Meta-analysis of randomised trials comparing thiopurines in childhood acute lymphoblastic leukaemia.

Mercaptopurine has been used in continuing treatment of childhood acute lymphoblastic leukaemia since the mid 1950s. Recent advances in the understanding of thiopurine pharmacology indicated that thioguanine (TG) might be more effective than mercaptopurine (MP). The US and UK cooperative groups began randomised thiopurine trials and agreed prospectively to a meta-analysis. All randomised trials of TG versus MP were sought, and data on individual patients were analysed by standard methods. Combining three trials (from US, UK and Germany), the overall event-free survival (EFS) was not significantly improved with TG (odds ratio (OR)=0.89; 95% confidence interval 0.78-1.03). Apparent differences in results between trials may be partly explained by the different types of patients studied. The larger treatment effect reported in males in the US trial was confirmed in the other trials. There was heterogeneity between sex/age subgroups (P=0.001), with significant EFS benefit of TG only observed for males aged <10 years old (OR=0.70; 0.58-0.84), although this did not result in a significant difference in overall survival (OR=0.83; 0.62-1.10). Additional toxicity occurs with TG. Mercaptopurine remains the standard thiopurine of choice, but further study of TG may be warranted to determine whether it could benefit particular subgroups.

Plasma pharmacokinetics and cerebrospinal fluid penetration of thioguanine in children with acute lymphoblastic leukemia: a collaborative Pediatric Oncology Branch, NCI, and Children's Cancer Group study.

PURPOSE: In preclinical studies, thioguanine (TG) has been shown to be more potent than the standard acute lymphoblastic leukemia (ALL) maintenance agent, mercaptopurine (MP), suggesting that TG may be more efficacious than MP in the treatment of childhood ALL. As part of a pilot trial in which TG was used in place of MP, we studied the plasma pharmacokinetics of oral TG and measured steady-state plasma and CSF TG concentrations during a continuous intravenous infusion (CIVI) in children with newly diagnosed standard-risk ALL. METHODS: Nine plasma samples were collected after each patient's first 60 mg/m2 oral TG dose during maintenance. CIVI TG (20 mg/m2/h over 24 h) was administered during the consolidation phase of therapy, and simultaneous plasma and CSF samples were collected near the end of the infusion. TG was measured by reverse-phase HPLC with ultraviolet detection. Erythrocyte TG nucleotide (TGN) concentrations were measured 7 days after a course of CIVI TG and prior to the start of each maintenance cycle. RESULTS: After oral TG (n = 35), the mean (+/- SD) peak plasma concentration was 0.46 +/- 0.68 microM and the AUC ranged from 0.18 to 9.5 microM.h (mean 1.5 microM.h). Mean steady-state plasma and CSF TG concentrations during CIVI (n = 33) were 2.7 and 0.5 microM, respectively. The mean (+/- SD) TG clearance was 935 +/- 463 ml/min per m2. Plasma TG concentrations did not correlate with erythrocyte TGN concentrations after oral or CIVI TG. The 8-OH-TG metabolite was detected in plasma and CSF. CONCLUSIONS: TG concentrations that are cytotoxic to human leukemia cell lines can be achieved in plasma after a 60 mg/m2 oral dose of TG and in plasma and CSF during CIVI of TG.

N-ras gene mutations in childhood acute non-lymphoblastic leukemia.

In view of the potential role for ras activation in leukemogenesis, we have screened a number of children with acute non-lymphoblastic leukemia (ANLL) for activating point mutations at codons 12, 13 and 61 of the N-ras proto-oncogene using panels of oligonucleotide probes in conjunction with polymerase chain reaction (PCR) gene amplification. In contrast to the frequent occurrence (approximately 30%) of N-ras mutation reported in adult ANLL, 6 of 46 cases (13%) at the time of diagnosis had N-ras mutations involving codons 12 and 13. In these patients we also determine whether presenting clinical symptoms, cellular pathology, karyotype, or eventual outcome distinguished them from the ras-negative group. N-ras activation tended to be associated with a higher white blood cell count at diagnosis (mean of 225,000/microliters vs 91,000/microliters) and fewer remissions obtained after 28 days of therapy (3/6, 50% vs 24/32, 75%). It is possible that activation of N-ras oncogene may be involved in the progression of some cases of childhood ANLL.

Changes in plasma methionine and total homocysteine levels in patients receiving methotrexate infusions.

Methotrexate reduces intracellular pools of 5-methyltetrahydrofolate and could result in reduced conversion of homocysteine to methionine by methionine synthetase. This study was designed to investigate the effects of moderate dose to very high dose methotrexate on methionine and total homocysteine as reflections of methotrexate induced intracellular events. Methionine and total homocysteine were measured prior to, during, and following twenty-six 24-h i.v. infusions of 33.6 g/m2 methotrexate (very high dose methotrexate) in 16 children with acute lymphocytic leukemia and seven 4-h i.v. infusions of 8 g/m2 methotrexate (high dose methotrexate) in 5 children with osteogenic sarcoma. Amino acids were measured by gas chromatography/mass spectrophotometry. Mean methionine levels decreased by 70.0 +/- 3.1% (SE) with very high dose methotrexate and 72.6 +/- 5.9% with high dose methotrexate at 24 and 4.5 h, respectively, after beginning methotrexate infusions. Mean total homocysteine levels increased by 61.7 +/- 3.1% with very high dose methotrexate and 55.6 +/- 17.5% with high dose methotrexate at 36 and 24 h, respectively, after beginning methotrexate infusions. No consistent or significant changes were noted in levels of total cysteine, leucine, isoleucine, or valine. Similar changes did not occur in patients receiving prednisone, vincristine, daunomycin, and intrathecal methotrexate as therapy for acute lymphocytic leukemia. These changes in homocysteine and methionine may reflect biological effects of methotrexate that may predict cytotoxicity of methotrexate.

Interleukin-1 enhances murine granulopoiesis in vivo.

Interleukin-1 (IL-1), a monokine involved in host response to infection and inflammation, has recently been shown to stimulate production of granulocyte-monocyte colony-stimulating factors (CSFs) from a variety of cell types in vitro. The purpose of this study was to investigate the effects of human IL-1 on granulopoiesis in vivo. CF1 female mice were injected with a single dose of either highly purified human IL-1 or recombinant human IL-1 alpha (rIL-1 alpha). Heat-inactivated IL-1 or rIL-1 alpha served as controls. Physiologic doses of the IL-1 preparations were initially established by evaluating neutrophil egress from bone marrow (BM). Significant peripheral neutrophilia developed 3 h after injection of 10 U (doubling units) purified IL-1, in association with decreased marrow neutrophils. Significant neutrophilia occurred 6 h after injection of 5 x 10(3) U (half-maximal units) rIL-1 alpha. Serum colony-stimulating activity (CSA) and BM colony formation (CFU-GM) were subsequently measured in standard agar culture at various times following injection. A significant rise in CSA occurred between 3 and 6 h after injection of purified IL-1, and a significant increase in BM CFU-GM developed 48 h after injection. Similar increases in CSA and CFU-GM occurred following injection of rIL-1 alpha. These results suggest that IL-1 may play an important role in the regulation of granulopoiesis in vivo by enhancing the production of CSFs required for myeloid proliferation.

Increased activity of the respiratory burst in cord blood neutrophils: kinetics of the NADPH oxidase enzyme system in subcellular fractions.

Previous studies with neutrophils from newborn infants compared to neutrophils from healthy adults have documented increased respiratory burst activity including enhanced superoxide anion (O2-) production, nitroblue tetrazolium dye reduction, and hexose monophosphate shunt activity. To investigate the biochemical basis for these observations, we examined oxidative metabolism in membrane-rich fractions of neutrophils. Neutrophils from cord blood of vaginally delivered term infants or healthy adults were disrupted by nitrogen cavitation and subcellular fractions collected on discontinuous sucrose density gradients. Subcellular fractions of newborn neutrophils separated in a fashion identical with samples from healthy adults. Activity of alkaline phosphatase, a plasma membrane marker, was increased 4- to 5-fold in disrupted cells free from nuclei (postnuclear supernatant) as well as plasma membrane fractions from newborn samples compared to those from healthy adults. Content of lactoferrin, a specific granule marker, was decreased in postnuclear supernatants but equivalent in specific granule fractions of newborn cells compared to those from adults. No differences were noted in myeloperoxidase content of postnuclear supernatants or any other subcellular fraction. Plasma membrane fractions from phorbol myristate acetate-stimulated cord blood neutrophils made significantly more O2- than samples from adults (newborn 32.9 +/- 8.1 nmol O2-/min/mg protein mean +/- SEM, n = 3 versus adult 10.8 +/- 4.2, n = 3; p less than 0.05). Plasma membrane-rich fractions were also collected by the technique of differential centrifugation and kinetic parameters of the NADPH-dependent oxidase enzyme(s) were measured for vaginally delivered newborn and adult samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Pancytopenia in propionic acidemia: hematologic evaluation and studies of hematopoiesis in vitro.

This study investigated the hematologic abnormalities of an infant with propionic acidemia and reversible pancytopenia. Light and electron microscopy of her bone marrow revealed severely disturbed cellular morphology with trilineage dysmyelopoiesis, hemophagocytosis, and numerous multinucleated histiocytes and megakaryocytes. The effects of her serum and of organic acids associated with propionic acidemia were studied on hematopoiesis in vitro. Mouse erythroid (CFU-E) and granulocyte-monocyte colonies (CFU-GM) were assayed by fibrin clot technique; human CFU-GM were grown in agar culture. The infant's serum reduced mouse CFU-E and CFU-GM by 43 and 32%, respectively, compared with normal human sera, but had no effect on human CFU-GM in our culture system. Buffered propionic acid caused concentration-dependent inhibition of mouse CFU-E and human CFU-GM over a range reported in sera of acutely ill infants with propionic acidemia. Neither cell viability nor subsequent colony formation was diminished by preincubation of bone marrow cells with propionic acid for 48 h. The three other organic acids studied, tiglic acid, 3-OH propionate, and glycine, did not inhibit growth of mouse CFU-E, CFU-GM, or human CFU-GM, and glycine significantly enhanced formation of the latter. Evaluation of the infant's hematologic abnormalities suggests that inhibition of bone marrow proliferation and maturation and, perhaps, shortened red blood cell survival were responsible for her pancytopenia. The studies performed in vitro implicate propionic acid in this hematopoietic dysfunction.