Deferoxamine inhibition of human neuroblastoma viability and proliferation.

Patients with widespread neuroblastoma (NB) frequently have elevated serum ferritin levels, and recently anti-NB effects of the iron chelator deferoxamine (DFO) have been reported. We have investigated the effect of DFO on human bone marrow NB cells from two untreated children with Evans Stage IV disease. DFO treatment caused dose- and time-dependent cytotoxicity of NB cells, with maximal killing at exposure to 50 micron DFO for 72 h. Cytotoxicity was prevented by cotreatment with stoichiometric amounts of iron salts and reversible by removal of DFO or addition of iron salts within 48 h of treatment. Additionally, DFO inhibited clonal growth of human bone marrow NB cells in methylcellulose in a time- and dose-dependent manner. These effects were also prevented by cotreatment with iron salts. Thus, DFO has potent antitumor effects on human NB cells which appear to be related to iron deprivation. DFO should be considered for further preclinical evaluation as an anti-NB agent.

Antileukemic effects of deferoxamine on human myeloid leukemia cell lines.

Deferoxamine (DFO) possesses antiproliferative activity against mitogen-stimulated lymphocytes, several tumor cell lines, and human leukemia and neuroblastoma cells. We have investigated its effects on the human myeloid leukemia lines HL-60, HEL, and U-937. In suspension culture, DFO causes a dose-dependent inhibition of proliferation of each cell line, with maximal inhibition observed at concentrations greater than 20 microM. These effects were prevented by cotreatment with iron salts and were at least partially reversible by removal of DFO from the culture system or addition of iron before 48 h of DFO exposure. Similar results were obtained in methylcellulose cultures of leukemic cells, with complete abolition of cell aggregates at day 7 in concentrations of 20 microM DFO or higher. DFO treatment caused a dose- and time-related decrease in DNA synthesis as measured by [3H]thymidine uptake, which was also reversed by treatment with iron salts. DFO caused slight reduction in RNA synthesis and did not affect protein synthesis. DFO caused significant antiproliferative effects on three myeloid leukemia cell lines, associated with inhibition of DNA synthesis, with in vitro effects observed at concentrations attainable in vivo. Evaluation of the antileukemic properties of DFO should continue.

Secondary acute myelogenous leukemia following safe exposure to etoposide.

PURPOSE: To present two patients as illustrations of the risk of developing secondary acute myelogenous leukemia (sAML) when theoretically safe doses of etoposide (VP-16) are used. PATIENTS AND METHODS: Patient no. 1 was a 15-year-old white girl diagnosed with stage IIa Hodgkin's disease. She was treated with a combination of vincristine, doxorubicin, bleomycin, and VP-16 (2 g/m2 total) over 4 months, followed by 25.5 Gy of involved-field radiotherapy. Patient no. 2 was an 11-year-old white boy diagnosed with virus-associated hemophagocytic syndrome (VAHS). He was treated with VP-16 intravenously (IV) and orally (0.3 g and 2.8 g/m2, respectively). RESULTS: Patient no. 1 developed AML 16 months from the diagnosis of Hodgkin's disease. Patient no. 2 developed AML 26 months from diagnosis. Both bone marrows were consistent with French-American-British (FAB) M4 disease. Both patients had abnormalities of the long arm of chromosome 11. CONCLUSION: The use of low-dose or oral VP-16 can be associated with the development of sAML. Clinicians should be cautious in the use of VP-16 in low-risk diseases.

Interleukin-12 (IL-12) enhances lysis of non-lymphoid leukemia cell lines in vitro.

IL-2 augments the ability of natural killer (NK) cells to kill myeloid leukemia cells in vitro, and may have a role in the eradication of minimal residual disease (MRD) in AML patients. The ability to enhance lysis of AML cells without the toxicity of IL-2 would be a significant improvement in the use of biologics against AML. Recent interest in IL-12 suggested that this cytokine might meet these criteria. The aim of this study was to evaluate the ability of IL-12 to enhance the in vitro lysis of the non-lymphoid leukemia cell lines in a standard 51Chromium release assay. Effector cells from normal volunteers were incubated with varying concentrations of IL-12 or IL-2 for 18-20 h, then the 51Cr-labeled target cells from five different cell lines of AML origin were added for 4 h. Percent lysis was determined and plotted over four effector:target (E:T) ratios. Our results indicated that IL-12 was able to enhance lysis of all cell lines tested at > or =5 units/ml. When IL-2 was added to the culture at a low dose along with IL-12, there appeared to be a synergistic effect. Although anti-gamma interferon was able to inhibit the cytolytic potential of effectors activated by IL-12, the lysis could not be completely blocked. Thus, it appears that IL-12 has the ability to stimulate NK lysis indirectly through the induction of gamma interferon as well as an alternate mechanism not related to gamma interferon. Thus, IL-12 may have a beneficial role in the treatment of non-lymphoid leukemia.

Pharmacokinetic interactions of cyclosporine with etoposide and mitoxantrone in children with acute myeloid leukemia.

The purpose of this study was to assess the effect of the multidrug resistance modulator cyclosporine (CsA) on the pharmacokinetics of etoposide and mitoxantrone in children with de novo acute myeloid leukemia (AML). Serial blood samples for pharmacokinetic studies were obtained in 38 children over a 24-h period following cytotoxin treatment with or without CsA on days 1 and 4. Drug concentrations were quantitated using validated HPLC methods, and pharmacokinetic parameters were determined using compartmental modeling with an iterative two-stage approach, implemented on ADAPT II software. Etoposide displayed a greater degree of interindividual variability in clearance and systemic exposure than mitoxantrone. With CsA treatment, etoposide and mitoxantrone mean clearance declined by 71% and 42%, respectively. These effects on clearance, in combination with the empiric 40% dose reduction for either cytotoxin, resulted in a 47% and 12% increases in the mean AUC for etoposide and mitoxantrone, respectively. There were no differences in the rates of stomatitis or infection between the two groups. CsA treatment resulted in an increased incidence of hyperbilrubinemia, which rapidly reversed upon conclusion of drug therapy. The variability observed in clearance, combined with the empiric 40% dose reduction of the cytotoxins, resulted in statistically similar systemic exposure and similar toxicity.

KRN5500 induces apoptosis (PCD) of myeloid leukemia cell lines and patient blasts.

The purpose of this study was to determine if KRN5500, a spicamycin derivative with a unique acyl tail, would induce programmed cell death (PCD) of myeloid leukemia cell lines and cryopreserved leukemic blasts from newly diagnosed children with acute leukemia (AL). Cells were incubated with varying concentrations (0-5 ng/ml) of KRN5500 and the percent PCD determined using a modified in situ end labeling (ISEL) technique with Klenow fragment. The percent PCD was calculated using the formula: Percent PCD (% PCD)=[number of apoptotic cells/(viable cells+apoptotic cells)]x100. DMSO (0.30% w/v) was added to the cells in culture as the positive control for PCD; the negative control was media or albumin. KRN5500 increased the amount of PCD significantly in all five of the tested cell lines; U937 41+/-1.8%, KG1a 40+/-0.3%, HEL 14+/-2.2%, HL-60 41+/-0. 9%, K562 36+/-2% (mean PCD+/-SD). Patient blasts exposed to KRN5500 had an increase in PCD when exposed to 2 ng/ml of agent from 2 to 8 h; acute myeloid leukemia patients 7.5+/-0.5% at 2 h to 43.5+/-1.6% at 8 h, and acute lymphocytic leukemia patients rose from 12.4+/-3.8% at 2 h to 29.9+/-11.6% after 8 h (mean+/-SE). Overall the PCD for the patient samples was 3.7 versus 28+/-4% at 2 and 8 h, respectively. PCD was proportional to the dose of KRN5500 and incubation time. Further pre-clinical and clinical studies are required.

Natural history of histiocytosis-X.

Histiocytosis-X can involve many organs and tissues in all age groups and in both sexes and occurs most often in caucasians. The signs and symptoms observed in the patient are directly related to the infiltration of Langerhans histiocytes, which compress or displace normal tissues and cause destruction of the tissues and organs involved. The disease tends to be benign and self-limiting when the involvement is limited to only one site. When more than one site is involved, the disease course can be chronic or acute. The chronic course tends to smolder for years, causing significant morbidity and disability but not death. The acute form tends to cause significant morbidity and early mortality.

Prophylactic treatment with activated prothrombin complex concentrate (FEIBA) reduces the frequency of bleeding episodes in paediatric patients with haemophilia A and inhibitors.

Orthopaedic complications are among the most disabling sequelae occurring in patients with haemophilia and inhibitors. Recurrent or refractory joint bleeds can lead to joint damage, limiting mobility and causing permanent disability. Activated prothrombin complex concentrates (aPCCs) are effective in controlling acute, intraoperative and postoperative bleeding in patients with haemophilia and inhibitors. The relatively long, dosing interval and safety profile distinguish aPCCs as a well-suited option for prophylaxis. Therefore, it is postulated that long-term routine aPCC administration will decrease the frequency of recurrent bleeds, prevent damage to normal joints, and slow the progression of existing joint disease in patients with inhibitors. To test this hypothesis, a retrospective chart audit was performed. In four treatment centres, five patients were identified who received aPCC [Factor Eight Inhibitor Bypassing Activity, Anti-Inhibitor Coagulant Complex (FEIBA); Baxter AG, Vienna, Austria] prophylactically for > or = 6 months to prevent or reduce further joint deterioration, reduce bleeding and prevent postsurgical bleeding. Median treatment duration was 15 months and included administration of >1300 doses of aPCC. Dosages ranged from 50 to 75 U kg(-1) three times per week in four patients; one patient received 100 U kg(-1) daily. Orthopaedic status was maintained in four patients and improved in one; the frequency of bleeding episodes was reduced in all patients. No adverse events or thrombotic complications were reported. This case series demonstrates that routine aPCC administration may be used safely and effectively to reduce the occurrence of bleeding episodes and to maintain or improve clinical joint status in some patients.

Use of factor VIII replacement during open heart surgery in a patient with haemophilia A.

We present a patient with haemophilia undergoing cardiac surgery treated with continuous infusion factor VIII.

Transient lupus anticoagulants associated with hemorrhage rather than thrombosis: the hemorrhagic lupus anticoagulant syndrome.

Lupus anticoagulants (LAs) represent a diverse group of antibodies directed against phospholipids. Patients with LAs may be free of symptoms but can have thrombotic complications including stroke, placental infarction, and fetal loss. Rarely hemorrhagic symptoms have been reported. We describe six previously healthy children who were first seen with clinical bleeding and prolonged activated partial thromboplastin time. Laboratory evaluation revealed positive results on mixing studies and evidence of phospholipid dependence of the anticoagulant, suggesting LAs. Four of six patients had anticardiolipin antibodies, and all four who were tested had reduced factor II activity levels. In all patients, bleeding symptoms resolved spontaneously within 3 months, and laboratory findings returned to normal within 6 months. The hemorrhagic LA syndrome should be considered in previously healthy children with new-onset bleeding and prolonged activated partial thromboplastin time. This clinical entity probably represents pathogenic mechanism distinct from thrombotic LA syndromes.

An infant girl with severe autoimmune hemolytic anemia: apparent anti-Vel specificity.

Autoantibodies directed against the common red-cell antigen Vel are unusual, with only 2 previous cases reported. This report describes an infant girl with steroid-resistant autoimmune hemolytic anemia whose serum contained an antibody with apparent specificity for Vel antigen. Transfusion with Vel-positive cells resulted in a massive hemolytic transfusion reaction. Survival of labelled Vel-negative cells was normal up to 8 h following transfusion, and no transfusion reaction followed subsequent use of Vel-negative cells. There was no evidence of a collagen vascular disease, an immunodeficiency syndrome, or a malignancy. Although rare, anti-Vel should be considered in unexplained autoimmune hemolytic anemia.

DDAVP therapy controls bleeding in Ehlers-Danlos syndrome.

PURPOSE: Patients with Ehlers-Danlos syndrome (EDS), especially types IV, VI, and VIII, are at increased risk of bleeding, and most do not have specific hemostatic deficiencies that would be amenable to replacement therapy. We have investigated the ability of DDAVP (desmopressin acetate) to control bleeding in EDS. PATIENTS AND METHODS: Two children with EDS, types VIII and VI, presented with hemorrhagic symptoms and scheduled surgical procedures. Ivy bleeding times (BTs) were measured before and after intravenous (i.v.) DDAVP challenge, and i.v. DDAVP was used prophylactically for their procedures. Laboratory testing was performed to rule out other hemostatic disorders. RESULTS: Both patients had prolonged BTs that corrected following i.v. DDAVP therapy; all other laboratory values were normal. Both patients had excellent clinical hemostasis with surgery, and one has continued to use intranasal DDAVP to control epistaxis and gingival bleeding. CONCLUSIONS: The bleeding time in both patients was corrected with DDAVP, and the patients did not have any postoperative bleeding. DDAVP should be considered in other patients who have EDS with bleeding tendencies.

Characterization of protein kinase C activity in interferon gamma treated murine peritoneal macrophages.

Macrophage activation for tumoricidal and microbicidal functions can be achieved in part by treatment with recombinant interferon gamma (IFN gamma) in vitro. We have previously demonstrated that IFN gamma treatment of murine peritoneal macrophages results in a two- to five-fold increase in the activity of Ca++, phospholipid dependent protein kinase C (Hamilton et al., J. Biol. Chem., 260:1378, 1985). We now report that this effect was not dependent upon continuing protein synthesis since treatment with cycloheximide under conditions where normal protein synthesis was inhibited by greater than 95% had no effect upon the development of increased enzyme activity. Examination of Ca++ and phospholipid requirements revealed no differences between enzyme isolated from control or IFN gamma treated cells. Similarly, protein kinase C from control and IFN gamma-treated cells could not be distinguished in terms of the diacylglycerol (DG) or phorbol diester (PMA) concentration required for stimulation of activity. Kinetic analysis of the ATP (as substrate) concentration dependence revealed that both control and treated enzyme preparations (either basal or stimulated) had comparable Km values. Maximum velocity (Vmax) was increased both by IFN gamma treatment and also by stimulation with DG or PMA. The major difference which could be discerned between protein kinase C derived from control versus IFN gamma-treated macrophages was the magnitude of the response to DG or PMA; IFN gamma treatment increased the stimulation index (i.e., ratio of basal to stimulated activity) by a factor of two to four fold. These results suggest that IFN gamma treatment leads to reversible modulation of existing protein kinase C resulting in increased catalytic efficiency when exposed to an appropriate stimulant.

Isolated vocal cord hematoma in a child with severe factor VIII deficiency.

We have reported the first case of isolated submucosal hematoma of the true vocal cord in a hemophilic patient. He was managed with factor VIII replacement therapy as an outpatient for five consecutive days, without complication. Similar but more serious cases usually require more aggressive intervention by the pediatrician and the otolaryngologist.

Single institution experience with high-dose cyclophosphamide, continuous infusion vincristine, escalating doses of VP-16-213, and total body irradiation with unpurged bone marrow rescue in children with neuroblastoma.

Seven consecutive autologous bone marrow transplants were performed in children with neuroblastoma with very good partial remission (VGPR). A combination of cyclophosphamide, escalating doses of VP-16-213, continuous infusion vincristine, and total body irradiation followed by infusion with unpurged bone marrow was used. The dose-limiting toxicity in this regimen was mucositis which occurred when the total dose of VP-16-213 was 2,400 mg/m2. The response rate to this regimen was 4/7 (-CR 48+, 21+, 21+, 35+ mo) 3/7 had a CP/PR post transplant with progressive disease between 1 and 4 months later (mean 2.6 mo). We conclude that this regimen is well tolerated when the maximum dose of VP-16-213 does not exceed 1,800 mg/m2. Further evaluation will be necessary with this regimen to determine its therapeutic value in a larger number of patients with neuroblastoma.