The Pharmacogenomics Journal (2015) 15, 385–390 © 2015 Macmillan Publishers Limited All rights reserved 1470-269X/15 www.nature.com/tpj ORIGINAL ARTICLE Impact of genetic variants of RFC1, DHFR and MTHFR in osteosarcoma patients treated with high-dose methotrexate S Jabeen1,2, L Holmboe3, GIG Alnæs2,4, AM Andersen5, KS Hall6 and VN Kristensen1,2,4 Osteosarcoma patients are commonly treated with high doses of methotrexate (MTX). MTX is an analog of folate, which is essential for DNA synthesis. Genetic polymorphism at single nucleotide can be indicative to the prognostic outcome in patients. Germ-line variants in candidate genes, coding for enzymes active in the metabolism of MTX, were studied in 62 osteosarcoma patients. Patients harboring the GG genotype in reduced folate carrier 1 (RFC1) rs1051266 had significantly better survival in comparison with patients having the AA genotype (P = 0.046). These patients also had a lower frequency of metastasis (15%, P = 0.029). Also patients homozygous for the G allele of rs1053129 in the dihydrofolate reductase (DHFR) gene were more likely to have a metastasis (45%, P = 0.005), and the methylenetetetrahydrofolate reductase (MTHFR) 677C allele was associated with higher degree of liver toxicity (88%, P = 0.007). The study suggests that germ-line variants in the MTX metabolic pathway are associated with survival and side effects in patients treated with MTX. The Pharmacogenomics Journal (2015) 15, 385–390; doi:10.1038/tpj.2015.11; published online 17 March 2015 INTRODUCTION Intracellular MTX is converted to polyanionic polyglutamates Methotrexate (MTX) is a folate analog used in the treatment of (PGMTX), which creates a gradient for continual cellular uptake of 11,12 autoimmune diseases and cancers such as osteosarcoma. Bone folates and folate analogs. Accumulated intracellular MTX and sarcomas such as osteosarcoma constitute approximately 0.2% of PGMTX interact with dihydrofolate reductase (DHFR), the enzyme all cancers. Of the approximately 40 new bone sarcomas that converts dihydrofolate (DHF) to tetrahydrofolate (THF), which 13,14 diagnosed each year in Norway 12–15 will be osteosarcoma.1 is essential for de novo purine synthesis. Osteosarcoma patients show a bimodal age distribution with a Treatment response mainly depends on the route of adminis- first peak during adolescence and a second peak in older tration, dose and duration of treatment. Response may also be adulthood.2 The second incidence peak in the elderly typically influenced by the activity of the proteins related to MTX uptake, appears in previously irradiated areas or is secondary to Paget’s and genetic variation in the genes coding for these proteins may disease.3 The current standard treatment for osteosarcoma is neo- affect their production or function. Therefore, we analyzed single- adjuvant chemotherapy given before surgery with a high-dose nucleotide polymorphisms (SNPs) and repeat polymorphisms in methotrexate (HDMTX) and subsequent leucovorin rescue, dox- key metabolic genes of the MTX pathway: RFC1, the major influx orubicin and/or cisplatin followed by surgical resection. A major pump; DHFR, the main target of MTX and PGMTX; methylene- challenge of HDMTX therapy is the great interpatient variability in tetetrahydrofolate reductase (MTHFR), en enzyme from the folate clinical response. Bielack et al.4 observed that over 40% of the metabolism pathway and ATP-binding cassette, sub-family G, patients showed poor response to chemotherapy-related treat- member 2 (ABCG2), one of the major MTX efflux pumps. ment, in a study where a good response was defined as less than 10% viable tumor. The wide spectrum of side effects during HDMTX treatment may include gastrointestinal disturbances, MATERIALS AND METHODS alopecia, elevated levels of liver enzymes, bone-marrow suppres- Patient material 5–7 sion and renal toxicity. Serum concentrations of MTX are Serum samples were available for 62 HDMTX-treated osteosarcoma monitored and used to adjust leucovorin rescue to prevent patients with detailed pharmacokinetic and genetic data (see below). toxicity. The patients were treated at The Norwegian Radium Hospital between MTX interacts with a number of enzymes of the folate metabolic 1994 and 2003. pathway. Folate is essential for the synthesis of DNA nucleotides, All treatment protocols in this study were multi-drug regimens most notably thymine, but also the purine bases. MTX enters cells consisting of HDMTX, cisplatin, doxorubicin and ifosfamide, and in some through three possible routes. The major route is via reduced patients, additional etoposide was administered. folate carrier 1 (RFC1).8–10 Reduced or altered expression of the proteins related to MTX uptake, commonly seen in malignant cells, Approvals may be another cause of altered transport kinetics of MTX into the The study protocol was approved by the Regional Ethics Committee. cells.9 Written informed consent was obtained from all participants. Studies on 1Division of Medicine, Department for Clinical Molecular Biology and Lab Science (EpiGen), Akershus University Hospital, Lørenskog, Norway; 2KG Jebsen Center for Breast Cancer Research, Department of Genetics, Institute for Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway; 3Department of Nuclear Medicine, Oslo University Hospital Ullevål, Oslo, Norway; 4Division of Cancer Surgery and Transplantation, Department of Genetics, Institute for Cancer Research, OUS HF, Radiumhospitalet, Oslo, Norway; 5Department of Pharmacology, Oslo University Hospital, Rikshospitalet, Oslo, Norway and 6Department of Oncology, Oslo University Hospital, Radiumhospitalet, Oslo, Norway. Correspondence: Professor V Kristensen, Clinical Molecular Biology, University of Oslo, P. B. 28, Lorenskog 1478, Norway. E-mail: [email protected] Received 22 August 2014; revised 13 January 2015; accepted 28 January 2015; published online 17 March 2015 SNPs and methotrexate treatment of osteosarcoma S Jabeen et al 386 human participants were approved according to the Declaration of Haploview was also used to estimate Hardy–Weinberg equilibrium for Helsinki (http://www.wma.net/e/policy/b3.htm). the genotypes, as a control for technical genotyping errors and biases in the selection of study populations. MTX treatment Genotype frequencies of SNPs and haplotypes were compared to binary variables with the Pearson’s chi square (χ2) test implemented in Patients were treated with MTX as a 4-h infusion with leucovorin rescue Haploview.17 The resulting associations were permuted 1000 times to started 24 h after initiation of MTX as described by Holmboe et al.15 obtain a measure of significance corrected for multiple testing. The results Evaluation of efficacy and toxicity was assessed by case note review were also verified by Pearson χ2 analysis in SPSS. We used statistical from three independent investigators (KRH, JRFC and DPJ), blinded for packages R version 2.5 (r-project.org) and SPSS (v13. CA, USA), unless genotyping results. otherwise stated. Since in the studied cases the actual underlying mode of inheritance of the causal allele was unknown, the co-dominant model, a DNA isolation single two degrees of freedom test, was shown to have a good overall DNA was isolated from serum samples using the QIAamp® DNA Mini kit performance19 and was applied here. ANOVA was applied to examine (Qiagen, Hilden, Germany) following suppliers specifications. The provided continuous versus categorical variables for clinical metabolic data versus spin-columns were loaded with 200 μl proteinase-treated serum, and DNA SNP frequencies. After Bonferroni correction for six tests (since two pairs of was cleaned with ethanol before eluted in 200 μl DNase-free water. SNPs were in LD), a P-value of 0.008 was considered as significant and presented in the paper. For survival analysis Kaplan–Meier analysis was Genotyping of SNP used to test association between three possible genotypes for each SNP and patient survival times. Log-rank test with P-value under 0.05 was SNPs: RCF1 rs1051266, DHFR rs1053129, DHFR rs1650723, DHFR rs1677666, considered as significant. MTHFR rs1801133, MTHFR rs1801131, ABCG2 rs2231135 and ABCG2 rs2231142 were genotyped on the Nanogen Molecular Biology Workstation 16 platform (Nanogen, San Diego, CA, USA) as described in Nordgard et al. RESULTS The accuracy of genotyping on Nanogen has previously been evaluated, and, with regard to both success and error rate, found comparable to the Polymorphism in RFC1 showed association with survival other platforms assayed, such as TaqMan, SNPstream and MassArray3. The major influx transporter of MTX, RFC1, is encoded by the fi Further, the genotypes obtained with Nanogen were veri ed by solute carrier family 19, member 1. Downregulation of RFC1 has sequencing and a perfect match (100%) to the genotype calls was been reported to be associated with impaired MTX transport and observed. resistance.20–22 We genotyped rs1051266 in exon 2, a well-studied, non-synonymous polymorphism (80G4A) that results in substitu- Metabolic data tion of a histidine for an arginine at residue 27 of the protein MTX and its major extracellular metabolite 7-OH-MTX were measured (Arg27His). A log-rank test for Kaplan–Meier analysis for GG versus in 8 serum samples per HDMTX cycle in 62 consecutive osteosarcoma AA showed that the patients homozygous for the G allele of RFC1 patients. Alanine aminotransferase (ALAT), alkaline phosphatase, gamma- fi glutamyltransferase and total bilirubin were measured in the same serum rs1051266