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Objectives: We aimed to identify predictive markers for metachronous gastric cancer (MGC) in early gastric cancer (EGC) patients curatively treated with endoscopic submucosal dissection (ESD)., Materials and Methods: From EGC patients who underwent ESD, bulk RNA sequencing was performed on non-cancerous gastric mucosa samples at the time of initial EGC diagnosis. This included 23 patients who developed MGC, and 23 control patients without additional gastric neoplasms for over 3 years (1:1 matched by age, sex, and Helicobacter pylori infection state). Candidate differentially-expressed genes were identified, from which biomarkers were selected using real-time quantitative polymerase chain reaction and cell viability assays using gastric cell lines. An independent validation cohort of 55 MGC patients and 125 controls was used for marker validation. We also examined the severity of gastric intestinal metaplasia, a known premalignant condition, at initial diagnosis., Results: From the discovery cohort, 86 candidate genes were identified of which KDF1 and CDK1 were selected as markers for MGC, which were confirmed in the validation cohort. CERB5 and AKT2 isoform were identified as markers related to intestinal metaplasia and were also highly expressed in MGC patients compared to controls (p < 0.01). Combining these markers with clinical data (age, sex, H. pylori and severity of intestinal metaplasia) yielded an area under the curve (AUC) of 0.91 (95% CI, 0.85-0.97) for MGC prediction., Conclusion: Assessing biomarkers in non-cancerous gastric mucosa may be a useful method for predicting MGC in EGC patients and identifying patients with a higher risk of developing MGC, who can benefit from rigorous surveillance., (© 2024 The Author(s). Cancer Medicine published by John Wiley & Sons Ltd.)

Background/aims: While DNA methylation and gastric microbiome are each associated with gastric cancer (GC), their combined role in predicting GC remains unclear. This study investigated the potential of a combined DNA methylation and gastric microbiome signature to predict Helicobacter pylori -negative GC., Methods: In this case-control study, we conducted quantitative methylation-specific polymerase chain reaction to measure the methylation levels of DKK3 , SFRP1 , EMX1 , NKX6-1 , MIR124-3 , and TWIST1 in the gastric mucosa from 75 H. pylori -negative patients, including chronic gastritis (CG), intestinal metaplasia (IM), and GC. A combined analysis of DNA methylation and gastric microbiome, using 16S rRNA gene sequencing, was performed in 30 of 75 patients., Results: The methylation levels of DKK3 , SFRP1 , EMX1 , MIR124-3 , and TWIST1 were significantly higher in patients with GC than in controls (all q<0.05). MIR124-3 and TWIST1 methylation levels were higher in patients with IM than those with CG and also in those with GC than in those with IM (all q<0.05). A higher methylation level of TWIST1 was an independent predictor for H. pylori -negative GC after adjusting for age, sex, and atrophy (odds ratio [OR], 15.15; 95% confidence interval [CI], 1.58 to 145.46; p=0.018). The combination of TWIST1 methylation and GC microbiome index (a microbiome marker) was significantly associated with H. pylori -negative GC after adjusting for age, sex, and atrophy (OR, 50.00; 95% CI, 1.69 to 1,476; p=0.024)., Conclusions: The combination of TWIST1 methylation and GC microbiome index may offer potential as a biomarker for predicting H. pylori -negative GC.

Background: Intestinal metaplasia (IM) is classified into complete intestinal metaplasia (CIM) and incomplete intestinal metaplasia (IIM). Patients diagnosed with IIM face an elevated susceptibility to the development of gastric cancer, underscoring the critical need for early screening measures. In addition to the complexities associated with diagnosis, the exact mechanisms driving the progression of gastric cancer in IIM patients remain poorly understood. OLFM4 is overexpressed in several types of tumors, including colorectal, gastric, pancreatic, and ovarian cancers, and its expression has been associated with tumor progression., Methods: In this study, we used pathological sections from two clinical centers, biopsies of IM tissues, precancerous lesions of gastric cancer (PLGC) cell models, animal models, and organoids to explore the role of OLFM4 in IIM., Results: Our results show that OLFM4 expression is highly increased in IIM, with superior diagnostic accuracy of IIM when compared to CDX2 and MUC2. OLFM4, along with MYH9, was overexpressed in IM organoids and PLGC animal models. Furthermore, OLFM4, in combination with Myosin heavy chain 9 (MYH9), accelerated the ubiquitination of GSK3β and resulted in increased β-catenin levels through the Wnt signaling pathway, promoting the proliferation and invasion abilities of PLGC cells., Conclusions: OLFM4 represents a novel biomarker for IIM and could be utilized as an important auxiliary means to delimit the key population for early gastric cancer screening. Finally, our study identifies cell signaling pathways involved in the progression of IM., (© 2024. The Author(s).)

Objective: To explore the potential pathogenic genes of intestinal metaplasia., Methods: Twenty-one patients with intestinal metaplasia admitted to the Department of Gastroenterology at the Second Affiliated Hospital of Anhui University of Chinese Medicine from January, 2022 to June, 2022, and 21 healthy subjects undergoing gastroscopic examination during the same period were enrolled in this study. All the participants underwent gastroscopy and pathological examination, and gastric tissue samples were collected for transcriptome sequencing to screen for differentially expressed genes (DEGs). The biological functions of the DEGs were analyzed using bioinformatics analysis, and qRT-PCR was used to validate the results., Results: Transcriptomic sequencing identified a total of 1373 DEGs, including 827 upregulated and 546 downregulated ones. The top 6 upregulated genes ( AGMAT , CCL25 , FABP1 , CDX1 , SPINK4 , and MUC2 ), ranked based on their significance and average expression level, were selected for validation, and qRT-PCR showed significant upregulation of their mRNAs in the gastric tissues of patients with intestinal metaplasia ( P < 0.05)., Conclusion: AGMAT , CCL25 , FABP1 , CDX1 , SPINK4 , and MUC2 participate in the occurrence and development of intestinal metaplasia, and may serve as potential biomarkers for diagnosing intestinal metaplasia.

Pancreatic ductal adenocarcinoma (PDAC) can originate from acinar-to-ductal metaplasia (ADM). Pancreatic acini harboring oncogenic Kras mutations are transdifferentiated to a duct-like phenotype that further progresses to become pancreatic intraepithelial neoplasia (PanIN) lesions, giving rise to PDAC. Although ADM formation is frequently observed in Kras G12D transgenic mouse models of PDAC, the exact mechanisms of how oncogenic Kras G12D regulates this process remain an enigma. Herein, we revealed a new downstream target of oncogenic Kras, cytokine CCL9, during ADM formation. Higher levels of CCL9 and its receptors, CCR1 and CCR3, were detected in ADM regions of the pancreas in p48 cre :Kras G12D mice and human PDAC patients. Knockdown of CCL9 in Kras G12D -expressed pancreatic acini reduced Kras G12D -induced ADM in a 3D organoid culture system. Moreover, exogenously added recombinant CCL9 and overexpression of CCL9 in primary pancreatic acini induced pancreatic ADM. We also showed that, functioning as a downstream target of Kras G12D , CCL9 promoted pancreatic ADM through upregulation of the intracellular levels of reactive oxygen species (ROS) and metalloproteinases (MMPs), including MMP14, MMP3 and MMP2. Blockade of MMPs via its generic inhibitor GM6001 or knockdown of specific MMP such as MMP14 and MMP3 decreased CCL9-induced pancreatic ADM. In p48 cre :Kras G12D transgenic mice, blockade of CCL9 through its specific neutralizing antibody attenuated pancreatic ADM structures and PanIN lesion formation. Furthermore, it also diminished infiltrating macrophages and expression of MMP14, MMP3 and MMP2 in the ADM areas. Altogether, our results provide novel mechanistic insight into how oncogenic Kras enhances pancreatic ADM through its new downstream target molecule, CCL9, to initiate PDAC.

Background: Gastric intestinal metaplasia(GIM) is an independent risk factor for GC, however, its pathogenesis is still unclear. Ferroptosis is a new type of programmed cell death, which may be involved in the process of GIM. The purpose of this study was to analyze the expression of ferroptosis-related genes (FRGs) in GIM tissues and to explore the relationship between ferroptosis and GIM., Method: The results of GIM tissue full transcriptome sequencing were downloaded from Gene Expression Omnibus(GEO) database. R software (V4.2.0) and R packages were used for screening and enrichment analysis of differentially expressed genes(DEGs). The key genes were screened by least absolute shrinkage and selection operator(LASSO) and support vector machine-recursive feature elimination(SVM-RFE) algorithm. Receiver operating characteristic(ROC) curve was used to evaluate the diagnostic efficacy of key genes in GIM. Clinical samples were used to further validate hub genes., Results: A total of 12 differentially expressed ferroptosis-related genes (DEFRGs) were identified. Using two machine learning algorithms, GOT1, ALDH3A2, ACSF2 and SESN2 were identified as key genes. The area under ROC curve (AUC) of GOT1, ALDH3A2, ACSF2 and SESN2 in the training set were 0.906, 0.955, 0.899 and 0.962 respectively, and the AUC in the verification set were 0.776, 0.676, 0.773 and 0.880, respectively. Clinical samples verified the differential expression of GOT1, ACSF2, and SESN2 in GIM., Conclusion: We found that there was a significant correlation between ferroptosis and GIM. GOT1, ACSF2 and SESN2 can be used as diagnostic markers to effectively identify GIM.

Background & Aims: Barrett's esophagus is the precursor of esophageal dysplasia and esophageal adenocarcinoma. CDKN2A-p16 deletions were reported in 34%-74% of patients with Barrett's esophagus who progressed to dysplasia and esophageal adenocarcinoma, suggesting that p16 loss may drive neoplastic progression. KRAS activation frequently occurs in esophageal adenocarcinoma and precancer lesions. LGR5 + stem cells in the squamocolumnar-junction (SCJ) of mouse stomach contribute as Barrett's esophagus progenitors. We aimed to determine the functional effects of p16 loss and KRAS activation in Barrett's-like metaplasia and dysplasia development., Methods: We established mouse models with conditional knockout of CDKN2A-p16 (p16KO) and/or activated KRAS G12D expression targeting SCJ LGR5 + cells in interleukin 1b transgenic mice and characterized histologic alterations (mucous-gland hyperplasia/metaplasia, inflammation, and dysplasia) in mouse SCJ. Gene expression was determined by microarray, RNA sequencing, and immunohistochemistry of SCJ tissues and cultured 3-dimensional organoids., Results: p16KO mice exhibited increased mucous-gland hyperplasia/metaplasia versus control mice (P = .0051). Combined p16KO+KRAS G12D resulted in more frequent dysplasia and higher dysplasia scores (P = .0036), with 82% of p16KO+KRAS G12D mice developing high-grade dysplasia. SCJ transcriptome analysis showed several activated pathways in p16KO versus control mice (apoptosis, tumor necrosis factor-α/nuclear factor-kB, proteasome degradation, p53 signaling, MAPK, KRAS, and G1-to-S transition)., Conclusions: p16 deletion in LGR5 + cell precursors triggers increased SCJ mucous-gland hyperplasia/metaplasia. KRAS G12D synergizes with p16 deletion resulting in higher grades of SCJ glandular dysplasia, mimicking Barrett's high-grade dysplasia. These genetically modified mouse models establish a functional role of p16 and activated KRAS in the progression of Barrett's-like lesions to dysplasia in mice, representing an in vivo model of esophageal adenocarcinoma precancer. Derived 3-dimensional organoid models further provide in vitro modeling opportunities of esophageal precancer stages., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Metaplastic breast carcinoma (MpBC) typically consists of carcinoma of no special type (NST) with various metaplastic components. Although previous transcriptomic and proteomic studies have reported subtype-related heterogeneity, the intracase transcriptomic alterations between metaplastic components and paired NST components, which are critical for understanding the pathogenesis underlying the metaplastic processes, remain unclear., Methods: Fifty-nine NST components and paired metaplastic components (spindle carcinomatous [SPS], matrix-producing, rhabdoid [RHA], and squamous carcinomatous [SQC] components) were microdissected from specimens obtained from 27 patients with MpBC for gene expression profiling using the NanoString Breast Cancer 360 Panel on a NanoString nCounter FLEX platform. BC360-defined signatures were scored using nSolver software., Results: Hierarchical clustering and principal component analysis revealed a heterogeneous gene expression profile (GEP) corresponding to the NST components, but the GEP of metaplastic components exhibited subtype dependence. Compared with the paired NST components, the SPS components demonstrated the upregulation of genes related to stem cells and epithelial-mesenchymal transition and displayed enrichment in claudin-low and macrophage signatures. Despite certain overlaps in the enriched functions and signatures between the RHA and SPS components, the specific differentially expressed genes differed. We observed the RHA-specific upregulation of genes associated with vascular endothelial growth factor signaling. The chondroid matrix-producing components demonstrated the upregulation of hypoxia-related genes and the downregulation of the immune-related MHC2 signature and the TIGIT gene. In the SQC components, TGF-β and genes associated with cell adhesion were upregulated. The differentially expressed genes among metaplastic components in the 22 MpBC cases with one or predominantly one metaplastic component clustered paired NST samples into clusters with correlation with their associated metaplastic types. These genes could be used to separate the 31 metaplastic components according to respective metaplastic types with an accuracy of 74.2%, suggesting that intrinsic signatures of NST may determine paired metaplastic type. Finally, the EMT activity and stem cell traits in the NST components were correlated with specimens displaying lymph node metastasis., Conclusions: We presented the distinct transcriptomic alterations underlying metaplasia into specific metaplastic components in MpBCs, which contributes to the understanding of the pathogenesis underlying morphologically distinct metaplasia in MpBCs., (© 2023. The Author(s).)

Background: Intestinal metaplasia (IM) of the gastric cardia is an important premalignant lesion. However, there is limited information concerning its epidemiological and molecular features. Herein, we aimed to provide an overview of the epidemiological data for gastric cardiac IM and evaluate the role of EYA transcriptional coactivator and phosphatase 4 (EYA4) as an epigenetic biomarker for gastric cardiac IM., Methods: The study was conducted in the context of the gastric cardiac precancerous lesion program in southern China, which included 718 non-cancer participants, who undertook endoscopic biopsy and pathological examination in three endoscopy centers, between November 2018 and November 2021. Pyrosequencing and immunohistochemistry were performed to examine the DNA methylation status and protein expression level of EYA4., Results: Gastric cardiac IM presented in 14.1% (101/718) of participants and was more common among older (>50 years; 22.0% [95% CI: 17.8-26.8]) than younger participants (≤50 years; 6.7% [95% CI: 4.5-9.9]; P < 0.001). IM was more common in male participants (16.9% [95% CI: 13.2-21.3] vs. 11.3% [95% CI: 8.3-15.1]; P = 0.04). Pyrosequencing revealed that IM tissues exhibited significantly higher DNA methylation levels in EYA4 gene than normal tissues (P = 0.016). Further, the protein expression level of EYA4 was reduced in IM and absent in intraepithelial neoplasia tissues compared to normal tissues (P < 0.001)., Conclusions: Detection rates of gastric cardiac IM increase with age and are higher in men. Our findings highlight the important role of promoter hypermethylation and downregulation of EYA4 in gastric cardiac IM development., Competing Interests: None

Intestinal metaplasia (IM) is a risk factor for gastric cancer following infection with Helicobacter pylori. To explore the susceptibility of pure gastric IM to cancer development, we investigated genetic alterations in single IM gastric glands. We isolated 50 single IM or non-IM glands from the inflamed gastric mucosa of 11 patients with intramucosal gastric carcinoma (IGC) and 4 patients without IGC; 19 single glands in the noninflamed gastric mucosa of 11 individuals from our cohort and previous dataset were also included as controls. Whole-exome sequencing of single glands revealed significantly higher accumulation of somatic mutations in various genes within IM glands compared with non-IM glands. Clonal ordering analysis showed that IM glands expanded to form clusters with shared mutations. In addition, targeted-capture deep sequencing and copy number (CN) analyses were performed in 96 clustered IM or non-IM gastric glands from 26 patients with IGC. CN analyses were also performed on 41 IGC samples and The Cancer Genome Atlas-Stomach Adenocarcinoma datasets. These analyses revealed that polyclonally expanded IM commonly acquired CN aberrations (CNA), including amplification of chromosomes 8, 20, and 2. A large portion of clustered IM glands typically consisted of common CNAs rather than other cancer-related mutations. Moreover, the CNA patterns of clustered IM glands were similar to those of IGC, indicative of precancerous conditions. Taken together, these findings suggest that, in the gastric mucosa inflamed with H. pylori infection, IM glands expand via acquisition of CNAs comparable with those of IGC, contributing to field cancerization., Significance: This study contributes to our understanding of gastric intestinal metaplasia as a risk factor for gastric adenocarcinoma via their multifocal expansion and acquisition of CNAs and somatic mutations., (©2022 American Association for Cancer Research.)

Introduction: High expression of HOTAIR promotes tumor growth and carries a dismal prognosis for the patient. We investigated the prognostic value of HOTAIR expression in gastric cancer (GC) and systematically delineate the expression in relation to Helicobacter pylori infection and preneoplastic changes., Methods: HOTAIR expression was analyzed in surgical paired tissue samples of patients with GC and biopsy samples from patients with atrophic gastritis and/or intestinal metaplasia (AG ± -IM), chronic nonatrophic gastritis, and controls. The cancer genome atlas (TCGA) data were used for validation. HOTAIR expression was evaluated in sera and ascites of patients with GC. Quantitative HOTAIR expression analysis was performed using quantitative polymerase chain reaction, and LINE-1 methylation was assessed by bisulfite pyrosequencing., Results: HOTAIR was more frequently detected in tumor tissues compared with adjacent gastric mucosa (65.4% vs 8.6%). HOTAIR expression was associated with depth of tumor invasion and tumor location and with shorter overall survival in patients with diffuse-type GC as confirmed in the TCGA cohort. HOTAIR was not detectable in controls but was found in 2.2% of patients with chronic nonatrophic gastritis and 18.3% of patients with AG ± IM, which was further associated with IM, grade of IM, and H. pylori positivity., Discussion: HOTAIR expression was associated with GC and preneoplastic changes of stomach mucosa. Although HOTAIR expression was strongly linked to IM, HOTAIR expression was only associated with worse prognosis in Lauren diffuse and not intestinal type of GC. Further studies are needed to evaluate the value of HOTAIR as diagnostic and predictive biomarker in IM and translational therapeutic relevance of HOTAIR in diffuse-type GC., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The American College of Gastroenterology.)

Background: Helicobacter pylori infection is the most important risk factor for gastric cancer (GC). Human gastric adenocarcinoma develops after long-term H. pylori infection via the Correa cascade. This carcinogenic pathway describes the progression from gastritis to atrophy, intestinal metaplasia (IM), dysplasia and GC. Patients with atrophy and intestinal metaplasia are considered to have precancerous lesions of GC (PLGC). H. pylori eradication and endoscopy surveillance are currently the only interventions for preventing GC. Better knowledge of the biology of human PLGC may help find stratification markers and contribute to better understanding of biological mechanisms. One way to achieve this is by using co-expression network analysis. Weighted gene co-expression network analysis (WGCNA) is often used to identify modules from co-expression networks and relate them to clinical traits. It also allows identification of driver genes that may be critical for PLGC., Aim: The purpose of this study was to identify co-expression modules and differential gene expression in dyspeptic patients at different stages of the Correa pathway., Methods: We studied 96 gastric biopsies from 78 patients that were clinically classified as: non-active (n = 10) and chronic-active gastritis (n = 20), atrophy (n = 12), and IM (n = 36). Gene expression of coding RNAs was determined by microarrays and non-coding RNAs by RNA-seq. The WGCNA package was used for network construction, module detection, module preservation and hub and driver gene selection., Results: WGCNA identified 20 modules for coding RNAs and 4 for each miRNA and small RNA class. Modules were associated with antrum and corpus gastric locations, chronic gastritis and IM. Notably, coding RNA modules correlated with the Correa cascade. One was associated with the presence of H. pylori. In three modules, the module eigengene (ME) gradually increased in the stages toward IM, while in three others the inverse relationship was found. One miRNA module was negatively correlated to IM and was used for a mRNA-miRNA integration analysis. WGCNA also uncovered driver genes. Driver genes show both high connectivity within a module and are significantly associated with clinical traits. Some of those genes have been previously involved in H. pylori carcinogenesis, but others are new. Lastly, using similar external transcriptomic data, we confirmed that the discovered mRNA modules were highly preserved., Conclusion: Our analysis captured co-expression modules that provide valuable information to understand the pathogenesis of the progression of PLGC., Competing Interests: Declaration of Competing Interest The authors declare no competing interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Cells recognize both foreign and host-derived double-stranded RNA (dsRNA) via a signaling pathway that is usually studied in the context of viral infection. It has become increasingly clear that the sensing and handling of endogenous dsRNA is also critical for cellular differentiation and development. The adenosine RNA deaminase, ADAR1, has been implicated as a central regulator of the dsRNA response, but how regulation of the dsRNA response might mediate cell fate during injury and whether such signaling is cell intrinsic remain unclear. Here, we show that the ADAR1-mediated response to dsRNA was dramatically induced in 2 distinct injury models of gastric metaplasia. Mouse organoid and in vivo genetic models showed that ADAR1 coordinated a cell-intrinsic, epithelium-autonomous, and interferon signaling-independent dsRNA response. In addition, dsRNA accumulated within a differentiated epithelial population (chief cells) in mouse and human stomachs as these cells reprogrammed to a proliferative, reparative (metaplastic) state. Finally, chief cells required ADAR1 to reenter the cell cycle during metaplasia. Thus, cell-intrinsic ADAR1 signaling is critical for the induction of metaplasia. Because metaplasia increases cancer risk, these findings support roles for ADAR1 and the response to dsRNA in oncogenesis.

Background & Aims: Acinar to ductal metaplasia (ADM) occurs in the pancreas in response to tissue injury and is a potential precursor for adenocarcinoma. The goal of these studies was to define the populations arising from ADM, the associated transcriptional changes, and markers of disease progression., Methods: Acinar cells were lineage-traced with enhanced yellow fluorescent protein (EYFP) to follow their fate post-injury. Transcripts of more than 13,000 EYFP+ cells were determined using single-cell RNA sequencing (scRNA-seq). Developmental trajectories were generated. Data were compared with gastric metaplasia, Kras G12D -induced neoplasia, and human pancreatitis. Results were confirmed by immunostaining and electron microscopy. Kras G12D was expressed in injury-induced ADM using several inducible Cre drivers. Surgical specimens of chronic pancreatitis from 15 patients were evaluated by immunostaining., Results: scRNA-seq of ADM revealed emergence of a mucin/ductal population resembling gastric pyloric metaplasia. Lineage trajectories suggest that some pyloric metaplasia cells can generate tuft and enteroendocrine cells (EECs). Comparison with Kras G12D -induced ADM identifies populations associated with disease progression. Activation of Kras G12D expression in HNF1B+ or POU2F3+ ADM populations leads to neoplastic transformation and formation of MUC5AC+ gastric-pit-like cells. Human pancreatitis samples also harbor pyloric metaplasia with a similar transcriptional phenotype., Conclusions: Under conditions of chronic injury, acinar cells undergo a pyloric-type metaplasia to mucinous progenitor-like populations, which seed disparate tuft cell and EEC lineages. ADM-derived EEC subtypes are diverse. Kras G12D expression is sufficient to drive neoplasia when targeted to injury-induced ADM populations and offers an alternative origin for tumorigenesis. This program is conserved in human pancreatitis, providing insight into early events in pancreas diseases., (Copyright © 2022 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background & Aims: RUNX transcription factors play pivotal roles in embryonic development and neoplasia. We previously identified the single missense mutation R122C in RUNX3 from human gastric cancer. However, how RUNX3 R122C mutation disrupts stem cell homeostasis and promotes gastric carcinogenesis remained unclear., Methods: To understand the oncogenic nature of this mutation in vivo, we generated the RUNX3 R122C knock-in mice. Stomach tissues were harvested, followed by histologic and immunofluorescence staining, organoid culture, flow cytometry to isolate gastric corpus isthmus and nonisthmus epithelial cells, and RNA extraction for transcriptomic analysis., Results: The corpus tissue of RUNX3 R122C/R122C homozygous mice showed a precancerous phenotype such as spasmolytic polypeptide-expressing metaplasia. We observed mucous neck cell hyperplasia; massive reduction of pit, parietal, and chief cell populations; as well as a dramatic increase in the number of rapidly proliferating isthmus stem/progenitor cells in the corpus of RUNX3 R122C/R122C mice. Transcriptomic analyses of the isolated epithelial cells showed that the cell-cycle-related MYC target gene signature was enriched in the corpus epithelial cells of RUNX3 R122C/R122C mice compared with the wild-type corpus. Mechanistically, RUNX3 R122C mutant protein disrupted the regulation of the restriction point where cells decide to enter either a proliferative or quiescent state, thereby driving stem cell expansion and limiting the ability of cells to terminally differentiate., Conclusions: RUNX3 R122C missense mutation is associated with the continuous cycling of isthmus stem/progenitor cells, maturation arrest, and development of a precancerous state. This work highlights the importance of RUNX3 in the prevention of metaplasia and gastric cancer., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Barrett's esophagus (BE) and gastric intestinal metaplasia are related premalignant conditions in which areas of human stomach epithelium express mixed gastric and intestinal features. Intestinal transcription factors (TFs) are expressed in both conditions, with unclear causal roles and cis -regulatory mechanisms. Ectopic CDX2 reprogrammed isogenic mouse stomach organoid lines to a hybrid stomach-intestinal state transcriptionally similar to clinical metaplasia; squamous esophageal organoids resisted this CDX2-mediated effect. Reprogramming was associated with induced activity at thousands of previously inaccessible intestine-restricted enhancers, where CDX2 occupied DNA directly. HNF4A, a TF recently implicated in BE pathogenesis, induced weaker intestinalization by binding a novel shadow Cdx2 enhancer and hence activating Cdx2 expression. CRISPR/Cas9-mediated germline deletion of that cis -element demonstrated its requirement in Cdx2 induction and in the resulting activation of intestinal genes in stomach cells. dCas9-conjugated KRAB repression mapped this activity to the shadow enhancer's HNF4A binding site. Altogether, we show extensive but selective recruitment of intestinal enhancers by CDX2 in gastric cells and that HNF4A-mediated ectopic CDX2 expression in the stomach occurs through a conserved shadow cis -element. These findings identify mechanisms for TF-driven intestinal metaplasia and a likely pathogenic TF hierarchy., (© 2022 Singh et al.; Published by Cold Spring Harbor Laboratory Press.)

This study aimed to screen the potential candidate genes and relevant biological markers associated with gastrointestinal metaplasia that progresses to gastric cancer (GIM-GC). Microarray datasets (GSE78523) were downloaded from the GEO database. Differentially expressed genes (DEGs) between GIM-GC samples and healthy controls were identified. GO and KEGG pathway enrichment analyses were performed. STRING and Cytoscape were used to identify significant module and hub genes. Survival analysis was applied to identify key genes. A Venn diagram was built to find hub DEGs that differed in all three relevant comparisons (GIM-GC vs. healthy controls vs. GIM-NoGC). The clinical characteristics of the hub DEGs were evaluated using the Cancer Genome Atlas dataset. The study found 257 DEGs (217 upregulated and 40 downregulated). The upregulated DEGs were enriched in regulation of microvillus length and phospholipid binding and were components of the apical plasma membrane. Downregulated DEGs were involved in digestion and hormone activity and were found in the extracellular space. Fat digestion and absorption as well as gastric acid secretion were the pathways enrichment. The most important gene modules related mainly to O-glycan processing, extracellular exosome, hormone activity, and vitamin and fat digestion and absorption. Eleven hub genes were identified, of which APOB, FABP1, CDX2, GCG, HNF4A, SLC26A3, CFTR, MUC5AC, OLFM4, and SI were related to the prognosis. Olfactomedin-4 (OLFM4) was the most relevant DEG to identify GIM-GC. In conclusion: DEGs and hub genes are helpful to understand the molecular mechanisms of GIM-GC. OLFM4 may be a biological marker for GIM-GC.

Differentiated cells across multiple species and organs can re-enter the cell cycle to aid in injury-induced tissue regeneration by a cellular program called paligenosis. Here, we show that activating transcription factor 3 (ATF3) is induced early during paligenosis in multiple cellular contexts, transcriptionally activating the lysosomal trafficking gene Rab7b. ATF3 and RAB7B are upregulated in gastric and pancreatic digestive-enzyme-secreting cells at the onset of paligenosis Stage 1, when cells massively induce autophagic and lysosomal machinery to dismantle differentiated cell morphological features. Their expression later ebbs before cells enter mitosis during Stage 3. Atf3 -/- mice fail to induce RAB7-positive autophagic and lysosomal vesicles, eventually causing increased death of cells en route to Stage 3. Finally, we observe that ATF3 is expressed in human gastric metaplasia and during paligenotic injury across multiple other organs and species. Thus, our findings indicate ATF3 is an evolutionarily conserved gene orchestrating the early paligenotic autodegradative events that must occur before cells are poised to proliferate and contribute to tissue repair., (© 2021 The Authors.)

Adenomatous Polyposis Coli (APC) is lost in approximately 70% of sporadic breast cancers, with an inclination towards triple negative breast cancer (TNBC). TNBC is treated with traditional chemotherapy, such as paclitaxel (PTX); however, tumors often develop drug resistance. We previously created APC knockdown cells (APC shRNA1) using the human TNBC cells, MDA-MB-157, and showed that APC loss induces PTX resistance. To understand the mechanisms behind APC-mediated PTX response, we performed cell cycle analysis and analyzed cell cycle related proteins. Cell cycle analysis indicated increased G2/M population in both PTX-treated APC shRNA1 and parental cells, suggesting that APC expression does not alter PTX-induced G2/M arrest. We further studied the subcellular localization of the G2/M transition proteins, cyclin B1 and CDK1. The APC shRNA1 cells had increased CDK1, which was preferentially localized to the cytoplasm, and increased baseline CDK6. RNA-sequencing was performed to gain a global understanding of changes downstream of APC loss and identified a broad mis-regulation of cell cycle-related genes in APC shRNA1 cells. Our studies are the first to show an interaction between APC and taxane response in breast cancer. The implications include designing combination therapy to re-sensitize APC-mutant breast cancers to taxanes using the specific cell cycle alterations., Competing Interests: The authors have declared that no competing interests exist.

Prognosis of gastric cancer is dramatically improved by early diagnosis. Correa's cascade correlates the expression of some molecular markers with the progression of preneoplastic lesions toward carcinoma. This article reviews the diagnostic and prognostic values of molecular markers in complete ( MUC2 ) and incomplete ( MUC2, MUC5AC , and MUC6 ) intestinal metaplasia, gastric dysplasia/intra-epithelial neoplasia, and early gastric cancer. In particular, considering preinvasive neoplasia and early gastric cancer, some studies have demonstrated a correlation between molecular alterations and prognosis, for example, mucins phenotype in gastric dysplasia, and GATA6, TP53 mutation/LOH and MUC6 in early gastric cancer. Moreover, this review considers novelties from the literature regarding the (immuno)histochemical characterization of diffuse-type/signet ring cell gastric cancer, with particular attention to clinical outcomes of patients. The aim of this review is the evaluation of the state of the art regarding suitable biomarkers used in the pre-surgical phase, which can distinguish patients with different prognoses and help decide the best therapeutic strategy.

Mucus-secreting goblet cells are the dominant cell type in pulmonary diseases, e.g., asthma and cystic fibrosis (CF), leading to pathologic mucus metaplasia and airway obstruction. Cytokines including IL-13 are the major players in the transdifferentiation of club cells into goblet cells. Unexpectedly, we have uncovered a previously undescribed pathway promoting mucous metaplasia that involves VEGFa and its receptor KDR. Single-cell RNA sequencing analysis coupled with genetic mouse modeling demonstrates that loss of epithelial VEGFa, KDR, or MEK/ERK kinase promotes excessive club-to-goblet transdifferentiation during development and regeneration. Sox9 is required for goblet cell differentiation following Kdr inhibition in both mouse and human club cells. Significantly, airway mucous metaplasia in asthmatic and CF patients is also associated with reduced KDR signaling and increased SOX9 expression. Together, these findings reveal an unexpected role for VEGFa/KDR signaling in the defense against mucous metaplasia, offering a potential therapeutic target for this common airway pathology., Competing Interests: Declaration of interests A patent application (CU21016, “SUPPRESSION OF KDR, VEGF OR THE DOWNSTREAM MEK/ERK KINASE PATHWAY”) related to this work has been filed by Columbia Technology Ventures. M.J. and J.Q. are listed as co-inventors on this application., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Gastric cancer (GC) remains one of the most common causes of mortality worldwide. Intestinal metaplasia (IM) is one of the preneoplastic gastric lesions and is considered an essential predisposing factor in GC development. Here we present a review of recent most relevant papers to summarize major findings on the molecular alterations in gastric IM. The latest progress in novel diagnostic methods allows scientists to identify various types of molecular alterations in IM, such as polymorphisms in various genes, changes in the expression of micro-RNAs and long noncoding RNAs, and altered microbiome profiles. The results have shown that some of these alterations have strong associations with IM and a potential to be used for screening, treatment, and prognostic purposes; however, one of the most important limiting factors is the inhomogeneity of the studies. Therefore, further large-scale studies and clinical trials with standardized methods designed by multicenter consortiums are needed. As of today, various molecular alterations in IM could become a part of personalized medicine in the near future, which would help us deliver a personalized approach for each patient and identify those at risk of progression to GC.

Background: Cytokines and their gene variants are proven to play a role in pathogenic gastritis and carcinogenesis. The study assesses associations of the cytokine gene polymorphisms with extension of atrophic gastritis/intestinal metaplasia (AGIM) in patients without Helicobacter pylori infection on immunohistochemistry study. Methods: 224 adult consecutive patients undergoing an upper digestive endoscopy were included and grouped according to localization of AGIM: 37 patients with antrum-limited AGIM, 21 corpus-limited AGIM, 15 extended-AGIM (antrum and corpus) and 151 patients had no AGIM. Medical records of the patients were checked and a structured direct interview was applied in order to collect clinical data, including digestive symptoms. In all cases, IFN-γ +874T>A, TGF-β1 +869T>C, TNF-α -308G>A and -238G>A, and IL-6 - 174C>G polymorphisms were genotyped. Results: The mean age was significantly higher in the AGIM group, while the comorbidies were similar among patients with different localization of lesions or in patients without AGIM. There were no significant differences in digestive symptoms, nor in the consumption of non-steroidal anti-inflammatory drugs or proton pump inhibitor with the different extensions of AGIM. There was a significant association between oral anticoagulant consumption and localization of AGIM ( P = 0.042), frequency being higher among patients with corpus-limited AGIM than those with no AGIM ( P = 0.007, adjusted P = 0.041). TGF-β1 +869T>C was less frequent among patients with corpus-limited AGIM ( n =7, 33.3%) and extended AGIM ( n =5, 33.3%) than in antrum-limited AGIM ( n =25, 67.6%). There were no other significant differences regarding variant and wild genotype frequencies of IFN-γ +874T>A (86.5%, 81.0%, 86.7%, p=0.814), TNF-α -308G>A (35.1%, 28.6%, 53.3%, p=0.48) and IL-6 - 174C>G (70.3%. 61.9%, 73.3% p=0.656) among patients with antrum-limited, corpus-limited or extended AGIM. TGF-β1 +869T>C was associated with a decreased risk for corpus-affected AGIM (adjusted odds ratio: 0.42, 95% confidence interval: 0.19-0.93, P = 0.032). The dominant inheritance models no revealed significant association for IFN-γ +874T>A, TNF-α -308G>A and IL-6 - 174C>G gene polymorphism and the risk of localization of AGIM. Conclusion: TGF-β1 +869T>C gene polymorphism is associated with a decreased risk for corporeal localization of premalignant lesions, while IFN-γ +874T>A, TNF-α -308G>A and IL-6 - 174C>G are not associated with the risk for AGIM in immunohistochemically H. pylori negative patients., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Intestinal metaplasias (IMs) are generally regarded as pre-neoplastic gastric lesions. However, molecular alterations including genetic and epigenetic changes occurring in individual IM glands are not well defined., Aims: We sought to identify DNA methylation status, microsatellite instability (MSI) and allelic imbalance (AI) occurring in individual IM glands and non-IM glands within the same mucosa., Methods: We divided examined isolated gland obtained from GC into 4 components: isolated cancer, antral isolated intestinal metaplastic tissue, antral isolated non-metaplastic gland and isolated non-metaplastic gland derived from the greater curvature of the most distant gastric body without mucosal atrophy. We examined AI and microsatellite instability statuses using PCR-based microsatellite analysis. Next, the DNA methylation status (high methylation epigenome [HME], intermediate methylation epigenome [IME], and low methylation epigenome [LME]) was investigated. DNA methylation analysis of CDKN2A, mir34-b/c and MLHI genes was also performed., Results: Although antral isolated IM glands were characterized by IME, isolated non-IM glands showed LME. In isolated cancer glands, HME was frequently found, compared with isolated non-IM glands. DNA methylation of mir34-b/c was common in isolated cancer and IM glands, whereas DNA methylation of CDKN2A was a rare event in isolated samples. The MLH1 gene was not methylated in isolated non-IM glands. Although multiple AIs were frequently found in isolated cancer glands, a few AIs were detected in isolated IM glands., Conclusions: We suggest that the DNA methylation status and the status of the mir34-b/c gene among isolated samples of IMs and isolated non-IM glands have an impact on IM development.

In the urinary tract, there is an uncertain relationship between intestinal metaplasia (IM), primary adenocarcinoma, and urothelial carcinoma. Although IM is usually found adjacent to concurrent urothelial carcinoma or adenocarcinoma, small retrospective series have shown that most bladder biopsies with only IM do not subsequently develop cancer. However, IM with dysplasia does seem to be associated with a higher risk of concurrent malignancy or progressing to cancer. Since the molecular landscape of these lesions has remained largely unexplored, there are significant uncertainties about the oncogenic potential of IM in the bladder and urethra. This study investigated the presence of potentially oncogenic genetic variants in cases of IM with and without dysplasia. Twenty-three (23) cases of IM (3 urethra, 20 bladder) were sequenced using a solid tumor next-generation sequencing panel. Of these, five contained IM with high-grade dysplasia (including a case with paired IM-adenocarcinoma and another with paired IM-urothelial carcinoma) and 18 lacked dysplasia. Oncogenic genetic variants were found in all cases of IM with high-grade dysplasia and in five non-dysplastic IM cases, including mutations and copy number variants commonly seen in primary adenocarcinoma of the bladder and urothelial carcinoma. This study demonstrates that IM can harbor potentially oncogenic genetic variants, suggesting that it might represent a cancer precursor or a marker of increased cancer risk in a subset of cases.

Background: Gastric intestinal metaplasia (IM) is considered a precancerous lesion, and bile acids (BA) play a critical role in the induction of IM. Ectopic expression of HNF4α was observed in a BA-induced IM cell model. However, the mechanisms underlying the upregulation of the protein in IM cells remains to be elucidated., Methods: The effects of HNF4α on gastric mucosal cells in vivo were identified by a transgenic mouse model and RNA-seq was used to screen downstream targets of deoxycholic acid (DCA). The expression of pivotal molecules and miR-1 was detected by immunohistochemistry and in situ hybridization in normal, gastritis and IM tissue slides or microarrays. The transcriptional regulation of HDAC6 was investigated by chromatin immunoprecipitation (ChIP) and luciferase reporter assays., Results: The transgenic mouse model validated that HNF4α stimulated the HDAC6 expression and mucin secretion in gastric mucosa. Increased HDAC6 and HNF4α expression was also detected in the gastric IM cell model and patient specimens. HNF4α could bind to and activate HDAC6 promoter. In turn, HDAC6 enhanced the HNF4α protein level in GES-1 cells. Furthermore, miR-1 suppressed the expression of downstream intestinal markers by targeting HDAC6 and HNF4α., Conclusions: Our findings show that the HDAC6/HNF4α loop regulated by miR-1 plays a critical role in gastric IM. Blocking the activation of this loop could be a potential approach to preventing BA-induced gastric IM or even gastric cancer (GC).

Background & Aims: Chronic atrophic gastritis can lead to gastric metaplasia and increase risk of gastric adenocarcinoma. Metaplasia is a precancerous lesion associated with an increased risk for carcinogenesis, but the mechanism(s) by which inflammation induces metaplasia are poorly understood. We investigated transcriptional programs in mucous neck cells and chief cells as they progress to metaplasia mice with chronic gastritis., Methods: We analyzed previously generated single-cell RNA-sequencing (scRNA-seq) data of gastric corpus epithelium to define transcriptomes of individual epithelial cells from healthy BALB/c mice (controls) and TxA23 mice, which have chronically inflamed stomachs with metaplasia. Chronic gastritis was induced in B6 mice by Helicobacter pylori infection. Gastric tissues from mice and human patients were analyzed by immunofluorescence to verify findings at the protein level. Pseudotime trajectory analysis of scRNA-seq data was used to predict differentiation of normal gastric epithelium to metaplastic epithelium in chronically inflamed stomachs., Results: Analyses of gastric epithelial transcriptomes revealed that gastrokine 3 (Gkn3) mRNA is a specific marker of mouse gastric corpus metaplasia (spasmolytic polypeptide expressing metaplasia, SPEM). Gkn3 mRNA was undetectable in healthy gastric corpus; its expression in chronically inflamed stomachs (from TxA23 mice and mice with Helicobacter pylori infection) identified more metaplastic cells throughout the corpus than previously recognized. Staining of healthy and diseased human gastric tissue samples paralleled these results. Although mucous neck cells and chief cells from healthy stomachs each had distinct transcriptomes, in chronically inflamed stomachs, these cells had distinct transcription patterns that converged upon a pre-metaplastic pattern, which lacked the metaplasia-associated transcripts. Finally, pseudotime trajectory analysis confirmed the convergence of mucous neck cells and chief cells into a pre-metaplastic phenotype that ultimately progressed to metaplasia., Conclusions: In analyses of tissues from chronically inflamed stomachs of mice and humans, we expanded the definition of gastric metaplasia to include Gkn3 mRNA and GKN3-positive cells in the corpus, allowing a more accurate assessment of SPEM. Under conditions of chronic inflammation, chief cells and mucous neck cells are plastic and converge into a pre-metaplastic cell type that progresses to metaplasia., (Copyright © 2020 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Severe injury to the lining of the stomach leads to changes in the epithelium (reprogramming) that protect and promote repair of the tissue, including development of spasmolytic polypeptide-expressing metaplasia (SPEM) and tuft and foveolar cell hyperplasia. Acute gastric damage elicits a type-2 inflammatory response that includes production of type-2 cytokines and infiltration by eosinophils and alternatively activated macrophages. Stomachs of mice that lack interleukin 33 (IL33) or interleukin 13 (IL13) did not undergo epithelial reprogramming after drug-induced injury. We investigated the role of group 2 innate lymphoid cells (ILC2s) in gastric epithelial repair., Methods: Acute gastric injury was induced in C57BL/6J mice (wild-type and RAG1 knockout) by administration of L635. We isolated ILC2s by flow cytometry from stomachs of mice that were and were not given L635 and performed single-cell RNA sequencing. ILC2s were depleted from wild-type and RAG1-knockout mice by administration of anti-CD90.2. We assessed gastric cell lineages, markers of metaplasia, inflammation, and proliferation. Gastric tissue microarrays from patients with gastric adenocarcinoma were analyzed by immunostaining., Results: There was a significant increase in the number of GATA3-positive ILC2s in stomach tissues from wild-type mice after L635-induced damage, but not in stomach tissues from IL33-knockout mice. We characterized a marker signature of gastric mucosal ILC2s and identified a transcription profile of metaplasia-associated ILC2s, which included changes in expression of Il5, Il13, Csf2, Pd1, and Ramp3; these changes were validated by quantitative polymerase chain reaction and immunocytochemistry. Depletion of ILC2s from mice blocked development of metaplasia after L635-induced injury in wild-type and RAG1-knockout mice and prevented foveolar and tuft cell hyperplasia and infiltration or activation of macrophages after injury. Numbers of ILC2s were increased in stomach tissues from patients with SPEM compared with patients with normal corpus mucosa., Conclusions: In analyses of stomach tissues from mice with gastric tissue damage and patients with SPEM, we found evidence of type 2 inflammation and increased numbers of ILC2s. Our results suggest that ILC2s coordinate the metaplastic response to severe gastric injury., (Copyright © 2020 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: People are at a high risk of gastric cancer if their first-degree relatives suffered from atrophic gastritis (AG), intestinal metaplasia (IM), intraepithelial neoplasia (IEN), dysplasia (DYS), or gastric cancer (GC). This study was performed to analyse the association between FDR-GC and GC precursors., Methods: A cross-sectional study was performed to screen the prevalence of GC precursors from November 2016 to September 2019. A total of 1329 participants with FDR-GC, 193 participants with a family history of non-gastric cancer in FDRs (FDR-nGC), and 860 participants without a family history of cancer in FDRs (FDR-nC) were recruited in this study. The logistic regression model was used in this study., Results: The prevalence of normal, Non-AG, AG/IM, IEN/DYS, and GC was 31.91, 44.21, 13.81, 8.73, and 1.34%, respectively. The prevalence of IEN/DYS was higher in people with FDR-GC and FDR-nGC (FDR-GC: odds ratio (OR) = 1.655; 95%CI, 1.153-2.376; FDR-nGC: OR = 1.984; 95%CI, 1.122-3.506) than those with FDR-nC. The younger the age at which FDRs were diagnosed with GC, the more likely the participants were to develop AG/IM (P trend  = 0.019). The risk of precursors to GC was higher in participants whose FDR-GC was the mother than in those whose FDR-GC was the father or sibling (OR, non-AG: 1.312 vs. 1.007, 1.274; AG/IM: 1.430 vs. 1.296, 1.378; IEN/DYS: 1.988 vs. 1.573, 1.542). There was no statistically significant difference in non-AG (OR = 1.700; 95%CI, 0.940-3.074), AG/IM (OR = 1.291; 95%CI, 0.579-2.877), and IEN/DYS (OR = 1.265; 95%CI, 0.517-3.096) between participants with one or more FDR-GC., Conclusion: People with FDR-GC and FDR-nGC are at a high risk of IEN/DYS. When an FDR was diagnosed at a younger age, the risk of AG/IM was higher. The risk of GC precursors was higher in people whose FDR-GC was the mother.

Metaplastic breast cancer (MpBC) is an exceedingly rare breast cancer variant that is therapeutically challenging and aggressive. MpBC is defined by the histological presence of at least two cellular types, typically epithelial and mesenchymal components. This variant harbors a triple-negative breast cancer (TNBC) phenotype, yet has a worse prognosis and decreased survival compared to TNBC. There are currently no standardized treatment guidelines specifically for MpBC. However, prior studies have found that MpBC typically has molecular alterations in epithelial-to-mesenchymal transition, amplification of epidermal growth factor receptor, PI3K/Akt signaling, nitric oxide signaling, Wnt/β-catenin signaling, altered immune response, and cell cycle dysregulation. Some of these molecular alterations have been studied as therapeutic targets, in both the preclinical and clinical setting. This current review discusses the histological organization and cellular origins of MpBC, molecular alterations, the role of radiation therapy, and current clinical trials for MpBC.

Spasmolytic polypeptide-expressing metaplasia (SPEM) and pyloric gland adenoma (PGA) in the stomach are metaplastic and neoplastic lesions, respectively, in which gastric body glands are replaced by pyloric glands. The aim of this study was to evaluate the genomic profile of SPEM and compare it with intestinal-type gastric cancer (GC) and PGA. Thirteen gastrectomies showing PGA with or without dysplasia, GC and SPEM were retrospectively selected. MUC5AC, MUC6, gastrin, and TFF2 IHC were performed. Lesions were subjected to laser capture microdissection followed by DNA extraction. Forty-three DNA samples were extracted from PGA without cytological dysplasia, PGA with low-grade and high-grade dysplasia and pyloric gland adenocarcinoma, GC, SPEM, and adjacent normal tissue from the body of the stomach and were subjected to exome sequencing for 49 genes that are commonly dysregulated in GC. Sanger sequencing was performed for confirmation. Twenty nonsynonymous mutations were identified in SPEM, and none of these were frameshifts or indels. PGA with or without cytological dysplasia showed a significantly higher number of mutations compared with SPEM. As cytological dysplasia increased from no dysplasia to dysplasia in PGA, the percentage of frameshift mutations, indels, and missense variations increased. Further missense or frameshift mutations were observed in the KRAS, APC, TP53, and CTNNB1 genes in the PGA group. In GC, mutations were observed in the TP53 gene (p.Arg248Gln). Missense mutations in the MUC5AC, KRAS, BRAF, and EZH2 genes were common between SPEM and GC. SPEM showed fewer genomic variations than GC and PGA, and was genomically distinct from the pyloric epithelium in PGA. Stepwise progression of PGA from PGA without dysplasia to PGA with dysplasia/adenocarcinoma was associated an increase in mutations. SPEM appears to be more genomically similar to GC than PGA.

Acinar metaplasia is an initial step in a series of events that can lead to pancreatic cancer. Here we perform single-cell RNA-sequencing of mouse pancreas during the progression from preinvasive stages to tumor formation. Using a reporter gene, we identify metaplastic cells that originated from acinar cells and express two transcription factors, Onecut2 and Foxq1. Further analyses of metaplastic acinar cell heterogeneity define six acinar metaplastic cell types and states, including stomach-specific cell types. Localization of metaplastic cell types and mixture of different metaplastic cell types in the same pre-malignant lesion is shown. Finally, single-cell transcriptome analyses of tumor-associated stromal, immune, endothelial and fibroblast cells identify signals that may support tumor development, as well as the recruitment and education of immune cells. Our findings are consistent with the early, premalignant formation of an immunosuppressive environment mediated by interactions between acinar metaplastic cells and other cells in the microenvironment.

Background: Previous reports indicate that the methylenetetrahydrofolate reductase (MTHFR) 677C > T polymorphism plays a role in gastric cancer. However, whether it influences the development and progression of atrophic gastritis remains ambiguous. We aimed to determine the possible relationship between MTHFR C677T polymorphism and the severity of atrophic gastritis., Methods: A total of 128 patients without Helicobacter pylori infection were included in the study. The severity of gastric atrophy was assessed by pathological diagnosis using OLGA and OLGIM Gastritis Staging System. MTHFR 677C > T genotyping was performed by digital fluorescence molecular hybridization. Categorical variables were analyzed by percentages using the χ 2 test., Results: In this study, the TT genotype was significantly more frequent among Helicobacter pylori-negative patients aged ≤44 years (age ≤ 44 years vs. > 44 years, P = 0.039). Patients with TT genotype showed a higher ratio of incisura with atrophy or intestinal metaplasia (TT vs. CC + CT, P = 0.02). Furthermore, TT genotype was associated with more severe lesions compared with the CC + CT genotypes (TT vs. CC + CT for atrophy: P = 0.07; for intestinal metaplasia: P = 0.01; for moderate-to-severe lesions: P = 0.01). OLGA and OLGIM stages III-IV were observed more frequently in patients with TT genotype compared with CC + CT genotypes (for OLGA: P = 0.003; for OLGIM: P = 0.036)., Conclusions: The MTHFR 677C > T TT genotype showed an increased risk of moderate-to-severe lesions by OLGA and OLGIM stages, and these results indicate that MTHFR C677T polymorphism may act as a predictive marker for precancerous gastric lesions, especially in Helicobacter pylori-negative patients aged ≤44 years.

Background: Measuring the DNA methylome may offer the opportunity to identify novel disease biomarkers and insights into disease mechanisms. Although aberrant DNA methylation has been investigated in many human cancers and precancerous lesions, the DNA methylation landscape of gastric cardiac intestinal metaplasia (IM) remains unknown. Therefore, we aimed to investigate the genome-wide DNA methylation landscape and to search for potential epigenetic biomarkers of gastric cardiac IM., Methods: Histopathologic profiling was performed on a total of 118 gastric cardiac biopsies from cancer-free individuals. Genome-wide DNA methylation analysis was performed on 11 gastric cardiac mucosal biopsies (IM = 7; normal = 4) using Illumina 850K microarrays. Transcriptional relevance of any candidate epigenetic biomarker was validated by qRT-PCR., Results: The detection rate of gastric cardiac IM was 23% (27/118) in cancer-free individuals. Genome-wide DNA methylation profiling showed a global decrease in methylation in IM compared with normal tissues (median methylation = 0.64 and 0.70 for gastric cardiac IM and normal tissues, respectively). Differential methylation analysis between gastric cardiac IM and normal tissues identified 38,237 differentially methylated probes (DMPs) with a majority of sites showing hypermethylation in IM compared with normal tissues (56.3% vs. 43.7%). Subsequent analysis revealed a significant enrichment of hypermethylated DMPs in promoter and CpG islands (p < 0.001 for both, Pearson χ 2 test). For DMPs located in promoter CpG islands showing extreme hypermethylation, the candidate gene with the largest number of DMPs (n = 7) was mapped to HOXA5. Accordingly, mRNA expression of HOXA5 was significantly reduced in IM compared to normal tissue., Conclusions: Our results suggest the implication of alterations in DNA methylation in gastric cardiac IM and highlight that HOXA5 hypermethylation may be a promising epigenetic biomarker, emphasizing the role of aberrant HOXA5 expression in the pathogenesis of gastric cardiac IM.

Pancreatic ductal adenocarcinoma (PDAC) is associated with high mortality and therapy resistance. Here, we show that low expression of κB-Ras GTPases is frequently detected in PDAC and correlates with higher histologic grade. In a model of KRas G12D -driven PDAC, loss of κB-Ras accelerates tumour development and shortens median survival. κB-Ras deficiency promotes acinar-to-ductal metaplasia (ADM) during tumour initiation as well as tumour progression through intrinsic effects on proliferation and invasion. κB-Ras proteins are also required for acinar regeneration after pancreatitis, demonstrating a general role in control of plasticity. Molecularly, upregulation of Ral GTPase activity and Sox9 expression underlies the observed phenotypes, identifying a previously unrecognized function of Ral signalling in ADM. Our results provide evidence for a tumour suppressive role of κB-Ras proteins and highlight low κB-Ras levels and consequent loss of Ral control as risk factors, thus emphasizing the necessity for therapeutic options that allow interference with Ral-driven signalling.

Background: Cancer subtype switching, which involves unclear cancer cell origin, cell fate decision, and transdifferentiation of cells within a confined tumor microenvironment, remains a major problem in pancreatic cancer (PDA)., Results: By analyzing PDA subtypes in The Cancer Genome Atlas, we identified that epigenetic silencing of apoptosis-associated tyrosine kinase (AATK) inversely was correlated with mRNA expression and was enriched in the quasi-mesenchymal cancer subtype. By comparing early mouse pancreatic lesions, the non-invasive regions showed AATK co-expression in cells with acinar-to-ductal metaplasia, nuclear VAV1 localization, and cell cycle suppression; but the invasive lesions conversely revealed diminished AATK expression in those with poorly differentiated histology, cytosolic VAV1 localization, and co-expression of p63 and HNF1α. Transiently activated AATK initiates acinar differentiation into a ductal cell fate to establish apical-basal polarization in acinar-to-ductal metaplasia. Silenced AATK and ectopically expressed p63 and HNF1α allow the proliferation of ductal PanINs in mice., Conclusion: Epigenetic silencing of AATK regulates the cellular transdifferentiation, proliferation, and cell cycle progression in converting PDA-subtypes.

Background: Spasmolytic polypeptide-expressing metaplasia (SPEM) is present in more than 90% of resected gastric cancer tissues. However, although widely regarded as a pre-cancerous tissue, its genetic characteristics have not been well studied., Methods: Immunohistochemistry using Trefoil factor 2 (TFF2) antibodies was used to identify TFF2-positive SPEM cells within SPEM glands in the stomach of Helicobacter felis (H. felis) -infected mice and human clinical samples. Laser microdissection was used to isolate specific cells from both the infected mice and the human samples. The genetic instability in these cells was examined by measuring the lengths of microsatellite (MS) markers using capillary electrophoresis. Also, genome-wide genetic variations in the SPEM cells from the clinical sample was examined using deep whole-exome sequencing., Results: SPEM cells indeed exhibit not only heightened MS instability (MSI), but also genetic instabilities that extend genome-wide. Furthermore, surprisingly, we found that morphologically normal, TFF2-negative cells also contain a comparable degree of genomic instabilities as the co-resident SPEM cells within the SPEM glands., Conclusion: These results, for the first time, clearly establish elevated genetic instability as a critical property of SPEM glands, which may provide a greater possibility for malignant clone selection. More importantly, these results indicate that SPEM cells may not be the sole origin of carcinogenesis in the stomach and strongly suggest the common progenitor of these cells, the stem cells, as the source of these genetic instabilities, and thus, potential key players in carcinogenesis., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Metaplastic breast carcinoma (MBC) is a highly aggressive form of triple-negative cancer (TNBC), defined by the presence of metaplastic components of spindle, squamous, or sarcomatoid histology. The protein profiles underpinning the pathological subtypes and metastatic behavior of MBC are unknown. Using multiplex quantitative tandem mass tag-based proteomics we quantify 5798 proteins in MBC, TNBC, and normal breast from 27 patients. Comparing MBC and TNBC protein profiles we show MBC-specific increases related to epithelial-to-mesenchymal transition and extracellular matrix, and reduced metabolic pathways. MBC subtypes exhibit distinct upregulated profiles, including translation and ribosomal events in spindle, inflammation- and apical junction-related proteins in squamous, and extracellular matrix proteins in sarcomatoid subtypes. Comparison of the proteomes of human spindle MBC with mouse spindle (CCN6 knockout) MBC tumors reveals a shared spindle-specific signature of 17 upregulated proteins involved in translation and 19 downregulated proteins with roles in cell metabolism. These data identify potential subtype specific MBC biomarkers and therapeutic targets.

Background/aim: Metaplastic breast carcinoma (MBC) is a rare malignancy, which is often triple-negative for the hormone receptors and human epidermal growth factor receptor 2, and thus, does not benefit from targeted therapy. In this study, we examined the expression of methylation and demethylation enzymes by immunostaining MBC and the adjacent normal tissues or triple-negative ductal carcinoma (TNDC), and identified alterations that may be used as therapeutic targets., Materials and Methods: We retrospectively studied surgical specimens from 15 patients who underwent surgery for MBC at Kanagawa Cancer Center between 2005 and 2016, and similarly from 14 patients with TNDC. The frequencies of high methylation/demethylation enzyme expression were compared among them., Results: The frequencies of high enhancer of zeste homolog 2 (EZH2) and multiple myeloma SET domain (MMSET) expression were significantly higher in both MBC and TNDC than in normal tissue., Conclusion: EZH2 and MMSET may be useful therapeutic targets in MBC., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background & Aims: E-cadherin (Cdh1) is a key molecule for adherence required for maintenance of structural homeostasis. Loss of E-cadherin leads to poor prognosis and the development of resistance to chemotherapy in pancreatic cancer. Here, we evaluated the physiological and pathologic roles of E-cadherin in the pancreas., Methods: We crossbred Ptf1a-Cre mice with Cdh1 f /f mice to examine the physiological roles of E-cadherin in the pancreas. In addition, we crossbred these mice with LSL-Kras G12D/+ mice (PKC) to investigate the pathologic roles of E-cadherin. We also generated a tamoxifen-inducible system (Ptf1a-Cre ERT model). Organoids derived from these models using lentiviral transduction were analyzed for immunohistochemical features. Established cell lines from these organoids were analyzed for migratory and invasive activities as well as gene expression by complementary DNA microarray analyses., Results: None of the Ptf1a-Cre mice crossbred with Cdh1 f/f mice survived for more than 28 days. We observed aberrant epithelial tubules that resembled the structure of acinar-to-ductal metaplasia after postnatal day 6, showing features of pancreatitis. All of the PKC mice died within 10 days. We observed tumorigenicity with increasing stroma-like aggressive tumors. Ptf1a-Cre ERT models showed that deletion of E-cadherin led to earlier pancreatic intraepithelial neoplasm formation. Cells established from PKC organoids had greater migratory and invasive activities, and these allograft tumors showed a poorly differentiated phenotype. Gene expression analysis indicated that Hdac1 was up-regulated in PKC cell lines and a histone deacetylase 1 inhibitor suppressed PKC cell proliferation., Conclusions: Under physiological conditions, E-cadherin is important for maintaining the tissue homeostasis of the pancreas. Under pathologic conditions with mutational Kras activation, E-cadherin plays an important role in tumor formation via the acquisition of tumorigenic activity., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Gastric chief cells differentiate from mucous neck cells and develop their mature state at the base of oxyntic glands with expression of secretory zymogen granules. After parietal cell loss, chief cells transdifferentiate into mucous cell metaplasia, designated spasmolytic polypeptide-expressing metaplasia (SPEM), which is considered a candidate precursor of gastric cancer. We examined the range of microRNA (miRNA) expression in chief cells and identified miRNAs involved in chief cell transdifferentiation into SPEM. Among them, miR-148a was strongly and specifically expressed in chief cells and significantly decreased during the process of chief cell transdifferentiation. Interestingly, suppression of miR-148a in a conditionally immortalized chief cell line induced up-regulation of CD44 variant 9 (CD44v9), one of the transcripts expressed at an early stage of SPEM development, and DNA methyltransferase 1 (Dnmt1), an established target of miR-148a. Immunostaining analyses showed that Dnmt1 was up-regulated in SPEM cells as well as in chief cells before the emergence of SPEM in mouse models of acute oxyntic atrophy using either DMP-777 or L635. In the cascade of events that leads to transdifferentiation, miR-148a was down-regulated after acute oxyntic atrophy either in xCT knockout mice or after sulfasalazine inhibition of xCT. These findings suggest that the alteration of miR-148a expression is an early event in the process of chief cell transdifferentiation into SPEM., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Gastric carcinoma is related mostly to CagA+-Helicobacter pylori infection, which disrupts the gastric mucosa turnover and elicits an epithelial-mesenchymal transition (EMT) and preneoplastic transdifferentiation. The tumor suppressor Hippo pathway controls stem cell homeostasis; its core, constituted by the large tumor suppressor 2 (LATS2) kinase and its substrate Yes-associated protein 1 (YAP1), was investigated in this context., Methods: Hippo, EMT, and intestinal metaplasia marker expression were investigated by transcriptomic and immunostaining analyses in human gastric AGS and MKN74 and nongastric immortalized RPE1 and HMLE epithelial cell lines challenged by H pylori, and on gastric tissues of infected patients and mice. LATS2 and YAP1 were silenced using small interfering RNAs. A transcriptional enhanced associated domain (TEAD) reporter assay was used. Cell proliferation and invasion were evaluated., Results: LATS2 and YAP1 appear co-overexpressed in the infected mucosa, especially in gastritis and intestinal metaplasia. H pylori via CagA stimulates LATS2 and YAP1 in a coordinated biphasic pattern, characterized by an early transient YAP1 nuclear accumulation and stimulated YAP1/TEAD transcription, followed by nuclear LATS2 up-regulation leading to YAP1 phosphorylation and targeting for degradation. LATS2 and YAP1 reciprocally positively regulate each other's expression. Loss-of-function experiments showed that LATS2 restricts H pylori-induced EMT marker expression, invasion, and intestinal metaplasia, supporting a role of LATS2 in maintaining the epithelial phenotype of gastric cells and constraining H pylori-induced preneoplastic changes., Conclusions: H pylori infection engages a number of signaling cascades that alienate mucosa homeostasis, including the Hippo LATS2/YAP1/TEAD pathway. In the host-pathogen conflict, which generates an inflammatory environment and perturbations of the epithelial turnover and differentiation, Hippo signaling appears as a protective pathway, limiting the loss of gastric epithelial cell identity that precedes gastric carcinoma development., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Context—: Metaplastic carcinoma is a rare, triple-negative carcinoma of the breast that exhibits transformation of part or all of its glandular carcinomatous component into a nonglandular, or metaplastic, component. The World Health Organization currently recognizes 5 variants of metaplastic carcinoma based on their histologic appearance., Objective—: To review the histologic classifications, differential diagnosis, prognosis, and recent laboratory studies of metaplastic breast carcinoma., Data Sources.—: We reviewed recently published studies that collectively examine metaplastic carcinomas, including results from our own research., Conclusions.—: Metaplastic breast carcinoma has a broad spectrum of histologic patterns, often leading to a broad differential diagnosis. Diagnosis can typically be rendered by a combination of morphology and immunohistochemical staining for high-molecular-weight cytokeratins and p63. Recent studies elucidate new genes and pathways involved in the pathogenesis of metaplastic carcinoma, including the downregulation of CCN6 and WNT pathway gene mutations, and provide a novel MMTV-Cre; Ccn6 fl/fl knockout disease-relevant mouse model to test new therapies.

The Cdx2 homeobox gene is important in assigning positional identity during the finely orchestrated process of embryogenesis. In adults, regenerative responses to tissues damage can require a replay of these same developmental pathways. Errors in reassigning positional identity during regeneration can cause metaplasias-normal tissue arising in an abnormal location-and this in turn, is a well-recognized cancer risk factor. In animal models, a gain of Cdx2 function can elicit a posterior shift in tissue identity, modeling intestinal-type metaplasias of the esophagus (Barrett's esophagus) and stomach. Conversely, loss of Cdx2 function can elicit an anterior shift in tissue identity, inducing serrated-type lesions expressing gastric markers in the colon. These metaplasias are major risk factors for the later development of esophageal, stomach and colon cancer. Leukemia, another cancer in which Cdx2 is ectopically expressed, may have mechanistic parallels with epithelial cancers in terms of stress-induced reprogramming. This review will address how animal models have refined our understanding of the role of Cdx2 in these common human cancers.

The five-year survival rate of patients diagnosed with advanced pancreatic ductal adenocarcinoma (PDAC) has remained static at <5% despite decades of research. With the exception of erlotinib, clinical trials have failed to demonstrate the benefit of any targeted therapy for PDAC despite promising results in preclinical animal studies. The development of more refined mouse models of PDAC which recapitulate the carcinogenic progression from non-neoplastic, adult exocrine subsets of pancreatic cells to invasive carcinoma in humans are needed to facilitate the accurate translation of therapies to the clinic. To study acinar cell-derived PDAC initiation, we developed a genetically engineered mouse model of PDAC, called KPT, utilizing a tamoxifen-inducible Cre recombinase/estrogen receptor (ESR1) fusion protein knocked into the Ptf1a locus to activate the expression of oncogenic KrasG12D and Trp53R270H alleles in mature pancreatic acinar cells. Oncogene-expressing acinar cells underwent acinar-to-ductal metaplasia, and formed pancreatic intraepithelial neoplasia lesions following the induction of oncogene expression. After a defined latency period, oncogene-expressing acinar cells initiated the formation of highly differentiated and fibrotic tumors, which metastasized to the lungs and liver. Whole-transcriptome analysis of microdissected regions of acinar-to-ductal metaplasia and histological validation experiments demonstrated that regions of acinar-to-ductal metaplasia are characterized by the deposition of the extracellular matrix component hyaluronan. These results indicate that acinar cells expressing KrasG12D and Trp53R270H can initiate PDAC development in young adult mice and implicate hyaluronan deposition in the formation of the earliest characterized PDAC precursor lesions (and the progression of pancreatic cancer). Further studies are necessary to provide a comprehensive characterization of PDAC progression and treatment response in KPT mice and to investigate whether the KPT model could be used as a tool to study translational aspects of acinar cell-derived PDAC tumorigenesis., Competing Interests: The authors have declared that no competing interests exist.

Activating mutations and amplification of Kras and, more frequently, signatures for Kras activation are noted in stomach cancer. Expression of mutant Kras G12D in the mouse gastric mucosa has been shown to induce hyperplasia and metaplasia. However, the mechanisms by which Kras activation leads to gastric metaplasia are not fully understood. Here we report that Kras LSL-G12D/+ ;Pdx1-cre, a mouse model known for pancreatic cancer, also mediates Kras G12D expression in the stomach, causing gastric hyperplasia and metaplasia prior to the pathologic changes in the pancreas. These mice exhibit ectopic cell proliferation at the base of gastric glands, whereas wild-type mice contain proliferating cells primarily at the isthmus/neck of the gastric glands. Notch signaling is decreased in the Kras LSL-G12D/+ ;Pdx1-cre gastric mucosa, as shown by lower levels of cleaved Notch intracellular domains and downregulation of Notch downstream target genes. Expression of a Notch ligand Jagged1 is downregulated at the base of the mutant gland, accompanied by loss of chief cell marker Mist1. We demonstrate that exogenous Jagged1 or overexpression of Notch intracellular domain stimulates Mist1 expression in gastric cancer cell lines, suggesting positive regulation of Mist1 by Notch signaling. Finally, deletion of Jagged1 or Notch3 in Kras LSL-G12D/+ ;Pdx1-cre mice promoted development of squamous cell carcinoma in the forestomach, albeit short of invasive adenocarcinoma in the glandular stomach. Taken together, these results reveal downregulation of Notch signaling and Mist1 expression during the initiation of Kras-driven gastric tumorigenesis and suggest a tumor-suppressive role for Notch in this context., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Most pancreatic ductal adenocarcinoma (PDAC) develops from pancreatic epithelial cells bearing activating mutant KRAS genes through precancerous lesions, i.e. acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN). During pancreatic tumorigenesis, Hes1 expression starts with the transition from acinar cells to ADM, and continues during PanIN and PDAC formation, but the role of Hes1 in pancreatic tumorigenesis is not fully elucidated. Here we show that Hes1 plays an essential role in the initiation and progression of KRAS-driven pancreatic tumorigenesis. In vitro, activation of MAPK signaling due to EGF or mutant KRAS activation induced sustained Hes1 expression in pancreatic acinar cells. In vivo, acinar cell-specific activation of mutant KRAS by Elastase1-CreERT2;Kras G12D induced ADM/PanIN formation with Hes1 expression in mice, and genetic ablation of Hes1 in these mice dramatically suppressed PanIN formation. Gene expression analysis and lineage tracing revealed that Hes1 regulates acinar-to-ductal reprogramming-related genes and, in a Hes1-deficient state, mutant Kras-induced ADM could not progress into PanIN, but re-differentiated into acinar cells. In the Elastase1-CreERT2;Kras G12D ;Trp53 R172H mouse PDAC model, genetic ablation of Hes1 completely blocked PDAC formation by keeping PanIN lesions in low-grade conditions, in addition to reducing the occurrence of PanIN. Together, these findings indicate that mutant KRAS-induced Hes1 plays an essential role in PDAC initiation and progression by regulating acinar-to-ductal reprogramming-related genes.

Background & Aims: Most patients with esophageal adenocarcinoma (EAC) present with de novo tumors. Although this could be due to inadequate screening strategies, the precise reason for this observation is not clear. We compared survival of patients with prevalent EAC with and without synchronous Barrett esophagus (BE) with intestinal metaplasia (IM) at the time of EAC diagnosis., Methods: Clinical data were studied using Cox proportional hazards regression to evaluate the effect of synchronous BE-IM on EAC survival independent of age, sex, TNM stage, and tumor location. We analyzed data from a cohort of patients with EAC from the Mayo Clinic (n=411; 203 with BE and IM) and a multicenter cohort from the United Kingdom (n=1417; 638 with BE and IM)., Results: In the Mayo cohort, BE with IM had a reduced risk of death compared to patients without BE and IM (hazard ratio [HR] 0.44; 95% CI, 0.34-0.57; P<.001). In a multivariable analysis, BE with IM was associated with longer survival independent of patient age or sex, tumor stage or location, and BE length (adjusted HR, 0.66; 95% CI, 0.5-0.88; P=.005). In the United Kingdom cohort, patients BE and IM had a reduced risk of death compared with those without (HR, 0.59; 95% CI, 0.5-0.69; P<.001), with continued significance in multivariable analysis that included patient age and sex and tumor stage and tumor location (adjusted HR, 0.77; 95% CI, 0.64-0.93; P=.006)., Conclusion: Two types of EAC can be characterized based on the presence or absence of BE. These findings could increase our understanding the etiology of EAC, and be used in management and prognosis of patients., (Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Leucine-rich repeats and immunoglobulin-like domains (LRIG)-1 is a transmembrane protein that antagonizes epidermal growth factor receptor signaling in epithelial tissues. LRIG1 is down-regulated in various epithelial cancers, including bladder, breast, and colorectal cancer, suggesting that it functions as a tumor suppressor. However, its role in gastric carcinogenesis is not well understood. Here, we investigated the changes in LRIG1 expression during the stages of gastric cancer. We used a DMP-777-induced spasmolytic polypeptide-expressing metaplasia mouse model and a tissue array of human gastric cancer lesions. The effects of LRIG1 knockdown were also assessed using the human gastric cancer cell line SNU638 in a xenograft model. LRIG1 expression varied over the course of gastric carcinogenesis, increasing in spasmolytic polypeptide-expressing metaplasia lesions but disappearing in intestinal metaplasia and cancer lesions, and the increase was concurrent with the up-regulation of epidermal growth factor receptor. In addition, LRIG1 knockdown promoted the tumorigenic potential in vitro, which was manifested as increased proliferation, invasiveness, and migration as well as increased tumor size in vivo in the xenograft model. Furthermore, LRIG1 expression was determined to be a positive prognostic biomarker for the survival of gastric cancer patients. Collectively, our findings indicate that LRIG1 expression is closely related wto gastric carcinogenesis and may play a vital role as a tumor suppressor through the modulation of epidermal growth factor receptor activity., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)