Int. J. Cancer: 124, 1907–1913 (2009) ' 2008 Wiley-Liss, Inc.

Green tea drinking, high tea temperature and esophageal cancer in high- and low-risk areas of Jiangsu Province, China: A population-based case–control study Ming Wu1, Ai-Min Liu2, Ellen Kampman3, Zuo-Feng Zhang4, Pieter van’t Veer3, De-Lin Wu5, Pei-Hua Wang1, Jie Yang1, Yu Qin1, Li-Na Mu6, Frans J. Kok3 and Jin-Kou Zhao1* 1 Jiangsu Provincial Center for Disease Control and Prevention, Nanjing, Jiangsu, China 2 Dafeng Center for Disease Control and Prevention, Dafeng, Jiangsu, China 3 Division of Human Nutrition, Wageningen University, Wageningen, The Netherlands 4 Department of Epidemiology, School of Public Health, University of California, Los Angeles, CA 5 Ganyu Center for Disease Control and Prevention, Ganyu, Jiangsu, China 6 Department of Social and Preventive Medicine, School of Public Health and Health Professions, University at Buffalo, The State University of New York, Buffalo, NY Epidemiological studies suggested drinking green tea is inversely associated with esophageal cancer but results remain inconclusive. Moreover, inconsistent observations found high temperature drinks are associated with esophageal cancer. A population-based case–control study was conducted in a high-risk area (Dafeng) and a low-risk area (Ganyu) of esophageal cancer in Jiangsu province China from 2003 to 2007. It aimed to explore green tea drinking and tea temperature with the risk of esophageal cancer, and to compare the difference between different risk regions. Using identical protocols, 1,520 cases and 3,879 healthy controls were recruited as study subjects in 2 regions. Detailed information was collected to assess green tea drinking habits. Unconditional logistic regression was used to obtain OR and 95% CI. Results showed that ever drinking green tea elevated OR in both counties (Dafeng OR 5 1.2, 95% CI 5 0.9–1.5; Ganyu: OR 5 1.9, 95% CI 5 1.4– 2.4). Drinking tea at high temperature was found to increase cancer risk in both areas (Dafeng: OR 5 1.9, 95% CI 5 1.2–2.9; Ganyu OR 5 3.1 95% CI 5 2.2–4.3). However, after further adjustment for tea temperature, ever drinking tea was not related to cancer in either county (Dafeng: OR 5 1.0, 95% CI 5 0.7–1.3; Ganyu: OR 5 1.3, 95% CI 5 0.9–1.7). For dose-response relationships, we observed positive relationship with monthly consumption of tea (p for trend 5 0.067) and tea concentration (p for trend 5 0.006) after further adjustment for tea temperature. In conclusion, green tea drinking was not inversely associated with esophageal cancer in this study. However, drinking tea at high temperatures significantly increased esophageal cancer risk. There was no obvious difference of green tea drinking between low- and high-risk areas. ' 2008 Wiley-Liss, Inc. Key words: green tea; hot drinking; esophageal cancer; case–control study; smoking; alcohol drinking

Esophageal cancer is the sixth most common cause of cancer mortality worldwide. The number of new esophageal cancer cases in China accounted for 53% of all new cases in the world in 2002. The incidence and mortality rates (per 100,000) in China are 27.4 and 21.6 for men and 12.0 and 9.6 for women, respectively.1 Jiangsu Province, located in South-Eastern China, is one of the highest incidence areas of the disease. According to the Second National Death Cause Retrospective Survey, the mortality of esophageal cancer was 30 of 100,000 from 1990 to 1992 in Jiangsu province, much higher than the national average of 17 of 100,000.2 Although the mortality of esophageal cancer is high in most counties in Jiangsu; it differs considerably between counties, despite their similar geographic characteristics and socioeconomic status.3 Numerous epidemiological studies have demonstrated that environmental and lifestyle factors such as tobacco smoking, alcohol drinking and dietary habits are associated with the development of esophageal cancer.4,5 It is also suggested that the consumption of green tea may help prevent esophageal cancer in humans.6,7 Tea is currently grown in at least 30 countries, and it is the most frequently consumed beverage worldwide after water, especially in Asian countries such as China, Japan and India.8 The per capita Publication of the International Union Against Cancer

worldwide consumption of tea is approximately 120 ml brewed tea per day.9 Depending on the manufacturing process, tea is classified into 3 major types: green tea (non-fermented), oolong tea (half-fermented) and black tea (fermented). Green tea is derived from Camellia sinesis, an evergreen shrub of the Theaceae family. It contains many polyphenols known as cathechins, including epigallo-cathechin-3 gallate (EGCG), epigallo-cathechin (EGC) and epicathechin-3 gallate (ECG).10 A number of studies have provided evidence that the polyphenolic antioxidants present in tea may be capable of affording protection against cancer.11–13 A few epidemiological studies have addressed the association between green tea and esophageal cancer, but results remain inconclusive.14–17 Moreover, inconsistent observations suggest that high-temperature drinks are associated with esophageal cancer.18 Since 2003, a population-based case–control study has been conducted in selected high- and low-risk areas of esophageal cancer in Jiangsu, China. In this analysis, we evaluate the association of esophageal cancer with green tea drinking and tea temperature in high- and low-risk areas. The results may help us improve the current understanding of the effects of green tea drinking and hightemperature drinking on the development of esophageal cancer. Material and methods Study areas A population-based case–control study has been conducted in 2 counties of Jiangsu province, Dafeng and Ganyu from 2003 to 2007. Both Dafeng and Ganyu are less developed, coastal, rural counties in northern Jiangsu province. The total population in Dafeng and Ganyu are approximately 0.7 million and 1.1 million, respectively. Dafeng has a higher mortality of esophageal cancer than Ganyu. From 1996 to 2002, the yearly average age-adjusted mortality of esophageal cancer was 36 of 100,000 in Dafeng, but was 24 of 100,000 in Ganyu during the same period.19 Study subjects All subjects were restricted to local inhabitants who have lived in either area for at least 5 years. Newly diagnosed primary esophageal cancer patients from local adult residents were recruited as cases, using the data from local population based cancer registry agencies. The cancer registry agencies in both counties were established in the late 1990s and are part of the local Center for Disease Control and Prevention (CDC). All cases were identified by International Classification of Diseases, 10th revision (ICD-10, Grant sponsor: Jiangsu Provincial Health Department; Grant number: RC 2003090. *Correspondence to: No. 172 Jiangsu Road, Nanjing 210009, China. Fax: 186-25-83759411. E-mail: [email protected] Received 23 June 2008; Accepted after revision 21 October 2008 DOI 10.1002/ijc.24142 Published online 6 November 2008 in Wiley InterScience (www.interscience. wiley.com).

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code C15). Second primary and recurrent cancers were excluded. A system of rapid case recognition was used in the study. All regional hospitals were required by the local health authorities to report new patients shortly after diagnosis. As the cancer registry agencies are attached to local CDC, investigators from local CDC could identify and interview the cases as quickly as possible. In this study, 68 and 75% of newly registered esophageal cancer cases were identified and interviewed in Dafeng and Ganyu, respectively. Because of the low proportion of pathological examination in less developed rural areas (Dafeng 61%, Ganyu 30%), patients who were diagnosed by other sophisticated methods such as endoscopic examination or radiology were also included. Controls were derived from the same county as cases. Eligible controls were randomly selected from the general population, using the data of the county demographic database. Controls and cases were frequency matched by gender and age (65 years). Individuals with history of cancer were not eligible as controls. The responding rate of control was 87% in Dafeng and 85% in Ganyu. By study design, 600 cases and 600 controls in each county were required for the study. For Dafeng and Ganyu, recruitment of cases and controls was finished in 2006 and in 2007, respectively. As identical case–control studies on stomach, liver and lung cancer were also conducted in these 2 counties at the same time, controls for all cancer sites were used in this analysis. In total, 1,520 cases (637 in Dafeng County and 883 in Ganyu County) and 3,879 controls (1,938 in Dafeng County and 1,941 in Ganyu County) were recruited for this study.

weight and obese: low weight (BMI < 18.5), overweight (BMI  24 and BMI < 28), obesity (BMI  28).20 Chi-square and Student t-tests were used to compare the distribution of potential risk or protective factors among control groups between the 2 counties. Unconditional logistic regression with a maximum likelihood estimation of parameters was applied for both univariate and multivariate analyses. The strength of the association was quantified as odds ratios (OR), and 95% confidence intervals (CI) around the OR were used to quantify precision. Dummy variables were used in the logistic regression to estimate OR for each exposure category. The trend test of ordered variables was performed by assigning scores to different exposure levels and treated the categorical variable as a continuous variable in the logistic regression model. Effect modification was analyzed by stratification. Statistical interaction was assessed by including main effect variables and their product terms in the logistic regression model. On the basis of prior knowledge and confounding assessment, the effect of green tea was evaluated adjusting for age (continuous), gender (female 5 0, male 5 1), education level (ordered), income 10 years before (continuous), cancer family history of first degree relatives (No 5 0, yes 5 1), BMI (continuous), pack-years of smoking (continuous) and alcohol drinking (never or seldom 5 0, often 5 1). The effect after further adjustment for tea temperature was also presented (never drinker and normal tea temperature 5 0, high tea temperature 5 1). Results

Data collection Using standard protocols and a pretested standardized epidemiologic questionnaire, with written informed consent, we collected epidemiological data by face-to-face interviews in both counties. Five milliliters of blood samples were collected at the time of interview. The questionnaire included detailed information on known or potential risk or protective factors for esophageal cancer, including demographic information, socio-economic status, living conditions, environmental exposure, tobacco smoking, alcohol drinking, dietary habits, disease history, etc. Ever drinking green tea was defined as drinking at least 1 cup of green tea per week for more than 6 months. We collected lifetime general consumption of green tea drinking, and change of drinking pattern one year before diagnosis for cases or one year before interview for controls. Details of tea drinking habits included drinking status (current, former or never drinking), age when the person began to drink tea regularly, number of years drinking tea, monthly consumption of tea (grams/month), tea concentration and temperature of the water (boiling or not boiling) used to brew tea at the time of drinking. To validate the variables above, the questionnaire also collected information about the number of new cups of tea made each day (times the person changed the leaves in the tea cup), subsequent brewing of each cup (times the person poured new water into each cup without changing leaves). In the rural areas of Jiangsu Province, China, seldom do people drink oolong tea or coffee, therefore, we did not include information on these 2 beverages in the questionnaire. Statistical analysis Data were entered into the computer by Epidata 2.1b, cleansed and analyzed using SAS v8.2 software. In the analysis, ever drinking green tea was further categorized into former drinking and current drinking; individuals who quit drinking because of health reasons but quit in less than 1 year at the time of interview were considered current drinkers. Smoking was categorized into ever smoking and never smoking. Pack-years of smoking were also calculated. Alcohol drinking was categorized into never or seldom drinking and often drinking. For body mass index (BMI), the Chinese recommended standard was used for the definition of over-

The demographic characteristics of cases and controls are shown by county in Table I, together with socio-economic related variables, cancer family history of first degree relatives, as well as smoking and alcohol drinking status. Comparing the 2 counties, Ganyu has a higher proportion of male cases than Dafeng. Ganyu also has a lower educational level and lower previous income than Dafeng, as well as a lower prevalence of cancer family history (p < 0.01). Prevalence of smoking and green tea drinking in Dafeng is much lower than in Ganyu (p < 0.01). Although alcohol drinking appears to be higher in Dafeng, the difference was not statistically significant (p 5 0.18). Within both counties, cases were older and more often male. The OR and 95% CI for esophageal cancer with socio-economic status, cancer family history, smoking and alcohol drinking were also shown in Table I. Cases more frequently occurred in the population with lower socio-economic statuses, i.e., lower education level, lower previous income, and lower BMI. Cancer family history in first-degree relatives was found to significantly increase the risk of esophageal cancer (OR: Dafeng 5 1.4, Ganyu 5 2.1). An increased risk was observed among smokers in both counties (OR: Dafeng 5 1.4, Ganyu 5 1.5) as compared to nonsmokers. An apparent dose-response relationship was also found between esophageal cancer and pack-years of smoking (p for trend < 0.05). Similar to smoking, people who often drink alcohol tend to have a higher risk of esophageal cancer (OR: Dafeng 5 1.3, Ganyu 5 1.6) as compared to those who never or seldom drink alcohol. Table II shows the association between esophageal cancer and green tea drinking in each county. After adjusting for potential confounders including age, gender, education level, previous income, cancer family history, BMI, pack-years of smoking and alcohol drinking, we found that ever drinking green tea significantly increased esophageal cancer risk in Ganyu (OR 5 1.9, 95% CI 5 1.4–2.4), but it was not significant in Dafeng (OR 5 1.2, 95%CI 5 0.9–1.5). Former drinking was observed to be strongly associated with increasing OR in both counties (Dafeng: OR 5 3.4, 95% CI 5 1.9–6.1; Ganyu: OR 5 6.4, 95% CI 5 3.6–11.5), whereas for current drinking, increased risk was found in Ganyu (OR 5 1.6, 95% CI 5 1.2–2.1) but not in Dafeng (OR 5 1.0, 95% CI 5 0.8–1.3). Tea temperature was found to be positively related

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GREEN TEA DRINKING, TEA TEMPERATURE, ESOPHAGEAL CANCER TABLE I – DEMOGRAPHIC INFORMATION AND EPIDEMIOLOGIC CHARACTERISTICS OF STUDY SUBJECTS IN HIGH AND LOW RISK AREAS Dafeng (High) Case (%) (N 5 637)

Control (%) (N 5 1,938)

Gender Males 426 (66.9) 1,368 (70.6) Females 211 (33.1) 570 (29.4) Age Mean 6 SD (years) 65.4 6 9.0 63.6 6 11.0