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Objective: The presence of intestinal metaplasia (IM) is a risk factor for gastric cancer. However, it is still controversial whether IM itself is precancerous or paracancerous. Here, we aimed to explore the precancerous nature of IM by analysing epigenetic alterations., Design: Genome-wide DNA methylation analysis was conducted by EPIC BeadArray using IM crypts isolated by Alcian blue staining. Chromatin immunoprecipitation sequencing for H3K27ac and single-cell assay for transposase-accessible chromatin by sequencing were conducted using IM mucosa. NOS2 was induced using Tet-on gene expression system in normal cells., Results: IM crypts had a methylation profile unique from non-IM crypts, showing extensive DNA hypermethylation in promoter CpG islands, including those of tumour-suppressor genes. Also, the IM-specific methylation profile, namely epigenetic footprint, was present in a fraction of gastric cancers with a higher frequency than expected, and suggested to be associated with good overall survival. IM organoids had remarkably high NOS2 expression, and NOS2 induction in normal cells led to accelerated induction of aberrant DNA methylation, namely epigenetic instability, by increasing DNA methyltransferase activity. IM mucosa showed dynamic enhancer reprogramming, including the regions involved in higher NOS2 expression. NOS2 had open chromatin in IM cells but not in gastric cells, and IM cells had frequent closed chromatin of tumour-suppressor genes, indicating their methylation-silencing. NOS2 expression in IM-derived organoids was upregulated by interleukin-17A, a cytokine secreted by extracellular bacterial infection., Conclusions: IM cells were considered to have a precancerous nature potentially with an increased chance of converting into cancer cells, and an accelerated DNA methylation induction due to abnormal NOS2 expression., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

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., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: SETD2, the sole histone H3K36 trimethyltransferase, is frequently mutated or deleted in human cancer, including pancreatic ductal adenocarcinoma (PDAC). However, whether SETD2/H3K36me3 alteration results in PDAC remains largely unknown., Design: TCGA(PAAD) public database and PDAC tissue array with SETD2/H3K36me3 staining were used to investigate the clinical relevance of SETD2 in PDAC. Furthermore, to define the role of SETD2 in the carcinogenesis of PDAC, we crossed conditional Setd2 knockout mice ( Pdx cre Setd2 flox/flox ) together with Kras G12D mice. Moreover, to examine the role of SETD2 after ductal metaplasia, Crisp/cas9 was used to deplete Setd2 in PDAC cells. RNA-seq and H3K36me3 ChIP-seq were performed to uncover the mechanism., Results: SETD2 mutant/low expression was correlated with poor prognosis in patients with PDAC. Next, we found that Setd2 acted as a putative tumour suppressor in Kras-driven pancreatic carcinogenesis. Mechanistically, Setd2 loss in acinar cells facilitated Kras-induced acinar-to-ductal reprogramming, mainly through epigenetic dysregulation of Fbxw7 . Moreover, Setd2 ablation in pancreatic cancer cells enhanced epithelia-mesenchymal transition (EMT) through impaired epigenetic regulation of Ctnna1 . In addition, Setd2 deficiency led to sustained Akt activation via inherent extracellular matrix (ECM) production, which would favour their metastasis., Conclusion: Together, our findings highlight the function of SETD2 during pancreatic carcinogenesis, which would advance our understanding of epigenetic dysregulation in PDAC. Moreover, it may also pave the way for development of targeted, patients-tailored therapies for PDAC patients with SETD2 deficiency., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

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., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

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., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Objectives: Previous studies have suggested an important role of the transcription factor Gata6 in endocrine pancreas, while GATA6 haploinsufficient inactivating mutations cause pancreatic agenesis in humans. We aimed to analyse the effects of Gata6 inactivation on pancreas development and function., Design: We deleted Gata6 in all epithelial cells in the murine pancreas at the onset of its development. Acinar proliferation, apoptosis, differentiation and exocrine functions were assessed using reverse transcriptase quantitative PCR (RT-qPCR), chromatin immunoprecipitation, immunohistochemistry and enzyme assays. Adipocyte transdifferentiation was assessed using electron microscopy and genetic lineage tracing., Results: Gata6 is expressed in all epithelial cells in the adult mouse pancreas but it is only essential for exocrine pancreas homeostasis: while dispensable for pancreatic development after e10.5, it is required for complete acinar differentiation, for establishment of polarity and for the maintenance of acinar cells in the adult. Gata6 regulates directly the promoter of genes coding for digestive enzymes and the transcription factors Rbpjl and Mist1. Upon pancreas-selective Gata6 inactivation, massive loss of acinar cells and fat replacement take place. This is accompanied by increased acinar apoptosis and proliferation, acinar-to-ductal metaplasia and adipocyte transdifferentiation. By contrast, the endocrine pancreas is spared., Conclusions: Our data show that Gata6 is required for the complete differentiation of acinar cells through multiple transcriptional regulatory mechanisms. In addition, it is required for the maintenance of the adult acinar cell compartment. Our studies suggest that GATA6 alterations may contribute to diseases of the human adult exocrine pancreas.

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.

Background: Little is known about the stem cell organisation of the normal oesophagus or Barrett's metaplastic oesophagus. Using non-pathogenic mitochondrial DNA mutations as clonal markers, the authors reveal the stem cell organisation of the human squamous oesophagus and of Barrett's metaplasia and determine the mechanism of clonal expansion of mutations., Methods: Mutated cells were identified using enzyme histochemistry to detect activity of cytochrome c oxidase (CCO). CCO-deficient cells were laser-captured and mutations confirmed by PCR sequencing. Cell lineages were identified using immunohistochemistry., Results: The normal squamous oesophagus contained CCO-deficient patches varying in size from around 30 μm up to about 1 mm. These patches were clonal as each area within a CCO-deficient patch contained an identical mitochondrial DNA mutation. In Barrett's metaplasia partially CCO-deficient glands indicate that glands are maintained by multiple stem cells. Wholly mutated Barrett's metaplasia glands containing all the expected differentiated cell lineages were seen, demonstrating multilineage differentiation from a clonal population of Barrett's metaplasia stem cells. Patches of clonally mutated Barrett's metaplasia glands were observed, indicating glands can divide to form patches. In one patient, both the regenerating squamous epithelium and the underlying glandular tissue shared a clonal mutation, indicating that they are derived from a common progenitor cell., Conclusion: In normal oesophageal squamous epithelium, a single stem cell clone can populate large areas of epithelium. Barrett's metaplasia glands are clonal units, contain multiple multipotential stem cells and most likely divide by fission. Furthermore, a single cell of origin can give rise to both squamous and glandular epithelium suggesting oesophageal plasticity.

Background and Aims: The mechanism of transformation to intestinal metaplasia in Barrett's oesophagus has not been clarified. We previously reported that bile acids activate the Cdx2 promoter via nuclear factor kappa B (NF-kappaB) and stimulate production of Cdx2 protein in oesophageal keratinocytes with a resulting production of intestinal type mucin. In addition to Cdx2, Cdx1 may play an important role in the development of Barrett's oesophagus. Therefore, we studied the direct effects of bile acids on the expression of Cdx1 as well as the precise mechanisms of Cdx1 expression in cultured oesophageal squamous epithelial cells. Furthermore, we investigated the relationship between Cdx1 and Cdx2 expression in cultured oesophageal squamous epithelial cells., Methods: A rat model of Barrett's oesophagus was produced by anastomosing the oesophagus and jejunum. The expression of Cdx1 was investigated by immunohistochemistry, while the response of that expression to bile acids was studied using a Cdx1 promoter luciferase assay. In addition, oesophageal squamous epithelial cells were transfected with a Cdx1 or Cdx2 expression vector, after which their possible transformation to intestinal-type epithelial cells was investigated., Results: In our Barrett's rat model, the metaplastic epithelium and adjoining squamous epithelium strongly expressed Cdx1. Further, the bile acids mixture dose-dependently increased Cdx1 promoter activity and Cdx1 protein in oesophageal epithelial cells. Transfection of the Cdx1 expression vector in cultured oesophageal epithelial cells induced production of Cdx2 protein., Conclusion: Bile acid-induced sequential expression of Cdx1 followed by Cdx2 may have an important role in the development of Barrett's epithelium.

Background and Aims: Recent studies linked cytokine gene polymorphisms to H pylori related gastric cancer development. The current study evaluated the role of cytokine gene polymorphisms for mucosal cytokine expression, the gastric inflammatory response, and bacterial colonisation during H pylori infection., Patients and Methods: In 207 H pylori infected patients with chronic gastritis, polymorphisms at different loci of the interleukin (IL)-10, IL-1B, IL-1 receptor antagonist (IL-1RN), tumour necrosis factor (TNF)-A, and interferon (IFN)-G genes were genotyped by polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) analysis, and allelic discriminating TaqMan PCR. Mucosal cytokine mRNA copy numbers were determined by real time quantitative PCR. Presence of bacterial virulence factors was investigated by cagA, vacAs1/2, and babA2 PCR. Biopsies were assessed with regard to the degrees of granulocytic/lymphocytic infiltration and the presence of intestinal metaplasia (IM) and atrophic gastritis (AG)., Results: Proinflammatory IL-1 polymorphisms (IL-1RN*2(+)/IL-1B-511T/-31C(+)) were associated with increased IL-1beta expression, more severe degrees of inflammation, and an increased prevalence of IM and AG. Carriers of the IL-10-1082G/-819C/-592C alleles (GCC haplotype) had higher mucosal IL-10 mRNA levels than ATA haplotype carriers and were associated with colonisation by more virulent cagA(+), vacAs1(+), and babA2(+) H pylori strains. The TNF-A-307(G/A) and IFN-G+874(A/T) polymorphisms did not influence mucosal cytokine expression or the inflammatory response to H pylori., Conclusions: Cytokine gene polymorphisms influence mucosal cytokine expression, gastric inflammation, and the long term development of precancerous lesions in H pylori infection. Host polymorphisms are associated with certain bacterial strain types, suggesting host specific colonisation or adaptation. These findings contribute to the understanding of the complex interplay between host and bacterial factors involved in the development of gastric pathology.

Background: Monoclonal precancerous cells undergo successive biochemical and genetic changes during the multistep process of carcinogenesis in the gastrointestinal tract. Despite a high association with intestinal-type stomach cancer (differentiated adenocarcinoma of the stomach), the role of intestinal metaplasia is unclear in stomach carcinogenesis., Aims: To study the clonality of intestinal metaplasia., Methods: The clonality of 86 single intestinal metaplastic glands isolated by EDTA treatment from gastrectomy specimens from patients with cancer were investigated. The methylation sensitive restriction enzyme HpaII and polymerase chain reaction (PCR) were used to detect a polymorphic human androgen receptor gene locus linked to an inactive X chromosome., Results: Forty one (48%) intestinal metaplastic glands were heterotypic (mixed cells of different allelic methylation) and 45 (52%) were homotypic (cell population of the same allelic methylation), while almost all the single pyloric glands were homotypic. Eleven of 13 intestinal metaplastic mucosae that were 6 mm in diameter contained glands that had originated from different cells. There were no strong relationships between clonal type and location or histological type of intestinal metaplasia., Conclusion: Intestinal metaplasia in general is not a lesion that arises or proceeds monoclonally.