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

Abstract

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.

Systematic Review of Pharmacogenetic Factors That Influence High-Dose Methotrexate Pharmacokinetics in Pediatric Malignancies

“…Several additional genes were sparsely examined and do not fit into neither the folate pathway nor transporter gene categories. ELMO1 [ 89 ], NALCN [ 89 ], NRII2 (SXR) [ 20 ], SULT1E1 [ 90 ], UGT1A1 [ 46 ], and four intergenic polymorphisms [ 89 ] (rs3862227, rs4888024, rs4905865, and rs1359645) were all found to significantly affect MTX PK. While significant, these genes have only been reported once each.…”

Thymoquinone Augments Methotrexate-Induced Apoptosis on Osteosarcoma Cells

“…This event results in preventing purine and thymidine synthesis and consequently cell apoptosis [26]. Although studies have investigated the application of MTX in OS therapy [27,28], the drug mechanism of action on OS cells is unclear. In the present study, we illustrated that MTX can reduce cell proliferation and induce cell apoptosis through the up-regulation of Bax and caspase-9 expression and the down-regulation of BCL-2 expression.…”

Pharmacogenetics of chemotherapy treatment response and -toxicities in patients with osteosarcoma: a systematic review

“…rs2273697 was associated with hepatotoxicity and thrombocytopenia by Hattinger et al, however, as Fig. 2 indicates, six studies that also related this variant to hepatotoxicity did not find a significant association [ 35 , 37 , 41 , 60 – 63 ], negative results were also found in four studies that related this variant to thrombocytopenia [ 35 , 37 , 61 – 63 ]. In addition, rs2273697 was found to be associated with leukopenia according to both Hattinger et al and Hegyi et al, but this association was not found in 4 other cohorts [ 35 , 37 , 60 – 63 ].…”

“…The gene has not been linked to methotrexate or thrombocyte count before. The underlying mechanisms of the associations with all three variants are still unclear [ 61 ]. These associations are not replicated in other patient cohorts yet, as the publication was recent.…”

“…Finally, the study by Hurkmans et al assessed a panel of 1936 variants in 231 genes involved in absorption, distribution, metabolism and excretion of medicines for multiple toxicities [ 61 ]. Three variants in the Cytochrome P450 family were significantly associated with thrombocyte count, namely CYP4F8 rs4808326, CYP2B6 rs4803418 and CYP2B6 rs4803419, and these remained significant after Bonferroni correction for multiple testing.…”