Effect of platelet-derived growth factor on the proliferation of blast progenitors from acute myeloblastic leukemia patients

The effect of platelet-derived growth factor (PDGF) on self-renewal and terminal divisions of blast progenitors of acute myeloblastic leukemia was studied. The terminal divisions of blast progenitors were determined as the primary blast colony formation in methylcellulose culture; self-renewal was assessed by secondary colony formation by replating in methylcellulose and the recovery of clonogenic cells in suspension culture. PDGF neither enhanced nor suppressed primary or secondary blast colony formation in methylcellulose culture nor recovery of clonogenic cells in suspension. The results show that PDGF does not affect the self-renewal or terminal divisions of leukemic blast progenitors.     

Effect of recombinant GM-CSF and recombinant G-CSF on colony formation of blast progenitors in acute myeloblastic leukemia

The colony-promoting activities of recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) and recombinant granulocyte colony-stimulating factor (rG-CSF) on primary and secondary colony formation by blast progenitors (leukemic colony-forming units [L-CFU]) from 21 patients with acute myeloblastic leukemia (AML) were examined using blast colony assay and compared to colony promotion stimulated by phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM). Recombinant GM-CSF stimulated blast colonies in 13 out of 20 cases examined (1 case not done). The magnitude of stimulation by rGM-CSF varied significantly according to the type of AML, but in general was lower than that of PHA-LCM. Blast cells of type M1 did not form any colonies with rGM-CSF, although numerous colonies were produced with PHA-LCM. Type M4 blasts formed fairly large numbers of colonies, though slightly less than those stimulated by PHA-LCM. Blasts of type M2 and M5 formed colonies with the stimulation of rGM-CSF, but the numbers were considerably smaller than type M4 and those stimulated with PHA-LCM. Recombinant G-CSF stimulated blast colonies in only 5 out of 21 cases, 3 of them being type M2. The number of cases responding to rG-CSF was significantly smaller than that responding to rGM-CSF, and even in cases in which colonies were formed, the magnitude of stimulation was minimal. From these results it seems likely that blast cells of different types of AML require a different kind of CSF for their optimal growth; type M4 blasts responded to the stimulation of rGM-CSF well, but blasts of other types of AML responded poorly. Thus, except for type M4, CSF(s) other than rGM-CSF seems to be required for the sufficient growth of L-CFU. Recombinant G-CSF is not likely to play an essential role in the proliferation of leukemic blasts of most types. Previous exposure to rGM-CSF and rG-CSF did not alter the self-renewal capacity, cellular phenotype, and morphology of colony cells, indicating that the direction and degree of differentiation of L-CFU stimulated by rGM-CSF or rG-CSF were not different from those stimulated with PHA-LCM.     

Characterization of granulocyte-macrophage colony-stimulating factor receptor on the blast cells of acute myeloblastic leukemia

Iodinated granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to document the specific binding of GM-CSF to all acute myeloblastic leukemia (AML) samples examined in the present study. There was some heterogeneity in the number of GM-CSF binding sites per cell. To determine whether the low level of binding to some patient samples may be attributed to receptor occupancy by an endogenous source of GM-CSF, we devised an acid wash procedure that could remove surface-bound GM-CSF without affecting receptor properties. We thus document that GM-CSF specific binding to AML blasts before or after acid wash was the same, indicating that the observed heterogeneity in binding is not the result of receptor occupancy by an endogeneous source of GM-CSF. Saturation analyses are in favor of the presence of two classes of binding sites on AML blasts: a high-affinity receptor that binds GM-CSF with a dissociation constant (kd) of 3 to 73 pmol/L and a second class of low-affinity receptor that binds GM-CSF with a kd of 1 to 10 nmol/L. Binding studies with two established cell lines KG-1, and IRCM-8 also showed the presence of two classes of binding sites with high and low affinities. Analysis of GM-CSF titration curves in culture indicate that the median effective concentration required for stimulation of blast colony formation (EC50 = 5-36 pmol/L) were in the range of the kd of the high-affinity binding site, suggesting that this high-affinity binding site mediates the proliferative response.     

The proliferation in suspension of the progenitors of the blast cells in acute myeloblastic leukemia

A minority of blast cells in acute myeloblastic leukemia (AML) form colonies in culture in methylcellulose when stimulated by media conditioned by normal leukocytes in the presence of phytohemagglutinin (PHA-LCM). Blast colonies can be replated successfully, either as pooled cells or suspensions from single colonies. However, the plating efficiency declines with repeated passages, and more than four subcultures have not been achieved. In this study, blast populations were cultured in suspension, with fetal calf serum, alpha-minimal essential medium and PHA-LCM. In cells from 17 of 18 patients, exponential growth of clonogenic blast cells was maintained for six to seven days without reculturing. Colonies obtained from progenitors taken from liquid culture and replated in methylcellulose were replated to obtain the secondary plating efficiency (PE2). In 14 cases, this value was maintained or increased. In three instances, PE2 fell following culture in methylcellulose. When cells in suspension were recultured, exponential growth continued. In nine instances, exponential growth was maintained for from seven to 70 days. During this time, PE2 was maintained. Results from experiments using velocity sedimentation separation and analysis of single colonies were consistent with the view that the increase in clonogenic cells in suspension was a manifestation of their self-renewal capacity. The observations also support a model of blast progenitor growth that contains the postulate that these are capable not only of self-renewal but also of determination-like events leading to loss of proliferative capacity.     

Binding characteristics and proliferative action of purified granulocyte colony-stimulating factor (G-CSF) on normal and leukemic human promyelocytes

The binding of purified 125I-labeled murine granulocyte colony stimulating factor (125I-G-CSF) to normal and leukemic human cells was examined. Normal neutrophils and their precursors demonstrated specific labeling with 125I-G-CSF, whereas eosinophils, lymphocytes, and erythroid cells did not. Normal human promyelocytes demonstrated the highest binding among hemopoietic cells. Human myeloid leukemic cells also demonstrated consistent specific labeling with 125I-G-CSF. Normal promyelocytes and chronic myeloid leukemia promyelocytes demonstrated only transient clonal proliferation in vitro when stimulated by G-CSF, but this was not always the case with acute promyelocytic leukemic cells. The qualitative responsiveness of normal and leukemic cells to G-CSF was very similar despite heterogeneity in receptor numbers on individual cells. A subset of acute promyelocytic leukemic cells appeared unresponsive to stimulation by GM-CSF.     

Absence of mutations in the granulocyte colony-stimulating factor (G-CSF) receptor gene in patients with myelodysplastic syndrome/acute myeloblastic leukaemia occurring after treatment of aplastic anaemia with G-CSF

The development of myelodysplastic syndrome/acute myeloblastic leukaemia (MDS/AML) has been reported in patients with aplastic anaemia (AA) after administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF). Similarly, patients with severe congenital neutropenia (SCN) have an increased risk of developing MDS/AML after treatment with rhG-CSF. Point mutations in the G-CSF receptor gene are found in about 20% of SCN patients who are predisposed to MDS/AML. We investigated the occurrence of mutations in the G-CSF receptor in eight patients with AA who developed MDS/AML. No mutations were detected around the cytoplasmic domain of the gene in our patients, indicating that the mechanisms of clonal evolution to MDS/AML in patients with AA might be different from those with SCN.     

Proliferative state of blast cell progenitors in acute myeloblastic leukemia (AML).

Peripheral blood from patients with acute myeloblastic leukemia (AML) contains cells capable of giving rise to colonies in culture when stimulated by media conditioned by leukocytes (LCM) in the presence of phytohemagglutinin (PHA). Two types of colonies are recognized with high frequency: The first grows in the presence of low concentrations of PHA LCM, have a blast-like morphology, and are numerically correlated with morphologically identified blast cells. The second requires either high PHA LCM concentrations or PHA alone with or without 2-mercaptoethanol and consists of cells capable of forming rossettes with sheep erythrocytes and resembles. T-lymphocyte colonies from normal blood. Precursors of blast cell colonies from 15 leukemic patients were tested for cycle state, using either the 3H-thymidine or hydroxyurea techniques. All were found to have a high proportion of cells in the S phase of the cycle. In contrast, T lymphocyte precursors from three normal individual were quiescent. The data are consistent with the maintenance of the leukemic blast cell populations by the proliferative activity of a small subpopulation of blasts.     

Sensitivity to 5-azacytidine of blast progenitors in acute myeloblastic leukemia

In a previous study, we showed that the blast stem cells of acute myeloblastic leukemia (AML) were more sensitive to cytosine arabinoside (ara-C) when growing in suspension culture than during colony formation in methylcellulose. We suggested that the difference might be explained by considering the cellular mechanisms responsible for growth in suspension and colony formation. In the former, the clonogenic cells increase in number (self-renewal); in the latter, most of the divisions are terminal. The increased sensitivity to ara-C in suspension might then be attributed to its ability to inhibit self-renewal to a greater degree than cell division generally. A test of this hypothesis would be to compare the survival curves in suspension and in methylcellulose using a drug that spared or stimulated self-renewal. Such an agent is 5- azacytidine (5-aza) and has the additional advantage that its analogue, 6-azacytidine (6-aza) has no effect on self renewal. The data supported the hypothesis, since clonogenic AML blasts were much less sensitive to 5-aza in suspension than in methylcellulose. The effect of 6-aza, while qualitatively similar, was much less marked. Controls showed that the difference in survival curves could not be explained on a kinetic basis or by the secretion of growth factors by 5-aza-treated cells. We suggest that a comparison of the effects of drugs in suspension and in methylcellulose may be useful in preclinical screening of putative anti- AML compounds.     

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) in serum during induction treatment of acute leukaemia

Granulocyte-macrophage colony stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) are increasingly used to stimulate granulopoiesis in neutropenic patients but in most cases without any knowledge of the endogenous CSF-levels. With the purpose to define serum levels of GM-CSF and G-CSF during induction chemotherapy and haematological reconstitution in patients with acute leukaemia we have used enzyme-linked immunosorbent assay (ELISA) techniques to measure these growth factors in 18 patients with acute myeloid leukaemia (AML) and eight patients with acute lymphoblastic or undifferentiated leukaemia (ALL/AUL). G-CSF above 0.05 ng/ml was detected in 54% of the analysed AML samples, median 0.29 (range 0.05-2.80) ng/ml; and in 40% of analysed ALL/AUL samples, median 0.09 (range 0.05-3.00) ng/ml. In patients with AML there was a clear correlation between an elevated serum concentration of G-CSF and documented infections. On the other hand, 15/18 of the patients with acute myeloid leukaemia and 8/8 patients with ALL/AUL had non-detectable levels of GM-CSF (less than 0.10 ng/ml). Two patients had measurable levels of GM-CSF in all samples, median 0.71 (range 0.26-1.18) ng/ml and in these patients the levels successively decreased during and after chemotherapy and did not increase in response to infections. In normals detectable levels of GM-CSF were found in 2/35 individuals and G-CSF in 0/10 individuals.     

Recombinant human granulocyte colony-stimulating factor (G-CSF) combined conditioning regimen for allogeneic bone marrow transplantation (BMT) in standard-risk myeloid leukemia

We previously suggested that using a combined conditioning regimen including rhG-CSF with allogeneic BMT in refractory AML and CML in blast crisis might reduce the rate of relapse and improve disease-free survival, without any major side effects. In this study, we used the same protocol for 10 AML patients in complete remission (CR) and 6 CML patients in the chronic phase (CP). We compared disease-free survival as well as toxic side effects of the regimen with 6 AML patients in CR and 6 CML patients in CP treated with chemoradiotherapy without G-CSF. The conditioning regimen consisted of TBI and high-dose AraC. RhG-CSF was infused continuously at a dose of 5 μg/kg/day, starting 24 hr before the initial dose of total body irradiation (TBI) until the end of AraC therapy. In all 28 cases, there were no early stage deaths due to regimen-related toxicity (RRT). None of the 10 AML cases treated with the G-CSF combined regime relapsed. In 6 AML cases treated conventionally without G-CSF, one patient died of infection and another relapsed. There were no relapses in either CML group. In the combined G-CSF group, one patient died of interstitial pneumonitis 48 days after BMT, while the rest of the CML cases are still alive. There were no relapses with rhG-CSF and no serious adverse effects in terms of RRT, acute graft vs. host disease (GVHD), or leukocyte recovery. Am. J. Hematol. 57:303–308, 1998. © 1998 Wiley-Liss, Inc.     

Simultaneous administration of granulocyte colony-stimulating factor (Filgrastim) and induction chemotherapy in acute lymphoblastic leukemia

Summary The present study was designed to determine whether Filgrastim, a neutrophil-specific hematopoietic growth factor, could be administered simultaneously with intensive induction chemotherapy for adult acute lymphoblastic leukemia (ALL). The effect of Filgrastim on the severity of chemotherapy-induced neutropenia, fever, and infections was assessed in 15 patients treated according to the protocol of the German multicenter ALL (GMALL) trial 04/89. Filgrastim (5g/kg/day) was given concurrently with successive cycles of cyclophosphamide, cytosine-arabinoside (ara-C), 6-mercaptopurine (6MP), prednisone (PRD), intrathecal methotrexate, and prophylactic cranial irradiation. During the study period the median total duration of severe neutropenia (<0.5×10⁹/l) in 13 evaluable patients was 8 days, individual periods of neutropenia typically were short. Infections occurred in six patients; seven patients remained fever-free during treatment with Filgrastim. We conclude that simultaneous treatment with Filgrastim and chemotherapy in this specific setting is feasible and well tolerated. The efficacy of this treatment approach in terms of overall treatment results requires further testing in a randomized trial.     

Effects of angiogenic regulators on in vitro proliferation and cytokine secretion by native human acute myelogenous leukemia blasts

Angiogenesis seems to be important in the pathogenesis of acute myelogenous leukemia (AML). The endothelial cell proliferation and microvessel formation are regulated by a wide range of soluble mediators, including angiogenin, angiopoietin-2, basic fibroblast growth factors, vascular endothelial growth factor (VEGF), VEGF-D, angiostatin and endostatin. In the present study, it has been investigated whether these mediators have an additional direct effect on the proliferation and cytokine release by native human AML blasts. AML cells derived from a large group of consecutive patients were investigated. All these mediators could alter the proliferation and cytokine release [interleukin (IL) 1beta, IL6, IL8, tumor necrosis factor alpha] for a minority of patients. Alteration of spontaneous proliferation by at least one mediator was detected in five of 38 patients; whereas, altered cytokine (Flt3-ligand, granulocyte-macrophage colony-stimulating factor, stem cell factor)-dependant proliferation was observed for 10 patients. Growth enhancement was most frequently observed, whereas growth inhibition was uncommon. The effects on AML blast proliferation were often dependant on or were modulated by the presence of the three hematopoietic growth factors. Based on the present results, it is concluded that angioregulatory mediators have additional growth-enhancing effects directly on the AML blasts for certain patients. However, based on the results from this investigation and previous studies it is suggested that their major contribution to the pathogenesis of AML is through their effects on regulation of bone marrow angiogenesis, and future studies of these mediators in AML should probably focus on these effects.     

Systemic capillary leak syndrome after granulocyte colony-stimulating factor (G-CSF)

Capillary leak syndrome (CLS) commonly occurs in the intensive care setting. CLS is seen in conditions such as septic shock or may result from conditions such as multitrauma and pancreatitis, which result in the systemic inflammatory response syndrome (SIRS). We present two cases in which both patients suffered with CLS, which we believe was caused following administration of granulocyte colony-stimulating factor, to our knowledge not described in the intensive care patient previously. We discuss how these patients management differs from other intensive care unit patients with CLS and how it is important to diagnose this condition early in haematological oncology cases.     

Effect of recombinant interferons on colony formation of blast progenitors in acute myeloblastic leukemia

The effect of recombinant interferons (rIFNs) on primary and secondary colony formation by blast progenitors (leukemic colony-forming units, L-CFU) from the peripheral blood of patients with acute myeloblastic leukemia (AML) was examined. rIFN-alpha, rIFN-beta, and rIFN-gamma inhibited L-CFU in a dose-dependent manner. When 3000 U/ml rIFN-alpha, -beta, and -gamma were added, L-CFU were suppressed to 20%, 12%, and 40% of the control cultures, respectively. The concentrations of rIFN-alpha, -beta, and -gamma required for 50% inhibition of colony formation were 63, 29, and 250 U/ml, respectively. The self-renewal capacity of L-CFU was inhibited by rIFNs in a dose-dependent manner, the degree of inhibition being the same for all three rIFNs. These rIFNs also inhibited normal granulocyte-macrophage progenitors to a similar degree as L-CFU. Taken together, these in vitro studies indicate that these rIFNs may be efficacious in the treatment of AML, though development of granulocytopenia may be observed as a complication of IFN therapy.     

The effect of granulocyte-macrophage colony-stimulating factor on undifferentiated and mature acute myelogenous leukemia blast progenitors.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been used recently to recruit undifferentiated acute myelogenous leukemia (AML) blasts into the S-phase of the cell cycle and increase the fraction of cells killed by cell cycle-specific drugs. Using three AML blast colony assays combined with a suspension culture (delta assay), we determined the in vitro effect of GM-CSF on mature and undifferentiated AML blast progenitors obtained from bone marrow aspirates of six AML patients. GM-CSF stimulated AML blast colony proliferation at a concentration of 5 ng/ml in the methylcellulose and the agar clonogenic assays in six of six AML marrow samples. However, in the delta assay, which selects for immature AML progenitors, GM-CSF did not affect AML blast colony-forming cells in five of six AML marrow samples at concentrations ranging from 5 to 300 ng/ml. Our data imply that GM-CSF stimulates mature but not undifferentiated AML blast progenitors. It is therefore possible that GM-CSF may not be beneficial as a recruiting agent in most AML patients.     

Autocrine growth mechanisms of the progenitors of blast cells in acute myeloblastic leukemia

Autocrine growth mechanisms of leukemic blast progenitors in acute myeloblastic leukemia (AML) were investigated. Colony formation of leukemic blast progenitors was observed in 14 of 14 patients tested when purified blast cell fraction depleted of both T cells and monocytes was plated in methylcellulose without any colony-stimulating factor (CSF). However, there existed a minimal cell density required to initiate blast progenitor growth with marked patient-to-patient variation. To clarify the role of cell density on the spontaneous growth of blast progenitors, we tested whether leukemic cells produced and secreted some stimulatory humoral factor(s). Production of colony- stimulating activity (CSA) by blast cells was observed in 17 of 18 patients tested. Following further depletion of monocytes, the CSA levels decreased markedly in 14 patients, indicating that blast cells with monocytoid differentiation were responsible for CSA production. We also confirmed granulocyte colony-stimulating factor (G-CSF) and/or granulocyte macrophage-colony-stimulating factor (GM-CSF) production by leukemic blasts using specific immunologic assays. When leukemic cells were divided into nonadherent nonphagocytic cell fraction and adherent cell fraction, only nonadherent nonphagocytic cells showed clonogenecity and adherent blast cells lacked the colony-forming capacity. The results indicate that there are at least two blast cell subpopulations in AML: one is proliferating subpopulation with self- renewal capacity and the other is supporting subpopulation with functions such as CSF production. The quite intimate relationship between these two blast cell subpopulations in AML may play an important role on the growth of leukemic blast progenitors in vitro.     

Granulocyte colony-stimulating factor (G-CSF) as an adjunct to induction chemotherapy of adult acute lymphoblastic leukemia (ALL)

Summary Our purpose was to evaluate the ability of re-combinant human granulocyte colony-stimulating factor (r-metHuG-CSF) as an adjunct to induction chemo-therapy of acute lymphoblastic leukemia (ALL) to ameliorate chemotherapy-induced neutropenia and thus allow patients to receive full doses of chemotherapy on time. Sixteen consecutive patients with adult ALL (13 de novo, three relapsed) were treated with induction chemo-therapy according to the BMFT protocol and received in addition r-metHuG-CSF (200g/m²/day). Patients who were treated with the same induction chemotherapy but without G-CSF between 1982 and 1990 served as controls. Fifteen of the 16 patients achieved complete hematological remission. One patient died because of fungal septicemia. Compared with historical controls, G-CSF-treated patients had a significantly faster neutrophil recovery in phase I, resulting in neutrophil counts > 1000/l at day 17 vs day 26 (in median) in controls. In phase II, the onset of severe leukocytopenia (< 1500/l) was significantly (p = 0.01) delayed and the degree of leukocytopenia less pronounced (mean nadir 3300/l) in G-CSF-treated patients compared with controls (1880/l). The number of days of febrile neutropenia was not different in phase I. In phase II it was lower in study patients (0 vs 1.1 days), but the difference did not reach statistical significance (p = 0.09). Full doses of chemo-therapy could be given on time to 11/13 (85%) G-CSF pa-tients but to only 7/30 (23%) controls. These data indicate that (a) G-CSF can be given along with chemotherapy in induction treatment of ALL without compromising efficacy; (b) the duration of neutropenia in phase I is markedly shortened and the degree of leukocytopenia in phase II ameliorated; (c) these beneficial effects allow patients to receive full doses of chemotherapy on time.     

Dose-dependent effects of a tumor promotor on blast cell progenitors in human myeloblastic leukemia

AML blast cell progenitors form colonies in culture when stimulated by a media conditioned by leukocytes in th presence of PHA. Two cellular processes occur during colony formation: self renewal generates new progenitors, while others undergo a change that leads to decreased proliferative potential. We tested the effect of the potent tumor promotor, 12-0-tetradecanoyl phorbol acetate (TPA) on these events. TPA was found to be toxic to blast cell colony formation; doses in excess of 1 ng per ml usually abolished it. At doses lower than this, self renewal, as determined by replating either individual or pooled colonies, was increased. At proliferation inhibiting TPA doses, surviving cells showed a spindle morphology, and had increased ANA esterase activity. We interpret the data to mean that TPA decreases blast cell maturation at low doses and may increase it at high doses.