Impact of Exogenous Growth Factors on Proliferation and Chemosensitivity of Minimal Residual Acute Myeloid Leukemia

The biological heterogeneity of AML makes growth factor augmentation of cell cycle-dependent chemotherapy unlikely to be successful for all patients. Patients whose leukemic cells empirically demonstrate cytokine-induced chemosensitization in vitro might benefit from the concurrent administration of growth factors during consolidation chemotherapy. We have explored the growth factor-dependence and response of primary bone marrow samples from patients with AML at diagnosis, remission, and relapse to determine whether minimal residual leukemia remains growth factor-responsive. Most cases of AML studied at all phases of treatment were growth factor-responsive. Growth factor response of occult remission clonogenic leukemic precursors (CFU-L) was usually concordant with their response at diagnosis. Occult CFU-L were markedly resistant to cytosine arabinoside (median LD99% 20 μM); preincubation with IL-3 or GM-CSF did not significantly improve their ara-C sensitivity. While occult remission CFU-L appear to remain growth factor-responsive, it appears unlikely that growth factor augmentation of consolidation chemotherapy will overcome the important problem of drug resistance of residual leukemia.     

Ex vivo elimination of lymphoblastic leukemia cells from human marrow by mafosfamid

Studies were performed to evaluate the anti-tumor activity of mafosfamid, a new synthetic derivative of cyclophosphamide. We tested its ability to eliminate lymphoblastic leukemia cells from autologous bone marrow grafts following a 30 min preincubation in a highly sensitive clonogenic assay. Treatment with 50-100 micrograms mafosfamid/ml eliminated more than 4 logs of contaminating clonogenic tumor cells from a 200-fold excess of normal bone marrow. Flow cytometric studies showed differences in cell cycle kinetics between mafosfamid-resistant and mafosfamid-susceptible tumor cell clones. Compared to drug susceptible clonogenic tumor cells, clones that resisted treatment with 100 micrograms mafosfamid/ml exhibited a smaller percentage of cells in S-phase, indicating that mafosfamid is mostly cytotoxic to rapidly cycling tumor cells. The combination of mafosfamid and a target cell selective immunotoxin containing pokeweed anti-viral protein was superior to mafosfamid alone or immunotoxin alone for purging mafosfamid-resistant leukemic cells from human marrow.     

In vitro purging of clonogenic leukemic cells from human bone marrow by heat: simulation experiments for autologous bone marrow transplantation.

In order to apply a simple purging method by heat to autologous bone marrow transplantation (ABMT), we have revaluated the ability to purge clonogenic leukemic cells from the simulated marrow mixture of normal marrow cells and leukemic cell lines (HL-60, Molt-3 and HEL) in vitro by heat, using two different clonogenic assays for normal granulocyte-macrophage progenitors (CFU-GM) and leukemic cell lines. It appeared that in vitro hyperthermia (42 degrees C for 120 min) is able to selectively remove clonogenic leukemic cells from simulated tumor cell-normal marrow mixtures even when leukemic cell concentrations are increased up to 3 x 10(6) cells/ml in vitro, and results in a 4-6 log destruction of clonogenic leukemic cells/ml according to a limiting dilution assay, while leaving half of normal CFU-GM surviving. The hyperthermic purging of clonogenic leukemic cells was not affected in the presence of normal marrow cells in vitro. This high level of clonogenic leukemic cell depletion by heat correlated with that of immunologic and pharmacologic studies. These results suggest that in vitro hyperthermia could be applied effectively and safely for the elimination of residual clonogenic leukemic cells in autologous marrow grafts before ABMT.     

Reinfusion of leukemic cells with the autologous marrow graft: preclinical studies on lodging and regrowth of leukemia

To evaluate in quantitative terms the contribution of leukemic cells present in the autologous marrow graft to the occurrence of leukemia relapse after autologous bone marrow transplantation, preclinical studies were performed in a rat model for human acute myelocytic leukemia (BNML). Firstly, the number of leukemic cells which--after intravenous transfer--cause death from leukemia in 50% of the recipient rats proved to be 24.7 cells. Secondly, it appeared that the regrowth of leukemic cells in rats heavily pretreated with high-dose cyclophosphamide and total body irradiation was significantly hampered as compared with non-pretreated controls as judged by survival times (37 and 31 days, respectively after 10(3) BNML cells i.v.). The most likely explanation is treatment-induced damage to the microenvironment. Differences in patterns of lodging of infused leukemic cells were ruled out by comparing the uptake of 51Cr-labeled BNML cells in various organs. Finally, extrapolated from the available rat data on log leukemic cell kill induced by high-dose chemoradiotherapy, an hypothesis is presented relating the total tumor load in man to the clinical outcome of autologous bone marrow transplantation. From this hypothesis it is derived that the minimal number of leukemic cells that causes leukemia upon intravenous transfer varies between 10(4) and 10(6).     

IDA对白血病骨髓基质培养体系中HL-60细胞杀伤效应的观察

目的 :研究细胞毒药物对体外残留白血病模型中HL 6 0细胞的杀伤效应。方法 :采用Dexter型骨髓培养体系形成白血病骨髓基质细胞贴壁层 ,接种HL 6 0细胞共培养 ,用去甲氧基柔红霉素 (IDA)处理后 ,动态观察HL 6 0细胞的活性变化。结果 :随着IDA剂量的增加及培养时间的延长 ,HL 6 0细胞活力逐渐减弱 ,去甲氧基柔红霉素 (IDA)与骨髓基质细胞层或单纯的培养基体外孵育对HL 6 0细胞杀伤能力减弱。结论 :急性白血病骨髓基质有助于HL 6 0细胞逃避化疗药物杀伤 ,对残留白血病的形成具有重要作用。     

Molecular biology in acute leukemia

Acute leukemia is a clonal expansion of tumoral cells in bone marrow, blood or other tissues. The acute leukemias are classified as myeloid or lymphoid based on the lineage of the blast cells. Over the past three decades, remarkable advances have been made in the classification and treatment of acute leukemias. In the last years, the research into the molecular pathogenesis of acute leukemia has progressed. The knowledge of chromosomal translocations breakpoints and possible candidate oncogenes and tumor suppressor genes has allowed the integration of all these events into multistep cascades that impact specific signal transduction pathways and lead to leukemic transformation. Supported by an unrestricted educational grant by Bristol-Myers Squibb.     

Selective Cytotoxicity to Human Leukemic Myeloblasts Produced by Oligodeoxyribonucleotide Phosphorothioates Complementary to p53 Nucleotide Sequences

Cells were treated in vitro with oligodeoxyribonucleotide phosphorothioates (ODNs) complementary to sites common to both wild-type and mutant p53 nucleotide sequences. Acute myelogenous leukemia (AML) blasts from peripheral blood were exposed to four different p53 ODNs and showed anti-leukemic effects in suspension culture. This effect continued after removal of the ODN from the medium. Blocking of self-renewal of the leukemic blast stem cells in secondary plating of cells from cloning assays by two of the p53 ODNs was also observed. Control ODNs had no effect on leukemic blasts. Treatment of normal bone marrow cells with the four p53 ODNs did not influence their growth, nor was there any effect by the p53 ODNs on the leukemic cell-line, HL60, that does not express p53. These data suggest that p53 ODNs are selectively toxic to primary myelogenous blasts and may be therapeutically useful in AML.     

Use of five-color staining improves the sensitivity of multiparameter flow cytomeric assessment of minimal residual disease in patients with acute myeloid leukemia

Application of five-color staining may improve quantification of minimal residual disease by multiparameter flow cytometry in acute myeloid leukemia. We analysed bone marrow samples in 139 cases using a comprehensive antibody panel with five-color combinations. Sensitivity was estimated by quantification of leukemia-associated aberrant immunophenotype (LAIP)-positive cells for each LAIP in 18 normal bone marrow (BM) samples. The logarithmic difference (LD) in LAIP-positive cells between leukemic and normal BM amounted to a median of 3.32 (range 1.76 - 4.89). Skipping one color resulted in an increase of LAIP-positive normal bone marrow cells while percentages of LAIP-positive leukemic cells changed only marginally (median gain in LD = 0.54; maximum gain = 3.30). Because regenerating bone marrow has not been used as control data are most important to post-therapy checkpoints. In 32 patients with clinical follow-up, a LD higher than the median (3.25) at the follow-up checkpoint corresponded to a longer event-free survival. These data suggest that the application of five-color staining significantly improves the sensitivity and accuracy of the method.     

Over-expression of angiotensin-converting enzyme (CD 143) on leukemic blasts as a clue for the activated local bone marrow RAS in AML

Local bone marrow renin-angiotensin system (RAS) is an autocrine-paracrine system affecting hematopoiesis. Angiotensin II type 1a (AT1a) receptors are present on the CD34+ hematopoietic stem cells. Angiotensin II stimulates the proliferation of bone marrow and umbilical cord blood hematopoietic progenitors. There are preliminary data that local RAS might also be involved in leukemogenesis. ACE hyper-function may lead to the acceleration of negative hematopoietic regulator peptide, AcSDKP, metabolism, which in turn lowers its level in the bone marrow micro-environment, finally removing the anti-proliferative effect of AcSDKP on the hematopoietic cells and blasts. Renin expression could have a role on the leukemia development and angiotensin may act as an autocrine growth factor for acute myeloid leukemia (AML) cells. The aim of this study is to search ACE (CD 143) surface antigen by flow-cytometric analyses on the leukemic blast cells taken from the bone marrow of the patients with AML. Bone marrow aspiration materials and peripheral blood samples were obtained from 11 patients with AML (eight males, three females; aged 46 (range 26-67) years) and six patients with non-malignant hematological disorders (four males, two females; aged 56 (range 22-71) years). ACE (CD 143) surface antigen was shown to be over-expressed in leukemic myeloid blast cells. ACE is positively correlated with bone marrow blast count. Elucidation of the pathological activity of the local RAS-mediated regulation of the leukemogenesis is both pathobiologically and clinically important, since the angiotensin peptides represent a molecular target in the disease management.     

Studies on chronic myeloid leukemia cell populations with colony-forming abilities in PHA-leukocyte feeder and robinson assays

Investigation of leukemic colony-forming cells (CFC) in PHA-supplemented cultures requires removal of T lymphocyte precursors prior to culture. Using a method of discontinuous density gradient centrifugation with concurrent depletion of E-rosette forming cells, T lymphocytes were effectively separated from light density CML bone marrow and blood cell fractions. Consequently, in light density fractions (1.056 and 1.059 g/ml) pure leukemic colony growth was obtained in the PHA-leukocyte feeder (PHA-l.f.) assay. Fraction 1.062 g/ml also yielded pure leukemic colonies in most experiments. Comparison of the density distributions of leukemic PHA-l.f. CFC and Robinson CFC revealed that both CFC populations had congruent density profiles in most patients. In others PHA-l.f. CFC were found to be of somewhat higher density than Robinson CFC. The most striking divergence was apparent in a patient in blast crisis. The findings suggest that different subsets of precursor cells within the CML population proliferate in PHA-l.f. and Robinson colony methods. Both colony techniques are thus potentially useful for discriminating subpopulations of colony-forming cells in chronic myeloid leukemia.     

Studies of myelopoiesis in vitro on blood and bone marrow cells of patients with acute leukemia in long-term remission

In vitro myelopoiesis in a group of ten patients with acute leukemia (6 AML, 1 APML, 3 ALL) in long-term remission has been studied. The patients remained in first stable remission for at least 4 years and maintenance therapy had been completed in all patients except one. In the patients, colony formation of bone marrow cells after 7 days of incubation was significantly increased compared to a representative control group. The proliferation of microclusters after 3 incubation days was also markedly enhanced, and accelerated proliferation of all aggregates (microclusters, macroclusters and colonies) could be demonstrated in culture with the patients' bone marrow cells. The use of autologous feeder layers and autologous serum showed no inhibitory effect on colony formation. The proportion of eosinophil colonies formed with the patients' bone marrow cells was in the normal range. In contrast to the high proliferation capacity of bone marrow precursor cells the CFU-c number of peripheral blood was significantly decreased in the patients' group. No significant correlation between CFU-c number of bone marrow and blood cells could be found. The colony stimulating activity of the patients' peripheral mononuclear cells was normal compared to healthy controls. We conclude from this study that even in long-term remission of acute leukemia certain in vitro abnormalities exist in myelopoietic proliferation and regulation.     

A monoclonal antibody reactive with normal and leukemic human myeloid progenitor cells

Anti-MY9 is an IgG2b murine monoclonal antibody selected for reactivity with immature normal human myeloid cells. The MY9 antigen is expressed by blasts, promyelocytes and myelocytes in the bone marrow, and by monocytes in the peripheral blood. Erythrocytes, lymphocytes and platelets are MY9 negative. All myeloid colony-forming cells (CFU-GM), a fraction of erythroid burst-forming cells (BFU-E) and multipotent progenitors (CFU-GEMM) are MY9 positive. This antigen is further expressed by the leukemic cells of a majority of patients with AML and myeloid CML-BC. Leukemic stem cells (leukemic colony-forming cells, L-CFC) from most patients tested were also MY9 positive. In contrast, MY9 was not detected on lymphocytic leukemias. Anti-MY9 may be a valuable reagent for the purification of hematopoietic colony-forming cells and for the diagnosis of myeloid-lineage leukemias.     

PHA-induced colony formation in acute non-lymphocytic and chronic myeloid leukemia

A method which has previously been introduced for growing colonies in vitro from T-lymphocytic cells in normal subjects, has been applied to studying the growth of colonies from the bone marrow and blood of patients with acute non-lymphocytic leukemia and chronic myeloid leukemia. The technique consists of an underlayer of agar containing irradiated leukocytes and a liquid overlayer containing PHA. In leukemic patients, the assay permitted the formation of T-lymphocytic as well as leukemic colonies. Removal of the E-rosette forming cells before culture resulted in the appearance of mainly leukemic colonies. Cytogenetic analysis demonstrated the presence of the acquired karyotypic changes characteristic of the leukemic cells of these patients in the colony cells. Study of eight cases of acute non-lymphocytic leukemia, four cases of blast crisis of CML, and seven cases of CML chronic phase revealed the assay to be efficient in growing large numbers of leukemic blast colonies, as compared to the Robinson culture assay where large colony formation is only found in chronic leukemia [10]. Removal of the progenitors of lymphocytic colonies was sometimes incomplete in acute leukemia but not in CML.     

Membrane Fluidity and Adherence to Extracellular Matrix Components Are Related to Blast Cell Count in Acute Myeloid Leukemia

The level of blast cells in peripheral blood in acute myeloid leukemia (AML) varies in individual patients. The bone marrow egress of hematopoietic cells is an unclear phenomenon in which cell deformability and cytoadhesion to the extracellular matrix are involved. One component of deformability is the membrane fluidity. Using fluorescence polarization, we have studied the fluidity of blast cell membranes from 22 AML patients. This membrane was found to be highly fluid and a statistically significant correlation was found between the increase in membrane fluidity and the number of blast cells in the blood. Studying interaction between blast cells and several components of bone marrow stroma, we found adhesion to fibronectin and fibroblastic extracellular matrix. Adhesion to the extracellular matrix was inversally correlated to the level of blast cells in the blood. The observed increase in membrane fluidity and reduction of adhesion to bone marrow stroma may result in an increase of blast cells egress in AML.     

Bone Marrow Purging in Acute Leukemia with Alkyl-Lysophospholipids: A New Family of Anticancer Drugs

The alkyl-lysophospholipids are a new family of anticancer drugs which target the cell membrane as their site of action. Enzymes involved in signal transduction (protein kinase C and phosphatidylinositol phospholipase C), phospholipid biosynthesis (lysophosphatidyl acyltrans-ferase and CTPcholinephosphate cytidylyltransferase) and maintenance of membrane integ rity (Na, K ATPase sodium pump) are inhibited.

A unique feature of the alkyl-lysophospholipids is their selective cytotoxicity to neoplastic cells. This suggests that the compound would be an excellent agent for purging residual leu-kemic cells from marrows of patients in remission prior to autologous bone marrow trans plantation. Preclinical studies in a murine leukemia model and in an in vitro human system demonstrated successful elimination of leukemic cells from a mixture of normal and leukemic marrows. Twenty-nine poor risk patients with acute leukemia underwent autologous bone marrow transplantation and were reinfused with marrow treated in vitro with edelfosine. Nine of these patients remain in remission free of leukemia from 368 to 1369 days. These en couraging results warrant further investigation.     

Study of differentiation of fresh myelogenous leukemic cells by compounds that induce a human promyelocytic leukemic line (HL-60) to differentiate

The human promyelocytic leukemia cell line known as HL-60 can be triggered to mature to functional granulocytes and/or macrophages after exposure to a variety of compounds. The findings have generated enthusiasm for possible therapy of leukemia using compounds that induce leukemic cell differentiation. We investigated whether five compounds known to trigger HL-60 differentiation to granulocytes could trigger the maturation of blast cells from 12 patients with myelogenous leukemia. Maturation was judged by morphology, superoxide production, phagocytosis, expression of Fc receptors, and development of alpha-napthyl acetate esterase activity. The blast cells from most patients showed little morphological, histological or functional maturation after exposure to the various compounds as compared to the blast cells cultured without the compounds. Actinomycin was able to induce significant maturation of leukemic cells of some patients when maturation was analyzed by several statistical methods. Our study suggests that many compounds which trigger differentiation of promyelocytic leukemia cells may not trigger differentiation of less mature myeloid leukemic cells.     

Differential sensitivity of AKR murine leukemia and normal bone marrow cells to hyperthermia

To determine if there is a differential effect of hyperthermia on AKR murine leukemia and AKR normal bone marrow cells incubated in vitro, the fractional survival of leukemic and of normal cells with proliferative potential as a function of heating exposure was estimated by evaluating spleen colony formation. Normal bone marrow colony-forming units were assayed in lethally irradiated (750 centigrays) mice; leukemic colony-forming units were assayed in nonirradiated mice. Electron micrographic studies of leukemic cells treated with 41.8 degrees hyperthermia found that structural damage to the cell, i.e., changes in the Golgi apparatus, was associated with the lack of ability to form colonies. AKR leukemia cells were more sensitive than normal cells to hyperthermic killing at 41.8 degrees and at 42.5 degrees. This differential was found whether cells of each type were heated separately or when mixed together. This model system demonstrates an inherently greater sensitivity of neoplastic cells, as compared to normal syngeneic stem cells, to thermal killing. This finding may have relevance to autologous bone marrow transplantation in humans.     

Elimination of leukemic cells by the combined use of ether lipids in vitro

Two ether lipids, CP-46,665-1 (4-aminomethyl-1-[2,3-(di-n-decyloxy)-n- propyl]-4-phenylpiperidine) and ET-18-OCH3 (racemic 1-O-octadecyl-2-O-methylglycero-3-phosphocholine) have been shown to possess antileukemic activity in vitro. To explore the possible use of these compounds for purging remission bone marrow cells of leukemic cells, we examined the cytotoxic effect of these compounds on normal hematopoietic progenitor cells and leukemic cell line cells (HL-60, K-562, KG-1a, KG-1, and Daudi) by using the clonogenic assay. When cells were treated with CP-46,665-1 or ET-18-OCH3 (50 micrograms/ml for 1 h), these compounds did not inhibit the growth of normal progenitors, whereas the growth of the clonogenic leukemic cells was inhibited with differences in their sensitivities to the cytotoxic effect of CP-46,665-1 and ET-18-OCH3. Incubation of leukemic cells (HL-60 and Daudi cells) with both CP-46,665-1 (50 micrograms/ml) and ET-18-OCH3 (50 micrograms/ml) for 1 h resulted in a greater reduction of clonogenic leukemic cells than treated with each compound alone. Approximately a 3 log killing of clonogenic HL-60 cells and a 5 log killing of Daudi cells was achieved; however, the combined treatment of normal bone marrow cells with CP-46,665-1 and ET-18-OCH3 did not alter the growth of normal progenitors. This combined treatment also selectively eliminated the leukemic cells (HL-60 and Daudi cells) from a mixture (1000:1) of normal bone marrow cells and leukemic cells. It is conceivable that the pronounced difference in sensitivity to this combined treatment can be exploited for the elimination of residual leukemic cells in autologous remission marrow grafts.     

A new set of monoclonal antibodies against acute lymphoblastic leukemia

Six monoclonal antibodies produced by immunization of Balb/c mice with common acute lymphoblastic leukemia (cALL) cells were tested against various types of normal and malignant tissues. ALB1 and ALB2 are directed to the cALL antigen (CALLA gp100); ALB6 recognizes a determinant of p24; ALB7, ALB8 and ALB9 have a pattern of reactivity similar to Ba1. None of these antibodies specifically identify cALL but they should be useful tools for diagnosis or depletion of bone marrow in autologous therapy in transplantation. In addition, the example of ALB6 which acts as a platelet aggregating agent, suggests that the study of other cell systems expressing the antigens associated with cALL may shed light on the function of these antigens and subsequently on the physiopathology of the leukemic cells.     

Cumulative Bone Marrow Tromal damage caused by X-Irradiation and Cytosine-Arabinoside in Leukemic mice

A study of treated murine acute myeioid leukemia (AML) with an emphasis on the bone marrow stromal function is reported. Leukemia was induced in C57BI mice through intraperitoneal (i.p.) inoculation of C-1498 myelogenous leukemic cells. The leukemic mice were administered: (1) total body lethal X-irradiation (t.b.i); (2) two i.p. cytosine-arabinoside (Ara-C) injections followed by X-irradiation. Control mice received similar regimens. Bone marrow of experimental and control mice was processed for stromal cell cultures (SCC) andin vitro engraftment of hematopoietic cells onto the cultures. The results of this study indicate that the bone marrow stromal deficiency which occurs in leukemia is aggravated by Ara-C and irradiation treatments. Moreover, SCC of treated leukemic mice sustainin vitro hematopoiesis only to a limited degree. Stromal deficiency, as possible cause for graft failure in bone marrow transplanted leukemic patients, is discussed.