Abstract
Asparaginase is a mainstay of treatment of childhood acute lymphoblastic leukemia. Pegylation of asparaginase extends its biological half-life and has been introduced in the newest treatment protocols aiming to further increase treatment success. Hyperammonemia is a recognized side effect of asparaginase treatment, but little is known about its incidence and clinical relevance. Alerted by a patient with severe hyperammonemia after introduction of the new acute lymphoblastic leukemia protocol, we analyzed blood samples and clinical data of eight consecutive patients receiving pegylated asparaginase (PEG-asparaginase) during their treatment of acute lymphoblastic leukemia. All patients showed hyperammonemia (>50 μmol/L) and seven patients (88 %) showed ammonia concentrations > 100 μmol/L. Maximum ammonia concentrations ranged from 89 to 400 μmol/L. Symptoms varied from mild anorexia and nausea to headache, vomiting, dizziness, and lethargy and led to early interruption of PEG-asparaginase in three patients. No evidence of urea cycle malfunction was found, so overproduction of ammonia through hydrolysis of plasma asparagine and glutamine seems to be the main cause. Interestingly, ammonia concentrations correlated with triglyceride values (r = 0.68, p < 0.0001), suggesting increased overall toxicity.
The prolonged half-life of PEG-asparaginase may be responsible for the high incidence of hyperammonemia and warrants future studies to define optimal dosing schedules based on ammonia concentrations and individual asparagine and asparaginase measurements.
Introduction
In the past four decades, overall survival of childhood acute lymphoblastic leukemia (ALL) has improved dramatically from 34 % in the early 1970s to 86 % in the year 2004 (Kamps et al. 2010). Addition of asparaginase to treatment protocols has contributed significantly to this improved outcome (Muller and Boos 1998).
Asparaginase depletes plasma of the nonessential amino acid asparagine by hydrolyzing it into aspartic acid and ammonia. Since leukemic cells possess insufficient asparagine synthetase activity, an intracellular deficiency of asparagine leads to inhibition of protein synthesis and subsequent cell death (Prager and Bachynsky 1968).
Three different preparations of asparaginase are used in current ALL treatment protocols: native Escherichia colil-asparaginase, Erwinia chrysanthemil-asparaginase, and pegylated asparaginase (PEG-asparaginase). Pegylation of l-asparaginase reduces immunogenic potential and extends the half-life of the enzyme activity from 1.3 days to 5.7 days (Asselin et al. 1993).
Despite its proven efficacy, several side effects of asparaginase treatment have been reported: hypersensitivity reactions, disturbed liver functions, coagulation disorders, pancreatitis, hyperlipidemia, and silent inactivation by antibodies (Muller and Boos 1998).
Hyperammonemia due to asparaginase therapy has been described in a number of case reports (Leonard and Kay 1986; Alvarez and Zimmerman 2000; Laterza et al. 2003; Jorck et al. 2011), but little is known about its incidence, causative mechanism, and clinical relevance. The influence of the prolonged half-life of PEG-asparaginase on the duration and severity of hyperammonemia is unknown as well. Therefore, we studied ammonia concentrations and other metabolic parameters in pediatric ALL patients receiving PEG-asparaginase and observed hyperammonemia (>50 μmol/L) in all patients and ammonia concentrations >100 μmol/L in seven out of eight patients.
Design and Methods
After presentation of the index case (patient A), we analyzed ammonia concentrations in seven consecutive patients treated in our hospital and assigned to the medium risk group of the Dutch Childhood Oncology Group (DCOG) ALL 10 protocol during their treatment with PEG-asparaginase. Measurements were conducted between 2007 and 2009.
The DCOG ALL 10 protocol is a protocol based on the pediatric ALL protocol of the German Berlin-Frankfurt-Münster (BFM) Group, consisting of induction, high-dose methotrexate, followed by minimal residual disease–based assignment to standard, medium, or high-risk intensification and continuation treatment. The outline of the intensification/continuation treatment for medium-risk-group patients is shown in Fig. 1.
Fig. 1.
Schematic drawing of the Dutch Childhood Oncology Group intensification/continuation of medium-risk patients. This part of therapy starts 20 weeks after diagnosis. DEXA dexamethasone, VCR vincristine, DOX doxorubicine, MTX methotrexate, PEG-ASP PEG-asparaginase; 6-MP 6 mercaptopurine, ARA-C cytarabine, DAF di-adreson-F, PO per os, IV intravenously, push rapid injection; 1 h infusion during 1 h, ITH intrathecal
Ammonia concentrations were analyzed before the first administration of PEG-asparaginase, at least once, 1 week after PEG-asparaginase when also dexamethasone was administered in the previous week (week 2, 8, 14, 20, or 26) as well as 1 week after PEG-administration when no dexamethasone was administered (week 4, 6, 10, 12, 16, 18, 22, 24, 28, or 30). Hyperammonemia was defined as ammonia concentrations >50 μmol/L and clinical significant hyperammonemia as ammonia concentrations >100 μmol/L (Laterza et al. 2003; Cohn and Roth 2004; Steiner et al. 2007). At the above-mentioned time points, we also measured liver enzymes (alanine transaminase, aspartate transaminase, gamma glutamyl transferase, and alkaline phosphatase), bilirubin, total amylase, lipase, triglycerides, and cholesterol. In case of elevated ammonia concentrations or symptoms or signs suggestive of hyperammonemia, repeated ammonia measurements were performed. Blood samples were transported to the lab, centrifuged in the cold, and immediately stored at −20°C to deactivate the effect of PEG-asparaginase. Blood samples for ammonia analysis were generally taken 3–4 h after the last meal and analyzed by standardized enzymatic-photometric assay using a DxC 800 analyzer (Beckman Coulter diagnostics division, Brea, CA, USA).
To rule out secondary abnormalities such as urea cycle disorders, organic acidurias or fatty acid oxidation disorders, plasma amino acids, acylcarnitine concentrations, and, in six patients, urine orotic acid excretion were measured. In order to deactivate the PEG asparaginase in vitro, the blood sample tubes for amino acids and ammonia were immediately put in ice water, rapidly transported to the lab, centrifuged in the cold and the plasma deproteinized before storing the supernatant at −20°C until analysis.
Symptoms and signs associated with hyperammonemia, like headache, lethargy, nausea, vomiting, dizziness, and neurological symptoms were documented in the patient files. A waiver of the requirement for informed consent was granted by the Institutional Review Board of the University Medical Center Utrecht, which reviewed the study.
Results and Discussion
Patient characteristics and results are shown in Tables 1 and 2. Hyperammonemia was detected in all patients and ammonia concentrations >100 μmol/L in seven out of eight consecutive patients with acute ALL during treatment with PEG-asparaginase. All patients with clinically significant hyperammonemia displayed symptoms. Although generally mild, symptoms were severe in three patients (A, C, and D), leading to hospital admission and discontinuation of PEG-asparaginase therapy.
Table 1.
Patient characteristics and description of toxicity. M/F male or female, ALL Acute Lymphoblastic Leukemia, PEG-asparaginase Pegylated asparaginase, CR complete remission
| Patient | M/F | Age at diagnosis (years) | Diagnosis | Maximum documented ammonia concentration (μmol/L) | Number of PEG-asparaginase doses before maximum ammonia concentration | Symptoms | Treatment for hyperammonemia | Other toxicities during PEG-asparaginase therapy | Outcome |
|---|---|---|---|---|---|---|---|---|---|
| A (index case) | F | 5 | Pre B ALL | 400 | 6 | Headache, nausea, vomiting, lethargy, leading to admission | Lactulose (1.8 mL/kg), protein restricted diet; Omission of eight doses of PEG-asparaginase | Severe hypertriglyceridemia (1,946 mg/dL), hypercholesterolemia (703 mg/dL), elevated gamma GT (2,163 U/L) | In CR, 33 months after end of therapy |
| B | F | 3 | Pre B ALL | 258 | 13 | Mild lethargy, anorexia, weakness | None | Moderate hypertriglyceridemia (690 mg/dL) | In CR, 30 months after end of therapy |
| C | M | 3 | Common ALL | 248 | 8 | Lethargy, nausea | Omission of 1 dose of PEG-asparaginase | Moderate hypertriglyceridemia (717 mg/dL) | In CR, 28 months after end of therapy |
| Mild hypercholesterolemia (286 mg/dL) | |||||||||
| D | F | 12 | Pre B ALL | 281 | 14 | Dizziness, lethargy, headache, leading to admission | Lactulose, protein restricted diet; omission of 1 dose of PEG-asparaginase | Severe hypertriglyceridemia (3,645 mg/dL), | In CR, 27 months after end of therapy |
| Hypercholesterolemia (792 mg/dL) | |||||||||
| thrombosis vena axillaris, elevated gamma GT (1,280 U/L), hypoglycemia (2.4 mmol/L) | |||||||||
| E | M | 3 | Common ALL | 366 | 5 | Mild nausea, headache, malaise | None | Severe hypertriglyceridemia (8,725 g/dL) | In CR, 21 months after end of therapy |
| Hypercholesterolemia (448 mg/dL) | |||||||||
| F | F | 9 | Pre B ALL | 320 | 13 | Mild lethargy, nausea | None | Severe hypertriglyceridemia (1,991 mg/dL) | In CR, 21 months after end of therapy |
| Hypercholesterolemia (471 mg/dL) | |||||||||
| G | M | 5 | Common ALL | 123 | 2 | Anorexia | None | None | In CR, 18 months after end of therapy |
| H | F | 4 | Common ALL | 89 | 1 | None | Not applicable | none | In CR, 17 months after end of therapy |
Table 2.
Results of metabolic studies in described patients. Reference values: asparagine 34–94 μmol/L; aspartate 1–17 μmol/L; glutamine 333–809 μmol/L; glutamate 14–78 μmol/L
| Patient | Asparagine and aspartate | Glutamine and glutamate | Urine orotic acid |
|---|---|---|---|
| A (index case) | Asparagine: not available | Glutamine: normal (812 μmol/L) | Normal |
| Aspartate elevated (21 μmol/L) | Glutamate: not available | ||
| B | Not available | Not available | Not available |
| C | Asparagine: complete depletion (0 μmol/L) | Glutamine: decreased (325 μmol/L) | Normal |
| Aspartate: elevated (30 μmol/L) | Glutamate: elevated (169 μmol/L) | ||
| D | Asparagine: complete depletion (0 μmol/L) | Glutamine: decreased (1,295 μmol/L) | Normal |
| Aspartate: elevated (30 μmol/L) | Glutamate: elevated (653 μmol/L) | ||
| E | Asparagine: complete depletion (0 μmol/L) | Glutamine: decreased (189 μmol/L) | Normal |
| Aspartate: normal (13 μmol/L) | Glutamate: elevated (441 μmol/L) | ||
| F | Asparagine: complete depletion (0 μmol/L) | Glutamine: decreased (102 μmol/L) | Not available |
| Aspartate: elevated (24 μmol/L) | Gutamate: elevated (733 μmol/L) | ||
| G | Asparagine: complete depletion (0 μmol/L) | Glutamine: decreased (116 μmol/L) | Normal |
| Aspartate: elevated (21 μmol/L) | Glutamate: elevated (463 μmol/L) | ||
| H | Asparagine: complete depletion (0 μmol/L) | Glutamine: decreased (54 μmol/L) | Normal |
| Glutamate: elevated (263 μmol/L) | |||
| Aspartate: normal (8 μmol/L) |
Hyperammonemia after asparaginase therapy was first reported by Leonard and Kay in 1986 (Leonard and Kay 1986), followed by a number of other cases (Alvarez and Zimmerman 2000; Laterza et al. 2003; Jorck et al. 2011). Although the exact incidence of symptomatic hyperammonemia after asparaginase therapy is unknown, the small number of cases reported suggests a low frequency. Indeed, symptomatic hyperammonemia is not mentioned in recent overviews of toxicity of asparaginase treatment (Muller and Boos 1998; Earl 2009; Rytting 2010). In contrast, our observations suggest that symptomatic hyperammonemia is the rule rather than the exception after PEG-asparaginase administration.
This discrepancy may in part be a consequence of the tendency to analyze ammonia levels only in case of severe symptoms suggestive of hyperammonemia. In support, the only study that measured ammonia concentrations regardless of symptoms observed a similar high frequency of hyperammonemia: 7 out of 10 patients described by Steiner et al. reached ammonia concentrations >100 μmol/L 1 day after l-asparaginase administration (Steiner et al. 2007). However, these patients all remained without symptoms, contrasting with the overt symptoms in our patients. One could argue that most symptoms in our patients were nonspecific and could be attributable, at least in part, to other chemotherapeutics being administered. However, patients themselves reported these symptoms to be more severe than they experienced during other parts of leukemia treatment. Moreover, all symptoms disappeared after cessation of PEG-asparaginase administration. We speculate that the higher incidence of clinical symptoms of hyperammonemia in this study is secondary to the use of PEG-asparaginase. Due to the prolonged half-life of PEG-asparaginase, ammonia concentrations may not return to normal before the next dose is administered and ammonia toxicity may accumulate. As shown in Fig. 2, patients B, D, and F show a pattern consistent with this hypothesis. The linear correlation between the number of received doses of PEG-asparaginase and ammonia concentrations (Fig. 3) also supports this hypothesis. The half-life of PEG-asparaginase is likely to be longer than the above-mentioned 5.7 days, since asparagine was still depleted 3–4 weeks after the last dose of PEG-asparaginase in three out of three patients with available data. Interestingly, ammonia concentrations correlated with triglyceride values (r = 0.68, p < 0.0001), suggesting increased overall toxicity.
Fig. 2.
Ammonia concentrations in each patient related to the week of treatment. Arrows denote PEG-asparaginase administration. In patient A, 8 gifts were omitted; in patients C and D, the last gift was omitted
Fig. 3.
Correlative analysis of ammonia concentrations 1 week after PEG-asparaginase administration and week of treatment
Little is known about the exact mechanism through which asparaginase causes hyperammonemia. Normally, ammonia is removed rapidly from the circulation by being incorporated to glutamine in brain and muscle after which it is converted to urea via the urea cycle in the liver (Lockwood et al. 1979). Most likely, the altered equilibrium between glutamine and glutamate in the patients studied, favoring glutamate, contributes to the expansion of the ammonia pool. Given the plasma concentrations of asparagine and glutamine, the production of ammonia may well exceed the detoxification capacity in the liver. Indeed, analysis of plasma amino acid concentrations showed a complete depletion of asparagine and a marked reduction in glutamine concentration. The concentrations of aspartate (range: 21–50 μmol/L, reference values: 3–15 μmol/L, five out of six patients) and glutamate (range: 169–733 μmol/L, reference values: 17–69 μmol/L, six out of six patients) were strongly increased. Plasma concentrations of all urea cycle intermediates and excretion of orotic acid remained normal during therapy in all samples analyzed, arguing against malfunction of the urea cycle. Malnutrition during chemotherapy and proteolysis may induce catabolism, theoretically further increasing the ammonia load. However, we found no deficits of other amino acids in plasma, as would be expected in malnourished patients. Moreover, in case of weight loss during chemotherapy, tube feeding is instituted instantly in our hospital. Plasma acylcarnitine concentrations were also normal in all samples analyzed, excluding other known causes of secondary hyperammonemia.
In conclusion, our data clearly indicate that the clinical and biochemical consequences of asparaginase treatment are likely to be more pronounced when using PEG-asparaginase because of its prolonged half-life. Future studies are indicated to define the exact incidence of symptomatic hyperammonemia in patients receiving PEG-asparaginase, to define the relationship between hyperammonemia and clinical symptoms and to elucidate the link between hyperammonemia and hypertriglyceridemia. Finally, therapeutic drug monitoring by measuring ammonia concentrations, asparagine concentrations, and asparaginase levels might be of clinical use to define if lower doses of or longer intervals between PEG-asparaginase gifts are feasible. In the next DCOG ALL treatment protocol (ALL 11), therapeutic drug monitoring to optimize dosing of PEG-asparaginase will be studied.
Take Home Message
Pegylation of asparaginase, used in the treatment of childhood acute lymphoblastic leukemia, seems to result in a high incidence of symptomatic hyperammonemia due to its prolonged half-life.
Authorship and Disclosures
KH designed the research, collected and analyzed data, and wrote the chapter. BP performed and analyzed laboratory studies and revised the manuscript critically. TK revised the manuscript critically. PH designed the research, analyzed data, and wrote the chapter. MB designed the research, analyzed data, and wrote the chapter. The authors declare no conflict of interest.
Footnotes
Both last authors contributed equally
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