Abstract
Background
Reduced folate carrier 1 (RFC1) is an important solute carrier transporter crucial for the uptake and transport of methotrexate (MTX). This meta-analysis aimed to systemically analyze the relationship between the RFC1 G80A polymorphism and MTX-induced toxicity in pediatric acute lymphoblastic leukemia (ALL) patients.
Methods
A systematic search for studies reporting the correlation between the RFC1 G80A polymorphism and MTX-induced toxicity in pediatric ALL patients was performed via the Web of Science, EMBASE, PubMed, Wan Fang, CNKI, and VIP databases until June 16, 2025, followed by pooled analysis. The Newcastle‒Ottawa scale, Egger’s test, and leave‒one-out sensitivity analysis were performed to assess the study quality, publication bias and stability of the pooled results.
Results
Thirteen eligible studies were included in this meta-analysis, with 2, 5, and 6 studies having Newcastle‒Ottawa Scale scores of 7 points, 8 points, and 9 points, respectively, indicating good study quality. The pooled prevalence of the RFC1 80 AA genotype was 28.29% (95% CI: 23.44%-33.15%) in pediatric ALL patients. The risks of overall toxicity (OR = 4.68, 95% CI: 1.96–11.15), myelosuppression (OR = 1.85, 95% CI: 1.30–2.65), hepatotoxicity (OR = 2.44, 95% CI: 1.14–5.21), and gastrointestinal toxicity (OR = 1.61, 95% CI: 1.03–2.52) were greater in pediatric ALL patients with the RFC1 80 AA genotype than in those with the RFC1 80 GG + GA genotype who underwent MTX treatment. However, neurotoxicity risk (OR = 0.98, 95% CI: 0.40–2.41) and mucositis risk (OR = 1.24, 95% CI: 0.81–1.89) were not different between the two groups. No publication bias existed, while some of the pooled results were not stable according to the sensitivity analysis.
Conclusion
The RFC1 G80A polymorphism is closely correlated with elevated MTX-induced toxicity in pediatric ALL patients.
Supplementary Information
The online version contains supplementary material available at 10.1186/s12957-026-04222-9.
Keywords: Pediatric acute lymphoblastic leukemia, Methotrexate, Reduced folate carrier 1, Polymorphism, Toxicity
Introduction
Pediatric acute lymphoblastic leukemia (ALL) is a type of hematological malignancy characterized by the uncontrolled proliferation of immature lymphocytes, with the main peak occurring between 1 and 4 years of age [1, 2]. Its pathogenesis is closely related to chromosomal abnormalities and genetic variations, which affect the differentiation and proliferation of lymphoid precursor cells and are key factors in the occurrence and prognosis of the disease [1, 3]. Currently, much progress has been made in improving the prognosis of patients with pediatric ALL, such as risk stratification therapy, multidrug combination chemotherapy, central nervous system (CNS) prophylaxis, and supportive treatment [4, 5]; however, treatment-related toxicity continues to be a major challenge in the clinical management of pediatric ALL [6, 7].
Methotrexate (MTX), a key chemotherapeutic agent, is a cornerstone in the treatment of pediatric ALL [5, 8]. It primarily acts by inhibiting dihydrofolate reductase, thereby blocking DNA synthesis and effectively targeting rapidly proliferating leukemia cells [9]. In pediatric ALL therapy, MTX is utilized not only during the induction phase to rapidly reduce the leukemic burden but also during the consolidation, delayed intensification, and maintenance phases to prevent disease relapse [5]. Nevertheless, MTX may cause various toxicities during the treatment of pediatric ALL, such as hepatotoxicity, mucositis, bone marrow suppression, nephrotoxicity, neurotoxicity and gastrointestinal toxicity [10, 11]. Among these, hepatotoxicity is one of the most common and serious adverse reactions, and its pathology involves the accumulation of toxic metabolites during MTX metabolism, depletion of intracellular glutathione, and mitochondrial dysfunction [12, 13]. Genetic factors contribute significantly to the development of MTX-related toxicities [10, 12–15]. Currently, several biomarkers are available to help identify or predict the potential toxicity of MTX, which can assist in adjusting treatment regimens, reducing severe adverse events, and thereby improving patients’ treatment tolerance and prognosis [16–18].
Reduced folate carrier 1 (RFC1), also known as solute carrier family 19 member 1 (SLC19A1), is a type of solute carrier transporter (SCT), which is crucial for the uptake and transport of MTX [19, 20]. RFC1 plays a central role in determining intracellular drug accumulation and sensitivity; its expression restores MTX transport in recombinant models, whereas its downregulation leads to impaired uptake and resistance [21, 22]. Clinical evidence confirms a strong correlation between RFC1 expression and MTX accumulation in acute lymphoblastic leukemia cells [23]. Some studies have revealed a connection between the RFC1 G80A polymorphism and MTX-induced toxicity in pediatric ALL patients; however, the results are not consistent, and the sample size was not large in each previous study, leading to the necessity of a pooled analysis [24–36]. Furthermore, a previous meta-analysis indicated that there is no association between the RFC1 G80A polymorphism and MTX toxicity in pediatric ALL patients; however, this meta-analysis was published in 2014, and more recent evidence might have affected the conclusion [37]. Therefore, a meta-analysis involving recent evidence is needed.
On the basis of the above information, this meta-analysis aimed to systemically analyze the correlation of the RFC1 G80A polymorphism with MTX-induced toxicity in pediatric ALL patients.
Materials and methods
Search strategy
A systematic search was performed through online databases, including Web of Science, EMBASE, PubMed, Wan Fang, CNKI, and the VIP Database. The search strategy incorporated controlled vocabulary (MeSH/Emtree terms) and free-text keywords: “RFC1” OR “reduced folate carrier protein” OR “SLC19A1” OR “solute carrier family 19 member 1” OR “rs1051266” in combination with “methotrexate” OR “MTX” AND “acute lymphoblastic leukemia” OR “ALL”. The final database search was executed on June 16, 2025, ensuring that the most current evidence was included. The detailed search strategy for each of the databases is listed in Supplementary Table 1. This study was registered on the PROSPERO registration with the registration ID of 1,251,329 (available at https://www.crd.york.ac.uk/PROSPERO/).
Eligibility criteria
Studies were deemed eligible if they (i) enrolled pediatric ALL patients; (ii) specifically examined the association between the RFC1 G80A polymorphism (AA vs. GG + GA) and MTX-induced toxicity; (iii) reported genotype-stratified toxicity data or provided adequate information for such analyses; (iv) represented original research articles published in peer-reviewed journals; and (v) were available in either English or Chinese. In addition, the exclusion criteria were as follows: (i) reviews, meta-analyses, case reports, or nonhuman studies; (ii) duplicate publications or overlapping patient cohorts; or (iii) studies lacking valid data. Two researchers independently conducted the search and screening of the studies on the basis of the eligibility criteria. Any disagreements were resolved through discussion or with a third researcher for adjudication if needed.
Data collection
For each eligible study, the following general information was systematically extracted: first author name, publication year, study design, nationality, MTX dosage, number of patients, demographic characteristics (mean/median age, sex distribution), and genotyping methods. The collected information was subsequently organized into an Excel with a unified template. In addition, data concerning the RFC1 G80A polymorphism and MTX-induced toxicity in pediatric ALL patients were collected. MTX-induced toxicity included overall toxicity, myelosuppression, hepatotoxicity, gastrointestinal toxicity, neurotoxicity, and mucositis. All the data extracted for this meta-analysis did not involve data conversion. Data from four studies, including both acute lymphoblastic leukemia patients and other types of patients, were extracted individually by sending e-mails to the corresponding author without receiving responses from them [38–41].
Evaluation of study quality
Study quality was rigorously assessed via the Newcastle‒Ottawa Scale, a validated instrument evaluating 3 methodological domains: (1) selection bias (assessing 4 items, maximum 4 points), (2) comparability (evaluating 1 key dimension, maximum 2 points), and (3) ascertainment of exposure (examining 3 aspects, maximum 3 points). Studies achieving a total score ≥ 7 points (out of 9 possible) were classified as high quality [42]. Two researchers independently conducted this part of the study. Any disagreements were resolved through discussion or adjudicated with a third researcher.
Statistics
Statistical analyses were performed via STATA v.17.0 (StataCorp LLC, USA). Pooled genotype frequencies were calculated with 95% confidence intervals (CIs). The association between the RFC1 G80A polymorphism and MTX-induced toxicity (AA vs. GG + GA) was quantified via odds ratios (ORs) with 95% CIs. Between-study heterogeneity was evaluated through Cochran’s Q statistic and the I² index, with I² values > 50% or Q test p values < 0.05 indicating substantial heterogeneity. On the basis of heterogeneity findings, either fixed- or random-effects models were applied accordingly. Potential publication bias was assessed via a trim-and-fill sensitivity analysis. To verify the stability of the results, leave-one-out sensitivity analyses were performed by sequentially excluding individual studies. During the sensitivity analysis, when the significance of the pooled results did not change, it was defined as “stable”; otherwise, it was defined as “fluctuation”. Subgroup analysis and multivariable meta-regression analysis were used to explore possible causes of heterogeneity among the study results. p values < 0.05 were considered significant.
Results
Flowchart of study inclusion
A total of 490 studies were identified from the databases, among which 157 studies were excluded for duplication, 310 studies were excluded by screening the title and abstract, and 10 studies were excluded by screening the full texts, leading to the final inclusion of 13 studies in this meta-analysis (Fig. 1). Data from four studies, including both acute lymphoblastic leukemia patients and other types of patients, were extracted individually by sending e-mails to the corresponding author; however, no response was received from these authors, and these four studies were ultimately excluded [38–41].
Fig. 1.
Flowchart. meta-analysis flowchart presenting the search and screening processes
General information and quality evaluation of the included studies
The 13 studies included in this meta-analysis were published between 2003 and 2025, with sample sizes ranging from 53 to 332 cases and mean/median ages ranging from 4 to 10.2 years (Table 1). A total of 2, 5, and 6 studies had Newcastle‒Ottawa Scale total scores of 7 points, 8 points, and 9 points, respectively, suggesting that the studies were of generally good quality (Table 2).
Table 1.
List of included studies
| First author (year) | Study design | Nationality | MTX dosage (g/m2) | N | Mean/median age (years) | Male | Genotyping methods | Assessments |
|---|---|---|---|---|---|---|---|---|
| Kishi S (2003) [24] | Observational | American | 2.5 or 5 | 53 | 6 | 23 | PCR-Sequencing | Neurotoxicity |
| Pakakasama S (2007) [25] | Observational | Thai | 1.5 | 76 | 5.7 | NR | PCR-RFLP | Myelosuppression |
| Xu J (2009) [26] | Observational | Chinese | 3 or 5 | 101 | 5.3 | 56 | PCR-RFLP | Mucositis, hepatotoxicity, myelosuppression, gastrointestinal toxicity |
| Xu KK (2009) [27] | Observational | Chinese | 3 | 53 | 6.7 | 30 | PCR-RFLP | Overall toxicity, mucositis, hepatotoxicity, myelosuppression, gastrointestinal toxicity |
| Faganel Kotnik B (2010) [28] | Observational | Slovenian | 5 | 60 | 6.2 | 23 | NR | Mucositis |
| Lopez-Lopez E (2011) [29] | Observational | Spanish | 3 or 5 | 115 | 5.5 | 62 | PCR-RFLP | Delayed MTX elimination |
| Li H (2014) [30] | Observational | Chinese | 3 or 5 | 68 | 5.9 | 44 | PCR-Sequencing | Hepatotoxicity, myelosuppression |
| Liu SG (2017) [31] | Observational | Chinese | 2, 3, or 5 | 332 | 4 | 195 | Mass spectrometry | Mucositis |
| Cui YJ (2019) [32] | Observational | Chinese | 3 | 90 | 10.2 | 49 | PCR-RFLP | Mucositis, hepatotoxicity, myelosuppression, gastrointestinal toxicity |
| Wang Y (2019) [33] | Observational | Chinese | 1–3 | 142 | 5.8 | 63 | NR | Mucositis, gastrointestinal toxicity |
| Esmaili MA (2020) [34] | Observational | Iranian | 2–4 | 74 | 6.5 | 46 | PCR-Sequencing | Hepatotoxicity, neurotoxicity |
| Ramalingam R (2022) [35] | Observational | Indian | 3 or 5 | 115 | 6.6 | 76 | PCR-RFLP | Overall toxicity |
| Li M (2025) [15] | Observational | Chinese | 2–4 | 148 | 6 | 89 | PCR-Sequencing | Gastrointestinal toxicity |
MTX Methotrexate, NR Not reported, PCR Polymerase chain reaction, RELP Restriction fragment length polymorphism
Table 2.
Quality evaluation of included studies by Newcastle-Ottawa scale
| First author (year) | Selection bias | Comparability | Ascertainment of exposure | Total score | |||||
|---|---|---|---|---|---|---|---|---|---|
| Item 1 | Item 2 | Item 3 | Item 4 | Item 5 | Item 6 | Item 7 | Item 8 | ||
| Kishi S (2003) [24] | ★ | ★ | ★ | ★ | ★★ | ★ | ★ | 8 | |
| Pakakasama S (2007) [25] | ★ | ★ | ★ | ★ | ★★ | ★ | ★ | ★ | 9 |
| Xu J (2009) [26] | ★ | ★ | ★ | ★ | ★★ | ★ | ★ | 8 | |
| Xu KK (2009) [27] | ★ | ★ | ★ | ★ | ★★ | ★ | ★ | ★ | 9 |
| Faganel Kotnik B (2010) [28] | ★ | ★ | ★ | ★ | ★★ | ★ | ★ | 8 | |
| Lopez-Lopez E (2011) [29] | ★ | ★ | ★ | ★ | ★★ | ★ | ★ | ★ | 9 |
| Li H (2014) [30] | ★ | ★ | ★ | ★ | ★★ | ★ | ★ | ★ | 9 |
| Liu SG (2017) [31] | ★ | ★ | ★ | ★ | ★★ | ★ | ★ | 8 | |
| Cui YJ (2019) [32] | ★ | ★ | ★ | ★ | ★ | ★ | ★ | 7 | |
| Wang Y (2019) [33] | ★ | ★ | ★ | ★ | ★★ | ★ | ★ | 8 | |
| Esmaili MA (2020) [34] | ★ | ★ | ★ | ★ | ★★ | ★ | ★ | ★ | 9 |
| Ramalingam R (2022) [35] | ★ | ★ | ★ | ★ | ★★ | ★ | ★ | ★ | 9 |
| Li M (2025) [15] | ★ | ★ | ★ | ★ | ★ | ★ | ★ | 7 | |
★indicated 1 point
Prevalence of the RFC1 80 AA genotype in pediatric ALL patients
The rates of the RFC1 80 AA genotype in pediatric ALL patients differed among studies, ranging from 18.33% to 43.57%, and pooled analysis revealed that the summarized rate of the RFC1 80 AA genotype was 28.29% (95% CI: 23.44%−33.15%) in pediatric ALL patients (Fig. 2). Owing to the heterogeneity in this finding, a subgroup analysis based on region was carried out to explore the potential source of heterogeneity. Subgroup analysis indicated that the heterogeneity decreased to I2 = 14.7% (P = 0.310) in the non-Asian subgroup, whereas it remained high (I2 = 81.4%; P < 0.001) in the Asian subgroup (Supplementary Table 2). These findings implied that the region might be the source of heterogeneity regarding the prevalence of the RFC1 80 AA genotype in pediatric ALL patients.
Fig. 2.
Prevalence of the RFC1 80 AA genotype. Forest plot presenting pooled analysis of the incidence of the RFC1 80 AA genotype in pediatric ALL patients. A total of 13 studies were included in the analysis
Correlation of the RFC1 G80A polymorphism with MTX-induced toxicity in pediatric ALL patients
Pooled analysis revealed that the overall toxicity risk (OR = 4.68, 95% CI: 1.96–11.15; Fig. 3A) and myelosuppression risk (OR = 1.85, 95% CI: 1.30–2.65; Fig. 3B) were both greater in pediatric ALL patients with the RFC1 80 AA genotype than in those with the RFC1 80 GG + GA genotype who underwent MTX-involved treatment. In addition, hepatotoxicity risk (OR = 2.44, 95% CI: 1.14–5.21; Fig. 4A) and gastrointestinal toxicity risk (OR = 1.61, 95% CI: 1.03–2.52; Fig. 4B) were also elevated in pediatric ALL patients with the RFC1 80 AA genotype compared with those with the RFC1 80 GG + GA genotype who underwent MTX-involved treatment. However, neurotoxicity risk (OR = 0.98, 95% CI: 0.40–2.41; Fig. 4C) and mucositis risk (OR = 1.24, 95% CI: 0.81–1.89; Fig. 4D) were not different between the two groups.
Fig. 3.
Correlation between the RFC1 G80A polymorphism and overall toxicity and myelosuppression. Forest plot presenting pooled analysis of overall toxicity risk (2 studies were included in the analysis) (A) and myelosuppression risk (5 studies were included in the analysis) (B) between cases with the RFC1 80 AA genotype and cases with the RFC1 80 GG + GA genotype in pediatric ALL patients receiving MTX treatment
Fig. 4.
Correlations between the RFC1 G80A polymorphism and hepatotoxicity, gastrointestinal toxicity, neurotoxicity, and mucositis. Forest plot presenting pooled analysis of hepatotoxicity risk (5 studies were included in the analysis) A, gastrointestinal toxicity risk (5 studies were included in the analysis) B, neurotoxicity risk (2 studies were included in the analysis) C, and mucositis risk (6 studies were included in the analysis) (D) between cases with the RFC1 80 AA genotype and cases with the RFC1 80 GG + GA genotype in pediatric ALL patients receiving MTX treatment
Publication bias and results stability
No publication bias existed in the included studies according to the trim-and-fill analysis (Table 3). The pooled results were stable regarding the RFC1 80 AA genotype rate in pediatric ALL patients, myelosuppression risk, and mucositis but fluctuated regarding hepatotoxicity risk and gastrointestinal toxicity (Table 4). Because only two studies were included in the analysis of overall toxicity risk and neurotoxicity risk, leave-one-out sensitivity analysis was not carried out for these two outcomes.
Table 3.
Trim-and-fill analysis of publication bias
| Items | Number of studies | OR (95% CI) |
|---|---|---|
| Overall toxicity | ||
| Observed + Imputed | 2 + 1 | 5.91 (2.82, 12.38) |
| Myelosuppression | ||
| Observed + Imputed | 5 + 0 | 1.85 (1.30, 2.65) |
| Hepatotoxicity | ||
| Observed + Imputed | 5 + 0 | 2.44 (1.14, 5.21) |
| Gastrointestinal toxicity | ||
| Observed + Imputed | 5 + 0 | 1.61 (1.03, 2.52) |
| Neurotoxicity | ||
| Observed + Imputed | 2 + 1 | 0.76 (0.35, 1.65) |
| Mucositis | ||
| Observed + Imputed | 6 + 1 | 1.17 (0.77, 1.77) |
OR Odds ratio, CI Confidence interval
Table 4.
Sensitivity analysis by removing the included study one by one
| Omitted studies | Pooled OR (95% CI) |
|---|---|
| RFC1 80 AA genotype rate in pediatric ALL patients | |
| Kishi S (2003) [24] | 28.61 (26.35, 30.86) |
| Pakakasama S (2007) [25] | 28.73 (26.46, 31.00) |
| Xu J (2009) [26] | 28.73 (26.44, 31.02) |
| Xu KK (2009) [27] | 28.46 (26.22, 30.71) |
| Faganel Kotnik B (2010) [28] | 29.00 (26.73, 31.27) |
| Lopez-Lopez E (2011) [29] | 28.46 (26.17, 30.75) |
| Li H (2014) [30] | 25.90 (23.51, 28.29) |
| Liu SG (2017) [31] | 29.84 (27.33, 32.34) |
| Cui YJ (2019) [32] | 28.57 (26.29, 30.85) |
| Wang Y (2019) [33] | 27.97 (25.71, 30.23) |
| Esmaili MA (2020) [34] | 28.77 (26.49, 31.04) |
| Ramalingam R (2022) [35] | 27.66 (25.38, 29.94) |
| Li M (2025) [15] | 29.31 (26.97, 31.66) |
| Myelosuppression | |
| Pakakasama S (2007) [25] | 1.88 (1.30, 2.73) |
| Xu J (2009) [26] | 1.97 (1.33, 2.90) |
| Xu KK (2009) [27] | 1.89 (1.30, 2.75) |
| Li H (2014) [30] | 1.98 (1.16, 3.36) |
| Cui YJ (2019) [32] | 1.62 (1.11, 2.38) |
| Hepatotoxicity | |
| Xu J (2009) [26] | 2.31 (0.83, 6.41) |
| Xu KK (2009) [27] | 2.05 (0.86, 4.90) |
| Li H (2014) [30] | 2.23 (0.72, 6.89) |
| Cui YJ (2019) [32] | 2.10 (0.83, 5.32) |
| Esmaili MA (2020) [34] | 3.28 (2.15, 5.00) |
| Gastrointestinal toxicity | |
| Xu J (2009) [26] | 1.55 (0.93, 2.59) |
| Xu KK (2009) [27] | 1.49 (0.92, 2.41) |
| Cui YJ (2019) [32] | 1.48 (0.90, 2.46) |
| Wang Y (2019) [33] | 1.79 (1.08, 2.96) |
| Li M (2025) [15] | 1.78 (1.07, 2.98) |
| Mucositis | |
| Xu J (2009) [26] | 1.15 (0.73, 1.80) |
| Xu KK (2009) [27] | 1.15 (0.72, 1.81) |
| Faganel Kotnik B (2010) [28] | 1.28 (0.79, 2.06) |
| Liu SG (2017) [31] | 1.53 (0.93, 2.52) |
| Cui YJ (2019) [32] | 1.15 (0.71, 1.84) |
| Wang Y (2019) [33] | 1.26 (0.80, 1.98) |
OR Odds ratio, CI Confidence interval
Multivariate regression analysis
Multivariate regression analysis was carried out for each type of MTX-induced toxicity. However, all these factors, including nationality, MTX dosage, age, and genotyping methods, had no effect on the association of the RFC1 G80A polymorphism with MTX-induced toxicity (all P > 0.05, Supplementary Table 3).
Discussion
RFC1 is an important SCT, and its gene polymorphism (especially the G80A polymorphism) is implicated in the development of various malignancies [43–49]. With respect to solid malignancies, the RFC1 G80A polymorphism is correlated with increased susceptibility to thyroid cancer, breast cancer, nephroblastoma, and neuroblastoma and reduced susceptibility to colorectal cancer and head and neck cancer [43–46]. With respect to hematological malignancies, the results of previous studies are largely different, and several meta-analyses have shown that the RFC1 G80A polymorphism may be correlated with increased susceptibility to pediatric ALL and acute myelogenous leukemia but not with lymphoma [47–49].
The RFC1 G80A polymorphism is not rare in pediatric ALL patients [24–36]. A previous study reported that the prevalence of the RFC1 80 AA genotype was 18.33% in pediatric ALL patients, and another study reported that the percentage of the RFC1 80 AA genotype was 28.44% in pediatric ALL patients [28, 29]. Moreover, a high percentage of the RFC1 80 AA genotype (43.57%) was identified in a previous study [30]. These data suggest a varied prevalence of the RFC1 G80A polymorphism in pediatric ALL patients from different studies, ranging from 18.33% to 43.57% [24–36]. This meta-analysis revealed that the pooled rate of the RFC1 80 AA genotype was 28.29% (95% CI: 23.44%−33.15%) in pediatric ALL patients, implying a relatively high prevalence and potential value in disease management. Furthermore, the subgroup analysis revealed that region might be the source of heterogeneity regarding the prevalence of the RFC1 80 AA genotype in pediatric ALL patients. In addition, heterogeneity occurred in the Asia subgroup but not in the non-Asia subgroup. These findings imply that population-specific genetic backgrounds, environmental factors, food intake, lifestyle factors, and healthcare practices might be potential impact factors for the RFC1 80 AA genotype and susceptibility to pediatric ALL. In addition, this heterogeneity reminds the investigator to consider ethnic diversity when conducting genetic association studies.
SCTs are membrane proteins that facilitate the transport of a diverse range of solutes (such as nutrients and pharmaceutical compounds) across biological membranes, which play critical roles in metabolic, immunological, and neurological processes [50–52]. Dysregulation of SCTs has been implicated in various diseases, such as cancer and metabolic disorders, and SCTs are known to promote the cellular uptake of anticancer drugs, including MTX [19, 53–55]. As a result, the expression and gene polymorphisms of SCTs have shown potential as biomarkers related to both treatment efficacy and susceptibility to MTX-induced toxicity [19, 36, 56, 57].
RFC1 is a key member of the SCTs closely implicated in MTX-induced toxicity in patients with several diseases, such as rheumatoid arthritis, osteosarcoma, and pediatric ALL [36, 58, 59]. Many studies have focused on the connection between the RFC1 G80A polymorphism and MTX-induced toxicity in pediatric ALL patients [24–36]; however, the results are inconsistent, and sample sizes ranging from 53 to 332 cases are limited. In detail, this meta-analysis revealed that, regarding overall toxicity, one study reported that the RFC1 80 AA genotype was correlated with a higher risk of overall toxicity, whereas another reported not [27, 35]; regarding myelosuppression, 2 studies reported a connection between the RFC1 80 AA genotype and an elevated risk of myelosuppression, but another 3 studies reported no relationship [25–27, 30, 32]; regarding hepatotoxicity, 4 studies disclosed an association between the RFC1 80 AA genotype and an increased risk of hepatotoxicity, whereas another study revealed a nearly opposite result [26, 27, 30, 32, 34] in pediatric ALL patients receiving MTX treatment. This meta-analysis summarized 13 studies [24–36] and revealed that the RFC1 80 AA genotype was correlated with increased risks of overall toxicity, myelosuppression, hepatotoxicity, and gastrointestinal toxicity but was not correlated with the risk of neurotoxicity or mucositis in pediatric ALL patients receiving MTX treatment. These pooled results demonstrated a close correlation between the RFC1 G80A polymorphism and MTX-induced toxicity in pediatric ALL patients. The reason may be that the RFC1 A allele reduces the affinity and transport efficiency of this transporter protein for MTX, resulting in a decrease in intracellular uptake of MTX and an increase in the drug concentration in serum. The increase in serum MTX level will prolong the exposure time of the drug in the body, thereby aggravating adverse reactions such as bone marrow suppression, gastrointestinal and liver toxicity [36, 60]; Meanwhile, the decrease in intracellular MTX concentration may weaken its killing effect on tumor cells, suggesting that the A allele may simultaneously affect the efficacy and toxicity of the drug. Furthermore, the A allele of RFC1 may also affect the expression level and tissue-specific distribution of wild-type RFC1, thereby causing differentiated pharmacokinetic and toxic manifestations in different organs [19, 60]. From a clinical perspective, the RFC1 G80A polymorphism suggests that individualized MTX administration regimens should be considered in pediatric patients with ALL. RFC1 genotyping before treatment is helpful for optimizing dosage, reducing drug toxicity and improving treatment compliance. Future research can combine the RFC1 genotype information with pharmacokinetic models to further clarify the specific mechanism of action of this polymorphism in the in vivo distribution and toxicity risk of MTX.
This meta-analysis has several limitations. First, sensitivity analysis revealed that the overall toxicity risk, hepatotoxicity risk, and gastrointestinal toxicity risk were not stable, possibly because of the limited number of included studies. Second, although not statistically significant, a margin tendency of publication bias existed in the included studies reporting mucositis. Third, most of the included studies (10/13) were published more than 5 years ago, and sufficient recent studies to support the pooled results are lacking. Fourth, the meta-analysis of overall toxicity and neurotoxicity included only 2 studies for each outcome, which indicated that the heterogeneity metrics were unreliable and that the pooled effect size was unstable. Therefore, these findings need further verification. Fifth, a small number of studies focused on several outcomes, such as overall toxicity risk and neurotoxicity risk, which limits the reliability of the pooled analyses and prevents meaningful assessment of publication bias. Sixth, data from four studies that included both acute lymphoblastic leukemia patients and other types of patients were extracted individually by sending e-mails to the corresponding author; however, no response was received from these authors, and these four studies were ultimately excluded. This situation might affect the integrity and reliability of the study findings.
Conclusions
In conclusion, this meta-analysis systemically demonstrated a correlation between the RFC1 G80A polymorphism and elevated MTX-induced toxicity in pediatric ALL patients. This information provides promising evidence that the RFC1 G80A polymorphism is a biomarker for predicting MTX-induced toxicity, assisting in the adjustment of treatment regimens, reducing adverse reactions, and improving the prognosis of pediatric ALL patients. However, caution should be taken when interpreting the correlation of the RFC1 G80A polymorphism with MTX-induced overall toxicity risk and neurotoxicity risk because of the small number of studies, and further studies are needed to verify these findings.
Supplementary Information
Acknowledgements
Not applicable.
Registration and protocol
This study was registered on the PROSPERO registration with the registration ID of CRD420251251329 (available at https://www.crd.york.ac.uk/PROSPERO/).
Abbreviations
- RFC1
Reduced folate carrier 1
- MTX
Methotrexate
- ALL
Acute lymphoblastic leukemia
- CNS
Central nervous system
- SLC19A1
Solute carrier family 19 member 1
- SCTs
Solute carrier transporters
- Ors
Odds ratios
Authors’ contributions
Yihuan Yue: Conceptualization, formal analysis, methodology, project administration, writing - original draft, writing - review & editing; Li Yao: Data curation, investigation, resources, writing - original draft, writing - review & editing; Fuchun Wang: Data curation, investigation, resources, writing - original draft, writing - review & editing; Dongchuan Lai: Data curation, investigation, resources, writing - original draft, writing - review & editing.
Funding
No funding is available for this work.
Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.