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.     

Nuclear topography and expression of the BCR/ABL fusion gene and its protein level influenced by cell differentiation and RNA interference

Nuclear topography, expression of the BCR/ABL fusion gene and its protein level/cellular pattern were studied in CML cell line K562 stimulated to differentiation, apoptosis and influenced by ABL-RNA interference (ABL-RNAi). Phorbol ester-induced maturation of K562 cells was accompanied by repositioning of down-regulated BCR/ABL genes closer to the nuclear membrane. This nuclear rearrangement could be connected with differentiation-related heterochromatinization of the amplified BCR-ABL locus, as demonstrated by increased histone H3(K9) dimethylation and decreased H3(K9) acetylation of B3A2 breakpoint. Topography of BCR/ABL in differentiated K562 cells was compared with other leukemic cell types: PMA-maturation of HL60 cells did not influence the nuclear positioning of individual BCR and ABL genes. Moreover, BCR and ABL genes in non-stimulated HL60 as well as in the bone marrow cells of CML patients, i.e. also BCR/ABL fusion genes, were positioned more interiorly in comparison with BCR/ABL multiple loci of K562 cells. Decreased expression of BCR/ABL gene was also found after cell stimulation by selectively pro-apoptotic agent etoposide and by ABL-RNAi leading to apoptosis. In order to compare the efficiency of selected experimental strategies, levels of Bcr/Abl and c-Abl proteins were determined and in all cases tested were reduced. In K562 cells the Bcr/Abl and c-Abl proteins were distributed homogeneously in both the cell nucleus and cytoplasm, while differentiation of K562 cells was characterized by a distinct pattern of Bcr/Abl and c-Abl proteins that were focally distributed rather in the cytoplasm while apoptotic population was completely absent of Bcr/Abl and c-Abl signals.     

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.     

BCR/ABL genes and leukemic phenotype: from molecular mechanisms to clinical correlations

The Philadelphia chromosome (Ph), a minute chromosome that derives from the balanced translocation between chromosomes 9 and 22, was first described in 1960 and was for a long time the only genetic lesion consistently associated with human cancer. This chromosomal translocation results in the fusion between the 5' part of BCR gene, normally located on chromosome 22, and the 3' part of the ABL gene on chromosome 9 giving origin to a BCR/ABL fusion gene which is transcribed and then translated into a hybrid protein. Three main variants of the BCR/ABL gene have been described, that, depending on the length of the sequence of the BCR gene included, encode for the p190(BCR/ABL), P210(BCR/ABL), and P230(BCR/ABL) proteins. These three main variants are associated with distinct clinical types of human leukemias. Herein we review the data on the correlations between the type of BCR/ABL gene and the corresponding leukemic clinical features. Lastly, drawing on experimental data, we provide insight into the different transforming power of the three hybrid BCR/ABL proteins.     

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).     

A rare case of Philadelphia chromosome-positive chronic myelogenous leukemia with inversion in chromosome 9 and t(10;17)

The reciprocal translocation t(9;22)(q34;q11), leading to the formation of two fusion genes, BCR/ABL and ABL/BCR, is found in 90-95% of cases with chronic myeloid leukemia (CML). ABL-BCR expression does not correlate with prognosis, as assessed by cytogenetic response, since the ABL/BCR gene is expressed in only a proportion of CML patients. This study examined an exceptional BCR/ABL-positive CML case with inversion in 9q22q34 leading to the absence of ABL/BCR. Moreover, an unbalanced translocation between chromosomes 10 and 17 which caused deletion of the TP53 gene was identified. The TP53 gene plays a potential role in CML progression, and loss of TP53 may be regarded as a poor prognostic factor.     

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.     

Adult precursor B-ALL with BCR/ABL gene rearrangements displays a unique immunophenotype based on the pattern of CD10, CD34, CD13 and CD38 expresssion.

The Philadelphia chromosome (Ph+) reflects a balanced reciprocal translocation between the long arms of chromosomes 9 and 22 [t(9;22)(q34;q11.2] involving the BCR and ABL genes. At present, detection of BCR/ABL gene rearrangements is mandatory in precursor-B-ALL patients at diagnosis for prognostic stratification and treatment decision. In spite of the clinical impact, no screening method, displaying a high sensitive and specificity, is available for the identification of BCR/ABL+ precursor-B-ALL cases. The aim of the present study was to explore the immunophenotypic characteristics of precursor B-ALL cases displaying BCR/ABL gene rearrangements using multiple stainings analyzed by quantitative flow cytometry in order to rapidly (<1 h) identify unique phenotypes associated with this translocation. From the 82 precursor-B-ALL cases included in the study 12 displayed BCR/ABL gene rearragements, all corresponding to adult patients, four of which also displayed DNA aneuploidy. Our results show that BCR/ABL+ precursor B-ALL cases constantly displayed a homogeneous expression of CD10 and CD34 but low and relatively heterogeneous CD38 expression, together with an aberrant reactivity for CD13. In contrast, this unique phenotype was only detected in three out of 70 BCR/ABL cases. Therefore, the combined use of staining patterns for CD34, CD38 and CD13 expression within CD10-positive blast cells is highly suggestive of BCR/ABL gene rearrangements in adults with precursor B-ALL.