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ObjectiveGastric cancer (GC) is a leading cause of cancer-related mortality. Although microbes besides Helicobacter pylori may also contribute to gastric carcinogenesis, wild-type germ-free (GF) mouse models investigating the role of human gastric microbiota in the process are not yet available. We aimed to evaluate the histopathological features of GF mouse stomachs transplanted with gastric microbiota from patients with different gastric disease states and their relationships with the microbiota.DesignMicrobiota profiles in corpus and antrum tissues and gastric fluid from 12 patients with gastric dysplasia or GC were analysed. Thereafter, biopsied corpus and antrum tissues and gastric fluid from patients (n=15 and n=12, respectively) with chronic superficial gastritis, intestinal metaplasia or GC were inoculated into 42 GF C57BL/6 mice. The gastric microbiota was analysed by amplicon sequencing. Histopathological features of mouse stomachs were analysed immunohistochemically at 1 month after inoculation. An independent set of an additional 15 GF mice was also analysed at 1 year.ResultsThe microbial community structures of patients with dysplasia or GC in the corpus and antrum were similar. The gastric microbiota from patients with intestinal metaplasia or GC selectively colonised the mouse stomachs and induced premalignant lesions: loss of parietal cells and increases in inflammation foci, in F4/80 and Ki-67 expression, and in CD44v9/GSII lectin expression. Marked dysplastic changes were noted at 1 year post inoculation.ConclusionMajor histopathological features of premalignant changes are reproducible in GF mice transplanted with gastric microbiota from patients with intestinal metaplasia or GC. Our results suggest that GF mice are useful for analysing the causality of associations reported in human gastric microbiome studies.

Background & Aims WAP 4-disulfide core domain protein 2 (WFDC2), also known as human epididymis protein 4, is a small secretory protein that is highly expressed in fibrosis and human cancers, particularly in the ovaries, lungs, and stomach. However, the role of WFDC2 in carcinogenesis is not fully understood. The present study aimed to investigate the role of WFDC2 in gastric carcinogenesis with the use of preneoplastic metaplasia models. Methods Three spasmolytic polypeptide–expressing metaplasia (SPEM) models were established in both wild-type and Wfdc2-knockout mice with DMP-777, L635, and high-dose tamoxifen, respectively. To reveal the functional role of WFDC2, we performed transcriptomic analysis with DMP-777–treated gastric corpus specimens. Results Wfdc2-knockout mice exhibited remarkable resistance against oxyntic atrophy, SPEM emergence, and accumulation of M2-type macrophages in all 3 SPEM models. Transcriptomic analysis revealed that Wfdc2-knockout prevented the up-regulation of interleukin-33 (IL33) expression in the injured mucosal region of SPEM models. Notably, supplementation of recombinant WFDC2 induced IL33 production and M2 macrophage polarization, and ultimately promoted SPEM development. Moreover, long-term treatment with recombinant WFDC2 was able to induce SPEM development. Conclusions WFDC2 expressed in response to gastric injury promotes SPEM through the up-regulation of IL33 expression. These findings provide novel insights into the role of WFDC2 in gastric carcinogenesis.

To accurately quantify mRNA and protein levels, it is critical to choose appropriate internal standards. As the expression of housekeeping genes is assumed to remain constant, they are often employed to normalize signals to correct for sample-to-sample variations. However, recent studies have documented that β-actin and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression levels change in response to various stimuli during proliferation, activation, and differentiation. We investigated levels of α-, β-, γ-tubulin, β-actin, and GAPDH vary across the gastrointestinal tract of mice. We found that different regions of the small intestines had dramatically different expression profiles, as measured by western blot, quantitative Reverse transcription polymerase chain reaction (RT-PCR), and immunohistochemical staining. These results revealed that the expression levels of tubulins and β-actin were dramatically lower in the proximal duodenum, relative to the rest of the small intestines. These varying levels of housekeeping genes may reflect differences in the activities of specialized tissues and suggest unique requirements for tubulins in these tissue types. We conclude that the use of a single housekeeping gene to normalize gene expression in the gastrointestinal tracts of mice may introduce errors, as measured differences in gene expression may reflect regulation of the internal control rather than the mRNA or protein under investigation.

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder involving changes in normal bowel movements. The pathophysiology of IBS is not clearly understood owing to the lack of identifiable pathological abnormalities and reliable biomarkers. The aim of this study was to discover the novel and reliable biomarker for IBS. In this study, neonatal maternal separation (NMS) stress model was used for the IBS mouse model. Further assessment was conducted with whole gastrointestinal transit test, quantitative RT-PCR, histological examination, and western blot. Male pups developed symptoms similar to those of human IBS with diarrhea (IBS-D), such as low-grade inflammation, stool irregularity, and increased bowel motility. NMS stress influenced to the interstitial cells of Cajal (ICC) and induced altered bowel motility, resulting in IBS-D-like symptoms. In addition, we found neuronal nitric oxide synthase (nNOS) to be a novel biomarker for ICC under NMS stress. nNOS expression was only observed in the ICC of the submucosal plexus of IBS-D mice, and the inhibition of nNOS changed the phenotype from IBS-D to IBS with constipation. Our study demonstrates that early-life stress can influence to ICC and modulate bowel activity and that nNOS might be used as a biomarker for ICC stimulation in IBS.

Objectives Spasmolytic polypeptide-expressing metaplasia (SPEM) develops as a preneoplastic lesion in the stomachs of mice and humans after parietal cell loss. To identify the commonalities and differences between phenotypic SPEM lineages, SPEM were studied from three different mouse models of parietal cell loss: with chronic inflammation with Helicobacter felis infection; with acute inflammation with L635 treatment; and without inflammation following DMP-777 treatment. Design RNA transcripts from laser capture microdissected normal chief cells and SPEM lineages were compared using gene microarray. Alterations in transcripts were validated by quantitative real-time PCR. Clusterin and cystic fibrosis transmembrane conductance regulator (CFTR) were selected for immunohistochemical analysis in all mouse models as well as in human SPEM, intestinal metaplasia and gastric cancer. Results Transcript expression patterns demonstrated differences among the phenotypic SPEM models. Clusterin expression was significantly upregulated in all three mouse SPEM models as well as in human SPEM. The highest clusterin expression in human gastric cancers correlated with poor survival. Conversely, CFTR expression was upregulated only in SPEM with inflammation in mice. In humans, intestinal metaplasia, but not SPEM, expressed CFTR. Conclusions While markers such as clusterin are expressed in all phenotypic SPEM lineages, distinct patterns of upregulated genes including CFTR are present in murine metaplasia associated with inflammation, indicative of progression of metaplasia towards a more intestinalised metaplastic phenotype.

Objective Metaplastic lineages in the oxyntic mucosa of the stomach are critical preneoplastic precursors of gastric cancer. Recent studies have demonstrated that spasmolytic polypeptide-expressing metaplasia (SPEM) in the mouse oxyntic mucosa arises from transdifferentiation of mature gastric chief cells. Other investigations of intestinal progenitor cells have shown that cells demonstrating transcriptional activity for leucine-rich repeat containing G-protein-coupled receptor 5 (Lgr5) in the intestine, colon and gastric antrum function as adult stem cells. We have now investigated whether cells demonstrating Lgr5 transcriptional activity in the oxyntic mucosa of mice might be responsible for development of metaplasia. Design Lgr5-EGFP-IRES-Cre ERT2/+ ;Rosa26R mice were used to examine the distribution of Lgr5 transcriptionally active cells in the normal oxyntic mucosa as well as after treatment with DMP-777 or L-635 to induce acute SPEM. Lineage mapping was performed to determine if Lgr5-expressing cells gave rise to SPEM. Results Cells expressing transcriptional activity for Lgr5 in the oxyntic mucosa were present as scattered rare cells only along the lesser curvature of the stomach. These cells also stained for markers of chief cells (intrinsic factor and pepsinogen) but never showed any staining for proliferative markers (Ki-67). In Lgr5-EGFP-IRES-Cre ERT2/+ ;Rosa26R mice induced with tamoxifen, treatment with either DMP-777 or L-635 to induce acute oxyntic atrophy caused induction of SPEM, but no lineage mapping into SPEM from Lgr5-expressing cells was observed. Conclusion The results indicate that, while chief cells with Lgr5 transcriptional activity are present along the lesser curvature of the gastric oxyntic mucosa, they are not responsible for production of metaplasia.

Our previous study indicated that gene expression profiling of intestinal metaplasia (IM) or spasmolytic polypeptide-expressing metaplasia (SPEM) can identify useful prognostic markers of early-stage gastric cancer, and seven metaplasia biomarkers (MUC13, CDH17, OLFM4, KRT20, LGALS4, MUC5AC, and REG4) were selectively expressed in 17–50% of gastric cancer tissues. We investigated whether the combined expression of these metaplasia biomarkers could predict the prognosis of advanced stage gastric cancer. The expression of seven metaplasia biomarkers was evaluated immunohistochemically using tissue microarrays comprised of 450 gastric cancer patients. The clinicopathologic correlations and the prognostic impact were analyzed according to the expression of multiple biomarkers. MUC13, CDH17, LGALS4, and REG4 were significant prognostic biomarkers in univariate analysis. No expression of four markers was found in 56 cases (14.2%); 1 marker was seen in 67 cases (17%), 2 in 106 cases (27%), 3 in 101 cases (25.7%), and 4 in 63 cases (16%). Patients in which two or fewer proteins were expressed (group B) showed younger age, undifferentiated or diffuse type cancer, larger tumor size, larger number of metastatic lymph nodes, and more advanced stage than those in which three or more proteins were expressed (group A). In undifferentiated or stage II/III gastric cancer, the prognosis of group B was significantly poorer than that of group A by multivariate analysis. The combined loss of expression of multiple metaplasia biomarkers is considered an independent prognostic indicator in undifferentiated or stage II/III gastric cancer.

Objective Vitamin D 3 upregulated protein 1 (VDUP1) is a potent tumour suppressor whose expression is dramatically reduced in various types of human cancers, including gastric cancer. However, the precise mechanisms underlying tumour development remain unclear. In the present study, the authors examined the effect of VDUP1 on Helicobacter pylori -induced gastric carcinogenesis in mice. Design Gastric cancer was generated in VDUP1 knockout (KO) and wild-type mice using a combination of N -methyl- N -nitrosourea treatment and H pylori infection. Fifty weeks after treatment, gastric tissues from both types of mice were examined by histopathology, immunohistochemistry and immunoblotting. In vitro tests on the human gastric cancer cell line, AGS, were also performed to identify the underlying mechanisms of cancer development. Results The overall incidence of gastric cancer was significantly higher in VDUP1 KO mice than in wild-type mice. Similarly, VDUP1 KO mice showed more severe chronic gastritis, glandular atrophy, foveolar hyperplasia, metaplasia and dysplasia. Although no differences in the apoptotic index were apparent, lack of VDUP1 increased the rate of gastric epithelial cell proliferation in non-cancerous stomachs, with corresponding increases in tumour necrosis factor alpha (TNFα) level, nuclear transcription factor kappa B (NF-κB) activation and cyclooxygenase-2 (COX-2) expression. An in vitro study showed that H pylori -associated cell proliferation and induction of TNFα, NF-κB and COX-2 were inhibited in cells transfected with VDUP1. In addition, overexpression of VDUP1 in AGS cells suppressed TNFα-induced NF-κB activation and COX-2 expression. Conclusion Our data show that VDUP1 negatively regulates H pylori -associated gastric carcinogenesis, in part by disrupting cell growth and inhibiting the induction of TNFα, NF-κB and COX-2. These findings provide important insights into the role of VDUP1 in H pylori -associated tumourigenesis.

Intercellular communication via gap junctions, composed of protein subunits called connexins (Cxs), plays a key role in controlling cell growth, differentiation and carcinogenesis. Impaired gap junctional intercellular communication has been reported in various cancers and diseases. We investigated Cx32 expression patterns and semiquantitatively assessed Cx32 expression in cancers and preneoplastic lesions. To determine if cell proliferation is correlated with Cx32 expression, we evaluated Ki67 expression in a gastric cancer mouse model. In human and mouse, normal stomach and gastric adenocarcinoma tissues were used for immunohistochemical analyses. Cx32 was detected at cell–cell (intercellular) contact points in normal cells and exhibited punctate intercellular and intracytoplasmic staining in cancer cells. The frequency of Cx32 loss of expression was significantly higher in human adenocarcinomas than in normal stomach. As tumor cells were less differentiated, Cx32 expression levels and intercellular and intracytoplasmic staining were also significantly lower. The Cx32 expression pattern in the mouse gastric cancer model was similar in several important respects to that of human. In mucous metaplasia of the mouse stomach, Cx32 was mainly expressed in the cytoplasm of epithelial cells. There was also an inverse correlation between Cx32 expression and cell proliferation in mouse tumors. However, there was no difference in the levels of Cx32 mRNA between normal and cancerous tissues. These findings suggest that altered Cx32 expression, a loss of intercellular Cx32 and a gain of intracytoplasmic Cx32 in the form of punctate “dot”, plays an important role in the formation of gastric adenocarcinomas.

Background & Aims: The loss of parietal cells from the gastric mucosa (oxyntic atrophy) is a critical step in the pathogenesis of chronic gastritis and gastric adenocarcinoma. Parietal cells are known to secrete epidermal growth factor receptor (EGFR) ligands, which are critical regulators of differentiation in the gastric mucosa. Although all of the actions of EGFR ligands are mediated through a common EGFR protein, individual ligands may produce different physiologic responses. Previous investigations have suggested that a deficit in EGFR signaling in waved-2 mice accelerates the emergence of metaplasia after induction of acute oxyntic atrophy. We sought to determine whether specific EGFR ligands regulate the metaplastic response to oxyntic atrophy. Methods: To induce spasmolytic polypeptide-expressing metaplasia (SPEM), amphiregulin (AR) and transforming growth factor-α–deficient mice and their wild-type littermates were treated with DMP-777 for 0–14 days and for 14 days followed by 14 days of recovery off drug. We evaluated the gastric mucosal response to oxyntic atrophy using cell lineage–specific markers. Results: Although loss of transforming growth factor-α did not influence the induction of SPEM, loss of AR caused an acceleration and amplification in the induction of SPEM after acute oxyntic atrophy. Trefoil factor family 2/spasmolytic polypeptide and intrinsic factor dual-immunostaining cells significantly increased in the SPEM of AR-deficient mice. At the bases of glands, intrinsic factor immunoreactive cells also were costained for 5-bromo-2'-deoxyuridine, suggesting their re-entry into the cell cycle. Conclusions: The absence of AR promoted the rapid emergence of SPEM in response to oxyntic atrophy.

Overproduction of nitric oxide via inducible nitric oxide synthase (iNOS) is suggested to be a significant pathogenic factor in Helicobacter pylori induced gastritis. The purpose of this study was to examine the role of iNOS in H pylori associated gastric carcinogenesis.Two types of mice were used in this study: iNOS deficient mice (iNOS-/-) and wild-type littermates. Gastric cancer was generated in mice using a combination treatment comprising N-methyl-N-nitrosourea administration and H pylori infection. Fifty weeks after treatment, tumours in gastric tissues from both types of mice were examined using histopathology, immunohistochemistry, and immunoblotting for iNOS and 3-nitrotyrosine.The overall incidence of gastric cancer at week 50 was significantly lower in iNOS-/- compared with iNOS wild-type mice (p0.05). When analysed according to tumour pathology, the incidence of gastric adenocarcinoma was significantly lower in iNOS-/- compared with iNOS wild-type mice (p0.05). Immunostaining for iNOS was clearly observed in adenocarcinoma cells of iNOS wild-type mice, and was characterised by a strong cytoplasmic expression pattern. 3-Nitrotyrosine was expressed mostly in the area of the lamina propria of gastritis and adenoma lesions in iNOS wild-type mice. Immunoblotting analyses showed that iNOS and 3-nitrotyrosine were also expressed in both adenoma and adenocarcinoma tissues from iNOS wild-type mice. iNOS and 3-nitrotyrosine expression was greater in tumour tissues than in non-tumour tissues.These findings suggest that iNOS contributes to H pylori associated gastric carcinogenesis in mice.

Objective Intestinal metaplasia and spasmolytic polypeptide-expressing metaplasia (SPEM) are considered neoplastic precursors of gastric adenocarcinoma and are both marked by gene expression alterations in comparison to normal stomach. Since miRNAs are important regulators of gene expression, we sought to investigate the role of miRNAs on the development of stomach metaplasias. Design We performed miRNA profiling using a quantitative reverse transcription-PCR approach on laser capture microdissected human intestinal metaplasia and SPEM. Data integration of the miRNA profile with a previous mRNA profile from the same samples was performed to detect potential miRNA-mRNA regulatory circuits. Transfection of gastric cancer cell lines with selected miRNA mimics and inhibitors was used to evaluate their effects on the expression of putative targets and additional metaplasia markers. Results We identified several genes as potential targets of miRNAs altered during metaplasia progression. We showed evidence that HNF4γ (upregulated in intestinal metaplasia) is targeted by miR-30 and that miR-194 targets a known co-regulator of HNF4 activity, NR2F2 (downregulated in intestinal metaplasia). Intestinal metaplasia markers such as VIL1, TFF2 and TFF3 were downregulated after overexpression of miR-30a in a HNF4γ-dependent manner. In addition, overexpression of HNF4γ was sufficient to induce the expression of VIL1 and this effect was potentiated by downregulation of NR2F2. Conclusions The interplay of the two transcription factors HNF4γ and NR2F2 and their coordinate regulation by miR-30 and miR-194, respectively, represent a miRNA to transcription factor network responsible for the expression of intestinal transcripts in stomach cell lineages during the development of intestinal metaplasia.

Objective The glands of the stomach body and antral mucosa contain a complex compendium of cell lineages. In lower mammals, the distribution of oxyntic glands and antral glands define the anatomical regions within the stomach. We examined in detail the distribution of the full range of cell lineages within the human stomach. Design We determined the distribution of gastric gland cell lineages with specific immunocytochemical markers in entire stomach specimens from three non-obese organ donors. Results The anatomical body and antrum of the human stomach were defined by the presence of ghrelin and gastrin cells, respectively. Concentrations of somatostatin cells were observed in the proximal stomach. Parietal cells were seen in all glands of the body of the stomach as well as in over 50% of antral glands. MIST1 expressing chief cells were predominantly observed in the body although individual glands of the antrum also showed MIST1 expressing chief cells. While classically described antral glands were observed with gastrin cells and deep antral mucous cells without any parietal cells, we also observed a substantial population of mixed type glands containing both parietal cells and G cells throughout the antrum. Conclusions Enteroendocrine cells show distinct patterns of localisation in the human stomach. The existence of antral glands with mixed cell lineages indicates that human antral glands may be functionally chimeric with glands assembled from multiple distinct stem cell populations.

Gastric cancer is the second most common cause of cancer-related death in the world. A growing body of evidence indicates that inflammation is closely associated with the initiation, progression, and metastasis of many tumors, including those of gastric cancer. In addition, approximately 60% of the world's population is colonized by Helicobacter pylori, which accounts for more than 50% of gastric cancers. While the role of inflammation in intestinal and colonic cancers is relatively well defined, its role in stomach neoplasia is still unclear because of the limited access of pathogens to the acidic environment and the technical difficulties isolating and characterizing immune cells in the stomach, especially in animal models. In this review, we will provide recent updates addressing how inflammation is involved in gastric malignancies, and what immune characteristics regulate the pathogenesis of stomach cancer. Also, we will discuss potential therapeutics that target the immune system for the efficient treatment of gastric cancer.

Background & Aims Loss of parietal cells causes the development of spasmolytic polypeptide-expressing metaplasia (SPEM) through transdifferentiation of chief cells. In the presence of inflammation, SPEM can advance into a more proliferative metaplasia with increased expression of intestine-specific transcripts. We used L635 to induce acute SPEM with inflammation in mice and investigated the roles of inflammatory cells in the development of SPEM. Methods To study the adaptive immune system, Rag1 knockout, interferon-γ–deficient, and wild-type (control) mice received L635 for 3 days. To study the innate immune system, macrophages were depleted by intraperitoneal injection of clodronate liposomes 2 days before and throughout L635 administration. Neutrophils were depleted by intraperitoneal injection of an antibody against Ly6G 2 days before and throughout L635 administration. Pathology and immunohistochemical analyses were used to determine depletion efficiency, metaplasia, and proliferation. To characterize SPEM in each model, gastric tissues were collected and levels of Cftr , Dmbt1 , and Gpx2 mRNAs were measured. Markers of macrophage polarization were used to identify subpopulations of macrophages recruited to the gastric mucosa. Results Administration of L635 to Rag1 knockout, interferon-γ–deficient, and neutrophil-depleted mice led to development of proliferative SPEM and up-regulation of intestine-specific transcripts in SPEM cells, similar to controls. However, macrophage-depleted mice given L635 showed significant reductions in numbers of SPEM cells, SPEM cell proliferation, and expression of intestine-specific transcripts, compared with control mice given L635. In mice given L635, as well as patients with intestinal metaplasia, M2 macrophages were the primary inflammatory component. Conclusions Results from studies of mouse models and human metaplastic tissues indicate that M2 macrophages promote the advancement of SPEM in the presence of inflammation.

Upper gastrointestinal neoplasia in the esophagus, stomach, and pancreas is associated with the formation of preneoplastic metaplasias. We have previously reported the up-regulation of human epididymis protein 4 (HE4) in all metaplasias in the stomach of humans and mice. We have now sought to evaluate the expression of HE4 in metaplasias/preneoplastic precursors and cancers of the human stomach, pancreas, and esophagus. Tissue microarrays for gastric cancers, pancreatic cancers, and esophageal adenocarcinoma were stained with antibodies against HE4. Immunostaining was quantified by digital imaging, and the results were evaluated to assess the expression in metaplasias, the expression in cancer pathological subtypes, and the effects of expression on survival in patients with cancer. In patients with gastric cancer from Korea, HE4 was detected in 74% of intestinal and 90% of diffuse cancers, whereas in a gastric cancer cohort from Johns Hopkins, HE4 was detected in 74% of intestinal-type and 92% of diffuse cancers. Nevertheless, in both cohorts, there was no impact of HE4 expression on overall survival. In the esophagus, we observed the expression of HE4 in scattered endocrine cells within Barrett esophagus samples, but Barrett columnar metaplasias and HE4 were detected in only 2% of esophageal adenocarcinomas. Finally, in the pancreas, HE4 expression was not observed in pancreatic intraepithelial neoplasia lesions, but 46.8% of pancreatic adenocarcinomas expressed HE4. Still, we did not observe any influence of HE4 expression on survival. The results suggest that HE4 is up-regulated during gastric and pancreatic carcinogenesis.

Menetrier’s disease is a rare premalignant disorder of the stomach with no proven effective medical therapy. Increased epidermal growth factor receptor signaling has been implicated in the pathogenesis of Menetrier’s disease. We conducted a single-arm clinical trial with cetuximab, a monoclonal antibody that blocks epidermal growth factor receptor signaling, in nine individuals with clinically and histologically documented severe Menetrier’s disease that impaired quality of life to the extent that gastrectomy was being considered. Of the seven patients who completed the 1-month course of treatment, all showed statistically significant improvement both clinically (quality-of-life indices) and biochemically (increased parietal cell mass and gastric acidity). Furthermore, all seven patients who completed the 1-month trial elected to continue treatment, and four subsequently showed near-complete histological remission. Cetuximab should be considered as first-line therapy for Menetrier’s disease.

Background & Aims The loss of parietal cells from the fundic mucosa leads to the emergence of metaplastic lineages associated with an increased susceptibility to neoplastic transformation. Both intestinal metaplasia (IM) and spasmolytic polypeptide (TFF2/SP) expressing metaplasia (SPEM) have been identified in human stomach, but only SPEM is present in most mouse models of gastric metaplasia. We previously determined that loss of amphiregulin (AR) promotes SPEM induced by acute oxyntic atrophy. We have now examined whether SPEM in the AR −/− mouse predisposes the stomach to gastric neoplasia. Methods Gross pathology of 18-month-old wild-type, AR −/− , and TGF-α −/− mice were examined. Ki-67, β-catenin, Pdx-1, TFF3, and TFF2/SP expression was analyzed by immunohistochemistry. Metaplastic gastric mucosa was analyzed by dual immunostaining for TFF2/SP with MUC2 or TFF3. Results By 18 months of age, more than 70% of AR −/− mice developed SPEM while 42% showed goblet cell IM labeled with MUC2, TFF3, and Pdx-1. A total of 28% had invasive gastric lesions in the fundus. No antral abnormalities were observed in AR −/− mice. Metaplastic cell lineages in AR −/− mice showed increases in cell proliferation and cytosolic β-catenin expression. Dual staining for TFF2/SP with MUC2 or TFF3 showed glands containing both SPEM and IM with intervening cells expressing both TFF2/SP and MUC2 or TFF2/SP and TFF3. Conclusions AR −/− mice develop SPEM, which gives rise to goblet cell IM and invasive fundic dysplastic lesions. The AR −/− mouse represents the first mouse model for spontaneous development of fundic SPEM with progression to IM.

Adenocarcinoma in the human stomach evolves in the setting of Helicobacter pylori-induced oxyntic atrophy and chronic mucous cell metaplasia. Two types of metaplasia associated with development of intestinal-type cancers are observed in the human stomach: intestinal metaplasia (IM; intestinal type goblet cells in the stomach) and Spasmolytic Polypeptide (TFF2) Expressing Metaplasia (SPEM; with the presence of antral type TFF2-expressing mucous cells in the oxyntic region). Our previous study showed that SPEM developed from transdifferentiation of mature chief cells, rather than originating from aberrant differentiation of resident progenitor cells. Recent investigations have noted evidence for increased Ras activity in up to a third of gastric cancers. Thus, we have hypothesized that activated Ras, specifically induced in mature chief cells, elicits transdifferentiation of chief cells into SPEM and progression of SPEM to more intestinalized metaplasia. To test this hypothesis, we have examined a novel mouse model of metaplasia in the stomach in Mist1-CreERT2Tg/+;LSLK-Ras(G12D)Tg/+;mT/mGTg/+ mice. Tamoxifen (TAM) treatment induces the expression of activated K-Ras specifically in mature chief cells and marks the K-Ras-induced chief cells with GFP as a lineage tracer. This mouse model rapidly develops SPEM and IM within 3 months, following TAM induction of activated K-Ras expression in mature chief cells. At 1 week post TAM treatment, we found several groups of metaplastic glands between normal oxyntic glands that were positive for SPEM markers. At 3 months post TAM treatment, oxyntic glands were replaced withmetaplastic glands that showed highly proliferative mucous cell populations at the bases and the cells in the metaplastic glands were strongly positive for clusterin and TFF3 with distinct goblet cell morphologies, and also positive for Pdx1. Some glands were positive for IM markers such as villin and Cdx1. The mice at 4 months post TAM treatment developed severe inflammation, metaplasia, dysplasia and the invasive adenocarcinoma. Consistent with active K-Ras expression, cells with nuclear expression of phospho-ERK1/2 were also observed at the bases of metaplastic glands. Interestingly, we observed that the metaplastic glands were positive for GFP, indicating that they were derived from transdifferentiation of Cre-induced chief cells. Therefore, the induction of activated Ras specifically in mature chief cells initially leads to transdifferentiation of chief cells into SPEM followed by evolution of goblet cell IM and eventual neoplastic lesions. These studies demonstrate that expression of activated Ras in Chief cells can lead to the development of the entire spectrum of metaplastic and neoplastic lineages leading to gastric cancer.

Gastric cancer is one of the most common neoplasms, ranking as the second leading cause of cancer-related deaths worldwide. The predominant factor associated to gastric cancer is Helicobacter pylori infection, which leads to a chronic inflammatory response and subsequent oxyntic atrophy. Parietal cell loss result in two types of metaplasia, the intestinal metaplasia (IM) characterized by the presence of cells with goblet cell morphology and spasmolytic polypeptide-expressing metaplasia (SPEM) that shows morphological characteristics of the deep antral glands and express trefoil factor 2 (spasmolytic polypeptide). Although metaplastic lesions are considered neoplastic precursors, their direct association to cancer is still in debate. Similar to other cancers, gastric cancers are marked by global gene expression alterations. MicroRNAs (miRNAs) are small noncoding RNAs involved in the post-transcriptional regulation of gene expression and an increasing number of studies has been showing their aberrant expression in cancer. In order to identify miRNAs involved in the early stages of gastric cancer we performed a miRNA profiling on laser capture micro-dissected IM and SPEM cells from patient lesions. Using a qRT-PCR approach for quantitation of 754 human miRNAs, we identified 77 miRNAs differentially expressed in the metaplastic samples (greater than two-fold) in comparison to normal chief cells. The highest number of dysregulated miRNAs was observed in IM,which showed 45 up-regulated and 25 down-regulatedmiRNAs. In SPEM, 28 miRNAs were found up-regulated (21 of them also up-regulated in IM), whereas no down-regulation was detected. Some of the up-regulated miRNAs have already been associated to cancer in general (miR-26-a, miR-191, miR-155), as well as specifically to gastric cancer (miR-18b, miR-196b and miR-106a). However, the regulation of some of the top up-regulated miRNAs revealed here including miR-802, miR-922 and miR-622 are still poorly characterized in cancer. In a comparison with a previous mRNA profiling study performed on a similar group of samples, we observed that 108 out of the 568 mRNAs found up-regulated in IM are predicted targets of the 26 microRNAs down-regulated in IM, revealing a potential mechanism for the regulation of those genes in gastric metaplasia. Particularly interesting are 4 members of the miR-30 family, with 31 predicted mRNA targets amongst those up-regulated in IM. Although miR-30 family members have been described as down-regulated in gastric cancer, no data on their targets in this neoplasia is currently available. An extended characterization of the microRNAs identified here will provide a better understanding of their function in gastric metaplasia and progression to neoplasia, as well as might reveal useful early stage biomarkers or therapeutic targets.

Background & Aims Gastric cancer evolves in the setting of a pathologic mucosal milieu characterized by both loss of acid-secreting parietal cells and mucous cell metaplasias. Indeed, mucous cell metaplasia is considered the critical preneoplastic lesion for gastric cancer. Previous investigations have shown that infection of mice with Helicobacter felis or induction of acute parietal cell loss with the drug DMP-777 leads to the emergence of a type of metaplasia designated spasmolytic polypeptide-expressing metaplasia (SPEM). We have hypothesized that SPEM arises from proliferating cells in gland bases, either from a cryptic progenitor cell or by transdifferentiation of mature chief cells. Methods Taking advantage of the chief cell-restricted expression of Mist1-Cre-ER T2 , we used lineage mapping to examine whether SPEM lineages were derived from chief cells in 3 independent models of induction by DMP-777 treatment, L-635 treatment, or H felis infection. Results Treatment of mice with L-635 for 3 days led to rapid parietal cell loss, induction of a prominent inflammatory infiltrate, and emergence of SPEM. In all 3 models, SPEM developed, at least in part, from transdifferentiation of chief cells. We further found that acute parietal cell loss in the setting of inflammation (L-635 treatment) led to more rapid induction and expansion of SPEM derived from transdifferentiation of chief cells. Conclusions These studies provide direct evidence by lineage tracing that SPEM evolves from differentiated chief cells. Thus, mature gastric chief cells have the ability to act as cryptic progenitors and reacquire proliferative capacity within the context of mucosal injury and inflammation.

Background & Aims Intestinal metaplasia (IM) and spasmolytic polypeptide-expressing metaplasia (SPEM) are precursors to gastric carcinogenesis. We sought to identify molecular biomarkers of gastric metaplasias and gastric cancer by gene expression profiling of metaplastic lesions from patients. Methods Complementary DNA microarray analysis was performed on IM and SPEM cells isolated from patient samples using laser capture microdissection. Up-regulated transcripts in metaplastic lesions were confirmed by immunostaining analysis in IM, SPEM, and gastric cancer tissues. Proteins that were highly expressed specifically in gastric cancer tissues were analyzed for their association with survival in a test set (n = 450) and a validation set (n = 502) of samples from gastric cancer patients. Results Compared with normal chief cells, 858 genes were differentially expressed in IM or SPEM samples. Immunostaining was detected for 12 proteins, including 3 new markers of IM ( ACE2 , LGALS4 , AKR1B10 ) and 3 of SPEM ( OLFM4 , LYZ , DPCR1 ). Of 13 proteins expressed in IM or SPEM, 8 were expressed by 17%–50% of human gastric cancer tissues ( MUC13 , OLFM4 , CDH17 , KRT20 , MUC5AC , LGALS4 , AKR1B10 , REG4 ). Expression of CDH17 or MUC13 correlated with patient survival in the test and validation sets. Multivariate analysis showed that CDH17 was an independent prognostic factor in patients with stage I or node-negative disease. Conclusions We identified several novel biomarkers for IM, SPEM, and gastric cancer using gene expression profiling of human metaplastic lesions. Expression of CDH17 and MUC13 was up-regulated in gastric cancer tissues. CDH17 is a promising prognostic marker for early stage gastric cancer.

Gastric adenocarcinoma of the intestinal type is believed to develop through a series of histopathologic stages, originally described by Correa, that include chronic gastritis, gastric atrophy, intestinal metaplasia, dysplasia and finally cancer (1). Intestinal metaplasia has been the focus of great fascination by many pathologists, since it was unexpected to find intestinal type mucosa containing goblet cells in the body or antrum of the stomach. Numerous studies have implicated intestinal metaplasia as a useful biomarker for gastric cancer risk, and even possibly a precursor to gastric malignancy. However, this evidence has been largely correlative. In patients with gastric cancer, intestinal metaplasia is frequently found in the stomach, even with early gastric cancer. Intestinal metaplasia is much more prevalent in high-risk areas for gastric cancer compared to low risk areas. Finally, in several studies where serial endoscopies and biopsies were performed, patients with intestinal metaplasia, particularly type III, frequently developed gastric cancer (2). Intestinal metaplasia has been used as the key biomarker in studies of H. pylori eradication or gastric cancer prevention, defining the preneoplastic lesion often considered the “point-of-no-return” (3). Indeed, since the gastric cancers that developed in this setting also showed intestinal differentiation, it seemed logical to conclude that the cancers arose directly from these intestinal metaplastic cells. Neverthless, in contrast with other neoplasms, little genetic mutational evidence exists to implicate intestinal metaplastic lineages as true direct precursors of gastric neoplasia. Recent investigations have also highlighted the existence of a second metaplastic lineage, Spasmolytic Polypeptide-Expressing Metaplasia (SPEM) (4). SPEM is a metaplastic mucous cell lineage with phenotypic characteristics of deep antral gland cells including strong expression of Trefoil Factor 2 (TFF2, previously designated as spasmolytic polypeptide). In the past, this underappreciated mucous lineage was described with various names including pseudopyloric metaplasia or mucous metaplasia or antralization of the corpus, but mostly the lineage was ignored. Indeed, it is now appreciated that atrophy of the corpus and body of the stomach is always associated with the development of SPEM. In addition, SPEM is at least as strongly associated with the development of gastric cancer as intestinal metaplasia (4). Moreover, while intestinal metaplasia tends to be spotty or multifocal, SPEM typically appears diffusely throughout the body and corpus of the stomach in patients that progress to gastric cancer (5). SPEM is not simply an expansion of antral type mucosa, since gastrin cells are not present and SPEM cells express a number of proteins, such as HE-4, that are not expressed in the antrum (6). However, both the precise connection between intestinal metaplasia and SPEM and the relationship between SPEM and gastric cancer remain unclear. Recent studies have examined the cellular origin of SPEM in mice. These investigations have demonstrated that SPEM develops in the fundus in the setting of parietal cell loss following chronic Helicobacter infection (7, 8). acute parietal cell loss due to treatment with DMP-777 (9), or chronic genetic parietal cell ablation by transgenically expressed toxin (10). These studies have led to accumulating evidence that SPEM may originate from transdifferentiation of mature chief cells into SPEM (6). In the case of H. felis infection, gastritis cystica profunda and dysplasia evolve from preexisting regions of SPEM (11). Still these studies have not been able to address intestinal metaplasia because Helicobacter-infected mice do not typically develop intestinal metaplasia. Intestinal goblet cells do develop in the fundus of transgenic mice where cdx2, a transcription factor, which is a master developmental regulator of intestinal differentiation, is targeted to the stomach using elements of the H+/K+-ATPase β subunit promoter (12, 13). Interestingly, amphiregulin-deficient mice developed SPEM after 6 months of age, and a subset of mice did go on to develop goblet cell intestinal metaplasia (14). In that case, compound glands containing both SPEM and intestinal metaplasia were clearly present. While murine studies have not been able to clarify the relationship between intestinal metaplasia and SPEM, studies in Mongolian gerbils have provided additional insights. Mongolian gerbils develop both SPEM and intestinal metaplasia in the fundus following infection with Helicobacter pylori (15). In Mongolian gerbils, intestinal metaplasia developed in pre-existing SPEM glands and mixed glands expressing both SPEM and intestinal metaplasia were clearly present (15). All of these studies have supported the notion that intestinal metaplasia may develop from pre-existing SPEM. No published studies in humans have addressed directly the question of the relationship of SPEM to intestinal metaplasia in humans. We have therefore examined the morphological characteristics of SPEM and intestinal metaplasia in gastric resections specimens exclusively from the fundus. These studies have uncovered several critical observations about the induction of metaplasias in the stomach. First, SPEM can develop as a very localized phenomenon. Figure 1 demonstrates that SPEM can develop in single or groups of glands surrounded by regions with normal appearing mucous neck cells in the fundic mucosa. At times these SPEM gland groups are associated with adjacent areas with the appearance of mucous neck cell hyperplasia. These regions do not appear to interact with regions of intestinal metaplasia. The focal phenotype of SPEM glands suggests that they may act as part of a normal local reparative mechanism for the gastric mucosa. Figure 1 Focal early lesions for SPEM induction We have also sought to determine if there is a relationship between SPEM glands and intestinal metaplasia in gastric resections. Examination of resection sections containing regions of both SPEM and intestinal metaplasia led to identification of regions with compound glands where SPEM cells were observed in the deep portions of the glands with intestinal metaplastic lineages in the luminal portions of the glands (Figure 2). Thus, goblet cells staining with either Alcian Blue or Muc2 were observed on the luminal aspects of glands that contain PAS-positive and TFF2-staining SPEM at their bases (Figure 2). It is important to emphasise that these were not residual pyloric type glands, since these sections were taken from areas surrounded by corpus mucosa. While we did observe scattered proliferative Ki67-positive cells within SPEM, in regions with intestinal metaplasia immediately adjacent to or overlying SPEM, we observed strong staining for Ki67 in cells within the region demarcating the zone between SPEM and intestinal metaplasia (Figure 2). Many of the Ki67-positive cells were also stained for MUC2. Thus, we observed clear evidence for the existence of intestinal metaplasia emanating from SPEM. The elevated proliferation in intestinal metaplasia adjacent to SPEM may indicate a transition or secondary differentiation of SPEM into intestinal metaplasia. Figure 2 Compound glands containing SPEM and Intestinal Metaplasia These data indicate that the standard concept proposed by Professor Correa now merits further expansion or modification (Figure 3). Work in mouse models and human tissue suggests that loss of parietal cells leads initially to the induction of SPEM. With chronic inflammation in the setting of parietal cell loss, SPEM may give rise to a further differentiation into intestinal metaplasia. This evolution of mucous cell metaplastic lineages has been noted previously for the Ulcer Associated Lineages (UACL) identified in patients with inflammatory bowel disease (16). While these findings create a strategy for the induction and progression of metaplastic lineages in humans, they do not address the actual origin of gastric adenocarcinoma. It is particularly exciting to consider that if SPEM is derived from chief cell transdifferentiation, then chief cells themselves, or a subset of chief cells, represent an unrecognized progenitor population that can be induced in the pathological stomach. Given the more differentiated nature of intestinal metaplasia, it now seems somewhat less likely that gastric cancer arises from a goblet cell-containing epithelium. Still, we must acknowledge that at present it remains uncertain whether either SPEM or intestinal metaplasia is a true precursor for cancer. Nevertheless, it seems likely that evolution of intestinal metaplasia from SPEM may lead to a hyperproliferative state in which the infected and inflamed mucosa may be more susceptible to establishment of deleterious mutations in stem or progenitor populations. Thus, SPEM and intestinal metaplasia may be commensals for the neoplastic process rather than true direct precursors. Taken together, this work suggests that a broader view of metaplastic initiation and pre-neoplastic progression is merited in evaluating gastric carcinogenesis. Figure 3 A revised model for the evolution of metaplasia in the stomach