Methylenetetrahydrofolate reductase C677T polymorphism and toxicity to 5-FU-based chemotherapy in colorectal cancer

Purpose: To investigate the toxicity of methylenetetrahydrofolate reductase (MTHFR) polymorphism in colorectal cancer patients treated with 5-fluorouracil (5-FU). Methods: A total of 105 patients with colorectal cancer who underwent 5-FU therapy were included in this study. MTHFR C677T polymorphisms were determined using direct sequencing. Physical examination and the results of blood and urine tests were used to evaluate the toxicities, including gastrointestinal toxicity, hematopoietic toxicity, hair-skin toxicity and hand-foot syndrome. Results: In 90.5 % of all patients, 5-FU toxicity was observed. With regard to MTHFR C677T mutation, 45.7 % heterozygote mutants and 19.0 % homozygote mutants were observed. MTHFR C677T polymorphism was statistically related to 5-FU toxicity (p = 0.000). In addition, MTHFR C677T mutation was closely related to hematopoietic toxicity (p = 0.005). Conclusion: MTHFR C677T can be used for the prediction of 5-FU toxicity, and can also predict hematopoietic toxicity in patients with colorectal cancer.


INTRODUCTION
-fluorouracil (5-FU) has been used in the treatment of various cancers, especially in colorectal cancer (CRC) [1]. 5-FU has no antitumor effect, however, it can be involved in reducing DNA synthesis [2].
Methylenetetrahydrofolate reductase (MTHFR) is the most critical enzyme in the metabolism of folate and 5-FU [3]. Therefore, the MTHFR activity may predict the clinical responses and toxicity to 5-FU. The grade and type of toxicities primarily depend on demographic factors including gender, age, dose of 5-FU and different methods of administration [4]. Individual differences in fluoropyrimidine-related toxicity are partly explained by genetic factors.
The MTHFR gene is highly polymorphic [5]. One of the most common functional mutations in the MTHFR, C677T (rs1801133, A222V) has been identified the main variants in reducing the activity of this enzyme [6]. Therefore, MTHFR C677T polymorphisms are considered a potential predictor of clinical responses and 5-FU toxicity. However, the evidence of genetic association is relatively weak and published results from previous studies are not consistent [7][8][9][10].
Therefore, the purpose of this study was to investigate the relationship between MTHFR C677T mutations and the clinical responses and toxicity of patients with CRC who underwent 5-FU therapy.

Study population
One hundred and five CRC patients who were treated with 5-FU between 2016 and 2018 at the Hubei Hospital were enrolled in this study. Patients were eligible if they were adults, with biopsy-proven clinical T3/T4. Patients were excluded if they underwent pretreatment with any chemotherapeutic regimen, previous pelvic radiotherapy, and were allergic to 5-FU [11]. This study was approved by the ethics committee of the Hubei Hospital (approval no. 20160102) and followed the guidelines of the Helsinki Declaration [12].

Evaluation of toxicity
All adverse drug reactions and toxicities were recorded. Gastrointestinal toxicity, hematopoietic toxicity, hair and skin toxicity and hand-foot syndrome were defined as previously published. Adverse reactions were classified as grade 1, 2, 3 and 4 as defined as previously published [13].
Genotyping DNA was extracted from peripheral blood. The DNA was diluted to 10 ng per well, and Polymerase Chain Reaction (PCR) was carried out and the PCR products were genotyped using direct sequencing (ABI 3100 DNA sequencer, Shanghai, China).

Statistical analysis
Data were analyzed using SPSS 19.0 (Chicago, IL, USA) and expressed as the mean ± SEM, or percentage (%). The relationship between genotypes and toxicity, and the Hardy-Weinberg equilibrium (HWE) were determined by Chisquare test. Statistical significance was defined as p < 0.05.

The types and grades of patients' toxicity
All patients were followed up in the first four cycles of treatment with 5-FU. Based on the evaluation criteria for adverse events, 90.5 % (n = 95) of patients suffered from toxicities, and the proportions of grades 1, 2, 3 and 4 were 31.4, 25.7, 35.3 and 10.4%, respectively. Details of toxicity classifications are presented in Table 2.

Patient genotypes
As shown in Table 3, no significant differences were observed in the Hardy-Weinberg equilibrium between groups. The genotype frequencies of CC, CT and TT were 35.2, 45.7, and 19.0 %, respectively, while allele frequencies of C and T were 58.1 and 41.9 %.

Gene polymorphism was associated with 5-FU toxicity
At least one of four types of toxicity was observed in patients with MTHFR C677T polymorphism (p = 0.000) ( Table 4). The MTHFR C677T mutation was related to hematopoietic toxicity (p = 0.005, Table 5). However, the

DISCUSSION
MTHFR is a key enzyme in the metabolism of 5-FU.and the MTHFR C677T polymorphism can reduce MTHFR enzyme activity [14]. Therefore, the MTHFR C677T polymorphism may be closely related to the efficacy of 5-FU treatment. In this study, 90.5 % patients had adverse reactions when treated with 5-FU, and the MTHFR C677T frequency (45.7 % heterozygote mutants, 19.0 % homozygote mutants) was significantly higher than presented in previous reports [13,15].
The most important finding is that the MTHFR C677T mutation was related to the 5-FU toxicities when 5-FU was used alone, especially for the hematopoietic toxicity. Afzal et al reported that the MTHFR C677T polymorphism was closely related to fluoropyrimidine-related toxicity in CRC patients, however, no specific types of toxicity were mentioned [16]. In addition, Noor et al found that MTHFR C677T polymorphism increased the tumor response to 5-FU and increased gastrointestinal toxicity in CRC patients [17]. Loganayagam et al found that the MTHFR C677T polymorphism was closely related to hand-foot syndrome in CRC patients [18].
Moreover, Lu et al showed that the MTHFR C677T polymorphism increased gastrointestinal toxicity in gastric cancer patients [19]. However, Capitain et al found that the MTHFR C677T polymorphism was not associated with the toxicity of 5-FU in advanced CRC patients [20]. Based on the above-mentioned results, the relationship between the MTHFR C677T mutation and 5-FU toxicity in CRC patients remains controversial, and the reasons may be as follows.   First, differences in sample size may lead to differences in results. Second, differences in study population and ethnic differences may have affected the frequency of gene mutations. Third, not only used 5-FU, but also combined with other drugs.
Genetic markers can predict either drug toxicity or efficacy, which is critical for patients who are undergoing treatment with cytotoxic agents. However, there are significant regional and ethnic diversities in genetic polymorphisms in MTHFR C677T, and the frequency of mutations varies widely worldwide. In addition, the results of the study are quite different, therefore, it is important in requiring a degree of homogeneity in the selection of patients and treatments.

Limitations of the study
There is a limitation of the present study that should be considered. The relationship between MTHFR C677T polymorphism and 5-FU-related toxicities in CRC patients needs to be confirmed in a larger sample size.

CONCLUSION
MTHFR C677T can be used for the prediction of both 5-FU toxicity and also hematopoietic toxicity in patients with CRC.

Conflict of interest
No conflict of interest is associated with this work.

Contribution of authors
We declare that this work was done by the authors named in this article and all liabilities pertaining to claims relating to the content of this article will be borne by the authors.

Open Access
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