Granulocyte colony-stimulating factor and differentiation-induction in myeloid leukemic cells

The granulocyte colony-stimulating factor (G-CSF) belongs to a family of hemopoietic growth factors regulating the production of granulocytes and macrophages. Murine G-CSF stimulates the proliferation and differentiation of precursors of neutrophilic granulocytes and is also able to stimulate the functional activities of mature neutrophils. Among the hemopoietic growth factors, G-CSF has an outstanding capacity to induce terminal differentiation and suppression of self-renewal in myeloid leukemic cells. Murine and human G-CSF's show complete biological cross-reactivity across species and bind equally well to G-CSF receptors of either species. Specific receptors for G-CSF exist on all normal neutrophilic cells and have not been lost in the generation of primary human myeloid leukemias. This data indicates that G-CSF may be a useful reagent in the treatment of myeloid leukemia, in hemopoietic regeneration and in increasing resistance against infections.     

Differentiation of myeloid cells in liquid culture: 2. Acute myelocytic leukemia cells

In a companion paper we demonstrated that normal peripheral blood granulocytic precursor cells differentiate after 2-3 weeks in suspension culture. In the studies described here leukemic blast cells obtained from 14 patients with acute myelocytic leukemia (AML) and two patients with chronic myelocytic leukemia in blastic crisis were cultured in McCoy's 5A medium containing 15 per cent fetal bovine serum for 2-3 weeks at 37 degrees C in an atmosphere of 5 per cent CO2-95 per cent room air. 'Spontaneous' myeloid differentiation (20 x 10(4) viable mature myeloid cells ml-1) occurred in the cultures of cells obtained from 8 pts. The differentiation was granulocytic in three cases, monocytic in four cases and of mixed type in one case. Differentiation was independent of the growth of the cells in culture and occurred in four cases after the first week. Monocytic differentiation was seen only in AML of the FAB M4 type whereas granulocytic or mixed differentiation were seen only in AML of the FAB M1 or M2 types. When PHA leucocyte conditioned medium (PHA-LCM) was added to the cultures monocytic/macrophage differentiation was favoured. Inducers of the differentiation of the HL-60 cell line (N-methylacetamide, cytosine arabinoside, or retinoic acid) had no consistent effect on the differentiation and were at times inhibitory. Three patients received therapy with low dose cytosine arabinoside and no correlation was observed between the outcome of the treatment and leukemic cell differentiation in culture in the presence of the drug.     

Human granulocyte-specific nuclear antigen(s). II. Detection of antigens in human proliferative syndromes

Antisera raised to dehistonized chromatin from isolated normal human granulocytes revealed the presence of chromatin-associated antigens specific for the human neutrophils that appear during late stages of myeloid cellular differentiation. Immunological specificity was demonstrated by C fixation, immunodiffusion, and immunocytochemical reactions. Chromatin prepared from both normal granulocytes and specimens of myeloid leukemia showed immunologic reactivity. Although the normal antigens were detected in a specimen of CML, the position of immunodiffusion precipitin lines was different from that obtained with normal granulocyte chromatin. In addition, chromatin prepared from the myeloid leukemic cell line HL-60 expressed only one of the three precipitin bands normally found in immunodiffusion. The immunocytochemical staining reaction was confined to the nucleus of mature neutrophils in normal peripheral blood smears. Greater than 90% of cells in peripheral blood specimens of CML showed positive immunocytochemical nuclear staining. In other types of leukaemia, the normal mature granulocyte reacted with antiserum, but the nonmyeloid leukemic cells in these specimens did not. The specificity of immunologic reactions described here suggests the usefulness of nuclear antigens as cell markers.     

Cholesterol, phospholipids, and fatty acids of normal immature neutrophils: comparison with acute myeloblastic leukemia cells and normal neutrophils.

The lipid composition of immature myeloid cells from the bone marrow of normal persons and myeloblasts from patients with acute myeloblastic leukemia was studied and compared with the lipid composition of normal mature human neutrophils. Total cholesterol, phospholipid, and fatty acid composition was determined on each cell type. The leukemic cells showed decreased total cholesterol and cholesterol-to-phospholipid ratio, increase phosphatidylcholine and phosphatidylinositol, decreased phosphatidylethanolamine, and an increased percentage of unsaturated fatty acids when compared to normal mature neutrophils. A nearly identical pattern was seen in the normal immature myeloid precursors from normal bone marrow. We conclude that the altered lipid composition of acute myeloblastic leukemia cells is related to unexplained factors related to cell age and not to malignancy per se.     

Control of normal differentiation of myeloid leukemic cells. VI. Inhibition of cell multiplication and the formation of macrophages.

D+ but not D- myeloid leukemic cells can be induced by the appropriate conditioned medium or by serum from endotoxin treated mice, to undergo cell migration in agar, cell attachment to the surface of a Petri dish and differentiation to mature macrophages and granulocytes. Inhibition of cell multiplication by cytosine arabinoside, hydroxyurea, mitomycin C, thymidine, 5-bromodeoxyuridine, 5-iododeoxyuridine, 5-fluorodeoxyuridine or actinomycin D, but not by vinblastine or cycloheximide, induced cell migration, cell attachment to the Petri dish and the formation of macrophages in D+ cells. There was no induction of cell migration or formation of macrophages and a much lower induction of cell attachment in D- cells. The induction of these changes in D+ cells required protein synthesis and the inhibitors showed the same toxicity for D+ and D- cells. The results indicate, that the inhibitors induced specific surface membrane changes in D+ but not in D- cells.     

Selective treatment of leukemia L1210 with combination of deoxycytidine and lethal doses of cytosine arabinoside]

Oral administration of deoxycytidine simultaneously with intraperitoneal injections of toxic doses of cytosine arabinosidetomice with advanced L1210 leukemia diminished the toxic effects preventing drug death of these mice. They developed a marked antitumor effect. The mean survival time of these mice was considerably extended as compared to that of untreated animals or those given one of these drugs alone. At the optimum schedule of treatment about 23% of the mice survived over 60 days. Deoxycytidine protection reduced the antileukemic effect of cytosine arabinoside administered in nontoxic doses. The deoxycytidine plus cytosine arabinose combination was ineffective in the treatment of transplantable myeloid leukemia in mice.     

Adjuvant immunotherapy in acute nonlymphocytic leukemia

Summary Of 112 patients (maximum age 70 years) with acute nonlymphocytic leukemia, 62 (55%) went into remission on an induction therapy of cytosine arabinoside and daunorubicin. 20 patients were randomized for maintenance treatment consisting of chemotherapy only and 22 patients for combined chemo-immunotherapy. The chemotherapy consisted in 5-day courses of daunorubicin and cytosine arabinoside and of thioguanine and cytosine arabinoside, alternating every month. The chemo-immunotherapy group also received weekly intracutaneous injections of 10⁹ allogeneic nonirradiated leukemic myeloblasts and 10⁶ BCG organisms (Glaxo) by Heaf gun.The median duration of the first remission was 164 days for the chemotherapy group and 464 days for the chemo-immunotherapy group. The corresponding median times of survival were 344 days for the first group and 734 days for the second group. The difference concerning median duration of survival is statistically significant. Thus immunotherapy seems to prolong survival.     

The efficiency of strict reverse isolation and antimicrobial decontamination in remission induction therapy of acute leukemia

The efficiency of strict reverse isolation and antimicrobial decontamination in remission induction therapy of acute leukemia was studied retrospectively in 47 patients who were treated with a standardized aggressive chemotherapy of daunorubicin and cytosine arabinoside. Twenty-two patients were treated in strict reverse isolation with antimicrobial decontamination and 25 patients in the open ward without any measures against infections. In the patients in isolation the incidence of new infections per patient was 0.77 compared to 1.42 in the control group. The rate of complete remissions was 77% in the patients in isolation vs. 56% in the control patients.     

Septicemia due to Streptococcus mitis in neutropenic patients with acute leukemia

Eight neutropenic patients with acute lymphocytic or nonlymphocytic leukemia had septicemia due to different strains of Streptococcus mitis (St. mitis), a microorganism not commonly recognized as a special pathogen in leukemic patients. Four of the patients had been treated with high-dose cytosine arabinoside as part of the cytostatic regimen, six had a central venous line and four patients had oral lesions prior to the infection. Selective gut decontamination consisted of co-trimoxazole/colistin in five patients and quinolones in three patients. The first three patients died, either due to interstitial pneumonia with the adult respiratory distress syndrome (ARDS), or due to infection-triggered disseminated intravascular coagulation despite prompt empiric antibiotic therapy including vancomycin. The other patients improved after empiric supplementation of penicillin G (30 Mega/day) to the antibiotic regimen. Beginning ARDS in two of these patients dramatically responded to high-dose steroids. We conclude that St. mitis is a major pathogen in neutropenic leukemic patients. Infection appears to occur independently of acute leukemic cell type, regimen of selective gut decontamination, venous access, visible oral lesions or treatment with high-dose cytosine arabinoside. The clinical course of our patients raises questions about the value of commonly recommended empiric antibiotic regimens, which were clearly ineffective to control infections with St. mitis in this patient group. Our data indicate that immediate antibiotic therapy with penicillin G is indicated and may be life-saving for suspected St. mitis infections in neutropenic leukemic patients.     

Remission induction and remission maintenance in adult acute nonlymphocytic leukemia employing a modified cytostatic (COAP) regimen.

Thirty adult patients suffering from acute nonlymphocytic leukemia (ANLL) were treated according to a modified COAP regimen. Vincristine, cyclophosphamide, and prednisone were given by push injection, while cytosine arabinoside was infused over periods of 8 h. Nineteen patients (63%) achieved complete remission. Remission maintenance therapy consisted of 6-mercaptopurine daily and methotrexate twice weekly. Later in the study, COAP consolidation and reinduction was added, which improved the median duration of complete remission from 7 to 24 months. Comparison of the results with the literature shows that the modified COAP regimen is one of the most effective treatment schedules for adult ANLL.     

Day-6 bone marrow aspirate for the prediction of response to remission induction therapy for acute myelogenous leukaemia

Seventy-two adults were treated for acute myelogenous leukaemia (AML). Forty-two had previously untreated AML and 30 had AML after a preleukaemic phase, refractory AML or relapsed AML. The previously untreated patients received a 7-day course of cytosine arabinoside (100 or 200 mg/m2 daily), daunorubicin and vincristine while the remaining patients received a 7-day course of cytosine-arabinoside (1 g/m2 q 12h for 6 days) and amsacrine (on day 7). The percentage of malignant cells and the reduction in the percentage of malignant cells were determined by means of bone marrow aspirates taken on day 6 of the chemotherapy course and at the time of diagnosis. Both variables correlated significantly with the ultimate treatment outcome; the reduction in the percentage of malignant cells correlated even more significantly than the absolute percentage malignant cells in the day-6 bone marrow. By means of multiple regression analysis it became possible to calculate the probability of achieving complete remission for the individual patient; this is given by the equation: probability = 1.9-0.009X (% malignant cell reduction). In addition, the mean percentage of malignant cells in the day-6 bone marrow was significantly higher for patients who failed to achieve than those who entered complete remission. Eighty-six per cent of the patients with less than 20% malignant cells on day 6 entered remission, while 75% of the patients with more than 21% malignant cells failed to achieve complete remission (p less than 0.001). Although all of these calculations support the predictive value of the day-6 bone marrow aspirate, the 95% confidence intervals are too large to allow reliable and safe predictions; therefore more patients must be studied to demonstrate the reliability of this test.     

Effect of immunosuppression or immunostimulation on the growth rate of a lymphoid and of a myeloid leukemia in mice

Summary The influence of immunosuppression or immunostimulation on the growth rate of a lymphatic and of a myeloid murine leukemia has been investigated in syngeneic host-tumour relation. Immunosuppression by chemotherapy or X-rays, induced before transplantation of leukemia cells, did not change the survival time of the animals. Treatment with the immunosuppressive drug cortisone which is not cytostatic for these leukemias, if instituted after the transplantation of malignant cells, enhanced the growth rate of the lymphatic leukemia. Nonspecific stimulation with Corynebacterium parvum induced resistance to a graft of lymphatic leukemia in a majority of the mice, but only slowed the growth rate of myeloid leukemia, without preventing death. Immunosuppressive treatment, given before C. parvum, completely blocked the induction of resistance, and if given after C. parvum, abolished the established resistance to lymphatic leukemia. Thus, the danger of immunosuppression accompanying chemotherapy may lie in its abrogating the protective effects of nonspecific immunostimulation.Abbreviations PFC = plaque-forming-cell - SRBC = sheep red blood cell - cort. = cortisone - cyclo. = cyclophosphamide - L-asp. = L-asparaginase - mtx. = methotrexate - ara C = cytosine arabinoside - C.p. = Corynebacterium parvum     

An overview of chronic myeloid leukemia and its animal models

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

Secondary leukemia after severe aplastic anemia

We describe a patient with acute myeloid leukemia (AML) occurring 5 years after successful treatment of severe aplastic anemia (SAA) with antilymphocyte globulin (ALG). Four years after ALG, SAA had relapsed. A second remission of SAA was achieved, but was followed by transformation of the myelodysplastic syndrome into overt AML. After 2 courses of high-dose cytosine arabinoside and VP-16 complete remission occurred. This case shows that chemotherapy of secondary leukemia after SAA is feasible, and that ex-aplastic bone marrow is capable of complete recovery from chemotherapy-induced aplasia. Morphological anomalies of bone marrow noticed early during remission of SAA might predict a late transformation in leukemia.     

Decreased expression of Fc gamma RIII mRNA in leukemic granulocytes.

Morphologically mature granulocytes from patients with chronic myeloid leukemia show significant impairment in their ability to internalize aggregated IgG, a ligand that is rapidly phagocytosed by normal human granulocytes. With a view to understand the molecular basis of this defect, normal and leukemic granulocytes were examined for the steady-state levels of mRNA for Fc gamma RIII, a membrane-associated receptor that initially binds and traps the IgG-opsonized antigens. Northern blot analyses revealed that the level of the specific mRNA in CML granulocytes was between 0.08 and 0.69 times that seen in the normal granulocytes. This could be one of the contributory factors for the observed endocytic defect in the leukemic granulocytes.     

A randomized clinical trial of remission induction,consolidation, and chemo-immunotherapy maintenance in adult acute myeloblastic leukemia

Summary The Southeastern Cancer Study Group conducted a randomized clinical trial in acute myeloblastic leukemia and the blastic phase of chronic myelocytic leukemia to compare: Two induction programs (Schedule A) cytosine arabinoside and 6-thioguanine or (Schedule B) cytosine arabinoside, 6-thioguanine and daunorubicin; two consolidation programs (Schedule C) continuation of induction programs at a reduced dose or (Schedule D) a combination of cyclophosphamide, methotrexate and vincristine; and two maintenance programs — (Schedule E) 1 month of BCG, followed by methotrexate or (Schedule F) methotrexate. Over a 3 year period 372 patients were entered and 295 were judged evaluable. None of 11 patients with blastic phase of chronic myelocytic leukemia responded. There were no significant differences between the schedules in the number of patients with acute myeloblastic leukemia achieving complete remissions (37%, Schedule A vs. 41% Schedule B). The relapse rates on consolidation were similar (43%, Schedule C and 39%, Schedule D). BCG significantly prolonged the duration of first remission following consolidation (P<0.05) from 13.0–23.9 weeks. Survival was not significantly prolonged (92.7 weeks vs. 71.7 weeks). There were no serious complications from BCG therapy. Contributors. The following members of the Southeastern Cancer Study Group participated in this study: John T. Carpenter, John R. Durant, Richard Gams, William J. Hammack, George A. Omura, Gayle Roberts, University of Alabama School of Medicine, Birmingham, Alabama; Harold Silberman, Donald S. Miller, Duke University School of Medicine, Durham, North Carolina; William B. Kremer, Durham Veterans Administration Hospital, Durham, North Carolina; Evert A. Bruckner, Lawrence E. Cooper, Charles C. Corley, Joseph E. Hardison, Charles M. Huguley, Jr., James Keller, Mason G. Robertson, John D. Schmale, Charles Vogel, W. R. Vogler, William H. Whaley, E. F. Winton, Emory University School of Medicine, Atlanta, Georgia; Chan Kon Chin, Guy Faguet, Claude-Starr Wright, Medical College of Georgia, Augusta, Georgia; Y. S. Ahn, Howard E. Lessner, University of Miami School of Medicine, Miami, Florida; Dov Gorshein, Scott Murphy, Presbyterian University of Pennsylvania Medical Center, Philadelphia, Pennsylvania; William E. Barry, Sharon P. Fischer, Rosaline R. Joseph, Richard V. Smalley, Temple University School of Medicine, Philadelphia, Pennsylvania; Virgil Loeb, Jr., Cary Presant, Edward Reinhard, Shabbir H. Safdar, Washington University School of Medicine, St. Louis, Missouri; Norman Maldonado, Enrique Velez-Garcia, University of Puerto Rico School of Medicine, San Juan, Puerto Rico; S. A. Gregory, William H. Knospe, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois; Stephen Krauss, University of Tennessee Memorial Research Center, Knoxville, Tennessee; Karl Tornyos, New Orleans Veterans Hospital, New Orleans, Louisiana; W. B. Forman, R. W. Kellermeyer, A. Rassiga, Case Western Reserve University School of Medicine, Cleveland, Ohio; William R. Arrowsmith, George Porter, Donald M. Samples, Ochsner Clinic, New Orleans, Louisiana; Lois W. Dow, Charles L. Neely, University of Tennessee School of Medicine, Memphis, Tennessee; G. O. Broun, Jr., St. Louis University School of Medicine, St. Louis, Missouri     

Combination Chemotherapy using L-Asparaginase,Daunorubicin, and Cytosine Arabinoside in Adults with Acute Myelogenous Leukaemia

Cytosine arabinoside and daunorubicin used in an intensive intermittent regimen have been shown to be an effective combination for the induction of complete remissions in 14 out of 23 adult patients with acute myelogenous leukaemia. This gives an overall complete remission rate of 60%. A further patient had a good partial remission. The addition of L-asparaginase to the regimen has not increased the incidence of remission and there were more side effects in the L-asparaginasetreated group. Of the 10 patients treated with L-asparaginase in addition to cytosine arabinoside and daunorubicin, five achieved a complete remission. Of the 13 patients treated with cytosine arabinoside and daunorubicin without L-asparaginase, nine achieved a complete remission and one a good partial remission.     

Simultaneous determination of cytosine arabinoside,daunorubicin and etoposide in human plasma

A method for simultaneous bioanalysis of the three cytotoxic drugs cytosine arabinoside, daunorubicin and etoposide in human plasma was developed and validated. A HPLC method with ultra-violet and fluorescence detection, preceded by mixed-mode cation-exchange solid phase extraction sample preparation, was used for the quantification of the analytes. The assay was used for the simultaneous measurement of cytosine arabinoside, daunorubicin and etoposide with linearity in the ranges of 13–1500 ng/mL, 15–1000 ng/mL and 52.5–3500 ng/mL, respectively. The chromatographic run-time was 15.5 min. The overall precision (% relative standard deviation) was within 0.2–13.5% and the recovery ranged between 86.1% and 110.1% for the three drugs at all concentrations tested. Plasma samples were stable for at least two months when stored at −20 °C. The method was successfully applied to quantification of the three drugs in blood samples from patients undergoing induction treatment for acute myeloid leukaemia, thus demonstrating its suitability for clinical studies.