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(American Journal of Pathology. 2001;158:647-654.)
© 2001 American Society for Investigative Pathology

Regular Article

Helicobacter pylori Genotypes May Determine Gastric Histopathology

Cristina Nogueira^*, Céu Figueiredo^*, Fátima Carneiro^*, António Taveira Gomes^§, Raul Barreira^§, Paulo Figueira^§, Céu Salgado^§, Luis Belo, António Peixoto^*, Juan C. Bravo^||, Luis E. Bravo^||, Jose L. Realpe^**, Anton P. Plaisier^**, Wim G. V. Quint, Bernardo Ruiz, Pelayo Correa and Leen-Jan van Doorn

From the Institute of Molecular Pathology and Immunology ^*
and the Faculty of Medicine,
University of Porto, Porto, Portugal; the Delft Diagnostic Laboratory,
Delft, The Netherlands; Hospital de S. João,^§
Porto, Portugal; ENVC,
Viana do Catelo, Portugal; the Universidad del Valle,^||
Cali, Colombia; the Medical Faculty,^**
Department of Public Health, Erasmus University Rotterdam, Rotterdam, The Netherlands; and the Department of Pathology,
Louisiana State University, New Orleans, Louisiana

	Abstract

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The outcome of Helicobacter pylori infection has been associated with specific virulence-associated bacterial genotypes. The present study aimed to investigate the gastric histopathology in Portuguese and Colombian patients infected with H. pylori and to assess its relationship with bacterial virulence-associated vacA, cagA, and iceA genotypes. A total of 370 patients from Portugal (n = 192) and Colombia (n = 178) were studied. Corpus and antrum biopsy specimens were collected from each individual. Histopathological features were recorded and graded according to the updated Sydney system. H. pylori vacA, cagA, and iceA genes were directly genotyped in the gastric biopsy specimens by polymerase chain reaction and reverse hybridization. Despite the significant differences between the Portuguese and Colombian patient groups, highly similar results were observed with respect to the relation between H. pylori genotypes and histopathology. H. pylori vacA s1, vacA m1, cagA+ genotypes were significantly associated with a higher H. pylori density, higher degrees of lymphocytic and neutrophilic infiltrates, atrophy, the type of intestinal metaplasia, and presence of epithelial damage. The iceA1 genotype was only associated with epithelial damage in Portuguese patients. These findings show that distinct H. pylori genotypes are strongly associated with histopathological findings in the stomach, confirming their relevance for the development of H. pylori-associated gastric pathology.

	Introduction

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	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Patients

A total of 370 H. pylori-infected patients from Portugal and Colombia were studied. Portuguese patients (n = 192, 173 males and 19 females; age 43.3 ± 6.9 years) were recruited from shipyard workers during a screening program for (pre)malignant lesions of the gastric mucosa. Patients provided informed consent and underwent standard gastroscopy in 1998 in Hospital de S. João (Porto, Portugal). None of the patients had gastric cancer, 21 (11.0%) had duodenal ulcer, and eight (4.2%) had a gastric ulcer. Biopsy specimens were taken from corpus and antrum.

Colombian patients (n = 178, 73 males and 105 females; age 55.9 ± 8.4 years) were from the Andean region of Nariño, a part of Colombia with an extremely high prevalence of gastric cancer. All patients were participants of a randomized 6-year chemoprevention trial of gastric dysplasia performed in the towns of Tuquerres and Pasto, Nariño. Only patients with atrophy were selected for this trial, which followed a factorial design comprising anti-H. pylori therapy, as well as vitamin C and {beta}-carotene treatment. One of the patients had peptic ulcer disease and none had gastric cancer. Biopsy specimens were obtained in 1998 by gastroscopy from the corpus and antrum of the stomach.

Histopathology

Two gastric biopsy specimens from the antrum (one from the greater curvature and one from the incisura angularis) and one from the corpus were immersed in 10% formalin and embedded in paraffin. Sections were stained by hematoxylin and eosin, Alcian blue-periodic acid Schiff, and modified Giemsa. Only cases with adequately sized biopsy specimens of both antral and corpus mucosa were accepted for histological assessment by experienced pathologists, who were blinded with respect to the clinical information of each patient.

The following histopathological parameters were scored on an ordinal scale (0 to 3) using the criteria as described in the updated Sydney classification system²¹ : H. pylori density, chronic inflammation, polymorphonuclear activity (neutrophil activity), and glandular atrophy. Whenever present, intestinal metaplasia was typed as complete, mixed (complete and incomplete), or incomplete. Surface epithelial degeneration was scored as present or absent. Results from the two antrum biopsy specimens were combined and whenever differences were observed, the highest score was considered for statistical analyses.

DNA Isolation from Gastric Biopsies and vacA, cagA, and iceA Genotyping

Antral biopsies were obtained, immediately placed in liquid nitrogen, and then transferred in dry ice to -80°C freezers. Total DNA was extracted from gastric biopsy specimens using the method described by Boom and colleagues.²² Briefly, biopsies were homogenized in guanidinium isothiocyanate, using a sterile micropestle (Merck). DNA was captured onto silica particles, washed, and eluted in 100 µl of 10 mmol/L Tris-HCl, pH 8.3. Two µl was used for each polymerase chain reaction (PCR). Subsequently, vacA and cagA genotypes were determined by multiplex PCRs followed by reverse hybridization on a line probe assay (LiPA) as described earlier^12,23 For amplification of the iceA1 and iceA2 genes, allele-specific PCR reactions were performed as described^18,24

Statistical Analyses

Data were analyzed in SPSS version 8.0. The Pearson chi-square test and Fisher’s exact test were used to assess the relationships between individual genotypes and the presence of epithelial damage, atrophy, and intestinal metaplasia. Only cases containing single genotypes were included, and cases containing multiple genotypes were excluded. The t-test was used to analyze the age distributions between the different patient groups. Histopathological parameters, scored on ordinal scales (from 0 to 3), were analyzed by the Mann-Whitney test. Logistic regression analysis was used to relate the vacA, cagA, and iceA genotypes of H. pylori. Age (in 5-year classes) and gender were included as possible confounders. Selection of variables was based on a likelihood ratio test, using a significance level of 0.05 for inclusion or elimination.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
A total of 370 patients from Portugal and Colombia were endoscoped and gastric biopsy specimens were obtained from corpus and antrum. Comparison of the Portuguese and Colombian patients (Table 1) revealed several differences between the two groups. The mean age of the Colombian patients was significantly higher than that of the Portuguese patients (P < 0.001). Also, the gender distribution was different, because 90.1% of the Portuguese patients were male, versus only 40.4% of the Colombian patients (P < 0.001).

The vacA, cagA, and iceA status was analyzed for all 370 H. pylori-infected patients (Table 1) . The prevalence of vacA subtypes was different between Colombian and Portuguese patients. Subtype s1b was less prevalent in Portuguese (37.5%) as compared to Colombian patients (84.3%). Conversely, type s2 was more often found in Portuguese patients (29.7%) than in Colombian patients (7.3%). Type m1 seemed to be more prevalent in Colombian patients (85.4%) than in Portuguese cases (32.9%). Similarly, the cagA-positivity rate was higher among Colombian patients (90.4%) than among Portuguese patients (60.4%). The prevalence of the different iceA genotypes seemed to be similar among the two patient groups.

The prevalence of cases containing multiple vacA or iceA genotypes was much higher among the Portuguese patients than among Colombian cases (P < 0.001). Multiple vacA as well as iceA genotypes were found in 30 (15.6%) of the 192 Portuguese cases, whereas of the Colombian patients, only three (1.6%) of the 178 cases contained multiple genotypes for vacA as well as iceA (P < 0.001).

H. pylori Genotypes and the Density of H. pylori Colonization

The density of H. pylori was scored in antral and corpus biopsy specimens and the results are shown in Figure 1 . In Portuguese patients, the average density scores in the antrum were significantly higher than in the corpus (P < 0.001). There also was a significant relationship between the density of H. pylori colonization in the corpus and vacA s1 (P = 0.003), cagA+ (P = 0.008), and iceA1 (P = 0.048) genotypes, but not with the vacA m type. No significant relationships were observed between vacA s or m region, cagA status, or iceA genotypes and the density of H. pylori in the antrum biopsy specimens. In a total of seven (3.6%) antral biopsies no bacteria were observed (density = 0), whereas they were found positive by PCR.

View larger version (52K): [in this window] [in a new window]   Figure 1. Density of H. pylori colonization in Portuguese and in Colombian patients, according to the updated Sydney system. White = absent, light gray = rare, dark gray = moderate, black = abundant. Figures at the left show results for antral biopsy specimens, figures at the right show results for corpus biopsy specimens. A single asterisk above a pair of bars marks a significant difference at P < 0.05; double asterisks above a pair of bars marks a significant difference at P < 0.01 (Mann-Whitney test). Only cases containing a single genotype were included in the statistical analysis. Cases containing multiple genotypes were excluded.

H. pylori Genotypes and Chronic Inflammation

Among the Portuguese patients (Figure 2A) , there was a significant association between vacA s1, m1, and cagA+ genotypes and higher inflammation scores in both corpus and antrum biopsy specimens. The iceA status was only associated to the degree of inflammation in the corpus. Among Colombian patients (Figure 3A) , vacA s1, m1, and cagA+ genotypes are associated to higher inflammation scores in the corpus, but not in the antrum. The iceA genotype was not associated to the degree of inflammation.

View larger version (56K): [in this window] [in a new window]   Figure 2. Histological parameters determined in gastric biopsy specimens from Portuguese patients, according to the updated Sydney system. Patterns for lymphocytic infiltrates (A), neutrophil activity (B), and glandular atrophy (C) are as follows: white = absent, light gray = mild, dark gray = moderate, black = severe. Statistical analyses were performed using the Mann-Whitney test. Asterisks are identical to those in Figure 1 .

View larger version (60K): [in this window] [in a new window]   Figure 3. Histological parameters determined in gastric biopsy specimens from Colombian patients, according to the updated Sydney system. Patterns are identical to those in Figure 2 and asterisks are identical to those in Figure 1 .

Neutrophilic activity was investigated and results are shown in Figures 2B and 3B . In both Portuguese and Colombian patients, vacA s1, vacA m1, and cagA+ genotypes were strongly associated with higher activity in the corpus and in the antrum. The iceA status was not significantly associated with the neutrophil infiltration scores.

H. pylori Genotypes and Glandular Atrophy

Overall, the prevalence of atrophy among the Portuguese patients was lower than among Colombian cases. It should be taken into account that the Colombian patients were selected for presence of atrophy at baseline. Therefore, the association between presence of atrophy and H. pylori genotypes could not be assessed in Colombian patients. Nevertheless, the association between the degree of atrophy and H. pylori genotypes could still be analyzed.

Among the Portuguese patients, the ages were not normally distributed, and patients with atrophic gastritis (45.8 ± 6.8 years) were significantly older than patients with nonatrophic gastritis (42.0 ± 6.6 years) (P < 0.001). There was a strong association between the presence of glandular atrophy in the antrum and the individual vacA s1, m1, and cagA+ genotypes, as compared to the vacA s2, m2, and cagA- genotypes, respectively, but not with the iceA genotype. The odds ratio of vacA s1 versus s2, vacA m1 versus m2, and cagA+ versus cagA- strains for glandular atrophy were 32.21 (95% CI, 7.3 to 141.7), 15.18 (95% CI, 5.7 to 40.3), and 13.12 (95% CI, 4.7 to 36.6), respectively.

For both patient groups, the associations between the H. pylori genotypes and the degree of atrophy were further analyzed, and results are shown in Figures 2C and 3C . In both Portuguese and Colombian patients, vacA s1, vacA m1, cagA+ (but not the iceA) genotypes were each strongly correlated with the degree of glandular atrophy in the antrum. In the corpus of the Portuguese patients atrophy was found only in five cases, thus not permitting reliable analyses. In the corpus of Colombian patients, only an association between glandular atrophy and the cagA genotype was found.

H. pylori Genotypes and Intestinal Metaplasia

Firstly, a highly significant association was observed between the presence of intestinal metaplasia in the antrum and H. pylori genotypes. The prevalence of vacA s1, m1, or cagA+ strains was much higher among patients with intestinal metaplasia than in cases without intestinal metaplasia. Among Portuguese patients, the odds ratios for intestinal metaplasia in the antrum of vacA s1 versus s2, vacA m1 versus m2, and cagA+ versus cagA- strains were 16.2 (95% CI, 4.7 to 56.4), 9.3 (95% CI, 3.7 to 23.4), and 8.1 (95% CI, 3.1 to 21.2), respectively. In the corpus, intestinal metaplasia was observed in only a small number of patients. Among Colombian patients, the presence of metaplasia in the antrum was only significantly associated with the vacA s genotype with and odds ratio of 3.5 (95% CI, 1.0 to 12.0). Presence of metaplasia in the corpus was not associated to any of the genotypes.

Secondly, the association between H. pylori genotypes and the type of intestinal metaplasia is shown in Figure 4 . There was a strong association between the vacA s1, m1, and cagA+ genotypes and the type of metaplasia in the antrum of the Portuguese patients. In Colombian patients, a similar trend was observed in the antrum, but this failed to reach statistical significance. In the corpus biopsy specimens, there were no significant associations observed, except for the vacA m type in Portuguese patients, although it should be noted that there were only five Portuguese patients with intestinal metaplasia in the corpus.

View larger version (40K): [in this window] [in a new window]   Figure 4. Type of intestinal metaplasia in Portuguese and in Colombian patients. White = absent, light gray = complete, dark gray = mixed, black = incomplete. Statistical analyses were performed using the Mann-Whitney test. Asterisks are identical to those in Figure 1 .

The presence or absence of epithelial damage was assessed and results are shown in Figure 5 . Presence of vacA s1, vacA m1, and cagA+ genotypes are strongly associated with the presence of epithelial damage in the corpus of both Portuguese and Colombian patients and in the antrum of the Portuguese patients. The iceA status was significantly associated with the presence of epithelial damage in the antrum and corpus of the Portuguese patients.

View larger version (46K): [in this window] [in a new window]   Figure 5. Epithelial damage in Portuguese and in Colombian patients. White = absent, light gray = present. Statistical analyses were performed using the chi-square test. Asterisks are identical to those in Figure 1 .

Separate analysis of a subgroup of 77 cagA-negative Portuguese cases revealed that in these patients, vacA s1 genotypes are significantly associated with the presence of atrophy (P = 0.008), the degree of atrophy (P = <0.001), presence of intestinal metaplasia (P = 0.016), the type of intestinal metaplasia (P < 0.001), and epithelial damage (P = 0.024) in the antrum. Similarly, the vacA m1 strains are associated with presence of atrophy (P = 0.003), the degree of atrophy (P < 0.001), the presence of intestinal metaplasia (P = 0.006), and the type of intestinal metaplasia (P = 0.001) in the antrum, but not with the presence of epithelial damage.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
H. pylori infection causes chronic gastritis and is associated with the development of peptic ulcer disease, gastric carcinoma, and MALT lymphoma.^2,25,26 Although H. pylori infection causes chronic gastritis in virtually all patients, not all individuals develop more severe gastric pathology. Thus, there is a marked discrepancy between the number of individuals colonized and those with clinical symptoms. Although host and environmental factors are considered important,³ there is also evidence for a role of specific H. pylori virulence-associated genotypes. Patients infected with less virulent genotypes are more likely to have mild gastritis throughout their entire life, whereas patients infected with more virulent genotypes have a higher probability of developing peptic ulcer disease, atrophic gastritis, and eventually gastric carcinoma. Gastric carcinogenesis is a complex, multifactorial process, in which persistent H. pylori infection can play an important role.²⁶ Prolonged infection may lead to the loss of glands, resulting in multifocal atrophic gastritis, which can be followed by the development of intestinal metaplasia and dysplasia.⁵ As compared to the general population, patients with duodenal ulcer disease may have a reduced risk for the development of gastric cancer.²⁷ This finding, as well as other epidemiological observations,²⁸ suggest the existence of at least two distinct pathways, one resulting in duodenal ulcer disease, and the other associated with gastric ulcer disease, atrophic gastritis, and possibly gastric carcinoma. Atrophy is the first recognizable step in the multifocal atrophic gastritis complex that in more advanced stages may lead to intestinal metaplasia and dysplasia. vacA s- and m-region mosaicism can play an important role in such a chain of events. Several studies have consistently shown that the vacA s genotype is associated with disease in patients from various countries^11,30-33 although others did not confirm these findings.¹⁹ The vacA m region genotype was not significantly associated with peptic ulcer disease.¹³ However, the present study revealed the strong significance of the vacA m1 genotype with respect to epithelial damage, neutrophilic and lymphocytic infiltrates, atrophic gastritis, and intestinal metaplasia in patients from two geographically distinct regions. Atherton and colleagues³⁴ also presented evidence that m1 strains were associated with greater gastric epithelial damage than m2 strains in North American patients. Increased epithelial damage may be explained by a higher level of cytotoxin production by vacA s1/m1 strains as compared to vacA s1/m2 strains.¹¹ Earlier studies³⁵ also revealed a significant relationship between the level of cytotoxin production and the severity of atrophy, confirming that the cytotoxin is a disease-associated factor. Because the production of cytotoxin primarily depends on the mosaic structure of the vacA gene, especially of the s and m regions,¹¹ the association between vacA genotypes and atrophy as presented here, is consistent with these earlier observations. Moreover, the present study showed that vacA and cagA genotypes not only are related to the presence, but also with the degree of atrophy and the type of intestinal metaplasia, suggesting that these genotypes may play an important role in the development of lesions considered as precursors of gastric carcinoma. Our findings that severe damage to the gastric epithelium is associated with vacA s1/m1 mosaicism suggests that such damage represents the primary effect of the cytotoxin¹⁰ and that leukocyte infiltrates and atrophy may be sequential in the cascade that characterizes the precancerous process. Furthermore, the m1 and m2 forms of the VacA cytotoxin may recognize different receptors on human gastric epithelial cells.³⁶

The iceA genotype, which has been associated with peptic ulcer disease in some populations^17,18 seems to be of limited use in the patient populations studied here. iceA1 was significantly associated to the degree of lymphocyte infiltrates and epithelial damage in Portuguese patients, but not with neutrophil activity, atrophy, and intestinal metaplasia. Therefore, the clinical relevance of iceA remains to be studied in more detail in patients from different geographic origins.

Several genes of the cag pathogenicity island encode proteins that enhance the virulence of the strain, eg, by increasing the production of interleukin-8 by gastric epithelial cells,³⁷ causing infiltration of polymorphonuclear neutrophils, resulting in more severe inflammatory activity of the gastric epithelium. The present study showed that the great majority of patients have lymphocytic infiltrates in the corpus and antrum but neutrophil activity is found less frequently, and is strongly associated with vacA-s1, vacA-m1, and cagA+ genotypes. Several European and North American studies have shown that infection withcagA-positive H. pylori strains also increases the risk for atrophic gastritis and gastric cancer.^15,38 However, several studies in Asian populations did not confirm these relationships,³⁹ indicating that there are important geographic differences. In general, the incidence of H. pylori-associated diseases, such as gastric cancer is not uniformly distributed over the globe and does not parallel the prevalence of H. pylori infection. The highest incidence of gastric cancer is found in East Asia (Japan and Korea), and in Central America and South America (especially the Andean countries).⁴⁰ Within Europe, the highest incidence is found in Portugal, Austria, Italy, and Spain.⁴¹ In contrast, high prevalence of infection is reported from such areas with low gastric cancer risk, such as Africa,⁴² India, and the coastal regions of Latin America.⁴³

We and others have recently shown that the vacA and cagA genotypes of H. pylori are not equally distributed over the world.^19,20,44-46 There are significant differences between patient populations from Asia, different parts of Europe, and North and South America. Given the geographic distribution of specific H. pylori genotypes, it could be speculated that there might be an epidemiological relationship with the incidence of gastric cancer. The present study confirmed earlier findings in Portuguese patients showing that a high proportion are infected with multiple H. pylori strains.^44,47

With respect to age, gender, distribution of genotypes, as well as the prevalence of atrophy, there were significant differences between the Portuguese and Colombian patients, reflecting the different strategy used for selection of these cases. However, despite these differences, the highly similar relationships between bacterial genotypes and histopathological findings in both populations indicate that our observations are accurate and reliable.

Our study has several limitations. The biopsy specimens from each patient group were analyzed by two different pathologists, although they used the same scoring system.²¹ Also, only biopsies from the antrum of the stomach were used for genotyping analysis in each patient, and the influence of sampling errors cannot be excluded. Therefore, there may not be an absolute concordance between the genotype of the colonizing strain and underlying pathological observations in the corpus, because patients may carry different strains in the antrum and corpus. However, the antrum is the predominant site of H. pylori colonization⁴⁸ and the chance of false-negative results is lower than in corpus biopsies.⁴⁹

Furthermore, other possibly relevant factors, such as smoking, acid output, HLA-status, and other bacterial virulence factors were not studied. Despite the limitations of the present study, analysis of the vacA, cagA, and iceA virulence-associated genes directly in gastric biopsies permitted clinically relevant discrimination between H. pylori strains.

In conclusion, distinct H. pylori genotypes seem to be associated to the outcome of the infection and may have important clinical and epidemiological implications.

	Acknowledgements

 
We thank Els Stet and Nathalie Nouhan for preparation of the LiPA strips; and Profs. M. Sobrinho-Simões, L. David, M. Cardoso de Oliveira, and L. Tomé Ribeiro for their support.

	Footnotes

 
Address reprint requests to L. J. van Doorn, Delft Diagnostic Laboratory, R. de Graafweg 7, 2625 AD, Delft, the Netherlands. E-mail: l.j.van.doorn{at}ddl.nl

Supported by a grant from the Luso-American Foundation (FLAD, project number 492/97); the PRAXIS XXI program from the Portuguese Foundation for Science and Technology (to C. F. and C. N.); and the Colombian work was supported by grant P01-CA-28842 of the National Cancer Institute, National Institutes of Health, United States Public Health Service. Part of the biopsies of the Portuguese patients were obtained under a protocol established between ARS-Norte, Estaleiros Navais de Viana do Castelo, Hospital de S. João and IPATIMUP.

C. G. and C. F. contributed equally to this article.

Accepted for publication October 17, 2000.

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