Chronic myelogenous leukaemia with p185(BCR/ABL) expression: characteristics and clinical significance

We investigated the significance of p185BCR/ABL expression in patients with chronic myelogenous leukaemia (CML) in relation to disease features, therapy and outcome. Results of Western blot analysis for 1384 patients referred with a diagnosis of CML to our institution from 1989 to 1997 were reviewed. Clinical characteristics, results of cytogenetic analysis and RT-PCR for BCR rearrangement were analysed. Five patients with Ph-positive CML expressing the p185BCR/ABL hybrid protein were identified. By RT-PCR, bone marrow specimens of these patients were confirmed to have an e1a2 junction. The median age at diagnosis of these patients was 55 years (range 43-76). All had elevated white cell counts at diagnosis (median 50 x 109/l, range 11.7-163 x 109/l). Four patients had monocytosis (range 10-16%) with a low neutrophil/monocyte ratio in the peripheral blood (range 3.4-5.7). Patients presented with various stages of the disease (two in chronic-phase CP, two in accelerated-phase AP, and one in blastic-phase BP). The clinical course and therapy of the patients varied, with one patient receiving hydroxyurea only, three patients receiving hydroxyurea followed by interferon-alpha based regimens and bone marrow transplantation. The patient presenting in BP was treated with combination chemotherapy. The clinical outcome of the patients was also varied with one patient alive and in complete remission (with complete cytogenetic remission after transplant) and four patients dead after progression to more advanced stages. We conclude that patients with Ph-positive p185BCR/ABL CML frequently present with monocytosis and a low neutrophil/monocyte ratio in the peripheral blood, aiding the speculation that the presence of the p185BCR/ABL hybrid protein may contribute to a phenotype intermediate between CML and CMML. Of interest, the only other specific clinical feature identified was the absence of splenomegaly in four of five patients. There was no definite association with transformation to lymphoid blast phase.     

Blood outgrowth endothelial cells from chronic myeloid leukaemia patients are BCR/ABL1 negative

The existence of adult haemangioblasts with dual haematopoietic and endothelial developmental potential was confirmed after detection of Ph⁺ vascular endothelial cells in chronic myeloid leukaemia (CML) patients. Blood outgrowth endothelial cells (OECs) from CML patients were found not to harbour the Philadelphia translocation and were thus not clonally related to BRC/ABL1 ⁺ hematopoietic progenitors, but comprised a distinct subfraction of endothelial cells. Remarkably, the frequency of CML-derived OECs was 9-fold higher as compared to healthy donors ( n  = 19 and n  = 300, respectively; P  <   0·0001) and these cells showed increased proliferative potential, possibly reflecting the mobilisation of OEC progenitors by pro-angiogenic cytokines.     

Elevated platelet count features the variant type of BCR/ABL junction in chronic myelogenous leukaemia

A variant form of BCR/ABL junction was identified in a patient with chronic myelogenous leukaemia (CML). The BCR/ABL fusion mRNA of this patient showed in-frame junction between BCR exon c3 and ABL exon 2. Although the diagnosis of CML was made, the patient showed clinical features of essential thrombocythaemia (ET) rather than that of typical CML. Treatment with interferon-α showed no cytogenetic response. The c3-a2 type of BCR/ABL junction seems to be associated with elevated platelet count and thus could form a novel clinical entity different from typical CML.     

AICDA expression in BCR/ABL1-positive acute lymphoblastic leukaemia is associated with a peculiar gene expression profile

Activation-induced cytidine deaminase (AICDA) initiates somatic hypermutation and class-switch recombination of immunoglobulin (Ig) genes and induces mutations also in non-Ig genes. AICDA aberrant expression was detected in B-lineage acute lymphoblastic leukaemia (B-ALL), particularly BCR/ABL1+ B-ALL; patients expressing AICDA carried more copy number alterations than 'AICDA-negative' cases. To determine the role of AICDA, AICDA expression and gene expression profiling were studied in adult BCR/ABL1+ B-ALL. Patients displaying the full-length isoform AICDA are characterized by up-regulation of DNA repair/replication and cell cycle genes, suggesting their involvement in the genetic instability of BCR/ABL1+ B-ALL.     

Mechanisms of Transformation by the BCR/ABL Oncogene

The Philadelphia chromosome generates a chimeric oncogene in which the BCR and c-ABL genes are fused. The product of this oncogene, BCR/ABL, has elevated ABL tyrosine kinase activity, relocates to the cytoskeleton, and phosphorylates multiple cellular substrates. BCR/ABL transforms hematopoietic cells and exerts a wide variety of biological effects, including reduction in growth factor dependence, enhanced viability, and altered adhesion of chronic myelocytic leukemia (CML) cells. Elevated tyrosine kinase activity of BCR/ABL is critical for activating downstream signal transduction and for all aspects of transformation. This review will describe mechanisms of transformation by the BCR/ABL oncogene and opportunities for clinical intervention with specific signal transduction inhibitors such as STI-571 in CML.     

慢性粒细胞白血病BCR/ABL融合基因及复杂变异的染色体核型分析

目的探讨慢性粒细胞白血病(CML)患者BCR/ABL融合基因及其复杂变异的染色体核型变化及临床意义。方法在常规细胞遗传学(CC)方法检测基础上,运用分子生物学方法——荧光原位杂交(FISH)技术,采用多种位点特异性DNA探针(染色体全染、特殊位点、双色易位融合探针),对2002年9月至2007年2月中国医科大学附属第一临床学院血液科56例门诊及住院CML患者(慢性期51例,加速及急变期5例)进行染色体核型分析。结果56例CML患者有5例为细胞培养失败,均为慢性期患者,该5例细胞培养失败病例经FISH技术证实均出现Ph 染色体,余46例慢性期患者中有42例出现Ph 染色体,其中2例为双Ph 染色体;3例合并有复杂的染色体变化,包括染色体数目及结构的异常,分别为-2,-6,-19,-16,-20,-Y, 8以及t(2;4)(p16;p15)。CML慢性期共有47例患者出现Ph 染色体,总阳性率为92.17%。5例加速及急变期患者均出现Ph 染色体,其中3例患者合并有复杂的染色体核型变化:-Y,del(10),I(9)。结论染色体核型分析对CML的疾病分期、进展、治疗及预后有重要的临床意义。FISH技术在CML染色体核型分析上可作为重要技术补充手段弥补CC检查的不足。     

Inhibition of the ABL Kinase Activity Blocks the Proliferation of BCR/ABLLeukemic Cells and Induces Apoptosis

ABSTRACT: The BCR/ABL fusion protein transforms myeloid stem cells. Both chronic myelogenous leukemias (CML) and a subset of acute lymphoblastic leukemias (ALL) are associated with the expression of BCR/ABL proteins. This knowledge has not yet been translated into any specific tool to control ABL driven neoplastic cells growth. CGP57148B is an ATP-competitive inhibitor of the ABL protein kinase; it has been shown to inhibit the kinase activity of ABL bothin vitroandin vivoand to inhibit the growth of v-abl and bcr/abl transfectants, as well as thein vitroformation of bone marrow (BM)-derived colonies in the presence of growth factors in some CML patients. These studies were performed to investigate the activity of CGP57148B on the spontaneous proliferation of both fresh and cultured, leukemic and normal, BCR/ABL positive and negative cells, and to study its mechanism of action. Six cell lines derived from BCR/ABL+ leukemias (K562, BV173, KCL22, KU812, MC3, LAMA84), thirteen BCR/ABL negative lines, both neoplastic (KG1, SU-DHL-1, U937, Daudi, NB4, NB4.306) and derived from normal cells (PHA blasts, LAK, fibroblasts, LCL, renal epithelial cells, endothelial cells, CD34⁺cells), and 14 fresh leukemic samples were tested using a tritiated thymidine uptake assay. Thein vivophosphorylation of the BCR/ABL protein was evaluated by western blot, while apoptosis was detected by the annexin V/propidium binding test. The induction of differentiation was assayed by immunofluorescence using multiple antibodies.All six BCR/ABL⁺lines showed a dose dependent inhibition of their spontaneous proliferative rate, which was not accompanied by differentiation. The treatment caused, within minutes, dephosphorylation of the BCR/ABL protein, followed in 16-24 hours by a decrease in cycling cells and induction of apoptosis. No significant inhibition of DNA synthesis was observed in any BCR/ABL negative normal or neoplastic line at concentrations ≤3 μM, with the exception of fibroblasts and CD34 cells. Proliferation inhibition was observed also when using fresh samples obtained from two Ph+ ALL and 12 consecutive CML patients. Induction of apoptosis was observed in these samples too.The activity of CGP57148B can be monitored inex vivoisolated or cultured cells using a simple and reproducible assay, without the need for exogenously added growth factors. This molecule possibly exerts its effects through the inhibition of the kinase activity of BCR/ABL and the subsequent initiation of apoptosis, without inducing cell differentiation. Some normal cells are also affected.These data support the use of CGP57148B in initial clinical studies; possible toxic effects on BM and fibroblast-derived cells will have to be closely monitored. Thein vivomonitoring of patients will have to be focused on the induction of apoptosis in leukemic cells.     

Secondary fusion proteins as a mechanism of BCR::ABL1 kinase-independent resistance in chronic myeloid leukaemia

Drug resistance in chronic myeloid leukaemia (CML) may occur via mutations in the causative BCR::ABL1 fusion or BCR::ABL1-independent mechanisms. We analysed 48 patients with BCR::ABL1-independent resistance for the presence of secondary fusion genes by RNA sequencing. We identified 10 of the most frequently detected secondary fusions in 21 patients. Validation studies, cell line models, gene expression analysis and drug screening revealed differences with respect to proliferation rate, differentiation and drug sensitivity. Notably, expression of RUNX1::MECOM led to resistance to ABL1 tyrosine kinase inhibitors in vitro. These results suggest secondary fusions contribute to BCR::ABL1-independent resistance and may be amenable to combined therapies.     

p185(BCR/ABL) has a lower sensitivity than p210(BCR/ABL) to the allosteric inhibitor GNF-2 in Philadelphia chromosome-positive acute lymphatic leukemia

Background

The t(9;22) translocation leads to the formation of the chimeric breakpoint cluster region/c-abl oncogene 1 (BCR/ABL) fusion gene on der22, the Philadelphia chromosome. The p185^BCR/ABL or the p210^BCR/ABL fusion proteins are encoded as a result of the translocation, depending on whether a “minor” or “major” breakpoint occurs, respectively. Both p185^BCR/ABL and p210^BCR/ABL exhibit constitutively activated ABL kinase activity. Through fusion to BCR the ABL kinase in p185^BCR/ABL and p210^BCR/ABL “escapes” the auto-inhibition mechanisms of c-ABL, such as allosteric inhibition. A novel class of compounds including GNF-2 restores allosteric inhibition of the kinase activity and the transformation potential of BCR/ABL. Here we investigated whether there are differences between p185^BCR/ABL and p210^BCR/ABL regarding their sensitivity towards allosteric inhibition by GNF-2 in models of Philadelphia chromosome-positive acute lymphatic leukemia.

Design and Methods

We investigated the anti-proliferative activity of GNF-2 in different Philadelphia chromosome-positive acute lymphatic leukemia models, such as cell lines, patient-derived long-term cultures and factor-dependent lymphatic Ba/F3 cells expressing either p185^BCR/ABL or p210^BCR/ABL and their resistance mutants.

Results

The inhibitory effects of GNF-2 differed constantly between p185^BCR/ABL and p210^BCR/ABL expressing cells. In all three Philadelphia chromosome-positive acute lymphatic leukemia models, p210^BCR/ABL-transformed cells were more sensitive to GNF-2 than were p185BCR/ABL-positive cells. Similar results were obtained for p185^BCR/ABL and the p210^BCR/ABL harboring resistance mutations.

Conclusions

Our data provide the first evidence of a differential response of p185^BCR/ABL- and p210^BCR/ABL- transformed cells to allosteric inhibition by GNF-2, which is of importance for the treatment of patients with Philadelphia chromosome-positive acute lymphatic leukemia.     

实时定量PCR监测造血干细胞移植后BCR/ABL融合基因的表达

目的监测慢性粒细胞白血病（CML）患者异基因造血干细胞移植（allo-HSCT）后BCR／ABL融合基因表达水平的动态变化,从而判断移植效果,指导临床治疗。方法应用Taqman实时定量RT-PCR方法,以B-actin作为内参照,检测10例初发CML患者及25例接受allo-HSCT治疗的CML患者在移植前、后不同时段外周血中BCR／ABL mRNA表达水平。结果15例接受移植的患者移植前、移植后1、2个月的NBCR／ABL（％）中位数分别为：6．57（0．14～38．83）、0．10（0～1．71）、0（0～0．52）,3个月后全部为0。移植后早期检测BCR／ABL转录本较移植前逐渐下降,移植后1、2个月NBCR／ABL（％）均较移植前下降（x^2均为13．07,P〈O．01）;移植后2个月较1个月下降（x^2=8．10,P〈0．01）。其余10例患者从移植后3～43个月不同时段起连续检测NBCR/ABL（％）也全部为0。结论实时定量RT-PCR方法检测CML患者的BCR／ABL融合基因的表达,操作迅速,灵敏度、特异性好,用于移植后微小残留病的检测对评估预后并指导临床治疗具有重要意义。