Patterns of BCR/ABL Gene Rearrangements in Chronic Myeloid Leukemia with Complex t(9;22) Using Fluorescence In Situ Hybridization (FISH)

Chronic myeloid leukemia (CML) is characterized by the reciprocal translocation t(9;22)(q34;q11.2) which fuses the ABL1 oncogene on chromosome 9 with the BCR gene on chromosome 22. It is the BCR/ABL protein that drives the neoplasm and the ABL/BCR is not necessary for the disease. In the majority of CML cases, the BCR/ABL fusion gene is cytogenetically recognizable as a small derivative chromosome 22(der 22), which is known as the Philadelphia (Ph) chromosome. However, approximately 2-10% of patients with CML involve cryptic or complex variant translocations with deletions on the der(9) and/or der(22) occuring in roughly 10-15% of CML cases. Fluorescence in situ hybridization (FISH) analysis can help identify deletions and complex or cryptic rearrangements. Various BCR/ABL FISH probes are available, which include dual color single fusion, dual color extra signal (ES), dual color dual fusion and tri color dual fusion probes. To test the utility of these probes, six patients diagnosed with CML carrying different complex variant Ph translocations were studied by G-banding and FISH analysis using the BCR/ABL ES, BCR/ABL dual color dual fusion, and BCR/ABL tricolor probes. There are differences among the probes in their ability to detect variant rearrangements, with or without accompanying chromoso me 9 and/or 22 deletions, and low level disease.     

ABL/BCR gene variant with two-step mechanism: Unusual localization and rare/novel chromosomal rearrangements in CML patients

Aims: Variant translocations involving 9q, 22q and at least one additional genomic locus occur in 5-10% of the patients with chronic myeloid leukemia (CML). The mechanisms for the formation of these variant translocations are not fully characterized. Here we report CML cases presenting a variant translocation indicating two-step mechanism with rare/novel chromosomal rearrangement. Methods: Karyotype analysis was performed on metaphases obtained through short-term cultures of bone marrow and blood. Detection of BCR-ABL fusion gene was performed using dual-color dual-fusion (D-FISH) and extra signal (ES) translocation probes. BAC-FISH was also carried out. Results: In Patient 1, the third partner chromosome was der(11)(p15) with a 2F2G1R signal pattern, which is an unusual signal pattern with the two-step mechanism. Patients 2 and 3 showed typical positive (2F1G1R) signal pattern. In Patient 2, both the chromosome 22s were involved in variant formation. The second fusion was observed below the BCR gene of the second homologue. In Patient 3 the third chromosome was der(13)(q14). The fourth patient showed a variant pattern with BCR/ABL-ES probe involving der(X)(q13) region. Conclusion: The presence of different rearrangements of both 9q34 and 22q11 regions highlights the genetic heterogeneity of this subgroup of CML. In each case with variants, further studies with FISH, BAC-FISH or more advanced technique such as microarray should be performed. Future studies should be performed to confirm the presence of true breakpoint hot spots and assess their implications in CML with variant Ph.     

Influence of BCR/ABL fusion proteins on the course of Ph leukemias

The hallmark of chronic myeloid leukemia (CML) and a subset of acute lymphoblastic leukemia (ALL) is the presence of the Philadelphia chromosome as a result of the t(9;22) translocation. This gene rearrangement results in the production of a novel oncoprotein, BCR/ABL, a constitutively active tyrosine kinase. There is compelling evidence that the malignant transformation by BCR/ABL is critically dependent on its Abl tyrosine kinase activity. Also the bcr part of the hybrid gene takes part in realization of the malignant phenotype. We supposed that additional mutations accumulate in this region of the BCR/ABL oncogene during the development of the malignant blast crisis in CML patients. In ALL patients having p210 fusion protein the mutations were supposed to be preexisting. Sequencing of PCR product of the BCR/ABL gene (Dbl, PH region) showed that along with single-nucleotide substitutions other mutations, mostly deletions, had occurred. In an ALL patient a deletion of the 5th exon was detected. The size of the deletions varied from 36 to 220 amino acids. For one case of blast crisis of CML changes in the character of actin organization were observed. Taking into account the functional role of these domains in the cell an etiological role of such mutations on the disease phenotype and leukemia progression is plausible.     

Highly precise breakpoint detection of chromosome balanced translocation in chronic myelogenous leukaemia: Case series

Chronic myelogenous leukaemia (CML) has a special phenomenon of chromosome translocation, which is called Philadelphia chromosome translocation. However, the detailed connection of this structure is troublesome and expensive to be identified. Low‐coverage whole genome sequencing (LCWGS) could not only detect the previously unknown chromosomal translocation, but also provide the breakpoint candidate small region (with an accuracy of ±200 bases). Importantly, the sequencing cost of LCWGS is about US$300. Then, with the Sanger DNA sequencing, the precise breakpoint can be determined at a single base level. In our project, with LCWGS, BCR and ABL1 are successfully identified to be disrupted in three CML patients (at chr22:23,632,356 and chr9:133,590,450; chr22:23,633,748 and chr9:133,635,781; chr22: 23,631,831 and chr9:133,598,513, respectively). Due to the reconnection after chromosome breakage, classical fusion gene (BCR::ABL1) was found in bone marrow and peripheral blood. The precise breakpoints were helpful to investigate the pathogenic mechanism of CML and could better guide the classification of CML subtypes. This LCWGS method is universal and can be used to detect all diseases related to chromosome variation, such as solid tumours, liquid tumours and birth defects.     

Deletion of ABL/BCR on der(9) associated with severe basophilia

Chronic basophilic leukemia is a rare form in chronic myeloid leukemia patients. Only limited number of reports are available. Herein, we describe a patient who presented with fatigue, weight loss, leucocytosis, prominent basophilia, and mild eosinophilia. On biopsy, bone marrow was hypercellular with marked basophils. The immunophenotype showed abnormal expression of CD7, which is suggestive of basophilic maturation. Chromosomal analysis from GTG-banded metaphases revealed Ph positivity, and fluorescence in situ hybridization (FISH) with BCR/ABL dual color, dual fusion probe showed single fusion on the der(22) chromosome and ABL/BCR fusion was deleted on the der(9) chromosome. The deletion (ABL/BCR) on der(9) may be associated with basophilia which may be also indicative of the transformation of CML to acute myeloid leukemia.     

A Case of ABL-BCR Negative, Philadelphia Positive CML with t(5;9)(q13;q34)

The Philadelphia chromosome is found in more than 90 percent of chronic myeloid leukemia (CML) patients. In most cases, it results from the reciprocal t(9;22)(q34;q11), with the ABL proto-oncogene from 9q34 fused to the breakpoint cluster region (BCR) locus on 22q11. In 5 to 10 percent of patients with CML, the Ph originates from variant translocations, involving various breakpoints in addition to 9q34 and 22q11. Here we report a rare case of a Philadelphia positive CML patient carrying t(5;9)(q13;q34) and deletion of ABL/BCR on der(9) as a separate event.     

多色-FISH技术的进展及其在分子生物医学上的应用

传统显带分析技术以每条染色体独特的显带带型为依据,提供染色体形态结构的基本信息,用于染色体核型的初步分析。然而有些染色体重排由于涉及的片断太小或具有相似的带型,用该方法难以探测或准确描绘。多元荧光原位杂交(M-FISH),光谱核型分析(SKY),FISH-显带分析技术是染色体特异的多色荧光原位杂交技术(mFISH)。它们能够探测出传统显带分析不能发现的染色体异常,提供更准确的核型。M-FISH和SKY均以组合标记的染色体涂染探针共杂交为基础,二者的不同在于观察仪器和分析方法上。它们可对中期染色体涂片进行快速准确分析,描绘复杂核型,确认标记染色体,主要用于恶性疾病的细胞遗传学诊断分析。FISH-显带分析技术以FISH技术为基础,能同时检测多条比染色体臂短的染色体亚区域。符合该定义的FISH-显带分析技术各有特点,其共同特点是都能产生DNA特异的染色体条带。这些条带有更多色彩,能提供更多信息。FISH-显带分析技术已经成功地被用于进化生物学、放射生物学以及核结构的研究,同时也被用于产前、产后以及肿瘤细胞遗传学诊断,是很有潜力的工具。     

Evolution of BCR/ABL Gene Mutation in CML Is Time Dependent and Dependent on the Pressure Exerted by Tyrosine Kinase Inhibitor

Background

Mutations in the ABL kinase domain and SH3-SH2 domain of the BCR/ABL gene and amplification of the Philadelphia chromosome are the two important BCR/ABL dependent mechanisms of imatinib resistance. Here, we intended to study the role played by TKI, imatinib, in selection of gene mutations and development of chromosomal abnormalities in Indian CML patients.

Methods

Direct sequencing methodology was employed to detect mutations and conventional cytogenetics was done to identify Philadelphia duplication.

Results

Among the different mechanisms of imatinib resistance, kinase domain mutations (39%) of the BCR/ABL gene were seen to be more prevalent, followed by mutations in the SH3-SH2 domain (4%) and then BCR/ABL amplification with the least frequency (1%). The median duration of occurrence of mutation was significantly shorter for patients with front line imatinib than those pre-treated with hydroxyurea. Patients with high Sokal score (p = 0.003) showed significantly higher incidence of mutations, as compared to patients with low/intermediate score. Impact of mutations on the clinical outcome in AP and BC was observed to be insignificant. Of the 94 imatinib resistant patients, only 1 patient exhibited duplication of Philadelphia chromosome, suggesting a less frequent occurrence of this abnormality in Indian CML patients.

Conclusion

Close monitoring at regular intervals and proper analysis of the disease resistance would facilitate early detection of resistance and thus aid in the selection of the most appropriate therapy.     

Characterization of cryptic rearrangements, deletion, complex variants of PML, RARA in acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is characterized by a reciprocal translocation t(15;17)(q22;q21) leading to the disruption of Promyelocytic leukemia (PML) and Retionic Acid Receptor Alpha (RARA) followed by reciprocal PML-RARA fusion in 90% of the cases. Fluorescence in situ hybridization (FISH) has overcome the hurdles of unavailability of abnormal and/or lack of metaphase cells, and detection of cryptic, submicroscopic rearrangements. In the present study, besides diagnostic approach we sought to analyze these cases for identification and characterization of cryptic rearrangements, deletion variants and unknown RARA translocation variants by application of D-FISH and RARA break-apart probe strategy on interphase and metaphase cells in a large series of 200 cases of APL. Forty cases (20%) had atypical PML-RARA and/or RARA variants. D-FISH with PML/RARA probe helped identification of RARA insertion to PML. By application of D-FISH on metaphase cells, we documented that translocation of 15 to 17 leads to 17q deletion which results in loss of reciprocal fusion and/or residual RARA on der(17). Among the complex variants of t(15;17), PML-RARA fusion followed by residual RARA insertion closed to PML-RARA on der(15) was unique and unusual. FISH with break-apart RARA probe on metaphase cells was found to be a very efficient strategy to detect unknown RARA variant translocations like t(11;17)(q23;q21), t(11;17)(q13;q21) and t(2;17)(p21;q21). These findings proved that D-FISH and break-apart probe strategy has potential to detect primary as well as secondary additional aberrations of PML, RARA and other additional loci. The long-term clinical follow-up is essential to evaluate the clinical importance of these findings.     

The Functional Interplay Between the t(9;22)-Associated Fusion Proteins BCR/ABL and ABL/BCR in Philadelphia Chromosome-Positive Acute Lymphatic Leukemia

The hallmark of Philadelphia chromosome positive (Ph⁺) leukemia is the BCR/ABL kinase, which is successfully targeted by selective ATP competitors. However, inhibition of BCR/ABL alone is unable to eradicate Ph⁺ leukemia. The t(9;22) is a reciprocal translocation which encodes not only for the der22 (Philadelphia chromosome) related BCR/ABL, but also for der9 related ABL/BCR fusion proteins, which can be detected in 65% of patients with chronic myeloid leukemia (CML) and 100% of patients with Ph⁺ acute lymphatic leukemia (ALL). ABL/BCRs are oncogenes able to influence the lineage commitment of hematopoietic progenitors. Aim of this study was to further disclose the role of p96^ABL/BCR for the pathogenesis of Ph⁺ ALL. The co-expression of p96^ABL/BCR enhanced the kinase activity and as a consequence, the transformation potential of p185^BCR/ABL. Targeting p96^ABL/BCR by RNAi inhibited growth of Ph⁺ ALL cell lines and Ph⁺ ALL patient-derived long-term cultures (PD-LTCs). Our in vitro and in vivo stem cell studies further revealed a functional hierarchy of p96^ABL/BCR and p185^BCR/ABL in hematopoietic stem cells. Co-expression of p96^ABL/BCR abolished the capacity of p185^BCR/ABL to induce a CML-like disease and led to the induction of ALL. Taken together our here presented data reveal an important role of p96^ABL/BCR for the pathogenesis of Ph⁺ ALL.     

Bcr/Abl interferes with the Fanconi anemia/BRCA pathway: implications in the chromosomal instability of chronic myeloid leukemia cells

Chronic myeloid leukemia (CML) is a malignant clonal disorder of the hematopoietic system caused by the expression of the BCR/ABL fusion oncogene. Although it is well known that CML cells are genetically unstable, the mechanisms accounting for this genomic instability are still poorly understood. Because the Fanconi anemia (FA) pathway is believed to control several mechanisms of DNA repair, we investigated whether this pathway was disrupted in CML cells. Our data show that CML cells have a defective capacity to generate FANCD2 nuclear foci, either in dividing cells or after DNA damage. Similarly, human cord blood CD34(+) cells transduced with BCR/ABL retroviral vectors showed impaired FANCD2 foci formation, whereas FANCD2 monoubiquitination in these cells was unaffected. Soon after the transduction of CD34(+) cells with BCR/ABL retroviral vectors a high proportion of cells with supernumerary centrosomes was observed. Similarly, BCR/ABL induced a high proportion of chromosomal abnormalities, while mediated a cell survival advantage after exposure to DNA cross-linking agents. Significantly, both the impaired formation of FANCD2 nuclear foci, and also the predisposition of BCR/ABL cells to develop centrosomal and chromosomal aberrations were reverted by the ectopic expression of BRCA1. Taken together, our data show for the first time a disruption of the FA/BRCA pathway in BCR/ABL cells, suggesting that this defective pathway should play an important role in the genomic instability of CML by the co-occurrence of centrosomal amplification and DNA repair deficiencies.     

Molecular definition of high-resolution multicolor banding probes: first within the human DNA sequence anchored FISH banding probe set.

Fluorescence in situ hybridization (FISH) banding approaches are standard for the exact characterization of simple, complex, and even cryptic chromosomal aberrations within the human genome. The most frequently applied FISH banding technique is the multicolor banding approach, also abbreviated as m-band, MCB, or in its whole genomic variant multitude MCB (mMCB). MCB allows the differentiation of chromosome region-specific areas at the GTG band and sub-band level and is based on region-specific microdissection libraries, producing changing fluorescence intensity ratios along the chromosomes. The latter are used to assign different pseudocolors to specific chromosomal regions. Here we present the first bacterial artificial chromosome (BAC) array comparative genomic hybridization (aCGH) mapped, comprehensive, genome-wide human MCB probe set. All 169 region-specific microdissection libraries were characterized in detail for their size and the regions of overlap. In summary, the unique possibilities of the MCB technique to characterize chromosomal breakpoints in one FISH experiment are now complemented by the feature of being anchored within the human DNA sequence at the BAC level.     

BCR/ABL Directly Inhibits Expression of SHIP, an SH2-Containing Polyinositol-5-Phosphatase Involved in the Regulation of Hematopoiesis

The BCR/ABL oncogene causes chronic myelogenous leukemia (CML), a myeloproliferative disorder characterized by clonal expansion of hematopoietic progenitor cells and granulocyte lineage cells. The SH2-containing inositol-5-phosphatase SHIP is a 145-kDa protein which has been shown to regulate hematopoiesis in mice. Targeted disruption of the murine SHIP gene results in a myeloproliferative syndrome characterized by a dramatic increase in numbers of granulocyte-macrophage progenitor cells in the marrow and spleen. Also, hematopoietic progenitor cells from SHIP(-/-) mice are hyperresponsive to certain hematopoietic growth factors, a phenotype very similar to the effects of BCR/ABL in murine cells. In a series of BCR/ABL-transformed hematopoietic cell lines, Philadelphia chromosome (Ph)-positive cell lines, and primary cells from patients with CML, the expression of SHIP was found to be absent or substantially reduced compared to untransformed cell lines or leukemia cells lacking BCR/ABL. Ba/F3 cells in which expression of BCR/ABL was under the control of a tetracycline-inducible promoter showed rapid loss of p145 SHIP, coincident with induction of BCR/ABL expression. Also, an ABL-specific tyrosine kinase inhibitor, CGP57148B (STI571), rapidly caused reexpression of SHIP, indicating that BCR/ABL directly, but reversibly, regulates the expression of SHIP protein. The estimated half-life of SHIP protein was reduced from 18 h to less than 3 h. However, SHIP mRNA also decreased in response to BCR/ABL, suggesting that SHIP protein levels could be affected by more than one mechanism. Reexpression of SHIP in BCR/ABL-transformed Ba/F3 cells altered the biological behavior of cells in culture. The reduction of SHIP due to BCR/ABL is likely to directly contribute to the pathogenesis of CML.     

Down-regulation of miR-199b associated with imatinib drug resistance in 9q34.1 deleted BCR/ABL positive CML patients

Chronic myeloid leukemia (CML) occurs due to t(9,22) (q34;q11) and molecularly BCR/ABL gene fusion. About 15–18% Philadelphia positive CML patients have gene deletions around the translocation breakpoints on 9q34.1. The microRNAs (miRNAs), namely miR-219-2 and miR-199b, centromeric to the ABL1 gene are frequently lost in CML patients. We have designed a study to determine miR-219-2 and miR-199b expression levels which would help to understand the prognosis of imatinib therapy. A total of 150 CML patients were analyzed to identify 9q deletion. Fluorescent in-situ hybridization (FISH) was performed using BCR/ABL dual color, dual fusion probe to study the signal pattern and BAC probes for miR-199b and miR-219-2 (RP11-339B21 and RP11-395P17) to study the miRNA deletions. The expression level of miRNA was analyzed by real-time polymerase chain reaction (RT-PCR). FISH analysis revealed 9q34.1 deletion in 34 (23%) CML patients. The deletions were not detected using BAC probes for miRNAs in 9q deleted patients. The expression analysis showed down-regulation of miR-199b and miR-219-2 in the 9q deleted patients (34 CML) as compared to a pool of patients without deletion. However, miR-199b (9q34.11) was significantly (p = 0.001) down-regulated compared to miR-219-2. The follow-up study showed that the miR-199b was found to be strongly associated with imatinib resistance, as 44.11% patients showed resistance to imatinib therapy. Hence, the deletion in 9q34.1 region (ABL) plays an important role in disease pathogenesis. Eventually, miRNAs can provide new therapeutic strategies and can be used as a prognostic indicator.     

Comparative study of DNA damage, cell cycle and apoptosis in human K562 and CCRF-CEM leukemia cells: role of BCR/ABL in therapeutic resistance

The Philadelphia translocation t(9;22) resulting in the bcr/abl fusion gene is the pathogenic principle of almost 95% of human chronic myelogenous leukemia (CML). Imatinib mesylate (STI571) is a specific inhibitor of the BCR/ABL fusion tyrosine kinase that exhibits potent antileukemic effects in CML. BCR/ABL-positive K562 and -negative CCRF-CEM human leukemia cells were investigated. MTT survival assay and clonogenic test of the cell proliferation ability were used to estimate resistance against idarubicin. DNA damage after cell treatment with the drug at the concentrations from 0.001 to 3 microM with or without STI571 pre-treatment were examined by the alkaline comet assay. We found that the level of DNA damages was lower in K562 cells after STI571 pre-treatment. It is suggested that BCR/ABL activity may promote genomic instability, moreover K562 cells were found to be resistant to the drug treatment. Further, we provided evidence of apoptosis inhibition in BCR/ABL-positive cells using caspase-3 activity colorimetric assay and DAPI nuclear staining for chromatin condensation. We suggest that these processes associated with cell cycle arrest in G2/M checkpoint detected in K562 BCR/ABL-positive compared to CCRF-CEM cells without BCR/ABL expression might promote clone selection resistance to drug treatment.     

On the Power of Additional and Complex Chromosomal Aberrations in CML

Unregulated proliferation of mainly myeloid bone marrow cells and genetic changes in the hematopoietic stem cell system are important features in Chronic Myeloid Leukemia (CML). In clinical diagnosis of CML, classical banding techniques, fluorescence in situ hybridization (FISH) probing for the Philadelphia chromosome (Ph) or polymerase chain reaction amplifying the fusion products of the BCR-ABL fusion are state of the art techniques. Nevertheless, the genome of CML patients harbors many more cytogenetic changes. These might be hidden in subpopulations due to clonal events or involved in extremely complex aberrations. To identify these additional changes, several cytogenetic and molecular genetic techniques could be applied. Nevertheless, it has been proposed that identifying these aberrations is time consuming and costly and since they cannot be converted into a benefit for the patients, the necessity to perform these investigations has been questioned. In the times where highly specialized medicine is advancing into several areas of cancer, this attitude needs to be reassessed. Therefore, we looked at the usefulness of a combination of different techniques to unravel the genetic changes in CML patients and to identify new chromosomal aberrations, which potentially can be correlated to different stages of the disease and the strength of therapy resistance. We are convinced that the combination of these techniques could be extremely useful in unraveling even the most complex karyotypes and in dissecting different clones contributing to the disease. We propose that by doing so, this would improve CML diagnostic and prognostic findings, especially with regard to CML resistance mechanisms and new therapeutic strategies.     

Selective induction of apoptosis in Philadelphia chromosome-positive chronic myelogenous leukemia cells by an inhibitor of BCR - ABL tyrosine kinase, CGP 57148

The BCR - ABL tyrosine kinase has been implicated as the cause of Philadelphia chromosome (Ph1)-positive leukemias. We report herein that CGP 57148, a selective inhibitor of the ABL tyrosine kinase, caused apoptosis specifically in bcr - abl-positive chronic myelogenous leukemia (CML) cells, K562 and KYO-1. Upon treatment with CGP 57148, CRKL, a specific substrate for BCR - ABL that propagates signals via phosphatidylinositol-3' kinase (PI3K), was dephosphorylated, indicating inhibition of BCR - ABL tyrosine kinase at the cellular level. Likewise, MAPK/ERK, a downstream mediator of Ras, was also dephosphorylated. Caspase activation and cleavage of retinoblastoma protein (pRB) accompanied the development of CGP 57148-induced apoptosis. Inhibition of caspase suppressed apoptosis and the cleavage of pRB, and in turn arrested cells in the G1 phase. These results indicate that CGP 57148 shows apoptogenic and anti-proliferative effects on bcr - abl-positive cells by blocking BCR - ABL-initiated signaling pathways.