Dig Dis Sci (2014) 59:631–637 DOI 10.1007/s10620-013-2961-x
ORIGINAL ARTICLE
Different Gastric Mucosa and CagA Status of Patients in India and Japan Infected with Helicobacter pylori Keiichi Fujiya • Naoyoshi Nagata • Tomohisa Uchida • Masao Kobayakawa • Naoki Asayama • Junichi Akiyama • Takuro Shimbo • Toru Igari • Rupa Banerjee • D. Nageshwar Reddy • Masashi Mizokami • Naomi Uemura
Received: 29 August 2013 / Accepted: 14 November 2013 / Published online: 27 November 2013 Ó Springer Science+Business Media New York 2013
Abstracts Background and Aim Despite similar incidence of Helicobacter pylori infection, the frequency of gastric cancer is sevenfold higher in Japan than in India. The objective of this work was to define differences in H. pylori-induced gastritis and to identify the bacterial virulence factors involved. Materials and Methods We prospectively enrolled 353 consecutive patients who underwent endoscopy and received three gastric biopsies in Tokyo, Japan, and Hyderabad, India. Immunohistochemistry against H. pylori and East Asian CagA and hematoxylin–eosin and Giemsa
stain were used to examine gastric mucosal biopsy specimens. Histological scores were assessed in accordance with the updated Sydney System. Subjects with H. pylori infection were matched by age and sex to compare histopathology and bacterial virulence. Results Sixty patients infected with H. pylori were prospectively selected. Median histological scores for neutrophil and mononuclear cell infiltration and for atrophy were significantly higher in Japan than in India (neutrophils 4.0 vs 3.0, p \ 0.01; mononuclear cells 5.0 vs 4.5, p = 0.03; atrophy 3.0 vs 2.0, p \ 0.01, respectively). Scores for H. pylori density and intestinal metaplasia were
K. Fujiya N. Nagata (&) M. Kobayakawa N. Asayama J. Akiyama Department of Gastroenterology and Hepatology, National Center for Global Health and Medicine, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan e-mail: [email protected]
T. Igari Department of Clinical Pathology, National Center for Global Health and Medicine, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan e-mail: [email protected]
M. Kobayakawa e-mail: [email protected]
R. Banerjee D. N. Reddy Department of Gastroenterology, Asian Institute of Gastroenterology, 6-3-661 Somajiguda, Hyderabad 500082, India e-mail: [email protected]
N. Asayama e-mail: [email protected]
D. N. Reddy e-mail: [email protected]
K. Fujiya e-mail: [email protected]
J. Akiyama e-mail: [email protected] T. Uchida Department of Molecular Pathology, Oita University, 1-1 Hazamacho, Yufu City, Oita 879-5593, Japan e-mail: [email protected] T. Shimbo Department of Clinical Research and Informatics, National Center for Global Health and Medicine, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan e-mail: [email protected]
M. Mizokami The Research Center for Hepatitis and Immunology, Kohnodai Hospital, National Center for Global Health and Medicine, 1-7-1 Kohnodai, Ichikawa City, Chiba 272-8516, Japan e-mail: [email protected] N. Uemura Department of Gastroenterology and Hepatology, Kohnodai Hospital, National Center for Global Health and Medicine, 1-7-1 Kohnodai, Ichikawa City, Chiba 272-8516, Japan e-mail: [email protected]
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also higher in Japan, albeit without statistical significance (H. pylori 5.0 vs 3.0, p = 0.08; intestinal metaplasia 0.0 vs 0.0, p = 0.08). Prevalence of East Asian CagA-positive H. pylori was significantly higher in Japan (73.3 vs 0.0 %, p \ 0.01). Conclusion The significantly higher prevalence of histologically severe gastritis and East Asian CagA in patients from Japan with H. pylori infection may be involved in the pathogenesis of gastric cancer. Keywords East Asian CagA Endoscopy Gastric cancer risk H. pylori-associated gastritis Updated Sydney System Abbreviations H. pylori Helicobacter pylori CagA Cytotoxin-associated antigen A NCGM National Center for Global Health and Medicine AIG Asian Institute of Gastroenterology
Introduction Gastric cancer is the second most frequent cause of cancer death, resulting in 600,000 deaths per year worldwide [1]. Death from gastric cancer is more common in Asia, particularly in China, Japan, and Korea, where the prevalence of H. pylori infection is high [2]. The different incidence of gastric cancer compared with other countries may be affected by several factors, including the prevalence of H. pylori infection [3], H. pylori-associated gastritis [4], bacterial virulence [5], host genetic factors, and diet [6]. Comparative studies to evaluate differences in the development of gastric cancer among countries have been conducted [7–11]. Here, we focus specifically on the differences between patients from India and Japan. Although the prevalence of H. pylori infection is similar in India and Japan, the prevalence of gastric cancer in Japan is approximately seven times higher than that in India [2, 3]. We hypothesized that the difference in the development of gastric cancer results from the difference in the severity of H. pylori -induced gastric mucosal inflammation. Inflammation of the gastric mucosa, which is usually assessed histologically by use of the updated Sydney System [12], is rare in subjects who are H. pylori-negative, and the incidence of gastric cancer in these patients is extremely low [4]. To compare gastric mucosal inflammation among patients residing in different countries, it is important to evaluate those infected with H. pylori. However, previous
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histological comparisons of gastritis among countries included subjects who were H. pylori-negative [9] or unmatched for age or sex [8, 10, 11]. Here, we endoscopically and histologically evaluated differences in gastric mucosal inflammation between subjects in India and Japan with H. pylori infection. Further, to assess gastric cancer risk, we also determined the presence of the gene encoding the H. pylori virulence factor cytotoxin-associated antigen A (CagA) [13].
Methods Subjects The study was conducted under a prospective cross-sectional two-center design for all adults undergoing diagnostic endoscopy between December 2010 and March 2011 at the Endoscopy Unit, National Center for Global Health and Medicine (NCGM), Tokyo, Japan, and the Asian Institute of Gastroenterology (AIG), Hyderabad, India. Inclusion criteria were age 25–75 years, presence of dyspepsia, consecutive scheduled upper gastrointestinal endoscopies, and Japanese or Indian ethnicity. We excluded patients with a history of H. pylori eradication and those with upper gastrointestinal surgery, concomitant severe illness, regular use of aspirin, treatment with nonsteroidal anti-inflammatory drugs, or treatment with proton-pump inhibitors within four weeks before enrollment. Endoscopic biopsy was performed for all patients ultimately enrolled. The institutional review boards of the NCGM and the AIG approved the study protocol. Informed consent was obtained from all subjects in accordance with the Declaration of Helsinki. Endoscopy All endoscopies were performed by experienced endoscopists. Endoscopic diagnosis included reflux esophagitis, hyperplastic polyp, gastric ulcer, and duodenal ulcer. Histology Biopsy specimens were taken from three sites of the gastric mucosa as follows: the greater curvature of the antrum, the incisura angulus, and the greater curvature of the upper body. Three biopsy specimens were taken from each patient. The biopsied specimens were fixed in 10 % (v/v) formalin, embedded in paraffin, stained with hematoxylin–eosin and Giemsa, and analyzed immunohistochemically as described elsewhere using a polyclonal anti-H. pylori antibody (Dako, Glostrup, Denmark) and an anti-East Asian CagA-specific antibody prepared in our laboratory (Fig. 1) [14]. The latter
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Fig. 1 Immunohistochemistry of gastric mucosa biopsy specimens with anti-Helicobacter pylori antibody (a) and anti-East Asian-specific antibody (b)
mouse monoclonal antibody was raised against the peptide AINRKIDRINKIASAGKG, which is specific to East Asian CagA. Tissue sections from patients with Western CagA or CagA-negative status do not react with this antibody. The sensitivity, specificity, and accuracy of this immunohistochemical method were 96.7, 97.9, and 97.1 %, respectively [15]. Slides were interpreted by an expert pathologist (T.U.) who was not informed of the patients’ clinical and endoscopic findings. Updated Sydney System Score In accordance with the Updated Sydney System, a score of 0–3 (0, absent; 1, mild; 2, moderate; 3, marked) was assigned for the variables as follows: H. pylori density, neutrophil and mononuclear cell infiltration, glandular atrophy, and intestinal metaplasia [16]. Scores for each of the three biopsy specimens were summed, giving a possible range in scores of 0–9. We calculated the corpus-predominant gastritis/antrum-predominant gastritis ratio from the scores for neutrophil infiltration from the antrum and from the upper body. Assessment of H. pylori Status Helicobacter pylori infection was evaluated by staining tissue sections with hematoxylin–eosin and Giemsa combined with immunostaining using antibodies against H. pylori. When all of these methods yielded negative results, H. pylori infection was considered negative. Statistical Analysis All statistical analysis was performed by use of SPSS statistical software version 17.0 (SPSS, Chicago, IL, USA). Patient characteristics, endoscopic diagnosis, and CagA status were compared by use of Fisher’s exact test. Total
histological scores between patients from India and Japan were compared by use of the Mann–Whitney test.
Results Patient Characteristics Three-hundred and fifty-three patients who met the initial set of inclusion criteria were examined by endoscopy (Fig. 2). At the end of the selection process, 30 patients from India were individually matched by age and sex with 30 from Japan. During the matching process, we regarded the Indian patients as cases and randomly selected controls from among Japanese patients according to sex and decennial age. The characteristics of the 30 patients from each country who met all of the inclusion criteria are summarized in Table 1. There were no significant age or sex differences between the two groups. The frequency of reflux esophagitis was significantly higher in the Indian group than in the Japanese group. There were no significant differences in the other endoscopic findings, for example gastric hyperplastic polyp, gastric ulcer, and duodenal ulcer. Severity of Gastritis Among the 60 patients, median scores for H. pylori, neutrophils, mononuclear cells, atrophy, and intestinal metaplasia were 4.0, 4.0, 5.0, 3.0, and 0.0, respectively. The scores for neutrophils, mononuclear cells, and atrophy were significantly higher for subjects from Japan (neutrophils 4.0 vs 3.0, p \ 0.01; mononuclear cells 5.0 vs 4.5, p = 0.03; atrophy 3.0 vs 2.0, p \ 0.01) (Fig. 3). The scores for H. pylori density and intestinal metaplasia were also higher in patients from Japan but without statistical significance (H. pylori 5.0 vs 3.0, p = 0.08; IM 0.0 vs 0.0, p = 0.08). The corpus-predominant gastritis/antrum-predominant
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Fig. 2 Study flow chart. Three-hundred and fifty-three patients who met the initial set of inclusion criteria were examined by endoscopy. At the end of the selection process, 30 patients from India were individually matched by age and sex with 30 from Japan
Table 1 Patient characteristics and endoscopic diagnosis India (n = 30)
Japan (n = 30)
p value
Age (median (IQR))
50.0 (36.5–59.0)
50 (38.3–54.3)
Sex (M/F)
16/14
16/14
Epigastric pain
7 (23.3)
7 (23.3)
Reflux symptoms
22 (73.3)
0 (0)
0.01*
Bloating
8 (26.7)
0 (0)
0.02* 0.13*
CagA Status
Symptoms n (%)
Other
0.54*
12 (40)
4 (13.3)
30 (100)
30 (100)
Reflux esophagitis
9 (30)
2 (6.7)
0.04*
Gastric hyperplastic polyps
0 (0)
0 (0)
N.A.
Endoscopic biopsy n (%)
gastritis ratio in Japan (0.75) was significantly higher than that in India (0.55) (p \ 0.01).
Biopsies were analyzed with anti-East Asian CagA-specific antibody against H. pylori. Twenty-two (73.3 %) samples from patients from Japan were positive. In contrast, biopsies of all patients from India were negative (p \ 0.01).
Discussion
Endoscopic findings n (%)
Active gastric ulcer
3 (10)
2 (6.7)
1.00*
Active duodenal ulcer
9 (30)
3 (10)
0.10*
Macroscopically normal
14 (46.7)
23 (76.7)
0.03*
IQR interquartile range, N.A. not applicable * Fisher’s exact test
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The incidence of gastric cancer in Africa (11.8/100,000) and South Asia (10.7/100,000) is very low, although the prevalence of H. pylori infection is high [2]. Even among Asian countries where the prevalence of H. pylori infection is high, the incidence of gastric cancer varies, and is higher in East Asia (37.5/100,000) than in South (26.5/100,000) and Central Asia (2.3/100,000) [3]. We speculated that this difference is associated with bacterial virulence and the host’s inflammatory response to H. pylori. Therefore, we
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Fig. 3 Median histology scores for H. pylori-infected patients and cumulative biopsy scores. a H. pylori density (p = 0.08), b neutrophil infiltration (p \ 0.01), c mononuclear cell infiltration (p \ 0.01), d glandular atrophy (p \ 0.01), and e intestinal metaplasia
(p = 0.08). The horizontal line inside each box indicates the median, and the top and bottom of each box indicate the 25th and 75th percentiles, respectively. The I bars indicate maximum and minimum values
assessed inflammation of the gastric mucosa by use of endoscopy and histopathology. Endoscopic assessment showed that the frequency of reflux esophagitis was significantly higher in study patients from India than in those from Japan. This suggests that reflux esophagitis correlates with the high prevalence of increased acid secretion by the gastric mucosa of patients from India. This assumption is supported by the finding that pathological atrophy in patients from India was mild, because severe atrophy resulted in hypoacidity, because of reduced numbers of parietal cells. In contrast, even though gastric ulcer correlates positively with gastric cancer and is more prevalent in Japan than in other Asian countries [17], our study showed no significant difference between countries in ulcer prevalence. These results might be because of differences in endoscopic diagnostic standards between the two countries. Despite standard endoscopic diagnosis defining gastric ulcer as a mucosal defect that is 3 mm or larger [18], lesions that are typically diagnosed as erosions in Japan might have been diagnosed as ulceration in India. Despite efforts to ensure that endoscopic atrophic gastritis was assessed in accordance with the classification of Kimura and Takemoto [19], endoscopists in India might not be
sufficiently familiar with the classification of atrophic borders to enable accurate assessment. Gastric cancer may develop through H. pylori-related chronic inflammation of the gastric mucosa followed by atrophic gastritis, intestinal metaplasia, and dysplasia [20]. The development of gastric cancer is associated with neutrophil infiltration [4], atrophy [21], and intestinal metaplasia [21]. Several studies have evaluated differences in the development of gastric cancer between countries, on the basis of histological inflammation of the gastric mucosa [7, 11, 22]. In their comparison of H. pylori-positive Taiwanese, Lithuanian, and Latvian subjects, Jonaitis et al. [10] found no significant differences in atrophy or intestinal metaplasia. In contrast, in a comparison of H. pyloripositive Japanese, Korean, and American subjects, Lee et al. [8] found that neutrophils, atrophy, and intestinal metaplasia in the antrum were significantly more frequent in Korea than in the United States. Naylor et al. [9] found that corpus-predominant gastritis, pangastritis, severe atrophy, and intestinal metaplasia were significantly more frequent in subjects in Japan than in those in the United Kingdom. Although the present study included a few H. pylori-negative subjects, it matched subjects by age and sex.
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Similar studies have also been conducted among Asia populations. In a comparison between Japanese and Indonesian subjects who were H. pylori-positive, Abdullah et al. [11] found that neutrophil and mononuclear cell infiltration, atrophy, and intestinal metaplasia were more frequent in Japanese patients. In a multilateral comparison among Japanese (n = 120), Chinese (n = 105), Thai (n = 145), and Vietnamese (n = 80) subjects matched by age, sex, and endoscopic diagnosis, Matsuhisa et al. [7] demonstrated that gastric ulcer, atrophy in the angulus, and intestinal metaplasia were significantly more frequent in Japan. These authors also demonstrated that the corpuspredominant/antrum-predominant gastritis ratio was higher in Japan than in China (Beijing), Thailand, and Vietnam [22]. This study included only subjects who were H. pyloripositive, and subjects were matched by age and sex. This might have led to a more accurate comparison of gastritis. Scores for inflammation of the gastric mucosa were significantly higher in Japanese patients than in Indian patients, which is consistent with the findings of other studies of patients in Asia [7, 11, 22]. The results suggest that this variable is associated with the high prevalence of gastric cancer in Japan and reflects similar comparisons between patients in Japan and those in other Asian countries. We show here that the prevalence of infection with East Asian CagA-positive H. pylori was 73.3 % in Japan and 0 % in India. The cagA gene is polymorphic and is primarily classified into East Asian and Western types according to sequences located in the 30 coding region [23]. In East Asia, at least 90 % of H. pylori-positive subjects are CagA-positive, versus approximately 40 % in the West and Africa [24]. East Asian-type H. pylori is particularly prevalent in Japan, Korea, and China, and less so in South and Central Asia [25]. Gastritis, atrophy, and peptic ulcer are more severe in subjects infected with East Asian CagApositive H. pylori than in subjects with Western CagApositive H. pylori or CagA-negative H. pylori, and mortality from gastric cancer is 2.7-times higher [26]. Therefore, East Asian CagA status may be an important determinant of the differences in histology scores between Indian and Japanese patients. The strengths of this study include its exclusion of patients with a history of H. pylori eradication [27], or with regular use of aspirin [28], nonsteroidal anti-inflammatory drugs, [29] or proton-pump inhibitors [30]. These drugs can affect the gastric mucosa and preclude accurate assessment of the inflammatory effects of H. pylori infection on the gastric mucosa. Moreover, Giemsa staining and immunohistochemistry using anti-H. pylori antibody, with hematoxylin–eosin staining, facilitated more accurate diagnosis of H. pylori infection. One limitation is that the inclusion of
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patients positive only for H. pylori reduced the number of subjects. Inclusion of a larger number of samples might have enabled the detection of significant differences in H. pylori density and intestinal metaplasia. Moreover, all examinations of gastritis were performed by a gastrointestinal pathologist in Japan, and reproducibility could therefore not be assessed. Another limitation is that molecular genetic tests were not used to detect H. pylori infection. In conclusion, this study clarifies differences in H. pylori-infected gastric mucosa between Indian and Japanese patients matched for age and sex. The frequency of reflux esophagitis was higher in patients from India. In contrast, histology scores for H. pylori, neutrophil and mononuclear cell infiltration, atrophy, and intestinal metaplasia were higher in patients from Japan. East Asian CagA-positive H. pylori was detected in patients from Japan only. These differences may account for the different incidence of gastric cancer in these two countries. Acknowledgments This work was supported by a Grant-in-Aid from the Ministry of Health Labor and Welfare of Japan and a Grantin-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (271000) and a grant from the National Center for Global Health and Medicine. The funders were not involved in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Hisae Kawashiro, Clinical Research Coordinator, NCGM, Tokyo, Japan, for assistance with data collection. We express our sincere gratitude to Drs Anuradha Sekaran and Mitnala Sasikala, AIG, Hyderabad, India for the pathological specimens and blood samples. Conflict of interest
None.
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Dig Dis Sci (2014) 59:631–637 8. Lee I, Lee H, Kim M, et al. Ethnic difference of helicobacter pylori gastritis: Korean and Japanese gastritis is characterized by male- and antrum-predominant acute foveolitis in comparison with American gastritis. World J Gastroenterol. 2005;11:94–98. 9. Naylor GM, Gotoda T, Dixon M, et al. Why does japan have a high incidence of gastric cancer? Comparison of gastritis between UK and japanese patients. Gut. 2006;55:1545–1552. 10. Jonaitis L, Ivanauskas A, Janciauskas D, et al. Precancerous gastric conditions in high helicobacter pylori prevalence areas: comparison between eastern European (lithuanian, latvian) and asian (taiwanese) patients. Medicina (Kaunas).. 2007;43:623–629. 11. Abdullah M, Ohtsuka H, Rani AA, Sato T, Syam AF, Fujino MA. Helicobacter pylori infection and gastropathy: a comparison between Indonesian and Japanese patients. World J Gastroenterol. 2009;15:4928–4931. 12. Dixon MF, Genta RM, Yardley JH, Correa P. Classification and grading of gastritis. The updated Sydney system. International workshop on the histopathology of gastritis, Houston 1994. Am J Surg Pathol. 1996;20:1161–1181. 13. Hatakeyama M. Helicobacter pylori and gastric carcinogenesis. J Gastroenterol. 2009;44:239–248. 14. Uchida T, Kanada R, Tsukamoto Y, et al. Immunohistochemical diagnosis of the cagA-gene genotype of helicobacter pylori with antieast Asian CagA-specific antibody. Cancer Sci. 2007;98:521–528. 15. Nguyen LT, Uchida T, Kuroda A, et al. Evaluation of the antieast Asian CagA-specific antibody for CagA phenotyping. Clin Vaccine Immunol. 2009;16:1687–1692. 16. Rugge M, Genta RM. Staging and grading of chronic gastritis. Hum Pathol. 2005;36:228–233. 17. Wong BC, Ching CK, Lam SK, et al. Differential north to south gastric cancer-duodenal ulcer gradient in China. China ulcer study group. J Gastroenterol Hepatol. 1998;13:1050–1057. 18. Yeomans ND, Naesdal J. Systematic review: ulcer definition in NSAID ulcer prevention trials. Aliment Pharmacol Ther. 2008; 27:465–472.
637 19. Kimura K, Takemoto T. Endoscopic atrophy border. Endoscopy.. 1969;1:1–3. 20. Correa P, Haenszel W, Cuello C, Tannenbaum S, Archer M. A model for gastric cancer epidemiology. Lancet. 1975;2:58–60. 21. Whiting JL, Sigurdsson A, Rowlands DC, Hallissey MT, Fielding JW. The long term results of endoscopic surveillance of premalignant gastric lesions. Gut. 2002;50:378–381. 22. Matsuhisa T, Matsukura N, Yamada N. Topography of chronic active gastritis in helicobacter pylori-positive Asian populations: age-, gender-, and endoscopic diagnosis-matched study. J Gastroenterol. 2004;39:324–328. 23. Yamaoka Y. Pathogenesis of helicobacter pylori-related gastroduodenal diseases from molecular epidemiological studies. Gastroenterol Res Pract.. 2012;2012:371503. 24. Yamaoka Y, Orito E, Mizokami M, et al. Helicobacter pylori in north and South America before columbus. FEBS Lett. 2002;517: 180–184. 25. Yamaoka Y. Mechanisms of disease: Helicobacter pylori virulence factors. Nat Rev Gastroenterol Hepatol.. 2010;7:629–641. 26. Azuma T. Helicobacter pylori CagA protein variation associated with gastric cancer in Asia. J Gastroenterol. 2004;39:97–103. 27. Satoh K. Does eradication of helicobacter pylori reverse atrophic gastritis or intestinal metaplasia? Data from japan. Gastroenterol Clin North Am. 2000;29:829–835. 28. Li GQ, Xia HH, Chen MH, et al. Effects of aspirin on the development of helicobacter pylori-induced gastric inflammation and heterotopic proliferative glands in mongolian gerbils. Helicobacter.. 2008;13:20–29. 29. Barkin J. The relation between helicobacter pylori and nonsteroidal anti-inflammatory drugs. Am J Med. 1998;105: 22S–27S. 30. Kuipers EJ, Lundell L, Klinkenberg-Knol EC, et al. Atrophic gastritis and helicobacter pylori infection in patients with reflux esophagitis treated with omeprazole or fundoplication. N Engl J Med. 1996;334:1018–1022.
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ORIGINAL ARTICLE
Different Gastric Mucosa and CagA Status of Patients in India and Japan Infected with Helicobacter pylori Keiichi Fujiya • Naoyoshi Nagata • Tomohisa Uchida • Masao Kobayakawa • Naoki Asayama • Junichi Akiyama • Takuro Shimbo • Toru Igari • Rupa Banerjee • D. Nageshwar Reddy • Masashi Mizokami • Naomi Uemura
Received: 29 August 2013 / Accepted: 14 November 2013 / Published online: 27 November 2013 Ó Springer Science+Business Media New York 2013
Abstracts Background and Aim Despite similar incidence of Helicobacter pylori infection, the frequency of gastric cancer is sevenfold higher in Japan than in India. The objective of this work was to define differences in H. pylori-induced gastritis and to identify the bacterial virulence factors involved. Materials and Methods We prospectively enrolled 353 consecutive patients who underwent endoscopy and received three gastric biopsies in Tokyo, Japan, and Hyderabad, India. Immunohistochemistry against H. pylori and East Asian CagA and hematoxylin–eosin and Giemsa
stain were used to examine gastric mucosal biopsy specimens. Histological scores were assessed in accordance with the updated Sydney System. Subjects with H. pylori infection were matched by age and sex to compare histopathology and bacterial virulence. Results Sixty patients infected with H. pylori were prospectively selected. Median histological scores for neutrophil and mononuclear cell infiltration and for atrophy were significantly higher in Japan than in India (neutrophils 4.0 vs 3.0, p \ 0.01; mononuclear cells 5.0 vs 4.5, p = 0.03; atrophy 3.0 vs 2.0, p \ 0.01, respectively). Scores for H. pylori density and intestinal metaplasia were
K. Fujiya N. Nagata (&) M. Kobayakawa N. Asayama J. Akiyama Department of Gastroenterology and Hepatology, National Center for Global Health and Medicine, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan e-mail: [email protected]
T. Igari Department of Clinical Pathology, National Center for Global Health and Medicine, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan e-mail: [email protected]
M. Kobayakawa e-mail: [email protected]
R. Banerjee D. N. Reddy Department of Gastroenterology, Asian Institute of Gastroenterology, 6-3-661 Somajiguda, Hyderabad 500082, India e-mail: [email protected]
N. Asayama e-mail: [email protected]
D. N. Reddy e-mail: [email protected]
K. Fujiya e-mail: [email protected]
J. Akiyama e-mail: [email protected] T. Uchida Department of Molecular Pathology, Oita University, 1-1 Hazamacho, Yufu City, Oita 879-5593, Japan e-mail: [email protected] T. Shimbo Department of Clinical Research and Informatics, National Center for Global Health and Medicine, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan e-mail: [email protected]
M. Mizokami The Research Center for Hepatitis and Immunology, Kohnodai Hospital, National Center for Global Health and Medicine, 1-7-1 Kohnodai, Ichikawa City, Chiba 272-8516, Japan e-mail: [email protected] N. Uemura Department of Gastroenterology and Hepatology, Kohnodai Hospital, National Center for Global Health and Medicine, 1-7-1 Kohnodai, Ichikawa City, Chiba 272-8516, Japan e-mail: [email protected]
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also higher in Japan, albeit without statistical significance (H. pylori 5.0 vs 3.0, p = 0.08; intestinal metaplasia 0.0 vs 0.0, p = 0.08). Prevalence of East Asian CagA-positive H. pylori was significantly higher in Japan (73.3 vs 0.0 %, p \ 0.01). Conclusion The significantly higher prevalence of histologically severe gastritis and East Asian CagA in patients from Japan with H. pylori infection may be involved in the pathogenesis of gastric cancer. Keywords East Asian CagA Endoscopy Gastric cancer risk H. pylori-associated gastritis Updated Sydney System Abbreviations H. pylori Helicobacter pylori CagA Cytotoxin-associated antigen A NCGM National Center for Global Health and Medicine AIG Asian Institute of Gastroenterology
Introduction Gastric cancer is the second most frequent cause of cancer death, resulting in 600,000 deaths per year worldwide [1]. Death from gastric cancer is more common in Asia, particularly in China, Japan, and Korea, where the prevalence of H. pylori infection is high [2]. The different incidence of gastric cancer compared with other countries may be affected by several factors, including the prevalence of H. pylori infection [3], H. pylori-associated gastritis [4], bacterial virulence [5], host genetic factors, and diet [6]. Comparative studies to evaluate differences in the development of gastric cancer among countries have been conducted [7–11]. Here, we focus specifically on the differences between patients from India and Japan. Although the prevalence of H. pylori infection is similar in India and Japan, the prevalence of gastric cancer in Japan is approximately seven times higher than that in India [2, 3]. We hypothesized that the difference in the development of gastric cancer results from the difference in the severity of H. pylori -induced gastric mucosal inflammation. Inflammation of the gastric mucosa, which is usually assessed histologically by use of the updated Sydney System [12], is rare in subjects who are H. pylori-negative, and the incidence of gastric cancer in these patients is extremely low [4]. To compare gastric mucosal inflammation among patients residing in different countries, it is important to evaluate those infected with H. pylori. However, previous
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histological comparisons of gastritis among countries included subjects who were H. pylori-negative [9] or unmatched for age or sex [8, 10, 11]. Here, we endoscopically and histologically evaluated differences in gastric mucosal inflammation between subjects in India and Japan with H. pylori infection. Further, to assess gastric cancer risk, we also determined the presence of the gene encoding the H. pylori virulence factor cytotoxin-associated antigen A (CagA) [13].
Methods Subjects The study was conducted under a prospective cross-sectional two-center design for all adults undergoing diagnostic endoscopy between December 2010 and March 2011 at the Endoscopy Unit, National Center for Global Health and Medicine (NCGM), Tokyo, Japan, and the Asian Institute of Gastroenterology (AIG), Hyderabad, India. Inclusion criteria were age 25–75 years, presence of dyspepsia, consecutive scheduled upper gastrointestinal endoscopies, and Japanese or Indian ethnicity. We excluded patients with a history of H. pylori eradication and those with upper gastrointestinal surgery, concomitant severe illness, regular use of aspirin, treatment with nonsteroidal anti-inflammatory drugs, or treatment with proton-pump inhibitors within four weeks before enrollment. Endoscopic biopsy was performed for all patients ultimately enrolled. The institutional review boards of the NCGM and the AIG approved the study protocol. Informed consent was obtained from all subjects in accordance with the Declaration of Helsinki. Endoscopy All endoscopies were performed by experienced endoscopists. Endoscopic diagnosis included reflux esophagitis, hyperplastic polyp, gastric ulcer, and duodenal ulcer. Histology Biopsy specimens were taken from three sites of the gastric mucosa as follows: the greater curvature of the antrum, the incisura angulus, and the greater curvature of the upper body. Three biopsy specimens were taken from each patient. The biopsied specimens were fixed in 10 % (v/v) formalin, embedded in paraffin, stained with hematoxylin–eosin and Giemsa, and analyzed immunohistochemically as described elsewhere using a polyclonal anti-H. pylori antibody (Dako, Glostrup, Denmark) and an anti-East Asian CagA-specific antibody prepared in our laboratory (Fig. 1) [14]. The latter
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Fig. 1 Immunohistochemistry of gastric mucosa biopsy specimens with anti-Helicobacter pylori antibody (a) and anti-East Asian-specific antibody (b)
mouse monoclonal antibody was raised against the peptide AINRKIDRINKIASAGKG, which is specific to East Asian CagA. Tissue sections from patients with Western CagA or CagA-negative status do not react with this antibody. The sensitivity, specificity, and accuracy of this immunohistochemical method were 96.7, 97.9, and 97.1 %, respectively [15]. Slides were interpreted by an expert pathologist (T.U.) who was not informed of the patients’ clinical and endoscopic findings. Updated Sydney System Score In accordance with the Updated Sydney System, a score of 0–3 (0, absent; 1, mild; 2, moderate; 3, marked) was assigned for the variables as follows: H. pylori density, neutrophil and mononuclear cell infiltration, glandular atrophy, and intestinal metaplasia [16]. Scores for each of the three biopsy specimens were summed, giving a possible range in scores of 0–9. We calculated the corpus-predominant gastritis/antrum-predominant gastritis ratio from the scores for neutrophil infiltration from the antrum and from the upper body. Assessment of H. pylori Status Helicobacter pylori infection was evaluated by staining tissue sections with hematoxylin–eosin and Giemsa combined with immunostaining using antibodies against H. pylori. When all of these methods yielded negative results, H. pylori infection was considered negative. Statistical Analysis All statistical analysis was performed by use of SPSS statistical software version 17.0 (SPSS, Chicago, IL, USA). Patient characteristics, endoscopic diagnosis, and CagA status were compared by use of Fisher’s exact test. Total
histological scores between patients from India and Japan were compared by use of the Mann–Whitney test.
Results Patient Characteristics Three-hundred and fifty-three patients who met the initial set of inclusion criteria were examined by endoscopy (Fig. 2). At the end of the selection process, 30 patients from India were individually matched by age and sex with 30 from Japan. During the matching process, we regarded the Indian patients as cases and randomly selected controls from among Japanese patients according to sex and decennial age. The characteristics of the 30 patients from each country who met all of the inclusion criteria are summarized in Table 1. There were no significant age or sex differences between the two groups. The frequency of reflux esophagitis was significantly higher in the Indian group than in the Japanese group. There were no significant differences in the other endoscopic findings, for example gastric hyperplastic polyp, gastric ulcer, and duodenal ulcer. Severity of Gastritis Among the 60 patients, median scores for H. pylori, neutrophils, mononuclear cells, atrophy, and intestinal metaplasia were 4.0, 4.0, 5.0, 3.0, and 0.0, respectively. The scores for neutrophils, mononuclear cells, and atrophy were significantly higher for subjects from Japan (neutrophils 4.0 vs 3.0, p \ 0.01; mononuclear cells 5.0 vs 4.5, p = 0.03; atrophy 3.0 vs 2.0, p \ 0.01) (Fig. 3). The scores for H. pylori density and intestinal metaplasia were also higher in patients from Japan but without statistical significance (H. pylori 5.0 vs 3.0, p = 0.08; IM 0.0 vs 0.0, p = 0.08). The corpus-predominant gastritis/antrum-predominant
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Fig. 2 Study flow chart. Three-hundred and fifty-three patients who met the initial set of inclusion criteria were examined by endoscopy. At the end of the selection process, 30 patients from India were individually matched by age and sex with 30 from Japan
Table 1 Patient characteristics and endoscopic diagnosis India (n = 30)
Japan (n = 30)
p value
Age (median (IQR))
50.0 (36.5–59.0)
50 (38.3–54.3)
Sex (M/F)
16/14
16/14
Epigastric pain
7 (23.3)
7 (23.3)
Reflux symptoms
22 (73.3)
0 (0)
0.01*
Bloating
8 (26.7)
0 (0)
0.02* 0.13*
CagA Status
Symptoms n (%)
Other
0.54*
12 (40)
4 (13.3)
30 (100)
30 (100)
Reflux esophagitis
9 (30)
2 (6.7)
0.04*
Gastric hyperplastic polyps
0 (0)
0 (0)
N.A.
Endoscopic biopsy n (%)
gastritis ratio in Japan (0.75) was significantly higher than that in India (0.55) (p \ 0.01).
Biopsies were analyzed with anti-East Asian CagA-specific antibody against H. pylori. Twenty-two (73.3 %) samples from patients from Japan were positive. In contrast, biopsies of all patients from India were negative (p \ 0.01).
Discussion
Endoscopic findings n (%)
Active gastric ulcer
3 (10)
2 (6.7)
1.00*
Active duodenal ulcer
9 (30)
3 (10)
0.10*
Macroscopically normal
14 (46.7)
23 (76.7)
0.03*
IQR interquartile range, N.A. not applicable * Fisher’s exact test
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The incidence of gastric cancer in Africa (11.8/100,000) and South Asia (10.7/100,000) is very low, although the prevalence of H. pylori infection is high [2]. Even among Asian countries where the prevalence of H. pylori infection is high, the incidence of gastric cancer varies, and is higher in East Asia (37.5/100,000) than in South (26.5/100,000) and Central Asia (2.3/100,000) [3]. We speculated that this difference is associated with bacterial virulence and the host’s inflammatory response to H. pylori. Therefore, we
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Fig. 3 Median histology scores for H. pylori-infected patients and cumulative biopsy scores. a H. pylori density (p = 0.08), b neutrophil infiltration (p \ 0.01), c mononuclear cell infiltration (p \ 0.01), d glandular atrophy (p \ 0.01), and e intestinal metaplasia
(p = 0.08). The horizontal line inside each box indicates the median, and the top and bottom of each box indicate the 25th and 75th percentiles, respectively. The I bars indicate maximum and minimum values
assessed inflammation of the gastric mucosa by use of endoscopy and histopathology. Endoscopic assessment showed that the frequency of reflux esophagitis was significantly higher in study patients from India than in those from Japan. This suggests that reflux esophagitis correlates with the high prevalence of increased acid secretion by the gastric mucosa of patients from India. This assumption is supported by the finding that pathological atrophy in patients from India was mild, because severe atrophy resulted in hypoacidity, because of reduced numbers of parietal cells. In contrast, even though gastric ulcer correlates positively with gastric cancer and is more prevalent in Japan than in other Asian countries [17], our study showed no significant difference between countries in ulcer prevalence. These results might be because of differences in endoscopic diagnostic standards between the two countries. Despite standard endoscopic diagnosis defining gastric ulcer as a mucosal defect that is 3 mm or larger [18], lesions that are typically diagnosed as erosions in Japan might have been diagnosed as ulceration in India. Despite efforts to ensure that endoscopic atrophic gastritis was assessed in accordance with the classification of Kimura and Takemoto [19], endoscopists in India might not be
sufficiently familiar with the classification of atrophic borders to enable accurate assessment. Gastric cancer may develop through H. pylori-related chronic inflammation of the gastric mucosa followed by atrophic gastritis, intestinal metaplasia, and dysplasia [20]. The development of gastric cancer is associated with neutrophil infiltration [4], atrophy [21], and intestinal metaplasia [21]. Several studies have evaluated differences in the development of gastric cancer between countries, on the basis of histological inflammation of the gastric mucosa [7, 11, 22]. In their comparison of H. pylori-positive Taiwanese, Lithuanian, and Latvian subjects, Jonaitis et al. [10] found no significant differences in atrophy or intestinal metaplasia. In contrast, in a comparison of H. pyloripositive Japanese, Korean, and American subjects, Lee et al. [8] found that neutrophils, atrophy, and intestinal metaplasia in the antrum were significantly more frequent in Korea than in the United States. Naylor et al. [9] found that corpus-predominant gastritis, pangastritis, severe atrophy, and intestinal metaplasia were significantly more frequent in subjects in Japan than in those in the United Kingdom. Although the present study included a few H. pylori-negative subjects, it matched subjects by age and sex.
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Similar studies have also been conducted among Asia populations. In a comparison between Japanese and Indonesian subjects who were H. pylori-positive, Abdullah et al. [11] found that neutrophil and mononuclear cell infiltration, atrophy, and intestinal metaplasia were more frequent in Japanese patients. In a multilateral comparison among Japanese (n = 120), Chinese (n = 105), Thai (n = 145), and Vietnamese (n = 80) subjects matched by age, sex, and endoscopic diagnosis, Matsuhisa et al. [7] demonstrated that gastric ulcer, atrophy in the angulus, and intestinal metaplasia were significantly more frequent in Japan. These authors also demonstrated that the corpuspredominant/antrum-predominant gastritis ratio was higher in Japan than in China (Beijing), Thailand, and Vietnam [22]. This study included only subjects who were H. pyloripositive, and subjects were matched by age and sex. This might have led to a more accurate comparison of gastritis. Scores for inflammation of the gastric mucosa were significantly higher in Japanese patients than in Indian patients, which is consistent with the findings of other studies of patients in Asia [7, 11, 22]. The results suggest that this variable is associated with the high prevalence of gastric cancer in Japan and reflects similar comparisons between patients in Japan and those in other Asian countries. We show here that the prevalence of infection with East Asian CagA-positive H. pylori was 73.3 % in Japan and 0 % in India. The cagA gene is polymorphic and is primarily classified into East Asian and Western types according to sequences located in the 30 coding region [23]. In East Asia, at least 90 % of H. pylori-positive subjects are CagA-positive, versus approximately 40 % in the West and Africa [24]. East Asian-type H. pylori is particularly prevalent in Japan, Korea, and China, and less so in South and Central Asia [25]. Gastritis, atrophy, and peptic ulcer are more severe in subjects infected with East Asian CagApositive H. pylori than in subjects with Western CagApositive H. pylori or CagA-negative H. pylori, and mortality from gastric cancer is 2.7-times higher [26]. Therefore, East Asian CagA status may be an important determinant of the differences in histology scores between Indian and Japanese patients. The strengths of this study include its exclusion of patients with a history of H. pylori eradication [27], or with regular use of aspirin [28], nonsteroidal anti-inflammatory drugs, [29] or proton-pump inhibitors [30]. These drugs can affect the gastric mucosa and preclude accurate assessment of the inflammatory effects of H. pylori infection on the gastric mucosa. Moreover, Giemsa staining and immunohistochemistry using anti-H. pylori antibody, with hematoxylin–eosin staining, facilitated more accurate diagnosis of H. pylori infection. One limitation is that the inclusion of
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patients positive only for H. pylori reduced the number of subjects. Inclusion of a larger number of samples might have enabled the detection of significant differences in H. pylori density and intestinal metaplasia. Moreover, all examinations of gastritis were performed by a gastrointestinal pathologist in Japan, and reproducibility could therefore not be assessed. Another limitation is that molecular genetic tests were not used to detect H. pylori infection. In conclusion, this study clarifies differences in H. pylori-infected gastric mucosa between Indian and Japanese patients matched for age and sex. The frequency of reflux esophagitis was higher in patients from India. In contrast, histology scores for H. pylori, neutrophil and mononuclear cell infiltration, atrophy, and intestinal metaplasia were higher in patients from Japan. East Asian CagA-positive H. pylori was detected in patients from Japan only. These differences may account for the different incidence of gastric cancer in these two countries. Acknowledgments This work was supported by a Grant-in-Aid from the Ministry of Health Labor and Welfare of Japan and a Grantin-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (271000) and a grant from the National Center for Global Health and Medicine. The funders were not involved in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Hisae Kawashiro, Clinical Research Coordinator, NCGM, Tokyo, Japan, for assistance with data collection. We express our sincere gratitude to Drs Anuradha Sekaran and Mitnala Sasikala, AIG, Hyderabad, India for the pathological specimens and blood samples. Conflict of interest
None.
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