UNVEILING CANCER-RELATED METAPLASTIC CELLS IN BOTH HELICOBACTER PYLORI INFECTION AND AUTOIMMUNE GASTRITIS.

BACKGROUND AND AIMS: Gastric metaplasia may arise as a consequence of chronic inflammation and is associated with an increased risk of gastric cancer development. While Helicobacter pylori (Hp) infection and autoimmune gastritis (AIG) both induce gastric metaplasia, possible distinctions in resulting metaplastic cells and their respective cancer risks requires further investigation. METHODS: Employing both mouse models and human subjects, we scrutinized the metaplasia originating from Hp infection and AIG. Gastric pathology and metaplasia were examined through histopathologic assessment. Molecular features of metaplastic cells were defined using single-cell transcriptomics in murine models of Hp infection and AIG, as well as in human biopsies from patients with Hp infection and AIG. Expression of a newly defined cancer-related metaplastic biomarker was confirmed through immunofluorescence. RESULTS: Metaplasia in Hp infection and AIG displayed comparable histopathological and transcriptional features. Diverse metaplastic subtypes were identified across both disease settings, with subtle differences in the prevalence of certain subtypes between inflammatory contexts. Notably, Hp infection did not drive a unique metaplastic cell phenotype. One metaplastic subtype, which resembled incomplete intestinal metaplasia and shared transcriptional features with gastric cancer was identified in both diseases. This cancer-like metaplastic subtype was characterized by expression of the cancer-associated biomarker ANPEP/CD13. CONCLUSION: Both Hp infection and AIG trigger a diverse array of metaplastic cell types. Identification of a cancer-related metaplastic cell uniquely expressing ANPEP/CD13, present in both Hp- and AIG-induced gastritis, indicates the carcinogenic capacity of both diseases. This discovery can guide early detection and risk stratification for patients with chronic gastritis.

Understanding the malignant potential of gastric metaplasia of the oesophagus and its relevance to Barrett's oesophagus surveillance: individual-level data analysis.

OBJECTIVE: Whether gastric metaplasia (GM) of the oesophagus should be considered as Barrett's oesophagus (BO) is controversial. Given concern intestinal metaplasia (IM) may be missed due to sampling, the UK guidelines include GM as a type of BO. Here, we investigated whether the risk of misdiagnosis and the malignant potential of GM warrant its place in the UK surveillance. DESIGN: We performed a thorough pathology and endoscopy review to follow clinical outcomes in a novel UK cohort of 244 patients, covering 1854 person years of follow-up. We complemented this with a comparative genomic analysis of 160 GM and IM specimens, focused on early molecular hallmarks of BO and oesophageal adenocarcinoma (OAC). RESULTS: We found that 58 of 77 short-segment (<3 cm) GM (SS-GM) cases (75%) continued to be observed as GM-only across a median of 4.4 years of follow-up. We observed that disease progression in GM-only cases and GM+IM cases (cases with reported GM on some occasions, IM on others) was significantly lower than in the IM-only cases (Kaplan-Meier, p=0.03). Genomic analysis revealed that the mutation burden in GM is significantly lower than in IM (p<0.01). Moreover, GM does not bear the mutational hallmarks of OAC, with an absence of associated signatures and driver gene mutations. Finally, we established that GM found adjacent to OAC is evolutionarily distant from cancer. CONCLUSION: SS-GM is a distinct entity from SS-IM and the malignant potential of GM is lower than IM. It is questionable whether SS-GM warrants inclusion in BO surveillance.

Autoimmune gastritis: long-term natural history in naïve Helicobacter pylori-negative patients.

OBJECTIVE: Autoimmune gastritis (AIG) is an immunomediated disease targeting parietal cells, eventually resulting in oxyntic-restricted atrophy. This long-term follow-up study aimed at elucidating the natural history, histological phenotype(s), and associated cancer risk of patients with AIG consistently tested H. pylori-negative (naïve H. pylori-negative subjects). DESIGN: Two-hundred eleven naïve H. pylori-negative patients (tested by serology, histology, molecular biology) with AIG (F:M=3.15:1; p<0.001) were prospectively followed up with paired biopsies (T1 vs T2; mean follow-up years:7.5 (SD:4.4); median:7). Histology distinguished non-atrophic versus atrophic AIG. Atrophy was further subtyped/scored as non-metaplastic versus metaplastic (pseudopyloric (PPM) and intestinal (IM)). Enterochromaffin-like-cell (ECL) status was categorised as diffuse versus adenomatoid hyperplasia/dysplasia, and type 1 neuroendocrine tumours (Type1-NETs). RESULTS: Over the long-term histological follow-up, AIG consistently featured oxyntic-predominant-mononuclear inflammation. At T1, PPM-score was greater than IM (200/211 vs 160/211, respectively); IM scores increased from T1 to T2 (160/211 to 179/211), with no changes in the PPM prevalence (T1=200/211; T2=201/211). At both T1/T2, the prevalence of OLGA-III-stage was <5%; no Operative Link on Gastritis Assessment (OLGA)-IV-stage occurred. ECL-cell-status progressed from diffuse to adenomatoid hyperplasia/dysplasia (T1=167/14 vs T2=151/25). Type1-NETs (T1=10; T2=11) always coexisted with extensive oxyntic-atrophy, and ECL adenomatoid-hyperplasia/dysplasia. No excess risk of gastric or other malignancies was found over a cumulative follow-up time of 10 541 person years, except for (marginally significant) thyroid cancer (SIR=3.09; 95% CI 1.001 to 7.20). CONCLUSIONS: Oxyntic-restricted inflammation, PPM (more than IM), and ECL-cell hyperplasia/neoplasia are the histological AIG hallmarks. Compared with the general population, corpus-restricted inflammation/atrophy does not increase the GC risk. The excess of GC risk reported in patients with AIG could plausibly result from unrecognised previous/current H. pylori comorbidity.

Alterations in p53, Microsatellite Stability and Lack of MUC5AC Expression as Molecular Features of Colorectal Carcinoma Associated with Inflammatory Bowel Disease.

Colitis-associated colorectal carcinoma (CAC) occurs in inflammatory bowel disease (IBD) because of the "chronic inflammation-dysplasia-cancer" carcinogenesis pathway characterized by p53 alterations in the early stages. Recently, gastric metaplasia (GM) has been described as the initial event of the serrated colorectal cancer (CRC) process, resulting from chronic stress on the colon mucosa. The aim of the study is to characterize CAC analyzing p53 alterations and microsatellite instability (MSI) to explore their relationship with GM using a series of CRC and the adjacent intestinal mucosa. Immunohistochemistry was performed to assess p53 alterations, MSI and MUC5AC expression as a surrogate for GM. The p53 mut-pattern was found in more than half of the CAC, most frequently stable (MSS) and MUC5AC negative. Only six tumors were unstable (MSI-H), being with p53 wt-pattern (p = 0.010) and MUC5AC positive (p = 0.005). MUC5AC staining was more frequently observed in intestinal mucosa, inflamed or with chronic changes, than in CAC, especially in those with p53 wt-pattern and MSS. Based on our results, we conclude that, as in the serrated pathway of CRC, in IBD GM occurs in inflamed mucosa, persists in those with chronic changes and disappears with the acquisition of p53 mutations.

Chief cell plasticity is the origin of metaplasia following acute injury in the stomach mucosa.

OBJECTIVE: Metaplasia arises from differentiated cell types in response to injury and is considered a precursor in many cancers. Heterogeneous cell lineages are present in the reparative metaplastic mucosa with response to injury, including foveolar cells, proliferating cells and spasmolytic polypeptide-expressing metaplasia (SPEM) cells, a key metaplastic cell population. Zymogen-secreting chief cells are long-lived cells in the stomach mucosa and have been considered the origin of SPEM cells; however, a conflicting paradigm has proposed isthmal progenitor cells as an origin for SPEM. DESIGN: Gastric intrinsic factor (GIF) is a stomach tissue-specific gene and exhibits protein expression unique to mature mouse chief cells. We generated a novel chief cell-specific driver mouse allele, GIF-rtTA. GIF-GFP reporter mice were used to validate specificity of GIF-rtTA driver in chief cells. GIF-Cre-RnTnG mice were used to perform lineage tracing during homoeostasis and acute metaplasia development. L635 treatment was used to induce acute mucosal injury and coimmunofluorescence staining was performed for various gastric lineage markers. RESULTS: We demonstrated that mature chief cells, rather than isthmal progenitor cells, serve as the predominant origin of SPEM cells during the metaplastic process after acute mucosal injury. Furthermore, we observed long-term label-retaining chief cells at 1 year after the GFP labelling in chief cells. However, only a very small subset of the long-term label-retaining chief cells displayed the reprogramming ability in homoeostasis. In contrast, we identified chief cell-originating SPEM cells as contributing to lineages within foveolar cell hyperplasia in response to the acute mucosal injury. CONCLUSION: Our study provides pivotal evidence for cell plasticity and lineage contributions from differentiated gastric chief cells during acute metaplasia development.

Single-cell transcriptional analyses of spasmolytic polypeptide-expressing metaplasia arising from acute drug injury and chronic inflammation in the stomach.

OBJECTIVE: Spasmolytic polypeptide-expressing metaplasia (SPEM) is a regenerative lesion in the gastric mucosa and is a potential precursor to intestinal metaplasia/gastric adenocarcinoma in a chronic inflammatory setting. The goal of these studies was to define the transcriptional changes associated with SPEM at the individual cell level in response to acute drug injury and chronic inflammatory damage in the gastric mucosa. DESIGN: Epithelial cells were isolated from the gastric corpus of healthy stomachs and stomachs with drug-induced and inflammation-induced SPEM lesions. Single cell RNA sequencing (scRNA-seq) was performed on tissue samples from each of these settings. The transcriptomes of individual epithelial cells from healthy, acutely damaged and chronically inflamed stomachs were analysed and compared. RESULTS: scRNA-seq revealed a population Mucin 6 (Muc6)+gastric intrinsic factor (Gif)+ cells in healthy tissue, but these cells did not express transcripts associated with SPEM. Furthermore, analyses of SPEM cells from drug injured and chronically inflamed corpus yielded two major findings: (1) SPEM and neck cell hyperplasia/hypertrophy are nearly identical in the expression of SPEM-associated transcripts and (2) SPEM programmes induced by drug-mediated parietal cell ablation and chronic inflammation are nearly identical, although the induction of transcripts involved in immunomodulation was unique to SPEM cells in the chronic inflammatory setting. CONCLUSIONS: These data necessitate an expansion of the definition of SPEM to include Tff2+Muc6+ cells that do not express mature chief cell transcripts such as Gif. Our data demonstrate that SPEM arises by a highly conserved cellular programme independent of aetiology and develops immunoregulatory capabilities in a setting of chronic inflammation.

DNA damage signalling as an anti-cancer barrier in gastric intestinal metaplasia.

OBJECTIVE: Intestinal metaplasia (IM) is a premalignant stage that poses a greater risk for subsequent gastric cancer (GC). However, factors regulating IM to GC progression remain unclear. Previously, activated DNA damage response (DDR) signalling factors were shown to engage tumour-suppressive networks in premalignant lesions. Here, we interrogate the relationship of DDR signalling to mutational accumulation in IM lesions. DESIGN: IM biopsies were procured from the gastric cancer epidemiology programme, an endoscopic surveillance programme where biopsies have been subjected to (epi)genomic characterisation. IM samples were classified as genome-stable or genome-unstable based on their mutational burden/somatic copy-number alteration (CNA) profiles. Samples were probed for DDR signalling and cell proliferation, using the markers γH2AX and MCM2, respectively. The expression of the gastric stem cell marker, CD44v9, was also assessed. Tissue microarrays representing the GC progression spectrum were included. RESULTS: MCM2-positivity increased during GC progression, while γH2AX-positivity showed modest increase from normal to gastritis and IM stages, with further increase in GC. γH2AX levels correlated with the extent of chronic inflammation. Interestingly, genome-stable IM lesions had higher γH2AX levels underscoring a protective anti-cancer role for DDR signalling. In contrast, genome-unstable IM lesions with higher mutational burden/CNAs had lower γH2AX levels, elevated CD44v9 expression and modest promoter hypermethylation of DNA repair genes WRN, MLH1 and RAD52. CONCLUSIONS: Our data suggest that IM lesions with active DDR will likely experience a longer latency at the premalignant state until additional hits that override DDR signalling clonally expand and promote progression. These observations provide insights on the factors governing IM progression.

MiR130b from Schlafen4+ MDSCs stimulates epithelial proliferation and correlates with preneoplastic changes prior to gastric cancer.

The myeloid differentiation factor Schlafen4 (Slfn4) marks a subset of myeloid-derived suppressor cells (MDSCs) in the stomach during Helicobacter-induced spasmolytic polypeptide-expressing metaplasia (SPEM). OBJECTIVE: To identify the gene products expressed by Slfn4+-MDSCs and to determine how they promote SPEM. DESIGN: We performed transcriptome analyses for both coding genes (mRNA by RNA-Seq) and non-coding genes (microRNAs using NanoString nCounter) using flow-sorted SLFN4+ and SLFN4- cells from Helicobacter-infected mice exhibiting metaplasia at 6 months postinfection. Thioglycollate-elicited myeloid cells from the peritoneum were cultured and treated with IFNα to induce the T cell suppressor phenotype, expression of MIR130b and SLFN4. MIR130b expression in human gastric tissue including gastric cancer and patient sera was determined by qPCR and in situ hybridisation. Knockdown of MiR130b in vivo in Helicobacter-infected mice was performed using Invivofectamine. Organoids from primary gastric cancers were used to generate xenografts. ChIP assay and Western blots were performed to demonstrate NFκb p65 activation by MIR130b. RESULTS: MicroRNA analysis identified an increase in MiR130b in gastric SLFN4+ cells. Moreover, MIR130b colocalised with SLFN12L, a human homologue of SLFN4, in gastric cancers. MiR130b was required for the T-cell suppressor phenotype exhibited by the SLFN4+ cells and promoted Helicobacter-induced metaplasia. Treating gastric organoids with the MIR130b mimic induced epithelial cell proliferation and promoted xenograft tumour growth. CONCLUSION: Taken together, MiR130b plays an essential role in MDSC function and supports metaplastic transformation.

MicroRNA-92a-1-5p increases CDX2 by targeting FOXD1 in bile acids-induced gastric intestinal metaplasia.

BACKGROUND AND AIMS: Gastric intestinal metaplasia (IM) is common in the gastric epithelium of patients with chronic atrophic gastritis. CDX2 activation in IM is driven by reflux of bile acids and following chronic inflammation. But the mechanism underlying how bile acids activate CDX2 in gastric epithelium has not been fully explored. METHODS: We performed microRNA (miRNA) and messenger RNA (mRNA) profiling using microarray in cells treated with bile acids. Data integration of the miRNA/mRNA profiles with gene ontology (GO) analysis and bioinformatics was performed to detect potential miRNA-mRNA regulatory circuits. Transfection of gastric cancer cell lines with miRNA mimics and inhibitors was used to evaluate their effects on the expression of candidate targets and functions. Immunohistochemistry and in situhybridisation were used to detect the expression of selected miRNAs and their targets in IM tissue microarrays. RESULTS: We demonstrate a bile acids-triggered pathway involving upregulation of miR-92a-1-5p and suppression of its target FOXD1 in gastric cells. We first found that miR-92a-1-5p was increased in IM tissues and induced by bile acids. Moreover, miR-92a-1-5p was found to activate CDX2 and downstream intestinal markers by targeting FOXD1/FOXJ1 axis and modulating activation of nuclear factor kappa B (NF-κB) pathway. Furthermore, these effects were found to be clinical relevant, as high miR-92a-1-5p levels were correlated with low FOXD1 levels and high CDX2 levels in IM tissues. CONCLUSION: These findings suggest a miR-92a-1-5p/FOXD1/NF-κB/CDX2 regulatory axis plays key roles in the generation of IM phenotype from gastric cells. Suppression of miR-92a-1-5p and restoration of FOXD1 may be a preventive approach for gastric IM in patients with bile regurgitation.

Clinicopathological features of duodenal bulb biopsies and their relationship with upper gastrointestinal diseases.

AIM: To assess the prevalence of the lesions in duodenal bulb mucosa and the relationship between duodenal lesions and upper gastrointestinal diseases, including helicobacter pylori infection. METHODS: Clinical, endoscopic and pathological data of the cases with duodenal bulb and gastric mucosal biopsy from January 2005 to May 2017 were analyzed retrospectively. RESULTS: A total of 3540 patients were enrolled. The biopsy from protuberant lesions with endoscopic morphology are mostly duodenal gastric heterotopia or adenoma. The biopsy from duodenal ulcers are often observed in inflammatory changes and gastric metaplasia. Patients with gastric heterotopia had a significantly lower prevalence of chronic atrophic gastritis, intestinal metaplasia, and gastric ulcer; and much higher prevalence of gastroesophageal reflux disease and gastric fundic polyps. Patients with gastric metaplasia had been positively associated with gastroesophageal reflux disease, and negatively associated with gastric fundic polyps. There were positive correlation between helicobacter pylori infection and duodenal active inflammation, Brunner gland hyperplasia, gastric metaplasia and duodenal ulcer. However, Patients with gastric heterotopia in bulb had been negatively associated with helicobacter pylori infection. CONCLUSIONS: The mucosa lesions in duodenal bulb were associated with concurrent gastric fundic gland polyps, gastroesophageal reflux disease, duodenal ulcer, and helicobacter pylori infection.

Serum Level of Trefoil Factor 2 can Predict the Extent of Gastric Spasmolytic Polypeptide-Expressing Metaplasia in the H. pylori-Infected Gastric Cancer Relatives.

BACKGROUND & AIMS: Gastric cancer has familial clustering in incidence, and the familial relatives of gastric cancer sufferers are prone to have spasmolytic polypeptide-expressing metaplasia (SPEM), and intestinal metaplasia (IM) after H. pylori infection. This study tested whether serum pepsinogen I/II and trefoil factor family (TFF) proteins can predict SPEM or IM in the H. pylori-infected relatives of patients with gastric cancer. METHODS: We prospectively enrolled 119 H. pylori-infected relatives of gastric cancer patients of noncardiac gastric cancer patients, who then received panendoscopy to obtain gastric biopsy to define the presence of corpus gastritis index (CGI), SPEM, and IM. The advanced SPEM in histology was defined by TFF2 immunohistochemistry. Each patient also had checkups of serum TFF2, TFF3, and pepsinogen I/II by enzyme-linked immunosorbent assay (ELISA). RESULTS: The 119 H. pylori-infected relatives included 61 with SPEM, and 34 with IM. The presence of either IM or SPEM was not related to the serum TFF2, TFF3, and pepsinogen I/II levels (p > .05). Serum TFF2 levels were higher in relatives with CGI who also had advanced SPEM (p = .032). For relatives without CGI, the elevated serum TFF2 levels correlated with higher H. pylori density and more severe gastritis in antrum (p = .001). CONCLUSION: The serum TFF2 level cannot predict SPEM or IM in H. pylori-infected relatives of patients with gastric cancer. For H. pylori-infected relatives with CGI, serum TFF2 levels may predict the advanced severity of SPEM. Elevated serum TFF2 levels may indicate severe H. pylori-related inflammation, at risk of development or progression of SPEM in relatives without CGI.

miR-30-HNF4γ and miR-194-NR2F2 regulatory networks contribute to the upregulation of metaplasia markers in the stomach.

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.