Introduction

Acute lymphoblastic leukemia (ALL) is a common malignancy in children.1 Methotrexate (MTX), which can induce a low level of folate, affects the intracellular folate pool and influences the activity of the enzyme methylenetetrahydrofolate reductase (MTHFR), is an important drug for treatment of children with ALL.2–5 Although high-dose MTX (HD-MTX) with enhanced antineoplastic activity has achieved remarkable clinical success in treatment of children with ALL, unpredictable toxicities continue to challenge its clinical use.6 Our previous study identified clinical predictors from among patient characteristics (eg, age, sex, body surface area, subtype of leukemia, complete blood counts and liver function tests) for the occurrence of severe toxicities during HD-MTX therapy in children with ALL.3 Some studies have also shown that chemotherapeutic agent-related toxicities vary with polymorphism frequency.7,8 Overall, the identification of genetic predictors for MTX-induced toxicities would be valuable for selecting patients who may benefit from personalized treatment strategies.

MTHFR gene polymorphism is critical in intracellular folate homeostasis and metabolism.9 There are two well-characterized MTHFR polymorphisms: (1) a transition from a C to a T at nucleotide 677 (C677T, rs1801133), which results in an increased level of homocysteine, decreasing enzymatic activity and altering folate distribution,10 and (2) a transition from an A to C nucleobase at position 1298 (A1298C, rs1801131), which results in reduced MTHFR activity.11 Several published clinical reports conclude that risks of HD-MTX toxicities in treatment of children with ALL are associated with MTHFR C677T or MTHFR A1298C.1,5,8,12–20 However, other studies have reported the opposite conclusions, namely, that there is no significant relationship between MTHFR C677T or MTHFR A1298C and HD-MTX toxicities.4,21–24 Therefore, the potential predictive role of MTHFR C677T and MTHFR A1298C genetic variants in HD-MTX toxicities remains unclear. Moreover, HD-MTX toxicities vary among different races and could significantly affect its clinical use.18

The membrane transport protein P-glycoprotein (P-gp), also known as multidrug resistance protein 1 (MDR1), is encoded by the ATP-binding cassette subfamily B1 (ABCB1) gene and facilitates entry and exit of many molecules into the cell, particularly influencing the metabolism and clearance of drugs,25,26 such as MTX.1 One of the well-characterized polymorphisms that occurs in the ABCB1 gene is the transition from a C to a T at nucleotide 3435 in exon 26 (C3435T).26 However, most studies to date have investigated influences of ABCB1 gene SNPs on risk of ALL development,27,28 whereas little is known about whether they affect HD-MTX toxicities in the treatment of children with ALL.1

In addition, whether serum MTX levels are associated with MTHFR C677T and MTHFR A1298C as well as ABCB1 C3435T in HD-MTX treatment of children with ALL has yet to be clearly determined.

To answer these clinical questions, we performed a retrospective study to analyze the predictive role of the MTHFR C677T, MTHFR A1298C and ABCB1 C3435T genetic variants in HD-MTX toxicities (hematological toxicities, transient liver dysfunctions, vomiting and oral mucositis) during HD-MTX therapy in children with ALL. In addition, this study aimed to analyze the relationship between MTHFR C677T, MTHFR A1298C and ABCB1 C3435T and MTX elimination delay.

Patients and MethodsPatients

We conducted a noninterventional retrospective study between May 2015 and May 2020 in children with ALL who were treated with HD-MTX and had adequate medical records available at the Affiliated Hospital of Qingdao University, China. Children with ALL (aged ≤ 14 years) who received HD-MTX treatment and for whom MTHFR C677T, MTHFR A1298C and ABCB1 C3435T genotype analyses were performed and 44-h plasma MTX levels were determined were enrolled in this study. This study was conducted on the basis of the ethical principles of the Declaration of Helsinki and approved by the Institute Medical Ethics Committee of the Affiliated Hospital of Qingdao University.

HD-MTX Administration and Calcium Folinate (CF) Rescue Regimen

All included patients were treated according to the 2015 Chinese children’s leukemia cooperation group-ALL protocol (CCCG-ALL-2015). Consolidation chemotherapy consisted of four cycles of HD-MTX; low-risk children received 3 g/m2 of MTX, and intermediate- and high-risk children received 5 g/m2 of MTX. The 1/10 of the full dose was intravenously transfused within 0.5 h, and the remainder was infused during the following 23.5 h. At 42 h after the beginning of MTX administration, CF rescue was started every 6 h for 3 to 5 doses. The rescue regimen was given on the basis of 44-h plasma MTX levels, as we previously reported.3 MTX elimination delay was defined as a 44-h plasma MTX level > 1 μmol/L. As the next MTX dose was adjusted based on the previous 44-h plasma MTX level, we analyzed only the data for the first dose of MTX.

Determination of Plasma MTX Level and Genotyping

Serum levels of MTX for children with ALL monitored by the enzyme multiplied immunoassay technique (EMIT) at our hospital from May 2015 to May 2020 were acquired via the Viva-E® system (Siemens Healthcare, Forchheim, Germany). The MTHFR (C677T and A1298C) and ABCB1 (C3435T) genotypes for children with ALL monitored at our hospital were acquired via a fluorescence detector (TL998A, Xi’an Tianlong Technology Co., Ltd., Xi’an, China) using chromosome fluorescence in situ hybridization. Serum levels of MTX and MTHFR (C677T and A1298C) and ABCB1 (C3435T) genotypes were retrospectively collected.

Data Collection and Toxicities

The clinical data collected by retrospective chart review before the first MTX infusion included the subtype of leukemia, white blood cell count (WBC), neutrophil count (NEUT), hemoglobin (Hb) level, platelet (PLT) count, aspartate transferase (AST) level, and alanine transferase (ALT) level. The 44-h plasma MTX level, WBC, NEUT, Hb, and PLT counts; and AST and ALT levels were also collected after MTX infusion. Hematological toxicity, transient liver dysfunction, vomiting and oral mucositis were assessed on the basis of the National Cancer Institute Common Toxicity Criteria version 5.0 (NCI-CTCAE version 5.0). Hematological toxicity included reductions in WBC, NEUT, Hb, and PLT counts. Hepatic toxicity included increases in AST and ALT.

Statistical Analysis

Allele frequencies of polymorphisms were tested for Hardy‒Weinberg equilibrium using the chi-square test. All data were analyzed with SPSS statistical software (version 19.0; SPSS Inc., Chicago, Illinois, USA). Toxicities are represented by a toxicity event that did occur =1 or a toxicity event that did not occur = 0 during the HD-MTX course. Statistical associations between toxicities and MTHFR C677T, MTHFR A1298C and ABCB1 C3435T were analyzed using logistic regression and are presented as odds ratios (ORs) and 95% confidence intervals (CIs). The correlation between polymorphisms and MTX elimination delay was assessed using the chi-square test. All tests were two-sided, and P < 0.05 was considered statistically significant.

ResultsPatient Characteristics

A total of 106 children with newly diagnosed ALL who received the first HD-MTX treatment and underwent MTHFR C677T, MTHFR A1298C and ABCB1 C3435T genotype analyses and plasma MTX level determination at 44 h after the start of treatment were enrolled in this study. The genotype frequencies were in Hardy-Weinberg equilibrium (C677T variant: x2=1.880, P=0.391; A1298C variant: x2=0.046, P=0.977; C3435T variant: x2=0.293, P=0.864). The remaining patient characteristics and frequencies of genetic polymorphisms are summarized in Table 1. All toxicities categorized by differential grade are listed in Table 2. Toxicities such as depressed PLT, vomiting and oral mucositis were rare, whereas toxicities such as leukopenia, anemia, neutropenia, elevated ALT and elevated AST were common. Anemia was the most common mild drug-induced toxicity (> grade 1) (69.8%), followed by leukopenia (50.0%) and neutropenia (42.5%) (Table 2).

Table 1 Patient Main Characteristics and Frequencies of Genetic Polymorphisms

Table 2 Number of Courses per Grade of Adverse Events (n=106)

Correlation Between Genotype and MTX Elimination Delay

The correlation between genotype and plasma MTX level was statistically analyzed to determine whether different genotypes are associated with 44-h plasma MTX levels. Eighty-six patients had a 44-h plasma MTX level ≤ 1 μmol/L, and 20 patients had a 44-h plasma MTX level > 1 μmol/L (MTX elimination delay). There was no significant difference in the number of patients with MTX elimination delay between the low-risk group and the intermediate- and high-risk groups; thus, they were analyzed together. The correlation between genotype and MTX elimination delay is shown in Table 3. MTX elimination delay did not significantly differ between MTHFR C677T and MTHFR A1298C or among ABCB1 C3435T genetic variants (P > 0.05).

Table 3 The Correlation Between Genotyping and MTX Elimination Delay

Correlation Between Genotype and High-Dose MTX-Induced Toxicity

As there was a difference in the number of patients developing anemia, depressed PLT and elevated ALT between the low-risk group and the intermediate- and high-risk group, they were analyzed separately in line with the degree of risk. The C677T, A1298C and C3435T genetic variants displayed no correlations with toxicities (anemia, depressed PLT and elevated ALT) in the low-risk group or in the intermediate- and high-risk group (Tables S1 and S2).

No significant difference in the number of patients developing other kinds of toxicities (leukopenia, neutropenia, elevated AST, vomiting and oral mucositis) in the groups was found; thus, the groups were analyzed together. The correlation between genotype and toxicities (leukopenia, neutropenia, elevated AST, vomiting and oral mucositis) is shown in Table 4, with leukopenia being 6.444-fold increased in patients with the ABCB1 C3435T homozygous genotype (TT) compared to patients with the wild-type genotype (CC) (P=0.028). Furthermore, neutropenia was 4.978-fold increased in patients with the homozygous TT genotype compared to wild-type (CC) patients (P=0.034), and oral mucositis was 9.643-fold increased in the former (P=0.023). Conversely, no significant association between the toxicities investigated and the MTHFR C677T or MTHFR A1298C polymorphisms was found (P > 0.05).

Table 4 Analysis of Genotyping and Methotrexate-Induced Toxicity (≥ Grade 1)

Correlation Between the Number of Wild-Type Homozygous Genes and MTX-Induced Toxicity

To analyze the relationship between the combination of three genotypes and MTX-induced toxicity, analysis of the number of wild-type homozygous genes and MTX-induced toxicity is shown in Table 5. There were 5 patients with 0 homozygosity for wild-type genotypes, 57 patients with 1 homozygosity for wild-type genotypes, 40 patients with 2 homozygosities for wild-type genotypes and 4 patients with 3 homozygosities for wild-type genotypes. Overall, there was no significant difference between the number of wild-type genes and MTX-induced toxicity (≥ grade 1) (P > 0.05).

Table 5 Analysis Among the Number of Wild Homozygous Genes and Methotrexate-Induced Toxicity (≥ Grade 1)

Discussion

MTX is an important drug used in treatment of children with ALL.3 However, drug-related toxicities continue to limit its clinical use.29 Response to MTX largely varies among patients receiving HD-MTX treatment.2,30 Research on genetic predictors for MTX-induced toxicities would be helpful for selecting patients who may benefit from personalized treatment strategies. Several SNPs involved in MTX transport and metabolism have been explored as predictors of the efficacy and toxicity of MTX in treatment of children with ALL.31,32 However, the conclusions drawn are inconsistent.6 MTHFR gene polymorphism is critical for intracellular folate homeostasis and metabolism.9 P-gp, encoded by the ABCB1 gene, may influence metabolism and clearance of MTX.1 Our retrospective study enrolled 106 children with newly diagnosed ALL who were treated following the CCCG-ALL-2015 protocol at our hospital. The MTX dose was adjusted based on the previous 44-h plasma MTX level; therefore, we analyzed only the data for the first dose of MTX. In this study, we sought to investigate whether the MTHFR C677T, MTHFR A1298C and ABCB1 C3435T polymorphisms correlate with MTX elimination delay or MTX-related toxicities in a Chinese cohort.

The allelic frequencies of homozygous MTHFR C677T and MTHFR A1298C were 42.4 and 0.9%, respectively, whereas that of homozygous ABCB1 C3435T was 9.4%. The genotype frequencies for each polymorphism were in Hardy-Weinberg equilibrium. MTHFR A1298C was predominantly present as homozygous wild-type, but MTHFR C677T and ABCB1 C3435T showed higher allelic frequencies of homozygosity and heterozygosity.

Our study showed that ABCB1 C3435T was not associated with MTX elimination delay in HD-MTX treatment of children with ALL, which was consistent with some studies1,33,34 that concluded that ABCB1 C3435T was not associated with MTX elimination delay, MTX plasma level or the MTX AUC in HD-MTX treatment of children with ALL. MTHFR C677T and MTHFR A1298C were also not associated with MTX elimination delay in HD-MTX treatment of children with ALL in our study, consistent with some studies on Chinese populations6,29,35 and Korean populations.18 However, other studies have indicated the opposite conclusions in HD-MTX treatment of children with ALL in Iran,36 Poland37 and Egypt.38 This deviation might be attributed to the difference in racial groups, toxicity criteria, and sample size between the present and previous studies.

The toxicities investigated in our study were found not to be related to the MTHFR C677T or MTHFR A1298C polymorphism. Although the relationship between MTHFR C677T and MTHFR A1298C and HD-MTX toxicities has been extensively studied, definitive conclusions remain unavailable.2,29 Nevertheless, the majority of published studies found no association between MTHFR polymorphisms and risk of HD-MTX toxicities.39,40 This discrepancy might be attributed to the difference in racial groups, sample size, treatment regimens used and time points for evaluating HD-MTX toxicities between the present and previous studies. Another important reason may be that there are many other enzymes or transporter proteins that have a combined impact on MTX pharmacokinetic variability in addition to MTHFR.29 Therefore, MTHFR C677T and MTHFR A1298C gene polymorphisms may not be the main factors for HD-MTX toxicities in Chinese patients, though there may be a limited relationship between them.6

We also assessed genotype of the most commonly studied ABCB1 gene polymorphism C3435T, which is associated with decreased exporter activity, and hence, both leukemic blasts and somatic cells are exposed to high doses of chemotherapy. This might lead to an increase in the number and severity of adverse reactions.1,41 This study showed that the ABCB1 C3435T homozygous genotype (TT) was associated with increased neutropenia, which is consistent with a previous study.1 We also found a strong and significant association between the ABCB1 C3435T homozygous genotype (TT) and increased rates of leukopenia and oral mucositis. Our results may be explained by the fact that the C3435T mutation in ABCB1 causes decreased expression of the transporter, leading to higher intracellular levels and hence more toxicity. However, no association was found between ABCB1 C3435T and HD-MTX toxicities in pediatric patients in some studies.34,36,42 These inconsistent results might be attributed to variable treatment protocols, different toxicity criteria, and racial differences. Our data suggest that for patients carrying ABCB1 C3435T, we should perhaps adopt a precautionary treatment to prevent toxicities in HD-MTX treatment of TT allele pediatric patients.

To analyze the relationship between the combination of three genotypes and MTX-induced toxicity, analysis of the number of wild-type homozygous genes and MTX-induced toxicity was performed, but no relationship was found.

The strengths of our study include unified diagnostic criteria, HD-MTX administration and a CF rescue regimen from a single center. Nonetheless, our study also has certain limitations. First, because this was a retrospective study, we were only able to analyze the potential predictive role of the MTHFR C677T, MTHFR A1298C and ABCB1 C3435T genetic variants in toxicities during HD-MTX therapy in children with ALL. We could not analyze other genetic variants that may affect MTX transport and metabolism, such as in SLCO1B1, SHMT1, and ABCG2. Second, the retrospective study was limited to MTX-related toxicities, and other side effects, such as renal dysfunction and neurotoxicity, were not evaluated. Third, our findings are limited to our center, as the study was based on a single center rather than multiple centers. Therefore, further research is necessary to include a larger study population from multiple centers and investigate the impact of many other genetic variants on HD-MTX toxicities.

Conclusion

Given the cumulative evidence, whether MTHFR C677T and MTHFR A1298C can be used as predictors of HD-MTX toxicities and MTX elimination delay remains unclear. This study showed the ABCB1 C3435T homozygous TT genotype was associated with increased MTX-related toxicities (leukopenia, neutropenia and oral mucositis). The current results indicate that ABCB1 C3435T can predict some MTX-related toxicities. These findings may help to explain the variability of MTX-related toxicities and distinguish pediatric ALL patients with a relatively high risk of such toxicities before HD-MTX infusion and to optimize MTX treatment.

Data Sharing Statement

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.

Ethics Approval

Approval was obtained from the Institute Medical Ethics Committee of the Affiliated Hospital of Qingdao University (number QYFY WZLL 27980). This study was conducted adhere to the ethical principles of the Declaration of Helsinki. The requirement for written informed consent was waived due to the retrospective nature of the study. We declare that we will maintain the confidentiality of the patients’ personal information.

Consent for Publication

All authors have agreed for the publication.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Funding

There is no funding associated with this work.

Disclosure

The authors report no conflicts of interest in this work.

References

1. Zgheib NK, Akra-Ismail M, Aridi C, et al. Genetic polymorphisms in candidate genes predict increased toxicity with methotrexate therapy in Lebanese children with acute lymphoblastic leukemia. Pharmacogenetics and Genomics. 2014;24(8):387–396. doi:10.1097/FPC.0000000000000069

2. Hao Q, Song Y, Fang Q, et al. Effects of genetic polymorphisms on methotrexate levels and toxicity in Chinese patients with acute lymphoblastic leukemia. Blood Science. 2023;5(1):32–38. doi:10.1097/BS9.0000000000000142

3. Li X, Sui Z, Jing F, et al. Identifying risk factors for high-dose methotrexate-induced toxicities in children with acute lymphoblastic leukemia. Cancer Manage Res. 2019;11:6265–6274. doi:10.2147/CMAR.S207959

4. den Hoed MA, Lopez-Lopez E, te Winkel ML, et al. Genetic and metabolic determinants of methotrexate-induced mucositis in pediatric acute lymphoblastic leukemia. Pharmacogenomics J. 2015;15(3):248–254. doi:10.1038/tpj.2014.63

5. D’Angelo V, Ramaglia M, Iannotta A, et al. Methotrexate toxicity and efficacy during the consolidation phase in paediatric acute lymphoblastic leukaemia and MTHFR polymorphisms as pharmacogenetic determinants. Cancer Chemother Pharmacol. 2011;68(5):1339–1346. doi:10.1007/s00280-011-1665-1

6. Shen Y, Wang Z, Zhou F, Jin R. The influence of MTHFR genetic polymorphisms on methotrexate therapy in pediatric acute lymphoblastic leukemia. Open Life Sci. 2021;16(1):1203–1212. doi:10.1515/biol-2021-0121

7. Gregers J, Christensen IJ, Dalhoff K, et al. The association of reduced folate carrier 80G>A polymorphism to outcome in childhood acute lymphoblastic leukemia interacts with chromosome 21 copy number. Blood. 2010;115(23):4671–4677. doi:10.1182/blood-2010-01-256958

8. Sepe DM, McWilliams T, Chen J, et al. Germline genetic variation and treatment response on CCG-1891. Pediatr Blood Cancer. 2012;58(5):695–700. doi:10.1002/pbc.23192

9. Zhu C, Liu YW, Wang SZ, et al. Associations between the C677T and A1298C polymorphisms of MTHFR and the toxicity of methotrexate in childhood malignancies: a meta-analysis. Pharmacogenomics J. 2018;18(3):450–459. doi:10.1038/tpj.2017.34

10. D’Angelo V, Ramaglia M, Iannotta A, et al. Influence of methylenetetrahydrofolate reductase gene polymorphisms on the outcome of pediatric patients with non-Hodgkin lymphoma treated with high-dose methotrexate. Leukemia Lymphoma. 2013;54(12):2639–2644. doi:10.3109/10428194.2013.784758

11. Bottiger AK, Hurtig-Wennlof A, Sjostrom M, Yngve A, Nilsson TK. Association of total plasma homocysteine with methylenetetrahydrofolate reductase genotypes 677C>T,1298A>C, and 1793G>A and the corresponding haplotypes in Swedish children and adolescents. IntJ Mol Med. 2007;19(4):659–665.

12. Liu SG, Li ZG, Cui L, et al. Effects of methylenetetrahydrofolate reductase gene polymorphisms on toxicities during consolidation therapy in pediatric acute lymphoblastic leukemia in a Chinese population. Leukemia Lymphoma. 2011;52(6):1030–1040. doi:10.3109/10428194.2011.563883

13. Tantawy AA, El-Bostany EA, Adly AA, Abou El Asrar M, El-Ghouroury EA, Abdulghaffar EE. Methylene tetrahydrofolate reductase gene polymorphism in Egyptian children with acute lymphoblastic leukemia. Blood Coagul Fibrinolysis. 2010;21(1):28–34. doi:10.1097/MBC.0b013e32833135e9

14. Karathanasis NV, Stiakaki E, Goulielmos GN, Kalmanti M. The role of the methylenetetrahydrofolate reductase 677 and 1298 polymorphisms in Cretan children with acute lymphoblastic leukemia. Genet Test Mol Bioma. 2011;15(1–2):5–10. doi:10.1089/gtmb.2010.0083

15. Frikha R, Rebai T, Lobna BM, et al. Comprehensive analysis of methylenetetrahydrofolate reductase C677T in younger acute lymphoblastic leukemia patients: a single-center experience. J Oncol Pharm Pract. 2019;25(5):1182–1186. doi:10.1177/1078155218818244

16. Ramirez-Pacheco A, Moreno-Guerrero S, Alamillo I, Medina-Sanson A, Lopez B, Moreno-Galvan M. Mexican childhood acute lymphoblastic leukemia: a pilot study of the MDR1 and MTHFR gene polymorphisms and their associations with clinical outcomes. Genet Test Mol Bioma. 2016;20(10):597–602. doi:10.1089/gtmb.2015.0287

17. Haase R, Elsner K, Merkel N, et al. High dose methotrexate treatment in childhood ALL: pilot study on the impact of the MTHFR 677C>T and 1298A>C polymorphisms on MTX-related toxicity. Klin Padiatrie. 2012;224(3):156–159. doi:10.1055/s-0032-1304623

18. Chae H, Kim M, Choi SH, et al. Influence of plasma methotrexate level and MTHFR genotype in Korean paediatric patients with acute lymphoblastic leukaemia. J Chemother. 2020;32(5):251–259. doi:10.1080/1120009X.2020.1764280

19. Faganel Kotnik B, Grabnar I, Bohanec Grabar P, Dolzan V, Jazbec J. Association of genetic polymorphism in the folate metabolic pathway with methotrexate pharmacokinetics and toxicity in childhood acute lymphoblastic leukaemia and malignant lymphoma. Eur J Clin Pharmacol. 2011;67(10):993–1006. doi:10.1007/s00228-011-1046-z

20. Mahmoud LB, Mdhaffar M, Frikha R, et al. Use of MTHFR C677T polymorphism and plasma pharmacokinetics to predict methotrexate toxicity in patients with acute lymphoblastic leukemia. Adv Clin Exp Med. 2018;27(8):1061–1068. doi:10.17219/acem/69802

21. Aplenc R, Thompson J, Han P, et al. Methylenetetrahydrofolate reductase polymorphisms and therapy response in pediatric acute lymphoblastic leukemia. Cancer Res. 2005;65(6):2482–2487. doi:10.1158/0008-5472.CAN-04-2606

22. Radtke S, Zolk O, Renner B, et al. Germline genetic variations in methotrexate candidate genes are associated with pharmacokinetics, toxicity, and outcome in childhood acute lymphoblastic leukemia. Blood. 2013;121(26):5145–5153. doi:10.1182/blood-2013-01-480335

23. Yazicioglu B, Kaya Z, Guntekin Ergun S, et al. Influence of folate-related gene polymorphisms on high-dose methotrexate-related toxicity and prognosis in Turkish children with acute lymphoblastic leukemia. Turk J Haematol. 2017;34(2):143–150. doi:10.4274/tjh.2016.0007

24. van Kooten Niekerk PB, Schmiegelow K, Schroeder H. Influence of methylene tetrahydrofolate reductase polymorphisms and coadministration of antimetabolites on toxicity after high dose methotrexate. Eur J Haematol. 2008;81(5):391–398. doi:10.1111/j.1600-0609.2008.01128.x

25. Ho GT, Moodie FM, Satsangi J. Multidrug resistance 1 gene (P-glycoprotein 170): an important determinant in gastrointestinal disease? Gut. 2003;52(5):759–766. doi:10.1136/gut.52.5.759

26. Maroofi F, Amini S, Roshani D, Ghaderi B, Abdi M. Different frequencies and effects of ABCB1 T3435C polymorphism on clinical and laboratory features of B cell chronic lymphocytic leukemia in kurdish patients. Tumour Biol. 2015;36(4):2863–2868. doi:10.1007/s13277-014-2914-9

27. Rao DN, Anuradha C, Vishnupriya S, et al. Association of an MDR1 gene (C3435T) polymorphism with acute leukemia in India. Asian Pac J Cancer Prev. 2010;11(4):1063–1066.

28. Pongstaporn W, Pakakasama S, Chaksangchaichote P, Pongtheerat T, Hongeng S, Permitr S. MDR1 C3435T and C1236T polymorphisms: association with high-risk childhood acute lymphoblastic leukemia. Asian Pac J Cancer Prev. 2015;16(7):2839–2843. doi:10.7314/APJCP.2015.16.7.2839

29. Tan Y, Kong Q, Li X, et al. Relationship between methylenetetrahydrofolate reductase gene polymorphisms and methotrexate drug metabolism and toxicity. Transl Pediatr. 2023;12(1):31–45. doi:10.21037/tp-22-671

30. Li M, Kong XY, Wang SM. Analysis of the frequency distribution of five single-nucleotide polymorphisms of the MTRRgene in a Chinese pediatric population with acute lymphoblastic leukemia. Pharmacotherapy. 2022;42(6):442–452. doi:10.1002/phar.2685

31. Li M, Kong XY, Wang SM. Effects of splicing-regulatory polymorphisms in ABCC2, ABCG2, and ABCB1 on methotrexate exposure in Chinese children with acute lymphoblastic leukemia. Cancer Chemother Pharmacol. 2023;91(1):77–87. doi:10.1007/s00280-022-04498-0

32. Hu YH, Zhou L, Wang SS, et al. Methotrexate disposition in pediatric patients with acute lymphoblastic leukemia: what have we learnt from the genetic variants of drug transporters. Curr Pharm Des. 2019;25(6):627–634. doi:10.2174/1381612825666190329141003

33. Zhang YX, Ma Y, Zhang H, Zhang WP, Yang XY. Genetic polymorphism in MDR1 C3435T is a determinant of methotrexate cerebrospinal fluid concentrations in Chinese children with acute lymphoblastic leukemia. Int J Clin Pharm Ther. 2020;58(5):254–260. doi:10.5414/CP203462

34. Lopez‐Lopez E, Martin‐Guerrero I, Ballesteros J, et al. Polymorphisms of the SLCO1B1 gene predict methotrexate-related toxicity in childhood acute lymphoblastic leukemia. Pediatr Blood Cancer. 2011;57(4):612–619. doi:10.1002/pbc.23074

35. Wang SM, Sun LL, Zeng WX, Wu WS, Zhang GL. Influence of genetic polymorphisms of FPGS, GGH, and MTHFR on serum methotrexate levels in Chinese children with acute lymphoblastic leukemia. Cancer Chemother Pharmacol. 2014;74(2):283–289. doi:10.1007/s00280-014-2507-8

36. Esmaili MA, Kazemi A, Faranoush M, et al. Polymorphisms within methotrexate pathway genes: relationship between plasma methotrexate levels, toxicity experienced and outcome in pediatric acute lymphoblastic leukemia. Iran J Basic Med Sci. 2020;23(6):800–809. doi:10.22038/ijbms.2020.41754.9858

37. Cwiklinska M, Czogala M, Kwiecinska K, et al. Polymorphisms of SLC19A1 80 G>A,MTHFR 677 C>T, and tandem ts repeats influence pharmacokinetics, acute liver toxicity, and vomiting in children with acute lymphoblastic leukemia treated with high doses of methotrexate. Frontiers in Pediatrics. 2020;8:307. doi:10.3389/fped.2020.00307

38. El-Khodary NM, El-Haggar SM, Eid MA, Ebeid EN. Study of the pharmacokinetic and pharmacogenetic contribution to the toxicity of high-dose methotrexate in children with acute lymphoblastic leukemia. Med Oncol. 2012;29(3):2053–2062. doi:10.1007/s12032-011-9997-6

39. Yao P, He X, Zhang R, Tong R, Xiao H. The influence of MTHFR genetic polymorphisms on adverse reactions after methotrexate in patients with hematological malignancies: a meta-analysis. Hematology. 2019;24(1):10–19. doi:10.1080/10245332.2018.1500750

40. Frikha R, Jemaa MB, Frikha F, et al. Involvement of C677T MTHFR variant but not A1298C in methotrexate-induced toxicity in acute lymphoblastic leukemia. J Oncol Pharm Pract. 2021;27(6):1382–1387. doi:10.1177/1078155220951898

41. Erdelyi DJ, Kamory E, Zalka A, et al. The role of ABC-transporter gene polymorphisms in chemotherapy induced immunosuppression, a retrospective study in childhood acute lymphoblastic leukaemia. Cell Immunol. 2006;244(2):121–124. doi:10.1016/j.cellimm.2007.02.007

42. Liu SG, Gao C, Zhang RD, et al. Polymorphisms in methotrexate transporters and their relationship to plasma methotrexate levels, toxicity of high-dose methotrexate, and outcome of pediatric acute lymphoblastic leukemia. Oncotarget. 2017;8(23):37761–37772. doi:10.18632/oncotarget.17781