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Interferons (IFNs) are pleiotropic cytokines that exhibit multiple biological effects on cells and tissues. IFN receptors are expressed widely in mammalian cells and virtually all different cell types express them on their surface. The Type I IFN receptor has a multichain structure, composed of at least two distinct receptor subunits, IFNalphaR1 and IFNalphaR2. Two Jak-kinases, Tyk-2 and Jak-1, associate with the different receptor subunits and are activated in response to IFNalpha or IFNbeta to regulate engagement of multiple downstream signaling cascades. These include the Stat-pathway, whose function is essential for transcriptional activation of IFN-sensitive genes, and the insulin receptor substrate pathway, which regulates downstream activation of the phosphatidyl-inositol-3' kinase. Members of the Map family of kinases are also activated by the Type I IFN receptor and participate in the generation of IFN signals. The p38 Map kinase pathway appears to play a very important role in the induction of IFN responses. p38 is rapidly activated during engagement of the Type I IFN receptor, and such an activation is regulated by the small G-protein Rac1, which functions as its upstream effector in a tyrosine kinase-dependent manner. The activated form of p38 regulates downstream activation of other serine kinases, notably MapKapK-2 and MapKapK-3, indicating the existence of Type I IFN-dependent signaling cascades activated downstream of p38. Extensive studies have shown that p38 plays a critical role in Type I IFN-dependent transcriptional regulation, without modifying activation of the Stat-pathway. It is now well established that the function of p38 is essential for gene transcription via ISRE or GAS elements, but has no effects on the phosphorylation of Stat-proteins, the formation of Stat-complexes, and their binding to the promoters of IFN-sensitive genes. As Type I IFNs regulate gene expression for proteins with antiviral properties, it is not surprising that pharmacological inhibition of the p38 pathway blocks induction of IFNalpha-antiviral responses. In addition, pharmacological inhibition of p38 abrogates the suppressive effects of Type I IFNs on normal human hematopoietic progenitors, indicating a critical role for this signaling cascade in the induction of the regulatory effects of Type I IFNs on hematopoiesis. p38 is also activated during IFNalpha-treatment of primary leukemia cells from patients with chronic myelogenous leukemia. Such activation is required for IFNalpha-dependent suppression of leukemic cell progenitor growth, indicating that this pathway plays a critical role in the induction of the antileukemic effects of IFNalpha.

Purpose: To report a case of peripheral retinal nonperfusion and chronic myeloid leukemia in a 23-year-old woman., Design: Observational case report., Methods: A complete ophthalmic and systemic evaluation was performed., Results: Ophthalmic examination revealed peripheral retinal nonperfusion with retinal neovascularization in both eyes. Fluorescein angiography of both eyes showed a marked midperipheral and peripheral avascular retina temporally with arteriovenous anastomosis and seafan neovascularizations. Blood work showed no abnormalities, although marked leucocytosis (up to 750 x 10(9)/l) and thrombocytosis (646 x 10(9)/l) were present in 1998 when the patient was diagnosed with leukemia. Following treatment, the patient has been in remission., Conclusions: Peripheral retinal nonperfusion with retinal neovascularization may occur as a complication of chronic myeloid leukemia. In contrast to other studies describing this association, our patient had a bilateral peripheral retinal nonperfusion with seafan neovascularizations without relapse of the myeloid leukemia and without any of the other retinal signs associated with chronic myeloid leukemia, such as tortuosity of veins, intraretinal or preretinal hemorrhages, and cotton-wool exudates., (Copyright 2003 by Elsevier Science Inc.)

The anemia in beta-thalassemia major is caused by a combination of hemolysis and ineffective erythropoiesis, with the latter being more important. Studies of the underlying cause of the hemolysis have indicated that oxidant injury to circulating red blood cells (RBCs) was of critical importance, with evidence of oxidant damage to RBC membrane proteins 4.1 and band 3. Therefore, it seemed reasonable that oxidant damage to thalassemic erythroid precursors would cause their accelerated apoptosis and ineffective erythropoiesis. However, direct analysis showed that the apoptotic programs turned on in thalassemics were not those triggered by oxidative damage but were dependent on activation of FAS/FAS-Ligand interaction. Thus, destruction of thalassemic erythroid precursors may involve different mechanisms from those that cause RBC hemolysis.

Shuttling hnRNPs control the fate of eukaryotic mRNAs throughout their journey from the active site of transcription to that of translation; thus, gain or loss of their function in hematopoietic cells might result in altered hematopoiesis and/or be associated with the process of leukemogenesis. In BCR/ABL-expressing cells, there is a marked increase in the protein levels FUS, hnRNP A1 and hnRNP E2, three RNA-binding proteins involved in the regulation of mRNA processing, nucleocytoplasmic export, and translation. Ectopic expression and/or inhibition of the activity of these RNA-binding proteins affects proliferation, survival, and differentiation of normal and BCR/ABL-expressing cells, suggesting that enhanced expression/activity of certain RNA-binding proteins plays an important, but as yet unrecognized, role in BCR/ABL leukemogenesis. The identification of the mRNA subsets associated with RNA-binding proteins upregulated in BCR/ABL-expressing cells should functionally link the process of leukemogenesis with alteration of mRNA metabolism.

Objective: To explore the relation of cytogenetic changes in patients with chronic myeloid leukemia (CML) to the diagnosis, clinical staging and therapy protocol of the disease., Methods: According to established diagnostic criteria, 155 CML patients were divided into 3 groups and 3 clinical phases were identified on the basis of their Sokal scores. The bone marrow was obtained for G banding and karyotype analysis., Results: It was found that 148 patients (95.5%) carried Ph1 chromosome. Among the other 7 cases without Ph1 chromosome, 4 were identified as being bcr/abl fusion gene positive. The ratio of additional cytogenetic abnormalities were higher in patients in blast crisis or accelerated phase than in patients in chronic phase., Conclusion: CML consists of a group of diseases with high heterogeneity, and the prognoses of the patients mostly depend on the malignancy of the tumor. The occurrence of additional chromosomal abnormality is highly correlative with the risk index and clinical staging of the patients, which may serve prognostic purposes. Conventional cytogenetic analysis may help evaluate the therapeutic effect and make subsequent clinical decisions, and may also facilitate new karyotype identification.

Objective: To study the expression of bcr/abl hybridized gene in chronic myeloid leukemia (CML), acute lymphatic leukemia (ALL) and polycythemia vera (PV), and its clinical significance., Methods: The bcr/abl hybridized gene of interphase metaphase cells of bone marrow in 67 such patients were investigated with a probe of dual color-dual fusion translocation fluorescence in situ hybridization (D-FISH)., Results: In 38 CML patients, 34 (89.5%) were positive, with one having a typical t (9; 22) at first, which changed into negative after having been treated with interferon for 38 months. In another patient, 60 days after post-allogeneic peripheral blood stem cell transplantation (PBSCT), the cytomorphology and cytogenetics were in completely remission. But 3% cells were bcr/abl positive as detected by D-FISH. Six (25%) of 24 ALL patients were positive for Bcr/abl fusion gene, which was negative in 2 PV patients. Three patients suspected of having CML were also negative and one of these three was finally diagnosed as suffering from primary thrombocythemia and one, acute myeloid leukemia (M(2a)) as detected by ETO/AML(1) gene, though the other one was still not confirmed. Two (67%) of the 3 bcr/abl negative CML patients and 5 (87%) of the 6 bcr/abl positive ALL patients had refractory leukemia., Conclusion: bcr/abl hybridized gene is accurately detected by a probe of dual color-dual fusion translocation fluorescence in situ hybridization, which can serve as an effective index for clinical diagnosis, estimation of prognosis and monitor of minimal residual disease in some hematopathies.

Pulmonary function in 42 patients with chronic myelogenous leukemia (CML) was tested before and after HLA-matched (39 related, 3 unrelated) allogeneic bone marrow transplantation (BMT) between 1985 and 1999. Pulmonary function tests (PFTs) including ventilatory capacity, lung volumes, and diffusion capacity for carbon monoxide (DLCO) were performed before and 3, 6, 12, and 24 months after BMT, and every 12 months thereafter. Possible pre- and post-BMT risk factors were evaluated for their influence on pulmonary function. Patients were divided into two groups according to their survival duration for more than 12 months or not. Pretransplant PFTs were essentially normal except for mild reduction in DLCO values in the short-term survival group. Overall pulmonary function changes revealed persistent and significant decrease of forced vital capacity (FVC) and DLCO values after BMT. The DLCO values reached abnormal levels (< 80%) and showed a trend of incomplete recovery. Decrease of forced expiratory volume in the first second (FEV1) and vital capacity were also noted but the FEV1/FVC ratio remained within normal limits after BMT. Transient fall of total lung capacity after BMT was noted. However, its values did not reach abnormal levels such as to cause restrictive ventilatory impairment. Possible risk factors including gender, smoking, bronchiolitis obliterans, acute and chronic graft-versus-host disease (GVHD) were found to have significant influences on posttransplant pulmonary function changes by multiple regression analysis. Most patients except those who developed bronchiolitis obliterans were clinically asymptomatic. Development of bronchiolitis obliterans was the most important factor to cause both clinical symptoms and impaired pulmonary function. In summary, pulmonary function changes before and after HLA-matched allogeneic BMT in long-term survivors of CML only showed modest dysfunction. The primary negative presentation with the development of oxygenation defect had no clinical significance in most patients. The influences on the impairment of pulmonary function were multifactorial.

We previously showed that hypoxia (1% O(2)) favors the self-renewal of murine and human normal hematopoietic stem cells. This study represents the first attempt to characterize the effects of hypoxia on the maintenance of chronic myeloid leukemia (CML) progenitors. CD34(+) cells isolated from apheresis products of CML patients were incubated in hypoxia (1% O(2)) and normoxia (20% O(2)). After 8 days of culture, their proliferation, capacity for colony-forming-cell (CFC) generation in secondary cultures (pre-CFC), and phenotype (CD34 and platelet-activating factor receptor [PAF-R]) were compared with those of normal cells, and tyrosine phosphorylation in CML cells was measured. Hypoxia inhibits the proliferation of CD34(+) cells and preserves the pre-CFC capacity and cell-surface CD34 expression of CML cells better than normoxia. The PAF-R expression, which was absent on freshly isolated cells, was detected at the cell surface in both populations after 8 days of culture, but with a lower percentage of positive cells in CML cell cultures. Incubation in hypoxia suppressed the PAF-R expression of normal cells and increased it in CML cells, resulting in a similar expression in the two populations. These effects could be linked to inhibition by hypoxia of the tyrosine hyperphosphorylation of cellular proteins, a major hallmark of CML cells.

The Ph chromosome has been genetically linked to CML and ALL. Its chimeric fusion gene product, BCR-ABL, can generate leukemia in mice. This review will discuss selected model systems developed to study BCR-ABL induced leukemia and focuses on what we have learned about the human disease from these models. Five main experimental approaches will be discussed including: (i) Reconstitution of mice with bone marrow cells retrovirally transduced with BCR-ABL; (ii) Transgenic mice expressing BCR-ABL; (iii) Knock-in mice with BCR-ABL expression driven from the endogenous bcr locus; (iv) Development of CML-like disease in mice with loss of function mutations in heterologous genes; and (v) ES in vitro hematopoietic differentiation coupled with regulated BCR-ABL expression.

A common approach to testing for differences between the survival rates of two therapies is to use a proportional hazards regression model which allows for an adjustment of the two survival functions for any imbalance in prognostic factors in the comparison. When the relative risk of one treatment to the other is not constant over time the question of which therapy has a survival advantage is difficult to determine from the Cox model. An alternative approach to this problem is to plot the difference between the two predicted survival functions with a confidence band that provides information about when these two treatments differ. Such a band will depend on the covariate values of a given patient. In this paper we show how to construct a confidence band for the difference of two survival functions based on the proportional hazards model. A simulation approach is used to generate the bands. This approach is used to compare the survival probabilities of chemotherapy and allogeneic bone marrow transplants for chronic leukemia.

Patients with chronic myeloid leukemia (CML) have been shown to present with apparent metabolic abnormalities, such as enhanced endogenous intoxication against the background of deterioration of the functional condition of the liver and decline in immune reactivity of the organism; the erythrocytic membrane permeability changes considerably. The data secured are discussed from the standpoint of using them as prognostic criteria of the course of CML.

Background: The mechanisms favoring the growth advantage of Philadelphia chromosome positive cells over normal cells in chronic myelogenous leukemia (CML) are not fully elucidated but could be due partly to altered apoptosis and longer survival of CML clones. Also, little is known about the biologic characteristics of disease progression in CML. Bcl-2 expression has been demonstrated to exert an antiapoptotic effect resulting in increased cell survival. Other proteins such as Bax and Bad are proapoptotic proteins. Fas, a cell surface protein, can be triggered by an appropriate death-promoting ligand (FasL) to activate downstream caspases pivotal in initiation of programmed cell death. Although the mechanisms underlying cellular proliferative and apoptotic pathways are complex, with involvement of multiple interlocking proteins, the relative expression of pro- and antiapoptotic proteins may have an influence on disease progression. This study aimed to determine whether the changes in the cellular expression of Bcl-2, Bax, and Fas correlate with caspase-3 activity and disease progression in CML, or with response to interferon (IFN)-alpha therapy and prognosis in early chronic phase CML., Methods: Bcl-2, Bax, and Fas expression were measured on whole cell lysates from bone marrow mononuclear cell fractions by Western blot analysis and quantitative radioimmunoassay. Caspase-3 activity was determined using the DEVD system. Specimens from 203 patients with CML were examined. These included 130 patients in early chronic phase disease (ECP; diagnosis to therapy, < or =12 months), 33 patients in late chronic phase (diagnosis to therapy, > 12 months), 27 patients in accelerated phase, and 13 patients in blastic phase. Correlations between apoptosis proteins and CML phases, risk groups in ECP, and response to IFN-alpha therapy and survival in ECP were investigated by standard statistical methods, and positive findings were assessed by multivariate analysis., Results: Levels of Bcl-2, Fas, Bax, and caspase-3 activity did not correlate with disease progression. Among patients in ECP, higher Fas levels correlated with poorer risk groups (P = 0.05) and higher caspase-3 activity correlated with better risk groups (P = 0.048). With IFN-alpha therapy, major cytogenetic responses were noted in 30% of patients with high Fas and 53% with low Fas (P = 0.016) and failure to achieve a complete hematologic response (CHR) in 25% versus 2% (P = 0.0001). Survival was shorter with high Fas levels (5-year rates, 71% vs. 52%; P = 0.002), and the independent poor prognostic significance of high Fas levels was confirmed by multivariate analysis (P = 0.014). Response to IFN-alpha therapy and survival were not significantly different by different levels of Bcl-2, Bax, or caspase-3 activity., Conclusions: High Fas levels were associated with intrinsically worse disease at diagnosis, whereas high caspase-3 activity was associated with good risk disease. In ECP CML, high Fas levels were associated with significantly worse response to IFN-alpha therapy and with significantly worse survival. The influence of these cellular proteins and caspase-3 activity on apoptosis in CML is complex and merits further investigation., (Copyright 2001 American Cancer Society.)

The functions of JunB during myelopoiesis were studied in vivo. Transgenic mice specifically lacking JunB expression in the myeloid lineage (junB(-/-)Ubi-junB mice) develop a transplantable myeloproliferative disease eventually progressing to blast crisis, which resembles human chronic myeloid leukemia. Similarly, mice reconstituted with ES cell-derived junB-/- fetal liver cells also develop a myeloproliferative disease. In both cases, the absence of JunB expression results in increased numbers of granulocyte progenitors, which display enhanced GM-CSF-mediated proliferation and extended survival, associated with changes in the expression levels of the GM-CSFalpha receptor, the anti-apoptotic proteins Bcl2 and Bclx, and the cell cycle regulators p16(INK4a) and c-Jun. Importantly, ectopic expression of JunB fully reverts the immature and hyperproliferative phenotype of JunB-deficient myeloid cells. These results identify JunB as a key transcriptional regulator of myelopoiesis and a potential tumor suppressor gene.

The granulocyte-derived hemoregulatory peptide pyroGlu-Glu-Asp-Cys-Lys = pEEDCK is known to keep hematopoietic cells quiescent. When oxidized to its dimeric form (pEEDCK)2, it activates growth of hematopoietic progenitors in association with stroma-derived cytokines. (pEEDCK)2 has a Cys-Cys motif which is also a typical feature of the macrophage inflammatory protein (MIP-1alpha). The present study was designed to analyze differences between the response of normal and leukemic progenitor cells to (pEEDCK)2 or MIP-1alpha. When long-term bone marrow cultures (LTBMCs) were incubated with (pEEDCK)2 or MIP-1alpha and/or cytokines, the stimulatory effect on colony-forming units-granulocyte/erythroid/macrophage/megakaryocyte of LTBMC from chronic myeloid leukemia (CML) patients was less than 50% compared to LTBMC from healthy humans. No difference in oncogene expression could be observed in LTBMC from CML patients regarding reduction of Philadelphia chromosome-associated transcription of the BCR-ABL gene. With respect to the expression of growth and differentiation-associated genes (Galpha16, 5-lipoxygenase, phospholipaseA2, c-kit, and CD34), which were analyzed from LTBMC by semiquantitative reverse transcriptase-polymerase chain reaction, the same transcription rate was observed in CML patients and healthy donors. However, two isoforms of a key enzyme of oxidative metabolism, carnitine palmitoyltransferase (CPT1A and CPT1B), showed 50-fold higher expression rates in LTBMC cells of healthy donors compared to CML patients. It is known that a decrease in oxidative metabolism is associated with an increase in redox equivalents in malignancy. This might result in a reduction of disulphide bonds in (pEEDCK)2 or MIP-1alpha, thus inducing a downregulation of these factors in bone marrow from CML patients.

Background: The effect of allogeneic hematopoietic cell transplantation (alloHCT) on hematologic malignancies is based on the graft-versus-malignancy effect. Obtaining this effect with reduced toxicity has been possible by non-myeloablative (NMA) alloHCT. Once mixed chimeric status, and host versus graft with graft versus host tolerance are achieved, further strengthening of chimerism and graft-versus-malignancy effect can be obtained by donor lymphocyte infusions (DLIs) when needed., Methods: The patient group consisted of 13 patients with advanced hematological malignancies: seven had CML, four of them in blastic-, two in chronic- and the remainder in accelerated-phase; four patients with AML, refractory or in second remission state; one patient with primary refractory secondary AML; and one patient with ALL relapsed after alloHCT. Conditioning regimen consisted of fludarabine 30 mg/m(2)/day for 6 days and anti-T-lymphocyte globulin (ATG) 10 mg/kg/day for 4 days as immunosuppressive. Ara-C or Bu or melphalan were used as the cytoreductive component. All transplants were performed using HLA-identical sibling donors' peripheral blood hematopoietic cells, after priming with filgrastim. Post-transplant GvHD prophylaxis was achieved with CsA alone in 10 patients, and with CsA plus mycophenolate mofetil in the last three patients., Results: Median follow-up is 3 months (range, 0-20) for all the patients and 6 months (range, 2-15) for the live patients. Donor chimerism was shown in 10 patients, not regarding any pretransplant feature. DLIs were performed in seven patients after transplantation and two of them achieved complete chimeric status and molecular remission. Two CML patients in blastic phase (CML-BP), and the primary refractory secondary AML patient did not respond to procedure. In four patients, drug therapy in conventional doses was added to post-transplant DLIs for their relapsed or refractory diseases. Two patients with AML in second CR, and another CML-BP patient, relapsed or progressed after transplantation. A patient with CML-BP achieved CR and full donor chimerism after transplantation, but developed refractory post-transplant lymphoproliferative disease in the 19th month. Two patients with refractory AML, one patient with relapsed ALL and two patients with CML in chronic phase were in complete chimeric status and free of disease signs. Acute GvHD, Grade II-III, was observed in five patients, and two of them developed secondary progressive chronic GvHD subsequently. We observed one early death in a platelet transfusion refractory blastic phase CML patient due to intracranial hemorrhage. Procedure-related severe toxicity was not observed, either in standard-risk patients or stem-cell donors., Discussion: Establishing engraftment with donor chimerism was the first successful step in this approach. The second step, which was the result of the graft-versus-malignancy effect, could be seen in most of the patients, but was not sustained in all of them because of the aggressiveness of their malignancy. It can be suggested that the immunotherapeutic efficacy of this approach could be more successful, and with acceptable toxicity, when performed in patients with minimal residual disease. The role of NMA conditioning, and of the treatment in standard disease indications, remains to be determined in further studies.

Interferon-alpha (IFN-alpha), a molecule with multiple biological actions, is widely used in the treatment of chronic myelogenous leukemia (CML) and the other myeloproliferative disorders. This glycoprotein belonging to the type I subfamily of interferons has been recombinantly manufactured and has been approved for the biotherapy of CML, now becoming the first line of treatment for CML patients in chronic phase who are not candidates for allogeneic hematopoietic stem cell or bone marrowtransplantation. Interferon-alpha action involves binding to its cell membrane receptor and initiation of an intracellular signal transduction cascade. Two major pathways mediate the biologic actions of IFN-alpha. The JAK-STAT pathway leads to phosphorylation and activation of STAT 1 and STAT 2 molecules and transcription of genes like p21 and caspase-1 resulting in cycle arrest and apoptosis. The PKR (protein kinase dsRNA-induced) kinase phosphorylates and inhibits the eukaryotic initiator of translation eIF-2alpha leading again to apoptosis. The PKR kinase cascade also leads to activation of the transcription factor NF-kappaB. The relevance of this activation is unclearand it is possiblethat NF-kappaB has not had the opportunity to transcribe its target genes as it is a substrate of effector caspases and is maybe cleaved by them before exerting any transcription activity. Through the JAK-STAT and the PKR kinase pathways IFN-alpha is able to modify the proliferative and antiapoptotic actions of the constitutively activated kinase bcr-abl, the product of the t(9;22) translocation present in CML, and has therapeutic effects in this disease.

Chronic myelogenous leukemia (CML) cells are highly resistant to apoptosis induced by chemotherapeutic drugs. The observation that production of Bcr-Abl is the initiating event in CML has focussed attention on the survival signals triggered by this oncogene. A number of signal transducers and transcription factors have been associated with the antiapoptotic phenotype of CML cells, some of which lead to the expression and/or activation of members of the Bcl-2 family of apoptosis modulators, such as Bcl-xL and Bad. In this article, recent advances in understanding the antiapoptotic pathways triggered by Bcr-Abl in CML cells, are discussed.

Chronic myeloid leukemia (CML), a malignant transformation of hematopoietic cells, accounts for one-fifth of all leukemias and will be diagnosed in 4,400 individuals in the United States this year. CML has three phases: chronic, accelerated, and blastic. Interferon, hydroxyurea, busulfan, and bone marrow and stem cell transplantation are used to treat CML, but individuals who are in the accelerated phases or blast crisis usually respond poorly to treatment. A new tyrosine kinase inhibitor, STI 571, is being evaluated in clinical trials. STI 571 is highly bioavailable in oral form and produced minimal toxicities in phase I studies. Further research is needed to determine the rate of response and survival data, but STI 571 holds great promise in the treatment of CML.

Natural killer (NK) cells are deficient in patients with chronic myelogenous leukemia (CML), but the mechanisms responsible for the dysfunction are not completely understood. This study reports that CML cells effectively inhibit the baseline and interleukin-2 (IL-2)-induced NK cell cytotoxicity against a CML cell-derived line (K562). A sizable fraction of NK cells subsequently acquired features characteristic of programmed cell death/apoptosis. The CML cell-mediated inhibition of NK cells required triggering of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated formation of reactive oxygen species (ROS) and was prevented by catalase, a scavenger of ROS, and by histamine, acting via H(2)-receptor-mediated inhibition of ROS production in CML cells. In contrast, nonmalignant neutrophilic granulocytes inhibited NK cells via ROS production without the requirement of exogenous NADPH oxidase-triggering stimuli. We propose that paracrine production of ROS may contribute to the dysfunction of NK cells in CML and that histamine may serve as an autocrine inhibitor of ROS formation in leukemic granulocytes. (Blood. 2000;96:1961-1968)

We present a 46-year-old patient with Ph-chromosome negative, bcr-negative chronic myeloid leukaemia (CML) in accelerated phase with a clonal trisomy 21 in the leukaemic blast cells. A rapid progress of disease with appearance of monocytosis is described, showing similar features to chronic myelomonocytic leukaemia (CMML). Heterogeneous characteristics and possible distinction of these two entities are discussed.

Objective: Transforming growth factor beta3 (TGF-beta3) is a potent suppressor of human hematopoietic progenitor cells. In this article, we compare the activity of TGF-beta3 on highly purified CD34+ cells and more immature CD34-DR(-) cells from chronic myelogenous leukemia (CML) patients in chronic phase and normal donors., Materials and Methods: Primitive hematopoietic progenitors were stimulated in liquid cultures and clonogenic assays by early-acting growth factors such as stem cell factor (SCF) and interleukin 11 (IL-11) and the intermediate-late-acting stimulating factors IL-3, granulocyte-macrophage colony-stimulating factor, and erythropoietin. Molecular analysis of bcr/abl mRNA was performed on single CML colonies by nested reverse transcriptase polymerase chain reaction. Moreover, cell cycle analysis and assessment of apoptosis of normal and leukemic CD34+ cells were performed by propidium iodide (PI) alone and simultaneous staining with annexin V and PI, respectively., Results: The colony-forming efficiency of CML CD34+ cells was generally inhibited by more than 90% regardless of whether the colony-stimulating factors were used alone or combined. When compared to normal CD34+ cells, leukemic cells were significantly more suppressed in 6 of 8 culture conditions. The inhibitory effect of TGF-beta3 on CD34+ cells was exerted within the first 24 hours of incubation as demonstrated by short-term preincubation followed by IL-3-and SCF-stimulated colony assays. Evaluation of bcr/abl transcript on residual CML colonies incubated with TGF-beta3 demonstrated a small subset of neoplastic CD34+ cells unresponsive to the inhibitory effect of the study cytokine. TGF-beta3 demonstrated a greater inhibitory activity on primitive CD34+DR cells than on more mature CD34+ cells. Again, CML CD34+DR(-) cells were significantly more inhibited by TGF-beta3 than their normal counterparts in 3 of 8 culture conditions. Kinetic analysis performed on CD34+ cells showed that TGF-beta induces cell cycle arrest in G(1) phase. However, this mechanism of action is shared by normal and leukemic cells. Conversely, TGF-beta3 preferentially triggered the programmed cell death of CML CD34-cells without increasing the proportion of leukemic cells coexpressing CD95 (Fas receptor), and this effect was not reversed by functional blockade of Fas receptor. Conclusion. We demonstrate that TGF-beta3 exerts a potent suppressive effect on CML cells that is partly mediated by Fas-independent apoptosis.

Patients with Ph+ chronic myelogenous leukemia who relapse after a first allogeneic stem cell transplant still have a possibility of long-term survival. To assess the value of the individual therapeutic options, the factors predicting outcome should be identified. We investigated data from 500 patients who relapsed before July 1996; follow-up was updated during 1998. The actuarial survival from relapse was 34.2% (95% confidence interval [CI]: 29. 9%-38.5%) at 5 years and 23.4% (95% CI: 18.9%-27.9%) at 10 years. Survival after relapse was significantly related to 5 factors: time from diagnosis to transplant (< 2 years vs >/= 2 years), disease phase at transplant (first chronic phase vs other), disease stage at relapse (cytogenetic or chronic phase vs advanced phase), time from transplant to relapse (< 1 year vs >/= 1 year), and donor type (HLA-identical sibling vs volunteer unrelated donor). The effects of individual adverse risk factors were cumulative: The probability of survival at 10 years decreased stepwise from 42% (0 factors), 32% (1 factor), 14% (2 factors), 3% (3 factors), to 0% (4 or 5 factors). Novel strategies for high-risk patients are warranted. We conclude that these 5 factors should be taken into account when comparing results of salvage therapies in patients with Ph+ chronic myeloid leukemia relapsing after allogeneic stem cell transplant.

Objective: To explore the relationship between the genetic instability of microsatellite linked with mismatch repair gene and the evolution of CML to blast crisis., Methods: The loss of heterozygosity (LOH) and microsatellite instability (MSI) of two polymorphic microsatellite markers, D2s123 and D3s1298, linked with mismatch repair gene hMSH2 and hMLH1 respectively, were detected by PCR-silver staining method on the bone marrow cells of 18 CML patients, who clinically progressed from the chronic phase to accelerated phase or blast crisis., Results: Differences in microsatellite D2s123 and D3s1298 at the CML accelerated phase or blast crisis in 5 (27.8%) of the 18 patients were demonstrated compared with chronic phase. For D2s123, MSI and LOH were observed in 1 of 8 patients with accelerated phase (12.5%) and 2 of 10 patients in blast crisis (20%). For D3s1298, MSI and LOH were observed in 2 of 10 patients in blast crisis (20%)., Conclusion: The genetic alteration of microsatellite D2s123 and D3s1298 may play a role in the progress of some CML cases.

Chemokines are a family of related proteins that regulate leukocyte infiltration into inflamed tissue and play important roles in disease processes. Among the biologic activities of chemokines is inhibition of proliferation of normal hematopoietic progenitors. However, chemokines that inhibit normal progenitors rarely inhibit proliferation of hematopoietic progenitors from patients with chronic myelogenous leukemia (CML). We and others recently cloned a subfamily of CC chemokines that share similar amino-terminal peptide sequences and a remarkable ability to chemoattract T cells. These chemokines, Exodus-1/LARC/MIP-3alpha, Exodus-2/SLC/6Ckine/TCA4, and Exodus-3/CKbeta11/MIP-3beta, were found to inhibit proliferation of normal human marrow progenitors. The study described here found that these chemokines also inhibited the proliferation of progenitors in every sample of marrow from patients with CML that was tested. This demonstration of consistent inhibition of CML progenitor proliferation makes the 3 Exodus chemokines unique among chemokines. (Blood. 2000;95:1506-1508)

The infusion of lymphocytes from the original marrow donor (donor lymphocyte infusions [DLI]) is remarkably effective in treating chronic myeloid leukemia in relapse after allogeneic stem cell transplantation. DLI are less effective in acute leukemia and other hematologic tumors, but the use of interleukin-2 in conjunction with DLI after allograft may increase the response rate. The use of DLI to treat certain solid tumors is under investigation. In contrast, the value of donor lymphocytes for treating infectious complications post-transplant and graft failure has been established. The major drawback of DLI remains graft-versus-host disease, but novel regimens of administration and/or selective manipulation of donor cells prior to infusion have reduced its incidence. Further progresses in this area will help to establish the role of nonmyeloablative conditioning for allografting.

A 40-year old patient with small cell lung cancer (SCLC) was treated with combined modalities including high-dose chemotherapy with subsequent autologous peripheral blood progenitor cell transplantation plus adjuvant radiotherapy. He achieved complete remission with regards to the primary disease. After an interval of 28 months, he was diagnosed with chronic myelogenous leukemia (CML). Analysis of graft samples at time of primary treatment for SCLC using polymerase chain reaction (PCR) did not show the bcr-abl transcript characteristic for CML. This case supports the observation that CML can develop as a treatment-related malignancy and gives insight in the length of the preclinical phase of the disease.

Philadelphia chromosome-positive chronic myelogenous leukemia was diagnosed in a now 37-year old woman 16 years ago. Induction therapy with hydroxyurea and busulphan led to hematological remission lasting for about 4 months without treatment. Then, intermittent busulphan over a 7 years' period, and subsequently, alpha-interferon was given, of which ever decreasing doses (currently 3.5 megaunits interferon-alpha-2c once every 14 days) have been required to keep leukocyte counts in the target range. Although no major cytogenetic response was achieved by maintenance therapy, the patient has now been in an ongoing chronic phase of disease for 16 years. This is a rare case of indolent chronic myelogenous leukemia, in which, for undefined reasons, the leukemic cells have not acquired the capacity to transform leading to disease acceleration, which usually is imminent after a few years.

Chronic myelogenous leukemia is a myeloproliferative disorder. It is characterized by a biphasic or triphasic clinical course in which a benign chronic phase is followed by transformation into an accelerated and blastic phase. On a cytogenetic and molecular level, most patients with chronic myelogenous leukemia demonstrate BCR-ABL fusion genes in hematopoietic progenitor cells, which result from a reciprocal translocation between chromosomes 9 and 22; this translocation leads to a shortened chromosome 22, called the Philadelphia chromosome. Translation of the fusion products yields chimeric proteins of variable size that have increased tyrosine kinase activity. Conventional chemotherapy with hydroxyurea or busulfan can achieve hematologic control but cannot modify the natural disease course, which inevitably terminates in a rapidly fatal blastic phase. Since its introduction in the 1980s, allogeneic stem-cell transplantation has provided the groundwork for a cure of chronic myelogenous leukemia. However, few patients are eligible for this treatment because of donor availability and age restrictions. Therapy with interferon-alpha alone or in combination with cytarabine suppresses the leukemic clone, produces cytogenetic remissions, and prolongs survival. It is an effective alternative first-line treatment for patients ineligible for transplantation. New drugs active against chronic myelogenous leukemia may show increased activity in the transformed phases of the disease. Novel therapies and concepts are developing rapidly; targeted molecules are tyrosine kinases, ras, and messenger RNA through antisense oligonucleotides. Alternative transplantation options, such as stem cells from autologous sources and matched unrelated donors, are expanding. Immunomodulation by adoptive immunotherapy and vaccine strategies hold significant promise for the cure of chronic myelogenous leukemia.

We report a new chromosomal reciprocal translocation t(5;10)(q33;q22) in a 49-year-old man with atypical chronic myeloid leukemia (a-CML) and history of occupational exposure to petroleum products including benzene and other hydrocarbons. The t(5;10) (q33;q22) was found in 94% and 84% of metaphases in peripheral blood and bone marrow cells, respectively. Cytogenetic analysis of single colonies derived from granulocyte-macrophage (CFU-GM), and erythroid (BFU-E) hematopoietic progenitors showed that 88% and 40% of CFU-GM and BFU-E, respectively, had the t(5;10)(q33;q22). In contrast, peripheral blood T-lymphocytes, and cutaneous fibroblasts had normal 46,XY karyotype. Molecular analysis of the t(5;10)(q33;q22) translocation breakpoint is currently underway in order to identify genes located in this region which might provide insights into the pathogenesis of atypical myeloproliferative disorders.

In the majority of newly diagnosed patients with chronic myeloid leukaemia (CML), the bone marrow contains consistent numbers of normal Ph-negative surrogate stem cells (LTC-IC) which seem to decline rapidly with time. This is confirmed by mobilization studies showing that early after diagnosis is the optimal time to collect Ph-negative progenitor to be utilized for restoring Ph-negative haematopoiesis. In the marrow of the majority of CML patients at diagnosis Ph-positive LTC-IC are found at a lower frequency than Ph-negative LTC-IC and, unexpectedly, they do not show a tendency to increase with time as long as patients remain in chronic phase. Therefore, the decline of normal haematopoiesis does not seem related to a parallel increase in Ph-positive stem cells.

To study the genomic abnormality underlying the acute transformation of chronic myeloid leukemia (CML), 15 CML patients in blast crisis (BC), 3 in accelerated phase (AP), and 20 in chronic phase (CP) were analyzed by conventional cytogenetics, comparative genomic hybridization (CGH), and dual-color chromosomal painting. Philadelphia (Ph) chromosome was identified in every case studied. Only 5 among 20 CP patients had additional abnormalities while 13 of 18 patients with disease progression (BC + AP) showed extra numerical and/or structural chromosomal aberrations. Cytogenetically, the most common chromosome gains during BC and AP were double or triple Ph chromosomes (5 of 14 cases) and trisomy 8 (5 of 14 cases). Trisomies 7 and 17 (1 of 14 cases each) were also observed. CGH analysis detected genetic imbalances in eight cases. Gains of chromosome 20 (3 cases) and 17q (2 cases) were observed, respectively. The recurrent chromosome loss was the deletion of the short arm of chromosome 17, seen in one case with i(17)(q10) and one case with an unbalanced translocation (1;17). In one case, a very complex chromosomal rearrangement, del(3),del(6),der(6)t(17;3;6),der(17)t(6;17), was seen. A novel finding of this work is the involvement of chromosome 1(q12-21qter) in CML disease progression. Overrepresentation of 1(q12-21qter) region was detected by CGH in one case which had a derivative chromosome 17. This abnormal chromosome was later confirmed by fluorescence in situ hybridization (FISH) painting to be a fusion between chromosome 1 and 17 to form the der(17)t(1;17) (q12-21;p11). Two other cases showed the same region being involved in translocations, t(1;10)(q12-21;q26) and t(1;11)(q12-21;p15). It is possible that one or more genes residing on chromosome 1q12-21 may be important in the acute transformation of CML. In conclusion, we find that the combined use of CGH, chromosome painting, and classic cytogenetic analysis allows a better evaluation of the genomic aberration involved in CML blastic transformation, and offers new directions for its further molecular investigations.

The viral Crk oncogene (v-Crk) is known to induce sarcomas in chicken and its cellular homologs c-Crk I, c-Crk II, and Crk-like (CRKL) have been implicated in many signal transduction events. These include cell differentiation, cell migration, and the induced nonresponsiveness of T-cells to stimulation of the T-cell receptor (TCR), a state known as anergy. CRKL is also the most prominent substrate of the Bcr-Abl oncoprotein which causes human chronic myelogenous leukemias (CML). The modular composition of the Crk family adapters which largely consist of Src homology (SH2 and SH3) domains has prompted an intensive search for physiological and pathological upstream and downstream signalling partners which selectively bind to these adapters. Upstream proteins include various receptors and large multisite docking proteins, while several protein kinases and guanine nucleotide release proteins (GNRPs) have been suggested to function downstream of c-Crk and CRKL. Most Crk/CRKL SH2- and SH3-binding proteins contain several docking sites with considerable sequence similarity. Thus the binding requirements of Crk/CRKL SH2 and SH3 domains are now well defined, providing a basis for the design of small inhibitory molecules to block the function of these adapter proteins. The enzymatic cascades activated through Crk family adapters are only partially known, but stress kinases (SAPKs/JNKs) and the GTPase Rap1, as well as the B-Raf isoform of the Raf protein kinases, are affected in some systems. Several yet unidentified, highly selective Crk interacting proteins detectable in specific cell types remain to be studied. More detailed analyses of the enzymatic activities triggered through Crk-type adapters will also be crucial to fully define the signalling pathways controlled by this protein family.