HD-MTX plays an important role in the treatment of childhood and adult cancers, such as ALL, lymphoma, and osteosarcoma (Treon and Chabner, 1996). It has been used in consolidation therapy for ALL to overcome resistance and prevent central nervous system (CNS) relapse (Larsen et al., 2016). However, the delayed clearance of HD-MTX induces multiple side effects, including nephrotoxicity, mucositis, hepatotoxicity, and neurotoxicity. To prevent toxicity, proper hydration with alkalinization and leucovorin rescue are administered, while nonsteroidal anti-inflammatory drugs, penicillin, and aspirin-containing medications should be avoided in combination with MTX. Studies emphasized the need to monitor serum MTX levels, perform liver and renal function tests, and decide whether to increase leucovorin or use glucarpidase (carboxypeptidase G2) at the right time (Ramsey et al., 2018). In South Korea, to reduce HD-MTX toxicity, the infusion time was shortened from 24 h to 4 h compared with the COG AALL0232 protocol (Larsen et al., 2016). Despite appropriate efforts, severe renal toxicity occurs in some patients, requiring dialysis and sometimes leading to chronic renal disease. Therefore, many studies have aimed to elucidate the interindividual variability in the pharmacokinetics explained by pharmacogenomics of MTX (Song et al., 2021; Taylor et al., 2021). However, most previous studies were conducted based on candidate gene panel analyses with a few genes or mutations related to the folate pathway or transporter genes. Very few studies have conducted whole-genome or whole exome sequencing (WES).

Racial and ethnic disparities were observed in adverse drug events, and studies involving Asians were fewer compared to other races, such as white people, black people, and Hispanics (Baehr et al., 2015). Despite the growing need to consider racial factors when determining MTX toxicities or responses (Torres et al., 2022; Xu et al., 2022; Liu et al., 2024), no genomic research has been performed on the pharmacokinetics of MTX in childhood ALL in South Korea. In this study, we aimed to investigate the genomic variants related to delayed clearance of HD-MTX using WES in pediatric patients with ALL.

We identified variants that potentially contribute to delayed HD-MTX clearance from serum measurements after HD-MTX infusion in pediatric patients with ALL. Delayed clearance of HD-MTX increases the risk of severe nephrotoxicity and consequently induces severe life-threatening complications in some cases (Howard et al., 2016). Our data also showed a strong correlation between the 24-h MTX level and the subsequent Cr levels. The incidence of delayed clearance was significantly higher in infusions showing max fold Cr than in total infusions (49% vs 23.5%; p = 0.0002). These findings suggest that the peak levels and delayed MTX clearance are associated with subsequent nephrotoxicity.

In this study, 23.5% of the total infusions (80/341 infusions) corresponded to delayed clearance of HD-MTX. AKI occurred in 12.9% of the infusions (44/341 infusions). Delayed MTX clearance has been described in several studies. In a study of pediatric and adult patients with osteosarcoma in China, the frequency of delayed clearance was reported to be 6.19% and 0.86% when delayed and severely delayed clearances were defined as concentration >5 μmol/L at 24 h and concentration >20 μmol/L at 24 h, respectively (Zhang et al., 2016). Another study reported that 22% of patients with lymphoma or patients with leukemia aged 16–84 years experienced delayed clearance of HD-MTX based on the definition of MTX concentration ≥1 μmol/L 48 h or ≥0.1 μmol/L 72 h after HD-MTX (Narita et al., 2017). The frequency of delayed clearance in our study was higher than that reported in other studies, partially because of the broad criteria used.

MTX-induced renal toxicity is caused by the precipitation of MTX and its metabolites in the renal tubules or by the direct toxic effects of MTX on the renal tubules (Garneau et al., 2015). In a meta-analysis of the toxicity of HD-MTX in osteosarcoma, the incidence of grade ≥ II MTX renal toxicity was reported to be 1.5% (range, 0.0%–12.4%) (Widemann et al., 2004). Amitai et al. reported that 13% of patients with hematological malignancies experienced AKI after HD-MTX (median dose, 1941; range, 743–5442 mg/m²) (Amitai et al., 2020). Another retrospective study reported that 38.6% of adult patients with hematological malignancies experience renal toxicity (Wiczer et al., 2016).

In particular, renal disorders can reduce renal function and hinder drug clearance (Garza et al., 2022). Renal clearance of drugs depends on the GFR, renal blood flow (RBF), and tubular function (Richard et al., 2020). MTX toxicity may occur unpredictably even without an underlying patient’s condition or external factors, and genetic polymorphisms, such as transporter-related polymorphisms, may be involved. Therefore, the genetic variants associated with delayed clearance and AKI after HD-MTX infusion must be identified.

In this study, ENG rs1800956 and PKD1L2 rs16954698 were identified as promising biomarkers for predicting delayed MTX clearance. These two variants satisfied at least two of the following criteria: deleterious (SIFT), probably damaging (PolyPhen-2), and deleteriousness (CADD) (Supplementary Table S7). Notably, all criteria were met for ENG rs1800956. Since SIFT, PolyPhen-2, and CADD are prediction tools that evaluate scores based on the protein’s structure, sequence conservation, and functional information (Eilbeck et al., 2017), these two variants could have both structural and functional impacts on the proteins they encode.

Endoglin (ENG) is a transmembrane glycoprotein expressed on endothelial cells that functions as a co-receptor for several ligands of the transforming growth factor beta (TGF-β) family (Rossi et al., 2019). The kidney contains various types of endothelial cells. Renal endothelial function plays a role in maintaining to RBF (Basile and Yoder, 2014) and common types of chronic kidney disease (CKD) are characterized by renal endothelial dysfunction (Jourde-Chiche et al., 2019). Specifically, the glomerular endothelium is highly fenestrated, covered by a rich glycocalyx, and plays a key role in the filtration properties of the glomerular barrier and the maintenance of podocyte structure. Capillary glomerular renal endothelial cells show enriched expression of genes associated with the TGF-β–BMP signaling pathway, including ENG, which is involved in glomerular capillary formation. Overexpression of TGF-β induces proteinuria and glomerulosclerosis (Dumas et al., 2021).

Previous studies have shown that TGF-β plays diverse roles in renal fibrosis and inflammation, and an infusion of TGF-β2 resulted in a gradual decline in renal medullary blood (Spurgeon et al., 2005; Gu et al., 2020). Although the most important mechanism of MTX-induced AKI is intratubular crystal precipitation, decreased RBF or urine flow can also affect drug elimination and AKI (Pentel and Benowitz, 1984; Perazella and Luciano, 2015; Garza et al., 2022). Cyclosporine (CSA), an immunosuppressant used in organ and bone marrow transplants as well as inflammatory conditions, stimulates renal and systemic productions of TGF-β, and experimental and clinical studies have suggested that TGF-β overexpression may be an important factor in the development of CSA nephrotoxicity (Burdmann et al., 2003).

Moreover, ENG protein is highly expressed in kidney (HPA, 2015), and its vascular expression is elevated in animal models of renal injury, potentially contributing to disease severity by promoting endothelial cell activation and inflammation (Bus et al., 2018). ENG is upregulated in various CKD, and interstitial ENG expression is correlated with eGFR (Gerrits et al., 2022). In addition, ENG was downregulated by MTX in monocyte-derived human macrophages (Rios et al., 2023). These imply the involvement of ENG in MTX elimination and nephrotoxicity. In our study, ENG rs1800956 also showed negative (−0.09) and positive (17.79) coefficients in association with MTX levels and 24 h MTX levels, respectively (Supplementary Table S4). This suggests that heterozygotes for this variant have a slower decline in blood MTX concentration. The allele frequency of ENG rs1800956 was higher in Koreans and East Asians than in Europeans with gnomAD (Korea 9.0%, East Asia 10.9%, and Europe 0.1%). The high allele frequency in Koreans might have contributed to the identification of variants as significant in our study.

The polycystic kidney disease 1-like 2 (PKD1L2) gene encodes a polycystin-1-like subfamily, and the protein has ion channel segments that are highly conserved among polycystin-2 family members, suggesting that they possess cation channel functions (Li et al., 2003; Yuasa et al., 2004). According to the Gene Ontology (GO) for PKD1L2, the GO classes included calcium ion (Ca²⁺) transmembrane transport and calcium channel activity (AmiGO, 2017). Ca²⁺ is a critical signaling molecule in kidney development, all kidney cellular functions, and emergence of kidney diseases (Cao et al., 2006). The activation of Ca²⁺ channels regulates renal function by controlling the contraction of the renal vasculature, and Ca²⁺ channel blockers significantly increase the RBF and GFR significantly (Cao et al., 2006). Besides, PKD1L2 was associated with resistance and sensitivity to MTX in the breast cancer and gastric cancer (Udagawa et al., 2020). These studies have indicated that the functions of PKD1L2 are not only relevant to RBF and drug elimination, but also to be related to MTX.

The polymerase iota (POLI, Polι), polymerases eta (Polη), kappa (Polκ), and Rev1 belong to the Y-family of polymerases (McIntyre, 2020). The activities of Polι and other noncanonical human polymerases also appear responsible for the toxicity of nucleoside analogs in human immunodeficiency virus and hepatitis B virus (McIntyre, 2020). In addition, a deficiency of Polι in human cell lines was found to result in an increased sensitivity to oxidative damage, and this evidence suggests a role for pol ι in the cellular response to DNA damage induced by oxidative stress (Parsons et al., 2013). Based on these findings, POLI may be involved in adverse drug reactions as a drug-related gene.

The hypoxic microenvironment, which downregulates calcium transporter proteins and inhibits apoptotic cell signaling, is considered a contributing factor that promotes DNA instability in prostate cancer cells by upregulating NSE1 Homolog, SMC5-SMC6 Complex Component (NSMCE1)-related E3 ubiquitin ligases and inhibiting efficient DNA repair (Luo et al., 2020). NSMCE1 is upregulated in hypoxic environments; however, evidence linking this to drug-induced nephrotoxicity is lacking.

Human myotubularin-related proteins (MTMRs) six and nine belong to the myotubularin family (Clague and Lorenzo, 2005). MTMR6 inhibits the activity of calcium-activated potassium channels (type KCa3.1) and is localized in the plasma membrane (Srivastava et al., 2005; Choudhury et al., 2006; Srivastava et al., 2006). A high MTMR6 expression level has been observed in B-cell lymphoid leukemia cells with increased resistance to irradiation-induced apoptosis, implicating MTMR6 as a regulator of apoptosis (Vallat et al., 2003). According to a previous study (Zou et al., 2009), MTMR9 influences MTMR6 function at multiple levels, such as by increasing its stability, co-localization, and co-expression with MTMR6. Furthermore, the co-expression of MTMR6 and MTMR9 decreased etoposide-induced apoptosis (Zou et al., 2009). MTMR6 and MTMR9 may influence the response to chemotherapeutic agents. However, the frequency of some candidate variants was very rare, so replication analysis could not be performed. Therefore, additional analysis is needed on large scale data for these.

ABCG2 rs2231142, MTHFD1 rs2236225, MTHFR rs1801133, SLCO1B1 rs2306283 and rs4149056, and SLC19A1 rs1051266, which were found to be significant in previous studies, were not statistically significant in our study (Cheng et al., 2021; Schulte et al., 2021; Taylor et al., 2021). Other variants in ABCB1, SLCO1B1, and ABCC2 did not appear to be associated with delayed MTX clearance in our analysis because they were located in introns, were synonymous variants, or were very rare in East Asians.

If MTX toxicity occurs, an immediate increase in leucovorin dose may be helpful (Ackland and Schilsky, 1987). In severe cases, MTX may be removed through hemodialysis. Recombinant bacterial enzyme glucarpidase has been approved by the US Food and Drug Administration for use in patients with delayed MTX clearance. Glucarpidase converts MTX into its inactive metabolites and provides a nonrenal pathway for MTX elimination. Recently, a clinical decision support tool for predicting delayed MTX clearance was introduced (Taylor et al., 2020). However, delayed clearance was not predicted when MTX was administered. Furthermore, glucarpidase are difficult to use in many countries. Pharmacogenetic findings can help predict MTX nephrotoxicity in advance and allow the use of glucarpidase or other alternative chemotherapeutic agents instead of MTX in patients with variants related to delayed clearance.

This study has some limitations. First, because the variants were relatively frequent in Asians, some were not detected in replication cohorts with few Asians and could not be validated. Therefore, further analysis is required with large-scale datasets involving diverse ethnic backgrounds. Second, future research should be conducted using multi-omics data, such as transcriptomic and epigenomic data, to achieve an integrated understanding. Third, experimental work is needed to how the variants or genes affect MTX levels and toxicities. Due to the low variant frequencies and small sample size in our cohort, identifying interactions between candidate variants was challenging. It is also possible that MTX clearance may be delayed by a related gene variant that is fully linked to the ENG rs1800956. Further studies with larger cohorts could help to more clearly elucidate the combined effects of multiple variants, but our study offers a meaningful contribution to understanding individual genetic variability in this context.

To the best of our knowledge, this is the first study to identify the variants associated with MTX nephrotoxicity in Korean pediatric patients with ALL. In conclusion, ENG rs1800956 and PKD1L2 rs16954698 were the most significantly associated with delayed MTX clearance, and other genetic variants were also found. These findings will help us understand HD-MTX-induced nephrotoxicity and reduce adverse reactions caused by treatment modification.