Acute lymphoblastic leukemia (ALL) is the most common malignancy among children. The enzyme asparaginase has been used for over 40 years to treat pediatric ALL and contributes to the dramatically improved overall survival outcome. Recently, there is renewed interest in using asparaginase in older adolescents and adults [4]. Asparaginase catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia. Lymphoblastic leukemic cells rely on an exogenous supply of asparagine for cellular growth, and treatment with asparaginase deprives tumor cells of this essential amino acid and leads to cell death. There are currently two asparaginase preparations approved by the FDA: pegylated asparaginase (pegasparase, Oncaspar®) derived from Escherichia coli and Erwinia asparaginase (Erwinase®) derived from Erwinia chrysanthemi.
As a foreign bacterial protein, asparaginase may elicit an immune reaction and form inactivating antibodies. The inactivation of asparaginase activity may either present with or without an allergic reaction (defined as clinical hypersensitivity and silent inactivation, respectively) [1]. Recent clinical trials have demonstrated that silent inactivation is relatively common with a reported incidence between 8-44%, and undetected silent inactivation negatively affected the outcome [2, 3]. Since there is no cross-reactivity between the pegylated E. coli and Erwinia asparaginase, therapy can be maintained by switching formulation.
Evaluation of therapy effectiveness can be determined 3 ways: 1) serum asparagine concentration, 2) anti-asparaginase antibodies concentration, and 3) asparaginase activity. However, there are practical challenges with the first two methods. The rapid ex vivo metabolism of asparagine in the presence of asparaginase demands stringent specimen collection and processing. Likewise, tests for anti-asparaginase antibodies do not discriminate inactivating and non-inactivating antibodies, and thus the diagnostic specificity and positive and negative predictive values for silent inactivation are poor. Expert consensus, therefore, recommends monitoring asparaginase activity [2].
Therapeutic drug monitoring of asparaginase activity is essential to identify patients with silent inactivation, and to guide the need to change formulation and maintain therapy. However, currently there is limited availability for clinically validated asparaginase activity assays. Some methods measure the ammonia formed by Nessler’s method or by enzyme-coupled colorimetric methods. For better analytical specificity, some methods utilize substrate analogs and measure enzyme activity by plate-based colorimetric methods. All current asparaginase activity methods are labor-intensive and require multiple reaction conditions and calibration curves to cover clinically relevant concentration ranges. Furthermore, there is little data on specimen stability, interferences, and dilution effects. A study at the Hospital for Sick Children in Toronto, Ontario focused on improving a labor-intensive colorimetric method for measuring asparaginase activity to be more amenable for the clinical laboratory. Briefly, asparaginase calibrators and quality control stock solutions were prepared by dissolving lyophilized pharmacological-grade asparaginase in saline and diluted with pooled blank serum. We have optimized the assay conditions to simplify the calibration and workflow processes, and validated this simplified and consolidated procedure. Additionally, we also investigated factors that affect test results including specimen stability, interferences, and use of diluents for specimen dilutions. Samples were demonstrated to be stable at room temperature over several hours as well as up to 3 freeze-thaw cycles. Importantly, we discovered interferences when using pooled blank serum as an absorbance blank and as a specimen diluent. Investigation of the cause of interference showed differences between neat and heat- or acid-inactivated serum, indicating potential endogenous asparaginase activity in pooled blank serum. Patient results can be affected and misinterpreted when using pooled blank serum for blanking or for dilutions. Thus, we have also investigated the effects of different diluents and dilution methods. We have demonstrated that both pooled blank serum and bovine serum albumin (BSA) have comparable results as a diluent. Strategies to better identify interferences for asparaginase activity assay include using inactivated serum as blank, monitor blank absorbance, screen pooled blank serum prior to calibrator and quality control preparations, and using BSA for specimen dilutions.
In summary, asparaginase therapy is indispensable for the treatment of pediatric ALL. Despite its improvement in outcomes, asparaginase toxicities remain an issue. More specific and robust asparaginase activity assays are needed for therapeutic drug monitoring and identification of silent inactivation for patients receiving asparaginase therapy. Alternate asparaginase preparation may reduce asparaginase-related toxicities and adverse effects.
References:
- Hijiya and van der Slus (2016) Asparaginase-associated toxicity n children with acute lymphoblastic leukemia. Leukemia and Lymphoma 57:4: 748-757.
- Van der Slus et al. (2016) Consensus expert recommendations for identification and management of asparaginase hypersensitivity and silent inactivation. Haematologica 101(3): 279-285.
- Vrooman et al. (2013) Postinduction dexamethasone and individualized dosing of E. coli L-Asparaginase each improve outcome of children and adolescents with newly diagnosed acute lymphoblastic leukemia: results from a randomized study – Dana-Farber Cancer Institute ALL Consortium Protocol 00-01. Journal of Clinical Oncology 31(9): 1202-1210.
- Stock W et al. (2011) Prevention and management of asparaginase/pegasparaginase-associated toxicities in adults and older adolescents: recommendations of an expert panel. Leuk Lymphoma 52(12): 2237-2253.