Genetic predisposition of Asparaginase-induced hypertriglyceridemia in children with acute lymphoblastic leukemia: Two case reports and review of literature

Abstract

BACKGROUND

Asparaginase (ASP) is an essential antitumor agent of acute lymphoblastic leukemia (ALL) in children. Hypertriglyceridemia (HTG) represents one of several ASP-associated toxicities, alongside with hypersensitivity reactions, pancreatitis, liver dysfunction, and thrombotic events. HTG may necessitate immediate therapy modification and can lead to life-threatening complications that contraindicate further anticancer treatment. However, the clinical and genetic mechanisms underlying this condition remain poorly understood.

CASE SUMMARY

We report two rare cases of severe HTG in pediatric patients with B-ALL following treatment with pegylated ASP (PEG-ASP). To investigate the genetic basis of this susceptibility, we performed genetic testing using a targeted gene panel for hyperlipidemia and identified several potential disease-related variants associated with inherited dyslipidemia. Although the functional and causal roles of these variants require further validation, we hypothesize that they contributed substantially to the development of HTG in these patients.

CONCLUSION

Genetic factors may predispose to HTG during PEG-ASP therapy; with further evidence, genetic testing might be considered before ASP treatment

Key Words: Children; Acute leukemia; Asparaginase; Toxicity; Hypertriglyceridemia; Genetics; Clinical testing; Case report

Core Tip: Pegylated (PEG) asparaginase (ASP) is a key chemotherapy agent used in pediatric acute lymphoblastic leukemia treatment protocols. This modified form of ASP exhibits prolonged activity and reduced immunogenicity, contributing to its therapeutic efficacy and dosing convenience. However, PEG-ASP therapy can lead to various toxicities, including hypertriglyceridemia (HTG). Several genes involved in triglyceride (TG) metabolism are known to influence susceptibility to this condition, with genetic variants potentially predisposing individuals to elevated TG levels. Based on the two cases presented here, we hypothesize that genetic factors may contribute to HTG in certain pediatric patients receiving PEG-ASP therapy.

INTRODUCTION

Acute lymphoblastic leukemia (ALL) is the most common oncohematological disease among pediatric patients, with an incidence of 20-35 cases per million in children aged 0-19 years[1,2]. B-cell and T-cell ALL, the two main forms of ALL, account for 75%-80% of cases of acute leukemia in children and adolescents[3,4]. ALL therapy protocols vary depending on the type and stage of the disease, but in most cases they are effective, and outcome are generally favorable, with the 5-year survival rate reaching 92% in children and 76% in adolescents aged 14-20 years[1-4].

Asparaginase (ASP) is a major component of chemotherapy protocols and has been used in combination with vincristine and glucocorticosteroids for the treatment of ALL for over 40 years[5,6]. ASP is an enzyme that catalyzes the cleavage of the essential amino acid asparagine, which is necessary for cell growth and development, into L-aspartic acid and ammonia. Unlike healthy cells that endogenously synthesize asparagine from glutamine using the enzyme asparagine synthetase, leukemic blast cells have insufficient asparagine synthetase activity and are therefore unable to synthesize the necessary amount of this amino acid to maintain their metabolism. Thus, administration of ASP depletes asparagine in tumor cells, leading to their starvation and death[7-10]. To date, four forms of the drug have been approved for clinical use: Native ASP derived from Escherichia coli, its pegylated (PEG) form, native ASP derived from Erwinia chrysanthemi, and recombinant ASP derived from Erwinia. Clinical studies have shown that native ASP has a short half-life and high immunogenicity. Conjugation of the enzyme with PEG molecules reduces these disadvantages while maintaining biological activity. As a result, the PEG-ASP is used as a first-line treatment for ALL, both at the initial stage and in cases of relapses[1,4,5,11]. Intensive pediatric chemotherapy regimens, including PEG-ASP, significantly improve overall survival not only in childhood but also in adolescence. However, in some cases, it is necessary to limit the dose of ASP or completely discontinue chemotherapy due to adverse toxic effects such as hypersensitivity, hepatotoxicity, thrombosis, hyperglycemia, osteonecrosis, pancreatitis, and hypertriglyceridemia (HTG)[11-13]. The latter complication is pathogenetically associated with the development of life-threatening conditions in patients, such as pancreatitis and vascular thrombosis, as well as cardiovascular diseases after completion of treatment.

HTG is a disorder of lipid metabolism in which the concentration of triglyceride (TG)-rich particles in the blood exceeds 1.7 mmol/L. The severe form of HTG (an increase in blood TG levels to 10 mmol/L or more) is the most clinically significant, as it can lead to life-threatening conditions such as acute pancreatitis. HTG is known to be a complex genetically determined pathology. Monogenic chylomicronemia syndrome is very rare, whereas multifactorial chylomicronemic syndrome is the most common. Increased blood TG levels may be associated with genetic variants in one of the five genes involved in TG metabolism [lipoprotein lipase (LPL), lipase maturation factor 1 (LMF1), apolipoprotein C-II (APOC2), apolipoprotein A-V (APOA5), and glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1] or with the presence of a high proportion of common alleles of single nucleotide polymorphisms (SNPs) with small effect, which collectively create a predisposition to the development of HTG[14-16]. Here, we report two clinical cases in which ALL chemotherapy with PEG-ASP in adolescents was complicated by the development of severe HTG. To investigate the inherited basis of this toxic effect, a genetic study was performed and revealed the presence of heterozygous variants in HTG-associated genes.

CASE PRESENTATION

Chief complaints

Case 1: A 16-year-old girl was hospitalized in the Department of Chemotherapy for Oncohematological Diseases at the Almazov National Medical Research Center. Upon admission, the main complaints were generalized bone pain and weakness.

Case 2: A 17-year-old boy was referred to the Almazov National Medical Research Center due to severe HTG while receiving chemotherapy, including PEG-ASP.

History of present illness

Case 1: The patient was diagnosed with a grade II relapse of ALL with CD13+ co-expression with no signs of extramedullary involvement. In accordance with the clinical guidelines for the children ALL approved by the Russian Ministry of Health, treatment of the isolated bone marrow relapse, which developed 3.5 years after completion of the primary ALL therapy, consisted of standard antitumor therapy regimens, including PEG-ASP. A second clinical and hematological minimal residual disease (MRD)-negative remission was achieved. Complications such as toxic hemolytic anemia, three-lineage aplasia of hematopoiesis, grade 2 anorexia, hepatotoxicity, perianal dermatitis and grade 2 hypoalbuminemia were promptly managed. The first part of block II- idarubicin[17] was performed within 22 days as a remission consolidation. On this stage a pronounced lipemia was visually detected in the test tube when venous blood was collected for laboratory examination.

Case 2: After achieving complete clinical and hematological, MRD-negative remission of BII-ALL, the patient was subjected to consolidation therapy. At the first stage, taking into account the severity of complications, the use of ASP drugs was excluded, and consolidation stages II-V were carried out without modifications according to the clinical guidelines for the children ALL. During consolidation stage IV, the patient developed an increase in blood TG concentration to 27.04 mmol/L, however, since the patient’s condition remained stable, chemotherapy was continued according to the treatment protocol. During routine biochemical testing at consolidation stage V, the TG level increased to 9.48 mmol/L without clinical manifestations; therefore, chemotherapy was continued without modification. Pronounced lipemia was observed during blood sampling three days after the last administration of PEG-ASP, and, the TG level reached 33.36 mmol/L. To identify the causes of the condition and adjust therapy, treatment was suspended, and the patient was referred to the Department of Chemotherapy for Oncohematological Diseases of the Almazov National Medical Research Center.

History of past illness

Case 1: At the age of 11, the patient was diagnosed with B-ALL CD13+ (immunophenotype SSClow CD45dim CD34+ CD38+ CD19+ CD79a+ CD10+ CD22+ CD20+ CD58+ CD13+; B-2 variant according to European Group for Immunophenotyping of Leukemias) and underwent treatment for the first active phase of ALL in accordance with the ALL-Moscow-Berlin 2015 protocol (therapeutic group A), which resulted in complete clinical and hematological MRD-negative remission. Tolerance of ASP drugs was satisfactory during the induction and consolidation periods. Severe osteoporosis with aseptic necrosis of the distal tibia and moderate infectious complications were recorded as complications of chemotherapy.

Case 2: The patient received chemotherapy in accordance with the ALL-Moscow-Berlin 2015 protocol (therapeutic group C) following the diagnosis of BII-ALL. At the induction stage, he developed complications in the form of subarachnoid hemorrhage in the left frontal and parietal lobes and sinus thrombosis, which were presumed to be associated with the administration of PEG-ASP. The clinical manifestations of these complications, including the right-sided hemiparesis and convulsive syndrome, were completely resolved. The patient achieved complete clinical and hematological MRD-negative remission as a result of induction chemotherapy.

Personal and family history

Case 1: According to the family history, there were no clinically pronounced or asymptomatic forms of dyslipidemia among the patient’s relatives.

Case 2: An additional biochemical blood test revealed grade 2 HTG in the patient’s mother.

Physical examination

Case 1 and case 2: Standard physical examination was performed with no remarkable findings.

Laboratory examinations

Case 1: Before the start of PEG-ASP therapy, the plasma TG concentration was 1.06 mmol/L, and total cholesterol was 4.4 mmol/L. No routine lipid level measurements were performed at the start of the therapy until the development of HTG. A decrease in the concentration of sodium and albumin in the patient’s blood (125 mmol/L and 26 g/L, respectively) and an increase in alanine aminotransferase activity to 53.4 U/L were observed on day 14 of PEG-ASP therapy. Severe dyslipidemia developed, with an increase in total cholesterol to 17.88 mmol/L, TG to 60.05 mmol/L, and very low-density lipoproteins to 27.55 mmol/L in the fasting lipid profile (Tables 1 and 2). To investigate the etiology of HTG and exclude the effect of hereditary factors we performed a targeted high throughput next-generation sequencing on Illumina MiSeq device (chemistry PE 2 × 75 bp) using SureSelect Target Enrichment kit (Agilent Technologies; Waldbronn, Germany). We applied a custom panel of genes associated with hereditary lipid metabolism disorders (LDLR,GPIHBP1, APOA5, LPL, APOB, APOE, APOA4, ABCG8, ABCG5, PCSK9, CETP, LDLRAP1, LMF1, LRP6). The targeted probe regions were extended by 100 bp in both directions to cover non-coding gene regions. After initial library analysis, the data were mapped to the GRCh38 version of the human genome assembly and processed according to GATK recommendations. Variants were assessed for their functional consequences using various prediction programs, such as Combined Annotation Dependent Depletion (CADD) and Sorting Intolerant from Tolerant. The identified genetic variants were confirmed by bidirectional Sanger sequencing. Genetic analysis revealed a heterozygous variant in the LPL gene (chr8:g.19948197G>A; NM_000237.3:c.106G>A; NP_000228.1:p. Asp36Asn, rs1801177), classified as likely benign according to the recommendations of the American College of Medical Genetics and Genomics[18]. The frequency of this variant in the population is 1.6% according to GnomAd database (Table 3).

Table 1 Cholesterol, triglycerides and high-density lipoproteins levels at different time points after asparaginase treatment of patient 1.

Patient 1	Reference interval	Before PEG-ASP therapy	PEG-ASP therapy
			Day 7	Day 14	Day 21	Day 28
CHOL (mmol/L)	3.10-5.20	3.16	15.90	17.80	2.29	5.50
TG (mmol/L)	0.00-1.69	1.06	NA	60.00	42.10	6.08
HDL (mmol/L)	1.17-1.55	1.67	0.31	1.44	1.31	1.89

PEG-ASP: Pegylated asparaginase; CHOL: Cholesterol; TG: Triglycerides; HDL: High-density lipoproteins; NA: Not available.

Table 2 Cholesterol, triglycerides and high-density lipoproteins levels at different time points after asparaginase treatment of patient 2.

Patient 2	Reference interval	Before PEG-ASP therapy	PEG-ASP therapy
			Day 12	Day 14	Day 21	Day 31
CHOL (mmol/L)	3.10-5.20	NA	13.39	3.97	4.87	4.99
TG (mmol/L)	0.00-1.69	NA	30.91	2.50	4.38	2.25
HDL (mmol/L)	1.17-1.55	NA	1.17	0.49	0.79	1.32

PEG-ASP: Pegylated asparaginase; CHOL: Cholesterol; TG: Triglycerides; HDL: High-density lipoproteins; NA: Not available.

Table 3 Genetic variants identified in patients with severe hypertriglyceridemia.

Patients	Genes	Gene functions	Ref.	Variants	ACMG classification	CADD	GnomAD
Patient 1	LPL	Encodes the main enzyme of triglyceride hydrolysis, lipoprotein lipase	[28,31]	Chr8:g.19948197G>A; NM_000237.3:c.106G>A; NP_000228.1:p.Asp36Asn, rs1801177	Likely benign	19.6	1.6%
Patient 2	LPL	Encodes the main enzyme of triglyceride hydrolysis, lipoprotein lipase	[28,31]	Chr8:g.19956018A>G; NM_000237.3:c.953A>G; NP_000228.1:p.Asn318Ser, rs268	Conflicting classifications of pathogenicity	21.3	1.97%
	APOA5	Encodes apolipoprotein A-V, which activates lipoprotein lipase and participates in the absorption of residual particles and the secretion of very-low-density lipoprotein by hepatocytes	[31]	Chr11:g.116790666T>C; NM_001371904.1:c.563A>G; NP_001358833.1:p.Lys188Arg rs1303929283	Variant of uncertain significance	21.5	0.0009%

ACMG: American College of Medical Genetics and Genomics; CADD: Combined Annotation Dependent Depletion; GnomAD: Genome Aggregation Database.

Case 2: The patient’s lipid profile was not tested before the start of PEG-ASP therapy. Upon hospitalization on the 12th day after administration of PEG-ASP, lipemia persisted; the TG level was 30.9 mmol/L without clinically significant manifestations (Table 1). To exclude an association with genetically determined forms of HTG, targeted sequencing was performed using a protocol as described for patient 1. The genetic analysis revealed a heterozygous missense variant in the APOA5 gene (chr11:g.116790666T>C; NM_001371904.1:c.563A>G; NP_001358833.1:p.Lys188Arg, rs1303929283) with a population frequency of 0.0009% and a CADD score of 21.5. This genetic variant is considered a variant of uncertain significance according to American College of Medical Genetics and Genomics recommendations[18], taking into account data from the ClinVar database. In addition, this patient had a heterozygous missense variant in LPL gene (chr8:G.19956018A>G; NM_000237.3:c.953A>G; NP_000228.1:p.Asn318Ser, rs268) with conflicting interpretation of pathogenicity. The frequency of this variant in the population was 1.97%, and the CADD score was 21.3 (Table 3).

Imaging examinations

Case 1 and case 2: No imaging was recorded.

FINAL DIAGNOSIS

Case 1

Second clinical and hematological MRD-negative remission of bone marrow relapse of B-II ALL with CD13+ co-expression and total bone marrow infiltration, complicated by severe HTG.

Case 2

Severe HTG.

TREATMENT

Case 1

To correct dyslipidemia, the patient was prescribed a low-fat diet, two cascade plasma filtration procedures with an interval of 48 hours to reduce elevated blood TG levels, infusion therapy (4000 mL/m²/day), and activated partial thromboplastin-controlled heparin therapy to prevent thrombosis.

Case 2

The patient was prescribed a low-fat diet to correct HTG and received infusion therapy in hyperhydration mode with potassium and calcium supplementation. Two plasma exchange procedures were performed with an interval of 48 hours to rapidly reduce TG levels. The TG levels in the patient’s blood decreased to 2.50 mmol/L after the first plasma exchange procedure, however, the following day, the TG levels increased up to 11.6 mmol/L. After the second procedure, the TG levels decreased to 2.02 mmol/L and did not rise significantly thereafter.

OUTCOME AND FOLLOW-UP

Case 1

Given the risk of recurrent HTG associated with the use of ASP, the treatment regimen was modified by adding blinatumomab immunotherapy.

Case 2

Lipid-lowering therapy led to normalization of TG levels, which allowed continuation of the planned chemotherapy in the form of reinduction therapy. Chemotherapy was tolerated satisfactorily, and no recurrent increase in TG levels was observed.

DISCUSSION

Severe HTG is a relatively rare toxic effect of ALL chemotherapy and, according to literature data, occurs in 11%-50% of patients receiving ASP[1,19]. In most cases, it is asymptomatic and occurs without clinical manifestations. Several published studies have noted that elevated blood TG levels are more frequently observed in children and adolescents older than 14 years[20-22]. This pattern may reflect differences in the pharmacokinetic properties of ASP in children of different ages[21]. Notably, in the clinical cases described here, both patients were adolescents (16 and 17 years old). However, to confirm the relationship between age and the risk of developing HTG, it is necessary to monitor a larger number of patients or perform a meta-analysis of previously published cases.

To date, it has not been precisely determined how many administrations of ASP are required to achieve a therapeutic effect before toxic effects appear. In both clinical cases, elevated TG levels were detected during the consolidation stage after several injections of ASP. Similar cases were described by Wang et al[23] and Lynggaard et al[21], who reported that a higher cumulative number of PEG-ASP doses may increase the risk of pancreatitis and other ASP-associated toxic effects.

Several cases of HTG have also been reported, with TG elevation detected at the time of ALL diagnosis before the initiation of chemotherapy. For example, Sonowal and Gupta[24] describe a 1.5-year-old boy diagnosed with B-ALL who simultaneously presented with high TG levels in the blood. In another study reported by Khera et al[25], lipemia was observed in a girl during hospitalization for B-ALL. In both cases, family history was unremarkable, the genetic cause of the condition was not established, and TG levels returned to normal a few days after the start of chemotherapy. The authors speculated that the transient nature of HTG in these patients could be explained by a high tumor burden[24,25].

The combined use of ASP and glucocorticosteroids in ALL therapy disrupts lipid metabolism; however, the exact pathogenetic mechanism of dyslipidemia remains unclear. It is assumed that glucocorticosteroids increase TG synthesis, mobilize fatty acids, and enhance the activity of LPL, which is responsible for the TG hydrolysis. At the same time, PEG-ASP may increase TG levels by inhibiting LPL activity, leading to an accumulation of exogenous chylomicrons and increased endogenous production of very low-density lipoproteins[26-28]. The reason for the selective development of HTG in a small proportion of patients remains unclear. We suggest that additional endogenous or exogenous factors, such as genetic predisposition, may contribute to the development of this condition. Five main genes are known to be associated with HTG. The LPL gene encodes the enzyme LPL, which regulates the TG levels in blood plasma. The APOC2, APOA5, LMF1, and GPIHBP1 genes encode proteins involved in the proper folding, activation, and functioning of this enzyme. Most described variants in these genes lead to loss of function of the encoded protein, and pathogenic or likely pathogenic variants in the LPL gene represent the most common genetic cause of HTG. In several studies, combined effect of common SNPs with small individual effects on TG concentration has been associated with a predisposition to HTG[14,15,27]. Thus, variants in HTG-associated genes may enhance the negative effects of PEG-ASP on LPL, further reducing its activity and thereby contributing to the development of HTG.

In the first clinical case, a rare heterozygous, likely benign variant in the LPL gene was identified. According to the study by Surendran et al[29], this amino acid substitution (NP_000228.1:p.Asp36Asn, rs1801177) is more common in patients with severe HTG compared with the control group and leads to loss of LPL function. Numerous genetic studies have identified more than 100 loss-of-function and functionally heterogeneous variants in the LPL gene (including this variant) that lead to LPL deficiency in patients with familial chylomicronemia syndrome[30]. In a large population study, carriers of both NP_000228.1:p.Asp36Asn, rs1801177 and c.-188-93T>G, rs1800590 variants in the LPL gene had higher TG levels than patients with only rs1800590[30]. However, the currently available information about this variant is still insufficient to draw definitive conclusions about its potential impact on the development of HTG.

In the second clinical case, the boy was found to carry a variant in the APOA5 gene (NP_001358833.1:p.Lys188Arg, rs1303929283). This gene encodes APOA5, which activates LPL and promotes lipolysis of TG, thereby enhancing their attachment to the cell surface[30]. A similar case was previously reported by Iannuzzi et al[22], describing a 28-year-old patient with ALL who developed HTG during the consolidation stage of PEG-ASP therapy. In that study, no pathogenic or likely pathogenic variants in the major HTG-associated genes were detected, except for a variant of uncertain significance in the APOC3 gene and four SNPs, three of which were located in the APOA5 gene. The APOA5 locus is separated from other apolipoprotein genes by a region of increased recombination, which makes it an independent risk gene influencing blood TG concentrations[22]. In addition to the variant in the APOA5 gene, our patient also carried a rare heterozygous variant with conflicting interpretation of pathogenicity in the LPL gene (NP_000228.1:p.Asn318Ser, rs268). In the study by Surendran et al[29], this variant was found more frequently in patients with severe HTG than in controls and was associated with decreased functional activity of LPL. This variant leads to LPL deficiency in patients with familial chylomicronemia syndrome. In almost all published case-control studies, the frequency of the NP_000228.1:p.Asn318Ser (rs268) variant was higher in individuals with HTG than in normolipidemic controls[30]. Therefore, we suggest that two variants identified in the APOA5 and LPL genes may, at least partly, contribute to the development of severe HTG in this patient.

Currently only four case reports are available in the literature regarding the genetic predisposition to PEG-ASP-associated HTG[22,23,31,32]. In addition to the case described by Iannuzzi et al[22], three other clinical cases demonstrated the importance of genetic components such as LPL pathogenic variants in a heterozygous state, polygenic risk score, and APOE3/4 genotype for the development of this complication. The scientific novelty of our work is supported by the additional attempt to establish genotype-phenotypic associations between severe HTG induced by PEG-ASP and the genetic factors associated with this condition. Since HTG still represents a rare complication of PEG-ASP treatment and such cases only occasionally undergo genotyping, every single described case adds scientific and clinical information on the potential genotype-phenotype associations and arouses interest in functional molecular studies of the rare variants described.

Taking into account all previously published cases along with two reported in this study, the potential clinical benefit of predictive genetic testing prior to PEG-ASP treatment initiation cannot be denied. However, the list of genes relevant for such screening, the identification of risk groups, baseline lipid levels, functional interpretation of the variants, timing and indications for genetic testing, as well as its potential impact on the therapy modification are yet to be identified. For example, if a patient is found to have genetic risk factors for HTG, it might be recommended to adjust the monitoring of TG levels during chemotherapy in a timely manner. Currently, additional genetic and biochemical studies are required to clarify the pathogenetic mechanisms underlying the development of HTG during ALL therapy in children and young adults carrying variants in HTG-associated genes.

An important limitation of our study is the small number of observations, as well as the absence of comparison of the identified variants in terms of their frequency in the control population. In addition, the current work does not include in-depth functional studies that would confirm the involvement of the identified genetic variants in the development of severe HTG in patients with ALL. Another important consideration is the need for polygenic risk assessment in combination with genotyping, which was not performed in the current study. Expanding the number of the described cases, conducting detailed molecular studies, and calculating a polygenic risk score are highly important in order to translate these observations into routine clinical practice.

CONCLUSION

HTG is one of the toxic effects of PEG-ASP therapy and can provoke life-threatening conditions, such as thrombosis and acute pancreatitis. Regular monitoring of blood biochemical parameters, modification of chemotherapy protocols, and lipid-lowering interventions are necessary in patients with HTG. To assess the potential risk of HTG during chemotherapy with ASP and to uncover its background in cases of already documented HTG, genetic testing for HTG-associated genes may be considered. However, the spectrum of genes to be included and the causative role of genetic variants still need to be determined. While we conclude that genetic determinants may act as predisposing factors for the development of HTG, they may not be solely responsible for this condition following PEG-ASP treatment. This approach may help to identify patients at risk and allow timely modification of chemotherapy protocols.

ACKNOWLEDGEMENTS

This work has been performed using the resources of Research Equipment Sharing Center “Preclinical Translational Research Centre” at Almazov National Medical Research Center.