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。     

荧光原位杂交及染色体核型分析在典型及非典型慢性粒细胞白血病的应用

慢性粒细胞白血病(CML)是一种起源于造血干细胞的恶性克隆性疾病,90%以上的CML可检测到Ph染色体或BCR/ABL融合基因,Ph染色体是其特征性的细胞遗传学改变。非典型慢性粒细胞白血病(aCML)属于骨髓增生异常/骨髓增生性疾病范畴,     

Ph+急性单核细胞白血病伴复杂核型病例并文献复习

Ph染色体是9号染色体长臂上的ABL基因与22号染色体长臂末端的BCR基因发生融合,形成融合基因BCR/ABL,常发生在慢性粒细胞白血病(chronic myelocytic leukemia,CML)患者中,也可见于2%~5%的儿童急性淋巴细胞白血病和20%~35%的成人急性淋巴细胞白血病[1]。然而在急性髓系白血病(acute myelocytic leukemia,AML)中,出现Ph染色体阳性(Ph+)的非常少见,检出率仅为0.9%~3%[2]。Ph+AML     

STI571治疗BCR/ABL融合基因阳性白血病观察

目的 :观察酪氨酸激酶抑制剂STI5 71在BCR/ABL融合基因阳性表达白血病的治疗效果和副作用。方法 :已确诊BCR/ABL 的 8例慢性粒细胞性白血病 (CML)和 1例急性淋巴细胞白血病 (ALL)患者采用STI5 71 Ara -C治疗。结果 :2例CML -慢性期患者均CR至今 ,6例CML -急粒变患者有 2例CR ,其中 1例CR至今已 3 7周 ,1例ALL在第三次血液学CR后经治疗维持CR2 8周后复发。结论 :STI5 71可诱导BCR/ABL 白血病患者完全缓解 ,降低骨髓BCR/ABL 白血病细胞 ,延长CR期 ,药物副作用轻微易耐受 ,药物作用的个体差异性较大     

荧光原位杂交在慢性粒细胞白血病细胞BCR/ABL融合基因检测中的应用及其意义

目的:探讨荧光原位杂交(FISH)技术检测慢性粒细胞白血病(CML)骨髓和外周血细胞的BCR/ABL融合基因的临床价值。方法:应用FISH对正常对照组及CML患者骨髓和外周血细胞BCR/ABL融合基因进行检测和分析。结果:慢性期CML患者骨髓和外周血细胞该融合基因阳性细胞率分别为(61.9±22.3)%和(68.4±19.8)%,加速期为(77.2±16.7)%和(86.8±12.1)%,急变期为(80.6±17.5)%和(81.4±18.0)%,两者细胞中BCR/ABL基因的阳性率未见统计学差异(P>0.05),且骨髓和外周血细胞之间融合基因阳性细胞比率呈直线正相关。同时发现在完全临床缓解患者中,经伊马替尼治疗的CML患者(71.4%)较经干扰素和羟基脲联合治疗者(10.0%)有更高的分子生物学缓解(P<0.05)。结论:通过FISH对CML患者骨髓和(或)外周血细胞融合基因进行监测,有助于CML的诊断、治疗及微小残留白血病的监测。     

BCR／ABL融合基因致白血病作用研究进展

BCR/ABL联合基因是一个重要的凋亡抑制基因，在大多数慢性粒细胞白血病(CML)，Ph^ 急性淋巴细胞白血病(ALL)及其他一些白血病均有该基因的表达。预示BCR/ABL融合基因可能是上述白血病发生的重要原因。     

EG5和BCR/ABL在慢性粒细胞白血病中表达及其临床意义

目的观察驱动蛋白EG5 mRNA在正常人外周血和骨髓中的表达情况,探讨其在各期慢性粒细胞白血病（CML）中的表达及与BCR/ABL mRNA表达的相关性,评估EG5 mRNA在CML诊断和预后分析中的应用价值。方法采用逆转录聚合酶链反应（RT-PCR）检测EG5 mRNA和筑巢式逆转录聚合酶链反应（Nested-RT-PCR）检测BCR/ABL mR-NA,分别对8例正常人外周血及骨髓样本和62例CML患者的外周血样本进行检测。结果正常外周血标本中未检测到EG5的表达,正常骨髓标本中可见EG5的低丰度表达;EG5辅助诊断CML的敏感度和特异性分别为89.6%（26/29）和84.8%（28/33）;BCR/ABL融合基因诊断CML的敏感度和特异性分别为96.5%（28/29）和90.9%（30/33）;CML中EG5和BCR/ABL表达的差异无统计学意义,且两者之间呈高度正相关。结论EG5和BCR/ABL在慢性粒细胞白血病疾病进展中的表达情况基本一致,EG5和BCR/ABL的联合检测及动态观察,可作为CML临床辅助诊断、疗效和预后判断的指标之一。     

1例BCR/ABL P230阳性的慢性粒细胞白血病特殊骨髓形态报道

骨髓增殖性肿瘤(MPN)是一类造血干细胞或结缔组织细胞出现异常克隆性增殖的疾病.临床上将有关联的4种疾病:真性红细胞增多症(PV)、原发性血小板增多症(ET)、原发性骨髓纤维化(PMF)和慢性粒细胞白血病(CML)统称为 MPN;世界卫生组织(WHO)在2001年将慢性中性粒细胞白血病(CNL)和慢性嗜酸性粒细胞白血病(NOS)划分到 MPN当中;在2016年,又将PMF进一步分为骨髓纤维化(MF)前或早期 PMF(pre PMF)和明显纤维化期PMF(overt PMF);根据Ph染色体或BCR/ABL是否阳性,又将MPN分为Ph染色体或BCR/ABL阳性CML和Ph染色体或BCR/ABL阴性的PV、ET和PMF[1].而伴P230阳性、血小板异常增高、骨髓形态原始细胞增多及病态造血明显的伴P230阳性的CML病例却较为罕见,且与其他类型MPN的骨髓形态较难鉴别,现报道如下.     

BCR/ABL P230阳性的慢性粒细胞白血病特殊外周血检查结果分析

目的分析1例BCR/ABL P230阳性慢性粒细胞白血病(CML)患者临床及实验室检查结果,与慢性中性粒细胞白血病(CNL)相鉴别,以提高对二者认识及诊治水平。方法收集患者临床资料及实验室检查,包括血象、骨髓象、组织化学染色、染色体检查、融合基因检测等进行综合分析。结果血常规:无贫血,白细胞、血小板增多,多次白细胞分类:中性粒细胞均0.86。骨髓象:有核细胞增生极度活跃,粒系增生,以中晚期粒细胞为主,嗜酸粒细胞及嗜碱粒细胞易见;巨核细胞增生,血小板成堆多见。组织化学碱磷酶染色阴性。Ph染色体阳性,t(9;22)(q34;q11),BCR断裂点在μ区,与μ-BCR相应的有e19a2,其编码蛋白为P230。结论这种Ph+BCRμ区基因重排应诊断为CML,而不应诊断为CNL,无Ph染色体和BCR/ABL融合基因才是真正的CNL。     

Bcr/Abl融合基因阳性、Flk1+CD34-慢性粒细胞白血病细胞体内生物学特性的研究

目的体内研究慢性粒细胞白血病(CML)患者骨髓中BCR/ABL+、Flk1+CD34-细胞的白血病干细胞特性。方法取CML患者骨髓,淋巴细胞分离液分离单个核细胞,常规接种培养,免疫磁珠分选CD45-、GlyA-及CD34-的贴壁细胞,流式细胞术鉴定其免疫表型,将其输入经亚致死量照射的NOD/SCID小鼠体内,检测人源细胞的植入及分化情况。结果植入的CML患者骨髓源BCR/ABL+、Flk1+CD34-细胞在受体小鼠体内分化为白血病细胞,受体小鼠骨髓细胞中存在Bcr/Abl融合基因阳性细胞。结论Bcr/Abl融合基因阳性Flk1+CD34-细胞具有白血病干细胞特性。     

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.     

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.     

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.     

Immunophenotypes on blast cells of chronic myelogenous leukemia]

Immunophenotypes of chronic myelogenous leukemia(CML) in chronic phase and in blastic crisis were reviewed. CML cells in chronic phase show a relatively mature immunophenotypes, such as CD13, CD33, CD15, and MPO, but not positive for CD34, CD117, TdT, and HLA-DR. When a CML transforms into blastic crisis, the blast cells demonstrate an immature myeloid(acute myelogenous leukemia(AML)-like) phenotypes in 60-70% of cases. The blast cells which have myeloid markers show CD13, CD33, MPO. In contrast to de novo AML, these myeloid blast cells often express megakaryocytic, erythroid markers or natural killer cell markers, and in some of the cases, the myeloid blast cells have complex phenotypes, with co-expression of markers from two or three lineages. The blast cells, in 25-30% of cases, demonstrate lymphoid blast phenotype characteristics similar to acute lymphoblastic leukemia(ALL), common ALL, or pre-B-ALL. In 60-80% of cases, the lymphoid blast cells co-express myeloid phenotype, fulfilling the criteria of biphenotypic leukemia.     

The P190, P210, and P230 forms of the BCR/ABL oncogene induce a similar chronic myeloid leukemia-like syndrome in mice but have different lymphoid leukemogenic activity

The product of the Philadelphia chromosome (Ph) translocation, the BCR/ABL oncogene, exists in three principal forms (P190, P210, and P230 BCR/ABL) that are found in distinct forms of Ph-positive leukemia, suggesting the three proteins have different leukemogenic activity. We have directly compared the tyrosine kinase activity, in vitro transformation properties, and in vivo leukemogenic activity of the P190, P210, and P230 forms of BCR/ABL. P230 exhibited lower intrinsic tyrosine kinase activity than P210 and P190. Although all three oncogenes transformed both myeloid (32D cl3) and lymphoid (Ba/F3) interleukin (IL)-3-dependent cell lines to become independent of IL-3 for survival and growth, their ability to stimulate proliferation of Ba/F3 lymphoid cells differed and correlated directly with tyrosine kinase activity. In a murine bone marrow transduction/transplantation model, the three forms of BCR/ABL were equally potent in the induction of a chronic myeloid leukemia (CML)-like myeloproliferative syndrome in recipient mice when 5-fluorouracil (5-FU)-treated donors were used. Analysis of proviral integration showed the CML-like disease to be polyclonal and to involve multiple myeloid and B lymphoid lineages, implicating a primitive multipotential target cell. Secondary transplantation revealed that only certain minor clones gave rise to day 12 spleen colonies and induced disease in secondary recipients, suggesting heterogeneity among the target cell population. In contrast, when marrow from non- 5-FU-treated donors was used, a mixture of CML-like disease, B lymphoid acute leukemia, and macrophage tumors was observed in recipients. P190 BCR/ABL induced lymphoid leukemia with shorter latency than P210 or P230. The lymphoid leukemias and macrophage tumors had provirus integration patterns that were oligo- or monoclonal and limited to the tumor cells, suggesting a lineage-restricted target cell with a requirement for additional events in addition to BCR/ABL transduction for full malignant transformation. These results do not support the hypothesis that P230 BCR/ABL induces a distinct and less aggressive form of CML in humans, and suggest that the rarity of P190 BCR/ABL in human CML may reflect infrequent BCR intron 1 breakpoints during the genesis of the Ph chromosome in stem cells, rather than intrinsic differences in myeloid leukemogenicity between P190 and P210.     

Immunogenotypes and surface marker analysis in Ph1 positive chronic myelogenous leukemia in the blastic crisis]

Immunophenotypic and immunogenotypic changes in 23 patients with Ph positive chronic myelogenous leukemia in blast crisis were determined using a panel of monoclonal antibodies and gene probes. According to the immunophenotypes, 9 patients were considered to be in lymphoid blast crisis, including 7 patients with lymphoblastic crisis and 2 patients with lymphoid/myeloid mixed blast crisis. Leukemia cells from the remaining 14 patients showed myeloid phenotypes and 10 of these had platelet-associated antigens. Rearrangement of the immunoglobulin (Ig) gene was observed in all the 9 patients with lymphoid blast crisis, and Ig gene rearrangement was associated with the expression of CD19 antigen. Two patients with myeloid blast crisis showed rearrangements of T-cell-receptor gene, but, dissociation between phenotypes and genotypes was not frequently observed in patients with blast crisis.     

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。     

慢性粒细胞白血病急粒变与急淋变骨髓细胞形态学、免疫表型、细胞遗传学特征及预后差异分析

本研究旨在观察慢性粒细胞白血病（c池）急粒变及急淋变患者骨髓细胞形态学、免疫表型、细胞遗传学特征及预后差异，为分层诊治和预后判断提供实验学基础。回顾性分析西安交通大学医学院第一附属医院血液内科2009年1月-2014年1月期间收治的31例CML急变期临床资料，其中24例急粒变及7例急淋变，对患者外周血及骨髓原始细胞比例、嗜酸及嗜碱细胞百分比、免疫分型、细胞遗传学特征及预后进行差异分析。结果表明，CML急粒变患者外周血及骨髓原始细胞比例无明显差别，在外周血及骨髓中易见嗜酸及嗜碱细胞。慢性粒细胞白血病急淋变患者骨髓原始细胞比例高于外周血，且外周血及骨髓中嗜酸及嗜碱细胞少见。7例CML急淋变患者均为B系ALL，均表达CD10、CD19、CD34、nA—DR，淋系积分≥1．5分；2例同时伴髓系CD13及CD33表达，髓系积分均为1分。24例慢粒急粒变患者主要表达CD33、CD13、CD38、CD34、CD11b及HLA-DR，髓系积分≥2分，其中2例伴淋系积分别为0．5、1分。31例CML患者初诊时检测Ph染色体100％阳性，其中CML急粒变患者有3例同时检出其它染色体畸变。CML急粒变及急淋变患者总生存率无明显差异，但应用酪氨酸激酶抑制剂治疗者的总生存率高于未用酪氨酸激酶抑制剂治疗者。结论：CML急淋变患者外周血嗜酸及嗜碱细胞较CML急粒变患者少见，B系ALL多见，主要表达cD10及cD19；CML急粒变患者主要表达CD33、CD／3、CD38、CD34、CD11b及HLA-DR；慢粒急变期患者均可伴有其他系抗原表达，但一般积分小于2；CML急粒变患者常伴有Ph染色体以外的畸变；CML急粒及急淋变患者总生存率无明显差异，但应用酪氨酸激酶抑制剂治疗者总生存率优于未用酪氨酸激酶抑制剂者。     

The spectrum of molecular alterations in the evolution of chronic myelocytic leukemia.

DNA from 135 patients with chronic myelogenous leukemia (CML) at various clinical stages and Philadelphia (Ph1) chromosome positive acute lymphoblastic leukemia was investigated for alterations in a variety of proto-oncogenes which have been implicated in the evolution of CML from its chronic phase to blast crisis. The most common genetic change found in the evolution of typical Ph1 chromosome positive CML to blast crisis was an alteration of the p53 gene involving either a rearrangement, a deletion, or a point mutation in the coding sequence of the gene. Alterations of the p53 gene were found in the myeloid and the rare megakaryocytic variant of blast crisis but were absent in the lymphoid leukemic transformants. Gross structural alterations were seen in 11 of 54 (20%) of myeloid or unknown phenotypes of blast crisis and in only 1 of 44 chronic phase cases. Eight examples of mutations in the open reading frame of the p53 gene at codons 49, 53, 60, 140, 202, 204, 238, and 239 were observed in blast crisis patients. Mutations in the N-RAS gene were rare in typical blast crisis (2 of 27 cases) but were found in megakaryocytic and Ph1 negative myeloid blast crisis. We concluded that heterogeneous alterations in the p53 gene and occasionally in the N-RAS genes accompany the evolution of chronic phase CML to blast crisis.