者Lyn的表达差异,分析Lyn与I临床特征、BCR／ABL融合基因和染色体的关系。结果表明,76例CML均表达Lyn;伊马替尼耐药组Lyn表达明显高于正常对照组、初治组及伊马替尼治疗有效组（P〈0．05）,初治组和伊马替尼治疗有效组的Lyn表达水平与正常对照组无明显差别（P〉0．05）。Lyn表达水平与性别、年龄、平均血红蛋白水平、平均血小板计数、外周血幼稚细胞比例和脾脏大小无明显相关（P〉0．05）,但与初诊时外周血白细胞计数增高相关（P〈0．05）。Lyn表达与BCR／ABL融合基因定量无明显相关性（P〉0．05）;10例伊马替尼耐药的CML中,1例患者存在t（6;22）和t（2;9）改变,与Ph染色体共存,其余9例患者仅存在Ph染色体改变。结论：CML患者和正常人骨髓细胞均表达Lyn,Lyn在伊马替尼耐药的CML中表达明显增高,Lyn高表达与CML患者初诊时白细胞计数明显增高相关。     

BCR/ABL探针在骨髓增殖性疾病中的临床运用

本研究通过回顾分析荧光原位杂交(FISH)检测BCR/ABL融合基因结果,探讨其在慢性骨髓增殖性疾病(CMPD)和Ph+急性淋巴细胞白血病(Ph+ALL)的鉴别诊断及治疗后微小残留病(MRD)动态监测中的运用价值。初诊和治疗后病例分别使用BCR/ABL(ES)和BCR/ABL(DF)探针检测BCR/ABL融合基因。结果表明:初诊CMPD 49例,形态学符合CML骨髓象28例,确诊CML 23例,形态符合率23/28(82.1%),BCR/ABL阳性23/23,敏感度、特异度均为100%。确诊病例BCR/ABL阳性细胞率为81.3%±17.7%;allo-HSCT 13例,9例长期无病生存,4例复发,经供者淋巴细胞输注(DLI)、伊马替尼或allo-HSCT治疗后多次监测BCR/ABL阴性;伊马替尼治疗16例,其中11例于1年后多次监测BCR/ABL阴性,5例分别于6、7、10年后监测BCR/ABL阳性,其中1例经allo-HSCT成功,BCR/ABL转阴。结论:FISH技术是一项敏感、特异的诊断技术,针对初诊和治疗后病例分别运用两种不同的探针检测BCR/ABL融合基因,有助于准确、快速鉴别诊断CML、Ph+ALL,动态监测酪氨酸激酶抑制剂治疗和allo-HSCT后MRD。     

荧光原位杂交技术检测BCR/ABL融合基因

目的建立荧光原位杂交(FISH)技术,直接原位检测间期细胞中BCR/ABL融合基因,辅助临床诊断和治疗慢性粒细胞性白血病(CML)。方法应用FISH技术检测15例CML患者骨髓培养细胞BCR/ABL融合基因,其中5例CML患者同时取骨髓直接涂片检测,5例CML患者同时取外周血浓缩单个核细胞直接涂片检测。以5例非CML患者骨髓培养细胞为阴性对照。结果15例患者骨髓标本成功培养10例,染色体分析检测到Ph1染色体8例。15例患者骨髓培养细胞FISH检测BCR/ABL融合基因阳性14例。5例CML患者同时做骨髓直接涂片FISH检测,结果阳性4例。5例CML患者同时做血标本浓缩单个核细胞FISH检测阳性2例。5例非CML患者FISH结果均阴性。结论FISH技术能直接原位检测间期细胞中BCR/ABL融合基因,且快速、可靠、成功率高,是诊断和监测CML的一项有效新技术。     

Differential diagnosis of chronic myeloid leukemia and the related disorders]

Chronic myeloid leukemia(CML) is a generic term that includes five subtypes; i.e. chronic granulocytic leukemia(CGL) (95% of all CML, 90% are Ph+, 5% are Ph-, BCR/ABL+), atypical CML(survival is worse than that of CGL), chronic myelomonocytic leukemia(a subtype of myelodysplastic syndrome), chronic neutrophilic leukemia (Ph-, BCR/ABL-) and juvenile CML(Ph-, BCR/ABL-). It is not so easy to make a diagnosis of Ph-negative CML. Also, about 25% of adult acute lymphoid leukemia(ALL) patients and some essential thrombocythemia patients have Ph chromosome. In addition, about a half of cases with Ph-positive ALL have the same size of BCR/ABL fusion protein as that in Ph-positive CML. It is necessary to distinguish them by the distinctive morphological, cytogenetical and immunological characteristics of these diseases.     

BCR/ABL双色双融-荧光原位杂交技术的信号模式探讨

目的研究BCR/ABL双色双融合探针荧光原位杂交技术(dual color dual fusion BCR/ABL probe-fluorescence in situ hy-bridization,DCDF-FISH)在白血病遗传学异常检测中的常见信号模式,探讨各种信号模式与染色体异常的关系。方法采用BCR/ABL的DCDF-FISH技术和染色体核型分析技术对初诊的40例慢性粒细胞白血病(chronic myelocytic leukemia,CML)、30例急性淋巴细胞白血病(acute lymphoblastic leukemia,ALL)以及10例其它(包括AML和骨髓增生性疾病)患者骨髓标本进行检测,比较DCDF-FISH各种信号模式与染色体关系。结果DCDF-FISH检测见11种信号模式:2R2G为正常细胞信号模式;2R3G、3R2G及3R3G为无BCR/ABL融合基因的异常信号模式;1R1G2F、1R1G1F、1R2G1F和2R1G1F模式的核型都为t(9;22);1R1G3F核型模式为t(9;22)伴有Ph+;2R2G1F为三条以上染色体易位t(9;22;V);1R2G2F为t(9;22)伴多一条22号染色体,这些信号模式的细胞内都存在着BCR/ABL融合基因。结论BCR/ABL DCDF-FISH检测时存在着多种信号模式,明确各种信号模式的意义,对临床疾病的诊断、预后有重要指导意义。     

慢性髓系白血病急变期分子遗传学研究进展

9号和22号染色体相互易位产生Ph染色体及BCR-ABL融合基因,几乎在所有慢性髓系白血病（CML）出现,BCR-ABL编码的蛋白具有持续增高的酪氨酸激酶活性,使白血病细胞异常增殖。急变期是CML的晚期,在此期间常常出现其它附加染色体和分子的改变。大量研究表明,BCR-ABL基因与其他失调的基因共同作用并异常激活下游的信号传导通路,促进了疾病的进展。酪氨酸激酶抑制剂伊马替尼对大多数慢性期CML患者治疗效果显著。IRIS5年的临床试验显示：用伊马替尼治疗的98%患者达血液学完全缓解,92%患者达主要细胞遗传学缓解,87%患者达完全细胞遗传学缓解。然而,仍有少数慢性期和大多数进展期患者用伊马替尼治疗疗效欠佳。在耐药机制的研究中发现ABL激酶区点突变与临床耐药关系密切。第二代酪氨酸激酶抑制剂可改善伊马替尼耐药,本文就急性变的分子机制、伊马替尼耐药等做一综述。     

Chronic myeloid leukemia: pathophysiology,diagnostic parameters,and current treatment concepts

Chronic myeloid leukemia (CML) is a stem cell disease characterized by excessive accumulation of clonal myeloid (precursor) cells in hematopoietic tissues. CML cells display the translocation t(9; 22) that creates the bcr/abl oncogene. The respective oncoprotein (= BCR/ABL) exhibits constitutive tyrosine kinase activity and promotes growth and survival in CML cells. Clinically, CML can be divided into three phases: the chronic phase (CP), the accelerated phase (AP), and the blast phase (BP) that resembles acute leukemia. Progression to AP and BP is associated with occurrence of additional genetic defects that cooperate with bcr/abl in leukemogenesis and lead to resistance against antileukemic drugs. The prognosis in CML is variable depending on the phase of disease, age, and response to therapy. The only curative approach available to date is stem cell transplantation. For those who cannot be transplanted, the BCR/ABL tyrosine kinase inhibitor STI571 (Glivec, Imatinib), interferon-alpha (with or without ARAC), or other cytoreductive drugs are prescribed. Currently available data show that STI571 is a superior compound compared to other drugs in producing complete cytogenetic and molecular responses. However, despite superior initial data and high expectations for an effect on survival, long term results are not available so far, and resistance against STI571 has been reported. Forthcoming strategies are therefore attempting to prevent or counteract STI571 resistance by co-administration of other antileukemic drugs. Whether these strategies will lead to curative drug therapy in CML in the future remains at present unknown.     

荧光原位杂交技术检测BCR/ABL融合基因的临床应用

本研究探讨应用荧光原位杂交技术( FISH)检测BCR/ABL融合基因在临床中的应用价值.对初诊考虑为慢性骨髓增殖性疾病或骨髓增生异常/骨髓增殖性疾病的患者、急性淋巴细胞白血病患者及异基因造血干细胞移植后的慢性髓系白血病(CML)患者,行骨髓细胞学检查并用FISH法检测BCR/ABL融合基因.结果表明:①46例在初诊时考虑为慢性骨髓增殖性疾病或骨髓增生异常/骨髓增殖性疾病的患者中,有22例患者诊断为CML,应用FISH法检测BCR/ABL融合基因均为阳性(100％),骨髓细胞学检查显示CML者占86.4％ (19/22);另24例患者诊断为非CML的慢性骨髓增殖性疾病及骨髓增生异常/骨髓增殖性疾病,FISH法检测BCR/ABL融合基因均为阴性(100％),而骨髓细胞学检查有3例支持CML诊断、1例支持MDS诊断;②7例急性淋巴细胞白血病患者,有3例FISH法检测BCR/ABL融合基因为阳性;③2例异基因造血干细胞移植后的CML患者,应用HSH法检测BCR/ ABL融合基因可检测到阳性细胞(分别为6.5％及1.2％).结论:FISH法检测BCR/ABL融合基因敏感、可靠,对慢性骨髓增殖性疾病及骨髓增生异常/骨髓增殖性疾病的诊断及鉴别诊断有重要价值;可明确Ph+急性淋巴细胞白血病患者的诊断;在CML患者行异基因造血干细胞移植后监测微小残留病灶方面也有重要意义.     

骨髓形态学染色联合荧光原位杂交技术在急性白血病鉴别诊断中的应用

目的 探讨联合骨髓形态学染色和荧光原位杂交(MGG-FISH)技术在Ph染色体阳性急性淋巴细胞白血病(Ph+ALL)和慢性粒细胞白血病急性淋巴细胞白血病变(CML-LBC)鉴别诊断中的临床应用价值.方法 应用BCR(绿色)和ABL(橘红色)双色双融合探针,通过MGG-FISH技术对4例Ph+ALL患者、4例CML-LBC患者、1例CML急性白血病变合并淋巴瘤、1例CML急性混合细胞白血病变患者的骨髓涂片标本进行检测,依据形态学分别检测10份标本红细胞系、粒细胞系和淋巴细胞系的BCR/ABL融合信号阳性细胞百分率.结果 通过MGG-FISH方法分析,4份Ph+ALL骨髓标本红细胞系未累及,BCR/ABL融合信号阳性细胞百分率均为0%;粒细胞系阳性率分别为11%(1/9)、8%(1/12)、0%(0/8)、10%(1/10);淋巴细胞系阳性率分别为97%(76/78)、98%(87/89)、98%(97/99)、97%(75/77).4份CML-LBC骨髓标本红细胞系阳性率分别为100%(8/8)、91%(10/11)、82%(9/11)、88%(7/8);粒细胞系阳性率分别为89%(8/9)、96%(94/98)、100%(47/47)、98%(40/41);淋巴细胞系阳性率分别为96%(78/81)、93%(52/56)、96%(68/71)、95%(58/61).其余2份标本阳性率均超过80%,且通过MGG-FISH分析鉴定了恶性克隆的来源.结论 MGG-FISH技术可以准确、快速地对原发性Ph+ALL和CML-LBC进行鉴别,并可进行克隆源性分析.     

BCR/ABL induces multiple abnormalities of cytoskeletal function.

The BCR/ABL oncogene causes human chronic myelogenous leukemia (CML), a myeloproliferative disease characterized by massive expansion of hematopoietic progenitor cells and cells of the granulocyte lineage. When transfected into murine hematopoietic cell lines, BCR/ABL causes cytokine-independence and enhances viability. There is also growing evidence that p210(BCR/ABL) affects cytoskeletal structure. p210(BCR/ABL) binds to actin, and several cytoskeletal proteins are tyrosine phosphorylated by this oncoprotein. Also, at least one aspect of cytoskeletal function is abnormal, in that the affinity of beta1 integrins for fibronectin is altered in CML cells. However, isolated changes in beta1 integrin function would be unlikely to explain the clinical phenotype of CML. We used time-lapse video microscopy to study cell motility and cell morphology on extracellular cell matrix protein-coated surfaces of a series of cell lines before and after transformation by BCR/ABL. BCR/ABL was associated with a striking increase in spontaneous motility, membrane ruffling, formation of long actin extensions (filopodia) and accelerated the rate of protrusion and retraction of pseudopodia on fibronectin-coated surfaces. Also, while untransformed cells were sessile for long periods, BCR/ABL-transformed cells exhibited persistent motility, except for brief periods during cell division. Using cell lines transformed by a temperature-sensitive mutant of BCR/ABL, these kinetic abnormalities of cytoskeletal function were shown to require BCR/ABL tyrosine kinase activity. Similar abnormalities of cytoskeletal function on fibronectin-coated surfaces were observed when hematopoietic progenitor cells purified by CD34 selection from patients with CML were compared with CD34 positive cells from normal individuals. Interestingly, alpha-interferon treatment was found to slowly revert the abnormal motility phenotype of BCR/ABL-transformed cells towards normal. The increase in spontaneous motility and other defects of cytoskeletal function described here will be useful biological markers of the functional effects of BCR/ABL in hematopoietic cells.     

The underestimated role of basophils in Ph+ chronic myeloid leukaemia

Chronic myeloid leukaemia (CML) is a hematopoietic neoplasm defined by the chromosome translocation t(9;22) and the related oncogene, BCR‐ABL1. In most patients, leukaemic cells can be kept under control using BCR‐ABL1‐targeting drugs. However, many patients relapse which remains a clinical challenge. In particular, patients with advanced (accelerated or blast phase) CML have a poor prognosis. So far, little is known about molecular and cellular interactions and features that contribute to disease progression and drug resistance in CML. One key prognostic factor at diagnosis is marked basophilia. However, although basophils are well‐known multifunctional effector cells, their impact in CML remains uncertain. In this article, we discuss the potential role of basophils as active contributors to disease evolution and progression in CML. In particular, basophils serve as a unique source of inflammatory, angiogenic and fibrogenic molecules, such as vascular endothelial growth factor or hepatocyte growth factor. In addition, basophils provide vasoactive substances, like histamine as well as the cytokine‐degrading enzyme dipeptidyl‐peptidase IV which may promote stem cell mobilization and the extramedullary spread of stem and progenitor cells. Finally, basophils may produce autocrine growth factors for myeloid cells. Understanding the role of basophils in CML evolution and progression may support the development of more effective treatment concepts.     

Imatinib-induced apoptosis: a possible link to topoisomerase enzyme inhibition

What is known and Objective: Imatinib is a specific BCR/ABL inhibitor, commonly used for the treatment of chronic myeloid leukaemia (CML), a hematological malignancy resulting from a chromosomal translocation that generates the BCR/ABL fusion protein. Recent studies showed that the imatinib has cytotoxic and apoptotic effects on many BCR/ABL‐negative cancers. Numerous compounds with cytotoxic potential exert their functions by interfering with the DNA topoisomerase. In this study, we examined the effects of imatinib on tumour cell‐killing in relation to DNA topoisomerase enzyme inhibition. Methods: We determined the cytotoxicity by cell proliferation assay (XTT; tetrazolium hydroxide), using the human K562 CML cells, and loss of mitochondrial membrane potential by monitoring the changes in caspase‐3 enzyme activity. Type I and II topoisomerase activities were measured by supercoiled plasmid relaxation and minicircle DNA decatenation assays respectively. Results and Discussion: Imatinib‐induced apoptosis and inhibited cell proliferation in a dose‐dependent manner. We also found that the imatinib was effective in both type I and type II topoisomerase reactions to a varying degree between 94% and 7% for the concentration range of 1 mm– 0·02 mm in a dose‐dependent manner. What is new and Conclusion: Our results suggest that the inhibition of topoisomerases may be a significant factor in imatinib‐induced apoptosis in CML.     

Disease-related gene and tumor progression]

Chronic myelogenous leukemia is a stem cell tumor characterized by the t(9; 22)(q34; 11) translocation generating the BCR/ABL chimeric gene. The BCR/ABL fusion gene shows several functions, including inhibition of adhesion to stroma cells and extracellular matrix, activation of mitogenic signalings, inhibition of apoptosis, and degradation of inhibitory proteins, and thereby causes transformation of hematopoietic progenitors. Among its functions, the signal transduction pathways activated by the fusion gene are Ras and MAP kinase pathways, Jak-Stat pathways, PI3 kinase pathways, and Myc pathways. Molecular mechanisms in blastic crisis remains largely unknown. However, loss of functions of tumor suppressor genes such as p53, RB, and p16, activation of oncogene Ras, overexpression of Evi-1 might be involved in disease progression.     

采用 DCDF-FISH 早期监测 BCR / ABL( +) ALL 患者耐药简

本研究探讨双色双融合荧光原位杂交技术（DCDF-FISH）对BCR/ABL阳性伴复杂染色体易位的急性淋巴细胞白血病（ALL）患者的应用价值.通过骨髓细胞形态学检查、染色体分析、流式细胞术和DCDF-FISH技术等方法观察1例急性淋巴细胞白血病的临床特征,并通过DCDF-FISH技术动态观察患者对治疗的反应及病情演变.结果表明：患者发病时呈现急性淋巴细胞白血病表现,流式细胞术发现患者表达幼稚B淋巴细胞分子标志CD10、CD19和CD34,染色体分析显示,患者骨髓细胞有46,XY,i（8）,ider（9）t（9;22）[23]/47,idem,＋der（22） t（9;22）[7]核型;FISH显示,患者初发病时83％细胞含有BCR/ABL融合基因,其中5％的肿瘤细胞显示1R1G2F信号模式、14％显示1R1G3F、64％显示1R1G4F;患者经格列卫联合VTLP化疗而完全缓解时,FISH显示肿瘤细胞降19％,但是1R1G2F信号模式的细胞却增加到18％;患者经过巩固治疗后复发,1R1G2F信号模式的细胞增加到38％,最后患者因耐药而死亡.结论：复杂易位的BCR/ABL（＋）的急性淋巴细胞白血病患者存在多个肿瘤细胞亚群,且不同的亚群对药物的反应性可能不同,因此通过DCDF-FISH技术的信号模式以及对不同亚群细胞动态变化观察,可以在早期监测患者对治疗的反应性以及耐药情况.     

BCR/ABL-mediated increased expression of multiple known and novel genes that may contribute to the pathogenesis of chronic myelogenous leukemia

The BCR/ABL chimeric protein plays a central role in the pathogenesis of chronic myelogenous leukemia (CML). Intensive research has elucidated many signal transduction pathways activated by BCR/ABL. However, few studies addressed BCR/ABL-dependent alterations in gene expression that may contribute to the pathobiology of CML. To additionally define such downstream genes, we performed a subtractive hybridization between cord blood (CB) CD34(+) cells transduced with an MSCV-retrovirus vector containing either enhanced green fluorescent protein (eGFP) alone or p210(BCR/ABL)-internal ribosome entry site-eGFP. Thirty-four subtracted clones expressed in p210-eGFP but not eGFP-transduced CD34(+) cells have been confirmed by Northern blot and sequenced. Fifty-nine percent represent novel proteins, and 41% are homologous to known genes. Quantitative real-time PCR analysis confirmed that 14 of 14 genes tested were also overexpressed in additional populations of p210(BCR/ABL)-transduced CB CD34(+) cells, as well as in CD34(+) cells from primary newly diagnosed CML patients versus GFP-transduced CB or samples from normal donors. Western blot analysis showed that the known sequences were also overexpressed at the protein level. Treatment of BCR/ABL(+) cells with the Abl-specific tyrosine kinase inhibitor STI571 decreased expression at the mRNA as well as protein level of some but not all of the gene products. This suggests that increased gene expression is in some cases tyrosine kinase-independent. Some of the overexpressed genes are implicated in cellular processes known to be disturbed in CML, including the mitogen-activated protein kinase or the ubiquitin pathway, whereas overexpression of other genes, including RAN and NUP98, may implicate new cellular pathways involved in CML. Additional characterization of downstream genes activated by BCR/ABL may lead to important new insights in the molecular mechanisms underlying CML and identify potentially novel therapeutic targets for CML.