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.

Subacromial volume and rotator cuff tears: Does an association exist?

BACKGROUND: Rotator cuff pathology occurs commonly and its cause is likely multifocal in origin. The development and progression of rotator cuff injury, especially in relation to extrinsic shoulder compression, remain unclear. Traditionally, certain acromial morphologies have been thought to contribute to rotator cuff injury by physically decreasing the subacromial space. The relationship between subacromial space volume and rotator cuff tears (RCT) has, however, never been experimentally confirmed. In this study, we retrospectively compared a control patient population to patients with partial or complete RCTs in an attempt to quantify the relationship between subacromial volume and tear type. MATERIALS AND METHODS: We retrospectively identified a total of 46 eligible patients who each had shoulder magnetic resonance imaging (MRI) performed from January to December of 2008. These patients were stratified into control, partial RCT, and full-thickness RCT groups. Subacromial volume was estimated for each patient by averaging five sequential MRI measurements of subacromial cross-sectional areas. These volumes were compared between control and experimental groups using the Student's t-test. RESULTS: With the numbers available, there was no statistically significant difference in subacromial volume measured between: the control group and patients diagnosed partial RCT (P > 0.339), the control group and patients with complete RCTs (P > 0.431). CONCLUSION: We conclude that subacromial volumes cannot be reliably used to predict RCT type.

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.

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.

Asparaginase antibody and asparaginase activity in children with higher-risk acute lymphoblastic leukemia: Children's Cancer Group Study CCG-1961.

We investigated the anti-asparaginase antibody (Ab) and asparaginase enzymatic activity in the sera of 1,001 patients (CCG-1961) with high-risk acute lymphoblastic leukemia (HR-ALL). Patients received nine doses of native Escherichia coli asparaginase during induction. Half of rapid early responders (RER) were randomly assigned to standard intensity arms and continued to receive native asparaginase. The other RER patients and all slow early responders received 6 or 10 doses of PEG-asparaginase. Serum samples (n = 3,193) were assayed for determination of asparaginase Ab titers and enzymatic activity. Three hundred ninety of 1,001 patients (39%) had no elevation of Ab among multiple evaluations-that is, were Ab-negative (<1.1 over negative control)-and 611 patients (61%) had an elevated Ab titer (>1.1). Among these 611 patients, 447 had no measurable asparaginase activity during therapy. Patients who were Ab-positive but had no clinical allergies continued to receive E. coli asparaginase, the activity of which declined precipitately. No detectable asparaginase activity was found in 81 of 88 Ab-positive patients shortly after asparaginase injections (94% neutralizing Ab). The Ab-positive patients with clinical allergies subsequently were given Erwinase and achieved substantial activity (0.1-0.4 IU/ml). An interim analysis of 280 patients who were followed for 30 months from induction demonstrated that the Ab-positive titers during interim maintenance-1 and in delayed intensification-1 were associated with an increased rate of events. The CCG-1961 treatment schedule was very immunogenic, plausibly due to initially administrated native asparaginase. Anti-asparaginase Ab was associated with undetectable asparaginase activity and may be correlated with adverse outcomes in HR ALL.

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.

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.