Blood Res 2016; 51(1):
Published online March 31, 2016
https://doi.org/10.5045/br.2016.51.1.50
© The Korean Society of Hematology
1Department of Pediatrics, Haeundae Paik Hospital, Inje University College of Medicine, Busan, Korea.
2Department of Pediatrics, Cancer Research Institute, Seoul National University Children's Hospital, Seoul National University College of Medicine, Seoul, Korea.
Correspondence to : Correspondence to Hee Young Shin, M.D., Ph.D. Department of Pediatrics, Cancer Research Institute, Seoul National University Children's Hospital, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul 03080, Korea. hyshin@snu.ac.kr
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methotrexate (MTX), one of the main drugs used to treat osteosarcoma, is a representative folic acid antagonist. Polymorphisms of various enzymes involved in the metabolism of MTX could contribute to differences in response to MTX in pediatric osteosarcoma patients.
Blood and tissue samples were obtained from 37 pediatric osteosarcoma patients who were treated with high-dose MTX therapy. The following 4 single nucleotide polymorphisms (SNPs) were analyzed:
Plasma MTX levels at 48 hours after high-dose MTX infusion were significantly associated with
This study suggests that plasma levels of MTX are associated with GI and renal toxicities after high-dose MTX therapy, and genetic polymorphisms that affect the metabolism of MTX may influence drug concentrations and development of significant side effects in pediatric patients treated with high-dose MTX.
Keywords Pediatric, Osteosarcoma, Methotrexate, Toxicity, Single nucleotide polymorphism
High-dose methotrexate (MTX) with leucovorin (5-formyltetrahydrofolic acid) rescue in combination with doxorubicin and a platinum agent has served as a cornerstone of neoadjuvant chemotherapy for osteosarcoma. High-dose MTX treatment is associated with various toxicities, including toxicities of central nervous system (CNS), liver, kidney, bone marrow and gastrointestinal system, particularly oral mucosa. Various studies have reported that several pharmacokinetic parameters, including a high plasma MTX concentration and prolonged exposure to high levels of MTX, were associated with the development of high-dose MTX-induced toxicities [1,2,3]. However, MTX levels exhibit significant inter-individual variability, and acute toxicity after high-dose MTX is often unexpected and not typically dose dependent; thus, it is difficult to predict who will develop a more serious adverse response to high-dose MTX. Folate pathway genes are involved in the metabolism of MTX and are very polymorphic. Numerous pharmacogenetic studies have reported that single nucleotide polymorphisms (SNPs) alter the activity or expression of folate pathway enzymes, which may influence the response to and toxicity caused by MTX in various malignancies and autoimmune diseases [4,5,6].
However, comparatively few data are available regarding associations between various genetic polymorphisms of the folate pathway genes and pediatric osteosarcoma. We hypothesized that polymorphisms of the folate pathway genes may influence plasma concentrations of MTX and MTX-induced toxicity in pediatric patients with osteosarcoma. Among various candidate SNPs in folate pathway genes, SNPs in solute carrier family 19, member1 (
The patients included in this study were Korean children with osteosarcoma who were treated with high-dose MTX-containing chemotherapeutic protocols between 1986 and 2010. This study was approved by the Ethics Committee of Institutional Review Board (IRB No. H-0906-067-284). In all cases, informed consent was obtained from the patients, their parents or both. The patients received high-dose MTX according to the following treatment protocols: CCG 7921A, CCG 7921B and COG AOST 0331 [7,8]. High-dose MTX at 12 g/m2 was administered separately by an interval of 1 week. Each infusion lasted for 4 hours. Intravenous hydration and alkalization were achieved 12 hours prior to the start of MTX therapy. High-dose MTX was started at a urine pH>7.0 to provide protection against MTX-induced renal dysfunction. Leucovorin rescue was initiated 24 hours after the start of MTX infusion at a dose rate of 15 mg/m2, and the dosages were adjusted based on plasma MTX concentrations and continued until plasma MTX levels reached <0.1 µmol/L. The patient characteristics and clinical data used in this study were collected retrospectively.
Therapeutic drug monitoring was performed at 24, 48 and 72 hours from the beginning of the infusion of high-dose MTX. Plasma MTX concentrations were measured by a fluorescence polarization immunoassay on a TDx system (Abbot Laboratories, Abbot Park, IL). The highest plasma levels of total bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and creatinine were used as criteria for liver and kidney toxicity. Grades of mucositis, liver, kidney and neurologic toxicities were assessed according to the CTCAE 3.0 [9].
Genomic DNA from peripheral blood and formalin-fixed, paraffin-embedded (FFPE) tumor tissues from the 37 patients were analyzed. Genomic DNA was extracted from peripheral blood lymphocytes and tumor tissues using a QIAamp DNA Mini Kit and a QIAamp DNA FFPE Tissue kit (QIAGEN, Hilden, Germany), respectively, according to the manufacturer's instructions. DNA yield, integrity and protein contamination were determined by spectrophotometry. Candidate SNPs of genes involved in the folate pathway and the transport of MTX included
PCR amplification was performed in a total volume of 20 µL with a mixture containing 5 µM forward and reverse sequencing by synthesis (SBS) primers (Table 1), 0.25 mM deoxynucleotide (dNTP) mix, 112.5 mM Tris HCl (pH 9.0), 3 mM MgCl2, 75 mM KCl, 30 mM (NH4)2SO4, and 1.5 U of Taq polymerase (Biotools, Madrid, Spain). The reactions were preheated to 94℃ for 2 minutes, followed by 10 amplification cycles, which consisted of the following: denaturation at 94℃ for 15 seconds, annealing at 50℃ for 30 seconds and extension at 72℃ for 1 minute. This protocol was followed by 40 amplification cycles using following parameters: denaturation at 94℃ for 15 seconds, annealing at 60℃ for 230 seconds and extension at 72℃ for 40 seconds in a 96-well thermal cycler (Applied Biosystems, CA, USA). Sixteen microliters of PCR product was mixed with 4 µL of an activation solution (YeBT, Seoul, Korea), and the resultant mixture was incubated at 37℃ for 60 minutes and 85℃ for 15 minutes.
Five µL of activated PCR product was added to 20 µL of an allele-specific primer extension (ASPE) reaction mixture containing 75 mM Tris HCl (pH 9.0), 2 mM MgCl2, 50 mM KCl, 20 mM (NH4)2SO4, 25 nM ASPE primer mix (Table 2), 6 mM biotin dCTP, 50 mM of dATP, dGTP, dTTP mix and 1.0 U of Taq polymerase (Biotools, Spain). The reactions were denatured at 94℃ for 5 minutes, followed by 35 amplification cycles to label the amplicons: 94℃ for 30 seconds, 55℃ for 1 minute, 72℃ for 2 minutes and a final extension at 72℃ for 7 minutes.
Microspheres (FlexMAP beads, Tm Bioscience, Toronto, Canada) with anti-tags for each allele-specific primer were added to 22 µL of the ASPE reaction product to a final volume of 42 µL in 2× Hybrisol (YeBT, Seoul, Korea). The mixtures were denatured for 10 min at 95℃, and then incubated for 30 min at 37℃. The microspheres were washed thrice in 160 µL of TM hybridization buffer (0.2 M NaCl, 0.1 M Tris (pH 8.0), 0.08% Triton X-100). Streptavidin-R-phycoerythrin (SAPE, MOSS, Pasadena, MD) was diluted 1:500 in TM hybridization buffer, and 100 µL was added to the microsphere-hybridized ASPE reaction products. The mixture was incubated for 15 min at room temperature.
Microsphere fluorescence was measured using a Luminex 200 cytometer (Luminex, Austin, TX). Data were collected from a minimum of 50 microspheres of each type. Masterplex GT software (Miraibio, San Francisco, CA) was used to analyze SNP genotypes. Homozygous alleles were discriminated by differences of 25% in median fluorescence intensity (MFI). The difference ratio was calculated by dividing the net MFI of one allele by the sum of the net MFI of all alleles and multiplying by 100.
The associations between SNPs and plasma MTX concentrations at 24, 48, and 72 hours after high-dose MTX infusion were evaluated using one way ANOVA, Kruskal-Wallis test and Mann-Whitney test. Analyses of plasma MTX concentrations and MTX-induced toxicities were performed using Mann-Whitney test. The frequencies of severe complications among SNPs were compared with a chi-square test and Fisher's exact test.
A total of 37 pediatric patients with osteosarcoma were enrolled in this study. Table 3 presents the characteristics of the patients. The median age was 11.8 years, and the femur was the most common site of primary tumor. One-third of the patients had distant metastasis at diagnosis. Each patient received a median of 10 cycles (range, 2-20) of high-dose MTX therapy. For some patients high-dose MTX was associated with serious toxicities resulting in treatment interruption or discontinuation. A total of 393 courses of high-dose MTX chemotherapy were evaluated in this study.
The distribution of the examined folate pathway gene SNPs is listed in Table 4. These SNPs consisted of common polymorphisms in
High-dose MTX-induced toxicity was recorded and graded according to Common Terminology Criteria for Adverse Events version (CTCAE) 3.0. At least one episode of grade 3-4 liver toxicity was experienced by 26 patients (70.3%). In addition, grade 3 to 4 kidney toxicity and neurotoxicity were observed in 4 and 1 patients (10.8% and 2.7%), respectively. Mucositis higher than grade 3 was experienced by 4 patients (10.8%). The results of the analysis on MTX-induced toxicity and plasma MTX concentrations at 48 and 72 hours after high-dose MTX infusion are displayed in Table 6. Severe liver, kidney, and neurologic toxicities and mucositis were as defined by the presence of grade 3 or higher symptoms. Higher plasma MTX levels at 48 hours were associated with severe mucositis and kidney toxicity (
Results of the analysis about the associations between the candidate gene SNPs and development of grade 3 or 4 toxicities are described in Table 7.
Identifying genetic predictors of MTX-related toxicity may help to determine individual dosage adjustment and to minimize adverse effects. This study analyzed polymorphisms of several candidate genes involved in the folate-MTX metabolic pathway and investigated possible associations between these SNPs and clinical data of osteosarcoma patients after high-dose MTX therapy. In clinical practice, plasma MTX monitoring is essential after high-dose MTX infusion to detect those who are at risk for MTX-related toxicity and to determine the dose and duration of leucovorin rescue. Various cutoff points based on the MTX plasma half-life have been used to determine the rate of MTX clearance and leucovorin rescue to prevent or minimize toxicity. Studies evaluating plasma MTX concentrations at 24, 48, and 72 hours have concluded that significant effects were observed at 48 hours, and plasma MTX levels at 48 hours after high-dose MTX infusion were found to be independent of both treatment protocol and patient age [10,11,12].
In this study, MTX plasma levels at 48 hours after high-dose MTX therapy showed an association with the
High-dose MTX therapy can cause significant side effects, including myelosuppression, mucositis, nausea, vomiting, diarrhea, hepatotoxicity, and reduced kidney function as well as neurotoxicity [17]. Prior to the routine monitoring of plasma MTX concentrations and dosing of leucovorin accordingly, the incidence of fatal toxicity after high-dose MTX reached approximately 5%, and early studies revealed that a high risk for bone marrow and gastrointestinal mucosal toxicities was related to MTX concentrations greater than 5 to 10 µmol/L at 24 hours, 1 µmol/L at 48 hours, and 0.1 µmol/L at 72 hours [18,19,20]. However, it has become mandatory to monitor plasma MTX during high-dose MTX therapy to adjust hydration, alkalization, and leucovorin rescue individually. Thus, in most cases, these toxicities can be prevented and ameliorated by the administration of leucovorin [21,22]. Nevertheless, this study showed that plasma MTX concentrations at 48 and 72 hours were significantly associated with MTX-related toxicity, especially mucositis and kidney toxicity.
Toxic response to high-dose MTX displays great inter-individual variability. Many studies have suggested that genetic factors contribute to the occurrence of MTX-related toxicity and that MTX toxicity can be modified by SNPs in genes involved in the metabolism, transport, and functions of folates and MTX [23,24,25]. This study also identified that the
In conclusion, our study demonstrated the associations between MTX plasma levels at 48 and 72 hours after MTX infusion and renal toxicity and mucositis. In addition, we identified the influence of
Plasma methotrexate levels at 48 hours after high-dose methotrexate infusion were significantly associated with the 80G>A variants of
Plasma methotrexate levels at 48 (
Blood Res 2016; 51(1): 50-57
Published online March 31, 2016 https://doi.org/10.5045/br.2016.51.1.50
Copyright © The Korean Society of Hematology.
Jeong A Park1, and Hee Young Shin2*
1Department of Pediatrics, Haeundae Paik Hospital, Inje University College of Medicine, Busan, Korea.
2Department of Pediatrics, Cancer Research Institute, Seoul National University Children's Hospital, Seoul National University College of Medicine, Seoul, Korea.
Correspondence to:Correspondence to Hee Young Shin, M.D., Ph.D. Department of Pediatrics, Cancer Research Institute, Seoul National University Children's Hospital, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul 03080, Korea. hyshin@snu.ac.kr
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methotrexate (MTX), one of the main drugs used to treat osteosarcoma, is a representative folic acid antagonist. Polymorphisms of various enzymes involved in the metabolism of MTX could contribute to differences in response to MTX in pediatric osteosarcoma patients.
Blood and tissue samples were obtained from 37 pediatric osteosarcoma patients who were treated with high-dose MTX therapy. The following 4 single nucleotide polymorphisms (SNPs) were analyzed:
Plasma MTX levels at 48 hours after high-dose MTX infusion were significantly associated with
This study suggests that plasma levels of MTX are associated with GI and renal toxicities after high-dose MTX therapy, and genetic polymorphisms that affect the metabolism of MTX may influence drug concentrations and development of significant side effects in pediatric patients treated with high-dose MTX.
Keywords: Pediatric, Osteosarcoma, Methotrexate, Toxicity, Single nucleotide polymorphism
High-dose methotrexate (MTX) with leucovorin (5-formyltetrahydrofolic acid) rescue in combination with doxorubicin and a platinum agent has served as a cornerstone of neoadjuvant chemotherapy for osteosarcoma. High-dose MTX treatment is associated with various toxicities, including toxicities of central nervous system (CNS), liver, kidney, bone marrow and gastrointestinal system, particularly oral mucosa. Various studies have reported that several pharmacokinetic parameters, including a high plasma MTX concentration and prolonged exposure to high levels of MTX, were associated with the development of high-dose MTX-induced toxicities [1,2,3]. However, MTX levels exhibit significant inter-individual variability, and acute toxicity after high-dose MTX is often unexpected and not typically dose dependent; thus, it is difficult to predict who will develop a more serious adverse response to high-dose MTX. Folate pathway genes are involved in the metabolism of MTX and are very polymorphic. Numerous pharmacogenetic studies have reported that single nucleotide polymorphisms (SNPs) alter the activity or expression of folate pathway enzymes, which may influence the response to and toxicity caused by MTX in various malignancies and autoimmune diseases [4,5,6].
However, comparatively few data are available regarding associations between various genetic polymorphisms of the folate pathway genes and pediatric osteosarcoma. We hypothesized that polymorphisms of the folate pathway genes may influence plasma concentrations of MTX and MTX-induced toxicity in pediatric patients with osteosarcoma. Among various candidate SNPs in folate pathway genes, SNPs in solute carrier family 19, member1 (
The patients included in this study were Korean children with osteosarcoma who were treated with high-dose MTX-containing chemotherapeutic protocols between 1986 and 2010. This study was approved by the Ethics Committee of Institutional Review Board (IRB No. H-0906-067-284). In all cases, informed consent was obtained from the patients, their parents or both. The patients received high-dose MTX according to the following treatment protocols: CCG 7921A, CCG 7921B and COG AOST 0331 [7,8]. High-dose MTX at 12 g/m2 was administered separately by an interval of 1 week. Each infusion lasted for 4 hours. Intravenous hydration and alkalization were achieved 12 hours prior to the start of MTX therapy. High-dose MTX was started at a urine pH>7.0 to provide protection against MTX-induced renal dysfunction. Leucovorin rescue was initiated 24 hours after the start of MTX infusion at a dose rate of 15 mg/m2, and the dosages were adjusted based on plasma MTX concentrations and continued until plasma MTX levels reached <0.1 µmol/L. The patient characteristics and clinical data used in this study were collected retrospectively.
Therapeutic drug monitoring was performed at 24, 48 and 72 hours from the beginning of the infusion of high-dose MTX. Plasma MTX concentrations were measured by a fluorescence polarization immunoassay on a TDx system (Abbot Laboratories, Abbot Park, IL). The highest plasma levels of total bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and creatinine were used as criteria for liver and kidney toxicity. Grades of mucositis, liver, kidney and neurologic toxicities were assessed according to the CTCAE 3.0 [9].
Genomic DNA from peripheral blood and formalin-fixed, paraffin-embedded (FFPE) tumor tissues from the 37 patients were analyzed. Genomic DNA was extracted from peripheral blood lymphocytes and tumor tissues using a QIAamp DNA Mini Kit and a QIAamp DNA FFPE Tissue kit (QIAGEN, Hilden, Germany), respectively, according to the manufacturer's instructions. DNA yield, integrity and protein contamination were determined by spectrophotometry. Candidate SNPs of genes involved in the folate pathway and the transport of MTX included
PCR amplification was performed in a total volume of 20 µL with a mixture containing 5 µM forward and reverse sequencing by synthesis (SBS) primers (Table 1), 0.25 mM deoxynucleotide (dNTP) mix, 112.5 mM Tris HCl (pH 9.0), 3 mM MgCl2, 75 mM KCl, 30 mM (NH4)2SO4, and 1.5 U of Taq polymerase (Biotools, Madrid, Spain). The reactions were preheated to 94℃ for 2 minutes, followed by 10 amplification cycles, which consisted of the following: denaturation at 94℃ for 15 seconds, annealing at 50℃ for 30 seconds and extension at 72℃ for 1 minute. This protocol was followed by 40 amplification cycles using following parameters: denaturation at 94℃ for 15 seconds, annealing at 60℃ for 230 seconds and extension at 72℃ for 40 seconds in a 96-well thermal cycler (Applied Biosystems, CA, USA). Sixteen microliters of PCR product was mixed with 4 µL of an activation solution (YeBT, Seoul, Korea), and the resultant mixture was incubated at 37℃ for 60 minutes and 85℃ for 15 minutes.
Five µL of activated PCR product was added to 20 µL of an allele-specific primer extension (ASPE) reaction mixture containing 75 mM Tris HCl (pH 9.0), 2 mM MgCl2, 50 mM KCl, 20 mM (NH4)2SO4, 25 nM ASPE primer mix (Table 2), 6 mM biotin dCTP, 50 mM of dATP, dGTP, dTTP mix and 1.0 U of Taq polymerase (Biotools, Spain). The reactions were denatured at 94℃ for 5 minutes, followed by 35 amplification cycles to label the amplicons: 94℃ for 30 seconds, 55℃ for 1 minute, 72℃ for 2 minutes and a final extension at 72℃ for 7 minutes.
Microspheres (FlexMAP beads, Tm Bioscience, Toronto, Canada) with anti-tags for each allele-specific primer were added to 22 µL of the ASPE reaction product to a final volume of 42 µL in 2× Hybrisol (YeBT, Seoul, Korea). The mixtures were denatured for 10 min at 95℃, and then incubated for 30 min at 37℃. The microspheres were washed thrice in 160 µL of TM hybridization buffer (0.2 M NaCl, 0.1 M Tris (pH 8.0), 0.08% Triton X-100). Streptavidin-R-phycoerythrin (SAPE, MOSS, Pasadena, MD) was diluted 1:500 in TM hybridization buffer, and 100 µL was added to the microsphere-hybridized ASPE reaction products. The mixture was incubated for 15 min at room temperature.
Microsphere fluorescence was measured using a Luminex 200 cytometer (Luminex, Austin, TX). Data were collected from a minimum of 50 microspheres of each type. Masterplex GT software (Miraibio, San Francisco, CA) was used to analyze SNP genotypes. Homozygous alleles were discriminated by differences of 25% in median fluorescence intensity (MFI). The difference ratio was calculated by dividing the net MFI of one allele by the sum of the net MFI of all alleles and multiplying by 100.
The associations between SNPs and plasma MTX concentrations at 24, 48, and 72 hours after high-dose MTX infusion were evaluated using one way ANOVA, Kruskal-Wallis test and Mann-Whitney test. Analyses of plasma MTX concentrations and MTX-induced toxicities were performed using Mann-Whitney test. The frequencies of severe complications among SNPs were compared with a chi-square test and Fisher's exact test.
A total of 37 pediatric patients with osteosarcoma were enrolled in this study. Table 3 presents the characteristics of the patients. The median age was 11.8 years, and the femur was the most common site of primary tumor. One-third of the patients had distant metastasis at diagnosis. Each patient received a median of 10 cycles (range, 2-20) of high-dose MTX therapy. For some patients high-dose MTX was associated with serious toxicities resulting in treatment interruption or discontinuation. A total of 393 courses of high-dose MTX chemotherapy were evaluated in this study.
The distribution of the examined folate pathway gene SNPs is listed in Table 4. These SNPs consisted of common polymorphisms in
High-dose MTX-induced toxicity was recorded and graded according to Common Terminology Criteria for Adverse Events version (CTCAE) 3.0. At least one episode of grade 3-4 liver toxicity was experienced by 26 patients (70.3%). In addition, grade 3 to 4 kidney toxicity and neurotoxicity were observed in 4 and 1 patients (10.8% and 2.7%), respectively. Mucositis higher than grade 3 was experienced by 4 patients (10.8%). The results of the analysis on MTX-induced toxicity and plasma MTX concentrations at 48 and 72 hours after high-dose MTX infusion are displayed in Table 6. Severe liver, kidney, and neurologic toxicities and mucositis were as defined by the presence of grade 3 or higher symptoms. Higher plasma MTX levels at 48 hours were associated with severe mucositis and kidney toxicity (
Results of the analysis about the associations between the candidate gene SNPs and development of grade 3 or 4 toxicities are described in Table 7.
Identifying genetic predictors of MTX-related toxicity may help to determine individual dosage adjustment and to minimize adverse effects. This study analyzed polymorphisms of several candidate genes involved in the folate-MTX metabolic pathway and investigated possible associations between these SNPs and clinical data of osteosarcoma patients after high-dose MTX therapy. In clinical practice, plasma MTX monitoring is essential after high-dose MTX infusion to detect those who are at risk for MTX-related toxicity and to determine the dose and duration of leucovorin rescue. Various cutoff points based on the MTX plasma half-life have been used to determine the rate of MTX clearance and leucovorin rescue to prevent or minimize toxicity. Studies evaluating plasma MTX concentrations at 24, 48, and 72 hours have concluded that significant effects were observed at 48 hours, and plasma MTX levels at 48 hours after high-dose MTX infusion were found to be independent of both treatment protocol and patient age [10,11,12].
In this study, MTX plasma levels at 48 hours after high-dose MTX therapy showed an association with the
High-dose MTX therapy can cause significant side effects, including myelosuppression, mucositis, nausea, vomiting, diarrhea, hepatotoxicity, and reduced kidney function as well as neurotoxicity [17]. Prior to the routine monitoring of plasma MTX concentrations and dosing of leucovorin accordingly, the incidence of fatal toxicity after high-dose MTX reached approximately 5%, and early studies revealed that a high risk for bone marrow and gastrointestinal mucosal toxicities was related to MTX concentrations greater than 5 to 10 µmol/L at 24 hours, 1 µmol/L at 48 hours, and 0.1 µmol/L at 72 hours [18,19,20]. However, it has become mandatory to monitor plasma MTX during high-dose MTX therapy to adjust hydration, alkalization, and leucovorin rescue individually. Thus, in most cases, these toxicities can be prevented and ameliorated by the administration of leucovorin [21,22]. Nevertheless, this study showed that plasma MTX concentrations at 48 and 72 hours were significantly associated with MTX-related toxicity, especially mucositis and kidney toxicity.
Toxic response to high-dose MTX displays great inter-individual variability. Many studies have suggested that genetic factors contribute to the occurrence of MTX-related toxicity and that MTX toxicity can be modified by SNPs in genes involved in the metabolism, transport, and functions of folates and MTX [23,24,25]. This study also identified that the
In conclusion, our study demonstrated the associations between MTX plasma levels at 48 and 72 hours after MTX infusion and renal toxicity and mucositis. In addition, we identified the influence of
Plasma methotrexate levels at 48 hours after high-dose methotrexate infusion were significantly associated with the 80G>A variants of
Plasma methotrexate levels at 48 (
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Plasma methotrexate levels at 48 hours after high-dose methotrexate infusion were significantly associated with the 80G>A variants of
Plasma methotrexate levels at 48 (