Philadelphia-negative chromosomal evolution during treatment for chronic myeloid leukemia

Chromosome evolution is one of the major mechanisms of disease progression and resistance in chronic myeloid leukemia (CML) patients. However, the clinical significance of chromosomal evolution in the Philadelphia (Ph)-negative clone during therapy is not fully understood. We evaluated 94 CML patients in the chronic phase of CML during treatment of the disease. Six of them had Ph-negative chromosome abnormalities during treatment. Four patients with a single abnormality and a good molecular response showed no obvious complications from the chromosomal changes, while two other patients who had complex abnormalities and previous treatment had poor outcomes. Our results highlight the need for close monitoring of this kind of patient, not only on a molecular level but also at the cytogenetic level.     

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.     

Regional mapping of two human immunoglobulin V lambda genes and analysis of the V lambda locus in chronic myeloid leukaemia.

The human immunoglobulin V lambda locus has been studied in relation to chromosomal translocations involving chromosome 22. DNA probes for two V lambda genes which belong to different subgroups and do not cross hybridize, were used to show that both V lambda genes are located on the Philadelphia chromosome in chronic myeloid leukaemia (CML). Both genes map in band 22q11 to a region that is bounded on the distal side by the breakpoints for CML 9:22 translocations and on the proximal side by the breakpoint for an X:22 translocation. We have found no evidence for rearrangements or amplification of either V lambda gene in CML, in either the chronic or acute phases of the disease. In K562 cells which are derived from the pleural effusion of a patient with Ph1-positive CML, there appears to be no rearrangement of the V lambda genes, but they are both amplified about four times. We have estimated that the minimum size for the amplification unit in K562 cells is 186 kb.     

bcr rearrangement and C-abl gene expression in Ph1-positive hybrid acute leukemia with simultaneous proliferation of lymphoid and myeloid blasts

bcr gene rearrangement and c-abl gene expression were analyzed in a patient with Philadelphia chromosome (Ph1)-positive hybrid acute leukemia with simultaneous proliferation of lymphoid and myeloid blasts. These data were compared with those from a patient with chronic myelogenous leukemia (CML) in mixed crisis. The leukemic cells of both patients showed immuno-phenotypic profiles such as non-T, non-B common ALL with some MPO-positive leukemic cells and rearranged JH genes. On analysis of molecular events associated with the Ph1 chromosome, the leukemic cells of a patient with CML in mixed crisis showed bcr rearrangement and an 8.5-kb bcr-abl chimeric mRNA, but those of a patient with Ph1-positive hybrid acute leukemia showed no 8.5-kb bcr-abl mRNA, as previously reported in a number of Ph1-positive acute lymphoblastic leukemia (ALL) cases. These results revealed that the molecular event found in Ph1-positive ALL is not only restricted to lymphoid lineage but may play an important role in the proliferation of the myeloid lineage.     

Cytogenetic findings in chronic myelogenous leukemia

Cytogenetic findings in chronic myeloic leukemia are represented in a survey. More than 90 per cent of CML are characterized by Ph1 chromosomes, with more than 90 per cent of the cases being involved in a translocation (9; 22). Further, non-incidental aberrations are +Ph1, isochromosome (17q) and +8 which particularly develop at the acute stage. Isochromosome 17q is assumed to be a marker for a straightly impending development of a blast crisis. Ph1-negative CML is connected with a comparatively bad prognosis for the patient. Partial trisomy 9q+ is indicated here as a marker chromosome. For the patient concerned congenital chromosome defects, such as the Down-syndrome, represent a higher risk of being affected with leukemia.     

The human catechol-O-methyltransferase (COMT) gene maps to band q11.2 of chromosome 22 and shows a frequent RFLP with BglI.

We have been able to assign the human catechol-O-methyltransferase gene (COMT) to chromosome 22q11.2 by using Southern blot analysis of panels of somatic cell hybrids and chromosomal in situ hybridization. Furthermore, Southern blot analysis of DNA from blood and bone marrow samples of a patient with chronic myeloid leukemia (CML), having an extra Philadelphia chromosome (Ph1) in addition to the one produced by the reciprocal translocation between chromosomes 9 and 22, showed increased COMT and BCR gene dosage as compared to DNAs originating from CML patients with only one Ph1 chromosome or from chromosomally normal individuals. Control hybridizations of the same blot with TCRG- and TCRA-specific probes showed corresponding signal intensities in all samples. A relatively frequent two-allele COMT gene RFLP (PIC = 0.37) was recognized in DNAs digested with BglI. Our gene mapping result is in concordance with that previously reported by Brahe et al. (1986), who used an autoradiozymogram assay on different somatic cell hybrids to map this gene to chromosome 22.     

Clonal evolution with isodicentric Ph1 chromosome in Ph1-positive CML: karyotypic conversion after bone marrow transplantation

Clonal chromosomal evolution was observed in a 16-year-old boy suffering from Ph1-positive CML. An isodicentric Ph1 chromosome appeared 20 weeks after the initial diagnosis. At that time an allogeneic bone marrow transplantation was performed. Thereafter, during an observation period of more than 13 months, chromosome analyses showed neither the Ph1 chromosome nor the abnormal isodicentric variant. Close cytogenetic monitoring is suggested to reveal early unfavorable prognostic signs of the onset of blast crisis before it becomes evident in the bone marrow morphology.     

Novel variant Ph translocation t(9;22;11)(q34;q11.2;p15)inv(9)(p13q34) in chronic myeloid leukemia involving a one-step mechanism

Chronic myeloid leukemia (CML) is a clonal malignant disorder of a pluripotent hematopoietic stem cell characterized by the presence of a Philadelphia (Ph) chromosome. Less than 10% of patients present variant Ph chromosomes involving 1 or more additional chromosomes, other than chromosomes 9 and 22, with uncertain prognosis. There are mainly 1- or 2-step mechanisms proposed to explain the genesis of variant Ph chromosomes depending on whether the involved chromosomes are simultaneously broken and rejoined or if a standard t(9;22) occurs first. By combined standard cytogenetic and FISH analysis we detected a novel variant Ph translocation among chromosomes 9, 11 and 22 in a patient with CML without progression to an accelerated phase of the disease after 7 years, with the derivative chromosome 9 also having an acquired pericentric inversion. This novel case illustrates the use of FISH in metaphase to confirm a new rearrangement not previously described in variant Ph formation and that the present karyotype could have originated by a 1-step mechanism with 4 simultaneous breakages without deletion of ABL1.     

TGF-beta1-induced PI3K/Akt/NF-kappaB/MMP9 signalling pathway is activated in Philadelphia chromosome-positive chronic myeloid leukaemia hemangioblasts

Overwhelming evidence from chronic myeloid leukaemia (CML) research indicates that patients harbour quiescent CML stem cells that are responsible for blast crisis. While the haematopoietic stem cell (HSC) origin of CML was first suggested over 30 years ago, recently CML-initiating cells beyond HSCs are also being investigated. We have previously isolated fetal liver kinase-1-positive (Flk1(+)) cells carrying the BCR/ABL fusion gene from the bone marrow of Philadelphia chromosome-positive (Ph(+)) patients with hemangioblast property. Here, we show that these cells behave abnormally comparing with the hemangioblasts in healthy donors. These Ph(+) putative CML hemangioblast up-regulated TGF-β1 and result in activating matrix metalloproteinase-9 to enhance s-KitL and s-ICAM-1 secretion. Further studies showed that phosphatidylinositol-3 kinase (PI3K)/Akt/nuclear factor-κB signalling pathway was involved in CML pathogenesis. These findings provide direct evidence for the first time that hemangioblasts beyond HSCs play a critical role in the progression of CML.     

Activation of the abl oncogene and its involvement in chromosomal translocations in human leukemia

Activation of the abl gene and its involvement in human leukemia is one of the most thoroughly characterized examples of the structural alterations of chromosomes associated with the conversion of a normal cell into a cancer cell. The abl oncogene as first identified on the Abelson murine leukemia virus (A-MuLV). Activation of the viral oncogene is associated, in part, with the truncation of the gene at its 5' end. As in studies with other retroviruses, results with A-MuLV presaged the mechanism of activation by abl in naturally occurring human malignancies. Thus, chronic myelogenous leukemia (CML) is consistently associated with a translocation of a piece of chromosome 9 onto chromosome 22 creating what is known as the Philadelphia chromosome (Ph1). The result of this translocation is the truncation of the 5' end of the cellular abl gene, which is located at the breakpoint of chromosome 9. The function of the abl gene product is poorly understood but is thought to participate in an, as yet, undefined pathway of growth control signals, which originate outside the cell, and traverse through the cell into its nucleus. The loss of the gene product's N-terminal amino acid sequences brought about by the truncation of the 5' portion of the gene is consistent with the hypothesis that the protein's growth-controlling activity is deregulated by the structural alterations which occur in the cancer cells. The abl gene and CML serve as a paradigm of the mechanism of activation of proto-oncogenes by chromosomal alterations. The case of CML and the Ph1 chromosome illustrates the findings we might expect as other chromosomal abnormalities are characterized at the molecular level.     

Identification of a melanoma antigen, PRAME, as a BCR/ABL-inducible gene

In order to elucidate molecular events in BCR/ABL-induced transformation, we adopted a polymerase chain reaction (PCR)-based technique of differential display and compared mRNA expression in human factor-dependent cells, TF-1, with that in factor-independent cells, ID-1, which were established from TF-1 cells by transfection of BCR/ABL. Cloning and sequencing of a gene which was upregulated in ID-1 cells revealed that the gene was identical to a melanoma antigen, PRAME. Our present study demonstrated that PRAME was markedly expressed in primary leukemic cells with chronic myeloid leukemia (CML) in blastic crisis and Philadelphia (Ph)+-acute lymphoblastic leukemia (ALL), in which BCR/ABL played an important role as a pathogenic gene. Moreover, comparison of PRAME expression among CD34+ cells with CML in blastic, accelerated, and chronic phases revealed a higher expression in CML in advanced phases. Thus PRAME was considered to be a good candidate for a marker of Ph+-leukemic blast cells as well as a new target antigen of leukemic blast cells that cytotoxic T cells can recognize.     

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.     

Clonal expansion after bone marrow transplantation in a patient with chronic myelogenous leukemia

A patient with chronic myelogenous leukemia (CML) having the standard [t(9;22), Ph] translocation is presented where the Philadelphia (Ph) chromosome disappeared following bone marrow transplantation (BMT). The Ph chromosome reappeared in host cells after one year of stable hematologic remission. Three additional cell lines, all possessing the Ph chromosome with other abnormalities were consistently present in her marrow cells. Two years after BMT, ninety percent of her dividing bone marrow cells had become leukemic. The patient's clinical status remains unchanged, despite complex cytogenetic findings. The high incidence of multiple aberrant leukemic clones present in this case remains intriguing. Possible mechanisms for this unique transformation after BMT are discussed.     

Rejoining between 9q+ and Philadelphia chromosomes results in normal-looking chromosomes 9 and 22 in Ph1-negative chronic myelocytic leukemia

Summary Rearrangement of the breakpoint cluster region (bcr) and the chromosomal location of c-abl and 3-bcr were studied in two patients with Philadelphia chromosome (Ph¹)-negative chronic myelocytic leukemia (CML). One patient (patient 1) had a normal karyotype and the other (patient 2), 46,XY,inv(3)(q21q26). Both patients showed the bcr rearrangement by Southern blot analysis with a 1.2 kb 3-bcr probe. In situ hybridization studies demonstrated the location of the homologous sequences of bcr on chromosome 22 in patient 1, and on chromosomes 9 and 22 in patient 2. These findings indicate that the morphologically normal-looking chromosomes 9 and 22 in patient 2 are the result of a retranslocation between chromosomes 9q+ and 22q-, abnormalities which were first formed by a standard Ph¹ translocation.     

The Chronic Leukemias: A Review of Disease Manifestations and the Aims of Therapy

Certain aspects of the chronic leukemias that may influence future therapeutic trials are reviewed. In chronic lymphocytic leukemia (CLL), there is minimal mitotic activity in lymphoid tissues; indolent, long-lived lymphocytes, unresponsive to antigenic or phytohemagglutinin (PHA) stimulation accumulate. In many patients, erythroid precursors fail to proliferate despite the stimulus of a severe anemia, but a proliferative response can be initiated by prednisone. We need to know how the normal proliferative responses of these cells are modified, because the correction of these abnormalities would relieve most of the disease manifestations. CLL may not be a neoplastic disorder. In chronic myelogenous leukemia (CML), the leukocyte doubling time shortens as the disease duration lengthens; a significant correlation between this time and survival is demonstrated. Before therapy designed to eliminate the Ph¹-positive (Philadelphia chromosome) stem cell is tried, we need to know whether a normal hematopoietic stem cell exists in Ph¹-positive CML.     

Philadelphia chromosomal breakpoints are clustered within a limited region, bcr, on chromosome 22

We have identified and molecularly cloned 46 kb of human DNA from chromosome 22 using a probe specific for the Philadelphia (Ph') translocation breakpoint domain of one chronic myelocytic leukemia (CML) patient. The DNAs of 19 CML patients were examined for rearrangements on chromosome 22 with probes isolated from this cloned region. In 17 patients, chromosomal breakpoints were found within a limited region of up to 5.8 kb, for which we propose the term "breakpoint cluster region" (bcr). The two patients having no rearrangements within bcr lacked the Ph' chromosome. The highly specific presence of a chromosomal breakpoint within bcr in Ph'-positive CML patients strongly suggests the involvement of bcr in this type of leukemia.     

An overview of chronic myeloid leukemia and its animal models

Chronic myeloid leukemia(CML) is a form of leukemia characterized by the presence of clonal bone marrow stem cells with the proliferation of mature granulocytes(neutrophils, eosinophils, and basophils) and their precursors. CML is a type of myeloproliferative disease associated with a characteristic chromosomal translocation called the Philadelphia(Ph) chromosome or t(9;22) translocation(BCR-ABL). CML is now usually treated with targeted drugs called tyrosine kinase inhibitors(TKIs). The mechanism and natural history of CML is still unclear. Here, we summarize the present CML animal disease models and compare them with each other. Meanwhile, we propose that it is a very wise choice to establish zebrafish(Danio rerio) CML model mimics clinical CML. This model could be used to learn more about the mechanism of CML, and to aid in the development of new drugs to treat CML.     

Evidence of ABL-kinase domain mutations in highly purified primitive stem cell populations of patients with chronic myelogenous leukemia

To study the hierarchical levels of stem cell targets for ABL-kinase domain mutations in CML, highly purified CD34+CD38- and CD34+CD38+ cell populations and their LTC-IC-derived progeny were analyzed in four patients at diagnosis (n=1) or in advanced phases (n=3) of their disease. In the single patient with early phase CML who later developed an Imatinib Mesylate-resistance and a Y253H mutation, no mutation was detectable in purified cell fractions analyzed at diagnosis nor in their LTC-IC-derived progeny. In contrast, in three patients in advanced phase CML, ABL-kinase mutations demonstrated in peripheral blood cells by sequencing (Q252E and M351T) were detectable in the FACS-sorted cells and became amplified in the LTC-IC-derived progeny of the primitive cells. These findings demonstrate that in late CP or advanced CML, ABL-kinase mutations occur as an intraclonal event in the primitive Ph1+ stem cell compartments with progression of this clone towards IM-resistant blast phase.