Int. J. Cancer: 71, 776–779 (1997) r 1997 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer SEROPREVALENCES OF HEPATITIS B AND C VIRUSES AND HELICOBACTER PYLORI INFECTION IN A SMALL, ISOLATED POPULATION AT HIGH RISK OF GASTRIC AND LIVER CANCER Shu-Yuan CHEN1, Tzeng-Ying LIU2, Meei-Jin CHEN2, Jaw-Town LIN3, Jin-Chuan SHEU3 and Chien-Jen CHEN1,* 1Graduate Institute of Epidemiology, College of Public Health, National Taiwan University, Taipei 10018, Taiwan 2Health Bureau of Lienkiang County, Matzu 3Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan (Republic of China) The objective of this study was to examine the seroprevalences of chronic infection with hepatitis B and C viruses and Helicobacter pylori in Matzu, a group of small islets with 5,566 civilian residents who have extremely high mortality from cancers of the stomach and liver. The standardized mortality ratios (SMR) of all cancer sites combined, liver cancer and stomach cancer in 1984–1993 were calculated using the general population in Taiwan as the referent (SMR 5 100). The SMRs (95% confidence interval) for all cancer sites combined, liver cancer and stomach cancer were 160 (131– 195), 252 (170–360) and 351 (229–516), respectively, in Matzu. A health survey was carried out with 1,485 civilian residents aged 30 years or more, giving a reponse rate of 69% among those who were eligible. Serum samples were tested for antibodies against Helicobacter pylori (anti-HP) by enzymelinked immunosorbent assay and hepatitis B surface antigen (HBsAg) and antibodies against hepatitis C virus (anti-HCV) by enzyme immunoassay. The seroprevalence was 61% for anti-HP, 24.7% for HBsAg and 1.8% for anti-HCV in Matzu. While mortality rates of liver and stomach cancers were significantly higher in Matzu than in Taiwan, the seroprevalences of anti-HP, HBsAg and anti-HCV in Matzu were similar to or even lower than those in Taiwan. These findings suggest the existence of risk factors other than microbial agents involved in the development of stomach and liver cancers. Int. J. Cancer 71:776–779, 1997. r 1997 Wiley-Liss, Inc. Stomach cancer is estimated to be the second most common cancer in the world, in spite of its declining secular trend in nearly all countries (Parkin et al., 1993) including Taiwan (Huang et al., 1988). The incidence and mortality of stomach cancer still ranked as sixth and third among leading cancer sites, respectively, in 1991 in Taiwan (Department of Health, 1995). Although several risk factors such as blood type A, chronic gastric diseases, cigarette smoking, alcohol drinking, green tea drinking, increased consumption of salted meat, cured meat, smoked food, fried food and fermented beans, and lower consumption of milk have been documented in Taiwan (Lee H.H. et al., 1990), their biological significance remains unknown. After the successful culture of a spiral-shaped organism known today as Helicobacter pylori (H. pylori) (Marshall, 1983), many studies were carried out to examine the association between H. pylori and stomach cancer. Previous prospective epidemiological studies have shown a significant association between H. pylori and stomach cancer, with a relative risk ranging from 2.8 to 6.0 (Forman et al., 1991; Parsonnet et al., 1991; Nomura et al., 1991). An ecological correlation of stomach cancer mortality with seroprevalence of antibodies against H. pylori (anti-HP) was also found in China (Forman et al., 1990). However, only a moderate association (p 5 0.3) was observed among 13 European populations (Eurogast Study Group, 1993; Giesecke, 1993; Forman et al., 1993). In spite of this body of evidence, the association between H. pylori infection and stomach cancer remains controversial. Liver cancer, chiefly hepatocellular carcinoma (HCC), is the leading cancer in Taiwan where hepatitis B virus (HBV) is hyperendemic with an infection rate of 85–90% and a chronic hepatitis B surface antigen (HBsAg) carrier rate of 15–20%. The HBsAg carrier status has been well documented as one of the most important risk factors for HCC. The attributable risk percentage of HBsAg carrier status has been estimated to be as high as 79% (Chen et al., 1991) and the multivariate-adjusted relative risk of developing liver cancer for HBsAg carrier status was 17.0 in a cohort study of 13,737 male adults in Taiwan (Chen et al., 1993). In addition to HBsAg, HCV is also an important viral agent for HCC. The population-attributable risk percentage of developing liver cancer for HCV was estimated as 3%, with a multivariate-adjusted relative risk as high as 23.7 (Yu et al., 1991). In a nested case-control study, after adjustment for HBsAg status and other risk factors, the anti-HCV was significantly associated with the development of liver cancer, showing a multivariate-adjusted relative risk of 88.2 (Chang et al., 1994). The Matzu islets are located near the north coast of Fukien Province of mainland China and are separated from Taiwan island by the Taiwan Strait. Being fortified islands on the frontier, they have been governed by military administration since 1949. Transport between Taiwan and Matzu and the principal supplies of foodstuffs from Taiwan were dependent on the military services up to 1992. The civilian population in Matzu was 5,566 in 1994, with a steady emigration to Taiwan island. Residents in Matzu are engaged in fishery, farming, storekeeping and the civil service. As the cultivable land is limited and natural resources are not abundant in Matzu, most foodstuffs are imported from Taiwan island. The residents are reported to have a high mortality from cancers of the stomach and liver; the reasons for this increased mortality remain to be elucidated. The specific aim of this study was to examine the mortality from stomach and liver cancers and the seroprevalences of HBsAg, anti-HCV and anti-HP in Matzu. Another aim was to compare the seroprevalences of anti-HP, HBsAg and anti-HCV in Matzu with those reported in Taiwan. MATERIAL AND METHODS Mortality analysis In Taiwan and Matzu, it is mandatory to register any event of birth, death, marriage, divorce, migration, education and occupation in the household registration offices, so the death certificate registry is quite complete. Death certificates of residents who died from cancers during 1984–1993 were collected from Matzu household registration offices. All the death certificates of Matzu were reviewed and coded for their underlying causes of death according to the Ninth Revision of the International Classifications of Diseases, Injuries and Causes of Death (ICD-9, World Health Organization, 1977), as in Taiwan. Mid-year populations by age and sex in Matzu were abstracted from demographic reports of the local household registration offices. The age-adjusted mortality Contract grant sponsor: Department of Health, Executive Yuan, Republic of China *Correspondence to: Chien-Jen Chen, Institute of Epidemiology, College of Public Health, National Taiwan University, 1 Jen-Ai Road Section 1, Taipei 10018, Taiwan. Fax: 886 2 3511955. E-mail: [email protected] Received 3 December 1996; revised 10 February 1997 HBV, HCV, H. PYLORI AND CANCERS OF STOMACH AND LIVER rates of all cancer sites combined, liver cancer and stomach cancer in Taiwan using the 1976 world population as standard population were derived from the national death certification profile and demographic data. In order to compare the cancer mortality rates in Taiwan and Matzu, age-standardized mortality ratios (SMR) from cancers in Matzu were also calculated using the general population in Taiwan as the standard population (SMR 5 100). The 95% confidence intervals for the SMR were obtained by the exact method (Breslow and Day, 1987). Seroprevalence survey A health survey was carried out from July to August 1995 in 5 islets of Matzu including Nankan, Peikan, Tunging, East Chukuang and West Chukuang. In this health survey, all civilian residents aged 30 or more years old and living in Matzu during the period of survey were invited to participate. No military personnel were recruited. In all, 2,158 eligible civilian residents were invited to participate, and 1,485 (69%) of them took part in the survey, including 759 men and 726 women. Their mean age (6 standard deviation) was 46.6 (6 12.8) years old. The sociodemographic characteristics of participants and non-participants were comparable. Blood samples were collected from study subjects by venepuncture and centrifuged to separate and aliquot the serum samples. They were shipped to the laboratory at National Taiwan University on dry ice and stored at 270°C until examination. Serum samples from Matzu were tested independently for the presence of IgG anti-HP by enzyme-linked immunosorbent assay using commercial kits (GAP-IgG ELISA test, Biomerica, Newport Beach, CA). The antigen was an inactivated native antigen of H. pylori. The serum samples were diluted 1:200. The secondary antibody was a sheep anti-human IgG, conjugated to horseradish peroxidase. The specimen was considered positive for IgG antibodies to H. pylori if its optical density value was greater than or equal to the upper cut-off value, i.e., 20 units/ml in the calibration curve. The IgG assay had a sensitivity of 99.4% and a specificity of 94.2% based on the manufacturer’s information. The anti-HP seroprevalence in Matzu was compared with those observed in 3 townships (Peinan, Potzu, and Chutung) of Taiwan island (Lin et al., 1995a). In this previous report, the anti-HP was also examined by enzyme-linked immunosorbent assay using commercial kits from a different manufacturer (HEL-p test, Amrad, Abbotsford, Australia). The test had a sensitivity of 96% and a specificity of 93%, according to the manufacturer’s information. Serum samples from Matzu were also tested for HBsAg and anti-HCV by enzyme immunoassay (EIA) using commercial kits (Abbott, North Chicago, IL). The second-generation kits were used for the EIA test of anti-HCV. All positive samples were re-tested by the same method. Only repeatedly positive samples were considered anti-HCV-positive. The seroprevalences of HBsAg and antiHCV in Matzu were compared with those observed in a study carried out in 7 townships (Paisa, Huhsi, Makung, Sanchi, Potzu, Kaohsu and Chutung) in Taiwan (Chen et al., 1995). Both HBsAg and anti-HCV in this previous report were tested by the same methods and commercial kits. The age-adjusted seroprevalences and their standard errors were calculated for anti-HP, anti-HCV and HBsAg, using the 1995 population in Taiwan area as the standard population. The Pearson correlation coefficients between the seroprevalences of HBsAg, anti-HCV and anti-HP of study subjects in Matzu were calculated. The significance levels are based on 2-sided significance tests. RESULTS Using the 1976 world population as the standard population, the age-adjusted mortality rate from cancers of all sites combined in Matzu, 1984–1993, was 169.6 per 100,000 as shown in Table I. The SMR (95% confidence interval, CI) for all cancer sites combined was 160 (131–195) compared with the general population in Taiwan (SMR 5 100). The age-adjusted mortality rates from liver 777 TABLE I – STANDARDIZED MORTALITY RATIOS WITH 95% CONFIDENCE INTERVALS FOR ALL CANCERS, LIVER AND STOMACH CANCER IN MATZU, 1984–1993 Cancer (ICD codes) Age-adjusted Age-adjusted rate in Taiwan1 rate in Matzu2 All sites combined (140–208) Liver (155) Stomach (151) Standardized mortality ratio3 Ratio (95% confidence interval) 102.5 169.6 160 (131–195) 19.9 11.9 53.2 43.0 252 351 (170–360) (229–516) 1Age-adjusted mortality rate in 1982–1991 per 100,000 using 1976 world population as standard population.–2Age-adjusted mortality rate in 1984–1993 per 100,000 using 1976 world population as standard population.–3Age-specific mortality rates in Taiwan, 1982–1991 were used as standard rates to calculate the standardized mortality ratio; i.e., SMR for each cancer in Taiwan is 100. TABLE II – COMPARISON OF THE AGE-ADJUSTED SEROPREVALENCES OF HELICOBACTER PYLORI INFECTION AMONG RESIDENTS AGED 30 OR MORE IN TAIWAN AND MATZU Area Matzu Taiwan Peinan Potzu Chutung Age-adjusted seroprevalence3 Age-adjusted stomach cancer mortality rate Prevalence (%) (95% confidence interval) 43.01 61.04 (57.5–64.5) 12.72 10.22 8.42 76.15 79.25 58.65 (64.5–87.8) (67.8–90.5) (45.1–72.1) 1Age-adjusted mortality rate in 1984–1993 per 100,000 using 1976 world population as standard population.–2Age-adjusted mortality rate in 1982–1991 per 100,000 using 1976 world population as standard population.–3Age-adjusted seroprevalences per 100 using 1995 population in Taiwan area, including Taiwan island, Penghu, Kinmen and Matzu islets, as standard population, which was weighted for the standard error of the age-adjusted seroprevalences.–4The age-adjusted seroprevalence per 100 was calculated from the age-specific seroprevalence of Helicobacter pylori among residents aged 30 or more in Matzu, 1995.–5The age-adjusted seroprevalence per 100 was calculated from the age-specific seroprevalence of Helicobacter pylori among residents aged 30 or more in the 3 townships of Taiwan island, according to data abstracted from Lin et al. (1995a). and stomach cancers were 53.2 and 43.0 per 100,000 with SMRs (95% CI) of 252 (170–360) and 351 (229–516), respectively. Table II illustrates the age-adjusted mortality rates from stomach cancer and age-adjusted seroprevalences of anti-HP in Matzu and 3 townships in Taiwan. The age-adjusted mortality rates from stomach cancer were significantly higher in Matzu than in 3 townships in Taiwan. The age-adjusted rates were 43.0, 12.7, 10.2 and 8.4 per 100,000, respectively, in Matzu, Peinan, Potzu and Chutung. However, the age-adjusted seroprevalence of anti-HP in Matzu (61.0%) was only slightly higher than that in Chutung (58.6%) and even lower than those in Peinan (76.1%) and Potzu (79.2%). Table III depicts the age-adjusted mortality rates from liver cancer and age-adjusted seroprevalences of HBsAg and anti-HCV among male residents in Matzu and 7 townships in Taiwan. The highest age-adjusted liver cancer mortality rate was observed in Matzu (90.0 per 100,000). The age-adjusted liver-cancer mortality rates ranged from 19.6 to 81.1 per 100,000 in Taiwan. The age-adjusted seroprevalence of HBsAg was significantly higher in Matzu (29.6%) and Paisa (38.3%) than in 6 other townships in Taiwan, ranging from 19.7% to 22.6%. While the ratio between the highest and lowest liver cancer mortality rates was as high as 4.6 (90.0 vs. 19.6 per 100,000), the ratio between the highest and lowest HBsAg seroprevalences was only 1.9 (38.3% vs. 19.7%). The age-adjusted seroprevalence of anti-HCV was low in Kaoshu (1.3%), Matzu (1.4%), and Makung (1.6%) and highest in Paisa CHEN ET AL. 778 TABLE III – COMPARISON OF THE AGE-ADJUSTED SEROPREVALENCES OF HEPATITIS B SURFACE ANTIGEN (HBsAg) AND ANTIBODY AGAINST HEPATITIS C VIRUS (ANTI-HCV) AMONG MALE RESIDENTS AGED 30–64 IN TAIWAN AND MATZU Area Matzu Taiwan Paisa Huhsi Makung Sanchi Potzu Kaoshu Chutung Age-adjusted liver cancer mortality rate Age-adjusted seroprevalence3 HBsAg Prevalence (%) Anti-HCV (95% CI) Prevalence (%) 90.01 29.64 (22.7–36.5) 1.44 81.12 62.52 57.52 40.92 32.12 23.52 19.62 38.3 5 19.75 21.85 20.75 22.65 20.95 20.05 (27.3–49.4) (13.4–25.9) (18.8–24.7) (14.2–27.3) (18.0–27.2) (16.6–25.2) (17.2–22.8) 18.35 5.45 1.65 11.85 7.35 1.35 2.35 HBsAg/Anti-HCV (95% CI) Prevalence (%) (95% CI) (0.0–3.2) 30.74 (23.7–37.7) (10.5–26.1) (1.3–9.5) (0.4–2.8) (5.0–18.5) (4.0–10.6) (0.0–2.5) (1.3–3.4) 48.85 23.55 23.15 29.65 28.95 22.05 22.15 (37.5–60.2) (16.6–30.5) (20.0–26.2) (21.4–37.9) (23.7–34.0) (17.5–26.4) (19.2–25.0) 1Age-adjusted mortality rate among males in 1984–1993 per 100,000 using 1976 world population as standard population.–2Age-adjusted mortality rate among males in 1982–1991 per 100,000 using 1976 world population as standard population.–3Age-adjusted seroprevalences per 100 using 1995 male population in Taiwan area, including Taiwan island, Penghu, Kinmen and Matzu islets, as standard population, which was weighted for the standard error of the age-adjusted seroprevalences.–4The age-adjusted seroprevalence per 100 was calculated from the age-specific seroprevalences of HBsAg, anti-HCV and seropositivity of HBsAg and/or anti-HCV among male residents aged 30–64 in Matzu, 1995.–5The age-adjusted seroprevalence per 100 in the 7 townships of Taiwan island was calculated from the age-specific seroprevalences of HBsAg, anti-HCV and seropositivity of HBsAg and/or anti-HCV among male residents aged 30–64, according to data abstracted from Chen et al. (1995). (18.3%). The highest prevalence of seropositivity of HBsAg and/or anti-HCV was in Paisa (48.8%). The ratio between the highest and lowest prevalence of seropositivity of HBsAg and/or anti-HCV was 2.2. The associations between the seroprevalences of HBsAg, antiHCV and anti-HP among civilian residents aged 30 or more in Matzu were analyzed, but no significant correlations were observed (r 5 20.002 for anti-HP vs. HBsAg; r 5 20.019 for anti-HP vs. anti-HCV; r 5 20.029 for HBsAg vs. anti-HCV). DISCUSSION Death certificates in Matzu were not included in the national vital statistics before 1994, but it is mandatory to register any death event in the household registration offices in Matzu, as in Taiwan. In this study, we found that cancers of the liver and stomach were the 2 leading cancers in Matzu. The age-adjusted mortality rates were significantly higher in Matzu than in Taiwan. The difference in mortality rates between Matzu and Taiwan could not be due to the survival difference, because the 2 cancers had a very poor prognosis in both Matzu and Taiwan (Lee C.S. et al., 1986; Wu et al., 1994). No association between H. pylori infection and mortality rates of stomach cancer was observed in this study. The lack of association in this study may be due to the high prevalence of H. pylori in Matzu and Taiwan. Recent prospective studies also showed an apparent lack of association in China (Webb et al., 1996). A nested case-control study also failed to observe a significant odds ratio for the association between H. pylori and stomach cancer (Lin et al., 1995b). Besides the Chinese, there are several populations with high H. pylori infection rates yet relatively low rates of gastric cancer in Africa (Holcombe, 1992). The stomach-cancer risk was quite different in various areas of Italy, but the seroprevalences of anti-HP among adults were similar (Palli et al., 1993). No correlation between stomach cancer risk and seroprevalence of anti-HP among children and adolescents was observed in Costa Rica (Sierra et al., 1992). These results suggest the existence of other major risk factors involved in the etiology of stomach cancer in addition to H. pylori infection in Matzu. Dietary factors, such as excessive salt intake, preserved food consumption, exposure to nitroso-compounds, and low consumption of fresh fruits and vegetables may play important roles in gastric carcinogenesis in Matzu, since cultivable land and natural resources are limited. Further elucidation of these risk factors for stomach cancer in Matzu and assessment of the interaction between these risk factors and H. pylori infection are important for elucidation of the possible mechanisms of gastric carcinogenesis. In this study, despite the strikingly higher liver cancer mortality rate in Matzu than in Taiwan, the seroprevalence of HBsAg was only slightly higher in Matzu than in Taiwan. The seroprevalence of anti-HCV in Matzu was even lower than in Taiwan. Combining the seroprevalence of HBsAg and anti-HCV, residents in Matzu still had only a slightly higher seropositive rate than those in Taiwan. This finding is consistent with those of previous studies. In Taiwan, the mortality rate of liver cancer is significantly higher in mountainous aboriginal areas, the Penghu islets, and the endemic area of arseniasis; but the HBsAg carrier rates were similar or slightly higher among residents in these areas than in the general population of Taiwan (Lu and Chen, 1991; Wang et al., 1993; Chen et al., 1995). Our findings indicate that HBV may be important in the etiology of liver cancer, but major risk factors other than HBV and HCV are responsible for the high liver-cancer mortality in Matzu. In addition to HBV and HCV, aflatoxin exposure, low consumption of vegetables and low serum levels of retinol, cigarette smoking and habitual alcohol drinking have been documented as major risk factors for liver cancer in Taiwan (Hatch et al., 1993; Chen et al., 1991, 1993, 1996b; Yu et al., 1995b). Foodstuffs in Matzu are either stored for months before consumption or preserved as salted, pickled, fermented or nitrated foods. As the supply of fresh vegetables and fruit in Matzu has been far from adequate, it is suspected that the intake of micronutrients may be low among residents in Matzu. A recent study has demonstrated the combined effects of aflatoxin exposure and HBsAg carrier status on the development of hepatocellular carcinoma in Taiwan (Wang et al., 1996). Another study has shown a dose-response relation between the level of serum aflatoxin-albumin adducts and risk of HCC among subjects with null genotypes of glutathione S-transferase (GST) M1 and/or T1, but not among those with non-null genotypes (Chen et al., 1996a). Whether residents in Matzu have a higher prevalence of null genotypes of GST M1 and T1 deserves further investigation. Genetic polymorphism of CYP2E1 was found recently to modify the risk of cigarette-smoking-related hepatocarcin- HBV, HCV, H. PYLORI AND CANCERS OF STOMACH AND LIVER ogenesis (Yu et al., 1995a). Whether genetic susceptibility is also involved in the development of HCC in Matzu remains to be elucidated. H. pylori may predispose residents in Matzu to a high risk of stomach cancer through its ability to induce chronic atrophic gastritis which may lead to subsequent gastrocarcinogenesis from intestinal metaplasia and dysplasia to stomach cancer. 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