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Introduction

Chemotherapy significantly improves the clinical outcome of patients with esophageal cancer. Preoperative chemotherapy may be considered as standard treatment for patients with clinical stage II or III esophageal cancer (1). Furthermore, chemoradiotherapy for esophageal cancer has been established as a treatment for stage I disease, as well as for advanced cases (2,3). The combination of 5-fluorouracil (5-FU) plus cisplatin (CDDP) is currently the standard chemotherapy for esophageal cancer. Although chemotherapy for esophageal cancer using 5-FU plus CDDP has become a widely applied treatment, chemotherapy-induced oral mucositis (OM) has been reported to complicate the course in ≤40% of patients receiving an 5-FU-based regimen (4,5). The discomfort and pain associated with OM may impair oral intake of fluids and calories, resulting in anorexia, cachexia, dehydration and overt malnutrition (6). Nishimura et al reported that the incidence of grade ≥1 OM according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 3 (NCI-CTCAE 3.0; https://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/ctcaev3.pdf), was the highest during chemotherapy for esophageal cancer (57.8%) (7). Predicting toxicities prior to chemotherapy is crucial for treatment selection. We previously reported that the TNF-α rs1799964 polymorphism was associated with susceptibility to OM in patients treated with 5-FU plus CDDP for gastrointestinal malignancies (8). Currently, 5-FU plus CDDP chemotherapy is mainly performed for esophageal cancer. In order to validate the reliability of our previous report, we investigated the association between TNF-α rs1799964 and chemotherapy-induced OM in esophageal cancer patients.

Patients and methods

Patients

This was a retrospective cohort study that included patients treated for esophageal cancer between 1997 and 2013. The participants comprised 170 consecutive Japanese patients (152 men and 18 women) with esophageal cancer who underwent chemotherapy at the Department of Gastroenterological, Breast and Endocrine Surgery of the Yamaguchi University Graduate School of Medicine (Ube, Japan). The experimental group comprised 64 patients between 1997 and 2004, who were reported in our previous article (8). The validation group comprised 106 patients who underwent chemotherapy between 2005 and 2013. Chemotherapy was performed as preoperative or postoperative treatment, or for progressive (unresectable) cases.

Chemotherapy

Chemotherapy was combined with radiotherapy to increase effectiveness for cases with progressive disease. Since patients with esophageal cancer who received preoperative chemotherapy exhibited superior overall survival compared with those who received postoperative chemotherapy in the Japan Clinical Oncology Group (JCOG) trial 9907, as was reported at the American Society of Clinical Oncology Annual Meeting in 2008 (1), preoperative chemotherapy was introduced from 2008 onwards. Up to 2007, a low-dose 5-FU plus CDDP chemotherapy regimen had been used, which consisted of 5-FU at a concentration of 330 mg/m2 administered by continuous intravenous infusion on days 1–7, and CDDP at a concentration of 6 mg/m2 administered intravenously for 2 h by drip infusion on days 1–5. This regimen was repeated for 4 weeks. In addition, the regimen of 5-FU plus CDDP chemotherapy reported in JCOG9907 was also used, which included standard-dose 5-FU plus CDDP chemotherapy, consisting of 5-FU at 800 mg/m2 administered by continuous intravenous infusion on days 1–5 and CDDP at 80 mg/m2 administered intravenously for 2 h by drip infusion on day 1. This regimen was repeated twice every 3 weeks. If radiation was used in combination with chemotherapy, the doses of 5-FU (700 mg/m2) and CDDP (70 mg/m2) were reduced (2). NCI-CTCAE 3.0 was used to evaluate chemotherapy-induced OM. Toxicity was evaluated when symptoms of the highest-grade OM appeared.

DNA specimens and genotyping

For DNA analysis, 7-ml samples of peripheral blood were obtained from all the patients. DNA was isolated by a conventional NaI method and stored at 4°C (9). All polymorphisms were identified with the tetra-primer amplification refractory mutation system polymerase chain reaction (PCR); the details of the primers and PCR conditions were previously described (10–12).

Statistical analysis

Continuous data were expressed as median and range and were analyzed using the Mann-Whitney U test. Categorical data were analyzed using the χ2 test or Fisher's exact test. Differences in genotype frequency were analyzed using the χ2 test or Fisher's exact test of independence, with which each of the genotype frequencies was evaluated to determine whether it was consistent with expected Hardy-Weinberg proportions. As there were too few homozygotes for the rare allele to perform a 2×3 χ2 test or Fisher's exact test, homozygotes of the dominant alleles and variant carriers were compared by a 2×2 test. Odds ratios (OR) and 95% confidence intervals (CI) were also calculated. A P-value of <0.05 was considered to indicate statistically significant differences. All statistical calculations were performed with the IBM SPSS Statistics version 22.0 software package (IBM Japan Inc., Tokyo, Japan).

Results

Patient characteristics

The patient characteristics of the experimental and the validation groups are summarized in Table I. The percentage of patients receiving postoperative adjuvant chemotherapy was significantly lower in the validation group compared with that in the experimental group (P<0.01). There was a significant difference between the experimental and validation groups regarding the chemotherapy regimen. The distribution of TNF-α genotypes was similar between the two groups (rs1799964; experimental group: TT 68.8%, CT 25.0% and CC 6.2%; validation group: TT 73.6%, CT 24.5% and CC 1.9%). The incidence of grade 1–4 OM in the experimental and validation groups was 40.6 and 37.7%, respectively. Although the treatment regimen was changed from low-dose 5-FU plus CDDP to standard-dose 5-FU plus CDDP from 2008 onwards, there were no differences in the frequency of OM between the two treatments (grade 1–4 OM: low dose, 42.5% vs. standard dose, 35.6%, P=0.35).

Table I. Patient characteristics.

Table I.

Patient characteristics.

Characteristics	Experimental group (n=64)	Validation group (n=106)	P-value
Age, years	62 (27–82)	64 (40–81)	0.18
Gender			0.15
Male	60 (93.8)	92 (86.8)
Female	4 (6.3)	14 (13.2)
BMI, kg/m2	20.2 (12.6–28.8)	19.2 (14.1–31.2)	0.21
Purpose of chemotherapy			<0.01
Postoperative	35 (54.7)	30 (28.3)
Preoperative	0 (0.0)	45 (42.5)
Progression/recurrence	29 (45.3)	31 (29.2)
Radiotherapy			0.59
Yes	18 (28.1)	34 (32.1)
No	46 (71.9)	72 (67.9)
Regimen of chemotherapy			<0.01
Low-dose FP	64 (100.0)	16 (15.1)
Standard-dose FP	0 (0.0)	90 (84.9)
Genotype of TNF-α rs1799964			0.32
TT	44 (68.8)	78 (73.6)
CT	16 (25.0)	26 (24.5)
CC	4 (6.2)	2 (1.9)
Oral mucositis			0.84
Grade 0	38 (59.4)	66 (62.3)
Grade 1	12 (18.7)	17 (16.0)
Grade 2	8 (12.5)	14 (13.2)
Grade 3	4 (6.3)	8 (7.6)
Grade 4	2 (3.1)	1 (0.9)

[i] Data are presented as median values (range) or as absolute numbers (%). BMI, body mass index; FP, 5-fluorouracil plus cisplatin; TNF, tumor necrosis factor.

Association between patient characteristics and OM

The associations between patient characteristics and grade 1–4 of OM by univariate analysis are shown in Table II. In the experimental group, the incidence of the TT genotype of TNF-α rs1799964 was significantly higher among patients with grade 1–4 OM compared with patients without OM (OR=4.0, 95% CI=1.2–13.9; P=0.029). In the validation group, the body mass index (BMI; OR=0.8, 95% CI=0.69–0.94; P<0.01) and the incidence of the TT genotype of TNF-α rs1799964 (OR=2.8, 95% CI=1.03–7.8; P=0.043) were significantly higher in patients with grade 1–4 OM compared with patients without OM. Radiotherapy tended to be associated with a high frequency of grade 1–4 OM (P=0.076). There was no significant difference in the frequency of OM by chemotherapy regimen. Regarding the association between the TNF-α allele of rs1799964 and OM, the results were similar for the experimental and validation groups.

Table II. Asssociation between patient characteristics and grade 1–4 oral mucositis by univariate analysis in the experimental and validation groups.

Table II.

Asssociation between patient characteristics and grade 1–4 oral mucositis by univariate analysis in the experimental and validation groups.

	Experimental group (n=64)			Validation group (n=106)
Characteristics	OR	95% CI	P-value	OR	95% CI	P-value
Age	1.02	0.97–1.1	0.49	0.98	0.93–1.03	0.33
Gender
Male vs. female	0.67	0.09–5.1	0.7	0.4	0.13–1.3	0.12
BMI	1.1	0.9–1.3	0.47	0.8	0.69–0.94	<0.01
Purpose of chemotherapy
Postoperative vs. preoperative/progression/recurrence	1.3	0.45–3.4	0.69	1.4	0.59–3.3	0.46
Radiotherapy
Yes vs. no	0.9	0.3–2.8	0.86	2.1	0.92–4.9	0.076
Regimen of chemotherapy
Low- vs. standard-dose FP				1.8	0.63–5.3	0.28
TNF-α rs1799964 genotype
TT vs. CT+CC	4.0	1.2–13.9	0.029	2.8	1.03–7.8	0.043

[i] OR, odds ratio; CI, confidence interval; BMI, body mass index; FP, 5-fluorouracil plus cisplatin; TNF, tumor necrosis factor.

Discussion

Our previous study demonstrated that patients with gastrointestinal malignancies who had the TT genotype of the TNF-α promoter gene polymorphism rs1799964 had a significantly higher risk of chemotherapy-induced OM compared with patients with other genotypes. Although several gastrointestinal malignancies were investigated in our previous study, in the present study only patients with esophageal cancer were investigated as the experimental group. We observed that the rs1799964 polymorphism of TNF-α was associated with chemotherapy-induced OM even in the experimental group, which only included patients with esophageal cancer. Furthermore, similar results were also demonstrated in the validation group.

TNF-α is a key pro-inflammatory cytokine that causes tissue damage, and release of TNF-α may initiate and accelerate the development of OM (13). It has been reported in several human studies that the levels of pro-inflammatory cytokines are increased in the blood and saliva of patients during cancer treatment (14–16). Other studies using animal models have shown evidence of changes in the serum level and tissue expression of nuclear factor κ-B and the pro-inflammatory cytokines [TNF-α, interleukin (IL)-1β and IL-6) following administration of chemotherapeutic drugs (14,17–19). The rs1800629 polymorphism of TNF-α has been reported to be associated with hypercytokinemia, such as sepsis (20–22); this polymorphism was also reported to be associated with enhanced TNF-α transcription in vitro (23). However, the frequency of TNF-α rs1800629 in the Japanese population was significantly lower compared with that in Caucasians (frequency of A allele: Japanese vs. Caucasian, 1.1–1.3 vs. 10–18%, respectively) (24). Cui et al reported an analysis of the association between TNF-α rs1799964, which was the subject of the present study, and the serum concentration of TNF-α (25); they found that the TC and CC genotypes of rs1799964 were associated with decreased serum TNF-α levels. The authors also performed a functional analysis to compare the activities of the rs1799964 T/C alleles. The gene expression of the C allele was significantly reduced compared with that of the T allele, which was defined as wild-type, when measured using a luciferase assay in cultured HepG2 cells. However, Higuchi et al reported that the transcriptional activity of the C allele of rs1799964 was twice as high as that of the dominant T allele in response to concanavalin A (26). There is a discrepancy between our results and the reported promoter activities of TNF-α rs1799964 polymorphisms. Further studies are required to confirm the association between TNF-α promoter polymorphism and OM.

Although it has been reported that the patient-related risk factors of chemotherapy-induced OM include age, gender, nutritional status, oral microbiota, status of oral health and hygiene, salivary secretory function and neutrophil count, there remains some controversy regarding risk factors (27). In this study, low BMI was one of the risk factors in the validation group, but it was not confirmed as a risk factor in the experimental group. Clinical practice guidelines have been drawn for the management of OM based on evidence and expert opinions (28–30). Oral care reduces the microbial load; therefore, maintenance of oral hygiene is a major factor in the prevention and mitigation of oral injury. Cryotherapy, keratinocyte growth factor-1 (palifermin), low-level laser therapy and benzydamine mouthwash are recommended as other preventive measures. Further clinical trials are required before drugs such as palifermin may be used in the general medical field, and their cost-effectiveness must also be discussed. It is considered that preventive treatments should be aggressively introduced to patients identified as being at high risk of OM prior to chemotherapy, including patients with the TT genotype of TNF-α rs1799964.

This study had several limitations, including its single-institution nature, the relatively small sample size, and the fact that the clinical data were collected and analyzed retrospectively. Therefore, our findings require validation in a prospective study including a larger number of patients treated at several institutions. The results of this study demonstrated that the rs1799964 polymorphism of TNF-α was associated with OM risk in patients with esophageal cancer treated with chemotherapy, and the result was validated in a separate cohort, suggesting that this polymorphism may be used as a predictive biomarker. Analysis of such polymorphisms may help individualize chemotherapeutic regimens to optimally treat esophageal cancer, while minimizing the risk of OM.

Acknowledgements

This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (no. 26461912).

References