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 front-line 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 ). In this regard, there was a relatively frequent occurrence of silent hypersensitivity during therapy (22%, 90 of 410 patients on the protocol). Based on 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. Because antibody levels tend to increase over time with successive asparaginase exposures, even in the absence of a reaction, comparisons between reacting and non-reacting 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 non-reacting 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 analysis to those reacting patients who had serum obtained 5–30 days before or after the allergy (n = 132 patients), the estimated true positive 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, falsely 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 ROC 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 (), 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 ().
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,31–35
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 metabolism,36, 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 front-line 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 to develop symptomatic osteonecrosis than those with low antibody AUC (). 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 (, 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 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 front-line 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.11–14
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 re-challenge, 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 with no allergy had positive 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 to 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 to those without) anti-Elspar antibodies who received Oncaspar (P
= 0.005), but not in those who received Erwinase (P
= 0.29), 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 front-line 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 pharmacologic 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.