There are two types of pyloric gland-like metaplasia in the corpus of stomach: pyloric and pseudopyloric metaplasias. They show the same morphology as the original pyloric glands in H&E staining. Pseudopyloric metaplasia is positive for pepsinogen (PG) I immunohistochemically, whereas pyloric metaplasia is negative. Recently, spasmolytic polypeptide-expressing metaplasia (SPEM) is proposed for pyloric gland-like metaplasia mainly in animal experiments. SPEM expresses trefoil factor family 2 (TFF2) and is often considered synonymous with pseudopyloric metaplasia. We reviewed consecutive 22 Japanese patients with autoimmune gastritis (AIG) to investigate TFF2 expression in pyloric and pseudopyloric metaplasias by counting all pyloric gland-like glands in biopsy specimens taken from greater curvature of the middle corpus according to the Updated Sydney System. Pyloric metaplasia was seen in all the 22 cases, and pseudopyloric metaplasia was found in 15 cases. Of 1567 pyloric gland-like glands in all the cases, 1381 (88.1%) glands were pyloric metaplasia glands, and the remaining 186 (11.9%) glands were pseudopyloric metaplasia glands. TFF2 expression was observed in pyloric or pseudopyloric metaplasia glands in 20 cases. TFF2 expression was recognized in 409 of 1381 (26.9%) pyloric metaplasia glands and 27 of 186 (14.5%) pseudopyloric metaplasia glands ( P <0.01, chi-square test). In conclusion, SPEM was not always the same as pseudopyloric metaplasia in human AIG, and the majority of metaplasia in AIG was not pseudopyloric but pyloric metaplasia.

We previously reported the development of pancreatic acinar cell metaplasia (PACM) in the glandular stomach of a duodenal contents reflux model (reflux model). We aimed to investigate the characteristics and histogenesis of PACM using a reflux model. A reflux model was created using 8-week-old male Wistar rats, which were killed up to 30 weeks postoperatively. Histological examination was performed to analyze the glandular stomach–jejunal anastomosis. Furthermore, electron microscopic images of PACM samples were compared with pancreatic and gastric glands removed from rats that had not undergone surgery. Immunostaining for α-amylase, HIK1083, TFF2, and Ki-67 was performed, and double fluorescent staining was carried out using antibodies against α-amylase and HIK1083, or α-amylase and TFF2. In all reflux model rats, PACM was observed proximal to the glandular stomach–jejunal anastomosis, surrounded by pseudopyloric metaplasia. The number of chief cells was decreased in the deep part of the gland, where PACM occurred. Electron microscopy showed that PACM cells had greater numbers of rough endoplasmic reticulum tubules than chief cells, and exhibited pancreatic acinar cell morphology. Upon immunochemical staining, the regenerative foveolar epithelium and part of the pseudopyloric glands stained strongly positive for TFF2, whereas PACM cells were only weakly positive. Double fluorescent staining identified early lesions of PACM in the neck, which were double positive for α-amylase and TFF2, but negative for HIK1083. PACM could be induced by duodenal contents reflux. PACM originates from stem cells located in the neck of oxyntic glands during gastric mucosal regeneration.

Abstract Background Ezrin, ERK, STAT3, and AKT are proteins that are overexpressed in various types of cancer, although their expressions in tongue cancer has received less focus. This study aimed to address associations between the expression levels of these proteins and with characteristics of the tumor and patient survival. Methods We performed immunohistochemical staining of ezrin, ERK, STAT3, and AKT in tumors from patients with tongue carcinoma in situ (CIS, n = 17) and tongue squamous cell carcinoma (SCC, n = 46). Statistical differences between the SCC versus the CIS cohorts were estimated by calculations of bivariate odds ratios of low versus high expression of the proteins. Fisher's exact tests were used to appraise interassociations between the proteins, as well as expression levels versus patient and tumor characteristics. Survival based on Kaplan–Meier statistics in combination log‐rank tests were used to address potential effects of the patient and tumor characteristics versus 5‐year survival rate. Results The relative high: low expression of all four proteins in the two cohorts differed, and particularly ERK was markedly overexpressed in the SCC versus the CIS cohort (odds ratio = 45.3, p < .01). The relative high: low expression each protein versus patient and tumor characteristics; showed associations between AKT expression and T stage (p = .002) plus node metastases (p = .12), and between ERK expression and drinking (p = .01) and smoking history (p = .01). There was no significant difference observed between ERK and the three other molecules, nor any significant difference between the degree of expression of each protein and the 5‐year disease‐specific survival rate. Conclusion Ezrin, ERK, STAT3, and AKT appear to be involved in the progress from carcinoma in situ in the tongue into squamous cell carcinoma. ERK in particular is overexpressed, suggesting that ERK may be a novel therapeutic target for preventing tongue cancer.

Metabolic programming of cancer cells is an essential step in transformation and tumor growth. We established two-dimensional (2D) monolayer and three-dimensional (3D) cultures, the latter called a "tissueoid cell culture system", using four types of tongue cancer cell lines. We also undertook a comprehensive metabolome analysis of three groups that included xenografts created by transplanting the cell lines into nude mice. In addition, we undertook a functional analysis of the mitochondria, which plays a key role in cancer metabolism. Principal component analysis revealed the plots of the four cell lines to be much narrower in 2D culture than in 3D culture and xenograft groups. Moreover, compared to xenografts, the 2D culture had significantly lower levels of most metabolites. These results suggest that the unique characteristics of each cell disappeared in 2D culture, and a type of metabolism unique to monolayer culture took over. Conversely, ATP production, biomass synthesis, and maintenance of redox balance were shown in 3D culture using sufficient nutrients, which closely resembled the metabolic activity in the xenografts. However, there were several differences between the metabolic activity in the 3D culture and xenografts. In vivo, the cancer tissue had blood flow with stromal cells present around the cancer cells. In the xenografts, we detected metabolized and degraded products in the liver and other organs of the host mice. Furthermore, the 3D system did not show impairment of mitochondrial function in the cancer cells, suggesting that cancer cells produce energy simultaneously through mitochondria, as well as aerobic glycolysis.

BACKGROUND:We developed a 3-dimensional (3D) culture system using a high-purity silica fiber scaffold of unwoven sheets called CellbedTM.METHODS:We used adherent colon and esophagogastric junction adenocarcinoma cells, tongue squamous cell carcinoma (SqCC) cells, and nonadherent gastric cancer cells. These cells were subjected to staining with various substances and observed by electron microscopy. To evaluate the effects of extracellular matrix in carcinoma tissues, SqCC cells were cultured in Cellbed coated with collagens I, III, and IV.RESULTS:Especially well-differentiated carcinoma cells cultured in this 3D system showed their own unique characteristics: luminal formation in adenocarcinoma cells and cell stratification and keratinization in SqCC cells. Scanning electron microscopy revealed the proliferation of cancer cells with cytoplasm entwined in Cellbed. Intercellular desmosomes in squamous epithelia were detected by transmission electron microscopy of vertical cross sections. SqCC cells cultured in Cellbed coated with collagen IV showed enhanced invasive and proliferative abilities.CONCLUSION:Because the morphology of cancer cells cultured in this 3D culture system is similar to that in living organisms, we called the system a "tissueoid cell culture system." Coating with collagen IV enables the modification of cell-matrix interactions as well as recapitulation of the in vivo microenvironment.

To evaluate the usefulness of forensic autopsies in determining latent prostate cancer (PC) prevalence, we examined latent PC prevalence from autopsies and compared our findings between decedents with and without cancer. Data from forensic autopsies performed in Japan from 2004 to 2014 were obtained. For each prostate, histopathological examinations were performed in both the base and the apex sections. Three hundred and seventeen Japanese decedents were selected for analysis. The mean age of decedents was 56.4 ± 17.8 years (range, 14–94 years). Among this population, 39.4% died suddenly of disease and 60.6% died of external causes. Latent PC was identified in 45 (14.2%) decedents, who ranged from 27 to 93 years old (mean, 71.1 ± 12.9 years). The prevalence of clinically significant PC with a Gleason score of 7 or more was 8.8%, and the rate increased with age. Fifteen males had cancers other than PC. The prevalence of overall latent PC was significantly higher for those with cancer compared with those without (40.0% vs. 12.9%; P = 0.003). In this study, the use of forensic autopsy materials provided the opportunity to obtain a more accurate natural history of PC, as the decedents in this situation would have been more likely to have died suddenly while behaving as normal prior to death, and less likely to have been impacted by long‐term medical interventions.

Esophageal squamous cell carcinomas (ESCCs) as well as adenocarcinomas (EACs) were developed in rat duodenal contents reflux models (reflux model). The present study aimed to shed light on the mechanism by which bile acid stimulation causes cancer onset and progression. Metabolomics analyses were performed on samples of neoplastic and nonneoplastic tissues from reflux models, and K14D, cultivated from a nonmetastatic, primary ESCC, and ESCC-DR, established from a metastatic thoracic lesion. ESCC-DRtca2M was prepared by treating ESCC-DR cells with taurocholic acid (TCA) to accelerate cancer progression. The lines were subjected to comprehensive genomic analyses. In addition, protein expression levels of glucose-6-phosphate dehydrogenase (G6PD), nuclear factor kappa B (NF-κB) (p65) and O-linked N-Acetylglucosamine (O-GlcNAc) were compared among lines. Cancers developed in the reflux models exhibited greater hexosamine biosynthesis pathway (HBP) activation compared with the nonneoplastic tissues. Expression of O-GlcNAc transferase (OGT) increased considerably in both ESCC and EAC compared with nonneoplastic squamous epithelium. Conversely, cell line-based experiments revealed the greater activation of the pentose phosphate pathway (PPP) at higher degrees of malignancy. G6PD overexpression in response to TCA exposure was observed. Both NF-κB (p65) and O-GlcNAc were expressed more highly in ESCC-DRtca2M than in the other cell lines. Moreover, ESCC-DRtca2M cells had additional chromosomal abnormalities in excess of ESCC-DR cells. Overall, glucose metabolism was upregulated in both esophageal cancer tissue and cell lines. While bile acids are not mutagenic, chronic exposure seems to trigger NF-κB(p65) activation, potentially inducing genetic mutations as well as facilitating carcinogenesis and cancer progression. Glucose metabolism was upregulated in both esophageal cancer tissue and cell lines, and the HBP was activated in the former. The cell line-based experiments demonstrated upregulation of the pentose phosphate pathway (PPP) at higher degrees of malignancy. While bile acids are not mutagenic, chronic exposure seems to trigger G6PD overexpression and NF-κB (p65) activation, potentially inducing genetic mutations as well as facilitating carcinogenesis and cancer progression.

AimsThe aim of this study was to clarify the histopathological features of fundic gland polyps (FGPs) in patients treated with proton pump inhibitors (PPIs) and to investigate the mechanism of enlargement of FGPs after PPI treatment.Methods and resultsA total of 196 biopsy specimens of FGPs, which consisted of 87 FGPs in patients treated with PPIs (PPI group) and 109 FGPs in patients treated without PPIs (non‐PPI group) were compared histologically using haematoxylin and eosin staining, Ki67 immunohistochemistry and multiplex immunohistochemical stain with Ki67, MUC5AC and MUC6. The significant histological features of FGPs in the PPI group were: larger size of dilated fundic gland cysts, larger number of foveolar and mixture type fundic gland cysts, foveolar cell hyperplasia, parietal cell protrusion, mononuclear cell infiltration and a higher percentage of Ki67‐positive cells in the deeper layers of the glands. Multiplex immunohistochemical stain showed that Ki67‐positive cells were also positive for MUC5AC, and the Ki67‐positive rate was significantly higher in MUC5AC‐positive cells of the PPI group than of the non‐PPI group. Gene mutations of β‐catenin were found in only 9.7% of FGPs in the PPI group.ConclusionsEnlargement of fundic gland cysts due to foveolar cell proliferation and parietal cell protrusion might promote the enlargement of FGPs in patients treated with PPIs. β‐catenin gene mutations might not be associated with these histological changes of FGPs after PPI treatment.

Epidemiological studies suggest that poor nutrition during pregnancy influences offspring predisposition to experience developmental and psychiatric disorders. Animal studies have shown that maternal undernutrition leads to behavioral impairment, which is linked to alterations in monoaminergic systems and inflammation in the brain. In this study, we focused on the ethanolamine plasmalogen of the brain as a possible contributor to behavioral disturbances observed in offspring exposed to maternal undernutrition. Maternal food or protein restriction between gestational day (GD) 5.5 and GD 10.5 resulted in hyperactivity of rat male adult offspring. Genes related to the phospholipid biosynthesis were found to be activated in the PFC, but not in the NAcc or striatum, in the offspring exposed to prenatal undernutrition. Corresponding to these gene activations, increased ethanolamine plasmalogen (18:0p-22:6) was observed in the PFC using mass spectrometry imaging. A high number of crossings and the long time spent in the center area were observed in the offspring exposed to prenatal undernutrition and were mimicked in adult rats via the intravenous injection of ethanolamine plasmalogen (18:0p-22:6) incorporated into the liposome. Additionally, plasmalogen (18:0p-22:6) increased only in the PFC, and not in the NAcc or striatum. These results suggest that brain plasmalogen is one of the key molecules to control behavior, and its injection using liposome is a potential therapeutic approach for cognitive impairment. SIGNIFICANCE STATEMENT Maternal undernutrition correlates to developmental and psychiatric disorders. Here, we found that maternal undernutrition in early pregnancy led to hyperactivity in rat male offspring and induced gene activation of phospholipid-synthesizing enzyme and elevation of ethanolamine plasmalogen (18:0p-22:6) level in the PFC. Intravenous injection of ethanolamine plasmalogen (18:0p-22:6) incorporated into the liposome maintained crossing activity and the activity was circumscribed to the center area for a long time period, as in prenatally undernourished offspring with aberrant behavior. Furthermore, the amount of ethanolamine plasmalogen (18:0p-22:6) increased in the PFC of the rat after injection. Our result suggests that brain plasmalogen is one of the key molecules to control behavior and that its injection using liposome is a potential therapeutic approach for cognitive impairment.

Barrett's esophagus is considered a precancerous lesion of esophageal adenocarcinoma (EAC). Long‐segment Barrett's esophagus, which is generally associated with intestinal metaplasia, has a higher rate of carcinogenesis than short‐segment Barrett's esophagus, which is mainly composed of cardiac‐type mucosa. However, a large number of cases reportedly develop EAC from the cardiac‐type mucosa which has the potential to involve intestinal phenotypes. There is no consensus regarding whether the definition of Barrett's epithelium should include intestinal metaplasia. Basic researches using rodent models have provided information regarding the origins of Barrett's epithelium. Nevertheless, it remains unclear whether differentiated gastric columnar epithelium or stratified esophageal squamous epithelium undergo transdifferentiation into the intestinal‐type columnar epithelium, transcommittment into the columnar epithelium, or whether the other pathways exist. Reflux of duodenal fluid including bile acids into the stomach may occur when an individual lies down after eating, which could cause the digestive juices to collect in the fornix of the stomach. N‐nitroso‐bile acids are produced with nitrites that are secreted from the salivary glands, and bile acids can drive expression of pro‐inflammatory cytokines via EGFR or the NF‐κB pathway. These steps may contribute significantly to carcinogenesis.

Background Rat gastroduodenal reflux models have been used for analyzing Barrett’s carcinogenesis. Mice seem to be more useful than rats for studies targeting genes. Methods We induced gastroduodenal contents reflux by esophagojejunostomy using C57BL/6J mice. Mice were divided into a standard diet and high-fat diet groups and kept for 60 weeks. Bile was sampled from the gallbladder to analyze bile acid fractions, and the esophagus was removed for a histological investigation. Human esophagogastric junction adenocarcinoma cells (OE19) were exposed to taurocholic acid (TCA), after which cell proliferative activity was measured. Rat esophageal cancer cell lines, ESCC-DR and ESCC-DRtca with higher malignant potential induced by continuous TCA exposure, were used to perform comprehensive genetic analysis (CGH). Results Barrett’s epithelium onset occurred in all mice, and no differences in histological changes were noted between the standard diet and high-fat diet groups. However, no development of adenocarcinoma was noted. Most of the mouse bile acid was taurine conjugates. In the experiment using OE-19 cells, TCA promotes cell proliferation in a dose-dependent manner. Array CGH analysis revealed a large number of chromosomal abnormalities in the ESCC-DR, in addition to genetic abnormalities such as in the UGT2B gene, the substrate of which is bile acid. TCA administration resulted in more chromosomal abnormalities being detected. Conclusions We showed the effects of TCA in cancer progression in vitro. However, Barrett’s adenocarcinoma onset rates differ between mice and rats despite undergoing similar reflux stimulation including taurine-conjugated bile acids being detected in mouse bile juice. These results suggest that host factors seem to influence Barrett’s carcinogenesis.

BACKGROUND:To examine whether gastric carcinoma (GC) with chromosomal instability (CIN-type GC), the largest category in the Cancer Genome Atlas classification, consists of a single genetic lineage, we conducted a multisampling analysis of genomic DNA copy-number profile. METHODS:We performed array-based comparative genomic hybridization using formalin-fixed paraffin-embedded tissues from 54 gland-forming GCs containing a total of 106 DNA samples from mucosal, extramucosal invasive, and lymph node lesions. Microarray data were analyzed by unsupervised hierarchical clustering and penetrance plots. Epstein-Barr virus infection status and mismatch repair (MMR) enzyme-silencing/p53/mucin expression were examined by in situ hybridization and immunohistochemistry, respectively. RESULTS:The samples examined were divided into gain-rich cluster A and loss-rich cluster B, which were different in tumor locus and patient age. The T1/T2-4 ratio, the frequency of small cancers (diameter ≤2-4 cm), and intestinal mucin expression were higher in cluster B than in cluster A, but there were no significant differences in the frequencies of MMR silencing, mutant p53 pattern, and lymph node metastasis between the 2 clusters. CONCLUSIONS:We demonstrated that CIN-type GC could be categorized into 2 genetic lineages which are different in terms of rapidity of local extension but similar in terms of nodal metastasis risk.

Phosphorylation of pyruvate dehydrogenase by pyruvate dehydrogenase kinase 4 (PDK4) 4 inhibits its ability to induce a glycolytic shift. PDK4 expression is frequently upregulated in various cancer tissues, with its elevation being critical for the induction of the Warburg effect. PDK4 is an attractive target for cancer therapy given its effect on shifting glucose metabolism. Previous research has highlighted the necessity of identifying a potent compound to suppress PDK4 activity at the submicromolar concentrations. Here we identified natural diterpene quinones (KIS compounds) that inhibit PDK4 at low micromolar concentrations. KIS37 (cryptotanshinone) inhibited anchorage-independent growth in three-dimensional spheroid and soft agar colony formation assays of KRAS-activated human pancreatic (MIAPaCa-2 and Panc-1) and colorectal (DLD-1 and HCT116) cancer cell lines. KIS37 also suppressed KRAS protein expression in such cell lines. Furthermore, KIS37 suppressed phosphorylation of Rb protein and cyclin D1 protein expression via the PI3K-Akt-mTOR signaling pathway under nonadherent culture conditions and suppressed the expression of cancer stem cell markers CD44, EpCAM, and ALDH1A1 in MIAPaCa-2 cells. KIS37 also suppressed pancreatic cancer cell growth in both subcutaneous xenograft and orthotopic pancreatic tumor models in nude mice at 40 mg/kg (intraperitoneal dose) without any evident toxicity. Reduced ALDH1A1 expression was observed in KIS37-treated pancreatic tumors, suggesting that cancer cell stemness was also suppressed in the orthotopic tumor model. The aforementioned results indicate that KIS37 administration is a novel therapeutic strategy for targeting PDK4 in KRAS-activated intractable human pancreatic cancer.

BACKGROUND:Ductal carcinoma in situ (DCIS) of the breast is heterogeneous in terms of the risk of progression to invasive ductal carcinoma (IDC). To treat DCIS appropriately for its progression risk, we classified individual DCIS by its profile of genomic changes into 2 groups and correlated them with clinicopathological progression factors.METHODS:We used surgically resected, formalin-fixed, paraffin-embedded tissues of 22 DCIS and 30 IDC lesions. We performed immunohistochemical intrinsic subtyping, array-based comparative genomic hybridization, and unsupervised clustering.RESULTS:The samples were divided into 2 major clusters, A and B. Cluster A showed a greater number of gene and chromosome copy number alterations, a larger IDC/DCIS ratio, a higher frequency of nonluminal subtype, a lower frequency of luminal subtype, and a higher nuclear grade, when compared with cluster B. However, there was no difference in the frequencies of lymph node metastasis between clusters A and B. We identified 9 breast-cancer-related genes, including TP53 and GATA3, that highly contributed to the discrimination of A and B clusters.CONCLUSION:Classification of breast tumors into rapidly progressive cluster A and the other (cluster B) may contribute to select the treatment appropriate for their progression risk.