Effects of gene polymorphisms on delayed MTX clearance, toxicity, and metabolomic changes after HD-MTX treatment in children with acute lymphoblastic leukemia

Effects of gene polymorphisms on delayed MTX clearance, toxicity, and metabolomic changes after HD-MTX treatment in children with acute lymphoblastic leukemia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effects of gene polymorphisms on delayed MTX clearance, toxicity, and metabolomic changes after HD-MTX treatment in children with acute lymphoblastic leukemia Yao Zhou, Haoping He, Luping Ding, Tianjiao Wang, Xiaomeng Liu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3151727/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Oct, 2023 Read the published version in European Journal of Pediatrics → Version 1 posted 11 You are reading this latest preprint version Abstract Objective To assess the role of methotrexate-related gene polymorphisms in children with acute lymphoblastic leukemia (ALL) during high-dose methotrexate (HD-MTX) therapy and to explore their effects on serum metabolites before and after HD-MTX treatment. Methods The MTHFR 677C > T, MTHFR 1298A > C, ABCB1 3435C > T, and GSTP1 313A > G genotypes of 189 children with ALL who received chemotherapy with the CCCG-ALL-2020 regimen from January 2020 to April 2023 were analyzed, and toxic effects were reported according to the Common Terminology Criteria for Adverse Events (CTCAE, version 5.0). Fasting peripheral blood serum samples were collected from 27 children before and after HD-MTX treatment, and plasma metabolites were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS). Results The results of univariate and multivariate analyses showed that MTHFR 677C > T and ABCB1 3435 C༞T gene polymorphisms were associated with the delayed MTX clearance ( P < 0.05 ) and lower platelet count after treatment in children with MTHFR 677 mutation compared with wild-type ones ( P < 0.05 ), and pure mutations in ABCB1 3435 were associated with higher serum creatinine levels (P T, MTHFR 1298A > C, ABCB1 3435 C > T, and GSTP1 313A > G genes and hepatotoxicity or nephrotoxicity ( P > 0.05 ). However, the serum metabolomic analysis indicated that the presence of the MTHFR 677C > T gene polymorphism could potentially contribute to delayed MTX clearance by influencing L-phenylalanine metabolism, leading to the occurrence of related toxic side effects. Conclusion MTHFR 677C > T and ABCB1 3435 C > T predicted the risk of delayed MTX clearance during HD-MTX treatment in children with ALL. Serum L-phenylalanine levels were significantly elevated after HD-MTX treatment in children with the MTHFR 677C > T mutation gene. Acute lymphoblastic leukemia HD-MTX Gene polymorphism Metabolomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1.Introduction Acute lymphoblastic leukemia (ALL) is one of the most common childhood malignancies, characterized by the abnormal proliferation of lymphoblasts. Over the years, advancements in combination chemotherapy regimens have significantly improved the prognosis for children with ALL. The survival rate has remarkably increased from less than 10% in the 1960s to more than 90% in the present era 1,2 . There are three main phases of treatment for ALL, induction, consolidation and maintenance 3 . Among them, high-dose methotrexate (HD-MTX) has proven to be an effective therapy for managing extramedullary infiltration and systemic consolidation in children with ALL. The utilization of HD-MTX has shown promising results in improving the long-term survival rate of children with ALL 4 . In recent decades, improvements in MTX treatment and dosing regimens have led to significant improvements in the survival rates of patients with ALL. Nonetheless, the mucosal toxicity, hematotoxicity, and hepatotoxicity produced et.al by high-dose MTX in children undergoing HD-MTX treatment may lead to serious complications and even interruption of treatment 5,6 . CCCG-ALL-2020 is a prospective, randomized and multicenter study conducted by the Chinese Pediatric Oncology Group. In this chemotherapy regimen of HD-MTX treatment, MTX-related toxicity and delayed clearance occurred in a large proportion of children despite dose adjustment during treatment based on creatinine clearance (CCR) and MTX concentrations at 44 hours (C 44h ) after the child's first HD-MTX chemotherapy 5 . MTX-related toxicity in patients is influenced by various factors, including the dose and duration of treatment as well as genetic factors 7,8 . A growing number of studies suggested that MTX-related genes play an important role in MTX pharmacokinetics and toxicities, while the results are unidentical across studies, which may be related to factors such as the ethnicity of the study population, sample size, and chemotherapy dose 9–11 . Nevertheless, genetic polymorphisms of MTX-related genes may be potential predictors of personalized HD-MTX therapy. Metabolomics is a valuable approach for investigating biochemical processes associated with metabolites. Given that metabolites can be easily detected in accessible biofluids, metabolomics has been extensively utilized for cancer diagnosis and prediction of adverse effects caused by anticancer drugs 12 , such as Lung cancer 13 , breast cancer 14 , lymphoma 15 , etc. In ALL, ssignificant alterations in metabolite profiles have been found pre- and post-induction and consolidation therapy 16 . The effects of MTX-related genes, contributing to patient-related toxicity and delayed clearance, on serum metabolites were further investigated through combined metabolomics and gene polymorphism analysis. In the present study, the effects of MTHFR 677C > T, MTHFR 1298A > C, ABCB1 3435 C > T, and GSTP1 313A > G gene polymorphisms on MTX-related toxicity and delayed MTX clearance during HD-MTX treatment were analyzed in 189 children with ALL. The potential metabolic pathways affected by genes influencing delayed MTX clearance and toxicity were explored by examining changes in associated plasma metabolites before and after HD-MTX treatment. 2.Methods 2.1 Patients This study included 189 patients who were hospitalized at Qilu Hospital of Shandong University (Jinan, China) from January 2020 to April 2023. All patients included in the study were diagnosed with ALL and fulfilled the inclusion criteria of the CCCG-ALL-2020 protocol, receiving treatment accordingly. Patients who died or discontinued treatment prior to HD-MTX chemotherapy were excluded. From the eligible patients, a random selection of 27 individuals was made, and serum samples were collected before and after HD-MTX chemotherapy for metabolomic analysis. Informed consent was obtained from patients’ legal guardians, and the study was approved by the Ethics Committee of Shandong University Qilu Hospital (Approval No. ChiCTR2000035264). Detailed HD-MTX specific data can be found in the supplementary materials. Detailed HD-MTX specific programs can be found in the supplementary materials. 2.2 Data Collection and Definition Patients’ demographic data, immunophenotype, MTX-related genes, risk stratification, HD-MTX chemotherapy details, and follow-up data were extracted from electronic medical records. The chemotherapy details for HD-MTX included the administered dose of MTX, C 20h , C 44h , C 68h , and relevant tests, such as neutrophil count (NEU), glutamic-pyruvic transaminase (ALT), glutamic oxalacetic transaminase (AST), and creatinine (Cr) during HD-MTX. Delayed clearance of MTX was defined as MTX concentration at C 44h > 1.0 µmol/L or at C 68h ≥ 0.2 µmol/L during any HD-MTX chemotherapy session. Other toxic effects were reported according to the Common Terminology Criteria for Adverse Events (CTCAE, version 5.0) 17 . 2.3 Serum sample collection and metabolite extraction and processing Fasting peripheral blood serum samples were collected before HDMTX treatment (day 1) and after HDMTX treatment (day 3). Samples were collected in EDTA tubes. The samples were thawed and vortexed for 30 s .For tissue homogenate preparation, the samples were gradually added to H2O and homogenized. Cells were added to water and sonicated for 10 min at 4°C. In the case of metabolites, sample volumes of 200 µL were extracted using MeOH:ACN (1:1, v/v). The samples were then vortexed for 30 sec and sonicated for 10 min. To precipitate proteins, the samples were incubated at -20°C for 1 h, followed by a 15-min centrifugation at 20,000 g and 4°C. The resulting supernatant was discarded, and the remaining solution was evaporated to dryness in a vacuum concentrator. The dry extracts were reconstituted in ACN:H2O (1:1, v/v) at a ratio of 40 µL per milligram of protein. The reconstituted extracts were vortexed for 30 sec and sonicated for 10 min. After centrifugation for 15 min at 20,000 rpm and 4°C to remove insoluble debris, the supernatants were transferred to HPLC vials and stored at -80°C until liquid chromatography-tandem mass spectrometry (LC-MS) analysis 2.4 Data Analysis Unconditional logistic regression was employed to calculate odds ratios (OR) and 95% confidence intervals (95% CI) for the incidence of delayed clearance and hematotoxicity following HD-MTX treatment. The Shapiro-Wilk test was employed to assess the normal distribution of methotrexate plasma concentration and other indicators related to hematology, liver function, and kidney function. For normally distributed data, one-way analysis of variance (ANOVA) was utilized to compare the differences between groups. For abnormally distributed data, the Kruskal-Wallis test was employed. A relevant trend was considered for P-value < 0.1, while P-value < 0.05 was considered statistically significant. SPSS 26.0 software (IBM, Armonk, NY, USA) was utilized to carry out statistical analysis, and GraphPad Prism 9.0 software (GraphPad Software Inc., San Diego, CA, USA) was used for image rendering, unless otherwise specified. 3. Results 3.1 Patients’ clinical characteristics Table 1 summarizes patients’ characteristics. The study group consisted of 189 children with a median age of 6.7 ( interquartile range, 3.7–9.4) years old. The risk classification of ALL was based on factors, such as age, leukocyte count, immunophenotype, central nervous system status, karyotype analysis, molecular status, and end induction minimal residual disease (MRD) level. Among patients, 89 (44.4%) were classified as standard risk, while 100 (55.6%) were classified as intermediate or high-risk. Fasting plasma samples were collected from 27 newly diagnosed children at two time points: ( 1 ) on the day before HD-MTX treatment and ( 2 ) after completion of HD-MTX treatment, specifically on day 3 of chemotherapy. The dose of MTX administered varied based on patients’ risk level, with the low-risk group receiving 3 g/m2 and the intermediate- or high-risk group receiving 5 g/m2 of MTX chemotherapy. The MTX-related genotypes of the children included in the study were analyzed, and all genotypes were found to be consistent with Hardy-Weinberg equilibrium. Table 1 Demographic and clinical characteristics of patients Patient characteristics Number (n = 189) Age (years): median [IQR * ] 6.7 [3.7–9.4] Sex M /F: n ( %) 110(58.2%)/79 (41.8%) MTHFR 677:n (%) CC 28(14.8%) CT 80(42.3%) TT 81(42.9%) MTHFR 1298:n (%) AA 149(78.8%) AC 35(18.5%) CC 5(2.7%) ABCB1 3435:n (%) GG 25(13.2%) AG 99(52.4%) AA 65(34.4%) GSTP1 313:n (%) AA 122(64.5%) GA 57(30.2%) GG 10(5.3%) Overall risk group n (%) Standard 89(47.1%) Intermediate or High 100(52.9%) * IQR: interquartile range. Table 2 Demographic and clinical characteristics of 27 patients undergoing metabolomic analysis Patient characteristics Number (n = 27) Age (years): median [IQR * ] 6.8 [3.5–11] Gender Male /female: n ( %) 12 (44.4%)/15 (55.6%) BMI ( kg/m 2 ): median [IQR] 17.2 [15.3–19] MTX metabolic gene (MTHFR 677) n (%) CC 12 (44.4%) CT 10 (37.0%) TT 5 (18.6%) Overall risk group n (%) Standard 12 (44.4%) Intermediate 15 (55.6%) * IQR: interquartile range. 3.2 Association of MTX-related gene polymorphisms with delayed MTX clearance and C 44h As depicted in Fig. 1 , the MTHFR 677CT/TT genotype exhibited a higher likelihood of delayed clearance compared with the wild-type ( OR = 3.056/3.456, P = 0.019/0.009 ). In the ABCB1 3435C > T gene, homozygous mutations were more prone to exhibit delayed MTX clearance compared with the wild-type ( OR = 2.82, P = 0.042 ), whereas no significant difference was noted in genotypes with heterozygous mutations. Similarly, in the C 44h study, higher MTX concentrations were found in children with the MTHFR 677CT/TT genotype ( P = 0.007/0.017 ). However, no significant differences were identified in MTHFR 1298A > C and GSTP1 313A > G genes regarding MTX clearance delay. Notably, children with pure mutations in the MTHFR1298 gene displayed lower C 44h levels, possibly due to the smaller sample size of this genotype in the present study ( Fig. 2 ) . To exclude the effects of confounding factors, multifactorial analysis was performed using logistic regression models for the four gene polymorphisms (Fig. 3) , with the MTHFR 677TT genotype having a higher risk of delayed MTX clearance compared with wild-type children ( OR = 2.841, P = 0.045 ) and the ABCB1 3435G > A pure mutation having a higher chance of delayed clearance compared with the heterozygous mutation ( OR = 3.136, P = 0.036 ). 3.3 Association of MTX-related gene polymorphisms with side effects of HDMTX treatment MTX-related toxicities, including myelosuppression, hepatotoxicity, and nephrotoxicity were evaluated during HD-MTX treatment in 188 children with ALL, as illustrated in Fig. 4 . Children with the MTHFR 677 mutation exhibited lower platelet levels after treatment compared with the wild-type ( CC vs CT + TT, P = 0.044 ), and those with a pure mutation in ABCB1 3435 showed higher serum creatinine levels ( P = 0.016 ). However, no significant differences were identified between genotypes in the analysis of transaminases and other indicators. Notably, when renal damage were evaluated as dichotomous variables, no significant effect of MTX-related gene polymorphisms was found, indicating that the elevated serum Cr levels caused by mutations in these genes are not sufficient to cause pathological changes in patients, but still need to be brought to the attention of clinicians. Unlike the results of single-factor analysis, children with the MTHFR1298A > C heterozygous mutation were found to have a higher likelihood of developing myelosuppression ( P = 0.049 ). However, this association was not observed in patients with the pure mutation, which could be attributed to the small number of children with the pure mutation included in the study (Fig. 5) . 3.4 MTX-related genes could affect serum metabolite changes before and after HD-MTX treatment A random selection of 27 children was made for the metabolomic analysis of peripheral blood serum before and after HD-MTX treatment (Table. 2) . The results revealed a significant increase in serum L-phenylalanine level in children with the MTHFR677 CT/TT mutation after treatment, whereas no similar alteration was found in other genotypes (Fig. 6) . 4. Discussion The survival rate of children with ALL has reached a high level; however, the long-term combination chemotherapy is accompanied by significant toxic side effects that can remarkably impact the quality of life. Therefore, there is a need to predict chemotherapy-related toxic effects prior to treatment and make timely adjustments to the chemotherapy dosage 18 . HD-MTX plays a crucial role in the treatment and prevention of extramedullary leukemia, while it may also lead to significant toxic reactions that should not be overlooked. The occurrence and severity of MTX-related toxicity may be influenced by various factors, including the dosage and duration of treatment, genetic factors, etc. 19 . Among them, genetic factors may be involved in the activities of MTX-associated enzymes, which may affect the metabolism and intracellular transport of the drug, thereby inducing the associated toxicity 20 . In the present study, the potential impact of MTX-related gene polymorphisms on MTX-related toxicity and clearance delay during HD-MTX treatment was analyzed in 189 Chinese children with ALL. Additionally, the Serum metabolomics impact underlying the toxic reactions and clearance delay associated with MTX-related gene polymorphisms were explored through metabolomics analysis. Methylenetetrahydrofolate reductase (MTHFR) is a folate-dependent enzyme that plays an important role in the conversion of homocysteine (Hcy) to methionine 21 . To date, several polymorphic sites have been reported in the MTHFR gene, with the most extensively studied SNP sites being primarily 677 C > T (rs1801133) and 1298 A > C (rs1801131). The substitution of C > T at MTHFR rs1801133 could result in an alanine to valine change, enhancing the enzyme's thermogenicity and disrupting flavin adenine dinucleotide (FAD) binding, thereby reducing catalytic activity 22 , and this missense mutation could result in approximately 70% and 35% reduction in normal MTHFR enzymatic activity in TT and CT genotype carriers, respectively 23 . In addition, the A > C substitution at MTHFR rs1801131 could lead to the substitution of glutamate with alanine in the regulatory binding domain of the enzyme. This alteration could reduce the binding affinity of the enzyme for S-adenosylmethionine (SAM) and subsequently result in the decreased enzymatic activity 24 . The findings of the present study revealed an association between the MTHFR 677C > T mutation and an elevated risk of delayed MTX clearance following HD-MTX treatment. These results are consistent with the findings reported by Hiroyuki Imanishi et al. 25 and Lobna Ben Mahmoud et al. 26 . There were also significant differences in C 44h concentrations, so increasing the dose of Calcium folinate rescue in children with MTHFR 677C > T mutation may help reduce the adverse reactions of HD-MTX. However, no such change was found in the case of the 1298A > C mutation. Notably, when evaluating C 44h as a continuous variable, it was revealed that children with the 1298A > C mutation gene had lower 44-hour MTX blood concentrations. This observation could be attributed to the smaller number of children included in the study with the 1298A > C mutation. Conducting larger clinical studies can provide further insights into this relationship. Ilan S. Weisberg et al. showed that the 1298A > C mutation could affects the enzyme to a lesser extent than 677C > T 27 , which could also be one of the possible reasons for the insignificant effect of 1298A > C on the delayed clearance of MTX. No significant correlation between MTHFR gene polymorphisms and MTX toxic response in patients was found in the most of studies 11 , which is similar to the findings of the present study and may be due to the fact that MTX-related toxicity depends on a combination of factors, including folate status, dosing regimen, dietary and environmental factors, etc. This problem may be eliminated by establishing a complex multi-factor prediction model. ABCB1 is a gene that encodes P-glycoprotein (P-gp) 28 . P-gp is an ATP-dependent membrane glycoprotein that transports intracellular xenobiotics to the extracellular compartment and acts as a pump for drug transport 29 . This gene play an important role in the bioavailability of toxic substances and metabolites and prevents their intracellular accumulation. The ABCB1 3435 C > T polymorphism (rs1045642) decreases the enzymatic activity of P-gp and reduces the number of carriers, leading to intracellular accumulation of drugs with associated toxic effects 30 . ABCB1 corresponds to a wide range of drug substrate actions that affects the toxic response of several antineoplastic drugs, such as carboplatin 31 , cisplatin 32 , MTX 33 , fluorouracil 34 , adriamycin 35 , etc. In the present study, children with ALL who were treated with HD-MTX were analyzed, and it was revealed that homozygous mutations in the ABCB1 3435 C > T gene were more likely to have delayed MTX clearance compared with wild-type ones and had higher serum Cr levels compared with heterozygous mutations. Previous research reported that ABCB1 3435C > T could increase the risk of MTX-related toxicity 36 , however, the present study showed no significant increase in toxicity in patients with both pure and heterozygous mutant genotypes, which is in accordance with Abdel-Hameed I M Ebid et al.’s findings 37 . This may be related to the ethnicity of the study population, MTX dosing regimen, etc. On the other hand, numerous studies confirmed that the P-gp was associated with other polymorphisms in the ABCB1 gene, such as ABCB1 1236C > T, 2677 G > T/A, 1199G > A, etc. 38 , so that simultaneous detection of multiple ABCB1 SNPs for multifactorial analysis will be necessary in the future studies. Glutathione S-transferases (GST) are a group of multifunctional drug metabolizing enzymes, of which GST Pi1 (GSTP1) is widely present in human tissues and is involved in cell repair and drug metabolism. This enzyme can bind to lipophilic cytotoxic drugs to enhance their water solubility and promote drug excretion 39 . The association between the GSTP1 genetic polymorphism (rs1695, 313A > G) and the toxic response to various chemotherapeutic agents has been extensively studied 40 . For instance, Woorim Kim et al. evaluated the relationship between the GSTP1 gene polymorphism and the toxicity of platinum-based chemotherapeutic agents and showed that the GSTP1 313A > G gene mutation was associated with a significant increase in gastrointestinal response and hematologic toxicity compared with wild-type mutations 41 . Similarly, JinYu Gong et al. found similar findings in their study of toxic reactions to cyclophosphamide chemotherapy, and their study also showed that patients with the GSTP1 313A > G gene mutation had an increased risk of infection when treated with cyclophosphamide 42 , while MTX-related toxicity was less studied. The significance of GSTP1 313A > G polymorphism in HD-MTX treatment was investigated for the first time in the present study. However, no relevant effects of GSTP1 313A > G on MTX clearance delay and associated toxicity was found. This could potentially be attributed to the extensive hydration and alkalinization administered during HD-MTX, counteracting the adverse effects of GSTP1 genes. To further understand the exact role of GSTP1 in HD-MTX treatment, additional in vivo and in vitro experiments are warranted. The changes in serum metabolites before and after HD-MTX treatment were studied in 27 ALL patients, and a combined analysis was conducted with MTX-related genes. The analysis revealed that patients with the MTHFR 677CT/TT genotype exhibited significantly higher serum L-phenylalanine levels after HD-MTX treatment, as well the increased risk of delayed MTX clearance and liver damage. However, no significant correlation was noted in relation to other genes. Amino acids play a crucial role in tumor growth, prognosis, and therapeutic response. They act as regulators of gene expression and are essential components in protein synthesis 43,44 . In drug synthesis, the introduction of amino acids into natural products is one of the structural modification strategies to increase water solubility, enhance pharmacological activity, and reduce toxicity 45 . Kleopatra H. et al. showed in patients with the MTHFR 677C > T mutation and elevated blood levels of Hcy, there was an increase in the activity of cell membrane Na+-K+-ATPase. Furthermore, incubation with L-phenylalanine demonstrated a protective effect on the enzyme, preventing its activation 46 , thereby reducing the occurrence of associated mucosal damage. Similarly, a study conducted by Lamia Heikal et al. indicated that L-phenylalanine intake improved vascular endothelial function in a mouse model of hypertension 47 . Elevated serum levels of L-phenylalanine were found in patients with the MTHFR 677CT/TT genotype after treatment. There was a tendency for delayed clearance of MTX and the increased risk of liver damage in these patients. This observation may be attributed to the effect of L-phenylalanine on cell membrane permeability, leading to the enhanced translocation of intracellular MTX to the extracellular compartment and resulting in higher concentrations of MTX in the serum. However, further validation through more robust in vivo and ex vivo experiments is necessary to confirm this finding. In order to mitigate the delayed clearance of MTX and associated toxic effects, it may be beneficial for patients to reduce their intake of L-phenylalanine-rich foods during HD-MTX treatment. 5. Conclusions In conclusion, the present study confirmed that both heterozygous and homozygous mutations in MTHFR 677C > T could lead to a significantly increased risk of delay in MTX clearance, and the results showed that L-phenylalanine has the potential to serve as a predictive marker for the metabolic effects of the MTHFR 677C > T polymorphism. Homozygous mutations of ABCB1 3435C > T also significantly increased the risk of delayed MTX clearance, while heterozygous mutations did not exhibit such effect, and the current evidence does not support the involvement of the GSTP1 313A > G gene in the reference value for HD-MTX treatment. Declarations Data availability statement The original data supporting the conclusions of this paper will be provided by the authors, where ethical requirements are met. Funding This work was supported by Shandong Provincial Natural Science Foundation（ZR202010220039） Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author Contributions Y. Zhou and H. He analyzed the results and wrote the manuscript. L. Ding, T. Wang, X. Liu and M. Zhang organized the data and revised the manuscript. A. Zhang and J. Fu conceived the topic and critically reviewed the manuscript. All the authors approved the manuscript for publication. # Y. Zhou and H. He contributed equally. Ethics statement This study was performed in line with the principles of the Declaration of Helsinki. The study involving human participants was reviewed and approved by the Institutional Review Board of Qilu Hospital, Shandong University. Written informed consent was obtained from all participants or their parents. References Lavasidis, G. et al. Therapeutic interventions for childhood cancer: An umbrella review of randomized evidence. 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Adv Clin Exp Med 27 , 1061-1068 (2018). https://doi.org:10.17219/acem/69802 Weisberg, I. S. et al. The 1298A-->C polymorphism in methylenetetrahydrofolate reductase (MTHFR): in vitro expression and association with homocysteine. Atherosclerosis 156 , 409-415 (2001). https://doi.org:10.1016/s0021-9150(00)00671-7 Cheema, Y., Kiani, Y. S., Linton, K. J. & Jabeen, I. Identification and Empiric Evaluation of New Inhibitors of the Multidrug Transporter P-Glycoprotein (ABCB1). Int J Mol Sci 24 (2023). https://doi.org:10.3390/ijms24065298 Engle, K. & Kumar, G. Cancer multidrug-resistance reversal by ABCB1 inhibition: A recent update. Eur J Med Chem 239 , 114542 (2022). https://doi.org:10.1016/j.ejmech.2022.114542 Ibrahim, M. A. A. et al. Potential drug candidates as P-glycoprotein inhibitors to reverse multidrug resistance in cancer: an in silico drug discovery study. J Biomol Struct Dyn , 1-16 (2023). https://doi.org:10.1080/07391102.2023.2176360 Ferracini, A. C. et al. GSTP1 and ABCB1 Polymorphisms Predicting Toxicities and Clinical Management on Carboplatin and Paclitaxel-Based Chemotherapy in Ovarian Cancer. Clin Transl Sci 14 , 720-728 (2021). https://doi.org:10.1111/cts.12937 Zheng, Z. et al. SORL1 stabilizes ABCB1 to promote cisplatin resistance in ovarian cancer. Funct Integr Genomics 23 , 147 (2023). https://doi.org:10.1007/s10142-023-01075-3 Li, M., Kong, X. Y. & Wang, S. M. Effects of splicing-regulatory polymorphisms in ABCC2, ABCG2, and ABCB1 on methotrexate exposure in Chinese children with acute lymphoblastic leukemia. Cancer Chemother Pharmacol 91 , 77-87 (2023). https://doi.org:10.1007/s00280-022-04498-0 Nomura, H. et al. ABCB1 and ABCC2 genetic polymorphism as risk factors for neutropenia in esophageal cancer patients treated with docetaxel, cisplatin, and 5-fluorouracil chemotherapy. Cancer Chemother Pharmacol 86 , 315-324 (2020). https://doi.org:10.1007/s00280-020-04118-9 Chen, Z. et al. The lncRNA-GAS5/miR-221-3p/DKK2 Axis Modulates ABCB1-Mediated Adriamycin Resistance of Breast Cancer via the Wnt/β-Catenin Signaling Pathway. Mol Ther Nucleic Acids 19 , 1434-1448 (2020). https://doi.org:10.1016/j.omtn.2020.01.030 Plaza-Plaza, J. C. et al. Pharmacogenetic polymorphisms contributing to toxicity induced by methotrexate in the southern Spanish population with rheumatoid arthritis. Omics 16 , 589-595 (2012). https://doi.org:10.1089/omi.2011.0142 Melikoglu, M. A. & Balkan, E. Can we predict unresponsiveness to methotrexate in rheumatoid arthritis? A pharmacogenetic study. Inflammopharmacology 30 , 193-197 (2022). https://doi.org:10.1007/s10787-021-00921-9 Megías-Vericat, J. E. et al. Positive impact of ABCB1 polymorphisms in overall survival and complete remission in acute myeloid leukemia: a systematic review and meta-analysis. Pharmacogenomics J 16 , 1-2 (2016). https://doi.org:10.1038/tpj.2015.79 Mazari, A. M. A. et al. The Multifaceted Role of Glutathione S-Transferases in Health and Disease. Biomolecules 13 (2023). https://doi.org:10.3390/biom13040688 Cui, J. et al. GSTP1 and cancer: Expression, methylation, polymorphisms and signaling (Review). Int J Oncol 56 , 867-878 (2020). https://doi.org:10.3892/ijo.2020.4979 Kim, W., Cho, Y. A., Kim, D. C. & Lee, K. E. Association between Genetic Polymorphism of GSTP1 and Toxicities in Patients Receiving Platinum-Based Chemotherapy: A Systematic Review and Meta-Analysis. Pharmaceuticals (Basel) 15 (2022). https://doi.org:10.3390/ph15040439 Gong, J. Y. et al. Evaluating the role of GSTP1 genetic polymorphism (rs1695, 313A>G) as a predictor in cyclophosphamide-induced toxicities. Medicine (Baltimore) 100 , e24423 (2021). https://doi.org:10.1097/md.0000000000024423 Häusler, R. E., Ludewig, F. & Krueger, S. Amino acids--a life between metabolism and signaling. Plant Sci 229 , 225-237 (2014). https://doi.org:10.1016/j.plantsci.2014.09.011 Li, X. & Zhang, H. S. Amino acid metabolism, redox balance and epigenetic regulation in cancer. Febs j (2023). https://doi.org:10.1111/febs.16803 Chen, X. H. et al. Transporter proteins knowledge graph construction and its application in drug development. Comput Struct Biotechnol J 21 , 2973-2984 (2023). https://doi.org:10.1016/j.csbj.2023.05.001 Schulpis, K. H. et al. Erythrocyte membrane Na+,K+-ATPase and Mg2+-ATPase activities in subjects with methylenetetrahydrofolate reductase (MTHFR) 677 C-->T genotype and moderate hyperhomocysteinaemia. The role of L-phenylalanine and L-alanine. Clin Chem Lab Med 44 , 423-427 (2006). https://doi.org:10.1515/cclm.2006.069 Heikal, L. et al. l-Phenylalanine Restores Vascular Function in Spontaneously Hypertensive Rats Through Activation of the GCH1-GFRP Complex. JACC Basic Transl Sci 3 , 366-377 (2018). https://doi.org:10.1016/j.jacbts.2018.01.015 Additional Declarations No competing interests reported. Supplementary Files Fig.S1.jpg Supplementarymaterials.HDMTXtreatmentprotocol.docx Cite Share Download PDF Status: Published Journal Publication published 18 Oct, 2023 Read the published version in European Journal of Pediatrics → Version 1 posted Editorial decision: Major revision 23 Sep, 2023 Reviews received at journal 19 Sep, 2023 Reviews received at journal 30 Aug, 2023 Reviewers agreed at journal 25 Aug, 2023 Reviewers agreed at journal 09 Aug, 2023 Reviewers agreed at journal 31 Jul, 2023 Reviewers agreed at journal 15 Jul, 2023 Reviewers invited by journal 12 Jul, 2023 Editor assigned by journal 11 Jul, 2023 Submission checks completed at journal 11 Jul, 2023 First submitted to journal 08 Jul, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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version.\u003c/p\u003e","description":"","filename":"Fig.6ChangesinserumLphenylalaninelevelsbeforeandafterHDMTXindifferentMTHFR677CTgenotypes.png","url":"https://assets-eu.researchsquare.com/files/rs-3151727/v1/c27142051846f7bb041f37bd.png"},{"id":45090675,"identity":"d3b71e99-bee5-4606-8f71-cbe86d1bf4c3","added_by":"auto","created_at":"2023-10-23 15:04:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1135937,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3151727/v1/e28a9a51-209e-4eed-8539-7511e0d2ae0e.pdf"},{"id":40045834,"identity":"a109eea9-a5d0-42c1-8f03-b2275f582b67","added_by":"auto","created_at":"2023-07-14 15:26:54","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5629709,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.S1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3151727/v1/1e7af7aa80e3aaf313e72e61.jpg"},{"id":40045829,"identity":"f479331e-35a2-4de4-979f-3a1b45f9b227","added_by":"auto","created_at":"2023-07-14 15:26:53","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":18036,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.HDMTXtreatmentprotocol.docx","url":"https://assets-eu.researchsquare.com/files/rs-3151727/v1/89c05cd22972b5b1fea8ab70.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of gene polymorphisms on delayed MTX clearance, toxicity, and metabolomic changes after HD-MTX treatment in children with acute lymphoblastic leukemia","fulltext":[{"header":"1.Introduction","content":"\u003cp\u003eAcute lymphoblastic leukemia (ALL) is one of the most common childhood malignancies, characterized by the abnormal proliferation of lymphoblasts. Over the years, advancements in combination chemotherapy regimens have significantly improved the prognosis for children with ALL. The survival rate has remarkably increased from less than 10% in the 1960s to more than 90% in the present era\u003csup\u003e1,2\u003c/sup\u003e. There are three main phases of treatment for ALL, induction, consolidation and maintenance\u003csup\u003e3\u003c/sup\u003e. Among them, high-dose methotrexate (HD-MTX) has proven to be an effective therapy for managing extramedullary infiltration and systemic consolidation in children with ALL. The utilization of HD-MTX has shown promising results in improving the long-term survival rate of children with ALL\u003csup\u003e4\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn recent decades, improvements in MTX treatment and dosing regimens have led to significant improvements in the survival rates of patients with ALL. Nonetheless, the mucosal toxicity, hematotoxicity, and hepatotoxicity produced et.al by high-dose MTX in children undergoing HD-MTX treatment may lead to serious complications and even interruption of treatment\u003csup\u003e5,6\u003c/sup\u003e. CCCG-ALL-2020 is a prospective, randomized and multicenter study conducted by the Chinese Pediatric Oncology Group. In this chemotherapy regimen of HD-MTX treatment, MTX-related toxicity and delayed clearance occurred in a large proportion of children despite dose adjustment during treatment based on creatinine clearance (CCR) and MTX concentrations at 44 hours (C\u003csub\u003e44h\u003c/sub\u003e) after the child's first HD-MTX chemotherapy\u003csup\u003e5\u003c/sup\u003e. MTX-related toxicity in patients is influenced by various factors, including the dose and duration of treatment as well as genetic factors\u003csup\u003e7,8\u003c/sup\u003e. A growing number of studies suggested that MTX-related genes play an important role in MTX pharmacokinetics and toxicities, while the results are unidentical across studies, which may be related to factors such as the ethnicity of the study population, sample size, and chemotherapy dose\u003csup\u003e9\u0026ndash;11\u003c/sup\u003e. Nevertheless, genetic polymorphisms of MTX-related genes may be potential predictors of personalized HD-MTX therapy.\u003c/p\u003e \u003cp\u003eMetabolomics is a valuable approach for investigating biochemical processes associated with metabolites. Given that metabolites can be easily detected in accessible biofluids, metabolomics has been extensively utilized for cancer diagnosis and prediction of adverse effects caused by anticancer drugs\u003csup\u003e12\u003c/sup\u003e, such as Lung cancer\u003csup\u003e13\u003c/sup\u003e, breast cancer\u003csup\u003e14\u003c/sup\u003e, lymphoma\u003csup\u003e15\u003c/sup\u003e, etc. In ALL, ssignificant alterations in metabolite profiles have been found pre- and post-induction and consolidation therapy \u003csup\u003e16\u003c/sup\u003e. The effects of MTX-related genes, contributing to patient-related toxicity and delayed clearance, on serum metabolites were further investigated through combined metabolomics and gene polymorphism analysis.\u003c/p\u003e \u003cp\u003eIn the present study, the effects of MTHFR 677C\u0026thinsp;\u0026gt;\u0026thinsp;T, MTHFR 1298A\u0026thinsp;\u0026gt;\u0026thinsp;C, ABCB1 3435 C\u0026thinsp;\u0026gt;\u0026thinsp;T, and GSTP1 313A\u0026thinsp;\u0026gt;\u0026thinsp;G gene polymorphisms on MTX-related toxicity and delayed MTX clearance during HD-MTX treatment were analyzed in 189 children with ALL. The potential metabolic pathways affected by genes influencing delayed MTX clearance and toxicity were explored by examining changes in associated plasma metabolites before and after HD-MTX treatment.\u003c/p\u003e"},{"header":"2.Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Patients\u003c/h2\u003e \u003cp\u003eThis study included 189 patients who were hospitalized at Qilu Hospital of Shandong University (Jinan, China) from January 2020 to April 2023. All patients included in the study were diagnosed with ALL and fulfilled the inclusion criteria of the CCCG-ALL-2020 protocol, receiving treatment accordingly. Patients who died or discontinued treatment prior to HD-MTX chemotherapy were excluded. From the eligible patients, a random selection of 27 individuals was made, and serum samples were collected before and after HD-MTX chemotherapy for metabolomic analysis. Informed consent was obtained from patients\u0026rsquo; legal guardians, and the study was approved by the Ethics Committee of Shandong University Qilu Hospital (Approval No. ChiCTR2000035264). Detailed HD-MTX specific data can be found in the supplementary materials. Detailed HD-MTX specific programs can be found in the supplementary materials.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Data Collection and Definition\u003c/h2\u003e \u003cp\u003ePatients\u0026rsquo; demographic data, immunophenotype, MTX-related genes, risk stratification, HD-MTX chemotherapy details, and follow-up data were extracted from electronic medical records. The chemotherapy details for HD-MTX included the administered dose of MTX, C\u003csub\u003e20h\u003c/sub\u003e, C\u003csub\u003e44h\u003c/sub\u003e, C\u003csub\u003e68h\u003c/sub\u003e, and relevant tests, such as neutrophil count (NEU), glutamic-pyruvic transaminase (ALT), glutamic oxalacetic transaminase (AST), and creatinine (Cr) during HD-MTX. Delayed clearance of MTX was defined as MTX concentration at C\u003csub\u003e44h\u003c/sub\u003e\u0026thinsp;\u0026gt;\u0026thinsp;1.0 \u0026micro;mol/L or at C\u003csub\u003e68h\u003c/sub\u003e\u0026thinsp;\u0026ge;\u0026thinsp;0.2 \u0026micro;mol/L during any HD-MTX chemotherapy session. Other toxic effects were reported according to the Common Terminology Criteria for Adverse Events (CTCAE, version 5.0)\u003csup\u003e17\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Serum sample collection and metabolite extraction and processing\u003c/h2\u003e \u003cp\u003eFasting peripheral blood serum samples were collected before HDMTX treatment (day 1) and after HDMTX treatment (day 3). Samples were collected in EDTA tubes. The samples were thawed and vortexed for 30 s .For tissue homogenate preparation, the samples were gradually added to H2O and homogenized. Cells were added to water and sonicated for 10 min at 4\u0026deg;C. In the case of metabolites, sample volumes of 200 \u0026micro;L were extracted using MeOH:ACN (1:1, v/v). The samples were then vortexed for 30 sec and sonicated for 10 min. To precipitate proteins, the samples were incubated at -20\u0026deg;C for 1 h, followed by a 15-min centrifugation at 20,000 g and 4\u0026deg;C. The resulting supernatant was discarded, and the remaining solution was evaporated to dryness in a vacuum concentrator. The dry extracts were reconstituted in ACN:H2O (1:1, v/v) at a ratio of 40 \u0026micro;L per milligram of protein. The reconstituted extracts were vortexed for 30 sec and sonicated for 10 min. After centrifugation for 15 min at 20,000 rpm and 4\u0026deg;C to remove insoluble debris, the supernatants were transferred to HPLC vials and stored at -80\u0026deg;C until liquid chromatography-tandem mass spectrometry (LC-MS) analysis\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Data Analysis\u003c/h2\u003e \u003cp\u003eUnconditional logistic regression was employed to calculate odds ratios (OR) and 95% confidence intervals (95% CI) for the incidence of delayed clearance and hematotoxicity following HD-MTX treatment. The Shapiro-Wilk test was employed to assess the normal distribution of methotrexate plasma concentration and other indicators related to hematology, liver function, and kidney function. For normally distributed data, one-way analysis of variance (ANOVA) was utilized to compare the differences between groups. For abnormally distributed data, the Kruskal-Wallis test was employed. A relevant trend was considered for P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.1, while P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. SPSS 26.0 software (IBM, Armonk, NY, USA) was utilized to carry out statistical analysis, and GraphPad Prism 9.0 software (GraphPad Software Inc., San Diego, CA, USA) was used for image rendering, unless otherwise specified.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Patients\u0026rsquo; clinical characteristics\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e summarizes patients\u0026rsquo; characteristics. The study group consisted of 189 children with a median age of 6.7 (\u003cem\u003einterquartile range, 3.7\u0026ndash;9.4)\u003c/em\u003e years old. The risk classification of ALL was based on factors, such as age, leukocyte count, immunophenotype, central nervous system status, karyotype analysis, molecular status, and end induction minimal residual disease (MRD) level. Among patients, 89 (44.4%) were classified as standard risk, while 100 (55.6%) were classified as intermediate or high-risk. Fasting plasma samples were collected from 27 newly diagnosed children at two time points: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) on the day before HD-MTX treatment and (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) after completion of HD-MTX treatment, specifically on day 3 of chemotherapy. The dose of MTX administered varied based on patients\u0026rsquo; risk level, with the low-risk group receiving 3 g/m2 and the intermediate- or high-risk group receiving 5 g/m2 of MTX chemotherapy. The MTX-related genotypes of the children included in the study were analyzed, and all genotypes were found to be consistent with Hardy-Weinberg equilibrium.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDemographic and clinical characteristics of patients\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient characteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNumber (n\u0026thinsp;=\u0026thinsp;189)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (years): median [IQR\u003csup\u003e*\u003c/sup\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.7 [3.7\u0026ndash;9.4]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSex M /F:\u003cem\u003en\u003c/em\u003e ( %)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e110(58.2%)/79 (41.8%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMTHFR 677:n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e28(14.8%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e80(42.3%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e81(42.9%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMTHFR 1298:n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e149(78.8%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e35(18.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5(2.7%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eABCB1 3435:n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e25(13.2%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e99(52.4%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e65(34.4%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGSTP1 313:n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e122(64.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e57(30.2%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10(5.3%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOverall risk group n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStandard\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e89(47.1%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIntermediate or High\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100(52.9%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003e* IQR: interquartile range.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDemographic and clinical characteristics of 27 patients undergoing metabolomic analysis\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient characteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNumber (n\u0026thinsp;=\u0026thinsp;27)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (years): median [IQR\u003csup\u003e*\u003c/sup\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.8 [3.5\u0026ndash;11]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGender\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale /female:\u003cem\u003en\u003c/em\u003e ( %)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12 (44.4%)/15 (55.6%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMI ( kg/m\u003csup\u003e2\u003c/sup\u003e): median [IQR]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e17.2 [15.3\u0026ndash;19]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMTX metabolic gene (MTHFR 677) n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12 (44.4%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10 (37.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5 (18.6%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOverall risk group n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStandard\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12 (44.4%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIntermediate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15 (55.6%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003e* IQR: interquartile range.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Association of MTX-related gene polymorphisms with delayed MTX clearance and C\u003csub\u003e44h\u003c/sub\u003e\u003c/h2\u003e \u003cp\u003eAs depicted in \u003cb\u003eFig.\u0026nbsp;1\u003c/b\u003e, the MTHFR 677CT/TT genotype exhibited a higher likelihood of delayed clearance compared with the wild-type (\u003cem\u003eOR\u0026thinsp;=\u0026thinsp;3.056/3.456, P\u0026thinsp;=\u0026thinsp;0.019/0.009\u003c/em\u003e). In the ABCB1 3435C\u0026thinsp;\u0026gt;\u0026thinsp;T gene, homozygous mutations were more prone to exhibit delayed MTX clearance compared with the wild-type (\u003cem\u003eOR\u0026thinsp;=\u0026thinsp;2.82, P\u0026thinsp;=\u0026thinsp;0.042\u003c/em\u003e), whereas no significant difference was noted in genotypes with heterozygous mutations. Similarly, in the C\u003csub\u003e44h\u003c/sub\u003e study, higher MTX concentrations were found in children with the MTHFR 677CT/TT genotype (\u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.007/0.017\u003c/em\u003e). However, no significant differences were identified in MTHFR 1298A\u0026thinsp;\u0026gt;\u0026thinsp;C and GSTP1 313A\u0026thinsp;\u0026gt;\u0026thinsp;G genes regarding MTX clearance delay. Notably, children with pure mutations in the MTHFR1298 gene displayed lower C\u003csub\u003e44h\u003c/sub\u003e levels, possibly due to the smaller sample size of this genotype in the present study \u003cb\u003e( Fig.\u0026nbsp;2 )\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eTo exclude the effects of confounding factors, multifactorial analysis was performed using logistic regression models for the four gene polymorphisms \u003cb\u003e(Fig.\u0026nbsp;3)\u003c/b\u003e, with the MTHFR 677TT genotype having a higher risk of delayed MTX clearance compared with wild-type children (\u003cem\u003eOR\u0026thinsp;=\u0026thinsp;2.841, P\u0026thinsp;=\u0026thinsp;0.045\u003c/em\u003e) and the ABCB1 3435G\u0026thinsp;\u0026gt;\u0026thinsp;A pure mutation having a higher chance of delayed clearance compared with the heterozygous mutation (\u003cem\u003eOR\u0026thinsp;=\u0026thinsp;3.136, P\u0026thinsp;=\u0026thinsp;0.036\u003c/em\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Association of MTX-related gene polymorphisms with side effects of HDMTX treatment\u003c/h2\u003e \u003cp\u003eMTX-related toxicities, including myelosuppression, hepatotoxicity, and nephrotoxicity were evaluated during HD-MTX treatment in 188 children with ALL, as illustrated in \u003cb\u003eFig.\u0026nbsp;4\u003c/b\u003e. Children with the MTHFR 677 mutation exhibited lower platelet levels after treatment compared with the wild-type (\u003cem\u003eCC vs CT\u0026thinsp;+\u0026thinsp;TT, P\u0026thinsp;=\u0026thinsp;0.044\u003c/em\u003e), and those with a pure mutation in ABCB1 3435 showed higher serum creatinine levels (\u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.016\u003c/em\u003e). However, no significant differences were identified between genotypes in the analysis of transaminases and other indicators.\u003c/p\u003e \u003cp\u003eNotably, when renal damage were evaluated as dichotomous variables, no significant effect of MTX-related gene polymorphisms was found, indicating that the elevated serum Cr levels caused by mutations in these genes are not sufficient to cause pathological changes in patients, but still need to be brought to the attention of clinicians. Unlike the results of single-factor analysis, children with the MTHFR1298A\u0026thinsp;\u0026gt;\u0026thinsp;C heterozygous mutation were found to have a higher likelihood of developing myelosuppression (\u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.049\u003c/em\u003e). However, this association was not observed in patients with the pure mutation, which could be attributed to the small number of children with the pure mutation included in the study \u003cb\u003e(Fig.\u0026nbsp;5)\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.4 MTX-related genes could affect serum metabolite changes before and after HD-MTX treatment\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eA random selection of 27 children was made for the metabolomic analysis of peripheral blood serum before and after HD-MTX treatment \u003cb\u003e(Table. 2)\u003c/b\u003e. The results revealed a significant increase in serum L-phenylalanine level in children with the MTHFR677 CT/TT mutation after treatment, whereas no similar alteration was found in other genotypes \u003cb\u003e(Fig.\u0026nbsp;6)\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe survival rate of children with ALL has reached a high level; however, the long-term combination chemotherapy is accompanied by significant toxic side effects that can remarkably impact the quality of life. Therefore, there is a need to predict chemotherapy-related toxic effects prior to treatment and make timely adjustments to the chemotherapy dosage\u003csup\u003e18\u003c/sup\u003e. HD-MTX plays a crucial role in the treatment and prevention of extramedullary leukemia, while it may also lead to significant toxic reactions that should not be overlooked. The occurrence and severity of MTX-related toxicity may be influenced by various factors, including the dosage and duration of treatment, genetic factors, etc. \u003csup\u003e19\u003c/sup\u003e. Among them, genetic factors may be involved in the activities of MTX-associated enzymes, which may affect the metabolism and intracellular transport of the drug, thereby inducing the associated toxicity\u003csup\u003e20\u003c/sup\u003e. In the present study, the potential impact of MTX-related gene polymorphisms on MTX-related toxicity and clearance delay during HD-MTX treatment was analyzed in 189 Chinese children with ALL. Additionally, the Serum metabolomics impact underlying the toxic reactions and clearance delay associated with MTX-related gene polymorphisms were explored through metabolomics analysis.\u003c/p\u003e \u003cp\u003eMethylenetetrahydrofolate reductase (MTHFR) is a folate-dependent enzyme that plays an important role in the conversion of homocysteine (Hcy) to methionine\u003csup\u003e21\u003c/sup\u003e. To date, several polymorphic sites have been reported in the MTHFR gene, with the most extensively studied SNP sites being primarily 677 C\u0026thinsp;\u0026gt;\u0026thinsp;T (rs1801133) and 1298 A\u0026thinsp;\u0026gt;\u0026thinsp;C (rs1801131). The substitution of C\u0026thinsp;\u0026gt;\u0026thinsp;T at MTHFR rs1801133 could result in an alanine to valine change, enhancing the enzyme's thermogenicity and disrupting flavin adenine dinucleotide (FAD) binding, thereby reducing catalytic activity\u003csup\u003e22\u003c/sup\u003e, and this missense mutation could result in approximately 70% and 35% reduction in normal MTHFR enzymatic activity in TT and CT genotype carriers, respectively \u003csup\u003e23\u003c/sup\u003e. In addition, the A\u0026thinsp;\u0026gt;\u0026thinsp;C substitution at MTHFR rs1801131 could lead to the substitution of glutamate with alanine in the regulatory binding domain of the enzyme. This alteration could reduce the binding affinity of the enzyme for S-adenosylmethionine (SAM) and subsequently result in the decreased enzymatic activity\u003csup\u003e24\u003c/sup\u003e. The findings of the present study revealed an association between the MTHFR 677C\u0026thinsp;\u0026gt;\u0026thinsp;T mutation and an elevated risk of delayed MTX clearance following HD-MTX treatment. These results are consistent with the findings reported by Hiroyuki Imanishi et al.\u003csup\u003e25\u003c/sup\u003e and Lobna Ben Mahmoud et al.\u003csup\u003e26\u003c/sup\u003e. There were also significant differences in C\u003csub\u003e44h\u003c/sub\u003e concentrations, so increasing the dose of Calcium folinate rescue in children with MTHFR 677C\u0026thinsp;\u0026gt;\u0026thinsp;T mutation may help reduce the adverse reactions of HD-MTX. However, no such change was found in the case of the 1298A\u0026thinsp;\u0026gt;\u0026thinsp;C mutation. Notably, when evaluating C\u003csub\u003e44h\u003c/sub\u003e as a continuous variable, it was revealed that children with the 1298A\u0026thinsp;\u0026gt;\u0026thinsp;C mutation gene had lower 44-hour MTX blood concentrations. This observation could be attributed to the smaller number of children included in the study with the 1298A\u0026thinsp;\u0026gt;\u0026thinsp;C mutation. Conducting larger clinical studies can provide further insights into this relationship. Ilan S. Weisberg et al. showed that the 1298A\u0026thinsp;\u0026gt;\u0026thinsp;C mutation could affects the enzyme to a lesser extent than 677C\u0026thinsp;\u0026gt;\u0026thinsp;T \u003csup\u003e27\u003c/sup\u003e, which could also be one of the possible reasons for the insignificant effect of 1298A\u0026thinsp;\u0026gt;\u0026thinsp;C on the delayed clearance of MTX. No significant correlation between MTHFR gene polymorphisms and MTX toxic response in patients was found in the most of studies \u003csup\u003e11\u003c/sup\u003e, which is similar to the findings of the present study and may be due to the fact that MTX-related toxicity depends on a combination of factors, including folate status, dosing regimen, dietary and environmental factors, etc. This problem may be eliminated by establishing a complex multi-factor prediction model.\u003c/p\u003e \u003cp\u003eABCB1 is a gene that encodes P-glycoprotein (P-gp)\u003csup\u003e28\u003c/sup\u003e. P-gp is an ATP-dependent membrane glycoprotein that transports intracellular xenobiotics to the extracellular compartment and acts as a pump for drug transport\u003csup\u003e29\u003c/sup\u003e. This gene play an important role in the bioavailability of toxic substances and metabolites and prevents their intracellular accumulation. The ABCB1 3435 C\u0026thinsp;\u0026gt;\u0026thinsp;T polymorphism (rs1045642) decreases the enzymatic activity of P-gp and reduces the number of carriers, leading to intracellular accumulation of drugs with associated toxic effects\u003csup\u003e30\u003c/sup\u003e. ABCB1 corresponds to a wide range of drug substrate actions that affects the toxic response of several antineoplastic drugs, such as carboplatin \u003csup\u003e31\u003c/sup\u003e, cisplatin \u003csup\u003e32\u003c/sup\u003e, MTX \u003csup\u003e33\u003c/sup\u003e, fluorouracil \u003csup\u003e34\u003c/sup\u003e, adriamycin \u003csup\u003e35\u003c/sup\u003e, etc. In the present study, children with ALL who were treated with HD-MTX were analyzed, and it was revealed that homozygous mutations in the ABCB1 3435 C\u0026thinsp;\u0026gt;\u0026thinsp;T gene were more likely to have delayed MTX clearance compared with wild-type ones and had higher serum Cr levels compared with heterozygous mutations. Previous research reported that ABCB1 3435C\u0026thinsp;\u0026gt;\u0026thinsp;T could increase the risk of MTX-related toxicity\u003csup\u003e36\u003c/sup\u003e, however, the present study showed no significant increase in toxicity in patients with both pure and heterozygous mutant genotypes, which is in accordance with Abdel-Hameed I M Ebid et al.\u0026rsquo;s findings\u003csup\u003e37\u003c/sup\u003e. This may be related to the ethnicity of the study population, MTX dosing regimen, etc. On the other hand, numerous studies confirmed that the P-gp was associated with other polymorphisms in the ABCB1 gene, such as ABCB1 1236C\u0026thinsp;\u0026gt;\u0026thinsp;T, 2677 G\u0026thinsp;\u0026gt;\u0026thinsp;T/A, 1199G\u0026thinsp;\u0026gt;\u0026thinsp;A, etc.\u003csup\u003e38\u003c/sup\u003e, so that simultaneous detection of multiple ABCB1 SNPs for multifactorial analysis will be necessary in the future studies.\u003c/p\u003e \u003cp\u003eGlutathione S-transferases (GST) are a group of multifunctional drug metabolizing enzymes, of which GST Pi1 (GSTP1) is widely present in human tissues and is involved in cell repair and drug metabolism. This enzyme can bind to lipophilic cytotoxic drugs to enhance their water solubility and promote drug excretion \u003csup\u003e39\u003c/sup\u003e. The association between the GSTP1 genetic polymorphism (rs1695, 313A\u0026thinsp;\u0026gt;\u0026thinsp;G) and the toxic response to various chemotherapeutic agents has been extensively studied\u003csup\u003e40\u003c/sup\u003e. For instance, Woorim Kim et al. evaluated the relationship between the GSTP1 gene polymorphism and the toxicity of platinum-based chemotherapeutic agents and showed that the GSTP1 313A\u0026thinsp;\u0026gt;\u0026thinsp;G gene mutation was associated with a significant increase in gastrointestinal response and hematologic toxicity compared with wild-type mutations\u003csup\u003e41\u003c/sup\u003e. Similarly, JinYu Gong et al. found similar findings in their study of toxic reactions to cyclophosphamide chemotherapy, and their study also showed that patients with the GSTP1 313A\u0026thinsp;\u0026gt;\u0026thinsp;G gene mutation had an increased risk of infection when treated with cyclophosphamide\u003csup\u003e42\u003c/sup\u003e, while MTX-related toxicity was less studied. The significance of GSTP1 313A\u0026thinsp;\u0026gt;\u0026thinsp;G polymorphism in HD-MTX treatment was investigated for the first time in the present study. However, no relevant effects of GSTP1 313A\u0026thinsp;\u0026gt;\u0026thinsp;G on MTX clearance delay and associated toxicity was found. This could potentially be attributed to the extensive hydration and alkalinization administered during HD-MTX, counteracting the adverse effects of GSTP1 genes. To further understand the exact role of GSTP1 in HD-MTX treatment, additional in \u003cem\u003evivo\u003c/em\u003e and in \u003cem\u003evitro\u003c/em\u003e experiments are warranted.\u003c/p\u003e \u003cp\u003eThe changes in serum metabolites before and after HD-MTX treatment were studied in 27 ALL patients, and a combined analysis was conducted with MTX-related genes. The analysis revealed that patients with the MTHFR 677CT/TT genotype exhibited significantly higher serum L-phenylalanine levels after HD-MTX treatment, as well the increased risk of delayed MTX clearance and liver damage. However, no significant correlation was noted in relation to other genes. Amino acids play a crucial role in tumor growth, prognosis, and therapeutic response. They act as regulators of gene expression and are essential components in protein synthesis\u003csup\u003e43,44\u003c/sup\u003e. In drug synthesis, the introduction of amino acids into natural products is one of the structural modification strategies to increase water solubility, enhance pharmacological activity, and reduce toxicity\u003csup\u003e45\u003c/sup\u003e. Kleopatra H. et al. showed in patients with the MTHFR 677C\u0026thinsp;\u0026gt;\u0026thinsp;T mutation and elevated blood levels of Hcy, there was an increase in the activity of cell membrane Na+-K+-ATPase. Furthermore, incubation with L-phenylalanine demonstrated a protective effect on the enzyme, preventing its activation \u003csup\u003e46\u003c/sup\u003e, thereby reducing the occurrence of associated mucosal damage. Similarly, a study conducted by Lamia Heikal et al. indicated that L-phenylalanine intake improved vascular endothelial function in a mouse model of hypertension\u003csup\u003e47\u003c/sup\u003e. Elevated serum levels of L-phenylalanine were found in patients with the MTHFR 677CT/TT genotype after treatment. There was a tendency for delayed clearance of MTX and the increased risk of liver damage in these patients. This observation may be attributed to the effect of L-phenylalanine on cell membrane permeability, leading to the enhanced translocation of intracellular MTX to the extracellular compartment and resulting in higher concentrations of MTX in the serum. However, further validation through more robust in vivo and ex vivo experiments is necessary to confirm this finding. In order to mitigate the delayed clearance of MTX and associated toxic effects, it may be beneficial for patients to reduce their intake of L-phenylalanine-rich foods during HD-MTX treatment.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn conclusion, the present study confirmed that both heterozygous and homozygous mutations in MTHFR 677C\u0026thinsp;\u0026gt;\u0026thinsp;T could lead to a significantly increased risk of delay in MTX clearance, and the results showed that L-phenylalanine has the potential to serve as a predictive marker for the metabolic effects of the MTHFR 677C\u0026thinsp;\u0026gt;\u0026thinsp;T polymorphism. Homozygous mutations of ABCB1 3435C\u0026thinsp;\u0026gt;\u0026thinsp;T also significantly increased the risk of delayed MTX clearance, while heterozygous mutations did not exhibit such effect, and the current evidence does not support the involvement of the GSTP1 313A\u0026thinsp;\u0026gt;\u0026thinsp;G gene in the reference value for HD-MTX treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original data supporting the conclusions of this paper will be provided by the authors, where ethical requirements are met.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Shandong Provincial Natural Science Foundation（ZR202010220039）\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY. Zhou and H. He analyzed the results and wrote the manuscript. L. Ding, T. Wang, X. Liu and M. Zhang organized the data and revised the manuscript. A. Zhang and J. Fu conceived the topic and critically reviewed the manuscript. All the authors approved the manuscript for publication. \u003cstrong\u003e\u003csup\u003e#\u003c/sup\u003e\u003c/strong\u003eY. Zhou and H. He contributed equally.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was performed in line with the principles of the Declaration of Helsinki. The study involving human participants was reviewed and approved by the Institutional Review Board of Qilu Hospital, Shandong University. 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Amino acid metabolism, redox balance and epigenetic regulation in cancer. \u003cem\u003eFebs j\u003c/em\u003e (2023). https://doi.org:10.1111/febs.16803\u003c/li\u003e\n\u003cli\u003eChen, X. H.\u003cem\u003e et al.\u003c/em\u003e Transporter proteins knowledge graph construction and its application in drug development. \u003cem\u003eComput Struct Biotechnol J\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 2973-2984 (2023). https://doi.org:10.1016/j.csbj.2023.05.001\u003c/li\u003e\n\u003cli\u003eSchulpis, K. H.\u003cem\u003e et al.\u003c/em\u003e Erythrocyte membrane Na+,K+-ATPase and Mg2+-ATPase activities in subjects with methylenetetrahydrofolate reductase (MTHFR) 677 C--\u0026gt;T genotype and moderate hyperhomocysteinaemia. The role of L-phenylalanine and L-alanine. \u003cem\u003eClin Chem Lab Med\u003c/em\u003e \u003cstrong\u003e44\u003c/strong\u003e, 423-427 (2006). https://doi.org:10.1515/cclm.2006.069\u003c/li\u003e\n\u003cli\u003eHeikal, L.\u003cem\u003e et al.\u003c/em\u003e l-Phenylalanine Restores Vascular Function in Spontaneously Hypertensive Rats Through Activation of the GCH1-GFRP Complex. \u003cem\u003eJACC Basic Transl Sci\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 366-377 (2018). https://doi.org:10.1016/j.jacbts.2018.01.015\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-pediatrics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejpe","sideBox":"Learn more about [European Journal of Pediatrics](https://www.springer.com/journal/431)","snPcode":"431","submissionUrl":"https://submission.nature.com/new-submission/431/3","title":"European Journal of Pediatrics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Acute lymphoblastic leukemia, HD-MTX, Gene polymorphism, Metabolomics","lastPublishedDoi":"10.21203/rs.3.rs-3151727/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3151727/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eTo assess the role of methotrexate-related gene polymorphisms in children with acute lymphoblastic leukemia (ALL) during high-dose methotrexate (HD-MTX) therapy and to explore their effects on serum metabolites before and after HD-MTX treatment.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe MTHFR 677C\u0026thinsp;\u0026gt;\u0026thinsp;T, MTHFR 1298A\u0026thinsp;\u0026gt;\u0026thinsp;C, ABCB1 3435C\u0026thinsp;\u0026gt;\u0026thinsp;T, and GSTP1 313A\u0026thinsp;\u0026gt;\u0026thinsp;G genotypes of 189 children with ALL who received chemotherapy with the CCCG-ALL-2020 regimen from January 2020 to April 2023 were analyzed, and toxic effects were reported according to the Common Terminology Criteria for Adverse Events (CTCAE, version 5.0). Fasting peripheral blood serum samples were collected from 27 children before and after HD-MTX treatment, and plasma metabolites were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe results of univariate and multivariate analyses showed that MTHFR 677C\u0026thinsp;\u0026gt;\u0026thinsp;T and ABCB1 3435 C༞T gene polymorphisms were associated with the delayed MTX clearance (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) and lower platelet count after treatment in children with MTHFR 677 mutation compared with wild-type ones (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), and pure mutations in ABCB1 3435 were associated with higher serum creatinine levels \u003cem\u003e(P\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). No significant association was identified between MTHFR 677C\u0026thinsp;\u0026gt;\u0026thinsp;T, MTHFR 1298A\u0026thinsp;\u0026gt;\u0026thinsp;C, ABCB1 3435 C\u0026thinsp;\u0026gt;\u0026thinsp;T, and GSTP1 313A\u0026thinsp;\u0026gt;\u0026thinsp;G genes and hepatotoxicity or nephrotoxicity (\u003cem\u003eP\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e). However, the serum metabolomic analysis indicated that the presence of the MTHFR 677C\u0026thinsp;\u0026gt;\u0026thinsp;T gene polymorphism could potentially contribute to delayed MTX clearance by influencing L-phenylalanine metabolism, leading to the occurrence of related toxic side effects.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eMTHFR 677C\u0026thinsp;\u0026gt;\u0026thinsp;T and ABCB1 3435 C\u0026thinsp;\u0026gt;\u0026thinsp;T predicted the risk of delayed MTX clearance during HD-MTX treatment in children with ALL. Serum L-phenylalanine levels were significantly elevated after HD-MTX treatment in children with the MTHFR 677C\u0026thinsp;\u0026gt;\u0026thinsp;T mutation gene.\u003c/p\u003e","manuscriptTitle":"Effects of gene polymorphisms on delayed MTX clearance, toxicity, and metabolomic changes after HD-MTX treatment in children with acute lymphoblastic leukemia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-07-14 15:26:49","doi":"10.21203/rs.3.rs-3151727/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-09-23T18:21:20+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-09-19T05:58:59+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-08-30T13:17:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"c2f26bb0-a459-4971-abaa-8757f7b31d2f","date":"2023-08-25T20:29:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"591a3042-02ee-421b-9201-b22fe44aefcb","date":"2023-08-09T19:54:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"a4ee87fc-0c24-44cd-99f8-c5aa66893dd3","date":"2023-07-31T07:30:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"0958bac2-ae4b-48fb-bb0f-e4a8d72b80cf","date":"2023-07-15T09:54:21+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-07-12T09:52:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-07-12T01:28:02+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-07-12T01:27:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Journal of Pediatrics","date":"2023-07-08T11:37:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-pediatrics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejpe","sideBox":"Learn more about [European Journal of Pediatrics](https://www.springer.com/journal/431)","snPcode":"431","submissionUrl":"https://submission.nature.com/new-submission/431/3","title":"European Journal of Pediatrics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"36efcefd-190c-4040-b938-a4bb16ed7368","owner":[],"postedDate":"July 14th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-23T15:02:14+00:00","versionOfRecord":{"articleIdentity":"rs-3151727","link":"https://doi.org/10.1007/s00431-023-05267-8","journal":{"identity":"european-journal-of-pediatrics","isVorOnly":false,"title":"European Journal of Pediatrics"},"publishedOn":"2023-10-18 15:00:38","publishedOnDateReadable":"October 18th, 2023"},"versionCreatedAt":"2023-07-14 15:26:49","video":"","vorDoi":"10.1007/s00431-023-05267-8","vorDoiUrl":"https://doi.org/10.1007/s00431-023-05267-8","workflowStages":[]},"version":"v1","identity":"rs-3151727","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3151727","identity":"rs-3151727","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}