Methylenetetrahydrofolate reductase polymorphism and capecitabine-induced toxicity in patients with gastric cancer

Purpose: To evaluate the effect of methylenetetrahydrofolate reductase (MTHFR) polymorphism on toxicity in gastric cancer (GC) patients treated with capecitabine. Methods: One hundred and twenty-six GC patients were treated with capecitabine in the study. DNA from GC patients was genotyped for MTHFR A1298C using direct sequencing. Toxicity evaluations were graded. Clinical response was assessed. Results: In 87.3 % of the patients, capecitabine toxicity was observed. As for MTHFR A1298C polymorphism, 55.6 % patients who exhibited it were associated with reduced MTHFR activity. MTHFR A1298C was associated with capecitabine-related toxicity (p = 0.008); in addition, MTHFR A1298C was significantly associated with gastrointestinal toxicity (p = 0.026), but not with other types of toxicity. Conclusion: The findings suggest that MTHFR A1298C may be useful for predicting toxicity in GC patients receiving capecitabine treatment, especially gastrointestinal toxicity.


INTRODUCTION
Gastric cancer (GC) is one of the main causes of cancer death all over the world [1,2]. GC accounts for about 35 -40 % of new cases and deaths each year in China [1,2]. Although earlystage and locally-advanced GC can be treated by surgery, many patients have local recurrence or distant metastasis after gastrectomy [3,4]. However, about a quarter of patients have lost the best time to perform surgery at the time of diagnosis [5]. It is generally believed that palliative chemotherapy for advanced diseases and perioperative chemotherapy can improve the survival rate and quality of life of GC patients [6]. Capecitabine, a prodrug of 5-fluorouracil (5-FU), is widely used to treat gastrointestinal cancer, including GC. However, chemotherapy may sometimes bring serious, unpredictable toxicity to patients without any tumor response. Genetic variation may play an important role in toxic and side effects induced by chemotherapeutic drugs. Increasing evidence suggests that individual variations in drug metabolizing enzymes may affect the efficacy of anticancer drugs [7].
Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in folate metabolism, and reduced activity of MTHFR could augment the cytotoxicity of fluoropyrimidines [8]. Therefore, mutations in MTHFR may cause enzyme deficiency resulting in toxicity with capecitabine therapy. Furthermore, a number of patients have been described as having reduced activity of the enzyme and severe toxicity of fluoropyrimidines including capecitabine [9,10]. A common SNP in MTHFR causes reduced enzyme activity in homozygous individuals: MTHFR 1298A>C (rs1801131) polymorphism results in a 30 -40% reduction in its activity [8].
However, the relationship between the rs1801131 mutation in MTHFR and the efficacy and toxicity of capecitabine in GC patients has not been reported in central China.
The aim of the present study was to evaluate the relationship between rs1801131 in MTHFR and the efficacy of capecitabine chemotherapy in GC patients to assist in therapeutic selection.

EXPERIMENTAL Samples
In total 126 GC patients treated with capecitabine were enrolled in the Hubei hospital from 2015 to 2018 who met the following criteria [2,11]: age > 18; histologically-confirmed GC; Eastern Cooperative Oncology Group was 0 -2; no malignant coexistence; adequate organ function; patients received capecitabine treatment; patients had completed at least two cycles of chemotherapy. Ethics approval was obtained from the ethics committee of Hubei Hospital (approval no. 20150501) and followed the guidelines of the Helsinki Declaration [12], and all the patients gave written informed consent.
Capecitabine (Roche, NJ, USA) was orally administered at a dose of 1,000 mg/m 2 twice daily from day1 to day14 for 3 weeks. Continue treatment until disease progression, unacceptable toxicity, patient refusal, or medical determination to stop treatment.

Assessment of efficacy and toxicity
Follow-up procedures including intermediate medical history, physical examination, hematology, and diagnostic imaging. Toxicity evaluations was graded as previously described [13]. Computed tomography was used to evaluate clinical response based on Response Evaluation Criteria in Solid Tumors [11]. The toxicities include hematopoietic, gastrointestinal, hand-foot syndrome and hair-skin toxicities. All reactions and toxicities were recorded.

MTHFR genotyping
Genomic DNA was extracted using the Kit (TIANGEN, Beijing, China). Primers for MTHFR 1298A>C were designed using Primer 5.0 (CA, USA). PCR reactions were performed in a volume of 25 μL. The sequence of the primers was shown in Table 1. The PCR products were determined using DNA sequencing (ABI 3100, Shanghai, China).

Statistical analysis
Analyses were done using SPSS 19.0. Allele frequency and number of adverse reactions were determined by counting. The relationships between polymorphism of MTHFR and the existence of toxicity was analyzed using Chi square test. A p < 0.05 was considered statistically significant.

Adverse reactions related to treatment
All patients were followed up after treatment with capecitabine. 87.3 % of the patients suffered from toxicities. Treatment was prematurely stopped in 10 % of the patients due to severe gastrointestinal or haematological adverse events, and no toxic death was reported. The most frequently observed toxicities were hematological and gastrointestinal, but hair and skin, and hand-foot syndrome were observed only rarely. Details of toxicity classification are shown in Table 3.

Genotype frequencies
Genotypes and allele frequencies are listed in Table 4. For the MTHFR 1298A>C locus, 56 (44.4 %) patients were wild type, 12 (9.5 %) patients were homozygote type and 58 (46.0 %) patients were heterozygote, respectively, and the allele frequencies of A and C were 67.5 and 32.5 % in this study.

Correlation between MTHFR genotypes and toxicity
Patients with MTHFR A/C + C/C genotypes had a significantly higher incidence of toxicities than patients with MTHFR A/A genotype (P = 0.008) ( Table 5). The MTHFR A1298C genotype was significantly associated with the incidence of gastrointestinal toxicity (p = 0.026) (Table 6), however, MTHFR A1298C genotype has no significant correlation with other types of toxicity.

DISCUSSION
It is generally believed that there are many factors influencing the outcome of drug treatment including mixed environmental factors, physiological factors, genetic variation of drugmetabolizing enzymes, drug transporters, and drug targets, therefore, genetic polymorphisms play a key role in determining toxicity. The MTHFR polymorphisms result in decreased enzyme activity and then low levels of plasma folate and high homocysteine. Folate takes part in various biochemical reactions to provide or accept one-carbon units [14]. Folate deficiency is likely to develop into cancer, possibly due to DNA structural defects [15].
The study is based on the MTHFR pharmacogenetics of capecitabine in the treatment of GC patient. MTHFR 1298A>C genotype was significantly correlated with the incidence of adverse events in patients with GC treated with capecitabine, especially in gastrointestinal toxicity, however, MTHFR A1298C genotype has no significant correlation with the other types of toxicity.
In this study, 87.3 % of the patients suffered from toxicities when treated with capecitabine, and the MTHFR A1298C frequency (46.0 % heterozygote mutants, 9.5 % homozygote mutants) was significantly higher than previous reports [13], but similar to another study [8], and the reasons are as follows, sample size, differences in race and region.
Several studies examined genetic variants related to capecitabine therapy, such as MTHFR and thymidylate synthase, but without conclusive results, and the role of pharmacological genetic markers in predicting the toxicity and efficacy of capecitabine remains to be explored. Liu et al found that MTHFR A1298C polymorphism had no significant association with GC patients' survival [16]. Zhao et al suggested that MTHFR 1298CC genotype showed protective effect for all GC patients, and MTHFR 1298A > C polymorphisms may be useful prognostic biomarkers for GC patients [17]. In addition, van Huis-Tanja et al concluded that MTHFR 677C>T and 1298A>C polymorphisms were not related to the occurrence of severe toxicity (and efficacy) of capecitabine-based chemotherapy in metastatic colorectal cancer [18]. Thus, the association of MTHFR A1298C remained unclear.
However, capecitabine is increasingly being included in the first-line treatment of solid tumors, and its efficacy is comparable to that of 5-FU single or combined [18]. The toxicity spectrum is different, capecitabine causes higher incidence of hand-foot syndrome than 5-FU injection [19], therefore, reducing drug adverse reactions requires better drug management.

Limitations of the study
This is the first study on the role of pharmacogenetics in capecitabine toxicity in GC patients in central China. There are some limitations of the present study that should be considered. (i) The present results need to be validated in a larger cohort. (ii) capecitabine was given as monotherapy (iii) folic acid is not added in the 5-FU protocol; therefore, the methylenetetrahydrofolate pool is directly controlled by MTHFR; (iv) the patients received chemotherapy, ruling out the possibility of acquired chemotherapy resistance. Therefore, it cannot be ruled out that the observed difference in MTHFR effect may be caused by other factors.

CONCLUSION
The results of this study suggest that MTHFR A1298C can be used for the prediction of capecitabine toxicities in patients with GC; in particular, it can predict gastrointestinal toxicity. These findings, as well as other supporting or opposing conclusions need to be validated in a larger cohort.

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.

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