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Related Collections Neoplasia Brief Reports Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 15 November 2002, Vol. 100, No. 10, pp. 3832-3834 BRIEF REPORT Polymorphism G80A in the reduced folate carrier gene and its relationship to methotrexate plasma levels and outcome of childhood acute lymphoblastic leukemia Caroline Laverdière, Sonia Chiasson, Irina Costea, Albert Moghrabi, and Maja Krajinovic From the Centre de Recherche, Hôpital Sainte-Justine, Montréal, QC, Canada.     Abstract Top Abstract Introduction Study design Results and discussion References Methotrexate (MTX) is a key compound of chemotherapeutic regimens used in the treatment of childhood acute lymphoblastic leukemia (ALL). Resistance to this drug may arise by, among other factors, altered cellular uptake that may hamper the efficacy of the treatment. Recently, a G80A polymorphism has been described in the reduced folate carrier gene (RFC1), which encodes the major MTX transporter. Here, we assessed the association between the genetic polymorphisms G80A and both MTX plasma levels and childhood ALL outcome. Children with the A80 variant had worse prognoses than patients with the GG genotype (P = .04), as shown by event-free survival estimates. Patients homozygous for A80 had higher levels of MTX (P = .004) than the other genotype groups. Possible explanations for observed associations are discussed; however, additional experiments are required to achieve understanding of the underlying mechanism. (Blood. 2002;100:3832-3834) © 2002 by The American Society of Hematology.     Introduction Top Abstract Introduction Study design Results and discussion References Methotrexate (MTX) is a key component in the treatment of childhood acute lymphoblastic leukemia (ALL), the most frequent malignancy in the pediatric population.1,2 The reduced folate carrier gene (RFC1) is a major MTX transporter whose impaired function was recognized as a frequent mechanism of antifolate resistance.3,4 Different gene alterations affecting RFC1 transport properties were found in cell lines selected for antifolate resistance and in patient lymphoblasts.5-8 Recently, a G80A polymorphism, which replaces His by Arg at position 27 of the RFC1 protein, was identified.9 The G variant correlated with lower plasma folate and higher homocysteine levels in healthy persons9 and was found at higher frequency in children with neural tube defects.10,11 Because folate and homocysteine homeostasis are affected by MTX action,12,13 it is possible that RFC1 G80A may also modulate the outcome in patients treated with this drug. In the present study, we analyzed the association between the RFC1 G80A polymorphism and both disease outcome and MTX plasma level in children with ALL.     Study design Top Abstract Introduction Study design Results and discussion References Patients The patients (n = 204) included in this study were children of French Canadian origin who had ALL and were treated at the Sainte-Justine Hospital in Montreal, QC, Canada between 1988 and 2000. Multiagent chemotherapeutic protocols used were DFCI 87-01, DFCI 91-01, and DFCI 95-01, all developed by the Dana-Farber Cancer Institute.14,15 Patient samples were obtained at diagnosis after informed consent was provided according to the Declaration of Helsinki. Approval for this study was obtained from the institutional review board of the Hôpital Sainte-Justine. Patient characteristics and clinical prognostic factors at diagnosis (sex, age, white blood cell count [WBC], ALL cell type, treatment protocol, risk group, and DNA index) are given in Table 1. Each patient enrolled in this retrospective study received a high MTX dose (4 g/m2) during the induction phase. MTX was also given intrathecally for central nervous system treatment (6-12 mg, depending on age) and at a once-a-week dose of 30 mg/m2 during the maintenance phase. Children who had relapses or fatal outcomes from the disease were defined as having an event. To reduce MTX toxicity, leucovorin rescue therapy (200 mg/m2 followed by 24 mg/m2 every 6 hours until routinely measured plasma MTX levels were 0.1 µM or lower) was given after high-dose MTX. MTX plasma levels were thus available at 3 time points (24, 36, and 48 hours) for the 73 children for whom RFC1 genotyping was also performed.                                View this table: [in this window] [in a new window]   Table 1. Characteristics of children with and without event following treatment for ALL Genotyping and MTX plasma levels RFC1 amplification of patient DNA isolated from buccal epithelial cells, peripheral blood, or bone marrow in remission was obtained as described elsewhere.9 Polymerase chain reaction-restriction fragment-length polymorphisms (PCR-RFLP; HaeII digestion) was used to optimize allele-specific oligonucleotide (ASO) hybridization assay (Figure 1A) subsequently used for the screening of patient DNA. Standard conditions were applied,16 except that ASOs specific for the G80 (5'gcacacgaggCgccg) or the A80 (5'gcacacgaggTgccg) RFC1 variant were designed and hybridized at 45°C with PCR products immobilized on a membrane. Measurement of MTX plasma levels was performed by fluorescence polarization immunoassay (TDx Abbott Laboratories, Chicago, IL) according to the manufacturer's instructions. View larger version (18K): [in this window] [in a new window]   Figure 1. RFC, G80A polymorphism: genotyping and its impact on ALL outcome and MTX levels. (A) ASO hybridization assay of RFC1 G80A polymorphism. PCR products of RFC1 gene obtained from the DNA of each patient are blotted in duplicate. The membranes are hybridized with ASO specific for G80 (left panel) or A80 (right panel). Hybridization signal in spots A1, A2, and C2 indicate homozygous patients with the GG genotype, in B2 a heterozygous patient with the AG genotype, and in B1 and C1 patients with the AA genotype. (B) Kaplan-Meier estimates of EFS for patients with and without the RFC1 A80 variant. EFS curves for patients positive (lower line) or negative (upper line) for the RFC1A80 variant are presented. The P value for the differences of survival between these patients groups is indicated. (C) Relationship between RFC1 G80A polymorphism and MTX plasma level. Lines represent log values of MTX levels (µM, measured at 3 time points) in GG, GA, and AA genotypes. MTX levels differ across RFC1 genotype groups (P = .02). When MTX levels in patients with the AA genotype are compared with those of the rest of patients, the difference is .004. Statistics Differences in the frequencies of genotypes dichotomized as carriers of RFC1 A80 variant versus noncarriers between children with and without an event, and those with or without the prognostic factors listed in Table 1, were assessed by 2 analysis. For event-free survival (EFS) analysis, survival time corresponded to the time between diagnosis and the event (n = 35). For patients without an event (censored cases, n = 169), it corresponded to the end of follow-up (5 years after treatment) or to February 2002 (patients who received treatment or who entered the follow-up period). Overall time to the event and of follow-up ranged from 1 to 84 months (interquartile range, 26-84 months); the median was 49.5 months. Differences between EFS obtained by Kaplan-Meier for the patients with and without the RFC1 A80 variant were determined using a log-rank test. The impact of the RFC1 A80 variant on the EFS probabilities was estimated by Cox regression analysis with the enclosure of prognostic factors that influenced ALL outcomes in this group of patients and by applying forced entry of all variables or stepwise routine. The influence of the patients' characteristics on EFS was assessed by Kaplan-Meier and Cox regression analyses. Repeated measures analysis of variance (ANOVA) was used to compare MTX levels (µM) based on the 3 time-points between children with and without event and among patients with different RFC1 genotype categories. For that purpose, log-transformed values of MTX level were used because of its skewed distribution. All analyses were performed by SPSS version 10.00.     Results and discussion Top Abstract Introduction Study design Results and discussion References A significant difference in the frequency of RFC1 genotypes was observed between children with and without an event (Table 2). Carriers of the RFC1 A80 variant had a higher risk for events than were those with the GG genotype (odds ratio [OR] = 3.0; 95% CI, 1.1-8.1; P = .03). Similarly, Kaplan-Meier analysis showed that carriers of the A80 variant had the worst ALL outcomes (P = .04; Figure 1B). In Cox regression analysis, hazard ratio (HR) estimates for patients with RFC1 A80 retained their significance (HR = 2.8; 95% CI, 1.0-8.1; P = .05) in the presence of other prognostic factors, which also influenced ALL outcome (age, WBC, type of protocol, and risk classes; Table 1). When the initial Cox regression model was applied further to stepwise analysis, RFC1 genotype and age appeared to have the highest predictive value for an event (P = .05 and P = .03 respectively). We did not find any correlation between RFC1 genotypes and patient characteristics listed in Table 1.                                View this table: [in this window] [in a new window]   Table 2. Distribution of RFC1 G80A polymorphism among ALL patients with and without event We next analyzed the influence of RFC1 polymorphism on MTX plasma levels (Figure 1C) and found a significant association (P = .02), which was mainly caused by higher MTX plasma levels in the patients with AA than in those with other genotypes (P = .004). However, we did not observe the association between MTX levels and disease outcome (P = .6, data not shown). Although modest, the association between RFC1 polymorphism and ALL outcome suggests that this variant might contribute to the estimation of ALL prognosis. The finding of this study is in agreement with the reports of others who suggest the functional impact of this polymorphism: G80A influenced folate/homocysteine levels9,10 and correlated with neural tube defects,10 especially among children whose mothers reported low folate intake.11 The amino acid change (strong to weak basic amino acid) in a first transmembrane domain (TMD1) caused by G80A substitution was expected to alter RFC1 transport properties.17 Several alterations in TMD1 in cell lines selected for MTX resistance were shown to change the ratio of RFC1 affinities of MTX versus other folate substrates.18,19 Likewise, the carriers of A might have lower MTX affinity (and, presumably, higher MTX levels, as shown in this study) and higher affinity for other folate substrates (and higher level of folate cofactors, as shown in other studies).9-11 On the other hand, MTX level, which shows high pharmacokinetic variability,13,20 may be influenced by different factors, such as hepatic or renal function, and is not a reliable indicator of the transport function. In addition recent in vitro studies in erythroleukemia cell lines showed no difference in MTX transport between the G and the A RFC180 variant, whereas only a minor (2-fold) difference in transport of 5' formyl tetrahydrofolate cofactor was found.17 Therefore, additional studies are needed to explain the underlying mechanism linking RFC1 polymorphism and ALL outcome. A prospective study assessing intracellular MTX levels and RFC1 substrate binding affinities in patients with and without RFC1 A80 variant is under way in our laboratory. It would also be important to assess the relative impact of RFC1 polymorphism on ALL outcome with regard to other variants relevant for MTX response.     Acknowledgments We thank our patients and their parents for their collaboration, our colleagues Daniel Sinnett and Damian Labuda for discussions and contributions of biologic material, and Mark Bernstein for facilitating access to clinical data.     Footnotes Submitted October 16, 2001; accepted July 3, 2002. Supported by the Canadian Institutes of Health Research, Leukemia Research Fund of Canada, and Centre de recherche, Hôpital Ste-Justine. M. K. is a scholar of the Fonds de la Recherche en Santé du Québec. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Maja Krajinovic, Centre de recherche, Hôpital Sainte-Justine, 3175 Côte Ste-Catherine, Montréal, QC, H3T 1C5, Canada; e-mail: maja.krajinovic{at}umontreal.ca.     References Top Abstract Introduction Study design Results and discussion References 1. Pui CH. Acute lymphoblastic leukemia in children. Curr Opin Oncol. 2000;12:3-12[CrossRef][Medline] [Order article via Infotrieve]. 2. Camitta BM, Pullen J, Murphy S. Biology and treatment of acute lymphocytic leukemia in children. Semin Oncol. 1997;24:83-91[Medline] [Order article via Infotrieve]. 3. Gorlick R, Goker E, Trippett T, Waltham M, Banerjee D, Bertino JR. Intrinsic and acquired resistance to methotrexate in acute leukemia. N Engl J Med. 1996;335:1041-1048[Free Full Text]. 4. Sierra EE, Goldman ID. Recent advances in the understanding of the mechanism of membrane transport of folates and antifolates. Semin Oncol. 1999;26:11-23[Medline] [Order article via Infotrieve]. 5. Zhang L, Taub JW, Williamson M, et al. Reduced folate carrier gene expression in childhood acute lymphoblastic leukemia: relationship to immunophenotype and ploidy. Clin Cancer Res. 1998;4:2169-2177[Abstract]. 6. Gorlick R, Goker E, Trippett T, et al. Defective transport is a common mechanism of acquired methotrexate resistance in acute lymphocytic leukemia and is associated with decreased reduced folate carrier expression. Blood. 1997;89:1013-1018[Abstract/Free Full Text]. 7. Moscow JA, Connolly T, Myers TG, Cheng CC, Paull K, Cowan KH. Reduced folate carrier gene (RFC1) expression and anti-folate resistance in transfected and non-selected cell lines. Int J Cancer. 1997;72:184-190[CrossRef][Medline] [Order article via Infotrieve]. 8. Matherly LH, Taub JW, Ravindranath Y, et al. Elevated dihydrofolate reductase and impaired methotrexate transport as elements in methotrexate resistance in childhood acute lymphoblastic leukemia. Blood. 1995;85:500-509[Abstract/Free Full Text]. 9. Chango A, Emery-Fillon N, de Courcy GP, et al. A polymorphism (80GA) in the reduced folate carrier gene and its associations with folate status and homocysteinemia. Mol Genet Metab. 2000;70:310-315[CrossRef][Medline] [Order article via Infotrieve]. 10. De Marco P, Calevo MG, Moroni A, et al. Polymorphisms in genes involved in folate metabolism as risk factors for NTDs. Eur J Pediatr Surg. 2001;11(suppl 1):S14-S17. 11. Shaw GM, Lammer EJ, Zhu H, Baker MW, Neri E, Finnell RH. Maternal periconceptional vitamin use, genetic variation of infant reduced folate carrier (A80G), and risk of spina bifida. Am J Med Genet. 2002;108:1-6[CrossRef][Medline] [Order article via Infotrieve]. 12. Refsum H, Wesenberg F, Ueland PM. Plasma homocysteine in children with acute lymphoblastic leukemia: changes during a chemotherapeutic regimen including methotrexate. Cancer Res. 1991;51:828-835[Abstract/Free Full Text]. 13. Seidel H, Andersen A, Kvaloy JT, et al. Variability in methotrexate serum and cerebrospinal fluid pharmacokinetics in children with acute lymphocytic leukemia: relation to assay methodology and physiological variables. Leuk Res. 2000;24:193-199[CrossRef][Medline] [Order article via Infotrieve]. 14. Silverman LB, Declerck L, Gelber RD, et al. Results of Dana-Farber Cancer Institute Consortium protocols for children with newly diagnosed acute lymphoblastic leukemia (1981-1995). Leukemia. 2000;14:2247-2256[CrossRef][Medline] [Order article via Infotrieve]. 15. Krajinovic M, Labuda D, Mathonnet G, et al. Polymorphisms in genes encoding drugs and xenobiotic metabolizing, DNA repair enzymes, and response to treatment in children with acute lymphoblastic leukemia. Clin Cancer Res. 2002;8:802-810[Abstract/Free Full Text]. 16. Labuda D, Krajinovic M, Richer C, et al. Rapid detection of CYP1A1, CYP2D6, and NAT variants by multiplex polymerase chain reaction and allele-specific oligonucleotide assay. Anal Biochem. 1999;275:84-92[CrossRef][Medline] [Order article via Infotrieve]. 17. Whetstine JR, Gifford AJ, Witt T, et al. Single nucleotide polymorphisms in the human reduced folate carrier: characterization of a high-frequency G/A variant at position 80 and transport properties of the His(27) and Arg(27) carriers. Clin Cancer Res. 2001;7:3416-3422[Abstract/Free Full Text]. 18. Jansen G, Mauritz R, Drori S, et al. A structurally altered human reduced folate carrier with increased folic acid transport mediates a novel mechanism of antifolate resistance. J Biol Chem. 1998;273:30189-30198[Abstract/Free Full Text]. 19. Drori S, Jansen G, Mauritz R, Peters GJ, Assaraf YG. Clustering of mutations in the first transmembrane domain of the human reduced folate carrier in GW1843U89-resistant leukemia cells with impaired antifolate transport and augmented folate uptake. J Biol Chem. 2000;275:30855-30863[Abstract/Free Full Text]. 20. Balis FM, Holcenberg JS, Poplack DG, et al. Pharmacokinetics and pharmacodynamics of oral methotrexate and mercaptopurine in children with lower risk acute lymphoblastic leukemia: a joint children's cancer group and pediatric oncology branch study. Blood. 1998;92:3569-3577[Abstract/Free Full Text]. © 2002 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: B. Baslund, J. Gregers, and C. H. Nielsen Reduced folate carrier polymorphism determines methotrexate uptake by B cells and CD4+ T cells Rheumatology, April 1, 2008; 47(4): 451 - 453. [Abstract] [Full Text] [PDF] S. L. Hider, I. N. Bruce, and W. Thomson The pharmacogenetics of methotrexate Rheumatology, October 1, 2007; 46(10): 1520 - 1524. [Abstract] [Full Text] [PDF] S. Kishi, C. Cheng, D. French, D. Pei, S. Das, E. H. Cook, N. Hijiya, C. Rizzari, G. L. Rosner, T. Frudakis, et al. 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