Introduction

Methotrexate (MTX) is an antitumor drug that is associated with folic acid metabolism used in the treatment of hematological malignancies, autoimmune disease, and others. HD-MTX is commonly employed in regimens for leukemia, lymphoma and osteosarcoma1,2. However, Unpredictable toxicity such as hematological, gastrointestinal, hepatic have also appeared while producing therapeutic effects, which continue to challenge its clinical use3,4,5. Therefore, it is particularly important to identify genetic predictors of MTX-induced toxicity based on pharmacogenetics.

The methylenetetrahydrofolate reductase (MTHFR), which is an essential enzyme in the folic acid cycle as it induces the conversion of 5, 10-methylenetetrahydrofolate into 5-methyltetrahydrofolate, which is the active and predominant form of folate in the circulation6. The gene polymorphism of MTHFR would result in reduced activity of the enzyme, thus affecting the metabolism process of folic acid. Since methotrexate’s mechanism of action is closely linked to folate metabolism, MTHFR gene polymorphism may indirectly influence the efficacy and toxicity of methotrexate7. The transport protein P-glycoprotein, a product of the ABCB1 gene, plays an important role in the absorption and distribution of MTX8. Several published clinical reports conclude that the safety of MTX is related to MTHFR C677T gene polymorphism. Han et al. 9considered that patients with MTHFR 677TT genotype had a significantly higher risk of hematopoietic toxicity compared with those with CC genotype. The meta-analysis revealed that the MTHFR C677T polymorphism was significantly associated with severer liver toxicity induced by HD-MTX treatment in the Asian population10. However, the report by Guo et al. indicated that no significant association was found between the MTX-related toxicities and MTHFR C677T or MTHFR A1298C polymorphisms. On the contrary, the ABCB1 3435TT genotype was associated with increased MTX-related toxicities (leukopenia, neutropenia and oral mucositis)11.

At present, researches has focused on the effect of MTHFR C677T polymorphism on MTX-related toxicity in patients and the results were controversial12,13. The relationship between MTHFR C677T, MTHFR A1298C and ABCB1 C3435T polymorphisms and MTX-related toxicity has not been clarified in the literature. In addition, it has not been clearly determined whether plasma MTX levels are associated with MTHFR C677T, MTHFR A1298C and ABCB1 C3435T polymorphisms in HD-MTX treatment of hematologic malignancies. Thus, our present research aimed to analyze the influence of MTHFR C677T, MTHFR A1298C and ABCB1 C3435T gene polymorphisms on MTX-related toxicity and delay elimination in adult with hematological malignancies during HD-MTX therapy, and to provide guidance for individualized medication during the application of HD-MTX.

Methods

Patients

We retrospectively analyzed the data of patients with hematologic malignancies treated with HD-MTX from January 2020 to December 2023 in Shaanxi Provincial People’s Hospital. The study included patients with acute lymphoblastic leukemia, primary central nervous system lymphoma, Burkitt’s lymphoma, diffuse large B-cell lymphoma, and extranodal NK/T-cell lymphoma. The patients included were: age ≥ 18-years-old, Han ethnicity, diagnosed as a hematological malignancies by clinical examination, don’t have any other malignant disorders, performed the genotype analyses of MTHFR C677T, MTHFR A1298C and ABCB1 C3435T, plasma MTX levels was determined after 48 h and 72 h at least. Exclude patients with severe complications or those using other immunosuppressive agents. Exclude patients who have used any other drugs known to cause similar toxic reactions to methotrexate within 2 weeks prior to the administration of methotrexate.

Ethics statement

This study was conducted in accordance with the Declaration of Helsinki and approved by the Institute Medical Ethics Committee of the Shaanxi Provincial People’s Hospital. The study subjects were informed about the study details and gave written consents to participate.

HD-MTX treatment protocols

All included patients were treated with HD-MTX-based chemotherapy(1 g/m2 ~ 5 g/m2). The medication regimens for the patients are as follows: acute lymphoblastic leukemia (HD-MTX 1 g/m2 + cytarabine 3 g/m22), primary central nervous system lymphoma (rituximab 375 mg/m²+ HD-MTX 3.5 g/m²+ cytarabine 2 g/m²+ thiotepa 30 mg/m², HD-MTX 3.5 g/m2 + cytarabine 2g/m2 ± rituximab 375mg/m2, HD-MTX 3.5 g/m2 + temozolomide 150mg/m2 + rituximab 375mg/m2), Burkitt’s lymphoma (HD-MTX 1 g/m2 + cytarabine 3 g/m2 + rituximab 375mg/m2), diffuse large B-cell lymphoma (rituximab 375 mg/m²+ cyclophosphamide 750 mg/m²+ doxorubicin 40-50 mg/m²+ vincristine 1.4 mg/m²+ prednisone 100 mg + HD-MTX 3–3.5 g/m2), extranodal NK/T-cell lymphoma (L-asparaginase 6000u/m²+ HD-MTX 3 g/m² + dexamethasone 40 mg/d).The 1/10 of the total MTX dose was administered intravenously within the first 30 min, and the remainder was administered at a constant rate within the next 23.5 h. Leucovorin rescue was initiated 12 h after the end of the HD-MTX infusion at a dose of 15 mg/m2, and given every 6 h until the plasma MTX concentration was below 0.1µmol/L. According to clinical guidelines and previous studies, MTX elimination delay was defined as within 48 h plasma MTX level ≥ 1 µmol/L or within 72 h level ≥ 0.1 µmol/L after the patient finishes the routine treatment with MTX in this research14,15. Hydration, alkalization of urine, and organ protection were given 24 h before MTX application, and the maintenance of urine pH > 7 was performed.

Determination of genotyping and serum MTX level

The plasma MTX level was determined by enzyme multiplied immunoassay technique(EMIT) using Siemens Automated blood concentration Monitoring analyzer (Siemens Healthcare, Forchheim, Germany). The gene polymorphisms of MTHFR C677T, MTHFR A1298C and ABCB1 C3435T were acquired via the Fluotech48E fluorescence quantitative analyzer (Tianlong Science and Technology, Xi’an, China) using chromosome fluorescence in situ hybridization.

Toxicity

Toxicity data were collected systematically, blinded to both patient genotype and MTX level. The following toxicities were analyzed: hematologic toxicity (hemoglobin, leukocytes, neutrophils and platelets), oral mucositis, hepatotoxicity (aspartate aminotransferase, alanine aminotransferase, and totalbilirubin), nephrotoxicity (serum creatinine), gastrointestinal toxicity (diarrhea, nausea, vomiting). Toxicity was graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 5.0 when appropriate. Specifically, 0 indicates no toxicity, 1 indicates mild, 2 indicates moderate, 3 indicates severe, 4 indicates life-threatening, and 5 indicates toxicity-related death. Since most grade 1 and a few grade 2 adverse events are reversible, toxicity was stratified into three groups during the analysis: no toxicity (CTCAE grade 0), mild toxicity (CTCAE grade 1–2), and severe toxicity (CTCAE grade 3–4).

Statistics

All data were analyzed by SPSS statistical software version 20.0 (SPSS Inc., Chicago, USA). Categorical data are presented in the form of frequency and percentage, and continuous data are presented in the form of mean ± standard deviation. Allele frequencies of gene polymorphisms were tested for Hardy-Weinberg equilibrium using the chi-square test. The Statistical associations between MTHFR C677T, MTHFR A1298C and ABCB1 C3435T gene polymorphisms and MTX-related toxicity was detected by logistic regression. Chi-square tests were used to evaluate the association between MTHFR C677T, MTHFR A1298C and ABCB1 C3435T gene polymorphisms and MTX elimination delay. Statistical significance was defined as P value <0.05.

Results

Patients

A total of 111 patients fulfilled the inclusion criteria, including 46 males (41.44%) and 65 females (58.56%), with an average age of 48 ± 20 years. Table 1 provides a summary of the characteristics of the patients, their clinical condition and the frequencies of genetic polymorphisms. 63.96% of the patients diagnosed with acute lymphoblastic leukemia, 22.53% of the patients diagnosed with primary central nervous system lymphoma, and the rest included Burkitt’s lymphoma(4.50%), diffuse large B-cell lymphoma(7.21%), extranodal NK/T-cell lymphoma(1.80%). The genotype frequencies were in Hardy-Weinberg equilibrium (C677T variant: x2 = 2.068, P = 0.555; A1298C variant: x2 = 2.592, P = 0.458; C3435T variant: x2 = 3.021, P = 0.388). 51 patients were complicated with endocrine system diseases, 44 patients with Cardiovascular system diseases and 28 patients with nervous system disease when diagnosed. Hepatic toxicity and renal toxicity was the most frequently observed, appearing in 77.48% of the patients, followed by hematopoietic toxicity (71.17%) and gastrointestinal toxicities (29.73%). Mucositis was the least commonly observed toxicity appearing in 19.82% of the patients. Hematopoietic toxicity occurred 5.3 ± 12.7 days after MTX treatment, hepatic toxicity and renal toxicity occurred 3.8 ± 7.6 days after MTX treatment, and gastrointestinal toxicity and mucositis occurred 3.1 ± 6.2 days and 6.0 ± 8.5 days respectively. These toxicities typically occured after 1-2 courses of HD-MTX treatment.

Table 1 Patient main characteristics.
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Association between the gene polymorphisms and toxicity

Association between the MTHFR C677T, MTHFR A1298C and ABCB1 C3435T gene polymorphisms and different toxicity outcomes experienced by the patients are shown in Table 2. There was a significant difference of grade 1–2 hematopoietic toxicity in patients when compared with the MTHFR 677CC genotype and CT genotype (P = 0.043). Significant differences in grade 1–2 hematopoietic toxicity were also observed when comparing MTHFR 1298AA with AC (P = 0.012) and CC (P<0.001), respectively. Although no significant differences were observed, patients carrying the T allele seemed to have a higher frequency of grade 3–4 hematopoietic toxicity. Among the hepatic toxicity and renal toxicity of grade 1–2, there was a significant difference between MTHFR 1298AA and CC(P = 0.004). Significant differences in gastrointestinal toxicities of grade 3–4 were observed when comparing the MTHFR 677CC genotype with the TT genotype (P = 0.027). The MTHFR 1298AA genotype confers greater risk of gastrointestinal toxicities of grade 1–2 when compared with CC (P<0.001). Compared with ABCB1 3435CC, the CT genotype showed a higher incidence rate in grade 3–4 mucositis（P=0.029）. The ABCB1 C3435T gene polymorphism displayed no correlations with hematopoietic toxicity, hepatic toxicity and renal toxicity, gastrointestinal toxicities.

Correlation between the gene polymorphisms and MTX elimination delay

We assessed the relationship between MTX plasma concentration at 48 h and 72 h and the MTHFR C677T, MTHFR A1298C and ABCB1 C3435T gene polymorphisms. As shown in Table 3, the MTX plasma concentration at 48 h(≥ 1µmol/L) of MTHFR CT genotype had higher rate than CC genotype (P = 0.031), suggesting there was a MTX elimination delay in this genotype. A significant effect was observed between MTHFR 677CC and TT genotype of the MTX plasma concentration at 72 h (P = 0.012). For MTHFR A1298C, significant difference were observed between AA and AC genotype at 48 h(P = 0.019). Similarly, a difference was also observed between MTHFR 1298AA and CC genotype at 72 h (P = 0.033). There was no significant difference between the ABCB1 C3435T gene polymorphism and MTX elimination delay.

We then evaluated risk factors for delayed MTX elimination at 48 h. Univariate analysis extracted the potential variables as risk factors for delayed MTX elimination. The results showed that age(≥ 60 years), gender(female), complication disease, AST level were the possible factor affecting MTX delay elimination at 48 h. (Table 4). Multiple logistic regression analysis were performed on these variables (including MTHFR 677CT and MTHFR 1298AC genotype) to identify independent risk factors. The results revealed that age(≥ 60 years) and MTHFR 1298AC genotype were identified as independent risk factors(Table 5).

Discussion

As an antifolate chemotherapeutic agent, MTX has been widely used in the treatment of hematological malignancies. However, toxicities of HD-MTX are considered as a notable concern. In this study, we investigated whether the MTHFR C677T, MTHFR A1298C and ABCB1 C3435T gene polymorphisms correlate with MTX-related toxicity or MTX elimination delay in adult with hematological malignancies during HD-MTX therapy.

Regarding MTHFR C677T, we found that MTHFR C677T polymorphism were associated with hematopoietic toxicity and gastrointestinal toxicity. This conclusion was consistent with some studies. Chang et al.12 showed that significant differences in anemia were observed when comparing the MTHFR 677 CC genotype with the CT + TT genotype. Similarly, Zhou et al.16 evaluated similar findings in their study of MTHFR C677T gene polymorphism. MTX can inhibit dihydrofolate reductase, thereby interfering with DNA synthesis and affecting cell growth and division, especially in rapidly dividing bone marrow cells, thus causing bone marrow suppression. The MTHFR C677T variant reduces the activity of the MTX enzyme, resulting in differences in hematopoietic toxicity17. However, this study did not reveal significant differences in hematopoietic toxicity when compared with the TT genotype. Perhaps because the mild toxicity are usually reversible18, or because effective treatment measures have been taken before a more severe toxicity occurred, there was no significant difference shown in the 3–4 level of toxicity. The results of the meta-analysis revealed that the MTHFR C677T gene polymorphism was correlated with gastrointestinal toxicity19. However, Ebid et al.20 proposed no significant association between the MTHFR C677T polymorphism and any studied toxicity outcomes in patients with hematological malignancies, with racial differences being a key factor for such result discrepancies. The racial difference could also lead to different results21. A study on the occurrence of MTX-related toxicity and MTHFR gene polymorphism in Korean with ALL also found inconsistencies with this article22. Some literature reported that the MTHFR C677T gene polymorphism was associated with the MTX-related toxicities in the Han ethnicity, and the polymorphism may increase the risk of renal and hematologic toxicities, though these results require further validation12.

We found that the The MTHFR 1298CC genotype showed significantly increased risk of hematopoietic, hepatic and renal, gastrointestinal toxicity when compared with AA genotype. Notably, all these correlations are related to mild toxicity, no patients experienced grade 3–4 toxic reactions in our study. This finding is similar to the study reported by Shen et al.23 and El Masri et al.24. The authors proposed that patients carrying the C allele may exhibit a reduced incidence of gastrointestinal and hepatic toxicities, a trend that was similarly identified in our study25. However, others weren’t in agreement with our results. Liu et al.19 and Zhou et al.16 have not found a significant correlation between the MTHFR A1298C gene polymorphism and adverse reactions caused by HD-MTX. The conclusions of previous studies in this field are inconsistent, which may have resulted from small sample sizes, different disease types and different chemotherapy regimens.

Our report indicated that ABCB1 C3435T gene polymorphism was significantly associated with mucositis. Guo et al.11 showed that ABCB1 3435TT was associated with increased MTX-related toxicities (leukopenia, neutropenia and oral mucositis). Han et al.9 identified an important influence of the ABCB1 and MTHFR on MTX-related hematopoietic toxicity in adults with hematological malignancies. Hepatic toxicity was associated with ABCB1 C3435T as reported by Suthandiram et al.26 Although our study found no significant association between ABCB1 C3435T gene polymorphism and other toxicities, we believe that expanding the sample size and subdividing this subgroup into different disease groups may yield statistically significant differences.

The division of oral mucosal cells requires folic acid. Therefore, the interference of MTX with folic acid synthesis may lead to mucosal damage and inflammation27. The previous studies reported that MTHFR and ABCB1 gene polymorphisms were association with MTX-induced mucositis, and even ABCB1 and MTHFR gene polymorphisms has been suggested as a risk predictor for MTX-induced mucositis28. However, our study found no correlation between MTHFR gene polymorphism and mucositis. Some researches came to similar conclusions26. The results may be related to the different diseases included and the small sample size, which is also the limitation of our study.

The present study identified that MTHFR 677CT genotype showed differences at 48 h and TT genotype showed differences at 72 h when compared with the CC genotype, which means patients carrying the T allele may have MTX elimination delay. The literature reported that the MTHFR 677TT genotype was associated with a risk of increased methotrexate plasma levels accumulation via a decreased MTHFR enzyme activity29. Ebid et al.20 found ten times higher risk for delayed elimination in MTHFR 677TT genotype versus the CC genotype. But the result was opposed to data documented by Cwiklinska et al. 30. Our research also indicates that MTHFR 1298AC and CC genotypes were associated with the elimination delay of methotrexate. Wang et al. expressed the same view, suggesting that this might increase the risk of adverse effects29. However, other research showed that there was no significant correlation between MTHFR A1298C gene polymorphism and MTX elimination delay11,23. Additionally, our results are in agreement with a few reports that demonstrated no significant association with MTX plasma levels for ABCB131. However, whether the ABCB1 C3435T gene polymorphism is related to MTX delay elimination is controversial. Zhou et al.16 found ABCB1 C3435T gene polymorphism predicted the risk of delayed MTX clearance in children with ALL. The ABCB1 gene encodes a membrane transporter protein called P-glycoprotein , which acts as an efflux transporter and functional barrier in many different tissues. Another reason may be that besides ABCB1, many other enzymes or transporter proteins jointly affect MTX pharmacokinetic variability.

In this context, we analyzed risk factors for delayed MTX elimination at 48 h. Data collected at 72 h was excluded from discussion and analysis due to absence and deviation, deemed unrepresentative. The results of univariate analysis showed that age(≥ 60 years), gender(female), complication and AST level were the influencing factors of MTX delay elimination. Age(≥ 60 years) and MTHFR 1298AC were identified as independent risk factors. Previous study have shown that age was independent risk factor for delayed MTX elimination32,33. This may be caused by the pharmacokinetic characteristics of MTX. Young patients have higher MTX clearance and lower delayed elimination incidence, while elderly patients have lower clearance and are more prone to delayed elimination. Thus, elderly patients should use HD-MTX more cautiously. Ranchon et al.’s33 study reported that patient’s age and gender were identified as the potential risk factors for delayed methotrexate elimination. Misaka et al.’s34 analysis also showed female gender is an independent risk factor for delayed MTX elimination in children, adolescents and young adults with osteosarcoma. In our study, the the proportion of patient with MTX delayed elimination at 48 h was significantly higher in female than in male. Considering these results, female gender may be a factor that causes a MTX delayed elimination. In consistent with prior studies, the present research also confirmed complication as a risk factor for MTX delayed elimination14,35,36. Patients with hematologic malignancies often have other complications and were treated with related drugs. It is likely that drug-drug interaction have an impact on metabolism of methotrexate, or potentiate methotrexate-induced toxicity. For example, the use of proton pump inhibitors has been reported to have an effect on the elimination of MTX36,37.

We have to admit that there are some limitations in our research that need to be addressed. Firstly, this was a retrospective analysis conducted in a single institution with a relatively small sample size, which may limit a comprehensive assessment of the potential significance of gene polymorphisms and lead to deviations in the research results. Secondly, although all included patients received HD-MTX(1 g/m2 ~ 5 g/m2), there were still variations in the MTX chemotherapy regimens. Different MTX doses, treatment duration may significantly affect the toxicity of MTX. Thirdly, it was often not a single treatment, but rather combined with other toxic drugs, which may affect the accuracy of the analysis results. Since MTX is the standard treatment drug for hematological malignancies in our study, for ethical reasons, we were unable to set up a placebo group to directly compare the incidence of toxicity without MTX. Additionally, previous studies have emphasized that the combined genotypes of MTHFR polymorphism (i.e., the 677CT/1298CC and 677CT/1298AC genotypes of the subjects) can enhance the MTX-related toxicity in ALL patients, and indicate that the MTHFR 677T-1298 C haplotype may be a promising biomarker in ALL38. Based on this perspective, the adverse reactions we observed in this study might be the combined result of the MTHFR polymorphism. Therefore, further studies with a larger sample size, multiple centers, and more detailed information should be conducted to better evaluate the impact of gene polymorphisms on risk.

Conclusion

In conclusion, the present study confirmed that MTHFR C677T, MTHFR A1298C and ABCB1 C3435T gene polymorphisms are associated with MTX-related toxicity in adult with hematological malignancies. Age(≥ 60 years) and MTHFR 1298AC were identified as independent risk factors for delayed elimination of MTX. This reminds us that the MTHFR C677T, MTHFR A1298C and ABCB1 C3435T gene polymorphisms are expected to be a useful tool in reducing toxicity and improving outcome in personalized HD-MTX therapy for patients with hematological malignancies. However, larger and better-designed studies and further evaluations are needed to verify our findings.

Table 2 The correlation between genotyping and toxicity.
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Table 2 (continue) the correlation between genotyping and toxicity.
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Table 3 The correlation between genotyping and MTX concentration.
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Table 4 Univariate logistic regression analysis associated with delayed MTX elimination at 48 h.
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Table 5 Multiple logistic regression analysis of risk factors for delayed MTX elimination at 48 h.
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