Skip to content
BY 4.0 license Open Access Published online by De Gruyter March 11, 2022

MTHFR c.665C>T guided fluoropyrimidine therapy in cancer: gender-dependent effect on dose requirements

Charalampia Ioannou, Georgia Ragia, Ioanna Balgkouranidou, Nikolaos Xenidis, Kyriakos Amarantidis, Triantafyllia Koukaki, Eirini Biziota, Stylianos Kakolyris and Vangelis G. Manolopoulos

Abstract

Objectives

The fluoropyrimidine derivatives 5-Fluorouracil and Capecitabine are widely used for the treatment of solid tumors. Fluoropyrimidine metabolism involves a cascade of different enzymes, including MTHFR enzyme. MTHFR c.665C>T polymorphism, leading to decreased MTHFR activity, is a potential pharmacogenomic marker for fluoropyrimidine drug response. The aim of the present study was to analyze the association of MTHFR c.665C>T polymorphism with fluoropyrimidine response in terms of therapy induced adverse events (AEs), requirement of dose reduction and delayed drug administration or therapy discontinuation.

Methods

The study group consisted of 313 fluoropyrimidine-treated cancer patients. PCR-RFLP was used to analyze MTHFR c.665C>T polymorphism.

Results

In female patients, MTHFR c.665 CT and TT genotypes were associated with dose reduction (p=0.029). In gender stratification, regression analysis adjusted for age of disease onset, body surface area and AE incidence, showed that MTHFR CT and TT genotypes increased both need for fluoropyrimidine dose reduction (OR 5.050, 95% CI 1.346–18.948, p=0.016) and percentage of dose reduction (β=3.318, 95% C.I. 1.056–5.580, p=0.004) in female patients. Such differences were not present in male patients. No other associations were found.

Conclusions

MTHFR c.665C>T polymorphism was associated with fluoropyrimidine dose reduction in female cancer patients. This gender*MTHFR interaction merits further investigation.

The fluoropyrimidine derivatives 5-Fluorouracil (5-FU) and Capecitabine (CAP) are widely used for the treatment of solid tumors, including gastrointestinal, breast, head and neck cancer [1]. Fluoropyrimidine metabolism involves a cascade of different enzymes, including dihydropyrimidine dehydrogenase (DPD) enzyme that catabolizes fluoropyrimidines to inactive metabolites, thymidylate synthase (TS) enzyme that methylates deoxyuridine monophosphate (dUMP) to form thymidine monophosphate (dTMP) and MTHFR enzyme that converts 5,10-Methyltetrahydrofolate (5,10-MTHF) to 5-MTHF. Fluoropyrimidine metabolism finally leads to the generation of the active nucleotide analogues 5-fluoro-2′-deoxyuridine-5′-monophosphate (FdUMP) and 5-fluorouridine-5′-triphosphate (FUTP) that are incorporated into both DNA and RNA, exerting thus the fluoropyrimidine antineoplasmic action. FdUMP forms a ternary complex with 5,10-MTHF and TS; this complex restrains the synthesis of DNA. Imbalance of fluoropyrimidine nucleotide analogues ratio is associated with fluoropyrimidine response and incidence of adverse events (AEs) that can be life-threatening or fatal and represent a major cause of reduced dosage, delayed drug administration and/or therapy discontinuation [1].

Substantial inter-individual variation in fluoropyrimidine-related AEs is partly explained by genetic factors. A detailed graphic representation of the candidate genes involved in fluoropyrimidine pharmacokinetics is described by Thorn et al. [1]. Currently, DPYD is the only pharmacogene applied in fluoropyrimidine clinical setting [2]. MTHFR is also a potential PGx marker for fluoropyrimidine drug response since it reduces the amount of 5,10-MTHF available for binding to FdUMP and TS. Among MTHFR gene polymorphisms, rs1801133 (c.665C>T, historically referred to as C677T, p. Ala222Val), is a common functional polymorphism which promotes the thermolability and diminishes the enzyme activity [3]. Therefore, the MTHFR c.665C>T polymorphism enhances the cytotoxic potential of fluoropyrimidines and may be closely related to dose requirements and the efficacy of fluoropyrimidine treatment.

In the present study, we have analyzed the potential association of MTHFR c.665C>T polymorphism with the clinical response of Greek cancer patients to 5-FU/CAP regiments in terms of 5-FU/CAP induced AEs and 5-FU/CAP dose reduction and delayed drug administration or therapy discontinuation. Patient cohort consists of 313 cancer patients treated with fluoropyrimidines, as described elsewhere in detail [4]. In brief, eligible were patients treated with 5-FU or CAP in monotherapy or in combination with other antineoplastic drugs. Toxicity was recorded by grade according to the common terminology criteria for adverse events v5.0. All patients were evaluated by the same team of oncologists who were responsible for clinical decisions on chemotherapeutic regiment, dosages, timeline of drug administration and therapy discontinuation [4]. In the same cohort, we have previously shown a gender driven association of TYMS-TSER 3R/2R polymorphism with incidence of AEs in female cancer patients [4]. Therefore, in the present study, we have further seeked for potential gene-gender and gene-gene interactions both in pooled sample of fluoropyrimidine treated cancer patients as well as in sub-groups after stratifying patients according to the type of cancer. All patients participated after being informed about the study by their attending clinician and giving written consent. The protocol of the study was approved by the Scientific Council and the Ethics Committee of the Academic General Hospital of Alexandroupolis (Greece) and was conducted according to the Declaration of Helsinki.

Main characteristics of patient cohort in total and stratified by gender are presented on Table 1. In stratification of patients in sub-groups for dose reduction (44 cases) and for having delayed drug administration or therapy discontinuation (59 cases), cases vs. controls were age-matched and no differences were observed in body weight, height or body surface area (BSA). As expected, cases in both dose reduction and delayed drug administration or therapy discontinuation groups had increased frequency of AE, lower dose intensity and greater % dose reduction (p<0.001 for all comparisons). Dose reduction cases had a 30% range of % dose reduction (minimum 20, maximum 50), whereas delayed drug administration or therapy discontinuation cases had a range of 10 cycles until therapy modification (minimum 1, maximum 11).

Table 1:

Main characteristics of patient cohort in total and stratified by gender.

Patient cohort (n=313) Male patients (n=160) Female patients (n=153) p-Value
Age, years (mean ± SD) 64.2 ± 10.6 66.1 ± 9.7 62.1 ± 11.1 0.001
Body weight, kg; median (25, 75 percentiles) 74.36 (64.00, 83.00) 76.00 (65.00, 87.00) 70.00 (63.00, 80.00) 0.006
Height, m (mean ± SD) 1.65 ± 0.01 1.71 ± 0.07 1.58 ± 0.07 <0.001
BSA; median (25, 75 percentiles) 1.79 (1.6, 1.9) 1.9 (1.7, 2.0) 1.7 (1.6, 1.8) <0.001
Smokers, n (%) 72 (23.0) 56 (35.0) 16 (10.5) <0.001
Adverse events, n (%) 208 102 (63.8%) 106 (69.3) 0.3
Dose reduction, n (%) 44 (14) 26 (16.2) 18 (11.8) 0.254
Fluoropyrimidine dose reduction, %; median (25, 75 percentiles) 20 (20, 25) 20 (20, 25) 20 (20, 25) 0.181
Delayed drug administration or therapy discontinuation, n (%) 59 (18.8%) 34 (21.3%) 25 (16.3%) 0.267
Relative dose intensity, %; median (25, 75 percentiles) 100 (96, 100) 100 (96, 100) 100 (100, 100) 0.173

  1. BSA, body surface area, SD, standard deviation. Normality of continuous variables was checked using Shapiro–Wilk test. All normally distributed continuous variables are expressed as mean ± SD, whereas variables with skewed distribution are expressed as median (25th, 75th percentiles). Comparisons for normally distributed continuous variables between two groups were performed with independent t-test. Non normally distributed continuous variables were compared by Mann–Whitney test. Comparisons for categorical data were performed by using chi-square (χ2)-test. The dose intensity was calculated as the total dose divided by the duration of dosing, while the planned dose intensity was calculated as the planned dose divided by the planned duration of dosing. The relative dose intensity (RDI) was calculated as (dose intensity/planned dose intensity)*100. A p-value less than 0.05 was considered statistically significant. The Statistical Package for Social Sciences (SPSS) for Windows, Version 17.0 (IBM Corp., NY, USA) was used for all analyses.

In patient cohort, the frequencies of MTHFR c.665C>T CC, CT and TT genotypes were 46.0, 45.4 and 8.6%, respectively, whereas the frequency of T allele was 31.3%. Genotype and allele frequencies were in Hardy-Weinberg equilibrium (χ2=0.94, p=0.33).

When comparing frequencies of MTHFR c.665C>T genotypes and alleles between AE cases and AE controls, no differences were observed (p=0.354 and 0.186, respectively) (Table 2). Similarly, no differences were observed when dominant and recessive models of inheritance were applied (p=0.301 and p=0.210, respectively, data not shown). When patients were stratified by gender, gene*gender interaction was not identified (p=0.175 and 0.097, for genotypes and alleles, respectively, in males; p=0.906 and 0.907, for genotypes and alleles, respectively, in females) (Table 2).

Table 2:

Association of MTHFR c.665C>T genotypes with AEs in patient cohort and stratified by gender.

MTHFR c.665C>T genotypes Patient cohort (n=313) Male patients (n=160) Female patients (n=153)
Without AEs (n=105) With AEs (n=208) p-Value Without AEs (n=58) With AEs (n=102) p-Value Without AEs (n=47) With AEs (n=106) p-Value
CC 44 (41.9%) 100 (48.1%) 0.354 23 (39.7%) 50 (49.0%) 0.175 21 (44.7%) 50 (47.2%) 0.906
CT 49 (46.7%) 93 (44.7%) 26 (44.8%) 45 (44.1%) 23 (48.9%) 48 (45.3%)
TT 12 (11.4%) 15 (7.2%) 9 (15.5%) 7 (6.9%) 3 (6.4%) 8 (7.5%)

  1. Comparisons for categorical data were performed by using chi-square (χ2)-test. A p-value less than 0.05 was considered statistically significant. The Statistical Package for Social Sciences (SPSS) for Windows, Version 17.0 (IBM Corp., NY, USA) was used for all analyses.

MTHFR c.665C>T genotypes and alleles were assessed as for their potential association with fluoropyrimidine dose reduction and delayed drug administration or therapy discontinuation. In pooled sample, when MTHFR c.665C>T genotypes were assessed as for their potential association with fluoropyrimidine dose reduction, no association was found (data not shown). Similarly, no association was present in the pooled sample when either dominant or recessive model of MTHFR c.665C>T genotypes was applied (Dominant model is displayed in Table 3). When patients were stratified by gender, MTHFR c.665T allele carriage (CT and TT genotypes) was associated with dose reduction in female patients (77.8% in female patients with dose reduction vs. 50.4% in female patients without dose reduction, p=0.029) (Table 3). Our study detected a statistically significant difference of 27.4% in the frequency of MTHFR CT+TT genotypes between female patients requiring dose reduction and those without with 60.6% power. No difference was present in male patients (p=0.953 and 0.682, for dominant model and alleles, respectively). In regression analysis adjusted for age of disease onset, BSA and AE incidence, MTHFR CT and TT genotypes were the sole determinants of increased need for fluoropyrimidine dose reduction in female patients (OR 5.050, 95% CI 1.346–18.948, p=0.016). In a similar linear model with percentage of dose reduction [(final dose-initial dose)*100/initial dose] as a dependent variable, AE incidence (β=3.774, 95% C.I. 1.314–6.235, p=0.003) and MTHFR CT and TT genotypes (β=3.318, 95% C.I. 1.056–5.580, p=0.004), were predictors of dose reduction only in female patients.

Table 3:

Association of MTHFR c.665C>T dominant model of inheritance with dose reduction in patient cohort and stratified by gender.

MTHFR c.665C>T genotypes Patient cohort (n=313) Male patients (n=160) Female patients (n=153)
Without dose reduction (n=269) With dose reduction (n=44) p-Value Without dose reduction (n=134) With dose reduction (n=26) p-Value Without dose reduction (n=135) With dose reduction (n=18) p-Value
CC 128 (47.6%) 16 (36.4%) 0.166 61 (45.5%) 12 (46.2%) 0.953 67 (49.6%) 4 (22.2%) 0.029
CT+TT 141 (52.4%) 28 (63.6%) 73 (54.5%) 14 (53.8%) 68 (50.4%) 14 (77.8%)

  1. Comparisons for categorical data were performed by using chi-square (χ2)-test. A p-value less than 0.05 was considered statistically significant. The Statistical Package for Social Sciences (SPSS) for Windows, Version 17.0 (IBM Corp., NY, USA) was used for all analyses.

Based on our previous findings of the association of TYMS-TSER 2R allele carriage with AEs in female patients [4] and considering that AE incidence is a main clinical factor for fluoropyrimidine dose adjustments, TYMS*MTHFR interaction was inserted as independent variable in binary regression model. This gene-gene interaction increased need for fluoropyrimidine dose reduction in female patients (OR 1.816, 95% CI 1.104–2.988, p=0.038 after Bonferroni correction).

No associations were found of MTHFR c.665C>T polymorphism with delayed drug administration or therapy discontinuation both in pooled sample [distinct genotypes (p=0.482), alleles (p=0.265), dominant model (p=0.230), recessive model (p=0.639)] and in gender stratification analysis (p=0.619 and 0.362, for genotypes and alleles, respectively, in males; p=0.782 and 0.544, for genotypes and alleles, respectively, in females) (Table 4).

Table 4:

Association of MTHFR c.665C>T genotypes and alleles with delayed drug administration or therapy discontinuation in patient cohort and stratified by gender.

MTHFR c.665C>T genotypes Patient cohort (n=313) Male patients (n=160) Female patients (n=153)
Without delayed drug administration or therapy discontinuation (n=254) With delayed drug administration or therapy discontinuation (n=59) p-Value Without delayed drug administration or therapy discontinuation (n=126) With delayed drug administration or therapy discontinuation (n=34) p-Value Without delayed drug administration or therapy discontinuation (n=128) With delayed drug administration or therapy discontinuation (n=25) p-Value
CC 121 (47.6%) 23 (39.0%) 0.482 60 (47.6%) 13 (38.2%) 0.619 61 (47.7%) 10 (40.0%) 0.782
CT 112 (44.1%) 30 (50.8%) 54 (42.9%) 17 (50.0%) 58 (45.3%) 13 (52.0%)
TT 21 (8.3%) 6 (10.2%) 12 (9.5%) 4 (11.8%) 9 (7.0%) 2 (8.0%)

  1. Comparisons for categorical data were performed by using chi-square (χ2)-test. A p-value less than 0.05 was considered statistically significant. The Statistical Package for Social Sciences (SPSS) for Windows, Version 17.0 (IBM Corp., NY, USA) was used for all analyses.

Dose intensity was also calculated in our patient cohort to assess the potential effect of MTHFR c.665C>T polymorphism in a variable depicting both delayed drug administration and dose reduction. The studied SNP was not associated with dose intensity either in pooled sample or in gender stratification analysis (data not shown).

DPYD polymorphisms are valuable predictors of the response to fluoropyrimidine chemotherapy [2]. Screening of additional DPYD polymorphisms could enhance the prevention of fluoropyrimidine toxicity. Towards this direction, implementation of other genetic variants, such as variants in TYMS and MTHFR, holds promise to improve fluoropyrimidine response in cancer patients. Studies assessing the effect of TYMS gene polymorphisms on CAP-5-FU response have been thoroughly discussed previously [4].

For MTHFR, results of several studies have shown an association of MTHFR polymorphisms with different outcomes of fluoropyrimidine response, mainly focusing on drug toxicity [5], [6], [7] or tumor recurrence and survival [8], [9], [10]. However, results are still inconclusive and are not yet supported from current meta-analyses [11, 12].

In the present study, we have found a gender-dependent association of MTHFR c.665C>T polymorphism with fluoropyrimidine dose reduction, whereas in pooled sample analyses MTHFR c.665T allele was not associated with fluoropyrimidine dose reduction or toxicity. Female carriers of MTHFR c.665T allele had significantly increased need (OR 3.392) for decreased dose requirements. Additionally, the gene*gene interaction of MTHFR c.665C>T and TYMS-TSER was also associated with fluoropyrimidine dose reduction in female cancer patients treated with 5-FU or CAP. A similar gender-driven association was present in a previous study of our team when assessing the association of TYMS-TSER polymorphism with fluoropyrimidine AE incidence in the same cancer patient cohort [4]. We thereby encourage gene*gender interaction analyses in cancer pharmacogenomic studies. Interestingly, we did not find an association of MTHFR c.665T allele with dose intensity of fluoropyrimidines. This is potentially attributed to the direct effect of MTHFR on dose requirements. Additionally, in our patient cohort a cancer heterogeneity is present both in terms of tumor site and fluoropyrimidine-based chemotherapeutic scheme.

MTHFR c.665C>T polymorphism is strongly associated with higher homocysteine levels. Gender dependent effects of MTHFR C677T polymorphism have been previously described in studies assessing the relation between the c.665C>T MTHFR polymorphism and fasting plasma total homocysteine concentrations [13, 14]. Both studies describe that male carriers of MTHFR c.665T allele have increased homocysteine levels. Interestingly, 5-FU activation interferes with folate–homocysteine cycle via TS inhibition [1, 15]. Thus, gender*MTHFR as well as TYMS*MTHFR interactions we have found in female patients in our study may be associated with homocysteine cycle. In the present manuscript, however, this assumption is not based on experimental data and should be therefore tested in different studies.

In conclusion, MTHFR c.665T allele is associated with fluoropyrimidine dose requirements in female cancer patients treated with 5-FU or CAP. We suggest that sub-group analyses are important and provide evidence for sex related pharmacogenomic markers that should be further investigated in larger patient cohorts.


Corresponding author: Prof. Vangelis G. Manolopoulos, Laboratory of Pharmacology, Medical School, Democritus University of Thrace, Dragana Campus, 68100 Alexandroupolis, Greece, Phone: +30 2551 030523, E-mail:

  1. Research funding: Financial support for project IMPReS (MIS 5047189) was provided by the Program “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014–2020) co-financed by Greece and the European Union (European Regional Development Fund).

  2. Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

  3. Competing interests: Authors state no conflict of interest.

  4. Informed consent: Informed consent was obtained from all individuals included in this study.

  5. Ethical approval: The study protocol was approved by the Scientific Council and the Ethics Committee of the Academic General Hospital of Alexandroupolis and was conducted according to Declaration of Helsinki.

References

1. Thorn, CF, Marsh, S, Carrillo, MW, McLeod, HL, Klein, TE, Altman, RB. PharmGKB summary: fluoropyrimidine pathways. Pharmacogenet Genomics 2011;21:237–42. https://doi.org/10.1097/fpc.0b013e32833c6107.Search in Google Scholar

2. Amstutz, U, Henricks, LM, Offer, SM, Barbarino, J, Schellens, JHM, Swen, JJ, et al.. Clinical pharmacogenetics implementation Consortium (CPIC) guideline for dihydropyrimidine dehydrogenase genotype and fluoropyrimidine dosing: 2017 update. Clin Pharmacol Ther 2018;103:210–6. https://doi.org/10.1002/cpt.911.Search in Google Scholar

3. Toffoli, G, De Mattia, E. Pharmacogenetic relevance of MTHFR polymorphisms. Pharmacogenomics 2008;9:1195–206. https://doi.org/10.2217/14622416.9.9.1195.Search in Google Scholar

4. Ioannou, C, Ragia, G, Balgkouranidou, I, Xenidis, N, Amarantidis, K, Koukaki, T, et al.. Gender-dependent association of TYMS-TSER polymorphism with 5-fluorouracil or capecitabine-based chemotherapy toxicity. Pharmacogenomics 2021;22:669–80. https://doi.org/10.2217/pgs-2021-0031.Search in Google Scholar

5. Loganayagam, A, Arenas Hernandez, M, Corrigan, A, Fairbanks, L, Lewis, CM, Harper, P, et al.. Pharmacogenetic variants in the DPYD, TYMS, CDA and MTHFR genes are clinically significant predictors of fluoropyrimidine toxicity. Br J Cancer 2013;108:2505–15. https://doi.org/10.1038/bjc.2013.262.Search in Google Scholar

6. van Huis-Tanja, LH, Gelderblom, H, Punt, CJ, Guchelaar, HJ. MTHFR polymorphisms and capecitabine-induced toxicity in patients with metastatic colorectal cancer. Pharmacogenet Genomics 2013;23:208–18. https://doi.org/10.1097/fpc.0b013e32835ee8e1.Search in Google Scholar

7. Fernandes, MR, Rodrigues, JCG, Dobbin, EAF, Pastana, LF, da Costa, DF, Barra, WF, et al.. Influence of FPGS, ABCC4, SLC29A1, and MTHFR genes on the pharmacogenomics of fluoropyrimidines in patients with gastrointestinal cancer from the Brazilian Amazon. Cancer Chemother Pharmacol 2021;88:837–44. https://doi.org/10.1007/s00280-021-04327-w.Search in Google Scholar

8. Custodio, A, Moreno-Rubio, J, Aparicio, J, Gallego-Plazas, J, Yaya, R, Maurel, J, et al.. Pharmacogenetic predictors of outcome in patients with stage II and III colon cancer treated with oxaliplatin and fluoropyrimidine-based adjuvant chemotherapy. Mol Cancer Ther 2014;13:2226–37. https://doi.org/10.1158/1535-7163.mct-13-1109.Search in Google Scholar

9. Zhao, J, Li, W, Zhu, D, Yu, Q, Zhang, Z, Sun, M, et al.. Association of single nucleotide polymorphisms in MTHFR and ABCG2 with the different efficacy of first-line chemotherapy in metastatic colorectal cancer. Med Oncol 2014;31:802. https://doi.org/10.1007/s12032-013-0802-6.Search in Google Scholar

10. Ramos-Esquivel, A, Chinchilla-Monge, R, Abbas, J, Valle, M. C677T and A1298C MTHFR gene polymorphisms and response to fluoropyrimidine-based chemotherapy in Mestizo patients with metastatic colorectal cancer. Pharmacogenet Genomics 2021;31:191–9. https://doi.org/10.1097/fpc.0000000000000440.Search in Google Scholar

11. Zhong, L, He, X, Zhang, Y, Chuan, JL, Chen, M, Zhu, SM, et al.. Relevance of methylenetetrahydrofolate reductase gene variants C677T and A1298C with response to fluoropyrimidine-based chemotherapy in colorectal cancer: a systematic review and meta-analysis. Oncotarget 2018;9:31291–301. https://doi.org/10.18632/oncotarget.24933.Search in Google Scholar

12. Zhong, L, Fu, Q, Zhou, S, Chen, L, Peng, Q. Relevance of MTHFR polymorphisms with response to fluoropyrimidine-based chemotherapy in oesophagogastric cancer: a meta-analysis. BMJ Open 2018;8: e020767. https://doi.org/10.1136/bmjopen-2017-020767.Search in Google Scholar

13. Papoutsakis, C, Yiannakouris, N, Manios, Y, Papaconstantinou, E, Magkos, F, Schulpis, KH, et al.. The effect of MTHFR(C677T) genotype on plasma homocysteine concentrations in healthy children is influenced by gender. Eur J Clin Nutr 2006;60:155–62. https://doi.org/10.1038/sj.ejcn.1602280.Search in Google Scholar

14. Russo, GT, Friso, S, Jacques, PF, Rogers, G, Cucinotta, D, Wilson, PW, et al.. Age and gender affect the relation between methylenetetrahydrofolate reductase C677T genotype and fasting plasma homocysteine concentrations in the Framingham Offspring Study Cohort. J Nutr 2003;133:3416–21. https://doi.org/10.1093/jn/133.11.3416.Search in Google Scholar

15. Longley, DB, Harkin, DP, Johnston, PG. 5-fluorouracil: mechanisms of action and clinical strategies. Nat Rev Cancer 2003;3:330–8. https://doi.org/10.1038/nrc1074.Search in Google Scholar

Received: 2021-11-19
Accepted: 2022-02-08
Published Online: 2022-03-11

© 2022 Charalampia Ioannou et al., published by De Gruyter, Berlin/Boston

This work is licensed under the Creative Commons Attribution 4.0 International License.