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.     

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.     

Complex translocation involving four chromosomes in a novel Philadelphia-positive chronic myeloid leukemia case

The so-called Philadelphia (Ph) chromosome is present in more than 90% of chronic myeloid leukemia (CML) cases. Approximately 5-10% of these patients show complex translocations involving a third chromosome in addition to chromosomes 9 and 22. Since the majority of CML cases are currently treated with imatinib, variant rearrangements in general have no specific prognostic significance, although the mechanisms involved in resistance to therapy have yet to be investigated. This study evalutated a CML case with complex chromosomal aberrations not previously observed. A four chromosome translocation involving chromosomal regions such as 12q24.2-24.31 and 16p11.2 besides 9q34 and 22q11 were characterized in detail by array-proven multicolor banding (aMCB). A beneficial response to imatinib was noted in the patient.     

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.     

BCR translocation to derivative chromosome 2: a new case of chronic myeloid leukemia with a complex variant translocation and Philadelphia chromosome

The well-known typical fusion gene BCR/ABL is observed in connection with a complex translocation event in 5-8% of cases of chronic myeloid leukemia (CML). The present study described an exceptional CML case with complex chromosomal aberrations not previously observed. Aberrations included a translocated BCR to the derivative chromosome 2 [der(2)] that also involved a four-chromosome translocation, implying chromosomal regions 1p32 and 2q21, besides 9q34 and 22q11.2, which were characterized by molecular cytogenetics.     

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).     

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.     

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.     

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.     

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).