Analyses Of The Prevalence Of Toxicities And Its Risk Factors Of High-dose Methotrexate Chemotherapy In Pediatric Acute Lymphoblastic Leukemia

Part 1 The Prevalence of Toxicities and its Risk Factors after High-dose Methotrexate Chemotherapy in Pediatric Acute Lymphoblastic LeukemiaBackground:Methotrexate（MTX）is a critical component of the successful treatment of pediatric acute lymphoblastic leukemia（ALL）.Toxicity is the main factor leading to the interruption of chemotherapy and even leads to death.This study aimed to analyze the prevalence of toxicities and its risk factors after receiving high-dose methotrexate chemo-therapy according to CCCG-ALL-2015 regimen in pediatric acute lymphoblastic leukemia.Methods:We included 294 patients who were diagnosed with acute lymphoblastic leuke-mia in the Pediatrics Department of Union Hospital,Tongji Medical College,Huazhong University of Science and Technology from September 2015 to September 2020 and treated following the Chinese Children’s Cancer Group Acute Lymphoblastic Leukemia Protocol（CCCG-ALL-2015 protocol,Chi CTR-IPR-14005706）.The MTX dose was 3g/m² for pa-tients in low-risk（LR）and 5g/m² for patients in intermediate/high-risk（IR/HR）.Toxicities were reported as per the Common Terminology Criteria for Adverse Events（CTCAE ver-sion 5.0）.Association between categorical data were evaluated by the?² test and continu-ous data were by nonparametric test.Logistics regression was used to analyze the risk fac-tors of delayed MTX clearance and toxicities.SPSS 25.0 statistical software was used for statistical analyses.Results:A total of 1172 cycles of high-dose methotrexate chemotherapy from 294 patients were collected.The median age was 4.8 years（range:0.4-14 years）;119 females and 175males;135 in the LR group and 159 in the IR/HR group;268 were B-ALL,and 26 were T-ALL.The incidence rate of delayed MTX clearance at 44 h and 68 h were 18%vs 33.1%in the LR group and 27.3%vs 45.6%in the IR/HR,respectively.Age>6 years,males and IR/HR group were risk factors of delayed MTX clearance(P<0.05,OR_age:1.583,95%CI:1.186-2.112;OR_sex=1.545,95%CI:1.162-2.055;OR_risk:1.950,95%CI:1.452-2.617).Myelosuppression was the most common kind of toxicity（18.9%of cases lasting for more than 7 days）.Hepatotoxicity,acute kidney injury and neurotoxicity in grade 3/4 were found in 5.2%,0.9%and 0.8%of cycles,respectively.Female,IR/HR,BSA≤0.69m²,and C_44h≥1.0μmol/L were risk factors for grade 3/4 toxicities（P<0.05）.The occurrence of de-layed MTX clearance and toxicities were decreased with the increased number of HD-MTX chemotherapy.The increase rate of s Cr after chemotherapy was positively corre-lated with MTX concentration（Spearman correlation coefficient:0.337、0.318）,but was not associated with toxicities.Conclusion:MTX-related toxicities after HD-MTX chemotherapy in CCCG-ALL-2015 regimen was generally mild and controllable.IR/HR group（MTX 5g/m²）was the main risk factor for delayed clearance of MTX and toxicities.The increase rate of s Cr could be used as a predictor of MTX concentration.Part 2 Comparison between Two Dose-adjustments of High-Dose Methotrexate and Analyses of the Relationship between Methotrexate Chemotherapy and Prognosis of Pediatric Acute Lymphoblastic LeukemiaBackground: MTX-induced toxicities are related to the exposure time of methotrexate.Dose adjustment is one of the main measures to reduce toxicities.This study aimed to compare the pros and cons of the two dosage adjustments provided in CCCG-ALL-2015 regimen and analyze the effect of HD-MTX chemotherapy on the prognosis of childhood acute lymphoblastic leukemia。Methods: 1172 cycles of high-dose MTX chemotherapy from 294 patients treated following the CCCG-ALL-2015 protocol were enrolled and the data of actual MTX dosage,MTX concentration,toxicities and prognosis were analyzed.We compared data from the dose adjustment Program 1（fixed 20% reduction in dose）and Program 2（dose individualization according to MTX concentration at 16h）applied if MTX clearance was delayed in the previous cycle.Cox regression model was used to analyze the relationship between HD-MTX chemotherapy and relapse-free survival.Statistical analyses was performed with SPSS 25.0 statistical software.Results: Of all the 1172 cycles evaluated,405 cycles were dose-adjusted by Program 1 and 118 cycles received Program 2 monitoring.Patients who used Program 2 for dose adjustmen had a higher actual MTX infusion dose and infusion rate than those use Program 2（P<0.001）.Patients in the IR/HR group and dose-adjusted by Program 2 had a higher leucovorin rescue dose（P=0.023）.The prevalence of delayed MTX clearance and toxicities in patients receiving the first high-dose methotrexate chemotherapy were 35.5% and 24.7%,respectively.Dose-adjusted by Program 2 were able to maintain C44 h at the established target value（0.5-1.0μmol/L）in subsequent chemotherapy（P<0.001）,while patients who used Program 1 were prone to have low concentrations（P<0.05）.In patients who were dose-adjusted by Program 2 and had taken a full dose of MTX,only 28.1% cycles experienced repeated MTX clearance delay and a large proportion of cases（30.2%）had C44 h below 0.5 μmol/L.No significant difference was found in toxicities between these two programs except that hypokalemia was more frequently observed in patients using Program 2（P<0.001）.Male,IR/HR group,C68h≥0.2μmol/L were risk factors for recurrence（P<0.05,ORsex: 2.669,ORrisk: 2.734,ORC68h: 1.561）.There was no significant correlation between C44 h,MTX infusion rate,body surface area and relapse-free survival.Conclusion: Both the two programs could effectively reduce the delay in MTX clearance and MTX-induced toxicities,while dose adjusted by Program 2 was more efficient in maintaining sufficient exposure to MTX without significantly increasing toxicity.MTX dose should be adjusted using Program 2 in every exposure to HD-MTX to prevent toxicities.Minor adjustment of MTX dose had no significant effect on the relapse-free survival in pediatric acute lymphoblastic leukemia.Part 3 Relationships between MTHFR polymorphisms on the Elimination and Toxicities of Methotrexate after High-dose Methotrexate Chemotherapy in pediatric acute lymphoblastic leukemiaBackground: Methylenetetrahydrofolate reductase（MTHFR）is a key enzyme in folate metabolism.Studies have suggested that MTHFR polymorphisms are related to the efficacy and toxicities of MTX,but the results were controversial.This study aimed to analyze the effects of MTHFR C677 T and A1298 C gene polymorphisms on MTX clearance and toxicities after high-dose methotrexate chemotherapy in children with acute lymphoblastic leukemia.Methods: This study enrolled 145 patients diagnosed with acute lymphoblastic leukemia from September 2015 to September 2020 were enrolled.They received chemotherapy following the CCCG-ALL-2015 protocol（clinical trial number: Chi CTR-IPR-14005706）.Venous blood was collected and tested for MTHFR C677 T and A1298 C polymorphisms.The effects of MTHFR C677 T and A1298 C polymorphism on MTX elimination and toxicities was analyzed by ?2 or non-parametric test.All data were collected in a computerized database and analyzed with SPSS version 25.0.Results: Data of 576 HD-MTX chemotherapy cycles from 145 patients have been collected.Hardi-Weinberg genetic balance test showed that the gene frequency in the sample population was complied with the genetic balance law,and the sample was representative（P > 0.05）.The MTX infusion rate（actual MTX dose/suggested MTX dose）in patients with MTHFR C677 T TT genotype was significantly higher than those with CC or CT genotype.Patients with TT genotypes were favoring an increased risk of hypokalemia（1.369 to CC and 1.409 to CT types）.No significant difference was found between the prevalence of other toxicities among MTHFR C677 T genotypes（P>0.05）.The MTX infusion rate of patients with MTHFR A1298 C AC type was slightly lower than those with CC or AA gen- otype.Patients with AA genotype had a 1.405-fold higher risk of hepatotoxicity than those with AC genotype（P>0.05）.There was no significant difference between the prevalence of other toxicities among MTHFR A1298 C genotypes（P>0.05）.Neither MTHFR C677 T nor A1298 C polymorphism was significantly associated with MTX clearance delay.Conclusion: MTHFR C677 T and A1298 C polymorphisms were correlated with delayed clearance of MTX and the occurrence of MTX-related toxicities to a certain extent.However,they were not good predictors and had little significance for HD-MTX chemotherapy in clinical practice.