Asparaginase Antibodies Research Articles

A Pharmacokinetic Study of Native E.coli Asparaginase for Acute Lymphoblastic Leukemia Treated with ThaiPOG Protocol.

Background:Asparaginase is one of the essential chemotherapies used to treat acute lymphoblastic leukemia (ALL). Asparaginase antibody production may cause a subtherapeutic level and result in an inferior outcome. The aim of this study was to prove the efficacy of current native E.coli asparaginase-based protocol. Moreover, does subtherapeutic result appeared in small group of the trial?. Methods:A prospective study of asparaginase activity among patients who received native E.coli asparaginase 10,000 IU/m2 intramuscularly according to The Thai Pediatric Oncology Group (ThaiPOG) protocol was done. The plasma asparaginase activity was measured by the coupled enzymatic reaction. Pharmacokinetic data including peak activity (Cmax), time to maximum concentration (Tmax), area under the curve (AUC0-48h) being elucidated. Results:Eight patients (five males and three females), median age 9.5 years, were enrolled. The median asparaginase activity of seven cases who were eligible for calculation reached Tmax within 24 hours (range 6-48 hours) with mean±SD of Cmax 3.60±0.34 (range 3.02-4.11) IU/ml. Mean±SD of AUC0-48h is 143.23±36.94 IU.h/mL (range 71.07 – 180.12 IU.h/mL). The post-48-hour activity showed a mean±SD of 3.19±0.24 IU/ml (range 2.77-3.51 IU/ml) which implied an adequacy of activity over 48 hours and proper for the 12-day period. One relapsed ALL patient showed an extremely low AUC of asparaginase activity which coincided with urticaria after asparaginase injection. Subsequently, the asparaginase antibody was demonstrated in this patient. Conclusion:Native E. coli asparaginase-based protocol provides a compelling pharmacokinetic effect. Asparaginase activity and/or antibody testing is recommended for all cases especially in a relapsed patient, history of high accumulative dose of asparaginase or suspected allergic reaction. Patients with low asparaginase activity or allergy may benefit from switching to an alternative form of asparaginase to maintain treatment efficacy.

Metagenomic discovery and functional validation of L-asparaginases with anti-leukemic effect from the Caspian Sea.

SummaryBy screening 27,000 publicly available prokaryotic genomes, we recovered ca. 6300 type I and ca. 5200 type II putative L-asparaginase highlighting the vast potential of prokaryotes. Caspian water with similar salt composition to the human serum was targeted for in silico L-asparaginase screening. We screened ca. three million predicted genes of its assembled metagenomes that resulted in annotation of 87 putative L-asparaginase genes. The L-asparagine hydrolysis was experimentally confirmed by synthesizing and cloning three selected genes in E. coli. Catalytic parameters of the purified enzymes were determined to be among the most desirable reported values. Two recombinant enzymes represented remarkable anti-proliferative activity (IC50 <1IU/ml) against leukemia cell line Jurkat while no cytotoxic effect on human erythrocytes or human umbilical vein endothelial cells was detected. Similar salinity and ionic concentration of the Caspian water to the human serum highlights the potential of secretory L-asparaginases recovered from these metagenomes as potential treatment agents.

PB1664 ASPARAGINASE ACTIVITY AND ANTI‐ E. COLI ASPARAGINASE ANTIBODY LEVEL MONITORING FOR DIFFERENT ASPARAGINASE PREPARATIONS IN HONG KONG CHILDHOOD ALL PATIENTS

Background:Asparaginase is effective for treating paediatric ALL, nonetheless, hypersensitivity reaction is the most prevalent form of adverse events in patients. Silent inactivation has been reported that some patients develop anti‐asparaginase antibodies against the preparations without showing any allergic symptoms.Currently, native E.Coli asparaginase (Leuanse) is the only licensed preparation in Hong Kong whereas pegylated E.Coli asparaginase (Oncaspar) and Erwinia asparaginase are the second‐line and third‐line treatment on name‐patient basis respectively. In Hong Kong, the switch of preparation is guided by the presence of clinical reaction, the efficacy of the Oncaspar and difference of effectiveness between the two available brands of Erwinia asparaginase are not known. Monitoring of asparaginase activity and anti‐asparaginase antibody for guiding the switch of asparaginase preparation was shown to improve patients’ outcome but have not been exercised in Hong Kong.Aims:We aim to evaluate the implementation of asparaginase activity measurement for monitoring the efficacy of different asparaginase preparations in clinical settings.Methods:28 patients who were undergoing asparaginase treatment were recruited including 19 newly diagnosed patients who were followed up from the start of chemotherapy. Serum was collected during induction, continuation and re‐induction phase with time points corresponding to the half‐life of different preparations and after allergic reaction. Serum asparaginase activity and anti‐E.Coli asparaginase antibody were measured, evaluated and analysed with the grading of severity of hypersensitivity reaction.Results:Among the 28 patients, 53.5% (N = 15) of them experienced allergic reaction to Leunase; 9 of them were checked for asparaginase activity and all displayed low enzyme activity (&lt;100U/L); anti‐E.Coli asparaginase antibody level was measured for 13 of them, 75.0% (N = 10) were antibody positive (&gt;0.625 ng/mL). Among the 12 patients without reaction,61.5%(N = 8) of them were positive for the antibody while all of them could maintain good activity. Most of the patients had reaction at post‐induction phase, they tolerated an average of 11.4 doses of Leunase. 15 patients allergic to Leunase were switched to Oncaspar but 73.3% (N = 11) were hypersensitive to the preparation at a mean dose of 1.67. Enzyme activity was quantified for 6 of them, all of them failed to maintain normal activity while 3 patients without clinical reaction and 2 of them showed suboptimal asparagianse activity. 11 patients after hypersensitivity reaction to Oncaspar were treated with two brands of Erwinia asparaginase from UK or China, namely Erwinase and Baiyun Erwinase, depending on their availability. Among them, 3 patients (27.3%) were clinically hypersensitive but all samples collected before reaction were with normal activity. Both preparation of Erwinia asparaginase achieved good anti‐asparaginase activities.Summary/Conclusion:Our results high incidence of allergy to Leunase in Chinese children. Patients allergic to Leunase were either clinically hypersensitive or silently inactivated to Oncaspar, suggesting that Oncaspar may not be a suitable alternative after Leunase allergy while the two brands of Erwinia asparaginase displayed satisfactory efficacy without triggering much hypersensitive response. Given the specificity and sensitivity, measurement of asparaginase activity should be part of the clinical monitoring to ensure adequate efficacy of different preparations.

Do immunoglobulin G and immunoglobulin E anti-l-asparaginase antibodies have distinct implications in children with acute lymphoblastic leukemia? A cross-sectional study.

Backgroundl-Asparaginase is essential in the treatment of childhood acute lymphoblastic leukemia. If immunoglobulin G anti-l-asparaginase antibodies develop, they can lead to faster plasma clearance and reduced efficiency as well as to hypersensitivity reactions, in which immunoglobulin E can also participate. This study investigated the presence of immunoglobulin G and immunoglobulin E anti-l-asparaginase antibodies and their clinical associations.MethodsUnder 16-year-old patients at diagnosis of B-cell acute lymphoblastic leukemia confirmed by flow cytometry and treated with a uniform l-asparaginase and chemotherapy protocol were studied. Immunoglobulin G anti-l-asparaginase antibodies were measured using an enzyme-linked immunosorbent assay. Intradermal and prick skin testing was performed to establish the presence of specific immunoglobulin E anti-l-asparaginase antibodies in vivo. Statistical analysis was used to investigate associations of these antibodies with relevant clinical events and outcomes.ResultsFifty-one children were studied with 42 (82.35%) having anti-l-asparaginase antibodies. In this group immunoglobulin G antibodies alone were documented in 10 (23.8%) compared to immunoglobulin E alone in 18 (42.8%) patients. Immunoglobulin G together with immunoglobulin E were simultaneously present in 14 patients. Children who produced exclusively immunoglobulin G or no antibodies had a lower event-free survival (p-value = 0.024). Eighteen children (35.3%) relapsed with five of nine of this group who had negative skin tests suffering additional relapses (range: 2–4), compared to none of the nine children who relapsed who had positive skin tests (p-value < 0.001).ConclusionChildren with acute lymphoblastic leukemia and isolated immunoglobulin G anti-l-asparaginase antibodies had a higher relapse rate, whereas no additional relapses developed in children with immunoglobulin E anti-l-asparaginase antibodies after the first relapse.

Asparaginase May Affect Mercaptopurine Tolerability in the Context of Multi-Agent Therapy for Acute Lymphoblastic Leukemia

▪Background: Mercaptopurine (MP) and asparaginase (ASP) are critical components in the treatment of acute lymphoblastic leukemia (ALL). Dose-limiting toxicities of the two drugs are common, resulting in therapy interruption, which has been associated with inferior treatment outcome in some studies. However, the interaction between these drugs has not been clearly identified. Merryman et al (Pediatr Blood Cancer 2012) reported that in DFCI ALL 05-01, patients had lower blood counts and more dosage reductions of MP during consolidation therapy (with concomitant ASP treatment) than during continuation therapy (identical treatment without concomitant ASP). Among groups of homogeneously treated patients with ALL, variability in ASP exposure due to inactivating antibodies can affect ASP pharmacodynamics: we have reported that ASP antibodies were associated with lower plasma ASP activity and higher dexamethasone (DEX) clearance, leading to a lower risk of osteonecrosis and a higher risk of CNS relapse (Liu, Leukemia 2012; Kawedia, Blood 2012). Here we studied the possible effect of ASP antibodies on MP tolerability in St. Jude Children's Research Hospital Total XV, a clinical trial featured intensive ASP treatment.Methods: A total of 390 children with ALL treated on St. Jude Total XV protocol were evaluable. TPMT genotype was used to guide starting doses of MP. During maintenance treatment, planned MP doses were higher on the low-risk arm (LR; n = 202) than on the standard/high-risk arm (SHR; n = 188). MP dose intensity was estimated as (prescribed dose)/(protocol dose) for weeks 1-146 (boys) or 1-120 (girls) for patients on the LR and SHR arms of maintenance therapy.Native E.coli-ASP (Elspar) was administered intramuscularly at 10000 U/m2 thrice weekly for 6 or 9 doses during remission induction. During maintenance therapy, patients on the LR arm received ASP only during reinductions I (weeks 7-9) and II (weeks 17-19), whereas those on the SHR arm received 19 weekly doses at 25000 U/m2 during weeks 1-19. Patients were tested for serum anti-Elspar antibodies at days 5, 19, 34 of remission induction, day 1 of reinduction I and day 1 of reinduction II, and were grouped based on whether they were ever positive for antibodies at any time during therapy or not. The area under the antibody concentration-time curve (AUC) for the entire period up to week 19 was also estimated in 360 patients.Result: Overall MP dose intensity was higher in those with vs without ASP antibodies in patients on the LR (median 83 vs 75%, P = 0.003) and SHR arms (median 86 vs 76%, P = 3.3 × 10-5; Figure 1A), and MP dose intensity was correlated with ASP antibody AUC in patients on both treatment arms (LR, P = 7.7 × 10-3 and SHR, P = 2.4 × 10-4; Figure 1B). In a multivariate model including age, sex, risk arm, ancestry, TPMT status, NUDT15 genotype and ASP antibody status, TPMT genotype was the strongest determinant of MP dose intensity (-17% in heterozygotes, P = 1.9 × 10-8), followed by ASP antibody positivity (+8.9% dose intensity in those with antibodies, P = 5.8 × 10-6). The model also confirmed previously identified associations of higher MP dose intensity with higher African ancestry (Bhatia et al. Blood 2014) (P = 1.8 × 10-4) and lower Asian ancestry (P = 0.05) (Yang et al. J Clin Oncol 2015).Conclusion: Interindividual differences in ASP systemic exposure, as reflected by ASP antibodies, had a strong impact on MP tolerance, especially in patients on the SHR arm who received intensive ASP therapy. We have previously shown that patients who are positive for ASP antibodies not only have lower exposure to ASP but also to dexamethasone (Kawedia, Blood 2012; Liu, Leukemia 2012). These data further emphasize the capacity for variation in ASP exposure to impact yet another critical component of ALL therapy. [Display omitted] DisclosuresEvans:Prometheus Labs: Patents & Royalties: Royalties from licensing TPMT genotyping. Relling:Prometheus Labs: Patents & Royalties: Royalties from licensing TPMT genotyping.

Desensitization protocol should not be used in acute lymphoblastic leukemia patients with silent inactivation of PEGasparaginase

Asparaginase antibodies (AAAs) neutralize asparaginase and are often accompanied by a clinically overt allergic reaction, but in a proportion of the patients these antibodies occur without any signs of hypersensitivity, termed as silent inactivation.[1][1],[2][2] Vrooman et al. and Panosyan et al.

A prospective study on drug monitoring of PEGasparaginase and Erwinia asparaginase and asparaginase antibodies in pediatric acute lymphoblastic leukemia

AbstractThis study prospectively analyzed the efficacy of very prolonged courses of pegylated Escherichia coli asparaginase (PEGasparaginase) and Erwinia asparaginase in pediatric acute lymphoblastic leukemia (ALL) patients. Patients received 15 PEGasparaginase infusions (2500 IU/m2 every 2 weeks) in intensification after receiving native E coli asparaginase in induction. In case of allergy to or silent inactivation of PEGasparaginase, Erwinia asparaginase (20 000 IU/m2 2-3 times weekly) was given. Eighty-nine patients were enrolled in the PEGasparaginase study. Twenty (22%) of the PEGasparaginase–treated patients developed an allergy; 7 (8%) showed silent inactivation. The PEGasparaginase level was 0 in all allergic patients (grade 1-4). Patients without hypersensitivity to PEGasparaginase had serum mean trough levels of 899 U/L. Fifty-nine patients were included in the Erwinia asparaginase study; 2 (3%) developed an allergy and none silent inactivation. Ninety-six percent had at least 1 trough level ≥100 U/L. The serum asparagine level was not always completely depleted with Erwinia asparaginase in contrast to PEGasparaginase. The presence of asparaginase antibodies was related to allergies and silent inactivation, but with low specificity (64%). Use of native E coli asparaginase in induction leads to high hypersensitivity rates to PEGasparaginase in intensification. Therefore, PEGasparaginase should be used upfront in induction, and we suggest that the dose could be lowered. Switching to Erwinia asparaginase leads to effective asparaginase levels in most patients. Therapeutic drug monitoring has been added to our ALL-11 protocol to individualize asparaginase therapy.

A Prospective Study On Drug Monitoring Of Pegasparaginase and Erwinia Asparaginase and Asparaginase Antibodies In Pediatric Acute Lymphoblastic Leukemia

PurposeA prospective drug monitoring study was performed to analyse the efficacy of very prolonged use of PEGasparaginase and Erwiniaasparaginase by assessing asparaginase activity, asparagine, glutamine levels and asparaginase antibodies in children with newly diagnosed acute lymphoblastic leukemia (ALL). Patients and MethodsChildren received 15 PEGasparaginase infusions (2,500 IU/m2 every other week) according to the Dutch Childhood Oncology Group (DCOG)-ALL-10 medium risk intensification protocol after having received native E.coli asparaginase (5,000 IU/m2 every 3 days, 8 doses in total) in the induction course. In case of an allergy to or silent inactivation of PEGasparaginase, Erwinia asparaginase (20,000 IU/m22x-3x per week) was given. All asparaginase preparations were administered intravenously in one hour. Serum asparaginase activity, asparagine, glutamine levels and asparaginase antibodies were measured. Results89 patients were enrolled in two centers to monitor the PEGasparaginase courses. 62/89 (70%) patients without clinical allergy to and without silent inactivation of PEGasparaginase had serum mean trough activity levels of 899 U/L which were much higher than requested. 20/89 (22%) of the patients showed an allergy and 7/89 (8%) silent inactivation in intensification. All 20 allergic patients (grade 1-4 Common Terminology Criteria Adverse Events) showed PEGasparaginase activity levels of zero. This was not due to the fact that the PEGasparaginase infusion was stopped, as 18 patients showed their allergic reactions at the second dose whereas the serum asparaginase activity level after the first full dose already appeared to be zero in all 18 cases. Moreover, in 4 patients with grade 1 allergy, the second full PEGasparaginase dose was given with pre-treatment of clemastine and hydrocortisone, also resulting in unmeasurable serum activity levels of PEGasparaginase.59 children from 7 centers with allergy to or silent inactivation of PEGasparaginase who were switched to Erwinia asparaginase were enrolled to monitor the Erwiniaasparaginase courses.Only 2/59 (3%) of the patients developed an allergy to Erwinia asparaginase. No patients with silent inactivation of Erwinia asparaginase were seen. Of the non-allergic Erwinia asparaginase patients, 55/57 (96%) had at least one serum Erwinia asparaginase trough activity level ≥ 100 U/L and 57/57 (100%) ≥ 50 U/L. In 65% and 85% of all samples had serum trough activity levels ≥ 100 U/L and ≥ 50 U/L, respectively. In 33% of patients, the administration frequency could be reduced from 3 times to 2 times per week based upon serum Erwinia asparaginase activity levels ≥ 100 U/L at 72 hours. Serum asparagine level was strongly depleted, but not always completely depleted in Erwinia asparaginase treated patients in contrast to PEGasparaginase. Serum glutamine level was slightly lowered by Erwiniaasparaginase, but no glutamine depletion was observed with both compounds. The presence of serum asparaginase antibodies is related to allergy to and silent inactivation of asparaginase, but predicting asparaginase allergy or silent inactivation is clinically not applicable because of the low specificity, 64% (95%-CI: 43%-82%). ConclusionThe use of native E.coli asparaginase in induction leads to 22% allergy and 8% silent inactivation rates of PEGasparaginase in intensification. Therefore, PEGasparaginase should be used upfront already in the induction course instead of native E.coli asparaginase. The dose of PEGasparaginase of 2,500 IU/m2 can be lowered. Switching to Erwinia asparaginase in case of allergy to or silent inactivation of PEGasparaginase leads to effective asparaginase activity levels in the majority of patients. Measuring serum asparaginase activity levels to monitor efficacy of asparaginase is preferred over serum asparagine levels and serum asparaginase antibodies. Therapeutic drug monitoring has now been implemented to individualize PEGasparaginase and Erwinia asparaginase dose and to detect silent inactivation in the current DCOG-ALL-11 protocol. Disclosures:No relevant conflicts of interest to declare.

Should We Use a Desensitization Protocol In Acute Lymphoblastic Leukemia Patients With Silent Inactivation Of Pegasparaginase?

Abstract Purpose Previous studies have shown that children with silent inactivation of asparaginase had poorer outcomes as they were not switched to another asparaginase preparation that retained its activity. Recently, a case report was published that described the successful use of desensitization courses in a patient with severe hypersensitivity reaction to asparaginase. We analyzed whether continuation of asparaginase in case of silent inactivation may result in desensitization, disappearance of asparaginase antibodies (AAA) and recovery of asparaginase activity levels in children with newly diagnosed acute lymphoblastic leukemia (ALL). Patients and Methods Children who received intensified PEGasparaginase or Erwinia asparaginase for 30 weeks according to the intensification phase of the Dutch Childhood Oncology Group-ALL-10 medium-risk protocol were studied. All children had received native E.coli asparaginase in induction and all asparaginase preparations were administered intravenously in one hour. AAA against native E.coli asparaginase (Coli-AAA), PEGasparaginase (PEG-AAA), and Erwinia asparaginase (Erwinia-AAA) and PEGasparaginase and Erwiniaasparaginase activity levels were analyzed in serum. Results 7/89 patients had silent inactivation of PEGasparaginase. Two were detected by real-time asparaginase activity measurements and were switched to Erwinia asparaginase. Five patients continued PEGasparaginase because no real-time asparaginase measurements were available at the starting phase of our drug monitoring program. Those patients with silent inactivation were, therefore, not recognized in time. PEGasparaginase activity levels recovered in all 5 patients after 2-7 PEGasparaginase infusions. In all 5 patients, Coli-AAA were present at start of the intensification phase which declined over time coinciding with the rise of PEGasparaginase activity levels. PEG-AAA were absent at the start of intensification but also increased after 1-2 doses of PEGasparaginase, and declined thereafter also coinciding with recovery of the PEGasparaginase activity levels. 29% of the PEGasparaginase patients without an allergy and without silent inactivation were positive for Coli-AAA. Also in this group, the Coli-AAA gradually decreased to undetectable levels after 5 PEGasparaginase courses. In a different cohort of 59 patients treated with Erwinia asparaginase, there were no cases of silent inactivation and two developed allergic reactions. In 50% of the non-allergic patients, the Erwinia-AAA were absent at start of therapy, gradually increased and decreased to absent baseline values during 30 weeks of Erwiniaasparaginase therapy. Conclusion This unintended desensitization program applied in five patients with silent inactivation of PEGasparaginase leads to recovery of PEGasparaginase activity levels. However, this takes an unpredictable and sometimes long time period. Therefore, we do not advise such desensitization approaches, but recommend switching to Erwinia asparaginase. A significant proportion of patients treated for prolonged period with PEGasparaginase or Erwinia asparaginase develops antibodies without influencing asparaginase activity levels that disappear with continued use of the same asparaginase product. Disclosures: No relevant conflicts of interest to declare.

Genome-Wide Association Study Identifies Germline Polymorphisms Associated with Relapse of Childhood Acute Lymphoblastic Leukemia

AbstractAbstract 878 Introduction:Treatment outcome of childhood acute lymphoblastic leukemia (ALL) has improved dramatically in the last 40 years thanks to risk-directed therapy. However, a substantial proportion of patients still experience relapse, many of whom have no known risk factors. Prior efforts to improve risk stratification have primarily focused on genetic variations of the tumor (e.g., cytogenetic abnormalities) or on assessment of early antileukemic response (e.g., upfront prednisone response, minimal residual disease [MRD] after remission induction). The role of inherited genetic variation on treatment response in children with ALL is poorly characterized. Methods:We performed a genome-wide association study to evaluate the association of genotypes at 444,044 germline SNPs with the risk of relapse in 2,535 children with newly diagnosed ALL enrolled on 5 frontline clinical trials: St. Jude Total Therapy XIIIB, XV, the Children's Oncology Group (COG) P9904, P9905, and P9906 protocols. The associations between SNP genotypes and ALL relapse were evaluated by Gray's test and by Fine and Gray's hazard regression model, adjusting for genetic ancestry and treatment regimens. To identify SNPs reproducibly associated with relapse across treatment regimens, we performed 100 rounds of discovery and replication, each round dividing patients into 2 cohorts at a 1:1 ratio. Each time, we used the discovery cohort to perform a genome-wide screen, and then filtered SNPs based on their association in the replication cohort. Finally, SNPs were prioritized on the basis of the number of times their association with relapse was replicated. Results:A total of 134 SNPs, representing 88 genomic loci, were replicated in at least 10 rounds of discovery-replication tests. Across the genome, the strongest association with relapse risk was observed at 14q22.1 in the PYGL gene (rs7142143). Each copy of the C allele at this PYGL intronic SNP (rs7142143) conferred a 3.6-fold increase in the hazard rate of relapse (P=6.7×10−9) and the association of this SNP with relapse was replicated in 79 of 100 rounds of discovery-replication tests. Of 134 relapse SNPs, 73 were associated with one or more known prognostic clinical features in childhood ALL: 32 SNPs were related to leukocyte count ≥50,000/μl at diagnosis; 19 were enriched in children older than 10 years of age; 16 were associated with hyperdiploid ALL (DNA index ≥ 1.16); and 61 were associated with ALL molecular subtype and/or lineage (MLL rearrangements, ETV6-RUNX1, TCF3-PBX1, BCR-ABL1, or T-cell ALL). Interestingly, 110 of the 134 relapse SNPs (82%) were prognostic even among MRD-negative patients, and 133 (99%) remained significantly associated with relapse after adjusting for all known risk factors, strongly indicating the potential value of germline genetic variations in ALL risk classification. To explore the mechanisms by which SNPs might influence treatment outcome of ALL, we examined the association of the 134 relapse SNPs with four pharmacokinetic and pharmacodynamic endophenotypes in the St. Jude Total XIIIB and XV cohorts: methotrexate plasma clearance, intracellular accumulation of polyglutamated (active) methotrexate, dexamethasone plasma clearance, and asparaginase antibody levels. Fourteen of the 134 relapse SNPs were significantly associated with at least one of the four pharmacologic phenotypes in a manner consistent with a pharmacokinetically intuitive association with relapse (i.e., lower drug exposure translated into a higher risk of relapse). Conclusion:In this genome-wide association study, we systematically identified host genetic variations related to treatment outcome of childhood ALL, most of which were prognostic independent of known risk factors for relapse, and some also influenced outcome by affecting host disposition of antileukemic drugs. Disclosures:No relevant conflicts of interest to declare.

Clinical utility and implications of asparaginase antibodies in acute lymphoblastic leukemia

Hypersensitivity to asparaginase is common, but the differential diagnosis can be challenging and the diagnostic utility of antibody tests is unclear. We studied allergic reactions and serum antibodies to E. coli asparaginase (Elspar) in 410 children treated on St. Jude Total XV protocol for acute lymphoblastic leukemia. Of 169 patients (41.2%) with clinical allergy, 147 (87.0%) were positive for anti-Elspar antibody. Of 241 patients without allergy, 89 (36.9%) had detectable antibody. Allergies (P=0.0002) and antibodies (P=6.6 × 10(-6)) were higher among patients treated on the low-risk arm than among those treated on the standard/high-risk arm. Among those positive for antibody, the antibody titers were higher in those who developed allergy than in those who did not (P<1 × 10(-15)). Antibody measures at week 7 of continuation therapy had a sensitivity of 87-88% and a specificity of 68-69% for predicting or confirming clinical reactions. The level of antibodies was inversely associated with serum asparaginase activity (P=7.0 × 10(-6)). High antibody levels were associated with a lower risk of osteonecrosis (odds ratio=0.83; 95% confidence interval, 0.78-0.89; P=0.007). Antibodies were related to clinical allergy and to low systemic exposure to asparaginase, leading to lower risk of some adverse effects of therapy.

Dexamethasone exposure and asparaginase antibodies affect relapse risk in acute lymphoblastic leukemia

AbstractWe have previously hypothesized that higher systemic exposure to asparaginase may cause increased exposure to dexamethasone, both critical chemotherapeutic agents for acute lymphoblastic leukemia. Whether interpatient pharmaco-kinetic differences in dexamethasone contribute to relapse risk has never been studied. The impact of plasma clearance of dexamethasone and anti–asparaginase antibody levels on risk of relapse was assessed in 410 children who were treated on a front-line clinical trial for acute lymphoblastic leukemia and were evaluable for all pharmacologic measures, using multivariate analyses, adjusting for standard clinical and biologic prognostic factors. Dexamethasone clearance (mean ± SD) was higher (P = 3 × 10−8) in patients whose sera was positive (17.7 ± 18.6 L/h per m2) versus nega-tive (10.6 ± 5.99 L/h per m2) for anti–asparaginase antibodies. In multivariate analyses, higher dexamethasone clearance was associated with a higher risk of any relapse (P = .01) and of central nervous system relapse (P = .014). Central nervous system relapse was also more common in patients with anti–asparaginase antibodies (P = .019). In conclusion, systemic clearance of dexamethasone is higher in patients with anti–asparaginase antibodies. Lower exposure to both drugs was associated with an increased risk of relapse.

Anti–Escherichia coli asparaginase antibody levels determine the activity of second-line treatment with pegylated E coli asparaginase: a retrospective analysis within the ALL-BFM trials

Anti–Escherichia coli asparaginase antibody levels determine the activity of second-line treatment with pegylated E coli asparaginase: a retrospective analysis within the ALL-BFM trials

Anti–Escherichia coli asparaginase antibody levels determine the activity of second-line treatment with pegylated E coli asparaginase: a retrospective analysis within the ALL-BFM trials

AbstractHypersensitivity reactions limit the use of the antileukemic enzyme asparaginase (ASE). We evaluated Ab levels against Escherichia coli ASE and ASE activity in 1221 serum samples from 329 patients with acute lymphoblastic leukemia who had received ASE treatment according to the ALL-BFM 2000 or the ALL-REZ BFM 2002 protocol for primary or relapsed disease. ASE activity during first-line treatment with native E coli ASE and second-line treatment with pegylated E coli ASE was inversely related to anti–E coli ASE Ab levels (P < .0001; Spearman rank order correlation). An effect on ASE activity during second-line treatment with pegylated E coli ASE was, however, only observed when anti–E coli ASE Ab levels were high (> 200 AU/mL). In the presence of moderate or intermediate Ab levels (6.25-200 AU/mL) the switch from native to pegylated E coli ASE resulted in a significant increase of ASE activity above the threshold of 100 U/L (P < .05). Erwinia chrysanthemi ASE activity was not correlated with anti–E coli ASE Ab levels. Erwinia ASE was found to be the best ASE alternative if Ab levels against E coli ASE exceed 200 AU/mL. This retrospective analysis is the first to describe the relationship between the level of anti–E coli ASE Abs and serum activity of pegylated E coli ASE used second-line after native E coli ASE. These studies are registered at http://clinicaltrials.org as NTC00430118 and NCT00114348.

Clinical Utility and Implications of Asparaginase Antibodies in Acute Lymphoblastic Leukemia

Abstract 1481Clinical manifestations of allergy to asparaginase can be subtle, making the diagnosis challenging. The usefulness of antibody tests in differential diagnosis is unclear. We investigated clinical allergic reactions and serum antibodies to native E. coli asparaginase (Elspar) (at 4–5 time points per patient) in 410 children with newly diagnosed acute lymphoblastic leukemia (ALL) treated on St. Jude’s Total XV study. Of the 169 patients (41.2%) who exhibited clinical allergy to Elspar, 147 (87.0%) were positive for anti-Elspar antibodies as measured by ELISA. Of the 241 patients who never developed a clinical allergy, 89 (36.9%) had detectable antibodies. Antibody levels were estimated to be at their highest ∼ 50 days after an allergic reaction (Figure). Patients in the low-risk treatment arm were more likely than those in the standard/high-risk arm to have a clinical allergy (51% vs. 32%, P =.0002) and detectable antibodies (69% vs. 47%, P = 6.6 ×10−6) to Elspar. Of those patients positive for antibodies, the antibody exposure (area-under-the-level vs. time curve; AUC) over the entire course of asparaginase therapy (average ± standard deviation) was higher in those who developed allergy (30.5 ± 16.5 optical density × days) than in those who did not (10.8 ± 13.3; P < 1 × 10−15). Serum antibody measurements at week 7 of continuation therapy had a sensitivity of 87–88% and a specificity of 68–69% for predicting subsequent clinical reactions (occurring at weeks 7–9) or for confirming prior clinical reactions (occurring at weeks 1–6 of treatment). The median serum asparaginase activity was lower in patients positive for antibodies (0.05 IU/mL) than in those negative for antibodies (0.2 IU/mL; P = 7.0 × 10−6) 7 days after Elspar administration. After adjusting for age and treatment arm, children with higher antibody AUCs had a lower risk of symptomatic osteonecrosis (odds ratio 0.83; 95% confidence interval, 0.73–0.95; P =.007), a finding which may be caused by higher clearance of dexamethasone due to lower asparaginase activity (Yang et al, J Clin Oncol; 26: 1932–9, 2008). Antibodies to Elspar were significantly related to clinical allergy and were useful as a clinical test to predict or confirm clinical allergy to the drug. Antibodies also were associated with low plasma exposure to active asparaginase, consistent with a lower risk of other adverse effects of ALL therapy. [Display omitted] Anti-Elspar optical density (OD) level in serum samples (dots) relative to the number of days elapsed before (negative) or after (positive) clinical reaction to Elspar. The trend lines of median (solid) and quartiles (dashed) are shown. Disclosures:Evans:St. Jude Children’s Research Hospital: Employment, Patents & Royalties; Aldagen: Membership on an entity’s Board of Directors or advisory committees; NCI, NIH: Research Funding. Relling:St. Jude Children’s Research Hospital: Employment, Patents & Royalties; Sigma-Tau Pharmaceuticals, Inc:; NIH: Research Funding.

Multiple Doses of Intravenous Pegylated Asparaginase with a “Pediatric-like” Protocol in Adults with Newly Diagnosed Acute Lymphoblastic Leukemia (ALL): Toxicity, Clinical Outcome and Low Rate of Anti Asparaginase Antibody Formation.

Abstract 3082Poster Board III-19Several, large randomized multi-agent trials, in pediatric ALL have shown that prolonged use of asparaginase (ASP) during remission resulted in better outcomes. Adequate serum asparagine depletion from ASP administration has been suggested to have a therapeutic impact. Historically, adult ALL regimens use less, shorter or no ASP compared to pediatrics, as the drug is considered more toxic. However, a recent Adult ALL study (Huguet et al, JCO 27:911, 2009), suggested an improved outcome (EFS 55%, OS 60%) using a “pediatric-inspired” protocol, which included longer duration and higher cumulative doses of E-coli ASP. PEG-asparaginase (PEG-ASP) is a modified E.coli ASP with a longer half life, resulting in sustained asparagine depletion. PEG-ASP is also considered to be less allergogenic, and in children causes fewer clinical hypersensitivity reactions and lower rate of anti-ASP neutralizing antibodies (“silent” hypersensitivity i.e. antibodies without clinical hypersensitivity) than the native E. coli ASP. We report, the use of multiple doses (up to 6) of PEG-ASP, given throughout the induction and the post-remission phases in a “pediatric-like” protocol, to newly diagnosed adult ALL. We also determined the rate of anti-ASP neutralizing antibody formation throughout our study; currently these rates are unknown in adults. MethodsThe backbone of our protocol is an augmented BFM pediatric ALL regimen consisting of 8 cycles of multi-agent chemotherapy, followed by two year maintenance. PEG-ASP (2000 U/m2/dose) is given intravenously (IV) once on day 15 of the following cycles (total 6 doses): induction phase I (cycle 1), induction phase II (cycle 2), two cycles of consolidation (cycles 3 and 6), and two cycles of delayed re-induction (cycles 5 and 8). Steroids are always given before and for at least 7 (dexamethasone) or 14 (prednisone) days after the PEG-ASP dose depending on the treatment cycle. Since December 2006 Ph+ patients receive imatinib 600mg/daily. Results46 patients, aged 18-57 (median 33) years, precursor B cell - 41, T cell-5, Ph+ 11, were studied. Median WBC at diagnosis - 14,500/cumm (range 1,900-512,000). CR rate: 44(96%) pts (43 after induction phase I). So far the total number of PEG-ASP doses given was 160: 6-16 pts, 5- 3pts 3- 6 pts., 2-10 pts., 1-11 pts. Eighteen patients could not complete all doses of PEG-ASP: allogeneic SCT -8, refusal -1, pancreatitis -5 (one pt. had pancreatitis and SCT), anaphylaxis -2, DVT-1. Two additional patients died in CR from neutropenic sepsis. Patients with elevated liver enzymes, high bilirubin, DVT (except for one pt.) or hyperglycemia continued on the study. Number of pts. with PEG-ASP related grade 3/4 toxicities (at least once of each): anaphylaxis -2 (4%) (one additional patient a had grade 2 rash); pancreatitis 6 (13%) (one pt. after last dose); thrombosis- 5 (11%) with 4 of the 5 catheter-related in upper extremity; elevated liver enzymes -30 (64%); hyperbilrubinema - 11 (24%); hyperglycemia - 12 (26 %); hypertriglyceridemia -3 (7%); fatigue-5 (11%). All toxicities were reversible. Neutralizing antibodies were measured in 247 serum samples from 34 patients (33, 11 and 10 pts. after PEG-ASP doses # 1, 4, and 6 respectively). An antibody was detected in only ONE patient (after dose #1); he had PEG-ASP related rash. We confirmed the very long half life of PEG-ASP: 7 ±4.7 days after the fist dose increasing to 11.9±5.9 days after dose 4 and 6. Peak serum levels did not change significantly between the cycles. Four year EFS, OS and CIR were 59%, 64% and 32%, respectively. The 16 patients who received all 6 doses of PEG-ASP (no BMT) had a 4 year EFS of 85% (only 3 relapsed at 19, 28, 55 months). ConclusionThis study suggests that multiple IV dosing with the long acting PEG-ASP is feasible in replacing the native E.coli asparaginase in the context of “pediatric-inspired” ALL protocols in adults (age <57 years), maintaining their reported favorable outcomes, adding convenience with less frequent dosing, and acceptable toxicity. Multiple doses of PEG-ASP given IV (in conjunction with steroids) to adults, is associated with a low rate of clinical hypersensitivity reactions and neutralizing antibody formation. The very low rate, so far, of “silent” hypersensitivity in adults, suggests that PEG-ASP can be continued IV, with less concern for drug inactivation. Such approach may benefit adults with ALL. DisclosuresDouer:Enzon Pharmaceuticals: Honoraria, Research Funding, Speakers Bureau.

Sustained and Prolonged Asparagine Depletion by Multiple Doses of Intravenous Pegylated Asparaginase in the Treatment of Adults with Newly Diagnosed Acute Lymphoblastic Leukemia.

The better outcome in pediatric ALL may not be solely related to the biology of the disease. Adult regimens are less intensive than used in children and include less, shorter or no use of asparaginase; the drug is often considered more toxic than in children. However, adequate serum asparagine depletion was shown to be associated with improved overall outcome. Further in several, multi-agent, large pediatric randomized trials, longer administration of asparaginase in the post remission phase and more sustained asparagine depletion resulted in better outcome. PEG-asparaginase (PEG-ASP) is a modified E.coli ASP, with less hypersensitivity reactions and longer half-life. In adults a single IV dose of PEG-ASP during induction produces long duration of asparagine depletion of up to 4 weeks, t½ = 7 days, with similar toxicity to equivalent multiple doses of E.coli ASP (Douer et al Blood 19:2744,2007). We now report the use of multiple doses of PEGASP, given during induction and throughout the post-remission phases in order to produce prolonged and sustained asparagine depletion in the context of an intensified pediatric protocol, in newly diagnosed, treatment naïve, adult ALL. Sustained asparaginase activity after the short acting E.coli ASP, can be impaired by development of neutralizing anti – asparaginase antibodies. We therefore measured the asparaginase enzymatic activity in a cohort of our patients who received PEG-ASP which in children was shown to be associated with less antibody formation (Avramis et al Blood 2002; 99:1986).Methods: The backbone of our protocol is an augmented BFM pediatric ALL regimen consisting of 8 cycles of multi-agent chemotherapy, followed by maintenance. PEG-ASP (2000 U/m2/dose) is given IV once on day 15 of the following cycles (total 6 doses): induction phase I (cycle 1), induction phase II (cycle 2), two cycles of consolidation (cycles 3 and 6), and two cycles of delayed re-induction (cycles 5 and 8). Since December 2006, Ph+ patients receive imatinib 600mg/daily. Asparaginase enzymatic activity was measured in samples obtained before and after PEG-ASP doses in cycles 1, 5 and 8.Results: 41 patients, aged 19–57 (median 33) years, with precursor B cell – 36, T cell-5, Ph+ 9, were studied. Median WBC at diagnosis - 15,900/cumm (range 1,900–512,000). CR rate: 37 (95%) pts. all after induction phase I. To date 15 patients received all 6 doses of PEG-ASP, having completed all consolidation cycles. The other patients are still being treated. So far the number of PEG-ASP doses given is: 6(15pts), 3(6 pts), 2(7 pts), 1(13 pts) Fifteen patients could not continue the protocol for: allogeneic stem-cell transplantation (8), refusal (1), pancreatitis (5), grade 3 DVT(1). Two additional patients died in CR from neutropenic sepsis. Patients with elevated liver enzymes, high bilirubin, or hyperglycemia continued on the study. Total number of PEG-ASP doses was 135. No pt had an allergic reaction. The number of pts with grade 3/4 toxicities during PEG-ASP cycles were: elevated liver enzymes 24 (59%), hyperbilrubinema 8 (20%), hyperglycemia 12 (29 %), pancreatitis 6(15%), fatigue 4(10%), hypertriglyceridemia 3 (7%), catheter thrombosis- 4 (10%), neuropathy-1. All toxicities were reversible. With a median follow up of 18 months, EFS at 3 and 4 yrs is 68 % (Ph- patients 74%) and CIR is 23%. Asparaginase enzymatic activity after each dose was very long in a cohort of 19 pts with a population average of t½ = 10.99 days (peak concentration 0.88 IU/ml). All 74 specimens analyzed from this cohort of patients had significant asparaginase activity after each PEG-ASP dose, highly suggesting that none of the patients analyzed had developed neutralizing anti asparaginase antibodies. Additional patients with enzymatic measurements will be presentedConclusions: Multiple doses of PEG-ASP IV in adults (ages 19–57 years) provide prolonged and sustained asparagine depletion and no apparent drug inactivating by antibody formation. In combination with an intensified BFM-based protocol this pediatriclike strategy is feasible, without allergic reactions, and with acceptable toxicity. Although the follow up is relatively short the EFS appears to be high. Such approach may benefit adults with ALL

Asparagine Depletion, Safety and Antibody Production after E Coli Asparaginase Treatment in Children with Newly Diagnosed Acute Lymphoblastic Leukaemia Treated with EORTC 58951 Protocol: A Single Center Report.

Randomised trials in pediatric ALL show that multiple doses of E Coli Asparaginase (ASP) are associated with better outcome. In the EORTC 58951 protocol, all the patients except those with high risk features (HR or VHR) had 8 IV doses of ASP during induction Ia and were randomized during induction Ib to receive or not ASP (8 IV doses vs 0) and protocol II (8 IV doses vs 4), respectively long arm and short arm. The aim of this study was to report on safety, asparagine (ASN) depletion and antibody production in 28 consecutive patients with newly diagnosed previously untreated ALL.Methods. Twenty-eight patients included in the EORTC 58951 protocol were analyzed: one had very low risk features, 22 were average risk patients and 6 had HR features. Thirteen patients were randomized in the short ASP arm and 9 in the long ASP arm. ASN levels were assessed on days 35 of Ia, 27of Ib and 22 of IIa. The amino acid assays employed the HPLC method. Serum samples were taken for antibody titers against ASP analyzed by ELISA on days 12 and 35 of Ia, 17 and 27 of Ib (long arm only) and 8 and 22 of IIa, and before the start of maintenance therapy.Results. Twenty-eight patients aged 1year 3 months to 14 years 6 months (median 6 years) with newly diagnosed ALL (precursor B-cell: 25 ; T-cell: 3) were assessable. All patients achieved CR after induction phase Ia. Four patients had allogeneic bone marrow transplantation in first CR because of VHR features. Two other patients with HR features had intensified chemotherapy. Preliminary data showed that ASN depletion was obtained in all patients at the end of phase Ia, in all the patients “long arm” at the end of Ib and in one patient ” short arm” at the end of Ib All patients had depletion of ASN after protocol IIa We observed very few severe clinical allergic reactions in this series of patients (10%). Two patients were switched to Erwinia asparaginase because of clinical hypersensitivity. Investigation on correlation between the antibody production against ASP, clinical allergic reaction and depletion of ASN are in progress.Conclusions. Multiple doses of ASP given intravenously were well tolerated in this series of 28 consecutive patients with previously untreated ALL. Preliminary results reveal that the rate of antibodies production was low and depletion of ASN was obtained in near all the patients. Some patients presented with clinical allergic reactions but had low levels of ASN and no appearance of anti ASP antibodies. Other had antibodies without clinical allergy and with depletion of ASN.

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

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. 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). 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. In this study, the development of anti-Erwinia asparaginase antibodies was rare and was unrelated to the risk of relapse.

Antibody formation during intravenous and intramuscular therapy with Erwinia asparaginase.

Determination of the frequency of antibody formation during first and second exposure to Erwinia asparaginase after i.v. and i.m. administration. Thirty-nine children with newly diagnosed acute lymphoblastic leukemia (ALL) were included in this prospective study. Antibodies were determined (ELISA method) in plasma from these patients on specific days during and after therapy with 30,000 IU/m(2) i.v. or i.m. every day for ten days during the induction phase (first exposure). For 19 children, antibodies were measured in plasma during and after the re-induction phase (second exposure) following treatment with 30,000 IU/m(2) i.v. or i.m. twice a week for two weeks (Mondays and Thursdays). On the same days of therapy, enzyme activity (spectrophotometric method) and the concentration of asparagine (HPLC) was determined. During the first exposure, none of the patients developed anti-Erwinia asparaginase antibodies. During the second exposure, one patient (1 of 8 patients) treated intravenously developed antibodies, which were associated with disappearance of enzyme activity and reappearance of asparagine. Three of eleven patients developed antibodies of pharmacokinetic importance after i.m. therapy. None of the children had any clinical symptoms of hypersensitivity. The formation of antibodies and subsequently altered pharmacokinetics of Erwinia asparaginase seemed to be of importance only during a second period of asparaginase therapy.