Asparaginase activity levels and monitoring in patients with acute lymphoblastic leukemia

Abstract

Asparaginase is an integral component of multiagent chemotherapy regimens for the treatment of acute lymphoblastic leukemia (ALL). Adequate asparagine depletion is believed to be an important factor in achieving optimal therapeutic outcomes. Measurement of asparaginase activity allows practitioners to evaluate the potential effectiveness of therapy in real time. Asparaginase activity levels can be used to identify patients with silent inactivation and modify therapy in these patients. Patients with silent inactivation to asparaginase who are switched to therapy with an immunologically distinct asparaginase exhibit outcomes similar to patients who never developed silent inactivation. Despite these benefits, there exists no universally agreed-upon guideline for treatment adjustments based on asparaginase activity levels. The goal of this manuscript is to review the clinical evidence linking asparaginase activity levels to outcomes in patients with ALL and to provide an overview of how asparaginase activity levels may be used to guide treatment.

Keywords:

• Acute lymphoblastic leukemia
• asparaginase
• hypersensitivity
• silent inactivation
• activity level monitoring

Introduction

The American Cancer Society estimates 6590 children and adults will be diagnosed with acute lymphoblastic leukemia (ALL) in the United States in 2016 [1]. The five-year overall survival (OS) for patients of all ages is 68.1%. While 57.2% of newly diagnosed patients are aged <20 years, only 15.6% of deaths occur in this age group [2]. Outcomes for children and young adults have steadily improved in recent years, with OS now approaching 90% [3,4]. Adults diagnosed with ALL have a poorer outcome with five-year survivals of 38.8%, 25.1%, 14.8%, and 4.5% in patients aged 20–49, 50–64, 65–74, and ≥75 years at diagnosis, respectively [2].

Reports have shown improved outcomes in adolescents and young adults (AYA) when treated with pediatric-based regimens, compared with adult-based regimens [5–11]. Several factors contribute to the improved outcomes in AYAs when treated with more intense pediatric-based regimens including an increased emphasis on asparaginase use compared with adult protocols. Asparaginase has been an essential component of pediatric-based multiagent chemotherapy for children and young adults with ALL since the 1970s and has demonstrated response rates as a single agent of 20–68% depending on the formulation, dose, and schedule employed [12–18]. Additionally, pediatric-based studies containing intensified and prolonged asparaginase dosing have demonstrated improved response rates and outcomes, highlighting its important role in the treatment of patients with ALL [19–22].

Asparaginase achieves its antileukemic effect by depleting circulating asparagine and depriving cancer cells of the amino acid [23–25]. Given this mechanism of action, sufficient and sustained levels of asparagine depletion are believed to be important in obtaining optimal leukemic-cell death and favorable patient outcomes [26,27]. Assays measuring asparagine concentrations during asparaginase therapy have significant technical limitations, and thus asparaginase-enzyme activity levels are commonly used to monitor patients receiving asparaginase therapy. Real-time monitoring of asparaginase activity allows practitioners to measure enzymatic activity during therapy and incorporate these data into treatment-management decisions [28]. Asparaginase activity monitoring is also the only evidenced-based method currently available to identify patients with silent inactivation (also referred to as subclinical hypersensitivity) [29–31].

Adequate asparaginase activity levels that are likely to result in asparagine depletion are of critical importance for patients with ALL undergoing asparaginase therapy. The goal of this manuscript is to review the clinical evidence linking asparaginase activity levels to outcomes in pediatric and adult patients with ALL and to highlight how asparaginase activity levels have been used to guide treatment. Specific recommendations on how to interpret asparaginase activity measurements in practice will be provided.

Asparaginase

Asparaginase catalyzes the hydrolysis of the amino acid asparagine into aspartic acid and ammonia [32,33]. Prolonged deprivation of asparagine leads to reduced protein synthesis and eventually the initiation of apoptosis [34–36]. A number of leukemic blasts lack or have very low levels of asparagine synthetase, an enzyme that is able to synthesize asparagine de novo [33]. While this can result in selective leukemic-cell kill, drug resistance may also develop following asparaginase exposure through a number of mechanisms, including a compensatory increase in the production of asparagine and glutamine [37–39].

There are currently three preparations of asparaginase approved by the United States Food and Drug Administration (FDA) for use in patients with ALL. Native Escherichia coli asparaginase and pegylated (PEG)-asparaginase share E. coli as their common bacterial source, while the third preparation, asparaginase Erwinia chrysanthemi, is derived from the bacterium E. chrysanthemi [40]. In the United States, the manufacturer of the native E. coli formulation of asparaginase ceased production in 2012, and PEG-asparaginase is now primarily used as first-line treatment [41]. Due to its distinct bacterial source, asparaginase E. chrysanthemi shows no cross-reactivity with antibodies generated from E. coli-derived asparaginase [42]. This lack of cross-reactivity is the basis for the approval of asparaginase E. chrysanthemi as a component of a multiagent chemotherapeutic regimen for the treatment of patients with ALL who have developed hypersensitivity to E. coli-derived asparaginase [43]. All three asparaginases share the same mechanism of action, the depletion of asparagine, yet differ significantly in their pharmacokinetics (PKs) [40,44,45]. These differences have important implications regarding the appropriate dose, schedule, and the interpretation of asparaginase activity measurements.

Resistance to asparaginase therapy can occur through several mechanisms and may significantly affect circulating levels of asparagine or asparaginase activity. Upregulation of asparagine synthetase in response to asparaginase therapy is considered the most common form of resistance [38,39,46,47]; however, studies have shown this mechanism to be variable and to not affect all cell lines [15,48]. Some patients will develop antiasparaginase antibodies during asparaginase treatment [29,49]. The development of antiasparaginase antibodies can rapidly neutralize the circulating asparaginase, leading to a rapid clearance and inactivation of the enzyme, thus allowing asparagine concentrations to rebound [49]. Finally, a few studies have highlighted the importance of glutamine depletion in the antileukemic effect of asparaginase [50,51]. These reports suggest greater activity levels of asparaginase are required to provide adequate depletion of asparagine and a greater degree of glutamine depletion, both of which may be necessary for optimal leukemic-cell death [51].

Asparagine depletion and asparaginase activity

Under normal physiologic conditions, plasma asparagine concentrations range from 40 to 80 µM [52]. Depletion of asparagine has been associated with in vitro leukemic-cell death, and improved outcomes have been reported in patients with lower asparagine concentrations [53]. However, accurate measurement of serum asparagine in patients undergoing asparaginase therapy can be difficult due to the continued hydrolyzation of asparagine by the enzyme [54,55]. Several studies have demonstrated that serum asparagine concentrations show an inverse relationship with asparaginase activity and measurement of asparaginase activity levels can be used as a surrogate for asparagine depletion during asparaginase therapy [52,56–58].

Substantial debate exists regarding to what extent glutamine depletion contributes to asparagine depletion and leukemic-cell death. It has been hypothesized that a higher degree of glutamine depletion is required for sufficient asparagine depletion in patients with lower asparaginase activity levels [50]. PK modeling of the Children’s Cancer Group 1961 study suggested asparaginase activity levels ≥0.4 IU/mL would be required to deplete both glutamine and asparagine [51]. In contrast, a recent report showed that a glutaminase-free mutant asparaginase continued to exhibit anticancer activity in asparagine synthetase-negative leukemic-cell lines despite showing no change in glutamine concentrations [59].

Several studies have investigated the relationship between asparaginase activity and asparagine depletion. A pharmacodynamic model identified a minimal PEG-asparaginase activity level of 0.2 IU/mL for optimal asparagine depletion [56]. Results from the Children’s Oncology Group (COG) AALL07P4 study of PEG-asparaginase and calaspargase pegol asparaginase found that asparagine levels began to rebound with plasma asparaginase activity levels somewhere between 0.1 and 0.4 IU/mL [60]. The investigators stated that asparaginase activity ≥0.4 IU/mL was associated with greater asparagine depletion and hypothesized that this could result in improved outcomes [60].

Some studies have reported sufficient asparagine depletion with reduced asparaginase activity levels [61–64]. Results from the Associazione Italiana Ematologia Oncologia Pediatrica ALL 95 study showed asparagine concentrations below the limit of detection in 11 out of 36 (31%) asparaginase E. chrysanthemi samples with asparaginase activity <0.05 IU/mL [61]. Phase 2 results of 32 children treated with a recombinant asparaginase formulation reported that asparaginase activity levels as low as 0.02 IU/mL were associated with complete asparagine depletion in some patients [62].

Early studies in monkeys and humans reported cerebrospinal fluid (CSF) asparagine was detectable with serum asparaginase activity <0.1 IU/mL [65]. Although these data support that an asparaginase activity level of ≥0.1 IU/mL achieves adequate CSF asparagine depletion [65,66], Rizarri et al. [67] demonstrated in pediatric patients with newly diagnosed ALL that while serum asparagine was adequately depleted at this level, CSF asparagine was not depleted. A target asparaginase activity level of ≥0.1 IU/mL was adopted in recently published consensus expert recommendations for management of asparaginase hypersensitivity and silent inactivation [68]. Other asparaginase activity criteria have been proposed in the literature, with a number of reports proposing a higher asparaginase activity cutoff [50,51,60,67,69]. The optimal asparaginase activity level indicative of therapeutic efficacy remains unknown, and higher levels have been associated with improved response rates [22].

Asparaginase activity and outcomes

The clinical goal of asparaginase therapy is improved outcomes for patients with ALL, and there exists evidence linking intense and consistent asparaginase therapy with improved survival. The Dana-Farber Cancer Institute (DFCI) 77-01 study was one of the earliest randomized clinical trials to compare patients treated with or without asparaginase therapy as part of a multiagent chemotherapy regimen [70,71]. Patients in the treatment arm that included native E. coli asparaginase during intensification therapy showed greater OS rates at 9.4-year follow-up compared with patients who did not receive asparaginase (71 ± 9% vs 31 ± 11%, respectively; p = .03) [70,71]. This improvement in outcomes with the inclusion of asparaginase has been supported by a number of other studies [72–74].

Later reports highlighted the importance of intensified and prolonged asparaginase therapy. In the Dana-Farber Consortium Protocol 91-01, investigators evaluated and compared outcomes in patients treated with 30 weeks vs the standard 20 weeks of asparaginase therapy [21]. A total of 377 children (aged 0–18 years) were randomized to receive 2500 IU/m² of PEG-asparaginase intramuscularly (IM) every other week for 15 doses or 25,000 IU/m² of native E. coli asparaginase IM every week for 30 doses during the intensification phase. At a median follow-up of five years, the investigators reported improved event-free survival (EFS) for patients who were able to tolerate ≥26 weeks of asparaginase therapy compared with patients who tolerated ≤25 weeks (90% vs 73%, respectively; p < .01) [21]. Reasons for discontinuation of asparaginase therapy included pancreatitis, hypersensitivity reaction, thrombosis, hyperglycemia, hyperlipidemia, and hepatitis [21]. These results highlight the importance of continued asparagainse treatment if toxicity can be adequately managed.

Results from the Cancer and Leukemia Group B Study 9511 highlight the relationship between asparaginase activity levels and outcomes in patients with ALL [26]. Wetzler et al. [26] reported improved outcomes in patients who maintained asparaginase activity levels >0.03 IU/mL for 14 days following at least one of four doses of PEG-asparaginase, compared with patients who showed activity levels <0.03 IU/mL following all four doses (OS, 31 vs 13 months, respectively; p = .01). Although the asparaginase level of >0.03 IU/mL at 14 days used in this study is substantially lower than current recommendations, the results do highlight the negative impact of lower asparaginase levels. A relationship between asparaginase activity and outcomes was also reported in patients with relapsed ALL treated in the Pediatric Oncology Group protocol 9310 study [22]. Abshire et al. [22] reported median asparaginase activity was higher in patients who achieved complete response compared with patients who did not (0.75 vs 0.45 IU/mL, respectively, p = .012).

Factors that impact asparaginase activity levels

There exists substantial interpatient variability in asparaginase activity levels due to several factors, including asparaginase formulation and dose, method of administration, and immune responses [52,69,75,76]. A report from Schrey et al. [76] highlights the variability of asparaginase activity measurements across asparaginase formulations. Regular measurements of asparaginase activity were obtained from 736 patients following the administration of 1000 IU/m² of PEG-asparaginase, 5000 IU/m² of native E. coli asparaginase, or 10,000 IU/m² of asparaginase E. chrysanthemi [76]. Asparaginase activity on day 7 following 1000 IU/m² of PEG-asparaginase (median 0.33 IU/mL, range 0–0.95 IU/mL) and on day 3 following 5000 IU/m² of native E. coli asparaginase (median 0.23 IU/mL, range 0–1.5 IU/mL) showed high interpatient variability. Interpatient variability was also seen two days following IM and intravenous (IV) administration of 10,000 IU/m² of asparaginase E. chrysanthemi (0.15 IU/mL, range 0–0.84 and 0.12 IU/mL, range 0–0.88 IU/mL, respectively).

Hypersensitivity reactions to asparaginase are the most recognizable factor that can impact enzyme activity, through the formation of neutralizing antibodies. Asparaginase is a bacterial-derived enzyme and has the potential to elicit an immunologic response in all patients. If patients develop clinical symptoms of hypersensitivity reaction during therapy, they may be switched to an alternate asparaginase. In patients who develop clinical hypersensitivity reactions, switching to alternative asparaginase preparations yields clinical outcomes similar to patients who did not experience a hypersensitivity reaction [77]. However, clinical hypersensitivity does not always correlate with the formation of antiasparaginase neutralizing antibodies; patients may develop neutralizing antibodies even in the absence of clinical hypersensitivity, and those with low-grade reactions may not develop neutralizing antibodies [29,31]. The development of antiasparaginase neutralizing antibodies in the absence of clinical symptoms has been termed ‘silent inactivation’ and is associated with a lack of asparaginase activity. As silent inactivation lacks clinical symptoms, exact prevalence can be difficult to estimate, but silent inactivation has been reported in 8–29% of patients treated with native E. coli asparaginase [29–31,77]. The development of silent inactivation of asparaginase has been associated with poorer outcomes when patients were not switched to a different asparaginase preparation [29,31,49,78].

The type of asparaginase and the method of administration have important implications for the expected asparaginase activity levels at any given point in time. The three asparaginase preparations have significantly different PK profiles. PEG-asparaginase has a reported half-life of 5.7 days following administration [79]. Asparaginase activity levels ≥0.1 IU/mL have been reported for approximately 3–4 weeks following a single IV dose of 2000–2500-IU/m² PEG-asparaginase, depending on interpatient variability [52,58]. The half-life of asparaginase E. chrysanthemi is 7.51 h following IV infusion and 15.6 h after IM administration [79,80]. Due to the shorter half-life of asparaginase E. chrysanthemi, patients who are switched from PEG-asparaginase due to hypersensitivity or silent inactivation must be administered asparaginase E. chrysanthemi at a greater frequency to maintain adequate activity levels [43].

Results of the COG AALL07P2 trial showed that 25,000 IU/m² of asparaginase E. chrysanthemi administered IM three times per week achieved an asparaginase activity ≥0.1 IU/mL in 93% of evaluable patients at 48 h postdose and in 88% of patients at 72 h postdose [81]. Recently published PK results with IV administration of asparaginase E. chrysanthemi highlight the rapid clearance associated with IV delivery [80]. Peak asparaginase activity levels are also greater with IV administration due to the absence of the slow absorptive phase that occurs with IM administration. Vrooman et al. [80] reported that 25,000 IU/m² of asparaginase E. chrysanthemi administered IV three times per week achieved asparaginase activity ≥0.1 IU/mL in 83% of evaluable patients at 48 h postdose and in 43% of patients 72 h postdose. The FDA approved the following replacement schedule for patients who experience hypersensitivity to PEG-asparaginase: 25,000 IU/m² of asparaginase E. chrysanthemi IV or IM three times per week (Monday/Wednesday/Friday [M/W/F]) for six doses for each remaining PEG-asparaginase dose remaining in their schedule [43].

Asparaginase activity monitoring

Asparaginase activity monitoring allows for the regular measurement of asparaginase activity levels throughout asparaginase therapy and can be a valuable tool to guide treatment-management decisions. Individualized (ID) dosing protocols using asparaginase activity monitoring to guide asparaginase dose adjustment have been associated with improved outcomes when compared with patients where asparaginase activity monitoring was not used [29]. In the DFCI ALL 00-01 study, 384 pediatric patients (aged 1–18 years) were treated with native E. coli asparaginase and randomized to an ID or fixed-dosing (FD) paradigm [29]. Patients in both groups were scheduled to receive 30 weekly doses of native E. coli asparaginase during intensification. Patients in the ID groups with consecutive asparaginase activity levels <0.1 IU/mL despite dose adjustment were considered to exhibit silent inactivation, and these patients were switched to an alternate asparaginase preparation. Activity levels were not used to guide treatment in the FD group; patients were not switched due to low activity levels and only switched to a different preparation of asparaginase due to clinical hypersensitivity. Patients in the ID group exhibited significantly greater five-year EFS compared with patients in the FD group (90% vs 82%, respectively; p = .04) [29]. Investigators reported that the observed improvement in survival was likely due to the detection of silent inactivation in 10% of patients in the ID group and switching these patients to an alternate asparaginase [29].

The Dutch Childhood Oncology Group ALL 10 evaluated asparaginase activity monitoring as a means of identifying patients with silent inactivation in children aged 1–18 years with newly diagnosed ALL [30]. Patients without clinical hypersensitivity and with PEG-asparaginase activity <0.1 IU/mL at day 7 ± 1 or <0.02 IU/mL at day 14 ± 1 after IV administration were defined as having silent inactivation. Clinical hypersensitivity reactions were experienced by 20 of 89 patients (22%), and silent inactivation was found in seven of 89 (8%) of patients [30]. Asparaginase activity levels in all patients with clinical hypersensitivity were undetectable compared with a median activity level of 0.899 IU/mL in patients without a hypersensitivity reaction [30]. This study highlights the usefulness of asparaginase activity monitoring in identifying patients with silent inactivation.

Recommendations for asparaginase activity monitoring

While the benefits of asparaginase activity monitoring are generally acknowledged, the optimal asparaginase activity level and appropriate use of monitoring in clinical practice remains the subject of debate [28,68,82,83]. There are two primary ways that asparaginase activity monitoring can be utilized in practice: (1) to identify patients with silent inactivation, and (2) to incrementally adjust asparaginase doses in individual patients to achieve ‘optimal’ asparaginase activity. The use of activity monitoring to identify silent inactivation is supported by a number of clinical trials; however, real-time dose modulation of asparaginase has been less studied, and the benefits due to dose adjustment are difficult to distinguish from the benefits due to the identification and treatment of patients with silent inactivation [29].

There are currently two commercially available asparaginase activity assays capable of returning results in ≤5 days [84,85]. With increasing access to asparaginase activity levels during therapy, the ability to accurately interpret activity levels will become increasingly important. In the following section, we provide a brief review of published recommendations and provide a treatment pathway for asparaginase activity monitoring. A 2015 report by Bleyer et al. provided an activity-monitoring algorithm based on a target asparaginase activity level of ≥0.05 IU/mL at days 4–7 following PEG-asparaginase administration [82]. The authors recommended testing asparaginase activity in patients who have experienced clinical hypersensitivity or in patients with previous asparaginase exposure and suggest premedication with corticosteroids if accelerated asparaginase clearance is suspected. The proposed target asparaginase activity level in these guidelines (≥0.05 IU/mL at days 4–7 postdose) is substantially lower than what would be predicted given PK data for PEG-asparaginase from clinical trials [56,60]. Due to the PK of PEG-asparaginase administered IV, activity levels of 0.6–1.2 IU/mL would be expected on days 4–7 postdose [60], and the majority of patients should not show activity <0.2 IU/mL until approximately day 21 [56,60]. Patients with PEG-asparaginase activity of 0.05 IU/mL on days 4–7 postdose would be expected to exhibit progressively reduced activity levels during days 8–14 prior to their subsequent dose.

Recently published consensus recommendations from van der Sluis et al. [68] provide different guidelines for asparaginase activity monitoring. These recommendations state that asparaginase activity should be evaluated in all patients receiving asparaginase and define silent inactivation as PEG-asparaginase activity <0.1 IU/mL on day 7 and/or below the lower limit of quantification on day 14 [68]. A similar criterion (<0.1 IU/mL) has been used to define silent inactivation in clinical trials [29,30]. In the DFCI ALL 00-01 study, silent inactivation was defined as two consecutive activity measurements <0.1 IU/mL [29]. The consensus recommendations also acknowledge that steroid or antihistamine premedication reduce the symptoms of the allergy but may not prevent antibody development [68]. Due to this risk, premedication should not be used without asparaginase activity monitoring to identify patients with silent inactivation [68].

In an effort to provide recommendations for measurement of asparaginase activity, we propose a treatment algorithm to guide the use of activity monitoring in patients receiving PEG-asparaginase therapy for ALL (Figure 1). Our recommendations are as follows:

Figure 1. Algorithm for PEG-asparaginase activity monitoring administration. PEG: pegylated.

PEG-asparaginase

• Patients who develop severe clinical hypersensitivity during asparaginase therapy should be switched to an alternate asparaginase preparation.

• When possible, asparaginase activity levels should be measured in all patients undergoing asparaginase therapy for ALL.

  •   ^ The first PEG-asparaginase activity measurement should be obtained at day 3–7 postdose from all patients (dependent on the patient’s schedule).

• Patients with day 3–7 PEG-asparaginase activity <0.1 IU/mL should be immediately retested using a new blood draw as soon as possible and switched to asparaginase E. chrysanthemi if a second test confirms PEG-asparaginase activity <0.1 IU/mL.

  •   ^ If immediately-retested asparaginase activity levels ≥0.1 IU/mL, clinicians should use their judgment to determine if an additional measurement of asparaginase activity at day 12–15 is warranted, or if activity should be reassessed on the subsequent dose.

• Patients with day 3–7 PEG-asparaginase activity of 0.1 IU/mL to 0.5 IU/mL should be retested using a new blood draw at day 12–15 (dependent on patient’s schedule) to check for accelerated clearance.

  •   ^ Patients with asparaginase activity <0.1 IU/mL at day 12–15 are candidates for treatment adjustment based on clinician’s decision. Clinicians should account for PEG-asparaginase dose when evaluating day 12–15 asparaginase activity levels.

  •   ^ Recommended treatment adjustments include:

    •    – Switching patients to asparaginase E. chrysanthemi for suspected silent inactivation.

    •    – Dose modification of PEG-asparaginase for patients with near-threshold PEG-asparaginase activity levels.

• Patients with day 3–7 PEG-asparaginase activity ≥0.5 IU/mL may continue therapy as scheduled. To minimize testing burden, therapy may be continued without the need for a second asparaginase activity measurement at days 12–15.

Asparaginase E. chrysanthemi

• The approved substitution dose for PEG-asparaginase is six doses of asparaginase E. chrysanthemi 25,000 IU/m² administered IM or IV on a M/W/F schedule for each remaining dose of PEG-asparaginase [43].

  •   ^ IV asparaginase E. chrysanthemi may be infused over a 1- to 2-h period to attenuate the rapid rise in ammonia levels associated with asparaginase [86]. Attenuating the asparaginase-associated spike in ammonia levels may reduce a patient’s risk of experiencing hyperammonemia symptoms [87–89].

Conclusions

Asparaginase is a cornerstone of multiagent chemotherapy for patients with ALL and has been a contributing factor to the improved outcomes in AYA patients treated on pediatric-based protocols. Optimizing the treatment intensity and duration of asparaginase therapy will potentially result in further improvements in outcomes. Although the asparaginase activity level required for maximal efficacy is unknown, the historical target and recent consensus recommendations support a trough level of ≥0.1 IU/mL. Future studies will be needed to define the optimal therapeutic asparaginase trough level. Regular monitoring of asparaginase activity levels allows practitioners to detect patients with silent inactivation and switch these patients to asparaginase E. chrysanthemi, ensuring the continued depletion of asparagine and allowing these patients to continue benefiting from asparaginase therapy.

Potential conflict of interest

Disclosure forms provided by the authors are available with the full text of this article online at https://doi.org/10.1080/10428194.2017.1386305.

Acknowledgements

Under the direction of the authors, Cory Hussar, PhD, of The Curry Rockefeller Group, LLC (CRG), Tarrytown, NY, provided medical writing assistance for this publication. Editorial assistance in formatting, proofreading, copy editing, and fact checking was also provided by CRG. Jazz Pharmaceuticals, Inc, provided funding to CRG for support in writing and editing of this publication.

Additional information

Funding

This publication was supported by Jazz Pharmaceuticals, Inc.