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Secondary Ph-positive CML with a minority monosomy 7 clone.

We report a case of Ph-positive chronic myelogenous leukemia (CML) secondary to previous treatment for a lymphoma. At the time of original diagnosis of lymphoblastic lymphoma, chromosome studies of blood and bone marrow were normal. Following therapy and a clinical remission complicated by CNS relapse, the patient presented 16 months after treatment was discontinued with a WBC of 110,000 mm-3, consistent with CML. Blood and marrow cytogenetic studies at this time showed a Ph chromosome, t(9;22)(q34;q11) translocation, without other karyotypic alteration. A separate small clone with the karyotype 45,XY, -7 was found in the blood. His disease followed an aggressive course and he died 3 months later. The autopsy findings indicated CML in blast crisis. Molecular studies performed on cells replacing a lymph node revealed a rearrangement of the breakpoint cluster region (bcr) of chromosome #22 and of the immunoglobulin heavy chain locus. Taken together, it seems most likely that the patient's CML developed as a second neoplasm following successful elimination of his lymphoblastic lymphoma by therapy.

Antineoplastic Combined Chemotherapy Protocols↗

Identification of masked and variant Ph (complex type) translocations in CML and classic Ph in AML and ALL by fluorescence in situ hybridization with the use of bcr/abl cosmid probes.

In the present study, three chronic myelogenous leukemia (CML) patients with variant Philadelphia (Ph) chromosomes (complex types), two CML patients with a masked Ph, one case with Ph positive acute lymphocytic leukemia (ALL), and one with Ph positive acute myelocytic leukemia (AML) were analyzed by standard cytogenetic techniques (G-banding), Southern blot studies, and fluorescence in situ hybridization (FISH) procedures using probes from portions of the bcr and abl genes. It has been previously shown that this FISH approach could detect the bcr/abl fusion event in CML patients with classic or variant (simple type) Ph. Our results demonstrate that this FISH assay can also detect the bcr/abl fusion status in CML patients with masked or variant (complex type) Ph chromosomes and in some patients with Ph positive ALL or AML.

Adult↗

Further evidence for the lack of correlation between the breakpoint site within M-BCR and CML prognosis and for the occasional involvement of p53 in transformation.

The actual significance of the type of BCR-ABL rearrangement in chronic myeloid leukemia (CML) prognosis remains controversial. Also, the molecular events that lead to CML progression are largely unknown. We analyzed the M-BCR breakpoint position in 64 CML patients by Southern blot and correlated the molecular findings with the cytogenetic, hematologic, and clinical data. No statistically significant differences were found with respect to the clinical and hematologic data presented at diagnosis or in the median duration of chronic phase (CP) and survival between the groups of patients with 5' and 3' breakpoints. We also studied by PCR-SSCP and direct sequencing the p53 gene in patients with specimens available in both chronic phase and blast crisis. We identified p53 mutations in 17% of the blast crisis samples analyzed, whereas no abnormalities were found in CP. This finding suggests that only in a minor fraction of cases are lesions in the p53 gene involved in transformation. Given the present findings, along with previous reports, we believe that a novel mechanism to explain the heterogeneity of CML should be postulated and actively pursued, as should the identification of secondary molecular events more consistently involved in progression.

Adolescent↗

Sterol dependent LDL-receptor gene transcription in lymphocytes from normal and CML patients.

Sterol regulatory element (SRE) has been recognized to regulate various key genes coding for especially low density lipoprotein (LDL)-receptor, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and HMG-CoA synthase known to play a crucial role in the cholesterol feedback mechanism. The deranged cholesterol feedback mechanism has been widely recognised in initiation as well as progression of various types of cancers including chronic myeloid leukaemia (CML). Consequently, the present study was addressed to understand this phenomenon and revealed the existence of a unique 47 kDa protein factor having affinity for this SRE sequence in lymphocytes from normal subjects as well as its absence in lymphocytes from untreated CML patients. However, this factor appeared when the CML patients achieved complete haematological remission (CHR) through alpha-interferon therapy. Further, an inverse relationship was also observed between sterol modulated LDL-receptor gene transcription and the binding affinity of this 47 kDa factor to the SRE sequence. Based upon these results we propose that alpha-interferon through its receptor initiates phosphatidic acid dependent signalling which in turn regulates the affinity of 47 kDa sterol regulatory element binding factor as well as LDL-receptor gene transcription in lymphocytes from CML patients.

Bucladesine↗

The achievement of durable complete cytogenetic remission in late chronic and accelerated phase patients with CML treated with Imatinib mesylate predicts for prolonged response at 6 years.

Despite the positive results achieved by Imatinib mesylate (Imatinib) in the treatment of chronic myeloid leukemia (CML), over the past several years, Imatinib does not eradicate the leukemic clone. The long-term duration of response to the drug is not known. Long-term follow-up of CML patients treated with Imatinib will ultimately define the durability of such treatment and the frequency of reemergence of progressive disease. We present the results of a 6-year follow-up of 40 CML patients either in chronic or accelerated phase who obtained a durable (>6 months) complete cytogenetic remission (CCyR) after treatment with Imatinib in a single center. In 34 cases CCyR was obtained at an Imatinib dose of 400-600 mg/day and in 6 cases after a dose increase to 600-800 mg/day. At a median follow-up of 68 months, 6 cytogenetic relapses (15%) were observed. No progressions to more advanced phases of disease have been detected during the follow-up period. Cytogenetic relapse was predicted by either a decrease in the amount of BCR-ABL transcript of less than 2 logs after the achievement of CCyR (p=0.0041) or a time-to-CCyR of more than 12 months (p<0.0001). This 6-year follow-up of the efficacy of Imatinib therapy in CML patients who obtained a durable CCyR indicates that the relapses rate is low over this period of observation and that the rate of relapse does not increase over time.

Adult↗

The tumor suppressor PP2A is functionally inactivated in blast crisis CML through the inhibitory activity of the BCR/ABL-regulated SET protein.

The oncogenic BCR/ABL kinase activity induces and maintains chronic myelogenous leukemia (CML). We show here that, in BCR/ABL-transformed cells and CML blast crisis (CML-BC) progenitors, the phosphatase activity of the tumor suppressor PP2A is inhibited by the BCR/ABL-induced expression of the PP2A inhibitor SET. In imatinib-sensitive and -resistant (T315I included) BCR/ABL+ cell lines and CML-BC progenitors, molecular and/or pharmacological activation of PP2A promotes dephosphorylation of key regulators of cell proliferation and survival, suppresses BCR/ABL activity, and induces BCR/ABL degradation. Furthermore, PP2A activation results in growth suppression, enhanced apoptosis, restored differentiation, impaired clonogenic potential, and decreased in vivo leukemogenesis of imatinib-sensitive and -resistant BCR/ABL+ cells. Thus, functional inactivation of PP2A is essential for BCR/ABL leukemogenesis and, perhaps, required for blastic transformation.

Animals↗

Novel targeted therapies to overcome imatinib mesylate resistance in chronic myeloid leukemia (CML).

Imatinib mesylate (Gleevec) was developed as the first molecularly targeted therapy that specifically inhibits the BCR-ABL tyrosine kinase activity in patients with Philadelphia chromosome positive (Ph+) chronic myeloid leukemia (CML). Due to its excellent hematologic and cytogenetic responses, particularly in patients with chronic phase CML, imatinib has moved towards first-line treatment for newly diagnosed CML. Nevertheless, resistance to the drug has been frequently reported and is attributed to the fact that transformation of hematopoietic stem cells by BCR-ABL is associated with genomic instability. Point mutations within the ABL tyrosine kinase of the BCR-ABL oncoprotein are the major cause of resistance, though overexpression of the BCR-ABL protein and novel acquired cytogenetic aberrations have also been reported. A variety of strategies derived from structural studies of the ABL-imatinib complex have been developed, resulting in the design of novel ABL inhibitors, including AMN107, BMS-354825, ON012380 and others. The major goal of these efforts is to create new drugs that are more potent than imatinib and/or more effective against imatinib-resistant BCR-ABL clones. Some of these drugs have already been successfully tested in preclinical studies where they show promising results. Additional approaches are geared towards targeting the expression or stability of the BCR-ABL kinase itself or targeting signaling pathways that are chronically activated and required for transformation. In this review, we will discuss the underlying mechanisms of resistance to imatinib and novel targeted approaches to overcome imatinib resistance in CML.

Antineoplastic Agents↗

Evaluation of CML model cell lines and imatinib mesylate response: determinants of signaling profiles.

Our understanding of the mechanisms by which BCR-ABL drives CML is based, in part, on the use of model cell lines such as the K562 cell line. However, the BCR-ABL translocation may occur via a number of different junction points. In addition, CML is a disease of hematopoietic stem cells and, as a result, can give rise to multiple lineages of tumor cells. In this study, we examined the cellular signaling profiles following imatinib mesylate treatment of eight model CML and ALL cell lines that encompass three BCR-ABL junction points and multiple lineages. We used phosphorylation-specific antibodies and flow cytometry to determine the kinase and pathway activation states with each of the cell lines before and after imatinib mesylate exposure. The comparisons of signaling response profiles, junction points and lineages indicate that cell line lineage rather than BCR-ABL junction point may determine cellular response to imatinib mesylate. The large amount of variation observed among the cell lines suggests that further analysis is required to understand the complex signaling profiles present in CML patients.

Animals↗

Expression of SOCS3 mRNA in bone marrow cells from CML patients associated with cytogenetic response to IFN-alpha.

Previously, we have demonstrated that constitutive expression of suppressor of cytokine signaling-3 (SOCS3) affects the sensitivity of chronic myelogenous leukemia (CML) cell lines to interferon-alpha (IFN-alpha). In the present study, we analyzed the expression of SOCS3 mRNA in bone marrow cells from patients with CML at diagnosis, with the aid of real-time polymerase chain reaction. SOCS3 mRNA expression in bone marrow cells from CML patients who responded well to IFN-alpha therapy was significantly lower than that in cells from healthy volunteers and patients who were resistant to IFN-alpha therapy. Methylation of SOCS3 promoter was absent in bone marrow cells from all CML patients examined. These results indicate that the expression of SOCS3 mRNA is inversely associated with the sensitivity to IFN-alpha both in vitro and in vivo and that differences in SOCS3 mRNA expression are not due to the methylation status of SOCS3 promoters.

Bone Marrow Cells↗

BCR-ABL mutant kinetics in CML patients treated with dasatinib.

Dasatinib is efficient in vitro against most of CML cells harboring ABL kinase domain mutations and induces high rates of response in imatinib-resistant CML patients. Here, we monitored the mutated BCR-ABL transcripts during the follow-up of 12 CML patients treated with dasatinib. We identified four groups of patients based on their sensitivity to dasatinib. Clinical responses were correlated to the in vitro sensitivity of BCR-ABL mutants to dasatinib, however, some discrepancies were observed in a subfraction of CML patients, suggesting subtle differences between in vitro observations and clinical entities and/or the onset of other mechanisms responsible for dasatinib resistance.

Adult↗

Comprehensive analysis of BCR-ABL transcript types in Korean CML patients using a newly developed multiplex RT-PCR.

Diagnosis of chronic myeloid leukemia (CML) is based on the detection of BCR-ABL gene or Philadelphia chromosome (Ph chromosome), and fusion proteins with different sizes are encoded depending on the breakpoint in the BCR gene. In general, 3 breakpoint cluster regions in the BCR gene have been described: major (M-bcr), minor (m-bcr), and micro (mu-bcr). This study was designed to determine the frequency of BCR-ABL transcripts using one-step multiplex reverse transcription polymerase chain reaction (RT-PCR). Bone marrow (BM) or peripheral blood (PB) samples at diagnosis from 548 patients were obtained with a referring diagnosis of Ph-positive (Ph+) CML, and multistep RT-PCR and newly developed one-step multiplex RT-PCR were applied on each sample. Compared with the previous multistep RT-PCR, one-step multiplex RT-PCR with the primers is the more rapid and accurate method to identify the BCR-ABL breakpoints. Most patients (538/548, 98.18%) were found to have b3a2 or b2a2, and total frequency of occurrence of c3a2, e1a2, b2a3, b1a1, and e1a3 or coexpression of b2a2 and b3a2 was less than 2.00%. No differences were observed between women and men. As the multiplex RT-PCR technique distinguishes BCR-ABL transcripts in all samples with high sensitivity and specificity, it easily could be applied at early stages of diagnosis. The incidence of one or the other rearrangement in CML patients varies in different reported series, and the frequency in each type of BCR-ABL transcript in Korean CML patients seems to be different from those of Western countries.

Base Sequence↗

Pathophysiology of CML: do defects in integrin function contribute to the premature circulation and massive expansion of the BCR/ABL positive clone?

Hematopoiesis takes place in close contact with the marrow microenvironment. Normal progenitors adhere through a variety of receptors to stroma and extracellular matrix components, including fibronectin. Adhesion through beta1-integrin receptors to fibronectin not only anchor progenitors to the stroma but also result in direct adhesion-mediated signaling that inhibits progenitor proliferation. In contrast to normal hematopoiesis, chronic myelogenous leukemia (CML) is characterized not only by abnormal, premature circulation of primitive progenitors in the blood but also by continuous progenitor proliferation. Although CML progenitors express the same integrin receptors as normal progenitors, they fail to adhere to stroma and fibronectin, suggesting structural or functional abnormalities of these receptors. Furthermore, CML cells present in contact with stroma or fibronectin continue to proliferate, suggesting that failure to adhere through integrin receptors may also underlie the abnormal proliferation of CML progenitors. The observation that integrin-mediated adhesion and proliferation-inhibitory signaling can be restored through treatment with interferon-alpha or an activating anti-beta1-integrin antibody suggests a functional rather than structural defect that may be related to the presence of the BCR/ABL gene rearrangement in these cells. Insights into the role of integrins as adhesion molecules but also receptors that instruct hematopoietic progenitors to survive, proliferate, and possibly differentiate will not only further our understanding of the normal hematopoietic process but also provide insights into diseases characterized by deranged adhesion and proliferation that may lead to novel therapeutic approaches.

Animals↗

Survival from acute non-lymphocytic leukaemia (ANLL) and chronic myeloid leukaemia (CML) in European children since 1978: a population-based study.

We used data supplied by population-based cancer registries, collected and quality controlled using a common protocol, to analyse survival from acute non-lymphocytic leukaemia (ANLL) and chronic myeloid leukaemia (CML) among children in 17 European countries. Variations in survival in relation to age, country, histologic subtype and period of diagnosis (1978--1992) were examined. These are rare malignancies and survival can be studied reliably only by examination of data from a very large population (in this case EUROCARE). 5 years after diagnosis, overall survival was 44% (95% CI 33--55) for CML and 37% (95% CI 32--43) for ANLL. For both types of leukaemia, survival was slightly better for girls and worse in children under 5 years of age. Consistent with clinical literature, the ANLL subtypes with poorer prognosis were monocytic, megakaryocytic and erythroleukaemia. For ANLL, 5-year survival was better in Finland, the UK, The Netherlands and Germany (> or =40%); for CML, 5-year survival was highest in Italy, although the 95% CI were wide. The risk of death from ANLL and CML fell by 7% per year and 5% per year, respectively, after adjustment for age, gender and country. Since these rare childhood malignancies were virtually untreatable until 1970, these are very welcome trends.

Adolescent↗

Therapy-related changes of CD20+ and CD45RO+ lymphocyte subsets in chronic myeloid leukemia (CML): an immunohistochemical and morphometric study on sequential trephine biopsies of the bone marrow.

Little information exists about the amount of CD45RO+-T- and CD20+-B-lymphocytes in the bone marrow of patients with Philadelphia chromosome-positive chronic myelogenous leukemia (Ph1+-CML) at presentation or regarding corresponding changes during therapy. On the other hand, quantification of this cell compartment seems to be imperative for two reasons: first, the presumed association of immunocompetent lymphocyte subsets in the expansion of the leukemic cell clone; and second, a speculated relationship with the complex generation of myelofibrosis. Therefore, an immunohistological and morphometric study was performed on 219 representative trephine biopsies of the bone marrow derived from 70 patients with repeated examinations during the course of Ph1+-CML. For the identification of the different lymphocyte populations, the monoclonal antibodies UCHL-1 (CD45RO) and L26 (CD20) were applied on formaldehyde-fixed and decalcified specimens. In comparison to a control group and calculated per hematopoietic cells, the CML bone marrow showed about a 50% decrease in the total amount of lymphocytes. Determination of CD45RO+ and CD20+ subsets revealed a significant enhancement during treatment. Because of the different intervals (range, 10 to 25 mo) between first and last biopsy in the various therapeutic groups, results had to be modified by considering dynamic features. This calculation included changes of the lymphocyte subpopulations related to time. Contrasting the CD45RO+ lymphocytes, a relevant increase in the CD20+ subset could be observed after interferon-a treatment or corresponding combination regimens. No significant correlations were found between fiber density at onset (first biopsy) or development of fibrosis and lymphocyte proliferations in the course of CML. Our results are in keeping with the finding that a proper immune response consistent with an increased lymphocyte growth seems to be associated with a regression of the clonally-transformed cell population. Opposed to a repeatedly discussed pathomechanism, we failed to demonstrate any quantitative relationships between the extent of lymphocyte proliferations and occurrence or progression of myelofibrosis.

Adult↗

Molecular remission of CML after autotransplantation followed by adoptive transfer of costimulated autologous T cells.

Four patients with chronic myelogenous leukemia (CML) that was refractory to interferon alpha (two patients) or imatinib mesylate (two patients), and who lacked donors for allogeneic stem cell transplantation, received autotransplants followed by infusions of ex vivo costimulated autologous T cells. At day +30 (about 14 days after T-cell infusion), the mean CD4+ cell count was 481 cells/microl (range 270-834) and the mean CD8+ count was 516 cells/microl (range 173-1261). One patient had a relative lymphocytosis at 3.5 months after T-cell infusion, with CD4 and CD8 levels of 750 and 1985 cells/microl, respectively. All the four patients had complete cytogenetic remissions early after transplantation, three of whom also became PCR negative for the bcr/abl fusion mRNA. One patient, who had experienced progressive CML while on interferon alpha therapy, became PCR- post transplant, and remained in a molecular CR at 3.0 years of follow-up. All the four patients survived at 6, 9, 40, and 44 months post transplant; the patient who remained PCR+ had a cytogenetic and hematologic relapse of CML, but entered a molecular remission on imatinib. Autotransplantation followed by costimulated autologous T cells is feasible for patients with chronic phase CML, who lack allogeneic donors and can be associated with molecular remissions.

Adoptive Transfer↗

New understanding of the pathogenesis of CML: a prototype of early neoplasia.

The 9;22 chromosomal translocation characteristic of CML results in a fused bcr/abl gene and an abnormal fusion protein, p210bcr/abl. Relative to normal c-abl, p210bc1/abl has elevated tyrosine kinase activity that is essential for its transforming activity. We recently reported a prominent 62 kDa GAP-associated P-tyr protein and five additional consistent but less prominent P-tyr proteins as well as five more minor P-tyr proteins that are constitutively tyrosine phosphorylated in primary primitive lineage negative (lin-) chronic phase CML blasts but not in comparable primary lin- normal blasts. The GAP-associated p62 protein has now been purified, sequenced and its gene has been cloned; it is a previously unidentified protein and is currently being characterized. In analyzing P-tyr proteins in primary lin- normal blasts in response to various hematopoietic cytokines, we found a striking similarity in the tyrosine phosphorylation of four major and three minor proteins after stimulation with c-kit ligand (KL) and the P-tyr proteins that are constitutively phosphorylated in primary primitive lin- chronic phase CML blasts. Other cytokines tested (ie GM-CSF, G-CSF, IL-3, FLT3 ligand, TPO, EPO) were much less active or stimulated phosphorylation of other proteins. KL/c-kit and bcr/abl have some similar activities including enhancing survival and expansion of hematopoietic progenitor cells, probably acting primarily on early progenitors at the time of lineage commitment rather than on self-renewing stem cells. Activation of growth factor receptors promote a cascade of protein phosphorylations that can ultimately result in a wide range of cellular responses. Sustained activation of discrete signaling pathways in some types of cells results in differentiation, whereas transient activation instead causes a proliferative response; in other cell types, the converse is true. It may be postulated that stem cells and primitive progenitors are at a particularly susceptible stage of development that renders them especially responsive to sustained bcr/abl-induced phorphorylation of a number of signaling proteins that are components of critical regulatory pathways, including c-kit. The affected pathways control and coordinate multiple diverse cell processes including proliferation, differentiation, maturation and apoptosis, processes that are normally tightly regulated and integrated. Perturbation of these key pathways in primitive progenitors would be expected to seriously disrupt orderly hematopoiesis and could also explain the multiple subtle pleiotropic biological abnormalities characteristically observed in later maturing CML compartments that we have collectively designated 'discordant maturation'. The true situation is undoubtedly very complex and involves interaction of multiple cytokines and signaling pathways that we are now trying to define. Constitutive downstream activation of critical pathways in susceptible early progenitors that normally require KL or other factors for activation could explain most if not all features of the disease.

Adolescent↗

Evidence for specific immune response against P210 BCR-ABL in long-term remission CML patients treated with interferon.

Interferon-alpha treatment induces complete cytogenetic remission in 25% of Philadelphia chromosome (Ph)-positive chronic myelogenous leukemia (CML) patients. These remissions are durable unlike remissions induced with other therapies and yet residual leukemia is detectable in most of these patients. Total peripheral blood mononuclear cells (PBMCs) from CML patients in long-term remission following interferon treatment exhibited significantly higher proliferative responses (four- to 15-fold over background) than normals directed against P210 BCR-ABL in extracts of transfected monkey fibroblast cells. Surprisingly, similar enhanced levels of specific proliferative responses were observed with extracts from cells expressing Bcr and/or Abl proteins. In contrast, extracts from vector only or v-Mos-expressing cells had background level responses. Control monkey fibroblast cells lacking BCR-ABL expression failed to induce proliferation over background levels. Normal individuals had no significant responses to Bcr/Abl extracts. On the other hand, peripheral blood mononuclear cells from allogeneic bone marrow transplant CML patients had proliferative responses to cell extracts independent of Bcr-Abl. These data indicate that patients in remission due to alpha-interferon treatment have significantly higher levels of specific cellular immunoreactivity against Bcr/Abl sequences than normal controls, which could play a role in maintaining cytogenetic remission in Ph-positive CML patients.

Adult↗

Simple competitive two-step RT-PCR assay to monitor minimal residual disease in CML patients after bone marrow transplantation.

Quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) assessing the amount of transcripts of the BCR/ABL gene, the molecular marker of chronic myeloid leukemia (CML), is the only method sensitive enough for monitoring of minimal residual disease (MRD) in CML patients after bone marrow transplantation (BMT). In this study we present a simple modification of competitive Q-RT-PCR using natural competitors from cell lines K562 and BV173. The competitors were used in the form of unpurified RNA in cell lysates which ensured their high stability. Mixing competitors and samples before RNA extraction eliminated problems with quantification of cDNA or RNA entering the competitive reaction and with checking for the RNA quality and reverse transcription (RT) efficiency. The bulk of the malignant clone was expressed as the number of leukemic cells in 10(6) leukocytes when the overproduction of the BCR/ABL mRNA in the cell lines we used as competitors was taken into account. It was found to be 82-fold and 14-fold in K562 and BV173, respectively, in comparison with 100% Ph-positive CML standard. The assay reliability was verified by comparison of results with the mathematical model of competitive PCR. The assay is highly reproducible and sensitive (10(-5)). Its accuracy was proved to be excellent in a wide range of malignant cell concentrations. The method is demonstrated on three CML patients suffering from MRD after BMT. In conclusion, this method fulfills all criteria of competitive Q-RT-PCR. Because of its simplicity it is suitable for clinical laboratories and due to the high stability of the lysates used it may serve in the standardization of results between different laboratories.

Binding, Competitive↗