Pharmacogenetic aspects of safety of high-dose methotrexate therapy for acute lymphoblastic leukemia in children

Introduction

Drug-induced toxicity during high-dose methotrexate (HD-MTX) therapy (1000–5000 mg/m²) for childhood acute lymphoblastic leukemia (ALL) is a significant focus of contemporary research. While substantial progress has been made in achieving long-term complete remissions, this success is accompanied by a high incidence of treatment-related adverse effects [1, 2]. The global literature emphasizes the multifactorial nature of severe adverse drug reactions during HD-MTX-containing regimens [3]. Current scientific evidence indicates considerable interindividual variability in drug toxicity, underscoring the important role of pharmacogenetics (PG) in identifying polymorphic variants of candidate genes to optimize therapeutic strategies [4].

Transporter proteins are expressed in various tissues and significantly influence the pharmacokinetics (PK) of methotrexate—its absorption, distribution, and elimination—making it a cornerstone of ALL treatment protocols. Among numerous candidate genes, this study selected genes encoding transporter proteins as biomarkers: the SLCO1B1 gene, which codes for the organic anion-transporting polypeptide 1B1, and the ABCB1 gene (ATP-binding cassette sub-family B member 1), which encodes an ATP-dependent efflux pump also known as the multidrug resistance gene (MDR1, MIM *171050). According to major randomized studies, these genes have been associated with the development of severe neutropenia and have impacted treatment safety and efficacy [5, 6]. A 2024 systematic review by Rahmayanti SU, et al. identified the most frequently studied genes in relation to MTX pharmacokinetics from 2021 to 2024 as MTHFR, ABCB1, ABCC2, and SLCO1B1 [7].

Managing the frequency of adverse drug reactions associated with high-dose methotrexate remains a challenge. Dose reduction or discontinuation of cytotoxic drugs due to toxic complications can compromise the overall efficacy of therapy. This is because individual tolerance to MTX varies and depends on sex, ethnicity, and genetic polymorphisms in transporters, metabolizing enzymes, and targets involved in the MTX cellular pathway [6].

Objective

To assess the influence of polymorphisms in the ABCB1 (C3435T rs1045642, C1236T rs1128503, 2677G>T/A rs2032582, С>T rs4148738) and SLCO1B1 T521C rs4149056 genes on the safety profile of methotrexate therapy in children with ALL.

Materials and Methods

The study protocol was approved by the Ethics Committee of the N.N. Blokhin National Medical Research Center of Oncology of the Ministry of Health of Russia. A prospective analysis of a pediatric ALL patient database was conducted as part of a single-center cohort study in the Department of Pediatric Oncology and Hematology (Chemotherapy for Hemoblastoses) No. 1 at the L.A. Durnov Scientific Research Institute of Pediatric Oncology and Hematology of the N.N. Blokhin National Medical Research Center of Oncology.

Inclusion criteria: age from 1 month to 18 years; confirmed diagnosis of acute lymphoblastic leukemia (ICD-10 C91.0); provision of informed voluntary consent by a legal guardian for participation in the study.

Exclusion criteria: severe somatic pathology (hepatic, renal, cardiovascular, or nervous system disorders) precluding standard chemotherapy; psychotic state or severe mental illness in medical history (schizophrenia, epilepsy, bipolar disorder, etc.); concurrent use of drugs affecting the pharmacokinetics and/or pharmacodynamics of methotrexate; refusal to sign informed consent or to continue participation in the study, formally documented in writing by the legal guardian.

The study included 124 children with a confirmed ALL diagnosis, treated according to the ALL IC-BFM 2009/ALL REZ BFM 2002 protocols with high-dose methotrexate (>1 g/m²) in the aforementioned department. Laboratory methods utilizing the NCI toxicity criteria (CTCAE v5.0, 2018) were applied to determine adverse reaction (AR) grades.

The study material was peripheral blood, with no specified time for collection. The identification of single-nucleotide genetic polymorphisms in the studied genes was performed using real-time allele-specific polymerase chain reaction (PCR) on a CFX96 Touch Real-Time System with CFX Manager software version 3.0 (BioRad, USA). Genotyping of polymorphic markers was determined using commercial reagent kits for the respective polymorphisms (Sintol LLC, Russia) and the commercial "TaqMan®SNP Genotyping Assays" kit with TaqMan Universal Master Mix II, no UNG (Applied Biosystems, USA).

Statistical analysis was performed using SPSS Statistics 26.0 (USA). Sample size calculation considered the following parameters: power: 80%, two-sided test, α=0.05; effect – observed proportions (p1 and p0). For binary outcomes (presence of severe toxicity), the sample size was estimated based on the difference in proportions between genotype groups using normal approximation via Cohen's h. For one clinically significant endpoint with a moderate effect (e.g., mucositis ≥ grade 3 for rs1128503: CC~0.66 vs CT/TT~0.45), 190–200 patients were required for 80% power at α=0.05. For multiple comparisons (several SNPs × toxicity), 300 patients were required for 80% power at α=0.05. For non-normally distributed data, quantitative measures were presented as median (Me) with interquartile range (25–75% Q1–Q3). Intergroup differences for non-normal distributions were assessed using the Mann-Whitney U test. Association analysis was conducted using 2×2 contingency tables, Pearson's χ², Fisher's exact test, and univariate logistic regression without covariates. Multiple comparison corrections employed the Bonferroni, Holm-Bonferroni, and Benjamini-Hochberg False Discovery Rate (FDR) procedures. The baseline significance level was α=0.05.

Results

The genotype frequencies of the studied polymorphic variants ABCB1 rs1045642, rs1128503, and SLCO1B1 T521C rs4149056 in the study population were in Hardy-Weinberg equilibrium (HWE), unlike rs2032582 and rs4148738 (p < 0.05), indicating incomplete representativeness of the current sample (Table 1).

Table 1. Distribution of genotypes of the studied polymorphic gene variants in patients with calculation of compliance with Hardy-Weinberg equilibrium (HWE), (n=124)

After Bonferroni and Holm-Bonferroni correction, the significance of the associations was not maintained (p ≥ 0.14). When controlling for FDR using the Benjamini-Hochberg method, all four associations remained significant (q=0.047). It should be noted that the association results are sensitive to the correction method. The stringent Bonferroni correction may lead to the loss of true signals with a limited sample size, whereas the Benjamini-Hochberg FDR (BH-FDR) controls the proportion of false positives and is more suitable for pharmacogenetic studies involving multiple SNPs and phenotypes.

Table 2. Multiple comparisons correction

The analysis of MTX therapy efficacy has been presented in our previously published articles [8, 9]. The clinical and therapeutic characteristics of the patients (n=124) included in this safety assessment study are presented in Table 3.

Table 3. Clinical and therapeutic characteristics of patients included in the study

Based on medical records and peripheral blood samples from 124 patients, the male-to-female ratio was 1.2/1 (n=70 vs. n=54), with a median age of 7 years. The B-lineage ALL immunophenotype was predominant (67.7%). The intermediate-risk group was the most common in the study sample (48.4%). The study population was dominated by severe adverse reactions (ARs) > grade 3, including: hematological toxicity (87.9%), oropharyngeal mucositis (51.6%), hepatotoxicity (46%), infectious complications (34.6%), and neurotoxicity (7.2%); nephrotoxicity manifested as mild ARs of grades 1–2 (100%). A higher incidence of severe hematological toxicity, hepatotoxicity, and oropharyngeal mucositis was associated with a higher frequency of infectious complications (p < 0.001), as shown in Table 4.

Table 4. Comparative analysis of the incidence of hematological toxicity, mucositis, hepatotoxicity and infectious complications in MTX therapy in children with ALL

Association analysis using Pearson's χ² test and contingency tables established that the ABCB1 1236C>T polymorphic variant is a significant predictor of oropharyngeal mucositis development during MTX therapy, with greater AR severity shown for CC homozygotes. Patients with the SLCO1B1 T521C rs4149056 TT genotype had a 2.7-fold increased risk of severe infectious complications. Patients with the ABCB1 C3435T TT genotype had an increased risk of nephrotoxicity (p=0.035, OR: 8.3 [95% CI: 0.83–82.2]) and neurotoxicity (p=0.041, OR: 2.3 [95% CI: 1.02–5.12]). However, given the extremely wide CI and the small number of patients with the ABCB1 C3435T TT genotype, the association with nephrotoxicity requires further analysis. Other types of toxic ARs to MTX in relation to ABCB1 and SLCO1B1 gene polymorphisms showed no significant differences (Table 5).

Table 5. Comparative analysis of the frequency of ARs during MTX therapy in children with ALL, depending on the polymorphic variants of the studied genes

In addition to the association analysis of ABCB1 and SLCO1B1 gene polymorphisms with ARs, an analysis was conducted to find associations between the studied gene polymorphisms and delayed MTX elimination at 54 hours or more; no statistically significant differences were obtained (Table 6). The MTX excretion time did not differ significantly between patients with different genotypes (medians were 48 hours).

Table 6. Results of the associative analysis of polymorphisms of the ABCB1 and SLCO1B1 genes with the presence or absence of MTX excretion at 54 h

The other studied polymorphic variants of the transporter protein genes showed no significant influence on the development of ARs during MTX therapy, which is attributed to insufficient study power and incomplete conformity with Hardy-Weinberg equilibrium.

Study Limitations

This study has several limitations that should be considered when interpreting the results. First, the contribution of factors such as polymorphic variants in genes of the folate and methionine pathways, involved in phase II metabolism and transport of MTX, was not accounted for. Also, the MTX dosing regimen was not analyzed in the context of severe AR development, as all patients received high-dose MTX (>1 g/m² via 24-hour intravenous infusion). Second, increasing the sample size could enhance the statistical significance of the analysis of the association between MTX and toxicity manifestations. The low frequency of rare genotypes and the limited overall sample size reduce the statistical power of individual comparisons. Third, the absence of a control group in our study precludes the interpretation of deviations from expected distributions. The interpretation of the Benjamini-Hochberg False Discovery Rate (FDR) procedure depends on the definition of the hypothesis family; when the family is expanded (including additional comparisons), the q-values increase. This study did not perform multivariate regression analysis accounting for covariates, which might have influenced the identified associations. Such an analysis is planned for a larger sample in the future.

Discussion

The ABCB1 gene encodes P-glycoprotein (P-Gp), which influences the bioavailability of toxic substances and drug metabolites, including MTX. Previous studies have shown that ABCB1 polymorphisms can affect the immune response and apoptosis of cells playing an important role in the development of various cancers, including breast, gastric, and lung cancer, as well as leukemia [10]. The rs1045642 polymorphisms are the most studied in relation to MTX pharmacokinetics; they reduce P-Gp activity and decrease the number of transporter proteins, leading to intracellular accumulation of drugs like MTX [7, 11, 12]. The C to T nucleotide change at position 3435 results in the accumulation of high intracellular concentrations of MTX metabolites and lower plasma levels due to reduced drug efflux via membrane P-Gp. The CC genotype is more strongly associated with increased MTX exposure and a higher likelihood of delayed clearance than the TT genotype [13, 14].
Guo Q, et al. showed that the rs1045642 polymorphisms did not alter MTX pharmacokinetics, but patients with the homozygous TT genotype were more likely to experience elevated MTX-related toxicity (leucopenia, neutropenia, and oropharyngeal mucositis) than patients with the CC genotype [15]. The findings are consistent with the theoretical mechanism by which this specific gene affects MTX levels and toxicity, but its clinical significance and utility for personalized treatment decisions have not yet been established [7].
Studies by Ramsey LB, et al. in 2013 confirmed that SLCO1B1 polymorphisms play a major role in MTX elimination from the body [16]. Radtke S, et al. confirmed that the rs4149056 polymorphism had a significant association with MTX elimination; with each copy of the C allele in rs4149056, MTX elimination decreased by 12 ml/min/m²; thus, patients with the CC genotype had approximately 13% lower elimination than patients with TT genotypes [17].
The cited international literature defines a relevant direction for pharmacogenetic research in the Russian pediatric patient population. Current limitations include insufficient sample size and heterogeneity of treatment protocols, which may vary in administration route, drug dosage, concomitant medications, and treatment duration. However, a well-considered and rigorous study design in a larger and more diverse patient population, along with conformity of the studied polymorphisms to Hardy-Weinberg equilibrium, could overcome these obstacles and facilitate the translation of pharmacogenetic research findings into clinical practice. The identification of biomarkers predicting treatment response and the severity of expected toxicities in ALL therapy, and the ensuing opportunities for therapy optimization based on these data, represent a promising and modern direction in clinical onco-hematology.

Conclusion

Polymorphic variants of the ABCB1 and SLCO1B1 genes are significant predictive factors for the safety of MTX use. The results of this safety analysis of high-dose MTX therapy indicate the necessity for large-scale pharmacogenetic testing prior to attempts at implementation in real clinical practice. To improve the quality of pharmacogenetic research, it is essential to study not only transporter protein genes but also enzymes that play a significant role in the pharmacokinetics and pharmacodynamics of MTX. Haplotype and combinatorial analysis of linked single-nucleotide polymorphisms in various participants of the transport and metabolic pathways could enhance the accuracy of gene-AR relationship analyses. This would be of greater significance for optimizing therapy for children with ALL in the future and warrants further clinical research aimed at personalizing chemotherapy based on individual patient characteristics.