Nasopharyngeal carcinoma (NPC) is a rare cancer in most countries around the world with an incidence rate generally less than 1 per 100,000 person-years. However, the NPC incidence is extremely high in Southern China (25–30 per 100,000 person-years) [1], [2]. In Taiwan, an intermediate-risk area, the annual incidence rates for males and females in 2007 were 8.41 and 2.93 per 100,000 person-years, respectively [3].

Infection with Epstein-Barr virus (EBV) is considered a necessary cause of NPC. Long-term cigarette smoking, occupational exposures such as formaldehyde and wood dust, and genetic factors have also been documented as risk factors for NPC [4]–[8]. The dietary factors have been hypothesized to be involved in the development of NPC, but the evidence remains unclear except for Cantonese salted fish consumption in early childhood. Ho first proposed that salted fish consumption might be a risk factor for NPC [9]. In subsequent studies, intake of salted fish in childhood and adulthood was found to be associated with an excess risk of NPC in high-risk areas such as Guandong, Guangxi, and Hong Kong [10]–[13]. Even among individuals seropositive for immunoglobulin A antibodies against viral capsid antigen of EBV (anti-EBV VCA IgA), salted fish consumption during adulthood was associated with a 2-fold increase in NPC risk for those who had the highest consumption compared with whom never consumed [12]. However, no significant association during childhood and adulthood was found in low-risk areas including Philippines, Singapore, and United States [14]–[16].

In addition to salted fish at young ages, the intake of preserved foods has been found to be an NPC risk factor in many populations. In a meta-analysis of six case-control studies on the associations between preserved vegetable consumption in adulthood and NPC risk, the pooled odds ratio (95% confidence interval [CI]) was 2.04 (1.43–2.92) for the highest intake of preserved vegetables compared to the lowest intake [17]. In our previous study, the nitrosamine and nitrite consumption in childhood was significantly associated with an increased NPC risk [18].

In addition to salted fish and preserved foods, several studies reported an inverse relationship between consumption of vegetables and fruits and risk of NPC [16], [17]. The highest intake of fresh vegetables intake was associated with a 36% decrease in the risk of NPC in a meta-analysis [17]. A large case-control study conducted in China also reported a decrease NPC risk associated with the consumption of herbal tea and slowly cooked herbal soups [13].

We have reported the associations with NPC for various food items previously, with an emphasis on early life exposures [18]. While consumption of salted fish could not be fully evaluated due to the rarity with which this food item was consumed in our population, we did observe a positive association between early life consumption of non-soy foods high in nitrate/nitrosamines and risk of NPC. With respect to adult diet, we previously reported (17) that individuals in the highest quartile of intake of salted eggs or hot chili were at a significantly elevated risk of NPC compared to the lowest quartile. No significant association with NPC was observed for the intake of fresh soybean products, cured meats, smoked meats, fermented soybean products, preserved vegetables and fruits. However, this previous analysis did not evaluate macronutrient consumption and did not adjust for effects of potential confounders. In present analysis, we examined the associations between a wide range of food groups and macronutrients and NPC risk after taking known risk factors for NPC into consideration.

A total of 371 NPC cases and 321 unaffected controls were included in the analyses. The male proportion was 69.5% and 69.2% for cases and controls, respectively. The mean age (standard deviation) was 45.6 (11.6) years for cases and 46.0 (11.7) years for controls. Compared with controls, the cases were more likely to be Fukienese in ethnicity. The educational level was lower and the proportion with a family history of NPC was higher in cases than controls.

Table 1 shows the consumption frequency of meat, egg, and seafood in NPC cases and controls. No significant association was observed between NPC risk and intakes of meat and egg. Compared to the reference group of taking fresh fish of ≤2 times/week, the OR_adj was 0.92 (95% CI, 0.61–1.40) and 0.56 (95% CI, 0.38–0.83) for those with the fresh fish intake of 2–6 and >6 times/week, respectively (p for trend<0.01) after adjustment for age, gender, ethnicity, educational level, NPC family history, total calories intake, years of cigarette smoking, and exposures to formaldehyde and wood dust. The OR_adj for the fresh fish intake remained similar in the analysis restricted to cases and controls seropositive for anti-EBV markers. No significant associations with NPC risk were observed for the intake of Cantonese-style salted fish (OR_adj, 0.88; 95% CI, 0.35–2.21), although our ability to evaluate this association was limited by the small number of individuals reporting consumption of this food item. The intake of other seafood was also not significantly associated with NPC.

The associations with NPC risk for the intake of vegetable and fruit are shown in Table 2. There was a weak negative association between NPC risk and intakes of all fresh vegetables (p for trend, 0.05). The intake of dark green vegetables was inversely associated with NPC risk after adjustment for other risk factors. Compared to the lowest intake, OR_adj for the highest intake was 0.65 (0.41–1.02) for all fresh vegetables and 0.67 (0.43–1.04) for dark green vegetables. No association with NPC risk was observed for the consumption of carrots, bean pods, gourds, preserved vegetables, fruit, and oranges/tangerines. Similar results were observed in analyses restricted to individuals seropositive for anti-EBV.

No significant association with NPC was observed for the consumption of milk, soybean milk as shown in Table 3. The consumption of fresh fruit juices and black tea was borderline significant with NPC risk (P for trend = 0.05). Significant reverse associations were found for the increasing intake of oolong tea (OR_adj, 0.66 for >3 vs. 0 time/week; 95% CI, 0.44–0.98) and green tea (OR_adj, 0.61 for 1+ vs. 0 time/week; 95% CI, 0.40–0.91). A significant inverse trend in risk was found for drinking coffee (OR_adj, 0.56 for 0.5+ vs. 0 time per week, 95% CI, 0.37–0.85; p for trend = 0.01). The reverse associations with NPC risk for green tea and coffee remained statistically significant in the analyses restricted to cases and controls seropositive for anti-EBV markers.

The associations with NPC risk for daily intake of selected macronutrients are shown in Table 4. No significant association with NPC was found for the intake of fat, carbohydrate, vitamin C, tocopherol and sodium. Compared with individuals with lowest tertile of protein intake, individuals with highest tertile of protein intake had a lower risk of NPC (OR_adj, 0.50; 95% CI, 0.29–0.86; p for trend<0.01). Intake of vitamin A, especially vitamin A from plant source, was associated with a decreased NPC risk (OR_adj, 0.62 for highest tertile vs. lowest tertile; 95% CI, 0.41–0.94; p for trend = 0.02)

The correlation coefficients among intakes of various dietary factors are shown in the Table S1. There were significant correlations between consumption frequencies of green tea and oolong tea drinking (correlation coefficient [r], 0.69; p<0.01) and between dark green vegetable and vitamin A from plant source (r, 0.66; p<0.01). The protein intake was also significantly correlated with the consumption of fresh fish (r, 0.28; p<0.01), dark green vegetables (r, 0.22; p<0.01), and coffee (r, 0.27; p<0.01). In the final logistic regression analysis, only the intake of fresh fish, green tea, coffee, and vitamin A from plant source was included in the regression model as shown in Table 5. After adjustment for age, gender, ethnicity, educational level, family NPC history, cigarette smoking, and exposures to formaldehyde and wood dust; significant associations with NPC risk were observed for the intake of fresh fish, green tea, coffee, and vitamin A from plant source. The OR_adj, remained similar when the analyses were restricted to individuals with anti-EBV seropositivity.

In this case-control study aimed to evaluate the associations with NPC risk for comprehensive food items during adult life, we found a significant reverse dose-response relationship between NPC risk and increasing consumption frequency of fresh fish, green tea, and coffee consumption. Salted fish intake and preserved foods have been identified as risk factors for NPC in many populations, especially during the weaning and childhood periods [10]–[13], [27]–[29]. These foods contain N-nitroso compounds which are considered carcinogenic to human. However, the associations with NPC for salted fish and preserved vegetables during adult life were not observed in the present study. One possible explanation may be very few cases and controls ever consumed Cantonese salted fish. Another explanation may be only the salted fish consumed in early childhood rather than adulthood is associated with the NPC risk.

Several studies reported a reverse association between vegetable consumption and NPC [30]–[34]. In our previous report, there were significant reverse associations between NPC and intake of dark green vegetable [18]. In the present analysis, there was a protective effect of vegetable consumption on NPC, but the association was not statistically significant after adjustment for other risk factors.

In contrast to another study [14], a reverse association between fresh fish consumption and NPC risk was observed in this study. The reverse associations between cancers and fish consumption had been observed in many epidemiological studies [35], [36]. The potential mechanism for the protective effect may be due to the nutrients such as ω-3 fatty acids in fish. Both docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) have been shown to inhibit cell growth or decrease the risk of progression through inflammatory pathways in animal and in vitro studies [37].

The present study showed a significant reverse association between green tea consumption and NPC. This is consistent with another case-control study conducted in southern China (OR, 0.62) [38]. The association between tea consumption and decreased NPC risk is biologically plausible. The possible protective effect of tea consumption on various cancers has been evaluated in recent years, especially for green tea. In most animal studies, the extracts of tea could inhibit the formation and development of tumors at different sites [39]. Epigallocatechin gallate, one of the isomers in catechin, is a major component of green tea. It has a potent anti-microbial activity against bacteria, fungi, and viruses. Epigallocatechin gallate at 50 µM was reported to completely block EBV infection-induced cytokine expression and the EBV-induced B lymphocyte transformation [40].

An interesting reverse association between NPC risk and coffee consumption was observed in the present study after adjustment for other risk factors. Coffee is abundant in antioxidants such as caffeinic acid and chlorogenic acid. The coffee diterpenes, cafestol and kahweal have been considered to reduce the genotoxicity of several carcinogens by modifying detoxification enzymes [41], [42]. Coffee extracts were found to inhibit the virus activities in vitro [43]. However, the protective effects of tea and coffee on human cancers have been inconclusive in epidemiological studies [44]. The reasons for the discrepancy may be due to the differences in types of tea and coffee, consumption habits in various populations, and/or the adjustment for confounders.

EBV is considered the most important risk factor for NPC. The reverse associations with NPC for the intake of fresh fish, green tea and coffees remained statistically significant in the analyses limited to individuals seropositive for anti-EBV markers. In other words, the protective effects were independent from anti-EBV seropositivity and unlikely to be mediated through their effects on EBV lytic replication and resultant anti-EBV antibody seroconversion. In previous studies, NPC risk might be more relevant to the diet in childhood than the diet in adulthood [11], [13], [18]. It is worthwhile to examine whether the protective effect on NPC of fresh fish, tea and coffee consumed in adulthood as observed in this study also exist for the dietary items consumed in childhood.

The etiology of NPC involves genetic susceptibility, EBV infection, environmental factors and gene-EBV-environment interaction [45]–[47]. Recent GWAS studies have shown evidence of the important role for genetic factors play in the development of NPC [48], [49]. The absorption and metabolism of dietary component could be influenced by genetic polymorphisms. Components from diet could alter the genetic expression through epigenetic mechanisms [50]. Genistein and other isoflavones from soy and tea polyphenol(-)-epigallocatechin-3-gallate have been documented to reactivate methylation-silenced genes in cancer cell lines and inhibit cancer growth [51], [52].

This study has strengths and limitations. First of strengths, the dietary questionnaire included very comprehensive Taiwan food items and major micronutrient estimated from the inquired food items. The other advantage of this study was the control of potential confounding effects of other risk factors. Conversely, case-control studies are likely to be influenced by recall bias because cases might be more likely to remember and report exposure to known or possible risk factors than controls. However, we interviewed all cases before their diagnosis was confirmed by biopsy. Other limitations include the low consumption frequency of some food items, especially Guangdong salted fish, and the lack of portion size information in the questionnaire interview. Chinese food is very diverse and most dishes have mixtures of vegetables and meat; it is thus difficult to quantify the intake of various food items. Nonetheless, such limitations are likely to lead to bias towards the null value. Under such a conservative circumstance, we still observed a protective effect on NPC for the intake of fresh fish, green tea and coffee. If confirmed, the protective dietary factors reported herein could be incorporated into efforts to reduce NPC risk through dietary changes during adult life.