Asparaginase has been a backbone of pediatric acute lymphoblastic leukemia (ALL) therapy for decades, and has recently become routinely used in pediatric-inspired regimens to treat older adolescent and young adult (AYA) patients.1 Unfortunately, asparaginase causes multiple severe toxicities, including hepatotoxicity. Asparaginase-associated hepatotoxicity is common and dose-limiting for concurrent hepatically-metabolized chemotherapy and can result in chemotherapy dose reductions and/or omissions. Severe hepatotoxicity during early chemotherapy is associated with 10-30% reductions in event-free survival.2,3 Obesity, which is present in 20-33% of newly diagnosed pediatric and adult ALL patients,4,5 is associated with a higher risk of hepatotoxicity,5,6 further complicating incorporation of asparaginase into frontline therapy.
Until recently, pegaspargase (Oncaspar; SS-PEG) constituted the frontline pegylated L-asparaginase product for pediatric and AYA ALL. However, in December 2022, SSPEG was removed from the US market for patients <22 years old in favor of calaspargase-pegol-mknl (Asparlas; SC-PEG). SC-PEG received regulatory approval in 2018 to replace SS-PEG based on two trials, DFCI 11-001 and COG AALL07P4.7,8 With a different linker molecule, SC-PEG has a longer half-life and an approximately 50% higher overall exposure (by area-under-the-curve).7 Though not completely elucidated, asparaginase-induced hepatotoxicity is associated with hepatosteatosis.9 Increased asparaginase exposure has therefore created concerns for correspondingly greater hepatotoxicity, particularly in those with obesity and in AYA. However, the two head-to-head trials of SC-PEG versus SS-PEG were neither designed nor able to conclusively address this question of increased hepatotoxicity. Both trials included a majority of patients less than 10 years old, who are at baseline lower risk of hepatotoxicity. Also, they neither assessed obesity, nor reported clinically-relevant thresholds, such as hyperbilirubinemia requiring dose-modification.7,8 In the COG AALL07P4 trial, a significantly increased risk for all-grade hepatotoxicity from SC-PEG was indeed found in a late treatment phase. This difference was not present when limited post hoc to grade ≥3, possibly due to the population and/or small numbers in the trial.7 Despite SC-PEG approval in 2018, frontline use was limited until the recent removal of SS-PEG from the market for patients <22 years of age. In the absence of rigorous data for comparable safety, we therefore sought to use in vivo models to investigate this concern for hepatosteatosis. We hypothesized that SC-PEG would exhibit greater hepatosteatosis in a mouse model with and without obesity. To test this, we performed a side-by-side comparison of SC-PEG versus SS-PEG in control and obese mice to evaluate differences in hepatosteatosis and hepatotoxicity. Eighteen 15-week-old male diet-induced obese (DIO) C57BL/6NTac mice (DIO-B6-M, Taconic, La Jolla, CA) and 18 control mice (DIO-B6-Control) received weekly intraperitoneal injections of 1,000 IU/kg SC-PEG, 1,000 IU/kg SS-PEG, or vehicle (N=5-6 per group). All mouse work was approved by the UCLA Institutional Animal Care and Use Committee (IACUC approval ARC-2017-053) and performed in accordance with the USPHS Policy on Humane Care and Use of Laboratory Animals. Asparaginase-treated mice exhibited significant weight loss, with obese mice losing ~8 grams and control mice losing ~4 grams of body weight during the 3-week treatment period (Figure 1A). There were no differences in the weight curves between the two asparaginase formulations. Mice were humanely euthanized with CO2 inhalation 7 days after the third injection. Liver weight decreased with asparaginase treatment, though when normalized to body weight there were no significant differences (Figure 1B). Grossly, livers from the DIO mice were more tan in appearance compared to control mice (Figure 2A). Both asparaginase formulations caused livers from control mice to take on a more tan appearance, and livers from DIO mice to become more pale yellow, with no visible difference between SS-PEG and SC-PEG. Sections of the right and left medial lobes were fixed and stained with hematoxylin and eosin (H&E) for evaluation of steatosis (Figure 2B). DIO mice showed baseline steatosis, which was increased by both asparaginase formulations. Images were examined by a board-certified pathologist (JJC) blinded to the treatment conditions and scored for steatosis and inflammation per an established murine scoring system.10 Vehicle-treated DIO mice showed higher macrovascular steatosis than control vehicle mice (Figure 2C). Asparaginase increased macrovascular steatosis in control mice, and microvascular steatosis in both diet groups, with no difference between the two asparaginase formulations. Steatosis scores increased in the control mice and showed a tendency to increase in DIO mice. Serum alanine transaminase (ALT) was not significantly increased by either asparaginase treatment. Serum albumin levels declined with treatment, more so with SC-PEG than SS-PEG (P<0.001 in controls, P<0.05 in obese, two-sided t test).
Figure 1.Pegaspargase and calaspargase-pegol-mknl have similar effects on body weight and liver weight in our mouse model. (A) Body weights of mice before and during asparaginase versus vehicle treatments. (B) Final body weights and liver weights in absolute and relative to body weight at sacrifice. *P<0.05, **P<0.01, ***P<0.001 by two-tailed, unpaired t test. Error bars show standard deviation. SSPEG: pegaspargase; SC-PEG: calaspargase-pegol-mknl.
Figure 2.Pegaspargase and calaspargase-pegol-mknl lead to similar hepatosteatosis in our mouse model. (A) Photos of livers in situ after asparaginase versus vehicle treatment. (B) Representative images of hematoxylin and eosin-stained liver sections, taken on an Olympus BX43 microscope at 20x magnification with a 0.4 numerical aperture at 22°C. Image was acquired using Olympus DP27 Acquisition software Olympus cellSens Standard 3.2 (Build 23706), with no further image processing. (C) Steatosis scores and serum measures from mice 3 days after asparaginase or vehicle injections. *P<0.05, ***P<0.001 as indicated. ††P<0.01 versus control vehicle condition. All comparisons by two-tailed, unpaired t test. Error bars show standard deviation. SS-PEG: pegaspargase; SC-PEG: calaspargase-pegol-mknl; ALT: alanine aminotransferase.
In the present study, we demonstrate that both SS-PEG and SC-PEG cause hepatosteatosis in our mouse model, but there were no clear differences between these two formulations at equivalent dosing. SS-PEG caused a further decrease in plasma albumin than SC-PEG, thought the net difference was modest. Neither formulation resulted in transaminitis in the present study. Though elevated liver enzymes and bilirubin have been reported after a dose of 1,500 IU/kg of SS-PEG,11 our findings are consistent with the majority of studies that reported no transaminitis in mice treated with native asparaginase12-14 or even after four doses of up to 5,400 IU/kg SS-PEG.15 While this preclinical in vivo assessment of relative hepatotoxicity is reassuring for ongoing use of SC-PEG, the use of an adult DIO mouse model cannot definitively predict effects in children and AYA patients. Future real-world clinical studies are needed to assess whether differences in hepatotoxicity arise due to the longer half-life of SC-PEG in patients. Current active trials investigating asparaginase hepatotoxicity (clinicaltrials gov. Identifier: NCT05602194), and the ongoing SC-PEG trial i n AYA (clinicaltrials gov. Identifier: NCT04817761), stratify by obesity and/or focus on dose-limiting hepatotoxicity, and will therefore be critical to further evaluate potential differences in clinically relevant hepatotoxicity between these two asparaginase formulations. Understanding potential differences in asparaginase metabolism both with and without obesity has important clinical implications for determining optimal dosing in children and AYA patients.
Footnotes
- Received August 27, 2024
- Accepted November 15, 2024
Correspondence
Disclosures
EO serves as a consultant for Jazz Pharmaceuticals. The other authors have no conflicts of interest to disclose.
Funding
This study was funded by a Development Research Pilot Grant from the NCI (P20CA262733, to EO and SDM) and a fellowship grant from the Jonsson Comprehensive Cancer Center (to MC).
Acknowledgments
The authors thank Praveen Bandaru for assistance with data acquisition, the UCLA Translational Pathology Core Laboratory for processing tissues and histological sectioning and Prof Vicente Micol Molina for supporting Dr. Ruiz-Torres’ research efforts.
References
- Curran E, Stock W. How I treat acute lymphoblastic leukemia in older adolescents and young adults. Blood. 2015; 125(24):3702-3710. https://doi.org/10.1182/blood-2014-11-551481|PubMed|PubMed Central|Google Scholar
- Junk SV, Schaeffeler E, Zimmermann M. Chemotherapyrelated hyperbilirubinemia in pediatric acute lymphoblastic leukemia: a genome-wide association study from the AIEOPBFM ALL study group. J Exp Clin Cancer Res. 2023; 42(1):21. https://doi.org/10.1186/s13046-022-02585-x|PubMed|PubMed Central|Google Scholar
- Schulte R, Hinson A, Huynh V. Levocarnitine for pegaspargase-induced hepatotoxicity in older children and young adults with acute lymphoblastic leukemia. Cancer Med. 2021; 10(21):7551-7560. https://doi.org/10.1002/cam4.4281|PubMed|PubMed Central|Google Scholar
- Ghosh T, Richardson M, Gordon PM, Ryder JR, Spector LG, Turcotte LM. Body mass index associated with childhood and adolescent high-risk B-cell acute lymphoblastic leukemia risk: a Children’s Oncology Group report. Cancer Med. 2020; 9(18):6825-6835. https://doi.org/10.1002/cam4.3334|PubMed|PubMed Central|Google Scholar
- Advani AS, Larsen E, Laumann K. Comparison of CALGB 10403 (Alliance) and COG AALL0232 toxicity results in young adults with acute lymphoblastic leukemia. Blood Adv. 2021; 5(2):504-512. https://doi.org/10.1182/bloodadvances.2020002439|PubMed|PubMed Central|Google Scholar
- Hashmi SK, Navai SA, Chambers TM. Incidence and predictors of treatment-related conjugated hyperbilirubinemia during early treatment phases for children with acute lymphoblastic leukemia. Pediatr Blood Cancer. 2020; 67(2):e28063. https://doi.org/10.1002/pbc.28063|PubMed|PubMed Central|Google Scholar
- Angiolillo AL, Schore RJ, Devidas M. Pharmacokinetic and pharmacodynamic properties of calaspargase pegol Escherichia coli L-asparaginase in the treatment of patients with acute lymphoblastic leukemia: results from Children’s Oncology Group Study AALL07P4. J Clin Oncol. 2014; 32(34):3874-3882. https://doi.org/10.1200/JCO.2014.55.5763|PubMed|PubMed Central|Google Scholar
- Vrooman LM, Blonquist TM, Stevenson KE. Efficacy and toxicity of pegaspargase and calaspargase pegol in childhood acute lymphoblastic leukemia: results of DFCI 11-001. J Clin Oncol. 2021; 39(31):3496-3505. https://doi.org/10.1200/JCO.20.03692|PubMed|Google Scholar
- Sahoo S, Hart J. Histopathological features of L-asparaginase-induced liver disease. Semin Liver Dis. 2003; 23(3):295-299. https://doi.org/10.1055/s-2003-42647|PubMed|Google Scholar
- Liang W, Menke AL, Driessen A. Establishment of a general NAFLD scoring system for rodent models and comparison to human liver pathology. PLoS One. 2014; 9(12):e115922. https://doi.org/10.1371/journal.pone.0115922|PubMed|PubMed Central|Google Scholar
- Kumar GVN, Hoshitsuki K, Rathod S. Mechanistic studies of PEG-asparaginase-induced liver injury and hepatic steatosis in mice. Acta Pharm Sin B. 2021; 11(12):3779-3790. https://doi.org/10.1016/j.apsb.2021.11.022|PubMed|PubMed Central|Google Scholar
- Wilson GJ, Bunpo P, Cundiff JK, Wek RC, Anthony TG. The eukaryotic initiation factor 2 kinase GCN2 protects against hepatotoxicity during asparaginase treatment. Am J Physiol Endocrinol Metab. 2013; 305(9):E1124-1133. https://doi.org/10.1152/ajpendo.00080.2013|PubMed|PubMed Central|Google Scholar
- Wilson GJ, Lennox BA, She P. GCN2 is required to increase fibroblast growth factor 21 and maintain hepatic triglyceride homeostasis during asparaginase treatment. Am J Physiol Endocrinol Metab. 2015; 308(4):E283-293. https://doi.org/10.1152/ajpendo.00361.2014|PubMed|PubMed Central|Google Scholar
- Nikonorova IA, Zhu Q, Signore CC. Age modulates liver responses to asparaginase-induced amino acid stress in mice. J Biol Chem. 2019; 294(38):13864-13875. https://doi.org/10.1074/jbc.RA119.009864|PubMed|PubMed Central|Google Scholar
- Ali Mahmoud Assar M, Hüffel M, Afify M. Effects of asparaginases and L-carnitine on Western-diet-induced hepatosteatosis in mice. F1000Res. 2022; 1(11):128. https://doi.org/10.12688/f1000research.75870.1|PubMed|PubMed Central|Google Scholar
Figures & Tables
Figure 1.Pegaspargase and calaspargase-pegol-mknl have similar effects on body weight and liver weight in our mouse model. (A) Body weights of mice before and during asparaginase versus vehicle treatments. (B) Final body weights and liver weights in absolute and relative to body weight at sacrifice. *P<0.05, **P<0.01, ***P<0.001 by two-tailed, unpaired t test. Error bars show standard deviation. SSPEG: pegaspargase; SC-PEG: calaspargase-pegol-mknl.
Figure 2.Pegaspargase and calaspargase-pegol-mknl lead to similar hepatosteatosis in our mouse model. (A) Photos of livers in situ after asparaginase versus vehicle treatment. (B) Representative images of hematoxylin and eosin-stained liver sections, taken on an Olympus BX43 microscope at 20x magnification with a 0.4 numerical aperture at 22°C. Image was acquired using Olympus DP27 Acquisition software Olympus cellSens Standard 3.2 (Build 23706), with no further image processing. (C) Steatosis scores and serum measures from mice 3 days after asparaginase or vehicle injections. *P<0.05, ***P<0.001 as indicated. ††P<0.01 versus control vehicle condition. All comparisons by two-tailed, unpaired t test. Error bars show standard deviation. SS-PEG: pegaspargase; SC-PEG: calaspargase-pegol-mknl; ALT: alanine aminotransferase.
Article Information
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.