Adherence of Helicobacter pylori to Areas of Incomplete Intestinal Metaplasia in the Gastric Mucosa

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1 GASTROENTEROLOGY 1996;111: Adherence of Helicobacter pylori to Areas of Incomplete Intestinal Metaplasia in the Gastric Mucosa ROBERT M. GENTA,*,, INANÇ E. GÜRER,*, DAVID Y. GRAHAM,,x BHUVANESWARI KRISHNAN,* ANA MARIA SEGURA,*, OSCAR GUTIERREZ, Ø JONG G. KIM, # and JAMES L. BURCHETTE, Jr.** Departments of *Pathology, Medicine, Microbiology and Immunology, and x Molecular Virology, Veterans Affairs Medical Center and Baylor College of Medicine, Houston, Texas; Ø Universidad Nacional de Colombia, Bogotá, Colombia; # Guro Hospital, Korea University College of Medicine, Seoul, Korea; and **Department of Pathology, Duke University Medical Center, Durham, North Carolina Background & Aims: Helicobacter pylori is not usually at the border between gastric and intestinalized epithefound in areas of intestinal metaplasia. Thus, the devel- lium, as if the mucus or some other product secreted by opment of intestinal metaplasia has been viewed as a the goblet cells rendered the area utterly inhospitable for mechanism by which the stomach eliminates H. pylori. the bacteria. The aim of this study was to evaluate the frequency of The histogenesis of intestinal metaplasia is unknown. H. pylori adherence to intestinal metaplasia in different Nevertheless, the observations that H. pylori seems to populations. Methods: Mapped gastric biopsy speciavoid metaplastic areas and that, in advanced atrophic mens from 378 H. pylori positive subjects from various geographical regions were examined. Intestinal metadespite metaplastic gastritis, bacteria are frequently undetectable plasia was typed by staining with periodic acid Schiff/ serological evidence of infection 6 have prompted alcian blue and high-iron diamine/alcian blue. Results: the teleological speculation that intestinal metaplasia In 32 patients, H. pylori was found in intimate contact may represent a mechanism through which the gastric with intestinal metaplasia. This was documented by mucosa eliminates H. pylori. In truth, epidemiological electron microscopy. All areas of intestinal metaplasia studies in populations with widely diverse prevalences showing adherence contained sulfomucins and had no of atrophic gastritis do not always suggest a causal relabrush border. Posttreatment biopsy specimens from 4 tionship between H. pylori and intestinal metaplasia. 3,7 9 patients whose infection was not cured showed persis- However, the metaphor of intestinal metaplasia as a mechtence of H. pylori in intestinal metaplasia. Conclusions: anism for self-cure is conceptually attractive and reconciles These patients may have a strain of H. pylori with unwell with a carcinogenic role for H. pylori. It is therefore usual adhesion characteristics, or their type of intestinot surprising that it has gained wide acceptance. nal metaplasia may have biochemical properties that make it hospitable for H. pylori. The exclusive associaallows visualization of H. pylori while simultaneously af- Recently we developed a staining technique that tion of H. pylori adherence with incomplete intestinal metaplasia (a putative precursor of carcinoma) and its fording an excellent view of the histological features of greater frequency in Koreans (a population at risk for the gastric mucosa, including intestinal metaplasia (Figgastric cancer) suggest that this phenomenon may play ure 1). 10 While conducting a study designed to compare a role in the hypothetical sequence metaplasia ú dys- the histopathologic features of H. pylori gastritis in popuplasia ú carcinoma. lations with different incidences of gastric carcinoma and peptic ulcer disease, we encountered a gastric biopsy specimen in which the gastric mucosa, entirely replaced nfection with Helicobacter pylori is the most important by intestinal-type epithelium, was lined by innumerable Icause of chronic active gastritis worldwide. One of H. pylori. The organisms were not only situated in the the possible evolutions of chronic active gastritis is the overlaying mucus, a common and insignificant finding development of atrophic gastritis, a condition almost unipossibly caused by the displacement of mucus and bacteversally associated with extensive intestinal metaplasia. 1 ria occurring during the endoscopic procedure;. rather, Many investigators, including ourselves, have believed they were located deep in the lumen of the metaplastic that H. pylori is not encountered in areas of intestinal foveae, intimately connected with the metaplastic epithemetaplasia, and photomicrographs have been published to support this contention. 2 5 Figure 1 is representative Abbreviation used in this paper: DAB, 3,3 -diaminobenzidinetetof this iconography: it shows a heavily infected gastric rahydrochloride. mucosa adjacent to a segment of intestinalized epithe by the American Gastroenterological Association lium with no bacteria. H. pylori appear to halt abruptly /96/$3.00

2 November 1996 ADHERENCE OF H. PYLORI TO INTESTINAL METAPLASIA 1207 Figure 1. Photomicrograph of two adjacent antral foveae from a pa- Figure 3. In this high-power photomicrograph, H. pylori appear to have tient with H. pylori induced gastritis. The epithelium of the fovea on entered the mucin compartment of metaplastic goblet cells (modifie the left consists entirely of intestinal metaplasia; the fovea on the Genta stain; original magnificatio 4001). right is lined by normal gastric epithelium. Innumerable H. pylori are present in the fovea lined by gastric epithelium, whereas no organisms are present in the metaplastic fovea. This type of figur has been used to show the abhorrence of H. pylori for intestinalized epithelium Korea, South Africa, Jordan, India, and Colombia. Gastric (Genta stain; original magnificatio 1001). biopsy specimens were examined from six groups of adult patients with endoscopically documented duodenal ulcer, a group of Colombian patients with gastric carcinoma, and a lium, and even appeared to have entered the mucin com- group of Colombian patients with other nonneoplastic gastric partment of goblet cells (Figures 2 and 3). Intrigued by disorders. H. pylori was documented in 55 North Americans, this observation, we reexamined all biopsy specimens 80 Koreans, 36 South African mixed-race subjects, 20 Jordaniavailable from this study to search for similar cases. This ans, 16 Northern Indians, 47 Colombians with gastric carci- noma, and 118 Colombians with other gastric disorders. article reports the morphological aspects of this finding, its prevalence, and its associations. Materials and Methods Study Populations This study was approved by the Institutional Human Research Review Committee at Baylor College of Medicine and by the appropriate human research boards at the sites in Biopsy Mapping Protocol Investigators at each site were instructed to obtain jumbo forceps biopsy specimens from at least 6 of the 13 sites described in our standard mapping protocol. 11 North American, Colombian, Jordanian, and Indian patients had a minimum of 8 biopsy specimens each, and Koreans and South Figure 2. Gastric mucosa entirely replaced by intestinal-type epithe- Figure 4. Immunocytochemical staining confirme that the infecting lium lined by innumerable H. pylori. The organisms are situated not organisms had the morphological and immunologic characteristics only in the overlying mucus but also deep in the lumen of the metaplas- of H. pylori. This photomicrograph shows immunoperoxidase/dabstained tic foveolae, intimately connected with the metaplastic epithelium H. pylori adherent to both gastric and intestinal type epithetic (modifie Genta stain; original magnificatio 1001). lium (original magnificatio 1001).

3 1208 GENTA ET AL. GASTROENTEROLOGY Vol. 111, No. 5 Africans had biopsy specimens taken from 6 sites (4 from the antrum and 2 from the corpus). Table 1. Percentages of Patients With Intestinal Metaplasia, With the Incomplete Type of Intestinal Metaplasia Containing Sulfated Mucins, and With Adherent H. pylori Histopathology Biopsy specimens were shipped to our laboratory fixed in 10% buffered formalin. They were processed, oriented on positive metaplasia intestinal metaplasia attached edge, embedded in paraffin, and cut in sequential 4-mm sections. Group subjects (%) (% of total) (%) Slides from each specimen (usually with 8 12 sections) North Americans (29) 9 (18) 3 (5.5) No. of Subjects with Subjects with H. pylori intestinal incomplete type of H. pylori Koreans (47.5) 28 (35) 24 (30) were stained using the Genta stain. 10 When any area of intesti- South Africans 36 5 (14) 3 (8) 0 nal metaplasia was identified on a slide, additional sections Jordanians 28 9 (32) 1 (3.5) 1 (3.5) Indians 16 5 (31) 2 (12.5) 0 were prepared and stained with alcian blue ph 2.5/periodic Colombians acid Schiff and high-iron diamine/alcian blue ph 2.5 to iden- Gastric cancer (95) 28 (62) 3 (6.5) Other (67) 34 (29) 1 (0.8) tify subtypes of intestinal metaplasia. Subtypes were classified Total (51) 105 (28) 32 (8.5) as described by Filipe et al. 12 and assessed independently by two observers. Briefly, type I is characterized by mature goblet cells secreting acid sialomucins and sometimes sulfomucins, nonsecreting absorptive cells, and a well-defined brush border. epoxy resin. The toluidine blue stained sections were exam- Paneth cells are often present at the crypt base. This type of ined, and ultrathin sections of areas with intestinal metaplasia metaplasia is also known as complete or small intestinal. and H. pylori were stained with uranyl acetate and lead citrate. Type II shows mild architectural distortion, few or absent These sections were examined and photographed using a JEOL absorptive cells, and columnar cells containing a mixture of 1200 electron microscope ( Jeol Ltd., Tokyo, Japan). neutral and acid sialomucins; goblet cells secrete sialomucins and occasionally sulfomucins. Paneth cells are rare or absent. Immunocytochemical Staining for H. pylori In type III, the metaplastic foveae are tortuous, the architecture is disorganized, and immature columnar cells are abundant. Sections were placed on positively charged glass slides Columnar cells secrete sulfomucin, and goblet cells contain and immunostained by established capillary action methodolsialomucins and sulfomucins. Paneth cells are absent. Types ogy. After paraffin removal and quenching of endogenous per- II and III are generally known as incomplete or colonic. oxidase activity, tissue sections were postfixed in 10% neutral- Because incomplete intestinal metaplasia is frequently found buffered formalin. Digestion with the proteolytic enzyme in association with gastric carcinoma, several studies have sugmined in previous experiments to be essential for the optimal pepsin (0.25%; ph 2.0) was performed because it was deter- gested, although not proven, that this type of metaplasia is a precursor of gastric cancer. More recently, Filipe et al. have demonstration of H. pylori in formalin-fixed tissue sections. described a type IV, similar in all features to type III but H. pylori were detected with rabbit antibacterium polyclonal containing sulfomucins in both columnar and goblet cells. In antibody (Dako Corp., Carpinteria, CA). Rabbit immunogloba study conducted in Slovenia, this type was associated with ulin (Dako Corp.) diluted at the same immunoglobulin con- the greatest risk of cancer. 13 centration was used as a negative control. The unlabeled bound For the purpose of this study, H. pylori were considered to primary antibody was linked with biotinylated goat anti-rabbit be attached to segments of intestinal metaplasia only when immunoglobulin G (Vector Laboratories, Burlingame, CA) and the following criteria were fulfilled. (1) Intestinal metaplasia detected with horseradish peroxidase labeled streptavidin was present at least in one entire fovea. Thus, isolated alcian (Jackson ImmunoResearch Inc., West Grove, PA). Visualizablue positive goblet cells frequently found on the surface of tion of the formed complex was accomplished with 3,3 -diamithe gastric mucosa were ignored. (2) H. pylori must be posihancement with cupric sulfate solution and a counterstain with nobenzidine tetrahydrochloride (DAB) chromogen. DAB en- tively identified by their characteristic shape and the two polar dark dots typically acquired with our stain. 11 Single bacteria modified Harris hematoxylin completed the staining protocol. or clusters of rods of uncertain nature were not considered. (3) Sections were dehydrated, cleared, and permanently mounted. H. pylori must be numerous, appear to be attached to the epithelium, and/or present within goblet cells. Single bacteria Major Blood Group Antigen Determination (even if they had the characteristic spiral shape) or bacteria Although the prevalence of H. pylori has been found scattered in the mucus but not clearly connected with the to be independent of the major blood groups, 14 it has been epithelial surface were ignored. suggested that the availability of H. pylori receptors may be reduced in individuals of blood group A and B phenotypes Electron-Microscopic Studies compared with individuals of blood group O. 15 To explore a Tissue for electron-microscopic examination was retrieved from paraffin blocks. The tissue was deparaffinized and blood groups, we tested for expected ABO antibodies in 11 possible relationship between unusual adherence patterns and fixed in 2.5% glutaraldehyde in cacodylate buffer. It was then Korean patients whose sera were still available at the time of postfixed in osmium tetroxide, dehydrated, and embedded in the study.

4 November 1996 ADHERENCE OF H. PYLORI TO INTESTINAL METAPLASIA 1209 Results Approximately 4200 separate biopsy specimens from 378 H. pylori infected patients were examined. The percentages of patients with intestinal metaplasia and with the incomplete type of intestinal metaplasia (containing sulfated mucins) in each group are summarized in Table 1. H. pylori attached to metaplastic epithelium and fulfilling the above criteria was found in 78 biopsy specimens from 32 patients: 24 patients were Koreans with peptic ulcer disease, 3 were North Americans, 3 were Colombians with gastric cancer, 1 was a Colombian with nonulcer dyspepsia, and 1 was a Jordanian with duodenal ulcer. No H. pylori adherent to intestinal metaplasia was detected in any South African or Indian suba Figure 5. Electron micrograph showing bacteria lining the surface of goblet cell. Bacteria are also present on the surface of the adjacent ject. cell and in the overlying mucus (original magnificatio 12,0001). Four Korean patients underwent three sequential endoscopic procedures with biopsy mapping after being unsuccessfully treated for H. pylori with various antibi- 6 patients were group O, 1 patient was group A, 2 otic proton pump inhibitor regimens. Areas of intestinal patients were group B, and 2 patients were group AB. metaplasia with adherent H. pylori were found at each Although this is a very small sample, it reflects the remapping in all of these patients. spective proportions of blood types found in the general H. pylori organisms were attached intimately to the Korean population. 16 metaplastic epithelium (Figure 2). These organisms were not simply lying in the mucus overlying the metaplastic Discussion portions of mucosa. Large aggregates of bacteria were One of the first questions that can be asked is observed in foveolar spaces completely replaced by inteswhether the observation of H. pylori attached to incomtinal-type epithelium apparently attached to the interme- plete intestinal metaplasia is indeed an important novel diate absorptive cells, and both attached to and within finding or rather a trivial observation that had been made the mucous vacuole of goblet cells (Figure 3). In all cases, before but was not deemed worth reporting by other bacteria were observed only adherent to either type II, III, investigators. In a 1988 letter to GASTROENTEROLOGY, or, in one case, type IV intestinal metaplasia (incomplete Steadman et al. described the presence of H. pylori in areas types). Bacteria were never observed attached to the brush of intestinal metaplasia in the antral biopsy specimens of border typically observed in type I (complete) intestinal 3 Australian patients. 17 Their brief report, accompanied metaplasia. In four specimens, the scanty amount of tis- by convincing photomicrographs, has remained virtually sue available in the paraffin block prevented the perfor- unquoted to date (personal communication, Institute For mance of histochemical studies to determine the type of Scientific Information, Philadelphia, PA, December intestinal metaplasia; however, the architectural distribu- 1995). Although the authors offered insightful speculation of the goblet cells and the absence of a brush border tions about the possible mechanisms and suggested furwas strongly suggestive of an incomplete type. ther studies to clarify the relationship between H. pylori Immunocytochemical staining was performed on one and intestinal metaplasia, no one seems to have taken up biopsy specimen from each of 16 subjects. In each case, the challenge. If we assume that this phenomenon has it was confirmed that the infecting organisms had the not been observed during the 7 years elapsed between morphological and immunologic characteristics of H. py- the report of Steadman et al. and our detection of these lori (Figure 4). cases, then it becomes important to determine why. Every Electron-microscopic examination of these areas day hundreds of competent pathologists around the showed multiple bacteria on the surface of goblet cells. world examine gastric biopsy specimens, and many of Some of the bacteria were attached to the epithelium these pathologists specifically seek to better understand in the region of intercellular junction, where there was the relationship between H. pylori and the gastric mucosa. associated loss of microvilli (Figure 5). Cultures of gastric mucosa performed in these patients showed typical H. pylori colonies. The results of blood typing showed that We suspect that two reasons may have cooperated to result in the failure of making this observation earlier. One may be the type of stain used. With H&E, H. pylori

5 1210 GENTA ET AL. GASTROENTEROLOGY Vol. 111, No. 5 do not stain well, and goblet cells are observed as empty adenocarcinoma had extensive areas of intestinal metaplasia, circles that do not immediately stand out. Thus, the and 62% of them had type III metaplasia, yet adhercircles intimate association between bacteria and goblet cells ent H. pylori were detected only in one biopsy specimen may easily be overlooked. When special stains (such as each from 3 of 47 patients. Similarly, we did not detect Giemsa or Warthin Starry) or immunocytochemical any intestinal metaplasia adherent H. pylori in a large techniques are used to visualize H. pylori, the details of series of Korean patients with gastric adenocarcinoma the gastric mucosal morphology become blurred by the and extensive areas of incomplete intestinal metaplasia. counterstain. Our use of a combined stain that permits This suggests that if the direct presence of H. pylori the simultaneous visualization of the bacteria and the plays a role in the progression from incomplete intestinal morphological background of the gastric mucosa and that metaplasia to cancer, this role may be limited to the specifically highlights the goblet cells with the bright initial steps of carcinogenesis. Once invasive carcinoma blue provided by alcian blue at ph 2.5 greatly simplifies has developed, the surrounding metaplastic milieu may the task of studying the relationships between H. pylori become as inhospitable for H. pylori as the neoplastic and its mucosal environment. 11 tissue itself. The other reason may be the relative rarity of this Four patients who underwent sequential endoscopic occurrence. Although we found H. pylori adherent to procedures several months apart had H. pylori adherent intestinal metaplasia in Ç8.5% of the 378 patients studgests to areas of intestinal metaplasia at each visit. This sugied, one must remember that only a few (2 or 3 on that, rather than a fortuitous event, this phenome- average) biopsy specimens from each subject (78 of non may represent an essential, if unusual, component Ç4200 specimens) showed this association, giving only of the relationship between a given host and a certain a õ2% chance of encountering this association in any strain of H. pylori. One possible explanation is that the given specimen. It must also be noted that the 80 Korean intestinal metaplastic cells of these patients, although patients included in this study (21% of the study suband histochemically and morphologically similar to types II jects) contributed 24 cases (or 75% of all cases of docuties III, may have some biochemical or structural proper- mented adherence). Since observing our first case in a that make them hospitable for H. pylori. Korean patient, we have examined several thousand non- Steadman et al. 17 suggested that H. pylori may not be mapped gastric biopsy specimens (on average 3 4 biopsy actually adherent to the metaplastic areas; rather, bacteria specimens per patient) from North American subjects could be carried to these areas by their own movements infected with H. pylori. All sections were stained with or by the flow of mucus. We agree that this may indeed our triple stain, and we have been particularly alert for be the case when H. pylori are observed in the mucus similar cases. However, we were able to find only two overlying the metaplastic epithelium; in our cases, howadditional cases with small numbers of H. pylori adhering ever, this seems an unlikely mechanism because our criteto metaplastic epithelium. Thus, the rarity of this finding ria for inclusion required that organisms be detected not combined with the use of suboptimal staining techniques only in the mucus but also in sheets and clumps attached for its detection may have been responsible for its belated to the epithelial surface as well as within the goblet cell rediscovery. mucous droplets. The most interesting and perhaps the most significant Having determined that the phenomenon exists and aspect of this observation may be the propensity of H. is probably rare in most populations, the next question pylori to attach only to areas of intestinal metaplasia of relates to its significance. Although our data are still the incomplete type. This type of metaplasia is common inadequate to serve as the foundation of a solid hypothein populations in which gastric carcinoma is highly prev- sis, its exclusive association with incomplete intestinal alent (in our study, most patients were Koreans and Co- metaplasia (a putative precursor of gastric carcinoma) and lombians) but rare in those parts of the world (e.g., most the fact that it seems much more likely to occur in some of Europe and North America) where the majority of populations at risk for gastric cancer suggest that, at studies on H. pylori are performed. H. pylori was found least in some geographic settings, adherence of H. pylori to be adherent exclusively to intestinal-type epithelium may play a role in the progression from metaplasia to without a brush border and with various combinations dysplasia and carcinoma. of sulfomucin-producing cells. The brush border itself may therefore represent the cellular structure that pre- References vents the adherence of H. pylori. However, the absence 1. Correa P. Chronic gastritis: a clinico-pathological classification of a brush border is not sufficient to guarantee that H. Am J Gastroenterol 1988; 83: pylori will adhere. Virtually all Colombian patients with 2. Craanen ME, Blok P, Dekker W, Ferwerda J, Tytgat GN. Subtypes

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