Allergic reactions and antiasparaginase antibodies in children with high-risk acute lymphoblastic leukemia: A children's oncology group report.

BACKGROUND: The objectives of this study were to assess the incidence of clinical allergy and end-induction antiasparaginase (anti-ASNase) antibodies in children with high-risk acute lymphoblastic leukemia treated with pegylated (PEG) Escherichia coli ASNase and to determine whether they carry any prognostic significance. METHODS: Of 2057 eligible patients, 1155 were allocated to augmented arms in which PEG ASNase replaced native ASNase postinduction. Erwinia chrysanthemi (Erwinia) ASNase could be used to replace native ASNase after allergy, if available. Allergy and survival data were complete for 990 patients. End-induction antibody titers were available for 600 patients. RESULTS: During the consolidation phase, 289 of 990 patients (29.2%) had an allergic reaction. There were fewer allergic reactions to Erwinia ASNase than to native ASNase (odds ratio, 4.33; P < .0001) or PEG ASNase (odds ratio, 3.08; P < .0001) only during phase 1 of interim maintenance. There was no significant difference in 5-year event-free survival (EFS) between patients who received PEG ASNase throughout the entire study postinduction versus those who developed an allergic reaction to PEG ASNase during consolidation phase and subsequently received Erwinia ASNase (80.8% ± 2.8% and 81.6% ± 3.8%, respectively; P = .66). Patients who had positive antibody titers postinduction were more likely to have an allergic reaction to PEG ASNase (odds ratio, 2.4; P < .001). The 5-year EFS rate between patients who had negative versus positive antibody titers (80% ± 2.6% and 77.7% ± 4.3%, respectively; P = .68) and between patients who did not receive any ASNase postconsolidation and those who received PEG ASNase throughout the study (P = .22) were significantly different. CONCLUSIONS: The current results demonstrate differences in the incidence rates of toxicity between ASNase preparations but not in EFS. The presence of anti-ASNase antibodies did not affect EFS.

Comparative Toxicity by Sex Among Children Treated for Acute Lymphoblastic Leukemia: A Report From the Children's Oncology Group.

BACKGROUND: Epidemiologic studies find sex-based differences in incidence, survival, and long-term outcomes for children with cancer. The purpose of this study was to determine whether male and female patients differ with regard to acute treatment-related toxicities. PROCEDURES: We reviewed data collected on the Children's cancer group (CCG) high-risk acute lymphoblastic leukemia (ALL-HR) study (CCG-1961), and compared male and female patients' toxicity incidence and related variables in the first four phases of treatment. Similar analyses were performed with standard-risk ALL (ALL-SR) patients enrolled in CCG-1991. RESULTS: Among ALL-HR patients, females had significantly more hospital days, delays in therapy, grade 3 or 4 toxicities (e.g., gastrointestinal, liver), and supportive care interventions (e.g., transfusions, intravenous antibiotics) than males. Females were significantly more likely to have died of treatment-related causes than males (Hazard ratio = 2.8, 95%CI = 1.5-5.3, P = 0.002). Five months after beginning the treatment, the cumulative incidence of treatment-related deaths was 2.6% for females and 1.2% for males. Similar disparities were found among ALL-SR patients, with females experiencing significantly more hospital days and treatment-related toxicities than males. CONCLUSIONS: This study complements cancer survivorship studies that also report an increase in treatment-related late effects among females. Risk profiles appear to be different for male and female patients, with females having greater risk of developing both acute and long-term treatment-related toxicities. The underlying biological mechanisms for these sex differences are poorly understood and warrant further study in order to determine how sex-based outcome disparities can be addressed in future clinical trials and practice.

Diet-induced obesity alters vincristine pharmacokinetics in blood and tissues of mice.

Obesity is associated with poorer outcome from many cancers, including leukemia. One possible contributor to this could be suboptimal chemotherapy dosing in obese patients. We have previously found that vincristine (VCR) is less effective in obese compared to non-obese mice with leukemia, despite weight-based dosing. In the present study, we administered (3)H-VCR to obese and control mice to determine whether obesity would cause suboptimal VCR exposure. Blood VCR concentrations were fitted with a three-compartment model using pharmacokinetic analysis (two-stage PK) in three subsets of VCR concentrations vs. time method. Tissue and blood VCR concentrations were also analyzed using non-compartmental modeling. Blood VCR concentrations showed a triexponential decay and tended to be slightly higher in the obese mice at all time-points. However, the t(1/2,beta) and t(1/2,gamma) were shorter in the obese mice (9.7 min vs. 44.5 min and 60.3h vs. 85.6h, respectively), resulting in a lower AUC(0-infinity) (13,099 ng/m Lh vs. 15,384 ng/mL h). Had the dose of VCR been "capped", as is done in clinical practice, the AUC(0-infinity) would have been 36% lower in the obese mice than the controls. Tissue disposition of VCR revealed a biexponential decay from spleen, liver, and adipose. Interestingly, VCR slowly accumulated in the bone marrow of control mice, but had a slow decay from the marrow in the obese mice. Thus, obesity alters VCR PK, causing a lower overall exposure in circulation and bone marrow. Given the high prevalence of obesity, additional PK studies should be performed in obese subjects to optimize chemotherapy dosing regimens.

Immunogenicity of native or pegylated E. coli and Erwinia asparaginases assessed by ELISA and surface plasmon resonance (SPR-biacore) assays of IgG antibodies (Ab) in sera from patients with acute lymphoblastic leukemia (ALL).

BACKGROUND: Therapeutic uses of asparaginases (ASNase) have been shown to elicit immune responses resulting in the development of potentially life-threatening human anti-bacterial antibodies (Ab). A robust screening enzyme-linked immunosorbent assay (ELISA) to detect binding Ab(+) against ASNase has been developed and validated for therapeutic monitoring to support clinical trials. Recently, a protein chip bioassay (Biacore) was developed for the Ab of these proteins. These methods were compared. MATERIALS AND METHODS: A Biacore T-100 analyzer using a protein bioassay and an ELISA assay were used to determine the IgG immmuboglobulin Ab against ASNase in sera from 84 acute lymphoblastic leukemia (ALL) patients plus 6 controls (n=121 samples). These samples were characterized for anti-ASNase Ab neutralizing activity. Human E. coli ASNase, pegaspargase and Erwinase proteins were covalently coupled to the carboxy-methylated dextran matrix of a CM5 sensor chip (surface plasmon resonance, SPR). In the course of a nested experimental design, a wide range of human sera from patients who had obvious clinical allergic reactions after either native or pegaspargase treatments were tested. The data were fitted by a parametric logistic equation (+/-95% confidence interval, CI), which ranged from <3.0% to <14%. RESULTS: The specificity of Ab(+) was evaluated using "spiked" human IgG antibodies. Both assays provide near excellent linearity and sensitivity of response (<0.8 to <500 ratio and 1-3000 resonance units [RU]) of anti-ASNase Ab in human sera with low variance. The bioassay method was ten times more sensitive than the ELISA Ab assay. The lowest limit of quantification of Ab(+) ratio for the SPR assay was 0.6 whereas the upper limit of quantification was 3000 RU. The SPR assay results were in excellent accord with both the Ab(-) and the Ab(+). Ab(-) by the ELISA method (<1.003 ratio) was related to a mean RU value of 8.1. Despite the narrow range of ambiguity around the 1.1 Ab(+) ratio values, the majority of the specimens (93.2%) were determined to be Ab(+) by either ELISA or SPR determination. CONCLUSION: The vast majority (81/84 = 96.4%) of the IgG Ab(+) were neutralizing. The SPR Ab determination technique is reliable, accurate and more sensitive than the ELISA method.

Pharmacoanalytical assays of Erwinia asparaginase (erwinase) and pharmacokinetic results in high-risk acute lymphoblastic leukemia (HR ALL) patients: simulations of erwinase population PK-PD models.

BACKGROUND: Asparaginases are the cornerstone therapy of many successful combination regimens for the treatment of acute lymphoblastic leukemia (ALL), the most common malignancy in children and adolescents. Currently, two asparaginase formulations are available in the US, native Escherichia coli asparaginase (ASNase) and pegaspargase. A third formulation native Erwinia asparaginase (Erwinase, ERW) has recently been made available under a licensing exception for personal use. We report here the development and validation process of ERW pharmacoanalytical assays and the results in a few patients. MATERIALS AND METHODS: We developed and systematically validated the ERW enzyme activity and ERW concentration, anti-ERW antibody and related assays. Pharmacokinetic and pharmacodynamic (PK-PD) studies were performed in a limited number of patients who received 6,000 IU/m2 x 3 per week x 2 courses, and 4 patients who received 25,000 IU/m2 x 3 per week x 2 courses of ERW. RESULTS: The linearity and range of the Erwinase calibration lines for the pharmacoanalytical assays were excellent. The accuracy and precision were better than the FDA limit allows for oncology biological products (<30%) coefficient of variation (%CV) and related parameters in the quantification of ERW concentration. The validation of these parameters was equal to or better than during the assay development. PK-PD analyses of ERW in a few patients yielded an average half-life of elimination of 15.8+/-1.64 hours. There was an excellent PD response post ERW administration resulting in an ERW concentration-dependent asparagine (ASN, <0.5 microM) and glutamine (GLN, <50 microM) deamination. Pharmacodynamic correlations demonstrated that 0.1 to 0.2 IU/ml of ERW in serum were sufficient for 90% GLN and/or ASN deamination for up to 2 weeks. No anti-ERW antibody [Ab(+)] was seen among those few patients. None of the other 5 patients had an adverse event. Based on these post hoc results, simulations on various doses and schedules of this drug have been made. CONCLUSION: The pharmacoanalytical assays were excellent tools to evaluate the PK and PD data of ERW in pediatric patients with HR ALL. However, this initial PK-PD evidence needs further validation in future clinical trials. Insights into the PD contributions of ERW in anti-E. coli ASNase Ab(+) patients will guide us in optimal design and use of ERW as part of combination chemotherapy regimens in future clinical trials.

Correlation between high vascular endothelial growth factor-A serum levels and treatment outcome in patients with standard-risk acute lymphoblastic leukemia: a report from Children's Oncology Group Study CCG-1962.

PURPOSE: Many molecular pathways, including cell cycle control, angiogenesis, and drug resistance, mediate tumor growth and survival. Vascular endothelial growth factor-A (VEGF-A) serum levels <40 and >100 pg/mL have been associated with good and poor prognoses, respectively. EXPERIMENTAL DESIGN: The hypothesis was that serum VEGF-A levels in standard-risk acute lymphoblastic leukemia pediatric patients at induction are predictive of event-free survival (EFS). One hundred seventeen patients were entered in CCG-1962 study and randomized into the native and polyethylene glycolated asparaginase arms. VEGF-A levels were quantified by an ELISA assay. RESULTS: All patients had a decrease in VEGF-A levels by day 14 of induction, but they later dichotomized; EFS group levels remained low and event group levels increased. A correlation exists between high VEGF-A levels at entry to induction and time to event. Moreover, 6-year EFS patients have lower end of induction VEGF-A levels (28 +/- 6 pg/mL) than event patients (>100 pg/mL; P < 0.01). Kaplan-Meier curves using various VEGF-A values were produced; with < or =30 at entry into induction (day 0) and < or =60 pg/mL at the end of induction (day 28), patients with low VEGF-A levels had superior EFS (P < 1e-4). Furthermore, patients who had an increase in VEGF-A during induction (DeltaVEGF-positive, days 0-28) were more likely to have an event (P < 1e-4). Bifurcation by asparaginase treatment arm did not alter these results. CONCLUSIONS: These observations strongly support that high VEGF-A levels in induction are an asparaginase treatment-independent predictive marker for EFS. Hence, an anti-VEGF-A therapy should be tested in acute lymphoblastic leukemia.

Pharmacokinetic/pharmacodynamic relationships of asparaginase formulations: the past, the present and recommendations for the future.

The discovery of the tumour-inhibitory properties of asparaginase began 50 years ago with the observation that guinea-pig serum-treated lymphoma-bearing mice underwent rapid and often complete regression. Soon afterwards, the asparaginase of bacterial origin was isolated. The asparaginases of bacterial origin induce anti-asparaginase neutralising antibodies in a large proportion of patients (44-60%), thus negating the specific enzymatic activity and resulting in failure of the target amino acid deamination in serum. There is immunological cross-reaction between the antibodies against various formulations of native Escherichia coli-asparaginase and polyethylene glycol (PEG)-asparaginases, but not to Erwinia asparaginase, as suggested by laboratory preclinical findings. This evidence was strongly inferred from the interim analyses in the Children's Cancer Group (CCG)-1961 study. Thus, anti-E. coli or PEG-asparaginase antibodies seropositive patients may benefit from the Erwinia asparaginase. The inter-relationships between asparaginase activity, asparagine (ASN) and glutamine deamination remain largely unexplored in patients. Studies have shown that ASN depletion is insufficient to induce apoptosis in T lymphoblasts in vitro and that the inhibitory concentration of CEM T-cell line is correlated with the asparaginase concentration responsible for 50% glutamine deamination. The optimal catalysis of ASN and glutamine deamination in serum by asparaginase induces apoptosis of leukaemic lymphoblasts. The percentage of ASN and glutamine deamination was predicted by asparaginase activity. Asparaginase activity of 0.1 IU/mL provided insufficient depletion of both amino acids in high-risk acute lymphoblastic leukaemia (ALL) patients. With increasing glutamine deamination, mean asparaginase activities and percentages of post-treatment samples with effective ASN depletion (<3 micromol/L) increase. Both glutamine and ASN deamination are predicted by asparaginase activity. Further population analyses resulted in identification of sigmoid relationships between asparaginase levels and post-treatment glutamine and ASN deamination.Furthermore, pharmacodynamic analyses strongly suggested that >/=90% deamination of glutamine must occur before optimal ASN deamination takes place, due to the de novo ASN biosynthesis by the liver. These pharmacodynamic results from the best-fit population pharmacokinetic/pharmacodynamic model obtained from nonlinear mixed effects model pharmacodynamic analyses for standard-risk ALL patients are similar. These analyses produced the following results: (i) asparaginase activity 0.4-0.7 IU/mL was required for optimal (90%) ASN and glutamine deamination; and (ii) deamination of glutamine is dependent on asparaginase activity and it correlates with enhanced serum ASN deamination. Thus, glutamine deamination enhances asparaginase efficacy in ALL patients. Deamination of ASN >/=90% of control or ASN concentration <3 micromol/L may be associated with improved survival in this subset of patients. Our findings support the pharmacodynamic mechanism of PEG-asparaginase for disease control in ALL patients. These results taken together strongly support new experimental approaches for application of population pharmacokinetic/pharmacodynamic analyses to further enhance survival of leukaemia patients.

Asparaginase pharmacokinetics after intensive polyethylene glycol-conjugated L-asparaginase therapy for children with relapsed acute lymphoblastic leukemia.

PURPOSE: Asparaginase therapy is an important component in the treatment of children with acute lymphoblastic leukemia. Polyethylene glycol-conjugated asparaginase (PEG-ASNase) has significant pharmacological advantages over native Escherichia coli asparaginase. We investigated the pharmacokinetics of PEG-ASNase, presence of antibodies to PEG-ASNase, and concentrations of asparagine in serum and cerebrospinal fluid (CSF) in combination chemotherapy for relapsed pediatric acute lymphoblastic leukemia. EXPERIMENTAL DESIGN: Twenty-eight pediatric patients with relapsed medullary (n = 16) and extramedullary (n = 11) acute lymphoblastic leukemia were enrolled at three pediatric institutions and had at least two serum and CSF samples obtained for analysis. Patients received induction therapy (including PEG-ASNase 2500 IU/m2 intramuscularly weekly on days 2, 9, 16, and 23) and intensification therapy (including PEG-ASNase 2500 IU/m2 intramuscularly once on day 7). Serum samples were obtained weekly during induction and intensification. CSF samples were obtained during therapeutic lumbar punctures during induction and intensification. RESULTS: Weekly PEG-ASNase therapy resulted in PEG-ASNase activity of >0.1 IU/ml in 91-100% of patients throughout induction. During intensification, PEG-ASNase on day 7 resulted in PEG-ASNase activity >0.1 IU/ml in 94% and 80% of patients on days 14 and 21, respectively. Serum and CSF asparagine depletion was observed and maintained during induction and intensification in the majority of samples. PEG-ASNase antibody was observed in only 3 patients. CONCLUSIONS: Intensive PEG-ASNase therapy in the treatment of relapsed acute lymphoblastic leukemia reliably results in high-level serum PEG-ASNase activity, and asparagine depletion in serum and CSF is usually achieved. Incorporation of intensive PEG-ASNase in future trials for recurrent acute lymphoblastic leukemia is warranted.

Asparagine synthetase polymorphisms and toxicity and efficacy of asparaginases.

Asparaginases develop innovative "tumor starvation" conditions for all antileukemia treatments; however, administrations are limited by the toxicities of this drug. Patients exhibiting moderate toxicity have optimal treatment outcomes. Certain asparagine synthetase polymorphisms may contribute to severe host toxicities in divergent subsets of patients, whereas others do not. Clinical correlations should be evaluated.

Glutaminase activity determines cytotoxicity of L-asparaginases on most leukemia cell lines.

L-Asparaginase (ASNase) is a front-line chemotherapy for acute lymphoblastic leukemia (ALL), which acts by deaminating asparagine and glutamine. To evaluate the importance of glutaminase activity, we exploited a recently developed mutant of Helicobacter pylori ASNase (dm HpA), with amino acid substitutions M121C/T169M. The mutant form has the same asparaginase activity as wild-type but lacks glutaminase activity. Wild-type and dm HpA were compared with the clinically used ASNases from Escherichia coli (l-ASP) and Erwinia chrysanthemi (ERWase). Asparaginase activity was similar for all isoforms, while glutaminase activity followed the rank order: ERWase>l-ASP>wild-type HpA>dm HpA. Cytotoxic efficacy of ASNases was tested on 11 human leukemia cell lines and two patient-derived ALL samples. Two cell lines which we had previously shown to be asparagine-dependent were equally sensitive to the asparaginase isoforms. The other nine lines and the two patient-derived samples were more sensitive to isoforms with higher glutaminase activities. ERWase was overall the most effective ASNase on all cell lines tested whereas dm HpA, having the lowest glutaminase activity, was the least effective. These data demonstrate that asparaginase activity alone may not be sufficient for ASNase cytotoxicity, and that glutaminase activity may be required for full anti-leukemic efficacy.

Determination of drug synergism between the tyrosine kinase inhibitors NSC 680410 (adaphostin) and/or STI571 (imatinib mesylate, Gleevec) with cytotoxic drugs against human leukemia cell lines.

The primary growth factor receptors involved in angiogenesis and lymphomagenesis can be grouped into the vascular endothelial growth factor (VEGF) receptors and related families. Inhibition of VEGF and other growth factors, including c-Abl, c-Kit, platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and insulin-like growth factor (IGF), or their receptors containing tyrosine kinase domains by antiangiogenesis drugs disrupts cell survival signal transduction pathways and may contribute to the proapoptotic pathways in malignant cells. However, clinical trials suggest that signal transduction inhibitors have considerable antitumor activity when used as single agents only for a short time, most likely due to the development of drug resistance by the host or by the tumor cells. In order to prevent this problem and to augment their antitumor efficacy, these agents could be administered in combination with cytotoxic antineoplastic drugs. We hypothesized that the combination of the antiangiogenesis tyrosine kinase inhibitors with cytotoxic drugs would produce synergistic drug regimens. Two human T-lymphoblastic leukemia cell lines that express VEGF-R1, CEM/0 (wild-type, WT) and the drug-resistant clone CEM/ara-C/I/ASNase-0.5-2, were utilized in the drug combination studies. NSC 680410, a tyrosine kinase inhibitor given at 0.1 to 1 microM for 72 h, inhibited VEGF secretion and leukemic cell growth at 90% of vehicle-treated control cultures with an IC50 value of less than 1 microM. The cytotoxic drugs idarubicin (IDA), fludarabine (Fludara), and cytosine arabinoside (ara-C) were used for the various drug combinations. One-, two-, three-, and four-drug treatments were tested. Cell viability was documented by the MTT assay and photomicrographic estimation of apoptotic cells. Both the combination index (CI) and isobologram evaluations demonstrated strong synergism between these drugs and the tyrosine kinase inhibitor. NSC 680410 was highly synergistic with IDA, IDA + ara-C, and IDA + Fludara + ara-C, over the respective cytotoxic drug regimens at concentrations easily achieved in patient plasma. NSC 680410 potentiated the activity of IDA in both leukemia cell lines by 17.8- and 221.4-fold in the WT and drug-resistant line, respectively. The activity of NSC 680410 + IDA + ara-C was also potentiated by 58.8-fold in the WT line, and the activity of NSC 680410 + IDA + Fludara + ara-C by 2.4- and 6.47x10(6)-fold in the WT and drug-resistant lines, respectively. The results suggest that IDA was not needed for optimal synergistic activity in the CEM/0 cells, but IDA was a necessary component to obtain drug synergism in the drug-resistant clone. Similarly, STI571 (imatinib mesylate, Gleevec), the p210(bcr/abl) tyrosine kinase inhibitor, demonstrated synergism with Fludara + ara-C or IDA + ara-C. Most importantly STI571 showed synergism with NSC 680410, suggesting that these drugs inhibit different tyrosine kinase domains in human leukemia cells. Lastly, pretreatment of leukemic cells with NSC 680410 showed additivity with gamma radiation in comparison to either treatment modality alone. The data, taken together, suggest that by inhibiting the pro-survival signal transduction pathway (VEGF-R1) and DNA replication by cytotoxic drugs, leukemic cells undergo apoptosis in a synergistic manner. In conclusion, the combinations of antiangiogenesis and DNA-damaging cytotoxic drugs are highly synergistic regimens in both WT and drug-resistant leukemic cell lines and they should be examined further.

Anti-Erwinia asparaginase antibodies during treatment of childhood acute lymphoblastic leukemia and their relationship to outcome: a case-control study.

PURPOSE: A case-control study was performed to determine whether patients who had been treated with Erwinia asparaginase as part of their treatment for childhood acute lymphoblastic leukemia (ALL) and who showed relapsed of their disease more often developed anti-asparaginase antibodies than patients who remained in remission. METHODS: A group of 13 patients who showed relapsed of their disease (median follow-up 35 months) were randomly matched with control patients of the same risk group (two control patients to each case), who had received therapy of the same intensity during the same period (median follow-up 70 months). Anti- Erwinia asparaginase antibodies were measured (ELISA method) during maintenance therapy after asparaginase treatment (30,000 IU/m(2) daily for 10 days in all patients plus twice weekly for 2 weeks in intermediate-risk and high-risk ALL patients). RESULTS: The overall incidence of anti- Erwinia asparaginase antibodies was 8% (3 of 39 patients). There was no statistically significant difference in the incidence of antibody formation between patients who had suffered relapse (1 of 13) and those who had not (2 of 26). In two of the three patients who developed antibodies, the antibodies disappeared after some time, whereas one patient had measurable antibody levels for more than a year after asparaginase therapy. CONCLUSIONS: In this study, the development of anti-Erwinia asparaginase antibodies was rare and was unrelated to the risk of relapse.

In vitro and in vivo evaluations of the tyrosine kinase inhibitor NSC 680410 against human leukemia and glioblastoma cell lines.

PURPOSE: NSC 680410, the novel adamantyl ester of AG957, an inhibitor of the p210bcr/abl tyrosine kinase (CML, Ph(+)) and possibly other kinases, was tested for antitumor activity in ten human leukemia and human glioblastoma cell lines. METHODS: CEM/0, seven ara-C- and/or ASNase-resistant clones, Jurkat/0, the myelomonocytic line U937 and U87 MG glioblastoma cell lines were used for these studies. The drug-resistant leukemic clones lack p53, express bcl-2 and VEGF-R1, and thus are refractory to apoptosis. Since tyrosine kinases drive many proliferative pathways and these activities are increased in many leukemic cells, we hypothesized that NSC 680410 may induce cytotoxicity in drug-resistant leukemia clones, independently of p210bcr/abl expression. RESULTS: NSC 680410 exhibited significant antileukemic activity in CEM/0, Jurkat E6-1, and in the drug-resistant leukemic cell lines. The IC(50) values in nine leukemia lines ranged from 17 to 216 n M. Western blot analyses after NSC 680410 treatment demonstrated caspase-3 cleavage and ELISAs showed a fivefold upregulation of its activity in cellular extracts. In addition, U87 MG human glioblastoma cells, which express VEGF-R1, were treated with the Flt-1/Fc chimera, a specific inhibitor of VEGF, and showed 30-43% cell kill in the MTT assay. Furthermore, the combination of NSC 680410 plus Flt-1/Fc chimera demonstrated an eightfold synergism against U87 MG cells in vitro. To verify this observation in vivo, athymic mice were inoculated orthotopically into the caudate putamen with 10(6) U87 MG cells. On day 3, five mice per group were treated i.p. with either 8.3 mg/kg NSC 680410 daily for three doses per week for 4 weeks alone or in combination with one dose of Flt-1/Fc chimera 100 mg/kg subcutaneously. Treatment with NSC 680410 alone produced no weight changes and increased the median survival to 133%, whereas treatment with NSC680410 plus Flt-1/Fc chimera increased survival to 142% over control. Control animals had large intra- and extracranial tumors while the NSC 680410-treated mice had small, only intracranial tumors with necrotic centers. The combination treatment resulted in small residual tumors around the needle track, indicating significant inhibition of tumor growth. CONCLUSIONS: These studies demonstrate that the tyrosine kinase inhibitor NSC 680410 has significant antileukemic activity in p53-null, drug-resistant human leukemia cell lines, as well as significant antitumor activity in combination with Flt-1/Fc chimera against U87 MG glioblastoma brain tumors implanted in situ in athymic mice.

Deamination of glutamine is a prerequisite for optimal asparagine deamination by asparaginases in vivo (CCG-1961).

BACKGROUND: Glutamine (Gln) deamination by asparaginase (ASNase) appears to contribute in the decrease of serum asparagine (Asn) levels and enhance leukemic cell apoptosis. The pharmacodynamic (PD) rationale is based on the role of Gln as the main amino group donor for Asn synthesis from aspartate by the enzyme asparagine synthetase (AS). MATERIALS AND METHODS: Relationships between ASNase enzymatic activity and Asn or Gln levels were examined in 274 pairs of pre- and post-ASNase serum specimens from 200 high-risk acute lymphoblastic leukemia (ALL) patients from the Children's Cancer Group (CCG-1961). Data were analyzed according to a novel PD model based on previous best-fit projections (NONMEM) from the CCG-1962 standard-risk ALL study. RESULTS: The PD results from high-risk and standard-risk ALL patients were superimposable. The percentages of Asn and Gln deamination were predicted by ASNase activity in patients' sera. Pharmacodynamic analyses strongly suggested that > 90% deamination of Gln must occur before optimal Asn deamination takes place in vivo. Asparaginase activity > or = 0.4 IU/ml yielded mean Gln and Asn % deamination values of 90%. Lower ASNase concentrations yielded lower Gln or Asn % deamination. This ASNase concentration coincides with the in vitro determined IC50 value on CEM/0 human T-lymphoblastic leukemia cells. CONCLUSION: Asparaginase activity of > or = 0.4 IU/ml provided optimal Asn and Gln deamination in high-risk ALL patients. Deamination of Gln correlates with enhanced serum Asn deamination in vivo. Therefore, deamination of Gln may enhance the antileukemic effect of ASNase.

Changes of amino acid serum levels in pediatric patients with higher-risk acute lymphoblastic leukemia (CCG-1961).

BACKGROUND: Deamination of asparagine (Asn) and glutamine (Gln) by asparaginases (ASNase) is associated with good prognosis in acute lymphoblastic leukemia (ALL). Chemotherapy drugs used for ALL may accelerate catabolism of other amino acids (AA). MATERIALS AND METHODS: We studied ASNase activity and changes of Asn, Gln, serine (Ser), threonine (Thr), histidine (His), proline (Pro) and arginine (Arg) levels and sought relationships in sera from 73 pediatric ALL patients, who received ASNase-containing chemotherapy. RESULTS: Asparaginase activity averaged 0.4+/-0.34 IU/ml (mean+/-SDEV) in all specimens. All AA decreased after treatment, ranging from 18.6%-82.6% of control. Asparaginase activity of 0.7 IU/ml provided 90% Asn and Gl deamination. The data were dichotomized in subsets of low ASNase (range 0.02-0.39 IU/ml, mean=0.17+/-0.09 IU/ml) and high ASNase (range 0.4-1.69 IU/ml and mean=0.72+/-0.32 IU/ml). Asparagine and Gln % deamination values were correlated with ASNase activity (p=0.0002 and p=0.0001). Similarly, decreases of Arg and Ser levels were also correlated, p =0.0009 and p=0.032, respectively. In the high ASNase subset, a 39% decrease of Arg and 26% of Ser was obtained. Low ASNase activity was correlated with lower Asn and Gln % deamination and with moderate decrease of Ser (14.6%) and Arg (19.6%). Threonine, Pro and His also decreased, but no correlations were obtained with ASNase activity. CONCLUSION: Asparagine, Gln and five other AA declined during ASNase treatment. Asparagine and Gln % deamination values are highly correlated with serum ASNase activity. Asparaginase may indirectly cause moderate depletion of serum Arg and Ser levels, providing an enhancement in leukemia blasts apoptosis. Toxicity from the ASNase and other drugs could enhance the decrease of AA serum levels. Further studies are needed to verify these findings and their potential clinical importance in the treatment of ALL patients.

Pharmacokinetics-based integration of multiple doses of intravenous pegaspargase in a pediatric regimen for adults with newly diagnosed acute lymphoblastic leukemia.

PURPOSE: Asparaginase treatment is standard in all pediatric acute lymphoblastic leukemia (ALL) regimens, whereas in adults, it is either excluded or administered for a shorter duration. Several adult ALL protocols are adapting pediatric regimens, but the optimal implementation of asparaginase is not well studied, considering its potential higher toxicity. We studied a pegaspargase dosing strategy based on its pharmacokinetic characteristics in adults. PATIENTS AND METHODS: Between 2004 and 2009, 51 adults age 18 to 57 years with newly diagnosed ALL were treated with a regimen adapted from a pediatric trial that included six doses of intravenous pegaspargase at 2,000 IU/m(2) per dose. Intervals between doses were longer than 4 weeks and rationally synchronized with other chemotherapy drugs to prevent overlapping toxicities. Pegaspargase was administered with steroids to reduce hypersensitivity. Asparaginase-related toxicities were monitored after 173 pegaspargase doses. RESULTS: The most common grade 3/4 asparaginase-related toxicities were lengthy hyperbilirubinemia and transaminitis, occasionally resulting in subsequent treatment delays. All toxicities resolved spontaneously. Forty-five percent of patients were able to receive all six doses of pegaspargase, and 61% received ≥ three doses. In only 20% of patients, the drug was discontinued after pegaspargase-related serious toxicity. Ninety-six percent achieved complete remission, almost all within 4 weeks, and a low induction death rate was seen. Seven-year disease-free and overall survival were 58% and 51%, respectively. CONCLUSION: Our dose and schedule of pegaspargase, based on its pharmacokinetics, and our detailed toxicity profile could be applied for safer adaptation of pediatric ALL protocols in adults.

Adipocytes cause leukemia cell resistance to L-asparaginase via release of glutamine.

Obesity is a significant risk factor for cancer. A link between obesity and a childhood cancer has been identified: obese children diagnosed with high-risk acute lymphoblastic leukemia (ALL) had a 50% greater risk of relapse than their lean counterparts. l-asparaginase (ASNase) is a first-line therapy for ALL that breaks down asparagine and glutamine, exploiting the fact that ALL cells are more dependent on these amino acids than other cells. In the present study, we investigated whether adipocytes, which produce significant quantities of glutamine, may counteract the effects of ASNase. In children being treated for high-risk ALL, obesity was not associated with altered plasma levels of asparagine or glutamine. However, glutamine synthetase was markedly increased in bone marrow adipocytes after induction chemotherapy. Obesity substantially impaired ASNase efficacy in mice transplanted with syngeneic ALL cells and, like in humans, without affecting plasma asparagine or glutamine levels. In coculture, adipocytes inhibited leukemic cell cytotoxicity induced by ASNase, and this protection was dependent on glutamine secretion. These findings suggest that adipocytes work in conjunction with other cells of the leukemia microenvironment to protect leukemia cells during ASNase treatment.

Asparaginases: biochemical pharmacology and modes of drug resistance.

This is an ambitious effort attempting to present as many aspects as possible in a review article on asparaginases (ASNase), and their use against acute lymphoblastic leukemia (ALL) and T-cell lymphomas. In the process, the modes of drug resistance are described both of the host and in the leukemia cells themselves. These modes of drug resistance, developed by the ALL cells, are an attempt to overcome the toxic insult this class of anti-leukemic drugs causes to them. It is expected that by reading this article one would obtain a better understanding of the initial events in the leukemia development, its microenvironment, and the many issues that a leukemia specialist has to deal with, especially in the treatment of refractory and relapsed patient populations. The specific issues addressed in this review deal with the importance of nutrients in tumor growth and progression of malignancies; the cytogenetics of ALL, as well as its chemotherapy, are also briefly presented. The emphasis will turn to ASNase, their mechanisms of action, the immune responses they cause in a significant percentage of the ALL patients, the significance of the up-regulation of glutamine synthetase and asparagine synthetase and the complexity of the elucidation of the mechanisms of action of ASNase. Additional details on the ASNase epitope mapping of anti-ASNase antibodies, the degradation of the protein, and the unmet needs in producing an optimal ASNase protein, will be also presented. Finally, a brief description of the toxicity, as well as the correlative factor of ALL treatment with ASNase is given.