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.     

Activation-induced cytidine deaminase acts as a mutator in BCR-ABL1-transformed acute lymphoblastic leukemia cells

The Philadelphia chromosome (Ph) encoding the oncogenic BCR-ABL1 kinase defines a subset of acute lymphoblastic leukemia (ALL) with a particularly unfavorable prognosis. ALL cells are derived from B cell precursors in most cases and typically carry rearranged immunoglobulin heavy chain (IGH) variable (V) region genes devoid of somatic mutations. Somatic hypermutation is restricted to mature germinal center B cells and depends on activation-induced cytidine deaminase (AID). Studying AID expression in 108 cases of ALL, we detected AID mRNA in 24 of 28 Ph(+) ALLs as compared with 6 of 80 Ph(-) ALLs. Forced expression of BCR-ABL1 in Ph(-) ALL cells and inhibition of the BCR-ABL1 kinase showed that aberrant expression of AID depends on BCR-ABL1 kinase activity. Consistent with aberrant AID expression in Ph(+) ALL, IGH V region genes and BCL6 were mutated in many Ph(+) but unmutated in most Ph(-) cases. In addition, AID introduced DNA single-strand breaks within the tumor suppressor gene CDKN2B in Ph(+) ALL cells, which was sensitive to BCR-ABL1 kinase inhibition and silencing of AID expression by RNA interference. These findings identify AID as a BCR-ABL1-induced mutator in Ph(+) ALL cells, which may be relevant with respect to the particularly unfavorable prognosis of this leukemia subset.     

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.     

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。     

BCR-ABL融合蛋白多样性及其与白血病表型的关系

本文报告了BCR-ABL融合蛋白及其与白血病表型的关系。首先报告BCR-ABL融合基因的结构及其转录本,其中包括这些融合基因可分为M-bcr,m-bcr和u-bcr三种类型及其见于那些白血病;其次报告了BCR断点位置的变化,ABL的特殊断点,BCR-ABL信息RNA的拼接,BCR-ABL融合蛋白的结构与白血病表型的关系;此外,还介绍了BCR/ABL细胞有染色体的其它异常和Ph染色体的变种等;最后论述了BCR/ABL融合蛋白起源于何种细胞系,何种成熟阶段。少数文献报告此蛋白起始于多能干细胞。     

Molecular methods to detect the Philadelphia chromosome

The Ph1 chromosome has two molecular subtypes: a bcr-positive seen in CML and some cases of ALL, and the bcr-negative subtype mainly seen in ALL. In CML, because of the restriction of chromosome 22 breakpoints to the bcr, Southern analysis to detect bcr rearrangements also can be used to detect the Ph1 chromosome. In contrast, the translocation breakpoints on the Ph1 chromosome are scattered in ALL, so that other methods such as PFGE and PCR are necessary to detect the Ph1 chromosome. In both CML and ALL, use of these methods to detect molecular abnormalities may be superior to cytogenetics in detecting chromosomal abnormalities. Southern analysis also can be used in CML to map breakpoint locations within the bcr. This may offer prognostic information as to the length of chronic phase, but there is conflicting information as to the validity of this approach. The modified PCR (using cDNA from mRNA) can be used to detect the Ph1 chromosome and to define which of the molecular subtypes are present. The exquisite sensitivity of this method, which is capable of detecting as little as a single abnormal molecule of RNA or DNA, makes it suited for the detection of minimal residual disease in both CML and ALL. This is particularly useful after intensive therapies, such as bone marrow transplantation. Whether these low levels of fusion gene expression are of prognostic significance is still unclear.     

bcr—abl融合基因及其检测

bcr-abl融合基因位于染色体t(9;22)(q34;q11),由位于9号染色体上的细胞原癌基因abl部分序列从其正常位置易位至22号染色体的BCR区而形成。在绝大多数病例中,断裂点集中在5.8 kb的M-BCR区,主要形成b2a2和b3a2二种形式的转录物及蛋白质,二者差异仅为75个碱基及25个氨基酸。bcr-abl融合基因及其表达的检测对CML有重要的诊断与预后价值。用Southern印迹杂交、间期或中期染色体原位杂交等方法检测DNA,发现Ph阴性或变异型Ph病人存在bcr-abl融合基因,检测到许多新的融合类型,并确认异基因BMT后白血病的复发是源于自身MRL。用优化的RT-PCR,定量RT-PCR或RNA原位杂交检测RNA,可精确定位罕见或混合的接合型,还发现正常人中bcr-abl基因有低量表达且有随年龄增长而增加的趋势。用Western印迹或酶联免疫反应检测,发现BCR-ABL蛋白与C-ABL蛋白之比在外周血与骨髓中相似且与Ph染色体重排率有线性关系,还发现如果P190继P210之后出现,则CML的预后很差。     

FTY720, a new alternative for treating blast crisis chronic myelogenous leukemia and Philadelphia chromosome-positive acute lymphocytic leukemia

Blast crisis chronic myelogenous leukemia (CML-BC) and Philadelphia chromosome-positive (Ph1-positive) acute lymphocytic leukemia (ALL) are 2 fatal BCR/ABL-driven leukemias against which Abl kinase inhibitors fail to induce a long-term response. We recently reported that functional loss of protein phosphatase 2A (PP2A) activity is important for CML blastic transformation. We assessed the therapeutic potential of the PP2A activator FTY720 (2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol hydrochloride), an immunomodulator in Phase III trials for patients with multiple sclerosis or undergoing organ transplantation, in CML-BC and Ph1 ALL patient cells and in in vitro and in vivo models of these BCR/ABL+ leukemias. Our data indicate that FTY720 induces apoptosis and impairs clonogenicity of imatinib/dasatinib-sensitive and -resistant p210/p190(BCR/ABL) myeloid and lymphoid cell lines and CML-BC(CD34+) and Ph1 ALL(CD34+/CD19+) progenitors but not of normal CD34+ and CD34+/CD19+ bone marrow cells. Furthermore, pharmacologic doses of FTY720 remarkably suppress in vivo p210/p190(BCR/ABL)-driven [including p210/p190(BCR/ABL)(T315I)] leukemogenesis without exerting any toxicity. Altogether, these results highlight the therapeutic relevance of rescuing PP2A tumor suppressor activity in Ph1 leukemias and strongly support the introduction of the PP2A activator FTY720 in the treatment of CML-BC and Ph1 ALL patients.     

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。     

多重RT-PCR联合毛细管电泳法检测BCR-ABL融合基因

目的建立多重逆转录-聚合酶链反应(RT-PCR)联合毛细管电泳法检测BCR-ABL融合基因,以期应用于慢性粒细胞白血病(CML)辅助诊断。方法采用重叠延伸法构建BCR-ABL融合基因阳性模板,设计并优化检测BCR-ABL融合基因的多重RT-PCR联合毛细管电泳体系,对检测体系进行初步的性能评估。结果多重RT-PCR技术检测BCR-ABL融合基因(e1a2、e13a2和e14a2)的最低检测限在102~103拷贝/μL;50例临床确诊CML的患者应用该法检测BCR-ABL融合基因的阳性率为86.0%,其中e13a2型(20.0%),e14a2型(66.0%);本方法与骨髓细胞培养染色体核型分析相比,阳性符合率为97.6%,阴性符合率为75.0%,总符合率为94.0%,2种方法具有较高的一致性(Kappa=0.765,P0.05)。结论成功建立检测BCR-ABL融合基因的多重RTPCR联合毛细管电泳方法。该方法操作简单快捷、特异性好、灵敏度高,适合于临床上CML的辅助诊断和分子分型。     

Ph1-positive leukemia: cytogenetic outline and prognosis]

Ph1-positive leukemias consist of acute leukemia (Ph1 AL) and CML. Cytogenetically, Ph1 AL is often associated with +6, -7, +8, +21, or +Ph1. CML is predominantly accompanied by +Ph1, +8, i (17q), +19 in myeloid crisis and +Ph1, +8, +21 in lymphoid crisis. Thus, i(17q) seems specific for myeloid crisis of CML. Ph1 constricts ABL/BCR within M-BCR in CML and in one half of the adult Ph1 AL. BCR breaks upstream to M-BCR in the other half of adult AL and in most of childhood AL. However, the breakpoint does not affect clinical and hematological features in AL. Consequently, there seems to be two types of Ph1 leukemia; one is AL representing m-BCR rearrangement and the other is CML and Ph1 AL showing M-BCR rearrangement.     

223例儿童白血病30种融合基因检测结果分析

目的通过对30种融合基因在儿童白血病中的结果分析,结合形态、免疫分型和细胞遗传学明确其在儿童白血病中的分布情况。方法选取2017年5月至2019年4月该院儿科223例初发或复发白血病肿瘤患儿作为研究对象,其中男124例,女99例,年龄1个月至14岁,采用实时荧光探针PCR进行融合基因检测,融合基因包括急性淋巴细胞白血病(ALL)和急性髓系白血病(AML)常见的如BCR-ABL1、AML1-ETO、CBFβ-MYH11、MLL和PML-RARα等30种融合基因。结果 223例患儿中诊断为B-ALL152例,T-ALL9例,AML49例,以及其他13例,包括B-NHL2例,T-NHL1例,混合型急性白血病1例,分类不明急性白血病2例,JMML1例,CML转AML1例,CML5例。30种融合基因筛查总阳性率为43.0%(96/223),其中B-ALL阳性率为32.2%(49/152),T-ALL阳性率为22.2%(2/9),AML阳性率为75.5%(37/49),其他病例阳性率为61.5%(8/13)。ALL中最常见的融合基因为TEL-AML1,占15.5%(25/161),BCR-ABL1占4.3%(7/161),E2A-PBX1占3.7%(6/161),其次为AML-MTG16,占1.9%(3/161),MLL-AF4占1.2%(2/161),MLL-AF10占1.2%(2/161),MLL-ENL占1.2%(2/161),TLS-ERG占1.2%(2/161),MLL-AF6占0.6%(1/161);AML中最常见的融合基因为AML1-ETO,占20.4%(10/49),PML-RARα占16.3%(8/49),CBFβ-MYH11占10.2%(5/49),DEK-CAN占6.1%(3/49),MLL-AF10占8.2%(4/49),其他包括MLL-AF4、MLL-AF9、MLL-AF1p、NPM-MLF、TEL-ABL、SET-CAN、BCR-ABL1各1例。结论白血病患儿白血病融合基因阳性率较高,加强对白血病30种融合基因的检测有助于提高儿童白血病的诊治水平。     

多重RT-PCR技术联合染色体核型分析在儿童急性淋巴细胞白血病诊断分型中的应用

目的　研究多重逆转录 聚合酶链反应 (RT PCR)技术联合染色体核型分析在急性淋巴细胞白血病 (ALL)诊断分型中的价值。方法　采用多重RT PCR技术 ,染色体R或G显带技术对 5 0例儿童ALL进行分析。结果　 5 0例ALL患儿中 18例 (36 .0 % )分别具有 11种融合基因 ,包括E2A/PBX1、TEL/AML1、TLS/ERG、MLL/AF4、MLL/AF9、MLL/AF10、MLL/AFX、MLL/AF6、MLL/ELL、TAL1D、HOX11。在接受染色体检查的 4 8例ALL患儿中 ,染色体异常有 2 4例 (5 7.1% ) ,其中染色体数目和缺失异常为 18例 ,染色体易位 6例。多重RT PCR和核型分析联合使ALL的遗传学异常检出率增至 70 % (5 0例中 35例 )。结论　多重RT PCR和染色体核型分析两种方法相结合 ,可以相互补充 ,从而提高了ALL患儿遗传学异常的检出率 ,为儿童ALL的诊断、分型和预后判断提供可靠依据。