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。     

Complex chromosome rearrangements may locate the bcr/abl fusion gene sites other than 22q11

BACKGROUND AND OBJECTIVE: From 5-8% of Philadelphia (Ph) positive patients with chronic myeloid leukemia (CML) show variant translocations in which at least a third chromosome in addition to 9q34 and 22q11 is involved. The formation mechanisms and clinical significance of variant Ph translocations are still unclear. The BCR/ABL chimeric gene encoding for chimeric proteins is always present and maps on the 22q- regardless of the type of translocation. We studied two apparently Ph negative CML patients with unusual karyotypes both showing a typical b3a2 rearrangement. DESIGN AND METHODS: Dual-color fluorescence in situ hybridization (FISH) can visualize BCR and ABL genes and localize the BCR/ABL fusion gene. We used FISH to study the formation mechanisms of variant Ph translocations in two patients. RESULTS: The chimeric BCR/ABL gene was located on a locus other than the expected 22q11 in both patients. In the first case the fusion signal was present on the 9q34 band whereas in the second patient it was detected on chromosome 8, involved in masked Ph formation. INTERPRETATION AND CONCLUSIONS: The location of the hybrid BCR/ABL gene on chromosomes other than 22q- is a rare event which can only be observed using the FISH technique. When these unusual translocations occur the hypothesis most often put forward is that several consecutive cytogenetic events have taken place. The factors which regulate the formation of these breakpoints have yet to be clarified. The FISH technique allows the identification of chromosome rearrangements that could not otherwise be detected by conventional banding procedures. The location of the hybrid BCR/ABL gene on sites other than 22q11 represents a rare type of variant Ph translocation.The real frequency and clinical significance of such rearrangements need to be investigated.     

Adaphostin-induced oxidative stress overcomes BCR/ABL mutation-dependent and -independent imatinib resistance

The BCR/ABL kinase has been targeted for the treatment of chronic myelogenous leukemia (CML) by imatinib mesylate. While imatinib has been extremely effective for chronic phase CML, blast crisis CML and Ph+ acute lymphoblastic leukemia (ALL) are often resistant. In particular, mutation of the T315 residue in the bcr/abl activation loop renders cells highly resistant to imatinib and to second-generation kinase inhibitors such as BMS-354825 or AMN107. Adaphostin is a tyrphostin that was originally intended to inhibit the BCR/ABL kinase by competing with its peptide substrates. Recent findings have in addition implicated reactive oxygen species (ROS) in the cytotoxic mechanism of adaphostin. In view of this unique mode of action, we examined the effects of adaphostin on numerous imatinib-resistant leukemia models, including imatinib-resistant CML and Ph+ ALL cell lines, cells harboring point mutations in BCR/ABL, and specimens from imatinib-resistant CML patients, using assays for intracellular ROS, apoptosis, and clonogenicity. Every model of imatinib resistance examined remained fully sensitive to adaphostin-induced cell death. Collectively, these data suggest that ROS generation by adaphostin overcomes even the most potent imatinib resistance in CML and Ph+ ALL.     

Patient perceptions of second transplants in myeloma: impact on recruitment in the British Society of Blood and Marrow Transplantation/UK Myeloma Forum Myeloma X Relapse (Intensive) Trial

The BCR/ABL1 fusion gene, usually carried by the Philadelphia chromosome (Ph) resulting from t(9;22)(q34;q11) or variants, is pathognomonic for chronic myeloid leukaemia (CML). It is also occasionally found in acute lymphoblastic leukaemia (ALL) mostly in adults and rarely in de novo acute myeloid leukaemia (AML). Array Comparative Genomic Hybridization (aCGH) was used to study six Ph(+)AML, three bi‐lineage and four Ph(+)ALL searching for specific genomic profiles. Surprisingly, loss of the IKZF1 and/or CDKN2A genes, the hallmark of Ph(+)ALL, were recurrent findings in Ph(+)AML and accompanied cryptic deletions within the immunoglobulin and T cell receptor genes. The latter two losses have been shown to be part of ‘hot spot’ genome imbalances associated with BCR/ABL1 positive pre‐B lymphoid phenotype in CML and Ph(+)ALL. We applied Significance Analysis of Microarrays (SAM) to data from the ‘hot spot’ regions to the Ph(+)AML and a further 40 BCR/ABL1(+) samples looking for differentiating features. After exclusion of the most dominant markers, SAM identified aberrations unique to de novo Ph(+)AML that involved relevant genes. While the biological and clinical significance of this specific genome signature remains to be uncovered, the unique loss within the immunoglobulin genes provides a simple test to enable the differentiation of clinically similar de novo Ph(+) AML and myeloid blast crisis of CML.     

Rearrangement of the breakpoint cluster region and expression of P210 BCR-ABL in a "masked" Philadelphia chromosome-positive acute myeloid leukemia

The Philadelphia (Ph) translocation t(9;22)(q34;q11) occurs frequently in chronic myeloid leukemia (CML) but is less common in acute lymphoblastic leukemia (ALL) and rare in acute myeloid leukemia (AML). In most cases of CML and some cases of Ph+ ALL the protooncogene ABL from 9q34 is translocated to the breakpoint cluster region (bcr) of the BCR gene at 22q11 to form a chimeric gene encoding a novel 210-kd protein (P210 BCR-ABL) with enhanced tyrosine kinase activity. In other patients with Ph+ ALL and Ph+ AML, the breakpoint probably occurs in the first intron of the BCR gene; this results in a smaller chimeric gene which encodes a P190 BCR-ABL. We studied a patient with AML (FAB M6) arising de novo who had a "masked" Ph chromosome in association with extensive karyotypic changes. The leukemic cells initially showed rearrangement of the bcr, presence of a hybrid mRNA, and expression of the P210 BCR-ABL. These changes were absent in remission. These results support the concept that the BCR-ABL chimeric gene plays a crucial role in leukemogenesis but suggest that factors other than the position of the breakpoint in the BCR gene determine the lineage of the target cell for malignant transformation.     

Efficiency of interphase fluorescence in situ hybridization for BCR/ABL on peripheral blood smears for monitoring of CML patients: a comparison with bone marrow findings

Conventional cytogenetic analysis (CCA) is the standard method for monitoring of the Philadelphia (Ph) chromosome in chronic myeloid leukemia (CML). Evaluation of breakpoint cluster region/abelson murine leukemia (BCR/ABL) fusion using interphase fluorescence in situ hybridization on peripheral blood smears (PB-FISH) might be another approach allowing more frequent and less invasive follow-up investigations. Herein, BCR/ABL fusion gene was assessed on 21 PB smears from 16 CML patients in chronic phase. Results of PB-FISH were compared with those of CCA and interphase FISH on bone marrow aspirates (BM-FISH). PB-FISH analysis was combined with CD3 immunophenotyping that allowed simultaneous investigation of the leukemic status of CD3(+) T lymphocytes and scoring CD3(-) cells for BCR/ABL fusion gene. Moreover, the frequency of BCR/ABL fusion in nonlymphoid PB cells was estimated according to the differential leukocyte counts. The incidence of BCR/ABL(+) fusion signals in CD3(+) T cells of CML patients was 5.3% (SD +/- 1.9) and did not exceed the normal cut-off value of 8%. A significant correlation (P < 0.001) was found between results of PB-FISH and methods of BM analysis (CCA or BM-FISH). Correction of PB-FISH results to include only nonlymphoid or CD3(-) cells reduced the mean of differences and improved agreement between PB-FISH and CCA or BM-FISH methods. The best agreement was noted between CCA and PB-FISH on nonlymphoid cells. On the other hand, results of BM-FISH agreed well with those of PB-FISH on CD3(-) cells. These findings imply that PB-FISH on nonlymphoid or CD3(-) cells is reliable and may replace BM analysis for monitoring of response to treatment in CML patients.     

High frequency of point mutations clustered within the adenosine triphosphate-binding region of BCR/ABL in patients with chronic myeloid leukemia or Ph-positive acute lymphoblastic leukemia who develop imatinib (STI571) resistance

Point mutations were found in the adenosine triphosphate (ATP) binding region of BCR/ABL in 12 of 18 patients with chronic myeloid leukemia (CML) or Ph-positive acute lymphoblastic leukemia (Ph(+) ALL) and imatinib resistance (defined as loss of established hematologic response), but they were found in only 1 of 10 patients with CML with imatinib refractoriness (failure to achieve cytogenetic response). In 10 of 10 patients for whom samples were available, the mutation was not detected before the initiation of imatinib therapy. Three mutations (T315I, Y253H, and F317L present in 3, 1, and 1 patients, respectively) have a predicted role in abrogating imatinib binding to BCR/ABL, whereas 3 other mutations (E255K, G250E, and M351T, present in 4, 2, and 2 patients, respectively) do not. Thus we confirm a high frequency of mutations clustered within the ATP-binding region of BCR/ABL in resistant patients. Screening may allow intervention before relapse by identifying emerging mutations with defined impacts on imatinib binding. Certain mutations may respond to higher doses of imatinib, whereas other mutations may mandate switching to another therapeutic strategy.     

Complex variant Philadelphia translocations involving the short arm of chromosome 6 in chronic myeloid leukemia

BACKGROUND AND OBJECTIVES: Around 5% of chronic myeloid leukemias (CML) are characterized by complex variant Philadelphia (Ph) translocations involving one or more chromosomal regions in addition to 9 and 22. The BCR/ABL1 fusion gene is usually found on der(22). The additional gene(s) involved in complex variant Ph rearrangements have not been characterized. DESIGN AND METHODS: We performed fluorescent in situ hybridization (FISH) in three complex variant Ph translocations involving the short arm of chromosome 6 in addition to 9 and 22. The BCR/ABL1 D-FISH probe was applied to localize the BCR/ABL1 fusion gene as well as the 5'ABL1 and the 3'BCR. Locus-specific probes were used to narrow the 6p breakpoint. RESULTS: In all cases the BCR/ABL1 fusion gene was located on the Ph chromosome whereas the reciprocal ABL1/BCR gene was detected only in patient #2. On 6p, breakpoints were narrowed to three different regions: centromeric to the human major histocompatibility complex (MHC), between PAC 524E15 and PAC162J16, in the first patient, and telomeric to the MHC, between PAC 329A5 and PAC 145H9, and between PAC 136B1 and PAC 206F19, in the second and third patients, respectively. In patients #2 and 3 a chromosomal rearrangement different from a true complex variant was discovered. In both cases, a classical t(9;22) was associated with an additional translocation involving the der(9)t(9;22). INTERPRETATION AND CONCLUSIONS: Rearrangements at 6p in complex Ph aberrations involve more than one gene/locus. Classical t(9;22), masked by additional chromosomal rearrangements, can resemble complex variant Ph translocations, and can be detected only using appropriate FISH probes.     

Clonal eosinophils are a morphologic hallmark of ETV6/ABL1 positive acute myeloid leukemia

BACKGROUND AND OBJECTIVES: The ETV6 gene undergoes rearrangements with tyrosine kinases in hematologic malignancies and solid tumors. ETV6/ABL1 chimeric proteins have been detected both in lymphoid and myeloid disorders. Our objective was to study two new cases of ETV6/ABL1-positive acute myeloid leukemia (AML) and to focus on bone marrow morphology and on molecular cytogenetics of eosinophilic cells. DESIGN AND METHODS: Fluorescence in situ hybridization (FISH) was performed in two AML cases with different translocations, i.e. t(8;12)(p21;p13) and t(9;12) (q34; p13). We used probes for the short arm of chromosome 12, for ABL1 and BCR, for centromeric regions, and for whole chromosome arms. Polymerase chain reaction (PCR) was carried out by applying primers selected for the ETV6 gene. RESULTS: In both cases, bone marrow morphology was characterized by trilineage dysplasia and increased abnormal eosinophils. FISH showed the 5'ETV6 translocated to chromosome 8 in patient #1, and to chromosome 9 in patient #2. A 3' PCR identified chimeric products resulting from fusion between ETV6 exon 4 or exon 5, and ABL1 exon 2. Accordingly, an ETV6/ABL1 fusion signal was detected on der(8) in patient #1, and on der(9) in patient #2. Using interphase FISH abnormal bone marrow eosinophils were proved to belong to the neoplastic clone, carrying the ETV6 rearrangement. INTERPRETATION AND CONCLUSIONS: Our findings provide new information on the heterogeneity of conventional cytogenetics in ETV6/ABL1 positive leukemias, and indicate the putative target cell in this AML is an immature precursor capable of terminally differentiating towards eosinophils.     

Location of the BCR/ABL Fusion Genes on Both Chromosomes 9 in Ph Negative Young CML Patients: An Indian Experience

The BCR/ABL gene rearrangement is cytogenetically visualized in most chronic myeloid leukemia (CML) cases. About 5–10 % of CML patients lack its cytogenetic evidence, however, shows BCR/ABL fusion by molecular methods. We describe two CML patients with Philadelphia (Ph) negative (−ve) and BCR/ABL positive by fluorescence in situ hybridization (FISH). Both the cases were in chronic phase at diagnosis. Conventional cytogenetics and different FISH assays were adopted using BCR/ABL probes. Home-brew FISH assay using bacterial artificial clone (BAC) for BAC-CTA/bk 299D3 for chromosomal region 22q13.31-q13.32 was performed in case 1. Both the patients were Ph-ve. In first case, dual color dual fusion (DCDF)-FISH studies revealed 1 Red (R) 2 Green (G) 1 Fusion (F) signal pattern in 80 % of cells indicating BCR/ABL fusion signals on chromosomes 9 instead of Ph and 2G2F signal pattern in 20 % of cells indicating two BCR/ABL fusions on both chromosomes 9q34 on presentation. In second case, FISH studies revealed the 1R1G1F signal pattern indicating BCR/ABL fusion signals on chromosomes 9 instead of Ph in 100 % of cells at presentation. During follow-up, both the patients exhibited 2G2F signal pattern indicating two BCR/ABL fusions on both chromosomes 9q34, which indicated a clonal evolution in 100 % cells. Both the patients did not achieve therapeutic response. Relocation of BCR/ABL fusion sequence on sites other than 22q11 represents a rare type of variant Ph, the present study highlights the hot spots involved in CML pathogenesis and signifies their implications in Ph−ve BCR/ABL positive CML. This study demonstrated the genetic heterogeneity of this subgroup of CML and strongly emphasized the role of metaphase FISH, especially in Ph−ve CML cases, as it detects variations of the classical t(9;22).     

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.     

Primary chromosomal rearrangements of leukemia are frequently accompanied by extensive submicroscopic deletions and may lead to altered prognosis

BCR/ABL fluorescent in situ hybridization study of chronic myeloid leukemia (CML) and Philadelphia(+) (Ph(+)) acute lymphoid leukemia (ALL) indicated that approximately 9% of patients exhibited an atypical hybridization pattern consistent with a submicroscopic deletion of the 5' region of ABL and the 3' region of the BCR genes on the 9q(+) chromosome. The CML patients with deletions had a shorter survival time and a high relapse rate following bone marrow transplant. Since deletions are associated with both Ph(+) CML and ALL, it seemed probable that other leukemia-associated genomic rearrangements may also have submicroscopic deletions. This hypothesis was confirmed by the detection of deletions of the 3' regions of the CBFB and the MLL genes in AML M4 patients with inv(16) and in patients with ALL and AML associated with MLL gene translocations, respectively. In contrast, analysis of the AML M3 group of patients and AML M2 showed that similar large deletions were not frequently associated with the t(15;17) or t(8;21) translocations. Analysis of sequence data from each of the breakpoint regions suggested that large submicroscopic deletions occur in regions with a high overall density of Alu sequence repeats. These findings are the first to show that the process of deletion formation is not disease specific in leukemia and also implicate that the presence of repetitive DNA in the vicinity of breakpoint regions may facilitate the generation of submicroscopic deletions. Such deletions could lead to the loss of one or more genes, and the associated haploinsufficiency may result in the observed differences in clinical behavior. (Blood. 2001;97:3581-3588)     

DETECTION OF REARRANGEMENT WITHIN THE BREAKPOINT CLUSTER REGION OF CHROMOSOME 22 IN THE DIAGNOSIS OF CHRONIC MYELOID LEUKEMIA

Chronic myeloid leukemia (CML) is characterised by the presence of a Philadelphia (Ph) chromosome in approximately 95% of patients. Molecular analysis has shown that the Ph chromosome translocation breakpoints are clustered within 5.8kb on chromosome 22 (breakpoint cluster region or bcr). This has facilitated the diagnosis of CML by nucleic acid hybridisation using probes specific for the bcr to detect DNA rearrangement in this region. Forty patients diagnosed with CML, including four with variant Ph chromosome translocations and three with normal karyotypes were analysed for rearrangement within the bcr. All except one patient with Ph negative CML had rearrangement within the bcr. In contrast, none of the patients diagnosed with other hematological disorders such as the myelodysplastic or myeloproliferative syndromes (16 patients), acute myeloid leukemia (AML) (six patients), acute lymphoblastic leukemia (ALL) (five patients), including Ph positive ALL (two patients), showed rearrangement within the bcr. Analysis for rearrangement within the bcr is useful in the diagnosis of CML, especially when cytogenetic analysis is unsuccessful or in patients with normal karyotypes or variant Ph chromosome translocations.     

A murine model of CML blast crisis induced by cooperation between BCR/ABL and NUP98/HOXA9

Constitutive activation of tyrosine kinases, such as the BCR/ABL fusion associated with t(9;22)(q34;q22), is a hallmark of chronic myeloid leukemia (CML) syndromes in humans. Expression of BCR/ABL is both necessary and sufficient to cause a chronic myeloproliferative syndrome in murine bone marrow transplantation models, and absolutely depends on kinase activity. Progression of CML to acute leukemia (blast crisis) in humans has been associated with acquisition of secondary chromosomal translocations, including the t(7;11)(p15;p15) resulting in the NUP98/HOXA9 fusion protein. We demonstrate that BCR/ABL cooperates with NUP98/HOXA9 to cause blast crisis in a murine model. The phenotype depends both on expression of BCR/ABL and NUP98/HOXA9, but tumors retain sensitivity to the ABL inhibitor STI571 in vitro and in vivo. This paradigm is applicable to other constitutively activated tyrosine kinases such as TEL/PDGFbetaR. These experiments document cooperative effects between constitutively activated tyrosine kinases, which confer proliferative and survival properties to hematopoietic cells, with mutations that impair differentiation, such as the NUP98/HOXA9, giving rise to the acute myeloid leukemia (AML) phenotype. Furthermore, these data indicate that despite acquisition of additional mutations, CML blast crisis cells retain their dependence on BCR/ABL for proliferation and survival.