Insertion of the 3' ABL region into the long arm of chromosome 1 in a Philadelphia chromosome-negative chronic myeloid leukemia case

Chronic myeloid leukemia (CML) is a pluripotent hematopoietic stem cell disorder almost always characterized by the presence of the Philadelphia chromosome (Ph), usually due to t(9;22)(q34;q11). The presence of Ph results in the formation of the BCR/ABL fusion gene, which is a constitutively activated tyrosine kinase. Approximately 1% of CML patients appear to have a Ph-negative karyotype but carry a cryptic BCR/ABL fusion that can be located by fluorescence in situ hybridization (FISH) at chromosome 22q11, 9q34 or a third chromosome. This study investigated a rare Ph-negative CML case with insertion of the 3' ABL region into the long arm of derivative chromosome 1 but lacking the 5' BCR region on der(22).     

Philadelphia chromosome-positive chronic myelogenous leukemia with deleted fusion of BCR and ABL genes

In the great majority of patients with chronic myelogenous leukemia (CML) the reciprocal translocation between chromosomes 9 and 22, t(9;22)(q34;q11), resulting in the Philadelphia (Ph) chromosome produces fusion DNA sequences consisting of the 5' part of the major breakpoint cluster region-1 (M-BCR-1) and the ABL protooncogene which encodes for the P210BCR-ABL phosphoprotein with tyrosine kinase activity implicated in the pathogenesis of CML. Molecular analysis was performed on 25 patients with Ph-positive CML using 2 breakpoint cluster region (bcr) probes within the M-BCR-1 DNA sequences, and two of them did not contain either detectable rearranged DNA homologous to the 5' side bcr probe or ABL-related fusion mRNA. The chromosomal in situ hybridization technique revealed that these two Ph-positive CML cases did not carry DNAs homologous to the 5' bcr or ABL probes on the Ph chromosome. Furthermore, one of the two Ph-positive CML cases did not show either rearranged DNA or regions homologous to the 3' bcr probe on a 9q+ chromosome, while the other CML case showed a rearrangement detected by the 3' bcr probe and transposition of the 3' bcr homologous to the 9q+ chromosome. Thus, the possibility is raised that the BCR/ABL fusion DNA has been deleted in rare CML cases, and that the deletion possibly occurred in a stepwise manner following the formation of the Ph chromosome at any stage of the disease.     

Philadelphia Chromosome-positive Chronic Myelogenous Leukemia with Deleted Fusion of BCR and ABL Genes

In the great majority of patients with chronic myelogenous leukemia (CML) the reciprocal translocation between chromosomes 9 and 22, t(9;22)(q34;q11), resulting in the Philadelphia (Ph) chromosome produces fusion DNA sequences consisting of the 5′ part of the major breakpoint cluster region-1 (M-BCR-1) and the ABL protooncogene which encodes for the P210^BCR-ABL phosphoprotein with tyrosine kinase activity implicated in the pathogenesis of CML. Molecular analysis was performed on 25 patients with Ph-positive CML using 2 breakpoint cluster region (bcr) probes within the M-BCR-1 DNA sequences, and two of them did not contain either detectable rearranged DNA homologous to the 5′ side bcr probe or ABL-related fusion mRNA. The chromosomal in situ hybridization technique revealed that these two Ph-positive CML cases did not carry DNAs homologous to the 5′bcr or ABL probes on the Ph chromosome. Furthermore, one of the two Ph-positive CML cases did not show either rearranged DNA or regions homologous to the 3′bcr probe on a 9q+ chromosome, while the other CML case showed a rearrangement detected by the 3′bcr probe and transposition of the 3′bcr homologous to the 9q+ chromosome. Thus, the possibility is raised that the BCR/ABL fusion DNA has been deleted in rare CML cases, and that the deletion possibly occurred in a stepwise manner following the formation of the Ph chromosome at any stage of the disease.     

Analysis of Philadelphia chromosome-negative BCR-ABL-positive chronic myelogenous leukemia by hypermetaphase fluorescence in situ hybridization.

Background: In 5%–10% of patients with of chronic myelogenous leukemia (CML), the Philadelphia chromosome (Ph) is not identified, despite the presence of the associated BCR–ABL molecular abnormality (Ph-negative, BCR–ABL-positive CML) because of sub-microscopic rearrangements.Patients and methods: Six patients with Ph-negative, BCR–ABL-positive CML were investigated. The Ph chromosome detection via fluorescence in situ hybridization after 24-hour mitotic arrest of bone marrow cultures resulting in several hundreds of metaphases (hypermetaphase FISH or HMF) was useful in explaining the nature of the six cases.Results: Four patients had a low frequency of Ph-positive cells by HMF (5.7%, 4.8%, 3.9%, 0.2%), i.e., a typical Ph translocation. However, two cases involved a 9q34 inserted into chromosome 22q11 (74.2% and 92%), without a deletion from chromosome 22 and reciprocal translocation onto 9, i.e., not a typical Ph translocation. The pattern of UBCR gene rearrangement was characterized by the same genomic recombination of 5-BCR and c-ABL, both in the four cases of typical translocation (9;22) and in the two cases of insertion of 9q34 into chromosome 22q11.Conclusions: The HMF identified two different bases for Ph-negative, BCR–ABL-positive cells in CML – presence of low frequency of cells with typical Ph translocations or presence of cells with ABL insertions into the BCR gene on chromosome 22.     

A complex translocation t(5;9;22) in Philadelphia cells involving the short arm of chromosome 5 in a case of chronic myelogenous leukemia

Chronic myelogenous leukemia (CML) is genetically characterized by the reciprocal translocation of chromosome 9 and 22, t(9;22)(q34;q11) which results in the fusion of BCR/ABL gene observed on the derivative chromosome 22 called Philadelphia (Ph') chromosome. About 5-8% of Philadelphia positive patients with CML show various complex translocations involving one or more other chromosomes, in addition to chromosome 9 and 22. In our report we discuss one case with CML, his cytogenetic study revealed a complex translocation t(5;9;22)(p15.1; q34; q11.2), del 5p15.1-->pter, translocation BCR(22q11.2-->qter) to der(5), positive Ph-chromosome and positive t(BCR\ABL). Further confirmation of complex translocation was done by FISH study using the LSI BCR/ABL dual color dual fusion (DF) translocation probe, chromosome 5 and 22 whole paint probes.     

Atypical BCR and ABL D-FISH patterns in chronic myeloid leukemia and their possible role in therapy.

D-FISH uses DNA probes with fluorescence in situ hybridization to detect two fusion signals in cells with a t(9;22)(q34;q11.2) from patients with chronic myeloid leukemia (CML). Using D-FISH, 147 patients with CML were studied and considerable macro genetic variation was observed among their Ph-chromosomes. Typical D-FISH signal patterns were observed for 81% of patients, but three different atypical patterns were seen in 19% of patients. Atypical patterns among Ph-chromosomes were consistent with loss of the 3' portion of BCR that is usually translocated to chromosome 9, or loss of the 5' segment of ABL that usually remains on chromosome 9, or loss of both the 3' translocated BCR and 5' residual ABL hybridization sites. Atypical patterns were associated with all forms of Ph-chromosomes including t(9;22)(q34;q1.2), complex translocations and masked. The normal range for 500 interphase nuclei for patients with typical patterns is < 1%. By comparison, the normal range for patients with either of two atypical patterns was < or = 1.8% and for patients with the other atypical pattern was < or = 23%. Thus, special scoring criteria are needed to detect and quantify nuclei with atypical patterns using D-FISH. The proportion of patients that responded to therapy with interferon alpha-2b or interferon alpha-2b and ara-C for 36 patients with typical patterns was similar to 7 patients with atypical patterns.     

Ph-Negative Chronic Myeloid Leukemia: The Nature of the Breakpoint Junctions and Mechanism of ABL Transposition

CML is a well-defined hematological and clinical entity which sooner or later progresses to blast crisis and death of the patient. Leukemic cells of about 95% of patients show the Ph chromosome which is evidence of the t(9;22)(q34;q11), the cytogenetic rearrangement whereby the BCR gene on chromosome 22 and the ABL gene on chromosome 9 are joined together. The juxtaposition of these genes deregulates the usual function of ABL and causes leukemia. Leukemic cells of the remaining 5% (approximate) of CML patients show complex translocations or have a normal karyotype. Complex translocations achieve the same juxtaposition of BCR and ABL as the standard t(9;22). The Ph chromosome is usually present but may be masked. The final result of a complex translocation can be an apparently normal karyotype and, although BCR and ABL are juxtaposed, there is no Ph chromosome. This is the origin of some cases of Ph-negative CML, in others, part of chromosome 9 including ABL is inserted within the BCR gene. We believe that simple and complex recombinations and insertions are each caused by a single concerted event which breaks a number of adjacent DNA strands at the same time, followed by their mismatched joining and consequent recombination.

The association of BCR-ABL is the essential etiological feature of CML and can be explained, in all cases investigated, by chromosomal rearrangement which takes the form of simple or complex translocations or chromosome insertion. Events that initiate chromosome rearrangement are therefore important in the etiology of CML. Chromosome rearrangement is the result of abnormal DNA recombination which may be caused by an error in normal recombination processes or by endogenous and exogenous mutagens such as chemicals or radiation. These can be regarded as the prime causes of CML.     

Absence in Ph-negative, M-BCR rearrangement-positive chronic myelogenous leukemia of linkage between 5' ABL and 3' M-BCR sequences in Philadelphia translocation.

The Philadelphia (Ph) chromosome can be detected in the vast majority of patients with chronic myelogenous leukemia (CML). We performed a long-range analysis of chromosomal translocation junction by pulsed-field gel electrophoresis (PFGE) techniques, to examine whether molecular evidence of a reciprocal Ph translocation exists in Ph-positive CML as well as Ph-negative, M-BCR rearrangement-positive CML. The rearrangement within M-BCR and ABL was detected in all patients including nine Ph-positive CML, and three Ph-negative CML. The rearranged 3'-abl fragments showed comigration with rearranged 5'-bcr fragment in rare-cutting restriction enzyme digests in all patients with Ph-positive CML. Thus, the physical linkage of the 3' part of ABL to the 5' side of M-BCR on 22q-chromosome was shown. The same linkage was also demonstrated in all three patients with Ph-negative CML. Meanwhile, the rearranged 3'-bcr fragments showed comigration with rearranged pHabl5' (or T39-1-2) fragments in all patients with Ph-positive CML, indicating the linkage of the 5' end of ABL to the 3' part of M-BCR on 9q+ chromosome. However, this linkage was absent in two Ph-negative CML patients who could be studied. The results suggest that a genomic insertion of 3' ABL into M-BCR in Ph-negative CML occurs by a single cytogenetic event rather than a two-translocation mechanism.     

Leukemia cell lines: in vitro models for the study of Philadelphia chromosome-positive leukemia

The Philadelphia (Ph) chromosome, the main product of the (9;22)(q34;q11) translocation, is the cytogenetic hallmark of chronic myeloid leukemia (CML), a clonal myeloproliferative disorder of the hematopoietic stem cell; the Ph chromosome is also found in a sizeable portion of acute lymphoblastic leukemia (ALL) patients and in a small number of acute myeloid leukemia (AML) cases. At the molecular level, the t(9;22) leads to the fusion of the BCR gene (on chromosome 22) to the ABL gene (translocated from chromosome 9); this fusion gene BCR-ABL with its elevated tyrosine kinase activity must to be central to the pathogenesis of these disorders. Three different breakpoint cluster regions are discerned within the BCR gene on chromosome 22: M-bcr, m-bcr, and mu-bcr. Ph + leukemia cell lines are important tools in this research area. More than 20 ALL-and more than 40 CML-derived Ph + leukemia cell lines have been described. Furthermore, three Ph + B-lymphoblastoid cell lines, established from patients with Ph + ALL or CML, are available. Molecular analysis has documented BCR-ABL fusion genes in three apparently Ph chromosome-negative cell lines, all three derived from CML. Nearly all Ph + ALL cell lines have the m-bcr e1-a2 fusion gene (only two ALL cell lines have a b3-a2 fusion) whereas all CML cell lines, but one carry the M-bcr b2-a2, b3-a2 or both hybrids. The mu-bcr e19-a2 has been detected in one CML cell line. Four cell lines display a three-way translocation involving chromosomes 9, 22 and a third chromosome. Additional Ph chromosomes (up to five) have been found in four Ph + ALL cell lines and in 18 CML cell lines; though in some cell lines the extra Ph chromosome(s) might be caused by the polyploidy (tri- and tetraploidy) of the cells. Another modus to acquire additional copies of the BCR-ABL fusion gene is the formation of tandem repeats of the BCR-ABL hybrid as seen in CML cell line K-562. Both mechanisms, selective multiplication of the der(22) chromosome and tandem replication of the fusion gene BCR-ABL, presumably lead to enhanced levels of the fusion protein and its tyrosine kinase activity (genetic dosage effect). The availability of a panel of Ph + cell lines as highly informative leukemia models offers the unique opportunity to analyze the pathobiology of these malignancies and the role of the Ph chromosome in leukemogenesis.     

Complexity of an apparently simple variant Ph translocation in chronic myeloid leukemia

A patient with chronic myeloid leukemia (CML) presented with an apparently simple Ph translocation t(19;22)(q13;q11). In-situ hybridization revealed movement of the c-abl oncogene from a cytogenetically normal chromosome 9 to the Ph. Bcr-3' and c-sis probes hybridized to distal 1p and not to the 19q+ chromosome as expected from the cytogenetic findings. We concluded that this patient had a complex translocation involving four chromosomes: t(1;9;19;22)(p36;q34;q13;q11).     

Molecular cytogenetic study of derivative chromosome 9 deletion in chronic myeloid leukemia patients

The aims of this study are to investigate the frequency of derivative chromosome 9 (der (9)) deletion in Tunisian patients with chronic myeloid leukemia (CML) and to assess the correlation between this deletion and the cytogenetic response for patients treated with hydroxyurea (HU) or imatinib (IM). Karyotype analysis of 336 patients with CML was performed with R-banding technique. Fluorescence in situ hybridization (FISH) was carried using home-brew probes 17L7 and 248J22 for detecting, respectively, adjacent 5??ABL and 3??BCR deletions on der(9). Cytogenetic study demonstrated typical t(9;22)(q34;q11) translocation in 89.6% and variant translocation in 10.4% of patients. Interphase FISH studies showed deletion of der(9) in 59 (17.6%) of the 336 patients, 23 (39%) of them had variant rearrangements. There are 19 patients with solely 5??ABL deletion and 40 with concomitant 5??ABL and 3??BCR deletions. Cytogenetic response was evaluated during 18?months with HU or IM therapy. Our results demonstrate that (a) 3??BCR deletion is associated with 5??ABL deletion in all patients with der(9) deletions, (b) the 5??ABL and 3??BCR deletions arise simultaneously with t(9;22), (c) deletions on der(9) chromosome were frequently encountered in older patients and in patients presenting variant rearrangements, (d) both 5??ABL and 3??BCR deletions were associated with cytogenetic response failure in patients treated with HU, however, patients treated with IM and carrying der(9) deletions presented better cytogenetic response.     

Characterization of the translocation breakpoint in a patient with Philadelphia positive, bcr negative acute lymphoblastic leukaemia

Approximately 5% of children and 10-20% of adults with acute lymphoblastic leukaemia (ALL) have a chromosome translocation t(9;22) which at the cytogenetic level appears identical to that in chronic myeloid leukaemia (CML). The t(9;22) translocation was first recognised in CML patients by its 22q- or Philadelphia (Ph) chromosome. While all Ph positive CML patients so far described have a chromosome 22 breakpoint within the breakpoint cluster region (bcr) located in the 3' part of the phl gene, only some Ph positive ALL patients have breakpoints in bcr. We have cloned the breakpoint of the 9q+ chromosome from the DNA of a Ph positive ALL patient in whom there is no breakpoint in the bcr. The non-chromosome 9 sequences of the breakpoint region are shown to be derived from chromosome 22. The breakpoint in chromosome 22 is shown to be the first intron of the phl gene about 66kb upstream of the bcr. Using probes from this intron, rearrangements were detected in the DNA of two out of twelve additional Ph positive, bcr negative ALL patients.     

A three way complex translocation (4; 9; 22) in two patients with chronic myelocytic leukemia

Chronic myeloid leukemia (CML) is genetically characterized by the reciprocal translocation of chromosome 9 and 22, t(9;22)(q34;q11) which results in the fusion of BCR/ABL gene observed on the derivative chromosome 22 called Philadelphia (Ph') chromosome. About 5-8% of Philadelphia positive patients with CML show various complex translocations involving third chromosome in addition to chromosome 9 and 22. In present report we discuss two cases with CML referred at our centre. At the time of initial diagnosis and after 9 months of treatment, one of the patients showed 100% presence of Philadelphia positive in bone marrow culture. During follow-up in an accelerated state, his cytogenetic study revealed a complex translocation (4;9;22)(q25;q34;q11) along with an additional Philadelphia and marker chromosome. The second patient showed a complex (4;9;22)(q25;q34;q11) translocation at the time of diagnosis. He was on hydroxyurea and his follow-up cytogenetic study after the course of chemotherapy showed no changes. Further confirmation of complex translocation was done by FISH study using bcr/abl and whole chromosome 9 probes. Though the additional genes involved in complex variant Ph' rearrangements have not been characterized, both patients are healthy till 3 to 5 years of initial diagnosis. This could be attributed to the benign effect resulted from reciprocal translocation with no loss or gain of the genetic material.     

Interstitial insertion of varying amounts of ABL-containing genetic material into chromosome 22 in Ph-negative CML

We studied the cells from three selected patients with Ph-chromosome-negative chronic myeloid leukemia (CML) by Southern blotting, polymerase chain reaction, and in situ hybridization of informative probes to metaphase chromosomes. All three patients had rearrangement of M-BCR sequences in the BCR gene and expression of one or other of the mRNA species characteristic of Ph-positive CML. Leukemic metaphases studied after trypsin-Giemsa banding were indistinguishable from normal. The ABL probe localized both to chromosome 9 and 22 in each case. A probe containing 3' M-BCR sequences localized only to chromosome 22, and not to chromosome 9 as would be expected in Ph-positive CML. Two new probes that recognize different polymorphic regions distal to the ABL gene on chromosome 9 in normal subjects localized exclusively to chromosome 9 in two patients and to both chromosomes 9 and 22 in one patient. These results show that Ph-negative CML with BCR rearrangement is associated with insertion of a variable quantity of chromosome 9 derived material into chromosome 22q11; there is no evidence for reciprocal translocation of material from chromosome 22 to chromosome 9.     

Detection of BCR/ABL Fusion Gene by Hematological and Cytogenetical Analysis in Chronic Myeloid Leukemia Patients in Quetta,Pakistan

Background: Chronic myeloid leukemia (CML) is a myeloproliferative disorder of pluripotent stem cells,caused by reciprocal translocation between the long arms of chromosomes 9 and 22, t(9;22)(q34;q11), known asthe Philadelphia chromosome. Materials and Methods: A total of 51 CML patients were recruited in this study.Complete blood counts of all CML patients were performed to find out their total leukocytes, hemoglobin andplatelets. FISH was performed for the detection of BCR-ABL fusion and cryptogenic tests using bone marrowsamples were performed for the conformation of Ph (9;22)(q34;q11) and variant translocation mechanisms.Results: In cytogenetic analysis we observed that out of 51 CML patients 40 (88.9%) were Ph positive and 4(8.88%) had Ph negative chromosomes. Mean values of WBC 134.5 103/μl, hemoglobin 10.44 mg/dl, and platelets288.6 103/μl were observed in this study. Conclusions: In this study, Ph positive translocation between chromosome(9:22)(q34;q11) were observed in 40 (88.9%) CML patients.