Clinical utility and implications of asparaginase antibodies in acute lymphoblastic leukemia

Abstract

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.

Introduction

Asparaginase is a critical treatment component for acute lymphoblastic leukemia (ALL);1, 2, 3, 4, 5 however, its use is complicated by the development of clinical hypersensitivity.6, 7, 8, 9, 10, 11 Allergic reactions typically require discontinuation of the offending formulation (e.g., native E. coli asparaginase, Elspar) and substitution with another formulations (e.g., Erwinase or the pegylated form of E. coli asparaginase, Oncaspar); however, differentiating allergy to asparaginase from other acute reactions can sometimes be challenging. Serum asparaginase antibody has been associated with clinical allergy, but few studies have focused on its utility as a diagnostic test.10

Some previous studies have indicated that serum antibodies, even in the absence of clinical allergy, may inhibit serum asparaginase activity12 and attenuate its anticancer effect,13 although there are conflicting data.14, 15, 16, 17, 18 Many studies lack properly timed control samples from patients whose asparaginase therapy is identical to that of patients who do develop antibodies.

Here, we prospectively measured IgG antibodies to asparaginase at predetermined time points among 410 pediatric patients treated on a frontline trial of ALL, St. Jude Total XV protocol, and evaluated the predictive utility of antibody measures for allergy, and their association with asparaginase activity and adverse effects.

Patients and Methods

Patients

Between 2000 and 2007, 498 patients with newly diagnosed childhood ALL were enrolled at St. Jude Children’s Research Hospital frontline Total XV protocol: 239 patients treated on the low-risk (LR) arm and 259 on the standard/high-risk (SHR) arm. All patients received asparaginase treatment, and 410 patients (197 LR and 213 SHR) had serum samples evaluable for anti-asparaginase antibodies (Supplemental Table S1). The informed consent, IRB approval, risk arm assignment and detailed treatment regimens have been described previously.19 Race/ethnicity groups were assigned using germline genomic variation from Affymetrix mapping arrays to assess ancestry, as described.20

Asparaginase regimen and sample collection

During remission induction, Elspar was administered intramuscularly at a dose of 10 000 U/m2 three times weekly, for a total of six doses (on days 6, 8, 10, 12, 14 and 16) or nine doses (additionally on days 19, 21, 23) in patients with high levels (i.e., 1% or more) of leukemic cells in bone marrow on day 19 of remission induction.

Patients on the LR arm received nine doses of 10 000 U/m2 Elspar during reinduction I (weeks 7–9 from start of continuation treatment) and 9 doses during reinduction II (weeks 17–19) (Supplemental Figure S1). Patients on the SHR arm received Elspar at 25 000 U/m2 weekly for 19 doses in continuation treatment (weeks 1–19). For SHR patients with Philadelphia chromosome-positive ALL or induction failure, an additional dose of 25 000 U/m2 Elspar was given in the reintensification phase (after consolidation or after reinduction I based on minimal residual disease status). Patients who exhibited clinical allergy to Elspar were subsequently given Erwinia asparaginase (Erwinase) or polyethylene glycol-conjugated Elspar (Oncaspar), based on their availability (Erwinase was used preferentially when both were available), which was influenced by manufacturer-related drug shortages. Erwinase was given at 20 000 U/m2 three times weekly during remission induction for both LR and SHR patients, 20 000 U/m2 three times weekly during reinduction for LR patients and 25 000 U/m2 two times weekly in weeks 1–19 of continuation therapy for the SHR patients. Oncaspar was given at a dose of 2500 U/m2 weekly according to treatment phase. All forms of asparaginase were administered intramuscularly. If clinical allergy was confirmed for all three forms, asparaginase was discontinued.

Blood was collected into tubes without anticoagulant for asparaginase measurements on day 5, 19 and 34 of remission induction, day 1 of reinduction I and day 1 of reinduction II. Samples were collected before administering the asparaginase injection if given on the same day of sampling. Serum was frozen at −80°C until analysis.

Phenotyping of clinical allergy to asparaginase

The allergic reactions to asparaginase were characterized by local (pain, swelling, erythema) and/or systemic manifestations (fever, urticaria or edema) and graded using the National Cancer Institute Common Toxicity Criteria (NCICTC version 3.0); only patients who developed grade 2 or higher reactions received drug substitution.21

Anti-asparaginase antibodies

Serum was available for assay of antibodies in 410 patients on Total XV (Supplemental Table S1). A total of 2010 serum samples (Supplemental Table S2) were measured by ELISA for total IgG antibodies to three forms of asparaginase (Elspar, Erwinase and Oncaspar); IgE and other classes of antibodies were not measured. The method was based on modification of a previously reported assay.22 Anti-asparaginase antibodies were analyzed as a continuous variable (optical density (OD) readings at 1:400 dilution after normalization across instruments) and also classified as positive or negative, using thresholds as defined in detail in the Supplement. Those with positive antibodies in the absence of clinical allergy were considered to have silent hypersensitivity to asparaginase.

To estimate the long-term exposure to antibodies, the area under the antibody concentration versus time curve was estimated using the method of trapezoids in the 360 patients who had at least four out of five scheduled antibody tests over the first 35 weeks of therapy. Analyses were performed using the sum of the induction AUC (antibody AUC between day 5 and day 34 of remission induction) and continuation AUC (antibody AUC between week 7 and week 17 of continuation) to Elspar (Supplemental Figure S1 and Supplemental methods). Patients were considered to have detectable antibody if the AUC was greater than 2.12 (OD value × days), which corresponds to 2.58 s.d. above the mean value of negative cases (i.e., the antibody AUCs of the patients negative for anti-Elspar antibody consistently during therapy).

Sensitivity, specificity and predictive values

Sensitivity, specificity, positive predictive value and negative predictive value were calculated using the numbers of patients with true positive (TP), false positive (FP), true negative (TN) and false negative (FN) antibody results. For this purpose, patients who had clinical reactions attributed to Elspar over a given time period (e.g., weeks 7–9) were classified as TP or FN, depending on whether they were positive for anti-Elspar at an adjacent time point (e.g., week 7); the patients who never had a clinical reaction were classified as TN or FP, depending on whether they were ever positive for anti-Elspar during therapy. Sensitivity was defined as the percentage of reacting patients who were positive for anti-Elspar (calculated as TP/ (TP+FN)); specificity was defined as the percentage of nonreacting patients who were negative for anti-Elspar (calculated as TN/ (TN+FP)); positive predictive value was defined as the likelihood that a patient who was positive for antibody had a clinical reaction (calculated as TP/ (TP+FP)); and negative predictive value was defined as the likelihood that a patient who was negative for antibody never had a clinical reaction (calculated as TN/ (TN+FN)).

Determining serum asparaginase activity

Serum asparaginase activity was determined in a subset of samples from patients in the SHR arm: 77 week−7 samples and 69 week−17 samples, which were collected 6–8 days after the last Elspar administration of 25 000 U/m2; 9 week−7 samples and 10 week−17 samples, which were collected 3–4 days after the last Erwinase administration of 25 000 U/m2; and 14 week−7 samples and 16 week−17 samples, which were collected 6–8 days after the last Oncaspar administration of 2500 U/m2. We used a kinetic spectrophotometric assay similar to a previously described assay23 based on an enzymatically coupled oxidation of reduced nicotinamide adenine dinucleotide. Details are described in the Supplement.

Asparaginase adverse effects

Other than allergy, the adverse effects possibly related to asparaginase include pancreatitis, thrombosis and osteonecrosis (ON), the exposures to which were uniformly assessed in all patients. Patients were prospectively screened by magnetic resonance imaging for osteonecrosis of the hips and knees, which was graded as 0 (absent), 1 (asymptomatic), 2 (symptomatic), 3 (severe) and 4 (disabling) based on the NCICTC Version 3.0, as previously reported.24 All cases of grade 2–4 thrombosis and pancreatitis before week 20 of continuation therapy were considered possible adverse effects of asparaginase.

Statistical analysis

Comparisons of continuous variables between groups were made with the Wilcoxon rank sum test. Fisher’s exact test was used to determine the difference in frequencies of categorical variables (e.g., allergy or antibody positivity). Nonlinear and linear regression analyses were used to describe the change of antibody OD readings with time or with the frequency of clinical reaction. The prognostic capacity of antibody tests at different threshold levels was evaluated in terms of the receiver-operating characteristic curve (see Supplement for details). Logistic regression was applied for multivariate analysis of risk factors of osteonecrosis, and the cumulative incidence of osteonecrosis was compared between groups by log-rank test. Pancreatitis and thrombosis were compared between groups using time-dependent analysis,25 a Cox proportional hazards regression model where antibody status was treated as a time-dependent covariate. Statistical analyses were performed using R 2.11.1 (R Development Core Team, http://www.r-project.org) and SAS 9.2 (SAS Institute Inc., Cary, NC, USA).

Results

Asparaginase allergy

Among 410 patients, 169 (41%) had clinical allergy to Elspar, of whom 72 were first rechallenged with Erwinase and 94 with Oncaspar, and three were switched to other drugs. Thirty-three patients experienced a second episode of allergy (9 to Erwinase and 24 to Oncaspar), and two patients developed allergy to all three preparations. Patients in the LR arm were more likely than those in the SHR arm to experience hypersensitivity to Elspar, with cumulative incidences of clinical allergy being 51% versus 32% (P=0.0002), a finding consistent with that of our prior report.21 Antibody against Elspar was also more common in patients in the LR arm than those in the SHR arm: 69% versus 47%, P=6.6 × 10−6 (Figure 1). The majority of allergic reactions occurred upon first reexposure to Elspar following a 10- to 16-week hiatus after the induction phase: 78% (54 of 69) of patients in the SHR arm who had clinical allergy reacted during continuation weeks 1–6, and 96% (96 of 100) of patients in the LR arm who had clinical allergy reacted at reinduction I (Supplemental Table S3).

Figure 1
figure1

Frequency of hypersensitivity to Elspar. The frequency of allergic reactions and anti-Elspar antibody (Ab) positivity in patients enrolled on the LR and SHR treatment arms. Compared with those on the SHR arm, patients on the LR arm had more reactions (P<0.00001) and anti-Elspar antibody (P=0.0002).

We also investigated whether other clinical features such as age, gender, race or ALL lineage were associated with the risk of developing antibodies or an allergic reaction to Elspar (Supplemental Tables S4 and S5). Age and gender were not associated with allergy or antibody status; age was not related to allergy (P=0.68) or antibody status (P=0.86) even when the analysis was restricted to those with B-lineage ALL (adjusting for treatment arm and race). Allergies differed by race: reactions occurred in 45% of white, 31% of black and 33% of other (Hispanic and Asian) patients (P=0.037), and the frequencies of positive antibodies during therapy were 62%, 55% and 40% in those patient groups (P=0.005), respectively. All patients with T-cell ALL were enrolled in the SHR arm, and they had significantly fewer clinical reactions (19% versus 39%, P=0.003) and lower antibodies (35% versus 52%, P=0.026) to Elspar than those with B-lineage ALL in the SHR arm.

Antibodies and allergy

Anti-Elspar antibodies were more frequent among the patients with clinical allergy to Elspar: 87.0% (147 of 169 reacting patients) compared with only 36.9% (89 of 241) among nonreacting patients (P<1 × 10−15). Among the 236 patients who were considered positive for anti-Elspar antibody, those who had reactions had higher levels of antibody (based on OD readings) at the end of induction (P=0.0002) and during the continuation phase (week 7, P=8 × 10−9; and week 17, P<1 × 10−15) and higher cumulative antibody titer based on anti-Elspar antibody AUC (27.8±17.9 versus 10.8±13.3, P<1 × 10−15) than those who did not have reactions (i.e., the group who would be classified as ‘silent hypersensitivity’; Figure 2). However, patients with more severe clinical toxicity (grade 3–4 reactions) against Elspar did not have higher cumulative anti-Elspar antibody AUC (22.8±23.0 versus 28.6±16.9, P=0.11) than those with mild toxicity (grade 2 reactions). For Oncaspar, similarly, we observed no difference in anti-Oncaspar antibody AUC in those with grade 3–4 versus grade 2 reactions (5.7±7.8 versus 5.4±4.8, P=0.83). For Erwinase, all allergic reactions were grade 2, and thus no comparisons are possible.

Figure 2
figure2

Antibody levels in patients with and without clinical reactions. Left: Anti-Elspar OD measures (natural log) for 410 patients at days 5, 19 and 34 of remission induction and weeks 7 and 17 of continuation therapy. At each time point, patients are grouped based on whether they ever (or never) experienced a clinical allergic reaction (Rxn) and whether they were ever (or never) positive for Ab at any time during therapy. Among the patients positive for anti-Elspar, those who had reactions (white bars) had significantly higher anti-Elspar levels than those who had silent hypersensitivity (light gray) at day 34, week 7 and week 17. Among the patients negative for anti-Elspar antibodies (as defined by the threshold in the Supplement), those who had allergic reactions (hatched) exhibited slightly elevated anti-Elspar levels compared with those without any allergic reactions (dark gray) at weeks 7 (P=0.05) and 17 (P=0.03). Right: cumulative anti-Elspar antibody AUC over the first 35 weeks of therapy for antibody-positive patients who had allergic reactions versus those who had silent hypersensitivity. Box plots: median, quartiles, non-outlier range.

This study’s sampling strategy was designed to have evaluable antibody samples at comparable times relative to asparaginase dosing for all patients (those who developed an allergy and those who did not). Because allergic reactions may have occurred at times during which no samples were obtained, maximal values may not have been captured if antibody levels are maximal at the time of reaction. However, serial samples that happened to be obtained before, during, and after reactions indicated that antibody levels were estimated to be maximal at about 50 days after an allergic reaction (Supplemental Figure S2).

We investigated the utility of serum anti-Elspar antibody positivity to predict the future (if sampling done just before) or to confirm (if sampling done just after) clinical allergic reactions. The sensitivity, specificity and predictive values are shown in Table 1. The greatest utility was evident for the week-7 serum samples, which happened to be obtained closest to the time period during which most reacting patients had their allergic reaction (continuation weeks 7–9 for those in the LR arm and weeks 1–6 for those in the SHR arm). The week 7 anti-Elspar antibody test had a sensitivity of 87–88% and a specificity of 68–69% for predicting clinical reactions to Elspar at weeks 7–9 for LR patients and for confirming clinical reactions to Elspar at weeks 1–6 for SHR patients. The prognostic utility of the antibody tests at day 34, week 7 and week 17, as evaluated by the AUC of the receiver-operating characteristic curves, ranged from 0.70–0.91 (Supplemental Figure S3). Antibody test obtained at day 34 of induction for future clinical reactions showed a lower prognostic capacity compared with those at week 7 and week 17 (Supplemental Figure S3).

Table 1 Sensitivity, specificity and predictive values of three anti-Elspar antibody tests for predicting or confirming clinical reactions to Elspar

Interestingly, we observed a strong positive linear relationship between the anti-Elspar level (natural log of OD reading) at week 7 and the probability of patients reacting to Elspar at weeks 7–9 on the LR arm (R-square=0.96, P=6 × 10−8) or weeks 1–6 on the SHR arm (R-square=0.83, P=4 × 10−5; Supplemental Figure S4), consistent with our finding that anti-Elspar level was higher in patients with allergy than those with silent hypersensitivity (Figure 2).

The frequency of secondary allergy to Oncaspar was higher than that to Erwinase (24/94 or 26% versus 9/72 or 13%, P=0.05). For secondary treatment with Erwinase and Oncaspar, there was less association between clinical reactions to these agents and corresponding antibodies. We measured anti-Erwinase and anti-Oncaspar antibodies in 63 of the 72 patients who went on to be treated with Erwinase and 85 of the 94 patients who went on to receive Oncaspar. Among the 63 patients receiving Erwinase, 9 had an allergy to it, of whom only four had detectable anti-Erwinase antibody. Among the 85 patients receiving Oncaspar, 23 had a clinical reaction, of whom 20 had detectable anti-Oncaspar antibody. In addition, silent hypersensitivity occurred among 8 of 63 (13%) patients treated with Erwinase and among 47 of 85 (55%) patients treated with Oncaspar (Supplemental Figure S5).

Antibodies and serum asparaginase activity

In addition to testing the clinical utility of asparaginase antibodies as a tool to predict or confirm clinical allergy, antibodies have been hypothesized to be important as indicators of lower systemic exposure to asparaginase. Indeed, we found that in vivo serum asparaginase activity after Elspar was lower in the samples positive for antibodies than in those negative for antibodies (P=7 × 10−6, Figure 3a), and was inversely related to the level of antibody (based on OD readings, P=7 × 10−11, Figure 3b).

Figure 3
figure3

Correlation between serum asparaginase activity and antibody level. Serum asparaginase activity (IU/ml) measured 6–8 days after the last Elspar dose of 25 000 U/m2. (a) Asparaginase activity was significantly lower in the samples positive than in those negative for anti-Elspar antibodies. Box plots: median, quartiles, non-outlier range. (b) Asparaginase activity inversely correlated with anti-Elspar OD reading of serum samples. Individual data points and the regression line are shown (n=146).

To determine whether antibody was also inversely related to asparaginase activity after secondary treatment with Erwinase and Oncaspar, we measured in vivo serum asparaginase activity in 19 patients on Erwinase and 30 patients on Oncaspar at the time of the anti-Elspar antibody measurement. Asparaginase activity did not differ by anti-Elspar antibody positivity in patients who received second-line treatment with Erwinase (P=0.29), but it was lower (median 0.03 IU/ ml, range 0–2.73 IU/ml) in those who were positive for anti-Elspar antibody and received second-line treatment with Oncaspar compared with those who were negative for the antibody (median 2.04 IU/ml, range 1.07–2.77 IU/ml; P=0.005).

Antibodies and adverse effects

As we have shown that asparaginase can potentiate exposure to dexamethasone,26 we investigated whether cumulative asparaginase antibody titer affected the risk of symptomatic (grade 2–4) osteonecrosis. After adjustment for age and treatment arm, which are risk factors for osteonecrosis, higher cumulative antibody titer (based on antibody AUC) was associated with lower risk of symptomatic osteonecrosis (odds ratio for log2 antibody AUC=0.83; 95% CI, 0.78–0.89; P=0.007), consistent with antibodies causing less asparaginase exposure.

We also compared the cumulative risk of symptomatic osteonecrosis between patients negative for anti-Elspar antibody (i.e., anti-Elspar antibody AUC2.12) and those with detectable antibody (i.e., anti-Elspar antibody AUC>2.12; Figure 4). There was a lower cumulative incidence of symptomatic osteonecrosis in those with a higher anti-Elspar antibody AUC who were older than 10 years of age (in the LR arm, P=0.048; and the SHR arm, P=0.037), and in younger patients (in the LR arm, P=0.047; but not on the SHR arm, P=1.0) (Figure 4).

Figure 4
figure4

Cumulative incidence of osteonecrosis based on anti-asparaginase antibody AUC. Patients were stratified by LR versus standard/high-risk treatment arm and age at diagnosis: (a) patients in the LR arm with age greater than 10 years; (b) patients in the SHR arm with age greater than 10 years; (c) patients in the LR arm with age under 10 years; (d) patients in the SHR arm with age under 10 years. Left: Cumulative incidence of symptomatic (grade 2–4) osteonecrosis (ON) in patients with anti-Elspar antibody AUC (OD × days) lower than 2.12 (blue) versus greater than 2.12 (red). Right: box plot of anti-Elspar antibody AUC in patients with (blue) versus without symptomatic ON (red). aP value based on log-rank test and bP value based on Wilcoxon rank sum test. *Fisher’s exact test (P=0.11) was also performed because the sample size is small.

Seven patients had pancreatitis before the end of reinduction II, and five of them were positive for antibody. Twenty-eight patients had thrombotic events before the end of reinduction II, and seven of them were positive for antibody. After adjusting for age and treatment arm, there was no apparent association between antibody positivity and pancreatitis (P=0.22) or thrombosis (P=0.20).

Discussion

Although the relationship between antibodies and allergic reactions to asparaginase has been observed in many studies,8, 10, 11, 23 this is the first report focusing on the diagnostic utility of asparaginase antibody measures to predict or to confirm clinical allergy to asparaginase. Particularly in children, differentiating allergy from other diagnoses can be challenging. In the context of an asparaginase-intensive frontline clinical trial and by dichotomizing samples as positive versus negative, the most informative sample (at week 7 of continuation phase) had reasonably high (88%) sensitivity to predict reactions in the next 3 weeks but less favorable specificity (68%) to confirm past reactions (see Table 1). In this regard, there was a relatively frequent occurrence of silent hypersensitivity during therapy (22%, 90 of 410 patients on the protocol). On the basis of this test performance, for every 100 patients with a reaction during weeks 1–9, 88 would be expected to have positive and 12 would have negative antibody tests during this time period (at week 7); for every 100 patients without an allergy, 32 would have positive and 68 would have negative antibody tests at that time (week 7). The diagnostic value of antibody test at this time point is similar to those of some widely used clinical laboratory tests such as the diagnosis of venous thromboembolism with d-dimer (sensitivity 80–95% and specificity 40–70%);27 the prediction of rheumatoid arthritis with rheumatoid factor (sensitivity 40–80%, specificity 70–90%)28 and the diagnosis of systemic lupus erythematosus with antinuclear antibody (sensitivity 93%, specificity 50–80%).29

We were unable to identify a more informative time point that yielded better antibody-test performance. The early antibody test at the end of induction lacked the sensitivity to predict future clinical allergy. The later antibody tests (i.e., week 17) did not confirm as well for patients in the LR arm; for those in the SHR arm, the week 17 result had slightly reduced sensitivity (72.7%) and increased specificity (88.2%), but clinically it would not be possible to use such a result for making therapeutic decisions. Although antibody levels tend to increase over time with successive asparaginase exposures, even in the absence of a reaction, comparisons between reacting and nonreacting patients should control for place-in-therapy. In our study, serum samples were timed uniformly relative to therapy but were not available at the exact time of the reaction in most cases (and control samples, of course, would not have been available in nonreacting patients at exactly comparable times); thus, we were not able to assess the specificity of the serum anti-Elspar antibody test at the time of clinical reaction to Elspar. However, even if we limited the analysis to those reacting patients who had serum obtained 5–30 days before or after the allergy (n=132 patients), the estimated TP rate was not higher than 80% (data not shown).

We also investigated whether the predictive utility of the antibody measures was improved by leaving the measure as a continuous measure (OD of anti-Elspar antibody titers) instead of dichotomizing the results as positive versus negative. We observed a linear correlation between the antibody OD at week 7 and the probability of patients having a clinical reaction in the subsequent 3-week asparaginase course, and a correlation between week 7 antibody OD and the proportion who had clinical reactions in the previous 6-week asparaginase course (Supplemental Figure S4). For the LR patients with anti-Elspar OD>0.39 at week 7, 90% of them are predicted to have an event in the subsequent course; for the SHR patients with anti-Elspar OD>1.06 at week 7, 80% of them would have had an event in the previous course. However, false-positive readings (high OD readings in patients with no known reaction) remained a limitation, and thus we were not able to define an alternative universal threshold for distinguishing positive from negative antibody status that resulted in better overall balanced-test performance (based on the receiver-operating characteristic curve of the antibody tests, see Supplemental Figure S3).

In addition to the possible utility of an anti-asparaginase antibody test to facilitate diagnosis of allergy, antibodies may be an indicator of reduced serum asparaginase exposure. In samples obtained at a uniform time (6–8 days) post dose, we found that serum asparaginase activity was indeed inversely related to antibody level (Figure 3), consistent with reports from other groups.12, 13, 23, 30 Whether this attenuation of serum asparaginase activity is due to a direct neutralizing effect of antibodies, or because the presence of antibodies is an indicator of other immune-based mechanisms that enhance drug clearance (e.g., via the reticuloendothelial system), is unknown; the latter may explain why a few samples had low asparaginase activity despite being low in asparaginase antibodies (Figure 3).

We explored whether the presence of antibodies, with its likely attendant reduced exposure to asparaginase over time, would reduce other adverse effects of asparaginase therapy. Even though dexamethasone is the major cause of osteonecrosis in children with ALL,31, 32, 33, 34, 35 we have shown that asparaginase can increase the risk of osteonecrosis,35 which we hypothesize could be because asparaginase inhibits protein synthesis, decreases dexamethasone clearance,26 alters lipid metabolism36, 37 and induces coagulopathy.38, 39 Because osteonecrosis is a long-term complication, we used antibody AUC as a measurement of long-term exposure to antibodies against Elspar, the frontline asparaginase preparation. We observed that, among younger patients on the LR arm and all older patients on either LR or SHR arm, those with high antibody AUC were at lower risk of developing symptomatic osteonecrosis than those with low antibody AUC (Figure 4). The difference was not apparent among the younger patients on the SHR arm, possibly because younger patients are less prone to osteonecrosis, and those on the SHR arm also had lower antibodies, perhaps too low to have any effect on the development of osteonecrosis (Figure 4d, right panel). In multivariate analysis, after adjusting for age and treatment arm, antibody AUC was significantly associated with grade 2–4 osteonecrosis. This is the first report that states that anti-asparaginase antibodies were inversely related to the risk of osteonecrosis, but is consistent with our hypothesis that asparaginase potentiates glucocorticoid effects.35 We recently reported that asparaginase antibodies were associated with a higher risk of central nervous system relapse,40 but did not find that asparaginase antibodies were related to pancreatitis or thrombosis, perhaps indicating that these latter two adverse events are less ‘dose related’ than others.

In the Total XV study, with Elspar as the frontline asparaginase preparation, the frequency of clinical hypersensitivity to Elspar was 41% (169/410) for all patients who started with Elspar, and the incidence of IgG antibodies to Elspar was 58% (236/410), similar to the average from other reported series.11, 12, 13, 14 The hypersensitivity appeared to differ by treatment arm and immunophenotype, consistent with the result from our previous ALL trial, Total XIII.11, 14 Patients on the LR arm exhibited more hypersensitivity to asparaginase than did those on the SHR arm, possibly because the LR arm included less immunosuppressive chemotherapy and included longer periods of asparaginase ‘holiday’ followed by rechallenge, a practice noted to be associated with allergy by others.9

There was a very high frequency of ‘silent hypersensitivity’ observed in patients who subsequently received Oncaspar (55% of patients treated with Oncaspar did not develop allergy but were positive for antibodies against Oncaspar). However, 24% of patients who never received Oncaspar had positive antibodies at week 7 and 17, which could be due to cross-reactivity between sera positive to both Elspar and Oncaspar in patients who are exposed initially to Elspar (Oncaspar is pegylated Elspar).41 Consistent with that idea, only 13% of patients who received Erwinase had silent hypersensitivity, compared with 6% of patients who never received Erwinase. Allergy was also more common (P=0.05) in those who received Oncaspar rather than Erwinase as their second-line agent, and serum asparaginase activity was lower in those with (compared with those without) anti-Elspar antibodies who received Oncaspar (P=0.005), but not in those who received Erwinase (P=0.29), which is consistent with recent reports.30 Together, these data suggest that Erwinase may be preferred over Oncaspar in those who receive primary treatment with Elspar and experience allergy, although it is possible that such a decision depends upon the dose of Oncaspar versus Erwinase and the absolute level of anti-Elspar antibodies.30

Interestingly, patients with T-cell ALL were less likely to develop asparaginase allergy and had lower antibody levels at post-asparaginase time points than those with B-lineage ALL. The mechanisms underlying this difference are unclear, but suggest an intriguing added value for asparaginase in T-cell ALL.

In summary, we comprehensively analyzed anti-Elspar antibody in an asparaginase-intensive frontline clinical trial. In this context, in which Elspar is given as the primary form of asparaginase, serum anti-Elspar antibodies have good utility as tools to help make the diagnosis of clinical hypersensitivity. Our data show that higher antibody levels are associated with greater clinical pharmacological effects including a higher risk of clinical allergy, and a greater attenuation of serum asparaginase enzyme activity with associated lower risk of osteonecrosis. Thus, measures of serum antibodies to asparaginase can be useful in patients with ALL.

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Acknowledgements

This work was supported by NCI grants CA 142665, CA 36401 and CA 21765 and the NIH/NIGMS Pharmacogenomics Research Network (U01 GM92666), and by the American Lebanese Syrian Associated Charities (ALSAC). We thank the clinical staff, research nurses, patients and their parents for participation; Dr Wenjian Yang, Nancy Kornegay and Mark Wilkinson for computational assistance and data preparation; May Chung and Natalya Lenchik for asparaginase antibody assays; Dr Laura Ramsey and Dr Colton Smith for insightful comments; and Dr David Armbruster for manuscript editing.

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Correspondence to M V Relling.

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Competing interests

MVR receives a portion of the income St Jude receives from licensing patent rights related to TPMT and GGH polymorphisms, and receives funding for investigator-initiated research on the pharmacology of asparaginase from Sigma-Tau Pharmaceuticals. WEE receives a portion of the income St Jude receives from licensing patent rights related to TPMT and GGH polymorphisms. All other authors declare no conflict of interest.

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Liu, C., Kawedia, J., Cheng, C. et al. Clinical utility and implications of asparaginase antibodies in acute lymphoblastic leukemia. Leukemia 26, 2303–2309 (2012). https://doi.org/10.1038/leu.2012.102

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Keywords

  • ALL
  • asparaginase
  • antibody
  • allergy
  • pharmacodynamics
  • osteonecrosis

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