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.     

Nucleotide sequence of both reciprocal translocation junction regions in a patient with Ph positive acute lymphoblastic leukaemia, with a breakpoint within the first intron of the BCR gene.

Breakpoints on chromosome 22 in the translocation t(9;22) found in Philadelphia positive acute lymphoblastic leukaemia patients fall within two categories. In the first the breakpoint is localized within the breakpoint cluster region of the BCR gene, analogous to the chromosome 22 breakpoint in chronic myeloid leukaemia. The second category has a breakpoint 5' of this area, but still within the BCR gene. We have previously shown that these breakpoints occur within the first intron of the BCR gene and cloned the 9q+ junction from such a patient. We have now determined the sequences around the breakpoints on both translocation partners from this patient as well as the germline regions. The chromosome 9 ABL sequence around the breakpoint shows homology to the consensus Alu sequence whereas the chromosome 22 BCR sequence does not. At the junction there is a 6 bp duplication of the chromosome 22 sequence which is present both in the 9q+ and in the 22q- translocation products. Possible mechanisms for the generation of the translocation are discussed.     

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.     

Reciprocal t(9;22) ABL/BCR Fusion Proteins: Leukemogenic Potential and Effects on B Cell Commitment

Background

t(9;22) is a balanced translocation, and the chromosome 22 breakpoints (Philadelphia chromosome – Ph⁺) determine formation of different fusion genes that are associated with either Ph⁺ acute lymphatic leukemia (Ph⁺ ALL) or chronic myeloid leukemia (CML). The “minor” breakpoint in Ph⁺ ALL encodes p185^BCR/ABL from der22 and p96^ABL/BCR from der9. The “major” breakpoint in CML encodes p210^BCR/ABL and p40^ABL/BCR. Herein, we investigated the leukemogenic potential of the der9-associated p96^ABL/BCR and p40^ABL/BCR fusion proteins and their roles in the lineage commitment of hematopoietic stem cells in comparison to BCR/ABL.

Methodology

All t(9;22) derived proteins were retrovirally expressed in murine hematopoietic stem cells (SL cells) and human umbilical cord blood cells (UCBC). Stem cell potential was determined by replating efficiency, colony forming - spleen and competitive repopulating assays. The leukemic potential of the ABL/BCR fusion proteins was assessed by in a transduction/transplantation model. Effects on the lineage commitment and differentiation were investigated by culturing the cells under conditions driving either myeloid or lymphoid commitment. Expression of key factors of the B-cell differentiation and components of the preB-cell receptor were determined by qRT-PCR.

Principal Findings

Both p96^ABL/BCR and p40^ABL/BCR increased proliferation of early progenitors and the short term stem cell capacity of SL-cells and exhibited own leukemogenic potential. Interestingly, BCR/ABL gave origin exclusively to a myeloid phenotype independently from the culture conditions whereas p96^ABL/BCR and to a minor extent p40^ABL/BCR forced the B-cell commitment of SL-cells and UCBC.

Conclusions/Significance

Our here presented data establish the reciprocal ABL/BCR fusion proteins as second oncogenes encoded by the t(9;22) in addition to BCR/ABL and suggest that ABL/BCR contribute to the determination of the leukemic phenotype through their influence on the lineage commitment.     

Atypical D-FISH patterns of BCR/ABL gene rearrangements in 169 chronic myeloid leukemia patients

The Philadelphia (Ph) chromosome, a hallmark chromosomal anomaly observed in 95 percent of chronic myeloid leukemia (CML) cases, is known to involve the Abelson (ABL) proto-oncogene on chromosome 9 and the breakpoint cluster region (BCR) gene on chromosome 22, producing BCR/ABL mRNA encoding an abnormal tyrosine kinase protein. In the process of generating BCR-ABL fusion, the deletion of residual BCR or ABL occurs in 15-30 percent of CML patients. In addition, some rearrangements are complex, and do not yield the ABL/BCR fusion due to the involvement of a third chromosome in the rearrangement. The possible role of these deletions and complex rearrangements in disease outcome is an ongoing topic of research. We report our results of cytogenetic analysis with GTG banding and fluorescence in situ hybridization using dual color dual fusion probe (D-FISH) from Vysis Inc, USA in 169 (109 male and 60 female) CML patients registered at The Gujarat Cancer and Research Institute (GC and RI) from April 2004 to December 2005. GTG banding was carried out in 123 cases having analyzable metaphases. Of these 123 cases, D-FISH revealed atypical signal patterns in 57 patients (46%), and 12 cases revealed additional complex translocations indicative of disease progression. Out of 57 cases with atypical FISH patterns, 22 included metaphase FISH results, and the rest had only interphase FISH performed. In addition to the hallmark Philadelphia chromosome, other chromosomal aberrations in CML revealed heterogeneity of molecular events. Pooling of more data may lead to identification of new CML sub-groups and hence help in the analysis of clinical trials. Patients enrolled in our prospective study of prognostic significance will be followed up for disease free and overall survival in correlation with ABL-BCR deletion status.     

The first BCR gene intron contains breakpoints in Philadelphia chromosome positive leukemia.

The hallmark of chronic myelogenous leukemia (CML) is a translocation between chromosomes 9 and 22 - the Philadelphia (Ph') translocation. The translocation is also found in acute lymphocytic leukemia (ALL) albeit in a lower percentage of patients. The breakpoint on chromosome 22 is located within the BCR gene: in CML, breakpoints are clustered within 5.8 kb of DNA, the major breakpoint cluster region (Mbcr). In ALL, breakpoints have been reported within the Mbcr but also in more 5' regions encompassing the BCR gene. To characterize the latter breakpoints, we have molecularly cloned and mapped the entire gene, which encompasses approximately 130 kb of DNA. Mbcr negative, Ph'-positive ALL breakpoints were not distributed at random within the gene but rather were found exclusively within the 3' half of the first BCR gene intron. In contrast to the Mbcr, which is limited to a region of 5.8 kb, this part of the intron has a size of 35 kb. Translocation breakpoints in this region appear to be specific for ALL, since it was not rearranged in clinically well-defined CML specimens nor in any other tumor DNA samples examined.     

Quantitative detection of BCR–ABL fusion gene and its application in monitoring chronic myeloid leukemia treatment

The BCR–ABL fusion gene in chromosome translocation, t (9; 22), and its product, p210BCR/ABL oncogenic tyrosine kinase, is the underlying molecular mechanism that leads to the development of CML. Quantitative detection of BCR–ABL fusion gene has become a reliable approach to diagnose and monitor CML. The aim of this study was to evaluate a Roche t (9; 22) kit in CML diagnosis, monitoring treatment responses, and identification of relapse. Using BCR–ABL fusion gene-expressing K562 cells, a series of standard samples were prepared and used to establish a curve for the calculation of BCR–ABL fusion gene expression in patient samples. Our results indicate that PCR detection system with aforementioned kit has good reproducibility. In addition, the relative concentration of BCR–ABL measured by PCR was in agreement with the patient’s response to the Imatinib treatment and bone marrow morphology remission. Furthermore, we found that the relative concentration of BCR–ABL fusion gene increased 1–3 months before CML relapse was clinically and cytogenetically diagnosed, suggesting that the PCR-based BCR–ABL fusion gene detection with t (9; 22) kit is able to diagnose the recurrence of CML at least 1 month earlier than the classic cytogenetic analysis. In conclusion, detection of BCR–ABL fusion gene expression in CML using Roche t (9; 22) kit has great clinical value in the primary diagnosis, monitoring treatment responses, and identification of relapse in CML patients.     

Characterization of a complex philadelphia translocation (1p-;9q+;22q-) by gene mapping

Summary Human-Chinese hamster somatic cell hybrids were obtained using circulating leucocytes from a chronic myeloid leukaemia (CML) patient carrying a complex Philadelphia (Ph¹) translocation (1p-; 9q+; 22q-). Hybrid clones which showed segregation of the translocation chromosomes were studied. The chromosome 22 markers ACO2, ARSA, and NAGA segregated with the 1p- derivative; and the chromosome 1 markers UMPK, PGD, and ENO1 segregated with the 9q+ derivative. Hence, molecular evidence has been obtained for the translocation of the distal part of 22q to chromosome 1 and for the translocation of the distal part of 1p to chromosome 9. No conclusions could be drawn either about translocation of chromosome 9 material or about a possible difference in breakpoint in chromosome 22 when compared with six cases of 9;22 translocations similarly studied and previously reported. In addition, a more precise mapping of PGM1 was obtained, the gene being proximal to UMPK and the breakpoint in 1p32.     

Molecular analysis of both translocation products of a Philadelphia-positive CML patient.

The breakpoint regions of both translocation products of the (9;22) Philadelphia translocation of CML patient 83-H84 and their normal chromosome 9 and 22 counterparts have been cloned and analysed. Southern blotting with bcr probes and DNA sequencing revealed that the breaks on chromosome 22 occurred 3' of bcr exon b3 and that the 88 nucleotides between the breakpoints in the chromosome 22 bcr region were deleted. Besides this small deletion of chromosome 22 sequences a large deletion of chromosome 9 sequences (greater than 70 kb) was observed. The chromosome 9 sequences remaining on the 9q+ chromosome (9q+ breakpoint) are located at least 100 kb upstream of the v-abl homologous c-abl exons whereas the translocated chromosome 9 sequences (22q-breakpoint) could be mapped 30 kb upstream of these c-abl sequences. The breakpoints were situated in Alu-repetitive sequences either on chromosome 22 or on chromosome 9, strengthening the hypothesis that Alu-repetitive sequences can be hot spots for recombination.     

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.     

aCGH local copy number aberrations associated with overall copy number genomic instability in colorectal cancer: coordinate involvement of the regions including BCR and ABL

In order to identify small regions of the genome whose specific copy number alteration is associated with high genomic instability in the form of overall genome-wide copy number aberrations, we have analyzed array-based comparative genomic hybridization (aCGH) data from 33 sporadic colorectal carcinomas. Copy number changes of a small number of specific regions were significantly correlated with elevated overall amplifications and deletions scattered throughout the entire genome. One significant region at 9q34 includes the c-ABL gene. Another region spanning 22q11-q13 includes the breakpoint cluster region (BCR) of the Philadelphia chromosome. Coordinate 22q11-q13 alterations were observed in 9 of 11 tumors with the 9q34 alteration. Additional regions on 1q and 14q were associated with overall genome-wide copy number changes, while copy number aberrations on chromosome 7p, 7q, and 13q21.1-q31.3 were found associated with this instability only in tumors from patients with a smoking history. Our analysis demonstrates there are a small number of regions of the genome where gain or loss is commonly associated with a tumor's overall level of copy number aberrations. Our finding BCR and ABL located within two of the instability-associated regions, and the involvement of these two regions occurring coordinately, suggests a system akin to the BCR-ABL translocation of CML may be involved in genomic instability in about one-third of human colorectal carcinomas.     

Localisation and distance between ABL and BCR genes in interphase nuclei of bone marrow cells of control donors and patients with chronic myeloid leukaemia

Quantitative measurements of the nuclear localisation of the ABL and BCR genes and the distance between them were performed in randomly oriented bone marrow cells of control donors and patients with chronic myeloid leukaemia (CML). Most ABL and BCR genes (75%) are located at a distance of 20–65% of the local radius from the nuclear centre to the nuclear membrane. A chimeric BCR-ABL gene located on a derivative chromosome 22 resulting from t(9;22)(q34;q11) [the so-called Philadelphia (Ph) chromosome] as well as the intact ABL and BCR genes of patients suffering from chronic myeloid leukaemia are also located mostly in this region, which has a mean thickness of 2 μm in bone marrow cells. We have not found any significant differences in the location of the two genes in the G₁ and G₂ phases of the cell cycle, nor between bone marrow cells and stimulated lymphocytes. Irradiation of lymphocytes with a dose of 5 Gy of γ-rays results in a shift of both genes to the central region of the nucleus (0–20% of the radius distant from the nuclear centre) in about 15% of the cells. The minimum distance between one ABL and one BCR gene is less than 1 μm in 47.5% of bone marrow cells of control donors. Such a small distance is found between homologous ABL and between homologous BCR genes in only 8.1% and 8.4% of cells, respectively. It is possible that the relative closeness of nonhomologous ABL and BCR genes in interphase nuclei of bone marrow cells could facilitate translocation between these genes. In 16.4% of bone marrow cells one ABL and one BCR gene are juxtaposed (the distance between them varies from 0–0.5 μm) and simulate the Ph chromosome. This juxtaposition is the result of the projection of two genes located one above another into a plane, as follows from the probability calculation. Received: 5 September 1996 / Accepted: 15 April 1997     

C-abl and bcr are rearranged in a Ph1-negative CML patient.

Chromosomal analysis of a patient with chronic myelocytic leukemia (CML) revealed a translocation (9;12) (q34;q21) without a detectable Philadelphia chromosome (Ph1). Using molecular approaches we demonstrate (i) a rearrangement within the CML breakpoint cluster region (bcr) on chromosome 22, and (ii) a joint translocation of bcr and c-abl oncogene sequences to the derivative chromosome 12. These observations support the view that sequences residing on both chromosome 9 (c-abl) and 22 (bcr) are involved in the generation of CML and suggest that a subset of Ph1-negative patients may in fact belong to the clinical entity of Ph1-positive CML.     

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.     

Alternative forms of the BCR-ABL oncogene have quantitatively different potencies for stimulation of immature lymphoid cells.

The Philadelphia chromosome (t9:22;q34:q11) is found in more than 90% of patients with chronic myelogenous leukemia, in 10 to 20% of patients with acute lymphocytic leukemia, and in 1 to 2% of patients with acute myelogenous leukemia. Alternative chimeric oncogenes are formed by splicing different sets of BCR gene exons on chromosome 22 across the translocation breakpoint to a common set of ABL oncogene sequences on chromosome 9. This results in an 8.7-kilobase mRNA that encodes the P210 BCR-ABL gene product commonly found in patients with chronic myelogenous leukemia or a 7.0-kilobase mRNA that produces the P185 BCR-ABL gene product found in most Philadelphia chromosome-positive patients with acute lymphocytic leukemia. To compare the efficiency of growth stimulation by these two proteins, we derived cDNA clones for each with identical 5' and 3' untranslated regions and expressed them from retrovirus vectors. Matched stocks were compared for potency to transform immature B-lymphoid lineage precursors. The growth-stimulating effects of P185 for this cell type were found to be significantly greater than those of P210. Structural changes in BCR may regulate the effectiveness of the ABL tyrosine kinase function, as monitored by lymphocyte growth response. Changes in mitogenic potency may help to explain the more acute leukemic presentation usually associated with expression of the P185 BCR-ABL oncogene.     

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.     

Chromatin structural elements and chromosomal translocations in leukemia

Recurring chromosome abnormalities are strongly associated with certain subtypes of leukemia, lymphoma and sarcomas. More recently, their potential involvement in carcinomas, i.e. prostate cancer, has been recognized. They are among the most important factors in determining disease prognosis, and in many cases, identification of these chromosome abnormalities is crucial in selecting appropriate treatment protocols. Chromosome translocations are frequently observed in both de novo and therapy-related acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). The mechanisms that result in such chromosome translocations in leukemia and other cancers are largely unknown. Genomic breakpoints in all the common chromosome translocations in leukemia, including t(4;11), t(9;11), t(8;21), inv(16), t(15;17), t(12;21), t(1;19) and t(9;22), have been cloned. Genomic breakpoints tend to cluster in certain intronic regions of the relevant genes including MLL, AF4, AF9, AML1, ETO, CBFB, MYHI1, PML, RARA, TEL, E2A, PBX1, BCR and ABL. However, whereas the genomic breakpoints in MLL tend to cluster in the 5' portion of the 8.3 kb breakpoint cluster region (BCR) in de novo and adult patients and in the 3' portion in infant leukemia patients and t-AML patients, those in both the AML1 and ETO genes occur in the same clustered regions in both de novo and t-AML patients. These differences may reflect differences in the mechanisms involved in the formation of the translocations. Specific chromatin structural elements, such as in vivo topoisomerase II (topo II) cleavage sites, DNase I hypersensitive sites and scaffold attachment regions (SARs) have been mapped in the breakpoint regions of the relevant genes. Strong in vivo topo II cleavage sites and DNase I hypersensitive sites often co-localize with each other and also with many of the BCRs in most of these genes, whereas SARs are associated with BCRs in MLL, AF4, AF9, AML1, ETO and ABL, but not in the BCR gene. In addition, the BCRs in MLL, AML1 and ETO have the lowest free energy level for unwinding double strand DNA. Virtually all chromosome translocations in leukemia that have been analyzed to date show no consistent homologous sequences at the breakpoints, whereas a strong non-homologous end joining (NHEJ) repair signature exists at all of these chromosome translocation breakpoint junctions; this includes small deletions and duplications in each breakpoint, and micro-homologies and non-template insertions at genomic junctions of each chromosome translocation. Surprisingly, the size of these deletions and duplications in the same translocation is much larger in de novo leukemia than in therapy-related leukemia. We propose a non-homologous chromosome recombination model as one of the mechanisms that results in chromosome translocations in leukemia. The topo II cleavage sites at open chromatin regions (DNase I hypersensitive sites), SARs or the regions with low energy level are vulnerable to certain genotoxic or other agents and become the initial breakage sites, which are followed by an excision end joining repair process.