Kim Robien, 1,5 Jeannette Bigler, 1 Yutaka Yasui, 6 John D. Potter, 1,4 Paul Martin, 2 Rainer Storb, 2 Cornelia M. Ulrich 1,3,4

Transcription

1 Biology of Blood and Marrow Transplantation 12: (2006) 2006 American Society for Blood and Marrow Transplantation /06/ $32.00/0 doi: /j.bbmt Methylenetetrahydrofolate Reductase and Thymidylate Synthase Genotypes and Risk of Acute Graft-versus-Host Disease Following Hematopoietic Cell Transplantation for Chronic Myelogenous Leukemia Kim Robien, 1,5 Jeannette Bigler, 1 Yutaka Yasui, 6 John D. Potter, 1,4 Paul Martin, 2 Rainer Storb, 2 Cornelia M. Ulrich 1,3,4 1 Cancer Prevention Program, Public Health Division, and 2 Clinical Research Division, Fred Hutchinson Cancer Research Center, and 3 Interdisciplinary Graduate Program in Nutritional Sciences and 4 Department of Epidemiology, University of Washington, Seattle, Washington; 5 Division of Epidemiology and Community Health, University of Minnesota, and University of Minnesota Cancer Center, Minneapolis, Minnesota; 6 Public Health Sciences, University of Alberta, Edmonton, Alberta, Canada Correspondence and reprint requests: Kim Robien, PhD, Division of Epidemiology and Community Health, University of Minnesota, 1300 S Second Street, Suite 300, Minneapolis, MN ( robien@epi.umn.edu). Received April 10, 2006; accepted May 30, 2006 ABSTRACT Methylenetetrahydrofolate reductase (MTHFR) and thymidylate synthase (TS) play key roles in intracellular folate metabolism. Polymorphisms in these enzymes have been shown to modify toxicity of methotrexate (MTX) after hematopoietic cell transplantation. In this study, we evaluated the risk of acute graft-versus-host disease (GVHD) associated with genetic variation in recipient and donor MTHFR and TS genotypes to assess whether genotype alters the efficacy of MTX in acute GVHD prophylaxis. Data on the transplantation course were abstracted from medical records for 304 adults who received allogeneic hematopoietic cell transplants. MTHFR (C677T and A1298C) and TS (enhancer-region 28-base pair repeat, TSER, and 1494del6) genotypes were determined using polymerase chain reaction/restriction fragment length polymorphism and TaqMan assays. Multivariable logistic regression was used to assess the associations between genotypes and risk of acute GVHD. Compared with recipients with the wild-type MTHFR 677CC genotype, those with the variant 677T allele showed a decreased risk of detectable acute GVHD (677CT: odds ratio, 0.8; 95% confidence interval, ; 677TT: odds ratio, 0.4; 95% confidence interval, ; P for trend.01). The variant MTHFR 1298C allele in recipients was associated with an increased risk of acute GVHD compared with the wild-type MTHFR 1298AA genotype (1298AC: odds ratio, 2.0; 95% confidence interval, ; 1298CC: odds ratio, 3.6; 95% confidence interval, ; P for trend <.01). No association with risk of acute GVHD was observed for donor MTHFR genotypes or for recipient or donor TS genotypes, with the exception of an increase in acute GVHD among recipients whose donors had the TSER 3R/2R genotype (odds ratio, 3.0; 95% confidence interval, ). These findings indicate that host, but not donor, MTHFR genotypes modify the risk of acute GVHD in recipients receiving MTX, in a manner consistent with our previously reported associations between MTHFR genotypes and MTX toxicity. A direct trade-off between drug toxicity and drug efficacy may play a role. Alternatively, the systemic folate environment, regulated by host tissues, might influence donor T-cell growth and activity American Society for Blood and Marrow Transplantation KEY WORDS Methylenetetrahydrofolate reductase Thymidylate synthase Acute graft-versus-host disease Methotrexate Chronic myelogenous leukemia 973

2 974 K. Robien et al. INTRODUCTION The antifolate drug methotrexate (MTX) is used as an immunosuppressive agent after hematopoietic cell transplantation (HCT) for acute graft-versus-host disease (GVHD) prophylaxis. Folate plays a key role as a methyl-group donor for DNA synthesis and thus is a vital nutrient for rapidly replicating cells, such as hematopoietic cells. We and others have found that some aspects of MTX treatment-related toxicity are modified by key polymorphisms in the folate pathway [1,2]. A critical question is whether this greater sensitivity to MTX corresponds to enhanced acute GVHD prophylaxis. Before consideration of genotype-based dose-adjustments to reduce toxicity in the HCT setting, it is important to assess whether drug efficacy changes by genotype. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the irreversible conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate and directs the flux of intracellular folate toward the conversion of homocysteine to methionine at the expense of nucleotide synthesis [3]. Two common non-synonymous genetic polymorphisms, C677T and A1298C, have been described for this enzyme. The homozygous 677TT genotype, which occurs in approximately 10%-15% of Caucasian and Asian populations [4], has been shown to have only 30%, and the heterozygous (CT) genotype approximately 60% of the MTHFR wild-type enzyme activity in vitro [5]. The 1298C allele has also been found to result in decreased in vitro activity, although to a lesser extent than that seen with the 677T allele [6,7]. Approximately 5%-10% of Caucasians carry the 1298CC genotype [8], which results in an enzyme with approximately 90% of wildtype MTHFR activity [9]. We previously reported that individuals with the MTHFR 677TT genotype are at increased risk of oral mucositis and delayed engraftment after HCT [10,11], which are likely to reflect MTX toxicity to some extent. Thymidylate synthase (TS) catalyzes the transfer of a methyl group from 5,10-methylenetetrahydrofolate to deoxyuridine monophosphate, creating deoxythymidine monophosphate for DNA synthesis. A polymorphic 28-base pair (bp) repeat in the 5=-untranslated region of the TS gene [12] functions as cis-acting transcriptional enhancer element (TSER). Double (2R) and triple (3R) repeats are the most common, but other numbers of repeats have been reported [13]. Individuals with the 3R/3R genotype have been found to have greater mrna expression compared with those with 2R/2R [12,14]. In addition, a functionally relevant 6-bp deletion polymorphism in the 3= untranslated region (1494del6) [15] has been associated with decreased mrna stability [16]. In this study, we evaluated the effect of recipient and donor MTHFR and TS genotypes on risk of acute GVHD among adults who underwent allogeneic HCT for chronic myelogenous leukemia. We hypothesized that recipient genotypes that confer a decreased ability to recycle folate (eg, MTHFR 677TT, MTHFR 1298CC, TSER 2R/2R, and TS 1494del6 / ) would be associated with lower risk of acute GVHD based on previous reports of greater MTX toxicity with these genotypes and thus presumably greater sensitivity to the drug [10,11,17,18]. We also hypothesized that a donor genotype associated with decreased ability to recycle folate would confer greater MTX efficacy and a lower incidence of acute GVHD. We expected that the donor genotype would be the more important determinant of risk of acute GVHD. METHODS Study Design and Recipient Population Subjects in this retrospective cohort study were adults undergoing allogeneic HCT at the Fred Hutchinson Cancer Research Center (FHCRC; Seattle, Wash) between 1992 and 2002 who (1) had a diagnosis of chronic myelogenous leukemia in chronic or accelerated phase before transplantation, (2) were 18 years of age at the time of transplantation, (3) received full myeloablative conditioning regimens with cyclophosphamide/total body irradiation or busulfan/cyclophosphamide, as previously described [19,20], (4) received all 4 scheduled doses of MTX, (5) did not receive leucovorin rescue, and (6) had DNA previously collected from pretransplantation peripheral blood and available for analysis. All study participants received MTX and cyclosporine for GVHD prophylaxis according to previously described protocols [21]. None of the bone marrow or peripheral blood stem cell infusions was depleted of T cells. Data collected referred to the first transplantation if the study participant received 1 hematopoietic cell transplant. This study was approved by the FHCRC institutional review board, and all study participants provided informed consent. Data Collection Medical records and patient databases were used to abstract study data for each study participant. Data collected from the medical record included height, weight, total dose of MTX received after transplantation, and total dose of leucovorin received. To verify reproducibility of chart abstraction data, 13 charts were randomly selected and re-abstracted (blinded to the results of the original abstraction); complete agreement between the 2 abstractions was confirmed. Data collected from patient databases included age, sex, race, conditioning regimen, average busulfan concentration at steady state, donor relationship, donor sex, and date of HCT.

3 MTHFR, TS, and Risk of Acute GVHD 975 Laboratory Analyses MTHFR and TS genotype frequencies were determined using polymerase chain reaction/restriction fragment length polymorphism (MTHFR C677T) or TaqMan assays (MTHFR C677T and A1298C, TSER, TS 1494del6) as reported previously [10,22]. All genotype determinations were performed blinded to acute GVHD status. Determination of Acute GVHD Acute GVHD was diagnosed and peak severity was graded according to standard criteria [23]. For this analysis, acute GVHD incidence was coded as a dichotomous variable (grade 1-4 acute GVHD versus no detectable acute GVHD) because the majority of the cohort had grade 2 acute GVHD, and there was insufficient variation of acute GVHD grades within the cohort to allow for stratification. GVHD data were locked for analysis on July 11, Statistical Analysis Multivariable logistic regression analysis was used to assess the association between genotypes and risk of acute GVHD. We first evaluated the risk of acute GVHD for each polymorphism (MTHFR C667T, MTHFR A1298C, TSER, and TS 1494del6) individually, with the homozygous wild-type genotype for each polymorphism as the reference. Then we evaluated risk of acute GVHD associated with the combined MTHFR (C677T and A1298C) or TS (TSER and TS 1494del6) genotype using individuals who were homozygous wild-type at both loci as the reference group for each analysis. Separate analyses were performed for recipient genotypes and for the genotypes of the corresponding donors as the exposures of interest. Potential confounding factors (including age, sex, race [Caucasian: yes/no], year of transplantation, stage of chronic myelogenous leukemia at transplantation, conditioning regimen, donor relationship, source of hematopoietic progenitor cells [bone marrow versus peripheral blood], age, and sex) were evaluated by adding each factor to the unadjusted model and then assessing its effect on the odds ratio (OR) associated with each genotype; a change of 10% was regarded as sufficient to include the variable in the final model. Age, donor relationship (related versus unrelated), and year of transplantation met the criteria for inclusion as covariates in the regression models. Restricting the analysis to Caucasians did not alter the overall trends in the observed associations; thus, the analyses include data from Caucasian and non-caucasian recipients. Analyses were performed with SAS 9.1 (SAS Institute, Cary, NC). A 95% confidence interval (CI) excluding 1.0 or a 2-sided P value.05 was considered statistically significant. RESULTS Selected characteristics of the study population are presented in Table 1. In total, 350 patients met the criteria for inclusion in the study. DNA was available for all eligible recipients, and 241 (69%) of their donors. The recipient MTHFR C677T genotypes were not in the Hardy-Weinberg equilibrium (HWE; P.03); however, this cohort did not meet the conditions required for HWE because it was a patient group rather than a random sample of the general population. Donor MTHFR C677T and recipient and donor MTHFR A1298C, TSER, and TS 1494del6 genotypes were in HWE. Duplication of a random 10% of the genotyping assays yielded 100% concordance. Detectable acute GVHD occurred in 290 (83%) of the study cohort. In most recipients, acute GVHD had grade 2 peak severity (Table 1). No relation between severity of oral mucositis and severity of acute GVHD was observed in this cohort. Oral mucositis was evaluated prospectively for approximately half of the cohort [10,11,24]; however, there were no statistically significant differences in Oral Mucositis Index scores [25] when stratified by peak acute GVHD Table 1. Characteristics of the Recipient Population (n 350)* Demographics Age (y) (18-67) Sex Male 170 (56) Female 134 (44) Weight (kg) ( ) Height (cm) ( ) Body mass index (kg/m 2 ) ( ) Race White 305 (87) Non-white 45 (13) Transplant information Stage of CML at transplant Chronic 305 (87) Accelerated 45 (13) Conditioning regimen Cyclophosphamide/total body irradiation 180 (51) Busulfan/cyclophosphamide 170 (49) Donor relationship Related donors 180 (51) HLA-matched 169 (94) HLA-mismatched 11 (6) Unrelated 1705 (49) Source of hematopoietic progenitor cells Bone marrow 334 (95) Peripheral blood 16 (5) Acute GVHD grade 0 60 (17) 1 31 (9) (57) 3 52 (15) 4 7 (2) CML indicates chronic myelogenous leukemia. *Mean SD (range) or number of patients (%).

4 976 K. Robien et al. Table 2. Risk of Acute GVHD by MTHFR Genotypes* Recipient Genotype OR (95% CI) Donor Genotype OR (95% CI) MTHFR C677T 677CC 1.0 (reference) 1.0 (reference) n 152 n CT 0.8 ( ) 0.7 ( ) n 143 n TT 0.4 ( ) 0.8 ( ) n 55 n 31 P for trend MTHFR A1298C 1298AA 1.0 (reference) 1.0 (reference) n 172 n AC 2.0 ( ) 0.8 ( ) n 127 n CC 3.6 ( ) 0.9 ( ) n 38 n 23 P for trend < *Adjusted for age, donor relationship (related versus unrelated), and year of transplantation. grade and adjusted for age and conditioning regimen (data not shown). The variant MTHFR 677T allele in recipients was associated with a statistically significant trend toward decreased risk of any detectable acute GVHD (MTHFR 677CT: OR, 0.8; 95% CI, ; MTHFR 677TT: OR, 0.4; 95% CI, ; P for trend.01; Table 2). The variant MTHFR 1298C allele in recipients was associated with a statistically significant increase in acute GVHD risk compared with the wildtype MTHFR 1298AA genotype (1298AC: OR, 2.0; 95% CI, ; 1298CC: OR, 3.6; 95% CI, ; P for trend.01). No association with acute GVHD risk was observed for donor MTHFR genotypes (Table 2). Recipient and donor TS genotypes showed no association with acute GVHD risk, with the exception of the donor TSER 3R/2R genotype, which showed a statistically significant increased risk of acute GVHD (OR, 3.0; 95% CI, ; Table 3). Although not statistically significant, analysis of the combined recipient MTHFR C677T/A1298C genotype showed similar patterns to the individual polymorphism analysis (Table 4). We were unable to assess accurately the risk of acute GVHD by combined donor MTHFR C677T/A1298C genotypes due to small numbers of certain haplotypes. Analysis of the recipient or donor combined TS genotypes by the number of variant alleles (0-1, 2, or 2 variant alleles) showed no statistically significant associations with risk of acute GVHD (Table 3). We previously reported no statistically significant associations between MTHFR C677T or A1298C genotype and overall survival in this cohort (adjusted for age, stage of disease before transplantation, and donor relationship) [26]. Because the inclusion criteria for this study required participants to have received all 4 scheduled doses of MTX and no leucovorin rescue (severe or life-threatening toxicity is an indication for withholding doses of MTX or rescuing the patient with leucovorin), there is a low likelihood of MTX toxicity-related early mortality in this study cohort. DISCUSSION This is the largest study to date to evaluate the effect of genetic variation in folate-related enzymes on risk of acute GVHD after HCT, and the first to evaluate the effect of the MTHFR A1298C, TSER, and TS 1494del6 genotypes. Strengths of our study design include homogeneity in the study participants pretransplantation diagnosis, treatment regimens, and acute GVHD prophylaxis. Another strength is that we used multivariable analyses, which allowed us to adjust for factors known to contribute to acute GVHD, such as donor relationship. In contrast to our findings, Pihusch et al [27] reported no association between MTHFR C677T genotype and risk of acute GVHD in their study of 89 allogeneic transplant recipients (compared with 304 reported in this study). However, it is difficult to compare their findings with ours because their research focused on genes related to thromboembolic complications rather than response to MTX, and only 38% of the recipients in their study received MTX as GVHD prophylaxis (most received mycophenolate). Table 3. Risk of Acute GVHD by TS Genotypes* Recipient Genotype OR (95% CI) Donor Genotype OR (95% CI) TSER TS 3R/3R 1.0 (reference) 1.0 (reference) n 97 n 71 TS 3R/2R 1.4 ( ) 3.0 ( ) n 159 n 108 TS 2R/2R 0.8 ( ) 1.5 ( ) n 73 n 53 P for trend TS 1494del6 6 /6 1.0 (reference) 1.0 (reference) n 149 n /6 1.1 ( ) 0.8 ( ) n 141 n /6 0.7 ( ) 0.9 ( ) n 39 n 26 P for trend TS functional genotype 0-1 variant allele 1.0 (reference) 1.0 (reference) n 150 n variant alleles 0.5 ( ) 1.1 ( ) n 141 n 110 >2 variant alleles 1.2 ( ) 1.0 ( ) n 38 n 21 P for trend *Adjusted for age, donor relationship (related versus unrelated), and year of transplantation.

5 MTHFR, TS, and Risk of Acute GVHD 977 Table 4. Risk of Acute GVHD by Combined Recipient MTHFR C677T/A1298C Genotype* 1298AA 1298AC 1298CC P for Trend 677CC 1.0 (reference) n ( ) n ( ) n CT 1.1 ( ) n ( ) n 64 Not observed 677TT 0.5 ( ) n 53 Not observed Not observed P for trend.19 *Adjusted for age, donor relationship (related versus unrelated), and year of transplantation. Their study also included adult and pediatric recipients with a variety of hematologic malignancies and conditioning regimens. A recent report by Murphy et al [28] of 193 patients found that having an HLA-matched donor with the MTHFR 677CT or TT genotype was associated with a decreased incidence of acute and chronic GVHD. No associations with GVHD risk were observed by recipient or HLA-mismatched donor MTHFR C677T genotype. This study included patients who received myeloablative (68%) or nonmyeloablative (32%) transplants, making comparisons difficult because the GVHD experience is considerably different between myeloablative and nonmyeloablative treatment regimens [29]. Their study cohort also included patients who had MTX dose reductions due to severe mucositis or hepatic dysfunction (27%) and patients who received leucovorin rescue (53%). We previously reported that the MTHFR 677TT recipient genotype is associated with increased early toxicity after transplantation [10,11] and the 1298CC recipient genotype may be associated with decreased early toxicity after transplantation [11] in response to MTX (although this is not apparent when the combined MTHFR genotypes are considered, which is probably a more meaningful analysis). We currently report that individuals with the MTHFR 677TT genotype appear to be at decreased risk of acute GVHD and individuals with the 1298CC genotype are at increased risk of acute GVHD, as would be expected if increased toxicity results from increased MTX activity and immunosuppression. Thus, the present results mirror, to some degree, the observed data on toxicity, associating the 677TT genotype with greater toxicity and enhanced efficacy (ie, reduced GVHD). The TS genotypes appear to have no association with risk of acute GVHD, but TS 1494del6 may confer increased toxicity [11]. In contrast to our original hypothesis that the MTHFR C677T and A1298C genotypes would have effects in similar directions with regard to acute GVHD risk, we found that the variants of each polymorphism were associated with opposite effects on risk of acute GVHD. The precise biological relevance of the MTHFR A1298C polymorphism is unclear, but its effects on in vitro enzyme activity have repeatedly been shown to be less pronounced than that of the C677T polymorphism [6,7,9]. In vivo, these findings have been supported by studies showing little difference in plasma homocysteine or plasma folate levels by MTHFR A1298C genotype [6,7,9,30,31], although red cell folate levels have been found to be significantly higher in individuals with the 1298CC genotype [31]. Several epidemiologic studies have reported stronger associations between the A1298C polymorphisms and various disease outcomes than those observed with the C677T polymorphism [26,32-35]. Thus, the functional relevance of the A1298C genotype may involve gene-environment or gene-gene interactions not captured in vitro, or the variant may result in alterations in enzyme transcription, translation, or protein degradation. A study using a baculovirus expression system failed to identify a biochemical phenotype associated with the 1298C variant protein, leading the investigators to suggest that the decreased activity of the variant protein is the result of differences in protein stability [36]. Our findings demonstrate the importance of evaluating both polymorphisms concurrently to understand fully the effect of genetic variability in the MTHFR gene and avoid erroneous conclusions. We did not observe an association between genetic variation in donor MTHFR and TS genotypes and acute GVHD risk. The systemic availability of folate metabolites (specifically 5-methyltetrahydrofolate) is regulated by MTHFR activity, and although the hematopoietic cells are of donor origin after HCT, the majority of systemic MTHFR activity remains that of the host tissues. Thus, recipient MTHFR genotypes may play a vital role in the overall availability of folate necessary for tissue repair and donor T-cell growth and activity. Decreased MTHFR activity associated with the 677TT genotype has been shown to result in lower levels of circulating 5-methyltetrahydrofolate in settings of low folate status [37-40], as would be expected during MTX treatment. Studies of the MTHFR A1298C polymorphism have failed to show any statistically significant differences in plasma 5-methyltetrahydrofolate levels by genotype [6,41,42]. Genetic variation may also modify other aspects of the host tissue response, including tissue response to inflammatory cytokines. MTHFR regulates intracellular concentrations of tetrahydrofolate derivatives, including 10-formyltetrahydrofolate, which is a vital 1-carbon group donor for purine biosynthesis; a reac-

6 978 K. Robien et al. tion catalyzed by 5-aminoimidazole-4-carboxamide ribonucleotide transformylase. Adenosine release into the extracellular space as a result of 5-aminoimidazole-4- carboxamide ribonucleotide transformylase inhibition and accumulation of purine biosynthesis intermediates has emerged as an important mechanism by which MTX exerts anti-inflammatory activity [43]. The findings of this study on the MTHFR C677T polymorphism support this hypothesis because decreased MTHFR activity among 677TT individuals would result in greater accumulation of purine intermediates, increased release of adenosine, decreased local tissue inflammation, and decreased acute GVHD. A better understanding of the functional relevance of the A1298C polymorphism is needed before attempting to explain how this polymorphism might relate to the adenosine release hypothesis. Several groups have demonstrated that polyglutamated MTX selectively inhibits TS activity and induces cell death in replicating cells due to a lack of thymine ( thymine-less death ) [44-46]. This TS inhibition may explain our findings of a general lack of association between TS genotype and risk of acute GVHD. However, one would expect TS inhibition to be dose-dependent as is seen with thymidylate synthase inhibitors such as 5-fluorouracil; individuals with the 3R/3R genotype (with greater TS expression) should exhibit less complete inhibition of TS than would be seen in individuals with the 2R/2R genotype. In this study, only the donor TSER 3R/2R genotype showed a statistically significant association with acute GVHD risk, which we are unable to explain. Our previous research found no difference in oral mucositis scores by TSER genotype; however, the homozygous variant TS 1494del6 6bp /6bp genotype was associated with a statistically significant increase in oral mucositis compared with the homozygous wildtype TS 1494del6 genotype [11]. This is the largest study to date on the effects of the MTHFR and TS genotypes on issues related to the immunosuppressive properties of MTX. However, despite the relatively large sample, statistical power to evaluate interaction between the MTHFR and TS genotypes or stratify by grade or site of acute GVHD was limited. Even larger studies are needed to evaluate the association between genotype and these details of the acute GVHD experience. The role of folate-mediated 1-carbon metabolism in the risk of acute GVHD after HCT deserves further investigation, as does further elucidation of the functional relevance of the MTHFR A1298C variant. Although this study was limited to individuals who received MTX for acute GVHD prophylaxis, the multifaceted role of folate in tissue replication and repair suggests that genetic variation in this pathway may have relevance in predicting outcomes in individuals who receive other forms of acute GVHD prophylaxis. ACKNOWLEDGMENTS Research Program Project Grant (P01) funding was provided by the National Heart, Lung and Blood Institute (HL36444) and the National Cancer Institute (CA18029) and a Cancer Center Support Grant (P30) from the National Cancer Institute (CA15704). Support for KR was provided by National Cancer Institute training grant R25 CA This project would not have been possible without the biologic material obtained with the help of Eric Mickelson and the FHCRC Human Immunogenetics Program; clinical data support from Gary Schoch and the FHCRC Clinical Computing group; and technical support from the FHCRC PHS Core Laboratory group. REFERENCES 1. Krajinovic M, Moghrabi A. 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