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Clinicopathological characteristics of invasive gastric Helicobacter pylori
Clinicopathologic characteristics of invasive gastric Helicobacter pylori Jonathan Dudley MD, Tad Wieczorek MD, Martin Selig BA, Hoiwan Cheung BA, Jeanne Shen MD, Robert Odze MD, Vikram Deshpande MBBS, Lawrence Zukerberg MD PII: DOI: Reference:
S0046-8177(16)30254-4 doi: 10.1016/j.humpath.2016.09.029 YHUPA 4031
To appear in:
Human Pathology
Received date: Revised date: Accepted date:
24 July 2016 14 September 2016 28 September 2016
Please cite this article as: Dudley Jonathan, Wieczorek Tad, Selig Martin, Cheung Hoiwan, Shen Jeanne, Odze Robert, Deshpande Vikram, Zukerberg Lawrence, Clinicopathologic characteristics of invasive gastric Helicobacter pylori, Human Pathology (2016), doi: 10.1016/j.humpath.2016.09.029
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Title: Clinicopathologic characteristics of invasive gastric Helicobacter pylori
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Brief title: Invasive H. pylori
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Jonathan Dudley, M.D.,* Tad Wieczorek, M.D.,† Martin Selig, B.A.,‡ Hoiwan Cheung, B.A.,* Jeanne Shen, M.D. † Robert Odze, M.D.,† Vikram Deshpande, M.B.B.S., ‡ Lawrence Zukerberg, M.D. ‡
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From the *Department of Pathology, Stanford University School of Medicine, Stanford, CA,
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USA; †Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA; ‡Department of Pathology, Massachusetts General Hospital and Harvard
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Medical School, Boston, MA, USA.
Address correspondence to: Jonathan Dudley, M.D., Stanford University School of
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Medicine, Department of Pathology, 300 Pasteur Drive, Lane 235, Stanford, CA 94305.
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Email: [email protected]. Phone: 650-723-5252. Fax: 650-725-6902. Conflicts of Interest and Source of Funding: None declared. Key words: Helicobacter pylori; gastritis; gastric ulcers
ACCEPTED MANUSCRIPT Objectives: Helicobacter pylori organisms have been observed deep within the stomach mucosa with an "intracellular" appearance, though the clinicopathologic characteristics of such cases remain poorly understood.
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Methods: We analyzed eighteen cases of deep mucosal H. pylori and associated clinical (sex, age, history of H. pylori infection, or proton pump inhibitor (PPI) use, medications, smoking, alcohol use, co-morbidities, treatment response treatment) and pathological (presence of lymphoid aggregates, intestinal metaplasia, PPI effect, active and/or chronic inflammation, quantity of invasive versus surface H. pylori) characteristics. Electron microscopy was performed on six cases with the highest burden of invasive H. pylori.
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Results: Within our sample, 3/16 had a history of prior H. pylori infection, 10/15 were receiving PPIs at the time of biopsy, and 12/13 had a negative post-treatment follow-up. Histology revealed invasive H. pylori were more commonly associated with chronic inflammation, in both the antrum (15/15 chronic; 8/15 acute) and fundus (17/18 chronic; 8/18 acute). Electron microscopy showed organisms within intercellular and luminal spaces, but no intracellular organisms.
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Conclusions: Deep mucosal H. pylori often have an intracellular appearance but are contained within intercellular and luminal spaces and are responsive to standard therapy.
ACCEPTED MANUSCRIPT Introduction More than half the world’s population is estimated to have gastric colonization by
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Helicobacter pylori [1]. The gram-negative bacterium is usually identified on the mucosal surface, where it stimulates a chronic and active inflammatory response. It is a major risk
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factor for peptic ulcer disease and increases the risk of developing gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma by 3- to 6-fold [2-3]. Despite
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these strong associations, only 15 to 20% of colonized individuals will ever develop gastric
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disease [4]. Additionally, while the organism is usually eradicated by a combination of antibiotics and inhibitors of acid secretion, a subset of patients fails to respond or
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experience recurrence [5-6]. In light of the worldwide burden of gastric H. pylori, these
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facts raise interest in identifying predictors of treatment-resistance and future pathology. Several mechanisms have been proposed to explain which patients are likely to
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suffer recurrence or future disease. H. pylori is a genetically diverse species, and strains
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carrying the 31-gene cag locus, the babA2 gene, and certain alleles of the vacA gene have all been associated with increased risk of gastric cancer [7]. Human polymorphisms also increase risk of disease; certain variants of the allele coding interleukin-1 beta, for example, result in increased expression of the protein and associated inflammation [8]. Additional host polymorphisms and dietary habits no doubt contribute in determining the baseline predisposition to gastric disease. The location of H. pylori in the stomach has also been postulated to determine its pathogenicity. Although the organism is usually confined to the mucosal surface, in a subset of patients, it can be observed deep within gastric pits, in intercellular spaces and the
ACCEPTED MANUSCRIPT lamina propria, and even inside vacuoles within epithelial and inflammatory cells [4, 9-12]. Some investigators have argued that such organisms, herein designated “invasive H. pylori,”
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are more resistant to treatment and significantly increase the likelihood of future disease
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[13]. Intracellular organisms, in particular, have been postulated to serve as a reservoir that can re-colonize the stomach after antibiotic treatment eliminates extracellular bacteria
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[9, 13]. As a result, some investigators have argued that antibiotics with intracellular
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activity should be used to ensure all organisms are eradicated [14-15].
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Despite the potential significance of invasive H. pylori, their clinicopathologic associations remain poorly understood. This paper surveys eighteen cases of invasive H.
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pylori identified during routine histological examination of gastric biopsies and resections within the Partners HealthCare system. We characterize the clinical characteristics of
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affected patients and the histological features of each specimen using the updated Sydney
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System for the classification and grading of gastritis [16]. Six cases with the highest quantity of invasive organisms were additionally subjected to electron microscopic
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evaluation. The goal of this paper is to evaluate the spectrum of clinical and pathologic associations of invasive H. pylori in order to clarify the significance of discovering these intracellular or invasive organisms in gastric specimens.
Materials and Methods Study Cohort
ACCEPTED MANUSCRIPT The study cohort consisted of 18 patients who had either endoscopic gastric biopsies (n=16) or resections (n=2) within the Partners HealthCare system between
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September 2010 and February 2014. Invasive organisms were generally discovered after
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ordering an H. pylori immunostain to evaluate for the presence of organisms. Patient records were reviewed to document age, sex, date of biopsy, history of H. pylori infection or
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proton-pump inhibitor use prior to biopsy, other past medical and medication history,
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history of tobacco or alcohol use, and H. pylori status after treatment. The average followup time was 38 months. The majority of the cases (n=12) were identified by two of the
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immunostain on gastric biopsies.
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authors (VD and TW) at an institution that routinely performs a Helicobacter pylori
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Histological Analysis
All hematoxylin and eosin (H&E) stained slides had an immunostain ordered for H.
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pylori. Both the H&E and immunostain were evaluated and graded by two pathologists (JD
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and LZ), one of whom has subspecialty training in gastrointestinal pathology (LZ). The updated Sydney System for the classification and grading of gastritis was applied separately to the antrum and fundus to describe as either “normal,” “mild,” “moderate,” or “marked” the following characteristics: quantity of H. pylori, atrophy, neutrophilic infiltrate, mononuclear cell infiltrate, and intestinal metaplasia [16]. Additional parameters assessed included the presence or absence of PPI effect, lymphoid aggregates, lymphoepithelial lesions, and the relative proportion of surface versus invasive organisms. One case of invasive Helicobacter heilmannii was also evaluated by the same criteria as well as by a Steiner stain.
ACCEPTED MANUSCRIPT Electron Microscopic Analysis The six cases with highest quantity of invasive organisms, as assessed by the H.
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pylori immunostain (rabbit monoclonal primary antibody, clone SP48, Ventana, Tuscon, AZ, USA), were microdissected from formalin-fixed, paraffin embedded tissue blocks, soaked in
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100% xylene overnight, rehydrated in a series of ethanol solutions, rinsed in sodium cacodylate buffer, and fixed for 1.5 hours with 2.5% glutaraldehyde, 2.0%
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paraformaldehyde, and 0.025% calcium chloride, in a 0.1 molar sodium cacodylate buffer
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with a pH of 7.4. Tissues were further processed in a Leica Lynx automatic tissue processor. Briefly, tissues were post fixed with osmium tetroxide, dehydrated in a series of ethanol
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solutions, en bloc stained during the 70% ethanol dehydration step for one hour, infiltrated with propylene oxide epoxy mixtures, embedded in pure epoxy, and polymerized over
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night at 60.1 degrees Celsius. Thin sections were stained with lead citrate and examined
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with an FEI Morgagni transmission electron microscope (FEI, Hillsboro, OR, USA). Images were captured with an AMT (Advanced Microscopy Techniques) digital CCD camera
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(Woburn, MA, USA). All cases were evaluated for the presence and location of H. pylori and exhaustively searched for potential intracellular organisms. The case of H. heilmannii was also evaluated using the above protocol. The study protocols were approved by the Institutional Review Board of Partners HealthCare.
Results
ACCEPTED MANUSCRIPT The average age of patients in the cohort was 58, with a range of 19-91 and an equal distribution of genders (Table 1). The average follow-up time was 38 months. Of the
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patients with H.pylori for whom the information was available, 3/16 had a history of prior
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H. pylori infection, 10/15 were receiving PPIs at the time of biopsy, and 11/12 had a negative post-treatment follow-up. A minority of patients had a history of using tobacco
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(5/15) and/or alcohol (4/16). The most common co-morbidities were gastroesophageal
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reflux (14/18) and hypertension (9/15). The majorities of cases in our series were discovered in the fall (8/18) and winter (7/18), versus the spring (1/18) and summer
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(2/18), raising the possibility of a seasonal association.
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Histologic findings are summarized in Table 2. Invasive H. pylori were found in both the body/fundus (18/18 cases) and antrum (12/18 cases) (Table 2). They were less often
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associated with acute inflammation, both in the antrum (15/15 chronic; 8/15 acute) and
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fundus (17/18 chronic; 8/18 acute). Lymphoid aggregates were more common in the antrum (8/15) than the fundus (4/18). A small minority of cases demonstrated atrophy
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(2/15 cases in antrum, 2/18 in fundus) and intestinal metaplasia (2/15). All cases had invasive organisms with a coccoid appearance, in contrast to the rod-shaped appearance of surface organisms. Immunohistochemistry for H. pylori revealed organisms ringing the base of glandular cells and within gastric pit lumina. Organisms also appeared immediately adjacent to, and in some cases, on top of cell nuclei, giving the appearance of an intracellular localization (Figure 1) and initial interpretation prior to electron microscopy was that these were intracellular H. pylori.
ACCEPTED MANUSCRIPT This possibility was evaluated with electron microscopy. Six cases with the highest quantity of invasive organisms revealed by the H. pylori immunostain were assessed,
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revealing organisms within surface mucin, deep within the lumina of gastric pits, and
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infiltrating between cells (Figure 2). Despite an exhaustive search, no intracellular
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organisms were identified in these six cases.
These results are usefully contrasted with a case of invasive gastric Helicobacter
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helmannii infection, which was separately identified and analyzed during this study. H.
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helmannii is rare in humans, with a prevalence estimated to be between 0.2 and 6%. It frequently colonizes the gastric mucosal surface of domestic animals, such as dogs and cats,
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which constitute a source for human infections [17-18]. It cross-reacts with the antibodies used for immunohistochemical identification of H. pylori, giving a similar appearance, and
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may also give false-positive results for H. pylori on serological and urease-breath tests. It is
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distinguished, however, by its long, cork-screw profile, noticeable on H&E but is most
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apparent on a Steiner stain (Figure 3, B). Notably, the individual case of H. helmannii identified in this series had a nearly identical appearance and localization pattern with the H. pylori immunostain as the cases of invasive H. pylori noted previously, with a distribution around the base of glandular cells and within the lumina of gastric pits (Figure 3, A). The H&E characteristics and grading pattern per the updated Sydney System for the classification and grading of gastritis were likewise similar (Supplementary Table 1 & 2). Unlike the invasive H. pylori examined in our series, however, a Steiner stain revealed the long, corkscrew profile typical of H. heilmannii (Figure 3, B) while the electron microscopy showed not only an intercellular (Figure 3, C),
ACCEPTED MANUSCRIPT but also a definitive intracellular localization for many organisms (Figure 3, D&E). Also unlike most of the H. pylori in our series, the patient was refractory to treatment, with a
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five-year history stomach pain and a failed response to triple therapy and subsequent
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proton-pump inhibitor treatment.
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Discussion
This study sought to evaluate routine cases of intracellular H. pylori and clarify the
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significance of discovering these H. pylori in routine gastric specimens. Electron microscopic evaluation of six cases revealed organisms infiltrating between cells and deep
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within gastric pits but no intracellular forms, indicating the organisms are invasive but not
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intracellular. In this series of cases, invasive H. pylori were less often associated with acute
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inflammation and were equally distributed between the antrum and fundus. Most of the invasive H. pylori in this series were not associated with a history of prior infection or
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treatment-resistance. Although this series should be evaluated in the context of other studies, by itself, it suggests that most patients with invasive H. pylori can be successfully treated with the same protocols used for H. pylori confined to the mucosal surface. The coincidence of an intracellular localization and treatment-failure in the case of invasive H. heilmannii is suggestive but must be interpreted with caution. Since H. heilmannii colonization is less common, the appropriate treatment regimen is not as well established as it is for H. pylori. It is possible that treatment failure occurred in this patient simply because the standard treatment regimen for H. pylori is less effective for H.
ACCEPTED MANUSCRIPT heilmannii, though reports have documented successful treatment of H. heilmannii species with triple therapy [19-20].
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Other studies have explored the significance of invasive H. pylori. The organism has been documented with increased prevalence in specimens from patients with gastric
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ulcers, gastric intestinal metaplasia, and gastric cancer [21-22]. Although these studies are limited by the specificity of H. pylori antibodies, that the organism can be invasive and
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intracellular is supported by several lines of evidence, including transmission electron
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microscopy with immunogold staining, in situ hybridization, and multilabel immunohistochemical methods [4, 11, 13, 23]. In this study, invasive organisms were
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always accompanied by at least some surface organisms with a similar appearance on immunohistochemistry. It is unclear whether PPI use promotes an invasive phenotype. One
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study surveying the relationship between PPI usage and H. pylori histology found a shift in
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location [24].
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organisms from the antrum to the fundus but failed to note a shift toward a deeper mucosal
The relevance of intracellular H. pylori for treatment failure is less established. Evidence for an association is primarily based on in vitro cell culture studies, analogy with other bacteria, and, in one study, persistence of intracellular forms in a proportion of a series of ten patients after treatment [4, 13]. The latter study demonstrated persistence of intracellular forms with immunohistochemistry. Our results demonstrate the difficulty of determining an organism’s intracellular status by light microscopy alone. Several studies have identified intracellular H. pylori in gastric biopsies using electron microscopy [12, 2526], but there is no direct evidence that H. pylori can replicate within cells, and one study,
ACCEPTED MANUSCRIPT using differential interference contrast video, demonstrated that intracellular H. pylori has a 24-hour half-life [27]. This raises doubts that intracellular forms of the organism could
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survive the typical 7- to 14-day regimen of triple therapy. Conclusions
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Our study evaluates the finding of "intracellular" H. pylori in routine gastric biopsies
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and resection specimens detected with H. pylori immunohistochemistry. Electron microscopy demonstrates that these are not intracellular in location but invasive into the
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deep mucosa and present in deep intercellular spaces and lumens. The incidence of these invasive organism is uncertain as our cases were collected prospectively. Such cases are
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more often associated with chronic inflammation and tend to involve both the antrum and
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fundus. Many of the patients were already taking proton pump inhibitor therapy at the time of diagnosis, which has been shown to drive H. pylori deeper into the mucosa. Most of
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the patients in our study responded to standard therapy, suggesting that patients with
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invasive H. pylori discovered in gastric biopsies can be treated with the same regimen used for patients with surface H. pylori. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Acknowledgements None. Authors’ contributions
ACCEPTED MANUSCRIPT JD, VD, LZ, and RO conceived of study design, gathered and analyzed data, and wrote the manuscript. Case material was provided by RO, TW, and JS. MS performed electron
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microscopy on relevant cases. HC analyzed the data, wrote, and edited the manuscript.
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References
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[1] Lacy BE, Rosemore J. Helicobacter pylori: ulcers and more: the beginning of an era. The Journal of nutrition 2001; 131, 2789S-2793S. [2] Forman D. Helicobacter pylori infection and cancer. British medical bulletin 1998; 54, 71-78. [3] Huang JQ, Sridhar S, Chen Y, Hunt RH. Meta-analysis of the relationship between Helicobacter pylori seropositivity and gastric cancer. Gastroenterology 1998; 114, 11691179. [4] Dubois A, Boren T. Helicobacter pylori is invasive and it may be a facultative intracellular organism. Cellular microbiology 2007; 9, 1108-1116. [5] Graham DY. Therapy of Helicobacter pylori: current status and issues. Gastroenterology 2000; 118, S2-8. [6] Knigge KL. The role of H pylori in gastrointestinal disease. A guide to identification and eradication. Postgraduate medicine 2001; 110, 71-72, 77-78, 81-72. [7] Peek RM, Jr., Blaser MJ. Helicobacter pylori and gastrointestinal tract adenocarcinomas. Nature reviews Cancer 2002; 2, 28-37. [8] El-Omar EM, Carrington M, Chow WH, McColl KE, Bream JH, Young HA, Herrera J, Lissowska J, Yuan CC, Rothman N, Lanyon G, Martin M, Fraumeni JF, Jr., Rabkin CS. Interleukin-1 polymorphisms associated with increased risk of gastric cancer. Nature 2000; 404, 398-402. [9] Dubois A. Intracellular Helicobacter pylori and gastric carcinogenesis: an "old" frontier worth revisiting. Gastroenterology 2007; 132, 1177-1180. [10] Jhala NC, Siegal GP, Klemm K, Atkinson BF, Jhala DN. Infiltration of Helicobacter pylori in the gastric mucosa. American journal of clinical pathology 2003; 119, 101-107. [11] Necchi V, Candusso ME, Tava F, Luinetti O, Ventura U, Fiocca R, Ricci V, Solcia E. Intracellular, intercellular, and stromal invasion of gastric mucosa, preneoplastic lesions, and cancer by Helicobacter pylori. Gastroenterology 2007; 132, 1009-1023. [12] Noach LA, Rolf TM, Tytgat GN. Electron microscopic study of association between Helicobacter pylori and gastric and duodenal mucosa. Journal of clinical pathology 1994; 47, 699-704. [13] Engstrand L, Graham D, Scheynius A, Genta RM, El-Zaatari F. Is the sanctuary where Helicobacter pylori avoids antibacterial treatment intracellular? American journal of clinical pathology 1997; 108, 504-509. [14] Fahey JW, Haristoy X, Dolan PM, Kensler TW, Scholtus I, Stephenson KK, Talalay P, Lozniewski A. Sulforaphane inhibits extracellular, intracellular, and antibiotic-resistant strains of Helicobacter pylori and prevents benzo[a]pyrene-induced stomach tumors. Proceedings of the National Academy of Sciences of the United States of America 2002; 99, 7610-7615. [15] Hulten K, Cars O, Hjelm E, Engstrand L. In-vitro activity of azithromycin in against intracellular Helicobacter pylori. The Journal of antimicrobial chemotherapy 1996; 37, 483489.
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[16] 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. The American journal of surgical pathology 1996; 20, 1161-1181. [17] Iwanczak B, Biernat M, Iwanczak F, Grabinska J, Matusiewicz K, Gosciniak G. The clinical aspects of Helicobacter heilmannii infection in children with dyspeptic symptoms. Journal of physiology and pharmacology : an official journal of the Polish Physiological Society 2012; 63, 133-136. [18] Singhal AV, Sepulveda AR. Helicobacter heilmannii gastritis: a case study with review of literature. The American journal of surgical pathology 2005; 29, 1537-1539. [19] Morgner A, Lehn N, Andersen LP, Thiede C, Bennedsen M, Trebesius K, Neubauer B, Neubauer A, Stolte M, Bayerdorffer E. Helicobacter heilmannii-associated primary gastric low-grade MALT lymphoma: complete remission after curing the infection. Gastroenterology 2000; 118, 821-828. [20] Schultz-Suchting F, Stallmach T, Braegger CP. Treatment of Helicobacter heilmanniiassociated gastritis in a 14-year-old boy. Journal of pediatric gastroenterology and nutrition 1999; 28, 341-342. [21] Semino-Mora C, Doi SQ, Marty A, Simko V, Carlstedt I, Dubois A. Intracellular and interstitial expression of Helicobacter pylori virulence genes in gastric precancerous intestinal metaplasia and adenocarcinoma. The Journal of infectious diseases 2003; 187, 1165-1177. [22] Chan WY, Hui PK, Leung KM, Thomas TM. Modes of Helicobacter colonization and gastric epithelial damage. Histopathology 1992; 21, 521-528. [23] Petersen AM, Krogfelt KA. Helicobacter pylori: an invading microorganism? A review. FEMS immunology and medical microbiology 2003; 36, 117-126. [24] Dickey W, Kenny BD, McConnell JB. Effect of proton pump inhibitors on the detection of Helicobacter pylori in gastric biopsies. Aliment Pharmacol Ther 1996; 10, 289293. [25] Ko GH, Kang SM, Kim YK, Lee JH, Park CK, Youn HS, Baik SC, Cho MJ, Lee WK, Rhee KH. Invasiveness of Helicobacter pylori into human gastric mucosa. Helicobacter 1999; 4, 77-81. [26] Papadogiannakis N, Willen R, Carlen B, Sjostedt S, Wadstrom T, Gad A. Modes of adherence of Helicobacter pylori to gastric surface epithelium in gastroduodenal disease: a possible sequence of events leading to internalisation. APMIS 2000; 108, 439-447. [27] Amieva MR, Salama NR, Tompkins LS, Falkow S. Helicobacter pylori enter and survive within multivesicular vacuoles of epithelial cells. Cellular microbiology 2002; 4, 677-690.
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9 9
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58 (19-91)
3 13 2
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Patient characteristics Age, years Mean (range) Gender Male Female History of H. pylori Yes No Unknown PPI treatment pre-biopsy Yes No Unknown Smoking history Yes No Unknown Alcohol use Yes No/Rare Unknown H. pylori test post-treatment? Negative Positive Unknown/not treated
10 5 3 5 10 3 4 12 2 11 1 6
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Table 1. Summary of clinical characteristics of 18 patients with invasive gastric H. pylori.
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7 0 3 4 13 8
7 4 9 7 2
1 11 3 2
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5 5 10 6 2
3 10 8 7
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Moderate Marked Present
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3 3 3 3 3 3
2 16 14 13 10
Mild
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None
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Gastric Antrum Acute inflammation Chronic inflammation Deep mucosal H. pylori Surface H. pylori Atrophy Lymphoid aggregates Gastric Fundus Acute inflammation Chronic inflammation Deep mucosal H. pylori Surface H. pylori Atrophy Lymphoid aggregates Intestinal Metaplasia PPI Effect
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2 3 4 2 8
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Table 2. Summary of histological findings in 18 patients with invasive gastric H. pylori. NA=Not available, PPI=Proton pump inhibitor.
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Figure 1. Histological appearance of invasive H. pylori. Patient 1 had invasive H. pylori in his antrum (A) and fundus (B), associated with chronic inflammation (C). Patient 2 had invasive and surface H. pylori (D and E), associated with mild active and chronic inflammation (F). (H. pylori immunostainon A, B (40x), D, E (60x); hematoxylin and eosin stain on C (20x) and F (20x).
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Figure 2. Electron microscopic appearance of invasive gastric H. pylori. The organisms were found infiltrating between cells (A-C) and deep in gastric pits (D), as indicated by arrows. No H. pylori organisms were identified in an intracellular location.
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Figure 3: Histological and electron microscopic appearance of H. heilmannii. H. pylori immunostainreveals a globular stain identical in appearance to that seen with intracellular H. pylori (A). The appearance of organisms on H&E prompted a Steiner stain, revealing the long, corkscrew profile typical of H. heilmannii(B). These are highlighted between cells (C) and inside of them (D&E) on electron microscopy. The arrows indicate the presence of H. heilmanniiorganisms. (H. pylori immunostainon A (20x), Steiner stain on B (40x), and electron microscopy on C, D, and E)
S0046-8177(16)30254-4 doi: 10.1016/j.humpath.2016.09.029 YHUPA 4031
To appear in:
Human Pathology
Received date: Revised date: Accepted date:
24 July 2016 14 September 2016 28 September 2016
Please cite this article as: Dudley Jonathan, Wieczorek Tad, Selig Martin, Cheung Hoiwan, Shen Jeanne, Odze Robert, Deshpande Vikram, Zukerberg Lawrence, Clinicopathologic characteristics of invasive gastric Helicobacter pylori, Human Pathology (2016), doi: 10.1016/j.humpath.2016.09.029
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Title: Clinicopathologic characteristics of invasive gastric Helicobacter pylori
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Brief title: Invasive H. pylori
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Jonathan Dudley, M.D.,* Tad Wieczorek, M.D.,† Martin Selig, B.A.,‡ Hoiwan Cheung, B.A.,* Jeanne Shen, M.D. † Robert Odze, M.D.,† Vikram Deshpande, M.B.B.S., ‡ Lawrence Zukerberg, M.D. ‡
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From the *Department of Pathology, Stanford University School of Medicine, Stanford, CA,
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USA; †Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA; ‡Department of Pathology, Massachusetts General Hospital and Harvard
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Medical School, Boston, MA, USA.
Address correspondence to: Jonathan Dudley, M.D., Stanford University School of
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Medicine, Department of Pathology, 300 Pasteur Drive, Lane 235, Stanford, CA 94305.
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Email: [email protected]. Phone: 650-723-5252. Fax: 650-725-6902. Conflicts of Interest and Source of Funding: None declared. Key words: Helicobacter pylori; gastritis; gastric ulcers
ACCEPTED MANUSCRIPT Objectives: Helicobacter pylori organisms have been observed deep within the stomach mucosa with an "intracellular" appearance, though the clinicopathologic characteristics of such cases remain poorly understood.
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Methods: We analyzed eighteen cases of deep mucosal H. pylori and associated clinical (sex, age, history of H. pylori infection, or proton pump inhibitor (PPI) use, medications, smoking, alcohol use, co-morbidities, treatment response treatment) and pathological (presence of lymphoid aggregates, intestinal metaplasia, PPI effect, active and/or chronic inflammation, quantity of invasive versus surface H. pylori) characteristics. Electron microscopy was performed on six cases with the highest burden of invasive H. pylori.
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Results: Within our sample, 3/16 had a history of prior H. pylori infection, 10/15 were receiving PPIs at the time of biopsy, and 12/13 had a negative post-treatment follow-up. Histology revealed invasive H. pylori were more commonly associated with chronic inflammation, in both the antrum (15/15 chronic; 8/15 acute) and fundus (17/18 chronic; 8/18 acute). Electron microscopy showed organisms within intercellular and luminal spaces, but no intracellular organisms.
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Conclusions: Deep mucosal H. pylori often have an intracellular appearance but are contained within intercellular and luminal spaces and are responsive to standard therapy.
ACCEPTED MANUSCRIPT Introduction More than half the world’s population is estimated to have gastric colonization by
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Helicobacter pylori [1]. The gram-negative bacterium is usually identified on the mucosal surface, where it stimulates a chronic and active inflammatory response. It is a major risk
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factor for peptic ulcer disease and increases the risk of developing gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma by 3- to 6-fold [2-3]. Despite
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these strong associations, only 15 to 20% of colonized individuals will ever develop gastric
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disease [4]. Additionally, while the organism is usually eradicated by a combination of antibiotics and inhibitors of acid secretion, a subset of patients fails to respond or
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experience recurrence [5-6]. In light of the worldwide burden of gastric H. pylori, these
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facts raise interest in identifying predictors of treatment-resistance and future pathology. Several mechanisms have been proposed to explain which patients are likely to
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suffer recurrence or future disease. H. pylori is a genetically diverse species, and strains
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carrying the 31-gene cag locus, the babA2 gene, and certain alleles of the vacA gene have all been associated with increased risk of gastric cancer [7]. Human polymorphisms also increase risk of disease; certain variants of the allele coding interleukin-1 beta, for example, result in increased expression of the protein and associated inflammation [8]. Additional host polymorphisms and dietary habits no doubt contribute in determining the baseline predisposition to gastric disease. The location of H. pylori in the stomach has also been postulated to determine its pathogenicity. Although the organism is usually confined to the mucosal surface, in a subset of patients, it can be observed deep within gastric pits, in intercellular spaces and the
ACCEPTED MANUSCRIPT lamina propria, and even inside vacuoles within epithelial and inflammatory cells [4, 9-12]. Some investigators have argued that such organisms, herein designated “invasive H. pylori,”
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are more resistant to treatment and significantly increase the likelihood of future disease
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[13]. Intracellular organisms, in particular, have been postulated to serve as a reservoir that can re-colonize the stomach after antibiotic treatment eliminates extracellular bacteria
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[9, 13]. As a result, some investigators have argued that antibiotics with intracellular
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activity should be used to ensure all organisms are eradicated [14-15].
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Despite the potential significance of invasive H. pylori, their clinicopathologic associations remain poorly understood. This paper surveys eighteen cases of invasive H.
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pylori identified during routine histological examination of gastric biopsies and resections within the Partners HealthCare system. We characterize the clinical characteristics of
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affected patients and the histological features of each specimen using the updated Sydney
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System for the classification and grading of gastritis [16]. Six cases with the highest quantity of invasive organisms were additionally subjected to electron microscopic
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evaluation. The goal of this paper is to evaluate the spectrum of clinical and pathologic associations of invasive H. pylori in order to clarify the significance of discovering these intracellular or invasive organisms in gastric specimens.
Materials and Methods Study Cohort
ACCEPTED MANUSCRIPT The study cohort consisted of 18 patients who had either endoscopic gastric biopsies (n=16) or resections (n=2) within the Partners HealthCare system between
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September 2010 and February 2014. Invasive organisms were generally discovered after
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ordering an H. pylori immunostain to evaluate for the presence of organisms. Patient records were reviewed to document age, sex, date of biopsy, history of H. pylori infection or
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proton-pump inhibitor use prior to biopsy, other past medical and medication history,
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history of tobacco or alcohol use, and H. pylori status after treatment. The average followup time was 38 months. The majority of the cases (n=12) were identified by two of the
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immunostain on gastric biopsies.
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authors (VD and TW) at an institution that routinely performs a Helicobacter pylori
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Histological Analysis
All hematoxylin and eosin (H&E) stained slides had an immunostain ordered for H.
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pylori. Both the H&E and immunostain were evaluated and graded by two pathologists (JD
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and LZ), one of whom has subspecialty training in gastrointestinal pathology (LZ). The updated Sydney System for the classification and grading of gastritis was applied separately to the antrum and fundus to describe as either “normal,” “mild,” “moderate,” or “marked” the following characteristics: quantity of H. pylori, atrophy, neutrophilic infiltrate, mononuclear cell infiltrate, and intestinal metaplasia [16]. Additional parameters assessed included the presence or absence of PPI effect, lymphoid aggregates, lymphoepithelial lesions, and the relative proportion of surface versus invasive organisms. One case of invasive Helicobacter heilmannii was also evaluated by the same criteria as well as by a Steiner stain.
ACCEPTED MANUSCRIPT Electron Microscopic Analysis The six cases with highest quantity of invasive organisms, as assessed by the H.
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pylori immunostain (rabbit monoclonal primary antibody, clone SP48, Ventana, Tuscon, AZ, USA), were microdissected from formalin-fixed, paraffin embedded tissue blocks, soaked in
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100% xylene overnight, rehydrated in a series of ethanol solutions, rinsed in sodium cacodylate buffer, and fixed for 1.5 hours with 2.5% glutaraldehyde, 2.0%
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paraformaldehyde, and 0.025% calcium chloride, in a 0.1 molar sodium cacodylate buffer
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with a pH of 7.4. Tissues were further processed in a Leica Lynx automatic tissue processor. Briefly, tissues were post fixed with osmium tetroxide, dehydrated in a series of ethanol
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solutions, en bloc stained during the 70% ethanol dehydration step for one hour, infiltrated with propylene oxide epoxy mixtures, embedded in pure epoxy, and polymerized over
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night at 60.1 degrees Celsius. Thin sections were stained with lead citrate and examined
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with an FEI Morgagni transmission electron microscope (FEI, Hillsboro, OR, USA). Images were captured with an AMT (Advanced Microscopy Techniques) digital CCD camera
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(Woburn, MA, USA). All cases were evaluated for the presence and location of H. pylori and exhaustively searched for potential intracellular organisms. The case of H. heilmannii was also evaluated using the above protocol. The study protocols were approved by the Institutional Review Board of Partners HealthCare.
Results
ACCEPTED MANUSCRIPT The average age of patients in the cohort was 58, with a range of 19-91 and an equal distribution of genders (Table 1). The average follow-up time was 38 months. Of the
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patients with H.pylori for whom the information was available, 3/16 had a history of prior
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H. pylori infection, 10/15 were receiving PPIs at the time of biopsy, and 11/12 had a negative post-treatment follow-up. A minority of patients had a history of using tobacco
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(5/15) and/or alcohol (4/16). The most common co-morbidities were gastroesophageal
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reflux (14/18) and hypertension (9/15). The majorities of cases in our series were discovered in the fall (8/18) and winter (7/18), versus the spring (1/18) and summer
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(2/18), raising the possibility of a seasonal association.
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Histologic findings are summarized in Table 2. Invasive H. pylori were found in both the body/fundus (18/18 cases) and antrum (12/18 cases) (Table 2). They were less often
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associated with acute inflammation, both in the antrum (15/15 chronic; 8/15 acute) and
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fundus (17/18 chronic; 8/18 acute). Lymphoid aggregates were more common in the antrum (8/15) than the fundus (4/18). A small minority of cases demonstrated atrophy
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(2/15 cases in antrum, 2/18 in fundus) and intestinal metaplasia (2/15). All cases had invasive organisms with a coccoid appearance, in contrast to the rod-shaped appearance of surface organisms. Immunohistochemistry for H. pylori revealed organisms ringing the base of glandular cells and within gastric pit lumina. Organisms also appeared immediately adjacent to, and in some cases, on top of cell nuclei, giving the appearance of an intracellular localization (Figure 1) and initial interpretation prior to electron microscopy was that these were intracellular H. pylori.
ACCEPTED MANUSCRIPT This possibility was evaluated with electron microscopy. Six cases with the highest quantity of invasive organisms revealed by the H. pylori immunostain were assessed,
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revealing organisms within surface mucin, deep within the lumina of gastric pits, and
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infiltrating between cells (Figure 2). Despite an exhaustive search, no intracellular
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organisms were identified in these six cases.
These results are usefully contrasted with a case of invasive gastric Helicobacter
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helmannii infection, which was separately identified and analyzed during this study. H.
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helmannii is rare in humans, with a prevalence estimated to be between 0.2 and 6%. It frequently colonizes the gastric mucosal surface of domestic animals, such as dogs and cats,
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which constitute a source for human infections [17-18]. It cross-reacts with the antibodies used for immunohistochemical identification of H. pylori, giving a similar appearance, and
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may also give false-positive results for H. pylori on serological and urease-breath tests. It is
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distinguished, however, by its long, cork-screw profile, noticeable on H&E but is most
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apparent on a Steiner stain (Figure 3, B). Notably, the individual case of H. helmannii identified in this series had a nearly identical appearance and localization pattern with the H. pylori immunostain as the cases of invasive H. pylori noted previously, with a distribution around the base of glandular cells and within the lumina of gastric pits (Figure 3, A). The H&E characteristics and grading pattern per the updated Sydney System for the classification and grading of gastritis were likewise similar (Supplementary Table 1 & 2). Unlike the invasive H. pylori examined in our series, however, a Steiner stain revealed the long, corkscrew profile typical of H. heilmannii (Figure 3, B) while the electron microscopy showed not only an intercellular (Figure 3, C),
ACCEPTED MANUSCRIPT but also a definitive intracellular localization for many organisms (Figure 3, D&E). Also unlike most of the H. pylori in our series, the patient was refractory to treatment, with a
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five-year history stomach pain and a failed response to triple therapy and subsequent
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proton-pump inhibitor treatment.
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Discussion
This study sought to evaluate routine cases of intracellular H. pylori and clarify the
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significance of discovering these H. pylori in routine gastric specimens. Electron microscopic evaluation of six cases revealed organisms infiltrating between cells and deep
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within gastric pits but no intracellular forms, indicating the organisms are invasive but not
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intracellular. In this series of cases, invasive H. pylori were less often associated with acute
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inflammation and were equally distributed between the antrum and fundus. Most of the invasive H. pylori in this series were not associated with a history of prior infection or
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treatment-resistance. Although this series should be evaluated in the context of other studies, by itself, it suggests that most patients with invasive H. pylori can be successfully treated with the same protocols used for H. pylori confined to the mucosal surface. The coincidence of an intracellular localization and treatment-failure in the case of invasive H. heilmannii is suggestive but must be interpreted with caution. Since H. heilmannii colonization is less common, the appropriate treatment regimen is not as well established as it is for H. pylori. It is possible that treatment failure occurred in this patient simply because the standard treatment regimen for H. pylori is less effective for H.
ACCEPTED MANUSCRIPT heilmannii, though reports have documented successful treatment of H. heilmannii species with triple therapy [19-20].
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Other studies have explored the significance of invasive H. pylori. The organism has been documented with increased prevalence in specimens from patients with gastric
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ulcers, gastric intestinal metaplasia, and gastric cancer [21-22]. Although these studies are limited by the specificity of H. pylori antibodies, that the organism can be invasive and
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intracellular is supported by several lines of evidence, including transmission electron
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microscopy with immunogold staining, in situ hybridization, and multilabel immunohistochemical methods [4, 11, 13, 23]. In this study, invasive organisms were
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always accompanied by at least some surface organisms with a similar appearance on immunohistochemistry. It is unclear whether PPI use promotes an invasive phenotype. One
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study surveying the relationship between PPI usage and H. pylori histology found a shift in
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location [24].
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organisms from the antrum to the fundus but failed to note a shift toward a deeper mucosal
The relevance of intracellular H. pylori for treatment failure is less established. Evidence for an association is primarily based on in vitro cell culture studies, analogy with other bacteria, and, in one study, persistence of intracellular forms in a proportion of a series of ten patients after treatment [4, 13]. The latter study demonstrated persistence of intracellular forms with immunohistochemistry. Our results demonstrate the difficulty of determining an organism’s intracellular status by light microscopy alone. Several studies have identified intracellular H. pylori in gastric biopsies using electron microscopy [12, 2526], but there is no direct evidence that H. pylori can replicate within cells, and one study,
ACCEPTED MANUSCRIPT using differential interference contrast video, demonstrated that intracellular H. pylori has a 24-hour half-life [27]. This raises doubts that intracellular forms of the organism could
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survive the typical 7- to 14-day regimen of triple therapy. Conclusions
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Our study evaluates the finding of "intracellular" H. pylori in routine gastric biopsies
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and resection specimens detected with H. pylori immunohistochemistry. Electron microscopy demonstrates that these are not intracellular in location but invasive into the
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deep mucosa and present in deep intercellular spaces and lumens. The incidence of these invasive organism is uncertain as our cases were collected prospectively. Such cases are
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more often associated with chronic inflammation and tend to involve both the antrum and
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fundus. Many of the patients were already taking proton pump inhibitor therapy at the time of diagnosis, which has been shown to drive H. pylori deeper into the mucosa. Most of
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the patients in our study responded to standard therapy, suggesting that patients with
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invasive H. pylori discovered in gastric biopsies can be treated with the same regimen used for patients with surface H. pylori. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Acknowledgements None. Authors’ contributions
ACCEPTED MANUSCRIPT JD, VD, LZ, and RO conceived of study design, gathered and analyzed data, and wrote the manuscript. Case material was provided by RO, TW, and JS. MS performed electron
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microscopy on relevant cases. HC analyzed the data, wrote, and edited the manuscript.
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References
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[1] Lacy BE, Rosemore J. Helicobacter pylori: ulcers and more: the beginning of an era. The Journal of nutrition 2001; 131, 2789S-2793S. [2] Forman D. Helicobacter pylori infection and cancer. British medical bulletin 1998; 54, 71-78. [3] Huang JQ, Sridhar S, Chen Y, Hunt RH. Meta-analysis of the relationship between Helicobacter pylori seropositivity and gastric cancer. Gastroenterology 1998; 114, 11691179. [4] Dubois A, Boren T. Helicobacter pylori is invasive and it may be a facultative intracellular organism. Cellular microbiology 2007; 9, 1108-1116. [5] Graham DY. Therapy of Helicobacter pylori: current status and issues. Gastroenterology 2000; 118, S2-8. [6] Knigge KL. The role of H pylori in gastrointestinal disease. A guide to identification and eradication. Postgraduate medicine 2001; 110, 71-72, 77-78, 81-72. [7] Peek RM, Jr., Blaser MJ. Helicobacter pylori and gastrointestinal tract adenocarcinomas. Nature reviews Cancer 2002; 2, 28-37. [8] El-Omar EM, Carrington M, Chow WH, McColl KE, Bream JH, Young HA, Herrera J, Lissowska J, Yuan CC, Rothman N, Lanyon G, Martin M, Fraumeni JF, Jr., Rabkin CS. Interleukin-1 polymorphisms associated with increased risk of gastric cancer. Nature 2000; 404, 398-402. [9] Dubois A. Intracellular Helicobacter pylori and gastric carcinogenesis: an "old" frontier worth revisiting. Gastroenterology 2007; 132, 1177-1180. [10] Jhala NC, Siegal GP, Klemm K, Atkinson BF, Jhala DN. Infiltration of Helicobacter pylori in the gastric mucosa. American journal of clinical pathology 2003; 119, 101-107. [11] Necchi V, Candusso ME, Tava F, Luinetti O, Ventura U, Fiocca R, Ricci V, Solcia E. Intracellular, intercellular, and stromal invasion of gastric mucosa, preneoplastic lesions, and cancer by Helicobacter pylori. Gastroenterology 2007; 132, 1009-1023. [12] Noach LA, Rolf TM, Tytgat GN. Electron microscopic study of association between Helicobacter pylori and gastric and duodenal mucosa. Journal of clinical pathology 1994; 47, 699-704. [13] Engstrand L, Graham D, Scheynius A, Genta RM, El-Zaatari F. Is the sanctuary where Helicobacter pylori avoids antibacterial treatment intracellular? American journal of clinical pathology 1997; 108, 504-509. [14] Fahey JW, Haristoy X, Dolan PM, Kensler TW, Scholtus I, Stephenson KK, Talalay P, Lozniewski A. Sulforaphane inhibits extracellular, intracellular, and antibiotic-resistant strains of Helicobacter pylori and prevents benzo[a]pyrene-induced stomach tumors. Proceedings of the National Academy of Sciences of the United States of America 2002; 99, 7610-7615. [15] Hulten K, Cars O, Hjelm E, Engstrand L. In-vitro activity of azithromycin in against intracellular Helicobacter pylori. The Journal of antimicrobial chemotherapy 1996; 37, 483489.
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[16] 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. The American journal of surgical pathology 1996; 20, 1161-1181. [17] Iwanczak B, Biernat M, Iwanczak F, Grabinska J, Matusiewicz K, Gosciniak G. The clinical aspects of Helicobacter heilmannii infection in children with dyspeptic symptoms. Journal of physiology and pharmacology : an official journal of the Polish Physiological Society 2012; 63, 133-136. [18] Singhal AV, Sepulveda AR. Helicobacter heilmannii gastritis: a case study with review of literature. The American journal of surgical pathology 2005; 29, 1537-1539. [19] Morgner A, Lehn N, Andersen LP, Thiede C, Bennedsen M, Trebesius K, Neubauer B, Neubauer A, Stolte M, Bayerdorffer E. Helicobacter heilmannii-associated primary gastric low-grade MALT lymphoma: complete remission after curing the infection. Gastroenterology 2000; 118, 821-828. [20] Schultz-Suchting F, Stallmach T, Braegger CP. Treatment of Helicobacter heilmanniiassociated gastritis in a 14-year-old boy. Journal of pediatric gastroenterology and nutrition 1999; 28, 341-342. [21] Semino-Mora C, Doi SQ, Marty A, Simko V, Carlstedt I, Dubois A. Intracellular and interstitial expression of Helicobacter pylori virulence genes in gastric precancerous intestinal metaplasia and adenocarcinoma. The Journal of infectious diseases 2003; 187, 1165-1177. [22] Chan WY, Hui PK, Leung KM, Thomas TM. Modes of Helicobacter colonization and gastric epithelial damage. Histopathology 1992; 21, 521-528. [23] Petersen AM, Krogfelt KA. Helicobacter pylori: an invading microorganism? A review. FEMS immunology and medical microbiology 2003; 36, 117-126. [24] Dickey W, Kenny BD, McConnell JB. Effect of proton pump inhibitors on the detection of Helicobacter pylori in gastric biopsies. Aliment Pharmacol Ther 1996; 10, 289293. [25] Ko GH, Kang SM, Kim YK, Lee JH, Park CK, Youn HS, Baik SC, Cho MJ, Lee WK, Rhee KH. Invasiveness of Helicobacter pylori into human gastric mucosa. Helicobacter 1999; 4, 77-81. [26] Papadogiannakis N, Willen R, Carlen B, Sjostedt S, Wadstrom T, Gad A. Modes of adherence of Helicobacter pylori to gastric surface epithelium in gastroduodenal disease: a possible sequence of events leading to internalisation. APMIS 2000; 108, 439-447. [27] Amieva MR, Salama NR, Tompkins LS, Falkow S. Helicobacter pylori enter and survive within multivesicular vacuoles of epithelial cells. Cellular microbiology 2002; 4, 677-690.
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58 (19-91)
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Patient characteristics Age, years Mean (range) Gender Male Female History of H. pylori Yes No Unknown PPI treatment pre-biopsy Yes No Unknown Smoking history Yes No Unknown Alcohol use Yes No/Rare Unknown H. pylori test post-treatment? Negative Positive Unknown/not treated
10 5 3 5 10 3 4 12 2 11 1 6
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Table 1. Summary of clinical characteristics of 18 patients with invasive gastric H. pylori.
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7 0 3 4 13 8
7 4 9 7 2
1 11 3 2
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3 10 8 7
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Moderate Marked Present
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3 3 3 3 3 3
2 16 14 13 10
Mild
2
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None
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Gastric Antrum Acute inflammation Chronic inflammation Deep mucosal H. pylori Surface H. pylori Atrophy Lymphoid aggregates Gastric Fundus Acute inflammation Chronic inflammation Deep mucosal H. pylori Surface H. pylori Atrophy Lymphoid aggregates Intestinal Metaplasia PPI Effect
NA
7
2 3 4 2 8
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Table 2. Summary of histological findings in 18 patients with invasive gastric H. pylori. NA=Not available, PPI=Proton pump inhibitor.
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Figure 1. Histological appearance of invasive H. pylori. Patient 1 had invasive H. pylori in his antrum (A) and fundus (B), associated with chronic inflammation (C). Patient 2 had invasive and surface H. pylori (D and E), associated with mild active and chronic inflammation (F). (H. pylori immunostainon A, B (40x), D, E (60x); hematoxylin and eosin stain on C (20x) and F (20x).
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Figure 2. Electron microscopic appearance of invasive gastric H. pylori. The organisms were found infiltrating between cells (A-C) and deep in gastric pits (D), as indicated by arrows. No H. pylori organisms were identified in an intracellular location.
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Figure 3: Histological and electron microscopic appearance of H. heilmannii. H. pylori immunostainreveals a globular stain identical in appearance to that seen with intracellular H. pylori (A). The appearance of organisms on H&E prompted a Steiner stain, revealing the long, corkscrew profile typical of H. heilmannii(B). These are highlighted between cells (C) and inside of them (D&E) on electron microscopy. The arrows indicate the presence of H. heilmanniiorganisms. (H. pylori immunostainon A (20x), Steiner stain on B (40x), and electron microscopy on C, D, and E)