Plasma Methotrexate Levels Research Articles

Involvement of the ABCB1 C3435T Variant but Not the MTHFR C677T or MTHFR A1298C Variant in High-Dose Methotrexate-Induced Toxicity in Pediatric Acute Lymphoblastic Leukemia Patients in China.

It remains unclear whether the MTHFR C677T, MTHFR A1298C and ABCB1 C3435T genetic variants are associated with methotrexate (MTX) elimination delay and high-dose MTX (HD-MTX) toxicities in the treatment of pediatric acute lymphoblastic leukemia (ALL). The aim of our study was to analyze the potential predictive role of MTHFR C677T, MTHFR A1298C and ABCB1 C3435T in toxicities and the relationship between these variants and MTX elimination delay during HD-MTX therapy in pediatric ALL patients. We conducted a retrospective study on ALL patients receiving HD-MTX treatment with available MTHFR C677T, MTHFR A1298C and ABCB1 C3435T genotype and 44-h plasma MTX levels. Logistic regression analyses and chi-square tests were used to assess the relationship between the variants and HD-MTX toxicities and MTX elimination delay. Genotype frequencies were in Hardy-Weinberg equilibrium. MTX elimination delay did not significantly differ between MTHFR C677T and MTHFR A1298C or ABCB1 C3435T. Leukopenia (P=0.028), neutropenia (P=0.034) and oral mucositis (P=0.023) were 6.444-fold, 4.978-fold and 9.643-fold increased, respectively, in ABCB1 C3435T homozygous genotype (TT) patients compared to wild-type (CC) patients. No significant association was found between the toxicities investigated and MTHFR C677T or MTHFR A1298C. This study showed that the ABCB1 C3435T homozygous allele genotype (TT) is associated with increased MTX-related toxicities (leukopenia, neutropenia and oral mucositis). These results may help to distinguish pediatric ALL patients with a relatively high risk of MTX-related toxicities before HD-MTX infusion and optimize MTX treatment.

Selective and reliable fluorometric quantitation of anti-cancer drug in real plasma samples using nitrogen-doped carbon dots after MMIPs solid phase microextraction: Monitoring methotrexate plasma level

A novel selective and reliable ratiometric fluorescence probe has been successfully synthesized for precise, sensitive, and simple quantitation of methotrexate (MTX). Hydrothermal method was employed to fabricate nitrogen-doped carbon dots using Annona squamosa seeds (AS-CDs) as a starting material, and their characteristics were confirmed using transmission electron microscopy (TEM), UV-Vis spectroscopy, fluorescence spectroscopy, X-ray diffractometry (XRD), and Fourier Transform Infrared Spectroscopy (FTIR). The ratiometric fluorometric assay, which is based on measuring the ratio of emissions (F355/F430), has a wide detection range of 5–2000 ng /mL and a limit of detection (LOD, S/N = 3) of 1.5 ng /mL. The developed sensing method was successfully applied to the quantification of MTX in rabbit plasma samples and parenteral formulations, achieving satisfactory recoveries %. Magnetic molecularly imprinted solid-phase microextraction was used for selective extraction of MTX from plasma samples. The pharmacokinetic parameters were successfully determined in real rabbit plasma samples after intravenous administration of MTX. The as-designed probe does not only improve the sensitivity, but also enhances the precision and accuracy of the proposed method. Overall, this study presents a promising approach for the detection of MTX in genuine samples with acceptable degree of selectivity and sensitivity.

Correlation between methylation level of the SLC19A1 promoter region and methotrexate metabolism in adult acute lymphoblastic leukemia.

Aims: To analyze the methylation level in the promoter region of SLC19A1 in adult acute lymphoblastic leukemia (ALL) patients, and explore the relationship between methotrexate (MTX) drug metabolism and SLC19A1 methylation. Methods: The methylation levels of the SLC19A1 promoter region in 52 adult ALL patients who received high-dose MTX chemotherapy were retrospectively analyzed in combination with clinical indicators and plasma MTX concentration. Results: Methylation levels of 17 CpG units were differently correlated with clinical parameters of ALL patients including gender, age, immunophenotype and Philadelphia chromosome status. Patients with delayed MTX drug excretion had higher methylation levels in the SLC19A1 promoter region. Conclusion: The methylation may affect the MTX plasma level and adverse reactions, which may predict patients at risk of adverse reactions after high-dose MTX therapy.

Association between high-dose methotrexate-induced toxicity and polymorphisms within methotrexate pathway genes in acute lymphoblastic leukemia.

Methotrexate (MTX) is a folic acid antagonist, the mechanism of action is to inhibit DNA synthesis, repair and cell proliferation by decreasing the activities of several folate-dependent enzymes. It is widely used as a chemotherapy drug for children and adults with malignant tumors. High-dose methotrexate (HD-MTX) is an effective treatment for extramedullary infiltration and systemic consolidation in children with acute lymphoblastic leukemia (ALL). However, significant toxicity results in most patients treated with HD-MTX, which limits its use. HD-MTX-induced toxicity is heterogeneous, and this heterogeneity may be related to gene polymorphisms in related enzymes of the MTX intracellular metabolic pathway. To gain a deeper understanding of the differences in toxicity induced by HD-MTX in individuals, the present review examines the correlation between HD-MTX-induced toxicity and the gene polymorphisms of related enzymes in the MTX metabolic pathway in ALL. In this review, we conclude that only the association of SLCO1B1 and ARID5B gene polymorphisms with plasma levels of MTX and MTX-related toxicity is clearly described. These results suggest that SLCO1B1 and ARID5B gene polymorphisms should be evaluated before HD-MTX treatment. In addition, considering factors such as age and race, the other exact predictor of MTX induced toxicity in ALL needs to be further determined.

Effects of SLCO1B1 on elimination and toxicities of high-dose methotrexate in pediatric acute lymphoblastic leukemia.

Aim: To evaluate the association between SLCO1B1 polymorphisms and elimination/toxicities of high-dose methotrexate (MTX). Methods: SLCO1B1 rs11045879 and rs4149056 polymorphisms were retrospectively genotyped in 301 children with newly diagnosed acute lymphoblastic leukemia. MTX concentration, doses of leucovorin rescueand toxicities were recorded. Results: SLCO1B1 rs11045879C carriers (CC+CT) had higher plasma MTX levels at 96 hr, and longer MTX elimination time. The number of leucovorin rescue doses in rs4149056C carriers (CC+CT) was more than those in TT ones. Moreover, SLCO1B1 polymorphisms were associated with HDMTX toxicities including thrombocytopenia, renal toxicityand anal mucositis, but not associated with MTX level at other time points or delayed elimination. Conclusions: Our data demonstrate that genotyping of SLCO1B1 might be useful to optimize MTX therapy.

Overview of Methotrexate Toxicity: A Comprehensive Literature Review.

Methotrexate (MTX) is significantly more effective than and has a considerable advantage over placebo in patients with severe and persistent rheumatoid arthritis (RA). The drug is used to treat a variety of malignant disorders (leukemia and cancer of the lung, breast, and uterus) and ectopic pregnancy. As its side effects are outweighed by its effectiveness, MTX is a first-line antirheumatic drug in many countries. MTX is found in extracellular compartments, such as the synovium, as well as other organs, such as the kidney and liver. To improve treatment, increase adherence, and decrease mortality in MTX therapy, it is essential to reduce its toxicity and understand its side effects. Therefore, this comprehensive review was conducted to assist physicians and researchers in better understanding the toxicity of MTX and how to deal with this toxicity. MTX is eliminated via the kidneys, which are capable of excretion and reabsorption within the renal tubules. Although higher doses of MTX (known as high-dose MTX (HD-MTX), defined as doses of 500 mg/m2 or greater) are often more beneficial, they can produce toxicity and side effects such as bone marrow suppression, pulmonary toxicity, nephrotoxicity, hematologic toxicity, and an increased risk of infections. Treatment of severe MTX toxicity has three main goals: clearance of MTX from the bloodstream, folinic acid therapy, and organ treatment. Leucovorin is highly beneficial in preventing myelosuppression, gastric toxicity, and neurotoxic effects after HD-MTX therapy. The preferred antidote for MTX poisoning is folinic acid. Glucarpidase has been licensed for the treatment of high plasma MTX levels of >1 μmol/L in patients with compromised renal function who have delayed MTX elimination. In patients with renal deficiency, a lower initial dose is considered with an estimated glomerular filtration rate (eGFR) between 30 and 59 mL/minute. These patients need to be monitored, and a more gradual dosage increase and a lower weekly maximum should be considered regarding their general health situation. MTX is contraindicated in patients with RA if the eGFR is <30 mL/minute.

Is hypoalbuminemia a risk factor for high-dose methotrexate toxicity in children with acute lymphoblastic leukemia?

BackgroundRepeated high-dose methotrexate (HDMTX) is a critical component of contemporary childhood acute lymphoblastic leukemia (ALL) treatment regimens. Serum albumin is considered a carrier of methotrexate (MTX) in the blood. Hypoalbuminemia is not a rare finding in children with leukemia. This study aimed to investigate the relationship between pre-infusion serum albumin and possible HDMTX toxicities.MethodsThirty Egyptian children with ALL were consecutively enrolled in the study between May 2018 and July 2020. They were prospectively followed up while receiving HDMTX during the consolidation phase of the TOTAL study XV protocol. HDMTX was administered intravenously as a 24-h infusion every 2 weeks. Doses of 2.5 g/m2 were used for low-risk patients and 5 g/m2 for standard/high-risk patients. The Common Terminology Criteria for Adverse Events (V.4.03) was used to report the observed toxicities after HDMTX cycles. Plasma MTX levels were estimated at 24 h (MTX24) from the beginning of HDMTX infusion in the first consolidation cycle. Serum albumin level was determined before HDMTX administration, and pre-infusion hypoalbuminemia was defined when serum albumin was <3.5 g/dL.ResultsThe patients’ age ranged from 2.3 to 13.3 years at diagnosis, and most of them had B cell ALL (86.7%). Overall, 120 HDMTX cycles were analyzed, equally distributed between low and standard/high risk. Grade 3–4 anemia, grades 3–4 thrombocytopenia, febrile neutropenia, and oral mucositis were significantly more frequent in HDMTX cycles with pre-infusion hypoalbuminemia than those with normal pre-infusion albumin (p=0.003, p=0.007, p=0.006, and p=0.001, respectively). In addition, pre-infusion hypoalbuminemia was significantly associated with additional hospitalization due to HDMTX toxicity (p=0.031). Most HDMTX toxicities were comparable irrespective of the MTX dose. Oral mucositis was more frequently encountered in the 2.5 g/m2 than the 5 g/m2 HDMTX cycles (46.7 vs. 26.7%, p=0.023). A significantly longer hospitalization (due to HDMTX toxicity) was observed in the 5 g/m2 HDMTX cycles (median= 7 days vs. 4 days, p=0.012).ConclusionsSerum albumin levels should be checked before starting HDMTX cycles, especially in resource-limited settings where malnutrition is common, and serum MTX monitoring may not be available. Optimizing serum albumin levels before HDMTX may help decrease the possibility of HDMTX toxicities.

Analysis of Drug Metabolizing Gene Panel in Osteosarcoma Patients Identifies Association Between Variants in SULT1E1, CYP2B6 and CYP4F8 and Methotrexate Levels and Toxicities.

High-dose methotrexate is a cornerstone agent in the chemotherapeutic treatment of patients with osteosarcoma. However, patients often develop methotrexate-induced toxicities. We aim to identify determinants of methotrexate-induced toxicities in osteosarcoma patients by investigating the relation between drug plasma levels, methotrexate-induced toxicities, and germline variants in genes related to drug absorption, distribution, metabolism, and elimination. A cohort of 114 osteosarcoma patients was genotyped for 1,931 variants in 231 genes using the Drug Metabolism Enzymes and Transporters Plus array. Methotrexate plasma levels and laboratory measurements during and after high-dose methotrexate treatment concerning renal function, liver damage, and myelopoiesis to reflect toxicity outcomes were obtained. One hundred and thirteen patients and a subset of 545 variants in 176 genes passed quality control checks. Methotrexate plasma levels showed associations with creatinine, alanine aminotransferase, and hemoglobin. Genetic variant rs3736599 in the 5’-untranslated region of SULT1E1 was associated with lower 48 hour methotrexate plasma levels [coef -0.313 (95% CI -0.459 – -0.167); p = 2.60 × 10-5]. Association with methotrexate-induced decreased thrombocyte counts was found for two intronic variants in CYP2B6 {rs4803418 [coef -0.187 (95% CI -0.275 – -0.099); p = 3.04 × 10-5] and rs4803419 [coef -0.186 (95% CI -0.278 – -0.093); p = 8.80 × 10-5]}. An association with increased thrombocyte counts was identified for the intronic variant rs4808326 in CYP4F8 [coef 0.193 (95% CI 0.099 – 0.287); p = 6.02 × 10-5]. Moreover, a secondary analysis with a binary approach using CTCAE toxicity criteria resulted in a nominal significant associations (p < 0.05) for two out of three variants (rs4803418 and rs4808326). This is the first study to identify genetic variants in SULT1E1, CYP2B6, and CYP4F8 to be associated with methotrexate pharmacokinetics and toxicities. Validation of these variants in an independent cohort and further functional investigation of variants in the identified genes is needed to determine if and how they affect methotrexate plasma levels and the development of methotrexate-induced toxicities.

MTXPK.org: A Clinical Decision Support Tool Evaluating High-Dose Methotrexate Pharmacokinetics to Inform Post-Infusion Care and Use of Glucarpidase.

Methotrexate (MTX), an antifolate, is administered at high doses to treat malignancies in children and adults. However, there is considerable interpatient variability in clearance of high‐dose (HD) MTX. Patients with delayed clearance are at an increased risk for severe nephrotoxicity and life‐threatening systemic MTX exposure. Glucarpidase is a rescue agent for severe MTX toxicity that reduces plasma MTX levels via hydrolysis of MTX into inactive metabolites, but is only indicated when MTX concentrations are > 2 SDs above the mean excretion curve specific for the given dose together with a significant creatinine increase (> 50%). Appropriate administration of glucarpidase is challenging due to the ambiguity in the labeled indication. A recent consensus guideline was published with an algorithm to provide clarity in when to administer glucarpidase, yet clinical interpretation of laboratory results that do not directly correspond to the algorithm prove to be a limitation of its use. The goal of our study was to develop a clinical decision support tool to optimize the administration of glucarpidase for patients receiving HD MTX. Here, we describe the development of a novel 3‐compartment MTX population pharmacokinetic (PK) model using 31,672 MTX plasma concentrations from 772 pediatric patients receiving HD MTX for the treatment of acute lymphoblastic leukemia and its integration into the online clinical decision support tool, MTXPK.org. This web‐based tool has the functionality to utilize individualized demographics, serum creatinine, and real‐time drug concentrations to predict the elimination profile and facilitate model‐informed administration of glucarpidase.

Evaluation of possible pharmacokinetic interaction between methotrexate and proton pump inhibitors in rats.

Methotrexate (MTX), an antifolate agent, is primarily eliminated by the kidney. Organic anion transporter 3 (OAT3) contributes to renal MTX clearance. Several studies have shown an association between co-administration of proton pump inhibitors (PPIs) and delayed elimination of MTX, but the findings are conflicting. In this study, we aimed to evaluate whether the differential inhibitory effects of PPIs on the OAT3-mediated transport of MTX are associated with the risks of delayed MTX elimination. We investigated the effects of PPIs on rat (r) OAT3-mediated MTX uptake using HEK293T cells expressing rOAT3. To examine whether PPIs could affect the pharmacokinetics of MTX, changes in plasma concentration-time profiles were assessed when MTX (50mg/kg, ip) and a range of PPIs (2mg/kg, iv) were administered to rats. In vitro studies demonstrated that PPIs inhibited rOAT3-mediated uptake of MTX, with estimated IC50 values of 2.1-5.2μM, and a rank order of esomeprazole ≈ lansoprazole ≈ omeprazole > rabeprazole. When MTX and esomeprazole were co-administered to rats, the plasma concentration of MTX 6h after administration and the t1/2 were significantly higher than those in the vehicle group. The effect of lansoprazole was not significant, but showed a tendency to prolong plasma MTX levels. Famotidine, a histamine H2-receptor antagonist, showed a weak inhibitory effect on rOAT3-mediated MTX uptake, although it did not affect plasma concentration-time profile of MTX in vivo. Esomeprazole increases the t1/2 of MTX in rats, which may be partially attributed to the inhibition of rOAT3.

Polymorphisms within methotrexate pathway genes: Relationship between plasma methotrexate levels, toxicity experienced and outcome in pediatric acute lymphoblastic leukemia.

Objective(s):The current study aimed to investigate the relationship of genetic polymorphism and plasma methotrexate (MTX) levels, toxicity experience and event free survival (EFS) in pediatric acute lymphoblastic leukemia (ALL). Materials and Methods:The study included 74 ALL patients. Polymerase chain reaction and genotyping of methylene tetrahydrofolate reductase (MTHFR) rs1801133, MTHFR rs1801131, ATP-binding cassette superfamily B1 (ABCB1) rs1045642, ATP-binding cassette superfamily G2 (ABCG2) rs2231142 and solute carrier 19A1 (SLC19A1) rs1051266 genetic variations were performed. The plasma MTX levels were investigated at 48 hr after the first dose of MTX infusion.Results:MTHFR rs1801133 TT genotype, ABCBa1 rs1045642 CT genotype and ABCG2 rs2231142 CA genotype revealed a statistically significant association with the MTX plasma levels (P<0.01, P<0.05, P<0.05, respectively). The MTHFR rs1801133 TT genotype had a statistically significant association with hematopoietic toxicity (P<0.01) and interventions (P<0.05). The MTHFR rs1801131 AC genotype was related to the decreased hepatic toxicity (P<0.05). The SLC19A1 rs 1051266 GA genotype was related to the increased hepatic toxicity (P<0.05). Only the ABCB1 rs1045642 CT and TT genotypes had a statistically significant correlation with EFS (P<0.05, P<0.05, respectively). Conclusion:Our findings showed that genetic polymorphism could be associated with plasma MTX levels, toxicity experienced and EFS in Iranian pediatric ALL.

Influence of plasma methotrexate level and MTHFR genotype in Korean paediatric patients with acute lymphoblastic leukaemia

Whether plasma MTX concentrations and MTHFR C677T and A1298C polymorphisms could be used as a predictor of occurrence of MTX-related toxicities in Korean paediatric patients with acute lymphoblastic leukaemia (ALL) were assessed. HD-MTX related toxicities, MTHFR polymorphisms and MTX plasma concentrations following 337 HD-MTX cycles to 117 children with ALL on maintenance therapy were analyzed. A significantly higher frequency of hyperbilirubinemia (P = 0.0443) and renal toxicity (P = 0.0107) were associated with high MTX concentrations by Fisher’s exact test. Moreover, high MTX concentrations at 24 h, 48, and 72 h were significantly associated with increased frequency of vomiting (P < 0.05) and hyperbilirubinemia (P < 0.05) by Mann-Whitney U test. There was a significantly higher frequency of mucositis in patients with the MTHFR 677 TT genotype (P = 0.0273) and a significantly higher frequency of MTX dose reduction in patients with the 677 TT genotype (P = 0.0217), compared to the CC/CT genotype. Independently, plasma MTX concentrations and MTHFR C677T genotype could be useful markers for tailoring MTX dosing and monitoring adverse effects in childhood ALL HD-MTX therapy in Korean patients.

Methotrexate-associated toxicity in children with Down syndrome and acute lymphoblastic leukemia during consolidation therapy with high dose methotrexate according to ALL-BFM treatment regimen.

Children with Down syndrome (DS) and acute lymphoblastic leukemia (ALL) often suffer from severe toxicities during treatment, especially with high-dose methotrexate (HD-MTX). Systematic data on methotrexate (MTX) toxicity in these patients are rare. We analyzed seven MTX-associated toxicities during consolidation therapy in 103 DS- and 1,109 non-DS-patients (NDS) with ALL (NDS-ALL) enrolled in ALL-Berlin–Frankfurt–Münster (ALL-BFM) trials between 1995–2016 and 1995–2007, respectively. Patients received four courses MTX (5 g/m2 each) plus intrathecal MTX and 6-mercaptopurine (6-MP). From 2004 onwards, a dose of 0.5 g/m2 in the first MTX course has been recommended for DS-patients. DS-patients showed higher rates of grade 3/4 toxicities after the first course with 5 g/m2 MTX compared to NDS-patients (grade 3/4 toxicities 62 in 45 DS-patients vs. 516 in 1,089 NDS-patients, P<0.001). The dose reduction (0.5 g/m2) in DS-patients has reduced toxicity (39 in 51 patients, P<0.001) without increasing the relapse risk (reduced dose, 5-year cumulative relapse incidence = 0.09±0.04 vs. high dose, 0.10±0.05, P=0.51). MTX dose escalation to 1.0 g/m2 for DS-patients who tolerated 0.5 g/m2 (n= 28 of 51 patients) did not result in an increased rate of grade 3/4 toxicities after the second course (P=0.285). Differences in MTX plasma levels at 42 and 48 hours after the start of the first methotrexate infusion did not explain higher toxicity rates in DS-patients treated with 0.5 g/m2 compared to NDS-patients treated with 5 g/m2. Within the DS cohort a higher MTX plasma level was associated with increased toxicity. In conclusion, dose reduction in the first MTX course reduced severe toxicities without increasing the risk of relapse. (ClinicalTrials.gov identifier: NTC00430118, NCT01117441).

Methotrexate Disposition in Pediatric Patients with Acute Lymphoblastic Leukemia: What Have We Learnt From the Genetic Variants of Drug Transporters.

Methotrexate (MTX) is one of the leading chemotherapeutic agents with the bestdemonstrated efficacies against childhood acute lymphoblastic leukemia (ALL). Due to the narrow therapeutic range, significant inter- and intra-patient variabilities of MTX, non-effectiveness and/or toxicity occur abruptly to cause chemotherapeutic interruption or discontinuation. The relationship between clinical outcome and the systemic concentration of MTX has been well established, making the monitoring of plasma MTX levels critical in the treatment of ALL. Besides metabolizing enzymes, multiple transporters are also involved in determining the intracellular drug levels. In this mini-review, we focused on the genetic polymorphisms of MTX-disposition related transporters and the potential association between the discussed genetic variants and MTX pharmacokinetics, efficacy, and toxicity in the context of MTX treatment. We searched PubMed for citations published in English using the terms "methotrexate", "transporter", "acute lymphoblastic leukemia", "polymorphisms", and "therapeutic drug monitoring". The retrieval papers were critically reviewed and summarized according to the aims of this mini-review. Solute carrier (SLC) transporters (SLC19A1, SLCO1A2, SLCO1B1, and SLC22A8) and ATP-binding cassette (ABC) transporters (ABCB1, ABCC2, ABCC3, ABCC4, ABCC5, and ABCG2) mediate MTX disposition. Of note, the influences of polymorphisms of SLC19A1, SLCO1B1 and ABCB1 genes on the clinical outcome of MTX have been extensively studied. Overall, the data critically reviewed in this mini-review article confirmed that polymorphisms in the genes encoding SLC and ABC transporters confer higher sensitivity to altered plasma levels, MTX-induced toxicity, and therapeutic response in pediatric patients with ALL. Pre-emptive determination may be helpful in individualizing treatment.

The effect of intravenous hydration strategy on plasma methotrexate clearance during intravenous high-dose methotrexate administration in pediatric oncology patients.

BackgroundHigh-dose methotrexate (HD-MTX) is widely used as a standard chemotherapeutic agent in pediatric cancers. Most research studies have confirmed the therapeutic efficacy of HD-MTX; however, strategies to prevent side effects vary among institutions, especially in developing countries, with limited monitoring of plasma methotrexate level.ObjectiveTo evaluate the effect of intravenous hydration during HD-MTX administration on plasma methotrexate clearance in pediatric oncology patients.Materials and methodsThis study retrospectively reviewed 165 courses of HD-MTX administered to children with acute lymphoblastic leukemia (ALL), non-Hodgkin’s lymphoma (NHL), or osteosarcoma. Demographic data of patients were collected. Adverse complications related to HD-MTX and 72-hour plasma methotrexate level were analyzed between patients receiving intravenous hydration ≥3,000 mL/m2/day and those receiving hydration <3,000 mL/m2/day.ResultsAmong 56 HD-MTX (1.5 g/m2) courses in ALL, delayed methotrexate clearance was only found in one course administered with hydration <3,000 mL/m2/day. However, no correlation was observed between adverse complications and methotrexate levels. Of 34 HD-MTX (1.5–3 g/m2) courses in NHL, no significant correlation was observed between methotrexate levels and intravenous hydration. However, increased adverse complications were found in the course with delayed methotrexate clearance. Interestingly, among 75 HD-MTX (10–12 g/m2) courses in osteosarcoma, normal methotrexate clearance was successfully achieved in all courses administered with hydration ≥3,000 mL/m2/day compared with those administered with hydration <3,000 mL/m2/day (P=0.007). Furthermore, the courses administered with hydration <3,000 mL/m2/day and had delayed methotrexate clearance were more likely to develop adverse complications.ConclusionIntravenous hydration of ≥3,000 mL/m2/day during HD-MTX administration is essentially required in osteosarcoma and can be considered as optional in ALL with HD-MTX <1.5 g/m2, especially in developing countries with limited monitoring of plasma methotrexate level.

Monitorización terapéutica de metotrexato en pacientes leucémicos mediante cromatografía líquida de alta resolución. Desarrollo, validación y aplicación clínica del método analítico

IntroductionTherapeutic drug monitoring of methotrexate (MTX) is a practice used during acute lymphoblastic leukaemias treatments in order to adjust the leucovorin rescue dose and avoid toxicity. The aim of this work was to evaluate the performance of a bioanalytical method based on liquid chromatography in the control of paediatric acute lymphoblastic leukaemia treatments. Materials and methodsMTX plasma concentrations were evaluated in a total of 61 patients, divided in 2 groups according to the treatment: 24h infusion and 36h infusion. The method used 250μL aliquots of pre-processed plasma, using a validated analytical method, in an Agilent 1260 chromatograph with a 5μm Zorbax Eclipse XDB-C18 column (4.6×150mm) and a mobile phase composed of acetonitrile and 0.05mol/L of a sodium acetate buffer solution at pH 3.5. The detection was performed at 305nm. ResultsThe method did not show interferences by metabolites or other drugs. The lower limit of quantitation was 0.05μmol/L. Inter- and intra-assay precision and accuracy were acceptable in the working range (0.05-5.0μmol/L), and linear behaviour was observed. Toxic plasma levels of MTX were observed in 29.4% and 60% of the patients from 24h infusion and 36h infusion, respectively. Therefore, leucovorin rescue treatment was either applied earlier or reinforced. ConclusionsThe chromatography method was efficient and accessible for the therapeutic drug monitoring of MTX in paediatric patients, allowing appropriate medical decision making within a short time of analysis.

The role of the MTHFR C677T polymorphism in methotrexate-induced toxicity in pediatric osteosarcoma patients.

Osteosarcoma patients receive high doses of methotrexate (MTX). However, pharmacogenetic information remains limited and has mainly been investigated in leukemia so far. We investigated the link between the MTHFR C677T genotype, toxicity levels (mucositis, MTX plasma level, hematological toxicity and hepatotoxicity) and survival of 48 pediatric osteosarcoma patients. The TT genotype did not show more toxicity compared with the CC/CT genotype. However, plasma MTX levels were related with mucositis, but not with hematological toxicity, nor hepatotoxicity. Survival rates did not differ between homozygous and non-homozygous patients. Yet, homozygous patients had higher relapse risk. The MTHFR C667T polymorphism is not predictive for toxicity or overall survival, but could be used for relapse risk stratification.

Polymorphisms in methotrexate transporters and their relationship to plasma methotrexate levels, toxicity of high-dose methotrexate, and outcome of pediatric acute lymphoblastic leukemia.

High-dose methotrexate (HDMTX) plays an important role in the treatment of acute lymphoblastic leukemia (ALL) although there is great inter-patient variability in the efficacy and toxicity of MTX. The relationship between polymorphisms in genes encoding MTX transporters and MTX response is controversial. In the present study, 322 Chinese children with standard- and intermediate-risk ALL were genotyped for 12 polymorphisms. SLCO1B1 rs10841753 showed a significant association with plasma MTX levels at 48 h (P = 0.017). Patients who had the ABCB1 rs1128503 C allele had longer duration of hospitalization than did those with the TT genotype (P = 0.006). No association was found between oral mucositis and any polymorphism. Long-term outcome was worse in patients with the SLCO1B1 rs4149056 CC genotype than in patients with TT or TC (5-year event-free survival [EFS] 33.3 ± 19.2% vs. 90.5 ± 1.7%, P < 0.001), and was worse in patients with the SCL19A1 rs2838958 AA genotype than in patients with AG or GG (5-year EFS 78.5 ± 4.6% vs. 92.2 ± 1.8%, P = 0.008). Multiple Cox regression analyses revealed associations of minimal residual disease (MRD) at day 33 (hazard ratio 3.458; P = 0.002), MRD at day 78 (hazard ratio 6.330; P = 0.001), SLCO1B1 rs4149056 (hazard ratio 12.242; P < 0.001), and SCL19A1 rs2838958 (hazard ratio 2.324; P = 0.019) with EFS. Our findings show that polymorphisms in genes encoding MTX transporters substantially influence the kinetics and response to HDMTX therapy in childhood ALL.

1398 - SNPS in microRNAs associated with methotrexate plasma levels in Spanish children with acute lymphoblastic leukemia

1398 - SNPS in microRNAs associated with methotrexate plasma levels in Spanish children with acute lymphoblastic leukemia

MiR-pharmacogenetics of methotrexate in childhood B-cell acute lymphoblastic leukemia.

Methotrexate (MTX), the key drug in childhood B-cell acute lymphoblastic leukemia (B-ALL) therapy, often causes toxicity. An association between genetic variants in MTX transport genes and toxicity has been found. It is known that these transporters are regulated by microRNAs (miRNAs), and miRNA single nucleotide polymorphisms (SNPs) interfere with miRNA levels or function. With regard to B-cell ALL, we have previously found rs56103835 in miR-323b that targets ABCC4 associated with MTX plasma levels. Despite these evidences and that nowadays a large amount of new miRNAs have been annotated, studies of miRNA polymorphisms and MTX toxicity are almost absent. Therefore, the aim of this study was to determine whether there are other variants in miRNAs associated with MTX levels. Blood samples of 167 Spanish patients with pediatric B-cell ALL treated with the LAL-SHOP protocol were analyzed. We selected all the SNPs described in pre-miRNAs with a minor allele frequency more than 1% (213 SNPs in 206 miRNAs) that could regulate MTX transporters because the miRNAs that target MTX transporter genes are not completely defined. Genotyping was performed with VeraCode GoldenGate platform. Among the most significant results, we found rs56292801 in miR-5189, rs4909237 in miR-595, and rs78790512 in miR-6083 to be associated with MTX plasma levels. These miRNAs were predicted, in silico, to regulate genes involved in MTX uptake: SLC46A1, SLC19A1, and SLCO1A2. In this study, we detected three SNPs in miR-5189, miR-595, and miR-6083 that might affect SLC46A1, SLC19A1, and SLCO1A2 MTX transport gene regulation and could affect MTX levels in patients with pediatric B-cell ALL.