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The Southeastern Cancer Study Group conducted a post-remission induction randomized trial in adult acute myelogenous leukemia to assess the efficacy of alternate drug therapy during consolidation and of immunotherapy during maintenance. Of 508 evaluable patients entered into the study, 335 (66%) achieved a complete remission treated with a 7-day infusion of cytosine arabinoside at a dose of 100 mg/sq m/day and 3 days of daunorubicin at a dose of 45 mg/sq m/day. Those in remission were randomized to receive 3 courses of 1 of 3 consolidation regimens: (A) a continuous infusion of 5-azacytidine, 150 mg/sq m/day for 5 days; (B) 5-azacytidine plus β-deoxythioguanosine, 300 mg/sq m/day for 5 days; or (C) cytosine arabinoside, 100 mg/sq m/day intravenously, and thioguanine, 100 mg/sq m orally every 12 hr, plus daunorubicin, 10 mg/sq m every 24 hr daily for 5 days. There was no difference in relapse rate among the 3 arms. Those completing consolidation and remaining in remission were randomized to 1 of 3 maintenance regimens: (D) chemotherapy, 5-day infusion of cytosine arabinoside and 2 days of daunorubicin (same doses as induction) given every 13 wk for 1 yr; (E) BCG given twice weekly for 1 mo and then monthly for 1 yr; or (F) the combination of regimens D and E. The median duration of remission was significantly better on regimen D (17.4 versus 9.4 and 9.5 mo), and median survival was 29 mo compared to 21 mo for the other regimens. Those given different drugs during consolidation than used for induction (regimens A and B) and subsequent chemotherapy for maintenance (regimen D) had the longest remission durations and survival. Immunotherpay was not as good as intensive chemotherapy for maintenance.

Alkyl-lysophospholipids (ALP) are analogues of 2-lysophos-phatidylcholine that have been reported to have selective antitumor activity. These compounds could potentially be useful in purging bone marrow of leukemic cells in autologous marrow transplantation in acute leukemia. To determine the efficacy of pharmacological purging by ALP, we have designed a human assay system to mimic the conditions expected in the clinical setting of autotransplantation using remission marrow. A simulated remission marrow (SRM) was prepared by mixing normal marrow cells and HL60 cells in a ratio of 1,000:1. The effect of cryopreserva-tion on ALP-treated normal, HL60, and SRM cells was examined. In separate experiments, ALP significantly reduced the number of clonogenic HL60 cells with no effect on normal marrow progenitors. The effect of ALP was more apparent after cryopreservation. Incubation of HL60 cells with 50 μ/mL ALP for four hours followed by cryopreservation resulted approximately in a 3 log reduction of clonogenic HL60 cells. ALP also selectively purged the small number of leukemic cells from SRM. In SRM, the data suggested that ALP had indirect cytotoxic activity on leukemic cells by enhancing the cytotoxic activity of monocytes in addition to its direct effect. We found no evidence that clonogenic HL60 cells decreased because of induction of differentiation by ALP. These data indicated that treatment of marrow cells with ALP offers an efficient means to eliminate leukemic cells from the graft.

The action of an alkyl-lysophospholipid (ALP), ET180CH3,* on clonogenicity, 3H-TdR uptake, and cell numbers was tested in two human leukemic cell lines, HL60 and K562, and short-term human leukemic bone marrow cultures. ALP eliminated clonogenicity in HL60 but not in K562 cultures; 3H-TdR uptake and cell numbers were depressed at low concentrations of ET180CH3in HL60, but not K562 cultures. The action of the lysophospholipid analog on human leukemic bone marrow short-term cultures at low concentrations was similar to its action on HL60 cultures; clonogenicity and 3H-TdR uptake were depressed, but cell numbers were not significantly affected. The demonstration of differential action of ALP on two cell lines should significantly simplify the investigation of the mechanism of the reported2'3differential action of ET180CH3on normal and leukemic cell membranes.

Human lactoferrin (LF) has been reported to inhibit in vitro granulopoiesis by means of decreasing colony-stimulating activity production by monocytes. We performed a series of experiments to determine if the reported experimental results could be replicated using highly purified murine LF and murine target cells. Three different types of experiments were performed. (1) Medium was conditioned by lung, femoral shaft, and adherent peritoneal cells in the presence and absence of LF, and the granulopoietic stimulating activity in the conditioned media was assayed by means of a 7-day agar colony assay and a 3-day liquid slide chamber assay, which quantitates 3H-TdR incorporation into DNA. (2) In cultures stimulated by an underlayer of adherent peritoneal cells, marrow cell colony formation in agar was determined after 7 days of culture in the presence or absence of LF. (3) LF was added to 3-day liquid marrow cell cultures that had been stimulated by lung or femoral shaft conditioned media. In all experimental situations, highly purified, iron-saturated LF in concentrations up to 107Mhad no effect on in vitro granulopoiesis. These results do not support LF’s reputed regulatory role in granulopoiesis.

Ether lipids (EL) and hyperthermia have been shown to possess a relatively selective cytotoxicity to leukemic cells. In this study, the combined effects of EL (ET-18-OCH3, ET-16-NHCOCH3, or BM 41.440) and hyperthermia on the growth of hematopoietic progenitors, myeloid leukemic cell lines, and leukemic cells obtained from patients with acute myeloid leukemia (AMD were examined to determine if this combination resulted in a greater selective killing of leukemic cells than that achieved by either EL or heat alone. When the cells were treated simultaneously with EL (50 µg/mL) and hyperthermia (42°C) for one hour, the killing of leukemic cell line cells was enhanced considerably. Among the three EL, however, the combination of ET-18-OCH3and heat seemed to be the most cytotoxic to leukemic cell line cells with no effect on the growth of hematopoietic progenitors. An increase in the duration of treatment with ET-18-OCH3to four hours with heat added during the last hour resulted in a further reduction of leukemic cell line cells while sparing 50% of hematopoietic progenitors after cryopreservation. The combined treatment with ET-18-OCH3and heat also inhibited the growth of leukemic progenitors obtained from AML patients by 97% to 100%. These data indicate that the combined treatment with EL and hyperthermia might offer an efficient means to eliminate myeloid leukemic cells in vitro.

The usefulness of granulocyte transfusions is in part dependent upon the number of granulocytes transfused. The invention of the continuous-flow cell separator has made it possible to obtain granulocytes from normal donors. Efforts to improve the yield are under study. This controlled study was undertaken to determine the effect of a single dose of hydrocortisone on granulocyte yield from volunteer donors and on granulocyte bactericidal function. Twenty-two normal volunteers were randomized between no therapy or a single intravenous injection of 120 mg/sq m of hydrocortisone 2 hr prior to initiation of a 4-hr leukapheresis using the Aminco cell separator operated at 750 rpm and a flow rate of 41 ml/min. Significant increases in granulocyte yield and reductions in lymphocyte and monocyte yields were obtained in the hydrocortisone-treated group. Granulocytes from each group were equally effective in the phagocytosis of yeast particles and in vitro bactericidal activity.

Kinetic measurements were performed on marrow aspirates in 62 patients with various types of leukemia prior to chemotherapy. Higher labeling indexes were seen in chronic myelocytic leukemia and the blastic phase of chronic myelocytic leukemia than in the acute leukemias. In acute nonlymphocytic leukemia, kinetic measurements were correlated with clinical features and response to a chemotherapy program designed to synchronize cells to render them more susceptible to cycle-active agents or to a program given in a more conventional fashion. Age, sex, initial white blood cell and platelet counts, and presence of infection at time of diagnosis did not correlate with kinetic measurements or response to chemotherapy, but some clinical features did positively correlate with response to chemotherapy. Kinetic measurements correlated to some degree with response to therapy on one schedule but not on the other schedule.

A liquid culture system employing slide chambers was developed to facilitate the study of proliferation and differentiation of mouse neutrophilic granulocytes and cells of the monocyte-macrophage series. Cultures were initiated with 1-2.5 × 104light density marrow cells which had been fractionated on Ficoll-Hypaque. In the presence of colony-stimulating activity (CSAHU), two types of clusters were observed. One was tight, spheroidal, and composed of neutrophilic granulocytes, while the other was a loose grouping of flattened cells of the monocyte-macrophage series. The slide chamber culture is adaptable to microscopic assay techniques (e.g., cluster size, autoradiography, immunofluorescence) as well as quantitative biochemical methods (e.g., rate of 3H-thymidine incorporation, DNA quantitation). We have demonstrated both a shortening of the generation time of granulocytes in tight clusters and increasing rates of 3H-thymidine incorporation into DNA, with a corresponding increase in total culture DNA as a function of CSAHUconcentration. Granulocytic differentiation in the tight spheroidal clusters has been demonstrated by histochemical stains and immunofluorescence of an antibody to a marker protein specific for the secondary granule of the neutrophil (lactoferrin).

Autologous bone marrow transplantation is potentially curative in the treatment of acute leukemia if residual leukemic cells in the marrow can be eliminated prior to transplantation. We studied the purging effects of a synthetic alkyl-lysophospholipid (ALP) on marrow containing leukemic cells from a transplantable myelomonocytic leukemia (WEHI-3B) in BALB/c mice. Simulated remission bone marrow containing 2% leukemic cells treated in vitro with 20 and 100 µg/mL of ET-18-OCH3(1-octadecyl-2-methyl-sn-glycerol-3-phosphocholine) significantly prolonged survival of lethally irradiated transplanted recipients. At a dose of 100 µg/mL, 88% of the mice survived for the duration of the experiment (approximately five months). Autopsies showed that 25% of these survivors had microscopic evidence of leukemia. Thus, in vitro treatment of marrow eliminated leukemic blasts and spared sufficient normal stem cells to allow hematologic reconstitution. The effect of ET-18-OCH3is not entirely selective for leukemic cells. A spleen colony assay showed that ALP has some cytotoxic effect on normal hematopoietic stem cells.

The pyrimidine analog, 5-azacytidine (NSC 102816), was administered by continuous intravenous infusion in Ringer’s lactate in increasing doses to sets of patients with metastatic cancer to establish a dose sufficient to produce mild toxicity. Twenty-one patients (23 trials) were treated with doses of 50-200 mg/sq m/ day for 5 days every 2-4 wk. Nausea and vomiting were moderate and easily preventable. Doses of 100-200 mg/sq m for 5 days every 14 days produced granulocytopenia, usually after two courses. Less toxicity was observed when courses were given every 21-28 days. Forty-five patients with previously treated and refractory acute myeloblastic leukemia were treated. The majority received doses of 150 mg/sq m for 5 days every 2 wk. Eleven (24%) complete remissions and four partial remissions were observed. The number of courses to achieve remission averaged three and required an average of 59 days. Nine patients with blastic crisis of chronic myeloblastic leukemia and four with refractory acute lymphoblastic leukemia failed to respond. 5-Azacytidine administered by continuous infusion is well tolerated and is an active compound in acute myeloblastic leukemia.

A randomized trial was conducted comparing ketoconazole and nystatin in the prevention of oral candidiasis and appearance of invasive fungal infections in 51 neutropenic leukemic patients undergoing induction chemotherapy. Ketoconazole was administered in a 200 mg dose twice daily. Nystatin oral suspension was given in doses of 500,000 units four times daily. Surveillance cultures of the throat and urine were obtained prior to treatment and conducted weekly. Patients were enrolled in the study if the absolute granulocyte count was less than 1500μl, if physical examination revealed no evidence of oral candidiasis, no evidence of urinary tract infection, and there was no pulmonary infiltrate on chest x-ray. Patients were continued on study until the absolute granulocyte count reached 1500μl, evidence of oral candidiasis appeared, or presumed or proven invasive fungal infections appeared. Of the 46 evaluable patients, 22 received ketoconazole, 3 (14) developed oral candidiasis, and 5 developed suspected systemic fungal infections (23). Of 24 patients who received nystatin, 4 (17) developed oral candidiasis and 8 (33) developed systemic fungal infections, 4 proven and 4 suspected. Significantly more patients on the nystatin arm progressed to invasive fungal infections. Ketoconazole was not superior to nystatin in reducing the frequency of oral candidiasis but possibly reduced the frequency of invasive fungal infections.

IT IS generally agreed that single daily doses of folic acid antagonists given by mouth for prolonged periods are usually ineffective in the treatment of solid tumors.1-5 Since the successful treatment of women with choriocarcinoma by Li et al and Hertz et al6-9 with large daily oral, intravenous or intramuscular doses of methotrexate, there has been renewed interest in dosage schedules and routes of administration.10-12 Repeated courses in large oral doses (up to 25 mg for five days),13 single intravenous injections (2.5-15 mg/kg),14 or continuous intra-arterial infusions (50 mg/24 hours)10 produced objective responses in solid tumors more frequently than previously noted.In addition to these clinical observations there has been considerable advance in knowledge of the pharmacology of methotrexate recently. It has been reviewed in detail by several authors.15-18 It seemed appropriate to us to attempt to apply this new pharmacologic knowledge to

Oral and intravenous ferrokinetic studies were carried out in 9 patients with gout or hyperuricemia to determine the effect of allopurinol on iron absorption, storage, and red cell incorporation. Two studies were carried out in each patient: one during the control period and the other while receiving allopurinol. Allopurinol had no measurable effect on iron metabolism.