Abstract

This study investigated whether a polymorphism in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene (C677T) modifies responses to methotrexate (MTX) in patients undergoing bone marrow transplantation. About 10% to 12% of the population carry the MTHFR TT genotype (enzyme activity, 30% of wild type [CC]). Patients (n = 220) with chronic myelogenous leukemia underwent marrow allografts and were given a short course of MTX. MTX toxicity measures included the oral mucositis index (OMI), speed of engraftment (platelet and granulocyte counts), and bilirubin. Patients with lower MTHFR activity (TT genotype) had 36% higher mean OMI during days 1 to 18 (+5.7, P = .046) and 20% higher OMI between days 6 and 12 (+3.8, P = .27). Platelet counts recovered more slowly among patients with the TT genotype compared to wild type (24% slower recovery to 10 000 platelets/μL, P = .23; 34% slower to 20 000/μL, P = .08). Patients with decreased MTHFR activity appear at risk of higher MTX toxicity. Because of the high prevalence of the TT genotype, these results may have implications for MTX dosage.

Introduction

Methotrexate (MTX) is an antifolate chemotherapeutic drug,1 and is used in patients undergoing marrow transplantation to prevent graft-versus-host disease (GVHD).2 Toxicities include mucositis and myelosuppression.1 We hypothesized that MTX sensitivity would vary with genetic variability in folate-metabolizing enzymes.

Folate is essential for nucleotide synthesis. The effectiveness of MTX is largely attributable to its role as an inhibitor of dihydrofolate reductase. Its metabolites also inhibit other folate enzymes, including 5,10-methylenetetrahydrofolate reductase (MTHFR),1,3,4 which converts 5,10-methylenetetrahydrofolate to 5,10-methyltetrahydrofolate.

A common MTHFR polymorphism (C677T) results in reduced activity.5 The variant TT genotype, associated with about 30% of wild-type (CC) activity, is present in about 10% to 12% of white and Asian populations. Heterozygotes (CT) (about 60% activity) constitute approximately 40% of the population. Variations are seen in risk of acute lymphocytic leukemia,6colorectal neoplasia,7-10 neural tube defects,11,12 and possibly cardiovascular disease,13 largely in the presence of low folate levels. Because MTX induces a low folate state, we hypothesized that toxicity would be aggravated among patients with the TT and, possibly, the CT genotype.

Study design

Study design and patient population

We undertook a study of patients undergoing marrow transplantation at the Fred Hutchinson Cancer Research Center (FHCRC) from 1992 to 1999. The following criteria applied: (1) first chronic phase of chronic myelogenous leukemia14; (2) first transplantation; (3) conditioning regimen: either busulfan/cyclophosphamide15 or cyclophosphamide/total body irradiation, as described15,16; (4) age at transplantation, at least 18 years; and (5) available DNA. All patients received 4 doses of MTX (intravenously, day 1, at 15 mg/m,2 and days 3, 6, and 11, at 10 mg/m2) and cyclosporine (as previously described15) for prevention of GVHD.2 All patients gave informed consent. The study was approved by the FHCRC Institutional Review Board.

Data collection

Data were abstracted by a single abstractor (blinded to genotypes) on previous interferon treatment, smoking, MTX administration, and, if applicable, MTX serum levels and leucovorin administration.

Data from the patient database included: (1) conditioning regimen; (2) donor/matching status; (3) demographics; (4) weight, height, and calculated body mass index and surface area; (5) platelet and granulocyte counts; (6) bilirubin and creatinine; and (7) survival status or day of last contact.

Mucositis is a major toxicity associated with folate antagonists. The oral mucositis index (OMI) was developed17 to measure severity of oral mucosal changes; patients were examined every 2 to 3 days. Oral mucositis usually peaks between days 7 and 11 and resolves by days 18 to 21 after transplantation. We assessed mean scores on days 6 to 12 (peak period) and days 1 to 18 (overall mucositis course).

MTHFR genotyping

Genotyping for the MTHFR C677T polymorphism was performed,9 blinded to outcome measures; 10% blinded duplicate samples yielded 100% concordance. Genotypes were in Hardy-Weinberg equilibrium.

Statistical data analysis

The primary outcome was oral mucositis, as measured by the OMI (mean, days 6-12, at least 2 assessments; and days 1-18, at least 4 assessments). Secondary outcomes included granulocyte engraftment, assessed as time to the first of 3 consecutive days of counts exceeding 100/μL and 500/μL; platelet engraftment, assessed as time to the first of 7 consecutive days of counts exceeding 10 000/μL and 20 000/μL without transfusion; and bilirubin, days 1 to 18. We were limited in our ability to investigate other measures of MTX toxicity: MTX serum levels, leucovorin administration as a rescue drug, and intestinal mucositis.

The original cohort consisted of 238 patients with chronic myelogenous leukemia (CML). Charts for 227 patients were abstracted. For 7 patients, we could not verify that 4 doses of MTX had been given; 136 and 124 patients, respectively, met inclusion criteria for OMI measurement during days 6-12 and 1-18.

All analyses were performed to investigate differences in outcomes by MTHFR genotype: individuals with the CT and TT genotypes were each compared to those with the CC genotype. Linear regression was used for continuous variables. Adjusted OMI means were calculated for all genotypes with covariates set at mean levels. Comparisons across genotypes were made using t tests. Measures of engraftment were analyzed using proportional hazards. Patients were censored on day 29 because surveillance was incomplete after this time. Patients who died before day 29 were censored on day of death (n = 2).

All analyses were adjusted for age, sex, conditioning regimen (corresponding to donor type), total delivered MTX dose, weight, and height. Engraftment analyses were also adjusted for creatinine. Other covariates that did not affect the relationship between genotype and outcome were race, smoking status, birthplace, and previous interferon treatment.

Results and discussion

Characteristics of the patients are shown in Table1. Patients with lower MTHFR activity had a higher OMI during days 1 to 18 (Figure1). The mean (95% confidence interval) for patients with the CC (wild type) genotype was 15.9 (13.2-18.6); for the CT genotype, 17.5 (14.8-20.3); and for the TT genotype, 21.6 (16.8-26.4) (P < .05 compared to CC). The increase across CT (+1.6) and TT genotypes (+5.7) corresponds to the approximate residual MTHFR enzyme activity among those who carry the variant allele (CT, 60% activity; TT, 30% activity). Similar trends were observed for days 6 to 12: OMI differences [compared to the mean for CC genotype = 18.4 (15.2-21.7)]: CT genotype: +2.7,P = .25; TT genotype: +3.8, P = .27). Higher levels of mucositis among patients with a TT genotype correspond to a 36% increase in the mean OMI during days 1 to 18.

Table 1.

Characteristics of the study population

Study cohort CML patients
N = 220 n (%)*
Demographic information  
Age (y) 39.8 ± 9.4 (18-61) 
Age groups (no. patients)  
 18-30 38 (17%)  
 31-40 78 (36%) 
 41-50 74 (34%)  
 > 50 30 (14%)  
Sex (no. patients)  
 Male 117 (53%)  
 Female 103 (47%) 
Race/Ethnicity (no. patients)  
 White 196 (89%) 
 Nonwhite 24 (11%)  
Birthplace (no. patients)  
 United States 204 (93%)  
 Non-United States 16 (7%)  
Weight (kg) 78.0 ± 15.7 (41.2-132.1) 
Height (cm) 171.2 ± 10.0 (123.4-194.5) 
Smoking status (no. patients)  
 Never 113 (51%)  
 Current 9 (4%) 
 Former 66 (30%)  
 No information 32 (15%) 
Transplant-related characteristics  
Conditioning regimen (no. patients)  
 Cy/TBI 112 (51%)  
 Bu/Cy 108 (49%) 
Donor type (no. patients)  
 Unrelated 112 (51%) 
 Related 108 (49%)  
  HLA-matched 101 (46%) 
  HLA-mismatched 6 (3%)  
  Unknown 1 (0.5%) 
Previous interferon treatment (no. patients)  
 Yes 98 (45%)  
 No 122 (56%) 
Survival status (no. patients)  
 Alive 170 (77%) 
 Dead 50 (23%)  
Oral mucositis index  
 OMI d 6-12 20.1 ± 12.2  (0.0-64.0) 
 OMI d 1-18 17.4 ± 9.8 (1.3-54.4) 
Granulocyte recovery  
 Median days to reach 100/μL 15 (6-38) 
 Median days to reach 500/μL 21 (11-51) 
Platelet recovery  
 Median days to reach 10 000/μL 18 (9-85) 
 Median days to reach 20 000/μL 18 (9-85) 
Bilirubin levels  
 Median level d 1-18 1.42 (0.30-6.64) 
 Highest level d 1-18 2.30 (0.50-17.5) 
 Difference between highest and baseline  level, d 1-18 1.48 (0-17.1) 
MTHFR genotype (no. patients)  
 CC (wild type) 92 (42%)  
 CT (heterozygous) 92 (42%)  
 TT (homozygous variant) 36 (16%) 
Study cohort CML patients
N = 220 n (%)*
Demographic information  
Age (y) 39.8 ± 9.4 (18-61) 
Age groups (no. patients)  
 18-30 38 (17%)  
 31-40 78 (36%) 
 41-50 74 (34%)  
 > 50 30 (14%)  
Sex (no. patients)  
 Male 117 (53%)  
 Female 103 (47%) 
Race/Ethnicity (no. patients)  
 White 196 (89%) 
 Nonwhite 24 (11%)  
Birthplace (no. patients)  
 United States 204 (93%)  
 Non-United States 16 (7%)  
Weight (kg) 78.0 ± 15.7 (41.2-132.1) 
Height (cm) 171.2 ± 10.0 (123.4-194.5) 
Smoking status (no. patients)  
 Never 113 (51%)  
 Current 9 (4%) 
 Former 66 (30%)  
 No information 32 (15%) 
Transplant-related characteristics  
Conditioning regimen (no. patients)  
 Cy/TBI 112 (51%)  
 Bu/Cy 108 (49%) 
Donor type (no. patients)  
 Unrelated 112 (51%) 
 Related 108 (49%)  
  HLA-matched 101 (46%) 
  HLA-mismatched 6 (3%)  
  Unknown 1 (0.5%) 
Previous interferon treatment (no. patients)  
 Yes 98 (45%)  
 No 122 (56%) 
Survival status (no. patients)  
 Alive 170 (77%) 
 Dead 50 (23%)  
Oral mucositis index  
 OMI d 6-12 20.1 ± 12.2  (0.0-64.0) 
 OMI d 1-18 17.4 ± 9.8 (1.3-54.4) 
Granulocyte recovery  
 Median days to reach 100/μL 15 (6-38) 
 Median days to reach 500/μL 21 (11-51) 
Platelet recovery  
 Median days to reach 10 000/μL 18 (9-85) 
 Median days to reach 20 000/μL 18 (9-85) 
Bilirubin levels  
 Median level d 1-18 1.42 (0.30-6.64) 
 Highest level d 1-18 2.30 (0.50-17.5) 
 Difference between highest and baseline  level, d 1-18 1.48 (0-17.1) 
MTHFR genotype (no. patients)  
 CC (wild type) 92 (42%)  
 CT (heterozygous) 92 (42%)  
 TT (homozygous variant) 36 (16%) 

CML indicates chronic myelogenous leukemia; Cy, cyclophosphamide; Bu, busulfan; OMI, oral mucositis index; TBI, total body irradiation; MTHFR, 5,10-methylenetetrahydrofolate reductase.

*

Because of rounding not all percentages total 100.

Mean ± SD (range).

Median (range).

Fig. 1.

Differences in oral mucositis index (OMI) days 6 to 12 and 1 to 18 after transplantation (mean ± SE).

Patients with the homozygous variant MTHFR TT genotype have higher OMI scores. Adjusted mean OMI scores in patients with the wild type CC genotype (reference group) are 18.4 for days 6 to 12 and 15.9 for days 1 to 18 after transplantation, corresponding to an increase of 20% and 36% in the OMI score, respectively, among patients with the TT genotype. All analyses are adjusted for patients' age, sex, conditioning regimen, total methotrexate dose, height, and body weight.

Fig. 1.

Differences in oral mucositis index (OMI) days 6 to 12 and 1 to 18 after transplantation (mean ± SE).

Patients with the homozygous variant MTHFR TT genotype have higher OMI scores. Adjusted mean OMI scores in patients with the wild type CC genotype (reference group) are 18.4 for days 6 to 12 and 15.9 for days 1 to 18 after transplantation, corresponding to an increase of 20% and 36% in the OMI score, respectively, among patients with the TT genotype. All analyses are adjusted for patients' age, sex, conditioning regimen, total methotrexate dose, height, and body weight.

Recovery of platelet counts was slower among patients with variant genotypes than among those with the CC genotype (Table2). Recovery to 10 000/μL was 24% slower for the TT genotype (P = .23), and recovery to 20 000/μL, 34% slower (P = .08). Recovery of granulocytes was slightly slower among individuals with variant genotypes, although neither was statistically significant nor showed a trend.

Table 2.

Indicators of engraftment by 5,10-methylenetetrahydrofolate reductase genotype

Measure of
MTX toxicity
MTHFR CC
genotype
(wild type)
MTHFR
CT genotype
MTHFR
TT genotype
Platelet recovery*    
Days to reach 10 000 platelets/μL 1.0  (ref) 1.00  (0.70-1.42) 0.76  (0.49-1.19)  
Days to reach 20 000 platelets/μL 1.0  (ref) 1.02  (0.71-1.45) 0.66  (0.42-1.05)  
Granulocyte recovery    
Days to reach 100 granulocytes/μL 1.0  (ref) 0.93  (0.68-1.30) 1.05  (0.68-1.60)  
Days to reach 500 granulocytes/μL 1.0  (ref) 0.88  (0.63-1.23) 0.93  (0.74-1.16) 
Measure of
MTX toxicity
MTHFR CC
genotype
(wild type)
MTHFR
CT genotype
MTHFR
TT genotype
Platelet recovery*    
Days to reach 10 000 platelets/μL 1.0  (ref) 1.00  (0.70-1.42) 0.76  (0.49-1.19)  
Days to reach 20 000 platelets/μL 1.0  (ref) 1.02  (0.71-1.45) 0.66  (0.42-1.05)  
Granulocyte recovery    
Days to reach 100 granulocytes/μL 1.0  (ref) 0.93  (0.68-1.30) 1.05  (0.68-1.60)  
Days to reach 500 granulocytes/μL 1.0  (ref) 0.88  (0.63-1.23) 0.93  (0.74-1.16) 

Rate ratios of recovery and 95% confidence intervals are presented. A rate ratio <1.0 indicates slower cell count recovery than in the wild-type reference group (CC genotype). MTX indicates methotrexate. See Table 1 for other abbreviation.

*

Cox regression analyses, adjusted for age (4 groups), sex, conditioning regimen, total MTX dose, weight (kg), height (cm), and mean creatinine levels (days 1-12).

Mean bilirubin levels and increases in bilirubin levels between days 1 and 18 were not related to genotype (data not shown).

To our knowledge, this is the first study illustrating a relationship between a folate-related variant and MTX toxicity. As hypothesized, patients with the MTHFR TT genotype experienced higher toxicity, evidenced by increased oral mucositis and a trend toward delayed platelet recovery. Imbalances in folate pools in those with the TT genotype are predictable18 and have been demonstrated.19 Low folate (dietary or MTX induced) may further decrease availability of folate for nucleotide synthesis.9 

A model of oral mucositis has recently been described.20In patients undergoing marrow transplantation, mucosal toxicity induced by the conditioning regimens predominates. We showed that patients with the MTHFR TT genotype experience higher OMI levels during days 1 to 18, with an effect size similar to that of cyclophosphamide/total body irradiation conditioning over busulfan/cyclophosphamide conditioning (> 30%). Among these patients, reduced DNA repair capacity, attributable to a decreased synthesis of nucleotides, probably results in delayed healing.

Engraftment of platelets was similarly affected. One might predict that the donor genotype would be more relevant for this outcome. However, the observed delay in recovery may indicate that the presence of the much larger number of recipient somatic cells affects folate pools. The product of MTHFR is 5′-methyl-tetrahydrofolate reductase, the transport form of folate. Thus, patients with a TT genotype may show a decreased availability of folate.19,21-23 For consent reasons we were not able to genotype the donors. Future studies should obtain both patient and donor genotypes to enhance understanding of this possible interaction and its impact.

The high prevalence of the MTHFR variant allele and the ability to perform genotyping for this single nucleotide polymorphism, rapidly and inexpensively, may render it a candidate for expanding tailored therapy24; genotyping may reduce MTX toxicity among a subgroup of patients. Although alternatives to MTX for GVHD prevention are limited, dose adjustment or newly evaluated drugs25may be appropriate.

We are indebted to the staff members who were involved in the conduct of this study: Lori Hubbard who performed the chart abstraction, Gary Schoch who provided support with the master patient database, and Sheryl Marks, Eileen van Hollebecke, and members of the FHCRC Collaborative Data Services who performed the data entry of the chart abstraction data. We thank Anajane Smith, Eric Mickelson, and Dr Effie Petersdorf for support in DNA retrieval, Michele Lloid for performing the OMI exams, Juanita Leija for technical assistance with the genotyping, and Mari Nakayoshi for graphical and word processing assistance.

Supported by the National Cancer Institute Cancer Center Support Grant Interdisciplinary Pilot Program, by the National Institutes of Health grants CA18029, CA18221, CA 15704 (Core), and HL 36444, and by the National Institutes of Environmental Health Sciences Center ES-07033.

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.

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Author notes

Cornelia Ulrich, Fred Hutchinson Cancer Research Center, Cancer Prevention Research Program, 1100 Fairview Ave N, MP-900, Seattle, WA 98109-1024; e-mail: nulrich@fhcrc.org.